Journal of Endodontics - PDFCOFFEE.COM (2024)

EDITOR Kenneth M. Hargreaves

ASSOCIATE EDITORS Anita Aminoshariae Amir Azarpazhooh Anibal R. Diogenes Ashraf F. Fouad Gerald N. Glickman Anil Kishen Ariadne M. Letra Linda Levin Robert S. Roda Frank C. Setzer Franklin R. Tay

AMERICAN ASSOCIATION OF ENDODONTISTS BOARD OF DIRECTORS President Alan S. Law President-Elect Stefan I. Zweig Vice President Craig S. Hirschberg Secretary Natasha M. Flake

You can now access the Journal of Endodontics online through the AAE Web site at www.aae.org. The site features: Online Table of Contents This link will take you to the table of contents for the most current issue of the JOE. Interested in what’s coming in the next issue? This site will have it first. Instructions for Authors Review an electronic version of the JOE instructions to authors. Guidelines for Publishing Papers in the JOE These guidelines provide a detailed and practical review of tips for preparing and editing manuscripts, figures and tables for publication. Scientific Advisory Board The editors have greatly expanded the Scientific Advisory Board to facilitate an electronic peer review of all manuscripts submitted to the JOE. We thank these colleagues who generously share their expertise. We will post the names of Scientific Advisory Board members throughout the year on the Web site and publish them annually in the December issue of the JOE. EndNoteTM Library The editors of the JOE are pleased to provide this free downloadable library of the titles and abstracts of papers published in the JOE since 1975. The software program permits searching by author, year or keyword, and EndNoteTM is also helpful in both designing research studies and in writing manuscripts. Moreover, the EndNoteTM program automatically formats references into the correct style for the JOE (to do this, select ‘‘Styles’’ from the menu, open the ‘‘Medicine’’ folder and click on ‘‘Vancouver’’ to run the correct reference style). The editors will update this JOE library periodically throughout the year. Users must purchase the EndNoteTM software program to use this feature (see www.endnote.com for more information).

Treasurer Steven J. Katz Immediate Past President Alan H. Gluskin AAE Foundation President Mary T. Pettiette Executive Director Kenneth J. Widelka

DIRECTORS District I Judy McIntyre Elizabeth S. Perry District II Paul A. Falcon Marcus D. Johnson District III Theodore D. Ravenel William D. Stanley District IV Michael J. Mintz Susan B. Paurazas District V Kirk A. Coury Bradley H. Gettleman District VI Kenneth B. Wiltbank Scott L. Doyle District VII Janice C. Chou Kenneth W. Tittle

The Journal of Endodontics (ISSN 0099-2399), copyright © 2021 by the American Association of Endodontists, is published monthly by Elsevier, 230 Park Avenue, Suite 800, New York, NY 10169-0901, USA. Annual subscription rate: Individual US: $488; Individual Non-US: $571. AAE members—annual dues include $50 for JOE subscription. Call (800) 654-2452 to subscribe. Periodicals postage paid at New York, NY and at additional mailing ofﬁces. POSTMASTER: Send address changes to Journal of Endodontics, Elsevier Health Sciences Division, Subscription Customer Service, 3251 Riverport Lane, Maryland Heights, MO 63043. Prices are subject to change. Indexed by Current Contents (Clinical Medicine, Science Citation Index, SciSearch, Research Alert [personalized search service], ISI/BioMed [online database], Biomedical Engineering Citation Index), and Index to Dental Literature/Medline.

‘‘The statements, opinions and advertisem*nts in the Journal of Endodontics are solely those of the individual authors, contributors, editors or advertisers, as indicated. Those statements, opinions and advertisem*nts do not reﬂect any endorsem*nt by the American Association of Endodontists or its agents, authors, contributors, editors, advertisers or the publisher. Unless otherwise speciﬁed, the American Association of Endodontists and the publisher disclaim any and all responsibility or liability for such material.’’

Association of German Certiﬁed Endodontists An Afﬁliated Society Sponsor of the Journal of Endodontics

September 2021 Volume 47 Number 9

EDITORIAL 1337

Insights into the September 2021 Issue of the JOE Anita Aminoshariae, Amir Azarpazhooh, Anibal R. Diogenes, Ashraf F. Fouad, Gerald N. Glickman, Anil Kishen, Ariadne M. Letra, Linda Levin, Robert S. Roda, Frank C. Setzer, Franklin R. Tay, and Kenneth M. Hargreaves

AAE POSITION STATEMENT 1340 CONSORT RANDOMIZED 1345 CLINICAL TRIAL

AAE Position Statement on Vital Pulp Therapy Effect of Apical Third Enlargement to Different Preparation Sizes and Tapers on Postoperative Pain and Outcome of Primary Endodontic Treatment: A Prospective Randomized Clinical Trial Shazra Fatima, Ashok Kumar, Syed Mukhtar Un Nisar Andrabi, Surendra Kumar Mishra, and Rajendra Kumar Tewari

REVIEW ARTICLE 1352

Artiﬁcial Intelligence in Endodontics: Current Applications and Future Directions Anita Aminoshariae, Jim Kulild, and Venkateshbabu Nagendrababu

1358

Uncertainty Bounds in Clinical Trials Published in Endodontic Journals within the Last 5 Years: Are We Conﬁdent on What We Read? Giorgos N. Tzanetakis, and Despina Koletsi

CLINICAL RESEARCH 1365

1376

Associations between Pain Severity, Clinical Findings, and Endodontic Disease: A Cross-Sectional Study Ozge Erdogan, Matthew Malek, and Jennifer L. Gibbs

1383

Oral Functional Behaviors and Tooth Factors Associated with Cracked Teeth in Asymptomatic Patients Pasinee Nuamwisudhi, and Thanomsuk Jearanaiphaisarn

1391

Inﬂuence of Voxel Size and Filter Application in Detecting Second Mesiobuccal Canals in Cone-beam Computed Tomographic Images Sâmia Mouzinho-Machado, Lucas de Paula Lopes Rosado, Fernanda Coelho-Silva, Frederico Sampaio Neves, Francisco Haiter-Neto, and Sergio Lins de-Azevedo-Vaz

1398

Factors Associated with Incomplete Endodontic Care Carla Y. Falcon, Anthony R. Arena, Rebecca Hublall, Craig S. Hirschberg, and Paul A. Falcon

REGENERATIVE ENDODONTICS 1402

Dental Pulp Autotransplantation: A New Modality of Endodontic Regenerative Therapy—Follow-Up of 3 Clinical Cases Victor Pinheiro Feitosa, Mara Natiere Gonçalves Mota, Lorena Vasconcelos Vieira, Diego Martins de Paula, Lívia Lisboa Ribeiro Gomes, Luzia Kelly Rios Solheiro, Manoel Asciton de Aguiar Neto, Diego Armando Leite Carvalho, and Francisbênia Alves Silvestre

1409

Activation of Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Channels Promotes Odontogenic Differentiation of Human Dental Pulp Cells Yaxin Lou, Yangqiu Liu, Jiange Zhao, Weiping Tian, Na Xu, Chengcheng Zang, and Kehua Que

BASIC RESEARCH – BIOLOGY 1417

Inﬂuence of Preoperative Pulp Inﬂammation in the Outcome of Full Pulpotomy Using a Dog Model João Miguel Santos, Joana A. Marques, Patrícia Diogo, Ana Messias, Vitor Sousa, Diana Sequeira, and Paulo J. Palma

1427

Fibroblasts Control Macrophage Differentiation during Pulp Inﬂammation Chloé Le Fournis, Charlotte Jeanneau, Thomas Giraud, Ikhlas El Karim, Fionnuala T. Lundy, and Imad About

September 2021 Volume 47 Number 9

1435

Engineered Chitosan-based Nanoparticles Modulate Macrophage–Periodontal Ligament Fibroblast Interactions in Bioﬁlm-mediated Inﬂammation Hebatullah Hussein, and Anil Kishen

BASIC RESEARCH – TECHNOLOGY 1445

Computer-Controlled CO2 Laser Ablation System for Cone-beam Computed Tomography and Digital Image Guided Endodontic Access: A Pilot Study Jacob C. Simon, Jason W. Kwok, Frank Vinculado, and Daniel Fried

1453

Accuracy and Efficiency of 3-dimensional Dynamic Navigation System for Removal of Fiber Post from Root Canal–Treated Teeth Anmar Janabi, Patricia A. Tordik, Ina L. Griffin, Behzad Mostoufi, Jeffey B. Price, Priya Chand, and Frederico C. Martinho

1461

The Osteogenic Assessment of Mineral Trioxide Aggregate–based Endodontic Sealers in an Organotypic Ex Vivo Bone Development Model Pedro S. Gomes, Bruna Pinheiro, Bruno Colaço, and Maria H. Fernandes

1467

The Effect of Root Canal Preparation Size and Taper of Middle Mesial Canals on Fracture Resistance of the Mandibular Molar Teeth: An In Vitro Study mur Kılıç, Emrah Karatas¸lıog lu, and Mehmet Emin Kaval Yag

1472

Impact of Canal Taper and Access Cavity Design on the Life Span of an Endodontically Treated Mandibular Molar: A Finite Element Analysis Mostafa M. A. Elkholy, Nawar Naguib Nawar, William Nguyen Ha, Shehabeldin Mohamed Saber, and Hyeon-Cheol Kim

1481

Analysis of Instrumentation Protocols Regarding the Quality of Mesial Canal Preparation in Mandibular Molars: A Micro–computed Tomographic Study Flavia Darius Vivacqua, Marco Antonio Hungaro Duarte, Rodrigo Ricci Vivan, Murilo Priori Alcalde, Renan Diego Furlan, and Clovis Monteiro Bramante

1487

Syringe Irrigation in Minimally Shaped Root Canals Using 3 Endodontic Needles: A Computational Fluid Dynamics Study Christos Boutsioukis, and Patricia Gutierrez Nova

1496

Inﬂuence of Minimally Invasive Access Cavity Designs on the Fracture Resistance of Endodontically Treated Mandibular Molars Subjected to Thermocycling and Dynamic Loading Sneha Susan Santosh, Suma Ballal, and Velmurugan Natanasabapathy

1501

Effects of Root Canal Curvature and Mechanical Properties of Nickel-Titanium Files on Torque Generation Sang Won Kwak, Jung-Hong Ha, Ya Shen, Markus Haapasalo, and Hyeon-Cheol Kim

CASE REPORT/ 1507 CLINICAL TECHNIQUES

Ingrowth of Mineralized Tissue into the Root Canal of Immature Permanent Teeth after a Traumatic Injury: A Report of 3 Cases Zameera Fida, Lucy Yu, Neeta Prabhu, and Bill Kahler

1515

Successful Pulp-Preserving Treatment for Peri-invagin*tion Periodontitis of Double Dens Invagin*tus With Oehlers Type IIIA and IIIB: A Case Report Naoto Kamio, Natsuko Gomyo, and Kiyoshi Matsushima

SOCIETY NEWS 1521 1527

Associate Registry Donor Honor Roll

GUIDELINES FOR AUTHORS AND REVIEWERS

Introduction The Journal of Endodontics is owned by the American Association of Endodontists. Submitted manuscripts must pertain to endodontics and may be original research (eg, clinical trails, basic science related to the biological aspects of endodontics, basic science related to endodontic techniques, case reports, or review articles related to the scientiﬁc or applied aspects of endodontics). Clinical studies using CONSORT methods (http://www.consort-statement.org/ consort-statement/) or systematic reviews using meta-analyses are particularly encouraged. Authors of potential review articles are encouraged to ﬁrst contact the Editor during their preliminary development via e-mail at [emailprotected]. Manuscripts submitted for publication must be submitted solely to JOE. They must not be submitted for consideration elsewhere or be published elsewhere.

Disclaimer The statements, opinions, and advertisem*nts in the Journal of Endodontics are solely those of the individual authors, contributors, editors, or advertisers, as indicated. Those statements, opinions, and advertisem*nts do not affect any endorsem*nt by the American Association of Endodontists or its agents, authors, contributors, editors, or advertisers, or the publisher. Unless otherwise speciﬁed, the American Association of Endodontists and the publisher disclaim any and all responsibility or liability for such material.

Before You Begin Ethics in Publishing. For information on Ethics in publishing and Ethical guidelines for journal publication see http://www.elsevier. com/publishingethics and http:// www.elsevier.com/journal-authors/ethics.

Human and Animal Rights. If the work involves the use of animal or human subjects, the author should ensure that the work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans http:// www.wma.net/en/30publications/10policies/ b3/index.html; EU Directive 2010/63/EU for animal experiments http://ec.europa.eu/ environment/chemicals/lab_animals/ legislation_en.htm; Uniform Requirements for manuscripts submitted to Biomedical journals http://www.icmje.org. Authors should include a statement in the manuscript that informed

consent was obtained for experimentation with human subjects. The privacy rights of human subjects must always be observed.

Conflict of Interest. All authors must disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. See also http://www.elsevier.com/ conflictsofinterest. Further information and an example of a Conflict of Interest form can be found at: http://help.elsevier.com/app/ answers/detail/a_id/286/p/7923.

Submission Declaration. Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a published lecture or academic thesis or as an electronic preprint, see http://www.elsevier.com/ postingpolicy), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyrightholder. Changes to Authorship. This policy concerns the addition, deletion, or rearrangement of author names in the authorship of accepted manuscripts: Before the accepted manuscript is published in an online issue: Requests to add or remove an author, or to rearrange the author names, must be sent to the Journal Manager from the corresponding author of the accepted manuscript and must include: (a) the reason the name should be added or removed, or the author names rearranged and (b) written conﬁrmation (e-mail, fax, letter) from all authors that they agree with the addition, removal or rearrangement. In the case of addition or removal of authors, this includes conﬁrmation from the author being added or removed. Requests that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author, who must follow the procedure as described above. Note that: (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication

of the accepted manuscript in an online issue is suspended until authorship has been agreed. After the accepted manuscript is published in an online issue: Any requests to add, delete, or rearrange author names in an article published in an online issue will follow the same policies as noted above and result in a corrigendum.

Reporting Clinical Trials. Randomized controlled trials should be presented according to the CONSORT guidelines. At manuscript submission, authors must provide the CONSORT checklist accompanied by a ﬂow diagram that illustrates the progress of patients through the trial, including recruitment, enrollment, randomization, withdrawal and completion, and a detailed description of the randomization procedure. The CONSORT checklist and template ﬂow diagram can be found on http://www. consort-statement.org.

Copyright. Upon acceptance of an article, authors will be asked to complete a Journal Publishing Agreement (for more information on this and copyright see http://www.elsevier. com/copyright). Acceptance of the agreement will ensure the widest possible dissemination of information. An e-mail will be sent to the corresponding author conﬁrming receipt of the manuscript together with a Journal Publishing Agreement form or a link to the online version of this agreement. Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal circulation within their institutions. Permission of the Publisher is required for resale or distribution outside the institution and for all other derivative works, including compilations and translations (please consult http://www.elsevier.com/permissions). If excerpts from other copyrighted works are included, the author(s) must obtain written permission from the copyright owners and credit the source(s) in the article. Elsevier has preprinted forms for use by authors in these cases: please consult http://www.elsevier.com/ permissions. Retained Author Rights As an author you (or your employer or institution) retain certain rights; for details you are referred to: http://www.elsevier.com/authorsrights.

Role of the Funding Source. You are requested to identify who provided ﬁnancial support for the conduct of the research and/or preparation of the article and to brieﬂy describe the role of the sponsor(s), if any, in study design; in the collection, analysis and

interpretation of data; in the writing of the report; and in the decision to submit the article for publication. If the funding source(s) had no such involvement then this should be stated.

Funding Body Agreements and Policies. Elsevier has established agreements and developed policies to allow authors whose articles appear in journals published by Elsevier, to comply with potential manuscript archiving requirements as speciﬁed as conditions of their grant awards. To learn more about existing agreements and policies please visit http://www.elsevier.com/ fundingbodies.

Language (Usage and Editing Services). Please write your text in good English (American or British usage is accepted, but not a mixture of these). Authors who feel their English language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientiﬁc English may wish to use the English Language Editing service available from Elsevier’s WebShop http://webshop. elsevier.com/languageediting/ or visit our customer support site http://support. elsevier.com for more information.

Submission Submission to this journal proceeds totally online and you will be guided stepwise through the creation and uploading of your files. The system automatically converts source files to a single PDF file of the article, which is used in the peer-review process. Please note that even though manuscript source files are converted to PDF files at submission for the review process, these source files are needed for further processing after acceptance. All correspondence, including notification of the Editor’s decision and requests for revision, takes place by e-mail removing the need for a paper trail. Submit Your Article Please submit your article via http://ees.elsevier.com/joe/.

Preparation General Points on Composition. Authors are strongly encouraged to analyze their final draft with both software (eg, spelling and grammar programs) and colleagues who have expertise in English grammar. References listed at the end of this section provide a more extensive review of rules of English grammar and guidelines for writing a scientific article. Always remember that clarity is the most important feature of scientific writing. Scientific articles must be clear and precise in their content and concise in their delivery because their purpose is to inform the reader. The Editor reserves the right to edit all manuscripts or to reject those manuscripts that lack clarity or precision or that have unacceptable grammar or syntax. The

following list represents common errors in manuscripts submitted to the Journal of Endodontics: a. The paragraph is the ideal unit of organization. Paragraphs typically start with an introductory sentence that is followed by sentences that describe additional detail or examples. The last sentence of the paragraph provides conclusions and forms a transition to the next paragraph. Common problems include one-sentence paragraphs, sentences that do not develop the theme of the paragraph (see also section “c,” below), or sentences with little to no transition within a paragraph. b. Keep to the point. The subject of the sentence should support the subject of the paragraph For example, the introduction of authors’ names in a sentence changes the subject and lengthens the text. In a paragraph on sodium hypochlorite, the sentence, “In 1983, Langeland et al, reported that sodium hypochlorite acts as a lubricating factor during instrumentation and helps to flush debris from the root canals” can be edited to: “Sodium hypochlorite acts as a lubricant during instrumentation and as a vehicle for flushing the generated debris (Langeland et al, 1983).” In this example, the paragraph’s subject is sodium hypochlorite and sentences should focus on this subject. c. Sentences are stronger when written in the active voice, that is, the subject performs the action. Passive sentences are identified by the use of passive verbs such as “was,” “were,” “could,” etc. For example: “Dexamethasone was found in this study to be a factor that was associated with reduced inflammation,” can be edited to: “Our results demonstrated that dexamethasone reduced inflammation.” Sentences written in a direct and active voice are generally more powerful and shorter than sentences written in the passive voice. d. Reduce verbiage. Short sentences are easier to understand. The inclusion of unnecessary words is often associated with the use of a passive voice, a lack of focus, or run-on sentences. This is not to imply that all sentences need be short or even the same length. Indeed, variation in sentence structure and length often helps to maintain reader interest. However, make all words count. A more formal way of stating this point is that the use of subordinate clauses adds variety and information when constructing a paragraph. (This section was written deliberately with sentences of varying length to illustrate this point.) e. Use parallel construction to express related ideas. For example, the sentence, “Formerly,

endodontics was taught by hand instrumentation, while now rotary instrumentation is the common method,” can be edited to “Formerly, endodontics was taught using hand instrumentation; now it is commonly taught using rotary instrumentation.” The use of parallel construction in sentences simply means that similar ideas are expressed in similar ways, and this helps the reader recognize that the ideas are related. f. Keep modifying phrases close to the word that they modify. This is a common problem in complex sentences that may confuse the reader. For example, the statement, “Accordingly, when conclusions are drawn from the results of this study, caution must be used,” can be edited to “Caution must be used when conclusions are drawn from the results of this study.” g. To summarize these points, effective sentences are clear and precise, and often are short, simple and focused on one key point that supports the paragraph’s theme. h. Authors should be aware that the JOE uses iThenticate, plagiarism detection software, to ensure originality and integrity of material published in the journal. The use of copied sentences, even when present within quotation marks, is highly discouraged. Instead, the information of the original research should be expressed by the new manuscript author’s own words, and a proper citation given at the end of the sentence. Plagiarism will not be tolerated and manuscripts will be rejected or papers withdrawn after publication based on unethical actions by the authors. In addition, authors may be sanctioned for future publication.

Use of Wordprocessing Software. It is important that the file be saved in the native format of the wordprocessor used. The text should be in single-column format. Keep the layout of the text as simple as possible. Most formatting codes will be removed and replaced on processing the article. In particular, do not use the wordprocessor’s options to justify text or to hyphenate words. However, do use bold face, italics, subscripts, superscripts etc. When preparing tables, if you are using a table grid, use only one grid for each individual table and not a grid for each row. If no grid is used, use tabs, not spaces, to align columns. The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also the Guide to Publishing with Elsevier: http://www.elsevier.com/guidepublication). Note that source files of figures, tables and text graphics will be required whether or not you embed your figures in the text. See also the section on Electronic artwork.

To avoid unnecessary errors you are strongly advised to use the spell-check and grammarcheck functions of your wordprocessor.

otherwise. List here those individuals who provided help during the research (e.g., providing language help, writing assistance or proof reading the article, etc.).

Essential Title Page Information Title. Concise and informative. Titles are often used in information-retrieval systems. Avoid abbreviations and formulae where possible. Author names and affiliations. Where the family name may be ambiguous (e.g., a double name), please indicate this clearly. Present the authors’ affiliation addresses (where the actual work was done) below the names. Indicate all affiliations with a lower-case superscript letter immediately after the author’s name and in front of the appropriate address. Provide the full postal address of each affiliation, including the country name and, if available, the e-mail address of each author. Corresponding author. Clearly indicate who will handle correspondence at all stages of refereeing and publication, also postpublication. Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the complete postal address. Contact details must be kept up to date by the corresponding author.

Structured Abstract A structured abstract, by means of appropriate headings, should provide the context or background for the research and should state its purpose, basic procedures (selection of study subjects or laboratory animals, observational and analytical methods), main findings (giving specific effect sizes and their statistical significance, if possible), and principal conclusions. It should emphasize new and important aspects of the study or observations.

Abstract Headings. Introduction, Methods, Results, Conclusions

Keywords Immediately after the abstract, provide a maximum of 6 keywords, using American spelling and avoiding general and plural terms and multiple concepts (avoid, for example, “and,” “of”). Be sparing with abbreviations: only abbreviations ﬁrmly established in the ﬁeld may be eligible. These keywords will be used for indexing purposes.

Acknowledgements Collate acknowledgements in a separate section at the end of the article before the references and do not, therefore, include them on the title page, as a footnote to the title or

The authors deny any conﬂicts of interest related to this study.

Original Research Article Guidelines Title Page. The title describes the major emphasis of the paper. It must be as short as possible without loss of clarity. Avoid abbreviations in the title because this may lead to imprecise coding by electronic citation programs such as PubMed (eg, use sodium hypochlorite rather than NaOCl). The author list must conform to published standards on authorship (see authorship criteria in the Uniform Requirements for Manuscripts Submitted to Biomedical Journals at www.icmje.org). Include the manuscript title; the names and affiliations of all authors; and the name, affiliation, and full mailing address (including e-mail) of the corresponding author. This author will be responsible for proofreading page proofs and ordering reprints when applicable. Also highlight the contribution of each author in the cover letter. Abstract. The Abstract concisely describes the purpose of the study in 250 or fewer words. It must be organized into sections: Introduction, Methods, Results, and Conclusions. The hypothesis is described in the Abstract Introduction. The Abstract describes the new contributions made by this study. The Abstract word limitation and its wide distribution (eg, PubMed) make it challenging to write clearly. This section is written last by many authors. Write the abstract in past tense because the study has been completed. Provide 3-5 keywords. Introduction. The introduction briefly reviews the pertinent literature in order to identify the gap in knowledge that the study is intended to address and the limitations of previous studies in the area. Clearly describe the purpose of the study, the tested hypothesis, and its scope. Many successful manuscripts require no more than a few paragraphs to accomplish these goals; therefore, do not perform extensive literature review or discuss the results of the study in this section. Materials and Methods. The Materials and Methods section is intended to permit other investigators to repeat your experiments. There are 4 components to this section: (1) detailed description of the materials used and their components, (2) experimental

design, (3) procedures employed, and (4) statistical tests used to analyze the results. Most manuscripts should cite prior studies that used similar methods and succinctly describe the essential aspects used in the present study. A “methods figure” will be rejected unless the procedure is novel and requires an illustration for comprehension. If the method is novel, then you must carefully describe the method and include validation experiments. If the study used a commercial product, the manuscript must either state that you followed manufacturer’s protocol or specify any changes made to the protocol. If the study used an in vitro model to simulate a clinical outcome, describe either experiments made to validate the model or previous literature that proved the clinical relevance of the model. The statistical analysis section must describe which tests were used to analyze which dependent measures; P values must be specified. Additional details may include randomization scheme, stratification (if any), power analysis as a basis for sample size computation, dropouts from clinical trials, the effects of important confounding variables, and bivariate versus multivariate analysis.

Results. Only experimental results are appropriate in this section; do not include methods, discussion, or conclusions. Include only those data that are critical for the study, as defined by the aim(s). Do not include all available data without justification; any repetitive findings will be rejected from publication. All Figures, Charts, and Tables must be cited in the text in numerical order and include a brief description of the major findings. Consider using Supplemental Figures, Tables, or Video clips that will be published online. Supplemental material often is used to provide additional information or control experiments that support the results section (eg, microarray data).

Figures. There are 2 general types of figures: type 1 includes photographs, radiographs, or micrographs; type 2 includes graphs. Type 1: Include only essential figures and use composite figures containing several panels of photographs, if possible. Each panel must be clearly identified with a letter (eg, A, B, C), and the parts must be defined in the figure legend. A figure that contains many panels counts as 1 figure. Type 2: Graphs (ie, line drawings including bar graphs) that plot a dependent measure (on the Y axis) as a function of an independent measure (usually plotted on the X axis). One example is a graph depicting pain scores over time. Use graphs when the overall trend of the results is more important than the exact numeric values of the results. A graph is a convenient way to report that an ibuprofentreated group reported less pain than a

placebo-treated group over the ﬁrst 24 hours, but pain reported was the same for both groups over the next 96 hours. In this case, the trend of the results is the primary ﬁnding; the actual pain scores are not as critical as the relative differences between the NSAID and placebo groups.

Tables. Tables are appropriate when it is critical to present exact numeric values; however, not all results need be placed in either a table or figure. Instead of a simple table, the results could state that there was no inhibition of growth from 0.001%-0.03% NaOCl, and a 100% inhibition of growth from 0.03%-3% NaOCl (N55/group). If the results are not significant, then it is probably not necessary to include the results in either a table or as a figure. Acknowledgments. All authors must affirm that they have no financial affiliation (eg, employment, direct payment, stock holdings, retainers, consultantships, patent licensing arrangements, or honoraria), or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past 3 years. Disclose any potential conflict of interest. Append a paragraph to the manuscript that fully discloses any financial or other interest that poses a conflict. Disclose all sources and attribute all grants, contracts, or donations that funded the study. Specific wording: “The authors deny any conflicts of interest related to this study.” References. The reference style can be learned from reading past issues of JOE. References are numbered in order of citation. Place text citation of the reference Arabic number in parentheses at the end of a sentence or at the end of a clause that requires a literature citation. Do not use superscript for references. Original reports are limited to 35 references. There are no limits in the number of references for review articles.

Other Article Types and Guidelines Manuscripts submitted to JOE that are not Original Articles must fall into one of the following categories. Abstract limit: 250 words. Note that word limits, listed by type, do not include ﬁgure legends or References. If you are not sure whether your manuscript falls within one of the categories listed or if you would like to request pre-approval to submit additional ﬁgures, contact the Editor at [emailprotected].

CONSORT Randomized Clinical Trial. Must strictly adhere to the Consolidated Standards of Reporting Trials—CONSORT— minimum guidelines for publication of randomized clinical trials (http://www.consortstatement.org). Word limit: 3500. Headings: Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgments. Maximum number of ﬁgures: 4. Maximum number of tables: 4.

Review Article. Either narrative articles or systemic reviews/meta-analyses. Case Report/Clinical Techniques articles, even when they include an extensive review of the literature, are categorized as Case Report/ Clinical Techniques. Word limit: 3500. Headings: Abstract, Introduction, Discussion, Acknowledgments. Maximum number of ﬁgures: 4. Maximum number of tables: 4. Clinical Research. Prospective or retrospective studies of patients or patient records, research on biopsies excluding the use of human teeth for technique studies. Word limit: 3500. Headings: Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgments. Maximum number of ﬁgures: 4. Maximum number of tables: 4.

Basic Research—Biology. Animal or culture studies of biological research on physiology, development, stem cell differentiation, inflammation, or pathology. Primary focus is on biology. Word limit: 2500. Headings: Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgments. Maximum number of figures: 4. Maximum number of tables: 4. Basic Research—Technology. Focus primarily on research related to techniques and materials used, or on potential clinical use, in endodontics. Word limit: 2500. Headings: Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgments. Maximum number of figures: 3. Maximum number of tables: 3. Case Report/Clinical Techniques. Reports of an unusual clinical case or use of a cutting edge technology in a clinical case. Word limit: 2500. Headings: Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgments. Maximum number of figures: 4. Maximum number of tables: 4.

Units Follow internationally accepted rules and conventions: use the international system of

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EDITORIAL

Insights into the September 2021 Issue of the JOE

Welcome to the September 2021 issue of the JOE. Here, we share some of our favorite articles that are published in this issue of the Journal. We hope you look forward to reading these and other articles in the JOE.

FEATURED PAPERS FROM THIS ISSUE This JOE issue features the latest American Association of Endodontists Position Statement on vital pulp therapy (VPT). The statement1 reviews the current knowledge base for VPT and highlights significant changes in application and techniques. Although VPT traditionally aimed at preserving the radicular pulp in immature permanent teeth to facilitate apexogenesis, this has shifted to a broader view of creating optimal conditions for pulp tissue repair and preservation. Evidence now exists that not only teeth with a pulpal diagnosis of “reversible pulpitis,” but also with “irreversible pulpitis” can be treated by VPT, given that direct visualization of the pulp demonstrates favorable conditions. Calcium silicate cements, such as MTA or “bioceramics,” are increasingly used and display advantageous properties and improved clinical success rates ranging from 85% to 100% at 1 to 2 years compared with the traditional materials, including calcium hydroxide, glass ionomers, or resin-based cements, with success rates between 43% and 92%. In addition, the statement provides insight into the future of VPT, anticipating chairside techniques that will use biomarkers for the assessment of pulpal viability. In this issue of the Journal, Fatima et al.2 report on a randomized controlled study evaluating the effect of apical third enlargement on postoperative pain and clinical outcomes. The study enrolled 120 participants who had asymptomatic mandibular molars with periapical radiolucencies and divided them into 2 groups for root canal therapy: group 1 had the canal diameter at the working length (WL) enlarged to 2 sizes greater than the first file to bind at WL (the initial apical binding file or IABF) and group 2 that had the canal diameter at the WL enlarged to 3 sizes greater than IABF. Each

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group was further subdivided into group A and B (4% taper and 6% taper preparations, respectively). Short-term postoperative pain evaluation and longer-term assessment of reductions in periapical index scores (up to 1 year) was performed showing no difference in postoperative pain scores between groups. However, the effect of preparation size and canal taper did have a significant effect on healing. The 4% taper preparations with only a 2-size increase in diameter over the IABF had a much lower rate of healing (57%) at 1 year compared with the other 3 groups (all .90%). The authors concluded that a 4% taper preparation that was only 2 sizes larger than the IABF was not sufficient to clean the canals well enough to allow consistent healing. In the Discussion, they stated that the most common IABF size was #15 so that “the minimum apical preparation size required to disinfect the canals adequately was #25/0.06 or #30/0.04 in the majority of the cases.” There were several limitations the reader must make when interpreting this study. The sample size was not large and the follow-up time was relatively short, and only 2-dimensional imaging was used for healing assessment. More research in this area is clearly necessary to refine these concepts, but this study does seem to support the idea that better healing comes with better cleaning of the canal space. Artificial intelligence (AI) has made significant progress in endodontics over the past few years. This issue of the JOE has a comprehensive review that identified all the endodontic articles that were published in AI applications and also discussed gaps in that literature.3 AI algorithms learn the relationship between the features and what they call “ground truth.” Ground truth is a term used in AI that compares the results of machine learning (ML) against the accuracy of the real clinical evaluation. ML is a part of AI where it is based on sample data and algorithms. It is further divided into deep learning, and is a component of ML, but takes it to the next level where it makes use of deep neural networks. Most of the articles have been published using ML. Altogether, the current article identified 7 areas in endodontic research that could benefit

from AI methods: (1) detection of periapical lesions, (2) detection of a periapical cyst or granuloma, (3) detection of root fractures, (4) WL determination, (5) morphology determination, (6) prediction of retreatment, and (7) prediction of stem cell viability. Although still in its infancy, AI has the capacity to significantly improve outcomes in endodontics in the future. Most of the past studies used synthetic data or were in vitro. Clinical studies with large sample sizes would be required to ascertain and improve this technology. Gaps in the literature and possible future directions in endodontics were also discussed. Randomized controlled trials (RCTs) are important for evidence-based clinical decision making. In trials, hypothesis testing with the report of P value level has been widely used, and yet largely criticized as being potentially misleading, as they do not provide insights to the clinical relevance or magnitude of a treatment effect. In this respect, the use of confidence interval (usually 95% CI) is of paramount value to establish how confident we are that the identified effect is real or whether the data are compatible with a clinically relevant effect. In this issue of JOE, Tzanetakis and Koletsi4 presented a review on the current state of evidence on CI reporting in clinical endodontic research. From their search of 141 RCTs published in the Journal of Endodontics, the International Endodontic Journal, and the Australian Endodontic Journal from January 2016 to December 2020, only one-third reported CI. This means that 70% of the remaining trials ignore the value and importance of uncertainty bounds for the presentation of the results on the effectiveness and/or safety of competing treatment modalities and relied more on P values. Future efforts should be made to highlight the value and importance of CIs in the reporting of the results of clinical trials. The identification of a “molecular signature” of an inflamed dental pulp is crucial for the development of more accurate pulp status diagnostic assessment tools. Fortunately, advancements in bioinformatics have allowed the generation of large data sets

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on the global changes in gene expression seen in pulpitis. Although robust, this methodology often leads to high variability among studies. In this issue of the JOE, Liu and colleagues5 used a robust rank data aggregation approach to combine and analyze messenger RNA (mRNA), micro RNA (miRNA), and long noncoding RNA (LnRNA) data sets from different studies. Several genes were found to be differentially expressed, in addition to multiple miRNA and LnRNA. Functional analyses identified at least 20 genes that were highly regulated in pulpitis. As expected, most of the upregulated genes were related to inflammatory responses, such as TLR2, C3, and CCR1. Conversely, many genes associated with odontogenesis or mineralization were downregulated, such as DSPP and RUNX2. Additional targets not previously emphasized were identified, including ITGAX and TREM1. The expression of these targets and some other relevant markers was validated by quantitative reversetranscriptase polymerase chain reaction in normal and inflamed human dental pulp. Collectively, this study combined existing large data sets from gene expression studies and identified a more robust set of markers for pulpitis, in addition to few miRNA and LnRNAs that could be associated with the epigenetic regulation of pulpitis. Cone-beam computed tomography (CBCT) has been recognized as an invaluable aid in endodontic retreatments. In addition to diagnostic information, these 3-dimensional images facilitate the surveillance of root canal anatomy preoperatively. This is particularly useful with complex anatomy where the failure to access the entire system during debridement can result in a compromised prognosis, but the clarity of the images used to evaluate internal tooth anatomy is crucial, and can be diminished by artifacts created by preexisting filling materials in retreatment cases. Various technical factors that impact image quality in CBCT have been identified and they include instrument settings like field of view and milliamperage, voxel size, and the software used to construct the images. The use of enhancement filters allows further manipulation of an image to optimize diagnostic accuracy. Here, Mouzinho-Machado and colleagues6 present an in vitro study that evaluates the impact of a combination of voxel sizes and enhancement filters on the detection of MB2 canals in previously instrumented and filled teeth. Twenty maxillary first molar teeth with confirmed MB2 canals and 20 control maxillary first molars without an MB2 were evaluated.

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Aminoshariae et al.

Both test and control teeth were scanned before and after various fill materials were placed in all but the MB2 canal. Various voxel sizes and enhancement filters were applied, and then blinded, calibrated observers evaluated the scans. The results indicated that the use or type of enhancement filter did not have a statistically significant effect on detection of the MB2 canal in previously filled teeth; however, voxel size did make a significant difference in the diagnostic acuity. The authors concluded that a voxel size of 80 mm on the machine they tested was the most accurate. Tooth autotransplantation has been performed with high degree of success for a long time. More recently, regenerative endodontic therapy has shown favorable outcomes, even in cases with mature teeth. In the case series by Feitosa and colleagues,7 3 cases are presented in which the novel technique of dental pulp autotransplantation is described. Three adults who had a single-canal tooth with pulp necrosis and apical periodontitis, as well as an intact third molar indicated for extraction, were included. In a single appointment for each patient, the third molar was extracted without resection, and stored in saline. The tooth with pulp necrosis was isolated, accessed, and debrided, including irrigation with ciprofloxacin, metronidazole, and minocycline, followed by 17% EDTA. The pulp from the third molar was then transplanted into the pulp space of the treated tooth and its spread was aided by disinfected gutta percha points. The tooth was then restored with Biodentine (Septodont, Lancaster, PA) and a resin restoration. Three-, 6-, and 12-month follow-up appointments showed absence of symptoms, continued healing, pulp responsiveness to electric pulp testing, and positive laser Doppler imaging in all cases. Although further research is warranted, this is a novel method that may contribute to regenerative endodontic treatments. The transient receptor potential (TRP) family of ligand-gated ion channels are evolutionarily conserved sensory channels, having first been discovered in the fruit fly. TRP channels detect many environmental changes, including temperature changes that are associated with dentinal hypersensitivity. On activation, many TRP channels gate inward calcium ions, leading to cellular changes, such as differentiation. Here, Lou and coworkers8 evaluated the role of the TRP ankyrin 1 (TRPA1) and the TRP vanilloid 1 (TRPV1) thermosensitive channels in the odontogenic differentiation of human dental pulp cells (HDPCs). The authors discovered that both

types of channels were upregulated during odontogenic differentiation of HDPCs. The channels also affected odontogenic differentiation by regulating intracellular Ca21 concentration. These findings may offer novel approaches to development of drugs that promote tissue regeneration. The impact of preoperative pulpal inflammation on tissue response to VPT procedures remains a concern. In this issue of the JOE, Santos and colleagues9 evaluated the influence of preoperative pulp inflammation on the outcome of full pulpotomy performed in permanent posterior teeth of beagle dogs. The tissue response to 4 commercially available biomaterials, including ProRoot MTA (DentsplySirona, Charlotte, NC), TotalFill BC Putty (Brassler, Savannah, GA), and Biodentine (Septodont, Lancaster, PA) and an experimental cement (PCM, pulp capping material) was also assessed. Dentin exposure was performed on 120 premolar roots to induce pulpal inflammation. After 1 week, pulp exposure and full pulpotomy were performed using 1 of the 4 biomaterials or experimental cement, and coronal restoration was performed using glass ionomer cement. Control teeth were extracted immediately before initiation of pulpotomy procedures. Animals were killed after 14 weeks, and mandible and maxilla dissected for histological processing. Pulp-dentin tissues were histologically and radiographically assessed with regard to calcified barrier thickness by 2 blinded observers. Results showed that hemostasis in teeth with previously exposed dentin was significantly delayed, confirming the pulp inflammation status. Interestingly, both the histologic and radiographic outcomes of full pulpotomy were not influenced by the preoperative vital pulp status, and no evidence of root resorption, periapical radiolucency, or microorganism progression was found in either control or experimental groups. Except for PCM, which resulted in a mild to moderate inflammation of the pulp tissue in most of the teeth, the choice of biomaterial did not affect histologic outcomes. Short-term preoperative pulp inflammation did not negatively influence radiographic and histologic outcomes of full pulpotomy, and the studied biomaterials may be considered suitable for use as pulp capping agents. Crosstalk between tissue-specific multipotent cells and immune cells regulate the pathophysiology of apical periodontitis. Macrophages (MQ) are functionally diversified immune cells that play critical roles in the regulation of homeostasis, inflammation, and healing in a tissue-specific and context-

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dependent manner. Exposure of MQ to different environmental cues results in their polarization into different functional phenotypes: M1 (proinflammatory) or M2 (regulatory/antiinflammatory/healing). In this article, Hussein and Kishen10 studied the influence of posttreatment residual biofilm on MQ– periodontal ligament fibroblast (PdLF) interactions, and evaluated the effect of engineered chitosan-based nanoparticles on modulating the MQ–PdLF crosstalk in biofilm-mediated inflammation. The findings demonstrated that PdLF actively interacted with macrophages and influenced the immune response to biofilm. The PdLF induced phenotypic maturation of macrophages, whereas macrophages promoted migration of PdLF via paracrine signaling. The engineered chitosan-based nanoparticles influenced MQ– PdLF crosstalk through modulatory effects on inflammatory mediators and transcription factors that facilitated phenotypic switch of MQ into M2-phenotypes and promoted PdLF migration. Further research on the immune modulatory properties of these nanoparticles on the cell signaling pathways and clinically relatable animal models was suggested for future investigations. Clinicians must understand canal curvature, file design, niti type, torque control, and a host of other key factors to minimize or prevent file separation and help produce a shape confluent with natural anatomy. In this month’s Journal of Endodontics, Kwak and colleagues11 compared the torque generated by 4 different niti files, including WaveOne Primary (Dentsply Sirona, Ballaigues, Switzerland) and WaveOne Gold (Dentsply Sirona), both reciprocating systems and made of superelastic niti. These were compared with ProTaper Next X2 (Dentsply Sirona) and ProTaper Universal F2 (Dentsply Sirona), both rotary systems and made of heat-treated niti. The study showed dynamic changes in torque when a file was rotated in artificial root canals with different canal curvature angles. Clinically, the amount of generated torque is affected by various canal conditions when a file cuts the root dentin. Despite the study limitations, it was concluded that factors such as a smaller angle of root canal curvature, the use of heat-treated niti files with fewer contact points, and the use of niti files with continuous rotation resulted in a lower amount of generated torque during instrumentation. Thus, from the perspective of torque generation, the use of ProTaper Next X2 may be advantageous for a highly curved canal.

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We hope you enjoy this issue of your Journal of Endodontics.

REFERENCES 1.

American Association of Endodontists Special Committee on Vital Pulp Therapy. AAE Position Statement on Vital Pulp Therapy. J Endod 2021;47:1340–4.

Fatima S, Kumar A, Andrabi S, et al. Effect of apical third enlargement to different preparation sizes and tapers on postoperative pain and outcome of primary endodontic treatment: a prospective randomized clinical trial. J Endod 2021;47:1345–51.

Aminoshariae A, Kulild J, Nagendrababu V. Artiﬁcial intelligence in endodontics: current applications and future directions. J Endod 2021;47:1352–7.

Tzanetakis G, Koletsi D. Uncertainty bounds in clinical trials published in endodontic journals within the last 5 years. Are we conﬁdent on what we read? J Endod 2021;47:1358–64.

Liu L, Wang T, Huang D, Song D. Comprehensive analysis of differentially expressed genes in clinically diagnosed irreversible pulpitis by multi-platform data integration using a robust rank aggregation approach. J Endod 2021;47:1365–75.

Mouzinho-Machado S, Rosado L, CoelhoSilva F, et al. Inﬂuence of voxel size and ﬁlter application in detecting second mesiobuccal canal in CBCT images. J Endod 2021;47:1391–7.

Feitosa V, Mota M, Vieira L, et al. Dental pulp auto transplantation: a new modality of endodontic regenerative therapy - followup of three clinical cases. J Endod 2021;47:1402–8.

Lou Y, Liu Y, Zhao J, et al. Activation of TRPA1 and TRPV1 channels promotes odontogenic differentiation of human dental pulp cells. J Endod 2021;47:1409–16.

Santos J, Marques J, Dioga P, et al. Inﬂuence of pre-operative pulp inﬂammation in the outcome of full pulpotomy using a dog model. J Endod 2021;47:1417–26.

Anita Aminoshariae, DDS, MS Case Western Reserve University School of Dentistry Cleveland, Ohio

Amir Azarpazhooh, DDS, MSc, PhD, FRCD(C) Faculty of Dentistry University of Toronto Toronto, Ontario, Canada

Anibal R. Diogenes, DDS, MS, PhD University of Texas Health San Antonio School of Dentistry San Antonio, Texas

Ashraf F. Fouad, DDS, MS University of Alabama at Birmingham Birmingham, Alabama

Gerald N. Glickman, DDS, MS, MBA, JD Texas A&M College of Dentistry College Station, Texas

Anil Kishen, BDS, MDS, PhD Faculty of Dentistry University of Toronto Toronto, Ontario, Canada

Ariadne M. Letra, DDS, MS, PhD University of Texas Health Science Center at Houston School of Dentistry Houston, Texas

Linda Levin, DDS, PhD Private Practice Durham, North Carolina

Robert S. Roda, DDS, MS Private Practice Scottsdale, Arizona

Frank C. Setzer, DMD, PhD, MS University of Pennsylvania School of Dental Medicine Philadelphia, Pennsylvania

Franklin R. Tay, BDSc(Hons), PhD The Dental College of Georgia Augusta University Augusta, Georgia

Kenneth M. Hargreaves, DDS, PhD* University of Texas Health San Antonio School of Dentistry San Antonio, Texas Address requests for reprints to Dr. Kenneth M. Hargreaves, Department of Endodontics, UT Health San Antonio, San Antonio, TX 78229. e-mail: [emailprotected] Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/j.joen.2021.07.019

10.

Hussein H, Kishen A. Engineered chitosan-based nanoparticles modulate macrophage – periodontal ligament fibroblast interactions in biofilm-mediated inflammation. J Endod 2021;47:1435–44.

11.

Kwak S, Ha J-H, Shen Y, et al. Effects of root canal curvature and mechanical properties of nickel-titanium ﬁles on torque generation. J Endod 2021;47:1501–6.

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AAE POSITION STATEMENT

AAE Position Statement on Vital Pulp Therapy The following statement was prepared by the AAE Special Committee on Vital Pulp Therapy: Craig S. Hirschberg, DDS, Chair; George Bogen, DDS; Johnah C. Galicia, DMD, MS, PhD; Ronald R. Lemon, DMD; Ove A. Peters, DMD, MS, PhD; Nikita B. Ruparel, DMD, MS, PhD; Franklin R. Tay, BDSc, PhD; and David E. Witherspoon, BDS, MS.

INTRODUCTION The American Association of Endodontists is dedicated to excellence in the art and science of endodontics and to the highest standards of patient care. The basis for endodontic treatment utilizes the best available evidence from scientiﬁc and clinical studies in concert with the accumulated clinical knowledge and judgment of the practitioner. Vital pulp therapy (VPT) techniques are means of preserving the vitality and function of the dental pulp after injury resulting from trauma, caries, or restorative procedures. VPT procedures have traditionally included indirect or direct pulp capping, and partial or complete pulpotomy1. For years, the focus of VPT was on the preservation of the radicular pulp in immature adult teeth, so as to assure completion of root formation (apexogenesis). Today, the focus of VPT is broader; practitioners may have treatment options to consider other than pulpectomy or root canal therapy (RCT) in mature teeth, including teeth previously thought to have irreversibly inﬂamed pulps. This position statement addresses diagnostic considerations, caries management, pulp management, placement of biomaterials, and restoration. The intent of the authors is to consider vital pulp therapy from the perspective of the practice of specialty endodontics. However, this statement may be of use to any practitioner in assessing whether they have the appropriate expertise and armamentarium to perform VPT procedures in appropriately selected cases.

DIAGNOSTIC CONSIDERATIONS FOR VPT A basic tenet for clinical dentistry is that treatment is recommended and performed

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after the formulation of a sound diagnosis. This has been considered of particular relevance when vital pulp therapy was to be considered. The current AAE diagnostic terminology assigns a vital pulp to one of three categories: “normal,” “reversible pulpitis” or “irreversible pulpitis” (which could be symptomatic or asymptomatic)2. Traditionally the designation of a pulpal diagnosis is based upon the clinician’s consideration of a patient’s pain history, and appropriate clinical testing to assess the status of the pulp including the application of cold stimulus and electric pulp testing. These tests would be best termed pulp sensibility tests, as definitive tests of pulp vitality, such as measures of pulp oxygen tension, are not currently available for clinical use3. The primary provoked response to pulp sensibility testing, indicating more severe pulpal inflammation is described as an exaggerated and “lingering” response to cold stimulus, with the underlying pathomechanisms of c-fiber sensitization and inflammation-induced hypersensitivity4,5. In addition to such pulp sensibility testing, percussion tests may infer pulpal conditions from the presence of symptomatic apical periodontitis; with the presence of percussion pain, i.e., mechanical allodynia, the pulp is considered to be in an irreversibly inflamed state6. Diagnostic quality intraoral radiographs of the suspected teeth are recommended to evaluate accurately the extent of root formation and other concomitant hard tissue changes7. Historically, there has been a widespread belief that, even in aggregate, clinical test results are not well correlated with histologic descriptions of the pulpal status8,9. The viewpoint that VPT is an option only for cases where testing results were consistent with “reversible pulpitis” has recently been challenged10–12. Based on clinical, biological and theoretical considerations, the irreversibility of the pulpal disease has come into question. Histologic evidence of the progression of pulpitis suggests that there is no discrete boundary that would render a pulp beyond repair11. Rather, pulpitis may be

interpreted as a temporally and spatially graded disease, with some suggesting the following terms for gradation: “initial”, “mild”, “moderate” and “severe pulpitis10,12.” Research is underway to understand the role of inflammatory mediators that better indicate pulpal status13,14. For example, point of care analysis could use dentinal fluid15 (without pulp exposure) or pulp blood16 (with pulp exposure) to determine markers associated with tissue degradation, such as matrix metalloproteinase-9. In the absence of clinically available molecular biologic tests, direct observation of the pulp (use of a surgical microscope is recommended) can give relevant information for determining the suitability of the case for VPT. First, a misdiagnosed necrotic pulp can be accurately identified. Secondly, direct observation of pulp tissue during and after achieving hemostasis offers additional diagnostic information about the condition of the pulpal tissue17. Utilizing direct visualization of the pulp, it appears that even symptomatic pulps may be candidates for VPT18.

CARIES MANAGEMENT Complete caries removal is essential to eliminate infected tissues and visualize pulp tissue conditions under magnification when pulpal exposures occur19,20. Residual caries compromises necessary observations of pulpal inflammation levels and areas of potential necrosis. Accordingly, predictable management of vital pulp tissue should not be performed without complete removal of both demineralized enamel and infected dentin. Hard or firm dentin and dentin below white spot enamel lesions is infected by bacteria in both active and arrested lesions. Specifically, histobacteriological studies have consistently shown the presence of chronic inflammatory cell infiltrates and subclinical pulp inflammation where carious tissues are retained, thus potentially compromising pulp vitality21,22. Additionally, adhesion of bonding resins to sound dentin has shown higher micro-tensile bond strengths compared to caries-affected dentin23,24.

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The use of caries detectors or laser ﬂuorescence during caries removal can be helpful adjuncts to assist the clinician in removing diseased tissues, particularly when close to the pulp cavity25–27. Therefore, the clinician can focus on complete removal of demineralized infected dentin, rather than avoiding pulp exposure, to improve the chances of pulpal repair28. Detectors can create an objective standard for all clinicians during caries removal without reliance exclusively on clinical philosophy or subjective judgement29.

USE OF SODIUM HYPOCHLORITE Sodium hypochlorite is an antimicrobial solution that provides hemostasis, disinfection of the dentin- pulp interface, biofilm removal, chemical removal of the blood clot and fibrin, and clearance of dentinal chips along with damaged cells at the mechanical exposure site30. Examination of pulp tissues after exposure with magnification is a critical step in pulp assessment. Hemorrhage must be controlled to allow clinical assessment of inflammatory levels and identify potential necrotic tissues that require removal before application of an appropriate biomaterial. Hemostasis for the pulp tissue is typically achieved by bathing the resected pulp tissue in sodium hypochlorite for 5 to 10 minutes, although recommended durations may vary, either via direct passive irrigation or on a sodium hypochlorite- soaked cotton pellet31–45. Although several hemostatic options are available, sodium hypochlorite can be used safely in direct contact with pulp tissue at various concentrations, from dilute solutions to full bottle strength, without compromising pulp integrity30,46–48. Sodium hypochlorite has not been shown to adversely alter pulp cell recruitment, cytodifferentiation, and hard tissue deposition49. Sodium hypochlorite also eliminates composite staining, addressing an aesthetic concern.

USE OF CONTEMPORARY MATERIALS IN VPT Calcium silicate cements (CSC) have gained momentum for use in vital pulp therapy (VPT) procedures50,51. CSCs are a class of materials that include tricalcium silicates, dicalcium silicates, hydraulic calcium silicate cements, and “bioceramics.” Clinical outcomes have demonstrated consistent success with these materials and mineral trioxide aggregate (MTA)

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is one of many tricalcium silicates that is widely used and the most extensively studied. When MTA and other CSCs are used for VPT procedures in permanent teeth with symptomatic or asymptomatic irreversible pulpitis, success rates range from 85-100% at 1-2 years26,35,38,42,45,52–54. However, it is noteworthy that calcium hydroxide, glass ionomer cements (GICs) and resin- based materials trail in clinical outcomes and demonstrate a lower range of success varying from 43%-92%42,55,56. Immunomodulatory effects of the new generation of biomaterials provide an added and much needed benefit to their biocompatible, osteogenic and bioactive properties13,57–67. The formation of mineralized barriers using CSCs show improved quality over calcium hydroxidebased materials50,68–70. Silicate materials also possess favorable physicochemical characteristics that include high alkalinity, intratubular mineralization, inhibition of biofilm formation, reduction of robust proinflammatory mediators and postoperative pain during dental pulp procedures57,58,63,70,71. The newer generations of CSCs do demonstrate improved setting times72–74 including modified compositions that reduce tooth discoloration61,71–73,75. The choice of a biomaterial must therefore be made on existing evidence with considerations for patient centered outcomes, reliable mineralized tissue formation and continued pulp vitality.

IMMEDIATE PLACEMENT OF PERMANENT RESTORATIVE MATERIAL Restoration of the teeth is a critical step in endodontic procedures. Immediate restoration should be a part of the restorative treatment plan for a tooth receiving VPT. Teeth undergoing VPT using CSCs as the primary sealing material and restored immediately with a long-term restoration have a high success rate19,41,76–82. Although studies have shown some success with delayed ﬁnal restoration in the short to medium term35,83, long-term assessments have demonstrated that a minimal time span84 between placement of a foundational restoration44,85 after vital pulp treatment is a strong predictor for successful outcomes32–34,36,42,43,51,52,86–94. Indicated advantages of immediate restoration include beneﬁts in the prevention of microleakage, protection of the biomaterial layer, reduction of post-operative sensitivity

and thermal conductivity, and establishment of a foundation for cuspal coverage restoration should it be required. No negative impacts of restoring the teeth immediately have been indicated. An appropriate waiting period is recommended prior to additional tooth preparation for deﬁnitive (cuspal coverage) restoration. A practitioner, using professional judgment and clinical expertise, should consider absence of signs and symptoms and susceptibility of the tooth to fracture to assess whether the tooth is ready for a deﬁnitive restoration after completion of VPT.

SUMMARY The primary goal of VPT procedures is the creation of optimal conditions for pulp tissue repair and preservation. The amount of pulp tissue removed or retained is dependent on tissue viability assessments based on access for visualization to evaluate hemorrhage control and clinical tissue appearance86. A pretreatment diagnosis of irreversible pulpitis is not necessarily an indication for pulpectomy, as more conservative treatment could be considered35,43,44,95,96. Procedural decisions for the amount of pulp tissue retention or removal should be based on operator assessments, clinical judgement, overall treatment plan, and the patient’s general oral and systemic health status. Authors would encourage additional clinical trials to assess long-term outcomes of vital pulp therapy and the development of chairside techniques utilizing biomarkers to assess pulpal viability. A review of the endodontic diagnostic terminology used to classify the severity of pulpal disease is also warranted. Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/j.joen.2021.07.015

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CONSORT RANDOMIZED CLINICAL TRIAL Shazra Fatima, MDS, Ashok Kumar, MDS, Syed Mukhtar Un Nisar Andrabi, MDS, Surendra Kumar Mishra, MDS, and Rajendra Kumar Tewari, MDS

Effect of Apical Third Enlargement to Different Preparation Sizes and Tapers on Postoperative Pain and Outcome of Primary Endodontic Treatment: A Prospective Randomized Clinical Trial

ABSTRACT Introduction: The purpose of this study was to evaluate the effect of apical preparation size and taper on postoperative pain and healing after primary endodontic treatment. Methods: One hundred and twenty patients with asymptomatic mandibular first molars with radiographic evidence of periapical pathology and with a periapical index (PAI) score 3 were randomly assigned to 2 groups, group 1 and 2, based on apical enlargement to 2 and 3 sizes larger than the initial apical binding file (IABF), respectively. Each group was further divided into subgroups A and B depending on the apical enlargement taper of 4% and 6%, respectively. Endodontic treatment was performed, and the final apical enlargement in all the groups was performed as follows: group 1A, 2 sizes larger than the IABF with a 4% taper; group 1B, 2 sizes larger than the IABF with a 6% taper; group 2A, 3 sizes larger than the IABF with a 4% taper; and group 2B, 3 sizes larger than the IABF with a 6% taper. Postoperative pain was assessed at 6, 12, 24, 48, and 72 hours. Clinical evaluation and the change in the PAI score on radiographs were assessed at the 3-, 6-, and 12-month follow-ups. Results: No significant difference in postoperative pain was found. The success rate was lowest (57.1%) in group 1 subgroup A as evidenced by the significant change in the PAI score between group 1 subgroup A and the rest of the groups at the 6- and 12-month follow-ups. Conclusions: Apical preparation to 2 sizes larger than the IABF with a 4% taper is insufficient and results in significantly lower success rates compared with larger preparation sizes and tapers. (J Endod 2021;47:1345–1351.)

KEY WORDS: Apical enlargement; apical periodontitis; periapical healing; postoperative pain; preparation taper

The role of intraradicular microorganisms in the pathogenesis of apical periodontitis is well established1. Endodontic therapy aims to eliminate microbial infection from the root canal system and prevent its reinfection. It is imperative to meticulously debride and disinfect the apical third because this area serves as a niche for the residual microorganisms2. Because of the presence of distinctive anatomic variations, such as isthmuses, ﬁns, ramiﬁcations, and lateral canals, this area has been considered to be a “critical zone”3. Many authors have reported that apical preparation to sizes larger than previously recommended is required to disinfect the canal4–6 adequately. On the other hand, it is

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SIGNIFICANCE Meticulous debridement and disinfection of the apical third of the root canal are critical to achieve success in endodontic treatment. A preparation size and taper that would effectively disinfect the apical third while conserving dentin is still subject to dispute.

From the Department of Conservative Dentistry and Endodontics, Dr Ziauddin Ahmad Dental College, Aligarh Muslim University, Aligarh, India Address requests for reprints to Dr. Syed Mukhtar Un Nisar Andrabi, Department of Conservative Dentistry and Endodontics, Dr Ziauddin Ahmad Dental College, Aligarh Muslim University, Aligarh 202001, India. E-mail address: mukhtarandrabi@gmail. com 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.05.010

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argued that a larger preparation size results in an unnecessary removal of dentin, leading to weakening of the tooth structure. Therefore, a preparation size and taper that would effectively disinfect the apical third of the root canal while conserving dentin are still subject to dispute. Various studies have been conducted to evaluate the effect of apical size enlargement on the bacterial load reduction4,7, cleanliness of the canals8,9, postoperative healing of apical periodontitis10,11, and postoperative pain12,13 after endodontic treatment. The proponents of a larger apical preparation size advocate that it allows better penetration of irrigants and a significant reduction in the number of residual bacteria in the root canal system14. However, Saini et al10 reported that apical enlargement to 3 sizes larger than the first apical binding file is adequate, and any further enlargement does not influence the treatment outcome. Buchanan et al15 also proposed minimal apical enlargement, citing the possibility of adverse procedural errors such as apical transportation and zipping due to extensive apical enlargement. Although apical enlargement offers many biological advantages, it has also been associated with an increased incidence of postoperative pain16. This has been attributed to direct irritation of the periapical tissue or extrusion of the debris, irrigant, and/ or sealer during mechanical instrumentation12. Conflicting results have been reported in this perspective; some related foraminal enlargement to a higher incidence of postoperative pain12, whereas others refuted any relationship between them13. Silva et al13 suggested that apical enlargement has little or no influence on postoperative pain, and it should be used in clinical procedures for better disinfection and treatment outcome. Currently, there is no consensus on the ideal apical preparation size and taper that would result in the least incidence of postoperative pain and promote optimum healing of the periapical tissues. Few randomized controlled trials have been conducted to evaluate the effect of apical enlargement on periapical healing and postoperative pain separately10,12,13,17. To the best of our knowledge, the combined effect of apical preparation size and preparation taper on the incidence of postoperative pain and the outcome of endodontic treatment has not been investigated. The present study was designed as a randomized clinical trial to evaluate the effect of apical preparation size and taper on postoperative pain and healing after primary endodontic treatment.

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MATERIALS AND METHODS This study was conducted at the postgraduate clinic of the Department of Conservative Dentistry and Endodontics, Dr Ziauddin Ahmad Dental College and Hospital, Aligarh Muslim University, Aligarh, India. The study was approved by the institutional ethics committee and subsequently registered at Clinical Trials Registry-India (www.ctri.nic.in, CTRI/2019/09/021135). Patients were recruited from the pool of patients reporting to the Department of Conservative Dentistry and Endodontics between September 2019 and February 2020. Healthy, American Society of Anesthesiologists I, patients of either sex and 14–65 years of age (Table 1) reporting with asymptomatic apical periodontitis in a mandibular first molar with radiographic evidence of periapical radiolucency corresponding to a periapical index (PAI) score 3 were selected for the study. Patients who were medically compromised, pregnant, or had uncontrolled diabetes; smokers; and patients with a history of analgesic intake in the past 1 week were excluded from the study. The study design, clinical procedure, and risk associated were explained to the patients, and an informed written consent was obtained for participating in the trial. An equal proportion randomization allocation ratio was used to randomly assign participants into 2 groups and 2 subgroups. A random number sequence was generated by the central administrative authority (not involved in the study) for particular groups and subgroups, which were assigned sequentially to the patients fulfilling the eligibility criteria. Neither the patient nor the primary investigator was aware of the allocated group until informed consent was obtained from the participant. With the study power 5 0.80, a confidence interval of 95%, standard deviation 6 0.510, and a minimum clinically important difference set at 0.5 for using the PAI and visual analog scale (VAS), a sample size of 50 patients was required per group. To compensate for dropout, a total of 60 patients were enrolled in each group.

Clinical Procedure The preliminary examinations, clinical and radiographic examination, and treatment procedure were performed by a single operator (S.F.). At the first appointment, local anesthesia containing 2% lignocaine with 1:80,000 epinephrine (LIGNOX 2%; Indoco Remedies Ltd, Mumbai, India) was administered, and caries was excavated under rubber dam isolation. The access cavity was prepared using a high-speed handpiece with sterile distilled water as a coolant, and patency was established using a #10 K stainless steel file (Dentsply Maillefer, Ballaigues, Switzerland). Coronal and middle third flaring was performed using Hyflex CM rotary files €tten, (Coltene/Whaledent AG, Altsta Switzerland) with an 8% taper held in an endomotor (Endomate-DT, NSK-Nakanishi Inc, Tochigi, Japan) operating at a speed of 500 rpm and a torque of 1.5 Ncm. Irrigation was performed with 3% sodium hypochlorite (Percan; Septodent, Healthcare Pvt India Ltd, Maharashtra, India) using a 27-G side-vented irrigation needle (S.S White Lakewood, New Jersey). The working length was determined using an electronic apex locator (Root ZX; J Morita MFG Corporation, Fushimi-Ku, Kyoto, Japan) and a diagnostic file radiograph. A small K-file was gently advanced in the root canal until the analog of the electronic apex locator (Root ZX) displayed “Apex.” The length was measured, and 0.5 mm was reduced from the measured length. This length was confirmed using straight and angled radiographs and was opted as the final working length. The first standardized hand K-file that snugly fit at the working length was considered as the initial apical binding file (IABF). Although the determination of the IABF based on tactile sensation has some limitations18, it is still considered to be the most practical method of estimation11–13. Biomechanical preparation was performed using Hyflex CM rotary files with the master apical file size set at 2 sizes and 3 sizes larger than the IABF, respectively, as follows: Group 1A: 2 sizes larger than the IABF with a 4% taper

TABLE 1 - Demographic Characteristics Groups 2 sizes .IABF with a 4% taper 2 sizes .IABF with a 6% taper 3 sizes .IABF with a 4% taper 3 sizes .IABF with a 6% taper Total

Male/female

Age (years)

13/15 16/13 14/15 13/16 56/59

30.8 (14–56) 29.5 (14–59) 30.8 (14–59) 32.3 (14–60) Mean age 5 30.85

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TABLE 2 - P Values of Postoperative Pain of All the Groups Showing an Insigniﬁcant Difference in Postoperative Pain at Different Time Intervals

Time interval P value

Pain at 6 hours

Pain at 12 hours

0.75

0.78

Group 1B: 2 sizes larger than the IABF with a 6% taper Group 2A: 3 sizes larger than the IABF with a 4 % taper Group 2B: 3 sizes larger than the IABF with a 6% taper Once the canals were prepared to the desired size and taper, irrigation with 5 mL 17% EDTA (Anabond Desmear; Deccan Dental Depot Private Limited, Hyderabad, India) followed by a final rinse with 5 mL 3% sodium hypochlorite was performed. Canals were dried with sterile absorbent paper points (Dentsply Maillefer), and calcium hydroxide (SRL Pvt Ltd, Mumbai, India) mixed with normal saline in a pastelike consistency was placed in the canals. The cavity was temporarily restored with an interim restorative material (Cavit; 3M, ESPE, Germany). All the participants were then briefed about the VAS and subsequently trained by a single instructor to mark the intensity of pain experienced by them at 6, 12, 24, 48, and 72 hours. Telephonic assistance was provided to the patients to record postoperative pain on the VAS at different intervals. Patients were instructed to report after 1 week. At the next appointment, patients were asked about any signs and symptoms and clinically verified by checking tenderness on percussion of the tooth. In the absence of any symptoms, the temporary restoration was removed under rubber dam isolation, and the canals were irrigated copiously with 3% sodium hypochlorite to flush out the intracanal medicament. After a thorough cleaning of the canals, irrigation with manual dynamic agitation using a gutta-percha cone was performed using 3% sodium hypochlorite followed by a final rinse with 5 mL normal

Pain at 24 hours 0 .78

Pain at 48 hours

Pain at 72 hours

0.77

0.93

saline. Obturation was performed using guttane/Whaledent AG) and zinc oxide percha (Colte eugenol–based sealer using the cold lateral compaction method. Postendodontic restoration was performed using dental amalgam. Using preset exposure parameters, a postoperative radiograph was taken with the paralleling technique, which served as the baseline radiograph.

Outcome Measurement The assessment of pain at the interval of 6, 12, 24, 48, and 72 hours and the change in periapical radiolucency at the 3-, 6-, and 12month follow-ups were measured as the primary outcome of the treatment. The secondary outcome measurement was based on the clinical success of the case determined by the absence of pain on follow-up visits, the absence of tenderness on percussion or palpation, no associated sinus tract or evidence of soft tissue swelling, lack of abnormal mobility of the tooth, and periodontal probing depth within normal limits (,3.5 mm). Postoperative pain was evaluated using the VAS. All the participants were thoroughly trained for using this scale by the same instructor. The participants were asked to place a line perpendicular to the 10-cm horizontal line at the point that best represented the intensity of their pain. Using a ruler, the distance on the 10-cm line between the “no pain” anchor and the marked line was measured, which provided the range of score from 1–4. The VAS scale was quantiﬁed from 1 (no symptom) to 4 (severe pain or swelling). A

distance measurement of 1–4 mm represented no pain (score 1), 5–44 mm mild pain (score 2), 45–74 mm moderate pain (score 3), and 75–100 mm severe pain (score 4). Patients were contacted telephonically for assistance in reporting pain on the VAS form. Periapical radiolucency was compared using the PAI score19. In case of dissimilar scores in individual roots of mandibular molars, the highest PAI score among the roots was assigned as the overall score. The radiographs were evaluated in a dimly lit room on a view box by 2 independent examiners blinded to the study aim. The radiographs were masked till the apical third of the root canal by placing an opaque cardboard sheet on the view box such that only the periapical area and the apical 2– 3 mm of the root canal were visible. The examiners were precalibrated to the scale using 100 periapical radiographs and the PAI calibration set. To conﬁrm the scores, the investigators were asked to perform the scoring again at an interval of 30 days. In case of a disagreement between the scores, the investigators were asked to meet and discuss the scores until a consensus was reached.

Statistical Analysis The statistical analysis was performed using SPSS software Version 26.0 (IBM Corp, Armonk, NY). The Shapiro-Wilk test was applied to assess the normality of the data. The assessment of the outcome of the treatment and postoperative pain was performed using the Kruskal-Wallis test and the Mann-Whitney U test. The intragroup comparison at different follow-up intervals was performed using the Friedman test. The correlation between age, sex, preoperative lesion size, and postoperative pain and outcome was calculated using the Spearman correlation test. Multinomial logistic regression was applied to determine the association between different independent variables (age, sex, size, and taper) and the outcome of the treatment.

TABLE 4 - Multinomial Logistic Regression to Determine the Association between Variables and the Outcome of the Treatment 95% CI for Exp (B)

TABLE 3 - P Values of the Periapical Index of All Groups at Different Follow-up Intervals Time interval 3 months 6 months 12 months P value

.067

.007*

.001*

*A statistically signiﬁcant difference was revealed in the change in the periapical index at the 6-month and 12month intervals.

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Variables

Sig

Exp (B)

Lower

Upper

Sex Age Size Taper Constant

0.184 0.041 21.627 21.673 2.464

0.564 0.026 0.634 0.638 0.982

0.744 0.110 0.010† 0.009† 0.012

1.202 1.042 0.197 0.188 11.756

0.398 0.991 0.057 0.054 0.991

3.634 1.095 0.681 0.655 1.095

CI, confidence interval; SE, standard error; Sig, significant difference. *Regression coefficient. † Significant difference.

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Enrollment

Assessed for eligibility (n= 120) Excluded (n=0) Randomized (n=120)

Allocation

Group 1 (APS – 2X IABF) Allocated for intervention = 60 Received intervention = 60 Did not receive intervention = 0 Subgroup A (4% taper) n= 30 Lost to followup = 2 (unable to contact)

Subgroup B (6% taper) n= 30 Lost to followup = 1 (unable to contact)

Analyzed (n= 57) Excluded from analysis (lost to follow-up) (n=3)

Group 2 (APS – 3 X IABF) Allocated for intervention = 60 Received intervention = 60 Did not receive intervention = 0 Subgroup A (4% taper) n= 30 Lost to followup = 1 (unable to contact)

Subgroup B (6% taper) n= 30 Lost to followup = 1 (unable to contact)

Analyzed (n= 58) Excluded from analysis (lost to follow-up) (n=2)

APS – Apical preparation size IABF – Initial apical binding file

FIGURE 1 – The Consolidated Standards of Reporting Trials ﬂowchart.

RESULTS One-hundred twenty patients were enrolled in the study. Five patients dropped out. One hundred ﬁfteen patients were evaluated for the assessment of postoperative pain and the outcome of the treatment.

Postoperative Pain The intergroup comparison revealed no significant difference (P . .05) in postoperative pain between both groups at the interval of 6, 12, 24, 48, and 72 hours (Table 2). The intragroup comparison revealed a significant difference in postoperative pain between 6 and 48 hours and between 6 and 72 hours (P , .05) in 2 sizes larger than the IABF with the 4% taper group. Age, sex, and preoperative lesion size did not reveal any significant correlation with postoperative pain.

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PAI Score The cases with a PAI score 2 and with the absence of clinical signs and symptoms at the 12-month follow-up were considered a success. The success rates were 57.1%, 92.8%, 93.1%, and 96.1% in group 1 subgroup A (2 sizes .IABF with a 4% taper), group 1 subgroup B (2 sizes .IABF with a 6% taper), group 2 subgroup A (3 sizes .IABF with a 4% taper), and group 2 subgroup B (3 sizes .IABF with a 6% taper), respectively. Three cases from group 1 subgroup A (2 sizes .IABF with a 4% taper) and 2 cases from group 1 subgroup B (2 sizes .IABF with a 6% taper) did not show a change in the PAI score from baseline to the 12-month follow-up. A signiﬁcant difference (P , .05) in the change in the PAI score was revealed between group 1 subgroup A (2 sizes .IABF with a 4% taper) and the rest of the groups at the 6- and 12month follow-ups (Table 3). The intragroup

comparison revealed a significant difference (P , .05) in the change in the PAI score from baseline to the 12-month follow-up in all of the groups. Multinomial logistic regression analysis revealed a significant association between the master apical file size and the preparation taper and the outcome of the treatment (Table 4). However, no association was revealed between other independent variables like age and sex and the outcome of the treatment.

DISCUSSION The present study was designed as a randomized prospective clinical trial (Fig. 1) evaluating the effect of apical preparation size and taper on postoperative pain and the outcome of primary endodontic treatment. The primary outcome measurement was analyzed in 2 phases.

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assessment of periapical healing was performed using PAI scoring on intraoral periapical radiographs by 2 independent examiners. The examiners were calibrated using PAI calibration set based on the methods of Wang et al23. Substantial agreement between the examiners was observed with an interrater reliability score 5 0.78. The validity of this scale is based on the use of reference radiographs with an established histologic diagnosis19. The objective nature of this scale allows accurate interpretation of the results provided that examiners are properly calibrated to the scale.

FIGURE 2 – Representative images for baseline and 12-month radiographs; (A, B ): 2 sizes . IABF with 6% taper, (C, D ): 3 sizes . IABF with 4% taper. (E, F ): 3 sizes . IABF with 6% taper. 12-month follow-up radiograph shows healing in the periapical lesion in all the groups. In the first phase, postoperative pain after biomechanical preparation was assessed. A statistically nonsignificant difference (P . .05) in postoperative pain was observed at time intervals of 6, 12, 24, 48, and 72 hours among various groups. Extrusion of infected debris, irrigants, or intracanal medicaments in the periapical region has been cited as one of the possible reasons for postoperative pain12. In this study, we limited canal preparation 0.5–1 mm short of the “radiographic apex” while maintaining canal patency with #10 K stainless steel files. This could be a reason for the nonsignificant difference in the incidence of postoperative pain among various groups. Other factors that might have contributed are the absence of preoperative pain, the placement of calcium hydroxide as an intracanal medicament13,20,

and the use of rotary instrumentation21–23. The patients can report a mild degree of pain for reasons other than the endodontic treatment, such as sensitivity due to clamp placement, sustained mouth opening, or local anesthetic injection13. By the end of 48 hours, pain reduced substantially in all the groups (74.6% of patients had no pain and 7.5% of patients had only mild pain). Therefore, a mild degree of pain is an expected outcome after endodontic intervention, and the intensity of pain reduces within the ﬁrst 2 days24. At 72 hours, only 1 patient complained of severe pain in group 1 subgroup A (2 sizes .IABF with a 4% taper), and an emergency intervention was scheduled. The second phase assessed periapical healing after endodontic treatment at follow-up intervals of 3, 6, and 12 months. The

TABLE 5 - Correlation between Age, Sex, and Preoperative Lesion Size and Periapical Healing at Different Time Intervals Variable Age Sex Preoperative lesion size

Correlation coefﬁcient

3 months 2.011 2.037 .250*

6 months .002 2.035 .081

12 months 2.116 .004 .012

In this study, a significantly high success rate was observed with apical preparation 2 sizes larger than the IABF and a 6% taper compared with the same preparation size with a 4% taper (92.8% vs 57.1%). However, the success rate was almost the same when the preparation size was increased to 3 sizes larger than the IABF with a 4% taper (92.8% vs 93.1%, Fig. 2A–F). In the majority of the cases (68.2%), the IABF, determined after coronal preflaring, was a #15 K-file. Therefore, in the present study, the minimum apical preparation size required to disinfect the canals adequately was #25/0.06 or #30/0.04 in the majority of the cases. Further enlargement in preparation size or taper did not significantly affect the outcome of the treatment. The importance of the penetration of irrigants in the apical third for efficient debridement and disinfection cannot be understated. In an in vitro study comparing the effect of apical enlargement on the intracanal bacterial count, Coldero et al (7) pointed out that removing dentin in the apical third may not be necessary if an adequate taper is achieved that allows satisfactory irrigation of the canals. In contrast, many in vitro studies have reported a reduced intracanal bacterial count and endotoxin level with an increased apical preparation size.9,25–28 However, caution must be exercised while applying these results in clinical practice because several additional factors come into play in an in vivo setup that may be absent in a laboratory setup. The significantly high success rate with minimal apical preparation and an increased taper attained in this study may be attributed to many reasons. First, the increased taper allows better penetration of the irrigation needle, permitting efficient debridement of the canals. The volume of irrigant delivered plays a pivotal role in the disinfection of the root canal system29. Second, treatment performed in multiple visits with interappointment antimicrobial dressing may have resulted in effective disinfection of the canals. In a classic histopathologic study, it was demonstrated

*Signiﬁcant difference.

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that an improved microbiological status was achieved by the root canal systems of teeth treated in multiple visits with 1-week interappointment calcium hydroxide dressing21. The anatomic complexities, such as isthmuses, fins, and dentinal tubules, have been shown to harbor bacteria that are inaccessible to the mechanical instrumentation30. In the present study, a greater taper enabled easy placement of intracanal medicament until the apical third of the root canal system, which could have been a contributing factor for the better outcome. Finally, minimal apical enlargement with an increased taper aids in the replenishment of irrigant in the apical third enabled by the loose positioning of the irrigation needle at the junction of the middle and apical third31. Boutsioukis et al31 demonstrated that the increased taper allows improved replacement of the irrigant and results in the distribution of shear stress of the wall apically to the needle tip, which reduces the risk of extrusion of the irrigant. The increased taper with minimal apical enlargement allows effective disruption of the vapor lock effect by mechanical agitation with gutta-percha cones that snugly fit at the apical foramen while at the same time providing space for the irrigants to escape coronally. Preoperative factors, such as the size of the periapical lesion, are known to affect the outcome of the root canal treatment10,32 (Table 5). In order to minimize the effect of lesion size on healing, only mandibular first molars with periapical pathology 2 mm and

5 mm were included in the study. Mandibular first molars were selected for the study because they are the most commonly involved teeth with pulpal and periradicular disease. The periradicular area of mandibular first molars can be predictably and repeatedly radiographed by the paralleling technique, making PAI scoring easier. Significantly better periapical healing has also been associated with certain postoperative factors, such as the extent and quality of obturation and optimum coronal restoration after endodontic treatment32. In this study, the obturation was terminated within 0.5–2 mm of the apex. The quality of the coronal restoration was verified postoperatively and at every follow-up interval by the operator as well as the supervisor of the study. Before deriving conclusions from the results, the limitations of this study must be taken into consideration. The smaller sample size, shorter follow-up period, use of periapical radiographs for determining success, and determination of apical enlargement size based on the IABF may be considered as the limitations of this study. Studies with a larger sample size and longer follow-up periods are required to prove the association between apical enlargement and the outcome of the treatment. Also, 3-dimensional imaging may be better for evaluating the change in periapical radiolucency. The IABF may not represent the actual diameter of the apical region because of the high anatomic variability of the apical third of the root canal system18. However, early coronal preflaring enhances the

tactile sensation of the apical constriction and diameter by reducing ﬁle contact with the canal walls and providing straight-line access to the coronal and middle thirds33. The determination of the IABF after coronal preﬂaring might provide a better estimation of the apical diameter, as done in the present study.

CONCLUSIONS Within the limits of this study, the following conclusions can be drawn: 1. Postoperative pain is not influenced by the apical preparation size and taper of the instrument. 2. Apical preparation to 2 sizes larger than the IABF with a 4% preparation taper is insufficient and results in a lower success rate compared with preparations done with larger sizes and tapers. 3. The minimum apical preparation size required to adequately disinfect the canals was #25/0.06 or 30/0.04 in the majority of the cases. 4. Further enlargement of the apical third to larger sizes and tapers does not result in a further significant improvement in the success rate of the treatment.

ACKNOWLEDGMENTS The authors deny any conﬂicts of interest related to this study.

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REVIEW ARTICLE Anita Aminoshariae, DDS, MS,* Jim Kulild, DDS, MS,† and Venkateshbabu Nagendrababu, BDS, MFDS RCPS(Glasg), MDS, PhD‡

SIGNIFICANCE AI can be used in clinical scenarios like appointments, scheduling, diagnosis, treatment, and disease prediction in endodontics. AI models are designed to provide guidance and support to a dentist in his or her clinical practice.

Artificial Intelligence in Endodontics: Current Applications and Future Directions ABSTRACT Introduction: Artificial intelligence (AI) has the potential to replicate human intelligence to perform prediction and complex decision making in health care and has significantly increased its presence and relevance in various tasks and applications in dentistry, especially endodontics. The aim of this review was to discuss the current endodontic applications of AI and potential future directions. Methods: Articles that have addressed the applications of AI in endodontics were evaluated for information pertinent to include in this narrative review. Results: AI models (eg, convolutional neural networks and/or artificial neural networks) have demonstrated various applications in endodontics such as studying root canal system anatomy, detecting periapical lesions and root fractures, determining working length measurements, predicting the viability of dental pulp stem cells, and predicting the success of retreatment procedures. The future of this technology was discussed in light of helping with scheduling, treating patients, drug-drug interactions, diagnosis with prognostic values, and robotic-assisted endodontic surgery. Conclusions: AI demonstrated accuracy and precision in terms of detection, determination, and disease prediction in endodontics. AI can contribute to the improvement of diagnosis and treatment that can lead to an increase in the success of endodontic treatment outcomes. However, it is still necessary to further verify the reliability, applicability, and cost-effectiveness of AI models before transferring these models into day-to-day clinical practice. (J Endod 2021;47:1352–1357.)

KEY WORDS Artiﬁcial intelligence; artiﬁcial neural networks; convolutional neural networks; endodontics

From the *Department of Endodontics, Case School of Dental Medicine, Cleveland, Ohio; †Department of Endodontics, University of MissouriKansas City School of Dentistry, Kansas City, Missouri; and ‡Department of Preventive and Restorative Dentistry, College of Dental Medicine, University of Sharjah, Sharjah, United Arab Emirates Address requests for reprints to Dr Anita Aminoshariae, Department of Endodontics, Case School of Dental Medicine, 10900 Euclid Avenue, Cleveland, OH 44124. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.003

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Artificial intelligence (AI) is a branch of applied computer science that was first described by John McCarthy in 19561. AI has been described as the "fourth industrial revolution," which uses computer technology to simulate intelligent behavior, critical thinking, and decision making similar to humans2,3. AI has been shown to improve efficiency, accuracy, and precision similar to medical professionals in a more timely manner at a lower cost3. In health care, 2 types of AI are available: virtual and physical (robotics)4. The virtual type deals with mathematical algorithms used for diagnosis and prognosis5, imaging and osteoporosis6, scheduling appointments7, drug dosage algorithms8, drug interactions9, and electronic health records4. The physical aspect includes robotic assistance in surgery10, telepresence11, rehabilitation12, and socially assistive robots in the care of the elderly13. AI can exist of expert systems whereby a system is built to follow rules, sometimes fuzzy rules, created by a domain expert2. In machine learning, software algorithms are used that can learn relationships from examples without explicit instructions14,15. Although there are unsupervised learning systems that can identify clusters (eg, a system to identify different patient phenotypes from data in endodontics), most applications in dentistry use supervised learning in which training data include many samples, each with a variety of characteristics or features (eg, images of a patient, sex, age, number of caries, and more) and a ground truth determination (eg, endodontic visit or no endodontic visit)2. The AI algorithm learns the relationship between the features and ground truth using standard approaches (eg, random forest or support vector machine) or artificial neural networks (ANNs), which mimic a system of biological neurons with many neural connections that are adapted in “learning.”

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With the advent of advanced graphics cards and algorithmic innovations, deep learning networks are now used. In deep learning, one uses ANNs with many layers of neurons, generating millions of neural connection weights to be adapted and enabling the system to learn very nonlinear relationships between features and ground truth determinations16. In imaging, because of the size of the data and because the actual feature of interest might be small (eg, the apex), convolutional neural networks (CNNs) are used whereby a network examines a subregion of the image that is moved about the image to make determinations17. In essence, CNNs process multiple images and use filtration to analyze those images18. Ethical challenges and potential risks are emerging legal considerations that add to the complexity of the system19. Applications of AI in dentistry have not been routine in dental practices20. However, the advent of these technologies has influenced imaging and pathology21, dental image diagnostics18,22, caries detection23, electronic records24, and robotic assistance25. AI systems have the potential to revolutionize the field of medicine and dentistry by identifying solutions in managing multiple clinical problems that have also made clinicians’ tasks easier. AI research in endodontics has grown in parallel to other specialties in dentistry. The knowledge of the endodontist has to be updated in regard to the application of AI. Hence, the aim of this review was to discuss the literature related to the applications of AI in terms of diagnosis, clinical decision making, and predicting successful treatment in endodontics and, additionally, to identify existing gaps in the application of AI.

CURRENT APPLICATIONS OF AI IN ENDODONTICS Detection AI has been used in endodontics mostly by virtual employment such as the detection of periapical lesions, crown, and root fractures; working length determination; and morphology detection. These procedures describe the virtual aspect of AI (Table 1).

Detection of Periapical Lesions The diagnosis and treatment planning of teeth with periapical lesions and/or symptoms can be challenging to clinicians. Apical periodontitis is a common disease that comprises approximately 75% of radiolucent jaw lesions26. Early detection might increase successful treatment outcomes27, avoid spreading of the disease to surrounding

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tissues, and minimize further complications28. The most common 2-dimensional diagnostic aids used to identify apical periodontitis in dayto-day clinical practice are intraoral periapical radiographs and panoramic radiographs. Periapical lesions are usually noted as radiolucencies in the radiographs. However, information obtained from periapical radiographs is not accurate because the actual 3-dimensional anatomy is converted into a 2-dimensional image29. Hence, cone-beam computed tomographic (CBCT) imaging, a 3-dimensional imaging technology, was developed and has been used to accurately detect periapical lesions as well as their size and location. A meta-analysis reported that the accuracy values for detecting periapical lesions were 0.96, 0.73, and 0.72 for CBCT imaging, conventional periapical radiography, and digital periapical radiography, respectively30. However, the use of CBCT imaging resulted in less accuracy in diagnosing apical periodontitis in root-ﬁlled teeth31. Endres et al32 reported that a deep learning algorithm model can match the diagnostic performance of 24 oral and maxillofacial surgeons in detecting periapical radiolucencies on panoramic radiographs. One study compared the ability of CNN models with 3 oral and maxillofacial radiologists in detecting simulated periapical lesions on intraoral radiographs. They concluded that the mean values of sensitivity, speciﬁcity, and area under the receiver operating characteristic curve per fold were greater in the CNN group compared with oral and maxillofacial radiologists’ interpretations33. Ekert et al34 reported that the potential of deep CNNs was accurate in the discriminatory ability to detect apical lesions on panoramic radiographs compared with dentists with more than 10 years of clinical experience. However, both the studies were conducted with a limited sample size and used panoramic radiography, a tool used very infrequently by endodontists for diagnosis33,34. Generally, the detection of a periapical lesion by a radiograph is subject to large variations between examiners, and discriminatory ability is based highly on an examiner’s experience34. The variations between the examiners and bias can be reduced by using AI systems27,34. Deep learning segmentation proved to have excellent accuracy in detecting a periapical lesion on CBCT images27. The reliability of correctly detecting a periapical lesion from CBCT images by a CNN system was around 92.8%. Some authors have also reported that volume measurements

conducted by a deep CNN system and humans were similar35, whereas the volume deviation of lesions has not been considered, which might affect the reliability of the study2. The application of AI systems in detecting a periapical lesion from radiographs and CBCT scans might improve reliability and aid clinicians to reach detection accuracies similar to, or superior to, experienced specialists27,32,34,35. Additionally, it may reduce the diagnostic efforts of the dentist by saving assessment time and allowing semiautomated documentation. However, the sensitivity of AI systems should be improved, and additional studies should be conducted before its clinical application34. Poswar Fde et al36 reported on different gene expressions for a periapical cyst versus a periapical granuloma. The authors analyzed the gene expression to differentiate between a cyst and a granuloma using a multilayer perceptron neural network for gene classification. The limitation of the study was that some data in the algorithm might not be present. The algorithm did not differentiate between physiological and inflammatory cytokines. However, the authors proposed that this methodology might be useful for distinguishing other biological processes (eg, biomarkers for cancers). Zheng et al37 compared an anatomically constrained Dense U-Net with existing biomedical image analysis algorithms regarding lesion detection accuracy and dice coefficient indices of a multilabel segmentation. Despite a small sample size, the authors reported that the novel deep learning algorithm allowed CBCT segmentation and the detection of pathosis with an increase in sensitivity and specificity.

Detection of Root Fractures Vertical root fractures (VRFs) represent 2%–5% of crown/root fractures and are considered a serious complication that could result in either root resection or tooth extraction38,39. Radiographs and CBCT imaging help in detecting a VRF that can be difficult to diagnose. The lack of a definitive diagnosis may result in an unnecessary surgical procedure or tooth extraction. The clinical presentation and low sensitivity of conventional radiographs in the detection of VRFs frequently pose a diagnostic dilemma for a clinician. In their meta-analysis, Talwar et al39 reported that CBCT imaging was better in detecting VRFs in unfilled teeth compared with radiographs, whereas radiographs were marginally better than CBCT imaging in root-filled teeth. Because of the inability of conventional methods to accurately detect VRFs, there has been a call for the development of alternative methods to improve

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TABLE 1 - Current Applications of Artiﬁcial Intelligence (AI) in Endodontics Aim

AI methods

Detection of periapical lesions

ML, CNN, DL

Detection of periapical cyst or granuloma

ML, ANN

Detection of root fractures

ML, CNN, PNN

Working length determination

ML, ANN

Determination of morphology

DL, ML, CNNs

Prediction of retreatment

ML, CBR

Prediction of stem cell viability

ML, ANN (NFIS)

Results The periapical lesion from radiographs and CBCT scans detected by AI might improve reliability and allow any clinician to reach diagnostic accuracies similar, or superior to, experienced specialists. The gene expression was analyzed to differentiate between a cyst and a granuloma using AI. AI might be helpful in the detection of root fractures. Research in this area is promising and ongoing. AI might be helpful as an adjunct in aiding clinicians with working length determination. The algorithm developed by AI and information analysis was reported to measure the root canal curvature and its 3-dimensional modiﬁcation after the instrumentation. The combination of methods was able to predict statistical probabilities for extraction. NFIS predicted cell viability after various regenerative protocols and challenges with microbial infection.

ANN, artiﬁcial neurons network; CBR, case-based reasoning; CNN, convolutional neurons network; DL, deep learning; ML, machine learning; NFIS, neuro-fuzzy inference system; PNN, probabilistic neural network.

the diagnosis of VRFs. f*ckuda et al40 reported that CNNs may be a promising tool to detect (recall 5 0.75 [sensitivity], precision 5 0.93 [positive predictive value], and F measure 5 0.83 [index used to evaluate machine learning performance]) VRFs on panoramic radiographs. Another study sought to develop a probabilistic neural network to diagnose VRFs in intact and root-filled teeth on periapical radiographs and CBCT images41. They concluded that the detection of a root fracture on CBCT images is better in terms of accuracy, sensitivity, and specificity compared with images from 2-dimensional radiographs. However, this conclusion was made from an analysis of single-rooted premolar teeth. Future research should be conducted to study the ability of a probabilistic neural network to detect vertical root fractures in multirooted teeth. Using synthetic data, Shah et al42 created cracks in second molars and analyzed them with wavelets. These mathematical functions allow weak signal recovery from noisy environments in a machine learning approach. Despite a small sample size, cracks were reliably detected with high-resolution CBCT images using steerable wavelets. The authors proposed that the validity of this technique needed to be confirmed by ex vivo and clinical methodologies. In an ex vivo

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experiment, Vicory et al43 simulated microfractures in 22 teeth with 14 teeth kept as the negative control group. Using wavelets and machine learning, the authors reported that a micro–computed tomographic image was more accurate than CBCT images in detecting fractured teeth. The authors reported that the positive predictive value of machine learning was superior to the observers’ interpretation, but there was room for improvement. The authors stated that future research should have a larger sample size.

Working Length Determination Correct working length determination is an important step in achieving success in root canal treatment outcomes. Various methods used for working length determination have included radiographic, digital tactile sense, the patient’s response to a ﬁle or paper point inserted into a root canal system, electronic apex locators, and CBCT imaging44–47. In general, radiography and electronic apex locators are the most common methods used routinely by clinicians in dental practice. In digital radiography, the quality of the images plays a key role in the accurate interpretation of root canal system morphology48. However, various other factors inﬂuence radiographic interpretations, which could result in an

incorrect diagnosis49. Hence, a need arises to develop computer-based methods to determine consistently accurate working lengths. Saghiri et al47 reported that ANNs can be used as a second opinion to locate the apical foramen on radiographs, which can ultimately improve the accuracy of working length determination. In another study, Saghiri et al50 investigated the accuracy of working length determination by an ANN in a human cadaver model to mimic the clinical situation. They found no difference in root length measurements when comparing an ANN with actual measurement after extraction. They also reported that the ANN (96%) performed superiorly in minor anatomic constriction determination compared with an endodontist (76%) using periapical radiographs. Hence, an ANN can be considered as an accurate method for working length determination.

Root and Root Canal System Morphology Knowledge of root and root canal system variations is an important factor that influences the success of nonsurgical root canal treatment. Generally, periapical radiographs and CBCT imaging have been used for this purpose. CBCT imaging has demonstrated higher accuracy to assess the root and root canal configurations compared with radiographs. However, because of radiation issues, it cannot be recommended in routine clinical practice. Hiraiwa et al17reported that the deep learning system on panoramic radiographs demonstrated high accuracy in the differential diagnosis of a single or multiple root(s) in the distal roots of mandibular first molars. Learning models were created by extracting image patches from panoramic radiographs and inputting them into deep learning systems. The algorithm developed by AI and information analysis demonstrated the ability to measure the root canal curvature and its 3-dimensional modification after the instrumentation51. However, more studies are required to confirm the results of this study. Lahoud et al52 reported an automated 3-dimensional tooth segmentation using the CNN approach. The authors evaluated 433 CBCT radiographic segmentations of teeth in a timely, accurate, and efficient clinical reference and reported that AI performed as good as a human operator but much faster. In another study but with panoramic radiography, the authors combined 2 deep CNNs and expert refinement53. Using 153 panoramic radiographs, the authors reported that the AI tool yielded a high sensitivity and specificity in a very fast performance for the detection and segmentation of teeth.

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Predictions Retreatment Predictions Campo et al54 reported on a case-based reasoning paradigm to predict the outcome of nonsurgical root canal retreatment with risks and beneﬁts. The system in essence reported whether one should perform retreatment or not. The system included data from areas such as performance, recall, and statistical probabilities. The strength of the system is that it might be able to accurately predict the outcome of retreatment. The limitation was that the system would be only as good as the information in the data. Case-based reasoning is the process of creating solutions to problems based on a previous encounter with similar past problems. By retrieving similar cases, important information and knowledge can be integrated. The issue with variability and the abundance of different approaches could create heterogeneity with this system55. It is important that future articles take this heterogeneity of a human approach under consideration and perhaps increase the sample size to achieve better sensitivity, speciﬁcity, and accuracy.

Predicting the Viability of Stem Cells Bindal et al56 evaluated the dental stem cells isolated from the dental pulp in various regenerative therapies using the neuro-fuzzy inference system. In a simulated clinical scenario, this system predicted the outcome by testing the viability of the stem cells after treatment with bacterial lipopolysaccharides. The neuro-fuzzy inference system was a tool to predict cell viability after various regenerative protocols that are challenged with microbial infection56. The authors measured the viability

of dental pulp stem cells after lipopolysaccharide treatment to induce an inﬂammatory reaction. The authors then analyzed the accuracy level of the outcome using adaptive neuro-fuzzy interferences to predict cell viability of these stem cells after microbial invasion.

Gaps in AI and Endodontic Treatment To date, there is no 1. Programming technology in appointment scheduling, patient management, and recall; as decisions on scheduling, appointment, delegation, and recall are constantly updated to meet the demands of the health care system, AI can schedule patient treatments based on the continuous needs and acquired medical information. 2. Procedure to inform the clinician about drug interactions and/or treatment adjustments based on the available electronic health record; as the population grows and more people live longer, they are taking more medications. If health care records can be made available, there is a potential for AI to predict patient-speciﬁc drug-drug complications. 3. Procedure to provide an accurate endodontic diagnosis based on medical, dental, and clinical ﬁndings; based on the acquired information and data collection, AI would be able to improve diagnosis and staging and to predict outcomes. This would include outcome prediction or prognostic risk determination. 4. Accurate physical and robotic microsurgical treatment available to the

endodontist; in implant technology, robotic-assisted dental surgery can help the surgeon with proper navigation of the implant placement57. It would be expected that the same system would assist endodontists in navigation in endodontic surgery. The authors reported that the mean deviations of the implant robotic placement were as accurate as both static and dynamic navigations, but to date no comparative cohort studies have been done comparing robotic technology with traditional endodontic surgery or treatment. Future studies should compare the different techniques in accuracy and safety with a robotically guided placement.

CONCLUSION In endodontics, AI might aid in clinical applications, particularly in the detection of periapical pathosis, root fractures, determination of working length, and prediction of disease. There is a need for highquality evidence to evaluate the performance of AI regarding its reliability, applicability, legal and ethical considerations, and costeffectiveness before widespread adoption into routine clinical practice.

ACKNOWLEDGMENTS The authors would like to thank Dr. David Wilson, Professor, Biomedical Engineering at CWRU for his invaluable input with this manuscript. The authors deny any conﬂicts of interest related to this study.

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REVIEW ARTICLE Giorgos N. Tzanetakis, DDS, MSc, MSc, PhD,* and Despina Koletsi, DDS, MSc, DMD, DLSHTM, PGCHEd†

SIGNIFICANCE Overreliance on P values to present the results of clinical trials may prove misleading and hamper the power calculations of further studies. Extensive reporting of uncertainty bounds such as conﬁdence intervals shall enhance the transparency and credibility of research ﬁndings.

Uncertainty Bounds in Clinical Trials Published in Endodontic Journals within the Last 5 Years: Are We Confident on What We Read? ABSTRACT Introduction: The aim of this study was to assess the prevalence of reporting of confidence intervals (CIs) in clinical trials in endodontics and discover any further associations with a range of publication characteristics. Methods: The electronic contents of the 3 leading endodontic journals with the highest impact factors (International Endodontic Journal, Journal of Endodontics, and Australian Endodontic Journal) were assessed from January 2016 to December 2020. The number and proportion of clinical trials reporting CIs for the difference in effectiveness/safety of competing interventions for the outcome of interest were recorded. Associations with journal, year of publication, study design, use of analyses for more complex sets of data, and others were assessed. Univariable and multivariable logistic regressions were used to identify significant predictor variables. Yearly linear trend effects were also sought. Results: A total of 141 reports of clinical trials were identified. The majority were published in the Journal of Endodontics (90/141, 63.8%) followed by the International Endodontic Journal (41/141, 29.1%). CI reporting was confirmed only for 29.1% of the sample of reports (41/141). There was strong evidence that reports of clinical trials including analyses of more complex sets of data presented 11.47 times higher odds for CI reporting (adjusted odds ratio 5 11.47; 95% CI, 4.19–31.41; P , .001). Conclusions: The inclusion of uncertainty measures as represented by the reporting of CIs is suboptimal within endodontic clinical trial reports. (J Endod 2021;47:1358–1364.)

KEY WORDS Clinical trials; conﬁdence interval; endodontic research; P value; randomized controlled trials

From the *Department of Endodontics, School of Dentistry, National and Kapodistrian University of Athens, Athens, Greece; and †Clinic of Orthodontics and Pediatric Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland Address requests for reprints to Dr Giorgos N. Tzanetakis, 421B Mesogeion Avenue, 15343 Agia Paraskevi, Athens, Greece. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.04.025

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Clinical trials are considered the cornerstone of field research and are designed to assess the effectiveness and/or safety of an intervention of interest; specifically, randomized controlled trials (RCTs), if designed, conducted, and reported adequately, bear the potential to represent the backbone of evidence-based clinical decision making1,2. The determination of whether a treatment delivered to patients is effective and safe largely depends on hypothesis testing and statistical inference, which are in turn dominated by the presence and selection of P values by the scientists up-to-date and across the entirety of biomedical clinical research. Hypothesis testing, initially described by Neyman and Pearson in 19333, has been extensively used by authors to examine a null hypothesis (H0) of no effect and to test the validity of an alternative hypothesis with some effect. The critical P value level is frequently reported as the cutoff point below which the authors may allow themselves to decide on whether an intervention, medication, or treatment is more effective than another and draw conclusions based solely on “statistical significance” of the results. A P value 5 .05, which is the most frequently used threshold, means that there is 5% probability to observe the recorded treatment effect/difference or a more extreme one between the sampled study groups when in reality no difference exists between the population groups. Such values and thresholds have been largely criticized as being potentially misleading4,5 with questionable use and aptitude to provide insights to the clinical relevance of a treatment effect, disregarding its size and range of possible values across similar studies6,7. P values are used to substantiate a statistically significant difference, whereas their

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importance and interpretation on the clinical relevance of a finding are limited. Thus, informing decision making for the effectiveness or safety of a treatment based on the perceived P value–derived statistical significance might be erroneous and misleading8,9. Essentially, P values are largely influenced and dependent on the studied sample size and the variance of an effect. In this respect, reporting of measures that would include an uncertainty estimate of the effect size in a specific clinical trial appears intuitively more straightforward; this pertains more directly and most importantly to clinical research, which is considered the gold standard when attempting to inform decisions grounded on the effectiveness of a treatment. The confidence interval (CI) is a measure of such an uncertainty about the true effect in a population. In other words, the CI reveals how confident we are that the identified effect is real or whether the data are compatible with a clinically relevant effect10. As with the arbitrary threshold of .05 for the P value, the 95% CI is usually considered, and the interpretation is derived as follows: if we draw 100 samples from the population of interest (or if we run a similar study 100 times), 95 of them (or 95% of them) would include the true population effect. Thus, the interpretation of the results through the CIs is not largely based on a binary decision about statistical significance; rather, it is based on the effect size of a treatment effect and a perceived limit of plausible values. The CI (also referred to as the frequentist CI) should be differentiated from the credible interval (CrI) (also known as the CI of the Bayesian statistical approach). As noted previously, the frequentist CI bases its interpretation on random sampling from a target population and the confidence we have to the true (but unknown) estimate. Similarly, the CrI is a measure of uncertainty in Bayesian inference, which is analogous to the CIs in the frequentist approach. In brief, the perceived superiority of the CrI relies on the more direct interpretation of the latter, where the values of the parameter of interest are derived directly from a posterior distribution that reflects the population distribution, which is aligned to evidence provided by the collected data. Notwithstanding their theoretical advantages, Bayesian statistical inference and approaches are not yet widely used in dentistry and beyond; thus, the scope and focus of the present study are on the frequentist CI11. Prior research within dentistry has revealed that the reporting of CIs in the results of clinical trials ranges between 7% and 29%, thus reflecting a rather bleak picture overall within the fields of orthodontics, prosthodontics, endodontics, oral and

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maxillofacial surgery, periodontology, and implantology. Apparently, the figures may vary slightly across different domains and years of publication of trial findings7,12–16. Evidence from the quality of statistical reporting in leading dental journals has also revealed that only about 10% of articles published in 2010 were free from any problem related to the reporting of statistics. However, CIs were not analyzed at that time17. Although orchestrated efforts have been attempted to enhance the quality of reporting of the existing literature in endodontics following meta-epidemiologic endeavors in dentistry18,19, there is currently scarce evidence on the reporting of CIs versus P values pertaining to endodontic clinical research specifically. Therefore, the aim of the present empirical study was to document the prevalence of the reporting of CIs in clinical trials in endodontics and also to determine associations with publication and study design characteristics.

MATERIALS AND METHODS The electronic contents of the 3 major journals with the highest impact factors in 2019 according to Journal Citation Reports (Clarivate Analytics) pertaining to endodontic research were searched between January 1, 2016, and December 31, 2020, to identify original publications of prospective clinical trials or RCTs and assess whether they included the reporting of CIs. These journals were the International Endodontic Journal (IEJ), Journal of Endodontics (JOE), and Australian Endodontic Journal (AEJ). A total of 2187 articles were screened for eligibility after a priori exclusion of editorials, commentaries, and letters to the editor. Designs other than interventional pertaining to clinical trials as described previously were excluded. Data extraction was performed in preformulated piloted forms, and calibration was conducted between the 2 assessors (G.N.T. and D.K.) after initial piloting with regard to outcome classification (ie, the reporting of CIs or otherwise). In addition, a range of publication characteristics was assessed including the journal, year of publication, study design (randomized or nonrandomized prospective clinical trials), continent of authorship based on the affiliation of the first author, number of participating researchers as identified in the author list, number of centers involved (single or multicenter based on affiliation details and additional details about the place of the study within the Materials and Methods section), statistical significance of the primary outcome of interest, and the use of more sophisticated

analytical techniques pertaining to complex sets of data, other than pair-wise or simple inferential statistics, to statistically analyze the ﬁndings of the clinical trial reports. Examples of such techniques comprise multivariable and multivariate statistics, including simultaneous assessments of multiple risk factors and/or outcomes as well as multilevel hierarchical models that incorporate data clustering and longitudinal/panel data.

Statistical Analysis Descriptive statistics were initially conducted to analyze the characteristics of the sample of trials identified with regard to the aforementioned variables. Cross-tabulations were constructed, and Pearson chi-square and Fisher exact tests were conducted to assess the association between the reporting of CIs and publication characteristics. Univariable and multivariable logistic regressions were performed to examine the effect of publication characteristics, including the journal, year of publication, study design, inclusion of sophisticated statistics, and others on the reporting of CIs for the effect estimate of the outcome of interest. Potential predictors were examined sequentially 1 at a time in the crude model and retained in the final multivariable logistic regression model if P was , .10. Odds ratios and respective 95% CIs were presented as relevant effect measures. A Wald test was used to record significant predictors. Departure from the linear trend was explored for the effect of the year on CI reporting through the likelihood ratio test. The Hosmer-Lemeshow test was used to check the model fit. The unweighted kappa statistic was used to assess interrater agreement on the outcome of interest (the reporting of CIs), and a perfect agreement was deemed (kappa 5 1.0) possible, specifically due to the “hard” nature of the examined outcome. Any preliminary disagreement at the piloting stage and before calibration was settled through discussion and the achievement of a consensus between the 2 investigators. The predefined level of significance was set at P , .05 (2-sided). All analyses were conducted with Stata version 15.1 (StataCorp, College Station, TX).

RESULTS One hundred forty-one prospective clinical trials were included in the present empirical report, both randomized and not (Fig. 1). JOE published the highest number of clinical trials (90/141, 63.8%) followed by IEJ (41/141, 29.1%) and AEJ (10/141, 7.1%). The largest number of clinical trial reports was published in 2018 (39/141, 27.7%), with a declining

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FIGURE 1 – A flowchart of the study selection. number in both 2019 (19/141, 13.5%) and 2020 (23/141, 16.3%). The vast majority of the clinical trials identified followed a randomized design (121/141, 85.8%); most were led by institutions originating outside America or Europe (78/141, 55.3%) and coauthored more frequently by more than 6 authors (60/141, 42.6%). Statistical significance for the primary outcome was confirmed for 84 of 141 reports (59.6%), whereas univariable statistics was the most prevalent pattern of analytical techniques described for data management within the published studies (98/141, 69.5%) (Table 1). Overall, less than one third of the clinical trials published during the last 5 years in major endodontic journals included the reporting of CIs for the effect estimate of the outcome under study (41/141, 29.1%), whereas the differences across the journals were not statistically significant (Table 1). The most recent 3 years (ie, 2018, 2019, and 2020) have demonstrated an approximately 2-fold or 3fold increase in the percentage of trials including the reporting of CIs compared with earlier reports from 2016 and 2017 (P 5 .04) (Table 1). A large number of researchers included in the author list (6) was associated

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with increased probabilities of CI reporting (P 5 .02). The use of more sophisticated analytical techniques pertaining to complex sets of data was more likely to include and report the respective CIs (P , .001) (Table 1). According to the multivariable model, reporting of the trial’s results after performing a more complex analytical plan ensued 11.47 times higher odds for the reporting of CIs (odds ratio 5 11.47; 95% CI, 4.19–31.41; P , .001) conditional on the year of publication, the continent of origin, and the number of authors involved (Table 2). Departure from the linear trend for the effect of the year of publication on CI reporting was conﬁrmed (likelihood ratio test, P 5 .03); thus, the results are presented across each year category.

DISCUSSION Findings in Context The ﬁndings of the present empirical study highlighted the current state of evidence on CI reporting in clinical endodontic research. Apparently, a large number of published reports, pertaining to approximately 70% of the identiﬁed research, ignore the value and

importance of uncertainty bounds for the presentation of the results on the effectiveness and/or safety of competing treatment modalities. Overreliance on P values in clinical trials within endodontic research was evident. To further elucidate P value use and interpretation, an example is presented as follows: let us assume that researchers are interested in the identification of the most effective apexification treatment procedure during a 12-month period by examining the number of failed cases of immature traumatized anterior teeth after assessing the findings of 2 trials comparing 2 different clinical procedures (A and B). In trial 1, only 5 of 100 patients with procedure A had failure versus 15 of 100 with procedure B (Fisher exact test: P 5 .03); in trial 2, which included 500 patients per group, 110 with procedure A versus 150 with procedure B were recorded to present failures (Fisher exact test: P 5 .03). We can see that both trials report a statistically significant difference between the 2 apexification treatment procedures at the same critical level (P 5 .03) and thus might be interpreted similarly. However, we cannot be confident that the clinical treatment effect is the same because an increase of just 1 failed event for the procedure A in trial 1 will result in a P value of .06, which would be considered nonsignificant and alter our previous interpretation. Instead, the same increase in the second trial would not alter our inference because the P value of the test statistic would remain nearly as is. The unraveling importance of presenting CIs along with the effect estimates for a perceived difference between interventions has been widely identified within biomedical literature for a long time10. Aligned with the principles of statistical inference, the CI is considered to provide an estimate of uncertainty and imprecision around the retrieved effect in a given study. If we conduct a number of similar studies under the exact same research question, then due to sampling variation, we shall get a “cloud” of effect sizes around the true population effect. However, the true effect is largely unknown because it is practically impossible to assess the entirety of the population, and so the CI represents a range of values among which we may be 95% confident that the true population value resides. The common selection of 95% interval follows the arbitrary 5% of significance for the P value threshold. To stress the importance and clinical relevance of CIs, let us consider the following example: a randomized trial comparing the effect of 2 different materials used for pulpotomy in mature teeth on successful treatment outcome has identified that material

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TABLE 1 - The Frequency Distribution for the Reporting of Conﬁdence Intervals (CIs) in Clinical Trials Pertaining to Endodontic Research across Article Characteristics Representing Background Variables (N 5 141) CI reporting

Journal AEJ IEJ JOE Year 2016 2017 2018 2019 2020 Study design RCT Nonrandomized PCT Continent America Europe Asia/other No. of authors 1–3 4–5 6 No. of centers Single center Multicenter Signiﬁcance No Yes Statistical analysis Univariable Complex/ multivariable Total

No, n (%)

Yes, n (%)

Total, N (%)

9 (90.0) 26 (63.4) 65 (72.2)

1 (10.0) 15 (36.6) 25 (27.8)

10 (100.0) 41 (100.0) 90 (100.0)

23 (76.7) 27 (90.0) 24 (61.5) 12 (63.2) 14 (60.9)

7 (23.3) 3 (10.0) 15 (38.5) 7 (36.8) 9 (39.1)

30 (100.0) 30 (100.0) 39 (100.0) 19 (100.0) 23 (100.0)

87 (71.9) 13 (65.0)

34 (28.1) 7 (35.0)

121 (100.0) 20 (100.0)

P value .26*

.04*

.53†

.04† 24 (63.2) 14 (56.0) 62 (79.5)

14 (36.8) 11 (44.0) 16 (20.5)

38 (100.0) 25 (100.0) 78 (100.0)

25 (89.3) 39 (73.6) 36 (60.0)

3 (10.7) 14 (26.4) 24 (40.0)

28 (100.0) 53 (100.0) 60 (100.0)

52 (73.2) 48 (68.6)

19 (26.8) 22 (31.4)

71 (100.0) 70 (100.0)

41 (71.9) 59 (70.2)

16 (28.1) 25 (29.8)

57 (100.0) 84 (100.0)

84 (85.7) 16 (37.2)

14 (14.3) 27 (62.8)

98 (100.0) 43 (100.0)

100 (70.9)

41 (29.1)

141 (100.0)

.02*

.54† .83† ,.001†

AEJ, Australian Endodontic Journal; IEJ, International Endodontic Journal; JOE, Journal of Endodontics; PCT, prospective clinical trial; RCT, randomized controlled trial. *Fisher exact test. † Pearson chi-square test.

A presents 2% (2/100) failures, whereas material B accounts for 12% (12/100) of failed teeth. The relative risk is 0.27 with 95% CI of 0.07–0.99. In this case, the interpretation is as follows: the risk for tooth failure is 73% lower in material A compared with material B, and the findings are also compatible with a risk reduction between 1%–93%, thus revealing high levels of imprecision and uncertainty with regard to the size and magnitude of the presumably beneficial effect of material A. If the sample is increased to 1000 and there are 20 failures for material A and 120 for material B, then the corresponding point estimate remains as is (ie, a 73% reduction for the risk of failure for material A); however, the 95% CI goes narrower (ie, 0.18–0.41). This removes some of the uncertainty about the effect of the compared materials to the overall tooth failure, denoting that the findings in 95% of identical

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trials would give a decreased risk for material A compared with material B, from 59% to 82%. This shows that unlike P values, the interpretation of the clinical finding based on a dichotomous measure of statistical significance is eliminated, whereas we can obtain useful information about the range of potential effectiveness of a treatment modality. Again, if the finding is related to a safety outcome, increased precision gained by a perceived narrowing of the confidence bounds based on the increased sample size is evidently particularly important, and clinical decision making is to be highly informed. Albeit no significant effect was confirmed for the journal and year of publication, it was evident that the leading journals in terms of impact factor ranking in 2019 (Clarivate Analytics) presented the highest proportion of clinical trials including the

reporting of CIs for the presentation of their findings (JOE and IEJ). Such practices of enhanced quality of reporting might indicate an improvement of publication policies over the years as well as the adoption of a more stringent editorial and review process after article submissions. In essence, an improvement potential has been identified by the present study for the most recent publications within the 3-year period of 2018– 2020, with an adoption of CIs approaching 40% of the published reports of the clinical trials (39.1% in the year 2020, Table 1). This finding might be associated with more active adherence to reporting guidelines by authors of the trial reports within the dental specialty20, following more closely the long-standing establishment of the Consolidated Standards of Reporting Trials (CONSORT) statement and its extensions21–23. The use of more complex data sets and analyses for the presentation of the findings of a clinical trial was identified as the sole strong predictor of the adoption of CI reporting in the present study. This potentially denotes the involvement of a statistician or a trial methodologist at the design and analysis stage of the trials whose educational, experience, and knowledge level might be accountable for the data presentation in a more transparent and rigorous manner. In turn, this might also designate the importance of multilevel collaborations when designing clinical trials to reduce research waste24,25.

Prior Research This is the first meta-epidemiologic study in endodontic research with a clear focus on CI adoption in clinical trials on a number of recently published reports and across a 5-year period. Evidence from the biomedical literature has revealed that less than a quarter of studies in the orthopedic literature reported CIs26, whereas more recent publications from surgical research are confirmatory of such low frequencies27. Previous studies on the quality of reporting of RCTs in endodontics as well as in other dentistry disciplines have focused on the compliance of RCTs in general with the CONSORT guidelines12 or with the recently developed Preferred Reporting Items for RAndomized Trials in Endodontics (PRIRATE) statement, which was specifically designed for endodontic research19. An overall suboptimal quality of RCTs published in endodontics has been identified in an earlier and more limited sample of studies, albeit covering a time span from 1997–2012, with a considerably lower proportion of those (15.7%) reporting a CI for the results of a primary or secondary outcome12. Along the same line, the

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TABLE 2 - Univariable and Multivariable Logistic Regression with Odds Ratios (ORs) and Associated 95% Conﬁdence Intervals (CIs) for the Effect of a Number of Publication Characteristics on the Reporting of CIs or Not (N 5 141) Univariable Category Journal JOE IEJ AEJ Year 2016 2017 2018 2019 2020 Study design RCT Nonrandomized PCT Continent Asia/other America Europe No. of authors 1–3 4–5 6 No. of centers Single center Multicenter Signiﬁcance No Yes Complex/ multivariable analysis No Yes

95% CI

Multivariable P value*

95% CI

P value*

.26 Reference 1.50 0.29

0.68–3.29 0.03–2.40 .09

Reference 0.37 2.05 1.91 2.11

.05 Reference 0.20 2.20 1.62 1.49

0.08–1.58 0.71–5.95 0.54–6.75 0.64–6.95

0.03–1.10 0.60–8.05 0.34–7.75 0.33–6.78

.53 Reference 1.38

0.51–3.75 .04

Reference 2.26 3.04

.78 Reference 0.95 1.50

0.96–5.33 1.16–7.97

0.30–2.97 0.40–5.70

.02 Reference 2.99 5.56

.49 Reference 2.12 2.65

0.78–11.47 1.51–20.47

0.45–9.94 0.53–13.09

.54 Reference 1.25

0.61–2.60 .83

Reference 1.09

0.52–2.28 ,.001

Reference 10.13

4.38–23.41

,.001

Reference 11.47

4.19–31.41

investigation of a more recent random sample of RCTs within the discipline published between 2015 and 2019 demonstrated that only a quarter of studies were rated as of high reporting quality overall. The adoption of CIs in the presentation and the interpretation of the results of this sample of RCTs were not straightforward19. Such ﬁndings have been corroborated by similar empirical studies within dentistry. Early reports from orthodontic research have elucidated the suboptimal reporting of conﬁdence bounds in all study designs, with a very limited amount of orthodontic publications in 2008–2009, approximating 7%, having acknowledged the value and reporting of CIs7. Similarly, a report from the same discipline revealed an overall suboptimal reporting quality of RCTs published in leading orthodontic

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journals from 2001–2013, with the inclusion of CIs merely in 29% of the sample13. Moreover, analogous findings have been identified in other disciplines, with identified figures within the range of 18% for prosthodontics and implantology research published between 2005 and 201214 and 25% for periodontal research published between 2011 and 201615. A more recent report from oral and maxillofacial surgery has revealed similar findings, with less than a third of studies published from 2009– 2019 having included the reporting of CIs for their research findings16.

Strengths and Limitations The present study constitutes an empirical report on a sample of clinical trials, reﬂecting the most recent publications of major

endodontic journals. We focused on published findings solely from clinical study designs in an attempt to follow the standards of current reporting practices, which may additionally have an effect on the clinical relevance of competing treatments. We followed a clear and transparent methodological plan, with all predefined variables within the methodology section being analyzed and reported, thus trying to eliminate selective reporting; we also followed established methodology within meta-epidemiologic literature to allow for direct comparisons with prior research within the dental specialty and beyond. Our search was confined to standard electronic contents of journals within the specialty and did not expand to nonspecialty journals and a free database search. As previously mentioned, only clinical trials, either randomized or not, were considered. However, this might in turn represent a best-case scenario and possibly overestimate the identified prevalence of reporting of uncertainty bounds in endodontic research; first, because core specialty journals as the ones assessed are more likely to publish reports of trials of endodontic interventions that would concurrently gain the interest of clinicians specialized in endodontics and, second, because prospective clinical research endeavors, either randomized or not, are considered to comprise the highest level of quality of conduct and reporting within the evidence pyramid. In all, based on the findings of the present study and if one would aim to frame future initiatives to ameliorate current practices, efforts should be made to highlight the value and importance of CIs in the reporting of the results of clinical research alongside with a closer monitoring of adherence to reporting guidelines by the journal editors and reviewers.

CONCLUSIONS The inclusion of uncertainty measures as represented by the reporting of CIs is suboptimal within endodontic clinical trial reports. The use of more complex data sets and analyses by the authors of clinical trials is considered a strong predictor of CI reporting.

ACKNOWLEDGMENTS The authors deny any conﬂicts of interest related to this study.

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CLINICAL RESEARCH

Comprehensive Analysis of Differentially Expressed Genes in Clinically Diagnosed Irreversible Pulpitis by Multiplatform Data Integration Using a Robust Rank Aggregation Approach ABSTRACT Introduction: Molecular diagnosis may overcome the limitations of clinical and histologic diagnosis in pulpitis, thereby benefiting many treatment techniques, such as vital pulp therapies. In this study, integrated microarray data on pulpitis were used to obtain a list of normalized differentially expressed (DE) genes for analyzing the molecular mechanisms underlying pulpitis and identifying potential diagnostic biomarkers. Methods: A systematic search of public microarray and sequencing databases was performed to obtain expression data of pulpitis. Robust rank aggregation (RRA) was used to obtain DE gene lists (RRA_DEmRNAs and RRA_DElncRNAs) between inflamed pulp and normal samples. DE genes were evaluated by functional enrichment analyses, correlation analyses for inflammation-related RRA_DEmRNAs, and protein-protein interaction and competing endogenous RNA network construction. Quantitative real-time polymerase chain reaction validation was applied in snap-frozen pulp tissues. Results: Using the GSE77459 and GSE92681 data sets, 280 RRA_DEmRNAs and 90 RRA_DElncRNAs were identified. RRA_DEmRNAs were significantly enriched in inflammation-related biological processes and osteoclast differentiation and tumor necrosis factor, chemokine, and B-cell receptor signaling pathways. The molecular complex detection and cytoHubba methods identified 2 clusters and 10 hub genes in the protein-protein interaction network. The competing endogenous RNA network was composed of 2 long noncoding RNAs (ADAMTS9-AS2 and LINC00290), 2 microRNAs (hsa-miR-30a-5p and hsa-miR-128-3p), and 3 messenger RNAs (ABCA1, FBLN5, and SOCS3). The expression between most top inflammation-related RRA_DEmRNAs in pulpitis showed positive correlations. Quantitative real-time polymerase chain reacation validated the expression trends of selected genes, including ITGAX, TREM1, CD86, FCGR2A, ADAMTS9-AS2, LINC00290, hsa-miR-30a-5p, hsa-miR-128-3p, RASGRP3, IL3RA, CCDC178, CRISPLD1, LINC01857, AC007991.2, ARHGEF26-AS1, and AL021408.1. Conclusions: The identified biomarkers provide insight into the pathology and may aid in the molecular diagnosis of pulpitis. (J Endod 2021;47:1365–1375.)

Liu Liu, DDS, MSD,*† Tianyi Wang, DDS,* Dingming Huang, DDS, PhD,*† and Dongzhe Song, DDS, PhD*†

SIGNIFICANCE We identiﬁed novel genes associated with clinically diagnosed irreversible pulpitis based on data generated from different microarray platforms. These results improve our understanding of the pathologic process and provide candidate loci for molecular diagnostics.

From the *State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases and †Department of Conservative Dentistry and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China

Dental pulp; diagnosis; integrated analysis; noncoding RNA; pulpitis

Address requests for reprints to Dr Dongzhe Song, West China Hospital of Stomatology, 14# Third Section, Renmin Nan Road, 610041 Chengdu, China. E-mail address: [emailprotected]. cn 0099-2399/$ - see front matter

Pulpitis is one of the most common endodontic diseases; in addition to the pain, distress, and economic burden associated with this disease, it may increase the risk of many systemic diseases if left untreated1,2. Advances in the development of new bioactive materials have provided multiple therapeutic solutions for pulpitis3. The long-term success of these therapeutic strategies, particularly the widely used

KEY WORDS

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vital pulp therapy techniques, typically requires accurate assessment of the degree of inflammation of the dental pulp3. Current guidelines for determining the pulp status, such as the American Association of Endodontists criteria for endodontic diagnosis4, focus on a combination of clinical objective and subjective findings, such as results of thermal, cold, and electric pulp tests and reports of pain. However, discrepancies may exist between the clinically and histologically diagnosed in situ pulp status5, A shift from tissue-level research to cellular and molecular studies has resulted in the identification of many mediators of pulpal inflammation with potential diagnostic value6. However, these studies have typically focused on 1 or a few genes or molecules, emphasizing the need for high-throughput gene expression profiling to identify additional diagnostic and therapeutic markers6,7. Recent studies showed that noncoding RNAs play important roles in epigenetic regulation of pulpitis8,9. Long noncoding RNAs (lncRNAs), which are noncoding RNAs over 200 nucleotides, can competitively bind microRNAs (miRNAs) to interrupt their original interaction with target messenger RNAs (mRNAs). This competitive endogenous (ce) RNA mechanism may also be involved in the inflammatory process of pulpitis10. Early gene expression profiles for inflamed human pulp tissues obtained using different microarray platforms are limited by inconsistencies in diagnostic criteria and a lack of access to raw data11. Accordingly, deposition of raw data from microarray analyses in public databases, such as Gene Expression Omnibus (http://www.ncbi.nlm. nih.gov/), has recently been emphasized12. However, the small sample sizes evaluated because of the difficulty of obtaining ex vivo inflamed pulp tissues combined with inconsistencies arising from different microarray platforms, data preprocessing methods, and data outliers led to heterogeneity between studies7,8,11. There is an increased need to integrate data from different data sets for pulpitis to obtain normalized results based on larger samples. Implemented as a GNU R package (R Core Team, Vienna, Austria), the robust rank aggregation (RRA) method has been used to integrate gene lists from different data sets13 and obtain results that are robust to noise. To the best of our knowledge, the RRA method has not been applied to pulpitis data sets. In this study, we integrated different microarray data sets for clinically diagnosed pulpitis and generated lists of differentially expressed (DE) mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) after cleaning raw data

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using the RRA method. Based on DEmRNAs and DElncRNAs, gene enrichment and pathway analyses were performed, proteinprotein interaction (PPI) and ceRNA networks were constructed, and significant PPI clusters and hub genes in pulpitis were identified. To further understand the inflammatory process in pulpitis and its implications for odontogenesis at the molecular level, correlations between inflammation-related DEmRNAs and odontogenesis-related mRNAs in the data sets were evaluated. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the expression of selected key genes in pulpitis and healthy pulp tissue samples.

MATERIALS AND METHODS Ethical Declaration Discarded teeth were collected for qRT-PCR validation from patients after obtaining written informed consent. The study protocols were approved by the Ethics Committee of West China Hospital of Stomatology, Sichuan University (approval number: WCHSIRB-D2020-368).

Workflow of the Study The workflow of the study is summarized in Figure 1. Details behind our workflow are provided in the Supplemental Materials and Methods (available online at www.jendodon. com).

Data Processing and Analysis Data processing and statistical analyses, unless otherwise stated, were performed in the R programming environment (R version 4.0.3, R Core Team, Vienna, Austria). P , .05 was deﬁned as statistically signiﬁcant.

RESULTS Summary of Samples and Data Sets The data sets included in the analyses are summarized in Table 1. Box plots for each data set showed acceptable normalization (Supplemental Fig. S1 is available online at www.jendodon.com). Based on the results of the principal component analysis, possible batch effects in GSE77459 were adjusted, and GSM2434473, GSM2434475, and GSM2434484 in GSE92681 were considered as outlier samples and manually removed (Supplemental Fig. S2 is available online at www.jendodon.com). We included 11 inﬂamed pulp samples and 10 normal pulp samples in the analyses.

Identiﬁcation and Integration of DEmRNAs and DElncRNAs in Pulpitis According to the established cutoff values, we identiﬁed DEmRNAs and DElncRNAs in each data set. Volcano plots showing the distributions of DEmRNAs and DElncRNAs in each data set were generated (Supplemental Fig. S3 is available online at www.jendodon. com). Figure 2A and B shows heat maps of the top 10 up-regulated and down-regulated mRNAs and lncRNAs. In total, 174 upregulated and 106 down-regulated RRA_DEmRNAs and 31 up-regulated and 59 downregulated RRA_DElncRNAs were screened after RRA. The full RRA_DEmRNA and RRA_DElncRNA lists are shown in Supplemental Tables S3 and S4 (available online at www.jendodon.com).

Functional Enrichment Analysis In our Gene Ontology (GO) analysis, T-cell activation (GO:0042110, P , .05) was the most significantly enriched biological process (BP) in inflamed pulp followed by leukocyte cell2cell adhesion (GO:0007159, P , .05) and neutrophil degranulation (GO:0043312, P , .05) (Fig. 2C). The top 10 terms in the molecular function and cellular component categories are summarized in Figure 2C. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, osteoclast differentiation (hsa04380, P , .05), the tumor necrosis factor (TNF) signaling pathway (hsa04668, P , .05), the chemokine signaling pathway (hsa04062, P , .05), and the B-cell receptor signaling pathway (hsa04662, P , .05) were significantly enriched (Fig. 2D). The detailed GO and KEGG pathway enrichment results are provided in Supplemental Tables S5 and S6 (available online at www.jendodon.com). Gene set enrichment analysis (GSEA) of DEmRNAs in each data set cross-validated the results of GO and KEGG analyses. HALLMARK_TNFA_SIGNALING_VIA_NFKB was significantly enriched in GSE77459, and HALLMARK_IL2_STAT5_SIGNALING, HALLMARK_INFLAMMATORY_RESPONSE, and HALLMARK_KRAS_SIGNALING_UP were significantly enriched in GSE92681 (Supplemental Fig. S4A and B is available online at www.jendodon.com). GSEA GO results for each data set are summarized in Supplemental Figure S4C and D (available online at www.jendodon.com).

Correlation Analyses In total, 14 RRA_DEmRNAs were included in the top inﬂammation-related gene list

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GSE92681

GSE77459

Batch-effect detection and removal Re-mapping of probe sets by Rsubread Extraction of mRNA and lncRNA list from each gene set seperately

Pulpitis vs. normal miRNA expression data suppl.file from 10.1016/j.joen.2012.02.020 p.value < 0.05, q.value ≤ 0.05 , abs(logFC) ≥ 1

Differential expression analysis by limma

DEmiRNAs miRDB

abs(logFC) > 0.5, p.Value < 0.05

DE77459mRNAs DE77459lncRNAs

× miRTarBase × Targetscan

DE92681mRNAs DE92681lncRNAs

RRA_DEmRNAs

Combining results by RobustRankAggreg score < 0.05

RRA_DEmRNAs

RRA_DElncRNAs miRcode abs(logFC) ≥ 1

GO/KEGG GSEA

RRA_DElncRNAs-miRNAs

Correlation analysis

DEmiRNAs-RRA_DEmRNAs intersect by DEmiRNAs

GeneCards

× Top inflammtion-related RRA_DEmRNAs ×

inflamation-related gene list odontogenesis-related gene list

ceRNA network

Top odontogenesis-related RRA_DEmRNAs PPI network(STRING)

Key genes qRT-PCR validation of selected genes

FIGURE 1 – The workflow adopted in the study. Different background colors reflect different processes in our study. Black, downloading data from databases. Deep blue, data cleaning. Red, differential expression analysis. Green, functional enrichment analysis. Purple, correlation analysis and PPI network construction. Orange, ceRNA network construction. Brown, qRT-PCR validation of selected genes. (relevance score .20). Correlation analyses of the expression levels in the GSE77459 and GSE92681 data sets revealed significant moderate to strong positive correlations between the top inflammation-related RRA_DEmRNAs (Fig. 3A and B). FCGR2A was not detected in GSE77459. Correlation analyses between the expression levels of the top inflammationrelated RRA_DEmRNAs and odontogenesisrelated genes revealed that most top inflammation-related RRA_DEmRNAs had significant moderate to strong negative correlations with RUNX2, KRT14, AMBN, and FGFR1 in GSE77459 (Fig. 3C). In GSE92681, most top inflammation-related RRA_DEmRNAs showed significant moderate to strong negative correlations with PTCH1 and TP53 and significant moderate to strong positive correlations with CTNNB1 (Fig. 3D).

PPI Network Supplemental Table S7 (available online at www.jendodon.com) shows the entire PPI list.

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The significant PPI clusters generated by the molecular complex detection (MCODE) and the hub gene network calculated by cytoHubba are shown in Figure 4A–C. Genes with high scores were likely to play important roles in irreversible pulpitis. ITGAX and TREM1 were considered as seed genes, with the highest scores in the top 2 MCODE clusters (Fig. 4A and B). Based on the rankings by the cytoHubba maximum clique centrality method, 10 hub genes were identified (Fig. 4C). CD86 and FCGR2A were the top 2 hub genes. Genes shown in Figure 4A–C are inflammation-related genes, except for FYB.

ceRNA Network A ceRNA network was constructed based on 2 lncRNAs (ADAMTS9-AS2 and LINC00290), 2 miRNAs (hsa-miR-30a-5p and hsa-miR128-3p), and 3 mRNAs (ABCA1, FBLN5, and SOCS3) (Fig. 4D). Six interaction targets (CXCL10, TLR10, TLR2, CCL2, CD86, and IDO1) of ABCA1 and SOCS3 were found in signiﬁcant PPI clusters and the hub gene

network. The numbers of target sites, seed match types, conservation scores, validation methods, and expression patterns of miRNA targets in the ceRNA network are summarized in Table 2.

qRT-PCR Validation of Selected Genes The top 2 hub genes CD86 and FCGR2A calculated by cytoHubba; the seed genes ITGAX and TREM1 from the signiﬁcant PPI clusters generated by MCODE; hsa-miR30a-5p, hsa-miR-128-3p, LINC00290, and ADAMTS9-AS2 from the ceRNA network; and the top 2 up- and down-regulated RRA_DEmRNAs and RRA_DElncRNAs from differential expression analysis (RASGRP3, IL3RA, CCDC178, CRISPLD1, LINC01857, AC007991.2, ARHGEF26AS1, and AL021408.1) were selected for qRT-PCR validation. All qRT-PCR results were signiﬁcant and showed the same trends as in microarray analyses after RRA

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Hematopoietic cell lineage Phagosome Tuberculosis Cytokine−cytokine receptor interaction Count

Leishmaniasis

5.0

Osteoclast differentiation

7.5 10.0

Cell adhesion molecules

12.5

Rheumatoid arthritis

15.0

Staphylococcus aureus infection

pvalue

TNF signaling pathway 0.001

Chemokine signaling pathway

0.002

B cell receptor signaling pathway Viral protein interaction with cytokine and cytokine receptor Th1 and Th2 cell differentiation Intestinal immune network for IgA production Malaria 0.04

0.06 GeneRatio

0.08

FIGURE 2 – RRA analysis and functional enrichment analysis. A heat map of the top 10 up- and down-regulated (A ) mRNAs and (B ) lncRNAs in RRA analysis. Red indicates upregulated genes in inflamed pulp samples, and blue represents down-regulated genes in inflamed pulp samples; log2 fold-changes of each gene in GSE77459 and GSE92681 are presented in the corresponding column. Visualization of (C ) enriched terms in the biological process, molecular function, and cellular component categories in GO analysis and (D ) enriched terms in KEGG analysis; the size of the round circles indicates the number of enriched genes. Colors in the round circles reflect P values, and all enriched terms are significant (P , .05).

(Fig. 4E; Supplemental Fig. S5 is available online at www.jendodon.com).

DISCUSSION Pulpitis is typically considered as a defensive reaction to irritants, such as invading microorganisms and chemical/mechanical stimuli. If the balance between irritants and defensive reactions is disrupted, pulpitis can become irreversible, and destructive biological processes may occur in the dental pulp5. In clinical settings, it is crucial to evaluate the severity of pulp inﬂammation to guide treatment, which may include vital pulp therapy or routine root canal therapy3. Unfortunately,

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inconsistencies between clinical and histologic ﬁndings using current diagnostic criteria for pulpitis may affect the treatment rationality and effectiveness. Various microarray data sets related to pulpitis are publicly available in databases. Identifying key genes involved in pulpitis based on microarray data is critical to obtain a global view of pulpitis as well as for molecular diagnosis and targeted therapy. Although we only included data sets in which samples were diagnosed using American Association of Endodontists criteria, there are concerns that these current diagnostic techniques can cause inaccuracy and heterogeneity, in turn impacting the ﬁnal quality of our analysis. To

address this issue, the normalization of the raw data was reviewed, and expression matrices were strictly cleaned by removing batch effects and deleting outlier samples that could not be corrected to ensure the hom*ogeneity of the samples in each group and the distinctiveness between groups (Supplemental Fig. S2 is available online at www.jendodon.com). The probe sets were remapped to retain annotated information obtained from different platforms as uniﬁed and up-to-date. Existing evidence shows that combining raw data from different platforms is not straightforward because there are many sources of inconsistencies and bias requiring cross-platform normalization and the removal of batch effects14. Based on the

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TABLE 1 - Characteristics of the Microarray Data Sets Included in the Study GEO accession number Diagnostic criteria Numbers of samples Sources of samples Normal pulp samples Inﬂamed pulp samples Pain intensity records Platform Year

GSE92681†

GSE77459* AAE 6 normal pulps vs 6 inﬂamed pulps

AAE 5 normal pulps vs 7 inﬂamed pulps

From teeth without irreversible pulpitis and extracted for various reasons From teeth diagnosed with irreversible pulpitis Recorded using visual analog scale GPL17692 2016

From healthy third molars or teeth extracted for orthodontic purpose From teeth diagnosed with irreversible pulpitis Not recorded GPL16956 2017

AAE, American Association of Endodontists; GEO, Gene Expression Omnibus. *Accession date: December 20, 2020 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE77459). † Accession date: December 20, 2020 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE92681).

summary-level data from individual data sets, the RRA method is robust to noise, can deal with incomplete rankings/lists, and is efficient13. In our study, 280 RRA_DEmRNAs and 90 RRA_DElncRNAs were identified after RRA analysis. GO and KEGG analyses indicated that RRA_DEmRNAs were involved in several processes related to inflammation and immunity, suggesting that they contribute to the pulp defense mechanism. The role of the TNF signaling pathway activation was crossvalidated by a GSEA, and the IL2–STAT5 and KRAS signaling pathways may also contribute to the inflammatory response in irreversible pulpitis. The GSEA of GO further corroborated that pulpitis is a defense mechanism involving inflammation and immune responses. In the PPI network, all mRNA nodes in significant clusters and hub genes were annotated as inflammation-related mRNAs according to the GeneCards database, except for FYB. ITGAX, also known as CD11c, is the seed gene in cluster 1; it encodes integrin alpha X chain protein. ITGAX-positive cells have been detected in rheumatoid arthritis joint synovial tissues and participate in inflammation in asthma15,16. Recent studies have emphasized the diagnostic and therapeutic value of the cluster 2 seed gene TREM1, which is an early serum inflammatory marker in patients who are overweight17. CD86 is considered as a biomarker of proinflammatory M1 macrophages and is expressed in the dental pulp18. FCGR2A encodes a number of immunoglobulin Fc receptors on many immune response cells and can be detected in the saliva19. We detected correlations between expression levels of the top inflammationrelated genes. Particularly, FCGR2A, C3, TLR2, CCL2, CCR1, S100A9, and CXCL10 were highly positively correlated and found in the top 2 clusters or were hub genes in the network. The expression levels of the top inflammation-related genes showed significant

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moderate to high positive correlations with CTNNB1 in GSE92681, which encodes betacatenin protein as part of the canonical Wnt signaling pathway20. However, in both data sets, there were more significant negative correlations than positive correlations, suggesting that inflammation of the pulp in irreversible pulpitis negatively affects odontogenesis. Noncoding RNAs play critical roles in dental pulp inflammation, and there is increasing evidence of the diagnostic value of noncoding RNAs8,9. We constructed a ceRNA network, which suggested that the lncRNA ADAMTS9-AS2 competitively binds to hsamir-30a-5p and hsa-miR-128-3p to regulate miRNA-related downstream gene silencing. hsa-mir-30a-5p is an inflammatory regulator in many biological and pathologic processes21,22. hsa-miR-128-3p can mediate the TNF-a inflammatory response by targeting sirtuin 123. ADAMTS9-AS2 can control chondrogenic differentiation24. LINC00290 is a potential inflammatory biomarker according to the genome-wide association studies catalog25 and may also competitively bind to hsa-mir-30a-5p. However, to the best of our knowledge, this is the first study providing evidence of the roles of hsa-mir-30a-5p, hsamiR-128-3p, LINC00290, and ADAMTS9-AS2 in irreversible pulpitis. Our analysis showed that SOCS3 and ABCA1, the downstream targets of hsa-mir-30a-5p and hsa-miR-1283p, respectively, may interact with genes in significant clusters and hub genes, including TLR2, CCL2, CXCL10, CD86, TLR10, and IDO1. Half of these genes (TLR2, CCL2, and CXCL10) are top inflammation-related RRA_DEmRNAs (relevance score .20), suggesting a relation between the ceRNA network and pulp inflammation. Overall robustness was observed in our miRNA target quality study, and all predicted miRNA-target interactions have been reported in miRTarBase to be validated using at least 1

method. The core downstream mRNAs in the ceRNA network, SOCS3 and ABCA1, showed distinct expression fold changes, with conserved 8mer-type sites for binding to corresponding miRNAs with good conservation scores. For lncRNA-miRNA interactions, target sites in ADAMTS9-AS2 are less conserved than the target site in LINC00290, which may reflect the functionality of the predicted miRNA targets. However, the match types of target sites and expression patterns still reflected promising interactions between lncRNAs and miRNAs in our study. In 2019, Lei et al10 generated a ceRNA network based on DElncRNAs from GSE92681, DEmiRNAs reported by Zhong et al,9 and DEmRNAs from GSE77459. The integrated DEmRNA and DElncRNA list obtained in our study by applying the RRA method to GSE77459 and GSE92681 benefited from the larger sample size and reduced biases generated from single data sets. Xi et al26 constructed a ceRNA network based on overlapping DEmRNAs from GSE77459 and GSE92681, DElncRNA from GSE92681, and miRNAs from several online databases. Compared with manually overlapping DEmRNAs, the RRA method can get a generate robust gene list without excessive sacrificing of candidate genes because of incomplete lists (ie, RRA can save significant genes with expression data available only in 1 data set based on their positions in the RRA method–generated rank list13). Purely predictive miRNA targets were used by Xi et al26 without considering the miRNA expression profile in pulpitis and healthy pulp tissue samples. The target quality of miRNAs was not investigated by Lei et al10 and Xi et al26, which can provide more information for determining robust miRNAtarget interaction relationships. In addition, Lei et al and Xi et al did not describe their dataprecleaning protocols despite the importance of quality control, batch-effect removal, and

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FIGURE 3 – Results of correlation analysis. Correlation analysis of expression levels between each pair of top inflammation-related RRA_DEmRNAs in (A ) GSE77459 and (B ) GSE92681. Correlations of expression levels between each pair of the top inflammation- and odontogenesis-related RRA_DEmRNAs in (C ) GSE77459 and (D ) GSE92681. Pearson correlation coefficients (r ) were calculated among expression levels of included genes and are presented in corresponding columns. The depth of the color reflects the Pearson correlation coefficient; red or blue color indicates positive or negative correlation, respectively. *P , .05. annotation revision. Chen et al27 merged raw data from GSE77459 and GSE92681 and used ComBat normalization to adjust for batch effects; however, the lncRNA probes were removed, and no lncRNA results were reported. Furthermore, there are issues with directly merging raw data from 2 different microarray platforms from different manufacturers, which may not be completely resolved using the ComBat method28. The methodological differences described previously may account for the discrepancies among DE gene lists in different studies and thus may have influenced PPI and ceRNA network construction. On the other hand,

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similarities still exist among the results of previous studies and the present study, and the overlapped pathways and genes may provide a more concrete understanding of the molecular mechanism of pulpitis. Enriched GO BP terms were mostly concentrated on inﬂammation and immune response, such as neutrophil degranulation, neutrophil activation, leukocyte chemotaxis, T-cell activation, and cell adhesion. KEGG analysis showed that cytokine–cytokine receptor interaction, cell adhesion molecules, the TNF signaling pathway, and the chemokine signaling pathway were signiﬁcantly enriched in all previous studies as well as our current study.

For PPI analyses, the comparability was better between the results from Chen et al27 and our study because both studies cleaned and merged the mRNA data of GSE77459 and GSE92681, and the common hub genes were CD86 and SPI1. In addition, FCGR2A, FCGR2B, and CCL2 were found in the hub gene list of one study and significant clusters of another. The ceRNA networks of the present study and the study by Lei et al10 focused on miRNA-SOCS3 interactions, and SOCS3 can interact with several targets from the hub gene network and significant clusters. The first core step toward the clinical molecular diagnosis of pulpitis is identifying

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CD1D

SELPLG

FCGR1A SELL

VAV1

TLR2

CD163

SLAMF1

CD53

CD74

CTSS

CD2 FCGR2B

CD48 CD44

FYB

HCK NCF4 PPBP

TNFSF13B

CCR2

CXCL10

PECAM1

FGL2

C3AR1

KIT

CD86

SPI1 ITGAX

ANPEP

PLEK

CCL2 TLR10

TREM1

MME

LAPTM5 CCL21 NCF2 C3

GPR183

ALOX5AP S1PR4

LYN FPR3

LILRB2

MNDA

CYBB IL3RA

LncRNA NPY5R

FCGR2A

MicroRNA

C5AR1

IDO1

Inflammation related mRNA OPRK1

CCR1

CLEC7A

S100A9

Other mRNA

ADAMTS9-AS2

ABCA1 hsa-miR-128-3p FBLN5

LINC00290

CD44

C3AR1

SOCS3

hsa-miR-30a-5p

Cluster 2

KIT

CD53

VAV1

MME

ANPEP

HCK

CD74 CXCL10

LAPTM5

CCR1

IDO1

FCGR2A

TLR10

CD1D GPR183

NCF4

PLEK

CLEC7A

TREM1 CD86

CCL2

TLR2

SPI1

LYN CD48

TNFSF13B

FCGR2A

OPRK1

CYBB

PECAM1

FCGR2B

CD163

HCK MNDA

CCL21

CD86

FCGR1A S1PR4

ITGAX

LILRB2

CCR2

NCF2 CD53

Hub genes

FPR3

NCF2

SELPLG IL3RA

CD2

FYB SLAMF1

LILRB2 PLEK

CCR1

S100A9

ALOX5AP FCGR2B

FGL2

PPBP SELL NPY5R

4 2 0

p al orm

ulp la In f

4 2 0

ulp dp N

p al orm

* 1.0 0.5 0.0

p al orm

ulp In f

la m

lp pu ed

ulp In f

la m

CD86 *

4 3 2 1 0

lp pu ed

p al orm

1.5

* 1.0 0.5 0.0

lp lp pu pu al ed rm lam f No n I

ulp l In f

FCGR2A 6 4 2 0

ulp dp No

1.5

* 1.0 0.5 0.0

p al orm

ulp l Inf

ulp dp me

ADAMTS9-AS2

hsa-miR-128-3p relative miRNA expression fold

relative miRNA expression fold

hsa-mir-30a-5p 1.5

relative mRNA expression fold

TREM1 18 16 14 12 10

relative mRNA expression fold

ITGAX 8

relative lncRNA expression fold

CTSS C5AR1

p al rm

ulp l Inf

lp pu

LINC00290 relative lncRNA expression fold

Cluster 1

1.5

* 1.0 0.5 0.0

lp lp pu pu al ed rm am l f No In

FIGURE 4 – Network construction and qRT-PCR validation. Circles represent inflammation-related mRNAs, squares represent other mRNAs, triangles represent lncRNAs, and hexagons represent miRNAs. (A ) Significant PPI cluster 1 and (B ) cluster 2 generated by MCODE with seed genes as ITGAX and TREM1, respectively. (C ) Hub genes in the PPI network generated by cytoHubba. The depth of color and size of nodes in A–C are positively correlated with the score for each gene generated by MCODE or cytoHubba. High scores indicate a more important role in irreversible pulpitis. (D ) The ceRNA network and its interactions with members in significant clusters and hub genes. The yellow color indicates genes in ceRNA network; the blue and green colors indicate genes in cluster 1 and 2, except hub genes; and the red color indicates hub genes. (E ) qRT-PCR validation of selected genes from significant clusters, hub genes, and the ceRNA network (*P , .05).

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TABLE 2 - Characteristics of microRNA Targets in the Competitive Endogenous RNA Network Number of target sites and match types* Conserved sites

Poorly conserved sites

Expression patterns

Target RNA

Target RNA class

Total

8mer

7mer-m8

7mer-A1

Total

8mer

7mer-m8

7mer-A1

6mer sites

Conservation score†

Validation methods

hsa-miR-30a-5p‡ hsa-miR-30a-5p hsa-miR-30a-5p hsa-miR-128-3p‡

SOCS3 ADAMTS9-AS2 LINC00290 ABCA1

mRNA lncRNA lncRNA mRNA

1 1 1 2

1 0 0 1

0 1 0 0

0 0 1 1

0 0 0 0

0 0 0 1

0.95 0.22 0.67 0.63

hsa-miR-128-3p hsa-miR-128-3p

FBLN5 ADAMTS9-AS2

mRNA lncRNA

1 1

0 0

0 1

1 0

0 0

1 0

0 0

0.62 0.22

NGS NGS NGS Reporter assay, Western blot, qPCR, microarray NGS NGS

microRNA

log2 FC of the target in GSE77459

log2 FC of the target in GSE92681

3.10 21.04 21.20 1.80

2.02 21.07 21.04 1.05

1.26 21.04

1.26 21.07

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FC, fold change; lncRNA, long noncoding RNA; mRNA, messenger RNA; NGS, next-generation sequencing; qPCR, quantitative polymerase chain reaction. *8mer means an exact match to the seed region (position 2–7) of the mature miRNA 1 position 8 followed by an ‘A’, 7mer-m8 means an exact match to the seed region of the mature miRNA 1 position 8, 7mer-1A means an exact match to the seed region of the mature miRNA followed by an ‘A’, and 6mer means an exact match to the seed region of the mature miRNA. † The higher the conservation score, the more likely the predicted sites are to be true target sites. ‡ Log2 FC of hsa-miR-30a-5p: 21.22; log2FC of hsa-miR-128-3p: 21.47.

and characterizing key inflammatory regulators in the pulp6. Second, the quality and quantity of samples affect diagnostic accuracy in pulpitis29,30. Two types of sampling are currently reported: atraumatic sampling using dentinal fluid, gingival cervical fluid, or even saliva29–31 or invasive sampling methods, such as pulp blood analyses32. Our results provide a comprehensive overview of candidate diagnostic biomarkers. Key mRNAs (CD86, FCGR2A, ITGAX, and TREM1) identified in our study have diagnostic value in several inflammatory conditions and may become good biomarkers for reflecting inflammation of pulp tissue. Furthermore, FCGR2A and TREM1 can be evaluated by less traumatic sampling methods (eg, using serum and saliva)17,19. Previous studies mainly focused on the diagnostic value of proteins and protein-coding RNAs29–32; however, the diagnostic value of noncoding RNAs for pulpitis should not be ignored8,9. The newly generated RRA_DElncRNA list and ceRNA network provide pulpitis-related noncoding RNA biomarker candidates, with hsa-miR-30a-5p, hsa-miR-128-3p, LINC00290, and ADAMTS9-AS2 considered as key biomarkers. With the application of portable high-speed polymerase chain reaction systems, in 10 minutes, clinicians can amplify sequences with a wide dynamic range, up to 800 bp, with simple laboratory requirements33; this equipment may become

ideal for the rapid chairside detection of noncoding RNAs. Our study had several limitations. First, because of the difficulty in collecting sufficient high-quality samples of inflamed pulp for highthroughput analyses, the sample size of our study was limited, even after a systematic search for available data sources and data integration. Additionally, although we validated our results by qRT-PCR, the 7 inflamed and normal pulp samples also comprised a small sample size; because of this, alpha and beta errors may exist, which decrease the prediction accuracy. Second, although we strictly precleaned data to remove intragroup discrepancies per study, interstudy differences may still exist because of the heterogeneity of inclusion criterion details in actual use because only limited baseline data are available from Gene Expression Omnibus (Table 1), which may have influenced the results of the RRA analysis. Third, the technological limitations of microarray may influence the final results, such as cross-hybridization, the inability to discover new transcripts, and the relatively narrow dynamic range34; Without using array hybridization technology, data from RNAsequencing platforms are considered as a good validation to our study with higher specificity and sensitivity and a wider dynamic range. Unfortunately, after our comprehensive search in online databases and published studies, no public RNA-sequencing data are

currently available. As stated previously, future studies (particularly RNA-sequencing studies) involving more samples and more detailed baseline data are needed to facilitate the development and validation of speciﬁc diagnostic gene signatures in pulpitis. In conclusion, our comprehensive analysis identiﬁed novel genes associated with clinically diagnosed irreversible pulpitis based on data generated from different microarray platforms. These results improve our understanding of the pathologic process and provide candidate loci for molecular diagnostics.

ACKNOWLEDGMENTS The authors thank the original contributors of the microarray data reanalyzed in our study. Supported by the National Natural Science Foundation of China (grant nos. 81900996 [D.Z.S] and 81771063[D.M.H]) and China Postdoctoral Science Foundation (grant no. 2019M653441 [D.Z.S]). The authors deny any conﬂicts of interest related to this study.

SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found in the online version at www.jendodon.com (10.1016/j.joen.2021.07. 007).

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Ricucci D, Siqueira JF Jr, Li Y, Tay FR. Vital pulp therapy: histopathology and histobacteriologybased guidelines to treat teeth with deep caries and pulp exposure. J Dent 2019;86:41–52.

AAE Consensus Conference Recommended Diagnostic Terminology. J Endod 2009;35:1634.

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Galicia JC, Henson BR, Parker JS, Khan AA. Gene expression proﬁle of pulpitis. Genes Immun 2016;17:239–43.

Huang X, Chen K. Differential expression of long noncoding RNAs in normal and inﬂamed human dental pulp. J Endod 2018;44:62–72.

Zhong S, Zhang S, Bair E, et al. Differential expression of microRNAs in normal and inﬂamed human pulps. J Endod 2012;38:746–52.

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Yoshiba N, Edanami N, Ohkura N, et al. M2 phenotype macrophages colocalize with Schwann cells in human dental pulp. J Dent Res 2020;99:329–38.

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van Schie RC, Wilson ME. Saliva: a convenient source of DNA for analysis of bi-allelic polymorphisms of Fc gamma receptor IIA (CD32) and Fc gamma receptor IIIB (CD16). J Immunol Methods 1997;208:91–101.

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Huang MJ, Zhao JY, Xu JJ, et al. lncRNA ADAMTS9-AS2 controls human mesenchymal stem cell chondrogenic differentiation and functions as a ceRNA. Mol Ther Nucleic Acids 2019;18:533–45.

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Xi X, Ma Y, Xu Y, et al. The genetic and epigenetic mechanisms involved in irreversible pulp neural inﬂammation. Dis Markers 2021;2021:8831948.

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CLINICAL RESEARCH Ozge Erdogan, DDS,*† Matthew Malek, DDS,† and Jennifer L. Gibbs, MAS, DDS, PhD*†

SIGNIFICANCE Percussion and palpation hypersensitivity may be capturing different aspects of endodontic pathology and odontogenic pain processing. The results of these tests should be interpreted carefully for accurate endodontic diagnosis.

Associations between Pain Severity, Clinical Findings, and Endodontic Disease: A Cross-Sectional Study ABSTRACT Introduction: Thorough pain assessment and thermal and mechanical testing are the primary diagnostic tools used to assess the status of pulp and periapical tissues in teeth with potential endodontic pathology. This study evaluated predictors of acute odontogenic pain to better understand the relationship between endodontic pain, clinical testing, endodontic disease, and diagnoses. Methods: Participants (N 5 228) presenting with acute odontogenic pain underwent standardized clinical testing and reported their pain intensity. Univariate and multiple regression analyses were performed to evaluate the predictors of acute endodontic pain. Chi-square tests with Bonferroni adjustments were conducted to measure the frequency of endodontic diagnostic test ﬁndings and clinical observations in patients with different pulpal diagnoses. Results: A negative response to cold stimulation on the causative tooth and percussion hypersensitivity on the healthy adjacent tooth were the strongest predictors of higher levels of acute endodontic pain. Percussion hypersensitivity on the healthy adjacent tooth was present in a quarter of the cohort and was reported with equal frequency in teeth diagnosed with irreversible pulpitis, necrotic pulp, and previously initiated/ treated teeth. Although painful percussion on the causative tooth was more frequently reported in teeth diagnosed with necrotic pulp, painful palpation was more frequently reported on teeth diagnosed with previously initiated/treated teeth. Conclusions: Percussion hypersensitivity on the healthy adjacent tooth may reveal a lowered pain threshold and heightened pain sensitization. It is also possible that the 2 commonly performed mechanical sensory tests, percussion and palpation hypersensitivity, may detect different aspects of endodontic pathophysiology and pain processing. (J Endod 2021;47:1376–1382.)

KEY WORDS Dental pain; endodontic diagnosis; pain; pulpitis; sensitization; sensory testing

From the *Division of Endodontics in Restorative Dentistry and Biomaterials Sciences, Harvard School of Dental Medicine, Boston, Massachusetts; and † Department of Endodontics, New York University, College of Dentistry, New York, New York Address requests for reprints to Ozge Erdogan, Restorative Dentistry and Biomaterials Sciences, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115. E-mail address: ozge_erdogan@hsdm. harvard.edu 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.07.004

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Dental pulp is elegantly confined within enamel and dentin. Similarly, the teeth are confined within the periodontal ligament and alveolar bone. Therefore, it is very challenging to assess the true status of endodontic pathology, including that of the pulp and the periapical tissues, during diagnosis. Endodontic disease, either when the pulp is severely inflamed or when it becomes necrotic, can produce severe pain or patients can present with very advanced disease without any history of painful symptoms1–4. Therefore, a thorough and careful assessment of how pain relates to other clinical findings can provide valuable insight toward better inferring the degree of pulpal and periapical inflammation and infection3,5. In addition to acquiring a detailed pain history when patients first present to the clinic, surrogate tests including thermal and mechanical assessment are critical for pulpal and periapical diagnosis. Cold sensibility testing is a reliable diagnostic tool with high specificity and sensitivity to differentiate vital from necrotic pulp6,7. On the other hand, the diagnostic utility of mechanical sensory testing, including percussion, palpation, and the bite test, is less clear8. An abnormal or painful response to mechanical sensory testing could be due to inflammation or infection of the periapical tissues9, and indeed this is the common interpretation. However, it is also possible that percussion hypersensitivity may be detecting peripheral and central sensitization–induced mechanical allodynia, originating from inflamed and hypersensitive pulpal neuronal afferents, while the periapical tissues remain free of any pathology9–11.

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A better understanding of what each variety of mechanical sensory testing captures and how they associate with overall pain can ameliorate our understanding of mechanical testing. There is a complex dynamic interplay between endodontic disease, the pulpal and periapical diagnosis, and the perception of pain. The complexity is amplified because pain is both an outcome of disease and an end point of diagnosis. Furthermore, the sensory testing, used as a diagnostic tool, is simultaneously quantifying the pain as an outcome of the test (Fig. 1). Understanding how patients with endodontic disease experience pain and how this relates to other clinical findings may help us better understand the intricacies of this complexity. In this study, we evaluated potential predictors of acute endodontic pain, specifically focusing on sensory testing. We also evaluated the relationship between acute endodontic pain, mechanical sensory testing (percussion, palpation, and bite), and the pulpal diagnosis.

MATERIALS AND METHODS This cohort has been described in a prior publication12. The previous analysis evaluated associations between pain quality descriptors (eg, throbbing and shooting) and clinical sensory testing on teeth (eg, cold responsive) in patients with acute odontogenic pain. This study was approved by the institutional review boards of New York University (#11-01567) and the University of California San Francisco

(#10-00111)12. This article includes a novel analysis with distinct findings and conclusions using data from this cross-sectional cohort. In this study, we investigated the predictors of acute odontogenic pain using a multivariable regression analysis as well as the associations between clinical sensory testing and pulpal diagnosis. Briefly, patients who visited emergency dental clinics due to dental pain with at least 3 out of 10 intensity on a numeric rating scale (NRS) were included in the study. Also, there had to be a clear endodontic pathology localized to a single tooth. Patients were excluded if there was another suspected source of chronic or acute orofacial pain. We verbally administered a questionnaire and collected patients’ demographic information and pain history. Patients’ current pain intensity was asked on an NRS between 0 and 10. Calibrated study personnel, who were either endodontic faculty or endodontic residents, performed standardized endodontic diagnostic testing procedures, including a cold test and mechanical sensory testing (bite, percussion, and palpation), as described in our previous publication12. For each test, first, a healthy intact contralateral tooth and a healthy intact adjacent tooth followed by the causative tooth in question were evaluated. For each test, the unpleasantness of the sensation was rated from 1 to 3, with 1 being not or mildly unpleasant, 2 moderately unpleasant, and 3 severely unpleasant. For analysis, this result

FIGURE 1 – The complex interplay between endodontic disease, diagnosis, pain, and thermal and mechanical testing. Pain is a result of endodontic disease including pulpitis and apical periodontitis. Clinicians use pain as a tool to assess the status of the pulp and periapical areas in determining an endodontic diagnosis. Neuronal and immunologic changes within the pulp and sometimes around the periapical tissues may result in thermal and mechanical hypersensitivity. These changes are captured by sensory testing to diagnose endodontic disease. The readouts for these valuable tests are also quantiﬁed by patient-reported pain and sensitivity. Therefore, there are bidirectional relationships between endodontic disease, diagnosis, pain experience, and sensory testing. Created with BioRender.com.

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was scored as either nonpainful (score of 1) or painful (score of 2 or 3; ie, hypersensitive) percussion. Periapical pathology and the presence of any visible swelling were also recorded. The pulpal and periapical diagnosis were ultimately determined by 2 endodontic faculty members using the American Association of Endodontists guidelines3,5.

Statistical Procedures The sample size was calculated and found to be 211 using StataVersion 16 (StataCorp, College Station, TX) for the multiple regression analysis by computing the required sample size for the 2-sided R2 test to achieve 80% power with a 5% significance level for 5 covariates. We assumed the reduced model with the control covariates will have an R2 of 0.15, and the full model with all 5 covariates has an R2 of 0.2. Data were exported from Google Forms (Google, Mountain View, CA) to Excel (Microsoft, Redmond, WA), and then imported into StataVersion 16. Descriptive analysis of variables was performed to identify frequency, mean, and standard deviation. Univariate analysis of possible predictors of preoperative pain intensity was first assessed with the 2-sample t test (Table 1). A P value ,.05 was considered statistically significant. Furthermore, 2 consecutive models (model 1 and model 2) were constructed for multiple regression analysis to test for the predictors of intensity of acute endodontic pain (Table 2). To construct the models among the variables listed in Table 1 as a measure of thermal sensory testing, which also determines the vitality of the tooth, cold testing (positive response 5 1, negative response 5 0) was included in the models. Because swelling and periapical radiolucency most likely were present when the cold response was negative, they would be collinear variables and hence not included in the models. Among mechanical testing, the bite test variable was dropped because it did not appear highly associated with preoperative pain in the univariate analysis. In the first model, palpation hypersensitivity on the causative tooth was included. Palpation hypersensitivity on the adjacent tooth was not included because of significant collinearity with palpation sensitivity on the causative tooth. As stepwise forward selection method suggested, we included percussion hypersensitivity on the adjacent tooth in the second model. Percussion hypersensitivity on the adjacent tooth showed stronger correlation with current pain intensity in the univariate analysis than percussion hypersensitivity on

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TABLE 1 - Univariate Analysis to Predict Pain Intensity Variables

n (%)

Current pain mean (95% CI)

P value

158 70 121 107 44 184 114 81 57 132 106 122 163 65 114 114 155 73 96 132 46 182 65

5.4 (4.9–5.9) 4.6 (3.9–5.2) 5.5 (5.0–6.0) 4.8 (4.2–5.3) 5.1 (4.2–5.9) 5.2 (4.8–5.6) 5.4 (4.8–5.9) 5.1 (4.5–5.7) 3.8 (3.1–4.7) 5.7 (5.3–6.2) 4.7 (4.1–5.2) 5.6 (5.1–6.1) 5.5 (5.0–5.9) 4.4 (3.8–5.1) 5.6 (5.1–6.2) 4.7 (4.2–5.2) 5.4 (4.9–5.8) 4.7 (4.0–5.4) 5.4 (4.9–6.0) 5.0 (4.5–5.5) 5.9 (5.1–6.8) 5.0 (4.5–5.4) 6.2 (5.5–6.9)

.05*

159

4.7 (4.3–5.1)

6.4 (5.6–7.2)

179

4.8 (4.4–5.3)

6.0 (4.9–7.2)

188

5.0 (4.6–5.5)

Age ,50 y Age .50 y Female Male Chronic pain No chronic pain Pain duration 1 week or less Pain duration .1 week Intermittent pain† Variable/constant pain Positive response to cold Negative response to cold Painful percussion hypersensitivity No painful percussion hypersensitivity Painful palpation hypersensitivity No painful palpation hypersensitivity Painful bite test hypersensitivity No painful bite test hypersensitivity Presence of PARL No presence of PARL Swelling No swelling Painful percussion hypersensitivity on adjacent tooth No painful percussion hypersensitivity on adjacent tooth Painful palpation hypersensitivity on adjacent tooth No painful palpation hypersensitivity on adjacent tooth Painful bite test hypersensitivity on adjacent tooth No painful bite test hypersensitivity on adjacent tooth

.06 .79 .49 .001* .02* .02* .02* .10 .21 .04* .001*

.001*

.08

CI, conﬁdence interval; PARL, periapical radiolucency. *P , .05. † Intermittent pain is pain that comes and goes with some periods with no pain, variable pain is constant pain but pain type and intensity change over time, and constant pain is pain that remains constant over time.13

the causative tooth, and we did not want to include both in the model because of collinearity. A chi-square test was used to assess the associations of categoric variables. Subgroup analyses were performed with appropriate Bonferroni adjustments. A P value ,.05 was used to deﬁne a statistically signiﬁcant effect.

RESULTS The study sample consisted of 228 subjects who attended a dental emergency clinic due to dental pain. The demographics of the study population as well as the tooth-related characteristics including tooth type, location of the tooth, etiology of endodontic disease, pulpal and periapical diagnosis, and painrelated reports such as the use of analgesics

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are presented in the previous publication12. Brieﬂy, the mean age was 41 years, and 53% of the cohort were females. The most common etiology of endodontic disease was caries (57%). The most common pulpal diagnoses were irreversible pulpitis (IP) (34%) followed by pulp necrosis (36%). The most common periapical diagnoses were symptomatic apical periodontitis (67%) followed by acute apical abscess (14%). The average pain intensity at the time of the evaluation was 5.2 on the NRS, and 72% reported using some type of medication for pain relief.

Prediction of Acute Endodontic Pain: Univariate Analysis Based on a univariate analysis, female patients and those below the age of 50 years, when presenting for an urgent endodontic visit, reported higher intensity overall pain. Also,

patients who described the temporal nature of their pain as variable or constant pain (variable pain, constant pain but pain type and intensity change over time; constant pain, pain that remains constant over time) as opposed to intermittent pain (pain that comes and goes with some periods with no pain)13 reported signiﬁcantly higher pain levels (P , .05). Interestingly, patients with existing chronic pain conditions did not report higher intensity pain levels (Table 1). We then determined the association between clinical testing and examination ﬁndings and the intensity of pain. Patients who had a tooth that responded to cold stimulation reported less severe pain intensity than those with a negative response, as also reported in the previous publication12. Patients who reported moderate or severe pain to the percussion hypersensitivity or palpation hypersensitivity test on the causative tooth or the adjacent tooth and those who had swelling on clinical examination also had higher levels of overall pain. Percussion and palpation hypersensitivity on the adjacent tooth showed the strongest association with higher pain intensity reporting. The presence of a periapical radiolucency was not associated with higher pain intensity (Table 1).

Prediction of Acute Endodontic Pain: Multiple Regression Analysis In order to better understand the relative contribution of predictors identified in the univariate analysis to pain intensity, 2 consecutive multivariate regression models were constructed (model 1 and model 2, Table 2). The method for variable selection for the 2 models is described in Statistical Procedures in the Methods section. We found that age and sex were not predictors of preoperative pain intensity in regression models. However, we should acknowledge the observable trend for females to have higher pain intensity, even though it was not statistically significant (P 5 .10). In the first model, in line with the univariate analysis, we found that patients with a positive cold response reported less severe pain (P 5 .034). On the other hand, painful palpation hypersensitivity did not predict higher pain intensity as was observed with the univariate analysis. In the second model, percussion hypersensitivity on the adjacent tooth was a strong predictor of higher pain intensity (P 5 .001). These results point out that percussion hypersensitivity on the healthy adjacent tooth strongly predicted higher acute endodontic pain intensity, whereas palpation hypersensitivity on the causative tooth did not predict higher pain intensity.

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TABLE 2 - Multiple Regression Analysis to Predict Pain Intensity Model 1 Variable Standard error b Positive cold response (causative tooth) 20.85 0.40 Age 20.02 0.01 Sex (female) 0.62 0.38 Painful palpation (causative tooth) 0.56 0.40 Constant 5.70 0.68

95% CI P value 21.63 to 20.06 .034* 20.04 to 0.01 .14 20.12 to 1.36 .10 20.22 to 1.34 .16 4.36–7.04

Model 2 Variable Standard error b Positive cold response (causative tooth) 20.88 0.40 Age 20.02 0.01 Sex (female) 0.68 0.37 Painful palpation (causative tooth) 0.14 0.40 Painful percussion (adjacent tooth) 0.72 0.21 Constant 4.79 0.72

95% CI P value 21.66 to 20.10 .027* 20.04 to 0.004 .10 20.01 to 1.41 .07 20.66 to 0.95 .72 0.31–1.14 .001* 3.37–6.21

b, slope/estimated coefﬁcient; CI, conﬁdence interval. Model 1 estimates that patients with a positive response to cold test had 0.85 points lower pain intensity on the numeric rating scale compared with patients with negative response to cold test when adjusted for other variables. This remains similar in model 2. Model 2 estimates that patients with a painful response to percussion test on the adjacent tooth had 0.72 points higher pain intensity on the numeric rating scale compared with patients with a nonpainful response to the test when adjusted for other variables. N 5 228. Dichotomous variables are coded in the models as follows: sex (male 5 0, female 5 1), cold response (negative cold response 5 0, positive cold response 5 1), percussion/palpation hypersensitivity (no/mild pain 5 0, moderate/severe pain 5 1). *P , .05.

Associations between Mechanical Sensory Testing and Pulpal Diagnosis We next evaluated the frequency of reporting of painful response to different mechanical sensory tests in teeth with varying pulpal diagnoses to investigate what these tests might capture about endodontic disease. We found that a painful response to percussion was frequently observed in teeth with IP (64%), necrotic pulp (91%), and previously initiated/ treated teeth (PIT) (68%). A subgroup analysis

with Bonferroni adjustment (P 5 .017) determined that painful percussion was more frequently reported by patients diagnosed with pulpal necrosis compared with PIT (P 5 .001) and IP (P 5 .001). The frequency of reporting of painful percussion on the adjacent tooth was equally frequent in these 3 groups (IP: 30%, necrotic pulp: 33%, and PIT: 32%). Variability was also observed in the frequency of observing painful palpation in teeth with different pulpal diagnoses (IP: 36%, necrotic pulp: 58%, and PIT: 79%) (Fig. 2).

Unlike percussion, painful palpation was more frequently reported by patients diagnosed with PIT compared with both necrotic pulp (P 5 .017) and IP (P 5 .001). Painful palpation was more frequently reported by patients diagnosed with necrotic pulp compared with IP (P 5 .005). Finally, to better understand these ﬁndings, we investigated whether the frequency of percussion and palpation pain in the necrotic pulp and PIT groups could be related to unequal distribution of teeth with frank periapical pathology in these groups. To assess this, we looked at the frequencies of swelling and periapical radiolucencies in these groups. Again, with a subgroup analysis with a Bonferroni adjustment (P 5 .025), we found there was no difference in the frequency of the presence of periapical radiolucency and swelling between these 2 groups (Table 3). Therefore, the variation in frequencies of painful percussion and palpation in teeth with pulpal necrosis or previously initiated/treated pulpal status were not because of an unequal distribution of teeth with swelling and periapical radiolucency in these groups.

DISCUSSION In endodontics, the relationship between the underlying pathophysiology of the endodontic condition and the experience of pain is not well understood3,4. Investigating this relationship can provide insight into the biological processes underlying endodontic disease. In this study, we found that the presence of periapical radiolucency was not associated with pain, whereas subjects with swelling and/or a negative response to cold testing

FIGURE 2 – The association between mechanical sensory testing and pulpal diagnosis. (A ) The frequency of reporting of painful percussion on the causative tooth. Subgroup analysis with Bonferroni adjustment showed that painful percussion was more frequently reported by patients diagnosed with necrotic pulp compared with PIT and teeth diagnosed with IP (P , .05). (B ) The frequency of reporting of painful palpation on the causative tooth. Subgroup analysis with Bonferroni adjustment showed that painful palpation was more frequently reported by patients diagnosed with PIT compared with the necrotic pulp and IP groups. Painful palpation was more frequently reported by patients diagnosed with necrotic pulp compared with IP (P , .05). (C ) The frequency of reporting of painful percussion on the healthy adjacent tooth was reported similarly in IP, necrotic pulp, and PIT. N. Pulp/R. Pulpitis, normal pulp/reversible pulpitis. P. Treated/Initiated, previously treated tooth/initiated root canal treatment.

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TABLE 3 - Associations between Mechanical Sensory Testing and Pulpal Diagnosis

Variables Painful percussion hypersensitivity No painful percussion hypersensitivity Painful palpation hypersensitivity No painful palpation hypersensitivity Painful bite test hypersensitivity No painful bite test hypersensitivity Painful percussion hypersensitivity on adjacent tooth No painful percussion hypersensitivity on adjacent tooth Painful palpation hypersensitivity on adjacent tooth No painful palpation hypersensitivity on adjacent tooth Periapical radiolucency No periapical radiolucency Swelling No swelling

Normal pulp/ reversible pulpitis, n (%)

Irreversible pulpitis, n (%)

Necrotic teeth, n (%)

Previously initiated/ treated teeth, n (%)

7 (31.8) 15 (68.2) 2 (9.1) 20 (90.9) 7 (31.8) 15 (68.2) 1 (4.5)

50 (64.1) 28 (35.9) 28 (35.9) 50 (64.1) 52 (66.7) 26 (33.3) 23 (30.3)

74 (91.4) 7 (8.6) 47 (58.0) 34 (42.0) 68 (83.9) 13 (16.1) 26 (32.9)

32 (68.1) 15 (31.9) 37 (78.7) 10 (21.3) 28 (59.6) 19 (40.4) 15 (31.9)

21 (95.5)

53 (69.7)

53 (67.1)

32 (68.1)

1 (4.5)

11 (14.5)

21 (26.9)

11 (23.4)

21 (95.5)

65 (85.5)

57 (73.1)

36 (76.6)

0 (0) 22 (100) 1 (4.5) 21 (95.5)

18 (23.1) 60 (76.9) 2 (2.6) 76 (97.4)

52 (64.2) 29 (35.8) 25 (30.9) 56 (69.1)

26 (55.3) 21 (44.7) 18 (38.3) 29 (61.7)

N 5 228.

reported higher levels of pain. Previous studies found that in patients who had an endodontic emergency visit, symptomatic pulpitis and symptomatic necrotic cases reported similar pain levels2,14. On the other hand, both of these studies also reported that patients who were diagnosed with symptomatic pulpitis were able to wait longer than patients who were diagnosed with symptomatic necrotic teeth before seeking treatment2,14, suggesting that pain became less tolerable more often in necrotic cases. Biologically, it is possible that patients with necrotic, previously initiated, and previously treated teeth may have prolonged and exaggerated neuronal and immune responses15–17. These prolonged responses correspond with more advanced disease, which may be correlated with higher levels of pain17. However, results should be carefully interpreted because patients in this cohort presented to the emergency clinic with a chief complaint of pain. In a broader patient population, a signiﬁcant number of patients with these diagnoses would present without any symptoms3,4. In clinical practice, we often use mechanical sensory testing including percussion, palpation, and bite hypersensitivity testing. However, previous studies have challenged the sensitivity and speciﬁcity of these tests, especially percussion hypersensitivity, to differentiate the degree or the presence of endodontic disease8,18. We found that in a multiple regression analysis, although a painful response to palpation did not predict higher levels of pain, a painful response to percussion on the adjacent

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healthy tooth was highly predictive of higher acute pain intensity. To evaluate further, we looked at the frequency of identifying painful percussion and palpation with different pulpal diagnoses. Interestingly, painful percussion was reported by the majority of patients who had necrotic pulp, whereas painful palpation was more frequently reported by patients who had PIT, even though there was no difference in the frequency of swelling or periapical radiolucency in these 2 groups. These findings suggest that percussion and palpation testing may be capturing different aspects of endodontic pathology. Perhaps they capture the activity of different types of sensory neurons or even in different tissues. For example, intact but inflamed hypersensitive neurons of the periapical tissues could be detected with palpation sensitivity while degenerating pulpal neurons that have died back due to progressing necrosis of the pulp might be detected with percussion19. It is likely that palpation testing captures the hypersensitivity of tissues surrounding the tooth rather than within the tooth, whereas percussion testing may be capturing both. Dental pulp is uniquely innervated by different types and proportions of A-delta and C fibers and low-threshold mechanoreceptors20. It is reported that the expression of transient receptor potential cation channel subfamily V member 2 ion channel, which has a role in stretch, is higher than transient receptor potential cation channel subfamily V member 1 neurons in the pulp21. More recently, it was found out that almost all neurons of the pulp express mechanosensitive ion channels called

PIEZO219,22. It was also found out that PIEZO2 mechanosensitive ion channels play a pivotal role in mechanical pain23,24. For example, it may be hypothesized that dieback of PIEZO2 or transient receptor potential cation channel subfamily V member 2-positive neurons cause mechanical allodynia, which may be captured by percussion testing. Further investigation is needed to explore what different mechanical sensory testing in fact reveal about the pathophysiology of endodontic disease. It has been proposed that percussion hypersensitivity, especially in teeth with vital pulp, may actually reflect peripheral or central sensitization rather than indicating inflammation of the periapical tissues9–11,25. This study demonstrated that painful percussion on the healthy adjacent tooth was a strong predictor of higher overall levels of pain. It is likely that pain on the adjacent tooth represents central sensitization because it is coming from a nonaffected site. We also found that reports of painful percussion on the adjacent tooth were equally present in groups diagnosed with IP, pulp necrosis, and PIT. Interestingly, in all 3 of these groups, about one third of the patients reported that percussion on the adjacent tooth was painful. Spreading infection in large abscess and cellulitis cases could potentially explain the increased percussion hypersensitivity on the adjacent tooth. However, finding that in the IP group percussion hypersensitivity on the adjacent tooth was equally present emphasizes that reports of pain on percussion of the adjacent tooth are often due to peripheral or central sensitization. These findings suggest that we

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need to be careful about interpreting the percussion hypersensitivity testing results. It may not always specifically identify the specific tooth with periapical inflammation or infection but rather is identifying a lowered pain threshold and/or heightened pain sensitivity due to peripheral or central sensitization11,26–28. Importantly, the identification of multiple teeth that have pain on percussion is indicative that patients are experiencing high levels of ongoing pain, and a standard analgesic regimen may be insufficient to provide pain relief. It also poses the interesting question whether centrally acting analgesics may be more efficacious in these situations. We found that in the univariate analysis, females reported higher levels of pain. Although not statistically significant, when modeled in multiple regression analysis, females seemed to report higher acute endodontic pain, which is in line with the literature reporting that females experience more severe pain29,30. Moreover, we found that age did not predict preoperative pain. A previous report found that pain intensity is inversely proportionate with age when teeth with vital pulp were tested31. The difference could be due to the fact that in our study population, only about half of the cases had vital pulp. In addition, in this study, chronic pain did not predict higher levels of preoperative pain, although it is generally accepted that sensitization of the pain modulation system due to chronic pain increases acute pain severity32. The reason why age, sex, and the presence of chronic pain did not unequivocally predict higher levels of odontalgia could be due to the characteristics of the study population. It could also be due to unique features of endodontic pain; when pain due to either

pulpal inflammation or endodontic infection is severe enough to make an emergency dental visit, factors such as sex, age, and the presence of chronic pain may not so clearly influence the degree of pain experienced. There are several limitations of our study. First, there was the potential variability in the performance of clinical testing in the study. In order to minimize provider variability, study personnel were trained and calibrated, and patients were given a visual scale to help specify their responses more accurately. However, because pain reporting is inherently variable, there certainly was variability in patient responses to mechanical testing. Another limitation of the study is that pulp vitality was inferred by vitality (cold) tests instead of a more direct method like direct visualization. We were not able to observe and record the intraoperative status of the pulp by direct visualization because after the patients had been triaged during the emergency visit, they were referred for various treatment modalities. However, we know that cold testing has been shown to have high sensitivity and specificity to differentiate pulpal vitality6.

CONCLUSIONS Percussion hypersensitivity on the healthy adjacent tooth is a frequent observation in teeth with different pulpal diagnoses, and it strongly predicted the overall severity of pain experienced. This suggested that percussion hypersensitivity may identify a lowered pain threshold and heightened pain sensitization and at times is primarily due to central sensitization. Also, 2 different mechanical sensory tests (percussion hypersensitivity and

palpation hypersensitivity) differentially predicted the severity of acute dental pain. Furthermore, they were identiﬁed with different frequencies in patients with different pulpal diagnosis, suggesting that these tests may be revealing different aspects of endodontic pathophysiology and dental nociception. Importantly, pain as a highly individualized, multifaceted phenomena may generally be a problematic measure for inferring an endodontic diagnosis.

ACKNOWLEDGMENTS The authors thank the dental students and postgraduate residents who helped with data collection including Sean Burlingame (New York University), Ariella Spodek (New York University), Maheen Kibria (New York University), SooYoung Kim (University of California San Francisco), and Jeffrey Lee (University of California Sand Francisco). We also thank Jacqueline Starr (Harvard School of Dental Medicine) who reviewed our data analysis plan. Supported by Harvard Catalyst | The Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, National Institutes of Health Award UL 1TR002541) and Harvard University and its affiliated academic healthcare centers. The content is solely the responsibility of the authors and does not necessarily represent the official views of Harvard Catalyst, Harvard University and its affiliated academic healthcare centers, or the National Institutes of Health. The author deny any conflicts of interest related to this study.

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Cachovan G, Phark JH, Schon G, et al. Odontogenic infections: an 8-year epidemiologic analysis in a dental emergency outpatient care unit. Acta Odontol Scand 2013;71:518–24.

Toure B, Kane AW, Diouf A, et al. Preoperative pain and medications used in emergency patients with irreversible acute pulpitis or acute apical periodontitis: a prospective comparative study. J Orofac Pain 2007;21:303–8.

Gutmann JL, Baumgartner JC, Gluskin AH, et al. Identify and deﬁne all diagnostic terms for periapical/periradicular health and disease states. J Endod 2009;35:1658–74.

Michaelson PL, Holland GR. Is pulpitis painful? Int Endod J 2002;35:829–32.

Levin LG, Law AS, Holland GR, et al. Identify and deﬁne all diagnostic terms for pulpal health and disease states. J Endod 2009;35:1645–57.

Jespersen JJ, Hellstein J, Williamson A, et al. Evaluation of dental pulp sensibility tests in a clinical setting. J Endod 2014;40:351–4.

Mainkar A, Kim SG. Diagnostic accuracy of 5 dental pulp tests: a systematic review and metaanalysis. J Endod 2018;44:694–702.

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Pigg M, Nixdorf DR, Nguyen RH, et al. Validity of preoperative clinical ﬁndings to identify dental pulp status: a National Dental Practice-Based Research Network study. J Endod 2016;42:935–42.

Owatz CB, Khan AA, Schindler WG, et al. The incidence of mechanical allodynia in patients with irreversible pulpitis. J Endod 2007;33:552–6.

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Law AS, Nixdorf DR, Aguirre AM, et al. Predicting severe pain after root canal therapy in the National Dental PBRN. J Dent Res 2015;94:37S–43S.

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Kayaoglu G, Ekici M, Altunkaynak B. Mechanical allodynia in healthy teeth adjacent and contralateral to endodontically diseased teeth: a clinical study. J Endod 2020;46:611–8.

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Erdogan O, Malek M, Janal MN, Gibbs JL. Sensory testing associates with pain quality descriptors during acute dental pain. Eur J Pain 2019;23:1701–11.

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Jensen MP, Dworkin RH, Gammaitoni AR, et al. Assessment of pain quality in chronic neuropathic and nociceptive pain clinical trials with the neuropathic pain scale. J Pain 2005;6:98–106.

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Nusstein JM, Beck M. Comparison of preoperative pain and medication use in emergency patients presenting with irreversible pulpitis or teeth with necrotic pulps. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:207–14.

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Renard E, Gaudin A, Bienvenu G, et al. Immune cells and molecular networks in experimentally induced pulpitis. J Dent Res 2016;95:196–205.

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Sasaki H, Furusho H, Rider DB, et al. Endodontic infection-induced inﬂammation resembling osteomyelitis of the jaws in Toll-like receptor 2/interleukin 10 double-knockout mice. J Endod 2019;45:181–8.

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Rossi HL, See LP, Foster W, et al. Evoked and spontaneous pain assessment during tooth pulp injury. Sci Rep 2020;10:2759.

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Rechenberg DK, Held U, Burgstaller JM, et al. Pain levels and typical symptoms of acute endodontic infections: a prospective, observational study. BMC Oral Health 2016;16:61.

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Emrick JJ, von Buchholtz LJ, Ryba NJ. Transcriptomic classiﬁcation of neurons innervating teeth. J Dent Res 2020;99:1478–85.

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Fried K, Sessle BJ, Devor M. The paradox of pain from tooth pulp: low-threshold "algoneurons"? Pain 2011;152:2685–9.

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Gibbs JL, Melnyk JL, Basbaum AI. Differential TRPV1 and TRPV2 channel expression in dental pulp. J Dent Res 2011;90:765–70.

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Park SK, Choi SK, Kim YG, et al. Parvalbumin-, substance P- and calcitonin gene-related peptide-immunopositive axons in the human dental pulp differ in their distribution of varicosities. Sci Rep 2020;10:10672.

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Murthy SE, Loud MC, Daou I, et al. The mechanosensitive ion channel Piezo2 mediates sensitivity to mechanical pain in mice. Sci Transl Med 2018;10:eaat9897.

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Szczot M, Liljencrantz J, Ghitani N, et al. PIEZO2 mediates injury-induced tactile pain in mice and humans. Sci Transl Med 2018;10:eaat9892.

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Alelyani AA, Azar PS, Khan AA, et al. Quantitative assessment of mechanical allodynia and central sensitization in endodontic patients. J Endod 2020;46:1841–8.

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Matsuda M, Huh Y, Ji RR. Roles of inflammation, neurogenic inflammation, and neuroinflammation in pain. J Anesth 2019;33:131–9.

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Cobos EJ, Nickerson CA, Gao F, et al. Mechanistic differences in neuropathic pain modalities revealed by correlating behavior with global expression proﬁling. Cell Rep 2018;22:1301–12.

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Latremoliere A, Woolf CJ. Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain 2009;10:895–926.

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Mogil JS. Sex differences in pain and pain inhibition: multiple explanations of a controversial phenomenon. Nat Rev Neurosci 2012;13:859–66.

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Farac RV, Morgental RD, Lima RK, et al. Pulp sensibility test in elderly patients. Gerodontology 2012;29:135–9.

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CLINICAL RESEARCH Pasinee Nuamwisudhi, DDS, and Thanomsuk Jearanaiphaisarn, DDS, MSc

Oral Functional Behaviors and Tooth Factors Associated with Cracked Teeth in Asymptomatic Patients ABSTRACT Introduction: A cracked tooth may occur due to excessive applied force or tooth weakness. However, there is scant information concerning the cracked tooth risk factors. This study aimed to explore the oral functional behaviors and tooth factors associated with posterior cracked teeth. Methods: Fifty-six patients underwent their oral functional behavior assessment via a questionnaire. The intraoral parameters at the patient level (remaining teeth, occluding tooth pairs, overbite, overjet, and occlusal guidance type) and tooth level (remaining marginal ridge number, restored surface number, restorative materials, and cuspal inclination) were examined. The posterior teeth were stained with methylene blue dye and inspected for cracks using a microscope. The correlations between each patient-level parameter and the cracked tooth number/subject were determined using linear regression analysis. The cracked teeth were matched with their contralateral noncracked teeth, and binary regression analysis was used to analyze the association between tooth-level parameters and a cracked tooth. Multivariate regression analysis was performed if more than 1 parameter had a P value .1. Results: One hundred thirty-five cracked teeth were found. Eating hard food was significantly related to the cracked tooth number (P , .05). In molars, the occlusal surface restoration and cuspal inclination were significantly related to a cracked tooth, except the mesiobuccal cusp. In the multivariate analysis, the distolingual cusp inclination significantly predicted a cracked tooth (P , .05). In premolars, the lingual cusp inclination was associated with a cracked tooth (P , .05). Conclusions: Eating hard food, occlusal surface restoration, and steep cuspal inclination were associated with posterior cracked teeth. (J Endod 2021;47:1383–1390.)

SIGNIFICANCE Eating hard food was a behavior that was positively related to posterior cracked teeth. High cuspal inclination and class I amalgam restoration might be the risk factors for a posterior cracked tooth.

KEY WORDS Cracked tooth; dental cavity preparations; dental ﬁlling; eating behavior; multivariate analysis; risk factors

A cracked tooth is an incomplete tooth fracture originating from the crown and extending along the proximal surface of the tooth and may include the root.1,2 A cracked tooth presents with different symptoms depending on the crack depth and involved tissues.1,3 The symptoms range from mild to severe spontaneous pain consistent with irreversible pulpitis, pulp necrosis, or apical periodontitis.1 Previous studies revealed 42.2%–67.4% of teeth diagnosed as cracked teeth underwent endodontic treatment, and 13.6%–22.9% required extraction.2,4,5 The etiology of cracked teeth has 2 hypotheses: excessive force on a healthy tooth and physiological force on a weakened tooth.6 Previous studies agreed that a crack frequently occurred in an intact tooth or minimally restored tooth, whereas a fractured cusp was common in a tooth with extensive caries or a large restoration.2,4,7,8 Thus, crack initiation might derive from excessive force on an intact tooth; however, it is also inﬂuenced by multifactorial conditions that vary among patients.6 Nevertheless, there is scant information concerning the cracked tooth risk factors. Only 1 study statistically analyzed the related factors of cracked teeth and focused on only oral habits.9 This study’s aim was to identify the association between cracked teeth and the following suspected factors: oral functional behaviors and intraoral parameters at the patient level related to occlusal force and the tooth level related to the tooth’s strength. Understanding the association between cracked teeth and these factors will be beneﬁcial for preventing crack formation, retarding crack

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From the Department of Operative Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand Address requests for reprints to Dr Thanomsuk Jearanaiphaisarn, Department of Operative Dentistry, Faculty of Dentistry, Chulalongkorn University, 34 Henri Dunant Road, Pathumwan, Bangkok 10330, Thailand. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.05.012

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propagation, and assessing the risk of a cracked tooth in patients. The null hypothesis was that none of the oral functional behaviors or intraoral parameters was associated with posterior cracked teeth.

For behaviors 1 to 3, the subjects chose the most typical frequency ( or ,4 times/ month or none), whereas behaviors 4 to 5 were answered yes or no.

Intraoral Examination

MATERIALS AND METHODS This cross-sectional study was approved by the Ethics Committee of the Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand (HREC-DCU 2019-046), and conformed to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines (Version 4 as published in October/ November 2007). Informed consent was obtained from all subjects.

The teeth were cleaned using a rubber cup and pumice. A third-year endodontic resident (examiner 1) evaluated the subjects’ intraoral parameters. The following details were recorded: 1. The remaining teeth and occluding tooth pairs in centric occlusion 2. Overbite, overjet, and occlusal guidance type 3. The number of remaining marginal ridges (each posterior tooth) 4. Restored surfaces and restorative materials (each posterior tooth)

Subject Selection The subjects were patients who were 30–70 years old and visited the Graduate Endodontic Clinic, Faculty of Dentistry, Chulalongkorn University from December 2018 to May 2019 for endodontic treatment follow-up. The subject exclusion criteria and study design are provided in Figure 1. The study sample size was determined based on the rule of thumb for multiple regression analysis—the subject to variable ratio should be at least 10:1.10 In a previous study,9 there were 5 factors suspected as being related to a cracked tooth. Therefore, 50 subjects were required for this study.

Oral Functional Behavior Assessment Oral functional behaviors were assessed by a Thai-modified questionnaire based on Hilton et al’s study.11 The questionnaire’s reliability was evaluated by the Cohen kappa coefficient in a pilot study that demonstrated kappa values of 0.72–0.99, indicating substantial to almost perfect agreement.12 The subjects completed the behavior assessment questionnaire before oral examination. The questionnaire consisted of 5 oral functional behaviors: 1. Eating hard food: commonly ate hard foods (eg, nuts, crispy pork, fried fish skin, or ice) 2. Thermal cycling eating: consistently ate hot food alternating with cold food (eg, drinking cold water after eating hot soup) 3. Clenching: tightly gnashing or intensely grinding the teeth 4. Unilateral chewing: usually chewing food on only 1 side of the mouth 5. Biting on hard objects: an experience of biting on hard objects (eg, beer bottle caps, stones, or bones in food)

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Cuspal Inclination Measurement Before cracked tooth assessment, full-arch alginate impressions (Jeltrate Fast Set; Dentsply Sirona, York, PA) were taken to fabricate tooth models. A model with any cracked teeth and their contralateral teeth were marked and trimmed to their mesial/distal marginal ridges. Buccolingual views of these models were captured using a dental operating microscope (5!). The cuspal inclination of each cusp was determined by the angle between a line from the cusp tip to the central groove and an imaginary line perpendicular to the long axis of the tooth crown13 using ImageJ 1.52a (Wayne Rasband, Madison, WI).

Cracked Tooth Assessment The posterior teeth were stained with 2% methylene blue dye (Chulalongkorn University, Bangkok, Thailand), and crack lines were assessed using a dental operating microscope (OPMI pico; Carl Zeiss, Oberkochen, Germany) at 8.5! by an experienced endodontist (examiner 2). The tooth was deﬁned as cracked if a stained crack line was found on the marginal ridge that could not be removed by a dental explorer. Auxiliary examinations composed of a biting test with a FracFinder (Denbur Inc, Westmont, IL) and a transillumination test using a dental curing light (Demi Plus; Kerr Co) were performed. Each test was performed twice.

Statistical Analysis Statistical analysis was performed with SPSS Statistics Version 20 (IBM Corp, Armonk, NY). The parameters were classiﬁed into 2 levels: 1. Patient-level parameters: the remaining tooth number, occluding tooth pair

number, overbite, overjet, occlusal guidance type, and oral functional behaviors; the associations between each parameter and the posterior cracked tooth number were analyzed using a univariate linear regression model 2. Tooth-level parameters: the cuspal inclination, presence of an opposing tooth, having a crack on an opposing tooth, having a restoration, remaining marginal ridge number, restored surface number, and restorative material; the cracked teeth and their noncracked contralateral teeth data were used to analyze the associations between each parameter and having a posterior cracked tooth by univariate binary logistic regression. A tooth pair was excluded if 1 tooth could not have its marginal ridge inspected (eg, the tooth was restored with Mesio-occluso-distal restoration or crown). The variables at P .1 were included in the multivariate regression analysis to adjust for the various independent variables. The significance level was .05. The estimated adjusted odds ratios (ORs) with their corresponding 95% confidence intervals were calculated. Moreover, the variance inflation factor was calculated to determine the multicollinearity in the multivariate regression analyses.

RESULTS The 56 selected subjects included 17 men and 39 women who were 30–61 years old (30–39 years, 12.5%; 40–49 years, 42.9%; 50–59 years, 39.3%; and .60 years, 5.4%). Forty-six subjects had 1 cracked tooth, with 135 cracked teeth found. The prevalences of cracked tooth subjects were highest in the .60-year-old group (100%) and lowest in the 40- to 49-year-old group (70.83%). A cracked tooth was most commonly in mandibular second molars (21.5%) followed by mandibular first molars (17%), maxillary second molars (14%), and maxillary second premolars (13.3%). The intraoral parameter data of a subject with no cracked tooth were omitted because of a lost record. We determined the patient-level parameters related to the number of posterior cracked teeth in a subject (Table 1). The univariate analysis results indicated that eating hard food was the only oral functional behavior significantly related to the posterior cracked tooth number (P , .05). However, no patientlevel intraoral parameter was significantly related to the posterior cracked tooth number (P . .05). Because only 1 patient-level

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Subjects (n=69)

Exclusion criteria • Dental structure anomalies • Generalized periodontitis • < 2 posterior occluding tooth pairs • Used removable prosthesis • History of facial trauma • History of orthodontic treatment • Temporomandibular disorder • History of cracked tooth management

Selected subjects (n=56)

Oral functional questionnaire (n=56)

Intraoral examination by Examiner 1 (n=56)

1) Eating hard food 2) Thermal cycling eating 3) Clenching 4) Unilateral chewing 5) Biting on hard object

1) Remaining teeth 2) Occluding tooth pairs 3) Overbite, Overjet 4) Occlusal guidance type 5) Remaining marginal ridges 6) Restored surfaces 7) Restorative material

Taking impression for making study model (n=56)

Cuspal inclination

Cracked tooth assessment by Examiner 2 (n=56)

FIGURE 1 – The study design including subject selection, oral examination, and data collection.

parameter had a P value .1, multivariate analysis was not indicated. Among the cracked tooth subjects, there were 64 matched tooth pairs (46 molar and 18 premolar pairs). According to the pooled data (premolars and molars), the parameters with a P value ,.1, a crack in an opposing tooth, restored surface number, and restorative material were included in the multivariate analysis. However, no tooth-level parameter was significantly associated with a posterior cracked tooth in both univariate and multivariate analysis (P . .05, Table 2). Compared with an intact tooth, the molar univariate analysis (Table 3) demonstrated that 1 surface restoration was significantly related to a cracked tooth with an odds ratio of 3.44 (P , .05), whereas a complex restoration was not (P . .05). Moreover, the high cuspal inclination, except for the mesiobuccal cusp, was significantly related to a cracked molar (P , .05). The parameters with a P value .1, the cuspal inclination, having a restoration, restored surface number, and restorative material were included in the multivariate analysis. Consequently, the distolingual cuspal inclination was the sole significant predictor of

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a cracked molar with an OR 5 1.085 (P , .05, Table 3). No premolar parameters were significantly related to a posterior cracked tooth in univariate analysis (Table 4). However, in the multivariate regression model, the lingual cuspal inclination was significantly related to a cracked premolar with an OR 5 1.215 (P , .05, Table 4). The variance inflation factor values were 1.011–5.492 in all models, indicating low multicollinearity.14

DISCUSSION Although several studies focused on managing symptomatic cracked teeth,15,16 few studies have evaluated the factors related to cracked teeth, especially asymptomatic cracked teeth.9,17 Although the prevalence of a cracked tooth in patients in previous studies was 8.9%– 9.7%4,18), most subjects (84%) in this study had at least 1 posterior cracked tooth. This higher percentage might be due to different methodology and inclusion criteria. Here, we examined all posterior teeth stained with methylene blue dye using a dental operating microscope, and most of the detected cracked teeth were asymptomatic. However,

transillumination and the bite test detected only 29% and 6% of the cracked teeth, respectively. The characteristics of the cracked teeth in this study were relatively similar to those in the National Dental Practice–Based Research Network’s study, which found that .50% of cracked teeth were asymptomatic and that only 16% of cracked teeth had pain on biting.19 Thus, cracked teeth might be common or result from some oral functional behaviors. With many more cracked tooth subjects than noncracked tooth subjects in this study, matching the subjects with and without a cracked tooth was unfeasible. Therefore, the analysis of each patient-level parameter was correlated to the number of posterior cracked teeth/subject. These results indicated which patient behavior or intraoral parameters were related to having a cracked tooth. Based on our results, the null hypothesis was rejected. There are several oral functional behaviors that relate to high occlusal force on a tooth;20,21 however, only eating hard food was positively related to the posterior cracked tooth number. If a person eats hard food more frequently, it is likely that he or she would increase the chances of

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TABLE 1 - The Associations between the Number of Posterior Cracked Teeth and Oral Functional Behaviors or Intraoral Parameters (Patient Level): A Univariate Linear Regression Model Analysis

Nuamwisudhi and Jearanaiphaisarn

The cracked tooth number/subject (the number of subjects) Patient-level parameters Behaviors Eating hard food 4 times/month (n 5 24) ,4 times/month (n 5 23) None (n 5 9) Thermal cycling eating 4 times/month (n 5 19) ,4 times/month (n 5 20) None (n 5 17) Clenching 4 times/month (n 5 8) ,4 times/month (n 5 9) None (n 5 39) Unilateral chewing Yes (n 5 35) No (n 5 21) Biting on hard objects Yes (n 5 30) No (n 5 26) Intraoral parameters Remaining teeth (mean 6 SD) Occluding tooth pairs (mean 6 SD) Overjet (mean 6 SD) Overbite (mean 6 SD) Occlusal guidance type (n) Canine guidance Anterior group function Group function Other

0 (10)*

1 (10)

2 (11)

3 (13)

‡4 (12)

3 5 2

4 4 2

4 5 2

5 6 2

8 3 1

3 5 2

3 2 5

4 2 5

4 6 3

5 5 2

1 3 6

0 1 9

1 1 9

5 2 6

1 2 9

6 4

6 5

6 7

11 1

4 6

2 8

9 2

8 5

7 5

28.33 6 2.23 6.56 6 2.12 1.55 6 1.72 1.33 6 1.71

26.40 6 4.52 6.90 6 2.07 1.90 6 2.25 2.50 6 2.44

26.36 6 6.50 7.55 6 1.69 2.55 6 1.30 2.09 6 1.30

27.62 6 1.80 6.69 6 1.37 3.26 6 2.40 2.23 6 1.99

27.92 6 2.35 7.00 6 2.00 2.45 6 2.32 2.16 6 2.45

1 0 6 2

0 3 7 0

0 2 9 0

1 4 8 0

0 5 6 1

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B, unstandardized beta coefficient; CI, confidence interval; SD, standard deviation; SE, standard error for the unstandardized beta. *Missing intraoral parameter data of 1 subject in noncracked subjects. † There was a statistically significant difference (P , .05).

95% CI

P value

0.804

0.376

0.050–1.558

.037†

0.448

0.346

20.246 to 1.143

.201

0.062

0.386

20.712 to 0.845

.874

0.638

0.575

20.515 to 1.791

.272

0.546

0.560

20.576 to 1.668

.334

0.025 0.054 0.144 0.065 20.317

0.075 0.158 0.139 0.143 0.457

20.126 to 0.175 20.263 to 0.370 20.135 to 0.424 20.221 to 0.352 21.234 to 0.600

.745 .736 .305 .650 .491

TABLE 2 - The Associations between a Posterior Cracked Tooth and Tooth-Level Parameters, Univariate/Multivariate Logistic Regression, and Pooled Data (64 Tooth Pairs) Univariate model Tooth-level parameter Remaining marginal ridge 2 (intact) 1 Opposing tooth Presence Absence Crack in opposing tooth Presence Absence Presence of restoration Presence Absence Restoration surface Complex (.1 surface) One surface (occlusal) No restoration Restorative material Amalgam Resin composite No restoration

Multivariate model

Crack

Noncrack

95% CI

P value

54 10

49 15

1.653 Ref

0.68–4.02

.268

61 3

62 2

0.656 Ref

0.10–4.06

.650

18 46

10 54

2.113 Ref

0.88–5.03

.091*

49 15

41 23

1.833 Ref

0.84–3.96

.124

16 33 15

18 23 23

1.363 2.200 Ref

0.53–3.47 0.94–5.09

.517 .066*

44 5 15

35 6 23

1.928 1.278 Ref

0.87–4.23 0.33–4.94

.102* .723

95% CI

P value

2.001

0.81–4.88

.128

1.597

0.39–6.46

.512

1.355

0.37–4.90

.643

CI, conﬁdence interval; OR, odds ratio. *There was no signiﬁcant difference; however, this variable was considered for inclusion in the multiple regression model (P .1)

TABLE 3 - The Associations between a Posterior Cracked Tooth and Tooth-Level Parameters, Univariate/Multivariate Logistic Regression, and Molars (46 Tooth Pairs) Univariate model Tooth-level parameter Remaining marginal ridge 2 (intact) 1 Opposing tooth Presence Absence Crack in opposing tooth Presence Absence Presence of restoration Presence Absence Restoration surface Complex (.1surface) One surface (Occlusal) No restoration Restorative material Amalgam Resin composite No restoration Degree of cuspal inclination (mean 6 SD) Mesiobuccal cusp Mesiolingual cusp Distobuccal cusp Distolingual cusp

Multivariate model

Crack

Noncrack

95% CI

P value

95% CI

P value

39 7

33 13

2.195 Ref

0.78–6.14

.134

45 1

1.000 Ref

0.06–16.48

1.000

13 33

9 37

1.620 Ref

0.61–4.27

.330

40 6

33 13

2.626 Ref

0.89–7.66

.077*

2.158

0.32–14.51

.429

13 27 6

16 17 13

1.760 3.441 Ref

0.52–5.92 1.09–10.78

.361 .034†

1.690

0.58–4.87

.332

36 4 6

28 5 13

2.786 1.733 Ref

0.94–8.25 0.39–8.86

.064* .449

1.155

0.25–5.29

.853

23.30 6 6.38 25.36 6 7.82 24.17 6 7.49 24.70 6 7.40

22.44 6 7.09 21.60 6 7.99 20.09 6 6.72 20.38 6 6.66

1.020 1.063 1.085 1.093

0.95–1.08 1.00–1.12 1.02–1.15 1.02–1.16

.536 .028† .010† .006†

1.011 1.059 1.085

0.94–1.08 0.98–1.13 1.00–1.17

.765 .108 .046†

CI, confidence interval; OR, odds ratio; SD, standard deviation. *There was no significant difference; however, this variable was considered for inclusion in the multiple regression model (P .1). † There was a significant difference (P , .05).

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TABLE 4 - The Associations between a Posterior Cracked Tooth and Tooth-Level Parameters, Univariate/Multivariate Logistic Regression, and Premolars (18 Tooth Pairs) Univariate model Crack

Noncrack

95% CI

P value

15 3

16 2

0.625 Ref

0.09–4.27

.632

16 2

17 1

0.471 Ref

0.03–5.70

.554

5 13

1 17

6.538 Ref

0.67–62.98

.104*

9 9

8 10

1.250 Ref

0.33–4.63

.739

3 6 9

2 6 10

1.667 1.111 Ref

0.22–12.35 0.26–4.71

.617 .886

8 1 9

7 1 10

1.270 1.111 Ref

0.32–4.93 0.06–20.48

.730 .944

31.87 6 8.33 29.09 6 5.62

28.56 6 7.87 25.69 6 4.31

1.055 1.152

0.96–1.15 0.99–1.33

.228 .060*

Tooth-level parameter Remaining marginal ridge 2 (intact) 1 Opposing tooth Presence Absence Crack in opposing tooth Presence Absence Presence of restoration Presence Absence Restoration surface Complex (.1 surface) One surface (occlusal) No restoration Restorative material Amalgam Resin composite No restoration Degree of cuspal inclination (mean 6 SD) Buccal cusp Lingual cusp

Multivariate model OR

95% CI

P value

13.529

0.95–192.11

.054

1.01–1.45

.034†

1.215

having cracked teeth. Similar to previous studies, chewing on hard objects was a risk factor for a cracked tooth, and .50% of the cracked tooth patients had this habit.9,22 Hard food might induce a person to masticate with excessive force.20,23 Tooth structure and restorative materials expand or contract when the oral temperature changes; therefore, thermal cycling eating may cause a cracked tooth.24,25 The relationship between thermal cycling eating behavior and a cracked tooth was previously reported.9 Unilateral chewing and biting on hard objects were also the behaviors related to a cracked tooth.9 However, no correlation between these behaviors and the number of posterior cracked teeth was found in our study. These inconsistent results might result from using different dependent variables (ie, the number of cracked teeth vs the presence of a cracked tooth). Moreover, there were differences in the crack inclusion criteria between studies. In our study, a cracked tooth was a tooth with a dye staining crack line regardless of any symptoms, whereas Qiao et al 9 deﬁned a cracked tooth as a tooth with discomfort or sharp pain upon mastication. Nevertheless, clenching had no relationship with the number of posterior cracked teeth, which was in agreement with the study by Qiao et al.9

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By matching each cracked tooth with its noncracked contralateral tooth, individual diversity (eg, sex, age, heredity, lifestyle, and habits) was controlled. This is the first observational clinical study about cracked teeth to use a matched tooth method. An occlusal surface restoration was a significantly related parameter of cracked molars. A class I restored molar was 3.4-fold more likely to have a crack compared with an intact tooth. Although the pooled data did not show a significant association, posterior class I restored teeth were 2.2-fold more likely to be cracked compared with an intact tooth, which corresponded with previous studies.7,17 Interestingly, 85.7% of posterior cracked teeth with a class I restoration were restored with amalgam. Although restorative material was not significantly related to a posterior cracked tooth, amalgam restored teeth were ~2-fold more likely to be cracked compared with intact teeth, especially in molars (2.7-fold). This was possibly due to the high modulus of elasticity of amalgam; therefore, applying force on a tooth restored with amalgam will result in stress on the tooth.26 Cracks in amalgam-filled teeth may be caused by amalgam expansion. Amalgam has a much higher coefficient of thermal expansion than dentin, and its thermal conductivity is 20fold higher than a natural tooth.27,28 Thus,

when the intraoral temperature increases, amalgam expands, whereas dentin does not; therefore, stress will occur on the cavity walls, especially in a class I cavity, leading to crack line initiation. Moreover, when a vertical load was applied on an amalgam-restored tooth, stress concentration occurred at the cavity ﬂoor and line angles, especially in a converging cavity.28,29 There were few cracked teeth that were restored with resin composite in the present study; thus, the effect of resin composite on cracked teeth needs further investigation. The multivariate model analysis revealed that after adjusting for confounding factors, a cracked tooth was related to the inclination of the distolingual cusp in molars and lingual cusp in premolars. Cracked teeth had an average 4.32 and 3.4 higher cusp inclination than noncracked molars and premolars, respectively. These results corresponded with a previous study that showed that cracked maxillary ﬁrst molars were 5.5 –6.7 steeper compared with intact teeth.13 When a force was applied to a maxillary molar, where the cuspal inclined plane was steeper, the tensile stress at the central groove and the lateral force were higher.13,30 Some authors suggested that the wedging effect from the mesiolingual cusp of the maxillary molar could affect the distolingual cusp of the mandibular

JOE Volume 47, Number 9, September 2021

molar prone to fracture,1,13 whereas the maxillary molar oblique ridge might provide structural strength.1 The cracked teeth in our study were found most often in mandibular molars, especially in second mandibular molars, which is similar to previous studies.16,18,19 Second molars were cracked more than other teeth because a high bite force occurred when the tooth was close to the fulcrum of the mandible.31 Although having an opposing cracked tooth was not a significant parameter in the univariate models, it had a P value that approached significance in the multivariate model of premolars (P 5 .054), with a high OR of 13.529. The occlusal force may have exceeded the strength of the premolars; therefore, both occluding teeth were cracked. This outcome may be affected by the deeper cusp-fossa relationship in premolars, corresponding to the significantly steeper lingual cuspal inclination of cracked premolars compared with noncracked premolars. Moreover, premolars are subjected to more shear force than molars that receive more vertical force.32 Clinicians should provide information about cracked tooth–related factors to patients because few people with cracked teeth have signs or symptoms.19,22 Our study affirmed previous findings that eating hard food was a crack-correlated factor that increased the probability of a crack developing9 and the number of cracked teeth. Furthermore, our study’s results should raise awareness among

dentists about appropriate cavity preparation, restorative material choice, and cusp steepness of restored teeth, especially in a class I cavity. Steep cusp inclination and amalgam may increase the chance of a cracked tooth. Because of ethical considerations, old restorations were not removed before examination; therefore, a crack line under a restoration may have been missed, and the number of cracked teeth might be underestimated. However, we controlled for this by excluding subjects with a history of a cracked tooth. Some of these subjects’ cracked teeth might have been extracted or covered with a crown; thus, the real number of cracked teeth they had could not be determined. This research was a cross-sectional study in a group of the faculty’s patients; therefore, the subjects might have limited diversity. Future studies should increase the number of subjects and should collect data from groups of people in different regions. During the intraoral examinations, the examiner noticed that many cracked teeth with restorations had marginal ridges less than 2 mm thick. This ﬁnding corresponded to previous studies in which marginal ridge thickness affected the fracture resistance of a tooth.33,34 Unfortunately, the marginal ridge thickness was not analyzed in this study because the tool and method were not appropriate to accurately measure its thickness. The type of restoration of an

opposing tooth (eg, a crown or implant) was also considered a potential risk factor for crack development.2,35 However, the opposing tooth restoration was not considered in this study because there was no crown or implant opposing to a cracked tooth in the subjects. These factors should be evaluated in the future. In conclusion, eating hard food was an oral functional behavior that was positively related to posterior cracked teeth. Among the tooth characteristics, a posterior cracked tooth was related to having an occlusal surface restoration and high cuspal inclination.

ACKNOWLEDGMENTS The authors thank the clinical staff and students of the Graduate Endodontic Clinic for facilitating the oral examinations, Assistant Professor Dr Soranan Chandrangsu for statistical consultation, Associate Professor Dr Veera Lertchirakarn and Assistant Professor Dr Supawadee Naorungroj for valuable and constructive suggestions, and Dr Kevin Tompkins for manuscript editing. Supported by the resident fund from the Royal College of Dental Surgeons of Thailand. The sponsor was not involved in any aspect of this study. The authors deny any conﬂicts of interest related to this study.

REFERENCES

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Rivera E, Walton R. Longitudinal tooth cracks and fractures: an update and review. Endod Topics 2015;33:14–42.

Roh BD, Lee YE. Analysis of 154 cases of teeth with cracks. Dent Traumatol 2006;22:118–23.

Kahler W. The cracked tooth conundrum: terminology, classiﬁcation, diagnosis, and management. Am J Dent 2008;21:275–82.

Kang SH, Kim BS, Kim Y. Cracked teeth: distribution, characteristics, and survival after root canal treatment. J Endod 2016;42:557–62.

Krell KV, Caplan DJ. 12-month success of cracked teeth treated with orthograde root canal treatment. J Endod 2018;44:543–8.

Geurtsen W. The cracked-tooth syndrome: clinical features and case reports. Int J Periodontics Restorative Dent 1992;12:395–405.

Hiatt WH. Incomplete crown-root fracture in pulpal-periodontal disease. J Periodontol 1973;44:369–79.

Abou-Rass M. Crack lines: the precursors of tooth fractures - their diagnosis and treatment. Quintessence Int Dent Dig 1983;14:437–47.

Qiao F, Chen M, Hu X, et al. Cracked teeth and poor oral masticatory habits: a matched casecontrol study in China. J Endod 2017;43:885–9.

10.

Hair JF Jr, Black WC, Babin BJ, et al. Multivariate Data Analysis: A Global Perspective. Upper Saddle River, NJ: Pearson Education; 2010.

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Hilton TJ, Funkhouser E, Ferracane JL, et al. Correlation between symptoms and external characteristics of cracked teeth: ﬁndings from the National Dental Practice-Based Research Network. J Am Dent Assoc 2017;148:246–256.e1.

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Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33:159–74.

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Qian Y, Zhou X, Yang J. Correlation between cuspal inclination and tooth cracked syndrome: a three-dimensional reconstruction measurement and ﬁnite element analysis. Dent Traumatol 2013;29:226–33.

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Kutner MJ, Nachtsheim CJ, Neter J, et al. Applied Linear Statistical Models. 5th ed. New York: McGraw-Hill; 2004.

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Abbott P, Leow N. Predictable management of cracked teeth with reversible pulpitis. Aust Dent J 2009;54:306–15.

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Kim SY, Kim SH, Cho SB, et al. Different treatment protocols for different pulpal and periapical diagnoses of 72 cracked teeth. J Endod 2013;39:449–52.

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Seo DG, Yi YA, Shin SJ, et al. Analysis of factors associated with cracked teeth. J Endod 2012;38:288–92.

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Krell KV, Rivera EM. A six year evaluation of cracked teeth diagnosed with reversible pulpitis: treatment and prognosis. J Endod 2007;33:1405–7.

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Hilton TJ, Funkhouser E, Ferracane JL, et al. Recommended treatment of cracked teeth: results from the National Dental Practice-Based Research Network. J Prosthet Dent 2020;123:71–8.

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Cameron CE. Cracked-tooth syndrome. J Am Dent Assoc 1964;68:405–11.

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Rosen H. Cracked tooth syndrome. J Prosthet Dent 1982;47:36–43.

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Khovidhunkit SP, Songmanee S. Prevalence of cracked tooth in a group of patients at the Faculty of Dentistry, Mahidol University. M Dent J 2014;34:234–42.

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Wright EF, Bartoloni JA. Diagnosing, managing, and preventing cracked tooth syndrome. Gen Dent 2012;60:e302–7.

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Brown WS, Jacobs HR, Thompson RE. Thermal fatigue in teeth. J Dent Res 1972;51:461–7.

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Srivastava B, Devi N, Gupta N, et al. Comparative evaluation of various temperature changes on stress distribution in class II mesial-occlusal-distal preparation restored with different restorative materials: a ﬁnite element analysis. Int J Clin Pediatr Dent 2018;11:167–70.

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Bryant RW, Mahler DB. Modulus of elasticity in bending of composites and amalgams. J Prosthet Dent 1986;56:243–8.

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Craig RG, Peyton FA. Thermal conductivity of tooth structure, dental cements, and amalgam. J Dent Res 1961;40:411–8.

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Danley BT, Hamilton BN, Tantbirojn D, et al. Cuspal ﬂexure and stress in restored teeth caused by amalgam expansion. Oper Dent 2018;43:E300–7.

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Teixeira ES, Rizzante FA, Ishikiriama SK, et al. Fracture strength of the remaining dental structure after different cavity preparation designs. Gen Dent 2016;64:33–6.

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Xie N, Wang P, Wu C, et al. Impact of cusp inclinations on dental fractures in cracked tooth syndrome model and relevant risk evaluation. Exp Ther Med 2017;14:6027–33.

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Sathyanarayana HP, Premkumar S, Manjula WS. Assessment of maximum voluntary bite force in adults with normal occlusion and different types of malocclusions. J Contemp Dent Pract 2012;13:534–8.

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Schwartz RS, Robbins JW. Post placement and restoration of endodontically treated teeth: a literature review. J Endod 2004;30:289–301.

33.

Kalburge V, Yakub SS, Kalburge J, et al. A comparative evaluation of fracture resistance of endodontically treated teeth, with variable marginal ridge thicknesses, restored with composite resin and composite resin reinforced with Ribbond: an in vitro study. Indian J Dent Res 2013;24:193–8.

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Shahrbaf S, Mirzakouchaki B, Oskoui SS, et al. The effect of marginal ridge thickness on the fracture resistance of endodontically-treated, composite restored maxillary premolars. Oper Dent 2007;32:285–90.

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Rosen E, Volmark Y, Beitlitum I, et al. Dental implant placement is a possible risk factor for the development of multiple cracks in non-endodontically treated teeth. Sci Rep 2020;10:8527.

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JOE Volume 47, Number 9, September 2021

CLINICAL RESEARCH

Influence of Voxel Size and Filter Application in Detecting Second Mesiobuccal Canals in Cone-beam Computed Tomographic Images ABSTRACT Introduction: This study assessed the influence of voxel size and filter application in detecting second mesiobuccal (MB2) canals in cone-beam computed tomographic (CBCT) images. Methods: Using the OP300 CBCT system (Instrumentarium, Tuusula, Finland) and 3 voxel size protocols (80 mm, 125 mm, and 200 mm), we scanned 40 first molars: 20 with an MB2 canal and 20 without. All molars received silver palladium pins on the palatal root, whereas the non-MB2 molars were also filled with gutta-percha. Five oral radiologists assessed the presence of an MB2 canal under 3 filter application conditions: without filter, with sharpen 1 ! filter, and with sharpen 2 ! filter. Intra- and interobserver reproducibility was evaluated using the weighted kappa index. We compared the area under the receiver operating characteristic curves with SPSS Statistics v.20.0 (IBM Corp, Armonk, NY) using 2-way analysis of variance and the Tukey post hoc test with 5% significance level. Results: Our analysis found median intra- and interobserver agreement values of 0.70 and 0.56, respectively. The 80-mm voxel with sharpen 1 ! filter image group had the highest sensitivity, accuracy, and negative predictive values. As for specificity and positive predictive, the 80-mm voxel group without filter application presented the highest values. The areas under the receiver operating characteristic curve were higher in the 80-mm groups than in the 125-mm and 200-mm voxel size groups (P , .05). We found no differences among the filters used (P 5 .22) or for the filter–voxel size interactions (P 5 .88). Conclusions: A smaller voxel size increased the accuracy in detecting MB2 canals, whereas the enhancement filters did not. (J Endod 2021;47:1391–1397.)

KEY WORDS Cone-beam computed tomography; diagnostic imaging; endodontics; molar; radiographic image enhancement

Proper knowledge regarding the root canal system morphology is essential to avoid incomplete instrumentation, 1 of the key factors leading to failed endodontic treatments. Such a failure can result in signs and symptoms related to microbial proliferation, such as pain and a sinus tract1–4. Maxillary molars, the teeth that undergo the most endodontic treatment, often require retreatment due to failure in second mesiobuccal (MB2) canal detection5–7. The MB2 canal is regularly present in ﬁrst maxillary molars, with a prevalence of 69.6%8. Besides the frequency of missed MB2 canals in initial endodontic treatment, rehabilitation of compromised teeth usually uses metallic materials, such as pins of different chemical elements, that can interfere in detecting a particular structure or condition9–12. In this context, the American Association of Endodontists recommends using cone-beam computed tomographic (CBCT) to analyze teeth with potential extra canals and for those that may require retreatment13. In turn, the European Society of Endodontology indicates CBCT imaging for relapsing cases with possible untreated canals14. In the context of CBCT image acquisition, a dexel is deﬁned as the smallest unit at the image receptor of a given machine. Before creating a 3-dimensional image, bidimensional images called basis

JOE Volume 47, Number 9, September 2021

S^ amia Mouzinho-Machado, MD,* Lucas de Paula Lopes Rosado, MD,* Fernanda Coelho-Silva, MD,* Frederico Sampaio Neves, PhD,*† Francisco Haiter-Neto, PhD,* and Sergio Lins deAzevedo-Vaz, PhD*‡

SIGNIFICANCE We believe our study can contribute to this clinical area by showing if there is a possibility of radiation dose reduction when using both voxel size and ﬁlter application variations in MB2 canal detection.

From the *Division of Oral Radiology, Department of Oral Diagnosis, Piracicaba Dental School, University of Campinas, Piracicaba, S~ao Paulo, Brazil; † Department of Propedeutics and Integrated Clinic, Division of Oral Radiology, Federal University of Bahia, Salvador, Bahia, Brazil; and ‡Department of Clinical Dentistry, Federal University of ria, Espírito Santo, Espírito Santo, Vito Brazil Address requests for reprints to Dr Sergio Lins de-Azevedo-Vaz, Division of Oral Radiology, Department of Oral Diagnosis, Piracicaba Dental School, University of Campinas, Av Limeira 901, CEP 13414903, Piracicaba, SP, Brazil. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.011

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images are generated by the system; these have the pixel as their smallest constituent unit. When reconstructing these images, the CBCT system produces a digital volume composed by voxels. In addition, CBCT software may have a thickness changing tool, which groups several slices with the originally acquired voxel size to form an image of a thicker slice. This technique usually allows a certain level of adjustment in protocol creation, such as changing the voxel size15. Research suggests that a smaller voxel size results in higher diagnostic accuracy for MB2 canal detection16,17. However, using a smaller voxel size generally requires higher X-ray radiation doses because of the automatic adjustment of parameters such as milliamperage or field of view15. The software used to analyze CBCT images usually allows the application of enhancement filters, which are tools that modify the images by smoothing (blurring of structure limits), sharpening (increased definition of anatomic boundaries), and changing color, among others. Some software and filters have shown higher accuracy in detecting transverse root fracture18 and measuring apical bone loss19. These findings suggest that filter application can be used in conjunction with larger voxel sizes to achieve similar diagnostic accuracy to that offered by smaller ones, effectively reducing the radiation dose. Such a hypothesis supports the newest principle of radiation dose optimization for imaging examinations, which is based on reducing the dose to “as low as diagnostically acceptable, being indication-oriented, and patientspecific” (ALADAIP)20. To the best of our knowledge, there are no studies on enhancement filters associated with different voxel sizes for MB2 canal detection. Thus, the aim in this study was to evaluate the influence of both these parameters (voxels and filters) on MB2 canal detection.

MATERIALS AND METHODS This was an experimental in vitro study conducted after approval by the local research ethics committee (CAAE 25098619.1.0000.5418).

Sample Size and Preparation Our study included a total of 40 maxillary ﬁrst molars, 20 with an MB2 canal (test group) and 20 without it (control group), whose collection, preparation, and phantom construction were conducted following the work of Rosado et al21. The reference standard adopted to

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determine the presence or absence of an MB2 canal were micro–computed tomographic images taken with the Skyscan 1174 (Bruker, Kontich, Belgium) using the following parameters: 31.03-mm voxel size, 50 kVp, 800 mA, 0.5-mm aluminum ﬁlter, 0.5 rotation step, 180 arch rotation, and 1 frame averaging. Silver palladium pins 20-mm long with the same thickness as the ﬁles used during instrumentation (025.07) were inserted into the palatal canals up to two thirds of the root length. Gutta-percha was inserted into the other instrumented canals, which excludes the MB2 canal. The control group also received gutta-percha up to the working length.

Image Acquisition Tomographic images of the teeth were taken with the OP300 Maxio CBCT system (Instrumentarium, Tuusula, Finland) to obtain images with 80-mm, 125-mm, and 200-mm voxel sizes. The kilovoltage and ﬁeld of view were 90 kVp and 50 ! 50 mm, respectively. Milliamperage and exposure time automatically varied according to the selected voxel size as follows: 5 mA and 8.7 seconds (80 mm), 6.3 mA and 6.09 seconds (125 mm), and 8 mA and 2.34 seconds (200 mm). Because of the difference in exposure parameters, the dose area products were, from smaller to largest voxel size protocols, 377, 332, and 162 mGycm2 (Instrumentarium).

Image Evaluation For evaluation, we exported the images in the Digital Imaging and Communications in Medicine format from CliniView (Instrumentarium) and imported them into OnDemand 3D (Cybermed Inc, Seoul, Republic of Korea). Each tomographic volume underwent 3 filter application conditions: no filter, sharpen 1 ! filter, and sharpen 2 ! filter. The combination of 3 voxel sizes and 3 filter application protocols generated 9 experimental groups (resulting in a total of 360 images): no filter, 80-mm voxel size (NF-80); no filter, 125-mm voxel size (NF-125); no filter, 200-mm voxel size (NF-200); sharpen 1 ! filter, 80-mm voxel size (S1-80); sharpen 1 ! filter, 125-mm voxel size (S1-125); sharpen 1 ! filter, 200-mm voxel size (S1-200); sharpen 2 ! filter, 80-mm voxel size (S2-80); sharpen 2 ! filter, 125-mm voxel size (S2-125); and sharpen 2 ! filter, 200-mm voxel size (S2-200) (Fig. 1). Five previously calibrated oral radiologists with a minimum of 2 years of experience with CBCT diagnosis blindly evaluated the images regarding the presence or absence of an MB2 canal on LCD monitors

in a dimly lit room. They were allowed to adjust zoom, brightness, and contrast at their discretion and used a 5-point scale to verify an MB2 canal: 1, definitely absent; 2, probably absent; 3, uncertain; 4, probably present; and 5, definitely present. Thirty days after the first evaluation, 40% of the sample was reevaluated to calculate intraobserver reproducibility.

Statistical Analysis Intra- and interobserver reproducibilities were measured using the weighted kappa index. We calculated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy values for each experimental group. These values had the following clinical interpretation herein: sensitivity, reflecting the rate at which the MB2 canal was correctly classified as “present”; specificity, referring to the correct “absent” classification; accuracy, concerning the precision with which the findings reflect the real situation; and PPV and NPV, reflecting the probability of the evaluator correctly classifying the MB2 canal as present or absent, respectively22. We also compared the areas under the receiver operating characteristic (ROC) curves among the experimental groups using 2-way factorial analysis of variance and the Tukey post hoc test. ROC curve analysis shows the sensitivity values on the y-axis and 1 2 specificity values on the x-axis; thus, the location of the ROC curves that represent higher accuracy in the diagnostic task studied is in the upper left region of the graph23. Analyses were performed using SPSS Statistics Version 20.0 (IBM SPSS Statistics for Windows; IBM Corp, Armonk, NY) at a 5% significance level. The hypothesis tested was that the experimental groups with 125-mm and 200-mm voxel sizes with filter application (S1-125, S2-125, S1-200, and S2-200) would show the same accuracy in detecting the MB2 canal as the 80-mm voxel size groups.

RESULTS Our analysis found median intra- and interobserver agreement values of 0.70 (ranging from 0.32–0.97) and 0.56 (ranging from 0.21–0.90), respectively. Table 1 presents the agreement value range per experimental group. Group S1-80 presented the highest values of sensitivity, accuracy, and NPV, whereas NF-80 had the highest values of speciﬁcity and PPV. We found the highest

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FIGURE 1 – Axial reconstructions showing images of the 9 experimental groups and acronyms in examples without and with the presence of an MB2 canal. NF, no ﬁlter; S1, sharpen 1!; S2, sharpen 2!; 80, 80-mm voxel size; 125, 125-mm voxel size; and 200, 200-mm voxel size. areas under the ROC curve values in the NF-80 (0.91), S1-80 (0.94), and S2-80 (0.93) groups. The 80-mm voxel size groups (NF-80, S1-80, and S2-80) showed higher values than the 125-mm (NF-125, S1-125, and S2-125) and 200-mm (NF-200, S1-200, and S2-200) voxel

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size groups (P , .05). We found no differences between the 125-mm and 200-mm groups (P . .05), and no statistically significant differences among the filters used and for the filter–voxel size interaction (P . .05) (Table 2, Fig. 2).

DISCUSSION The presence of an MB2 canal can interfere with the endodontic treatment success as observed by the high frequency of maxillary molars that need retreatment due to failure in

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TABLE 1 - The Minimum, Maximum, and Median Kappa Values for Intra- and Interobserver Reproducibility Intraobserver

Interobserver

Protocols

Minimum

Maximum

Median

Minimum

Maximum

Median

NF-80 S1-80 S2-80 NF-125 S1-125 S2-125 NF-200 S1-200 S2-200 All groups

0.33 0.36 0.80 0.57 0.44 0.32 0.55 0.61 0.49 0.32

0.64 0.77 0.97 0.89 0.90 0.55 0.84 0.90 0.87 0.97

0.58 0.72 0.90 0.77 0.65 0.42 0.69 0.73 0.83 0.70

0.30 0.50 0.42 0.25 0.38 0.42 0.28 0.37 0.21 0.21

0.70 0.78 0.77 0.67 0.90 0.74 0.74 0.76 0.67 0.90

0.57 0.65 0.70 0.49 0.63 0.61 0.46 0.52 0.40 0.56

NF-80, 80-mm voxel size, no filter; NF-125, 125-mm voxel size, no filter; NF-200, 200-mm voxel size, no filter; S1-80, 80-mm voxel size, Sharpen 1!; S2-80, 80-mm voxel size, Sharpen 2!; S1-125, 125-mm voxel size, Sharpen 1!; S2-125, 125mm voxel size, Sharpen 2!; S1-200, 200-mm voxel size, Sharpen 1!; S2-200, 200-mm voxel size, Sharpen 2!.

detecting the MB2 canal7. Thus, our study aimed to use different combinations of enhancement filters and voxel sizes to assess the best protocol to detect an MB2 canal in maxillary molars with endodontic and prosthetic treatment. Rehabilitation of compromised teeth often requires the use of metallic materials, such as extra- and intracanal posts of different chemical elements, a variable that can affect diagnostic tasks9–12. One of the many issues relative to this metallic material influence is the beam hardening effect, in which materials of high atomic number, such as metals, absorb the lower-energy rays from the polychromatic spectrum emitted by the X-ray machine. Such absorption results in the recording of lower mean beam energy and subsequent dark streak formation (ie, artifacts) because of an error in the 3dimensional reconstruction24. Although the formation of artifacts was noticeable in the images under analysis, studying the metallic material influence in MB2 canal detection

was not our goal; rather, we aimed to simulate a common clinical scenario in which endodontically treated teeth restoration includes intracanal pins. Because Rosado et al21 found no influence of different intracanal materials in MB2 canal detection, we selected only 1 type of material—the silver palladium pins. Our findings showed higher accuracy for MB2 canal detection in the experimental groups with smaller voxel sizes, probably due to greater imaging detail and differentiation of smaller structures15. Other studies have tested the influence of voxel size on different diagnostic tasks, with the results varying according to the task and the CBCT systems used10,11,25–31. Despite the fact that the general influence of voxel size remains inconclusive, Bauman et al16 and Vizzotto et al17 also found higher accuracy in MB2 canal detection while using smaller voxel sizes in some of the study groups although without applying filters or considering their influence on this diagnostic task.

TABLE 2 - Diagnostic Values and Comparison of the Area Under the Receiver Operating Characteristic Curve (AUC) Values among the Experimental Groups Protocols NF-80 S1-80 S2-80 NF-125 S1-125 S2-125 NF-200 S1-200 S2-200

Sensitivity

Speciﬁcity

Accuracy

PPV

NPV

AUC

87.0 95.0 90.0 79.0 86.0 82.0 84.0 89.0 86.0

90.0 86.0 89.0 85.0 85.0 88.0 79.0 74.0 76.0

88.5 90.5 89.5 82.0 85.5 85.0 81.5 81.5 81.0

89.6 87.1 89.1 84.0 85.1 87.2 80.0 77.3 78.1

87.3 94.5 89.9 80.1 85.8 83.0 83.1 87.0 84.4

0.91* 0.94* 0.93* 0.85 0.90 0.88 0.84 0.87 0.87

NF-80, 80-mm voxel size, no filter; NF-125, 125-mm voxel size, no filter; NF-200, 200-mm voxel size, no filter; NPV, negative predictive value; PPV, positive predictive value; S1-80, 80-mm voxel size, Sharpen 1!; S2-80, 80-mm voxel size, Sharpen 2!; S1-125, 125-mm voxel size, Sharpen 1!; S2-125, 125-mm voxel size, Sharpen 2!; S1-200, 200-mm voxel size, Sharpen 1!; S2-200, 200-mm voxel size, Sharpen 2!. *A statistically significant difference (P , .05) among the given voxel size protocol and the others, considering the same filter condition. We found no statistically significant differences among filters or for the filter–voxel size interaction (P . .05).

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Sharpening filters, a variety of high-pass filters, are tools used to enhance highfrequency image content while reducing lowfrequency ones. Applying these filters leads to a voxel output with an altered gray scale relative to the original values. Because sharpening filters generally increase image contrast and highlight structure boundaries, we applied the Sharpen 1! and Sharpen 2! filters to increase MB2 canal detection, with the latter having a more intense effect on the image32,33. However, contrary to our initial hypothesis, the application of those filters had no influence on MB2 canal detection in images with 80-mm, 125-mm, and 200-mm voxel sizes. Although some studies also suggest no influence of filter application on tasks related to root and root canal conditions9,10,34–36, other research shows that filter application significantly increased the accuracy for detecting transverse root fracture and apical bone loss18,19. These studies show a wide variety of diagnostic tasks, visualization software, filters, and a general lack of detailed information on image processing, perhaps due to copyright, hence the difficulty in comparing results among studies using different CBCT systems and software. Nonetheless, the literature suggests that filters can potentially influence specific diagnostic tasks. Our results showed that the 80-mm voxel size groups had the highest diagnostic values—specificity and PPV in NF-80 and sensitivity, accuracy, and NPV in S1-80. Because the differences among the NF-80 and S1-80 diagnostic values were low, this finding suggests that applying a sharpening filter slightly increased MB2 canal detection, thus increasing the sensitivity values but also the image noise. Higher image noise values can lead to extra false-positive diagnoses and consequently to lower PPV and higher NPV. NF-125, in turn, had the lowest sensitivity and NPV values; S1-200 the lowest specificity and PPV values; and S2-200 the lowest accuracy. In the S1-200 group, similar to what may have happened to the S1-80 group, a higher amount of noise may have increased MB2 canal detection and falsepositive diagnoses, decreasing PPV and increasing NPV. Applying the Sharpen 2! filter to the 200-mm voxel group (S2-200) may have degraded the image resolution, decreasing the accuracy values. These results did not corroborate our hypothesis; we expected that applying sharpening filters would increase diagnostic values for MB2 canal detection using larger voxel sizes. While analyzing the areas under the ROC curves, we found no statistically significant differences among the filters used and for the filter–voxel size interaction.

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CBCT system, no impact of ﬁlter application on OnDemand 3D, and no interaction among ﬁlters and voxel sizes, studies using other systems and software may yield different results.

CONCLUSION A smaller voxel size increased the accuracy in detecting the MB2 canal, whereas enhancement filters had no influence on the accuracy for this diagnostic task. Consequently, CBCT images with 125-mm and 200-mm voxel sizes with filter application did not show the same accuracy in MB2 canal detection as images with 80-mm voxel size.

CREDIT AUTHORSHIP CONTRIBUTION STATEMENT

FIGURE 2 – ROC curves generated in the study. The green, blue, and orange lines represent the 80-mm, 125-mm, and 200-mm voxel groups, whereas the continuous dark, dashed dark, and continuous light lines represent the no ﬁlter, Sharpen 1! , and Sharpen 2! groups, respectively.

Regarding voxel size, the 80-mm groups had the highest areas under the ROC curve values (ie, NF-80 [0.91], S1-80 [0.94], and S2-80 [0.93]). Although the 80-mm voxel size groups (NF-80, S1-80, and S2-80) showed signiﬁcantly higher values than the 125-mm (NF-125, S1-125, and S2-125) and 200-mm (NF-200, S1-200, and S2-200) groups, the lowest area under the ROC curve was 0.84 (NF-200), meaning that all values can at least be considered excellent37. We also consider the possibility that the selected study design overestimates the observed diagnostic values.

As in vitro research, our study design lacks clinical information, signs, symptoms, and patient characteristics such as intrinsic movements, which could aid or hinder the diagnostic task. Despite these limitations, the design presented a valuable option, allowing the multiple and successive CBCT acquisitions necessary to achieve our goals, which are unfeasible in a clinical study because of ethical issues. A controlled in vitro study also allows us to reduce variables unrelated to the study objectives and the risk of bias. Although our study showed interference of different voxel sizes with MB2 canal detection on the OP300

^mia Mouzinho-Machado: Sa Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing - original draft. Lucas de Paula Lopes Rosado: Conceptualization, Investigation, Methodology, Resources, Writing - original draft. Fernanda Coelho-Silva: Data curation, Formal analysis, Methodology, Investigation, Writing - original draft. Frederico Sampaio Neves: Conceptualization, Methodology, Resources, Supervision, Writing - review & editing. Francisco Haiter-Neto: Conceptualization, Methodology, Supervision, Writing - review & editing. Sergio Lins deAzevedo-Vaz: Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Visualization, Project administration, Writing - review & editing.

ACKNOWLEDGMENTS The authors thank Espaço da Escrita–Pro Reitoria de Pesquisa for the language services provided. ~o de Funding: Coordenaça Aperfeiçoamento de Pessoal de Nível Superior (ﬁnance code 001). The authors deny any conﬂicts of interest related to this study.

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CLINICAL RESEARCH Carla Y. Falcon, DMD, MDS, Anthony R. Arena, DMD, MDS, Rebecca Hublall, DMD, Craig S. Hirschberg, DDS, and Paul A. Falcon, DMD, MS

SIGNIFICANCE Demographic and treatment factors are associated with incomplete endodontic treatment. Patients receiving palliative relief of pain as the initial treatment are less likely to complete endodontic treatment.

Factors Associated with Incomplete Endodontic Care ABSTRACT Introduction: Incomplete endodontic treatment has been associated with detrimental health outcomes. Methods: This retrospective study reviewed charts of patients receiving endodontic care over a 1-year period at the Postgraduate Endodontic Clinic at Rutgers School of Dental Medicine, Newark, NJ, to assess whether factors such as receipt of palliative endodontic care and demographic factors were associated with completion, or noncompletion, of initial nonsurgical root canal therapy (RCT). Results: A total of 1806 patient charts met the study inclusion criteria. With descriptive statistics and bivariate analysis, the variables of palliative care, Medicaid recipient, age group, and distance from the clinic were significantly associated with RCT completion (P , .05). In the binary logistic regression with all independent variables, palliative care and age group variables were the significant factors (P , .05). Patients who had no palliative care had 8.5 times the odds of completing RCT than patients who had received palliative care. The age group of 18–35 years had 0.59 times the odds of complete RCT than the age group ,18 years. Conclusions: Incomplete nonsurgical endodontic treatment is highly associated with the receipt of prior palliative care. Further research is indicated to investigate additional factors that may influence patient completion of endodontic care and opportunities to improve public health care program design to obtain optimal patient-centered outcomes. (J Endod 2021;47:1398–1401.)

KEY WORDS Delayed root canal therapy; incomplete endodontic treatment; Medicaid

Department of Endodontics, Rutgers School of Dental Medicine, Rutgers, The State University of New Jersey, Newark, New Jersey Address requests for reprints to Dr Carla Y. Falcon, Rutgers School of Dental Medicine, 110 Bergen Street, Newark, NJ 07103. E-mail address: [emailprotected]. edu 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.012

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Pain is a powerful motivator for seeking dental care. A recent study indicated 76% of dental-related visits to a hospital dental emergency department during ofﬁce hours were to address dental pain1. Palliative endodontic procedures, such as pulpotomy and pulpectomy, are effective means of managing and relieving endodontic pain2,3. However, once pain is alleviated, many individuals do not follow through in addressing the source of their pain. Incomplete endodontic treatment has been associated with detrimental health outcomes including pain, swelling, tooth loss due to caries or fracture, and progression of cystic lesions as well as systemic health issues such as maxillofacial infections requiring hospital care, pneumonia, and cardiovascular disease4–8. Demographic factors have been associated with dental utilization and completion of care, including insurance status, clinic location, age, access, race/ethnicity, and poverty status9–12. This retrospective study reviewed the charts of patients receiving endodontic care over a 1-year period in the Postgraduate Endodontic Clinic at Rutgers School of Dental Medicine, Newark, NJ. The study assessed whether several factors were associated with completion, or noncompletion, of endodontic therapy. The factors assessed included treatment factors (receipt of palliative endodontic care) and demographic factors (age, sex, distance to the clinic, and whether the patient was a Medicaid beneﬁt recipient).

METHODS AND MATERIALS This study was approved by the Newark Health Sciences Institutional Review Board of Rutgers University. AxiUm electronic health records (Exan Software, Coquitlam, BC, Canada) of patients treated in the Postgraduate Department of Endodontics at Rutgers School of Dental Medicine were reviewed for a 1-year period of time, July 1, 2018, to June 30, 2019. The review included all patients who had initial nonsurgical root canal therapy (RCT) codes D3310, D3320, or D3330 planned, in process, or completed during the speciﬁed time period. The following study variables were recorded in a deidentiﬁed data sheet: palliative care received (yes/no), RCT status (none/incomplete/complete), age

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DISCUSSION

(years), sex (male/female), zip code of home address, and Medicaid recipient (yes/no). The zip code of the home address was used to calculate the distance from the clinic using Google Maps (Google, Mountain View, CA). If patients required 2 visits of palliative care on the same tooth, only the initial visit was included as a matter of convention. A palliative visit midtreatment was not included in the data set.

Patients experiencing pain typically achieve resolution of symptoms with the initiation of endodontic therapy; most patients experience significant pain relief after pulpotomy or pulpectomy2,13,14. However, motivation to continue with dental treatment often wanes after the initial crisis intervention1,15. At least 2 longitudinal studies have followed adults with Medicaid benefits presenting with a dental emergency to a hospital emergency department. It was found that 41.5%–52.4% of patients did not visit a dentist within 6 months of the emergency department visit1,15. In the current study, 66% of patients who received palliative care at the Postgraduate Endodontic Clinic at Rutgers School of Dental Medicine did not complete their planned endodontic treatment. A study from the University Dental School and Hospital in Cork, Ireland, had a similar finding, with 61% of patients failing to complete root endodontic therapy after emergency pulpectomy16. Completion rates of endodontic therapy have been previously studied and vary by clinical practice setting and demographic factors. The University of Queensland performed an analysis of 783 initiated endodontic therapies. Eighty-six percent were completed, with maxillary first molars and patients requiring an additional visit for irrigation and/or dressing more likely to have an incomplete endodontic treatment17. In

RESULTS A total of 1806 patient charts met the study inclusion criteria. Descriptive statistics and the result of bivariate analysis are included in Table 1. The variables of palliative care received, Medicaid recipient, age group, and distance from the clinic are signiﬁcantly associated with RCT completion in this bivariate analysis; however, the independent variables were not controlling each other. A binary logistic regression was also conducted with all independent variables (Table 2). The variables palliative care received and age group were the signiﬁcant factors in this model. Patients who did not receive palliative care had 8.5 times the odds of completing RCT than patients who had received palliative care. The age group of 18– 35 years had 0.59 times the odds of completing RCT than the age group ,18 years.

TABLE 1 - Descriptive Statistics and the Result of Bivariate Analysis

Palliative care received Yes No Medicaid recipient Yes No Sex Female Male Age (y) 1: ,18 2: 18–35 3: 36–50 4: 50–65 5: .65 Distance from the clinic (mile)

RCT complete

RCT not performed/ in process

Total

P value

471 1335

158 1104

33.5 82.7

313 231

82.7 17.3

,.0001*

951 855

570 692

59.9 80.9

381 163

40.1 19.1

,.0001*

1070 736

736 526

68.8 71.5

334 210

31.2 28.5

.2221

258 589 428 335 196

184 71.3 385 65.4 294 68.7 240 71.6 159 81.1 n 5 1262, mean 5 16.5, SD 5 18.0

74 28.7 204 34.6 134 31.3 95 28.4 37 18.9 n 5 544, mean 5 12.4, SD 5 14.5

.0010*

,.0018*

RCT, root canal therapy; SD, standard deviation. The variables of palliative care, Medicaid recipient, age group, and distance from the clinic are signiﬁcantly associated with RCT completion; however, the independent variables were not controlling each other in the bivariate analysis. *Statistically signiﬁcantly different at P , .05.

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contrast to the findings in our current study, prior receipt of emergency or palliative care was not significantly associated with completion of RCT. A study of 898 teeth that received RCT in the US military dental care system exhibited an overall completion rate of 77%3. Teeth with incomplete RCT presented with a higher incidence of preoperative pain at 70%3. It was also noted that 56% of teeth with incomplete RCT were eventually extracted, whereas only 2%–3% of teeth with completed endodontic treatment were extracted3. The present study at the Postgraduate Endodontic Clinic at Rutgers School of Dental Medicine found an endodontic therapy completion rate of 77%. The receipt of palliative care was positively associated with the completion of endodontic treatment with an odds ratio of 8.5. Demographic factors have also been associated with the completion of dental care. The present study at the Postgraduate Endodontic Clinic at Rutgers School of Dental Medicine found Medicaid recipient status, age group ,18 years, and increased distance from the clinic to be significantly associated with the completion of RCT in the bivariate analysis. However, in the binary logistic regression of study data, only age group was found to be significant. Dental attendance for minors has been associated with race, level of education, income status, and parental marital status18. Insurance status has also been demonstrated to be associated with the completion of dental care for adults and children10,11,18,19. Passage of the Affordable Care Act in 2010 affected the Medicaid dental patient and provider population by expanding enrollee eligibility and provided dental insurance and access to dental care for many communities that have been shown to have unmet dental needs. Nationally, an estimated 9.8 million adults gained dental benefits through Medicaid expansion or Medicaid policy changes after passage of the Affordable Care Act through 201720. Funding for Medicaid is provided by both state governments and the federal government. Federal guidelines for adult dental benefits are broad, providing states significant flexibility in creating their own dental benefit packages. There are no minimum requirements for adult dental coverage21. Most states provide at least emergency dental services for adults; however, less than half of states currently provide comprehensive dental services21. At Rutgers School of Dental Medicine, Medicaid dental insurance entities require prior authorization for definitive endodontic therapy, including nonsurgical RCT. This typically necessitates an initial visit for evaluation for Medicaid enrollees, separate from definitive

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TABLE 2 - Binary Logistic Regressions with all Independent Variables

Palliative care received Yes No Medicaid recipient Yes (reference) No Sex Female Male (reference) Age (y) 1: ,18 (reference) 2: 18–35 3: 36–50 4: 50–65 5: .65 Distance from the clinic (mile)

Odds ratio

95% CI

P value

8.56

6.54–11.20

,.0001*

1.21

0.93–1.60

.162

0.80

0.63–1.01

.059

0.59 0.71 0.79 0.89 1.01

0.41–0.86 0.48–1.05 0.52–1.20 0.53–1.50 1.00–1.01

.006* .082 .276 .672 .156

CI, confidence interval. Palliative care received and age group variables are the significant factors in this model. Patients who did not receive palliative care had 8.5 times the odds of completing root canal therapy than patients who had received palliative care. The age group of 18–35 years had 0.59 times the odds of completing root canal therapy than the age group ,18 years. *Statistically significantly different at P , .05.

endodontic treatment. If a Medicaid enrollee presents in acute endodontic pain, the provider may perform palliative care without prior authorization, typically a pulpotomy or pulpectomy. Simultaneously, the requisite documentation is submitted to Medicaid insurance entities for review and adjudication, leading to denial or authorization of deﬁnitive endodontic therapy. The preauthorization process typically takes several weeks. Upon

the successful receipt of insurance preauthorization, the patient must return to complete the deﬁnitive endodontic treatment. This preauthorization requirement effectively imposes a multivisit endodontic treatment for the subset of patients who have Medicaid dental insurance with an initial presentation of acute pain. Nonsurgical endodontic therapy has long been demonstrated to be effective when completed

in either 1 or multiple visits22–25. However, the multiple visits effectively imposed on the Medicaid study population may create an inherent difference in the treatment time line and the number of patient encounters. Several studies have shown an increased treatment time frame and an increased number of patient visits are associated with incomplete endodontic treatment3,16,17. This may explain the finding in the descriptive analysis that Medicaid patients were less likely to complete endodontic treatment. Further research is indicated to investigate additional factors that may influence patient completion of endodontic care including the number of patient visits to complete the procedure and the time from the first to the last encounter for treatment. In turn, health care outcomes can be further elucidated to assist in the improvement of public health care program design and obtain optimal patient-centered outcomes.

ACKNOWLEDGMENTS The authors thank Ms Shuying Jiang, Research Associate, Rutgers School of Dental Medicine, for her assistance in the statistical analysis of the data. Supported by the American Association of Endodontists Foundation Resident Research Grant. The authors deny any conﬂicts of interest related to this study.

REFERENCES 1.

Guivarc’h M, Saliba-Serre B, Le Coz P, Bukiet F. A cross-sectional analysis of patient care pathways and proﬁles in a dental emergency department. Int Dent J 2020;70:21–8.

Hasselgren G, Reit C. Emergency pulpotomy: pain relieving effect with and without the use of sedative dressings. J Endod 1989;15:254–6.

Wong M, Shelley JJ, Bodey T, Hall R. Delayed root canal therapy: an analysis of treatment over time. J Endod 1992;18:387–90.

Huh JK, Yang DK, Jeon KJ, Shin SJ. Progression of periapical cystic lesion after incomplete endodontic treatment. Restor Dent Endod 2016;41:137–42.

Lin PY, Chien KL, Chang HJ, Chi LY. Unﬁnished root canal treatments and the risk of cardiovascular disease. J Endod 2015;41:1991–6.

€nholm L, Lemberg KK, Tj€aderhane L, et al. The role of unﬁnished root canal treatment in Gro odontogenic maxillofacial infections requiring hospital care. Clin Oral Investig 2013;17:113–21.

Lin PY, Chiang YC, Chou YJ, et al. Association of unﬁnished root canal treatments with the risk of pneumonia hospitalization. J Endod 2017;43:29–35.

Pitiphat W. Limited evidence suggested that unﬁnished root canal treatments may increase the risk of cardiovascular disease. J Evid Based Dent Pract 2016;16:249–50.

Yarbrough C, Nasseh K, Vujicic M. Key differences in dental care seeking behavior between Medicaid and non-Medicaid adults and children. Health Policy Institute Research Brief. American Dental Association; 2014. Available at: https://www.ada.org/w/media/ADA/Science%20and% 20Research/HPI/Files/HPIBrief_0814_4.pdf?la5en. Accessed December 9, 2020.

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10.

Sohn W, Ismail A, Amaya A, Lepkowski J. Determinants of dental care visits among low-income African-American children. J Am Dent Assoc 2007;138:309–98.

11.

Cruz GD, Chen Y, Salazar CR, et al. Determinants of oral health care utilization among diverse groups of immigrants in New York City. J Am Dent Assoc 2010;141:871–8.

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Pajewski NM, Okunseri C. Patterns of dental service utilization following nontraumatic dental condition visits to the emergency department in Wisconsin Medicaid. J Public Health Dent 2014;74:34–41.

13.

Eren B, Onay EO, Ungor M. Assessment of alternative emergency treatments for symptomatic irreversible pulpitis: a randomized clinical trial. Int Endod J 2018;51(Suppl 3):e227–37.

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Oguntebi BR, DeSchepper EJ, Taylor TS, et al. Postoperative pain incidence related to the type of emergency treatment of symptomatic pulpitis. Oral Surg Oral Med Oral Pathol 1992;73:479–83.

15.

Singhal A, Momany ET, Jones MP, et al. Dental care after an emergency department visit for dental problems among adults enrolled in Medicaid. J Am Dent Assoc 2016;147:111–9.

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in R, Hannigan A. Endodontic treatment completion following Lynch CD, Burke FM, Ní Ríorda emergency pulpectomy. Community Dent Health 2010;27:114–7.

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Krishnan U, Huang HJ, Moule A, Lalloo R. An assessment of endodontic treatment completion rate in a university-based student clinic and the factors associated with incomplete treatment. Aust Endod J 2019;45:305–10.

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Badri P, Saltaji H, Flores-Mir C, Amin M. Factors affecting children’s adherence to regular dental attendance: a systematic review. J Am Dent Assoc 2014;145:817–28.

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Lee W, Kim SJ, Albert JM, Nelson S. Community factors predicting dental care utilization among older adults. J Am Dent Assoc 2014;145:150–8.

20.

Health Policy Institute. American Dental Association. Medicaid expansion and dental beneﬁts coverage. Available at: https://www.ada.org/w/media/ADA/Science%20and%20Research/ HPI/Files/HPIgraphic_1218_3.pdf?la5en. Accessed November 4, 2020.

21.

Medicaid.gov. Dental care. Available at: https://www.medicaid.gov/medicaid/beneﬁts/dentalcare/index.html. Accessed October 22, 2020.

22.

Moreira MS, Anuar ASN, Tedesco TK, et al. Endodontic treatment in single and multiple visits: an overview of systematic reviews. J Endod 2017;43:864–70.

23.

€stemeyer G. Single-visit or multiple-visit root canal treatment: systematic Schwendicke F, Go review, meta-analysis and trial sequential analysis. BMJ Open 2017;7:e013115.

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Wang C, Xu P, Ren L, et al. Comparison of post-obturation pain experience following one-visit and two-visit root canal treatment on teeth with vital pulps: a randomized controlled trial. Int Endod J 2010;43:692–7.

25.

Soltanoff W. A comparative study of the single-visit and the multiple-visit endodontic procedure. J Endod 1978;4:278–81.

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REGENERATIVE ENDODONTICS Victor Pinheiro Feitosa, DDS, MSc, PhD, Mara Natiere Gonçalves Mota, DDS, Lorena Vasconcelos Vieira, DDS, MSc, Diego Martins de Paula, DDS, MSc, PhD, Lívia Lisboa Ribeiro Gomes, DDS, Luzia Kelly Rios Solheiro, DDS, Manoel Asciton de Aguiar Neto, DDS, Diego Armando Leite Carvalho, DDS, and ^nia Alves Silvestre Francisbe

SIGNIFICANCE This study paves the way for clinical endodontic regenerative therapy by means of pulp autotransplantation from an extracted third molar to a further tooth requiring root canal treatment in the same patient.

Dental Pulp Autotransplantation: A New Modality of Endodontic Regenerative Therapy— Follow-Up of 3 Clinical Cases ABSTRACT The aim of this study was to develop a novel method of endodontic therapy, which we refer to as dental pulp autotransplantation. Three patients (2 males and 1 female) were selected for endodontic treatment of a uniradicular premolar and extraction of a third molar (without odontosection). Electric assessment of pulp vitality and computed tomographic imaging were undertaken followed by endodontic access and instrumentation using triantibiotic solution for irrigation in the host tooth. A few minutes before the transplant procedure, the third molar was extracted, the tooth was sectioned with a diamond blade in a low-speed handpiece, and the pulp was carefully removed. After premolar instrumentation, the harvested and preserved pulp tissue was reinserted into the root canal followed by direct pulp capping performed using s, France), a liner of resin-modiﬁed glass Biodentine (Septodont, Saint-Maur-des-Fosse ionomer cement and composite resin restoration. The teeth were followed up for at least 12 months after the procedures and were analyzed using computed tomographic imaging, electric pulp vitality testing, and Doppler ultrasound examination. At the 3- and 6-month follow-ups, positive pulp vitality and regression of periapical lesions were veriﬁed. After 9– 12 months, all teeth were revascularized as determined by Doppler imaging, and the tooth vitality was reestablished with no signs of endodontic/periodontal radiolucency or complications. Within the limitations of the study, considering that it was a case series with only 3 patients, we described a highly innovative procedure of pulp autotransplantation, which appears to be feasible, highlighting the potential for clinical application of pulp regeneration using this new modality of endodontic therapy. (J Endod 2021;47:1402–1408.)

KEY WORDS Pulp; regeneration; transplant

From the Paulo Picanço School of , Brazil Dentistry, Fortaleza, Ceara Address requests for reprints to Prof Victor Pinheiro Feitosa, Research Division, Paulo Picanço School of Dentistry, 900 Street, Fortaleza, Ceara , Joaquim Sa Brazil 60135-218. E-mail address: victorpfeitosa@hotmail. com 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.014

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Restorative dentistry and endodontics are trending toward a regenerative approach for the pulp/dentin complex, particularly after the recent advances on mesenchymal stem cell–based tissue engineering technologies1. Indeed, several animal models have been developed comprising different strategies to induce pulp regeneration and reconstruction in full length2,3. In order to aid the regeneration process, growth factors, cytokines, and several microenvironments were introduced2,4,5. However, little of these technologies actually reached the clinical case series. For the clinical scenario, pulp revascularization of immature teeth affords a threshold for the survey of novel regenerative insights6. Recently, a clinical trial7 compared induced bleeding with platelet-rich ﬁbrin and platelet-rich plasma for open apex necrotic teeth. Moreover, other clinical case series8,9 have shown the use of platelet-rich ﬁbrin to achieve pulp regeneration with reasonable outcomes. Nevertheless, animal studies10 revealed a lack of generation of proper sound pulp tissue by means of platelet concentrates, which rather form bonelike tissue in the root canal. A further strategy to attain successful pulp regeneration in the clinical outlook is the transplant of dental pulp stem cells (DPSCs) harvested from teeth requiring root canal treatment diagnosed with irreversible pulpitis11. Such cells were expanded in vitro before transplantation to the host teeth. More recently12, seeded minced pulp

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mesenchymal stem cells achieved fast replication and odontogenic differentiation with noteworthy potential for clinical application. The transplantation of the entire pulp might yield the optimal “scaffold” for the differentiation of DPSCs in their natural environment. Furthermore, most nerves and blood vessels are formed, thereby facilitating the revascularization and connective process of transplanted pulp. Altogether, this modality of the pulp regeneration technique could be obtained in clinical procedures without the need for in vitro DPSC laboratorial expansion. Dental pulp autotransplantation of pulp harvested from a third molar requiring extraction to a host tooth needing root canal treatment may aggregate a clinically feasible procedure with striking potential for true pulp regeneration, avoiding body rejection and inﬂammation. However, to the best of our knowledge, dental pulp autotransplantation has not been investigated thus far. Therefore, this case series aimed to describe the clinical procedures for dental pulp autotransplantation and to conduct a clinical follow-up for at least 9 months of 3 patients treated with this modality of regenerative endodontic treatment. Despite being a promising technique, there are still some points to be improved, such as the time in which the pulp remains with its vital cells to reconnect after extractions. The issue to be considered is that the pulps need to be removed only from vital teeth that have not suffered any type of trauma before, during, or after the extractions for the sake of a better autotransplantation prognosis.

extraction without odontosection and without the presence of a carious cavity. Initial panoramic radiography (Fig. 1A– C) and computed tomographic imaging of the tooth requiring root canal treatment were performed. All teeth were diagnosed with pulp necrosis depicting periapical radiolucency. Third molar extraction was undertaken without odontosectioning and with minimal injury to the tooth after local anesthesia using 1.8 mL 2% lidocaine (1:100,000 epinephrine) with the nerve block technique. The extracted teeth were brieﬂy stored in sterilized saline solution, and the site was sutured. The premolars requiring root canal treatment were anesthetized as mentioned previously and isolated with a rubber dam, and pulp chamber access was executed with diamond burs in a high-speed handpiece under running water after caries or defective restoration removal. Canal instrumentation was undertaken using

rotary files (WaveOne Gold; Dentsply Sirona, York, PA) with irrigation using triantibiotic solution (ciprofloxacin, minocycline, and metronidazole)13,14. Before the rotary files, 1 #10 manual K-file (Dentsply Sirona) was used to perform the patency. No apical bleeding was performed because the clinical protocol was not similar to revascularization15. The residue of irrigating solution was thoroughly washed with sterile saline solution. EDTA at 17% concentration for 5 minutes was used for the dentin conditioning protocol16. This solution was twice rinsed with sterilized saline solution, and the root canal was slightly dried with an absorbent paper cone. Meanwhile, the extracted third molar was initially cut with a small diamond saw in a lowspeed handpiece (Fig. 2A) running water washing with sterilized saline solution. This first cut penetrated only 1–2 mm around the entire mesial and distal surfaces and 3–4 mm on the

MATERIALS AND METHODS Three patients with a need for root canal treatment in a single-rooted premolar and third molar extraction were recruited from the Dental Clinics of Paulo Picanço School of Dentistry, , Brazil. The protocol and Fortaleza, Ceara project were evaluated and approved by the institutional ethics committee (protocol 3585776). All patients discussed the treatment options and signed informed consent forms that explained the aim of the study and the potential complications; they conformed with the clinical/radiographic follow-up periods of 1, 3, 6, 9, and 12 months. The inclusion criteria were patients 18–40 years old with no sex predilection and irreversible pulpitis or pulp necrosis signs with spontaneous pain or periapical radiolucency in single-rooted tooth, thereby requiring root canal treatment, with a periodontal pocket depth ,3 mm. The presence of tooth discoloration was not an exclusion criterion. Furthermore, the patients needed to possess a third molar prone to

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FIGURE 1 – (A–C ) Panoramic radiographs of the initial visit of 3 patients. The black circles indicate the tooth requiring root canal treatment (receptor tooth), whereas the asterisks indicate the third molars planned for extraction (pulp donator tooth). (A ) Patient 1, (B ) patient 2, and (C ) patient 3.

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etching technique with 37% phosphoric acid gel (Condac 37; FGM, Joinville, Brazil) only at the enamel borders for 15 seconds, which was rinsed with distilled water for 30 seconds. Both dentin and enamel were air-dried for 30 seconds before the active application of primer for 20 seconds with a slight air blast to evaporate solvent and active application of adhesive for 20 seconds. Bonding agent light curing was performed for 40 seconds with the Valo LED unit. Resin composite restoration was incrementally built with 2-mm-thick increments (Opallis, FGM) individually light cured for 20 seconds. After the occlusal check/adjustments, the patient went home with pharmacologic receipt of 600-mg ibuprofen pills to be taken every 8 hours for 3 days. No antibiotics were prescribed for any patient. Receptor teeth underwent computed tomographic imaging at this ﬁrst clinical visit (Fig. 3A1–3C1). Patients were scheduled for follow-up return visits every 3 months after the autotransplantation clinical procedure. At each return visit, they were evaluated by computed tomographic imaging (at 6 and 12 months) or periapical radiography (at 3 and 9 months), electric pulp vitality testing (Pulp Tester Digital; Odous de Deus, Belo Horizonte, Brazil), occlusal check/adjustments of restorations, and Doppler ultrasonic imaging (only at the 1year follow-up). Patients were warned that in case of unsuccessful autotransplantation, the transplanted pulp would be removed, and traditional endodontic treatment would be performed by ﬁlling the canal with gutta-percha and endodontic sealer.

RESULTS

FIGURE 2 – The method for minimally invasive pulp removal and insertion toward pulp transplantation. (A ) The initial mesiodistal cut of the donator tooth with a small diamond disc in a low-speed handpiece to create a notch around the entire surface of the tooth, which is the triggering zone to break the tooth into 2 parts. The notch was forced with a syndesmotome, and the tooth was fractured to expose the pulp. (B ) The fractured tooth after careful removal of the pulp with small tweezers. (C ) The careful insertion of the harvested pulp with the aid of a gutta-percha cone inside the previously cleaned and instrumented root canal at the receptor tooth.

occlusal surface of the molar in order to create a notch without touching the pulp tissue. The notch was pressed with a sharp straight syndesmotome to section the teeth into 2 halves, allowing careful pulp removal with small tweezers (Fig. 2B). Pulp tissue (from donator tooth) was inserted into the root canal of the receptor tooth with the aid of disinfected guttapercha cones (Fig. 2C) to avoid damaging the tissue. Direct pulp capping was performed in the receptor tooth using Biodentine (Septodont, Saint-Maur-des-Fosses, France)

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calcium silicate cement, which was manipulated according to the manufacturer’s instructions. After 10 minutes to allow complete setting of Biodentine, resin-modiﬁed glass ionomer cement (Riva Light-Cure; SDI, Bayswater, Australia) was applied as a liner and light cured for 20 seconds with an LED Valo unit (1200 mW/cm2; Ultradent, South Jordan, UT) to avoid a negative reaction between Biodentine and adhesive restoration17. The 2-step self-etch adhesive Clearﬁl SE Bond (Kuraray Medical, Kurashiki, Japan) was applied using a selective enamel

The 3 patients reported no symptoms within the first 3 months after the procedures. At the 3-month follow-up, all patients reported slight twinges at the periapical region of the receptor tooth, which started after 40 days and occurred during the initial 2–3 months after the procedure. No response to electric pulp vitality testing was detected at the 3-month follow-up. The patients depicted a reduction of periapical radiolucency as highlighted in computed tomographic imaging at the 6-month follow-up (Fig. 3A2–3C2) of the teeth submitted to the autotransplantation procedure. Moreover, the electric pulp vitality tests resulted in a positive vitality response with similar amperage to those of intact teeth from the same patient. After 1 year from autotransplantation, complete regression of periapical lesions was verified for patients 1 (Fig. 3A3) and 2 (Fig. 3B3), whereas the radiolucency in patient 3 was almost entirely diminished (Fig. 3C3). Positive pulp vitality was confirmed, and revascularization was further proved by

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FIGURE 3 – Computed tomographic images of teeth receiving pulp autotransplantation. A1–A3 are related to the ﬁrst patient’s lower second premolar, which (A1 ) depicted periapical radiolucency initially, (A2 ) was notably reduced at the 6-month follow-up, and (A3 ) disappeared after 1 year of the treatment. (B1–B3 ) The same trend occurred in patient 2 as well as in patient 3, with (C1–C3 ) a striking reduction of periapical lesions over time, although the 1-year period was not enough to demonstrate total absence of periapical radiolucency. At the 1-year follow-up, all teeth showed positive pulpal vitality in the electric test.

Doppler imaging (Fig. 4) at the periapical region of each tooth subjected to the pulp autotransplantation procedure. No signs of endodontic/periodontal complications were observed for all patients at the 1-year followup. The 3 cases are still being monitored.

DISCUSSION Entire tooth autotransplantation of the third molar is a well-known and established procedure for the replacement of defective molars requiring extraction in modern clinical dentistry. More recently, 3-dimensional printing technology aided such a procedure by allowing the training of operators and the

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simulation of a clinical scenario18. Even allogeneic tooth transplantation of the entire tooth from the daughter to the mother was successfully demonstrated with the support of 3-dimensional printing in order to achieve an optimal clinical outcome19. These clinical procedures triggered the concept and idea of the present report by means of using the tissue from an extracted third molar to replace a defective tissue in a further area of the oral environment. Several advantages are included when performing pulp tissue autotransplantation, such as a lack of transplant rejection, the same DNA and RNA in all cells, completely mature connective tissue, a formed neuronal network,

and previously built vascularization. Indeed, these factors may enhance the success of pulp regeneration treatments and the strategy12, which used minced pulp as a source of mesenchymal stem cells in order to provide odontogenic differentiation with high potential for protein and alkaline phosphatase expression. Furthermore, in vivo clinical regeneration of pulp tissue would be faster than using only scaffolds, mesenchymal stem cells, and growth factors once all structures are already formed. For a successful clinical procedure of pulp autotransplantation, the extraction of a third molar needs to occur with minimal damage to the tooth, and tooth sectioning

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FIGURE 4 – Doppler ultrasonic assessment of periapical revascularization in teeth subjected to pulp autotransplantation. (A1 and A2 ) Patient 1 and (C1 and C2 ) patient 3 demonstrated high blood ﬂux. The blood ﬂux of (B1 and B2 ) patient 2 was slightly lower than other patients (1 and 2) but was detected and positive in both arterial and venous directions.

cannot be used, excluding the possibility of using teeth that are not well positioned. Herein, the third molars extracted (asterisks in Fig. 1) were likely to be extracted with minimal injury because they were vertically inclined. Besides, the tooth must be stored for the shortest period in sterilized saline solution once complete disinfection before pulp removal and transplantation is mandatory20. The section of the extracted tooth is undertaken only after root canal instrumentation in the receptor tooth, and the entire process demands a sterilized handpiece, diamond disc, and gloves (Fig. 2A). Pulp removal is also removed with sterilized tweezers (Fig. 2B) and inserted sterilized gutta-percha cones (Fig. 2C). Altogether, such care might promote minimal contamination during the process, thereby increasing the likelihood for bacterial-free endodontic regeneration. In this regard, root canal instrumentation and irrigation were performed using rotary ﬁles and triantibiotic solution21, respectively. Although a recent report22 suggests clindamycin is less cytotoxic and promotes higher angiogenic potential than minocycline, the present case series employed the traditional solution used for revascularization procedures. Another drug feasible to replace minocycline would be doxycycline23, which is approved by the US Food and Drug Administration and does not induce tooth staining due to oxidation over time24. However, doxycycline was rarely investigated in pulp regeneration studies, and the triantibiotic solution was used only as

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irrigant, thereby diminishing the possibility of tooth staining. Another traditional procedure commonly performed is the final irrigation with 17% EDTA16 before tissue transplantation. By opening dentinal tubules and conditioning superficial dentin, several growth factors are released such as brain-derived neurotrophic and growth/differentiation factor 158. Particularly, these neuronal growth factors participated in the regeneration process of transplanted pulp in order to obtain vitality regain25. Concerning the few clinical studies about actual pulp regeneration, the majority dealt with teeth diagnosed with irreversible pulpitis8,9,11. With initial conditions depicting the absence of periapical radiolucency, the optimal vascularization and lack of inflammatory mediators in bone may facilitate the revascularization and reinnervation of transplanted tissue/cells. Conversely, in the present clinical cases, all 3 patients presented periapical radiolucency at the initial condition (Fig. 3A1–3C1), which may possess lower blood irrigation and likely bacterial contamination. These factors diminish the feasibility of the treatment. However, successful outcomes were attained in a relatively short period, also achieving a reduction of periapical lesions (Fig. 3). Autologous mixtures were proposed for transplantation to the root canal for the sake of endodontic regeneration, such as minced pulp12, extracted/expanded DPSCs11, platelet-rich fibrin8, and leukocyte

platelet-rich fibrin9. Nevertheless, these treatments require laboratory processes to obtain platelet concentrates or stem cell expansion/replication20. Herein, no laboratory intervention is demanded for pulp autotransplantation, which represents a significant benefit in comparison with previous clinical strategies. In other words, pulp autotransplantation as highlighted in the present report is entirely performed in a clinical dental office without the need for further experiments outside the office. In the clinical steps of the present case series, pulp capping after autotransplantation was performed with the gold standard calcium silicate cement (Biodentine), which attains the optimal pulpal response yielding the release of odontogenic growth factors, alkaline phosphatase release, and dentin bridge formation9,25. Afterward, resin-modified glass ionomer cement was applied, and the gold standard26 2-step self-etch adhesive Clearfil SE Bond was used with the selective enamel etching technique27 in order to afford optimal sealing of the transplanted pulp and cavity margins. In fact, these procedures for pulp capping might play an important role on the clinical long-term outcome of the autotransplanted pulps. To confirm apical revascularization, the 3 patients underwent Doppler ultrasonic evaluation (Fig. 4A1–C2), which confirmed blood perfusion with a characteristic pulse image (Fig. 4A1–4A3), thereby proving revascularization. This report is the threshold

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for the autotransplantation of pulp and likely the application of allogeneic pulp transplantation within relatives. The main limitations of the present treatment protocol rely on patients without the presence of third molars prone to pulp donation or in cases with teeth possessing an abscess or purulence or even when the third molars require odontosection. Indeed, the clinical procedure should be improved, surveying the most suitable irrigation protocols and antibiotics, the optimal root canal instrumentation, and the digitalization of the process in order to ease the ﬁtting of pulps into different shapes

and sizes of root canals. Future studies should focus on the possible assessment of pulp transplantation among people from different families and a feasible test to conﬁrm the gene compatibility for such a novel sort of transplantation procedure. Because the present report is a series of clinical cases, there is a limitation concerning the small sample size. Therefore, further studies should focus on a higher number of patients in a randomized clinical trial setup because it is a promising procedure when you have the conditions suitable to perform autotransplantation.

ACKNOWLEDGMENTS The authors thank Dr David Araujo for the Doppler ultrasonic surveys. Supported by the Brazilian Ministry of Education (Coordenaç~ao de Aperfeiçoamento de Pessoal do Nível Superior grant 23038.006958/2014-96, Principal Investigator Victor Pinheiro Feitosa). The authors deny any conﬂicts of interest related to this study.

REFERENCES

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Nakashima M, Iohara K, Bottino MC, et al. Animal models for stem cell-based pulp regeneration: foundation for human clinical applications. Tissue Eng Part B Rev 2019;25:100–13.

Li X, Ma C, Xie X, et al. Pulp regeneration in a full-length human tooth root using a hierarchical nanoﬁbrous microsphere system. Acta Biomater 2016;35:57–67.

Athirasala A, Lins F, Tahayeri A, et al. A novel strategy to engineer pre-vascularized full-length dental pulp-like tissue constructs. Sci Rep 2017;7:3323.

Nakashima M, Reddi AH. The application of bone morphogenetic proteins to dental tissue engineering. Nat Biotechnol 2003;21:1025–32.

Khayat A, Monteiro N, Smith EE, et al. GelMA-Encapsulated hDPSCs and HUVECs for dental pulp regeneration. J Dent Res 2016;96:192–9.

Nagata JY, Gomes BP, Lima TF, et al. Traumatized immature teeth treated with 2 protocols of pulp revascularization. J Endod 2014;40:606–12.

Shivashankar VY, Johns DA, Maroli RK, et al. Comparison of the effect of PRP, PRF and induced bleeding in the revascularization of teeth with necrotic pulp and open apex: a triple blind randomized clinical trial. J Clin Diagn Res 2017;11:ZC34–9.

Nageh M, Ahmed GM, El-Baz AA. Assessment of regaining pulp sensibility in mature necrotic teeth using a modiﬁed revascularization technique with platelet-rich ﬁbrin: a clinical study. J Endod 2018;44:1526–33.

Meza G, Urrejola D, Saint Jean N, et al. Personalized cell therapy for pulpitis using autologous dental pulp stem cells and leukocyte platelet-rich ﬁbrin: a case report. J Endod 2019;45:144–9.

10.

Del Fabbro M, Lolato A, Bucchi C, et al. Autologous platelet concentrates for pulp and dentin regeneration: a literature review of animal studies. J Endod 2016;42:250–7.

11.

Nakashima M, Iohara K, Murakami M, et al. Pulp regeneration by transplantation of dental pulp stem cells in pulpitis: a pilot clinical study. Stem Cell Res Ther 2017;8:61.

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Liang Z, Kawano S, Chen W, et al. Minced pulp as source of pulpal mesenchymal stem cells with odontogenic differentiation capacity. J Endod 2018;44:80–6.

13.

Miltiadous ME, Floratos SG. Regenerative endodontic treatment as a retreatment option for a tooth with open apex - a case report. Braz Dent J 2015;26:552–6.

14.

Ruparel NB, Teixeira FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012;38:1372–5.

15.

Estefan BS, El Batouty KM, Nagy MM, Diogenes A. Inﬂuence of age and apical diameter on the success of endodontic regeneration procedures. J Endod 2016;42:1620–5.

16.

Aksel H, Albanyan H, Bosaid F, et al. Dentin conditioning protocol for regenerative endodontic procedures. J Endod 2020;46:1099–104.

17.

Palma PJ, Marques JA, Antunes M, et al. Effect of restorative timing on shear bond strength of composite resin/calcium silicate–based cements adhesive interfaces. Clin Oral Investig 2020;25:3131–9.

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Kamio T, Kato H. Autotransplantation of impacted third molar using 3D printing technology: a case report. Bull Tokyo Dent Coll 2019;60:193–9.

19.

Xu HD, Miron RJ, Zhang XX, et al. Allogenic tooth transplantation using 3D printing: a case report and review of the literature. World J Clin Cases 2019;7:2587–96.

20.

Nakashima M, Iohara K, Zayed M. Pulp regeneration: current approaches, challenges, and novel rejuvenating strategies for an aging population. J Endod 2020;46:S135–42.

21.

Santiago CN, Pinto SS, Sassone LM, et al. Revascularization technique for the treatment of external inﬂammatory root resorption: a report of 3 cases. J Endod 2015;41:1560–4.

22.

Dubey N, Xu J, Zhang Z, et al. Comparative evaluation of the cytotoxic and angiogenic effects of minocycline and clindamycin: an in vitro study. J Endod 2019;45:882–9.

23.

Santos LG, Chisini LA, Springmann CG, et al. Alternative to avoid tooth discoloration after regenerative endodontic procedure: a systematic review. Braz Dent J 2018;29:409–18.

24.

Palasuk J, Windsor LJ, Platt JA, et al. Doxycycline-loaded nanotube-modiﬁed adhesives inhibit MMP in a dose-dependent fashion. Clin Oral Investig 2018;22:1243–52.

25.

Pedano MS, Li X, Yoshihara K, et al. Cytotoxicity and bioactivity of dental pulp-capping agents towards human tooth-pulp cells: a systematic review of in-vitro studies and meta-analysis of randomized and controlled clinical trials. Materials (Basel) 2020;13:2670.

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Feitosa VP, Sauro S, Zenobi W, et al. Degradation of adhesive-dentin interfaces created using different bonding strategies after ﬁve-year simulated pulpal pressure. J Adhes Dent 2019;21:199–207.

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Van Meerbeek B, Yoshihara K, Van Landuyt K, et al. From Buonocore’s pioneering acid-etch technique to self-adhering restoratives. A status perspective of rapidly advancing dental adhesive technology. J Adhes Dent 2020;22:7–34.

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REGENERATIVE ENDODONTICS Yaxin Lou, BM,* Yangqiu Liu, MM,* Jiange Zhao, BM,*

Activation of Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Channels Promotes Odontogenic Differentiation of Human Dental Pulp Cells

Weiping Tian, PhD,† Na Xu, MM,‡ Chengcheng Zang, PhD,§ and Kehua Que, PhD*

ABSTRACT Introduction: Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are thermosensitive channels that play an important role in thermal sensation or tooth pain by regulating intracellular Ca21 concentration that is essential for pulp tissue repair. The aim of this study was to evaluate the role of TRPA1 and TRPV1 channels in the odontogenic differentiation of human dental pulp cells (HDPCs). Methods: HDPCs were isolated from healthy human intact third molars and cultured in odontogenic differentiation medium. Gene and protein expression levels of TRPA1 and TRPV1 channels during the odontogenic differentiation of HDPCs were evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. HDPCs were then treated with channel agonists or antagonists, and the expression levels of odontogenic markers dentin sialophosphoprotein (DSPP) and osteopontin (OPN) were examined. Alkaline phosphatase activity and alizarin red staining were also conducted to detect mineralization levels. Results: Consistent with the mineralization degree and DSPP and OPN expression, messenger RNA and protein expression of TRPA1 and TRPV1 channels was up-regulated during the odontogenic differentiation of HDPCs. The application of TRPA1 or TRPV1 agonists increased the mineralized nodules of alizarin red staining and alkaline phosphatase activity and up-regulated the messenger RNA and protein expression of DSPP and OPN, respectively, with the highest values reached on the seventh day (P , .05). On the contrary, the mineralization level and DSPP and OPN expression could be suppressed by using the antagonists of these 2 channels. Conclusions: TRPA1 and TRPV1 channels not only showed up-regulated expression along with the odontogenic differentiation of HDPCs but also could affect the odontogenic differentiation by regulating intracellular Ca21 concentration. (J Endod 2021;47:1409–1416.)

KEY WORDS Dental pulp; odontogenic differentiation; transient receptor potential ankyrin 1 channel; Ttransient receptor potential vanilloid 1 channel

Dental pulp is a unique tissue surrounded by a rigid chamber that provides strong mechanical support and protection from the microbe-rich oral environment1. As the main components of dental pulp, human dental pulp cells (HDPCs) have the potential to differentiate into odontoblasts when perceiving external stimuli, such as cold, caries, mechanical trauma, or exposure to chemicals, thus resulting in tertiary dentin formation2. Under exposure to these stimuli, the odontogenic differentiation potential of HDPCs is important for subsequent pulp tissue repair to prevent further damage and encourage healing. Transient receptor potential (TRP) channels play critical roles in sensory physiology, including contributions to vision, taste, touch, and thermo- and osmosensation3. Some members of the transient receptor potential ankyrin (TRPA), transient receptor potential melastatin (TRPM), and transient receptor potential vanilloid (TRPV) subfamilies act as thermo-TRP channels in the mammalian peripheral nervous

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SIGNIFICANCE TRPA1 and TRPV1 channels in HDPCs could affect the odontogenic differentiation progression by regulating the intracellular Ca21 concentration. Related ﬁndings could show a new light on the development of novel drugs or materials for reparative pulp tissue.

From the Departments of *Endodontics and §Prosthodontics, College of Stomatology and †Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin, China; and ‡ Department of Paediatric Dentistry, Hospital of Stomatology, NanKai University, Tianjin, China Address requests for reprints to Dr Kehua Que, Department of Endodontics, College of Stomatology, Tianjin Medical University, Num22, Road Qixiangtai, Heping District, Tianjin 300070, China, or Dr Chengcheng Zang, Department of Prosthodontics, College of Stomatology, Tianjin Medical University, Num22, Road Qixiangtai, Heping District, Tianjin 300070, China. E-mail addresses: [emailprotected] or [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.007

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system by responding to moderate or noxious changes in the external temperature in a calcium-dependent way4. Among them, TRPA1 and TRPV1 channels have emerged as candidate thermosensitive channels that play a pivotal role in dental hypersensitivity or tooth pain5 and are activated at noxious cold temperatures below 17 C and high temperatures over 43 C, respectively6. These channels may have complex action modes and inﬂuence each other by structurally forming a complex7. As Ca21-permeable channels that responded to various stimuli, TRP channels could directly initiate cellular Ca21 signals and thus induce tissue damage and repair, cell stress, or pain sensation8. As 1 of the main elements in pulp-capping materials, Ca21 plays a regulatory role as an osteo/ odontoinductive component during a number of cellular events leading to tertiary dentinogenesis9. In vitro experiments revealed that Ca21-enriched materials such as mineral trioxide aggregate can induce the odontoblast differentiation of HDPCs10,11, and products derived from such materials received increased attention because of their good performance in vital pulp therapy with a mean success rate of 92%12. Recent evidence suggested that several Ca21-permeable receptors or channels, such as calcium-sensing receptor13 and TRPM7 channels14, are associated with the odontogenic differentiation of HDPCs and could aid in improving the current understanding of the basic mechanism underlying clinically successful dentin regeneration. Thermal or mechanical stimuli often happen when suffering caries or pulp exposure and could activate related TRP channels, which would function in the subsequent pulp tissue repair by allowing Ca21 ﬂow. Although emerging Ca21-enriched biomaterials have been widely used to promote pulp tissue repair, the functions of TRPA1 and TRPV1 channels in this aspect are unclear. As Ca21-permeable and thermal-sensitive channels, the TRPV1 channel is associated with nociceptive signal processing in pulpitis and high sensitivity of dental pulp15, and the TRPA1 channel is upregulated in the odontoblasts of caries teeth16. Therefore, their possible functions in the odontogenic differentiation of HDPCs need to be determined. This study examined the changes in the expression levels of TRPA1 and TRPV1 channels during the odontogenic differentiation of HDPCs and investigated the effect of their functional state on HDPC mineralization to illustrate the role of these 2 channels in the odontogenic differentiation of HDPCs.

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MATERIALS AND METHODS Chemicals and Solutions Cinnamaldehyde, cetylpyridinium chloride, b-glycerophosphate, hexadecadrol, and ascorbic acid were obtained from SigmaAldrich (St Louis, MO); HC030031, capsazepine, and capsaicin were from MedChemExpress (Monmouth Junction, NJ). Hanks’ balanced salt solution contained 140 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L D-glucose, 1.3 mmol/L MgCl2, 2 mmol/L CaCl2, and 10 mmol/L 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (free acid) at a pH of 7.4.

Cell Cultures and Treatments HDPCs were obtained from impacted third molars (N 5 10) from 10 healthy subjects after providing informed consent. The inclusion criteria for the donors were as follows: (1) 18–24 years old, (2) healthy third molars to be extracted for impacted reasons, (3) informed consent provided, (4) no systematic diseases, and (5) the teeth of the donors were intact and suffered no root fracture when extracted. The study was approved by the ethics committee of the Stomatological Hospital of Tianjin Medical University, Tianjin, China (TMUhMEC2020117). Pulp tissue was isolated within 1 hour after tooth extraction and digested by 3 mg/mL collagenase type I (Sigma-Aldrich) at 37 C for 0.5 hours. Cells derived from dental pulp explants were cultured in Dulbecco modiﬁed Eagle medium (Hyclone, Logan, UT) supplemented with 15% fetal bovine serum (Gibco, Carlsbad, CA), 100 UI/mL penicillin, and 100 mg/mL streptomycin (Solarbio, Beijing, China) at 37 C with 5% CO2 in a humidiﬁed incubator. The medium was changed every 3 days. Cells between passages 3 and 6 were used in all experiments. Odontogenic differentiation medium was prepared by adding 10 mmol/L b-glycerophosphate, 1027 mol/L hexadecadrol, and 50 mg/mL ascorbic acid to the culture medium described earlier. Human odontoblastlike cells were HDPCs cultured in the odontogenic differentiation medium for 2 weeks. To investigate the effect of TRPA1 or TRPV1 channels on the odontogenic differentiation of HDPCs, their agonists cinnamaldehyde (0.5 mg/mL) and capsaicin (10 mmol/L) or antagonists HC030031 (10 mmol/L) and capsazepine (10 mmol/L) were supplemented in the odontogenic differentiation medium, respectively. Cells

cultured in the odontogenic differentiation medium were used as controls.

Immunofluorescence Staining Cells were passaged to a culture dish with a glass bottom and incubated for 24 hours to obtain adherent cells; 4% paraformaldehyde (Sparkjade, Shandong, China) was applied for 15 minutes at room temperature to fix the cells. After cells were permeated for 5 minutes with 1% Triton X-100 (SigmaAldrich), nonspecific binding sites were blocked with 10% bovine serum albumin for 0.5 hours at 37 C. Cells were incubated overnight at 4 C with mouse polyclonal antibody against human dentin sialophosphoprotein (DSPP) (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and TRPA1 (1:500; Novus Bio, Shanghai, China). They were then incubated for 30 minutes at 37 C with secondary antibody, and cells stained with TRPA1 were counterstained with TRPV1 antibody (1:200; Alomone Labs, Jerusalem, Israel) using a multitarget immunofluorescence staining kit (Panevue, Beijing, China). Nuclei were stained blue with 4’,6diamidino-2-phenylindole. Immunofluorescence was observed under a laser confocal microscope (LSM900; Carl Zeiss, Oberkochen, Germany).

Immunohistochemistry Collected healthy human teeth were fixed with 4% paraformaldehyde and decalcified in a 10% EDTA 2Na solution for 2– 3 months. Then, the teeth were dehydrated, embedded in paraffin, and cut at 4 mm. The slices were dewaxed in xylene, rehydrated through a graded alcohol series, washed 3 times with ultrapure water, and then treated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase. After antigen retrieval in sodium citrate buffer (pH 5 6.0), the slices were blocked with normal goat serum for 20 minutes at room temperature. This was followed by incubation overnight at 4 C using mouse polyclonal antibody against human DSPP (1:100, Santa Cruz Biotechnology), TRPA1 (1:1000, Santa Cruz Biotechnology), and TRPV1 (1:1000, Novus Bio). They were then incubated for 10 minutes at room temperature with secondary antibody, and staining was detected with 3,30 diaminobenzidine and counterstained with hematoxylin. Images were captured with an Olympus DP72 microscope (Olympus, Tokyo, Japan).

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Calcium Imaging

Western Blot Analysis

Cultured human odontoblastlike cells were grown in a culture dish with a glass bottom to 70% confluence and then loaded with 3.33 mmol/L fluo-3-acetoxymethyl ester (fluo3AM; Beyotime, Jiangsu, China) for 40 minutes at 37 C in darkness. Fluo-3AM working fluid was removed from the sample, and freshly prepared culture medium was added. After incubation at 37 C for 1 hour, the culture medium was changed to Hanks’ balanced salt solution. Cells were stimulated with TRPA1 channel agonist cinnamaldehyde (10 mmol/L) and antagonists HC030031 (20 mmol/L), TRPV1 channel agonist capsaicin (10 mmol/L), and antagonist capsazepine (10 mmol/L). Fluorescence images were taken under a laser confocal microscope (LSM900, Carl Zeiss) at different time points. Data analysis of fluorescence intensity was perfomed by Image-Pro Plus software (Media Cybernetics, Rockville, MD).

Cells were counted, and the same amount of cells (approximately 5 ! 105) in each sample were lysed using 500 mL RIPA lysis buffer (Boster, Hubei, China) and 5 mL phenylmethanesulfonyl fluoride (Boster) on ice for 30 minutes. A protein sample of the same amount (3 mL) was separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel and electrotransferred to a polyvinylidene difluoride membrane. After blocking with 5% nonfat milk for 2 hours at room temperature, the membrane was incubated with their respective specific primary antibodies TRPA1 (1:500, Novus Bio), TRPV1 (1:200, Alomone Labs), DSPP (1:1000, Bioss, Boston, MA), and OPN (1:1000, Novus Bio) at 4 C overnight. GAPDH (1:40000, proteintech) was regarded as an internal control. After incubation with horseradish peroxidase–conjugated antimouse or antirabbit secondary antibodies (1:1000, Bioss), the membrane was exposed with chemiluminescence solution (Sparkjade). Quantitative analysis of protein was performed by ImageJ (National Insitutes of Health, Bethesda, MD) software.

Real-time Quantitative Polymerase Chain Reaction Total RNA was extracted using the Eastep Super Total RNA Extraction Kit (Promega, Shanghai, China) from cells and then converted to complementary DNA using the All-in-One First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD) in accordance with the manufacturer’s guidelines. Real-time quantitative polymerase chain reaction was performed using 2 ! TsingKe Master qPCR Mix (SYBR Green I; Tsingke Biotechnology, Beijing, China) on the CFX96 Real Time System (Bio-Rad, Hercules, CA). Relative gene expression data were normalized to housekeeping gene GADPH and analyzed using the 2-DDCt method. Primers sequences were TRPA1, forward 50 TGGTGCACAAATAGACCCAGT-30 and reverse 50 -TGGGCACCTTTAGAGAGTAGC3’; TRPV1, forward 50 CAGGCTCTATGATCGCAGGAG-30 , and reverse 50 -TTTGAACTCGTTGTCTGTGAGG3’; DSPP, forward 50 CCTAAAGAAAATGAAGATAATT-30 and reverse 50 -TAGAAAAACTCTTCCCTCCTAC3’; osteopontin (OPN), forward 50 ATAAGCGGAAAGCCAATGATGAGAG-30 and reverse 50 GGGTCTACAACCAGCATATCTTCA-3’; and GAPDH, forward 50 GGAGCGAGATCCCTCCAAAAT-30 and reverse 50 GGCTGTTGTCATACTTCTCATGG-3’.

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Alkaline Phosphatase Activity Assay HDPCs were cultured in 6-well culture plates. The alkaline phosphatase (ALP) activity was measured using an ALP activity kit (Beyotime) following the manufacturer’s instructions. In brief, 200 mL cell lysis buffer without inhibitors (Beyotime) were added per well followed by centrifugation at 10,000g for 10 minutes to obtain supernatant. Samples were incubated at 37 C for 8 minutes with 50 mL chromogenic substrate. After termination of reaction, the absorbance was determined at 405 nm.

NY). The Student t test or 1-way analysis of variance with the Bonferroni post hoc test was used to identify statistical signiﬁcance. Whitney and Kruskal-Wallis tests were used where the data were not normally distributed. Differences with P , .05 were considered statistically signiﬁcant. Each experiment was conducted 3 times to ensure repeatability.

RESULTS Functional Expression of TRPA1 and TRPV1 Channels in Pulp Slice Tissues and Human Odontoblastlike Cells As shown in Figure 1, the blank control of immunohistochemistry and immunofluorescence staining showed no obvious positive staining (Supplemental Figure S1 is available online at www.jendodon.com). The odontoblast layer was labeled by the immunohistochemical staining of DSPP (Fig. 1A). The TRPA1 and TRPV1 channels were expressed in the odontoblast layer of human dental pulp and were only slightly expressed in the inner pulp tissues (Fig. 1B and C). The cultured human odontoblastlike cells were positive for DSPP (green) (Fig. 1D–F) immunofluorescence, indicating that their functions were similar to those of odontoblasts. Double immunofluorescence staining showed the coexpression of TRPA1 (green) and TRPV1 (red) channels in human odontoblastlike cells (Fig. 1G–J). With extracellular Ca21, the activation of TRPA1 and TRPV1 channels with their agonist cinnamaldehyde (10 mmol/L) or capsaicin (10 mmol/L) rapidly increased the Ca21-dependent fluorescence intensity to peak values followed by a rapid decline and partial inhibition due to the addition of TRPA1 channel antagonists HC030031 (20 mmol/L) or TRPV1 channel antagonist capsazepine (10 mmol/L) (Fig. 1K and L).

Alizarin Red Staining Cells were ﬁxed with 4% paraformaldehyde and dyed with 1% alizarin red (pH 5 4.2, Sigma-Aldrich) for 30 minutes to evaluate mineralization. The excess dye was removed by washing with phosphate-buffered saline buffer, and images were captured for analysis. Then, the staining was destained for 30 minutes with 10% cetylpyridinium chloride. The absorbance at 562 nm was measured.

Statistical Analysis Statistical analysis was performed using SPSS software (Version 22.0; IBM Corp, Armonk,

Expression Levels of TRPA1 and TRPV1 Channels during the Odontogenic Differentiation of HDPCs The odontogenic differentiation of HDPCs can be induced by using an odontogenic differentiation medium and can be conﬁrmed by the gradually increased protein expression of DSPP and OPN (Fig. 2E). Alizarin red staining revealed the enhancement of HDPC mineralization with time, and the increased ALP activity proved the odontogenic differentiation of HDPCs. Alizarin red staining also showed

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FIGURE 1 – Functional expression of TRPA1 and TRPV1 channels in pulp slice tissues and human odontoblastlike cells. (A–C ) Immunohistochemistry of DSPP, TRPA1, and TRPV1 channels. (D–F ) Immunoﬂuorescence staining for (E and F ) DSPP (green ) and (D ) nuclei (blue ). (G–J ) Double immunoﬂuorescence staining for (H ) TRPA1 channel (green ), (I ) TRPV1 channel (red ), and (G ) nuclei (blue ). (K and L ) Representative traces of real-time Ca21 imaging. (A–F ) Scale bars 5 50 mm. (G–J ) Scale bars 5 20 mm.

mineralized nodule formation on day 21 and a signiﬁcant difference between days 14 and 21 and day 0 (P , .01) (Fig. 2A). The ALP activity was signiﬁcantly increased from day 3 (P , .01) and continuously increased until day 14

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(Fig. 2B). The messenger RNA (mRNA) and protein expression levels of TRPA1 and TRPV1 channels were generally up-regulated with time during this differentiation. In particular, TRPA1 mRNA expression showed no signiﬁcant

difference from day 0 until day 14 (P , .01), whereas TRPV1 mRNA level showed an increase on day 3 (P , .05) and peaked on day 7 (P , .01) (Fig. 2C). The protein level of TRPA1 and TRPV1 channels was signiﬁcantly

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FIGURE 2 – The expression of TRPA1 and TRPV1 channels during the odontogenic differentiation of HDPCs. (A ) Alizarin red staining. Scale bars 5 100 mm. (B ) ALP activity assay. (C ) Real-time quantitative polymerase chain reaction. (D ) Western blot analysis. (E ) Images of Western blot analysis of 4 proteins with DSPP and OPN. Data were presented as the mean 6 standard deviation. *P , .05 and **P , .01 compared with day 0. increased from day 3 to day 14 compared with that on day 0 (P , .01). A peak occurred on day 7 for TRPA1 channel, and TRPV1 channel maintained a high protein expression after a sharp increase on day 3 (Fig. 2D).

Activation of TRPA1 or TRPV1 Channels Promoted the Odontogenic Differentiation of HDPCs Treatment with TRPA1 channel agonist cinnamaldehyde (0.5 mg/mL) or TRPV1 channel agonist capsaicin (10 mmol/L) for 3 or 7 days up-regulated the mRNA and protein expression levels of DSPP and OPN compared with those of the control. Peak values were achieved on day 7 (P , .01), and the protein expression levels showed no statistical difference on day 3 (Fig. 3A–D). The ALP activity signiﬁcantly increased with time, peaked similarly on day 7 (P , .01), and decreased on day 14 (Fig. 3E). The mineralized nodule formation also increased with time and reached its peak on day 14 (Fig. 3F).

Inhibition of TRPA1 or TRPV1 Channels Suppressed the Odontogenic Differentiation of HDPCs The application of selective TRPA1 channel antagonist HC030031 (10 mmol/L) or TRPV1 channel antagonist capsazepine (10 mmol/L) for 7 and 14 days signiﬁcantly decreased the protein expression of DSPP and OPN compared with that of the control, and the

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lowest value was observed on day 7 (P , .01) (Fig. 4A and B). The ALP activity was reduced dramatically on day 14 (P , .01) with no statistical differences on days 3 and 7 (Fig. 4C). Alizarin red staining did not reveal any remarkable difference among the groups after the culture for 7 days, and the mineralized nodule formation on day 14 was decreased when the TRPA1 or TRPV1 channel antagonist was applied (Fig. 4D).

DISSCUSION This study investigated the possible functions of TRPA1 and TRPV1 channels in the odontogenic differentiation of HDPCs. Alizarin red staining and ALP activity assay were conducted to detect mineralization and verify the odontogenic differentiation of cultured cells. DSPP as a marker of odontoblastic differentiation and OPN as a middle-stage marker for hard tissue formation were selected to characterize the mineralization process. The mRNA and protein expression levels of TRPA1 and TRPV1 channels in HDPCs were generally up-regulated during the odontogenic differentiation of HDPCs induced by the odontogenic differentiation medium on day 7 or 14. The agonists and antagonists of the 2 channels were added to the odontogenic differentiation medium for different cultural periods and further showed positive effects on the odontogenic differentiation of HDPCs at the functional level. These results provide new insights into the reparative procedure of dental pulp.

TRPA1 and TRPV1 channels are involved in acute and chronic pain, inflammation, and intracellular Ca21 homeostasis and play key roles in the pathophysiology of nearly all organ systems17,18. Most TRPA1 channel–positive dorsal root ganglia neurons coexpress the TRPV1 channel7,19. Immunohistochemistry revealed that TRPA1 and TRPV1 channels were expressed in the odontoblast layer at the interface between dental pulp and dentin, thus implying their possible involvement in pulp repair20. Calcium imaging further revealed that the related agonists or antagonists of TRPA1 and TRPV1 channels could regulate intracellular Ca21 concentration to affect the functions of these channels. Although no significant differences were found in TRPA1 mRNA expression on day 3 or day 7, the mRNA expression of TRPA1 and TRPV1 channels tended to up-regulate on the whole, and the protein expression was significantly up-regulated during the odontogenic differentiation of HDPCs. This finding is consistent with previous studies showing that several TRP channels such as TRPC1 and TRPM7 are up-regulated and related to mineralization14,21. A previous work evaluating pulpal healing after cavity preparation in mice had reported positive DSPP and nestin expression from newly differentiated odontoblasts from days 3–5 and found reparative dentin formation on day 1422. This period of odontoblast differentiation coincided with the up-regulated expression of the previously mentioned TRP channels on days

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FIGURE 3 – The effect of activating TRPA1 and TRPV1 channels on the odontogenic differentiation of HDPCs. (A and B ) Real-time quantitative polymerase chain reaction for DSPP and OPN. (C and D ) Western blot analysis for DSPP and OPN. (E ) ALP activity assay. (F ) Alizarin red staining. Black arrows indicated mineralized nodules. Scale bars 5 50 mm. Data were presented as the mean 6 standard deviation. *P , .05 and **P , .01 compared with the control group at each time point.

the role of related channels or receptors by gene knockdown. By contrast, the present work explored the functions of TRPA1 and TRPV1 channels through intervention at the functional level under the activation and inhibition of these 2 channels by using agonists or antagonists. EI Karim et al5 reported that cultured and native human odontoblasts

DSPP

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express functional TRP channels including TRPV1 and TRPA1 channels that might mediate thermal sensation in teeth. Our study showed the effects of the 2 channels without artiﬁcially altering their expression levels to simulate the state of the 2 channels during odontogenic differentiation and revealed the reactions of HDPCs when these channels were

OPN

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7–14 with different degrees of effect on various stages. Emerging research has focused on the odontogenic differentiation of HDPCs promoted by the activation of Ca21-related channels or receptors such as the L-type calcium channel23 and calcium-sensing receptor13,24. Most of these studies explored

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FIGURE 4 – The effect of inhibiting TRPA1 and TRPV1 channels on the odontogenic differentiation of HDPCs. (A and B ) Western blot analysis for DSPP and OPN. (C ) ALP activity assay. (D ) Alizarin red staining. Scale bars 5 100 mm. Data were presented as the mean 6 standard deviation. *P , .05 and **P , .01 compared with the control group at each time point.

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activated. This work also provided conditions similar to the clinically affected TRP channels by external stimuli such as thermal changes. Thus, the possibility of functional intervention measures to facilitate pulp tissue repair need to be considered. Compared with TRPM7 and TRPC1 channels as mechanoreceptors in odontoblasts, TRPA1 and TRPV1 channels are mainly considered as the thermoreceptors and sense pulpal sensations of noxious stimuli, to which the response of HDPCs was illustrated in the present study. The increased intracellular Ca21 concentration by TRPV1 activation in odontoblasts is extruded by Na1 -Ca21 exchangers to aid in protective dentin formation, and this phenomenon might partly explain the increased mineralization25. The expression of DSPP and OPN on day 14 after treatment with TRPA1 or TRPV1 agonists was lower compared with that on day 7. This ﬁnding might be attributed to the peak protein expression levels of TRPA1 and TRPV1 channels on day 7. Higher protein levels of TRPA1 and TRPV1 meant that more channels could be activated when applying agonists

compared with day 14, thereby leading to a stronger effect for odontogenic differentiation. Another possible reason is the desensitization of cells after the continuous application of agonists26. The results also revealed for the ﬁrst time that the inhibition of TRPA1 or TRPV1 channels for 14 days could suppress the mineralization of HDPCs. This ﬁnding is in agreement with the previous observations by Muramatsu et al27, who reported that cementoblasts show inhibited mineralization when treated with TRPA1 channel antagonist HC030031, and Idris et al28, who stated that TRPV1 channel antagonist capsazepine inhibits osteoblast differentiation in vitro. In summary, the up-regulation of TRPA1 and TRPV1 channels led to the activation of additional channels. Such functional activation strongly promotes odontogenic differentiation and could be applied to enhance pulp tissue repair. In conclusion, the TRPA1 and TRPV1 channels in HDPCs could affect the odontogenic differentiation progression by regulating intracellular Ca21 concentration.

The ﬁndings shed new light on the development of novel drugs or materials for reparative or regenerative endodontic procedures.

ACKNOWLEDGMENTS Yaxin Lou and Yangqiu Liu contributed equally to this study. Supported by Tianjin Municipal Natural Science Foundation (grant no. 19JCYBJC29000), Tianjin Municipal Natural Science Foundation (grant no. 18JCQNJC78400), and Tianjin Municipal Natural Science Foundation (grant no. 19JCYBJC29000). The authors deny any conﬂicts of interest related to this study.

SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found in the online version at www.jendodon.com (https://doi.org/10.1016/ j.joen.2021.06.007).

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Yu C, Abbott PV. An overview of the dental pulp: its functions and responses to injury. Aust Dent J 2007;52:S4–16.

Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81:531–5.

Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 2007;76:387–417.

McKemy DD, Neuhausser WM, Julius D. Identiﬁcation of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 2002;416:52–8.

El Karim IA, Linden GJ, Curtis TM, et al. Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity. Pain 2011;152:2211–23.

Lee K, Lee BM, Park CK, et al. Ion channels involved in tooth pain. Int J Mol Sci 2019;20:2266.

Weyer AD, Stucky CL. Loosening pain’s grip by tightening TRPV1-TRPA1 interactions. Neuron 2015;85:661–3.

Vangeel L, Voets T. Transient receptor potential channels and calcium signaling. Cold Spring Harb Perspect Biol 2019;11:a035048.

An S. The emerging role of extracellular Ca21 in osteo/odontogenic differentiation and the involvement of intracellular Ca 21 signaling: from osteoblastic cells to dental pulp cells and odontoblasts. J Cell Physiol 2019;234:2169–93.

10.

Prati C, Gandolﬁ MG. Calcium silicate bioactive cements: Biological perspectives and clinical applications. Dent Mater 2015;31:351–70.

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s-Catala CJ, Collado-Gonz Toma alez M, García-Bernal D, et al. Biocompatibility of new pulpcapping materials NeoMTA Plus, MTA Repair HP, and Biodentine on human dental pulp stem cells. J Endod 2018;44:126–32.

12.

Alqaderi H, Lee CT, Borzangy S, et al. Coronal pulpotomy for cariously exposed permanent posterior teeth with closed apices: a systematic review and meta-analysis. J Dent 2016;44:1–7.

13.

Mizumachi H, Yoshida S, Tomokiyo A, et al. Calcium-sensing receptor-ERK signaling promotes odontoblastic differentiation of human dental pulp cells. Bone 2017;101:191–201.

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Cui L, Xu SM, Ma DD, et al. The effect of TRPM7 suppression on the proliferation, migration and osteogenic differentiation of human dental pulp stem cells. Int Endod J 2014;47:583–93.

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Cha M, Sallem I, Jang HW, et al. Role of transient receptor potential vanilloid type 1 in the trigeminal ganglion and brain stem following dental pulp inﬂammation. Int Endod J 2020;53:62–71.

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El Karim IA, McCrudden MT, McGahon MK, et al. Biodentine reduces tumor necrosis factor alpha-induced TRPA1 expression in odontoblastlike cells. J Endod 2016;42:589–95.

17.

Talavera K, Startek JB, Alvarez-Collazo J, et al. Mammalian transient receptor potential TRPA1 channels: from structure to disease. Physiol Rev 2020;100:725–803.

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Zhao R, Tsang SY. Versatile roles of intracellularly located TRPV1 channel. J Cell Physiol 2017;232:1957–65.

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Weng HJ, Patel KN, Jeske NA, et al. Tmem100 is a regulator of TRPA1-TRPV1 complex and contributes to persistent pain. Neuron 2015;85:833–46.

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Bleicher F. Odontoblast physiology. Exp Cell Res 2014;325:65–71.

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Song Z, Chen L, Guo J, et al. The role of transient receptor potential cation channel, subfamily C, member 1 in the odontoblast-like differentiation of human dental pulp cells. J Endod 2017;43:315–20.

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Saito K, Nakatomi M, Ohshima H. Dynamics of bromodeoxyuridine label-retaining dental pulp cells during pulpal healing after cavity preparation in mice. J Endod 2013;39:1250–5.

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Ju Y, Ge J, Ren X, et al. Cav1.2 of L-type calcium channel is a key factor for the differentiation of dental pulp stem cells. J Endod 2015;41:1048–55.

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Chen Y, Gao Y, Tao Y, et al. Identiﬁcation of a calcium-sensing receptor in human dental pulp cells that regulates mineral trioxide aggregate–induced mineralization. J Endod 2019;45:907–16.

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Tsumura M, Sobhan U, Muramatsu T, et al. TRPV1-mediated calcium signal couples with cannabinoid receptors and sodium-calcium exchangers in rat odontoblasts. Cell Calcium 2012;52:124–36.

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Ruparel NB, Patwardhan AM, Akopian AN, et al. hom*ologous and heterologous desensitization of capsaicin and mustard oil responses utilize different cellular pathways in nociceptors. Pain 2008;135:271–9.

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Muramatsu T, Kashiwagi S, Ishizuka H, et al. Alkaline extracellular conditions promote the proliferation and mineralization of a human cementoblast cell line. Int Endod J 2019;52:639–45.

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BASIC RESEARCH – BIOLOGY

Inﬂuence of Preoperative Pulp Inﬂammation in the Outcome of Full Pulpotomy Using a Dog Model

ABSTRACT Introduction: This study aimed to evaluate the impact of preoperative pulp inflammation on the histologic outcome of full pulpotomy performed in mature permanent posterior teeth using 4 different biomaterials. Methods: Five beagle dogs (providing a total of 120 roots) were selected. Dentin exposure was performed in teeth from the second and third quadrants. One week later, full pulpotomy procedures were performed using 4 different bioactive materials (ProRoot MTA [MTA], TotalFill BC Putty [BC], Biodentine [BIO], and an experimental cement [ie, pulp capping material]). The hemostasis time was registered. After 14 weeks, the animals were killed. Pulp-dentin tissues were histologically and radiographically assessed. The significance level was set at .05. Results: Teeth with previously exposed dentin revealed a statistically significant increase in the time required to achieve hemostasis (P , .001), therefore confirming the pulp inflammation status induced by 1-week exposure of occlusal dentin before performing full pulpotomy. There was no radiographic evidence of root resorption, periapical radiolucency, or lamina dura alterations. No statistically significant differences were observed between normal and inflamed pulp regardless of the evaluated histologic parameters. Moreover, histologic data concerning calcified barrier formation and the pulp tissue response show better results for BIO without statistical differences compared with MTA or BC (P . .05). The pulp capping material presented a lower performance, with statistically significant differences being detected in regard to the remaining 3 tested materials (P , .001). Conclusions: Radiographic and histologic outcomes of full pulpotomy are not jeopardized by short-term preoperative pulp inflammation. Moreover, BIO, MTA, and BC cements present suitable alternatives to be used as pulp capping agents. (J Endod 2021;47:1417–1426.)

~o Miguel Santos, DMD, Joa PhD,*†‡ Joana A. Marques, DMD,* Patrícia Diogo, DMD, PhD,*† Ana Messias, DMD, PhD,†§ Vitor Sousa, MD, PhD,k Diana Sequeira, DMD,*‡ and Paulo J. Palma, DMD, PhD*†

SIGNIFICANCE The time required for hemostasis does not inﬂuence the outcome of full pulpotomy in permanent teeth. Biodentine, ProRoot MTA, and TotalFill BC Putty present suitable alternatives to be used as pulp capping materials.

KEY WORDS Animal models; calcium silicate–based cements; histology; inﬂammation; pulpotomy; vital pulp therapy

Recently, considerable developments were made in vital pulp therapy (VPT) after the introduction of calcium silicate–based cements. The purpose of VPT, including direct pulp capping and partial and full pulpotomy, is to maintain pulp viability and function1,2. The etiology of pulp exposure has been specified as mechanical (either traumatic or iatrogenic) or carious. The former is associated with sound dentin surrounding the exposure site, whereas potentially infected peripheral dentin is frequently found in the latter scenario. Therefore, the nature of the exposure might differently influence the subsequent pulpal response3. In both clinical circ*mstances, VPT has been recommended, especially when immature permanent teeth are involved1. Pulpotomy is a commonly performed procedure with a sound evidence-based foundation attesting its clinical efficacy in the treatment of both primary4 and immature permanent teeth5. Nowadays, improved knowledge about pulp biology and the development of new bioactive materials increased the interest in pulpotomy regarding the management of permanent posterior teeth with irreversible pulpitis as an alternative to conventional endodontic treatment2,6–8. However, there is a

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From the *Institute of Endodontics, Center for Innovation and Research in Oral Sciences, ‡Center for Neuroscience and Cell Biology, §Institute of Oral Implantology and Prosthodontics, and k Institute of Pathological Anatomy, Faculty of Medicine, University of Coimbra, Coimbra, Portugal †

~o Address requests for reprints to Prof Joa Miguel Santos, Institute of Endodontics, Faculty of Medicine, University of Coimbra, Av. Bissaya Barreto, 3000-075 Coimbra, Portugal. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.018

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gap on the existing knowledge concerning the physiological basis for this clinical approach, which needs to be further clarified to enhance the predictability of treatment protocols. The impact of previous pulp inflammation on the tissue response to the pulpotomy procedure is still a concern1,9. Ricucci et al10 found that teeth with a clinical diagnosis of reversible pulpitis and irreversible pulpitis only exhibited focal areas of necrosis near the pulp horn, with the remaining coronal and radicular pulp tissue displaying normal architecture. Additionally, although moderate to severe inflammation may have a negative impact on the final outcome, low-grade inflammation may be an important prelude to early reparative or regenerative events11. Therefore, it is important to challenge the dogma that pulp must be free of inflammation before being capped in order to successfully introduce new regenerative procedures12,13. Chronic and acute inflammatory responses also involve the recruitment of different cellular and molecular processes, the outcomes of which may significantly differ. The resolution of acute inflammation may lead either to the restoration of normal tissue function and clearance of the injurious stimuli, mediators, and inflammatory cells or, conversely, to abscess formation and tissue necrosis14. The quantity of calcified tissue deposited and the quality of the dentin bridge formed in contact with the capping material may also be influenced by such factors9.

One important concern and criticism regarding the published animal1 and human15 histologic studies is the fact that the response to pulpotomy is evaluated on sound intact pulps, and it cannot be assumed that the same response will be exhibited by pulps with some degree of preoperative inflammation. Furthermore, similar reparative dentin formation, including a predentin-like pattern, has been reported among human, dog, and rat/mouse tooth models16. Thus, the main objective of this study was to evaluate the impact of preoperative pulp inflammation on the histologic outcome of full pulpotomy performed in mature permanent posterior teeth using a dog model. Additionally, the pulp tissue response to 4 different biomaterials (ProRoot MTA [MTA; Dentsply Maillefer, Tulsa, OK], TotalFill BC Putty [BC; FKG, La Chaux-des-Fonds, Switzerland], Biodentine [BIO; Septodont, Saint-Maur-des-Fosses, France], and an experimental cement [ie, pulp capping material (PCM); Coltene/Whaledent, Altst€atten, Switzerland]) was assessed. To our knowledge, this is the first study conducted in a large-animal model that intends to clarify the role played by inflammation in the histologic outcome of total pulpotomy. The tested null hypothesis states there are no statistically significant differences between normal and inflamed pulps on the histologic outcome of full pulpotomy with a similar dentin-pulp complex response regardless of the tested biomaterial.

MATERIALS AND METHODS The study design was approved by the Animal ~o Geral de Welfare Commission of the Direça ~o e Veterina ria (IRB0421/000/2020) Alimentaça and complies with EU Directive 2010/63/EU for animal experiments. The present study is based on the protocol recommended by ISO standard 7405:2008 and followed the Animal Research: Reporting of In Vivo Experiments guidelines17.

Experimental Procedures Sample size calculations were based on the primary null hypothesis of there being no difference on the histologic outcome of full pulpotomy between normal and inflamed pulps, measured as the continuity of the calcified barrier. The study was dimensioned to detect a minimum difference of 20% in the proportion of continuous calcified barriers between pulps status, considering 80% power and a significance level of .05. Power calculations determined a total of 96 samples, 48 per group (normal and inflamed pulps). In order to comply with the preposition that no animal life would be wasted and that the minimum number of animals to obtain valid and meaningful results would be used, each root of the maxillary and mandibular premolars was considered the statistical unit. All premolars (except fourth maxillary premolars) with closed apical foramina of 5 female beagle dogs (9 6 1 months old and weight of 10 6 1 kg) were selected for the study, providing a total of 120 roots.

FIGURE 1 – (A ) Intact tooth. (B ) After dentin exposure (approximately 2 mm2 area). (C ) Bleeding control after coronal pulp removal. (D ) Bioactive capping material in place.

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TABLE 1 - Distribution of the Roots among the Experimental Groups Pulp status Biomaterial

Normal

Inﬂamed

ProRoot MTA (MTA) TotalFill BC Putty (BC) Biodentine (BIO) Pulp capping material (PCM) Control groups Total

12 roots 12 roots 12 roots 14 roots 10 roots 60 roots

Biradicular premolars were assigned to experimental groups and first premolars to control groups. All animals were healthy without a history of previous procedures and were provided by Isoquimen Research Models and Services (Barcelona, Spain) (a Good Laboratory Practice-certified preclinical contract research organization). All experimental procedures were conducted in a controlled environment at 21 C room temperature. For the first intervention, the animals were sedated, and anesthesia was maintained by inhalation

(10 mg/kg thiopental and the inhalation of 2% isoflurane). Standardized periapical radiographs were taken using custom-made film-holding devices. Subsequently, dentin exposure was performed in teeth from the second and third quadrants by removing the cusp tip with a tapered high-speed diamond bur (FG ML 200442AA ISO198016, Diatech; Coltene/Whaledent, Altst€atten, Switzerland) in order to induce pulp inflammation by mechanically exposing an area of approximately 2 mm2 (Fig. 1A and B).

TABLE 2 - The Scores for Histologic Analysis of Calciﬁed Barriers and Dental Pulp Inﬂammation Scores

Parameter/description

Calcified barrier continuity 1 Complete bridge formation 2 Partial/incomplete dentin bridge formation extending to more than one half of the exposure 3 Initial dentin bridge formation extending to less than one half of the exposure 4 No dentin bridge formation Calcified barrier morphology 1 Dentin or dentin associated with irregular hard tissue 2 Only irregular hard tissue deposition 3 Only a thin layer of hard tissue deposition 4 No hard tissue deposition Calcified barrier thickness 1 ..25 mm 2 .1-–.25 mm 3 ,.1 mm 4 Partial or absent Inflammation intensity 1 Absent or very few inflammatory cells 2 Mild (an average of ,10 inflammatory cells) 3 Moderate (an average of 10–25 inflammatory cells) 4 Severe (an average .25 inflammatory cells) Inflammation extensity 1 Absent 2 Mild (inflammatory cells next to the dentin bridge area of exposure only) 3 Moderate (inflammatory cells observed in one third or more of the coronal pulp 4 Severe (all radicular pulp infiltrated or necrotic) Bacterial penetration 1 Absence of stained bacteria 2 Presence of stained bacteria in the coronal aspect of dentin 3 Presence of stained bacteria in all root dentin 4 Presence of stained bacteria inside the pulp This classification system attributes qualitative ordinal scores, with a score of “1” associated with the best expected outcome and a score of “4” corresponding to the worst expected histologic outcome.

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One week later, the animals were sedated, and anesthesia was maintained by inhalation with isoflurane. Root scaling and prophylaxis with polishing paste, rubber dam placement, and operative field disinfection with a povidone-iodine solution (Betadine Oral; Betadine, Lisbon, Portugal) were performed. After pulp exposure with a highspeed diamond bur, complete deroofing of the pulp chamber and coronal pulp removal were performed using a new sterile highspeed diamond bur (Endo Access Bur; Dentsply Maillefer, Ballaigues, Switzerland). Hemostasis was then promoted through irrigation with 3 mL 3% sodium hypochlorite (CanalPro NaOCl 3%; Coltene/Whaledent) s, and sterile Teflon (Merck Life Science, Alge Portugal) pellet compression. During the first 2 minutes of compression, the pulp chamber was irrigated with 3 mL sterile saline. If hemostasis was not achieved, additional 2minute compression periods were applied, and hemostasis was reevaluated until obtained (Fig. 1C). The total amount of time necessary to achieve hemostasis was registered, starting immediately after complete removal of the coronal pulp. The bioactive materials were randomly and evenly distributed among each tooth type (randomized block design). Five different charts were sealed in envelopes and randomly assigned to each dog. These were opened by a third investigator at the second intervention, who informed the operators about the material to be applied in each tooth, after complete removal of the coronal pulp (Table 1). Biomaterials were placed over the remaining radicular pulp tissue at the amputation site (Fig. 1D) according to the manufacturer’s instructions (Supplemental Table S1 is available online at www.jendodon.com) and gently compacted with a moistened cotton ne/ pellet (Roeko Cotton Pellets, Colte Whaledent). A glass ionomer cement was then used to perform the coronal restoration (Ketac Fill Plus Aplicap; 3M, St Paul, MN). A final periapical radiograph was taken. After 14 weeks, postoperative periapical radiographs were taken, and the animals were killed by an anesthetic overdose of thiopental followed by bilateral perfusion with 10% phosphate-buffered formalin. Regarding the controls, 2 teeth were extracted in the second intervention immediately before initiating full pulpotomy procedures as follows: 1 premolar exhibiting previous dentin exposure to histologically confirm the presence of pulp inflammation and 1 intact premolar as a reference for normal pulp architecture histology. Two additional controls remained until the last observation

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thickest, thinnest, and midmost point areas of the bridge. The mean of the 3 values of a representative slide was calculated. Preoperative, immediate postoperative, and follow-up radiographs were evaluated by a blind examiner and scored regarding root resorption, periapical radiolucency, and lamina dura according to a previous study20.

Statistical Analysis

FIGURE 2 – Relative frequencies of bleeding control time distribution in teeth with or without preoperative 1-week dentin exposure.

period at 14 weeks for subsequent histologic pulp status assessment. Therefore, each animal yielded a total of 4 controls.

Histologic and Radiographic Analysis Jaw and maxillary blocks were separated, dissected from the first premolar to the first molar, postfixed in phosphate-buffered formalin, and decalcified with Morse solution. Each block was divided between each tooth and trimmed, embedded in paraffin, and

serially sectioned every 6 mm along the mesiodistal plane, parallel to the canal long axis18. One of every 10 sections was stained with hematoxylin-eosin and the modiﬁed Brown and Brenn technique. Pulp-dentin tissues from each root were histologically assessed under a light microscope (Nikon Eclipse E60; Nikon, Tokyo, Japan). Two blind observers evaluated all specimens according to the parameters and scores (Table 2) adapted from Nowicka et al19. The calciﬁed barrier thickness was measured at the

Statistical analysis was performed using SPSS Statistics 27.0 MacOS version software (IBM Corp, Armonk, NY). Data are presented as counts and relative frequencies and statistically analyzed with the chi-square test considering Monte Carlo estimation whenever the assumptions for asymptotic signiﬁcance were not met. Kruskal-Wallis tests with all pair-wise comparisons and Bonferroni correction were used to compare the biomaterials. The signiﬁcance level was set at .05.

RESULTS All animals reached the end of the study without signs of distress. Only 1 tooth from the BC group was lost during histologic processing, and all remaining teeth were histologically assessed.

Clinical Findings Teeth with previously exposed dentin revealed a statistically signiﬁcant increase in the time

FIGURE 3 – Sequential radiographic records throughout the experimental protocol for normal and preoperative inﬂamed pulp groups (asterisk, intact tooth; arrow, exposed dentin).

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TABLE 3 - The Scores of the 4 Tested Materials Regarding the Evaluated Histologic Features Material, n (%) Parameter

Score

Calciﬁed barrier continuity

1 2

Calciﬁed barrier morphology

4 Total 1 2 3

Calciﬁed barrier thickness

Inﬂammation intensity

Inﬂammation extensity

4 Total 1 2 3 4 Total 1 2 3 4 Total 1 2 3 4 Total

Description

MTA

BIO

PCM

Total, N (%)

22 (91.7) 0 (0)

22 (91.7) 2 (8.3)

19 (86.4) 1 (4.5)

1 (3.6) 5 (17.9)

64 (65.3) 8 (8.2)

2 (8.3)

0 (0)

1 (4.5)

12 (42.9)

15 (15.3)

0 (0) 24 (100) 20 (83.3)

0 (0) 24 (100) 22 (91.7)

1 (4.5) 22 (100) 20 (90.9)

10 (35.7) 28 (100) 0 (0)

11 (11.2) 98 (100) 62 (63.3)

2 (8.3) 2 (8.3)

2 (8.3) 0 (0)

0 (0) 1 (4.5)

8 (28.6) 10 (35.7)

12 (12.2) 13 (13.3)

0 (0) 24 (100) 10 (41.7) 9 (37.5) 5 (20.8) 0 (0) 24 (100) 23 (95.8)

0 (0) 24 (100) 11 (45.8) 7 (29.2) 6 (25) 0 (0) 24 (100) 23 (95.8)

1 (4.5) 22 (100) 11 (50) 8 (36.4) 1 (4.5) 2 (9.1) 22 (100) 19 (86.4)

10 (35.7) 28 (100) 0 (0) 1 (3.6) 5 (35.7) 22 (78.6) 28 (100) 7 (25)

11 (11.2) 98 (100) 32 (32.7) 25 (25.5) 17 (17.3) 24 (24.5) 98 (100) 72 (73.5)

1 (4.2) 0 (0) 0 (0) 24 (100) 23 (95.8) 1 (4.2) 0 (0) 0 (0) 24 (100)

2 (9.1) 1 (4.5) 0 (0) 22 (100) 19 (86.4) 2 (9.1) 1 (4.5) 0 (0) 22 (100)

17 (60.7) 2 (7.1) 2 (7.1) 28 (100) 7 (25) 9 (32.1) 10 (35.7) 2 (7.1) 28 (100)

21 (21.4) 3 (3.1) 24 (24.5) 98 (100) 72 (73.5) 13 (13.3) 11 (11.2) 2 (2.0) 98 (100)

Dentin or dentin associated with irregular hard tissue Only irregular hard tissue deposition Only a thin layer of hard tissue deposition No hard tissue deposition .0.25 mm 0.1–0.25 mm ,0.1 mm Partial or absent Absent or very few inﬂammatory cells Mild Moderate Severe Absent Mild Moderate Severe

BC, TotalFill BC Putty; BIO, Biodentine; MTA, ProRoot MTA; PCM, pulp capping material.

required to achieve hemostasis (P , .001), therefore confirming the pulp inflammation status induced by 1-week exposure of occlusal dentin before performing full pulpotomy (Fig. 2). No experimental teeth presented signs of inflammation or infection at the end of the study.

Radiographic Findings The follow-up radiographs (Fig. 3) show the biomaterial was restricted to the radicular pulp’s entrance oriﬁce, except in 13% of cases in which the capping biomaterials slightly invaded the cervical third of the radicular pulp canal space (3/24 MTA, 1/24 BIO, 7/24 BC, and 4/28 PCM). No evidence of root resorption, periapical radiolucency, or lamina dura alterations was noted.

Histologic Findings Table 3 shows the scores obtained for each material regarding the evaluated parameters related to dentin bridge formation and pulp tissue inﬂammation.

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Concerning mineralized tissue formation, a complete bridge was observed in 91.7% of the teeth capped either with BIO or MTA (Figs. 4A–H and 5A–H). Similarly, BC showed complete bridge formation in 86.4% of the specimens. In contrast, PCM was associated with initial or no dentin bridge formation in 78.6% of the cases. Dentin deposition, with or without irregular hard tissue associated, was higher in BIO (91.7%) followed by BC (90.9%) and MTA (83.3%), whereas PCM teeth mainly exhibited the absence of or solely a thin layer of hard tissue deposition. BC, BIO, and MTA allowed the formation of bridges over 0.25-mm thick in 50%, 45.8%, and 41.7% of the teeth, respectively. Conversely, unfavorable results of the PCM group reveal that in 78.6% of the cases, the formed bridge was partial or even absent. Inﬂammation of the pulp tissue was primarily absent regardless of the material, except for PCM, which led to a mild inﬂammatory state in the majority of teeth.

Regarding the extensity, 67.8% of the teeth in the PCM group showed either mild or moderate inflammation (Fig. 4G and H). Additionally, 2 cases of severe inflammation were recorded in the PCM group. MTA, BIO, and BC mostly exhibited the absence of inflammatory cells (95.8% for both MTA and BIO and 86.4% for BC). No microorganism progression was detected with the Brown and Brenn staining technique regardless of the experimental group. In synthesis, analysis of histologic data concerning calcified barrier formation and the pulp tissue response shows better results for BIO without statistical differences compared with MTA or BC (P . .05). The experimental material (ie, PCM) presented a lower performance with statistically significant differences being detected in regard to the remaining 3 tested materials (P , .001). Moreover, no statistically significant differences were observed between normal and inflamed pulps regardless of the evaluated histologic parameters (Table 4).

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FIGURE 4 – The histologic response to full pulpotomy in teeth with preoperative normal pulp status (white scale bar 5 500 mm, hematoxylin-eosin) to the tested biomaterials (white star ). (A ) An overview of the biomaterial, complete bridge formation, and pulp tissue. (B ) Higher magnification with an early calcified barrier showing cellular inclusions (white arrowhead ) and fibrous tissue with dense collagen fibrils (yellow arrow ). (C ) The dentin bridge in continuity with root canal wall tertiary dentin with clear demarcation of the intervention moment by the calciotraumatic line (white arrowhead ). (D ) Higher magnification showing initial irregular hard tissue deposition (blue arrow ) in contact with the capping biomaterial followed by tubular tertiary dentin (white arrow ) deposited by elongated flattened cells resembling fibroblasts. (E ) Calcified barrier thickness scored 1 (.0.25 mm). (F ) Higher magnification with initial irregular hard tissue deposition (blue arrow ) beneath the biomaterial followed by tubular dentin and pulp tissue (white arrow ) with dense collagen and collagen septa (yellow arrow ). (G ) The absence of dentin bridge formation. (H ) Higher magnification exhibiting a thin layer of hard tissue deposition and pulp tissue with hyperemic blood vessels (yellow arrow ).

At 1 week, immediately before performing full pulpotomy procedures, histologic evaluation of the exposed dentin controls revealed vascular hyperemia but no signs of changes in the odontoblastic layer. At the 14-week observation period, teeth associated with dentin exposure exhibited higher reactionary dentinogenesis and hyperemia compared with the intact dentin controls.

DISCUSSION This study aimed to evaluate the inﬂuence of preoperative pulp inﬂammation on the outcome of full pulpotomy performed in mature canine posterior permanent teeth as well as to assess the pulpal response to 4 different bioactive materials. An insight on this topic is of uttermost importance because our results may encourage the expansion of clinical indications for VPT; desirable biological goals are out of

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reach for the traditional root canal treatment recommended for irreversible pulpitis management. Furthermore, the observed histologic response of the pulp tissue may provide guidance in regard to the selection of the biomaterial to be used as the pulpotomy agent. Pulpotomy presents a minimally invasive procedure that allows for maintaining pulp vitality2,13. Although randomized trials are scarce6,21, emerging evidence highlights the successful treatment outcomes attained in cases of teeth with signs and symptoms of irreversible pulpitis through coronal pulpotomy2,8,22. Hemostasis time, as an indicator of the ability to control bleeding, presents itself as a surrogate variable of inflammation23,24. In the present study, a strong and statistically significant association was found between previous dentinal exposure and increased hemostasis time (P , .001), thus reflecting the

role played by 7-day dentin exposure as a strategy capable to trigger inflammatory phenomena, which is in agreement with previous findings25. Although pulpal inflammation was induced, with no signs of microabscesses or infection being found9, the inability of discriminating between reversible or irreversible pulpitis represents 1 of this study’s limitations. Nowadays, besides being based on subjective and qualitative judgments of bleeding time, volume, and color of blood26, the diagnosis of pulp condition results from a decision-making process that mainly relies on clinical criteria that cannot be simulated in an animal model. Our findings suggest that both the histologic and radiographic outcomes of full pulpotomy are not influenced by the preoperative vital pulp status, although a statistically significant variation is verified according to the biomaterial used. Therefore, the null hypothesis is rejected.

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FIGURE 5 – The histologic response to pulpotomy in teeth with preoperative pulp inflammation status (white scale bar 5 500 mm, hematoxylin-eosin) to the tested biomaterials (white star ). (A ) A complete dentin bridge with only irregular hard tissue deposition and projection into the interior of the pulp canal space. (B ) Higher magnification showing cellular inclusions inside the hard tissue (blue arrow ) and pulp tissue presenting signs of fibrosis (yellow arrow ). (C ) Biomaterial slightly invading the radicular canal. (D ) Higher magnification exhibiting tubular tertiary dentin (white arrow ) and uninflamed pulp tissue with fibroblasts and abundant collagen bundles with a mild-degree edema (white oval ). (E ) The dentin bridge in continuity with root canal wall tertiary dentin and a calciotraumatic line (white arrowhead ). (F ) Higher magnification showing tubular tertiary dentin (white arrow ) and the pulp tissue with collagen fibers deposition showing some retraction artifacts (white oval ). (G ) Complete bridge formation with moderate intensity pulp inflammation (score 3). (H ) Higher magnification showing a predentin mineralization front with calcospherites (white arrowhead ) as well as polymorphic inflammatory infiltrate with lymphocytes and neutrophile predominance (yellow oval ).

Microleakage prevention is a determinant factor for pulpotomy success. Although this may be provided by the material itself20, the long-term success for pulpotomy depends on continuous bridge formation27,28, which provides additional pulp protection. In this study, all hydraulic calcium silicate cements induced the formation of a high percentage of dentinal bridges at the interface with the pulp tissue and controlled the level of inﬂammation underneath, which is in agreement with in vivo data from previous reports29–31. The dentin bridge at the site of injury was mainly hom*ogeneous with BIO followed by MTA and BC, with no statistical differences detected between these 3 biomaterials. PCM was associated with a higher percentage of samples with incomplete dentinal bridge formation and therefore was less reliable for long-term protection of the pulp than the other tested cements. Taken

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together, our data do not support the use of PCM in total pulpotomy. According to our results, although the presence of preoperative inflammation did not jeopardize the outcome of the treatment, an additional effort was required to adequately perform the clinical procedure. Overall, the increased technical difficulty was associated with superior vascular hyperemia, and, consequently, the time required to achieve hemostasis was longer. It is also important to clarify that all procedures were performed without prior administration of local anesthesia, with the ultimate goal of not masking the previously induced pulp inflammation. This represents the worst-case scenario for bleeding control, and it is important to underline that in the clinical context both pharmacologic strategies26 and cryotherapy32 present potential adjunct tools in vasoconstriction promotion. Additionally,

considering that preoperative inflammation does not affect the outcome of full pulpotomy, we hypothesize the possibility of avoiding inflammation induction in future animal studies, thus allowing the reduction of potential animal distress and costs. Within the limitations of the present study, it can be concluded that both radiographic and histologic outcomes of full pulpotomy are not jeopardized by the shortterm preoperative pulp inflammation. Moreover, BIO, MTA, and BC cements present suitable alternatives to be used as pulp capping agents. Further prospective clinical studies should be conducted to confirm these results.

CREDIT AUTHORSHIP CONTRIBUTION STATEMENT ~ o Miguel Santos: Conceptualization, Joa Methodology, Validation, Formal analysis,

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TABLE 4 - The Overall Results Concerning the Comparison between Normal and Inﬂamed Pulps in Regard to the Evaluated Histologic Features Preoperative dentin exposure, n (%) Parameter Calciﬁed barrier continuity

Score

Description

Yes

P value

1 2

Complete bridge formation Partial/incomplete dentin bridge formation extending to more than one half of the exposure Initial dentin bridge formation extending to less than one half of the exposure No dentin bridge formation Dentin or dentin associated with irregular hard tissue Only irregular hard tissue deposition Only a thin layer of hard tissue deposition No hard tissue deposition .0.25 mm 0.1–0.25 mm ,0.1 mm Partial or absent Absent or very few inﬂammatory cells Mild Moderate Severe Absent Mild Moderate Severe

32 (61.5) 3 (5.8)

32 (69.6) 5 (10.9)

..05

8 (15.4)

7 (15.2)

9 (17.3) 32 (62.5)

2 (4.3) 30 (65.2)

5 (9.6) 6 (11.5) 9 (17.3) 13 (25.0) 16 (30.8) 8 (15.4) 15 (28.8) 39 (75.0) 10 (19.2) 1 (1.9) 2 (3.8) 39 (75.0) 4 (7.7) 7 (13.5) 2 (3.8)

7 (15.2) 7 (15.2) 2 (4.3) 19 (41.3) 9 (19.6) 9 (19.6) 9 (19.6) 33 (71.7) 11 (23.9) 2 (4.3) 0 (0) 33 (71.7) 9 (19.6) 4 (8.7) 0 (0)

Calciﬁed barrier morphology

Calciﬁed barrier thickness

Inﬂammation intensity

Inﬂammation extensity

4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

..05

Statistically signiﬁcant difference (P , .05).

Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Joana A. Marques: Software, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Patrícia Diogo: Data curation, Writing – review & editing. Ana Messias: Software, Formal analysis, Visualization. Vitor Sousa: Resources, Visualization. Diana Sequeira: Methodology, Formal analysis, Investigation, Writing – review & editing. Paulo J. Palma: Methodology, Investigation, Writing – review & editing, Visualization.

ACKNOWLEDGMENTS ~o Maria Nobre, The authors thank Dr Joa Prof Ramiro Mascarenhas, and Mr Paulo cnica Nacional, Dias from the Estaç~ao Zoote ria e Instituto Nacional de Investigaç~ao Agra ria, Santare m, for the zealous care, Veterina monitoring, and housing of the animals as well as for ensuring all the adequate conditions to perform the experimental procedures. The authors also thank the Hard Tissues Histology Laboratory, Faculty of Medicine, University of Coimbra, for assisting with the histological processing (Ms. Claudia Brites).

ne/ This work was supported by Colte Whaledent AG (grant no. PEP:PS0639). The funding source had no involvement in the conduct of the research and/or preparation of the article. The authors deny any conﬂicts of interest related to this study.

SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found in the online version at www.jendodon.com (https://doi.org/10.1016/ j.joen.2021.06.018).

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Parirokh M, Torabinejad M, Dummer PM. Mineral trioxide aggregate and other bioactive endodontic cements: an updated overview - part I: vital pulp therapy. Int Endod J 2018;51:177– 205.

Santos JM, Pereira JF, Marques A, et al. Vital pulp therapy in permanent mature posterior teeth with symptomatic irreversible pulpitis: a systematic review of treatment outcomes. Medicina (Kaunas) 2021;57:573.

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Schwendicke F, Brouwer F, Schwendicke A, Paris S. Different materials for direct pulp capping: systematic review and meta-analysis and trial sequential analysis. Clin Oral Investig 2016;20:1121–32.

Cohenca N, Paranjpe A, Berg J. Vital pulp therapy. Dent Clin North Am 2013;57:59–73.

Aguilar P, Linsuwanont P. Vital pulp therapy in vital permanent teeth with cariously exposed pulp: a systematic review. J Endod 2011;37:581–7.

Taha NA, Khazali MA. Partial pulpotomy in mature permanent teeth with clinical signs indicative of irreversible pulpitis: a randomized clinical trial. J Endod 2017;43:1417–21.

Asgary S, Eghbal MJ, Bagheban AA. Long-term outcomes of pulpotomy in permanent teeth with irreversible pulpitis: a multi-center randomized controlled trial. Am J Dent 2017;30:151–5.

Cushley S, Duncan HF, Lappin MJ, et al. Pulpotomy for mature carious teeth with symptoms of irreversible pulpitis: a systematic review. J Dent 2019;88:103158.

Lin LM, Ricucci D, Saoud TM, et al. Vital pulp therapy of mature permanent teeth with irreversible pulpitis from the perspective of pulp biology. Aust Endod J 2020;46:154–66.

10.

Ricucci D, Loghin S, Siqueira JF Jr. Correlation between clinical and histologic pulp diagnoses. J Endod 2014;40:1932–9.

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Ozdemir Y, Kutukculer N, Topaloglu-Ak A, et al. Comparative evaluation of pro-inﬂammatory cytokine levels in pulpotomized primary molars. J Oral Sci 2015;57:145–50.

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Simon S, Perard M, Zanini M, et al. Should pulp chamber pulpotomy be seen as a permanent treatment? Some preliminary thoughts. Int Endod J 2013;46:79–87.

13.

Goldberg M, Njeh A, Uzunoglu E. Is pulp inﬂammation a prerequisite for pulp healing and regeneration? Mediators Inﬂamm 2015;2015:347649.

14.

Giraud T, Jeanneau C, Rombouts C, et al. Pulp capping materials modulate the balance between inﬂammation and regeneration. Dent Mater 2019;35:24–35.

15.

Bakhtiar H, Nekoofar MH, Aminishakib P, et al. Human pulp responses to partial pulpotomy treatment with TheraCal as compared with Biodentine and ProRoot MTA: a clinical trial. J Endod 2017;43:1786–91.

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Tziafas D. Characterization of odontoblast-like cell phenotype and reparative dentin formation in vivo: a comprehensive literature review. J Endod 2019;45:241–9.

17.

Kilkenny C, Browne W, Cuthill IC, et al. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 2010;160:1577–9.

18.

Santos JM, Palma PJ, Ramos JC, et al. Periapical inﬂammation subsequent to coronal inoculation of dog teeth root ﬁlled with resilon/epiphany in 1 or 2 treatment sessions with chlorhexidine medication. J Endod 2014;40:837–41.

19.

Nowicka A, Lipski M, Paraﬁniuk M, et al. Response of human dental pulp capped with biodentine and mineral trioxide aggregate. J Endod 2013;39:743–7.

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De Rossi A, Silva LA, Gaton-Hernandez P, et al. Comparison of pulpal responses to pulpotomy and pulp capping with biodentine and mineral trioxide aggregate in dogs. J Endod 2014;40:1362–9.

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Asgary S, Eghbal MJ, Fazlyab M, et al. Five-year results of vital pulp therapy in permanent molars with irreversible pulpitis: a non-inferiority multicenter randomized clinical trial. Clin Oral Investig 2015;19:335–41.

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Uesrichai N, Nirunsittirat A, Chuveera P, et al. Partial pulpotomy with two bioactive cements in permanent teeth of 6- to 18-year-old patients with signs and symptoms indicative of irreversible pulpitis: a noninferiority randomized controlled trial. Int Endod J 2019;52:749–59.

23.

Matsuo T, Nakanishi T, Shimizu H, Ebisu S. A clinical study of direct pulp capping applied to carious-exposed pulps. J Endod 1996;22:551–6.

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Taha NA, Abdelkhader SZ. Outcome of full pulpotomy using Biodentine in adult patients with symptoms indicative of irreversible pulpitis. Int Endod J 2018;51:819–28.

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Taylor PE, Byers MR. An immunocytochemical study of the morphological reaction of nerves containing calcitonin gene-related peptide to microabscess formation and healing in rat molars. Arch Oral Biol 1990;35:629–38.

26.

Ricucci D, Siqueira JF, Li YY, Tay FR. Vital pulp therapy: histopathology and histobacteriologybased guidelines to treat teeth with deep caries and pulp exposure. J Dent 2019;86:41–52.

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27.

Iwamoto CE, Adachi E, Pameijer CH, et al. Clinical and histological evaluation of white ProRoot MTA in direct pulp capping. Am J Dent 2006;19:85–90.

28.

Zanini M, Hennequin M, Cousson PY. A review of criteria for the evaluation of pulpotomy outcomes in mature permanent teeth. J Endod 2016;42:1167–74.

29.

Ricucci D, Grande NM, Plotino G, Tay FR. Histologic response of human pulp and periapical tissues to tricalcium silicate-based materials: a series of successfully treated cases. J Endod 2020;46:307–17.

30.

Woodmansey KF, Kohout GD, Primus CM, et al. Histologic assessment of quick-set and mineral trioxide aggregate pulpotomies in a canine model. J Endod 2015;41:1626–30.

31.

Dominguez MS, Witherspoon DE, Gutmann JL, Opperman LA. Histological and scanning electron microscopy assessment of various vital pulp-therapy materials. J Endod 2003;29:324– 33.

32.

Fayyad DM, Abdelsalam N, Hashem N. Cryotherapy: a new paradigm of treatment in endodontics. J Endod 2020;46:936–42.

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BASIC RESEARCH – BIOLOGY Le Fournis, PhD,* Chloe Charlotte Jeanneau, PhD,* Thomas Giraud, PhD,*†

Fibroblasts Control Macrophage Differentiation during Pulp Inﬂammation

Ikhlas El Karim, PhD,‡ Fionnuala T. Lundy, PhD,‡ and Imad About, PhD*

ABSTRACT Introduction: During pulp inflammation, recruited macrophages can differentiate into 2 phenotypes: proinflammatory M1 and anti-inflammatory M2. Pulp fibroblasts have previously been shown to regulate pulp inflammation via cytokine and growth factor secretion. We hypothesized that upon carious injury, pulp fibroblasts interact with macrophages and modulate their differentiation. Methods: Cultures of pulp fibroblasts were physically injured and incubated with lipoteichoic acid (LTA) to mimic the pulp environment underlying a carious lesion. Physical injuries without LTA were performed on cultured fibroblasts to simulate the surrounding pulp tissue. Fibroblast supernatants were collected and added to undifferentiated macrophages to study their differentiation into M1 or M2 phenotypes by investigating cytokine secretion profiles and phagocytosis capacity. Histologic staining and immunofluorescence were performed on healthy and carious human tooth sections to localize the 2 macrophage phenotypes. Results: LTA-stimulated fibroblasts induced macrophage differentiation into the M1 phenotype with a significant increase both in tumor necrosis factor alpha secretion and phagocytosis capacity. By contrast, injured fibroblasts without LTA led to M2 differentiation with a significant increase in interleukin 10 secretion and low phagocytosis capacity. In carious teeth, M1 macrophages were detected mainly in the pulp zone underlying caries, whereas M2 macrophages were detected in the peripheral inflammatory zone. Conclusions: Fibroblasts induced macrophage differentiation to proinflammatory M1 with high bacteria phagocytosis capacity to control infection at the carious front. Fibroblasts located at the periphery of the inflammatory zone induced macrophage differentiation to antiinflammatory M2. The fine balance between the 2 phenotypes may represent a prerequisite for initiating the healing process. (J Endod 2021;47:1427–1434.)

SIGNIFICANCE Depending on the stimulation type, pulp fibroblasts induce macrophage differentiation into proinflammatory M1 or antiinflammatory M2 occupying distinct locations within the pulp inflammation zone. This differentiation is essential for bacteria elimination and pulp protection during the carious process.

KEY WORDS Caries; macrophage differentiation; pulp biology; pulp ﬁbroblast; pulp inﬂammation control

When cariogenic bacteria infiltrate the dentin-pulp tissue during the carious process, resident cells such as odontoblasts, fibroblasts, macrophages, mast cells, and dendritic cells produce signals to initiate inflammation1–3. White blood cells such as neutrophils and monocytes are recruited to the infected pulp zone, and this immune cell migration is guided by chemokines such as interleukin (IL)-8 secreted by odontoblasts or Complement C5a by fibroblasts4,5. Circulating monocytes migrate toward the infected pulp tissue and are activated/differentiated into macrophages. Macrophages represent the major pulp immunocompetent cells in fighting cariogenic bacteria6,7. Depending on their environment, macrophages can adopt 2 phenotypes: M1 and M2. M1 differentiation occurs in response to intracellular pathogens, bacterial lipopolysaccharides (LPSs), and cytokines such as interferon gamma (IFN-g) and tumor necrosis factor alpha (TNF-a) secreted by Th1/natural killer cells8. M1 macrophages mediate the host defense against pathogens secreting proinflammatory cytokines (IL1b, TNF-a, and IL-6), reactive oxygen species, nitric oxide, and phagocyte foreign materials9. By contrast, M2 macrophages are activated by cytokines secreted by Th2/mast cells, such as IL-4, IL-13, IL-10, or transforming growth factor beta (TGF-b)8. M2 macrophages contribute to tissue repair by producing antiinflammatory cytokines such as IL-10, TGF-b, and vascular endothelial growth factor10. Thus, M1 and M2 macrophages have the potential to contribute to pulpal inflammation and healing processes. Previous studies reported that TNF-a, IL-1b, CXC chemokine ligand 10, and macrophage inflammatory protein 3a cytokines were detected in inflamed pulp, suggesting a predominance of M1 macrophages in response

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From the *Aix Marseille University, CNRS, Institute of Movement Sciences, Marseille, ^pital Timone, Service France; †APHM, Ho d’Odontologie, Marseille, France; and ‡ Wellcome-Wolfson for Experimental Medicine, Queen’s University Belfast, Belfast, United Kingdom Address requests for reprints to Prof Imad About, Institut des Sciences du Mouvement, UMR 7287 CNRS, Universite d’Aix-Marseille, Faculte d’Odontologie, 27 BD Jean Moulin, 13385 Marseille Cedex 5, France. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.015

Fibroblasts Control Macrophage Differentiation

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to caries progression11–13. M2 macrophages have been associated with dentin bridge formation; indeed, TGF-b secreted by M2 macrophages increase the alkaline phosphatase activity of dental pulp cells involved in new dentin synthesis after pulp exposure14. Similarly, a transient M2 accumulation is correlated with a thin layer of dentin formation in contact with mineral trioxide aggregate after pulpotomy15. Recent work showed colocalization of M2 macrophages with Schwann cells in healthy dental pulp, under caries, and during the wound healing/mineralized bridge formation after pulp capping with mineral trioxide aggregate16. Therefore, M1 macrophages regulate the inflammatory reaction in the dental pulp, whereas M2 macrophages appear to be involved in wound healing. M1 and M2 polarization is governed mainly by immune cell secretion, but recently nonimmune cells such as cardiac fibroblasts were shown to play a significant role in modulating macrophage polarization17. Indeed, LPS-stimulated cardiac fibroblasts promoted the macrophage M1 phenotype, whereas TGF-b1 stimulation of these cells induced the M2 phenotype17. This macrophage polarization is essential for cardiac remodeling after injury, evolving from an initial accumulation of proinflammatory M1 macrophages to a greater balance of antiinflammatory M2 macrophages. Dental pulp fibroblasts have unique properties, and recent data have shown that these cells play important functions in controlling pulp vascularization, infection, and regeneration18– 23 . Thus, although cardiac fibroblast stimulation affects macrophage polarization and the subsequent inflammation/healing process, the effect of fibroblasts on macrophage differentiation has never been investigated in pulp tissue. This work aimed to study the role of pulp fibroblasts on macrophage differentiation during the carious process. We investigated the location of M1 and M2 macrophages in healthy and carious tooth sections and hypothesized that during the carious process pulp fibroblasts could be subjected to 2 types of stimulation depending on their location. Those located directly beneath the carious site could be exposed to cariogenic bacteria or macromolecules such as lipoteichoic acid (LTA), whereas fibroblasts located at the periphery of the inflammatory zone may be modulated by the inflammatory process without any contact with bacteria or their components. Cells in this zone receive signals from the surrounding environment because of the inflammatory reaction including damageassociated molecular pattern molecules

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(DAMPs) and immunostimulatory molecules of tissue repair24,25. To simulate the fibroblast environment beneath the carious site, fibroblasts were physically injured and incubated with LTA while the cell environment at the periphery of the inflammatory zone was simulated by physical injury to fibroblasts (without LTA). Fibroblast supernatants were then collected and incubated with undifferentiated macrophages. The subsequent macrophage differentiation was studied by investigating proinflammatory TNF-a and anti-inflammatory IL-10 cytokine secretion, measuring their bacterial phagocytic capacity and comparing them with differentiated M1 and M2 as reference phenotypes.

MATERIALS AND METHODS Reagents Cell culture reagents were purchased from Dominique Dutscher (Brumath, France), primary antibodies from R&D Systems (Lille, France) and Alexa Fluor secondary antibodies from Life Technologies (Saint-Aubin, France).

Teeth Immature human third molars freshly extracted for orthodontics reasons and carious teeth were obtained in compliance with French legislation (informed patient consent and institutional review board approval of the protocol used).

Bacterial Culture Streptococcus mutans (American Type Culture Collection 31383) was obtained from American Type Culture Collection (Manassas, VA) and cultured at 37 C under aerobic condition in brain-heart infusion broth (Conda/ Pronadisa, Madrid, Spain).

Pulp Fibroblast Cell Culture and Treatment Primary pulp cells were prepared from immature third molars by the explant outgrowth method. Pulp fibroblasts were isolated from pulp cell cultures and characterized as previously described18,26. These cells were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, L-glutamine 2 mmol/L, penicillin/streptomycin 50 mg/mL, and amphotericin B 0.25 mg/mL at 37 C in a 5% CO2 atmosphere. To simulate the zone under the carious lesion, confluent pulp fibroblasts were incubated with 1 mg/mL LTA (InvivoGen, San Diego, CA) for 4 hours. Then, physical injuries

were performed with sterile scalpels in vertical and horizontal directions (10 in each direction in 6-well plates) in serum-free MEM. To simulate the cell environment at a distance from the caries front, confluent fibroblasts were physically injured and incubated in serum-free MEM. The fibroblast supernatants were harvested after 24 hours and used for subsequent macrophage differentiation experiments as “LTA 1 injury” or “injury.” Fibroblast cultures without physical injury or LTA were used as controls and referred to as “untreated.”

THP-1 Cell Culture THP-1 cells, a human monocytic cell line (Sigma-Aldrich, St Louis, MO), were cultured in Roswell Park Memorial Institute (RPMI) medium as described previously27.

Macrophage Differentiation Undifferentiated M0 macrophages were obtained by incubating THP-1 cells with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, InvivoGen) for 48 hours. M1 macrophages were obtained by incubating M0 in serum-free RPMI with a combination of PMA (100 ng/mL) and LPS (100 ng/mL, R&D Systems)/IFN-g (20 ng/mL, R&D Systems) for 24 hours. M2 macrophages were obtained by incubating M0 in serum-free RPMI with a combination of PMA (100 ng/mL) and IL-4 (20 ng/mL, R&D Systems) for 48 hours. This differentiation protocol was established based on recommendations by Murray et al28. To study fibroblast impact on macrophage differentiation, M0 macrophages were incubated for 48 hours with stimulated fibroblast supernatants containing PMA (100 ng/mL). M0 macrophages were incubated with injured fibroblast supernatant “injury” or LTA and injured fibroblast supernatant “LTA 1 injury.” The macrophage differentiation protocol is summarized in Figure 1.

Cytokine Secretion by Macrophages After M0 macrophage incubation with ﬁbroblast supernatants, cells were washed 3 times with phosphate-buffered saline. Serumfree RPMI (2 mL in 6-well plate) was added to macrophages for 24 hours. TNF-a and IL-10 were quantiﬁed in macrophage supernatants by enzyme-linked immunosorbent assay in 96well plates (Nunc Maxisorp, Dominique Dutscher) using the Duoset human TNF-a or IL-10 kits (R&D Systems) according to the manufacturer’s instructions. All experiments were performed in triplicate.

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72h

48h

THP1 +PMA

M0 M

THP1 +PMA

+LPS+IFN-γ

THP1 +PMA THP1 +PMA

secreted a higher level of IL-10 than M0. When M0 macrophages were incubated with untreated fibroblast supernatants, no change in TNF-a or IL-10 levels was observed. After incubation of M0 with injured fibroblast supernatants, the TNF-a secretion level was lower than that of M0, whereas the level of IL10 was comparable with that of M2. Incubation of M0 with injured and LTA-stimulated fibroblast supernatants led to a significant increase in TNF-a levels equivalent to M1, whereas IL-10 levels were significantly lower than M2 (Fig. 2A and B).

96h

M1 M M2 M

+ IL-4 + Fibroblast supernatant

M1/M2 Diﬀeren a on?

Pulp Fibroblasts untreated / injury / LTA + injury

FIGURE 1 – The macrophage differentiation protocol. Undifferentiated M0 macrophages were obtained by incubating THP-1 monocytes with PMA (100 ng/mL). M0 macrophages were incubated with PMA (100 ng/mL)/LPS (100 ng/mL)/ IFN-g (20 ng/mL) for 24 hours to obtain M1 or PMA (100 ng/mL)/IL-4 (20 ng/mL) for 48 hours to obtain M2 differentiation. The impact of pulp fibroblasts on macrophage differentiation was studied by incubating M0 macrophages with a combination of PMA (100 ng/mL) and fibroblast supernatants after different stimulation conditions for 48 hours. To quantify cytokine secretion by macrophages, the fibroblast supernatants incubated with M0 macrophages were discarded. Macrophages were washed 3 times with phosphate-buffered saline, and then they were incubated with a fresh serum-free medium for cytokine quantification.

Quantiﬁcation of Cariogenic Bacteria Phagocytosis by Macrophages Bacterial ingestion by macrophages was measured using a gentamicin protection assay29. S. mutans suspension (107 bacteria/ well) in serum-free RPMI was added to macrophages differentiated under the previously described conditions for 2 hours. Extracellular bacteria were killed with 200 mg/ mL gentamicin for 1 hour at 37 C, and intracellular bacteria were released by cell lysis using 0.1% Triton X-100 (Sigma-Aldrich) for 5 minutes at room temperature. Intracellular bacteria were then diluted, and 100 mL was spread on the agar plate (brain-heart infusion agar, Conda/Pronadisa) in triplicate. After incubation for 24 hours at 37 C, colonyforming units (CFUs) were counted, and the average was calculated. Results were expressed in CFU/mL using the following equation: number of counted colonies CFU=ml 5 volume of spread suspension !Dilution factor Negative controls were performed by incubating macrophages in each condition with Cytochalasin D (Sigma-Aldrich) at a nontoxic concentration (10 mmol/mL).

Histology and Immunoﬂuorescence Carious and intact teeth were ﬁxed and routinely processed to obtain tooth sections as described previously30. After rehydration,

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nonspeciﬁc binding sites were blocked with phosphate-buffered saline/3% bovine serum albumin for 1 hour at room temperature. Sections were then incubated overnight at 4 C with mouse immunoglobulin (Ig) G antihuman CD86 (5 mg/mL) or mouse IgG antihuman CD206 (5 mg/mL) followed by secondary antibody Alexa Fluor 488 goat antimouse IgG (2 mg/mL) or Alexa Fluor 594 donkey antimouse IgG (2 mg/mL), respectively, for 45 minutes. Isotype controls were performed with control IgG followed by the secondary antibodies. Nuclei were counterstained with 1 mg/mL 40 ,6-diamidino-2-phenylindole (DAPI) for 45 minutes. Some sections were stained with hematoxylin-eosin (H&E, Sigma-Aldrich) according to the manufacturer’s instructions.

Statistical Analysis All experiments were repeated at least 3 times, and statistical signiﬁcance (P , .05) was determined after checking normal distribution using the Student t test to compare the different incubation conditions. Data are expressed as means 6 standard deviation.

RESULTS Fibroblasts Affect TNF-a and IL-10 Secretion by Macrophages Undifferentiated M0 macrophages secreted a moderate level of TNF-a but did not secrete IL10. M1 macrophages secreted a higher TNF-a level than M0 but did not secrete the antiinﬂammatory IL-10 (Fig. 2A and B). M2 macrophages did not secrete TNF-a but

Pulp Fibroblasts Affect Macrophage Phagocytosis Capacity Undifferentiated (M0) as well as differentiated M1 and M2 macrophages phagocytosed S. mutans (Fig. 2C). However, phagocytosis by M1 and M2 was significantly higher than that of M0. Moreover, M1 phagocytosis capacity was 40 times higher than M2. When M0 macrophages were incubated with untreated fibroblast supernatants, their phagocytic capacity did not change. In contrast, M0 incubated with injured fibroblast supernatants had comparable phagocytosis capacity with M2 with no statistically significant difference (Fig. 2C). Moreover, when M0 macrophages were incubated with injured and LTA-induced fibroblast supernatants, their phagocytosis capacity was comparable with M1. In the presence of cytochalasin D, an inhibitor of F-actin–dependent phagocytosis, a drastic decrease of CFUs was observed, indicating that the counted bacteria were engulfed by phagocytosis and present intracellularly (Fig. 2C).

M1 Macrophages Are Mainly Located at the Central Inflammatory Area, Whereas M2 Macrophages Are Mostly Located in the Underlying Tissue at the Periphery of the Inflammatory Area H&E staining (Fig. 3A and B) on healthy tooth sections showed a well-organized pulp tissue. No expression of CD86 or CD206 was observed by immunofluorescence (Fig. 3C and D), indicating that M1 and M2 macrophages were not detected. H&E-stained carious tooth sections showed partial dentin destruction (black arrow, Fig. 3F) by cariogenic bacteria and pulp inflammation (limited by red dotted line, Fig. 3K). The expression of CD86 (red) and CD206 (green) was investigated in 2 areas: the center (Fig. 3G, magenta frame) and the periphery of the inflammation zone (Fig. 3L, green frame). The majority of M1 macrophages expressing CD86 were observed within the

Fibroblasts Control Macrophage Differentiation

1429

2500

TNF-α (pg/mL)

inﬂammatory area (Fig. 3H and M), whereas M2 macrophages expressing CD206 were mainly detected at the periphery of the inﬂammatory zone (Fig. 3I and N). No labeling was observed with the control isotypes (Fig. 3E, J, and O).

3000

2000 1500 1000

# *

500 0

1 M0

M2 3

2 M1

4 untreated

DISCUSSION

5 injury

LTA+injury 6

Fibroblast supernatant

350

IL-10 (pg/mL)

300

250 200 150

100 50 0

* $

$ 1

2 M1

3 M2

4 untreated

5 injury

6 LTA+injury

Fibroblast supernatant

C S.mutans phagocytosis by macrophages (CFU/mL)

1.E+06

Cytochalasin D

1.E+05

1.E+03

* #

1.E+04

1.E+02 1.E+01

& &

1.E+00 1 M0

2 M1

3 M2

4 untreated

5 injury

6 LTA+injury

Fibroblast supernatant FIGURE 2 – The effect of fibroblasts on macrophage cytokine secretion and phagocytosis capacity. (A ) Proinflammatory TNF-a cytokine. Undifferentiated M0 macrophages and M0 macrophages incubated with untreated fibroblast supernatants secrete moderate levels of TNF-a. M1 macrophages and macrophages incubated with injured and LTAinduced fibroblast supernatants secrete a high level of TNF-a. A low level of TNF-a is observed with injured fibroblast supernatants, whereas no TNF-a was secreted by M2 macrophages. (B ) Anti-inflammatory IL-10 cytokine. M2 macrophages and M0 macrophages incubated with injured fibroblast supernatants secrete a high level of IL-10 levels with no significant difference between the 2 conditions. Undifferentiated M0 macrophages, M0 macrophages incubated with untreated fibroblast supernatants, and M1 macrophages do not secrete IL-10. M0 macrophages incubated with injured and LTA-induced fibroblast secrete a low level of IL-10. (C ) Macrophage phagocytosis capacity. Phagocytosis of S. mutans by M1 macrophages and M0 macrophages incubated with injured and LTA-induced fibroblast supernatants showed the highest phagocytosis capacity compared with the M0 control. Phagocytosis of S. mutans by M2 macrophages and M0 macrophages incubated with injured fibroblast supernatants was high but 40 times less than M1 macrophages. When M0 macrophages were incubated with untreated fibroblast supernatants, their phagocytic capacity did not change. A very low number of viable intracellular bacteria was observed when macrophages were treated with cytochalasin D, indicating a drastic inhibition of phagocytosis. The results are represented in a logarithmic scale. *A statistical difference when a condition is compared with M0 (P , .05). #A statistical difference when a condition is compared with M1 (P , .05). $A statistical difference when a condition is compared with M2 (P , .05). &A statistical difference when a condition is compared with cytochalasin D treatment (P , .05). Bars represent mean values 6 standard deviation.

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The main outcome of this study is that, depending on their stimulation conditions, pulp fibroblasts induce macrophage differentiation into proinflammatory M1 or anti-inflammatory M2 macrophages that occupy distinct locations within the inflammation zone. This macrophage differentiation is essential for cariogenic bacteria elimination as well as for pulp tissue protection from the extension of the inflammation during the carious process. For this study, undifferentiated (M0) and differentiated (M1 and M2) macrophages were used as reference phenotypes to investigate the effect of fibroblast stimulation on macrophage cytokine secretion and phagocytic activity; M0 macrophages were incubated with differentiation media to obtain M1 and M2 populations. The characterization of M1 and M2 in our study was based on surface markers, cytokine secretion, and phagocytic capacity. Although M1 and M2 express several surface markers, M1 macrophages have been shown to strongly express CD86 and secrete a high level of TNFa and IL-12, whereas M2 macrophages have been shown to strongly express CD206 and secrete a high level of IL-10 and TGF-b131. Although a wide range of surface markers and cytokines can be used, we selected CD86/ TNF-a to identify M1 and IL-10/CD206 to identify M2 because these are the most widely used in the characterization of the 2 phenotypes31–33. Thus, studying TNF-a and IL-10 secretion in our work allows us to associate CD86 expression with M1 differentiation and CD206 expression with M2 differentiation. In agreement with previously published data8, our results show that M1 macrophages secrete high TNF-a levels and have high phagocytosis capacity, whereas M2 macrophages secrete high IL-10 levels and have moderate phagocytosis capacity. These profiles are clearly different from undifferentiated macrophages that secrete low TNF-a levels and no IL-10 and have limited phagocytosis capacity. In this work, phagocytosis was investigated using S. mutans as a well-recognized representative of cariogenic bacteria. Based on our earlier investigations demonstrating that macrophage phagocytosis varies in function of the bacterial species20, S. mutans was selected as an

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FIGURE 3 – M1 and M2 macrophage localization in human tooth sections. (A–E ) Sound and (F–O ) carious human tooth sections were stained with (A, B, F, G, K, and L ) H&E. Immunofluorescence did not show any (C ) CD86 or (D ) CD206 and highlighted the absence of M1 and M2 macrophage in sound teeth. In carious tooth sections stained with H&E, (F ) the black arrow indicates the carious lesion progression in the dentin and (K ) the red dots limit the resulting inflammatory reaction zone. (G–J ) The magenta frame shows the central zone of the inflammatory area, and (L–O ) the green frame shows the inflammatory reaction peripheral zone. Immunostaining of CD86 (in red ) indicates an intense labeling in the central inflammatory zone highlighting the presence of (H ) numerous M1 macrophages and (M ) much less in the peripheral zone. Immunostaining of CD206 (in green ) shows an intense labeling at the peripheral zone indicating (N ) M2 localization more in the peripheral than in (I ) the central area of the inflammatory reaction zone. (E, J, and O ) Isotypes controls were negative. Nuclei were counterstained with DAPI (blue ). Scale bars: A and F 5 500 mm; K 5 200 mm; and B, C, D, E, G, H, I, J, L, M, N, and O 5 50 mm

example to study the effect of fibroblast treatment conditions on the differentiated macrophage phagocytic capacity of the same strain. Interestingly, distinct cytokine secretion profiles were obtained depending on the type of fibroblast stimulation. When M0 macrophages were incubated with injured fibroblast supernatants (without LTA), they showed a high IL-10 secretion level and a moderate phagocytosis capacity. This profile is very similar to M2 macrophages. However, when they were incubated with supernatants of injured and LTA-stimulated fibroblasts, M0 macrophages secreted a high level of TNF-a and a low level of IL-10 and had high phagocytic capacity. This profile is very similar to M1 macrophages. However, it should be noted that when M0 macrophages were incubated with injured fibroblast supernatants, they secreted a low level of TNF-a, whereas after incubation with injured and LTA-induced fibroblast supernatants, they secreted a low level of IL-10. This secretion level was very low compared with M1 and M2 reference macrophages. This indicates that we do not obtain a hom*ogenous differentiation of cells into M1 or M2 but rather a balance of the 2 cell

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types with a predominant phenotype. These data demonstrate that injured and LTAstimulated fibroblasts, simulating both the presence of bacteria and cell damage, induce differentiation into M1, whereas the surrounding fibroblasts without a direct contact with bacteria induce macrophage differentiation into M2. The reason why fibroblasts enhance M2 polarization after injury without exposure to LTA is still unclear. Injured pulp cells have been shown to release DAMPs. DAMPs are not only danger signals but also central players in tissue inflammation and repair. Indeed, after sterile injury to pulp cells, recent data have demonstrated that DAMPs induced an NLRP3 inflammasome–dependent sterile inflammatory response that enhanced dental pulp cell migration, proliferation, and odontogenic differentiation25. It could be hypothesized that upon cariogenic bacteria invasion, 2 zones can be distinguished. The first zone that can be directly exposed to cariogenic bacteria is located beneath the carious dentin. It is simulated in vitro by injuring the cells physically and incubating them with LTA. The second zone is located at the periphery of this zone. This zone receives DAMPs secreted from injured/damaged cells

without being subjected to bacteria directly. The absence of direct contact between fibroblasts and bacteria in this condition is favorable to M2 differentiation. The pulp fibroblast-released cytokines can mediate this differentiation as demonstrated in cardiac tissue. Indeed, upon LPS stimulation, the release of TNF-a and monocyte chemoattractant protein 1 by cardiac fibroblasts allow monocyte differentiation into M1, whereas IL-10 and IL-5 secretion by the same cells allow M2 differentiation17. Our data clearly demonstrate that pulp fibroblasts interact with macrophages and actively influence their differentiation and that this is highly dependent on the exposure of pulp fibroblasts to injury or bacterial infection. Thus, macrophage differentiation seems to be rather reversible and dynamic and can be modulated by the environment as demonstrated in cocultures of macrophages with Schwann cells, which induced their differentiation into M216. During the inflammatory process, it has long been believed that macrophages are strictly of the M1 phenotype, whereas during the healing process they are strictly of the M2 phenotype. A previous study demonstrated

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Carious lesion

Den n

Central zone: M1 diﬀeren a on • Bacteria phagocytosis •

Inﬂamma on zone

TNF-α secre on

Periphery: M2 diﬀeren a on • IL-10 secre on

Cariogenic bacteria M1 Macrophage M2 Macrophage Fibroblast

Pulp

FIGURE 4 – A schematic representation of differentiated macrophage localization in the inflammatory zone under caries. During the carious process, cariogenic bacteria (in orange ) invade the dental pulp through the dentin tubules. Pulp fibroblasts close to the carious front are in contact with both damaged cell signals released by odontoblasts and pulp cells and bacteria. To reflect this situation in vitro, pulp fibroblasts were stimulated by LTA and injured. The macrophages recruited to this area are exposed to fibroblast secretion and differentiate into M1 macrophages involved in cariogenic bacteria phagocytosis, as demonstrated in the gentamycin assay, and proinflammatory TNF-a secretion. More distant fibroblasts, which are not subjected to bacterial invasion directly, differentiate into M2 macrophages involved in IL-10 anti-inflammatory cytokine secretion, limiting the immune response and preventing the underlying tissue damage. M1 macrophages allow the control and elimination of bacteria, whereas M2 macrophages at the periphery of the inflammatory zone dampen the inflammation reaction and limit pulp necrosis. the presence of M1 in the inflammation zone of teeth with deep caries, whereas M2 macrophages were detected in teeth with superficial caries, under the carious dentin, as well as after pulp capping with mineral trioxide aggregate along the nerve fibers in the wounded tissue16. A novel aspect of our study is the demonstration that both M1 and M2 are present in the inflammation area of carious teeth; M1 cells were predominant in the center of the inflammatory zone. They occupy the area beneath the invading cariogenic bacteria and represent the first macrophage defense line in the pulp, whereas M2 mainly occupied the peripheral zone. These cells have an anti-inflammatory activity, which limits tissue damage during the inflammation. However, it should be noted

that a mixed M1/M2 population is present in both zones. The presence of both phenotypes in central and peripheral zones is important to regulate the balance of pulp inflammation and repair. The phenotype plasticity is essential during the inflammation process to avoid significant cell damage and chronic inflammation34,35. Our results reporting the presence of a mixed M1/M2 population are in line with these data. Overall, taking into consideration both macrophage differentiation in vitro and their localization in vivo allows us to advance the following hypothesis on fibroblast interaction with macrophages: during the carious process, the pulp fibroblasts subjected to cariogenic bacteria and sensing cell damage signals stimulate M1 macrophage

differentiation. These cells have a high phagocytic capacity and lead to bacterial elimination by phagocytosis. The surrounding fibroblasts that are not in direct contact with bacteria induce M2 differentiation with antiinflammatory activity, limiting pulp tissue damage (Fig. 4). It can be assumed that this M1/M2 balance allows pathogen elimination and provides a control of pulpal inflammation that is required for initiating the healing process.

ACKNOWLEDGMENTS Supported by Aix-Marseille University and CNRS. The authors deny any conﬂicts of interest related to this study.

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Farges JC, Alliot-Licht B, Renard E, et al. Dental pulp defence and repair mechanisms in dental caries. Mediators Inﬂamm 2015;2015:1–16.

Giraud T, Jeanneau C, Bergmann M, et al. Tricalcium silicate capping materials modulate pulp healing and inﬂammatory activity in vitro. J Endod 2018;44:1686–91.

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Yumoto H, Hirao K, Hosokawa Y, et al. The roles of odontoblasts in dental pulp innate immunity. Jpn Dent Sci Rev 2018;54:105–17.

Farges JC, Keller JF, Carrouel F, et al. Odontoblasts in the dental pulp immune response. J Exp Zool B Mol Dev Evol 2009;312B:425–36.

Giraud T, Rufas P, Chmilewsky F, et al. Complement activation by pulp capping materials plays a signiﬁcant role in both inﬂammatory and pulp stem cells’ recruitment. J Endod 2017;43:1104–10.

Nakakura-Ohshima K. Possible role of immunocompetent cells and the expression of heat shock protein-25 in the process of pulpal regeneration after tooth injury in rat molars. J Electron Microsc (Tokyo) 2003;52:581–91.

Zhang J. The existence of CD11c1 sentinel and F4/801 interstitial dendritic cells in dental pulp and their dynamics and functional properties. Int Immunol 2006;18:1375–84.

Martinez FO. Macrophage activation and polarization. Front Biosci 2008;13:453.

Mantovani A, Sica A, Sozzani S, et al. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 2004;25:677–86.

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Novak ML, Koh TJ. Macrophage phenotypes during tissue repair. J Leukoc Biol 2013;93:875– 81.

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Nakanishi T, Takahashi K, Hosokawa Y, et al. Expression of macrophage inﬂammatory protein 3alpha in human inﬂamed dental pulp tissue. J Endod 2005;31:84–7.

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Adachi T, Nakanishi T, Yumoto H, et al. Caries-related bacteria and cytokines induce CXCL10 in dental pulp. J Dent Res 2007;86:1217–22.

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Cooper PR, Takahashi Y, Graham LW, et al. Inﬂammation-regeneration interplay in the dentinepulp complex. J Dent 2010;38:687–97.

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Park HC, Quan H, Zhu T, et al. The effects of M1 and M2 macrophages on odontogenic differentiation of human dental pulp cells. J Endod 2017;43:596–601.

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Takei E, Shigetani Y, Yoshiba K, et al. Initial transient accumulation of M2 macrophage– associated molecule-expressing cells after pulpotomy with mineral trioxide aggregate in rat molars. J Endod 2014;40:1983–8.

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Yoshiba N, Edanami N, Ohkura N, et al. M2 phenotype macrophages colocalize with Schwann cells in human dental pulp. J Dent Res 2020;99:329–38.

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Humeres C, Vivar R, Boza P, et al. Cardiac fibroblast cytokine profiles induced by proinflammatory or profibrotic stimuli promote monocyte recruitment and modulate macrophage M1/M2 balance in vitro. J Mol Cell Cardiol 2016;101:69–80.

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Chmilewsky F, Jeanneau C, Laurent P, About I. Pulp ﬁbroblasts synthesize functional complement proteins involved in initiating dentin-pulp regeneration. Am J Pathol 2014;184:1991– 2000.

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Coil J, Tam E, Waterfield JD. Proinflammatory cytokine profiles in pulp fibroblasts stimulated with lipopolysaccharide and methyl mercaptan. J Endod 2004;30:88–91.

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Le Fournis C, Hadjichristou C, Jeanneau C, About I. Human pulp ﬁbroblast implication in phagocytosis via complement activation. J Endod 2019;45:584–90.

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Tran-Hung L, Mathieu S, About I. Role of human pulp ﬁbroblasts in angiogenesis. J Dent Res 2006;85:819–23.

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Chmilewsky F, About I, Chung SH. Pulp ﬁbroblasts control nerve regeneration through complement activation. J Dent Res 2016;95:913–22.

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Jeanneau C, Lundy FT, El Karim IA, About I. Potential therapeutic strategy of targeting pulp ﬁbroblasts in dentin-pulp regeneration. J Endod 2017;43:S17–24.

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Cooper PR, Smith AJ. Molecular mediators of pulp inﬂammation and regeneration. Endod Topics 2013;28:90–105.

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Al Natour B, Lundy FT, Moynah PN, et al. Odontoblast cell death induces NLRP3 inﬂammasomedependent sterile inﬂammation and regulates dental pulp cell migration, proliferation and differentiation. Int Endod J 2021;54:941–50.

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Rufas P, Jeanneau C, Rombouts C, et al. Complement C3a mobilizes dental pulp stem cells and speciﬁcally guides pulp ﬁbroblast recruitment. J Endod 2016;42:1377–84.

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Tsuchiya S, Yamabe M, Yamaguchi Y, et al. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980;26:171–6.

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Murray PJ, Allen JE, Biswas S, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 2014;41:14–20.

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Laroux FS, Romero X, Wetzler L, et al. Cutting edge: MyD88 controls phagocyte NADPH oxidase function and killing of gram-negative bacteria. J Immunol 2005;175:5596–600.

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cl Te es O, Laurent P, Zygouritsas S, et al. Activation of human dental pulp progenitor/stem cells in response to odontoblast injury. Arch Oral Biol 2005;50:103–8.

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Tan HY, Wang N, Man K, et al. Autophagy-induced RelB/p52 activation mediates tumourassociated macrophage repolarisation and suppression of hepatocellular carcinoma by natural compound baicalin. Cell Death Dis 2015;6:e1942.

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vez-Gala n L, Ocan ~a-Guzma n R, Torre-Bouscoulet L, et al. Exposure of monocytes to Cha lipoarabinomannan promotes their differentiation into functionally and phenotypically immature macrophages. J Immunol Res 2015;2015:984973.

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Neves VC, Yianni V, Sharpe PT. Macrophage modulation of dental pulp stem cell activity during tertiary dentinogenesis. Sci Rep 2020;10:20216.

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Porcheray F, Viaud S, Rimaniol AC, et al. Macrophage activation switching: an asset for the resolution of inﬂammation. Clin Exp Immunol 2005;142:481–9.

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Benoit M, Desnues B, Mege JL. Macrophage polarization in bacterial infections. J Immunol 2008;181:3733–9.

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BASIC RESEARCH – BIOLOGY

Engineered Chitosan-based Nanoparticles Modulate Macrophage–Periodontal Ligament Fibroblast Interactions in Biofilmmediated Inflammation ABSTRACT Introduction: Crosstalk between immune cells and tissue-resident cells regulates the pathophysiology and posttreatment healing of apical periodontitis. This investigation aimed to understand the influence of residual root canal biofilm on macrophage (MQ)–periodontal ligament fibroblast (PdLF) interaction and evaluate the effect of engineered chitosan-based nanoparticles (CSnp) on MQ-PdLF interactions in residual biofilm-mediated inflammation. Methods: Six-week-old Enterococcus faecalis biofilms in root canal models were disinfected conventionally using sodium hypochlorite alone or followed by calcium hydroxide medication or CSnp dispersed in carboxymethylated chitosan (CMCS). The effect of the treated biofilms (n 5 25/group) on the inflammatory response of THP-1–differentiated MQ monoculture versus coculture with PdLF was evaluated for cell viability, MQ morphometric characterization, inflammatory mediators (nitric oxide, tumor necrosis factor alpha, interleukin [IL]-1 beta, IL1RA, IL-6, transforming growth factor beta 1 [TGF-b1], and IL-10), and the expression of transcription factors (pSTAT1/pSTAT6)/cluster of differentiation markers (CD80/206) after 24, 48, and 72 hours of interaction. PdLF transwell migration was evaluated after 8 and 24 hours. Unstimulated cells served as the negative control, whereas untreated biofilm was the positive control. Results: Biofilm increased nitric oxide and IL-1b but suppressed IL-10, IL-1RA, and PdLF migration with significant cytotoxic effects. CSnp/CMCS reduced nitric oxide and IL-1b (P , .01) while maintaining 90% cell survival up to 72 hours with evident M2-like MQ phenotypic changes in coculture. CSnp/CMCS also increased the IL-1RA/IL-1b ratio and enhanced TGF-b1 production over time (P , .05, 72 hours). In coculture, CSnp/CMCS showed the highest IL-10 level at 72 hours (P , .01), reduced the pSTAT1/pSTAT6 ratio, and enhanced PdLF migration (P , .01, 24 hours). Conclusions: CSnp/CMCS medication facilitated MQ switch toward M2 (regulatory/anti-inflammatory) phenotype and PdLF migration via paracrine signaling. (J Endod 2021;47:1435–1444.)

KEY WORDS Biofilm; chitosan nanoparticles; immunomodulation; inflammation; macrophage polarization The persistence of endodontic biofilms results in chronic inflammation and periradicular tissue destruction1. A substantial part of tissue damage that characterizes apical periodontitis is attributed to the host response to infection. Posttreatment tissue healing involves the orchestration of complex cellular and molecular biological processes to reconstitute the original architecture and function of the injured tissue2. However, the prevalence of posttreatment apical periodontitis is reported in cross-sectional studies to be 25%–50%3, with no clear evidence to view it as an isolated event that has no influence on an individual’s overall health4. Crosstalk between tissue-specific multipotent cells and immune cells via inflammatory mediators regulates the pathophysiology of apical periodontitis5. Macrophages (MQs) are functionally diversified

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Hebatullah Hussein, BDS, MDS,*†‡ and Anil Kishen, BDS, MDS, PhD*†§

SIGNIFICANCE An antibiofilm strategy using engineered chitosan nanoparticles highlighted its potential to rescue proinflammatory macrophage and periodontal ligament fibroblast crosstalk by modulating a macrophage switch into an antiinflammatory phenotype while promoting periodontal ligament fibroblast migration.

From the *The Kishen Lab, Dental Research Institute and †Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; ‡Faculty of Dentistry, Endodontics Department, Ain Shams University, Cairo, Egypt; and §Department of Dentistry, Mount Sinai Health System, Mount Sinai Hospital, Toronto, Ontario, Canada Address requests for reprints to Dr Anil Kishen, Dental Research Institute, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario, Canada M5G 1G6. E-mail address: anil.kishen@dentistry. utoronto.ca 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.017

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immune cells that play critical roles in the regulation of homeostasis, inflammation, and healing in a tissue-specific and contextdependent manner. The exposure of MQs to different environmental cues results in their polarization into different functional phenotypes—M1 (proinflammatory) or M2 (regulatory/anti-inflammatory/healing)6. MQs produce several cytokines that are involved in the pathogenesis of apical periodontitis as well as periapical healing7. Microenvironmental cues that allow the timely transition of M1 to the M2 phenotype while balancing the proand anti-inflammatory mediators are critical for the resolution of inflammation8. Periodontal ligament fibroblasts (PdLFs) are multipotent cells displaying distinct functional characteristics with an integral role in the normal turnover, repair, and regeneration of periodontal tissue9, in addition to active participation in the immunologic events in periodontitis5. Therefore, the regulation of host reactions requires the coordination of both MQs and fibroblasts10. To date, no treatment has been applied to modulate the interaction of MQs and PdLFs in endodontic infection– mediated inflammation for a predictable cellular level healing. Immunomodulatory biomaterials in the form of nanoparticles and nanofibers provide a wide array of tools for modulation of the immune system for tissue regeneration and wound healing applications11. Engineered bioactive chitosan-based nanoparticles (CSnp) dispersed in a water-soluble derivative of chitosan, carboxymethyl chitosan (CMCS), have been shown to significantly reduce residual biofilm in a root canal model and proinflammatory mediators of MQs while maintaining their viability12. Furthermore, CSnp have been reported in earlier investigations to retain their effective antibiofilm activity for up to 90 days of aging13 and sustained antibacterial action even in the presence of tissue inhibitors such as dentin matrix and bacterial remnants14. In this study, we aimed to understand the influence of posttreatment residual biofilm on MQ-PdLF interactions and to evaluate the effect of engineered bioactive CSnp on modulating the MQ-PdLF crosstalk in residual biofilm-mediated inflammation.

syntheses and their characteristics were described earlier13,15–17.

Biofilm Formation in Root Canal Models Root canals (N 5 110) were prepared after decoronation using ProTaper Universal rotary instruments (Dentsply Tulsa Dental Specialties, Tulsa, OK) until size F3. Canals were irrigated with 3 mL 3% sodium hypochlorite (NaOCl) delivered at each change of file, 1 mL 17% EDTA for smear layer removal, and then saline as the final flush. Specimens were individually assembled and autoclaved at 121 C for 20 minutes. Root canals were inoculated with an overnight culture of Enterococcus faecalis (ATCC 29212; American Type Culture Collection, Manassas, VA) in brain-heart infusion broth (optical density600nm 5 1, colony-forming units 5 108), centrifuged (1400g, 5 minutes), and then incubated at 37 C for 6 weeks. The culture medium was replenished every 48 hours. The negative control specimens (n 5 10) were not inoculated. Untreated biofilms served as the positive control (n 5 25).

Disinfection Procedures The specimens (n 5 25/group) were divided as follows: 1. NaOCl/EDTA: irrigated with 3 mL 6% NaOCl delivered using a 27-G needle 1 mm short of the working length and neutralized with 3 mL 5% sodium thiosulfate, 2 mL 17% EDTA irrigation for 1 minute, and a final flush with sterile saline 2. Calcium hydroxide (Ca[OH]2): as in NaOCl/ EDTA followed by the application of calcium hydroxide paste (UltraCal XS; Ultradent Products Inc, South Jordan, UT) for 7 days 3. CSnp/CMCS: CSnp dispersed in 1% CMCS (1 mg/mL) were applied intracanal for 72 hours after NaOCl/EDTA Posttreatment residual biofilms were characterized via microbiological analysis of pulverized dentin12. Microbial cultures showed a residual bacterial load ranging from 3.9 6 0.62 logs (NaOCl/EDTA), 1.65 6 1.01 logs (Ca[OH]2), and 1.32 6 0.78 logs (CSnp/ CMCS) versus 7.27 6 0.71 logs in untreated biofilm.

MATERIALS AND METHODS The analytical grade chemicals (purity 95%) used in this study were purchased from Sigma-Aldrich (St Louis, MO) unless otherwise stated. Extracted anonymous human singlerooted, single-canal teeth were used (university ethics review board approval #35228). CSnp were synthesized using the ionic gelation method. The details of CSnp

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Cell Cultures Cells of the third to fifth passage were used in the experiments. Cells were incubated in a humidified incubator at 37 C, 5% CO2. THP-1 monocytes (ATCC TIB-202, American Type Culture Collection) grown in RPMI 1640 medium (ATCC modification; Gibco, Grand Island, NY) supplemented with

10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and 0.05 mmol/L 2mercaptoethanol were differentiated (2.5 ! 10^5 cells/mL) with 100 nmol/L phorbol 12myristate 13-acetate for 24 hours. Cell attachment, MQ-like morphology, and CD68 staining confirmed the differentiation. Cells were incubated for 16 hours to rest in phorbol 12-myristate 13-acetate–free culture media without antibiotics. PdLFs18 were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. PdLFs were harvested after 85% confluence with trypsin/EDTA 0.05% (Gibco, Waltham, MA) for 3 minutes at 37 C.

Interaction of Treated Biofilm and MQs/PdLFs Treated root canal models were used to assess the cell interaction with residual biofilm. The apical 5 mm of root specimens was resected and outer surface disinfected with 70% ethanol. Each specimen was incubated in 2 mL antibiotic-free media (DMEM 1 10% fetal bovine serum) at 37 C for 30 minutes. Media conditioned with treated specimens were centrifuged, diluted 2-fold, and added to monocultured MQ (2 ! 10^5/mL) and PdLFs (1 ! 10^5/mL). Coculture experiments were performed using 24-transwell plates; MQ (2 ! 10^5 cells/mL) was seeded at the bottom and PdLF (2 ! 10^4 cells/100 mL) cultured on the insert (6.5-mm diameter, 8-mm pore size polycarbonate membrane; Corning, Lowell, MA). Conditioned medium was introduced to the cells and assessed after 24, 48, and 72 hours of interaction. Unstimulated cells served as the negative control. The following parameters were assessed in triplicate to characterize posttreatment inflammatory response.

Cell Viability MQs and PdLFs were incubated for 20 minutes with 200 mL calcein-AM (Invitrogen/Molecular Probes) and then observed under a ﬂuorescent inverted microscope (Vert.A1; Carl Zeiss, Jena, Germany) at 10! magniﬁcation. Acquired images were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD). Cell survival was expressed as the percentage relative to the negative control (100% viable). PdLF viability in a transwell setup was quantitatively evaluated using trypan blue exclusion assay after PdLF trypsinization from the inserts to count the viable cells.

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MQ Morphometric Descriptors

Transwell Migration Assay

Calcein-AM–stained images of MQ at 72 hours were ﬁrst scaled into microns. Nine frames/ image were analyzed, and 3 randomly selected cells per frame were measured regarding the cell area, perimeter, circularity, and aspect ratio using ImageJ software.

After 8 and 24 hours in serum–free conditioned media, transwell inserts were washed twice in PBS, and PdLFs on the membrane topside were removed using cotton tips. PdLFs migrated to the membrane underside were ﬁxed in 10% neutral buffered formalin for 10 minutes, washed in PBS, and stained with 0.5% (w/v) crystal violet dye in methanol for 10 minutes and then washed in PBS. Five images/insert were captured with an inverted microscope to count the migrating PdLFs. To evaluate the inﬂuence of MQs on PdLF migration, the number of migrating cells when PdLFs were cultured alone were subtracted from those migrated in coculture.

Assessment of Inﬂammatory Mediators Cell culture supernatants of MQ monoculture and coculture with PdLFs were collected, centrifuged (10,000 ! g, 5 minutes), aliquoted, and frozen at 280 C until analysis. Nitric oxide was assessed using the Griess reagent system (Promega, Madison, WI). A multiplex immunoassay for simultaneous analysis of inﬂammatory cytokines (tumor necrosis factor alpha [TNF-a], interleukin [IL]1b, IL-1RA, IL-6, and IL-10) was performed using the HCYTOMAG-60K Milliplex MAP Human cytokine magnetic bead panel ((Millipore, Billerica, MA). The ratio of IL-1RA to IL-1b was calculated. Transforming growth factor beta 1 (TGF-b1) production was analyzed using the Human TGF-b1 assay kit (Quantikine ELISA, Human TGF-b1 Immunoassay; R&D, Minneapolis, MN).

Immunofluorescence Analysis MQs were immunocytochemically stained to assess the relative expression of the transcription factors pSTAT1 and pSTAT6 as well as the surface markers CD80 and CD206 along with CD68 after 24 and 72 hours. At room temperature, cells were washed twice with 1! phosphate-buffered saline (PBS) and fixed with 10% neutral buffered formalin for 30 minutes. After washing twice, cells were permeabilized with 0.1% Triton X-100 in 1! PBS for 5 minutes, washed, and then blocked with sea block serum free-PBS (Abcam Inc, Cambridge, MA) for 1 hour. Cells were incubated overnight at 4 C with Alexa Flour (AF)-conjugated antibodies (BioLegend, San Diego, CA) diluted in a blocking reagent (AF594 anti-CD68 diluted 1:150), in addition to 1 of these 2 combinations: AF488 antipSTAT1 and AF647 anti-pSTAT6 antibodies diluted 1:200 or AF647 anti-CD80 and AF488 anti-CD206 diluted 1:150. After 3 washes in PBS, cells were counterstained with 40 ,6diamidino-2-phenylindole and examined under a confocal laser scanning microscope (Zeiss LSM 800; Carl Zeiss, Jena, Germany) using a 10!/0.45 objective. For image analysis, the ratios of pSTAT1/pSTAT6 and CD80/CD206 were determined based on their relative area after applying standard fluorescence intensity for thresholding using Volocity software (Quorum Technologies, San Jose, CA).

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and coculture (P , .01). All groups, except Ca(OH)2, expressed significantly higher IL-6 levels in coculture compared with MQ monoculture. The untreated biofilm and Ca(OH)2 resulted in initially high TGF-b1 in coculture, which decreased considerably after 48 and 72 hours of interaction. CSnp/CMCS treatment demonstrated a gradual increase in TGF-b1 production, which was significantly pronounced after 72 hours in mono- and cocultures (P , .05). IL-10 was detected in low levels in MQ monoculture supernatant of all groups. Coculture of MQ with PdLFs enhanced IL-10 secretion, and CSnp/CMCS increased its concentration progressively over time, resulting in the highest levels at 72 hours (P , .01, Fig. 2A–H).

Statistical Analysis Statistical analyses were performed using 1way analysis of variance and the post hoc Tukey test in case of signiﬁcance. To compare coculture with MQ monoculture, 2-way analysis of variance was used. The 2-tailed Student t test was used to compare PdLF migration in each group at 8 versus 24 hours. P , .05 was considered statistically signiﬁcant.

Immunoﬂuorescence Analysis CSnp/CMCS treatment resulted in reduced pSTAT1/pSTAT6 expression compared with other treatments at 24 and 72 hours (P , .05). CD80/CD206 showed no signiﬁcant differences between treatment groups (P . .05, Fig. 3A–D).

Transwell Migration Assay

RESULTS Cell Viability The untreated biofilm demonstrated early significant cytotoxic effects on MQs at 24 hours followed by NaOCl/EDTA and Ca(OH)2, whereas their cytotoxic effects on PdLFs were observed later at 48 hours. Cell survival decreased over time, and no difference was found at 72 hours (P . .05). CSnp/CMCS treatment resulted in significantly higher cell viability than other treatments at 72 hours (P , .01). Comparable effects of CSnp on PdLF viability versus other groups were observed in monoculture and coculture (Fig. 1A).

MQ Morphometric Descriptors MQs in coculture showed different phenotypic features than in monocultures (Fig. 1B–F). CSnp/CMCS resulted in more spread and a less circular and signiﬁcantly larger cell size (P , .01).

Assessment of Inﬂammatory Mediators The positive control and NaOCl/EDTA resulted in high levels of nitric oxide and IL-1b levels compared with Ca(OH)2 (P , .01). TNF-a showed consistently high levels in cocultures of CSnp/CMCS and the negative control. CSnp/CMCS concurrently suppressed nitric oxide and IL-1b (P , .001) and showed a signiﬁcantly high ratio of IL-1RA/IL-1b in mono-

After 8 hours, the experimental groups showed higher PdLF migration compared with the negative control without differences between them. NaOCl/EDTA, Ca(OH)2, and untreated bioﬁlms resulted in less migrating PdLFs with evident cell morphologic changes after 24 hours (Fig. 4 A and B), whereas CSnp/CMCS demonstrated the highest PdLF migration (P , .01).

DISCUSSION The application of engineered CSnp as an intracanal medication in a clinical setting would affect not only the residual root canal (apical) bioﬁlm but also the host tissues in the periradicular region associated with apical periodontitis. Apical root resorption is a common ﬁnding noticed in periapical pathologies, and the occurrence of extrusion of materials during root canal procedures is inevitable even under good clinical practice19. Thus, CSnp/CMCS medication could communicate with periapical tissues through the apical foramen or the dentinal tubules, particularly when cementum is resorbed, resulting in the modulation of the host cells’ response. Herein, to mimic the clinical setting, we conditioned the apical 5 mm of the root with the culture media to study cell interactions. The coculture setup aided in studying the bidirectional interaction between MQs and PdLFs simultaneously. CSnp, a cationic bioactive polymer, interacts electrostatically with negatively

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FIGURE 1 – (A ) Cell viability of MQs and PdLFs in monoculture and coculture setups under different treatment conditions relative to the negative control at 24, 48, and 72 hours. MQ morphometric descriptors in monoculture versus coculture at 72 hours. (B ) Cell area, (C ) cell perimeter, (D ) cell circularity, and (E ) cell aspect ratio. Data represent the mean 6 standard deviation. (A ) Different lowercase letters represent significant differences between groups at each time point; (B–E ) mean single-cell values for each parameter were used for statistics; different lowercase and uppercase letters represent significant differences between groups in mono- and cocultures, respectively. *A significant difference between mono- and coculture in each group, P , .05. (F ) Images of calcein-AM stained macrophages in monoculture and coculture (magnification 5 10!, scale bar 5 50 mm) with zoomed-in portions from coculture images showing analysis of cell morphology.

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FIGURE 1 – Continued

charged bacterial cells leading to altered cell permeability, leakage of intracellular components, and bacterial death15. Conversely, electrostatic interaction between positively charged nanoparticles and negatively charged eukaryotic cells is followed by invagin*tions of plasmalemma membranes and recruitment of cell surface receptors for intracellular uptake20. MQs uptake CSnp intracellularly through clathrin-mediated endocytosis and phagocytosis21. The formulation of CSnp dispersed in watersoluble CMCS has been characterized earlier16. They are stable in acidic microenvironment; thus, their bioactivity would not be affected by the inflammatory acidic pH. Their nanosized and spherical-shaped structure would facilitate favorable interaction with MQs. Furthermore, the hydrophilicity of their components, which hold structural similarity to proteoglycans and glycosaminoglycans, would be conducive to cell adhesion, growth, and spreading22. Chemical modification of chitosan chains using carboxymethyl moieties could influence the inflammatory reaction; carboxyl and hydroxyl groups have been reported to induce antiinflammatory cytokines10. These combined properties of CSnp/CMCS contributed to modulated inflammatory responses. The characterization of M1 and M2 MQ functional subsets could be performed by analyzing morphometric parameters, cytokine profiles, transcription factors, and cell surface markers. Previous analysis of

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MQ morphologies by visual inspection of cytoskeletal staining revealed differences in the cell size of M1 and M2 phenotypes; M1 demonstrated smaller and more rounded morphology, whereas M2 exhibited larger and more irregular cell bodies23. In our coculture setup, CSnp attributed to phenotypic changes of MQ exhibiting significantly larger cell size, less circularity, and more elongated morphology, suggestive of the M2 phenotype. M1 MQs display proinflammatory function by the secretion of mediators such as nitric oxide, IL-1, and TNF-a, which are critical in the development of apical periodontitis. IL1b is the prevailing form of IL-1 found in human periapical lesions/exudates, possessing the most potent stimulus for bone resorption while hindering reparative osteogenesis24. CSnp/ CMCS dampened both nitric oxide and IL-1b. Alternatively, M2 MQs have anti-inflammatory function and promote wound healing by the secretion of cytokines such as IL-1RA, IL-10, and TGF-b125. IL-1RA regulates IL-1b activity via binding to its receptor (IL-1RI) but without signal transmission, consequently blocking IL1 activity, as an essential mechanism for host defense against excessive inflammation26. CSnp/CMCS significantly elevated the IL-1RA/ IL-1b ratio throughout the interaction time. IL10 is a potent anti-inflammatory cytokine that suppresses bone resorption, facilitates wound healing, and restores hemostasis, whereas TGF-b is a pro–wound healing cytokine that regulates cellular processes critical in collagen

production27, both of which were increased in CSnp/CMCS. Ca(OH)2 was used in this study because of its application as an intracanal medication; however, it lacks a modulatory effect on periapical inflammation and possesses cytotoxic effects on MQs28. A similar cytotoxic effect of Ca(OH)2 was observed on MQs and PdLFs in our study. This effect might be attributed to the release of reactive hydroxyl ions, which account for its broad-spectrum antimicrobial activity29. Furthermore, in the current investigation, Ca(OH)2 not only reduced proinflammatory but also antiinflammatory mediators, which is in accordance with previous clinical study findings30, and it might be due to its protein denaturing effects31. PdLFs actively influence immune responses by interacting with innate immune cells, besides modulating cytokine production by MQs9. Our data showed a low level of IL-6 in MQ monocultures, whereas a significantly higher level of IL-6 was observed in cocultures of all groups, even without biofilm stimulation, except in the Ca(OH)2 group. PdLFs promoted IL-10 production by MQs, which could be explained in part to be IL-6 mediated, which was augmented in the presence of CSnp/ CMCS. These results were in accordance with a previous study in which IL-10 was not produced by PdLF or MQ monocultures, either stimulated or unstimulated with Porphyromonas gingivalis, whereas coculture of MQ with PdLFs or exposure to PdLF

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FIGURE 2 – Inflammatory mediators produced by MQ monoculture versus MQ-PdLF coculture after 24, 48, and 72 hours of interaction under different treatment conditions. (A ) Nitric oxide, (B ) TNF-a, (C ) IL-1b, (D ) IL-1RA, (E ) ratio of IL-1RA/IL-1b, (F ) IL-6, (G ) TGF-b1, and (H ) IL-10. Data represent mean 6 standard deviation; different lowercase and uppercase letters indicate significant differences between groups at each time point in mono- and cocultures, respectively. *A significant difference between mono- and coculture within each group at each time point, P , .05.

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FIGURE 3 – The expression of (A ) transcription factors pSTAT1/pSTAT6 and (B ) surface markers CD80/CD206 in MQ monoculture versus MQ–PdLF coculture after 24 and 72 hours of interaction under different treatment conditions. Data represent the mean 6 standard deviation; different lowercase and uppercase letters indicate significant differences between groups at each time point in mono- and cocultures, respectively. *A significant difference between mono- and coculture within each group at each time point, P , .05. Representative images of pSTAT1/pSTAT6-stained macrophages at 72 hours in (C ) monocultures and (D ) cocultures. MQs were also stained with anti-CD68 and nuclei counterstained with 40 ,6-diamidino-2-phenylindole, magnification 5 10!, scale bar 5 28 mm.

preconditioned-media induced IL-10 secretion32. The engagement of toll-like receptors results in STAT1 signal transduction, consequently initiating the expression of proinﬂammatory mediators. Phosphorylation of STAT1 was used as the M1 marker for THP-

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1 cells33. PAFR/JAK/STAT1 pathways have been shown to be involved in the inﬂammatory immune response induced by lipoteichoic acid of E. faecalis34. CSnp/CMCS reduced the pSTAT1/pTAT6 ratio, suggesting its regulatory role on STAT1 signaling to block the M1 MQ polarization. This observation could be

explained in part by the inhibitory effect of IL-10 on STAT1 phosphorylation35. CD80 and CD206 have been used as markers for M1 and M2, respectively; however, there is an overlap between different MQ subsets based on the expression of surface markers in the literature. For example, CD206 showed no differentiated

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FIGURE 3 – Continued expression between M1 and M2 THP-1 macrophages33, whereas CD80 expression increased relative to M0 irrespective of the various stimuli36. PdLFs showed increased migration in the coculture system compared with PdLFs alone (data not shown), indicating the importance of MQ in recruiting ﬁbroblasts. Higher PdLF migration prompted by CSnp/ CMCS could be attributed to higher levels of TGF-b1 relative to unstimulated cells. The untreated bioﬁlm, NaOCl/EDTA, and Ca(OH)2 groups demonstrated lower PdLF migration despite the initial high levels of TGF-b1, which could be attributed to concurrent cytotoxic effects. Repair and regeneration of periodontal tissue derive from the coordinated interactions between cells, communicating with each other via secreted molecules that bind to the receptors expressed on adjacent effector cells or by direct cell-to-cell contact37. Herein, we focused on the paracrine mode of cell communication using a transwell setup

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because of its crucial role in the amplification and control of the inflammatory response38. Nevertheless, juxtacrine or contact-dependent signaling through cell-cell and cell–extracellular matrix communication is essential. The complementary effect of these mechanisms adds to the complexity of the biologic systems and warrants developing more complex experimental models that mimics different signals while investigating cell-level responses in periapical tissues. In summary, our findings confirmed that PdLFs actively interact with MQs and influence the immune response to biofilm. PdLFs induced phenotypic maturation of MQs, whereas MQs promoted the migration of PdLFs via paracrine signaling. CSnp/CMCS antibiofilm medication influenced the MQ-PdLF crosstalk through its modulatory effect on inflammatory mediators and transcription factors that facilitated the phenotypic switch of MQ into M2 phenotype subsets and promoted PdLF migration, hence emphasizing the

potential of engineered CSnp to modulate the immune response promoting tissue healing. The effect of engineered CSnp on MQs’ signaling pathways that modulate periapical inﬂammation warrants further investigation.

ACKNOWLEDGMENTS Supported in part by research grant from the American Association of Endodontists Foundation (grant no. 509641), the Natural Sciences and Engineering Research Council of Canada: Discovery Grant (A.K. [RGPIN-202005844]), and the Egyptian Bureau of Cultural and Educational Affairs in Canada (scholarship for H.H.). Luminex Multiplex immunoassays were performed at the Analytical Facility of Bioactive Molecules at The Hospital for Sick Children, Toronto, ON, Canada. The authors deny any conﬂicts of interest related to this study.

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FIGURE 4 – Transwell migration assay of PdLFs. (A ) Quantitative analysis of migrating cells per region of interest (ROI) among different groups. Data represent the mean 6 standard deviation; different lowercase and uppercase letters indicate signiﬁcant differences between groups after 8 and 24 hours, respectively. *A signiﬁcant difference between 8 and 24 hours within each group, P , .01. (B ) Representative images showing crystal violet–stained PdLFs migrated to the underside of the membrane after 8 and 24 hours of interaction.

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Sundqvist G, Figdor D. Life as an endodontic pathogen. Endod Topics 2003;6:3–28.

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Hussein H, Kishen A. Antibioﬁlm and immune response of engineered bioactive nanoparticles for endodontic disinfection. J Clin Med 2020;9:730.

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BASIC RESEARCH – TECHNOLOGY Jacob C. Simon, DDS, PhD,* Jason W. Kwok, DDS,† Frank Vinculado,* and Daniel Fried, PhD*

Computer-Controlled CO2 Laser Ablation System for Cone-beam Computed Tomography and Digital Image Guided Endodontic Access: A Pilot Study ABSTRACT Introduction: Ideal endodontic access provides unobstructed entry to the pulp chamber and visualization of the canal orifices while preserving the maximum amount of tooth structure. The aim of this study was to implement the use of lasers to accurately and predictably access teeth to follow the principles of minimally invasive endodontics. Methods: Traditional, conservative, ultraconservative, bridge, truss, and orifice-directed accesses were performed. A computer-controlled 9.3-mm CO2 laser ablation system was assembled and coupled with custom software capable of combining cone-beam computed tomographic (CBCT) volumetric data with spatially calibrated digital images of teeth to provide an augmented reality environment for designing and preparing endodontic accesses. Twenty (N 5 20) sound posterior teeth with fully developed root canal systems were imaged with CBCT scans and accessed via laser ablation in vitro. Results: All 20 (20/20) teeth were successfully accessed without iatrogenic errors. Volumetric renderings from post-access CBCT scans were used to verify the access and determine accuracy qualitatively. The volumetric measurements of hard tissue removed were as follows: traditional 5 39.41 mm3, conservative 5 9.76 mm3, ultraconservative 5 7.1 mm3, bridge 5 11.53 mm3, truss 5 19.21 mm3, and orifice directed 5 16.86 mm3. Conclusions: Digital image guidance based on feature recognition and registration with CBCT data is a viable method to address the challenge of dynamic navigation for accessing the pulp chamber. Modern lasers with high pulse repetition rates integrated with computer-controlled scanning systems are suitable for the efficient cutting of dental hard tissues. (J Endod 2021;47:1445–1452.)

This study presents a laserbased dynamic navigation system designed to execute guided endodontic access for minimally invasive endodontics. This instrument has potential for in vivo clinical use in the future to perform the duties demonstrated in this article.

From the *Department of Preventive and Restorative Dental Sciences, University of California San Francisco, San Francisco, California; and †Department of Endodontology, University of Connecticut, Farmington, Connecticut

KEY WORDS Automated; dynamic navigation system; guided; endodontic access; laser

Ideal endodontic access provides unobstructed opening to the pulp chamber and visualization of the canal orifices while preserving the maximum amount of tooth structure1,2. The practice of minimally invasive endodontics is important to preserve coronal and pericervical tooth structure3. The retention of coronal and pericervical hard tissue distributes coronal stress over a larger volume of natural tooth structure, therefore minimizing stress concentration and predisposition to coronal and root fracture4–8. It is generally well understood that the cumulative loss of tooth structure leads to the overall detriment of the tooth and reduced resistance to fracture9–15. In order to minimize the cumulative loss of tooth structure, it would be beneficial to implement a system that can accurately and predictably access teeth in a minimally invasive manner. Computer-aided dynamic navigation systems (DNSs) have been adapted from guided implant systems to address this need16–22. DNS technologies use thermoplastic molds with radiopaque fiducial markers to register cone-beam computed tomographic (CBCT) scans with tooth anatomy. In this study, we present an alternative method to perform a minimally invasive access by using a computer-controlled, 9.3-mm CO2 laser ablation system to access teeth based on spatially calibrated

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SIGNIFICANCE

Address requests for reprints to Jacob C. Simon, Department of Preventive and Restorative Dental Sciences, University of California San Francisco, 707 Parnassus Avenue, San Francisco, CA 94142. E-mail address: [emailprotected] 0099-2399 Published by Elsevier Inc. on behalf of American Association of Endodontists. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/ 4.0/). https://doi.org/10.1016/ j.joen.2021.06.004

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digital image guidance and CBCT scans. The samples in this pilot study were chosen at random from a pool of posterior teeth with fully developed roots and closed apices and then were screened with periapical radiographs to ensure mature pulpal anatomy. Distinct, invariant tooth anatomy is extracted from volumetric data from CBCT scans and then overlayed and registered with digital images of the tooth crown. An interactive augmented reality user interface computer generates the precise location of the internal anatomy and displays it through graphic projections in the digital image. A toolbox of drawing mechanisms is used by the clinician to design the access of choice through the software. In cases in which CBCT scans are unavailable, the clinician can design access using only the digital image, just as a clinician does with his or her eyes and hands currently. The access design is transformed into an algorithm that scans the CO2 laser over the tooth, cutting the desired shape in a stepwise routine.

MATERIALS AND METHODS Sample Preparation Twenty (N 5 20) posterior teeth with developed roots and closed apices were screened with periapical radiographs to ensure mature pulpal anatomy from a pool of extracted teeth collected from patients in the San Francisco Bay Area with approval from the University of California San Francisco Committee on Human Research. Using the American Association of Endodontists endodontic case difficulty assessment form and guidelines, samples qualifying as minimal and moderate difficulty for radiographic appearance of canal(s) criteria were included for participation in this study. A sample size of 20 teeth was chosen as a significant number of samples to demonstrate a variety of access designs in molar and premolar teeth. Samples were sterilized using gamma radiation by the University of California San Francisco central sterilization facility and stored in 0.1% thymol solution to maintain tissue hydration and prevent bacterial growth. Teeth were mounted in orthodontic resin blocks extending from the cementoenamel junction to 3–5 mm beyond the apices, leaving the crown exposed for ease of mounting in the sample holder. In this study, different access designs are demonstrated and defined as follows: traditional (T), straight-line access; conservative (C), a contracted traditional access; ultraconservative (U), a centralized circular access of a 2-mm diameter or less; truss (Tr), a mandibular molar access design with separate mesial and distal preparations

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leaving a full-thickness connection of enamel and dentin between the buccal and lingual walls; bridge (B), a truss design with only a dentin connection between the buccal and lingual wall12,23,24; and orifice directed (O), circular or ovoid preparations less than a 2-mm diameter positioned directly coronal to the location of the canal orifices at the level of the pulpal floor.

CO2 Laser Parameters A Diamond J5-V CO2 laser (Coherent, Santa Clara, CA) was integrated with a 3-dimensional computer-controlled ablation system25. The J5-V in this study produced a Gaussian output beam and operated at l 5 9.3 mm with a pulse duration of 26 microseconds and a pulse repetition rate of 100 Hz when cutting dentin or 200 Hz when cutting enamel. The laser energy output was monitored using a power/energy meter (ED-200; Gentec, Quebec, Canada), the beam profile was imaged using a Pyrocam Beam profilometer (Ophir-Spiricon, Logan, UT), and the laser pulse temporal profile was measured with a room temperature HgCdTe detector (PF-10.6-3; Boston Electronics, Boston, MA) with a response time of a few nanoseconds. The laser beam was focused with a (f 5 100 mm) ZnSe scanning lens to a waist diameter of 200 mm measured by scanning a razor blade across the beam to determine the diameter (1/e2). The incident fluence was 30 J/cm2 and removed ~30 mm sound tissue structure per pulse confirmed through cross-polarized optical coherence tomographic scans (Model IVS-300-CP; Santec Corp, Komaki, Aichi, Japan) of ablated calibration samples.

Digital Images Digital images were acquired with a digital microscope (Model AM2111; AnMO Electronics Corp, New Taipei City, Taiwan), and the 640 ! 480 pixel array was calibrated with a lens distortion correctional algorithm in LabView Vision Assistant (National Instruments, Austin, TX) yielding an (x, y) accuracy of 23.7 mm/pixel. Fiducial points ablated into the occlusal surface of each sample were used to modify the calibration matrix from relative positional measurements to real-world coordinates.

CBCT Imaging A CBCT scan was acquired of each sample using the K9000 CBCT unit (Kodak/ Carestream, Rochester, NY) operating at 60 kV, 2.0 mA, a 24-second scan duration, and 33.1 mGy.cm2 with 76.5-mm pixel spacing and 76.5-mm slice thickness over a limited ﬁeld of view (5 cm ! 7 cm). Scans were acquired

before treatment, after laser access, and after crown-down ﬁling.

Ablation System The PXIe-8840 embedded controller (National Instruments, Austin, TX) and PXI-6259 dataacquisition module (National Instruments) in the PXIe-1062Q chassis (National Instruments) served as the computer control hardware for the subcomponents of the ablation system. Custom software was authored in LabView that integrated spatially calibrated digital images and CBCT Digital Imaging and Communications in Medicine (DICOM) data; provided an augmented reality user interface for access design; produced ablation algorithms; and coordinated the laser output with x, y, z motion control stages VP-25XL and UTM50PPIHL from Newport (Irvine, CA) to cut guided endodontic accesses into the samples. CBCT DICOM data are loaded into the LabView software and viewed as conventional axial, sagittal, and coronal orthoslices for treatment planning. The program was authored to permit 3-dimensional rotational correction of the slices in order to match the volumetric data to the true sample position in the digital image. The operator is permitted to angle the access design in 1 dimension, and the real-world cutting axis of the sample is adjusted to accommodate using a single-axis goniometer stage from ThorLabs (Newton, NJ). Using 3-dimensional image processing algorithms26, subregions are manually selected and transformed into 2-dimensional compressed axial slices representing the cusp locations, height of contour, pulp chamber perimeter, and orifice location and shape. Additionally, a surface topographic rendering is produced. The CBCT extracts can be modified by manual selection using the “magic wand” feature, allowing control over exact contours and features to be used for registration verification and access design. The selected features are converted into region of interest variables and overlaid with the digital image of the sample to verify the height of contour and cusp locations confirming registration. Outlines of the pulp chamber and orifices then represent their true anatomic location relative to the current vantage point, and a suite of drawing tools and preprogrammed shapes are used to design the final access shape. Confirmation of the final design produces 2 scanning patterns; the first uses the topographic rendering to first guide the laser to reduce the slope of cusps, and the second scans the entire preparation area in a raster. Samples are mounted in the laser path, and the program cuts the designed access using the 3 single-axis motorized stages. The

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FIGURE 1 – (T ) Traditional, (C ) conservative, (U ) ultraconservative, (B ) bridge, and (O ) oriﬁce-directed (O ) accesses cut with a CO2 laser. Visible microscopic images showing (1 ) preablation, (2 ) post–laser access, and (3 ) post–crown-down treatments. Volumetric renderings showing (4 ) mesiodistal and (5 ) buccolingual views.

ﬁrst scanning pattern for cusp slope reduction is performed at 200 Hz, and the second for dentin preparation is performed at 100 Hz. All ablation is performed with water coolant, air spray, and a passive vacuum for the removal of debris accumulation. Sample access was planned and executed in 1 sequence without modiﬁcation in order to demonstrate the simple effectiveness of the CO2 laser for accurate ablation.

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Endodontic Treatment After the access was executed, pulp chambers were irrigated with 4% hypochlorite and a #10 K-file, and an endodontic explorer was used to confirm canal presence and coronal patency. After CBCT imaging of the laser-produced access, a true crown-down preparation was performed to approximately two thirds of the root length using .10 and .08 taper K3 files. Canals were then prepared up to a #40/.06

Vortex Blue (Dentsply Sirona, Charlotte, NC) rotary ﬁle to approximately two thirds the canal length to demonstrate feasibility in treating the canal systems with all aforementioned accesses.

CBCT Image Analysis DICOM ﬁles were analyzed using 3D Slicer, an open source software package for scientiﬁc visualization27. Pre- and post-laser and post–

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FIGURE 2 – A mandibular molar accessed with a truss design. Visible microscopic images showing (A ) preablation, (B ) post–laser access, and (C ) post–crown-down treatment. Volumetric renderings of the sample occlusal surface indicating positioning of preparations (yellow ) in relation to oriﬁce location (red ) from the occlusal viewpoint (D ) after cutting with the CO2 laser and (E ) post–crown-down procedure. Volumetric renderings showing (F ) mesiodistal and (G ) buccolingual views. crown-down CBCT scans were segmented and 3-dimensionally rendered for visual/ qualitative comparison. Volumetric data were measured for segmented sections to determine the amount of hard tissue removed in the preparations.

RESULTS A variety of endodontic access cavities were successfully cut into 20 samples using a CO2 laser ablation system without any iatrogenic misalignment, gouging, or perforation. Representative samples from the population demonstrating a range of designs are shown in Figure 1. Traditional (T), conservative (C), ultraconservative (U), bridge (B) or truss (Tr), and oriﬁce-directed (O) accesses are presented with visible images 1–3 showing preablation, post–laser access, and post– crown-down treatments accompanied with volumetric renderings 4 and 5 showing the mesiodistal and buccolingual shape and angles. The volume of hard tissue removed from each sample was as follows: traditional (T) 5 39.41 mm3, conservative (C) 5 9.76 mm3, ultraconservative (U) 5 7.1 mm3, bridge (B) 5 11.53 mm3, and oriﬁce directed (O) 5 16.86 mm3. Figure 2 shows the results from the access of a mandibular molar prepared with a truss design centered over the mesial and distal radicular systems of before ablation (Fig. 2A), after laser access (Fig. 2B), and after the crown-down procedure (Fig. 2C). Volumetric renderings (Fig. 2D and E) are presented showing the positioning of

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preparations (yellow) in relation to orifice location (red) from the occlusal viewpoint after cutting with the CO2 laser (Fig. 2D) and after the crown-down procedure (Fig. 2E). Volumetric renderings (Fig. 2F and G) illustrate the mesiodistal and buccolingual shape and angles. The truss access in this sample has a volume of 19.21 mm3 hard tissue removed. Two maxillary molars are presented in Figure 3 prepared with a conservative preparation (C) and an orifice-directed access (O). Figure 3C1–C3 and 3O1–O3 are preablation, post–laser access, and post– crown-down treatment. Figure 3C4 and C5 and 3O4 and O5 show the positioning of preparations (yellow) in relation to orifice location (red) from the occlusal viewpoint after cutting with the CO2 laser (Fig. 3C4) and after crown-down treatment (Fig. 3C5). Figure 3O4 and O5 are post–crown-down renderings illustrating clear visualization of the buccal (Fig. 3O4) and palatal (Fig. 3O5) systems. Figure 3C6 and C7 and 3O6 and O7 demonstrate the mesiodistal and buccolingual shape and angles. The maxillary accesses presented have a volume of C 5 24.46 mm3 and O 5 19.65 mm3 hard tissue removed.

DISCUSSION The potential of a laser-based approach for selective caries removal and endodontic access preparation deserves renewed attention because of the improved performance and characteristics of newly developed infrared lasers compared with those researched over 20 years ago28. Carious and

sound hard tissues can be selectively ablated in precise stepwise increments of ~30-mm depths. Ejected infected tissue and toxins are heated to high temperatures, reducing the contamination of more apical layers of hard or soft tissue29. When integrated with computercontrolled systems, laser pulses can be focused and directed with precision and accuracy to produce preparations at or smaller than the fundamental limit of what is treatable with the current endodontic armamentarium. The results demonstrated in this study show that a computer-controlled 9.3-mm CO2 laser system can prepare a variety of endodontic access designs and can act as a dynamic navigation system by integrating CBCT volumetric data to produce an augmented reality environment permitting oriﬁce-directed access. Unlike other dynamic navigation systems used in endodontics such as X-NAV (X-Nav Technologies, Lansdale, PA), this system can also operate without CBCT data due to the fact that this DNS integrates a digital image of the tooth into the computer interface. The clinician can digitally design the access based solely on the image of the occlusal surface, and the preparation is automatically performed by the computercontrolled laser. Therefore, emergency treatment or cases in which CBCT is unavailable can still be automatically performed and guided by the clinician. In this pilot study, all of the access designs were preplanned using CBCT data and executed in 1 sequence without modiﬁcation in order to demonstrate the simple effectiveness of the CO2 laser for

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FIGURE 3 – Two maxillary molars prepared with a (C ) conservative preparation and an (O ) oriﬁce-directed access. (C1–C3 and O1–O3 ) Visible images (1 ) preablation, (2 ) post–laser access, and (3 ) post–crown-down treatment. Volumetric renderings showing the positioning of preparations (yellow ) in relation to oriﬁce location (red ) from the occlusal viewpoint (C4 ) after cutting with the CO2 laser and (C5 ) post–crown-down treatment. Post–crown-down renderings illustrating visualization of the (O4 ) buccal and (O5 ) palatal systems. (C6 and C7 and O6 and O7 ) Volumetric renderings and demonstrate the (6 ) mesiodistal and (7 ) buccolingual views.

accurate ablation. Each scan of the laser ablates ~30 mm hard tissue, and a simple division of the desired cutting depth measured from the CBCT data by the ablation depth per scan was predetermined to automatically cut to and stop at the desired depth. There are other possible methods to control access depth when the true necessary depth is unknown. One exciting method is spectral feedback, which would be useful in teeth with existing pulp tissue. This method analyzes

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component wavelengths of the ablation plume to discriminate between hard and soft tissue by measuring the presence of calcium from its strong spectral emission peak at 605 nm30,31. A system based on this method could scan the laser without predetermining the depth and be programmed to automatically stop when the soft tissue of the pulp chamber has been reached. In a previous in vivo study, we demonstrated a CO2 laser system that automatically ablated composites from teeth

using spectral feedback30. That study delivered laser light through an articulating arm and bite block onto the occlusal tooth surface. That same mechanism can be adapted to be secured to the rubber dam clamp with 3dimensional printing. Another method to control the access depth would be to use a coaxial imaging and ablation design analogous to one we constructed for a study we performed demonstrating short-wave infrared imaging–

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guided caries ablation using a low-cost 9.3-mm laser (DL-500; Access Laser, Everett, WA)32. In this method, the CO2 laser system can be scanned to a conservative set depth (ie, ablate 1 mm at a time), imaged with the coaxial beam and then presented to the clinician to verify the preparation location and shape, permitting alterations to the original design if necessary. The DL-500 CO2 laser we used has similar characteristics to the one used here and would be suitable for a clinical system. Currently, both CO2 and erbium lasers have Food and Drug Administration clearance for soft and hard tissue treatment. A clinical computer-controlled laser system is ideally suited for image-guided or spectral-guided caries removal followed by endodontic therapy. Because of the fact that lasers operate in a sterile ﬁeld, eject tissue, and provide hemostasis, pulpotomy procedures have the potential for a better prognosis. In particular, pediatric endodontics may ﬁnd these systems especially useful because of patient pain tolerance and the higher success rate of pulpotomy procedures of immature teeth33–37. When a complete root canal is planned, a computer-controlled laser system

has the potential to reduce off-target and offaxis access preparations. CBCT-guided lasers may also be useful for accessing calcified canals by drilling very small and accurate orifices on a guided path. In preliminary attempts in simulated calcified incisors, we have been able to reach depths between 8–12 mm apical to the pulpal floor. We plan to investigate this further in future research studies. Lastly, if erbium lasers can ablate tissue at a clinically satisfactory standard relative to the CO2 tested here, then clinicians who used the system could then employ the same laser for laser-activated irrigation. The limitations of the laser ablation system as constructed is the reliance on x, y, z motion stages that limits the laser scanning rate. Scanning the laser over a 2-mm diameter circle takes 30 seconds, yielding a preparation time of 6 minutes to prepare a 2-mm diameter ! 4-mm deep preparation. More complex access designs in this study took approximately 20 minutes to execute. Another limitation is that only tapered cylinder access shapes could be achieved because of the fact that the laser beam is stationary, and the sample is scanned in this in vitro setup.

Avoiding excessive heat accumulation in the tooth will be the limiting variable for preparation speed in such systems. Figure 1T1–T5 demonstrates charred dentin at the perimeter of the access due to overheating. Excessive heat accumulation during laser irradiation can cause crack formation and the charring of dentin. Several studies have explored the laser parameters that cause peripheral thermal damage with pulsed CO2 lasers38–40. Both limitations in preparation shape and heat distribution can be overcome by using a high-speed galvanometer, voice coil, or microelectromechanical scanning systems. Such scanning systems will also enable the use of angled incident laser beams allowing multiaxis angled beam delivery that will enable the cutting of inverted cones and hourglassshaped access forms.

ACKNOWLEDGMENTS The authors would like to acknowledge the support of NIDCR/NIH grants R01-DE019631 and F30-DE026052.

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Krapez J, Fidler A. Location and dimensions of access cavity in permanent incisors, canines, and premolars. J Conserv Dent 2013;16:404–7.

Patel S, Rhodes J. A practical guide to endodontic access cavity preparation in molar teeth. Br Dent J 2007;203:133–40.

Lin CY, Lin D, He WH. Impacts of 3 different endodontic access cavity designs on dentin removal and point of entry in 3-dimensional digital models. J Endod 2020;46:524–30.

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Rover G, Belladonna FG, Bortoluzzi EA, et al. Inﬂuence of access cavity design on root canal detection, instrumentation efﬁcacy, and fracture resistance assessed in maxillary molars. J Endod 2017;43:1657–62.

Krishan R, Paque F, Ossareh A, et al. Impacts of conservative endodontic cavity on root canal instrumentation efﬁcacy and resistance to fracture assessed in incisors, premolars, and molars. J Endod 2014;40:1160–6.

Zhang Y, Liu Y, She Y, et al. The effect of endodontic access cavities on fracture resistance of ﬁrst maxillary molar using the extended ﬁnite element method. J Endod 2019;45:316–21.

Jiang Q, Huang Y, Tu X, et al. Biomechanical properties of ﬁrst maxillary molars with different endodontic cavities: a ﬁnite element analysis. J Endod 2018;44:1283–8.

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Corsentino G, Pedulla E, Castelli L, et al. Inﬂuence of access cavity preparation and remaining tooth substance on fracture strength of endodontically treated teeth. J Endod 2018;44:1416–21.

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Saber SM, Hayaty DM, Nawar NN, Kim HC. The effect of access cavity designs and sizes of root canal preparations on the biomechanical behavior of an endodontically treated mandibular ﬁrst molar: a ﬁnite element analysis. J Endod 2020 Jul 5. https://doi.org/10.1016/j.joen.2020.06.040 [Epub ahead of print].

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Zubizarreta-Macho A, Munoz AP, Deglow ER, et al. Accuracy of computer-aided dynamic navigation compared to computer-aided static procedure for endodontic access cavities: an in vitro study. J Clin Med 2020;9:129.

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Mediavilla Guzman A, Riad Deglow E, Zubizarreta-Macho A, et al. Accuracy of computer-aided dynamic navigation compared to computer-aided static navigation for dental implant placement: an in vitro study. J Clin Med 2019;8:2123.

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Chong BS, Dhesi M, Makdissi J. Computer-aided dynamic navigation: a novel method for guided endodontics. Quintessence Int 2019;50:196–202.

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Connert T, Zehnder MS, Weiger R, et al. Microguided endodontics: accuracy of a miniaturized technique for apically extended access cavity preparation in anterior teeth. J Endod 2017;43:787–90.

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Krastl G, Zehnder MS, Connert T, et al. Guided endodontics: a novel treatment approach for teeth with pulp canal calciﬁcation and apical pathology. Dent Traumatol 2016;32:240–6.

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Connert T, Zehnder MS, Amato M, et al. Microguided endodontics: a method to achieve minimally invasive access cavity preparation and root canal location in mandibular incisors using a novel computer-guided technique. Int Endod J 2018;51:247–55.

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Zehnder MS, Connert T, Weiger R, et al. Guided endodontics: accuracy of a novel method for guided access cavity preparation and root canal location. Int Endod J 2016;49:966–72.

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Abou-Elnaga MY, Alkhawas MA, Kim HC, Refai AS. Effect of truss access and artiﬁcial truss restoration on the fracture resistance of endodontically treated mandibular ﬁrst molars. J Endod 2019;45:813–7.

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Silva E, Pinto KP, Ferreira CM, et al. Current status on minimal access cavity preparations: a critical analysis and a proposal for a universal nomenclature. Int Endod J 2020;53:1618–35.

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Hibst R, Stock K, Gall R, Keller U. Controlled tooth surface heating and sterilization by Er:YAG laser radiation. Proc SPIE 1996;2922:119–61.

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Simon JC, Choi JH, Jang A, Fried D. In vivo spectral guided removal of composite from tooth surfaces with a CO2 laser. Proc SPIE Int Soc Opt Eng 2020;11217:112170K.

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Yi I, Chan KH, Tsuji GH, et al. Selective removal of esthetic composite restorations with spectral guided laser ablation. Proc SPIE Int Soc Opt Eng 2016;9692:96920U.

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Chan KH, Fried D. Selective ablation of dental caries using coaxial CO2 (9.3-mm) and near-IR (1880-nm) lasers. Lasers Surg Med 2019;51:176–84.

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Nazemisalman B, Farsadeghi M, Sokhansanj M. Types of lasers and their applications in pediatric dentistry. J Lasers Med Sci 2015;6:96–101.

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Zhang S, Chen T, Ge LH. [Evaluation of clinical outcomes for Er:YAG laser application in caries therapy of children]. Beijing Da Xue Xue Bao Yi Xue Ban 2013;45:87–91.

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Keller U, Hibst R, Geurtsen W, et al. Erbium:YAG laser application in caries therapy. Evaluation of patient perception and acceptance. J Dent 1998;26:649–56.

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Ngoc VT, Van Nga TD, Chu DT, Anh LQ. Pulpotomy management using laser diode in pediatric patient with severe hemophilia A under general anesthesia-a case report. Spec Care Dentist 2018;38:155–9.

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Walker LA, Sanders BJ, Jones JE, et al. Current trends in pulp therapy: a survey analyzing pulpotomy techniques taught in pediatric dental residency programs. J Dent Child (Chic) 2013;80:31–5.

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Dela Rosa A, Sarma AV, Le CQ, et al. Peripheral thermal and mechanical damage to dentin with microsecond and sub-microsecond 9.6 microm, 2.79 microm, and 0.355 microm laser pulses. Lasers Surg Med 2004;35:214–28.

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Fried D, Zuerlein MJ, Le CQ, Featherstone J. Thermal and chemical modiﬁcation of dentin by 911 mm CO2 laser pulses of 5-100-ms duration. Lasers Surg Med 2002;3:275–82.

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Nguyen D, Chang K, Hedayatollahnajaﬁ S, et al. High-speed scanning ablation of dental hard tissues with a l59.3 mm CO2 laser: adhesion, mechanical strength, heat accumulation, and peripheral thermal damage. J Biomed Opt 2011;16:071410.

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BASIC RESEARCH – TECHNOLOGY Anmar Janabi, DDS,* Patricia A. Tordik, DMD, FICD,* Ina L. Grifﬁn, DMD,*

Accuracy and Efﬁciency of 3-dimensional Dynamic Navigation System for Removal of Fiber Post from Root Canal–Treated Teeth

Behzad Mostouﬁ, DDS, MDS,† Jeffey B. Price, DDS, MS,‡ Priya Chand, DDS,* and Frederico C. Martinho, DDS, MSc, PhD*

ABSTRACT Introduction: The purpose of this study was to investigate the accuracy and efficiency of the 3-dimensional dynamic navigation system (DNS) compared with the freehand technique (FH) when removing fiber posts from root canal–treated teeth. Methods: Twenty-six maxillary teeth were included. Teeth were root canal treated and restored with Parapost Taper Lux (Coltene/Whaledent, Altst€atten, Switzerland) luted with RelyX Unicem (3M ESPE, St Paul, MN). A core buildup was then performed using Paracore (Coltene/Whaledent). Teeth were mounted in tissue-denuded cadaver maxillae. Teeth were divided into 2 groups: the DNS group (n 5 13) and the FH group (n 5 13). Cone-beam computed tomographic scans were taken pre- and postoperatively. The drilling path and depth were planned virtually using X-guide software (X-Nav Technologies, Lansdale, PA) in both groups. For the DNS group, drilling was guided with X-Nav software and the FH group under a dental operating microscope. Global coronal and apical deviations, angular deflection, operation time, and the number of mishaps were compared between the groups to determine the accuracy and efficiency. The 3-dimensional volume (mm3) of all teeth was calculated before and after post removal using the Mimics Innovation Suite (Materialise NV, Leuven, Belgium). The ShapiroWilk, 1-way analysis of variance, and Fisher exact tests were used (P , .05). Results: The DNS group showed significantly less global coronal and apical deviations and angular deflection than the FH group (P , .05). DNS required less operation time than FH. Moreover, the DNS technique had significantly less volumetric loss of tooth structure than the FH technique (P , .05). Conclusions: The DNS was more accurate and efficient in removing fiber posts from root canal–treated teeth than the FH technique. (J Endod 2021;47:1453– 1460.)

KEY WORDS Dynamic navigation system; endodontics; guided endodontics; mimics; post removal

Post removal from root canal–treated teeth is often necessary for teeth with root canal failure1. However, the management of persistent or developed posttreatment apical periodontitis presents a dilemma in teeth restored with posts. Post removal is frequently a challenge to clinicians, and removing posts from root canals poses risks2–4. Mitigating these risks requires avoiding procedural errors such as unnecessary removal of sound root dentin, deviations from the root axis, microcrack, or root fracture5,6. These procedure errors can worsen the prognosis and threaten success of endodontic retreatment5. Various post removal systems and techniques have been evaluated2–5. However, the most common approach is grinding the post with burs or ultrasonic tips. This freehand technique (FH) has several disadvantages3,7,8. It is time-consuming and requires substantial removal of the coronal tooth structure to access the ﬁber post to allow proper visualization under the dental operating microscope (DOM)4. Additionally, estimating the angle of the post and determining the drilling trajectory is challenging, requiring signiﬁcant clinician experience3,9.

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SIGNIFICANCE The DNS is a 3-dimensional, real-time motion tracking technology. The DNS demonstrated accuracy and efficiency in removing fiber posts from endodontically treated teeth. It has the potential to be used in endodontics for fiber post removal.

From the *Division of Endodontics, Department of Advanced Oral Sciences and Therapeutics, †Department of Oral and Maxillofacial Surgery, and ‡ Department of Oncology and Diagnostic Sciences, School of Dentistry, University of Maryland, Baltimore, Maryland Address requests for reprints to Dr Frederico C. Martinho, Division of Endodontics, Department of Department of Advanced Oral Sciences and Therapeutics, University of Maryland, School of Dentistry, 650 West Baltimore Street, 6th Floor, Suite 6253, Baltimore, MD 21201. E-mail address: fmartinho@umaryland. edu 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.07.002

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Recently, the concept of “guided endodontics” has been explored8,10–13. This concept uses a preoperative cone-beam computed tomographic (CBCT) image to virtually plan and guide the bur during the procedure. A newer technique uses a 3-dimensional (3D)–printed endodontic guide to remove posts from root canal–treated teeth8,11–13. This static computer-assisted technique has been shown to reduce procedural errors during the root post removal, preventing the removal of excessive tooth structure, root perforation, or root weakening8,13. However, this approach has several limitations as described elsewhere8,14. More recently, following the same concept of guided endodontics, the 3D dynamic navigation system (DNS) has been introduced to endodontics15. This DNS technology, endorsed in implant dentistry16, has been adopted in endodontics. The DNS is a 3D real-time motion tracking technology. It is based on overhead tracking cameras, which capture the patient’s jaw position and the operator’s bur movement. Such a computerbased system has shown promising results for guided access cavity preparation17,18, locating calcified canals19,20, endodontic microsurgery21,22, and intraosseous anesthesia23. One significant advantage of the DNS is to enable real-time visualization of the position and angulation of the drill24, which allows altering the plan during the procedure if needed. To date, there is no study evaluating the performance of the 3D DNS for fiber post removal from root canal–treated teeth. In this study, we successfully explored a new post removal technique using the 3D DNS. We investigated the accuracy and efficiency of the 3D DNS compared with the FH technique when removing fiber posts from root canal–treated teeth. The null hypothesis tested here was that neither the DNS nor the FH technique can efficiently remove fiber posts from root canal–treated teeth.

MATERIALS AND METHODS The local institutional review board at the University of Maryland, Baltimore, MD (HP#00088609) approved this study. The sample size calculation with an alpha level of 5% and power of 80% indicated a total of 13 teeth per group. Twenty-six freshly extracted human maxillary single-rooted teeth (maxillary canines and incisors) obtained from the oral surgery department at the University of Maryland were included in this study. The inclusion criteria were as follows:

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(1) sound teeth (slightly decayed or restored), (2) single-rooted teeth with straight canals, (3) the presence of a single canal veriﬁed radiographically, (4) complete root development, and (5) teeth with at least a 15-mm root length. Teeth previously root canal treated with extensive caries or restorations, crowns, cracks, or fractures were excluded. All teeth were sterilized using steam autoclave and kept hydrated in distilled water.

Root Canal Preparation, Post Luting, and Core Buildup After accessing the cavity, a size #15 K-file (Dentsply Sirona, Ballaigues, Switzerland) was used to ensure patency and determine the working length. The working length was established at 1 mm short of the root apex (visually determined). Root canal preparation was performed with a crown-down technique using HyFlex Rotary Files (Coltene/Whaledent, Allst€atten, Switzerland). The canals were instrumented up to a size #40/.06 and irrigated with 5 mL 2.5% sodium hypochlorite between the files. The canals were irrigated with 5 mL 17% EDTA. The canal remained soaked with 17% EDTA for 1 minute, and the solution was renewed 3 times for a total of 3 minutes. Then, the canal was flushed with 5 mL 2.5% sodium hypochlorite, and final irrigation was performed with saline solution. The root canals were dried with paper points (Dentsply Sirona) and obturated with a corresponding 40/.06 guttapercha point (Coltene/Whaledent) and AH Plus sealer (Dentsply DeTrey GmbH, Konstanz, Germany) using a single-cone technique. The teeth were stored in 100% humidity at 98.6 F (~37 C) for 1 week to allow the sealer to set. A post space preparation was created to 12– 13 mm from the access opening using a 1.1mm ParaPost drill size 5.5 (Coltene/ Whaledent). A 1.1-mm wide parallel fiber post (ParaPost Fiber Lux, Coltene/Whaledent) was fitted in the canal and cut 2 mm below the access opening level. The posts were luted in the canal using RelyX Unicem (3M ESPE, St Paul, MN), and core buildup was performed using Paracore (Coltene/Whaledent) according to the manufacturer’s instructions.

(teeth #6, 7, 8, 9, 10, 11, and 12) experimental teeth were mounted in the maxilla using Examix NDS (GC America, Alsip, IL). A total of 4 tissue-denuded cadaver maxilla models were used. Two extracted human maxillary molars (#14 and #15) were mounted on the left side, and teeth #2 and 3 were mounted in the right side of the maxilla to fix an X-clip (X-Nav Technologies, Lansdale, PA). Before the preoperative CBCT scan, the X-clip with 3 radiopaque fiducials was molded to molar areas according to the manufacturer’s instructions for the DNS group. The X-clip was left in position on the opposite side from the procedure in the opposing arch. A single-arch CBCT scan (CS 93000; Carestream, Atlanta, GA) was taken at 0.120-mm3 voxel size resolution. After the preoperative CBCT scan, teeth were assigned and divided into 2 groups: the DNS group (n 5 13) and the FH group (n 5 13). Figure 1 illustrates the workflow used in the present study.

The DNS Group A single operator who was an experienced endodontist performed all the procedures for the DNS and FH groups. The operator was trained and calibrated in the DNS by performing 20 procedures. The Digital Imaging for Communication in Medicine data set was uploaded into the X-guide software (X-Nav Technologies) and entered into the DNS planning system. The drilling entry point, angle, trajectory, and depth needed to remove the ﬁber post were planned. To standardize the drilling procedures for the 2 groups, we ﬁrst used a size #2 (1.1 mm wide) Munce bur (CJM Engineering Inc, Ojai, CA) to drill 9 mm. Then, a size #1 (0.9 mm wide) Munce bur (CJM Engineering Inc) was used to complete the drilling at 12–13 mm. New burs were used for each tooth. After system calibration and drilling trajectory was planned, drilling through the post was done under the complete guidance of the navigation of the X-guide system (X-Nav Technologies) on a slow-speed handpiece at 5000 rpm (WS-56L; W&H, Ontario, Canada). Drilling was stopped when the drill reached the end of the preplanned path indicated in the system display. Figure 2 shows the X-guide software frame indicating the correct position and angulation of the bur during the drilling process.

Tissue-Denuded Cadaver Maxilla Model and Preoperative CBCT Scan

The FH Group

Teeth were mounted in a tissue-denuded cadaver maxilla obtained from the Maryland Anatomy Board, which was ﬁxed on a typodont base (ModuPROEndo, Acadental, KS). Six (teeth #6, 7, 8, 9, 10, and 11) to seven

All the steps used to plan the procedure for the DNS group previously listed were virtually the same for the FH group. The drilling through the ﬁber post was performed FH under a DOM (Global Surgical Corporation, St Louis, MO).

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the drilling until the visualization of guttapercha under the DOM. The number of mishaps (perforations) was recorded in both groups. In addition, to determine the amount of tooth structure lost, we measured the volume (mm3) of the tooth structure before and after the post removal using the using Mimics Innovation Suite (Materialise NV, Leuven, Belgium).

26 extracted teeth

Root canal therapy performed

Post and core buildup performed

Statistical Analysis

Pre-op CBCT scan obtained

FH-group

DNS-group

(n=13)

X-Nav drill path planned on the CBCT scan

Drilled out the post under the DOM

Drilled out the post under the X-Nav

Opera on me recorded

Post-opera ve CBCT scan obtained

Pre- and postopera ve

CBCT superimposed

Global coronal and apical devia ons, and algular deﬂec on measured

Quantitative data were recorded as the mean 6 standard deviation, and normality was tested using Shapiro-Wilk tests. After confirming normal distribution, the mean global coronal and apical deviations, the angular deflection from the drilling trajectory preplanned in the preoperative CBCT scan, the volume of the tooth structure, and the operation time were compared between the DNS and FH groups using 1-way analysis of variance. The frequency of perforation in each group was compared using the Fisher exact test. All statistical tests were performed at a significance level of 5% (P , .05).

RESULTS

FIGURE 1 – A ﬂowchart illustrating the workﬂow for the experimental methodology.

The drilling was stopped when gutta-percha was visualized in the canal under the DOM.

Accuracy and Efﬁciency Determination The accuracy was determined using superimposition software (X-Nav Technologies). The pre- and postoperative scans were superimposed. The accuracy in both groups was measured by the global coronal and apical deviations and angular deﬂection from the drilling trajectory preplanned in the preoperative CBCT scan to the one obtained in the postoperative CBCT scan (Fig. 3). The global coronal deviation is the overall difference in coronal position between the trajectory path and the actual drill path. The

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global apical deviation is the overall difference in apical position between the trajectory path and the actual drill path. The angular deflection is the largest angle between the center axes of the trajectory planned and the actual drill. The deviations of the postoperative drilling trajectory from the preplanned one were measured in 3 dimensions (X, Y, and Z). These measurements were performed by a blinded technician engaged in the planning and procedure of drilling out the fiber post. Efficiency was determined by the time of the operation in minutes. The time required to remove the post was measured using a digital stopwatch. For the DNS group, the time was recorded from the start of the drilling until the end of the preplanned path. For the FH group, the time was recorded from the beginning of

Table 1 shows the mean 6 standard deviation values for the global coronal and apical deviations, angular deflection, and operation time. The DNS group showed significantly less global coronal and apical deviations and angular deflection than the FH group (P , .05). The average time for the post removal in the FH group (8.30 6 4.65 minutes) was 2! greater than the DNS group (4.03 6 0.43 minutes) (P , .05) (Table 1). Additionally, the DNS group had a significantly less volumetric (mm3) loss of tooth structure than the FH technique (P , .05). The total volume (mm3) of tooth structure encountered before and after post removal for the DNS and FH groups is shown in Table 1. No perforation was detected in the DNS and FH groups. Figure 4A–D shows the drilling trajectory obtained in the postoperative CBCT scan in the FH and DNS groups.

DISCUSSION This study successfully investigated the accuracy and efficiency of the DNS technique, a 3D real-time motion tracking technique, compared with the FH technique when removing fiber posts from root canal–treated teeth. Our results showed that the DNS is more accurate and efficient in removing fiber posts from root canal–treated teeth than the FH technique. The DNS required less operation time than the FH technique. Moreover, the DNS technique had significantly

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FIGURE 2 – The X-guide software frame indicating the correct position and angulation of the bur during the drilling process.

less volumetric loss of tooth structure than the FH technique. Over the last decade, fiber posts have gained popularity6,11. Its modulus of elasticity is close to that of dentin, and its availability in tooth color makes it a better option in restoring a tooth in the esthetic zone than metal posts25. Nevertheless, the strong adhesive bond between the fiber post and dentin, along with the nature of the post material, makes it challenging to debond the post from dentin walls11. Indeed, the most common approach to removing fiber posts from root canals is grinding the post with burs or an ultrasonic tip2,3. Despite the number of different postremoval systems available in the market, most of them share the same concept of grinding the post using different burs. To the best of our knowledge, this is the first study to evaluate the accuracy and efficiency of the DNS technique to remove fiber

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posts from root canal–treated teeth. Although the concept of “guided endodontics” has been recently explored for post removal8,11–13, these previous studies relied on the static computer-assisted technique. In such a method, the trajectory of the bur is virtually planned in a preoperative CBCT scan using software, and a 3D-printed endodontic tray is made to guide the bur during surgery. Despite the advantages of this method8,11–13, this approach has several limitations14. Every step in the digital workflow can introduce inaccuracies14. It requires the fabrication of a 3D-printed endodontic guide, which complicates the workflow. Additionally, this guide is difficult to use in posterior areas with limited interocclusal or interdental spaces. Another critical disadvantage of this technique is the lack of 3D real-time visualization when drilling through the post, thus not allowing intraoperative changes in the predetermined

drill trajectory. However, this critical disadvantage is overcome in the dynamic computer-assisted method tested here, which allows a 3D real-time motion tracking to drill out the post15. In addition, the workflow implemented here for the DNS technique is straightforward, not requiring the creation of a 3D-printed guide as in the static computerassisted technique. To determine the accuracy of the DNS and FH technique, we measured the global coronal and apical deviations and the angular deflection in 3 dimensions (X, Y, and Z) from the drilling trajectory preplanned in the preoperative CBCT scan and the one obtained in the postoperative CBCT scan. Our data showed that the DNS group had significantly less global coronal and apical deviations than the FH technique. Additionally, we found a mean angular deflection of 1.75 for the DNS group and 4.49 for the FH group. Previous

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FIGURE 3 – A schematic view of the global coronal deviation (mm), global apical deviation (mm), and angular deﬂection

( ) between the trajectory path and actual drill path. Angular deﬂection is the largest angle between the center axes of the trajectory planned and the actual drill. Global coronal deviation is the overall difference in the coronal position between the trajectory path and the actual drill path. Global apical deviation is the overall difference in the apical position between the trajectory path and the actual drill path.

studies investigating the accuracy of DNS in intraosseous anesthesia23 and locating calcified canals20 found an angular deflection of 1.36 and 1.69 , respectively. More recently, Dianat et al21 evaluated the accuracy of DNS in root-end resection and revealed an angular deflection of 2.54 in the DNS group and 12.38 in the FH group. Data obtained in the present study, together with previous findings17–23, indicate a high level of accuracy of the DNS in endodontic procedures. It is worth pointing out that even though the DNS is a guided approach, the success of such a technique also depends on the operator’s skill19,26. The DNS has a perception of

0.25 mm and 0.5 with bur and handpiece tracking26. Accuracy can be affected by small amounts of hand trembling. Post removal can consume a large amount of the operator’s time during a retreatment session. Therefore, an accurate and fast post removal technique is desirable. In this study, the average time required for the post removal in the FH group (8.30 minutes) under the DOM was 2! greater than the DNS group (4.03 minutes). As with any other new technique, the DNS has a learning curve. However, the straightforward workﬂow required for the X-Nav system decreases this learning curve. In this study, it took 20 guided

attempts to calibrate a nonexperienced operator in the X-Nav system. Similar investigations also indicated 20 attempts to calibrate a novice operator19,21. Previous studies revealed that inexperienced clinicians showed a significant improvement of implant placement with as low as 3 attempts and up to 20 attempts27,28. It can be speculated that the average time taken to remove a fiber post found in this study might be reduced as the clinician gained experience in navigating the DNS. Overall, it appears that skilled and less experienced clinicians alike can benefit from this computer-aided technology for post removal. In this study, no root canal perforation was detected in the DNS and FH groups. The endodontist’s experience in removing posts from root canal–treated teeth may have contributed to this result. Previous studies investigated the risk of mishaps, including perforation during fiber post removal using different techniques2–5,29,30. In particular, Capar et al30 revealed that fiber post removal with drills does not significantly impact crack propagation, further supporting no occurrence of root fractures found in this study. Our results also indicate that an endodontist can remove a fiber post from the root canal under the DOM using a CBCT scan. However, this FH technique under the DOM resulted in a significant volumetric loss of tooth structure and required greater operation time than the DNS. It is worth highlighting that the postoperative CBCT scan also revealed a suboptimal trajectory of the bur with substantial removal of tooth structure to the apex for the FH group (Fig. 4). This substantial removal of coronal tooth structure was needed to access the fiber post and to allow proper visualization under the DOM. Despite the accuracy of the DNS found in this study, the DNS has limitations. This technique relies on a preoperative CBCT scan; therefore, the quality of the CBCT scan is crucial for accurate preoperative planning31,32. Artifacts from amalgam restoration or metal posts in the site of the procedure can be a limitation. Also, the

TABLE 1 - The Mean 6 Standard Deviation Values for the Global Coronal and Apical Deviations, Angular Deﬂection, Operation Time, and Volume of Tooth Structure Dynamic navigation DNS group

Freehand FH group

P value

0.91 6 0.65 mm 1.17 6 0.64 mm 1.75 6 0.63 4.03 6 0.43 min Before 5 542.50 6 81.97 mm3

1.13 6 0.84 mm 1.68 6 0.85 mm 4.49 6 2.10 8.30 6 4.65 min Before 5 571.34 6 132.05 mm3

,.05 ,.05 ,.05 ,.05 ,.05

After 5 487.87 6 74.70 mm3

After 5 533.16 6 133.12 mm3

Measurement Global coronal deviation Global apical deviation Angular deﬂection Operation time Volume of tooth structure

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FIGURE 4 – The drilling trajectory obtained in the postoperative CBCT scan in the FH group in the (A ) sagittal and (B ) coronal planes and in the DNS group in the (C ) sagittal and coronal (D ) planes. operator should always verify the CBCT scan for patient motion distortion. Another limitation of the DNS technique is the X-clip with 3 radiopaque fiducials must be left in position on the opposite side from the procedure in the opposing arch. Patients who are either edentulous in that region or who have severe tooth mobility may not be candidates for this procedure. Finally, the DNS requires the operator to look at the system display while working rather than directly visualizing the operating field under magnification. This can make the operator

feel uncomfortable during the first attempts. However, looking at the system display can be more ergonomic. Overall, the DNS has shown promise in clinical practice. Here, we demonstrate that the DNS is safe and can expedite fiber post removal without compromising tooth structure. The development of software for endodontic use is desired to improve endodontists’ experience. Future randomized clinical trials should compare the accuracy and efficiency of the dynamic and static navigation system using 3D-printed guides when

removing fiber posts from root canal–treated teeth. In conclusion, the DNS was more accurate and efficient in removing fiber posts from endodontically treated teeth than the FH technique.

ACKNOWLEDGMENTS Supported by the American Association of Endodontists, Foundation for Endodontics. The authors deny any conﬂicts of interest related to this study.

REFERENCES 1.

Schwartz RS, Robbins JW. Post placement and restoration of endodontically treated teeth: a literature review. J Endod 2004;30:289–301.

€lsmann M. A comparative in vitro study of different techniques for removal Haupt F, Pﬁtzner J, Hu of ﬁbre posts from root canals. Aust Endod J 2018;44:245–50.

Scotti N, Bergantin E, Alovisi M, et al. Evaluation of a simpliﬁed ﬁber post removal system. J Endod 2013;39:1431–4.

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Lindemann M, Yaman P, Dennison JB, et al. Comparison of the efﬁciency and effectiveness of various techniques for removal of ﬁber posts. J Endod 2005;31:520–2.

Abbott PV. Incidence of root fractures and methods used for post removal. Int Endod J 2002;35:63–7.

Parisi C, Valandro LF, Ciocca L, et al. Clinical outcomes and success rates of quartz ﬁber post restorations: a retrospective study. J Prosthet Dent 2015;114:367–72.

Peciuliene V, Rimkuviene J, Maneliene R, et al. Factors inﬂuencing the removal of posts. Stomatologija 2005;7:21–3.

Schwindling FS, Tasaka A, Hilgenfeld T, et al. Three-dimensional-guided removal and preparation of dental root posts-concept and feasibility. J Prosthodont Res 2020;64:104–8.

Chee W, Aloum A. Restoration of the anterior maxilla after thermal trauma as a sequela to post removal: a clinical report. J Prosthet Dent 2011;106:141–4.

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Valle Castan ~o S, Montiel-Company JM, et al. Effect of computer-aided Zubizarreta-Macho A, navigation techniques on the accuracy of endodontic access cavities: a systematic review and meta-analysis. Biology (Basel) 2021;10:212.

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Perez C, Finelle G, Couvrechel C. Optimisation of a guided endodontics protocol for removal of ﬁbre-reinforced posts. Aust Endod J 2020;46:107–14.

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nior G, Albuquerque RC, et al. Three-dimensional endodontic guide for Maia LM, Moreira Ju adhesive ﬁber post removal: a dental technique. J Prosthet Dent 2019;121:387–90.

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nior W, Toubes KM, et al. Endodontic guide for the conservative removal of Maia LM, Bambirra Ju a ﬁber-reinforced composite resin post. J Prosthet Dent 2021.

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Tahmaseb A, Wismeijer D, Coucke W, et al. Computer technology applications in surgical implant dentistry: a systematic review. Int J Oral Maxillofac Implants 2014;29:25–42.

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Chong BS, Dhesi M, Makdissi J. Computer-aided dynamic navigation: a novel method for guided endodontics. Quintessence Int 2019;50:196–202.

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Edelmann C, Wetzel M, Knipper A, et al. Accuracy of computer-assisted dynamic navigation in implant placement with a fully digital approach: a prospective clinical trial. J Clin Med 2021;10:1808.

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Dianat O, Gupta S, Price JB, et al. Guided endodontic access in a maxillary molar using a dynamic navigation system. J Endod 2021;47:658–62.

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Gambarini G, Galli M, Morese A, et al. Precision of dynamic navigation to perform endodontic ultraconservative access cavities: a preliminary in vitro analysis. J Endod 2020;46:1286–90.

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Jain SD, Carrico CK, Bermanis I. 3-Dimensional accuracy of dynamic navigation technology in locating calciﬁed canals. J Endod 2020;46:839–45.

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Dianat O, Nosrat A, Tordik PA, et al. Accuracy and efﬁciency of a dynamic navigation system for locating calciﬁed canals. J Endod 2020.

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Dianat O, Nosrat A, Mostouﬁ B, et al. Accuracy and efﬁciency of guided root-end resection using a dynamic navigation system: a human cadaver study. Int Endod J 2021;54:793–801.

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Gambarini G, Galli M, Stefanelli LV, et al. Endodontic microsurgery using dynamic navigation system: a case report. J Endod 2019;45:1397–402.

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Jain SD, Carrico CK, Bermanis I, et al. Intraosseous anesthesia using dynamic navigation technology. J Endod 2020;46:1894–900.

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Block MS, Emery RW, Cullum DR, et al. Implant placement is more accurate using dynamic navigation. J Oral Maxillofac Surg 2017;75:1377–86.

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Naumann M, Koelpin M, Beuer F, et al. 10-year survival evaluation for glass-ﬁber-supported postendodontic restoration: a prospective observational clinical study. J Endod 2012;38:432–5.

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Schermeier O, Lueth T, Cho C, et al. The precision of the RoboDent system — an in vitro study. In: Lemke HU, Inamura K, Doi K, editors. CARS 2002 Computer Assisted Radiology Surgery. Berlin, Heidelberg:: Springer Berlin Heidelberg; 2002. p. 947–52.

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Block MS, Emery RW, Lank K, et al. Implant placement accuracy using dynamic navigation. Int J Oral Maxillofac Implants 2017;32:92–9.

28.

Golob Deeb J, Bencharit S, Carrico CK, et al. Exploring training dental implant placement using computer-guided implant navigation system for predoctoral students: a pilot study. Eur J Dent Educ 2019;23:415–23.

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29.

Arukaslan G, Aydemir S. Comparison of the efﬁcacies of two different ﬁber post-removal systems: a micro-computed tomography study. Microsc Res Tech 2019;82:394–401.

30.

Capar ID, Saygili G, Ergun H, et al. Effects of root canal preparation, various ﬁlling techniques and retreatment after ﬁlling on vertical root fracture and crack formation. Dent Traumatol 2015;31:302–7.

31.

Buchgreitz J, Buchgreitz M, Mortensen D, et al. Guided access cavity preparation using cone-beam computed tomography and optical surface scans - an ex vivo study. Int Endod J 2016;49:790–5.

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Zehnder MS, Connert T, Weiger R, et al. Guided endodontics: accuracy of a novel method for guided access cavity preparation and root canal location. Int Endod J 2016;49:966–72.

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BASIC RESEARCH – TECHNOLOGY

The Osteogenic Assessment of Mineral Trioxide Aggregate– based Endodontic Sealers in an Organotypic Ex Vivo Bone Development Model ABSTRACT Introduction: Mineral trioxide aggregate (MTA)-based sealers are endodontic materials with widespread success in distinct clinical applications, potentially embracing direct contact with the bone tissue. Bone response to these materials has been traditionally addressed in vitro. Nonetheless, translational data are limited by the absence of native cell-to-cell and cell-tomatrix interactions that hinder the representativeness of the analysis. Ex vivo organotypic systems, relying on the culture of explanted biological tissues, preserve the cell/tissue composition, reproducing the spatial and organizational in situ complexity. This study was grounded on an innovative research approach, relying on the assessment of an ex vivo organotypic bone tissue culture system to address the osteogenic response to 3 distinct MTA-based sealers. Methods: Embryonic chick femurs were isolated and grown ex vivo for 11 days in the presence of MTA Plus (Avalon Biomed Inc, Bradenton, FL), ProRoot MTA (Dentsply Tulsa Dental, Hohnson City, Germany), Biodentine (Septodont, Saint Maurdes Fosses, France), or AH Plus (Dentsply Sirona, Konstanz, Germany); the latter was used as a control material. Femurs were characterized by histologic, histochemical, and histomorphometric analysis. Gene expression assessment of relevant osteogenic markers was conducted by quantitative polymerase chain reaction. Results: All MTA-based sealers presented an enhanced osteogenic performance compared with AH Plus. Histochemical and histomorphometric analyses support the increased activation of the osteogenic program by MTA-based sealers, with enhanced collagenous matrix deposition and tissue mineralization. Gene expression analysis supported the enhanced activation of the osteogenic program. Comparatively, ProRoot MTA induced the highest osteogenic functionality on the characterized femurs. Conclusions: MTA-based sealers enhanced the osteogenic activity within the assayed organotypic bone model, which was found to be a sensitive system for the assessment of osteogenic modulation mediated by endodontic sealers. (J Endod 2021;47:1461–1466.)

KEY WORDS Bone; ex vivo; mineral trioxide aggregate; osteogenesis

Mineral trioxide aggregate (MTA)-based sealers are endodontic materials with a composition based on calcium silicate that have found widespread clinical success in modern dentistry1. Given the adequate sealing capability, biocompatibility, and bioactivity with dental and periapical tissues, MTA-based sealers have been used for distinct clinical applications including apexiﬁcation, root canal and root-end ﬁlling, and repair of perforations and root fractures, broadly embracing direct contact with the bone tissue2. The setting of MTA-based materials is initiated in a moist environment, forming a hydrated matrix with embedded calcium hydroxide and leading to a pH increase3. Calcium, silicate, and hydroxide ions are released, potentially modulating the local cellular milieu3. The alkaline pH, which nurtures antibacterial and antifungal activity, also promotes the precipitation of phosphate ions and the deposition of an apatiterich layer that provides an effective biological seal4, further modulating the osteogenic response5.

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Pedro S. Gomes, DDS, MSc, PhD,*† Bruna Pinheiro, BSc, MSc,* Bruno Colaço, DVM, PhD,‡§ and Maria H. Fernandes, BSc, PharmD, PhD*†

SIGNIFICANCE MTA-based sealers enhanced the osteogenic tissue activity, increasing the gene expression, collagenous matrix deposition, and extracellular matrix mineralization, with ProRoot MTA inducing the highest functionality compared with a resin-based sealer.

From the *BoneLab–Laboratory for Bone Metabolism and Regeneration, Faculty of Dental Medicine and †Associated Laboratory for Green Chemistry/Network of Chemistry and Technology (LAQV/ REQUIMTE), University of Porto, Porto, Portugal; ‡Department of Zootechnics, University of Tras-os-Montes e Alto Douro, Vila Real, Portugal; and §Center for the Research and Technology of AgroEnvironmental and Biological Sciences, University of Tras-os-Montes e Alto Douro, Vila Real, Portugal Address requests for reprints to Dr Pedro S. Gomes, BoneLab – Laboratory for Bone Metabolism and Regeneration, Faculty of Dental Medicine, U. Porto R. Dr. Manuel Pereira da Silva, 4200-393 Porto, Portugal. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.006

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Bone response to MTA-based sealers has been thoroughly addressed in vitro, as recently reviewed5,6. These materials induce an adequate cell/tissue response, stimulating proliferation and enhancing the osteogenic differentiation of distinct precursor populations7,8. Nonetheless, and even if cell culture studies have been recognized as a standard to provide relevant information on the functionality of cell populations, translational data are limited by the absence of native 3dimensional cell-to-cell and cell-to-matrix interactions that hinder the representativeness of the system9. Ex vivo organotypic systems, relying on the culture of explanted tissues, were developed as transition models between in vitro and in vivo research, offering a relatively cost-effective and ethically acceptable option10. Regarding bone, cultured explants preserve the tissue composition, reproducing the spatial and organizational in situ complexity and allowing the study of tissue growth and remodeling11,12. Ex vivo organotypic bone models have been used with success for the biological evaluation of biomaterials and tissue engineering strategies, highly correlating with in vivo data13–15. In this frame, and to the best of the authors’ knowledge, no studies have previously addressed the biological response of endodontic sealers within ex vivo systems. In this study, we established an innovative research approach, relying on an ex vivo organotypic bone tissue system (ie, the embryonic chick femur) to assess the osteogenic response to 3 distinct MTA-based sealers, focusing on the characterization of the cellular functionality, cell-matrix interactions, and matrix mineralization through histochemical, histomorphometric, and gene expression analysis. A resin-based sealer was used as a reference control.

MATERIALS AND METHODS Endodontic Sealer Preparation The following sealers were used: MTA Plus, ProRoot MTA, Biodentine, and AH Plus, the later as a reference control. All sealers were manipulated in accordance with the manufacturers’ instructions under sterile conditions. After mixing, a mass of 2 g was hom*ogeneously distributed over the surface of 6-well tissue culture plates (Corning, Corning, NY) and incubated in a humidiﬁed environment with 5% CO2 at 37 C for 24 hours.

Embryonic Chick Femur Isolation Fertilized chick eggs (Gallus domesticus) were incubated in an Octagon 40 ECO rotating egg incubator (Brinsea, Weston-Super-Mare, UK)

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at 37.5 C and 50% humidity. On embryonic day 11, embryos were euthanized, and femurs were dissected upon the removal of soft tissue.

Organotypic Culture of Femurs in the Presence of Endodontic Sealers Isolated femurs were washed in alphaminimum essential culture medium (Minimum Essential Media; Gibco, Dublin, Ireland) and transferred into culture inserts (Netwell Insert, Corning). Inserts were placed in culture plates in which the sealers were distributed and allowed to set for 24 hours. Afterward, 1 mL complete culture medium (alpha-minimum essential medium supplemented with 100 U/ mL penicillin, 100 mg/mL streptomycin, and 50 mg/mL ascorbic acid; all from Gibco) was added to each well, and femurs were incubated at 37 C in a humidiﬁed atmosphere of 5% CO2 at the air/liquid interphase for 11 days, with daily changes of culture medium.

Histologic Preparation, Histochemical Staining, and Tissue Imaging At the end of the culture period, femurs were washed and fixed in 4% paraformaldehyde for 24 hours. Femurs were dehydrated in graded alcohols, cleared in Histo-clear (National Diagnostics, Hull, United Kingdom), and embedded in paraffin in an automated tissue processor (STP 120 Spin Tissue Processor; Thermo Scientific, Waltham, MA). Samples were sectioned (7 mm thickness) with a micrometer (Microm HM 335 E, Thermo Scientific) and transferred to glass slides. Tissue sections were stained with alcian blue/ sirius red, Masson trichrome, or von Kossa staining as described previously16,17. Images were captured using an Axiolab5 microscope and Axiocam208c imaging system (Zeiss, Oberkochen, Germany) (n 5 8 per condition, with a total of 6 sections from each femur/ histologic stain).

Histomorphometric Measurements The quantiﬁcation of sirius red– and von Kossa–positive structures in histologic sections was calculated as a proportion of the total diaphysis area based on color thresholds on ImageJ software (version 1.53 b; National Institutes of Health, Bethesda, MD). Fluorescent imaging of sirius red staining was performed in a Celena S digital imaging system with a mCherry ﬁlter cube (Ex580/25, Em645/ 75; Logos Biosystems, Gyeonggi-do, South Korea). Imaging of von Kossa staining was conducted as previously described.

Assessment of the Osteogenic Gene Expression Femurs were ground to powder in the presence of liquid nitrogen. Total RNA was isolated from the tissue with Trizol reagent (Invitrogen, San Diego, CA) according to the manufacturer’s protocol. The total RNA concentration and purity were assessed by ultraviolet spectrophotometry in the Synergy HT ELISA reader (Biotek, Winooski, VT). RNA was reverse transcribed into complementary DNA with the iScript Adv cDNA Kit (BioRad, Hercules, CA) in accordance with the manufacturer’s instructions. Quantitative polymerase chain reaction analysis was conducted in the Bio-Rad iQ5 real-time polymerase chain reaction system (Bio-Rad) using the SYBR Premix Ex Taq kit (Takara, Otsu, Shiga, Japan). The following optimized primers were acquired from BioRad: Gapdh (Unique Assay ID: qGgaCED0029996), Runx2 (Unique Assay ID: qGgaCID0019198), Col1a2 (Unique Assay ID: qGgaCED0025365), Spp1 (Unique Assay ID: qGgaCED0023869), Bmp2 (Unique Assay ID: qGgaCID0027472), and Sost (Unique Assay ID: qGgaCED0029174). The relative gene expression level was normalized to the internal control (Gapdh) based on the 22DDCt method, and the results were presented as the fold change relative to the control.

Statistical Analysis Four independent experiments were performed. In quantitative assays, each point represents the mean 6 standard error of 6 replicates. The normality of the data was evaluated by the Shapiro-Wilk test. For normal data sets, 1-way analysis of variance was performed followed by multiple comparisons using the Tukey test. For nonparametric data sets, the Kruskal-Wallis test was performed followed by multiple comparisons using Dunn tests. P values .05 were considered signiﬁcant.

RESULTS The biologic response to the 3 MTA-based sealers was assessed within the organotypic embryonic chick femur culture system for 11 days. AH Plus was used as a control reference material. At the end of the culture period, assessment of the tissue response was evaluated by histochemical, histomorphometric, and gene expression analysis. Femurs grown in the presence of AH Plus presented a response similar to the negative control (femurs grown in the absence of sealers, data not shown). An effective osteogenic activation, marked by the

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FIGURE 1 – Histochemical staining of chick femurs grown for 11 days in the presence of endodontic sealers. (A ) Micrographs of alcian blue/sirius red (left ) and Masson trichrome staining (right ), at low and high magniﬁcation. (B ) A micrograph of the ﬂuorescent assessment of the sirius red stain and (left ) morphometric determination of the stained area (right ). Histomorphometric determination of the percentage of collagen-positive area. *P , .05 compared with AH Plus. Images are representative of the indicated conditions and depict the mid-diaphysis region of the femurs.

deposition of a collagenous matrix at the periosteal regions, was evidenced by the red coloration within the alcian blue/sirius red

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staining and corresponding blue coloration within the Masson trichrome staining. Femurs grown in the presence of MTA-based sealers

(ie, MTA Plus, ProRoot, and Biodentine) presented an increased osteogenic activation as sustained by the increased area of

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FIGURE 2 – (Left ) Von Kossa histochemical staining of chick femurs grown for 11 days in the presence of endodontic sealers and morphometric determination of the stained area. (Right ) Histomorphometric determination of the percentage of the mineralized area. *P , .05 compared with AH Plus; **P , .05 compared with MTA Plus and Biodentine. Images are representative of the indicated conditions and depict the mid-diaphyseal region of the femur.

collagenous matrix deposition demonstrated by both histochemical stains. Additionally, a thicker and more developed trabecular organization, characterized by an open cell porous network arrangement composed of rods and plates interspersed with spaces and voids, can be identified (Fig. 1, insets). Comparatively, femurs developing in the presence of ProRoot MTA presented an increased area of collagenous deposition as well as a more mature trabecular organization compared with femurs grown in the presence of MTA Plus and Biodentine. Additionally, sirius red-positive regions were identified on fluorescence imaging. Comparatively, femurs grown in the presence of MTA-based sealers presented a significantly higher area of collagenous matrix deposition. Whether no differences were found between MTA-based

sealers, a trend for an increased value was identified with ProRoot. The ability to form a mineralized extracellular matrix was evaluated by von Kossa staining (Fig. 2). In femurs grown in the presence of AH Plus, a discrete layer of mineralized tissue was identified, with femurs grown in the presence of MTA-based sealers presenting a significantly higher mineralized area and a more mature trabecular organization. Histomorphometric determination revealed significantly higher levels of mineralized tissue for femurs grown in the presence of MTA-based sealers, with ProRoot MTA presenting the highest values. Femurs were further analyzed for the expression of relevant genes of the osteogenic program (Fig. 3). Femurs grown in the presence of MTA-based sealers presented

FIGURE 3 – Osteogenic gene expression of chick femurs grown for 11 days in the presence of endodontic sealers. Values are presented as mean 6 standard deviation. The expression of the reference control condition (AH Plus) was set at 1. *P , .05 compared with AH Plus; **P , .05 compared with MTA Plus and Biodentine.

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signiﬁcantly higher levels of Runx2, Col1a1, Spp1, and Bmp2 and signiﬁcantly lower levels of Sost than femurs of the control condition. Comparatively, cultured femurs in the presence of ProRoot presented the highest Runx2, Col1a1, Spp1, and Bmp2 levels and the lowest Sost expression level.

DISCUSSION In the present study, the osteogenic potential of distinct MTA-based sealers was assessed in an organotypic embryonic chick bone development model. Ex vivo systems allow the study of the cells’ functionality and cell-matrix interactions as found in the native tissue structure, preserving its physical and spatial complexity and improving the translatability between in vitro and in vivo research18. To the best of the authors’ knowledge, the osteogenic potential of endodontic sealers has not been previously addressed on bone organotypic models. All MTA-based sealers presented enhanced osteogenic performance compared with AH Plus. Histochemical and histomorphometric analyses support the increased activation of the osteogenic program by the MTA-based sealers, with enhanced collagenous matrix deposition and tissue mineralization. Comparatively, ProRoot MTA induced the highest osteogenic functionality. The osteoinductive ability of these materials is presumably related to the interfacial dissolution, precipitation, and ionexchange reactions that occur in the presence of a ﬂuid, increasing the availability of calcium and silicate ions into the microenvironment19. Both calcium20 and

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silicon21 induce a positive effect on osteoblastic functionality, synergizing to an enhanced cellular differentiation, matrix deposition, and mineralization22,23. Accordingly, previous in vitro studies support a high biocompatibility of MTA-based sealers and the ability to induce osteogenic functionality as assessed by the alkaline phosphatase activity, mineralized nodule formation, and osteogenic gene expression compared with resin-based sealers24–26. To further detail the osteogenic modulation capability, the expression of relevant genes was assayed. Runx2 is a transcription factor and a master regulator of the osteogenic program; Col1a1 codes for the major structural protein of the bone matrix; Spp1 codes for osteopontin, a major regulator of the mineralization process; Bmp2 codes for the hom*onymous protein with a potent osteogenic activity; and Sost codes for sclerostin an inhibitor of the bone formation27,28. Again, MTA-based sealers induced, transversely, the expression of Runx2, Col1a1, Spp1, and Bmp2 and decreased the Sost level compared with AH

Plus, with ProRoot inducing the most significant differences. The attained profile sustains an enhanced activation of the osteogenic gene program by the MTA-based sealers29. This process, as previously demonstrated in vitro, is presumably mediated by the activation of the mitogen-activated protein kinase cascades30,31, signaling pathways sensitive to the variation of calcium32 and silicon33 ionic levels, in the microenvironment. Despite the sustained osteogenic enhancement of the MTA-based sealers, differences between materials were verified. Dissimilarities may be attributed to the compositional differences of the sealers as well as their dissolution rate in the experimental microenvironment. Whether the sealers share most of the compositional elements, differences exist namely on the liquid component of the systems. MTA Plus and Biodentine include water-soluble polymers, and Biodentine includes calcium chloride– hydration accelerators, which, as previously demonstrated in vitro, may hinder the biological response34.

Overall, MTA-based sealers enhanced the osteogenic activity within the assayed organotypic bone development system, inducing the deposition of the collagenous matrix and enhancing tissue mineralization. The osteogenic gene expression proﬁle was induced in agreement. ProRoot MTA displayed the highest osteogenic induction compared with Biodentine and MTA Plus. The organotypic embryonic chick femur model was recognized as a sensitive system for the assessment of the osteogenic modulation mediated by endodontic sealers, supporting its usability as a cost-considerate, easily manipulated, and representative system, further ensuring the in situ dynamics of the cellular and matrix bone components.

ACKNOWLEDGMENTS ~o Supported by PT national funds (Fundaça rio da para a Ciência e Tecnologia and Ministe Ciência, Tecnologia e Ensino Superior) through the project UIDB/50006/2020. The authors deny any conflicts of interest related to this study.

REFERENCES

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Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature review—part III: clinical applications, drawbacks, and mechanism of action. J Endod 2010;36:400–13.

Torabinejad M, Parirokh M, Dummer P. Mineral trioxide aggregate and other bioactive endodontic cements: an updated overview–part II: other clinical applications and complications. Int Endod J 2018;51:284–317.

Chen Y, Ho C, Huang T, et al. The ionic products from mineral trioxide aggregate–induced odontogenic differentiation of dental pulp cells via activation of the Wnt/b-catenin signaling pathway. J Endod 2016;42:1062–9.

Gandolﬁ M, Taddei P, Tinti A, Prati C. Apatite-forming ability (bioactivity) of ProRoot MTA. Int Endod J 2010;43:917–29.

Babaki D, Yaghoubi S, Matin M. The effects of mineral trioxide aggregate on osteo/odontogenic potential of mesenchymal stem cells: a comprehensive and systematic literature review. Biomater Investig Dent 2020;7:175–85.

jo P, da Silveira M, et al. Comparison of the biocompatibility of de Oliveira N, de Souza Arau calcium silicate-based materials to mineral trioxide aggregate: systematic review. Eur J Dent 2018;12:317–26.

Wang Y, Li J, Song W, Yu J. Mineral trioxide aggregate upregulates odonto/osteogenic capacity of bone marrow stromal cells from craniofacial bones via JNK and ERK MAPK signalling pathways. Cell Prolif 2014;47:241–8.

Youssef A, Emara R, Taher M, et al. Effects of mineral trioxide aggregate, calcium hydroxide, Biodentine and Emdogain on osteogenesis, odontogenesis, angiogenesis and cell viability of dental pulp stem cells. BMC Oral Health 2019;19:133.

Bellido T, Delgado-Calle J. Ex vivo organ cultures as models to study bone biology. JBMR Plus 2020;4. https://doi.org/10.1002/jbm4.10345.

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Staines K, Brown G, Farquharson C. The ex vivo organ culture of bone. In: Idris A, editor. Bone Research Protocols. New York: Humana Press; 2019. p. 199–215.

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Ehnert S, Rinderknecht H, Aspera-Werz R, et al. Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models. Arch Toxicol 2020;94:3937–58.

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Marino S, Staines K, Brown G, et al. Models of ex vivo explant cultures: applications in bone research. Bonekey Rep 2016;5:818.

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Marshall K, Kanczler J, Oreffo R. Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering. J Tissue Eng 2020;11:1–25.

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Iordachescu A, Williams R, Hulley P, Grover L. Organotypic culture of bone-like structures using composite ceramic-ﬁbrin scaffolds. Curr Protoc Stem Cell Biol 2019;48:e79.

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Srinivasaiah S, Musumeci G, Mohan T, et al. A 300 mm organotypic bone slice culture model for temporal investigation of endochondral osteogenesis. Tissue Eng Part C Methods 2019;25:197– 212.

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Barros J, Ferraz M, Azeredo J, et al. Alginate-nanohydroxyapatite hydrogel system: optimizing the formulation for enhanced bone regeneration. Mater Sci Eng C Mater Biol Appl 2019;105:109985.

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Barros JA, Rodrigues de Melo LD, Reis da Silva RA, et al. Encapsulated bacteriophages in alginate-nanohydroxyapatite hydrogel as a novel delivery system to prevent orthopedic implantassociated infections. Nanomedicine 2020;24:102145.

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Cramer E, Ito K, Hofmann S. Ex vivo bone models and their potential in preclinical evaluation. Curr Osteoporos Rep 2021;19:75–87.

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Prati C, Gandolﬁ M. Calcium silicate bioactive cements: biological perspectives and clinical applications. Dent Mater 2015;31:351–70.

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Viti F, Landini M, Mezzelani A, et al. Osteogenic differentiation of MSC through calcium signaling activation: transcriptomics and functional analysis. PLoS One 2016;11:0148173.

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Kim E-J, Bu S-Y, Sung M-K, Choi M-K. Effects of silicon on osteoblast activity and bone mineralization of MC3T3-E1 cells. Biol Trace Elem Res 2013;152:105–12.

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Moorthi A, Parihar P, Saravanan S, et al. Effects of silica and calcium levels in nanobioglass ceramic particles on osteoblast proliferation. Mater Sci Eng C Mater Biol Appl 2014;43:458–64.

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Khan A, Saleem M, Afzal A, et al. Bioactive behavior of silicon substituted calcium phosphate based bioceramics for bone regeneration. Mater Sci Eng C Mater Biol Appl 2014;35:245–52.

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Lee B-N, Hong J-U, Kim S-M, et al. Anti-inﬂammatory and osteogenic effects of calcium silicate– based root canal sealers. J Endod 2019;45:73–8.

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M, Paula-Silva F, Faccioli L, et al. Expression of mineralization markers during pulp Daltoe response to Biodentine and mineral trioxide aggregate. J Endod 2016;42:596–603.

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Costa F, Gomes P, Fernandes M. Osteogenic and angiogenic response to calcium silicate– based endodontic sealers. J Endod 2016;42:113–9.

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Komori T. Regulation of proliferation, differentiation and functions of osteoblasts by Runx2. Int J Mol Sci 2019;20:1694.

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Jensen E, Gopalakrishnan R, Westendorf J. Regulation of gene expression in osteoblasts. Biofactors 2010;36:25–32.

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Salhotra A, Shah H, Levi B, Longaker M. Mechanisms of bone development and repair. Nat Rev Mol Cell Biol 2020;8:1–6.

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Rathinam E, Rajasekharan S, Chitturi R, et al. Gene expression proﬁling and molecular signaling of dental pulp cells in response to tricalcium silicate cements: a systematic review. J Endod 2015;41:1805–17.

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Rathinam E, Rajasekharan S, Chitturi R, et al. Gene expression proﬁling and molecular signaling of various cells in response to tricalcium silicate cements: a systematic review. J Endod 2016;42:1713–25.

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Woo S-M, Hwang Y-C, Lim H-S, et al. Effect of nifedipine on the differentiation of human dental pulp cells cultured with mineral trioxide aggregate. J Endod 2013;39:801–5.

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Odatsu T, Azimaie T, Velten M, et al. Human periosteum cell osteogenic differentiation enhanced by ionic silicon release from porous amorphous silica ﬁbrous scaffolds. J Biomed Mater Res A 2015;103:2797–806.

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Kang J-Y, Lee B-N, Son H-J, et al. Biocompatibility of mineral trioxide aggregate mixed with hydration accelerators. J Endod 2013;39:497–500.

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BASIC RESEARCH – TECHNOLOGY mur Kılıç, DDS,* Yag lu, DDS, Emrah Karatasxlıog PhD,* and Mehmet Emin Kaval, DDS, PhD†

The Effect of Root Canal Preparation Size and Taper of Middle Mesial Canals on Fracture Resistance of the Mandibular Molar Teeth: An In Vitro Study ABSTRACT Introduction: The aim of the present study was to investigate the influence of root canal preparation size and taper of middle mesial (MM) canals on fracture resistance of mandibular molars. Methods: Fifty-five mandibular molar teeth having an MM canal were selected based on the cone-beam computed tomographic analysis. After the decoronation and distal root separation procedure, the lengths of the mesial roots were standardized to 13 mm. The specimens were randomly distributed into 5 groups (n 5 11). Mesiobuccal and mesiolingual canals were prepared up to size 30.06 using VDW.ROTATE rotary files (VDW, Munich, Germany). The MM canal was prepared up to size 25.04, 25.06, 30.04, and 30.06, respectively. No preparation was done in the MM canal in the control group. After the irrigation protocol, the canals were obturated with the single-cone technique. A thin layer of siliconecoated specimens was embedded in acrylic resin and subjected to a fracture strength test by a universal testing machine. A vertical force was applied to the roots until they fracture. Statistical analysis was performed with 1-way analysis of variance and post hoc Duncan tests (P 5 .05). Results: There was no significant difference between group 25.04 and the control group, but the fracture strengths of these groups were found to be significantly higher than that of groups 25.06, 30.04, and 30.06 (P , .05). Conclusions: Within the limitations of this study, we concluded that increasing the apical diameter and taper in the MM canal reduces the fracture strength of mandibular molar teeth. Among the tested instrumentation sizes, fracture strength decreased significantly when greater than 25.04 instrumentation sizes were chosen. (J Endod 2021;47:1467–1471.)

SIGNIFICANCE This study investigated the inﬂuence of the instrumentation size of middle mesial canals on the fracture strength of mandibular molars. Among the experimental groups, instrumentation of the middle mesial canals greater than #25.04 signiﬁcantly decreased the fracture strength.

KEY WORDS Fracture resistance; instrumentation; middle mesial canal; root fracture

Clinicians should be able to determine the ideal root canal preparation size that will allow for both adequate disinfection and successful obturation without weakening the tooth or root due to excessive dentin loss. There is no consensus in the literature regarding the optimal apical preparation size and taper. Prior studies have shown that by increasing the apical size and taper, debris and bacteria can be removed from the root canal more easily, irrigation solutions prove more effective in relation to the working length, and the lateral and vertical forces generated during ﬁlling can be better distributed1–3. In addition, the use of high tapered rotary instruments can cause changes in both the canal volume and geometry, which increases the risk of vertical root fracture due to the removal of excessive dentin from the middle and coronal third of the root canal4–6. A number of studies have concluded that microcracks and fractures are associated with excessive root canal preparation7–9. The microcracks that occur during root canal preparation can lead to root fractures due to the impact of the occlusal forces10. These root fractures are considered 1 of the most serious complications associated with root canal treatment, and they can necessitate tooth extraction11–15. Thus, the use of less tapered instruments that allow the apical size to be

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From the *Department of Endodontology, Faculty of Dentistry, Izmir Katip Çelebi University, Izmir, Turkey; and †Department of Endodontology, Faculty of Dentistry, Ege University, Izmir, Turkey Address requests for reprints to Dr Mehmet Emin Kaval, Department of Endodontology, School of Dentistry, Ege University, 35100 Izmir, Turkey. E-mail address: mehmetkaval@hotmail. com 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.002

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increased while protecting the dentin has been recommended, especially in terms of the coronal part of the canal8,16,17. The presence of a third canal in the mesial root of mandibular ﬁrst molars has been reported in many studies18–22. The MM canal (Fig. 1) could be frequently detected in recent years because of developments in both endodontic microscopes and scanning systems23,24. To the best of our knowledge, in the literature, the effect of the presence and preparation of the MM canal on the fracture strength of mandibular molars was investigated only by Kelesx et al24; however, the relationship between the preparation size and taper in the middle mesial (MM) canals and the fracture strength of the tooth has not been evaluated previously. Therefore, the aim of the present study was to investigate the inﬂuence of root canal preparation size and taper of MM canals on fracture resistance of the mesial roots of mandibular molar teeth as well as to minimize the effects of all other relevant factors under in vitro experimental conditions.

MATERIALS AND METHODS Sample Size Calculation Based on data obtained in a previous study5, a power analysis comprising the chi-square test and variance analysis (G*Power 3.1 software; Heinrich Heine University, Dusseldorf, Germany) was performed wherein a 5 .05 and b 5 .95. The calculation revealed the minimum sample size for each group to be 8.

Sample Selection The protocol for the present study was approved by the local university ethics committee (R&D no. 2019/225). Four hundred forty-three mandibular molar teeth with complete and separate root development and without curvature (,10 ) that had been extracted for periodontal and prosthetic reasons were collected. After the extraction procedure, the teeth were stored in 0.1% thymol solution. All the samples were scanned using a cone-beam computed tomographic unit (New Tom 5G; QR Srl, Verona, Italy). The voxel size and the slice thickness were 0.2 mm and 0.1 mm, respectively. The imaging parameters were as follows: 110 kVp, 1–20 mA, and a 6 ! 6 mm ﬁeld of view. The images were reviewed using computer software (NNT, QR Srl) on a medical monitor (Radiforce MX270 W; Eizo Radiforce, Ishikawa, Japan) in a dark room. A total of 55 mandibular molar teeth with MM canals were selected and examined under a stereomicroscope at !16 magniﬁcation in

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order to exclude any teeth with cracks or root fractures.

Sample Preparation and Grouping The distal root and the coronal portion of all samples were separated from the mesial root under water cooling. The mesial root length was standardized to 13 mm. After the separation, all samples were reexamined using a stereomicroscope (!16 magnification) to check for any fractures that may have occurred during the separation process. Before grouping, the mesiodistal (MD) and buccolingual (BL) diameters of roots were measured at 13 mm from the anatomic apex using a digital caliper. In addition, each root was weighed using a sensitive precision balance. Four experimental groups (ie, group 25.04, group 25.06, group 30.04, and group 30.06) and 1 control group, which each contained 11 samples, were created as follows: Group 25.04 (n 5 11): the mesiobuccal (MB) and mesiolingual (ML) root canals were enlarged up to #30.06, whereas the MM canal was enlarged up to #25.04. Group 25.06 (n 5 11): the MB and ML root canals were enlarged up to #30.06, whereas the MM canal was enlarged up to #25.06. Group 30.04 (n 5 11): the MB and ML root canals were enlarged up to #30.06, whereas the MM canal was enlarged up to #30.04. Group 30.06 (n 5 11): the MB and ML root canals were enlarged up to #30.06, whereas the MM canal was enlarged up to #30.06. Control group (n 5 11): the MB and ML root canals were enlarged up to #30.06. No preparation was performed with regard to the MM canal. In all of the groups, the mechanical preparation of the root canals was performed using hand and rotary files. Initially, the root canals were prepared using a size 06 K-file (Dentsply Sirona, York, PA), and patency was established up to a size 10 K-file (Dentsply Sirona). The working length was determined 1 mm short of the apical foramen. The root canals were then sequentially instrumented using VDW.ROTATE nickel-titanium rotary systems (VDW, Munich, Germany) according to the manufacturer’s instructions (#15.04, #20.05, #25.04, #25.06, #30.04, and #30.06). Each file was only used 3 times. During the mechanical preparation, a total of 10 mL 2.5% sodium hypochlorite was used as an irrigant. The final flush was performed with 2 mL 2.5% sodium

hypochlorite, 2 mL 17% EDTA, and 2 mL distilled water using a 27-G irrigation needle (NaviTip 27-G needle; Ultradent, South Jordan, UT). The root canals were dried and ﬁlled according to the single-cone method using AH Plus canal sealer (Dentsply Maillefer, Ballaigues, Switzerland) and gutta-percha. All the root canal preparations and obturations were performed by an experienced endodontist.

Fracture Test As in the previous study11, the root surfaces of all samples were covered with 0.2-mm-thick aluminum foil and then embedded into autopolymerizing acrylic (Imicryl, Konya, Turkey) blocks (15 mm in height and 20 mm in diameter). Any samples that were not suitable for vertical positioning were removed and embedded once again in acrylic blocks. After the polymerization process, the roots were removed from the acrylic blocks. The aluminum foil was cleaned, and a thin layer of silicone material was used to simulate the periodontal ligament. Samples were stored in appropriate conditions and then tested using a Universal Test Machine (AG-5 kNG; Shimadzu, Tokyo, Japan). The fracture resistance test applied a 0.5-mm/min constant acceleration force from the central point of the axial area, parallel to the long axis of the root. The forces that caused the fracture were recorded as newtons.

Statistical Analysis Data were analyzed using the SPSS 20.0 software package (IBM Corp, Armonk, NY) with a signiﬁcance threshold set at 5%. The Levene test and the Shapiro-Wilk test were performed to investigate the hom*ogeneity of the variances and the normality of the distributions, respectively, for all the gathered data (MD-BL dimensions, weight, and fracture resistance). One-way analysis of variance was used to evaluate the difference among the MD and BL dimensions and the weights of the samples. Statistical analysis for fracture resistance was performed using 1-way analysis of variance followed by the Duncan post hoc test (a 5 .05).

RESULTS Table 1 presents the mean values of the MDBL dimensions and the weights of the samples. The result of the test conﬁrmed the standardization of roots among groups in terms of the BL-MD dimensions and the weight (P . .05). The means of the forces that caused fracture in the samples are shown in Table 2. Comparisons using the Duncan test showed that there was no signiﬁcant difference

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FIGURE 1 – The axial and sagittal section of the MM canal in a mandibular molar. Yellow arrows dictate MM canals on the axial sections. MB, MM, and ML canals are shown with green arrows on sagittal sections.

between group 25.04 and the control group, but the fracture strength of both groups was found to be signiﬁcantly higher than that of groups 25.06, 30.04, and 30.06 (P , .05).

DISCUSSION In recent years, preparation with minimally invasive techniques has been recommended in root canal treatment. This approach aims to avoid errors caused by excess dentin removal during root canal preparation16. Studies in the literature have demonstrated the important role of dentin in the pericervical area in transferring forces to the alveolar bone25. Therefore, it is risky to lose dentin in the coronal and apical regions as a result of excessive enlargements. In the present study, to investigate the effect of MM canal preparation size and taper on fracture strength, a static loading method was used similarly to previous studies9,11,26,27. This method is reliable for measuring fracture strength because it is comparable9,11,26,27. It is known that trauma due to the extraction, dryness, storage environment, and storage time of the teeth affects the fracture strength of the extracted teeth4,27. In order to minimize the negative effects of the previously mentioned factors, the teeth were stored in 0.1% thymol solution after the extraction, and all specimens were examined under a stereomicroscope to

conﬁrm the absence of any crack or defect formation. The correct vertical position of the specimen in the acrylic block is important for the hom*ogeneous distribution of the applied force generated from the test machines in studies with static loading28. In this study, control of the vertical position of samples in acrylic blocks was provided with precision by using template models. Additionally, similar to previous studies29–32, the periodontal ligament was simulated with a thin layer of silicone impression material. Yared and Dagher33 reported that increasing the apical size from #25 to #40 does not reduce residual infected dentin. Besides, it is known that preparation with #25.06 results in a reduction of intracanal bacteria and creates clean dentin walls in the middle and coronal third of the root canal34. In another previous study, it was shown that there was no signiﬁcant difference in the residual infected dentin and smear layer at the apical third of root canals prepared with .04 and .06 taper in the same apical size5,35. In this study, the narrowest preparation size was chosen as #25.04 for the MM canal, which is known to be narrower than the MB and ML canals. According to the study by Kelesx et al24, the presence of the MM canal is not a predisposing factor in the decrease of fracture strength, but enlargement of the MM canal

signiﬁcantly decreased fracture strength. Keles et al35 reported in another study that the preoperative dentin thickness of the MM canal was thinner than the MB and ML root canals. The preparation in the MM canal could cause the already thin dentin walls to become thinner. In order to minimize the risk of strip perforation and vertical root fracture, a balance should be established between the preparation size and the dentin thickness. Sabeti et al9 and Zangbiari et al11, who evaluated the effect of root canals prepared with different apical sizes and conicity on fracture strength, reported that an increased taper decreased fracture strength. The results of the present study are consistent with the results of the aforementioned studies. However, our results cannot be compared with other studies because of the lack of information in the literature about the effect of the preparation size of the mandibular molar teeth on the MM canals on fracture strength. The results of the present study contradict those of Lam et al12, who reported that an increase in apical size does not decrease the fracture strength of the roots. It is thought that the use of spreaders to apply force may have made a difference in the results. Harvey et al3 reported that prepared canals with high conicity distributed the forces better in the apical third of the root. Although different approaches such as photoelastic

TABLE 1 - Mean Values of Mesiodistal (MD)-Buccolingual (BL) Dimensions and the Weights of the Samples Groups Control group (n 5 11) 25.04 (n 5 11) 25.06 (n 5 11) 30.04 (n 5 11) 30.06 (n 5 11)

Weight (means in g)

MD dimensions (means in mm)

BL dimensions (means in mm)

0.54 6 0.16 0.55a 6 0.14 0.54a 6 0.1 0.57a 6 0.18 0.53a 6 0.14

4.49 6 0.65 4.3b 6 0.58 4.24b 6 0.28 4.12b 6 0.5 4.04b 6 0.43

8.96c 6 0.51 8.85c 6 0.75 9.05c 6 0.63 9.24c 6 0.74 9.03c 6 0.51

The same superscript letters indicate statistical similarity (P . .05).

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TABLE 2 - The Maximum, Minimum, and Mean Values of the Forces that Cause Fracture on the Samples in Newtons Fracture resistance (n) Groups #Control group (n 5 11) #25.04 (n 5 11) #25.06 (n 5 11) #30.04 (n 5 11) #30.06 (n 5 11)

Maximum

Minimum

Mean ± standard deviation

910.19 814.92 680.33 609.98 681.65

383.76 388.13 143.62 115.33 301.72

563.42x 6 127.66 579.78x 6 139.62 445.37y 6159.10 418.68y 6 151.15 427.23y 6 127.66

CONCLUSION Within the limitations of this study, we concluded that increasing the apical diameter and taper in the MM canal reduces the fracture strength of mandibular molar teeth. Among the tested instrumentation sizes, fracture strength decreased signiﬁcantly when greater than 25.04 instrumentation sizes were chosen.

Different superscript letters indicate a statistically signiﬁcant difference (P , .05).

model analysis are useful in investigating the fracture strength of teeth, the limited similarity of the model material to tooth tissues may lead to differences in results. Because it is not possible to remove the crowns and distal roots of teeth during the clinical procedure, the fracture load data obtained in this study are not absolute and

can change. In addition, teeth are exposed to thermal, chemical, and physical variables in the oral environment. Therefore, results obtained in in vivo conditions may be adversely affected. With all of these limitations, it should be kept in mind that the aim of this study was to compare different groups in standard conditions.

ACKNOWLEDGMENTS The authors thank Professor Beyser Pisxkin for the editorial assistance, Dr Ferhan Elmalı for the assistance in the statistical analysis, and VDW Dental (Munich, Germany) for providing the instruments used in this study. The authors deny any conﬂicts of interest related to this study.

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Krikeli E, Mikrogeorgis G, Lyroudia K. In vitro comparative study of the inﬂuence of instrument taper on the fracture resistance of endodontically treated teeth: an integrative approach–based analysis. J Endod 2018;44:1407–11.

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Uzunoglu E, Aktemur S, Uyanik MO, et al. Effect of ethylenediaminetetraacetic acid on root fracture with respect to concentration at different time exposures. J Endod 2012;38:1110–3.

29.

Singla M, Aggarwal V, Logani A, Shah N. Comparative evaluation of rotary ProTaper, Proﬁle, and conventional stepback technique on reduction in Enterococcus faecalis colony-forming units and vertical root fracture resistance of root canals. J Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:105–10.

30.

Prado M, De Lima N, De Lima C, et al. Resistance to vertical root fracture of root ﬁlled teeth using different conceptual approaches to canal preparation. Int Endod J 2016;49:898–904.

31.

Souza EM, Calixto AM, Lima CN, et al. Similar inﬂuence of stabilized alkaline and neutral sodium hypochlorite solutions on the fracture resistance of root canal–treated bovine teeth. J Endod 2014;40:1600–3.

32.

Adorno CG, Yoshioka T, Suda H. The effect of root preparation technique and instrumentation length on the development of apical root cracks. J Endod 2009;35:389–92.

33.

Yared GM, Dagher FEB. Inﬂuence of apical enlargement on bacterial infection during treatment of apical periodontitis. J Endod 1994;20:535–7.

34.

Torabinejad M, Khademi AA, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170–5.

35.

Keles A, Keskin C, Alqawasmi R, et al. Evaluation of dentine thickness of middle mesial canals of mandibular molars prepared with rotary instruments: a micro-CT study. Int Endod J 2020;53:519–28.

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BASIC RESEARCH – TECHNOLOGY Mostafa M. A. Elkholy, BDS, MSc, DDSc,* Nawar Naguib Nawar, BDS, MSc, DDSc,* William Nguyen Ha, BDSc, PhD,† Shehabeldin Mohamed Saber, BDS, MSc, PhD,*‡ and HyeonCheol Kim, DDS, MS, PhD§

SIGNIFICANCE Conservative and truss access cavity may prolong the life span of endodontically treated teeth, whereas the canal taper has minimal inﬂuence on the life span. Stress distribution patterns suggest that a crack might be initiated on any part of the root surface.

From the *Department of Endodontics, Faculty of Dentistry, The British University in Egypt, Cairo, Egypt; †University of Adelaide, Adelaide Dental School, Adelaide, South Australia, Australia; ‡ Department of Endodontics, Ain Shams University, Cairo, Egypt; and §Department of Conservative Dentistry, Pusan National University School of Dentistry, Dental Research Institute, Dental and Life Science Institute, Gyeongnam, Korea Address requests for reprints to Dr Hyeon-Cheol Kim, Department of Conservative Dentistry, School of Dentistry, Pusan National University Geumo-ro 20, Mulgeum, Yangsan, Gyeongnam 50612, Korea., or Dr Mostafa M.A. Elkholy, Departement of Endodontics, Faculty of Dentistry, The British University in Egypt. El Sherouk City, Misr-Ismalia Road, Cairo, Egypt. E-mail addresses: mostafa.elkholy@bue. edu.eg or [emailprotected] or [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.009

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Impact of Canal Taper and Access Cavity Design on the Life Span of an Endodontically Treated Mandibular Molar: A Finite Element Analysis ABSTRACT Introduction: This study investigated the impact of different canal tapers and access cavity designs on the life span of endodontically treated mandibular first molars using the finite element method. Methods: Finite element analysis was performed on simulated models with 3 access cavity designs (traditional, conservative, and truss). The mesial canals were prepared to either constant tapers of 25/.04 and 25/.06 or a variable taper corresponding to the cumulative canal preparation shapes of TruNatomy Prime (Dentsply Sirona, Charlotte, NC) and ProTaper Gold F2 (Dentsply Sirona). The distal canals in all models had a 40/.04 preparation. Using occlusal fingerprint analysis, all models were subjected to cyclic occlusal loading until model failure. The number of cycles until failure, the location of failure, stress distribution patterns, and the maximum von Mises stresses were assessed. Results: The traditional access models showed a lower life span than the conservative and truss models regardless of the canal taper, whereas there was not a notable difference in the conservative and truss models. The stresses migrated apically along the root surface and remarkably on the mesial aspect of the mesial root and the furcation area’s outer surface. After root canal preparation with different tapers, there were no evident changes in the pattern and magnitude of the stresses distributed along the root surface. Conclusions: The life span of the tooth is affected more significantly by the access cavity design than the root canal preparation taper. Because stress patterns migrate apically rather than concentrate in the pericervical area, crack initiation and propagation might occur anywhere on the root surface. (J Endod 2021;47:1472–1480.)

KEY WORDS Canal taper; conservative access; ﬁnite element analysis; life span; stress concentration; truss access

Tooth fracture in endodontically treated teeth (ETT) has been identified as a significant cause of tooth loss1. When ETT are exposed to masticatory forces, the cumulative process of crack initiation and propagation may develop over time, leading to fatigue failure2. It has been shown that the long-term retention of a tooth and its resistance to fracture are affected by the amount of remaining sound tooth structure3,4. As such, the concept of minimally invasive endodontics was proposed to preserve the tooth’s structural integrity and ensure its long-term survival5. Minimally invasive endodontics focuses on preserving the pericervical dentin (PCD) by minimizing the extension of both the access cavity and the degree of root canal preparation5. Several conservative access cavity designs have been proposed6. The traditional access cavity (TRD) requires complete removal of the pulp chamber roof followed by achieving straight-line access to the canal orifices with smooth and divergent axial walls so that all the orifices are seen through the outline form6. The conservative access cavity (CON) usually starts at the central fossa of the occlusal surface and extends only as far as necessary to detect the canal orifices7. The axial walls are smooth and convergent to the occlusal surface, and part of the pulp chamber roof is preserved, resembling a soffit7. The truss

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FIGURE 1 – Different access cavity designs used in the simulated models. TRD with a surface area of 22.5 mm2, CON with a surface area of 8.7 mm2, and TUS with a surface area of 4.9 mm2.

access cavity (TUS) is an ultraconservative, orifice-directed access cavity that aims to preserve a bridge of enamel and dentin between 2 or more small cavities prepared to access the canal orifice(s) in each root of multirooted teeth8. The significance of preserving adequate PCD during access cavity preparation as well as canal instrumentation was emphasized as being critical for the ferrule, fracture resistance, long-term retention7, and stress distribution patterns of ETT9. Previous finite element analysis (FEA) studies reported a dramatic increase in von Mises (VM) stress when the access cavity margin approached the occlusal load points9–11. It was reported that larger canal tapers were associated with higher stress levels on the external tooth surface11, higher fracture susceptibility12, and greater deviation of stress distribution patterns from normal on the root surface9. Sufficient understanding about the interaction between different access cavity designs and constant or variable canal tapers on the life span or longevity of ETT is lacking. Therefore, this study aimed to investigate, using the FEA method, the impact of different canal tapers and access cavity designs on the life span of a simulated endodontically treated mandibular molar.

MATERIALS AND METHODS FEA Model Generation An intact, sound, mature human mandibular ﬁrst molar was scanned in a high-resolution cone-beam computed tomographic machine (Planmeca ProMax 3d MID; Planmeca, Helsinki, Finland). The images were processed using the materialize interactive medical image control system (MIMICS 19.0; Materialise, Leuven, Belgium) to identify enamel and dentin. This produced a 3-dimensional model by forming masks and automatically growing

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threshold regions. Data were then optimized using the 3-Matic Medical 11.0 software (Materialise). The software SolidWorks (Dassault Systems, Cedex, France) combined enamel and dentin and was used to establish the surrounding periodontal ligaments and bone.

Access Cavity Design The method used in a previous study9 was duplicated to produce the intact (IT) model and 3 other models with different access cavity designs: (1) a TRD model with a totally deroofed pulp chamber, (2) a CON model formed by connecting the projections of the canal’s long axis on the occlusal surface, and

TABLE 1 - The Instrument Diameters (mm) along the Length of the Root Canal Preparation Instruments D(x) 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

25/.04

TNP

25/.06

PTF2

0.89 0.85 0.81 0.77 0.73 0.69 0.65 0.61 0.57 0.53 0.49 0.45 0.41 0.37 0.33 0.29 0.25

0.80 0.79 0.78 0.76 0.73 0.69 0.65 0.61 0.57 0.53 0.49 0.46 0.43 0.40 0.37 0.34 0.26

1.20 1.15 1.09 1.03 0.97 0.91 0.85 0.79 0.73 0.67 0.61 0.55 0.49 0.43 0.37 0.31 0.25

1.20 1.19 1.19 1.08 0.97 0.90 0.86 0.82 0.77 0.72 0.67 0.62 0.56 0.49 0.41 0.33 0.25

D(x), the diameter of an instrument at x mm from the apical end of an instrument; PTF2, ProTaper Gold F2; TNP, TruNatomy Primary.

(3) an ultraconservative TUS model in which slot and oval cavities were performed over the mesial and distal canals of the model (Fig. 1).

Root Canal Preparation For each access cavity design, root canal preparation was simulated by drawing a line in the central axis of the root canal and then creating a symmetrical conical shape around it with the following simulated root canal dimensions for the mesial canals: constant tapers of 25/.04 and 25/.06 and variable tapers corresponding to the cumulative canal preparation with TruNatomy Prime (TNP; Dentsply Sirona, Charlotte, NC) and ProTaper Gold F2 (PTF2, Dentsply Sirona). The TNP has an apical size of 26 with a regressive variable taper producing a 0.8-mm maximum flute diameter. The ProTaper Gold system (Dentsply Sirona) requires the use of multiple shaping and finishing files to reach PTF2. The overall created shape has an apical size of 25 with a regressive variable taper producing a 1.2-mm maximum flute diameter. Table 1 presents the canal diameter from D0–D16, and Figure 2 graphically illustrates the dimensions of the 4 canal preparations. These were used to create a total of 15 experimental models. The distal canal in all models had a simulated preparation of 40/.04. The dimensions of PTF2 were acquired using data from 3D Endo (Dentsply Sirona). The dimensions of TNP were estimated using marketing material alongside data released from the manufacturer (Dentsply Sirona) where the dimensions at D0, D6, D12, and D16 were listed. Simulated gutta-percha was used to fill the prepared root canals 0.5 mm short from the apex of roots up to 2 mm from the canals’ orifices. The access cavities were filled with simulated composite resin. The volumes of the

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the S-N curve of enamel and dentin was derived from previous testing15,16.

Occlusal Fingerprint Analysis Occlusal ﬁngerprint analysis was performed to determine the loading areas on the subjected model, simulating clinical conditions20. The original mandibular molar was digitally scanned with an occlusal antagonist using an intraoral scanner (Medit i500; Medit, Seoul, Korea). Data were imported to Exocad (Darmstadt, Germany) software in which wear facets and occlusal contacts were digitally mapped and marked. These areas were transferred to the FEA model on which loading was performed simultaneously on all these areas (Fig. 3A–C).

FEA All models were subjected to simulated cyclic occlusal loading with a magnitude of 50 N to resemble a clinically relevant situation21,22. Load application was simulated in 3 stages: ﬁrst to the sound model, second after access cavity preparations, and third after root canal preparations were simulated. Every model was subjected to simulated repeated cycles of loading until failure. The number of cycles until failure (NCF) was then registered as well as the failure location. The life span was calculated as the percentage decrease of the NCF of each model compared with the solid model. Mathematical analysis of the stress distribution patterns and maximum VM stresses were also assessed using the Cosmos software package.

RESULTS

FIGURE 2 – A schematic graph representing the sizes of the canal lumen after preparation according to the taper of the ﬁles. The graph has mirrored plots to resemble the overall shape of engine-driven instruments.

composite resin used to restore the access cavities were 133.63 mm3 for TRD, 67.79 mm3 for CON, and 52.97 mm3 for TUS.

Meshing and Set Material Properties All models were imported into the Cosmos software package (SolidWorks; Dassault

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mes, Cedex, France) for meshing. After Syste meshing of the models, the element size ranged from 0.47212–2.3606 mm according to the complexity of the models. All the materials, including the dental structures, were considered linear, isotropic, and hom*ogeneous8,13,14. The elastic modulus and the Poisson ratio of structures used to set up the FEA models are listed in Table 2, whereas

The increase (%) in stress within each model and the decrease (%) in the NCF after each intervention compared with the IT model are presented in Table 3. The IT model had the least maximum stress with a value of 3.02 MPa and survived 5.92 ! 1012 cycles before failure. After access cavity preparation, the maximum increase in stress was observed with the TRD (191.78%) followed by the CON (137.45%) and the TUS (133.50%). The increase in stress was associated with a decrease in the NCF by 24.99% (TRD), 12.38% (CON), and 10.73% (TUS). After root canal preparation, only the TRD model displayed a noticeable increase in stress and decreased NCF regardless of the preparation taper. Regarding the effect of the root canal taper, minor or no differences were observed after varying the preparation taper within each model. Stress distribution patterns and maximum VM are shown in Figures 4–6. In the IT model, the stress distribution pattern

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TABLE 2 - The Mechanical Properties of the Materials for Finite Element Analysis17–19

CON, conservative access cavity; PTF2, ProTaper Gold F2; TNP, TruNatomy Primary; TRD, traditional access cavity; TUS, truss access cavity. The life span was calculated as the percentage decrease of NCF of each model compared with the solid model.

ending with tooth failure in ETT has been strongly linked to cyclic loading in the form of mastication over time25,26. Another limitation of previous studies is that researchers usually simplify occlusal forces to a point load placed (eg, in the central fossa of the crown or on the cusp tip). However, all occlusal contact areas should be considered and loaded in different directions and to different extents27. An approach to simulate a more realistic loading scenario that combines individual occlusal kinematic data in our study was derived from occlusal fingerprint analysis. This approach provides an individual 3-dimensional dental occlusal compass that indicates the major pathways of interaction between antagonists20. This study showed that the highest stress values correspond to the location of the simulated failure, which was always found on the occlusal surface, regardless of the access design. The trend that the highest stress value was located on the occlusal surface is supported by previous studies9–11,28 that reported a dramatic increase in principal stress when the access cavity margin approached load points. The TRD models displayed marked confinement of occlusal stresses within the composite. This is probably attributed to a broader tooth-to-composite interface that hinders a smooth transition of stresses, as suggested by Jiang et al10, who highlighted the importance of considering the distance between the biting point and the cavity margin to avoid stress concentration. This study also presented that access cavity designs have more impact on the life span of ETT compared with the canal taper, whereas the taper change of canal preparation did not appear to make a substantial mechanical difference with all access designs. The argument that access cavities have more impact than the taper is supported by Wang et al28. However, our results contrast those of Smoljan et al29, who found on preaccessed mandibular molars by FEA that wider progressive taper preparations have less fracture resistance than narrow progressive taper canal preparations. TRD was associated with more stress buildup and less NCF than CON and TUS, whereas there was no noticeable difference between the CON and TUS. Similar findings were concluded in previous studies9,28,30. Although other studies found no difference in fracture resistance between TRD and CON31–33, the present results suggest that the life span of an ETT may outlast the patient’s life span. Because 50 N for 1.2 ! 106 cycles simulated 5 years of oral service34,35, the shortest life span expected for TRD was 2.86 ! 109 cycles until failure, which is

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Life Span of Endodontically Treated Teeth

Material

Elastic modulus (MPa)

Poisson ratio

84,100 18,600 68.90 0.69 13,700 12,500

0.33 0.31 0.45 0.45 0.30 0.30

Enamel Dentin Periodontal ligaments Gutta-percha Alveolar bone Composite resin

showed the crown architecture’s ability to house the burden of occlusal loads. The maximum stress was recorded on the occlusal surface near the central fossa with a value of 3.02 MPa. Stresses on the root were limited to the cervical belt above the furcation area, especially on the mesial aspect of the mesial root with a peak value of 1.76 MPa. After access cavity preparation, the stresses migrated apically along the root surface, notably on the mesial aspect of the mesial root and on the outer surface of the furcation area. This pattern was observed for all access designs. After root canal preparation with different tapers, there were no noticeable changes in the pattern or magnitude of stresses distributed along the root surface, except for a slight increase in stress magnitude at the apical foramen in the CON and TUS models.

DISCUSSION Minimally invasive endodontics has been suggested to increase the life span of ETT

through minimal dentin removal and preservation of the PCD without compromising the biological objectives of root canal treatment7. This study offered the first evaluation of different canal tapers that directly affect the PCD area, especially the recently introduced TNP, which claims to support the minimally invasive endodontics concept with minimal dimensions on the coronal part of the file23. The study also investigated how the interaction between access cavity design and root canal preparation ultimately affects a functioning tooth’s life span. This study sought to overcome some limitations of previous FEA studies; one is the application of a continuous and increasing static occlusal load while evaluating the tooth response to the increasing stress9,11,12. This method measures the load capacity of the model rather than its fatigue failure, which is more relevant from a clinical standpoint. By definition, fatigue failure occurs because of the application of fluctuating stresses, much lower than that required to cause a catastrophic failure24. Crack development propagation

TABLE 3 - The Stress, Number of Cycles before Failure (NCF), and the Life Span of Various Access and Preparation Models in a First Mandibular Molar

Intact tooth TUS Access only 25/.04 TNP 25/.06 PTF2 CON Access only 25/.04 TNP 25/.06 PTF2 TRD Access only 25/.04 TNP 25/.06 PTF2

Maximum stress (MPa)

Stress increase (%)

NCF

Life span decrease (%)

3.020 4.032 4.054 4.097 4.423 4.233 4.151 4.319 4.296 4.157 4.300 5.792 6.890 6.851 6.803 6.737

100.00 133.50 134.24 135.66 146.44 140.16 137.45 143.01 142.25 137.64 142.38 191.78 228.14 226.85 225.26 233.08

5.92 ! 1012 1.57 ! 1012 1.45 ! 1012 1.41 ! 1012 3.01 ! 1011 8.11 ! 1011 9.34 ! 1011 7.13 ! 1011 7.80 ! 1011 8.23 ! 1011 7.71 ! 1011 1.77 ! 1010 2.86 ! 109 3.03 ! 109 3.25 ! 109 3.55 ! 109

100.00 10.73 10.98 11.07 15.98 12.83 12.38 13.24 12.96 12.79 12.99 24.99 30.79 30.60 30.38 30.10

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FIGURE 3 – (A ) The occlusal view of the original tooth. (B ) The occlusal contacts and wear facets mapping. (C ) Occlusal ﬁngerprint analysis transferred to the ﬁnite model.

approximately equivalent to 477 years. However, postendodontic tooth fractures have generally been attributed to a combination of noncontrollable and controllable factors that weaken the tooth structure2,36.

Stress analysis of the models compared with the IT model displayed apical migration of stresses on the root surface and within it to the apical foramen, especially with the CON and TUS models.

The apical migration of stresses suggests that crack initiation and propagation in ETT might begin anywhere on the root surface, not only at the PCD area. This phenomenon requires further investigation

FIGURE 4 – An isometric view of the stress distribution pattern. The variation of the stress distribution pattern is attributed solely to the access cavity design rather than the canal taper. Conﬁned stresses within a cervical belt in the IT model (red arrows ). Stresses are conﬁned within the composite in TRD models (white arrows ). The highest stress concentration was registered within the TRD model (6.89 MPa), whereas the IT model showed the least (3.02 MPa).

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FIGURE 5 – A buccal view of the stress distribution pattern. The composite in the TRD models conﬁned the stresses internally. Thus, stresses reaching the buccal surface are markedly different from the patterns registered with either the TUS or the CON model. The highest stress concentration was registered within the TRD model (6.89 MPa), whereas the IT model showed the least (3.02 MPa).

because, in this simulated study, these areas were stress-free areas in the IT model. There would be a possibility that stresses present in the naturally prepared teeth are rendered risk free by the continuously remodeling apical cementum37. Furthermore, another limitation of this study was that it was performed on a speciﬁc mandibular molar model. It is unknown whether changes in root canal taper may affect long and slender roots, where it is anticipated that larger tapered preparations would have a more pronounced effect on the remaining dentin. Also, the use of a chewing simulator to mimic normal function might provide additional information.

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CONCLUSIONS Within the limitations of this study, it can be concluded that the tooth’s life span is affected significantly by the access cavity design rather than the root canal preparation taper. CON and TUS access designs appear to have comparable structural integrity. It is the clinician’s choice as to which is the more justified approach. A custom access outline is suggested for each tooth based on its occlusal fingerprint analysis. This outline should preferably exclude or include excessive load points to avoid stress concentration and clinical failure. Because stress patterns migrated apically rather than concentrating in the

pericervical area, this suggests that crack initiation and propagation might occur anywhere on the root surface.

ACKNOWLEDGMENTS Mostafa M.A. Elkholy and Hyeon-Cheol Kim contributed equally to this study. The authors thank the Digital Dentistry Hub at the Center of Innovative Dental Sciences at the British University in Egypt for providing the intraoral scanner and software used to scan the natural molar used in this study. The authors deny any conﬂicts of interest related to this study.

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FIGURE 6 – A proximal view of the stress distribution pattern. The loss of tooth structure in the access cavity preparation caused occlusal stresses to migrate apically along the root surface rather than stay conﬁned in the cervical belt below the crown and just above the furcation area (yellow arrows ). Note the increased stresses at the apical foramen area after canal shaping (red circles ). The highest stress concentration was registered within the TRD model (6.89 MPa), whereas the IT model showed the least (3.02 MPa).

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BASIC RESEARCH – TECHNOLOGY Flavia Darius Vivacqua, DDS, MS,* Marco Antonio Hungaro Duarte, DDS, MS, PhD,*

Analysis of Instrumentation Protocols Regarding the Quality of Mesial Canal Preparation in Mandibular Molars: A Micro–computed Tomographic Study

Rodrigo Ricci Vivan, DDS, MS, PhD,* Murilo Priori Alcalde, DDS, MS, PhD,† Renan Diego Furlan, DDS, MS,* and Clovis Monteiro Bramante, DDS, PhD*

ABSTRACT Introduction: This study aimed to use micro–computed tomographic imaging to analyze the quality of the endodontic preparation of mesial canals in mandibular molars provided by 3 instrumentation protocols. Methods: Forty-five extracted mandibular molars with 2 independent mesial canals were selected, and the initial micro–computed tomographic imaging was performed. The initial volume values of the canals were submitted to statistical analysis for paired division. The groups were determined according to the final enlargement of the canal and the working length adopted (ie, G25.06/11 mm, G35.05/foramen, and G50.01/ 21 mm). At the end of each instrumentation sequence, the root canals were scanned and analyzed with regard to the increase in the total and apical volume, centralization, and preparation transportation and the percentage of the total and apical uninstrumented walls. Results: For the intragroup comparison, the Wilcoxon test was used, and for the intergroup analysis, the Kruskal-Wallis and Dunn tests were used (P , .05). In the analysis of the canal total volume, a statistical difference was found between G25.06/11 mm and the remaining groups (P , .05). In the apical third, a statistical difference was observed between G25.06/ 11 mm and G50.01/21 mm (P , .05). No statistical difference was found between the groups in terms of centralization and transportation of the preparation or in terms of the percentage of the total or apical uninstrumented walls. Conclusions: The preparation of the mesial canals of mandibular molars up to larger tip files but with a lower taper at 1 mm before the foramen resulted in a larger volume of apical preparation, kept the preparation centralized, and provided safe apical dentin wear without excessive cervical wear. (J Endod 2021;47:1481–1486.)

KEY WORDS Endodontics; increased apical enlargement; micro–computed tomography; root canal preparation

In endodontics, there have been important technological advancements; however, treatment protocols that favor better cleaning and efficient reduction of microorganisms tend to be more successful and reduce the risk of reinfection1. According to some studies, the instruments with thermal treatment, which are used attached to electrical motors, have several advantages compared with the stainless steel files previously used because they contribute to the procedures due to their greater flexibility, high cutting efficacy, and improved fracture resistance2–4. Therefore, these instruments provide better maintenance of the root canal morphology, resulting in a more centralized preparation and consequent preservation of the apical foramen2,4. More efficient microbial reduction is still a challenge, mainly because of the anatomy of the root canal. Even in the main canal, the instruments have not been able to reach all the walls5,6. This probably occurs because preparation of the canal is being performed with final instruments that have a smaller

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SIGNIFICANCE Enlargement of the apical portion of the root canals is recommended. The apical limit and the correct choice of ﬁnal instruments are still being speculated. This choice aims at greater decontamination; however, it should not cause accidents in endodontics.

From the *Department of Dentistry, Endodontics and Dental Materials, Bauru ~o School of Dentistry, University of Sa ~o Paulo, Brazil; and Paulo, Bauru, Sa † Department of Health Science, University ~o Paulo, of the Sacred Heart, Bauru, Sa Brazil Address requests for reprints to Dr Flavia Darius Vivacqua, Department of Dentistry, Endodontics and Dental Materials, Bauru vio Pinheiro School of Dentistry, Al Octa Brisolla, 9-75, 17012-901 Bauru, SP, Brazil. E-mail address: ﬂ[emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.008

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diameter than that of the initial canal (before instrumentation) (ie, very conservative preparations result in the bacterial biofilm not being removed and in deficient cleaning)1. The hypotheses that the size of the apical preparation and determination of the ideal apical third enlargement might be important to achieve this improvement have been studied7–10. Studies evaluating the influence of the apical size on penetration of the irrigating solution, reduction in microorganisms, and wall treatment have demonstrated significant results in relation to the enlargement of this area8,10–12. Some studies in which the enlargement of the foramen was performed have demonstrated the occurrence of postoperative pain in the first few hours after treatment13. There is still some lack of consensus regarding the limit and extension of apical enlargement that could result in a lower percentage of uninstrumented walls and a larger quantity of dentin removal in the apical third5,6,10,14. This study aimed to evaluate the quality of 3 distinct instrumentation protocols using micro–computed tomographic (micro-CT) imaging. The variables taken into consideration were the apical diameter and the working length adopted. The null hypothesis tested was that the instrumentation protocols would show the same value of increase in canal volume throughout its total extension and in the apical third, the same centralization capacity in the cervical and apical third, and a similar percentage of walls untouched by the instrument.

MATERIALS AND METHODS For the sample calculation, G*Power v3.1 for €sseldorf, Mac (Heinrich Heine, Universit€at Du €sseldorf, Germany) was used, and the Du Wilcoxon–Mann-Whitney test of the t test family was selected. The data of a previous study that had evaluated the root canal preparation with curved canals15 were used, and the effect size in the present study was established (50.88). The alpha type error of 0.05, a beta power of 0.95, and an N2/N1 ratio

of 1 were also stipulated. A total of 30 canals per group were indicated as the ideal size required for noting significant differences. After approval by the research ethics committee, 90 human permanent mesial canals of 45 mandibular molars were selected. The specimens had complete root formation, mesial canals categorized as Vertucci type IV16, and canal anatomy with a curvature of up to 30 17 without extensive coronal destruction. The molars were scanned whole using a micro-CT system (SkyScan 1174v2; Bruker MicroCT, Kontich, Belgium) to confirm the type of anatomy. The access was achieved by using a high-speed spherical diamond tip (FG 1014; KG Sorensen, Cotia, Brazil) with the teeth included in a dental dummy, and the coronal access was conservatively performed by removing the entire pulp chamber ceiling with the use of an ENDO ZK (Beavers Dental, ) bur (JET, France), Morrisburg, Canada maintaining the occlusal convergence of the surrounding walls. The crown accessed was inserted into a silicone mold to maintain the same position from the first until the last scan according to a previous study14.

Micro-CT Analysis and Sample Division The teeth were scanned using the following parameters: a 19-mm voxel size, 50 kV, 800 mA, and a 0.8 step size rotation using a 1024 ! 1304 resolution. The images were reconstructed using a speciﬁc program (NREcon v.1.6.9, Bruker MicroCT) and stored with the axial section in the BMP format. The initial volumetric analysis was performed using CTAn v1.12 (Bruker MicroCT). The initial volumes were submitted to the Kruskal-Wallis and Dunn statistical tests for sample pairing and random division of the 3 groups (n 5 30); no statistical difference was found between the initial volume values of the 3 groups (P . .05).

Sample Preparation and Group Division The canal patency and length were established with a C-pilot #15.02 ﬁle (Dentsply VDW,

Munich, Germany) using a surgical microscope (ALLIANCE UNIQUE, S~ao Carlos, Brazil). The real working length of the canals varied from 19–21 mm. Each tooth was numbered after its length was determined, and then they were distributed so that a similar mean length of the teeth was maintained between all the groups. Three instrumentation protocols were used, considering the apical enlargement and working length used. The following ProDesign Logic (EASY-BASSI Equipamentos gicos, Belo Horizonte, Brazil) single Odontolo ﬁles were used: Logic #25/.06, #35/.05, and #50/.01. Each instrument was used to prepare 6 canals. The full length of the canals up to the foramen, 1 mm beyond the foramen (11 mm), and 1 mm short of the foramen (21 mm) determined the 3 groups: G25.06/11 mm, G35.05/foramen, and G50.01/21 mm.

Root Canal Preparation In the G25.06/11 mm group, only #25/.06 was used. The instrument was used 3 times: ﬁrst in the cervical preparation, then 3 mm short of the tooth length, and lastly 1 mm beyond the length of the tooth. In the G35.05/ foramen group, #25/.06 was used in a similar way as in G25.06/11 mm; however, for the last steps, the #25/.06 was used at the tooth length and then ended with the #35/.05 at the tooth length. In the G50.01/21 mm group, #25/.06 was used according to the G25.06/ 11 mm procedure but short of the tooth length; #25.06, #35/.05, and #50/.01 were used 1 mm short of the tooth length. At the end of each instrumentation procedure, a new scan was performed, and images were reconstructed using the same parameters as those previously described. The reconstructed images captured before and after each instrumentation were geometrically aligned in the sagittal, coronal, and axial planes using the DataViewer v.1.5.1 software (Bruker MicroCT) according to a previous study14. The entire instrumentation procedure was performed by a single operator using motor ENDO SI (EASY-BASSI Equipamentos gicos) operating at 600 rpm 2 N for Odontolo the #25/.06 and #35/.05 ﬁles and 350 rpm and

TABLE 1 - The Median, Minimum, and Maximum Values of the Initial, Final Volume and Percentage of Increase in the Total and Apical Volume of the canal According to the Instrumentation Protocol Total volume (mm3) Group G25.06/11 mm G35.05/foramen G50.01/21 mm

Apical volume (mm3)

Initial

Final

% Increase

Initial

Final

% Increase

1.98A (0.8–3.9) 1.76A (0.7–3.5) 1.97A (1.0–4.3)

3.18B (1.6–4.7) 3.50B (1.7–6.3) 4.10B (2.3–5.9)

48.50b (1.0–190) 87.10a (32.9–218.0) 95.15a (0.0–296.7)

0.45A (0.0–0.9) 0.43A (0.1–1.1) 0.39A (0.1–1.6)

0.78B (0.3–1.2) 0.95B (0.6–1.7) 1.24B (0.7–1.9)

59.0b (0–591.0) 125.0a,b (30.30–564.9) 190.2a (0.1–113.0)

Different lowercase letters indicate statistically signiﬁcant differences between groups (Kruskal-Wallis test, P , .05), and capital letters indicate signiﬁcant differences within groups (Wilcoxon test, P , .05).

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The centralization and transport measurements considered the apical 4 mm, from the foramen in the coronal direction, with 1-mm intervals, and the cervical 4 mm, from the furcation area in the apical direction, with the same intervals. The formulas used for calculation were as follows: (m1 2 m2) 2 (d1 2 d2) and (m1 2 m2)/(d1 2 d2) or (d1 2 d2)/ (m1 2 m2)18.

Percentage of Uninstrumented Walls In this analysis, the entire length of the canal and the apical 4 mm were considered, which were measured in the same way as previously described in the volume analysis, and analyses were performed with the same program.

Statistical Analysis

No signiﬁcant difference was found at all tested levels (P . .05).

Apical

43.63 (0.0–98.0) 53.38 (0.0–99.8) 18.88 (0.0–90.3)

Total

27.86 (0.0–81.9) 29.46 (0.0–94.1) 26.21 (0.0– 90.1)

0.43 (0.0–0.9) 0.45 (0.0–0.9) 0.55 (0.0–1.0)

Canal Transportation and Centering Ability

0.45 (0.0–1.0) 0.49 (0.0–0.9) 0.45 (0.1–0.8)

3 2

Volume Analysis The mesial canals were analyzed relative to the percentage of the total increase in volume, which was measured from the foramen up to 1 mm short of the furcation area, and in the apical 5 mm, which was considered starting from the foramen in a coronal direction in the CTAn v1.12 software (Bruker MicroCT).

0.48 (0.0–1.0) 0,35 (0.0–0.9) 0.51 (0.0–0.9)

0.32 (0.0–0.9) 0.51 (0.0–0.9) 0.27 (0.0–0.9)

0.56 (0.0–1.0) 0.56 (0.0–0.9) 0.63 (0.1–0.9)

0.36 (0.0–0.9) 0.38 (0.0–0.9) 0.51 (0.0–0.9)

0.50 (0.0–1.0) 0.41 (0.0–0.9) 0.37 (0.0–0.9)

0.48 (0.0–0.9) 0.54 (0.0–0.9) 0.37 (0.0–0.9) G25.06/11 mm G35.05/foramen G50.01/21 mm

Group

Uninstrumented area (%) Apical centralization (mm from foramen) Cervical centralization (mm from furcation)

TABLE 2 - The Median, Minimum, and Maximum Values of 4-mm Cervical and 4-mm Apical Centralization and the Total and Apical Uninstrumented Area (%) of Each Group

1.5 N for the #50/.01 ﬁle. After 3 pecking movements, the root canals were irrigated with 5 mL 5.25% sodium hypochlorite, and the instrument was cleaned and examined.

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The volume, centralization, transportation, and percentage of the uninstrumented area results obtained were evaluated regarding their normality using the Kolmogorov-Smirnov test, which revealed an abnormal distribution. In the intragroup comparison, the Wilcoxon test was used, and in the intergroup analysis, the Kruskal-Wallis and Dunn tests were used. The signiﬁcance level was 5% (P , .05), and the statistical program used was GraphPad Prism version 8.4.3 (GraphPad Software, San Diego, CA).

RESULTS Considering the initial total volume and the volume after each preparation protocol (dentin removal) within the same group, a statistically signiﬁcant difference (P , .05) was observed in all groups. This occurred in the same way with the apical volume and intragroup comparison (Table 1). In the intergroup analysis of the total volume, a statistical difference (P , .05) was observed between the G25.06/11 mm group and the other groups. There was no difference

between the G35.05/foramen and G50.01/ 21 mm groups (Table 1). In the intergroup analysis of the apical volume, a statistically signiﬁcant difference was found in the comparison between the G25.06/11 mm and G50.01/21 mm groups (P , .05). No statistical difference was observed in relation to the preparation centralization or to transport between groups in the cervical 4 mm or in the apical 4 mm (Table 2). For the percentage of uninstrumented walls during instrumentation, no statistically signiﬁcant difference (P . .05) was observed in the entire extension of the canal or in the apical third between the groups under analysis (Table 2). Figure 1 shows the representative micro-CT images of the groups studied.

DISCUSSION In this micro-CT study, the quality of 3 different instrumentation protocols was analyzed in mesial mandibular molars. The null hypothesis was partially rejected because there was a difference in relation to the percentage of the increase in canal volume between the groups analyzed. The mesial canals of mandibular molars are the root canals most commonly observed clinically and used in studies19,20; they have a challenging anatomy, causing difficulty in choosing the final apical file21. Because of anatomic variation, the diameter of the canal must be measured properly and before chemical-mechanical preparation, with the aim of personalizing the instrumentation and avoiding excessive wear22. Canal debridement or enlargement techniques do not guarantee the elimination of bacteria during root canal treatment10. However, larger apical dilations have been suggested as a way of increasing the efficacy of the canal cleaning and disinfection7,12. The inefficacy of the preparation might result from conservative apical instrumentation procedures, of which the consequence is the large quantity of bacteria and necrotic tissue in the canal1. Recent studies have pointed out the importance of larger apical preparations, with the aim of more effective cleaning, disinfection, and penetration of cement12,22–24, in addition to better removal of contaminated dentin and an increase in the action of irrigants in the apical region7,25. In this study, 3 working lengths and 3 final apical enlargements were considered: G25.06/11 mm, G35.05/foramen, and 50.01/ 21 mm. In the total and apical volume analysis, within the same group, a relevant statistical difference was observed in all instrumentation

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FIGURE 1 – Representative images of superimposed micro-CT images of the mesial canals of mandibular molars taken before (red ) and after (green ) the instrumentation protocols of each group. A–C represent the mesial canals along their entire length, and a–c represent the apical third.

procedures, indicating dentin removal and increased canal diameter in all phases. In the total volume, the protocol 35.05/ foramen and the protocol 50.01/21 mm obtained a signiﬁcantly higher percentage increase in volume than that found in the protocol 25.06/11 mm. Previous studies have demonstrated that apical enlargement exceeding the diameter #35 1 mm short of the foramen favors a higher percentage of instrumented areas and more thorough decontamination of the root canal2,12. The literature has suggested that apical enlargement with diameters exceeding #35 may improve the cleaning and disinfection of the root canal system22,24,26. However, the ideal apical dilation and the instrumentation limit continue to be widely discussed13,27. Some authors have suggested that determining the initial apical diameter and enlarging it with 3 to 4 larger instruments would be ideal2,7,8,15. Other studies have also observed that apical enlargement exceeding #35 reduced the total quantity of contaminated debris by

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around 30%25,26. The fact that the greater apical enlargement provided less debris may be related to better irrigation of the apical third. In 1 study with apical patency, file #10, and enlargement up to 40/.06, the irrigant reached the apical portion even more effectively28. Enlargements with files between #40 and #50 have also been reported, promoting better removal of contaminated material, apical cleaning8,15,26, and improvements in irrigation12. A statistical difference was found in the removal of the canal dentin (the total volume) between the G25.06/11 mm group and the other groups. No difference was found between the G35.05/foramen and G50.01/ 21 mm groups. This might be explained because the canals in the G50.01/21 mm group had previously been instrumented with the 35.05 file, which resulted in a #40 diameter at 1 mm short of the foramen. In the apical third, a difference in volume was observed between the G25.06/11 mm and G50.01/21 mm groups. Demonstrating that working with instruments with a smaller tip and a larger taper 1 mm beyond the foramen

did not remove more dentin than using the same instruments 1 mm short of the foramen. The enlargement of the foramen does not provide more effective cleaning of the apical third and may increase the risk of extravasation of irrigating solutions and filling material27. Similar results were found by other authors using the same working length3,10,24. Anatomic studies have shown that root canals are mostly irregular and with an oval cross section, and this might result in irregular preparation, removing dentin only from 1 of the sides29. Analyzing the cervical and the apical third regarding deviation and the transport of canals, no statistical difference was found in the groups tested. Because of the heat treatment of the memory control type found in the files used in the study, they are very flexible3,4, which contributes to the centralization of the preparation because the increase in the instrument tip was compensated with taper reduction that prevented canal deviation or transport. These factors are relevant because differentiated thermal treatments and a smaller taper and tip

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diameter aim at allowing better adaptation of the instrument to the canal anatomy9,30, preserving the thickness of the dentin in the cervical region14,30,31. There was no statistical difference in the percentage of the noninstrumented area among the 3 protocols in relation to the total canal or the apical third. Similar results were found in other studies with the same methodology and dental group6,19,32. Some authors have observed a reduction in the uninstrumented areas with the increase of the apical diameter19,33. Although the result was not signiﬁcant, the group 50.01/21 mm

reached a 24.7% larger portion of canal walls than the group 25.06/11 mm and exhibited a 34.5% larger surface touched compared with the 35.05/foramen, which should clinically result in more effective cleaning and decontamination. Within the limitations of this study and its application in the clinical environment, the authors emphasize that apical enlargement with thermically treated nickel-titanium ﬁles with larger tip diameters and a smaller taper 1 mm short of the foramen showed great potential to promote removal of the contaminated dentin in the apical region.

CONCLUSIONS The preparation of the mesial canals of the mandibular molars with ﬁles that have larger tips and a smaller taper 1 mm short of the foramen resulted in safe apical tooth wear and maintained the preparation centralized and without excessive cervical wear.

ACKNOWLEDGMENTS The authors deny any conﬂicts of interest related to this study.

REFERENCES

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^ças IN, Marceliano-Alves MF, et al. Unprepared root canal surface areas: Siqueira Junior JF, Ro causes, clinical implications, and therapeutic strategies. Braz Oral Res 2018;18:e65.

Fornari VJ, Silva-Sousa YTC, Vanni JR, et al. Histological evaluation of the effectiveness of increased apical enlargement for cleaning the apical third of curved canals. Int Endod J 2010;43:988–94.

Rodrigues CT, Duarte MA, Almeida MM, et al. Efﬁcacy of CM-Wire, M-Wire, and nickel-titanium instruments for removing ﬁlling material from curved root canals: a micro–computed tomography study. J Endod 2017;42:1651–5.

Pereira ES, Viana AC, Buono VT, et al. Behavior of nickel-titanium instruments manufactured with different thermal treatments. J Endod 2015;41:67–71.

Vieira ML, Dantas HV, Sousa FB, et al. Morphologic changes of apical foramen and microcrack formation after foraminal enlargement: a scanning electron microscopic and micro–computed tomographic analysis. J Endod 2020;46:17.

Paque F, Balmer M, Attin T, Peters OA. Preparation of oval-shaped root canals in mandibular molars using nickel-titanium rotay instruments: a micro-computed tomography study. J Endod 2010;36:703–7.

Saini HR, Tewari S, Sangwan P, et al. Effect of different apical preparation sizes on outcome of primary endodontic treatment: a randomized controlled trial. J Endod 2012;38:1309–15.

Perez AR, Alves FR, Marceliano-Alves MF, et al. Effects of increased apical enlargement on the amount of unprepared areas and coronal dentine removal: a micro-computed tomography study. Int Endod J 2018;51:684–90.

Lima CO, Barbosa AF, Ferreira CM, et al. The impact of minimally invasive root canal preparation strategies on the ability to shape root canals of mandibular molars. Int Endod J 2020;53:1680–8.

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Yadav SS, Shah N, Logani A, et al. Effect of “apical clearing” and “apical foramen widening” on apical ramiﬁcations and bacterial load in root canals: an ex-vivo stereomicroscopic study. Bull Tokyo Dental Coll 2014;55:67–75.

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Akhlaghi NM, Rahidmifar N, Moshari A, et al. The effect of size and taper of apical preparation in reducing intra-canal bacteria: a quantitative SEM study. Iran Endod J 2014;9:61–5.

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Butcher S, Mansoura A, Ibrahim M. Inﬂuence of apical preparation size on effective conventional irrigation in the apical third: a scanning electron microscopic study. Eur Endod J 2019;4:9–14.

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Yaylali IE, Teke A, Tunca YM. The effect of foraminal enlargement of necrotic teeth with a continuous rotary system on postoperative pain: a randomized controlled trial. J Endod 2017;43:359–63.

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Sant’Anna Junior A, Cavenago BC, Ordinola-Zapata R, et al. The effect of larger apical preparations in the danger zone of lower molars prepared using the Mtwo and Reciproc systems. J Endod 2014;40:1855–9.

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ElAyouti A, Dima E, Judenhofer MS, et al. Increased apical enlargement contributes to excessive dentin removal in curved root canals: a stepwise microcomputed tomography study. J Endod 2011;37:1580–4.

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Vertucci FJ. Root canal anatomy of the human permanent teeth. Oral Surg Oral Med Oral Pathol 1984;58:589–99.

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Schneider SW. A comparison of canal preparations in straight and curved canals. Oral Surg Oral Med Oral Pathol 1971;32:271–5.

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Gambill JM, Alder M, del Rio CE. Comparison of nickel-titanium and stainless steel hand-ﬁle instrumentation using computed tomography. J Endod 1996;22:369–75.

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Brasil SC, Marceliano-Alves MF, Marques ML, et al. Canal transportation, unprepared areas, and dentin removal after preparation with bt-race and protaper next systems. J Endod 2017;43:1683–7.

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F. A micro–computed tomographic assessment of root canal Peters OA, Arias A, Paque preparation with a novel instrument, TRUShape, in mesial roots of mandibular molars. J Endod 2015;41:1545–55.

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€ u €rek T, et al. Microcomputed assessment of transportation, centering Aydın ZU, Keskin NB, Ozy ratio, canal area, and volume increase after single-ﬁle rotary and reciprocating glide path instrumentation in curved root canals: a laboratory study. J Endod 2019;45:791–6.

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Campello AF, Alves MF, SiqueiraJr JF, et al. Determination of the initial apical canal diameter by the ﬁrst ﬁle to bind or cone-beam computed tomographic measurements using micro-computed tomography as the gold standard: an ex vivo study in human cadavers. J Endod 2019;45:619– 22.

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Rodrigues RC, Zandi H, Kristoffersen AK, et al. Inﬂuence of the apical preparation size and the irrigant type on bacterial reduction in root canal-treated teeth with apical periodontitis. J Endod 2017;43:1058–63.

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Laslami K, Dhoum S, El Harchi A, Benkiran I. Relationship between the apical preparation diameter and the apical seal: an in vitro study. Int J Dent 2018;10:1–5.

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De-Deus G, Marins J, Silva EJ, Souza E, et al. Accumulated hard tissue debris produced during reciprocating and rotary nickel-titanium canal preparation. J Endod 2015;41:676–81.

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Xu K, Wang J, Wang K, et al. Micro-computed tomographic evaluation of the effect of the final apical size prepared by rotary nickel-titanium files on the removal efficacy of hard-tissue debris. J Int Med Res 2018;46:2219–29.

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Albuquerque PP, Duarte MA, Pelegrine RA, et al. Inﬂuence of foraminal enlargement on the apical extrusion of ﬁlling material: volumetric analysis using micro-computed tomography. Aust Endod J 2020;46:210–6.

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Vera J, Siqueira JF Jr, Ricucci D, et al. One- versus two-visit endodontic treatment of teeth with apical periodontitis: a histobacteriologic study. J Endod 2012;38:1040–52.

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Martins JN, Marques D, Silva EJ, et al. Prevalence studies on root canal anatomy using conebeam computed tomographic imaging: a systematic review. J Endod 2019;45:372–86.

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Plotino G, Ozyurek T, Grande NM, Gundogar M. Inﬂuence of size and taper of basic root canal preparation on root canal cleanliness: a scanning electron microscopy study. Int Endod J 2019;52:343–51.

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Ng YL, Mann V, Gulabivala K. A prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. Int Endod J 2011;44:583–609.

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Duque JA, Vivan RR, Cavenago BC, et al. Inﬂuence of NiTi alloy on the root canal shaping capabilities of the ProTaper Universal and ProTaper Gold rotary instrument systems. J Appl Oral Sci 2017;25:27–33.

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BASIC RESEARCH – TECHNOLOGY Christos Boutsioukis, DDS, MSc, PhD, and Patricia Gutierrez Nova, DDS

Syringe Irrigation in Minimally Shaped Root Canals Using 3 Endodontic Needles: A Computational Fluid Dynamics Study ABSTRACT Introduction: The aim of this study was to compare the irrigant flow in curved root canals prepared to various apical sizes by constant-taper or variable-taper instruments during syringe irrigation with 3 endodontic needles at 2 different flow rates. Methods: Two matched curved mesial root canals of human mandibular molars were imaged by micro–computed tomographic imaging after preparation to apical size 20, 25, and 30/.06 taper either by constanttaper or variable-taper instruments. A Computational Fluid Dynamics model was used to simulate the irrigant flow in the 2 root canals prepared to each apical size during syringe irrigation with a 30-G open-ended needle and 30-G and 31-G closed-ended needles at 0.05 and 0.15 mL/s. Results: The irrigant could not penetrate up to the working length in root canals prepared to apical size 20 or 25/.06 taper. The 30-G open-ended needle combined with the low flow rate allowed the irrigant to reach the working length in size 30/.06 taper root canals while maintaining a relatively low apical pressure, but the wall shear stress was very low. The 31-G closed-ended needle combined with the high flow rate also delivered the irrigant to the working length in size 30 root canals and developed higher wall shear stress, but the apical pressure was also higher. Conclusions: Syringe irrigation using 30-G and 31-G needles was compromised in minimally shaped root canals. (J Endod 2021;47:1487–1495.)

SIGNIFICANCE Irrigant flow is a prerequisite for the cleaning and disinfection of the root canal. The flow created by syringe irrigation could not reach the working length in minimally shaped root canals irrespective of the needle type and size and the flow rate.

KEY WORDS Flow; irrigation; minimally shaped root canal; needle; syringe

Minimally invasive endodontics promotes the preservation of as much healthy hard dental tissue as possible in an effort to maintain the strength and function of the tooth1,2. This concept is mainly applied to access cavity preparation3–5, but it has also been extended to root canal instrumentation1,2,4,6. Minimal shaping of root canals to apical size 20–25 is advocated by instrument manufacturers,7–9 and it has been applied in several in vitro studies3,5,10. However, the limited space available inside a minimally shaped root canal may compromise the effectiveness of syringe irrigation1,2,4, the most widely used irrigation method11. Apical enlargement at least to size 30–35 is considered necessary in order to achieve debridement and disinfection of the root canal system because it allows 27- to 30-G irrigation needles to be inserted closer to the working length (WL) and the irrigant to overcome viscosity-related effects that limit its penetration12–20; 31-G irrigation needles have been proposed as a way to overcome these limitations21, but the flow created by these needles has not been evaluated so far. Furthermore, numerical models have been used in the past to investigate root canal irrigation17–19,22,23, but previous studies adopted a univariate approach. Thus the effect of any interactions between the irrigant flow rate; the apical root canal size and taper; and the needle type, size, and insertion depth remains largely unexplored. Therefore, the aim of this study was to compare the irrigant flow in curved root canals prepared to various apical sizes by constant-taper or variable-taper instruments during syringe irrigation with a 30-G open-ended and 30-G and 31-G closed-ended irrigation needles at 2 different flow rates using a Computational Fluid Dynamics (CFD) model.

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From the Department of Endodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, The Netherlands Address requests for reprints to Dr Christos Boutsioukis, Department of Endodontology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and Vrije Universiteit Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands. E-mail address: [emailprotected] 0099-2399 Copyright © 2021 The Authors. Published by Elsevier Inc. on behalf of American Association of Endodontists. This is an open access article under the CC BY-NCND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). https://doi.org/10.1016/ j.joen.2021.06.001

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MATERIALS AND METHODS Geometry of the Root Canals and Irrigation Needles The use of extracted teeth was approved by the institutional ethics committee (2018042). Two human mandibular molars with separate, moderately curved (20 –40 )24 mesial root canals were obtained from a pool of extracted teeth. Two of these mesial root canals were matched based on their length, curvature, and diameter in the apical 5 mm, so they were as similar as possible. These specimens were debrided and stored in a 0.05% thymol solution. The specimens were scanned by a micro–computed tomographic scanner €ttisellen, (mCT 40; Scanco Medical AG, Bru Switzerland) operating at 70 kV and 114 mA using a 10-mm voxel size before any intervention (initial scan). The scans were reconstructed, filtered, and segmented using Fiji 1.49m25 to obtain 3-dimensional renderings of the 2 root canals. Standard access cavities were prepared in both specimens. The working length (WL) was set 0.5 mm short of the major apical foramen. The apical 5 mm of the roots was sealed with cyanoacrylate (Pattex; Henkel, Dusseldorf, Germany). A glide path was prepared using stainless steel K-files of size 08– 15 (Dentsply Maillefer, Ballaigues, Switzerland). One of the 2 matched root canals was randomly allocated (www.randomizer.org) to the constant-taper protocol, and it was prepared by Mtwo rotary nickel-titanium files (VDW, Munich, Germany) size 15/.05 taper, 20/ .06, 25/.06, and 30/.06. The second matched root canal was prepared by variable-taper VTaper 2H rotary nickel-titanium files (17/.04, 20/.06, 25/.06, 30/.06; SS White Dental, Lakewood, NJ), which have a decreasing taper from their tip toward their shaft in order to remove less dentin in the middle and coronal third of the root canal9. After every instrument, the root canal was rinsed with 2% sodium hypochlorite (Orphi Farma, Lage Zwaluwe, The Netherlands) and distilled water. The distal root canals were left unprepared. The selection and chemomechanical preparation protocol is described in more detail in Supplemental Appendix S1 (available online at www. jendodon.com). Each specimen was scanned 3 more times by the micro–computed tomographic scanner after preparation of the matched root canals to apical size 20, 25, and 30. The geometry of 30-G irrigation needles has been described previously22,26,27. The geometry of the 31-G closed-ended doubleside-vented needle (Navitip; Ultradent Products Inc, South Jordan, UT) was obtained

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through examination under a stereoscopic €ttingen, microscope (Stemi SV-6; Zeiss, Go Germany).

CFD Model A previously validated CFD model27,28 was used in this study. In order to cope with the irregular geometry of real root canals, the hexahedral mesh originally used in the root canal was replaced by a hybrid mesh. The effect of this modification on the predictions of the model was examined in a preliminary study as detailed in Supplemental Appendix S2 (available online at www.jendodon.com). The comparison of the time-averaged velocity magnitude and vectors along the root canal between the new case with the modified mesh, the original case used for the validation of the model, and experimental particle image velocimetry measurements27 revealed a close agreement, thereby confirming that the mesh modification did not affect the results. The 3-dimensional geometry of the 2 matched root canals after preparation to apical size 20, 25, and 30 was imported into ANSYS Design Modeler 14.5 (ANSYS Inc, Canonsburg, PA). The flow domain (Fig. 1) included the complete root canal from the orifice until the apical end point of instrumentation (WL), where a wall was defined. The length of the root canals was standardized to 8.9 mm by the removal of excess coronal structure. The 3 selected needles, a 30-G flat open-ended needle and 30-G and 31-G closed-ended double-sidevented needles, were modeled using the actual needles as references (Navitip [30 G], Ultradent Products Inc; Endo-Irrigation Needle €nster, Germany; [30 G], Transcodent, Neumu and Navitip [31 G], Ultradent Products Inc). The external and internal diameters were standardized to 308 mm and 196 mm for the 30-G needles and 254 mm and 156 mm for the 31-G needles, respectively. The length of all needles was set to 31 mm. The positioning and bending of each needle was based on a preliminary in vitro experiment that determined the maximum attainable insertion depth (until binding) for each needle in curved molar root canals prepared by constant-taper or variable-taper instruments to apical size 20, 25, and 30 (Supplemental Appendix S1 is available online at www.jendodon.com). The maximum attainable insertion depth was converted to the minimum attainable distance from the WL. The average values per case were increased by 1 mm in order to prevent needle binding during irrigation, and they were used to define the fixed position of each needle inside the modeled root

canals. The needles were positioned in the root canals as centered as possible. ANSYS Mesh 14.5 (ANSYS Inc) was used to create the hybrid mesh of the flow domain (1.1–1.8 million cells). The mesh was refined near the walls and in areas where high velocity gradients were anticipated. Grid independence of the results was verified. Noslip boundary conditions were applied to all the walls, which were assumed to be rigid, smooth, and impermeable. A velocity inlet boundary condition was applied at the inlet of the needle, and flat velocity profiles corresponding to a flow rate of 0.05 or 0.15 mL/s were prescribed. Atmospheric pressure was imposed at the root canal orifice. Sodium hypochlorite 2.5% was used as irrigant (density 5 1060 kg/m3; viscosity 5 1.073$1023 Pa$s29), and it was modeled as an incompressible Newtonian fluid. The needle and the root canal were filled with the irrigant. Gravity was defined so as to mimic the orientation of the mesial root canals of a mandibular molar when the patient is lying horizontally during treatment. ANSYS Fluent 14.5 (ANSYS Inc), a finite volume solver, was used to solve the time-dependent Navier-Stokes equations in 3 dimensions. An unsteady isothermal flow was assumed, and no turbulence model was used. A steady-state solution was obtained first and then used as the initial condition for the unsteady simulations. All transport equations were discretized to be at least second-order accurate. For temporal discretization, a first-order implicit formulation was used. The convergence criterion was set to 1024 of the maximum scaled residuals. Pressure, velocity, and vorticity were also monitored to ensure adequate convergence in every time step. A time step of 1026 seconds was used throughout the calculations, which were carried out for a real flow time of 20 milliseconds on a workstation with a 14-core Intel Xeon 2.5 GHz processor (Intel, Santa Clara, CA) and 32 GB of RAM. The flow fields calculated for each of the 36 cases were compared in terms of irrigant velocity, wall shear stress, and apical pressure.

RESULTS Root Canal Anatomy The initial total length of the matched root canals assigned to the constant- and variabletaper preparation protocols was 11.1 and 9.8 mm, respectively. This was reduced to 10.4 and 9.3 mm after preparation to apical size 30. The curvature of the root canals was 37.0 and 36.7 24 before preparation, and it

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FIGURE 1 – (Left ) Geometry of the matched root canals after preparation to apical size 20, 25, and 30 and (right ) charts depicting the diameter of the root canals in the apical 5 mm. The slope of each line represents the root canal taper. A dashed line indicating a constant 0.06 taper has been added to the charts as a reference. decreased to 29.6 and 31.8 after preparation, respectively. Details on the diameter of the root canals in the apical 5 mm are provided in Figure 1.

Flow Pattern A steady ﬂow was developed by all needles in low ﬂow rate cases and also by the 30-G

closed-ended needle in the high flow rate cases. The flow was unsteady in the high flow rate cases that involved the 30-G open-ended or the 31-G closed-ended needle. The 30-G open-ended needle created a high-velocity irrigant jet directed apically with a slight diversion toward the outside of the root canal curvature. The closed-ended needles created

smaller jets at both outlets directed toward the apex at an angle of almost 45 to the needle axis. Most of the irrigant ﬂowed through the outlet most proximal to the tip (30-G needle, 0.05 mL/s: 61.0% [1.3%] and 0.15 mL/s: 70.4% [3.2%]; 31-G needle, 0.05 mL/s: 59.1% [2.5%] and 0.15 mL/s: 72.1% [1.5%]; data presented as time average [standard

FIGURE 2 – Triads of time-averaged irrigant velocity contours (top ) and vectors (middle ) along the z-y plane and streamlines indicating the route of massless particles released downstream from the needle inlet and colored according to time-averaged velocity magnitude (bottom ) in the root canal prepared with Mtwo ﬁles to apical size 25. Particle trajectories provide visualization of the main fresh irrigant ﬂow in 3 dimensions. Needles are colored in red.

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FIGURE 3 – Maximum time-averaged axial irrigant velocity in the apical 5 mm of the root canal as a function of distance from the WL for each needle type, instrument type, apical preparation size, and flow rate, indicating the irrigant penetration front. The scale of the vertical axis has been adjusted to 0–0.5 m/s to highlight differences in the area apically to the needles. Colored dots on the horizontal axis indicate the position of the needle tip for each root canal size. Velocities higher than 0.1 m/s (dotted horizontal line ) were considered to indicate clinically relevant penetration. deviation]). The jet velocity in the 31-G needle cases was higher than in the 30-G needle cases. All jets were more intense in high flow rate cases. The main flow pattern was not affected by the apical preparation size or the instrument type (Fig. 2).

Irrigant Penetration None of the needles was able to deliver the irrigant to the WL in size 20 or 25 root canals irrespective of the ﬂow rate (Fig. 3). The 30-G open-ended and the 31-G closed-ended needles performed similarly, and both were more effective than the 30-G closed-ended needle in these canals. The irrigant penetrated up to the WL in size 30 root canals when the 30-G open-ended needle was used regardless of the ﬂow rate. The same was noted for the 31-G closed-ended needle but only at the

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higher flow rate; a stagnation area was evident apically when the lower flow rate was used. To the contrary, the 30G closed-ended needle could not deliver the irrigant until WL even in the size 30/.06 taper root canals. The higher flow rate improved irrigant penetration when the closed-ended needles were used but not in the case of the open-ended needle. Irrigant penetration was comparable in root canals prepared with either type of instruments with minor differences that were attributed to the slightly different needle insertion depths.

small area within 0.5 mm apically to its tip. The 2 closed-ended needles developed a different pattern with 2 local maxima next to their outlets on the 2 opposite sides of the root canal wall. The 30-G open-ended and the 31-G closedended needles developed higher wall shear stress in the apical part of the root canals compared with the 30-G closed-ended needle. The wall shear stress was considerably higher in the high ﬂow rate cases compared with the low ﬂow rate ones, but it was not affected by the instrument type.

Wall Shear Stress

Apical Pressure

Low wall shear stress was developed in the apical 1–2 mm of root canals prepared to size 20 or 25. Slightly higher values were calculated in size 30 root canals (Fig. 4). The 30-G openended needle developed high shear stress in a

The irrigant pressure at the apical end of the root canal increased from size 20 to size 25, but in most cases, it decreased from size 25 to 30 (Fig. 5). The maximum values were similar for the 30-G open-ended needle and the 31-G

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FIGURE 4 – The maximum time-averaged irrigant shear stress applied on the root canal wall as a function of distance from the WL for the various needle types, instrument types, apical preparation sizes, and ﬂow rates. Colored dots on the horizontal axis indicate the position of the needle tip for each root canal size. Examples of contours of time-averaged irrigant shear stress on the root canal wall of size 25 root canals irrigated at 0.15 mL/s are provided above each graph to illustrate the shear stress pattern. A shadow of the needle is shown behind the root canal wall. The color map on the right side refers to the contours.

closed-ended needle; the 30-G closed-ended needle developed lower maximum pressure. High ﬂow rate cases resulted in higher apical pressure than the low ﬂow rate ones. The pressure was similar in root canals prepared by either type of instruments.

DISCUSSION The main research hypothesis tested in this study was that irrigant penetration and the wall shear stress developed in the apical third during syringe irrigation are compromised in

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minimally shaped root canals when 30-G or 31-G needles are used. The results of the computer simulations confirmed this hypothesis. Curved mesial root canals of mandibular molars were selected as a sufficient challenge for root canal preparation and needle placement in the apical third. Modeling the irregular geometry of real root canals required modifications of the mesh that could affect the validity of the model. Therefore, the predictions of the current model were compared both to the predictions of the original model and to the

experimental measurements used for its validation27 to confirm that the modified meshing strategy did not introduce additional error to the results. Previous research has shown that different subtypes of open-ended needles create very similar irrigant flow, and the same applies to closed-ended needles22. Consequently, only one 30-G open-ended and one 30-G closed-ended needle were included in this study to represent each type and also to allow comparisons with the 31-G closedended needle that had not been evaluated before. The selected irrigant flow rates fall within

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FIGURE 5 – Time-averaged irrigant pressure at the apical end of the root canal as a function of needle distance from the WL for the various needle types, instrument types, apical preparation sizes, and ﬂow rates.

the range applied by clinicians when 30-G needles are used30. Because there was no such information concerning 31-G needles, the same ﬂow rates were used in order to facilitate comparisons. Fresh irrigant is carried apically to the needle only by the axial component of the irrigant velocity (the component in the direction of the root canal axis at each level), so the magnitude of this component was used to determine the extent of irrigant penetration in the apical third, similarly to earlier studies16,22. Irrigant penetration and exchange are related to the chemical effect of irrigation31. The shear

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stress developed on the root canal wall was used as a surrogate for the mechanical cleaning effect of irrigation19,22,23. Recently, the CFD-calculated magnitude of the total irrigant velocity, which contains information about both the chemical and mechanical effects of irrigation, was correlated to bioﬁlm removal from artiﬁcial isthmuses and lateral canals in vitro32. The irrigant pressure at the WL was used as an indicator of the relative risk for inadvertent irrigant extrusion toward the periapical tissues18,19,22,23. Nevertheless, it should be emphasized that without a validated pressure threshold for sodium hypochlorite

accidents, it is not possible to determine in which of these cases an accident would actually occur. Several interesting results were generated by the numerical model. First, the irrigant could not penetrate until the WL in root canals prepared to apical size 20 or 25 irrespective of the type and size of the needle and the flow rate. Clinically, a small amount of irrigant may still be transported along the files during instrumentation, but both the chemical and mechanical effects will be very limited in this area. Thus, it is very unlikely that the apical third of minimally shaped root canals can be sufficiently cleaned and disinfected by syringe irrigation even when a 31-G needle is used. A minimum apical size 30 was required in order for the irrigant to reach the WL. These findings are in agreement with earlier studies that modeled simplified root canals and 30-G needles17,33 and are also consistent with the trend reported by in vitro studies that evaluated 27- to 30-G needles regarding the effect of apical size on irrigant penetration, root canal cleaning, and disinfection12–14,20. Overall, the 30-G closed-ended needle was the least effective type of needle. It could not be placed within 1 mm from the WL without binding16,22, and the irrigant did not penetrate until the WL in any of the simulated cases. The flow developed by the 30-G openended needle could reach the WL in size 30 root canals even at the lower flow rate while maintaining a relatively low apical pressure, but the wall shear stress was also very low. Under these conditions, the 30-G openended needle appeared to be less likely to extrude irrigant through the apical foramen than the closed-ended needles examined here, a finding that contradicted earlier studies17,18,22,34. Nonetheless, only a single flow rate was evaluated in these earlier studies, and all needles were inserted at the same distance from the WL. This contradiction highlighted the importance of the concurrent evaluation of several parameters in the same study and accounting for differences in the attainable insertion depth between the needles. The 31-G closed-ended needle combined with the higher flow rate was the most effective option when considering both the chemical and mechanical effects. In this case, the irrigant penetrated until the WL in size 30 root canals, and the wall shear stress was maximized. However, the apical pressure was higher than any of the low flow rate cases, which may indicate an increased risk of irrigant extrusion through the apical foramen.

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An interaction was found between the flow rate and the needle type regarding irrigant penetration. A higher flow rate improved irrigant penetration for the closed-ended needles but not for the open-ended one, which is in agreement with an earlier in vitro study35. However, increasing the flow rate from 0.05 to 0.15 mL/s led to a large increase in the wall shear stress in all cases; thus, a higher flow rate may still provide an advantage even when an open-ended needle is used. It is noteworthy that irrigant flow rate had a significant effect on biofilm removal from artificial isthmuses and lateral canals during syringe irrigation with an open-ended needle in vitro32. The flow was not distributed equally between the 2 side vents of the closed-ended needles. Most of the irrigant passed through the vent closer to the tip, which is in line with earlier findings22. Still approximately 30%– 40% flowed through the second vent, with more irrigant flowing through in the low flow rate cases. This was considerably higher than the previously reported 6.5%22. The difference could be attributed to the lower flow rate used in this study and to the smaller root canals that resisted the flow through the vent most proximal to the tip, thereby favoring the path through the second vent. The apical pressure decreased in size 30 root canals compared with size 25 when the 31-G closed-ended needle was used. This decrease could be due to the 25%–50% additional space that was available around the tip of the needle for the backflow of the irrigant17. A similar yet less pronounced decrease was also noted in the root canal prepared with variable-taper instruments when a 30-G needle was used.

One of the strengths of this study compared with earlier work was that the needles were inserted as close as possible to the WL without binding based on in vitro measurements rather than assumptions in order to study irrigation under optimum clinically realistic conditions. As a result, they were placed up to 0.5 mm closer to the WL in the root canal prepared with constant-taper instruments compared with the one prepared with variable-taper instruments. This difference may have improved irrigant penetration slightly in these cases16, but the current study design could not provide conclusive evidence about the effect of the insertion depth on the flow because other parameters were varied at the same time. Constant-taper and variable-taper instruments created similar shapes in the apical third of the matched root canals. Size 20 and 25 variable-taper instruments were slightly more conservative than the corresponding constant-taper instruments, but the opposite was true for size 30 instruments. These observations should be interpreted with caution because only 2 root canals were evaluated, but they may indicate that using an instrument of variable taper does not always result in less dentin removal or a variable-taper root canal shape, at least in the apical third. However, it should be noted that the variable-taper instruments used in this study had a constant 0.06 taper near their tip9, which could explain the similarities in the shape of the apical third. The lack of significant differences in the flow between the 2 root canals could also be attributed to this fact because the shape of the apical third determines irrigant penetration to a large extent17,33 and the

current analysis of the ﬂow focused on that area. On the other hand, the variable-taper instruments were more conservative 5 mm away from the WL. This did not seem to affect the ﬂow, but it could explain the differences in the attainable needle insertion depth between the 2 root canals.

CONCLUSIONS Preparation of curved root canals to apical size 20 or 25/.06 taper did not allow sodium hypochlorite to reach the WL. When the apical size was increased to 30/.06 taper, a 30-G open-ended needle allowed the irrigant to reach the WL even when irrigation took place at a low ﬂow rate (0.05 mL/s). The 31-G closed-ended needle combined with the high ﬂow rate (0.15 mL/s) also delivered the irrigant up to the WL in size 30 root canals. Although this gave rise to higher wall shear stress, it also developed higher apical pressure.

ACKNOWLEDGMENTS Supported by the European Society of Endodontology Annual Research Grant in 2017. The authors deny any conﬂicts of interest related to this study.

SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found in the online version at www.jendodon.com (https://doi.org/10.1016/ j.joen.2021.06.001).

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BASIC RESEARCH – TECHNOLOGY Sneha Susan Santosh, MDS, Suma Ballal, MDS, and Velmurugan Natanasabapathy, MDS

SIGNIFICANCE Minimally invasive access cavity designs (ConsAC and TrecAC) showed improved fracture resistance with lesser unfavorable fractures and hence would allow for longer survival of endodontically treated teeth.

Influence of Minimally Invasive Access Cavity Designs on the Fracture Resistance of Endodontically Treated Mandibular Molars Subjected to Thermocycling and Dynamic Loading ABSTRACT Introduction: The aim of this study was to investigate the fracture resistance of endodontically treated and restored permanent mandibular molars with minimally invasive access cavities subjected to thermocycling and dynamic loading. Methods: Forty first and second mandibular molars were randomly assigned to 4 groups (n 5 10/group) as follows: group 1, control (intact teeth); group 2, traditional access cavity (TradAC); group 3, conservative access cavity (ConsAC); and group 4, truss access cavity (TrecAC). After endodontic treatment, teeth were restored with SDR core (Dentsply Caulk, Milford, DE) and subjected to thermocycling followed by dynamic and static loading with a multiaxial fatigue testing machine (Instron, Canton, MA). The maximum load to fracture and pattern of failure (restorable/unrestorable) were recorded. Data were evaluated with analysis of variance and the Tukey post hoc test for multiple comparisons. Results: Fracture resistance of the samples in the control group were higher than those in the experimental groups (P , .005). TradAC exhibited the least resistance to fracture (P , .005). There was no statistically significant difference in the fracture resistance of ConsAC and TrecAC (P 5 .361) Unrestorable fractures were more frequent in the TradAC group compared with all other groups. Conclusions: Mandibular molars with ConsAC and TrecAC exhibited superior fracture resistance compared with TradAC. TradAC had the highest number of unrestorable fractures. (J Endod 2021;47:1496–1500.)

From the Department of Conservative Dentistry and Endodontics, Faculty of Dentistry, Meenakshi Ammal Dental College and Hospital, Meenakshi Academy of Higher Education and Research, Chennai, Tamil Nadu, India

KEY WORDS

Address requests for reprints to Dr Velmurugan Natanasabapathy, Department of Conservative Dentistry and Endodontics, Faculty of Dentistry, Meenakshi Academy of Higher Education and Research University, No 1, Alapakkam Main Road, Maduravoyal, Chennai 600 095, Tamil Nadu, India. E-mail address: [emailprotected] 0099-2399/$ - see front matter

Fracture of endodontically treated teeth (ETT) is 1 of the most undesirable phenomena in clinical practice. A retrospective clinical study that examined the 10-year survival of ETT observed root fractures in 36% of the extracted teeth.1 According to a ﬁnite element–based analysis, an increase in size of the access cavity increased the stresses in the cervical region.2,3 The authors concluded that access cavity designs that preserve a larger amount of coronal hard tissue may improve the fracture resistance of ETT.2,3 A prospective study also concluded that the percentage of extractions in ETT was 3 times higher when the volume of residual coronal tooth structure was less than 29.5%.4 Hence, a conservative endodontic access could improve the prognosis of ETT. The traditional access cavity (TradAC) allows for the liberal removal of sound dentin to achieve straight-line access, thereby increasing the susceptibility of ETT to fracture.5 Currently, with greater emphasis on dentin conservation, there is a need for minimally invasive access cavity (MiAC) designs in endodontics.6 The conservative access cavity (ConsAC) aims to preserve pericervical dentin (PCD) and

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Conservative access cavity; dynamic loading; fracture resistance; thermocycling; truss access cavity

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soffit. It involves accessing the teeth at the central fossa and extending the preparation only as necessary to detect canal orifices while maintaining a portion of the chamber roof.6 Preservation of this critical PCD has been shown to reduce cuspal flexure and improve fracture resistance.6–8 The truss access cavity (TrecAC) involves an orifice-directed approach, conserving the central part of the roof of the pulp chamber.9,10 Studies regarding the benefits of TrecAC with respect to improved fracture resistance are limited. An earlier study suggested that in ETT restoring the access cavity may improve fracture resistance up to 72% that of an intact tooth.11 SDR composite (Dentsply Caulk, Milford, DE) can be bulk filled in layers up to 4 mm and is being used commonly for replacing lost dentin in ETT.12–14 The flexible polymer technology prevents the translation of shrinkage stresses, thereby improving fracture resistance.13 Most of the previous studies evaluated the impact of MiAC on fracture resistance of ETT when subjected to a static load.7,8,10,13,15 However, laboratory tests performed under static loading do not simulate clinical conditions.7,16 Ordinola-Zapata and Fok et al 16 observed that most failures in a clinical setting occurred as a result of cyclic fatigue. Fatigue was a principal contributor to fracture in ETT, and measures of static strength used for testing MiAC were not an accurate reflection of its clinical performance.16 Aging by thermocycling is also necessary in order to best mimic the clinical scenario and simulate thermal alterations in the oral cavity.17,18 To date, there have been no studies comparatively evaluating the fracture resistance of ETT with various MiACs restored with bulk fill composite subjected to thermocycling and dynamic loading. Hence, the aim of this laboratory study was to evaluate the fracture resistance of endodontically treated and restored permanent mandibular molars with TradAC, ConsAC, and TrecAC subjected to thermocycling and dynamic loading.

MATERIALS AND METHODS Specimen Selection and Preparation Ethical clearance was obtained from the Institutional Review Board of Meenakshi Ammal Dental College, Meenakshi Academy of Higher Education and Research University, Chennai, Tamil Nadu, India (institutional review board number MADC/IRB-XXV/2018/390). A total of 40 freshly extracted, human permanent ﬁrst and second mandibular molars (n 5 40) were selected and stored in 0.1% thymol

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solution at room temperature. Teeth devoid of caries, attrition, cracks, and an intact crown with mature root apices were selected for the study. Samples of similar height (the distance between the occlusal surface and the apex of the tooth) and width (the mesiodistal distance at the cementoenamel junction) were selected for the study. A preoperative cone-beam computed tomographic unit (ProMax 3D Mid; Planmeca Oy, Helsinki, Finland) with a voxel size of 75 mm was used to assess the morphology and number of root canals. The specimens (n 5 40) were randomly divided into the following 4 groups with a sample size of 10 per group (n 5 10/group):

Group 1: Intact teeth (the control group) Group 2: TradAC Group 3: ConsAC Group 4: TrecAC

Access Cavity Preparation TradAc In the TradAC group, the access cavity was prepared with an 856 diamond bur (Komet Italia SRL, Milan, Italy) and an Endo Z bur (Dentsply Sirona, York, PA) mounted on a high-speed handpiece with water coolant. TradAC involved complete derooﬁng with exposure of the pulp horns and straight-line access to the root canals 19,20 (Fig. 1A).

ConsAC In the ConsAC group, access was prepared with an 856 diamond bur mounted on a highspeed handpiece with water coolant under a dental operating microscope (Labomed Dental Microscope Prima DNT). Access cavity preparation was initiated at the central fossa and extended only as necessary to detect canal oriﬁces while maintaining a portion of the chamber roof and preserving the PCD 6,21 (Fig. 1B).

TrecAC In the TrecAC group, separate cavities were prepared to approach the different roots of the molars without removal of the central part of the pulp chamber roof (ie, a single access to the mesial canals in the buccolingual direction and another access to reach the distal canal oriﬁce) 9,10,22 (Fig. 1C). Endodontic access was prepared using an 856 diamond bur mounted on a high-speed handpiece with coolant under the dental operating microscope (Labomed Dental Microscope Prima DNT).

Endodontic Treatment Root canals were negotiated 0.5 mm short of the apex using a size 10 K-ﬁle (Dentsply

Maillefer, Ballaigues, Switzerland) and instrumented with Mtwo nickel-titanium rotary ﬁles (Sweden & Martina, Padova, Italy) up to #25 tip size and a 0.06 taper. During instrumentation, 2 mL 3% sodium hypochlorite was delivered between each instrument. Final irrigation was performed using 2 mL 3% sodium hypochlorite, saline, and 2 mL 17% EDTA. Irrigants were activated with passive ultrasonic irrigation using a 20 size Irrisafe tip (Satelec, Acteon, Paris, France) at 40 kHz for 1 minute at 1 mm from the working length. The canals were then dried with absorbent points and obturated with gutta-percha and AH Plus sealer (Dentsply DeTrey, Konstanz, Germany).

Teeth Restoration Endodontic access cavities of the test groups were conditioned with 37% phosphoric acid (Etching Gel; Prime Dental Products, Thane, India) for 15 seconds in dentin and 30 seconds in enamel, rinsed with water, and gently airdried. Using microapplicator tips, the bonding agent (Prime & Bond Elect, Dentsply Sirona) was applied according to the manufacturer’s instructions. Subsequently, SDR was used as the bulk ﬁll core material, and the occlusal surface (2 mm) was restored using Ceram X duo composite (Dentsply Sirona).

Periodontal Ligament Simulation All the samples were mounted 2 mm apical to the cementoenamel junction in customized cylinders fabricated using self-cure resin with a 0.2-mm-thick lining of polyvinyl siloxane (Aquasil Ultra Monophase Regular Set, Dentsply Sirona) to simulate the periodontal ligament.23

Thermocycling The samples were placed in water baths of different temperatures, and thermocycling was performed in the following manner: 35 C for 28 seconds, 15 C for 2 seconds, 35 C for 28 seconds, and 45 C for 2 seconds for 5000 cycles, approximately simulating 6 months of thermal changes occurring in the oral cavity.24

Fracture Testing After thermocycling, the samples were subjected to a dynamic load of 125,000 cycles to simulate 6 months of aging using a multiaxial fatigue testing machine (Instron, Canton, MA) between 5 N and 50 N at 15 Hz.25 After dynamic loading, the teeth were subjected to a static load at the central fossa at an angle of 30 to the long axis of the tooth. A continuous compressive force at a crosshead speed of 1 mm/min was applied using a 5-mmdiameter, ball-ended steel compressive head.25 The loads at which the teeth fractured

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FIGURE 1 – Access cavity preparation. (A ) TradAC, (B ) ConsAC, and (C ) TrecAC.

were recorded in newtons.25 The fractured specimens were examined under a stereomicroscope (SZR- 10; Optika, Bergamo, Italy) in order to determine the level at which failure occurred. The specimens were classiﬁed as ‘‘restorable’’ when the site of fracture extended above the acrylic resin (above the level of bone simulation) and ‘‘unrestorable’’ when it extended below the acrylic resin (below the level of bone simulation).7

Statistical Analysis Data were analyzed using SPSS software (Version 21; IBM Corp, Armonk, NY), and a P value ,0.05 was considered statistically signiﬁcant. The normality of distribution was checked using the Shapiro-Wilk and Skewness-Kurtosis tests. The difference in compressive loads between groups was assessed using 1-way analysis of variance. Tukey post hoc analysis was used to evaluate multiple comparisons between various groups. The Fisher exact test followed by post hoc analysis (via standardized cell residuals) was used to assess the difference among the 4 groups with respect to the pattern of failure.26

RESULTS The values of the mean load to fracture for all groups are depicted in Table 1. One-way analysis of variance showed statistically significant differences in the fracture resistance between all the groups (P , .005). Tukey post hoc analysis for pair-wise comparisons (mean load to fracture) revealed significantly higher fracture resistance for the control group compared with all of the other test groups (P , .005). The mean load to fracture for TradAC was significantly lower than that of ConsAC and TrecAC (P , .005). There was no statistically significant difference in the fracture resistance of ConsAC and TrecAC (P 5 .361) (Table 1). Intact teeth exhibited the highest number of restorable fractures, whereas TradAC exhibited the highest number of unrestorable fractures (P , .05) (Table 2). There was no significant difference in the number of restorable or unrestorable fractures between ConsAC and TrecAC (P . .05).

DISCUSSION ETT undergo loss of coronal and radicular tooth structure as a result of prior pathology,

access cavity preparation, and restorative procedures.27,28 Current evidence suggests that fracture of ETT is multifactorial. A longterm retrospective cohort study reported that the volume of coronal dentin lost plays a signiﬁcant role in determining the survivability of ETT.29 Hence, conservation of tooth structure during access cavity preparation is of paramount importance.6 Two such recently advocated minimally invasive designs are ConsAC and TrecAC.7–10 In the present study, fracture resistance of TradAC, ConsAC, and TrecAC on endodontically treated mandibular molars was evaluated. Mandibular molars were selected because they exhibit the highest incidence of vertical root fractures among endodontically treated posterior teeth.30 A literature review suggests that restoration of ETT increases the fracture resistance up to 72% as that of an intact tooth.11 According to Garlapati et al,31 the use of a reinforcing restorative material (Ever X Posterior) could improve the fracture resistance of ETT. However, a more recent study reported SDR bulk ﬁll to be superior to Ever X Posterior (everX Posterior GC EUROPE, Leuven, Belgium) for core buildup in posterior

TABLE 1 - The Load to Fracture (Mean and Standard Deviation [SD]) for the Control and Intervention Groups

Groups

Mean load to fracture (N/mm2)

P value (1-way ANOVA)

Control

898.8 (96.4)

,.005

TradAC

421.7 (13.2)

ConsAC TrecAC

633.42 (47.9) 582.05 (90.6)

Multiple group comparison

P value (Tukey post hoc analysis) ,.005 ,.005 ,.005 ,.005 ,.005 .361

TradAC ConsAC TrecAC ConsAC TrecAC TrecAC

ANOVA, analysis of variance; ConsAC, conservative access cavity; TradAC, traditional access cavity; TrecAC, truss access cavity.

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TABLE 2 - The Type of Fracture (Restorable or Unrestorable) for the Control and Intervention Groups Type of fracture Group TradAC ConsAC TrecAC Control

Restorable a

1 5a 4a 8b

Unrestorable 9b 5a 6a 2a

ConsAC, conservative access cavity; TradAC, traditional access cavity; TrecAC, truss access cavity. Similar superscript lowercase letters in the same row indicate no statistically signiﬁcant differences (P . .05).

teeth.13 This could be attributed to its patented flexible polymer that prevents the translation of shrinkage stresses onto the tooth.13 In our study, before fracture testing, root canals were obturated and restored with SDR bulk fill base and Ceram X duo layering composite. A previous study has reported that a bilayered restoration can act as an effective stress absorber to reduce shrinkage stresses.32 Minimally invasive designs could lead to compromised pulp chamber debridement.9 Therefore, in the present study, irrigant activation was performed using passive ultrasonic irrigation. In this study, thermocycling was performed to simulate thermal alterations in the oral cavity and mimic the clinical condition.17,18 Saberi et al 18 reported that in the absence of thermal stresses, there was no significant difference among the 2 experimental groups (TrecAC and TradAC).

However, after thermocycling, TrecAC demonstrated higher fracture resistance, which was comparable with the control group (intact tooth).18 In the present study, after thermocycling, teeth were subjected to dynamic loading. In vitro tests performed under static loading do not accurately reflect intraoral conditions wherein loads are dynamic and failure primarily occurs due to fatigue.16 To the best of our knowledge, this is the first study of its kind to determine the fracture resistance of endodontically treated mandibular molars with TradAC, ConsAC, and TrecAC restored with SDR bulk fill base and Ceram X duo nanohybrid composite subjected to thermocycling (5000 cycles) and dynamic loading (125,000 cycles). Among the experimental groups, ConsAC and TrecAC had a higher fracture resistance compared with TradAC. Previous studies have exhibited improved fracture resistance with MiAC,7,8 whereas others have reported contrasting results.10,13,15 A recent critical analysis has concluded no clear scientific evidence regarding the benefits of MiAC over TradAC in terms of improved fracture resistance of ETT.17 This could be due to differences in methodology, the restorative materials used, the absence of thermocycling, and the methods used to assess fracture resistance.17 In the present study, these limitations were overcome, which could be a plausible explanation for the lower mean load to fracture values compared with other studies.7,8

The results of the current study revealed that TradAC had a higher number of unrestorable fractures compared with ConsAC and TrecAC. These ﬁndings are in agreement with previous reports.7,13 There was no signiﬁcant difference among the pattern of failure in the ConsAC or TrecAC group. Complete debridement of the pulp chamber and total polymerization of composites under the preserved central roof of TrecAC are issues that have to be addressed in future studies. The present study provides insight on the fracture resistance of 2 different MiACs (ConsAC and TrecAC) that best simulate a clinical scenario. However, there is a need for further clinical studies evaluating the impact of various MiACs on the fracture resistance of ETT with long-term follow-up.

CONCLUSION Within the limitations of this study, it can be concluded that ConsAC and TrecAC exhibited improved fracture resistance compared with TradAC in mandibular ﬁrst and second molars restored with SDR bulk ﬁll subjected to thermocycling and dynamic loading. MiAC designs presented with a higher number of restorable fractures compared with TradAC.

ACKNOWLEDGMENTS The authors deny any conﬂicts of interest related to this study.

REFERENCES

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Landys Boren D, Jonasson P, Kvist T. Long-term survival of endodontically treated teeth at a public dental specialist clinic. J Endod 2015;41:176–81.

Wang Q, Liu Y, Wang Z, et al. The effect of access cavities and canal enlargement on biomechanics of endodontically treated teeth: a ﬁnite element analysis. J Endod 2020;46:1501–7.

Zhang Y, Liu Y, She Y, et al. The effect of endodontic access cavities on fracture resistance of ﬁrst maxillary molar using the extended ﬁnite element method. J Endod 2019;45:316–21.

Al-Nuaimi N, Ciapryna S, Chia M, et al. A prospective study on the effect of coronal tooth structure loss on the 4-year clinical survival of root canal retreated teeth, and retrospective validation of the Dental Practicality. Int Endod J 2020;53:1040–9.

Boveda C, Kishen A. Contracted endodontic cavities: the foundation for less invasive alternatives in the management of apical periodontitis. Endod Topics 2015;33:169–86.

Clark D, Khademi J. Modern molar endodontic access and directed dentin conservation. Dent Clin North Am 2010;54:249–73.

Plotino G, Grande NM, Isuﬁ A, et al. Fracture strength of endodontically treated teeth with different access cavity designs. J Endod 2017;43:995–1000.

Krishan R, Paqu e F, Ossareh A, et al. Impacts of conservative endodontic cavity on root canal instrumentation efﬁcacy and resistance to fracture assessed in incisors, premolars, and molars. J Endod 2014;40:1160–6.

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Corsentino G, Pedulla E, Castelli L, et al. Inﬂuence of access cavity preparation and remaining tooth substance on fracture strength of endodontically treated teeth. J Endod 2018;44:1416– 21.

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Hamouda IM, Shehata SH. Fracture resistance of posterior teeth restored with modern restorative materials. J Biomed Res 2011;25:418–24.

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Isufi A, Plotino G, Grande NM, et al. Fracture resistance of endodontically treated teeth restored with a bulkfill flowable material and a resin composite. Ann Stomatol (Roma) 2016;7:4–10.

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€ Demiryu € et al. The effects of endodontic access cavity preparation € €rek T, Ulker €rek EO, Ozyu O, design on the fracture strength of endodontically treated teeth: traditional versus conservative preparation. J Endod 2018;44:800–5.

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Pedalino I, Hartup GR, Vandewalle KS. Depth of cure of bulk-ﬁll ﬂowable composite resins. Gen Dent 2015;63:e28–34.

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Ordinola-Zapata R, Fok AS. Research that matters: debunking the myth of the "fracture resistance" of root ﬁlled teeth. Int Endod J 2021;54:297–300.

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Silva EJ, Pinto KP, Ferreira CM, et al. Current status on minimal access cavity preparations: a critical analysis and a proposal for a universal nomenclature. Int Endod J 2020;53:1618–35.

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Saberi EA, Pirhaji A, Zabetivan F. Effects of endodontic access cavity design and thermocycling on fracture strength of endodontically treated teeth. Clin Cosmet Investig Dent 2020;23:149–56.

19.

Ingle JI. Endodontic cavity preparation. In: Ingle J, Tamber J, editors. Endodontics. 3rd ed. Philadelphia: Lea & Febiger; 1985. p. 102–67.

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Patel S, Rhodes J. A practical guide to endodontic access cavity preparation in molar teeth. Br Dent J 2007;203:133–40.

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Clark D, Khademi JA. Case studies in modern molar endodontic access and directed dentin conservation. Dent Clin North Am 2010;54:275–89.

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Narayana P. Access cavity preparations. In: Schwartz RS, Canakapalli V, editors. Best Practices in Endodontics: A Desk Reference. 1st ed. Chicago: Quintessence Publishing; 2015. p. 89–104.

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Soros C, Zinelis S, Lambrianidis T, et al. Spreader load required for vertical root fracture during lateral compaction ex vivo: evaluation of periodontal simulation and fracture load information. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:e64–70.

24.

Gale MS, Darvell BW. Thermal cycling procedures for laboratory testing of dental restorations. J Dent 1999;27:89–99.

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Moore B, Verdelis K, Kishen A, et al. Impacts of contracted endodontic cavities on instrumentation efﬁcacy and biomechanical responses in maxillary molars. J Endod 2016;42:1779–83.

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Shan G, Gerstenberger S. Fisher’s exact approach for post hoc analysis of a chi-squared test. PLoS One 2017;12:e0188709.

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Kishen A. Mechanisms and risk factors for fracture predilection in endodontically treated teeth. Endod Topics 2006;13:57–83.

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Kishen A. Biomechanics of fractures in endodontically treated teeth. Endod Topics 2015;33:3–13.

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Nagasiri R, Chitmongkolsuk S. Long-term survival of endodontically treated molars without crown coverage: a retrospective cohort study. J Prosthet Dent 2005;93:164–70.

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PradeepKumar AR, Shemesh H, Jothilatha S, et al. Diagnosis of vertical root fractures in restored endodontically treated teeth: a time-dependent retrospective cohort study. J Endod 2016;42:1175–80.

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Garlapati TG, Krithikadatta J, Natanasabapathy V. Fracture resistance of endodontically treated teeth restored with short ﬁbre composite used as a core material-an in vitro study. J Prosthodont Res 2017;61:464–70.

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Tiu J, Belli R, Lohbauer U. Rising R–curves in particulate/ﬁber-reinforced resin composite layered systems. J Mech Behav Biomed Mater 2020;103:103537.

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BASIC RESEARCH – TECHNOLOGY Sang Won Kwak, DDS, MS, PhD,* Jung-Hong Ha, DDS, MS,

Effects of Root Canal Curvature and Mechanical Properties of Nickel-Titanium Files on Torque Generation

PhD,† Ya Shen, DDS, PhD,‡ Markus Haapasalo, DDS, PhD,‡ and Hyeon-Cheol Kim, DDS, MS, PhD*

ABSTRACT Introduction: This study aimed to compare the torque generated by 4 different files in root canals with 4 different curvature angles. Methods: Four brands of nickel-titanium (NiTi) endodontic files were selected: WaveOne Primary (Dentsply Sirona, Ballaigues, Switzerland), WaveOne Gold Primary (Dentsply Sirona), ProTaper Universal F2 (Dentsply Sirona), and ProTaper Next X2 (Dentsply Sirona). A tempered steel block containing artificial canals with 4 different canal curvatures (15 , 25 , 35 , and 45 ) was constructed. Each file was used according to the manufacturer’s instructions in the dynamic model, with an added 15 axial upand-down movements of 4 mm at the end of the canal. The generated torque was recorded, and the total and maximum torque values were measured. Two-way analysis of variance and the Duncan post hoc comparison test were performed at a significance level of 95%. Results: A significant correlation between the curvature angle and the type of file system was observed (P , .05). As the degree of canal curvature increased, the generated total and maximum torque increased. At 15 and 25 , the NiTi files with reciprocating motion generated a higher total and maximum torque than files with continuous rotation. ProTaper Universal of conventional NiTi alloy showed the steepest increase in the generated total and maximum torque with the increasing curvature angle. The ProTaper Next file had the lowest torque values at the higher canal angles of 35 and 45 (P , .05). Conclusions: Despite the study limitations, it can be concluded that root canal curvature, design, and heat treatment of NiTi files and file kinematics affect the generated torque during instrumentation. (J Endod 2021;47:1501–1506.)

SIGNIFICANCE Under the study limitations, the factors such as a smaller angle of root canal curvature, the use of heat-treated NiTi ﬁles, and the use of NiTi ﬁles with continuous rotation result in a lower amount of generated torque during instrumentation.

KEY WORDS Curvature angle; heat-treated file; nickel-titanium file; reciprocation; torque Since nickel-titanium (NiTi) files were first introduced by the endodontic industry in 1988,1 NiTi files have become popular because of their greater flexibility and superior mechanical properties compared with conventional stainless steel files.2–4 However, unpredictable breakage has been a major concern during the clinical use of engine-driven NiTi files.5 Two types of fracture mechanisms of rotary instruments are well-known: torsional and cyclic fatigue fractures.6 Cyclic fatigue fracture occurs due to repetitive compressive and tensile stresses when the NiTi file rotates in a curved canal and exhibits no clinical signs of fracture or deformation.6 Several factors are related to file fracture, including the cross-sectional design of the NiTi file, heat treatment, rotational speed, torque, root canal configuration, and operator proficiency.7–13 Heat treatment makes NiTi files more flexible and resistant to cyclic fatigue.8 Controlled memory (CM) NiTi files have increased austenite temperatures to keep them in a martensite condition at body temperature, which is characterized by softness and ductility.13 The type of movement also affects cyclic fatigue resistance. The reciprocating movement was shown to increase cyclic fatigue resistance.14,15 The torque is defined as a measure of the force that tends to cause rotation around an axis. In clinical situations, the workload (generated reaction stress) when the file rotates could be shown as torque in an endodontic motor. During root canal instrumentation with engine-driven NiTi files, the endodontic motor inevitably generates torque to rotate the NiTi file. Although the generated torque has been reported to indicate the force to remove the root dentin,16 it also has been shown that stress is

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From the *Department of Conservative Dentistry, School of Dentistry, Dental and Life Science Institute, Dental Research Institute, Pusan National University, Yangsan, South Korea; †Department of Conservative Dentistry, School of Dentistry, Kyungpook National University, Daegu, South Korea; and ‡Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, The University of British Columbia, Vancouver, British Columbia, Canada Address requests for reprints to Dr Hyeon-Cheol Kim, Department of Conservative Dentistry, School of Dentistry, Pusan National University, Geumo-ro 20, Mulgeum, Yangsan, Gyeongnam 50612, South Korea. E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.06.019

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transmitted to the root dentin or the file.17 Several factors, such as the contact area between the canal wall and the NiTi file, the operator’s apical handling force, and preoperative canal volume, have an impact on torque generation.18 The generated torque increases as the contact area related to the geometry and taper of the NiTi file system increases.19 The intensity of stress on the instrument and dentin may also increase with the increasing curvature angle. The torque generated during root canal shaping may be affected by not only anatomic features, such as the degree of calcification and curvature angle of the root canal, but also by the features of the NiTi file, such as the cross-sectional design, heat treatment, and the type of movement. Therefore, this study aimed to compare the torque generated by 4 different NiTi files in canal curvatures with 4 different curvature angles. The null hypothesis was that the type of file movement (rotating vs reciprocating), the type of NiTi metal (superelastic vs heat treated), and the magnitude of the angle of the root canal curvature do not affect the amount of torque generated.

MATERIALS AND METHODS NiTi Files Four brands of size #25 NiTi endodontic files were selected for this experiment: the WaveOne Primary file (WO; Dentsply Sirona, Ballaigues, Switzerland), the WaveOne Gold Primary file (WOG, Dentsply Sirona), ProTaper Universal F2 (PTU, Dentsply Sirona), and ProTaper Next X2 (PTN, Dentsply Sirona). The 4 files had the same tip size (ISO #25) but different heat-treated alloy, cross-sectional geometries, and kinematics. The tapers of the apical 4 mm of WO, WOG, PTU, and PTN were .08, .07, .08, and .06, respectively, and they have a gradually diminishing coronal taper. Before the experiment, each file was examined under an operating microscope (Leica S6D; Leica Microsystems, Wetzlar, Germany) at 10! magnification, and files with any defects or deformities were discarded.

Experimental Model To prevent any changes in the dimensions of the canal during the instrumentation procedures, a tempered steel block containing artiﬁcial canals with 1 of the 4 curvatures (15 , 25 , 35 , or 45 ) was constructed (Fig. 1A and B). The curvature angles of the artiﬁcial canal were calculated using the Schneider method.20 The radii of curvature for 15 , 25 , 35 , and 45 were 18.0, 11.9, 7.8, and 6.1 mm, respectively. The length of the simulated canals was 16.5 mm, and the

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coronal and apical diameters of the canals were 1.63 and 0.6 mm, respectively. Sixty unused instruments of each NiTi file system were used (n 5 15 for each canal curvature and file system). The instrumentation procedure was performed using a torquecontrolled motor (X-smart, Dentsply Sirona) with the designated kinematics following the manufacturer’s recommendations at room temperature. The artificial canal was sprayed with synthetic oil (WD-40; WD-40 Company, San Diego, CA) as a lubricant to reduce friction and heat generation. Each file tip was located 4 mm from the end of the canal and driven in the dynamic model with 4 mm axial up-anddown movements (repetitive movement downward 4 mm first and then upward 4 mm) (Fig. 1). In total, 15 up-and-down movements, each 2 seconds, were applied using a custommade device for reproducible simulation of file movement within a root canal. During instrumentation, the generated torque from the endodontic motor was recorded at a rate of 100 Hz using the acquisition module and coupling customized software (Auto Endo Shaper 1071; DMJ System, Busan, Korea). The acquired torque data were analyzed using Origin 6.0 Professional (OriginLab Corp, Northampton, MA) to produce a scatterplot. The total torque value was calculated by summing the torque values of all data points, and the maximum torque value was measured at the highest point of the plot.

Statistical Evaluation The collected torque data were analyzed using the Kolmogorov-Smirnov test to evaluate the normality of the distribution. The data showed a normal distribution, and 2-way analysis of variance and the Duncan post hoc comparison test were performed to identify the influence of the 2 factors and their interactions on torque generation. The 2 independent variables were the degree of canal curvature and file type, and the dependent variable was the change in torque during file movement. The significance level was set at 95%. Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp, Armonk, NY).

RESULTS Tables 1 and 2 show the total and maximum torques generated during instrumentation in canals with each curvature angle. Two-way analysis of variance revealed that the generated total and maximum torques were affected by the curvature angles and the type of ﬁle system (P , .05). In addition, there was a signiﬁcant relationship between the curvature

angles and the file systems (P , .05, interaction ,.05). The total and maximum torques were significantly lower during instrumentation in canals with 15 and 25 curvature angles than those with 35 and 45 curvature angles (P , .05). As the degree of canal curvature increased, the generated total and maximum torque increased. PTN had the lowest maximum and total torque in all 4 curvatures; in the 2 highest curvatures, the difference to all other files was significant. PTU had an equally low torque as PTN at 15o and 25o canals, but the torque for PTU increased rapidly at higher curvatures; at 45o, it recorded the highest value together with the WO file. The ratio of the total torque values from instrumentation in canals with a 45 angle to that obtained from instrumentation in canals with a 15 angle were !6.58, !3.21, !2.04, and !1.50 for PTU, PTN, WO, and WOG, respectively. In canals with 15 and 25 angles, the NiTi files rotating with reciprocation motion (WO and WOG) generated higher total and maximum torques than those rotating with continuous motion (PTU and PTN) (P , .05). The total torque of PTU, PTN, and WO showed a statistically significant increase when the canal curvature angle increased from 25 to 35 and 35 to 45 (P , .05). In contrast, WOG showed a significant increase in the total torque only when the canal curvature angle increased from 35 to 45 (P , .05).

DISCUSSION The generated torque is considered to be the power needed to keep the file at a constant rotational speed and is essential for removing the root dentin. Torque is inevitably generated during root canal procedures with enginedriven NiTi files, even if the file rotates freely in the air.21 The geometry of the root canal, such as the canal curvature and the degree of calcification, is the cause of the fracture of the NiTi file.22 To date, several studies have reported that the cyclic fatigue resistance is affected by the parameters of the geometry of root canal curvatures, such as the radius length, the arc length, and the arc location.23– 25 However, the present study is the first to investigate torque changes during instrumentation in canals with different angles of curvature. A tempered steel metal block was used in our study to evaluate torque generation according to the variation in the canal curvature. To compare the results in different canals, the diameter and working length of the 4 artificial root canals were standardized. The sizes of the artificial canals were larger than the overall file diameter to eliminate additional

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FIGURE 1 – (A ) A custom-made tempered steel block containing artiﬁcial canals. (B ) The computer-aided design for the fabrication of the simulated canal block with different canal curvatures (15 , 25 , 35 , and 45 ).

torque generation while the files remove dentin (metal component) during instrumentation. In addition, 15 pecking movements of approximately 4 mm in depth, each lasting for 2 seconds, were performed during the instrumentation procedure. According to a previous study, using a longer pecking depth generates larger internal stress on the instruments and against the root.26 The rotational movement of the file without cutting the root dentin generates a small amount of torque. Therefore, a 4-mm-deep, up-anddown movement starting from 4 mm above the apical foramen was applied in our study to observe the changes in torque. The generated torque was affected by the characteristics of the files used and the curvature angle of the artificial canal (Table 1). Therefore, the null hypothesis was rejected. In our study, PTU and PTN, which are designed to rotate in a continuous clockwise motion, generated a lower total and maximum torque at curvature angles of 15 and 25 than WO and WOG, which rotate in reciprocation motion. The differences in torque may be

explained by the differences in the specific movement of the files. Reciprocation files alternate between clockwise and counterclockwise rotations during instrumentation. When the rotation changes from one direction to another, a momentary torque is generated to change the rotational direction (Fig. 2A and B). Therefore, the amplitude of the generated torque in the continuous rotation files was lower than that in the reciprocation files during movement. When the curvature angle increased from 15 to 45 , the ratio of the highest (45 ) to lowest (15 ) torque values by the reciprocating instruments (WO [!2.04] and WOG [!1.50]) was smaller than those of the rotary instruments (PTU [!6.58] and PTN [!3.21]). In other words, the increase in the generated torque was relatively smaller for the reciprocating files because they generated a relatively high torque compared with the continuous rotation files already at small angles of curvature (15 and 25 ). Compared with WO, the more flexible WOG generated lower values of total torque at a canal curvature angle

of 45 (Table 1). At an angle of 15 , both WO and WOG showed similar total torque values. However, the total torque of WO showed a sharp increase, especially when the angle increased from 35 to 45 . This result seems to have been influenced by the geometry, taper, and heat treatment methods of the WO file. WO may have 3 points of contact with the canal wall, whereas WOG files have 1 or 2 because of their cross sections (ie, the parallelogram shape of WOG and the convex triangular shape of WO).27 WOG is made from a martensite dominant alloy (so-called gold alloy), which has similar characteristics to CM NiTi files and is more flexible than WO, which is made of M-Wire.28 The conventional NiTi file has a shape memory effect in which a file recovers to its original shape when heated above a certain characteristic transformation temperature or when the external stress is removed.29 As the angle of curvature increases, files try to return to their original shape, and the friction between the file and canal wall increases. In contrast, the heat-treated files demonstrate martensitic

TABLE 1 - The Total Torque (Ncm) Generated during Instrumentation with 15 Pecking Strokes for Each Nickel-Titanium File System (Mean 6 Standard Deviation) (n 5 15) Curvature angle File system ProTaper Universal ProTaper Next WaveOne WaveOne Gold

.064 6 .012 (x) .061 6 .013Aa (y) .200 6 .026Ba (z) .207 6 .017Ba (w) Aa

.084 6 .020 (1.31 x) .062 6 .008Aa (1.01 y) .224 6 .033Ba (1.12 z) .216 6 .035Ba (1.04 w)

.235 6 .014 (3.67 x) .145 6 .011Ab (2.38 y) .272 6 .041Bb (1.36 z) .243 6 .056Ba (1.17 w)

.421 6 .051Cc (6.58 x) .196 6 .033Ac (3.21 y) .409 6 .030Cc (2.04 z) .311 6 .045Bb (1.50 w)

Different superscript capital letters indicate significant differences among the groups in the columns (file systems) (P , .05). Different superscript lowercase letters indicate significant differences among the groups in rows (curvature angles) (P , .05). xyzw is the total torque value of each file system at 15 angle. It shows the increased ratio of torque values at each angle compared with the torque values at the 15 angle.

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TABLE 2 - The Maximum Torque (Ncm) Generated during Instrumentation with 15 Pecking Strokes for Each Nickel-Titanium File System (Mean 6 Standard Deviation) (n 5 15) Curvature angle File system ProTaper Universal ProTaper Next WaveOne WaveOne Gold

.005 6 .001 (x) .005 6 .001Aa (y) .017 6 .003Ba (z) .020 6 .004Ca (w) Aa

.006 6 .001 (1.20 x) .005 6 .001Aa (1.00 y) .025 6 .008Cb (1.47 z) .016 6 .003Ba (0.80 w)

.025 6 .004 (5.00 x) .020 6 .001Ab (4.00 y) .029 6 .007Bb (1.71 z) .027 6 .009ABb (1.35 w)

.045 6 .010Cc (9.00 x) .026 6 .004Ac (5.20 y) .046 6 .007Cc (2.71 z) .036 6 .005Bc (1.80 w)

FIGURE 2 – The generated torque pattern from (A ) PTU and (B ) WO in 35 curvature. Note that the amplitude of the generated torque in continuous rotation files and reciprocation files shows a different pattern. properties at room temperature.8 Because heat-treated files have a much weaker shape memory effect at room temperature or body temperature, these files are more flexible in adapting to canal anatomy. Therefore, PTU files made from conventional NiTi alloys were most affected by the curvature angle, and WOG made from gold wire (a type of CM) was the least affected. The effect of file kinematics seemed to decrease as the canal curvature angle increased. PTU showed a lower generated torque at curvature angles of 15 and 25 , but the largest increase in the total and maximum torque generated was observed when the angle increased from 25 to 35 . The PTU is made from a conventional NiTi alloy. PTU also has a larger cross-sectional

area with 3 points of contact with the canal wall, which means contact with more surface area to the canal wall. The relatively higher stiffness of the PTU is thought to have affected these results. In contrast, PTN had the lowest total and maximum torque generated in all canal curvature angles. This was probably because of its continuous rotation, lower number of contact points with the canal wall, smaller cross-sectional area and taper, and swaggering motion. This study showed dynamic changes in torque when a file was rotated in artificial root canals with different canal curvature angles. Clinically, the amount of generated torque is affected by various canal conditions when a file cuts the root dentin. However, despite the

study limitations, it can be concluded that factors such as a smaller angle of root canal curvature, the use of heat-treated NiTi ﬁles with fewer contact points, and the use of NiTi ﬁles with continuous rotation results in a lower amount of generated torque during instrumentation. Thus, from the perspective of torque generation, the use of ProTaper Next (Dentsply Sirona) may be advantageous for a highly curved canal.

ACKNOWLEDGMENTS Supported by a 2-year research grant from Pusan National University. The authors deny any conﬂicts of interest related to this study.

REFERENCES 1.

Walia H, Brantley WA, Gerstein H. An initial investigation of the bending and torsional properties of nitinol root canal ﬁles. J Endod 1988;14:346–51.

Bird DC, Chambers D, Peters OA. Usage parameters of nickel-titanium rotary instruments: a survey of endodontists in the United States. J Endod 2009;35:1193–7.

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Cheung GS, Liu CS. A retrospective study of endodontic treatment outcome between nickeltitanium rotary and stainless steel hand ﬁling techniques. J Endod 2009;35:938–43.

Gergi R, Rjeily JA, Sader J, et al. Comparison of canal transportation and centering ability of twisted files, Pathfile-ProTaper system, and stainless steel hand K-files by using computed tomography. J Endod 2010;36:904–7.

Parashos P, Gordon I, Messer HH. Factors inﬂuencing defects of rotary nickel-titanium endodontic instruments after clinical use. J Endod 2004;30:722–5.

Sattapan B, Nervo GJ, Palamara JE, et al. Defects in rotary nickel-titanium ﬁles after clinical use. J Endod 2000;26:161–5.

Baek SH, Lee CJ, Versluis A, et al. Comparison of torsional stiffness of nickel-titanium rotary ﬁles with different geometric characteristics. J Endod 2011;37:1283–6.

Shen Y, Zhou H, Zheng Y, et al. Current challenges and concepts of the thermomechanical treatment of nickel-titanium instruments. J Endod 2013;39:163–72.

Kitchens GG Jr, Liewehr FR, Moon PC. The effect of operational speed on the fracture of nickeltitanium rotary instruments. J Endod 2007;33:52–4.

10.

Lee JY, Kwak SW, Ha JH, et al. Ex-vivo comparison of torsional stress on nickel-titanium instruments activated by continuous rotation or adaptive motion. Materials (Basel) 2020;13:1900.

11.

Martin B, Zelada G, Varela P, et al. Factors inﬂuencing the fracture of nickel-titanium rotary instruments. Int Endod J 2003;36:262–6.

12.

Parashos P, Gordon I, Messer HH. Factors inﬂuencing defects of rotary nickel-titanium endodontic instruments after clinical use. J Endod 2004;30:722–5.

13.

Shen Y, Coil JM, Zhou H, et al. HyFlex nickel-titanium rotary instruments after clinical use: metallurgical properties. Int Endod J 2013;4:720–9.

14.

Gambarini G, Gergi R, Naaman A, et al. Cyclic fatigue analysis of Twisted File rotary NiTi instruments used in reciprocating motion. Int Endod J 2012;45:802–6.

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De-Deus G, Moreira EJ, Lopes HP, et al. Extended cyclic fatigue life of F2 ProTaper instruments used in reciprocating movement. Int Endod J 2010;43:1063–8.

16.

Berutti E, Alovisi M, Pastorelli MA, et al. Energy consumption of ProTaper Next X1 after glide path with PathFiles and ProGlider. J Endod 2014;40:2015–8.

17.

Jamleh A, Adorno CG, Ebihara A, et al. Effect of nickel titanium ﬁle design on the root surface strain and apical microcracks. Aust Endod J 2016;42:25–31.

18.

Schrader C, Peters OA. Analysis of torque and force with differently tapered rotary endodontic instruments in vitro. J Endod 2005;31:120–3.

19.

Kwak SW, Ha JH, Cheung GS, et al. Effect of the glide path establishment on the torque generation to the ﬁles during instrumentation: an in vitro measurement. J Endod 2018;44:496–500.

20.

Schneider SW. A comparison of canal preparations in straight and curved root canals. Oral Surg Oral Med Oral Pathol 1971;32:271–5.

21.

Kwak SW, Ha JH, Cheung GS, et al. Comparison of in vitro torque generation during instrumentation with adaptive versus continuous movement. J Endod 2019;45:803–7.

22.

Lee MH, Versluis A, Kim BM, et al. Correlation between experimental cyclic fatigue resistance and numerical stress analysis for nickel-titanium rotary ﬁles. J Endod 2011;37:1152–7.

23.

Lopes HP, Chiesa WM, Correia NR, et al. Inﬂuence of curvature location along an artiﬁcial canal on cyclic fatigue of a rotary nickel-titanium endodontic instrument. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:792–6.

24.

Gao Y, Cheung GS, Shen Y, et al. Mechanical behavior of ProTaper universal F2 finishing file under various curvature conditions: a finite element analysis study. J Endod 2011;37:1446–50.

25.

Lopes HP, Vieira MV, Elias CN, et al. Inﬂuence of the geometry of curved artiﬁcial canals on the fracture of rotary nickel-titanium instruments subjected to cyclic fatigue tests. J Endod 2013;39:704–7.

26.

Ha JH, Kwak SW, Sigurdsson A, et al. Stress generation during pecking motion of rotary nickeltitanium instruments with different pecking depth. J Endod 2017;43:1688–91.

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27. 28. 29.

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Kwak SW, Lee CJ, Kim SK, et al. Comparison of screw-in forces during movement of endodontic ﬁles with different geometries, alloys, and kinetics. Materials (Basel) 2019;8(12):1506. € u €rek T. Cyclic fatigue resistance of Reciproc, WaveOne, and WaveOne Gold nickel-titanium Ozy instruments. J Endod 2016;42:1536–9. Ninan E, Berzins DW. Torsion and bending properties of shape memory and superelastic nickeltitanium rotary instruments. J Endod 2013;39:101–4.

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CASE REPORT/CLINICAL TECHNIQUES Zameera Fida, DMD,* Lucy Yu, BDSc (Hons),† Neeta Prabhu, BDS, MDS, MPhil,† and Bill Kahler, DClinDent, PhD‡

Ingrowth of Mineralized Tissue into the Root Canal of Immature Permanent Teeth after a Traumatic Injury: A Report of 3 Cases ABSTRACT The aim of this case series was to describe the endodontic management of 3 immature permanent teeth that sustained traumatic injuries and subsequently presented with complete ingrowth of mineralized tissue into the canal space. Ingrowth of bone/mineralized tissue into the canal has been considered a poor long-term outcome with an inherent risk of ankylosis. In cases 1 and 2, no endodontic treatment was undertaken, except for emergency management requiring splinting. The cases were followed for 36 and 23 months, respectively. No ankylosis was evident over the review period, and normal teeth eruption was apparent. In case 1, the tooth was treated orthodontically and was responsive to pulp sensibility testing. In both cases, there was an appearance of an internal periodontal ligament–like space on the inner root wall of the canal. In case 3, 2 years postinjury, pulp necrosis and apical periodontitis occurred, and the tooth was managed with regenerative endodontic treatment consistent with the European Society of Endodontology and the American Association of Endodontists guidelines/ recommendations for a regenerative procedure. The case was followed for 8 years after regenerative endodontic treatment. No ankylosis was noted with normal eruption of the teeth. The tooth was responsive to pulp sensibility testing despite the ingrowth of mineralized tissue, which was conﬁrmed clinically. (J Endod 2021;47:1507–1514.)

SIGNIFICANCE Ingrowth of bonelike or mineralized tissue into the canals of permanent teeth after trauma or treatment with regenerative protocols does not necessarily result in the poor outcomes of ankylosis and infraposition as demonstrated in 3 cases in this report.

KEY WORDS Ankylosis; dental trauma; eruption; ingrowth of bone; regenerative endodontic treatment; treatment outcomes

Ingrowth of bonelike or mineralized tissue ingrowth in permanent teeth with immature roots is considered an unfavorable complication1–3. Ingrowth of bone and the formation of an internal periodontal ligament (PDL) have been reported in cases of avulsed immature permanent incisors1. Intrusive luxation injuries to permanent incisors may develop pulp necrosis and ankylosis-related resorption, thereby necessitating extraction because of increasing infraposition3. The reported literature demonstrates a poor long-term outcome for such teeth. Without a gold standard histologic examination, the ingrowth of bone and PDL as described in previous studies can only be assumed based on radiographic interpretation of the teeth1–3. It can be inferred that the ingrowth was either mineralized tissue (bone) or soft tissue (PDL) because these ﬁndings have been corroborated by animal studies. The ingrowth of bonelike tissue has also been shown in histologic studies of traumatized permanent immature teeth after regenerative endodontic treatment (RET)5–8. Andreasen and Bakland9 listed 4 categories of tissue formation within canals with different prognostic outcomes treated with RET. One of these categories included the ingrowth of cementum, PDL, and bone. The long-term prognosis is only partly known, but cases developing internal ankylosis have been described3. This observation was not reported in 2 systematic reviews of teeth treated with RET10,11 because neither reported the formation of mineralized tissue inside the root canal space that led to ankylosis. Therefore, the suggested potential for ankylosis after RET and/or the ingrowth of mineralized tissue into the canal as observed by Andreasen and Bakland9 remains unclear.

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From *Boston Children’s Hospital/ Department of Developmental Biology, Harvard School of Dental Medicine, Boston, Massachusetts; †Westmead Centre for Oral Health, Department of Paediatric Dentistry and Orthodontics, Westmead, New South Wales, Australia; and ‡The University of Queensland Oral Health Centre, Herston, Queensland, Australia Address requests for reprints to Dr Bill Kahler, Endodontics Only, 9th Floor, 141 Queen St, Brisbane 4000, Australia E-mail address: [emailprotected] 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.05.011

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The ingrowth of mineralized tissue into the pulp space after avulsion is a common finding. Mineralized tissue within the pulp space can be found in 54%–58% of cases of avulsed teeth that underwent revascularization; however, the incidence of ankylosis for this healing pattern has not been specifically reported12,13. The ingrowth of mineralized tissues, which is different from intracanal obliteration, may be a less common finding after luxation injuries because this healing pattern has not been described in prior studies14,15. The long-term prognosis of the ingrowth of bonelike/mineralized tissue and PDL-like tissue into the canal of teeth that have sustained a traumatic injury has not been widely reported. The purpose of this case series was to present the outcome of 3 luxated teeth in which the ingrowth of mineralized tissue completely filled the canals. In 2 of the cases, no endodontic treatment was required. The third tooth was treated with RET, and a 10-year follow-up postinjury showed normal eruption without orthodontic intervention.

CASE 1 An 8-year-old female presented to the Boston Children’s Hospital Department of Dentistry, Boston, MA, in 2016 after sustaining a fall the previous day. Tooth #9 was intruded

approximately two thirds of its crown height (Fig. 1A). Periapical (PA) radiographs taken demonstrated immature root development of teeth #8 and #9 (Fig. 2A). It was decided to allow for tooth #9 to erupt spontaneously based on the International Association of Dental Traumatology guidelines for immature intruded teeth16. At the 2-week follow-up, tooth #9 had minimal re-eruption (Fig. 1B) and was nonresponsive to Endo Ice (Coltene/ Whaledent Inc., Cuyahoga Falls, OH; cold test with 1,1,1,2-tetrafluoroethane) (Fig. 2B). Orthodontic extrusion of tooth #9 was initiated (Fig. 2C). At 2 months postinjury, all sensitivity, percussion, and mobility tests were within normal limits for teeth #7, #8, and #10, whereas tooth #9 had a negative response to cold. Three months postinjury, tooth #9 presented with an approximately 1-mm discrepancy in height from tooth #8. A PA radiograph showed intracanal and apical calcifications (Fig. 2C). All endodontic testing remained normal, except tooth #9, which continued to be nonresponsive to cold. Given the patient’s anxiety, the presence of an open apex for tooth #9, the lack of definitive signs of pain/infection, and the proximity to the time of injury, a decision was made to defer endodontic treatment. Orthodontic movement was halted, and a 0.016 stainless steel passive wire was placed. Four months after trauma,

tooth #9 continued to be asymptomatic and nonresponsive to cold. The orthodontic wire was removed, and the mobility of tooth #9 was assessed. Eight months postinjury, teeth #8 and #9 presented with good alignment (Fig. 1D). The orthodontic appliance was removed, and an Essix retainer (Dentsply Sirona, Charlotte, NC) was fabricated for the patient. A PA radiograph revealed no periapical pathosis, and the root of tooth #8 was maturing. Intracanal calciﬁcations were noted for tooth #9, and a discrepancy in root length between teeth #8 and #9 was evident (Fig. 2D). The teeth were reviewed again at 9, 14, 20, 24 (Fig. 2E), and 36 months when tooth #9 revealed a delayed response to cold pulp sensibility testing. A PA radiograph revealed intracanal mineralized tissue and no pathology. At the 20-month review, the entire root appeared to be obliterated with mineralized tissue, and there was the appearance of an internal PDL space as described by Andreasen et al1. The root length for tooth #9 had not increased (Fig. 2F).

CASE 2 An 8-year-old female presented to the Children’s Hospital Westmead Department of Dentistry, Westmead, NSW, Australia, in 2018 after a fall a few hours prior. Clinical

FIGURE 1 – (A ) A clinical photograph at the time of injury in which a severe intrusion injury of tooth #9 is evident. (B ) A clinical photograph of the teeth 2 weeks postinjury. (C ) A clinical photograph at the preorthodontic assessment. (D ) A clinical photograph taken at the 36-month review showing successful eruption of the tooth, indicating ankylosis had not occurred.

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FIGURE 2 – (A ) A PA radiograph taken at the time of injury showing extrusion of tooth #9 and a wide-open apex. (B ) A PA radiograph taken 2 weeks postinjury. (C ) A PA radiograph taken 3 months postinjury showing a splint. (D ) A PA taken 14 months postinjury showing complete obliteration of the canal space with a bonelike appearance. The cessation of root development for tooth #9 is noted with normal root development occurring in tooth #8. (E ) A PA radiograph taken at the 24-month review. The presence of an internal PDL-like space can be observed. (F ) A PA radiograph taken at the 36-month review.

examination revealed concussion of tooth #8 and a mild labial and intrusive luxation of tooth #9 (Fig. 3A). A PA radiograph showed immature root development of teeth #8 and #9 (Fig. 4A). There was evidence of PDL space widening in the distal and apical aspects of tooth #9. In accordance with the International Association of Dental Traumatology guidelines16, the decision was made to reposition tooth #9 and splint it with composite resin and a 0.14-inch nickel-titanium wire. Tooth G was extracted because of the possible risk of aspiration, and the laceration was sutured (Fig. 3B). At the 2-week review, tooth #9 was still mobile; thus, the splint was left in situ. The splint was removed at the 4-week follow-up. At

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this stage, tooth #8 responded positively to cold sensibility testing, whereas no response was elicited from tooth #9 (an Endo Frost ne/Whaledent Inc., Cuyahoga Falls, [Colte OH]–propane/butane/isobutane gas mixture was used). Two months postinjury, tooth #9 remained asymptomatic, and a PA radiograph showed possible signs of root resorption on the distal root surface of tooth #9 (Fig. 4B). At the 8-month visit, the PA radiograph taken revealed ongoing root development with tooth #8 only (Fig. 4C). Of note was the increased radiopacity within the walls of the pulp canal of tooth #9, with a distinct trabecular pattern. Additionally, there appeared to be narrowing of the pulp chamber

in the coronal aspect. No changes to tooth #9 were noted at the 14-month follow-up appointment (Fig. 4D). Twenty months after the ﬁrst injury, the patient presented 2 days after sustaining another fall. Tooth #9 was tender to percussion; however, there was no excessive mobility. Surprisingly, the tooth had responded positively to cold testing. The PA radiograph taken showed no sign of root fractures or widening of the PDL space. The presence of an internal PDL-like space was noted (Fig. 4E). The patient was reviewed at 23 months postinjury, and tooth #9 remained asymptomatic and responded within normal limits to percussion and mobility tests (Fig. 4F). The patient was due

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FIGURE 3 – (A ) A clinical photograph taken at the time of injury. (B) A clinical photograph showing the splint in place. (C and D ) Clinical photographs taken at 23 months as a preorthodontic workup for crowding. No infraposition of the maxillary central incisors is evident with no ankylosis and normal eruption having occurred.

to commence orthodontic treatment to correct her class II malocclusion (Fig. 3C and D).

CASE 3 A 10-year-old male was referred in 2012 with a discolored tooth #9 and hyperplasia in the attached gingiva (Fig. 5A). A PA radiograph revealed the cessation of root development had occurred in tooth #9 compared with tooth #8. There was no pathosis evident, and the root apex was wide open with thin roots (Fig. 6A). There was a history of teeth #8 and #9 sustaining uncomplicated crown fractures at age 8, which were restored with composite resin. Tooth #9 was diagnosed with pulp necrosis, and it was decided to treat the tooth with an RET procedure17. The tooth was reviewed 1 month later; the gingival hyperplasia had resolved. The RET procedure was completed (Fig. 6B). Tooth #9 was reviewed at 6 months and was nonresponsive to pulp sensibility testing. A further review occurred 2 years postinjury where discoloration was noted, and all teeth were responsive to electric pulp testing. A PA radiograph revealed calciﬁc material with a bonelike appearance completely obliterating the canal (Fig. 6C). However, tooth #9 remained nonresponsive to cold pulp sensibility testing. The patient returned 5 years after treatment because the tooth had considerably

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discolored (Fig. 5B). The tooth remained responsive to electric pulp testing. A PA radiograph revealed no pathosis, but there had been no further root maturation (Fig. 6D). The canal appeared to be completely obliterated with calcific material presumed to be bone. An internal PDL-type space as described by Andreasen et al1 was not evident (Fig. 6D). A decision was made to perform internal bleaching to improve the esthetic appearance of the tooth. Because the intracanal barrier was amorphous, it was decided to replace the mineral trioxide aggregate (MTA). Under a rubber dam with anesthesia, the MTA was removed to reveal calcific tissue with minimal bleeding (Fig. 5C). MTA was more hom*ogenously and densely placed within the canal (Fig. 6E). Bleaching was commenced with Opalescence Endo (Ultradent Products Inc, Cologne, Germany). After 3 appointments, a satisfactory appearance was noted. Normal eruption had occurred with all maxillary incisors in a regular occlusal plane, indicating normal eruption and facial growth over the preceding 5 years since the RET despite calcific material within the canal (Fig. 5D). The patient was reviewed 8 years after treatment with RET and 10 years after the initial injury, and the tooth retained a pleasing esthetic appearance and normal appearance. A PA radiograph revealed no pathosis and an unchanged appearance compared with the review and replacement of the MTA 3 years earlier (Fig. 6F).

DISCUSSION Postinjury complications are expected on occasion after dental trauma and can include pulp necrosis and infection, cessation of root development, ankylosis, and resorption. Although the ingrowth of bone within the canal space has been considered a likely poor outcome1–3, this case series shows 3 teeth in which the ingrowth of mineralized tissue after trauma resulted in normal facial growth and eruption patterns, including orthodontic intervention in 2 cases. Therefore, the poor long-term outcome suggested by Andreasen and Bakland9 was not conﬁrmed in these 3 cases. In cases 1 and 2, no treatment was necessary despite the loss of pulp tissue and replacement with calciﬁc tissue within the canal because the teeth remained asymptomatic. Furthermore, in the third case in which RET was undertaken and the canal became obliterated, infection did not occur over an 8-year follow-up period. The absence of signs and symptoms of infection is considered a primary outcome goal for teeth treated with RET by the American Association of Endodontists (AAE)17 and the European Society of Endodontology18. Thickening of the dentin, an increase in the root length, and apical closure are all considered secondary outcome goals according to the AAE17. In case 3, there was no further root maturation after treatment with RET. However, a long-term follow-up of

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FIGURE 4 – (A ) A PA radiograph of teeth #8 and #9 taken at the time of injury showing a wide-open apex. (B ) A PA radiograph taken 2 months postinjury. (C ) A PA radiograph taken 4 months postinjury. (D ) A PA radiograph taken 12 months postinjury showing calciﬁc tissue with mineralized tissue within the canal. The cessation of root development for tooth #9 is noted with normal root development occurring in tooth #8. (E ) A periapical radiograph taken 20 months postinjury in which the presence of an internal PDL-like space can be observed. (F ) A PA taken 23 months postinjury.

FIGURE 5 – (A ) A clinical radiograph showing discoloration of tooth #9 and gingival proliferation between the central maxillary incisors. (B ) A clinical photograph taken 5 years postinjury showing a discolored tooth #9 and normal eruption of the maxillary incisors. (C ) A clinical photograph taken after the removal of the MTA intracanal barrier showing calciﬁc tissue and bleeding from mineralized tissue. (D ) A clinical radiograph taken after internal bleaching of tooth #9.

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FIGURE 6 – (A ) A PA radiograph taken 2 years postinjury showing a wide-open apex of tooth #9 in which the cessation of root development is evident, indicating pulp necrosis. (B ) A PA radiograph taken at the completion of RET. (C ) A PA radiograph taken 2 years postinjury showing complete obliteration of the canal space with mineralized tissue with a bonelike appearance. No further root maturation is evident. (D ) A PA radiograph taken 5 years post-RET in which no internal PDL-like space is evident. (E ) A PA radiograph taken 5 years post-RET in which the amorphous MTA intracanal barrier is replaced with a denser and more hom*ogenous MTA intracanal barrier so the internal bleaching would be contained in the coronal portion of the access cavity. (F ) A PA radiograph taken 10 years postinjury and 8 years post-RET.

8 years posttreatment with RET indicated a favorable outcome despite the cessation of root maturation. The reasons for the lack of root development after RET have been examined and discussed extensively in the past19–22. The AAE considers a positive response to pulp testing a tertiary goal for RET, which, if achieved, could indicate a more organized vital pulp tissue17. Thermal pulp tests with either CO2 or diﬂuorodichloromethane have been shown to be more reliable than electric pulp testing for immature teeth23,24. Interpreting pulp sensibility testing in young patients can also

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be unreliable because psychological factors like fear may influence the results25,26. In cases 1 and 3, with the longest follow-up, pulp sensibility tests remained responsive over time. For case 3 in which the ingrowth of bone was observed after RET, this cannot be considered a “more organized vital pulp tissue.” However, a comprehensive review of RET suggested that a positive response to pulp sensibility testing did not necessarily indicate regeneration of the pulp21. For case 3, which was accessed to replace the MTA intracanal barrier, the presence of calcific material/mineralized tissue and vascularity were confirmed clinically. Presumably, this

tissue accounted for the positive response to electric pulp testing. Andreasen et al4 stated that damage to the Hertwig epithelial root sheath explained the concomitant ﬁndings of bony invasion into the root canal and disrupted root growth. Damage to the Hertwig epithelial root sheath at the time of injury implies the loss of undifferentiated cells, which could give rise to further hard tissue formation within the root canal, and the only tissues available were “periodontally derived tissues”1. However, in case 3 in which RET was undertaken, the undifferentiated cells may have also originated from bone marrow stem cells27.

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Intracanal calciﬁcation observation is a common response after luxation injuries of permanent teeth and is believed to be related to the loss and reestablishment of the pulpal neural supply28. It has also been frequently associated with immature teeth treated with RET29. Similarity in mechanisms suggests that the ingrowth of bone or mineralized tissue is a variant of intracanal calciﬁcation that occurs less frequently. In cases 1 and 2, there was the appearance of an internal PDL space, as has been previously described1 (Figs. 2E, F and 4E, F). In both of these teeth, endodontic treatment was not required, and the presence of this internal ligamentlike space presumably infers the ingrowth of cementum and alveolar bone into the canal space. An internal PDL space was also observed in an animal study after revascularization30. The PDL is inserted into cementum and alveolar bone. For bone or cementum to grow into the canal, the pulp has to become necrotic. Bone and cementum are the product of osteoblasts and cementoblasts, respectively. From a cell biology perspective, osteoblasts and cementoblasts are mature differentiated cells and are not capable of continuous cell division to produce bone and cementum. Therefore, the mesenchymal stem

cells from the PDL and bone marrow have to migrate into the canal and differentiate into osteoblasts and cementoblasts to produce bone and cementum. This phenomenon should be considered a reparative procedure in regenerative endodontics. It is also of interest that the European Society of Endodontology position statement on revitalization procedures considers a radiographic detection of a new PDL along the inner wall of the root canal indicative of a successful RET outcome18. This criterion for a successful outcome is a point of difference with the AAE17. An internal PDL space was observed in an animal study after revascularization26. However, histologic studies on human teeth that have undergone RET have not described an internal PDL space5–7,31,32. Furthermore, it is not described in a radiographic study in which 5 different radiographic responses to RET were reported33. Therefore, the presence of an internal PDL space is not a predictable outcome in revitalization/regenerative procedures. In case 3, ligament space is not evident on the internal root canal walls (Fig. 6D and F). Despite the formation of a likely cellular attachment between the root wall surface and bone, evidenced by the absence of a PDL-like

space, ankylosis did not occur over an 8-year review. Therefore, the poor outcome described by Andreasen and Bakland9 when the ingrowth of mineralized tissue occurs in the canal space may be overstated.

CONCLUSION This case series depicts 3 traumatized teeth in which the ingrowth of bone into the canal space was observed without the deleterious effect of ankylosis. In the 2 cases with longerterm follow-ups, pulp sensibility and normal eruption of the teeth were observed. In the case treated with RET after necrosis, an 8-year posttreatment and 10-year postinjury followup attest to a longer-term successful outcome despite no further root maturation. The longterm prognoses of these teeth remain uncertain, and clinicians should always consider options for the replacement of extracted teeth as part of long-term treatment planning.

ACKNOWLEDGMENTS The authors deny any conﬂicts of interest related to this study.

REFERENCES

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Andreasen JO, Borum MK, Andreasen FM. Replantation of 400 avulsed permanent incisors. 3. Factors related to root growth. Endod Dent Traumatol 1995;11:69–75.

Heling I, slu*tzky-Goldberg I, Lustmann J, et al. Bone-like tissue growth in the root canal of immature permanent teeth after traumatic injuries. Endod Dent Traumatol 2000;16:298–303.

Tsilingaridis G, Malmgren B, Andreasen JO, Malmgren O. Intrusive luxation of 60 permanent incisors: a retrospective study of treatment and outcome. Dent Traumatol 2012;28:416–22.

Andreasen JO, Kristterson L, Andreasen FM. Damage of the Hertwig’s epithelial root sheath: effect upon root growth after autotransplantation of teeth in monkeys. Endod Dent Traumatol 1988;4:145–51.

Shimizu E, Ricucci D, Albert J, et al. Clinical, radiographic, and histological observation of a human immature permanent teeth with chronic apical abscess after revitalization treatment. J Endod 2013;39:1078–83.

Becerra P, Ricucci D, Loghin S, et al. Histological study of a human immature permanent premolar with chronic apical abscess after revascularization/revitalization. J Endod 2014;40:133–9.

Nosrat A, Kolahdouzan A, Hosseini F, et al. Histologic outcomes of uninfected human immature teeth treated with regenerative endodontics: 2 case reports. J Endod 2015;41:1725–9.

Meschi N, EzEldeen M, Torres Garcia AE, et al. A retrospective case series in regenerative endodontics: trend analysis based on clinical evaluation and 2- and 3-dimensional radiology. J Endod 2018;44:1517–25.

Andreasen JO, Bakland LK. Pulp regeneration after non-infected and infected necrosis, what type of tissue do we want? A review. Dent Traumatol 2012;28:3–8.

10.

Torabinejad M, Nosrat A, Verma P, Udochukwu O. Regenerative endodontic treatment or mineral trioxide aggregate apical plug in teeth with necrotic pulps and open apices: a systematic review and meta-analysis. J Endod 2017;43:1806–20.

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Tong HJ, Rajan S, Bhujel N, et al. Regenerative endodontic therapy in the management of nonvital immature permanent teeth: a systematic review-outcome evaluation and meta-analysis. J Endod 2017;43:1453–64.

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Kling M, Cvek M, Mejare I. Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol 1986;2:83–9.

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Coste SC, Silva EF, Santos LC, et al. Survival of replanted teeth after traumatic avulsion. J Endod 2020;46:370–5.

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Andreasen FM, Zhijie Y, Thomsen BL, Andersen PK. Occurrence of pulp canal obliteration after luxation injuries in the permanent dentition. Endod Dent Traumatol 1987;3:103–15.

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Andreasen JO, Bakland LK, Andreasen FM. Traumatic intrusion of permanent teeth. Part 3. A clinical study of the effect of treatment variables such as treatment delay, method of repositioning, type of splint, length of splinting and antibiotics on 140 teeth. Dent Traumatol 2006;22:99–111.

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Bourguignon C, Cohenca N, Lauridsen E, et al. International Association of Dental Traumatology guidelines for the management of traumatic dental injuries: 1. Fractures and luxations. Dent Traumatol 2020;36:314–30.

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American Association of Endodontists. Clinical considerations for a regenerative procedure. Revised 2018. https://f3f142zs0k2w1kg84k5p9i1o-wpengine.netdna-ssl.com/specialty/wpcontent/uploads/sites/2/2018/06/ConsiderationsForRegEndo_AsOfApril2018.pdf. Accessed July 21 2020.

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Galler KM, Krastl G, Simon S. European Society of Endodontology position statement: revitalization procedures. Int Endod J 2016;49:717–23.

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Nosrat A, Homayounfar N, Oloomi K. Drawbacks and unfavorable outcomes of regenerative endodontic treatments of necrotic immature teeth: a literature review and report of a case. J Endod 2012;38:1428–34.

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Verma P, Nosrat A, Kim JR, et al. Effect of residual bacteria on the outcome of pulp regeneration in vivo. J Dent Res 2017;96:100–6.

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Kim SG, Malek M, Sigurdsson A, et al. Regenerative endodontics: a comprehensive review. Int Endod J 2018;51:1367–88.

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Kahler B, Lin LM. Regenerative endodontics. In: Andreasen JO, editor. Textbook and Color Atlas of Traumatic Injuries to the Teeth. Oxford, UK:: Wiley Blackwell; 2019. p. 718–38.

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Fulling H-J, Andreasen JO. Inﬂuence of maturation status and tooth type of permanent teeth upon electrometric and thermal pulp testing. Scand J Dent Res 1976;84:286–90.

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Fuss Z, Trowbridge H, Bender IB, et al. Assessment of reliability of electric and thermal pulp sensibility testing agents. J Endod 1986;12:301–5.

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Levin LG. Pulp and periradicular testing. Pediatr Dent 2013;35:113–9.

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Andreasen FM, Kahler B. Diagnosis of acute dental trauma: the importance of standardized documentation: a review. Dent Traumatol 2015;31:340–9.

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Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res 2009;88:792– 806.

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Andreasen FM. Pulpal healing after luxation injuries and root fracture in the permanent dentition. Endod Dent Traumatol 1989;5:111–31.

29.

Song M, Cao Y, Shin SJ, et al. Revascularization-associated intracanal calciﬁcation: assessment of prevalence and contributing factors. J Endod 2017;43:2025–33.

30.

Wang X, Thibodeau B, Trope M, et al. Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 2010;36:56–63.

31.

Lui JN, Lim WY, Ricucci D. An immunoﬂuorescence study to analyse wound healing outcomes of regenerative endodontics in an immature premolar with chronic apical abscess. J Endod 2020;46:627–40.

32.

Saoud TM, Zaazou A, Nabil A, et al. Histological observations of pulpal replacement tissue in immature dog teeth after revascularization of infected pulps. Dent Traumatol 2015;31:243–9.

33.

Chen MY, Chen K-L, Chen C-A, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. Int Endod J 2012;45:294–305.

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CASE REPORT/CLINICAL TECHNIQUES

Successful Pulp-Preserving Treatment for Peri-invagin*tion Periodontitis of Double Dens Invagin*tus With Oehlers Type IIIA and IIIB: A Case Report ABSTRACT Dens invagin*tus (DI), which often occurs in the maxillary lateral incisor, is an important issue in endodontics because the treatment complexity increases depending on the degree of invagin*tion and the vitality or nonvitality of the pulp. An 11-year-old female patient with a sinus tract in the gingiva of the maxillary lateral incisors showed peri-invagin*tion periodontitis and double DI on radiography. Cone-beam computed tomographic imaging was used to examine the structure in the root canal in detail, and Oehlers type IIIA and IIIB DI was found. It was also shown that the patient’s bone defect was caused by type IIIA. Because a healthy reaction was observed in the pulp test, the final diagnosis was peri-invagin*tion periodontitis associated with type IIIA of the double DI with vital pulp. We expected the lesion to heal by treating only the type IIIA invagin*ted pseudo–root canal while preserving the healthy pulp. The invagin*ted root canal was cleaned under a microscope using ultrasonic instruments and nickel-titanium files to minimize irritation to the pulp. Because the lesion shrinkage was confirmed by conebeam computed tomographic imaging taken 3 months after the start of treatment, vertical compaction of the warm gutta-percha technique was performed. At the 6-month postoperative recall, the pulp was normal, and the lesions were further improved. Treatment of the main root canal of double DI is complicated. However, proper diagnosis and careful cleaning of the invagin*ted root canal are essential for healing while preserving the pulp. (J Endod 2021;47:1515–1520.)

Naoto Kamio, DDS, PhD, Natsuko Gomyo, DDS, and Kiyoshi Matsushima, DDS, PhD

SIGNIFICANCE Pulp preservation is possible even in double dens invagin*tus with periinvagin*tion periodontitis.

KEY WORDS Cone-beam computed tomography; double dens invagin*tus; invagin*ted root canal; periinvagin*tion periodontitis; vital pulp

Dens invagin*tus (DI) is a morphologic abnormality in which the enamel on the surface of the crown is folded and invades the inside of the pulp chamber before calcification of the tooth1. This folding phenomenon can cause bacterial invasion, stagnation, and proliferation. DI is easily overlooked because of the lack of abnormal clinical signs but can lead to endodontic or periodontal disease2. The incidence of DI varies depending on the definition and measurement methods of researchers, but it is reported to be about 0.04%210%, and the most susceptible tooth is the maxillary lateral incisor1–3. There are several classifications of DI according to the degree of invagin*tion, but Oehlers classification system is generally accepted4. The classes and the prevalence rate for each class are as follows:5 Type I (69.8%–93.8%): the invagin*tion is minimal and enamel lined. It is confined within the crown of the tooth and does not extend beyond the level of the external amelocemental junction. Type II (3.1%–26.6%): the invagin*tion is enamel lined and extends into the pulp chamber but remains within the root canal with no communication with the periodontal ligament. Type III (3.0%–12.5%): for type IIIA, the invagin*tion extends through the root and communicates laterally with the periodontal ligament through a pseudoforamen, and, for IIIB, the invagin*tion extends

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From the Department of Endodontics, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan Address requests for reprints to Naoto Kamio, Department of Endodontics, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan. E-mail address: kamio.naoto@nihon-u. ac.jp 0099-2399/$ - see front matter Copyright © 2021 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2021.05.013

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through the root and communicates with the periodontal ligament at the apical foramen. There is usually no communication with the pulp. There are reports of nonsurgical endodontic treatment6,7, surgical endodontic treatment8, and intentional reimplantation9 for type III DI treatment, but if the tooth is vital, it is recommended to ﬁrst perform root canal treatment of the invagin*ted canal in the DI10. Still, the thin dentin of the DI is susceptible to mechanical and bacteriologic effects. The invagin*ted enamel is also susceptible to pulp infection by recognizing dehiscence2. In recent years, endodontic treatment has enabled accurate preoperative diagnoses and minimal treatment of curved root canals using conebeam computed tomographic (CBCT) imaging and nickel-titanium (NiTi) ﬁles. In this case report, we introduce a case of peri-invagin*tion periodontitis that occurred in double DI with Oehlers type IIIA and IIIB. In the literature, there are about 20 reports on the treatment of double DI and 2 reports on triple DI. However, there are no reports of the prevalence; only the term “rarely” has been used. Furthermore, all reports mainly treat apical lesions due to pulp necrosis, and there are no reports of treatment for periinvagin*tion periodontitis that preserves the pulp of double DI. Herein we performed minimal treatment only on the invagin*ted root canal while retaining the healthy pulp.

CASE REPORT An 11-year-old girl presented to her family dentist with the complaint that the gums of her upper left anterior tooth were swollen. She was prescribed antibiotics and referred to Nihon University Hospital at Matsudo, Matsudo, Chiba, Japan, because of the complex morphology of the root of tooth #22 (World Dental Federation), which was thought to be the causative tooth. Tooth #22 had no history of trauma or past dental treatment, and teeth #12 and #47 were congenitally missing. Clinical examination revealed a spindle-shaped crown, and a sinus tract was found in the distal gingiva (Fig. 1A, B). There was discomfort with the percussion and tenderness due to palpations. The tooth was caries free, and an electric pulp sensitivity test showed a vital reaction. The gingival pocket depths were within normal limits. The radiographic examination revealed double DI with dead space in the pulp chamber (Fig. 1C). The mesial DI appeared to be invagin*ted from two thirds of the root to the apex, and the distal DI was invagin*ted to about one half of the root. The apical lesions were unclear, but bone

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defects were found in the distal alveolar bone (Fig. 1C). CBCT imaging (KaVo 3D eXam 1; KaVo Dental Systems Co, Ltd, Tokyo, Japan) was performed with the patient’s consent to obtain more detailed anatomic information. The mesial DI reached the apical foramen, which also appeared to be open. The distal DI was curved toward the distal palate, the invagin*ted root canal (pseudo–root canal) communicated to the periodontal ligament space, and the opening coincided with the center of the radiographic lesion (Fig. 1D–L). In addition, the main root canal was compressed, and there was no contact with the invagin*ted root canal (Fig. 1K). The final diagnosis was periinvagin*tion periodontitis derived from Oehlers type IIIA of the double DI type IIIA and IIIB with vital pulp. The treatment plan consisted of an invagin*ted root canal and preventive treatment of mesial DI while maintaining the vitality of the pulp. First, after discussing the treatment options and considerations, such as risk, informed consent was obtained from the patient and her caregiver. Local anesthesia was administered using 0.9 mL lidocaine with 1:80,000 adrenaline bitartrate (ORA injection; Showa Yakuhin Kako, Tokyo, Japan). Under rubber dam isolation, the access cavity preparation of the distal invagin*tion site was carefully performed using a dental operating microscope (OPMI pico; Carl Zeiss, Jena, Germany) in order to prevent perforation of the pulp chamber. The infected invagin*ted root canal was exposed (Fig. 3A), and the narrowed part of the covered enamel interfered with the expansion of the pseudoapex. Therefore, the enamel was shaped in advance with ultrasonic instruments (Diamond-coated ultrasonic tip; J Morita MFG Corp, Kyoto, Japan). Thermal stimulation could damage the pulp through thin dentin; hence, it was used in the presence of 2.5% sodium hypochlorite. After cleaning the enamel on the inner wall, patency was confirmed with a #10 K-file (Zipperer; VDW, Munich, Germany) up to the pseudo–apical foramen. Calcium hydroxide paste (Calcipex; Nippon Sika-Yakuhin, Shimonoseki, Japan) was applied, and a temporary blockade (Caviton; GC Corp, Tokyo, Japan) was performed. A sinus tract was also present at the second visit (2 weeks after her first visit). Local anesthesia was administered, and rubber dam isolation was performed as described earlier. The working length of the invagin*ted root canal was measured (DentaPort, J Morita MFG Corp) and instrumented with ProTaper Gold (Dentsply Sirona, York, PA) to size F1 (#20). Calcipex was also applied, and a temporary blockade was performed.

At her third visit (1 month after her first visit), she complained that the sinus tract became larger or smaller repeatedly. We expanded the invagin*ted root canal to ProTaper Gold size F2 (#25) and applied calcium hydroxide paste (Vitapex; Neo Dental Chemical Products Co, Tokyo, Japan). After a few days, the sinus tract disappeared, and the patient was followed up after 2 months. At the fourth visit (3 months from the first visit), sinus tract was not found and CBCT imaging was performed to confirm the reduction of the lesion (Figs. 2 and 4A). Vitapex was thoroughly removed and disinfected using 2.5% sodium hypochlorite and ultrasonic equipment irrigation (Fig. 3B). The smear layer was removed with a 15% EDTA solution, washed with physiological saline, and then the invagin*ted root canal was dried using a paper point. We thought that the insertion of a heated plugger, such as the continuous wave of condensation technique, was not preferable and tried filling with the warm backfill technique (vertical compaction of the warm gutta-percha technique; ie, after thinly applying zinc oxide eugenol sealer [CANALS; Showa Yakuhin Kako Co, Ltd, Tokyo, Japan] to the canal, gutta-percha warmed at 180 C was charged [SuperEndo Beta; Pentron Clinical Technologies, Wallingford, CT], and appropriate pressure was applied to fill the root canal) (Fig. 3C). After confirming that it was tightly packed (Fig. 4B), the access cavity and mesial invagin*tion site (type IIIB) were closed using composite resin. X-rays taken at the follow-up after 6 months showed the lesion further shrinking, and she remained asymptomatic (Fig. 4C).

DISCUSSION The deﬁnitive cause of DI is unclear. However, this case had a missing lateral incisor on the opposite side, suggesting a genetic effect during tooth development, as previously reported11,12. Some studies reported that treatment of the invagin*ted root canal of Oehlers type III with vital pulp led to healing6,7,13. On the other hand, there is also a report that the pulp should be treated positively when considering the mutual relationship between the main root canal and the existence of the communication path14. Based on these facts, Bishop and Alani10 stated that strict follow-up should be performed when only the invagin*ted root canal was treated. Therefore, in this case, the ﬁrst choice was to treat only the invagin*ted root canal; consequently, it was realized that the healing of the periinvagin*tion disease was achieved while maintaining the vitality of the pulp. This is more advantageous in terms of tooth strength than

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FIGURE 1 – Preoperative intraoral photography, 2-dimensional radiographs, and CBCT imaging of DI) (tooth #22). A spindle-shaped crown of the maxillary lateral incisor with double DI with enamel. (A ) Lateral incisor gingiva with a sinus tract (arrow). (B ) The occlusal view showing a structure with a slight oriﬁce. (C ) Double DI with a radiolucency image inside of the root. (D–H ) Sagittal CBCT images from mesial to distal. (D ) The mesial DI reaches the apical foramen, and (F ) the distal DI bends and opens into the periodontal ligament (arrow ). (I ) A schematic representation of root canal morphology. P, pulp; E, enamel; IIIA, Oehlers type IIIA DI; IIIB, Oehlers type IIIB DI; PF, pseudo–apicalforamen. (J–L ) Axial CBCT sections of the apical third to the coronal third. (K ) Distal DI opens in the center of the bone defect (arrow ). JOE Volume 47, Number 9, September 2021

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FIGURE 2 – A CBCT image taken at the time of the fourth visit (3 months from the ﬁrst visit). (A–D ) Sagittal CBCT images from mesial to distal. Bone defects due to peri-invagin*tion periodontitis tend to improve. (C ) A radiopaque image due to calcium hydroxide paste is observed in the invagin*ted root canal. (E–G ) Axial CBCT sections of the apical third to the coronal third.

aggressive root canal treatment (cutting the DI) and can be expressed as reducing the risk of future root fracture. On the other hand, it is necessary for patients to fully understand that there is a possibility of pulp necrosis in the future. There are many reports that CBCT imaging is useful for the treatment of DI. By type, most of them were used for type III15,16 and, conversely, less for type II and I17. In the present case, it was not clear whether the bone loss was due to an apical lesion or periinvagin*tion periodontitis by 2-dimensional radiographic imaging. However, CBCT imaging revealed that there was a pseudo– apical opening at the center of the lesion.

Ultrasound instruments were recommended to treat the invagin*ted root canal because the inner surface is made of enamel, and the rotating instruments used for root canals can be damaged10. We considered using NiTi files because of the possibility of invagin*ted root canal curvature and minimal enlargement2. Therefore, after sufficiently expanding the enamel with an ultrasonic instrument, it was possible to use an NiTi file and clean it without excessive filing. In addition to normal root canal filling, filling with mineral trioxide aggregate has also been reported for root canal filling of the invagin*ted root canal18. We thought that root canal filling using gutta-percha was more

useful than mineral trioxide aggregate for thinly curved root canals like this one. However, the continuous wave of condensation technique is concerned about the harmful effects of heat19 on the pulp, and lateral condensation6,7 is considered to require further apical enlargement. Therefore, in this case, the filling was performed using only the warm backfill technique; consequently, a good prognosis was obtained. Several cases of double DI have been reported20,21, 1 of which was cleaned using the self-adjusting file (SAF; ReDentNova, Ra’anana, Israel) due to the complexity of its main root canal and the invagin*ted root canal.21 However, no cases were found in healthy pulp with Oehlers type IIIA and IIIB.

FIGURE 3 – An intraoral photograph during treatment of type IIIA DI. (A ) Access preparation to the invagin*ted root canal. (B ) Ultrasonic irrigation with sodium hypochlorite ﬁlling the root canal. (C ) Vertical compaction of warm gutta-percha.

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FIGURE 4 – An intraoral photograph during root canal filling and radiographs at the time of root canal filling and after 6 months. (A ) The sinus tract has disappeared. Postoperative radiographs (B ) after gutta-percha filling and (C ) at 6 months. Achieving pulp preservation in the present case was impossible without a proper diagnosis by CBCT imaging and minimal intervention using a dental microscope and NiTi file. Because DI dentin is often thin, preserving the pulp is thought to contribute

biologically and physically to preserving the tooth. Type IIIB invagin*tion (mesial DI) was prophylactically treated because aggressive root canal treatment was not recommended;10 however, strict follow-up may be required.

ACKNOWLEDGMENTS The authors thank Editage (www.editage.com) for English language editing. The authors deny any conﬂicts of interest related to this study.

REFERENCES

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€lsmann M. Dens invagin*tus: aetiology, classiﬁcation, prevalence, diagnosis, and treatment Hu considerations. Int Endod J 1997;30:79–90.

Alani A, Bishop K. Dens invagin*tus. Part 1: classiﬁcation, prevalence and aetiology. Int Endod J 2008;41:1123–36.

Hovland EJ, Block RM. Nonrecognition and subsequent endodontic treatment of dens invagin*tus. J Endod 1977;3:360–2.

Oehlers FA. Dens invagin*tus (dilated composite odontome). I. Variations of the invagin*tion process and associated anterior crown forms. Oral Surg Oral Med Oral Pathol 1957;10:1204–18.

Capar ID, Ertas H, Arslan H, Ertas ET. A retrospective comparative study of cone-beam computed tomography versus rendered panoramic images in identifying the presence, types, and characteristics of dens invagin*tus in a Turkish population. J Endod 2015;41:473–8.

Schwartz SA, Schindler WG. Management of a maxillary canine with dens invagin*tus and a vital pulp. J Endod 1996;22:493–6.

Tsurumachi T. Endodontic treatment of an invagin*ted maxillary lateral incisor with a periradicular lesion and a healthy pulp. Int Endod J 2004;37:717–23.

Hata G, Toda T. Treatment of dens invagin*tus by endodontic therapy, apicocurettage, and retroﬁlling. J Endod 1987;13:469–72.

Lindner C, Messer HH, Tyas MJ. A complex treatment of dens invagin*tus. Endod Dent Traumatol 1995;11:153–5.

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Bishop K, Alani A. Dens invagin*tus. Part 2: clinical, radiographic features and management options. Int Endod J 2008;41:1137–54.

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Peck S, Peck L, Kataja M. Prevalence of tooth agenesis and peg-shaped maxillary lateral incisor associated with palatally displaced canine (PDC) anomaly. Am J Orthod Dentofacial Orthop 1996;110:441–3.

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€ € yu €kbayram I, Ozalp Kaya-Bu S x , Aytugar E, Aydemir S. Regenerative endodontic treatment of an infected immature dens invagin*tus with the aid of cone-beam computed tomography. Case Rep Dent 2014;2014:403045. M, Abella F, Duran-Sindreu F, et al. The use of cone-beam computed tomography in the Teixido preservation of pulp vitality in a maxillary canine with type 3 dens invagin*tus and an associated periradicular lesion. J Endod 2014;40:1501–4. Ferreiroa A, Rico-Romano C, et al. Diagnosis and endodontic treatment of type II Macho AZ, dens invagin*tus by using cone-beam computed tomography and splint guides for cavity access: a case report. J Am Dent Assoc 2015;146:266–70.

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JOE Volume 47, Number 9, September 2021

ASSOCIATE REGISTRY To place a notice: Associate Registry listings are offered as add-ons to online postings in the AAE Career Center. Visit careercenter.aae.org for more information and rates. Because there is a two-month lead time for the Journal of Endodontics print issue, listings must be submitted on or before the 10th of each

month to appear in the issue two months later (e.g., a submission received on March 10 will appear in the May issue). If the ad is submitted after the 10th of the month, it will appear in the JOE three months later (e.g., a submission received on March 11 will appear in the June issue).

Notices must ﬁt into one of the following categories: endodontic practice, multispecialty group practice, associate to partner or practice for sale. Each notice must advertise a speciﬁc position, practice or opportunity.

Endodontic Practice

Currently the practice has four operatories – two equipped and two plumbed and an expansion opportunity for three additional operatories is possible in the adjacent space. (That’s a total of seven possible operatories!) AN OVERVIEW OF THIS ENDODONTIC PRACTICE ON THE FRONT RANGE: 4 operatories (2 equipped and 2 plumbed; Expansion into adjacent space with three plumbed ops; 570 cases in the last 12 months; Collections of $865,000; EBITDA (LTM) $140,000. THE CURRENT DOCTOR IS INTERESTED IN SELLING TO ANOTHER DENTIST AND EXPLORING RETIREMENT OPTIONS. They are open to staying on for a smooth transition as well. Located on the edge of the Rocky Mountains, Colorado Springs offers affordable living, great access to the outdoors and plenty of job opportunities, along with lots of culture. For those seeking a healthy worklife balance – including epic outdoor adventure – you can have it all in Olympic City USA! To learn more about this practice and review the prospectus, please contact Sam Schoenecker with Professional Transition Strategies: SAM@ PROFESSIONALTRANSITION.COM or give us a call: 719.694.8320. We look forward to hearing from you!

offices in the Chicago suburbs and 2 downtown city offices. The Renovo culture is one of collaboration, support, and teamwork. We enjoy spending time with each other—in the office- and outside of it! About the Practice: The first RES office was started in 2011 in Schaumburg, IL. We have grown to include 8 convenient locations (100+ mile geographic coverage) within the Chicago area – Schaumburg, Elgin, Downers Grove, Frankfort, Rockford, Roscoe VillageChicago, Water Tower Place-Chicago – that allows us to provides increased market access to patients. We accept most PPO insurances and every location has an established referral base for consistent patient flow. Our online visibility and word of mouth reputation also contributes to our patient population. What sets us apart: Full patient load from the start—we preferentially book associate schedules; Highly competitive compensation; No marketing requirement—we already have established referral bases at all the offices; Mentor-ship if desired; All locations are equipped with the most up-to-date technology— 3D CBCT, Nomad, intraoral cameras, surgical microscopes; Clinical Autonomy: Our providers diagnose and design their own treatment plans. However, our group practice setting allows for a collaborative environment. We often discuss cases and treatment plans together on a daily basis and we offer as much support as needed and desired; Location—Chicago is an amazing metropolitan city full of amazing food, culture, history, arts, and beaches. With two international airports, travelling in/out of Chicago is also convenient and easy; Flexible partnership opportunities for interested doctors. Job Responsibilities: Examine, diagnose, prescribe treatment, and provide endodontic services. Pay Rate: Starting compensation is 45% of collections. Employment Type: Full-Time. Skills and Qualifications: An Illinois dental license and specialty license in endodontics are required to practice in the state of IL. A 4 year dental degree and completion of an endodontic residency is required to practice as an endodontist in the state of IL. Applicants will need to apply for an Illinois dental license and a specialty license in endodontics. The ideal candidate should demonstrate high clinical, surgical, and diagnostic skills. Candidates should also be comfortable communicating with both patients and referring doctor. Please reply to [emailprotected].

Alaska, Fairbanks—Excellent endodontic practice opportunity, no floods, no hurricanes, just an awesome practice in a wonderful town. If interested, please contact Dr. Joseph Vargas at (907) 456-3636 or (907) 460-2054. CV to: [emailprotected] or docjwv54@aol. com. Arizona, Phoenix—We are a well established, NON-CORPORATE, specialty-only practice whose growth makes this ad necessary. Work alongside another down-to-earth endodontistmentor where collaboration is enhanced. Now, if you want to interview with a “talent acquisition specialist”, work out of a different office everyday, not have microscopes and CBCT scanners at your disposal and possibly needing to answer to a general dentist and/or a private equity firm, then this opportunity is NOT for you! Benefits second to none. Extremely Generous School loan payback program! Let’s meet sooner rather than scrambling a couple of months before your program ends! Send CV & letter of interest to [emailprotected] Arizona, Prescott—Endodontic office with two locations in North/Central Arizona seeking Endodontist associate. We are a well- established office with a broad referral base for the last 25 plus years. Digital/scopes/CBCT. Two locations. Board certified mentor looking for an associate/ associate to partner to help with our way too busy schedule. Full time or part time to transition to full time. Must have certificate from an accredited endodontic program. Please reply to [emailprotected]. California, Oakland—We are currently seeking a full or part-time Associate Endodontist. Great opportunity for a motivated, hardworking endodontist looking for associateship to partnership position. We are a state of the art private specialty practice with multiple locations in the most coveted Bay Area in CA. Top compensation. New graduates welcomed to apply. Please submit CV. Please reply to [emailprotected]. Colorado, Colorado Springs—Busy, state-of-the-art, private endodontic practice in Colorado Springs, Colorado, seeking a full-time endodontist. Opportunity for future buy-in/ownership for the right person/skill set/ personality. Licensed Endodontist or upcoming successful completion of Endo Residency. Please reply to [emailprotected]. Colorado, Colorado Springs—New to the market is an exciting opportunity to own your own endodontic practice in Colorado Springs, Colorado! Located in an office building with easy access to the interstate in a rapidly growing area of town – the location can’t be beaten. JOE • Volume 47, Number 9, September 2021

Colorado, Denver—We are looking for a motivated, patient-focused associate to join our private endodontic practice in the Denver area. Our modern office is located in a highly desirable and growing area. The area is known for its top ranked local schools and its access to some of the best outdoor recreation in the world. Be busy right away. The office includes: Dexis sensors, Zeiss microscopes, CBCT, ASI carts, PBS Endo management software, and spacious operatories. Please reply to [emailprotected]. Florida, Brandon—Endodontic Associate— Tampa Bay Area. Well established, busy Endodontic group practice seeking energetic, quality Endodontist. Modern office with latest technology including microscopes, digital radiography, CBCT, and experienced support staff. Excellent earning potential and benefits. Recent graduates encouraged to apply. Please reply to [emailprotected]. Georgia, Atlanta—Well established, busy group specialty practice seeks an exceptional Endodontist to join our endodontic practices. Great opportunity for an experienced Endodontist looking to practice in modern offices with the latest endodontic technologies surrounded by a personable and dedicated endodontic team. Please reply to [emailprotected]. Illinois, Rockford—Renovo Endodontic Studio is a leading, privately-owned, specialty group that focuses on microsurgical endodontics and dental implants. Our offices are comprised of 6

Illinois, Worth—30 Minute Commute from Downtown Chicago, located in Southwest Suburbs. Serving: Palos Heights, Worth, Oak Lawn, Orland Park, Tinley Park, Chicago Ridge, Bridgeview, etc. just to name some of the surrounding areas. PRIVATE ENDODONTIC PRACTICE seeking a quality and patient care driven, Part Time to Full Time Associate. The practice has everything you could possibly want to 1521

ASSOCIATE REGISTRY, continued allow you to deliver the highest quality of care, with the least strain on you as a practitioner. Ground up practice built with Dexis Sensors, NOMAD X-ray machine, Zeiss Extaro microscopes, ASI CARTS, Integrated photography, and J MORITA CBCT. Endodontist must be dedicated to patient care and quality. Hours are ﬂexible, and we honor and respect a work life balance. Please reply to info@ axisendo.com.

compensation. New graduates welcome to apply. Must be comfortable with simple apiciectomies and all aspects of root canal therapy. Benefits include a 401 K matching / defined benefit plan. Website www.hverootcanal.com. Looking for a caring Endodontist with an emphasis on quality patient care and personalized care. Please submit resume. Opportunity to live and practice in a wonderful area and enjoy life.

Maine, Augusta—Endodontist position available in Central Maine. Enjoy the outdoor lifestyle Maine has to offer while treating some of the best people (patients) in the country. Practice where your service is appreciated and your off-hours respected. Ample patients from the start. Very understanding and cooperative referring base. Zeiss Mics and CBCT in ofﬁce. Please reply to [emailprotected].

North Carolina, Greenville—Well-established two-associate, four-owner Endodontic group is looking for an Associate Endodontist. We are self-owned and managed, with three offices in Eastern North Carolina, each approximately 45-90 minutes from Raleigh/Cary/Chapel Hill and 60-90 minutes to the Outer Banks. We strive to create a friendly, professional and caring atmosphere for our patients, who are equally selfpay and insured. We allow Associates freedom in creating their own schedule and pace with unlimited earning potential at a high collection percentage. Generous benefits package offered. All offices are outfitted with Microscopes, CareStream CBCT, and experienced staff. Position requires practicing in two locations approximately 50 minutes apart and call rotates among our providers allowing many nights and weekends free. Our offices operate 8-5 Monday-Thursday and 8-12 on Friday (optional). Please respond in confidence with a CV to: [emailprotected]. Requirements: Certificate in Endodontics from an US accredited program and a willingness to provide friendly, quality endodontic care. Applicant must be familiar with contemporary treatment techniques, including Microscopy and CBCT.

Michigan, Southeast—We are currently looking for full or part time endodontists to join our growing practice. Endodontic Associates is a well-established, quality-oriented group practice limited to endodontics, located in upscale communities in Southeast Michigan including Ann Arbor. Our objective is to provide endodontic services promptly, efficiently and consistent with the highest standards of the profession. We are searching for personable, self-motivated and productive endodontists who share those same goals. Partnership opportunities. We utilize operating microscopes, digital radiography and limited view cone beam CT imaging to treat the most complicated cases providing a positive patient experience with predictable, effective results. We have a very wide referral base that supports our practice and therefore offers an exceptional income potential. We have a welltrained support staff throughout our organization including central management. Benefits for Associate Drs. include malpractice insurance, paid membership dues, marketing expense, clinical needs, scrubs. We have established 401k /Roth plans, profit sharing plan and several health insurance plans to choose from. Other benefits include collegiality, mentor-ship, immediate supply of patients and a flexible schedule to enjoy a favorable work-life balance. Endodontic Associates also utilizes the highest level of PPE and infection control standards as recommended by CDC, ADA, and the AAE. Please find out more at our website https://www.endodonticassoc. com/ New Jersey, Sparta—I have a thriving endodontic practice with offices in Sparta and Randolph, New Jersey. I am searching for a professional individual with advanced evidence-based skills as my practice relies on quality endodontic care. This individual must be trained to render care using a microscope for both adults and children. Both offices have Zeiss Microscopes, use TDO practice management software, digital radiography, Carestream CBCT units and have the latest devices and instruments. Please reply to dra@ appelbaumendo.com. New York, Kingston—Hudson Valley Endodontics (Kingston, NY) is currently seeking a full-time Endodontist to join our thriving practice. Modern technology with CBCT scan and Zeiss surgical microscopes in every operatory. Top

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Ohio, Worthington—Central Ohio Endodontics is a well-established and respected practice in the vibrant, growing community of Columbus, OH. The pace among our three locations is fast and busy and provides ample financial opportunity for the right doctor. Known for our compassion and engagement with our patients, we provide personal touches throughout their entire experience and give them the treatment they deserve while delivering efficient, effective therapy. With our large team of highly trained endodontic professionals, you will have an abundance of support dedicated to your personal success. We are looking for an associate who can step into our busy practice and participate in the growth and development of our referral relationships. As a team of professionals who are in constant pursuit of excellence, an associate who is eager to learn and develop their clinical and business skills will thrive in this environment. This associate will work out of all three suburban locations, all of which have the latest technologies and innovations. Our doctors work four days a week and take turns rotating on-call weekends. We offer competitive compensation packages and benefits, including a personal CFO who will help you strategize how to reduce debt and fast-track your financial success, putting you on a pathway to partnership. Requirements: Strong clinical skills; Growthminded; Communication skills; Ethics; Values development and mentorship. Please visit https:// www.centralohioendodontics.com/ for more information on our practice. To learn more about our city, visit https://www.purpose.jobs/blog/

reasons-love-living-working-columbus. Oregon, Bend—This opportunity provides a wonderful opportunity for immediate patient flow in a practice designed to facilitate multiple provider care. SHORT TERM: We have had some COVID setbacks in our office and I would be interested if there is a practitioner that may want to join us for a few weeks-months to help us get caught back up on our patient care. LONG TERM: I am seeking an associate/partner that will share my very personable, patient oriented approach to care. The office features microscopes and ASI carts in all 7 operatories, a Kodak 9000 Cone Beam CT, TDO office software, Kodak digital sensors, oral/nitrous and IV sedation. I have been practicing in Oregon for 16 years and have developed a rapport with a large number of general dentists who provide high quality patient care. This provides my practice with patients who are confident that their general dentist has made a well-informed professional decision to refer them to our office. Our city in Oregon is a family friendly community known for its recreational opportunities and consistently appears on lists of the best towns for those who enjoy the outdoors. Hiking, fishing, hunting, golfing, Kayaking, Skiing, Snowmobiling, great restaurants and an amazing community to raise a family are just some of what one can find here. Please contact me at 541-390-5415. Texas, Plano—We are seeking an energetic fulltime/part time Endodontist to join our dynamic team. This position will encompass a full scope of Endodontic related treatments at our Dallas/ Fort Worth location. Experienced Endodontist as well as new graduates are welcome to apply! We have a state of the art office equipped with microscopes and a CBCT machine. This is also a well-established office that is highly productive with a high potential income. Compensation package will include: Paid Continued Education courses; Paid license fee; Reimbursem*nt for marketing expenses with referring doctors; Your responsibilities and requirements will consist of: Assuring the highest quality of care for patients while adhering to the highest standard of ethics and professionalism; Diagnosing and performing a variety of treatments; Marketing with referring doctors; State dental license; Degree from accredited dental school; and Endodontic certificate from a U.S. accredited Endodontic program. Please reply to [emailprotected]. Utah, Murray—Established group practice is seeking a full time associate. Our office is equipped with state-of-the-art equipment and a well trained staff. We are flexible regarding your workdays, vacation time, and patient schedule (one of the many benefits of a group practice!) We do not operate a ‘root canal mill’ we focus on individual care. We encourage a pathway to partnership for those interested in becoming a part owner. Please feel free to reach out if you are interested in living and working amongst the beautiful Rocky Mountains! You can also check out our website at greaterendodontics.com. Please reply to [emailprotected]. Virginia, Alexandria—Our multi-location practice located in Northern Virginia is seeking an associate. The practice is well-established with a reputation that has been positively maintained

JOE • Volume 47, Number 9, September 2021

ASSOCIATE REGISTRY, continued with our referring doctors as far back as 1962 and it continues to grow. Our three offices are outfitted with state-of-the-art equipment including Zeiss microscopes mounted in each operatory, Kodak limited-field CBCT, and intraoral sensors. We are looking for a motivated endodontist with at least three years of private practice experience. If interested, please email your cover letter and resume. Preferable 3 years of private practice experience. Please reply to nova.endo3@gmail. com. Wisconsin, Madison—Madison Endodontic Associates, S.C. (www.madisonendo.com). Positive office culture; Large referral base; Generous compensation; Beautiful view from operatories; Newly designed office with spa-like feel; First endodontic practice established in Madison, WI (1973); Well-respected, highquality endodontic practice; 10 spacious treatment operatories; TDO software; ASI carts; Microscopes (Global and Zeiss) in all operatories; Schick digital radiography; 3D-CBCT; Teaching/ lecturing opportunities; Benefits available include healthcare insurance and a 401(k) retirement plan; Full-time: 4 day work week—no weekends. Part-time: negotiable; Capitol and university city consistently ranked among the top places to live in the U.S.; Madison, WI offers a wonderful quality of life for individuals and families. Please send your resume/cover letter and reply to: awright@ madisonendo.com. Wisconsin, Metro Area – South—Unique fulltime opportunity with excellent compensation for the right quality-minded, personable endodontist with a heart to serve and a soft touch. Private practice in Southern Wisconsin metropolitan area. Strong patient flow in a well-established practice, with an exemplary reputation in the public as well as with an expansive referral base. Seeking well-trained endodontic team that exudes comfort and compassion. Top of the line technology and software with Zeiss microscopes (video/imaging ready), digital radiography, CBCT and more. We are holding the position for just the right endodontist that will thrive personally, professionally and financially with this opportunity. Please reply to [emailprotected].

Associate to Partner California, Roseville—Great opportunity to practice and live in fabulous Northern California. Roseville, CA is known as one of the best places to live and work. In fact, Roseville was the only CA city to make Money Magazine’s annual Best Places to Live in 2020. Roseville is a densely populated suburb of Sacramento, located within a 2-hour drive or either San Francisco or Lake Tahoe. The area is growing rapidly with both young families and business professionals resulting in an immediate need for an associate within our practice. The practice is well established, known for quality patient care, and has over 25 years of service within the community. Equipped with modern operatories, microscopes, ASI carts, and CBCT. Our support team are highly trained and focused on delivering excellent patient experiences. Looking for a caring and compassionate individual who is team oriented, has excellent clinical skills, and operates with high integrity. This opportunity will be a great match for the clinician who wants to be busy

JOE • Volume 47, Number 9, September 2021

immediately. We offer full benefits and competitive compensation. Benefit package includes a Pathway to Partnership, mentorship, and a fast track to success. Please reply to endo.op916@ gmail.com. Colorado, Colorado Springs—New to the market is an exciting opportunity to own your own endodontic practice in Colorado Springs, Colorado! Located in an office building with easy access to the interstate in a rapidly growing area of town – the location can’t be beaten. Currently the practice has four operatories – two equipped and two plumbed and an expansion opportunity for three additional operatories is possible in the adjacent space. (That’s a total of seven possible operatories!) AN OVERVIEW OF THIS ENDODONTIC PRACTICE ON THE FRONT RANGE: 4 operatories (2 equipped and 2 plumbed; Expansion into adjacent space with three plumbed ops; 570 cases in the last 12 months; Collections of $865,000; EBITDA (LTM) $140,000. THE CURRENT DOCTOR IS INTERESTED IN SELLING TO ANOTHER DENTIST AND EXPLORING RETIREMENT OPTIONS. They are open to staying on for a smooth transition as well. Located on the edge of the Rocky Mountains, Colorado Springs offers affordable living, great access to the outdoors and plenty of job opportunities, along with lots of culture. For those seeking a healthy worklife balance – including epic outdoor adventure – you can have it all in Olympic City USA! To learn more about this practice and review the prospectus, please contact Sam Schoenecker with Professional Transition Strategies: SAM@ PROFESSIONALTRANSITION.COM or give us a call: 719.694.8320. We look forward to hearing from you! Georgia, Atlanta—Atlanta Suburb: Premier private practice with multi-doctors and multilocations in desirable suburbs of Atlanta. Seeking an energetic and patient-oriented associate for a 4 day a week full-time position. This best in class, highly respected practice with a stellar reputation and great demographics is looking for the right match. Candidate must be a team player; experience is preferred but not a requirement. Must have a Georgia license or have the requirements for licensure by credentials (if not, practice will assist associate in obtaining GA licensure); a graduate of an accredited dental school and endodontic graduate program. Board certification is encouraged and preferred (practice will assist associate in obtaining Diplomate status). Looking for someone who is personable and willing to establish and nurture new referral relationships. Work-life balance is extremely important to this practice. Additional advantages: latest technology, wonderful staff, collegial partners, competitive compensation, full benefits, with the real draw being the high quality people you will be working with. Opportunities in existing and new office locations; associateship leading to partnership. If interested, please contact Dr. Daniel Price @ (615) 945-0105 and/or [emailprotected]. Iowa, Cedar Rapids—Busy endodontic group practice is looking for a driven endodontic resident or established endodontist. Board certification is encouraged. We serve a growing referral base and are adding new referrals weekly.

New building, modern dental equipment, and dedicated CBCT use. All treatment rooms are spacious with exterior facing windows along with wall mounted microscopes. All doctors have their own private office with covered heated parking. We pride ourselves on providing every Iowan with the dental care they deserve by accepting all major insurance plans. Our team is efficient, motivated to stay busy, and mindful that every patient leaves knowing that Root Canals Are Good! Want to know more, please visit eiendo. com, our Instagram page @rootcanalsaregood, or read what our patients have to say on Google. Interested applicants should send their curriculum vitae to Dr. Derek Peek at [emailprotected]. Kansas, Topeka—Solo practice of 26 years looking for an associate/partner immediately. Five full operatories. Four microscopes. CBCT. Board certified/eligible endodontist. Please reply to [emailprotected]. Nevada, Reno—A modern state-of-the-art endodontic practice located in beautiful Reno, Nevada is searching for full-time endodontic associate who has a passion for microsurgery or has a desire in learning endodontic microsurgery and implant placement techniques. We are a well-established fee for service, 20+ year endodontic practice with a strong referral base. Our team works closely with the doctors and are highly skilled and caring. We place high values on integrity and communication with the referring doctors and strive to be leaders in our dental community providing excellent endodontic and microsurgical care. Please send resume’, salary requirements and references to Cheryl at Cheryl@ renoendo.com. New York, Kingston—Hudson Valley Endodontics (Kingston, NY) is currently seeking a full-time Endodontist to join our thriving practice. Modern technology with CBCT scan and Zeiss surgical microscopes in every operatory. Top compensation. New graduates welcome to apply. Must be comfortable with simple apiciectomies and all aspects of root canal therapy. Benefits include a 401 K matching / defined benefit plan. Website www.hverootcanal.com. Looking for a caring Endodontist with an emphasis on quality patient care and personalized care. Please submit resume. Opportunity to live and practice in a wonderful area and enjoy life. New York, Syracuse—There is an excellent opportunity for an endodontist to join our growing, successful practice in Syracuse, NY. The group consists of 6 full-time practitioners who enjoy working together as a team. The offices are fully modernized, including cone beams, Zeiss microscopes, and many other new technologies. Our practice prides itself in quality surgical and conventional endodontics. There is also an opportunity to learn implantology within the practice. If you are a first or secondyear student, now is the time to start exploring career options. We are offering a very attractive financial package with benefits that include health insurance, health club membership, expense account, pension plan and more. The area offers a great quality of life at a very affordable cost. That, along with some of the finest public and private schools in the state, makes it a wonderful place to raise a family. There is a wealth of culture

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ASSOCIATE REGISTRY, continued here supported by fine museums, symphony and stage productions. Central New York is ideal for outdoor recreation, including some of the finest lakes in the country. There are three ski resorts within 30 minutes of downtown. The Adirondacks, Thousand Islands and Canada are approximately one hour away. The major metropolitan areas-NYC, Boston and Philadelphia can be reached in about 4 hours by car. Syracuse University and Lemoyne College bring much to the community including nationally recognized sports programs. All of the above, combined with fine restaurants and a fun nightlife, make Syracuse a great place to live and practice. If you are interested in applying for this position, please call Jeffrey S. Maloff at (315)413-1100 or send your resume to my email address at drjsm4life@ gmail.com. Pennsylvania, Eastern—A full time (or part time) position is available for a quality minded, energetic person to join our established and growing practice. Partnership timeframe is entirely up to you. All 5 operatories have microscopes, digital radiography and all the technological advances you are accustomed to. Equipped with CBCT as well. There is a well trained support team to facilitate your work flow, so you can solely focus on endodontic care. 401k, health insurance and other benefits available. Relocation bonus for full time position available. Please reply to [emailprotected].

Multispecialty Group Practice Colorado, Denver—We are seeking a fellowship-trained Endodontist to join our group practices. Candidates must be proficient in molar root canals. Clinicians must be patient and service oriented. Candidates must be able to handle high case volume or have a desire to do so. Our network providers value subspecialty care and offer consistent referrals. Proficiency in endodontic retreatments and apicoectomies is preferred, but not required. Our mentorship program will provide adequate training for the demands of the position. Excellent compensation; Daily minimum, based upon experience; State of the art facilities. Job Requirements: Candidates must be fellowshiptrained in Endodontics (recent or near graduating) or Board Certified with excellent interpersonal and communication skills.—Dental License in Colorado—Certificate of Endodontology. Contact: [emailprotected] Georgia, Atlanta—Located in beautiful historic downtown Marietta, GA, 20 minutes from Atlanta. The area offers a great quality of life at a very affordable cost. State of art, established, FEE FOR SERVICE Endodontic practice seeking a Full time or Part time quality oriented compassionate associate. Opportunity to grown into a partnership. We have a dedicated team that strives to give our patients a personable and caring experience. We have an incredible working environment with the latest technology including Carestream 8100 CBCT, digital radiography, Zeiss and global microscopes, TDO software, ASI carts and Kavo handpieces. Please respond with CV to [emailprotected]. Hawaii, Oahu—Part-time Endodontist Opportunity within our growing Dental practices

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in Aiea, Hawaii. *$7,500 Sign-on Bonus* For more information, please reach out to Jeff Farrell, Talent Acquisition Manager at FarrellJ@ interdent.com or 925.719.1462. Gentle Dental is completing full remodels in all of our Hawaii offices in the first Quarter of 2020. Beyond improving the look and feel, we’re investing heavily in the latest technology and equipment to be on the forefront of patient care. Our offices already feature Google Review ratings of 4.6 or higher from our patients who continue to rave about our patient care, teams, and culture. At Gentle Dental, our culture is based on innovation and providing a wellness approach to Dentistry to improve clinical results and consistently generate predictable treatment outcomes. We focus on providing the highest quality, multi-disciplinary, holistic, and comprehensive oral health services with clinicians who precisely diagnose, document, educate, and treat our patient populations. Massachusetts, Boston—Endodontist needed for full and part-time opportunities in the Greater Boston area. 42 North Dental has been providing dental care in New England for over 40 years. In our established, multi-specialty group practice we welcome an abundance of new patients each month. Our state of the art facility allows optimal patient care, a comprehensive approach to full service dentistry along with a strong focus on quality, service and patient satisfaction. Our specialists enjoy the benefit of internal referrals and group practice support. Each practice has a highly trained and experienced team of professionals to assist you along with digital xray, microscope and stateof-the-art equipment. Please reply to priyanki. [emailprotected]. Oklahoma, Midwest City—We are seeking an energetic full-time/part time Endodontist to join our dynamic team. This position will encompass a full scope of Endodontic related treatments at our Midwest City, Oklahoma location. Experienced Endodontist as well as new graduates are welcome to apply! We have a state of the art office equipped with microscopes and a CBCT machine. This is also a well-established office that is highly productive with a high potential income. Compensation package will include: Paid Continued Education courses; Paid license fee; Reimbursem*nt for marketing expenses with referring doctors; Your responsibilities and requirements will consist of: Assuring the highest quality of care for patients while adhering to the highest standard of ethics and professionalism; Diagnosing and performing a variety of treatments; Marketing with referring doctors; State dental license; Degree from accredited dental school; Endodontic certificate from a U.S. accredited Endodontic program. Please reply to [emailprotected]. Utah, Salt Lake City—We are seeking a Fellowship-Trained Endodontist to join our group practices. Candidates must be proficient in molar root canals. Clinicians must be patient and service oriented. Candidates must be able to handle high case volume or have a desire to do so. Our network providers value subspecialty care and offer consistent referrals. Proficiency in endodontic retreatments and apicoectomies is preferred, but not required. Our mentorship program will provide adequate

training for the demands of the position. Excellent compensation; Daily minimum, based upon experience; State of the art facilities. Job Requirements: Candidates must be fellowship trained in Endodontics (recent or near graduating) or Board Certified with excellent interpersonal and communication skills. Dental License in Utah; Certificate of Endodontology. Contact: [emailprotected] Wisconsin, Milwaukee—Are you an ENDODONTIST looking to start or continue your career with a company that not only offers you a job, but can offer you a DENTAL HOME to call your own on a FULL-TIME or PART-TIME basis? We are currently looking for an Endodontist to join our MILWAUKEE, APPLETON, and GREEN BAY locations! Consider joining a dentist-owned, family practice that started as a 1-chair clinic and has since grown to 14 locations throughout Wisconsin. Dental Associates fosters a culture that invites our patients into an environment where they are treated like family; with a caring and empathetic approach to dentistry. When you join our team, you can expect…PRACTICE WITHIN A PRACTICE. Our dedicated doctor teams and unique practice within a practice model and ensures the highest level of care while you to build rapport and grow within your team. TOTAL COMPENSATION PACKAGE. Our salary guarantee gives you stability while you get established. Not to mention, our robust patient base, full schedules, higher earning potential through the Doctor Incentive Program, full benefits package, vacation and 401k with vested match. Not to mention relocation allowance and bonuses availability in some locations. FOCUSED DENTISTRY & LEADERSHIP. Devote your time, energy and focus toward providing excellent oral health care rather than running the business side of things. Direct your team, your schedule and your treatment plans. Focus on providing the best care for your patients and lead your team without a committee. INNOVATION. As the world of dentistry evolves, so do we. We collaborate with our surgeons to stay at the forefront of the industry and each of our clinic are outfitted with state-of-the-art equipment. GROW YOUR PERSONAL BRAND. Our internal teams build and grow your personal brand so patients can find you and get to know you. INTERNAL SUPPORT. You focus on providing the best dental care and our support teams handle the rest: IT, marketing, staffing, scheduling, accounting, compliance, continuing education, patient financial services and more. CONTINUING EDUCATION. Prove yourself and you can grow as much as you want. Access to ongoing training and educational opportunities to expand your knowledge and experience. REPUTATION MANAGEMENT. Don’t worry about those pesky online reviews — we’ll take care of those distractions. Interested? Contact Katie Herman at [emailprotected]. Check out our current openings: https://www.dentalassociates. com/careers

Practice for Sale California, San Luis Obispo—Thriving endodontic ofﬁce in the beautiful town of San Luis Obispo, CA looking for buyer of practice and/or building. The ofﬁce includes 2100 square

JOE • Volume 47, Number 9, September 2021

ASSOCIATE REGISTRY, continued feet of space; with four operatories, CBCT designated room, staff room, beautiful views from every window and a couple minute walk to downtown! Dr. wanting to retire after practicing for over 30 years and very motivated. Come in and let us host you so you can see what an amazing opportunity this is. We are an efficient office, with up-to-date software and equipment; including CBCT and digital radiographs. If you are interested, please email rootfi[emailprotected] and attach CV and required licenses to practice. California, Solano County—Opportunity to purchase an established practice in a growing town. The 2,500 sq ft office has 6 large operatories. 5 fully digitized and equipped with 5 surgical microscopes, a CBCT, central N2O unit, and TDO software. Seller owns the real estate. Option to purchase both the business and the property. Owner willing to stay on to help with the transition. For details, contact the practice directly through email by clicking here at: [emailprotected]. Colorado, Colorado Springs—New to the market is an exciting opportunity to own your own endodontic practice in Colorado Springs, Colorado! Located in an office building with easy access to the interstate in a rapidly growing

area of town – the location can’t be beaten. Currently the practice has four operatories – two equipped and two plumbed and an expansion opportunity for three additional operatories is possible in the adjacent space. (That’s a total of seven possible operatories!) AN OVERVIEW OF THIS ENDODONTIC PRACTICE ON THE FRONT RANGE: 4 operatories (2 equipped and 2 plumbed; Expansion into adjacent space with three plumbed ops; 570 cases in the last 12 months; Collections of $865,000; EBITDA (LTM) $140,000. THE CURRENT DOCTOR IS INTERESTED IN SELLING TO ANOTHER DENTIST AND EXPLORING RETIREMENT OPTIONS. They are open to staying on for a smooth transition as well. Located on the edge of the Rocky Mountains, Colorado Springs offers affordable living, great access to the outdoors and plenty of job opportunities, along with lots of culture. For those seeking a healthy worklife balance – including epic outdoor adventure – you can have it all in Olympic City USA! To learn more about this practice and review the prospectus, please contact Sam Schoenecker with Professional Transition Strategies: SAM@ PROFESSIONALTRANSITION.COM or give us a call: 719.694.8320. We look forward to hearing

from you! New York, Brooklyn—Looking to transfer a long established, successful, fee for service Endodontic practice. Owner retiring due to medical issues but available to assist in transition. Endodontist associate of 23 years willing to stay on. Four fully equipped state of the art operatories including digital X-ray, microscopes with video out and CBCT. Practice occupies two floors of a brownstone style building on a traditional doctors row, tree lined wide street. Onsite parking for patients is a real plus in area. Easy access via mass transit. Front office staff have each been with the practice for more than 20 years. Bay Ridge is a thriving residential area within a short commute to Manhattan by public transportation or car. Wonderful place to live and has small town feeling in a big city. Inquiries: [emailprotected]. United States—Your Value Might Shock you! Call us to learn more at (855) 533-4689 Email: classifi[emailprotected] www. LargePracticeSales.com

The interactive, online career center provides members with the tools they need to plan their next career step or ﬁnd their next great hire. Job Seeker Benefits • Free Access • Personalized Job Search • Easy Job Application • Résumé Search Employer Benefits • Competitive Pricing • Easy Management • Company Awareness • Candidate Alerts

Explore more at www.aae.org/careercenter

JOE • Volume 47, Number 9, September 2021

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The Foundation proudly thanks the following companies for their generous contributions to support endodontic research, education and outreach. $

2,700,000

* $1,000,000 from Tulsa Dental Products

740,000

400,000

190,000

177,500

80,000 Coltene

75,000

Acadental ACTEON North America ASI Medical, Inc.

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245,000

200,000

117,000

110,000

100,000

50,000

American Dental Partners Foundation SdflĽf#Ghqwdo#Vhuylfhv XDR Radiology $

30,000

Endodontic Practice Partners Septodont, Inc. Vista Apex

25,000

Dydorq#Elrphg Legally Mine PreXion Seiler Instrument and Manufacturing $

5,000

Jordco, Inc.

JOE • Volume 47, Number 9, September 2021

Donor Honor Roll

* Indicates this pledge is paid in full.

∞ Indicates this member has joined the Perpetuity Plan. Δ Indicates this member has moved to a higher pledge level. State totals include voting members only

GIVING RECOGNITION LEVELS

Named Award $500,000

Platinum $250,000

AAE Alliance $415,700 College of Diplomates $25,000

Alabama Founder Paul D. Eleazer* John S. McMurphy*

Titanium $100,000

Gold $50,000

Silver $35,000

Diamond $25,000

Emerald $20,000

Topaz $15,000

Founder $10,000

Benefactor $5,000

Patron $2,000 Each year, the Foundation distributes over $1 million in donations and investment income to fund grants and programs which advance endodontic research, education support, and access to care—placing the specialty at the forefront of dentistry in investment back into our profession.

Benefactor Deborah S. BishopΔ Timothy A. Kerper*Δ John P. Lightfoot* J. Roland PorterfieldΔ S. Craig Rhodes Patron Larry W. Alley* James Rhett Baker Frances Ballagas Stanford Ballard Robert D. Barfield* Joe Breig H. John Caprara Jr.* Vimal Chedda Dyeus Chung Darin V. Cissell David W. Clanton* Kenan D. Clinton* T. Madelyn Coar* M. Shannon Daugherty* Charles Earnhardt Mark Essher Joel P. Hearn* Robert L. Hill* Schuyler F. Hunter* Kathy L. Jefferson* Jennifer E. Key B. Franklin Kimbell Jr.* Thomas P. King* Mark P. Kuglitsch John Lindell William B. Looney* Tres H. Manasco* Rebecca L. Martin* William Paul McCrary* Christopher S. McManus* Lawrence G. Miller Jr. James V. Mills Jr.* John C. Mills S. Park Mims* Richard W. Morgan* Frank B. Morris* Taylor Nelson Nicholas Netzel

JOE • Volume 47, Number 9, September 2021

Stephen R. Price*Δ Nikhil Reddy Benjamin L. Schrock Brandon Schultz Kendall Slaton E. Wilder Smith Jr.* James A. Smith Jr.* Terry J. Smorang Donald W. Sutton* John R. Tschudin* Gerard G. Weinacker* Jenny Whatley*Δ Tameika Wheeler Gary A. Wolanek* Nelson G. Woo

Alaska Topaz John K. JeppsonΔ Founder Douglas J. Luiten* Heather Sulte* Benefactor Daniel M. Keir* Richard A. Siry* John J. Stropko Patron Jane Bleuel* Orest M. Harkacz* Michael L. Mark Michael J. Mauger

Arizona Silver Robert S. Roda Diamond Edward H. Carlson*Δ∞ Joseph S. DovganΔ Glen A. Eisenhuth* Laurence D. Johns*Δ Hung Quoc Nguyen*Δ Charles L. Siroky* Emerald Bradley H. Gettleman* Topaz Jacqueline S. AllenΔ Christopher J. DouvilleΔ Steven L. FrostΔ* Howard Sorenson*Δ Susan L. Wood*Δ Founder Tung B. BuiΔ

Giving in Perpetuity D. Wayne Acheson Robert B. Amato Robert A. Augsburger Nona I. Breeland Edward H. Carlson Noah Chivian Philip W. Cohen Gerald C. Dietz Sr. Tevyah Dines Reid S. ElAttrache William F. Freccia Kenneth J. Frick Michael A. Glass Ronald C. Hansen Robert W. Hawkinson Jr. Martin D. Hickey Allan Jacobs Jeffrey M. Javelet Kevin M. Keating Alan S. Law Thomas A. Levy Kimberly A.D. Lindquist Dennis G. Longwill David L. Maddox Sandra Madison Scott B. McClanahan David G. Burros*Δ Thomas J. Cipriano* John W. GillanΔ Scott Morrison & Karen Panietz*Δ Kenneth D. Tyler* Benefactor Rodney J. Brimhall* Ryan H. Engelberg*Δ Jason J. HalesΔ James D. HamberlinΔ William J. HookerΔ Robert S. Kane* Mark A. KerrΔ Robert A. Lees II*Δ William B. Leibow* Philip M. Mahoney* Michael A. Markson* Thomas V. McClammy* Oscar M. Peña*Δ John P. Smith Ronald L. Steinbrunner*Δ Judy VanGheluwe*Δ Patron Nathan E. Baker* Justin W. Batz A. Dean Campbell*

Phillip L. Michaelson James M. Musselwhite Mark A. Odom Samuel W. Oglesby John S. Olmsted Terry W. Ott Roberta Pileggi Jerome V. Pisano William D. Powell Martha E. Proctor Robert L. Reames Louis E. Rossman William C. Roth Paula Russo Denis E. Simon III Charles L. Siroky Clara M. Spatafore Douglas W. Stewart Patrick E. Taylor Mahmoud Torabinejad Terrence V. Turner Christopher S. Wenckus David D. Whitaker Darrell W. Zenk Kenneth J. Zucker

Francisco J. Castano Steven L. Coleman* Shaun R. Cullimore* Glen E. Doyon* David B. Foley* Daniel B. Funk* Dennis E. Hanna* Micheal S. Harrison Dean M. Hauseman III* Justin S. Hughes* Vikash S. Huliyar Steven Hymovitch* E. Gus Khalifa* Andrew J. Krygier* John R. Kurtz* Thomas P. Lammot* I. Robert Matloff* Michael Mauger* Mark D. Melde* Michael J. Melde* Aundra L. Murphy Michael S. O’Connell* Steven Richardson* Gary K. Silver* Michael Simpson* Steven R. Sluyk* Kip M. Sterling* Barney D. Streit* Steven T. SwagerΔ

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DONOR ROLL, continued

Douglas E. Thomas* Matthew B. Tonioli* Nader Vafaie* Raymond van der Werf Francine Vickers*

James G. Schulze* James E. StichΔ Charles V. Tatosian* Mahmoud Torabinejad*Δ∞ Frank J. Wilkinson*

Arkansas

Emerald Joseph H. Schulz*Δ

Diamond Harvey E. MathenyΔ James A. Penney IIIΔ Founder Paul M. Curtis Jr.Δ* James M. Tinnin* Benefactor O. William Reeder Jr.* Richard H. Yamanaka Patron Gerald R. Avillion* Katherine E. Behrents* Nathaniel E. Behrents* Claudia Colorado Larry A. Gartman* Robert Gatti* James Hiatt Luke B. Kauffman* George B. Morledge III James P. Orahood Kenneth E. Pearson* S. Bryan Whitaker*

California Charitable Trust Alfred & Terri Frank Titanium Wayne G. Bemis*Δ Gold L. Stephen Buchanan*Δ Silver Kevin M. KeatingΔ* Diamond James A. Abbott*Δ George Bogen* Gary B. Carr* Frank Casanova* Sudha S. Chinta Janice C. ChouΔ Nava Fathi*Δ Alan H. GluskinΔ Marshall E. GomesΔ Ray Kuwahara* David L. Mayeda*Δ Steve N. McNicholas*Δ W. Craig NoblettΔ John E. Pratte* Robert J. Rosenberg* Clifford J. Ruddle*

1528

Topaz Lynne A. Baldassari-CruzΔ* Jeffery A. Daughenbaugh* Alan H. Gluskin*Δ Raymond S. ScottΔ Bruce C. Smith*Δ Stefan I. ZweigΔ Founder Leif K. Bakland*Δ Sheri L. Bernadett Richard C. Burns* Peter D. Cancellier* Gilman W. Carr* Nadia ChugalΔ Stephen Cohen* Mark L. Ellis Bruce R. Harkins* Roy M. Hayashi* Charles M. Holman*Δ Jeffrey H. JanianΔ Mo KangΔ Mark LaramoreΔ Dennis G. Longwill*∞ Lloyd Marcum* Douglas J. McKendry* William J. Mussone Ramin Noghreian*Δ Geoffrey T. Okada*Δ Kevin J. O’Neill*Δ Arthur S. Panella* Randolph D. Rush*Δ Michael J. Scianamblo*Δ Wyatt D. SimonsΔ Fred S. Tsutsui* Peter R. VanderslootΔ Gary T. Wuchenich* Benefactor Hamid R. Abedi* John Aivaz* James V. Anderson* Donald D. Antrim* Charles M. Arita*Δ Shahriyar BanihashemiΔ Emad Bassali*Δ Samir K. Batniji* David C. Beachler* James D. Beller* Robert D. Bernie* Rajiv Bhagat* Gordon J. Block* Keith R. BoyerΔ Robert V. Bravin* David C. BrownΔ Michael D. Brown

Mindy L. Buoncristiani* Manuel A. BustamanteΔ* Ian Chang*Δ Carolyn M. ChongΔ* Richard Chong* David Y. ChowΔ William B. Claussen* William C. Cliff*Δ A. Scott Cohen* Darrell A. Dang*Δ Stephen B. Davis*Δ Douglas A. Daws* Michael E. Dent* Thomas G. Dwyer* Samer M. Ebeid Hiri EtessamiΔ* John C. Fat*Δ Patrick J. Ferrillo Mehran Fotovatjah* William C. Francis*Δ Robert J. Frank* Johnah Galicia Cindy E. Geers* Douglas K. Greenwald* Joseph J. Grenn Leo Grudin* Wendy S. GuldenΔ Duane B. Gustafson* Ronald C. Hansen* Eugene C. Hanson Robert K. Higa* Gabriel Holley Brian E. Hornberger*Δ John M. Hoskins* John J. JaberΔ J. Michael Jann* Jeffrey M. Javelet*∞ Joanne Jensen-HawkinsΔ James R. Jespersen* Kathryn Jurosky & Kurtis Finley* Steven Kallman*Δ Hemant K. M. Kapadia Moon-Hee Kor William G. Kuhn Kevin D. Less*Δ Arthur J. LeClaire*Δ Lawrence M. LeVine* Thomas A. Levy*Δ Gerald C.H. Lim* Tanya Machnick* Craig J. Malin* Maria B. Manaloto* Bryan F. Mansour*Δ Scott L. MarcumΔ* Clark A. MartinΔ Debra A. Meadows* Jeffrey A. Meckler*Δ M. Sadegh Namazikhah* Dwayne L. Nash* Matthew J. Nealon* Nguyen T. Nguyen* Sean Noorvash* Priya O’Callaghan* Josanne M. O’Dell*Δ

updated as of 7/26/2021

Ronald J. Oleson* Meryl H. OlsenΔ Michelle C. Olsen* Lawrence H. Ota* William J. Palank* Ove A. Peters*Δ Jimmy Pham Lauren T.V. Phan-Vandersloot Daniel J. PierreΔ John P. Pruett*Δ Lester J. Quan*Δ Ali Rezai* James R. SanfilippoΔ Erick Y. Sato* John G. Schoeffel* Louis E. Schwarzbach* Bradley G. SetoΔ* Brandon Seto* James R. Silverman* Daniel J. Simon* Jay A. Solnit*Δ Mark D. StevensonΔ* Louis Z. Stromberg* Jerome H. Stroumza* Eugene I. Sugita* Sue W. Suh* Midori Tachibana* Kenneth W. TittleΔ James J. Vogel* William A. Walker Jr.* Phillip A. Waterman Jr.* Richard Weinstein* Leslie A. Werksman* Robert L. Willey* David J. Wolfe* Ralan D. WongΔ Jeremy M. YoungΔ Calif. State Assn. of Endodontists* Northern Calif. Acad. of Endodontics Southern Calif. Acad. of Endodontics* Patron Thomas G. Acierno* Rowshan Ahani* David F. Aimar* Mohammad Alduraibi Abdullah Alfreihi Sam Almassi Rayan Alrehili Gary M. Altenburg* Greg An* Craig W. Anderson* Robert E. Anderson Anastasios Angelidis* Paul Anstey* Andrey Antonenko Erol S. Apaydin* Mohammad Arif Hira Assad* Charlene M. Auld Marina F. Awad Joyce L. Awramik*

William S. Bachicha* Paymon Bahrami Tyler F. Baker Aneet S. Bal* Erik P. Balinghasay Orest Balytsky Ashley B. Barrineau Sarah Barzanji Amit Batheja Nermine Batniji Ron C. Bell* Kenneth A. Benjamin* Yaara Berdan Emily Berg Craig R. Bergquist Jeff S. Berlin* David S. Berrios Anu Bhalla Paul Bianchi* Thomas Bianchi* Joseph L. Bigas* James E. Bollinger* Robert D. Brennan* J. Daniel Brian Jr.* David C. Brown* Matthew R. Buhrley Jack I. Brown* Ronald Brown* David F. Browning* David S. Brunell*Pour David M. Butsumyo* Jason Calvert* Yangpei Cao Poppy Carlig John E. Carman* Jennifer Castellanos Sylvia Cecchini Youngsook Chae Peter A. Chalmers* Susan Chan* Donald K. Chang Insoon Chang Peipei Chang* Alex Chavez Robert A. Cheron Kevin Chiang Mary M. Chien* Yvonne F. Chiu* Esther S. Cho Frederick R. Cho* Byung K. Scott Choi Daniel Choi Jawoong Choi* Kevin Choi* Tina Chou Amber T. Chu Christopher S. Chun Christine L. Chung* Bradley P. Clark* Karen L. Coffman* Charles Marc Cohler* Brett H. Cole* Christine S. Cook* Robert P. Corr Dominic Cote

JOE • Volume 47, Number 9, September 2021

Ma Elena Courrech Martin Laurence Cree* Randolph R. Cross G. Reed Cummings* Tyson Curtis Maurice Cutler* Branden Dailey* Michelle K. Dang* Hoa T. Dao* Vi Dao Jon P. Dean* Gina Deeb* Joseph E. Dehmer Fred S. Dias* Stephen T. Dixon Anh Q. Do David M. Dobin* Ramsey Y.S. Doo* Kenneth E. Doty* Seng Douangpanya James R. Dow* Daryl Dudum* Arthur A. Dugoni* James A. Eberhardt Sanya Ebrahimzadeh Dennis Eguchi* Ali Ehsan Yuto Endo John T. Evans* Ronald J. Fair* Victor S.C. Fan* Denny Y. Fang* Hadi H. Faras C. Leonard Fath Nava Fathi* Brian Feiger* Cami E. Ferris* Amir M. Fesharaki* Daniel S. Fife Ronald W. Filice* SooHyung Kim Flakes Bruce Fogel* Robert B. Folk* Jared R. Fortman* Barry L. Fong* Zachary L. Freer Arthur D. Gage* Deepika Ganesh Randy W. Garland* Patrick J. Garrett Glenn B. Garvin* Michael K. Gavin* James D. Gearing* James J. Ghafourpour* Dudley H. Glick* Harold E. Goodis* Joshua C. Gorman Frederick J. Grassin* Melvin A. Greenspan* Joseph H. Greiner* Gery C. Grey* Garrett M. Guess* Rylan N. Gustafson Abeer Hafez Hadjir Haghparast*

Enborg B. Halle* Evan S. Halpern* Reza Hamedy Hamid Reza Hamid David C. Han* Terry G. Han* Philip J. Hankins*Δ Robert J. Hanlon Jr. Steven R. Hansen* Ashraf I. Harhash Armen A. Hartoonian* David W. Hartwick Spyridon Hasiakos* Ralph D. Hawkins*Δ Alvin K. Hayashi* Terrence W. Hayes* Samer Hejlawy A. Stephen Heller* Ronald P. Hempel* Scott K. Hetz Brent Hiebert James Y.S. Ho* Dao Hoang Barry Hoch* Sue Hoch* John Q. Holcomb* Stephen P. Holifield* Bruce M. Holt* Nathan Horwitz Angela Hsiao* G. Robert Hsu* George Huang David A. Hudson* Troy E. Hull* Rita Hung Sarah Hussain William S. Hwang* Lee R. Ingersoll* John I. Ingle* Samuel Ip James H. Isaacson* Harold L. Jacobson III* Avery Jaffe Robert J. Jensen* Charles E. Jerome* Ronald C.K. Jew*Δ Aileen E. Jitsumyo* Philip D. Jo Tejpaul S. Johl Edward A. Johnson* Reuben Johnson Taoheed Johnson* Elana Justice* Barbara J. Kabes* Rambod Kamrava* Hyun Ji Sophia Kang Mahmoud Karimipour Rahim Karmali Nooshin Katebzadeh* Jeffrey Kawilarang Kevin M. Keane* David M. Keating David W. Kelliny* Tom Kennedy Ankit Keshav

JOE • Volume 47, Number 9, September 2021

James D. Kettering* Ghazala R. Khan Noor F. Khouqeer Catherine Kim Daniel D. Kim* Yong C. Kim* Soohyung Kim Flakes James L. Kloss* Anne L. Knuut* Bernice T. Ko* Kenneth A. Koch Kimberly A. Kochis* Ravi S. Koka* Gregory J. Kolber* Daniel J. Kolzet Anthony L. Korbar* Edward D. Kosakoski Robert S. Koshiyama* Jeffrey M. Kotsubo* Jeffery D. Krupp* Abrar S. Kutbi Diana Kutsenko Florence Kwo Henry S. Kwon Hwee Y. Kwon Timothy H. Kwon* Sharon M. Kyomen* Jan M. LaCombe* Lise Laflamme & Richard Massoth* Gordon Lai Keyvan Lazar Laurie D. Lazarou* Khang T. Le* Matt LeCheminant Michael G. LeCheminant* Chaehwan Lee Christina N. Lee Darin C. Lee Edward S. Lee* Frances S. Lee* John Lee Serena Lee Rush A. Lenroot* Zhangrui Liang John M. Lies* Doo Yong Lim Ella T. Lim Jung Lim* Jerry H. Lin* Sean Lin* Loong C. Lin* Howard Liu* Philip M. Livingood* John D. Logsdon Victor Luikham Robert H. Lund* Tanya K. Machnick Suha S. Maddah Thomas P. Mack* A. Lee Maddox* Matthew Magar Karim Malek* Jose M. Malfaz James A. Malouf*

Thank you for your generosity to the Foundation for Endodontics! The Honor Roll represents cumulative giving, not necessarily the amounts of individual donations. Donors move up recognition levels with additional individual gifts and/or pledge commitments. Optional contributions made during the AAE membership process are tallied separately. The Honor Roll goes to print two months prior to arrival of the JOE by USPS mail. We sincerely apologize for any errors or omissions—please email [emailprotected] or call 312-517-2159 about any discrepancies. Victor R. Mancuso Jr.* William J. Marweg* Gary R. Massa* Barzin T. Massarat* John W. Masters* Aeshna Mathur Charles T. Maupin James McIntosh Robert B. McWilliams* R. Cary MeadΔ Brent A. Medema Miguel E. Mego Steven J. Merchant* Marshall Michaelian Marites O. Milan* Aye Min* Phillip S. Min* Fred Monempour Jay J. Monsef* Babak Monzavi* Victoria E. Moore* Robert S. Morrison* Steven G. Morrow* Shervin Moshashaei Gary W. Moss* Harvey D. Moss* Unes Nabipour* Lushen M. Naidoo Dan M. Nakamura* Todd T. Nakata* Brandon L. Nash* John F. Nelson* Roy NesariΔ Betsy H. Newman* Brian Nguyen Jan B. Trang Nguyen* Thien P. Nguyen* Dominic J. Niccoli* Nina A. Nielsen* Craig Nii* Kevin T. Nii* Marna Nii* Nooshin Noghreian* Douglas C. Norman* Kevin M. O’Dea* John Oganesyan* Samuel W. Oglesby*∞ Patrick K. Ohara* Micah M. Oller* Jenessa Sin-Mar Oo* L. Cary Orton* Lee E. Osnas* Lewis H. Overbey* Rodney L. Owen* Joseph L. Packer Margaret C. Pan* Terrell F. Pannkuk* Pejman Parsa Alex Parsi* James E. Pastor*

Jaymin J. Patel Niyati Patel Rajiv G. Patel Steven E. Penn* Christine Peters* Eric A. Pettersen* Lauren T. Phan Phuoc H. PhanΔ Corene J. Poelman* David Pokras Anthony P. Potente* Karen S. Potter* Nidhi Prakash* Reid V. Pullen* Nasreen S. Qader* Phuong Quang* Richard J. Rauth* Thomas J. Rauth* Yasaman Ravandoust Christopher J. Redd Ernest A. RillmanΔ Darron R. Rishwain* David W. Rising* Joy A. Rivero Sean A. Robertson* Gregory Roda* Rafael A. Roges* Roberto Roges* David D. Roland* Gary E. Romain* Ascella J. Ronson Mark J. Roper Daniel L. Rose* Richard H. Roth* Ilan Rotstein* Thomas R. Russell Steven Ryan* K.R. Buzz Ryskamp* Deema M. Saad* Sharon Sabet* Mohammad Sabeti Ahmad Sadeghein* Seyes Moein Sadrkhani Dongjin Sah Cyrus A. Salehi Robert Salehrabi Ahmed B. Salman Yuliya Salmeron Paulene K. Salter* Anthony H. Savage* Daniel S. Schechter* Benjamin Schein* Merrill E. Schmidt* Richard E. Schmidt* Thomas P. Serene Madhavi Setty* Farshad Seyedein* Avisha Shah* Rupen Shah* Robert H. Sharp*

1529

DONOR ROLL, continued

Thomas D. Sharples* Hassan Talat Shawli Michael G. Sherman Steven Shimazu* Tota Shimizu* Nicole A. Shinbori* Ramiar Shirani* Taha Shoreibah Adrian E. Silberman* Tory L. Silvestrin James H. Simon* Erik E. Singer* Mohammad Sirjani Gabriel Smith Jason O. Smith* Robert A. Smith* Douglas H. Snider* James S. Socoloske* Gregory Sprague Gordon J. Steuck* Kevin Stewart Larry C. Stoops* Jack A. Sturm* Sue W. Suh Jennifer Sun Jay J. SungΔ Tad T. Suzuki* Steven T. Swager Larry A. Sylva Douglas Szeto* Darsh*ta Talim Jaydeep Talim* David Tancreto* Radhika Tandon* Franco A. Tarm Mohammed Tarrosh Cynthia T. Tatsuta* Marshall J. Taylor* Yeow Teh Tee* James A. Thomas Jr.* Sony Thomas Timothy S. Tobias Ashi Torabinejad* Kenneth C. Trabert* Norman H. Tracy* Christine D. Tran Dan Q. Tran Kenny T. Tran Loc Tran* Henry O. Trowbridge* Patrick Tsai Jessy I. Tseng* Polymni Tsotsis Ray Tsuyuki Jr.*Δ Michael J. Turman Arjang Vahidnia Ali Vaziri* Rashi Vohra Keely Walgama Fengming Wang Roger A. Webster* Allen Wei* John A. Weisenseel Jr.* Damon J. Westwood* Clinton E. Weaver

1530

Djavan Wharton-Lake* John D. Williams* Anne Wiseman* Richard Wittenauer* Wenfei Wang Clifford T. Wong* Derrick Wong* Jason G. Wong* Justin Wong Marston Wong* Perry D. Wong* Timothy A. Wong* Winifred S. Wong* Dennis J. Wourms* Francesca Yacaman Ryan Yamanaka Bexter Yang* Kiarash Yeganegi Eric T. Yokota* Scott Young Sung-Ming S. Young Shatha Zahran Parisa Zakizadeh* Ladan Ziaiematin

Colorado Diamond David C. Funderburk* Gary G. GoodellΔ* John A. Khademi* Founder David J. HoltzmannΔ Sanjay N. PatelΔ Edward F. Rosenfeld*Δ William C. Roth*∞ Benefactor Bradley LeValleyΔ Omar A. Macaraeg* Lamont G. McMurtrey* James P. Ryan* Edward P. Theiss* Patron Amber D. Amelang-Severin Kevin Andrus* Robert E. Averbach* Daniel J. Barton* Brian Frutchey* James Burquest* David O. Carbone* Perri L. Carnes* Joseph G. Carr Jr.* G. Garo Chalian* Shane R. Christensen Matthew S. Davis Brock F. Deal*Δ Dale Denio G. Bruce Douglas* Stephen D. Fante* Brian Frutchey Rebecca Funderburk* Anthony F. Girardi

John C. Hansell* Anita A. Hoelscher David J. Ishley* Eric M. Jahde* Scott A. Johnson* Kelly B. Jones*Δ Jed S. Jultak* Wade A. Kennedy* Donald J. Kleier*Δ Steven L. Klyn* Deborah C. Knaup Mark L. Kochevar* Kerri Lawlor* Mark E. Levine* Scott D. Lowry* Michael B. McKee* Peter J. Mecham* James E. Miller III* Paul W. Northup* Heath Parry* Gary P. Pascoe* Donald D. Peters* Chad M. Reader* Jamie D. Ring* Ellen Sachs* Donald S. Safer* Manpreet S. Sarao* Carl C. Skulski* Demetrios C. Syrpes* Janet E. Tucker* Baljit Singh Uppal Judy K. Van Gheluwe* Mark D. Wood*

Alejandro Carrasco Philip Chang* Kenneth S. Chasen*Δ Trisha Charland* Nauman Chatha Henry P. Cohen* Alan H. Cooper* Sam P. DeMartino* Walter J. Doblecki* Andrew Fossum John F. Gell* Jeremiah M. Granados Robert P. Indyk* Homan Javaheri Jin Jiang Seth L. Kabakoff Carolyn Kilbride Christopher A. Lento* Anshul Mainkar Mark Melnick* Seymour Melnick* Ricardo D. Morant* Dennis L. Pipher* Stephen R. Quatrocelli Yousef Redhai Kevon E. Rennie Robert A. Rose* John M. Russo*Δ Kamran Safavi* Maksim Serebro Aniuska Tobin Thomas J. Turek* Martin J. Ungar* Qiang Zhu*

Connecticut Delaware Topaz Bruce Y. ChaΔ Founder Mark B. DerosiersΔ Kenneth P. Sunshine*Δ Benefactor Laurie R. Fleisher* Edmund G. HohmannΔ* Philip R. MasciaΔ Michael H. Rutberg*Δ Larz Spangberg* Connecticut Association of Endodontists Patron Sharefa Al-Asfour Luis Alberto Alcalde-Presedo Saleh Ashkanani Robert A. Balla* John Barrett Philip J. Bauer* Christopher Beus Jonathan Blacher Alan H. Brugg* Adolph Bushell* Alexandria Butler John C. Calhoun

updated as of 7/26/2021

Patron Michael T. Aloe* Vincent T. Cammarato* Robert C. Director* Daniel R. Kreshtool* Donald T. Liu* Debra J. Pace* George A. Zurkow*

District of Columbia Gold Paula Russo*Δ∞ Diamond Angela P. NogueraΔ* Topaz Julian R.D. MoiseiwitschΔ Benefactor Nelson J. GoodmanΔ Michael I. Pascal*Δ Michael J. RiberaΔ Patron David Bowers-Evangelista*

Gael M. Delany* Andrea D. Lisell* Kim A. Menhinick*Δ David M. Melrod Jessica A. Russo Robert N. Smyth

Federal Dental Health Services Topaz John Jeppson–VAΔ Founder Susan E. Hinman–NavyΔ Benefactor Michael J. Apicella–Army*Δ Stephen Davis–VA*Δ John R. Lundstrom–NavyΔ* Angela M. Montellano–Air ForceΔ Kelli J. Swenson–NavyΔ Patron Arezoo Barani–Air Force Rami Barfuss–Army Ann Michele Blake–Air Force Kenneth J. Boone* David J. Bowers–Air Force Carrie L. Burger–Navy* Daniel Brown–Navy Jared W. Cardon–Air Force Rhett B. Casper–Air Force* Thomas G. Cooper–Navy* Michael E. Crabtree*–Air Force Gretchen B. Dalrymple–Air Force Alexander K. Desta–Navy Jeffrey Domark–Navy John Dominici–VA* David M. Dow–Navy Michael D. Ferreira–Navy Jay Geistkemper Paul B. Hilfer–Air Force* Anthony P. Joyce–Army Gerald P. Kaban–Air Force Jered B. King–Air Force* Timothy C. Kirkpatrcik–Air Force Steven L. Klyn–Air Force* Michael A. Koch–Air Force* Bernard Lichtenstein–VA* Paul A. Lindauer–Navy* Harvey D. Moss–Navy Nancy Osborn Aric Petersen–Navy Nasreen Qadar–Navy Verne F. Reed–Navy* Melissa Ruff–Navy Mark D. Roberts–Air Force Kent A. Sabey–Air Force* Paulene Salter–VA* Allan Sandor–Navy

JOE • Volume 47, Number 9, September 2021

Maria Santos–Air Force Kyle J. Schmidt–Navy* Scott A. Schwartz–VA* Rodney V. Scott* Stephanie J. Sidow–Army* Michael R. Suhler–Air Force* Mark B. Sweet* Duane Van Niewenhuyzen– Navy Richard VanderWeele–Air Force* Brian Watkins–Navy* Sterling J. Whipple–Air Force Ross A. Yost–Air Force*

Florida Gold Vivian M. Cohen, Gregory Cohen, Jason Cohen, Kristin Cohen* In Memory of Philip W. Cohen Diamond Raed S. Kasem* Timothy J. Temple*Δ Topaz Gayle Obermayr*Δ Founder Philip W. Cohen* Joseph R. Cwikla* Malcolm S. Davis* Kimberly A. DetorriΔ* Marion C. Eldridge*Δ Kathleen Gaboardi* Randall T. Hedrick*Δ Ramon Hernandez Stanley M. Levin*Δ John P. LundgrenΔ Sheldon R. Mann*Δ Peter E. Murray*Δ Kenneth N. Namerow* Elizabeth C. NixonΔ Daneilla S. PeinadoΔ Roberta PileggiΔ Randy W. Griffin*Δ Bradley L. Schiff* Robert L. ShermanΔ Javier E. Simons* Fla. Asssociation of EndodontistsΔ Benefactor Sunghee Ahn* William H. Aippersbach*Δ Laurent W. Belanger*Δ Ericka A. Bennett* Eric W. Bludau* Jack P. CampbellΔ Charles J. Cunningham* Andrew R. D’Amelio*Δ Bethany Douglas*Δ L. Carl De Jongh*

Mark S. DeNunzio*Δ Wayne M. Dubin* Rita Echevarria* Faustino D. Garcia Edward N. Green* Robert H. Haller* John Thomas Hanco*ck* Gretchen HeinsenΔ* Edward A. Kotz Jr.*Δ Robert W. Ladley Gary A. Layton*Δ Demetrick W. LeCorn*Δ Stanley M. Levin* Vincent C. Lovetto Jr.* Melissa Marchesan Rory E. Mortman* Glenn L. Paulk*Δ Peter A. Pullon* Karla C. Ring Juan P. RodriguezΔ Hans R. Salheiser*Δ Michael P. Sardzinski*Δ Danni SaymanΔ Bruce D. Schulman* Craig Shapiro*Δ Jaime J. Silberman* Kenneth C. Sprechman* Barry H. StevensΔ Joel B. SlingbaumΔ* Timothy M. TalbottΔ* Robert A. Uchin* Claudio H. Varella Raymond T. Webber* Sy Weiner* John F. Whitt Jr.*Δ B. Harvey Wiener* Stephanie S. Williams*Δ J. Dennis Yavorsky*Δ Patron Heather Adu-Sarkodie* Briano Allen*Δ Joshua Allore Mobeen Alvi Kevin Andrus Juan C. Anguita James A. Aurelio* Sondra L. Avant* James R. Baker* William E. Barkins*Δ Jose F. Barros David A. Beach Bryan M. Beebe* Monique R. Belin Yehuda J. Benjamin Frank P. Berdos Brooke Berson Maryse Bertrand Douglas P. Bethoney Steven Black* Rachel Blakeley Yamilet Blanco James M. Blaney* Robert E. Borer* Carolina Botero

JOE • Volume 47, Number 9, September 2021

Kimberly A. Brackett* Kenneth W. Bradshaw* Greg Braunstein* Ryan Brennan Leandro R. Britto* Mauricia N. Brown Thomas A. Brown* Shaun S. Bullard Christian Cain Vince T. Cammarato IIIΔ Frank J. Cervone* Shiju Cherian Grace T. Chu Leeann Chu Margaret Cielecki Jill Clifford Kenneth P. Coffae* Robert A. Comora* Jose F. CostasΔ Frederick L. Cox*Δ Thomas P. Currie* Jaclyn G. Dam Laute* Alexander L. Dean Glenn S. Dean* R. Heath Deason* Frank Delgado* Dennis K. Demirjian Vincent DeNitto* Rolin Desir Nathan C. Dewsnup Robin B. Dodd* Patti C. Dowling* Mitchell R. Edlund Melania Elsner David D. Fabre* Edwin E. Fabrega Jr.* Romi M. Farber Thomas A. Ferretti* Michael D. Flax* Jason F. Foreman James L. Frazier* Martin H. Frost* Gustavo E. Fuentes* John H. Galberry* Eric R. Gallatin* Juliane Gallatin* Jacqueline Garcia Francisco N. Gari Robert D. Gear* Elizabeth Geisler Richard Gelman* Alexandre Ghazal* J. Mauricio Giraldo* Robert A. Gittess Charles R. Glosson* Michael Gonsky Wanda Goodreau* Gerard T. Grassi* Andrew E. Graves* Jay C. Green* Kenneth E. Grossman* James Guttuso* David Hatch Darlene Hachmeister* Mikilena Hall

Thank you for your generosity to the Foundation for Endodontics! The Honor Roll represents cumulative giving, not necessarily the amounts of individual donations. Donors move up recognition levels with additional individual gifts and/or pledge commitments. Optional contributions made during the AAE membership process are tallied separately. The Honor Roll goes to print two months prior to arrival of the JOE by USPS mail. We sincerely apologize for any errors or omissions—please email [emailprotected] or call 312-517-2159 about any discrepancies. Jeanette Pena Hall Jeff D. Hanzon Terrance Hayes* Brett Hill Adam Hinkley Audrey Hsin Aaron E. Isler Joslyn A. Jenkins John J. Jimenez* Hope Johnson Carl T. Jones* Elena Kan* Keith G. Kanter* Valerie M. Kanter Wayne W. Kearney* David Kellogg Julie A. KennedyΔ Alana M. Keough* Kenneth J. Kerman* Carolyn S. Kerr* Kunhyung Kim David J. Knight* Jay Ko Sergio Kuttler* Arthur J. LaneΔ Carlos D. LeCroy* Sandra Lee* Yoon Haeng Lee Stanley M. LevinΔ* Mark A. Limosani Michael R. Lindsay Chen Liu Glorimar Llavona Adam Lloyd John W. Loeffelholz Eileen M. Lujan Roderick M. MacIntyre* Jonathan J. Madras Ronak Makadia Bruce D. Manne* Sidni Manne Yosef Marder Sara Markovic Claire Martucci Mark A. Massey* Joseph C. Mavec* Allen D. McCall* Sean McCall* John T. McCann* William D. McGrady Steven Mcnu*tt* Alex Mehler Kevin B. Melker* Carol Beaton Merkle* Matthew Miller Steven Brock Miller Mani Mirpourian Glen B. Mitchell* Lauren H. Mitchell* Charles F. Mohaupt*

Ivan Moldauer* William H. Montes* Barbara G. Morgan*Δ Stephen E. Morrow* Jurssar Morsani* Nicklaus A. Morton Allan Moskow* Robert S. Mullaney* Frank S. Munaco* Umadevi P. Nair* Robin M. Nguyen Dennis G. Nielson* William B. Nipper* Thomas P. O’Connell Sandra Ogando Bamiduro R. Oguntebi* Seymour Oliet* Michael Orrantia* Jacob S. Otis Patricia Paparcuri Gary P. Pascoe Joseph W. Patnik* Alexander D. Paul* Russell A. Paul* Kelli Paxton* Ernesto Paz* Sonia I. Pena*Δ Lawrence J. PfeilΔ Jeff Phan Donald L. Pink* Valeria Pizzini Robert E. Powell Howard L. Pranikoff* Alexander Raiken Jonathan S. Rapp Jon L. Rauch* Richard L. Raymond Jamie D. Ring Jose L. Roig-Greene* David B. Rosenberg* Jeffrey B. Rosenberg* Craig A. Roth* Taras Roud* Alberto A. Ruiz* J. Tim Russin* Heather L. Ryan* Steven L. Ryan Mohamed H. Saber William L. Sainsbury Fredericka Salbo Heberto M. Salgueiro* Kira L. Santos Harkeet Sappal Jeffrey Sarmiento* Geoffrey L. Sas James B. Satovsky* Taner Cem Sayin Jon-Michael J. Scalercio Jordan Schapiro* W. Theodore Schwartz II*

1531

DONOR ROLL, continued

Osman N. Soliman* Paul A. Scott* Seth J. Shapiro* Sang Y. Shin* Scott Shwedel* Phillip A. Simon* Darren W. Sinopoli* Harold S. Sinrod* Mary E. Sorrentino Jenna Hart Starkey John C. Steffensen Russell B. Stoch* Adam P. Strimer* John M. Sullivan* Eric J. Sunderman* Martin A. Swartz* Robert B. Swindle* Laura Taiman Liwen Tao* Kayla Tavares Matthew Thomas Robert R. Thousand III Ryan M. Tigrett Lauren Tink Mark W. Todd Philip L. Topcik* Emily Tyler* Borasmy N. Ung* Sakib Vahora Jeffrey Paul Westra David D. Whitaker*Δ∞ Christopher A. Widmer Kenneth M. Wortman* Jiann-Jang Wu* Michelle Yang* Margaret H. Young* Robert Zelikow* John D. Zongker*

Georgia Diamond David J. JunnΔ Topaz Thomas E. Day Founder Clyde H. Andrews* Rodrick Barden*Δ Mark Barr* Eldon P. Carman*Δ David J. Dickey*Δ William H. RousseauΔ William F. Schroeder Jr.* Benefactor Mark A. Barr*Δ Brian E. BergeronΔ George T. Brown Jr.* Christopher GlassΔ Alan T. Goodman* Cameron M. Howard* Allan R. Malamy*Δ Thomas W. McDonald*

1532

Robert J. MichelichΔ* Emmanuel C. Ngoh Randolph P. O’Connor*Δ Albert L. Amato Richardson M. Odum*Δ James W. Riggans Jr.* Beth A. SheridanΔ* Rico D. ShortΔ Stephanie J. SidowΔ* Randolph M. Stevens* James S. Tate* Patron Matthew R. Anderson Jimmy W. Arnold* Randan L. Ashmore* J. Brian Baker* Fred E. Beall Sr.*Δ Thomas D. Blaney* Henry T. Bradford III* Hans A. Burg* Melanie W. Burns* John Camba Daniel J. Carlon* Randall A. Coggins* Christian Dahl Darlene Davis* David K. fa*gundes*Δ William E. Gandy* Daniel Garabadian Pinkney Gilchrist* Richard E. Golden Jr.* Nicholas A. Golubow* Joel D. Greenhill* Timothy A. Grubb* John Hayco*ck* Wm. Frank Kimbrough* Les H. Kravitz* Christopher Lea* Amy Z. Lee* Robert J. Loushine* Waheed A. Malik* Annika Marschall* Thomas A. Masters* Donald B. McGinty* Dale A. Miles* Derek V. Miles* Randall W. Miller* Samuel J. Mumpower W. Joel Newsom III David W. Norrington* H. Edward Paris* Wiley L. Purcell Ross A. Rickoff Steven Roberts* Carol S. Rohde* Timothy R. Rohde*Δ Rahul Saraf* James F. Schopler* Helen N. Sempira* Bhavik P. Shah* Richard S. Shapiro* Katherine Jie Shi Bradford P. Smith*Δ Andy W. Suresh*

Franklin R. Tay* Fucong Tian Jeffery E. Turner* Aurelia Vanderburg* John D. Welch* J. Kenneth Weldon Jr.* Jon Willison Lara Yeghiasarian

Guam Patron William C. Hightower* Jongsung Kim*

Hawaii Diamond Gary S. Yonemoto* Benefactor J. Craig Baumgartner*Δ Carl S. Haga Gary I. Kondo* Irma S. Kudo* Terry S. Matsumoto*Δ William L. Stevens* Michael S. TajimaΔ Randall D.J. YeeΔ Patron Ramie K. Barfuss* Glenn M. Biven* Brad B. H. Ching*Δ Zachary Dodson* Derrick C. Fu* Staphe T. Fujimoto* John M. Kurahara* H. Stanley Maerki, Jr. Robert K. Miura* Patrick J. Munley Mark D. Sakurai* Nozomu Yamauchi* Michael D. Yuen*

Idaho Diamond Bart B. Morrison* Benefactor Timothy W. Penberthy Adam J. Shipp Patron Val H. Bingham* William S. Boggs* Louis J. Buhrley Katherine A. Divine*Δ Stanton D. Widmer John M. Kurahara* H. Stanley Maerki Jr. Robert K. Miura* Patrick J. Munley Wade K. NobuharaΔ

updated as of 7/26/2021

Mark D. Sakurai* Michael D. Yuen*

Illinois Diamond Jerome V. Pisano* Martha E. Proctor*Δ Founder Paul J. Ashkenaz*Δ John B. Dale*Δ James M. Drinan*Δ Larry R. FarsakianΔ* Robert W. Hawkinson Jr.* Jeffrey H. Hembrough* Joseph D. Maggio* Donald A. Miller*Δ Cindy R. RauschenbergerΔ D. Lance Taylor*Δ Gene Z. Walchirk*Δ Christopher S. Wenckus*Δ Benefactor Anonymous Alan T. Azar*Δ Joseph V. Baldassano*Δ Ellen M. BarnesΔ Stephen M. Benjamin*Δ Brooke D. Benson Paul F. Bery Gary W. BrankinΔ Agnieszka ChruszczykΔ* Luz Cua* Matthew C. DavisΔ Michael Elasaad* Norman L. Eskoz*Δ Mohamed I. Fayad* John S. Fox*Δ Maria J. Fournier*Δ Nikolaos G. Gavrilis James F. Gianakakis* Brett E. GilbertΔ Nader GillΔ Robert A. Goldberg* Heath A. GroteΔ* John F. HattonΔ Fabian G. Hosein* Bradford R. Johnson* Kevin T. King* Darmon D. Kuntz* Cathy E. Longos*Δ Thomas K. Maloney* James E. McCormick*Δ Darlene C. Melton*Δ Richard A. Munaretto*Δ Charles R. Neach* Peter A. Paesani* Gregory J. ParsonsΔ* Vince Penesis* John E. StuparitzΔ* Taisa L. Szeremeta-Browar & Andrew Browar*Δ Frank J. Woolman* Ill. Association of

EndodontistsΔ Patron Satish B. Alapati* Thomas E. Allaway* Lauren Allegretti Moayad S. Alomar Dale Marshall Anderson* Ali Alsuhail Nabeel M. Atassi Vladana Babcic* Mark C. Baker* James J. Baldi*Δ Gordon L. Barkley* Michael J. Barrows* Andok Barseghyam* Doris Basali Donald C. Beard* Roxanne P. Benison* Scott K. Bentkover* Jhujhar S. Bhambra Marcella S. Borgman Robert F. Brandys* Gary W. Brankin Bruna M. Burgener* Young Joon Byun Andrew J. Calhoun Julianne Carrara* Lee M. Ceresa* William Cheng Lester B. Chernick* Robert J. Ceisel* Jill E. Cochran* Raymond R. Copeland Ashley R. Coulter Cynthia H. Czaperacker* Beth Damas* Michael F. Dani* Milton L. Davenport* Sundeep Dhawan Carlos Diaz-Albertini* Matthew K. Dietz* John W. Distel* Scott A. Drancik* Claron S. Edwards* Michael G. Elasaad*Δ Bryan T. Eslinger Keith A. Evans* Richard A. Felt* Stephen N. Ferraro Douglas L. Fillak Patrick I. Fitzgerald* Angelique R. Ford* Saman R. Gharib* Milica M. Golubovich Amanda A. Goodman* Harley B. Grandin Gerald D. Gray* Robert S. Greenberg* Bernard J. Grothaus* Alyson Hall James G. Heffernan* Mark A. Henry* Keven S. Herold Michael A. Heuer*

JOE • Volume 47, Number 9, September 2021

Mark A. Holman* Jeffrey H. Hopkins* David J. HurtΔ Noel R. Ianno* Egill L. Jacobsen* Daniel M. Janowski* Nisreen Jaweesh Lars B. Jonsson Sheetal S. Kalokhe* Daniel E. Kaplan* Louis G. Karras* Eiman Khalili-Araghi* Patrick F. King Richard A. Kohn* Jenny G. Kopp Annette Kugelmann Robert Lee Marcella Leonard* John E. Levin Garrick Liang Rosemary Linzer Neuhaus* Jeff Linden* Stephanie Liss* Paul Lundine Yolanda A. Madison* Terence Mah* Ronald C. Markarian* Natanya Marracino Alexandra Martella Robert F. McGarry* Geoffrey McMurray* Xiaoxian Meng* Amber A. Miller* Ronald M. Milnarik* Michael J. Mintz* Nermeen M. Moussa* Raymond F. Munaretto* Steven J. Muraski* Parth Nanavati Barry M. Nathanson* Julia C. Nguyen Vinh-Thy H. Nguyen John Nowak Jr.*Δ Andrea V. Nunney Mark A. O’Banion* Robert E. O’Donnell* Patrick O’Hara* Daniel Oh Cristina T. Olarov Ashley Orth Richard A. Pasiewicz* Ryan J. Pasiewicz Samir M. Patel Yagnik Patel John W. Pawluk* Antoinette M. Pettitt* Glenn R. Pouleson Manila Nuchhe Pradhan Rebecca S. Prescott Sebastian Adam Przybylo Chiara Quick Kermit M. Radke* Nijole A. Remeikis* Adam K. Remm Linda Ricks-WilliamsonΔ

Debby Rice, AAE staff Martin J. Rogers*Δ Edward T. Rose* Evan B. Rumack* Thomas Sarna* Robert S. Schmidt Suhaila Shariff Rajan Sharma* Steven M. Sieraski* Masood Sirjani Keith Sommers* Edward H. Song Trina Steinberg Nathanial B. Swaar* Kenan Tarabishy Gail MB Tischke William M. Todd* Elizabeth Uhrich* Joseph E. Van Cura* Shawn M. Velez Jeffrey T. Walker* Leslie F. Walker Brian S. Wardell Brian D. Watkins* Peter S. Weber Stephen M. Weeks* Franklin S. Weine Howard Weisbart* Joseph R. Wells* Jonathan W. Wong* Qian Xie* John M. Yaccino Helen Yang

Indiana Diamond E. Michael Feltman* Carl W. Newton* Kathryn G. Stuart*Δ Founder Donald E. Arens Charles L. Steffel*Δ Benefactor Robert J. Beck-Coon James W. Blackburn*Δ Krie Brasseale*Δ Edward J. Fischer*Δ Michael S. GideonΔ* Jason P. Glassley Brent R. Grafe* Michael B. Griffee* Michael E. Keller* Mark D. MaguraΔ* John E. Marosky* Allen Meier* David H. PfotenhauerΔ* Scott E. Shuler*Δ Mindy M. Van*Δ Curt A. Warren*Δ Patron Benjamin H. AdamsΔ

JOE • Volume 47, Number 9, September 2021

William R. Adams* Ronald K. Allen* Donald G. Anderson* Theodoros Aneziris Ronald L. Armstrong* Michael P. Aslin* Jason P. Barney Svetlana Berman* Ross T. Biggerstaff Steven W. Binkley Beau J. Brasseale Kara M. Brothers Cecil E. Brown* Robert A. Corns* Kevin A. Deardorf* James E. Duncan* Adam Everhart James W. Fisher* Harry G. Frank* Sean P. Hart* Charles F. Hine* Carolin F. Ibrahim Haris Iqbal Kiwan Kim Justin Kindler Daniel R. Kozlowski* John J. Kussmaul* Beverly J. Leddy* Joseph J. Legan* Paul J. Ley* Jacob D. Long Robert T. Mabry James Malooley Jr.* Patrick W. McIntyre Elizabeth A. Miller Tod R. Moretton* Thomas K. Nasser* Joyce Nazzal Thomas G. Oldag* McKay B. Packer* David P. Pflum* Blake T. Prather Philip K. Radtke* Edmund L. Rapp* Jonathan W. Reitz* Benjamin P. Ricketts Scott E. Risser* Paul S. Sahni John H. Slavens* Joseph D. Spires* Kenneth J. Spolnik* Brian P. Tate* Craig B. Thiessen Mychel M. Vail* Dennis J. Zent*

Iowa Gold Ty EricksonΔ William T. Johnson*Δ Keith V. KrellΔ* Diamond David C. HansenΔ

Thank you for your generosity to the Foundation for Endodontics! The Honor Roll represents cumulative giving, not necessarily the amounts of individual donations. Donors move up recognition levels with additional individual gifts and/or pledge commitments. Optional contributions made during the AAE membership process are tallied separately. The Honor Roll goes to print two months prior to arrival of the JOE by USPS mail. We sincerely apologize for any errors or omissions—please email [emailprotected] or call 312-517-2159 about any discrepancies. Jon J. Juhlin*Δ Topaz Richard E. WaltonΔ* Anne E. WilliamsonΔ Iowa Association of EndodontistsΔ Founder Lance W. Crawford* Bruce C. JustmanΔ Jeffrey P. LillyΔ* Michelle A. Schaeffer*Δ Benefactor Conrad L. Jungmann*Δ Michael H. Leuck*Δ Jack C. Liu*Δ John R. LundstromΔ* Randall Madsen Gary D. Matt* Kimberly A. Morio* Brian A. MyklebyΔ* Ricky D. NelsonΔ* Frank H. Sargent*Δ Jose D. VelaΔ Patron Heather Adu-Sarkodie Kyle J. Barry Daniel Bartling Thomas D. Becker* Arne M. Bjorndal* Jason A. Bouska Andrew J. Brasser Randall L. Breeden* Chad A. Campanelli Jared W. Cardon J. Brandon Carroll Emily Case Andrew W. Chan* Jeff Clam Jane A. Chiappinelli* Jonathan F. Crawford* Joseph E. D’Souza Jamal R. Flowers Christopher Friedl* Q. Michael Fuhs* Elliott Glenn Zachary Goettsche Manuel R. Gomez* Abeer A. Hafez* Luke J. Hamann Jennie Harris Sally Hays Mikaela Hazard Cody Heslington* Christopher Hogden Lauren Jensen Ben C. Jorgenson*

Lanny V. Kampfe* Mohamed A. Khowassah* Jay F. Leer Kristy L. Marker* Lindsey J. Meder-Cowherd* Olivia Meier Benjamin Nashleanas Kellie Paxton Kelsie Pittel Linda Ricks-Williamson Christopher L. Rowe Mathew J. Royal Allan J. Sandor Lulu Schaefer Kathleen Schaetzel Garrett Schultz Marjorie A. Taylor* Ryan Teahen David M. Thuet Amy Vermeer Jered Vislisel Brandon J. Vos Chase Wicker Lisa R. Wilcox* Gregory C. Witt*

Kansas Gold Anthony C. Leung Diamond William R. Watson Jr.* Topaz Grant W. Merritt*Δ Benefactor John E. Dietrich* Paul A. Jones* Daniel R. Loftus* Kristy L. MarkerΔ* Donald R. Mayer* Barton W. Putnam* Patron Robert H. Altomare* Rodney C. Brown* Mary Ann Chang* Jeffrey M. Clark* Kevin P. Cunningham* Steven P. Gish* Sara A. Hodges* Kerri L. Lawlor Donald H. Lemire Jr.* Andy V. Luong Randy S. Metzler* Edward A. Nelson* Casey L. Noble Kerry M. O’Neal*

1533

DONOR ROLL, continued

Ronald R. Riley* Alan G, Thibault* Dean A. Troyer* Terrence V. Turner*∞

Kentucky Gold Anthony C. Leung Diamond William S. Hopkins* Harvey E. MathenyΔ Founder Stephen D. Cox Peter G. Fotos*Δ Benefactor Paul L. Abbott* David Abide Mark W. Bothwell* Christopher J. Cook*Δ Roland C. Duell* Scott D. SeitzingerΔ Patron Amjad Ansari Michael S. Austin* Herman A. Blair* Richard P. Broering* Barry L. Burkett* Ricardo Caicedo* Gregory A. Carman* Austin R. Carr John Eric Cercek Christopher S. Cheuvront* Michael A. Childers* Mathilde Alexandra Clairet Stephen J. Clark* Bill S. Cook* Brian Cook* Hilary I. Dunstan Abigail C. Edds* Matthew S. Emerson* Lisa B. Foster Carmel Price Gleis Roycelyn L. Gray* William S. Hopkins Samantha Johnson Donald L. Kelley* Emily E. Kelly Christopher M. Kersey Jennifer I. Kron* Robert W. Mendel* Thomas P. Mullaney* Scott A. Norton* Brady J. Olsen Christopher A. Olson Matthew J. Palazzolo* E. Daniel Patterson* David P. Pflum Joseph R. Platt Jolanta N. Sauer* Brian Shaughnessy

1534

Bindu A. Soni Andrea J. Tory-Godlew Kimberly Petkovich Vaglio* David W. Vaughn* Jonathan L. Vlahos J. Eric Walden* Earl D. Walker* Kelly M. Walker* Matthew J. Walker Craig L. Walsh* Alfred H. Wiemann III*

Louisiana Silver Denis E. Simon III*Δ∞ Diamond Mark A. OdomΔ∞ Founder Lisa P. GermainΔ* Gary M. Shetler* Benefactor Michael S. BondΔ Gwendolyn D. Corbett*Δ H. Lance Donald*Δ Darren H. Hess* Eric J. HovlandΔ* Albert F. McMullen III* Kenneth W. Parks*Δ Anthony Rainwater* Charles O. Roy*Δ Marty J. Saltzman*Δ Alan D. SandiferΔ Jerald F. Turner Paul L. Wood*Δ Patron Kathryn L. Aasen Chelsea P. Accardo* Arezoo Barani Joshua D. Beaver Benjamin S. Ber* Bryan S. Berteaux Steven W. Black Ann Michele Blake* Bryan P. Bohning* Scott E. Bonson* Joseph P. Braud Jr.* Charles E. Brown III Kayla Byrne* Joseph L. Caldwell* Barry Cazaubon* Brent M. Chauvin William C. Chisholm* Yass Dastmalchi* Arthur W. Dickerson* Lori Anna Dees Thomas M. Flint* Jared R. Fortman K. Shane Fowler* Chad C. Fulmer Reynold L. Gaubert Jr.*

Steven M. Gaudet Jr. Pinkney H. Gilchrist IV John M. Gilmer Jr.* Blair P. Gremillion Ina Laurie Griffin* George W. Harrison IV* Catherine A. Hebert* Paul B. Hilfer* Van T. Himel* Rodney Isolani Billie G. Jeansonne* Joseph D. Kirn* Ronald R. Lemon* Daniel G. Lester* Sara Macway Quinton Miner* Quinn M. Mitchell Angela M. Montellano* Garrett B. Morris Terrell L. Murphy* Jeremy W. Parker Terryl A. Petropoulos Mark D. Roberts Kristopher Ruebsamen Kent A. Sabey* Alan R. Simmons Michelle B. Speier* David J. Toca* Percy B. Twine Lauren S. Vedros William H. Wayman* James M. Youn

Founder Louis H. BermanΔ* Richard A. FeinΔ* Ralph W. Niemann* Melvin J. Weissburg*Δ Edward C. Penick Endodontic Study ClubΔ Juheon SeungΔ

Maine

Patron Sunia Abdula Maha Alghofaily Dominick J. Alongi Theeb A. Alquria Mason Bahador Christin M. Baker Yaakov Barak Bryan Behm Ian G. Bennett Christopher D. Bradley Carolyn M. Brown* Kweli K. Carson* Kelvin Chou Howard M. Cohen* Jerry M. Cook Thomas G. Cooper Chayne Coston W. Gene Crooks* Gail M. Davis* Alexander K. Desta Seyed O. Dianat Kim Do David M. Dow Eda Elbirlik* Fazel Fakhari Reza Fardshisheh* Cassandra Feveb Edward K. Gamson Marc G. Geballa Pegah Ghiasi* Michael J. Goode*

Emerald Christopher D. Dorr*Δ Topaz Michelle L. Mazur-Kary*Δ Founder Peter R. Mellin*Δ Benefactor Takashi KomabayashiΔ Todd P. Mellin Patron Jason A. GarlockΔ Joseph C. Kehoe* Sadhana Prasad* Edward G. Sebok*

Maryland Gold Patricia A. TordikΔ Silver James O. RoahenΔ Diamond Badri JureidiniΔ*

updated as of 7/26/2021

Benefactor John D. Allemang Jr.* Ali Behnia* - In honor of parents Mr. and Mrs. Behnia, with gratitude Wing F. Chan*Δ Steven C. Cohen* Reza Fardshisheh* Kathleen T. Frankle* Charles P. Herbert Tony H. Hsu Robert S. KaneΔ Martin D. Levin* Jeffery O. Luzader Charles MannΔ Raksha Mirchandani*Δ Francis R. ParreiraΔ Michael D. Peterson* John F. Patterson* Amir A. Sarkarzadeh*Δ S. Craig Schneider*Δ Ronald C. Taylor*Δ Terry D. WebbΔ

Allan J. Goodfriend* John H. Greiner Saurabh Gupta Brian D. Hall J. Frederick Heaton* M. Lamar Hicks* Anmar Janabi Omar J. Jones III Barry L. Jurist* Binait Kabir Shahryar Nasir Khaliq Eunice Kim* Stanley H. Klein* Douglas J. Koch* Mahshid Majlessi Koopaeei Molly Kopacz* Grant Layton* Liang Li Jeffrey A. Lieberman* Kat Ligon* Brian T. Lu Kenneth Mangano* Saurabh Mannan Howard Martin Kathleen McNally* Raksha Mirchandani* Afua Mireku* Sasan Moghaddame-Jafari* James Morris Jr. John G. Mullally* Ghulam Murtaza* Kyle Nelson Nguyen T. Nguyen Ralph W. Niemann* Ali Nosrat Seth L. Perrins* Brian A. Pugh Bartley T. Quilin* Michelle K. Rampulla Theodore D. Ravenel Andrew M. Reff* David Roahen Alvin S. Ro* Howard L. Rosov* Glenn C. Schermer* Betty Schindler Ronald O. Segall* Fredric H. Simon* Derek A. Slosser Langston D. Smith* John I. Tifford* Candice L. Trasatti Bradley A. Trattner* Lawrence S. Vazzana* Johnathan Velardi Ian K. Walker* William Seay Walker III Curt A. Warren* Burton M. Waxman* Michael H. Webber* Lauren A. Wronsky Brian T. Wycall* Wing-Yee Yeung Zuwu Zhou Pirooz A. Zia*

JOE • Volume 47, Number 9, September 2021

Massachusetts Charitable Trust Herbert & Joan Schilder Diamond Jeffrey W. Hutter Jay Marlin* Peter A. MorganΔ Herbert & Joan Schilder* Emerald Shepard S. Goldstein*Δ Topaz Pat MachalinskiΔ* Leslie I. Miller*Δ Yuri ShamritskyΔ Fiza SinghΔ Founder Robert B. Amato*Δ Joel L. Dunsky* Marvin A. Eichner*Δ Stephen T. GallaΔ Roy M. Hayashi Barry M. Jaye*Δ Alvin Arlen Krakow* Thomas F. Winkler III* Benefactor Simon Beylin Anthony T. Borgia*Δ Dennis M. Byrne* Philip J. CabreraΔ* Sami Chogle*Δ Roger P. Desilets Jr.* Tevyah J. Dines*Δ John V. DolbecΔ* Timothy M. Gabe*Δ Yanling Jiang*Δ Zachary U. Kano* Robert L. Kittredge* Theodore S. Kyros* Roger R. Lacoste* Rudolph L. LantelmeΔ F. Graham Locke Nicholas J. Manzoli* Steve P. Murphy H. Robert Nagel* Lawrence M. Rubin*Δ Ramzi Sarkis Paul B. TalkovΔ Joseph I. Tenca* The Endodontic Group - Drs. Gregg Nagel, Lisa Wendell and Eugene Choi Robert R. White* Patron Farah Abbassi Ashraf Abd-Elmeguid Acdulaziz Abdulwahed Shadi Abedin

David V. Abdelmalak Anitha AbdulRahiman Mario E. Abdennour* Ayman Aboushala* Stacey Abrams Louay Abrass Jeremy Aczon Fereidoun Agha-Razi Mohmaed J. Ahmed Ivy Ahluwalia Zena A. Al-Adeeb Jacob Al-Hashemi Sam Alborz Mona Alenezi April L. Alford Mey A. Alhabib Rami Alhomrany Kathy Alikhani* Omar Alkhattab Munirah Almershed Arwa Alnoury Yousef A. Alnowailaty Samaher Khalid Alotaibi Mohammed A. AlShahrani Mashael Al-Qallaf Heba M. Al-Qatami Mohammed A. Alsakran* Yimar Angell Adela Apolli William Arden* Farah Assadipour Rayan Hassan Bahabri Adriana Baiz David L. Baker James Baker Kelly Barbera Anne-Marie Barussaud Adam Becker Peter J. Bellingham Samuel A. Black Jr. Christopher Bissada Sherry M. Bloomfield Eric Bludau Mahna Bose Pillai Coy M. Boyd Jr. George A. Bruder III Michael F. Buckley* Saitah Bufersen Richard H. Bush* Ne’eel Cajee Emanouela D. Carlson Timothy P. Carter* Cindy Castillo Paul Chae Alexios Charissis Loubna Y. Chehab Peter Chen* Yen-Tung Chen Kevin W. Choi Jacquelyn Chou Brian A. Christopherson Brian Chuang Hannah Cohen Jason T. Conn Taylor Cook

JOE • Volume 47, Number 9, September 2021

Michael V. Cristani Eduardo A. Cruz Sahar Dadvand Karen DaSilva Veronica DeArmas Christina DiBona Pastan* Eric D. Dionne Mark E. DohertyΔ* Daryl J. Dudum Roohi Ebtahaj* Mona El-Sheikh Barmack Emami* Peter T. Esposito* George K. Etre* Yulia Eve Farnaz Fadavi Reza Fardshisheh Usman Sibtain Fazli Irene Fernandes Zameera Fida* Jennifer M. Fong Steven Forgione* Luis Franco Elisa E. Fulton* Tamanna Gandhi Karen Gear* Sean M. Geary Samuel Gegamian* Jack Z. Gilad Ian B. Glick* C. Robert Goldberg* Gregory R. Goldfaden Marie Gosselin Clifton A. Grayer* Ian C. Grayson Daniel B. Green* Tyler Guinn Mary E. Guzek* Samantha R. Haas Mona Haghani Luke Harden Negar Harraji Hanna Heck Brett R. Henson Nedda Y. Hifeda Sara Hoge* Jennifer J. Hong Maria T. Hoyo* Tun-Yi Hsu* Dina Husein* Dan Ingel Tumare Iqbal Jacqueline Y. Jacobson* Scott C. Johnson Luz Marina D. Jutras Laila Kafi Lauren Kai Suneel C. Kandru David Kang Michael J. Kang Sung Woo Kang Bassel Kano* Mohamed Kayali Alana M. Keough Hussein Khimani

Thank you for your generosity to the Foundation for Endodontics! The Honor Roll represents cumulative giving, not necessarily the amounts of individual donations. Donors move up recognition levels with additional individual gifts and/or pledge commitments. Optional contributions made during the AAE membership process are tallied separately. The Honor Roll goes to print two months prior to arrival of the JOE by USPS mail. We sincerely apologize for any errors or omissions—please email [emailprotected] or call 312-517-2159 about any discrepancies. Matthew T. Kiebish Jonathan Kim Sun Ho Kim Timothy J. Kim Jack Klecker Jennifer M. Klein* George Koch Emma J. Koukol* Kevin Kuo Iman S. Labib* Katherine C. Lacoste* Hans Langara Jessica Langella Jungha Lee Sarah L. Lennan Harold J. Levin*Δ Steven P. Levine* Clayton Liesen Hongsheng Liu* Dieu T. Ly* P. Quinn Lybbert Arnold I. Maloff* Mark Manoukian Jay Marlin Alexa Martin Sarah E. Martinelli* Lauren Marzouca Brent J. Mayer Alex McClure Rachel A. McKee Amir H. Mehrabi Siddarth Mehta Christine Melito* Scott Micallef Andrew J. Miller John L. Miller Fardad Mobed* Kathleen Molgaard Philip J. Molloy Diana L. Montagu Cline Alexis K. Moore Mani Moulazadeh* Gregg Nagel* Matthew J. Nealon Niusha Nikkholgh Evan Novick Natali Nunez Peter S. Ok* Ibukun O. Olagbemi* Marta Orrego-Rafla David G. Oser Anuja Panda Pamela Pappas* HaBin Park James Pastore Ami Patel Trusha Patel Christina Penn Austin L. Perera Elizabeth S. Perry*Δ

Steven F. Pinto Surbmi Puri Neda Rajablou Shawn M. Record* Jessie Reisig Tina Ren Peter C. Rider* Allison G. Ritch-Glick* Carolina Rodriguez-Rad Miguel A. Roque Robert Jay Rosenkranz* Jan B. Rozen* Courtney Russell Michael P. RussoΔ Hayrapet Sahakyants Nidhu Saini Fadhela Salah Babar Saleem Ali Salehpour Arman Samad-Zadeh Ashkan Samadzadeh Sajini Sasthri Juan Savelli Iejaz Shadid Andrea Shah Hassan Shahabinejad Bilal Shammont Kanika Sharma Tammam Sheabar Natalie Shlosman* Jason M. Shroyer Wyatt D. Simons* Varon Singh Stephanie R. Slate Chad A. Spears Karyn Stern* Douglas W. Stewart*Δ∞ Michelle C. Stoffa* Andrew R. Stubbs Pallavi Suhag David M. Sullivan* Joyce Sun Samantha Synenberg Demetrios C. Syrpes Kristel B. Tabet Sowjanya Tadakapali Tadros M. Tadros Gabriel C. Tagher* Ramon Tancioco Nayrouz Talua Alan T. Tang* Vanessa Thai David Tran Jamie Tran Raina A. Trilokekar*Δ Hai Truong Dimitris Tsatsas Deborah E. Tung* Peter D. Tziros Ryan F. VanMoorlehem

1535

DONOR ROLL, continued

Nadia Virani Kurtis L. Wadsworth Bing C. Wan Mark Wang Robert K. Watts Joseph J. Williams*Δ Michael R. Wolov* Angela Wu Stephanie Wu Yean C. Wu-Young* Zhu Xiaofei Qi Xiong Deborah Yeh Howard Yoon Monique Yuan Emina Zaganjori* Jason Zeim Vangel R. Zissi*Δ Maan Zuaitar Massachusetts Association of Endodontists* Harvard School of Dental Medicine Class of 2006

Michigan Gold George T. GoodisΔ Allan JacobsΔ∞ Silver Gerald C. Dietz, Sr.Δ John W. Willoughby* Diamond Robert A. Coleman*Δ Gerald C. Dietz Jr.* Richard J. GardnerΔ* Richard A. Rubinstein* Topaz Michael B. Lindemann*Δ Founder James Howard* Richard L. Johnson*Δ Ryan B. McMahanΔ* Robert M. Rybicki* Benefactor James S. Allen*Δ Jeffrey L. Ash* John P. Braud Jr.Δ Brian J. Buurma*Δ John Cohen* A. Timothy DeConinck* Derik P. DeConinck*Δ Jeffrey J. Dwan*Δ Mark J. Dylewski*Δ Melvyn Eder* Steven Z. Edlund Steven E. Fegan*Δ Edward L. Fitzpatrick*Δ John E. Galsterer*Δ John G. Galsterer*

1536

Arnold H. Gartner* Daniel P. Gilliland* Michael A. Glass*∞ Kenneth Grove*Δ Jeffrey H. Haag*Δ Gerald J. HalkΔ Jeffrey P. Halvorson*Δ Louis E. HirschmanΔ Scott J. Hodges* Michael M. Hoen*Δ Sandra A.L. LaTurno*Δ Steven L. LiptonΔ* Lawrence R. Marcotte* Ronald E. Michaelson*Δ Susan L. MicklowΔ George Moricz* Kevin T. Mullin Mark V. NearingΔ* Susan B. PaurazasΔ Mark J. Robinson*Δ Douglas J. Schippers*Δ Louis M. Shoha*Δ Ronald R. Shoha* Steven D. Shoha*Δ Kevin A. Shugars*Δ Aric C. Smith*Δ Richard G. Somerlott* H. Robert Steiman* Michael A. Sulfaro*Δ Gary E. Tasch* Martin J. Tuck* Gail WoolhiserΔ* Eugene Wu*Δ Patron Khalid M. Ahmed* Dalia Al-Alfe Mohamad Almaaz Turki Y. Alhazzazi Christopher M. Allen Alireza Aminlari Thomas F. Armstrong* Mauricio L. Basso Patricia Bauer* Young Bin Bok Krista Bortnick* Tatiana M. Botero-Duque Carl BotvinickΔ Ryan G. Brandt Josef S. Bringas Jacob K. Butler Mauricio Chavarriaga Joseph D. Christensen Sam J. Ciacco* Carolina Cucco Darya Dabiri Beth A. Damas Nils E. Danielson Gianne S. DeCarolis* Anne DeGroft-Johnson Vincent M. DeNitto Erik DeYoung* Anthony E. Dietz* Erich A. Dittmar* Arthur Doerig

Andrew R. Drerup* Jason M. Duggan Craig F. Duhaime* John J. Dylewski* Jeffrey N. Dzingle Steven Z. Edlund John R. English* Samuel R. Epley* Todd V. Ester* Alayne S. Evans* Michael J. Gallagher* Bret Gargasz Ben Garagozloo Jorge Garcia Eduardo S. Ghaname Simon M. Ghattas Alessandro Giovanardi* Martin R. Goode* Daniel Goodman* Franklin L. Gordon Jr.*Δ Brad G. Griffin Theodore R. Grigg* Diogo Guerreiro Anthony J. Guinn Dennis M. GutΔ Paul E. Gutt* Taryn Kratz Harreld* Michael Hembrough Dana Hemisdottir Erlyn P. Hernandez James Herrington Bradley A. Hirschman* Wade Hirschman Graham Holland* Spencer Holmes Wesley R. Ichesco* Rutuja Jadhav Rachel James Sangeetha Jayaraman Ruma Kajwadkar Charles Kass* Steven D. Kesler Heidi G. Korn Paul F. Korte* Jeremy M. Kott Tanaya Kumar Lindsey LaLonde Arthur F. Lamia* Jay W. Lang* Joan Elena Lanier* John S. Lee Indaia Soares Leibovitch Brian J. Licari*Δ Edward M. Mack Mahshid Majlessi Sahil Manhas Jeffery C. Marderosian* Stephen T. McInerney* Daniel G. McLaughlin* Brian M. Meade* Kimberly K. Melegari* Jeremy H. Michaelson* Hamza Mir Sasan Moghaddame-Jafari Angela S. Mudie*

updated as of 7/26/2021

Patrick J. Mullally* Eoin Mullane Stephen W. NavarreΔ Eric J. Palte* Yashika Pande Yun Kyoung Park Kristopher J. Pfotenhauer Clare Quinlan Hooman Rabiee Mary Rafter* Ronald W. Reoch* Brandon S. Rogers Nahid Roghani* Maikel Roque Ruano Ross C. Ryan* Ahmed M. Sarhan Timur A. Sekercioglu David S. Selis Alexander Sevo Michael Shapiro Dominick N. Shoha* Allen Shorr* Michael A. Smith*Δ Edward F. Snella* Ryan I. Soden Joseph S. Son Rebecca Steffens Thomas J. Stein* Joseph B. Suffridge Lee A. TaitoΔ Xianli Tang* John E. Thomas* Robert J. Tironi* Michael C. Transtrum Yancy Tygesen* Robert J. Valice* Dan J. Vander Meulen* Gerald L. Vanderwall* Dmitry Vodopyanov Thomas C. Vokal Mayank D. Vora* Viraj R. Vora Lawrence C. Walsh* Daron S. Yarjanian Hassan M. Yehia Robert S. Yeung* Manal Zaibak Richard M. Zillich* Martha P. Zinderman* Edgardo Zuniga Michael Zuroff*

Minnesota Gold Darrell W. Zenk*∞Δ Alan S. LawΔ Diamond Scott L. DoyleΔ Scott B. McClanahan*∞Δ Kenneth J. Zucker*Δ∞ Topaz Robert L. KaufmanΔ*

Founder Walter R. Bowles*Δ Stephen G. Hunter*Δ Kris D. JohnsonΔ Stella M. Kitzenberg* Kimberly A.D. LindquistΔ∞ Mitchell L. PageΔ* Scott B. PetersΔ* Mark T. Phillips*Δ Michael Reynolds* Edward J. StecΔ Catherine E. WurmΔ Benefactor Roger J. BurkeΔ Andrew M. Doroschak*Δ Mahmoud E. El Deeb* Todd GeislerΔ Matthew M. Grau*Δ Eric H. Grutzner*Δ Mark Richard Jensen* Jerald H. Lyng*Δ Dennis M. McMahon* Susan G. Penniston*Δ Matthew J. Royal* Jeffrey L. RyanΔ Dennis M. Tucker*Δ Michael J. Tulkki*Δ Thomas J. ZbarackiΔ Patron Ramon Aguirre* Nicholas Anders Gerald C. Anderson* Michael W. Reagan Anderson Ashley E.P. Asano Deborah D. Baird* Michael K. Baisden* Brian D. Barsness Sara A. Barsness* James Alan Benson* John H. Bogle Scott A. Brezinsky Alexander M. Brown Mary Ann Bunczak-Reeh* Susan L. Camp* Melissa Chapman* Rhett B. Casper* Olga I. Castro* Safni Chiona Joseph Crepps Jonas G. Dale Colby Dimond Brock A. Droll* Kirk A. DuLac* Robert S. Edmunds* Gary Eggleston Leon A. Ernster* Rhett C. Finley Gerald J. Gray* Daryl Grigsby Samantha P. Harris Jeffrey D Heyse, Jr. Jessica B. Johnson Lisa Kandella

JOE • Volume 47, Number 9, September 2021

Thomas A. Karn* Jess A. Kelly* Levi T. Kinsey* Deborah C. Knaup Kristine A. Knoll Harish Koratkar* Richard L. Kronzer*Δ Chris R. Lai Emily Lammers* Sanaz Lavasani Michael J. Matwychuk* Brian M. Meade Laura Milroy Thomas E. Neafus* Tom H. Nguy John D. Nydahl* Brandon J. Penaz* Tyler Peterson Michael A. Pham Prathibha Rugmini Pillai Richard L. Rajacich* Jason Read Ernest S. Reeh* Samantha Roach* Carolina Rodriguez-Figueroa* Brent D. Rundquist* Stephen H. Sands* Christopher J. Saylor* Kathleen Schaetzel Tyler J. Schuurmans Cale A. StraitΔ Mark P. Sullivan Casey Turner Duane A. Van Nieuwenhuyzen Dustin Weitz Andrew T. Wiswall John C. Withrow* Amber Zedler

Mississippi Benefactor B. Craig Anderson* Mark W. Moore*Δ D. Clark Strange* Patron Andrew E. Abide Jr.* Brian Bergeron Joe G. Collins* Benjamin J. Fravel R. Scott Gatewood* Thomas F. Gerrets Jr.* Buford O. Gilbert* Carey A. Johnston* L. Ronald Martin* Jeffery B. Pride* Richard E. Rutledge J. Thad Strange*

Missouri Diamond H. Groves Cooke III* Kenneth J. Frick*Δ∞

Founder James A. Dryden* Benefactor Susan S. Adams* Lisa D. Castleman Charles W. Clarke* Daniel G. Ehrich* John Hatton* John T. Lask Dan L. Lavitt* Richard Orrick Ronald R. Riley Shelly L. Sarich*Δ Lane Stephenson* Mickey L. Unsell*Δ Rebeca Weisleder UrowΔ Ronald R. Wollard* Patron Anthony Altomare Thomas D. Becker Gregory T. Berg* Geoffrey R. Clive Gilbert J. Cyr* Benjamin F. Davis III* Tyler Davis Saman Delgoli Mathew R. Dennison Herad Divine Daniel A. Dunbar* Spencer J. Elmore* Colin H. Eliot* Michael C. Foreman Eric M. Foss Marileana Garcia Corretjer Brian J. Habas Ryan Harris M. Stephen Harrison, Jr. Jess Fowler Havron* Brian Hermanson Johnny Huynh Charles Kendall Katherine Kuntz Jakuc* Bradley S. Laird* Winnie Lam Zane M. Lambert* Warren A. Lawson* Parham Mansouri Carol A. McCallΔ Aliraad Moattar Craig M. Mulherin Scott D. Newlin* Jamie Paul* Rodrick E. Pearline* Candice H. Perry Aric M. Petersen Charles F. Poeschel* Edmond Rainey* Steven B. Raphael* B. Patrick Roach* Mark Roberts* Jay C. Rutz Norman A. Smith Jr.* Daniel G. Stamos*

JOE • Volume 47, Number 9, September 2021

David E. Stamos* Kate Stamos Pat Stamos Fariba Tahmasebi-Moshiri* Katherine E. Toole Elizabeth A. Uhrich David L. Vreeland* R. Joseph Weibert* Andrew Wirtz Chad C. Wollard* Jonathan E. Young

Nevada Montana Founder Joseph PetrinoΔ Benefactor Scott S. Dickson*Δ Wayne L. Hansen*Δ Stephen M. Lyon* Michael G. Stevens*Δ Karl F. WoodmanseyΔ Patron Thomas R. Bigelow Brett Bruggeman* Jeffery M. Hamling* Amber J. Osiecki* Russ P. Read*

Nebraska Topaz Thomas J. BeesonΔ Benefactor Toby L. Comer*Δ William C. Corcoran*Δ Corey K. Karimjee*Δ Merlyn W. Vogt*Δ Patron Fahd Alsalleeh* Hunter A. Bennett Stephen G. Biggs* Bryce W. Bonness* Kenneth J. Boone* Jeffrey Burroughs* Brett J. Cawley Alan Chee Dylan C. Downs Tobin Drake* Larry J. Ellison*Δ Tom G. Gound* Jose Luis Ibarrola* Kenneth I. Knowles* John A. Little* Nick J. Lyons Hany M. Makkawy* David G. Meier* Gary Middleton Gregory J. Nielsen Stephen P. Pryor* Jaclyn F. Rivera*

Topaz Ben B. Maze*Δ Benefactor Margaret M. Ashe William D. BrizzeeΔ Russel K. Christensen*Δ Matthew O. CoxΔ* Randall J. Iwasiuk* James E. Jones* Corey KarimjeeΔ Douglas R. Rakich* Patron Eric C. Applelin Kristen L. Beling & William J. Dougherty Jr.* W. Craig Bell* Francesca Cardinale David C. Fife Rod A. Gray Brandon Griffin Bryce Haslam Brian D. Haymore Darin J. Kajioka* David A. Maixner*Δ Bradley J. Nelson* Vinh-Thy H. Nguyen* Saro Oknaian Daniel I. Shalev* Michael L. Squitieri* Michael J. Walker

New Hampshire Founder Howard J. LudingtonΔ Douglas J. Katz*Δ Benefactor Andrew BradleyΔ Marilyn V. Steinert-Lyons*Δ Patron Aneesa Al-Khalidi* Andrew Bradley* Elliot R. Goldberg* Douglas & Pamela Moll* John W. Tucker*

New Jersey Silver Craig S. HirschbergΔ* Diamond Marc Balson* Noah Chivian*∞ Mitchell H. DavichΔ Harmon R. Katz* Topaz Marc P. GimbelΔ Founder Ronald I. Deblinger Paul FalconΔ* Mario R. Gebbia*Δ Jin Hahn*Δ Lee Meadvin*Δ Stephen J. TsoucarisΔ* Benefactor Aaron AueΔ Stanley M. Baer*Δ Arthur Berger* Kenneth M. Blumberg* Annmarie B. BrennanΔ* David Y. Chow* Edward B. DrozdΔ Gayle B. Elbaum* Leslie R. Elfenbein*Δ Jamie S. Gartenberg Vivian Graham Ryan K. Graver Donald H. Kahn Sahng Gyoon Kim*Δ Charles T. Loo Konstantin B. Maltsen*Δ John PanzarinoΔ Dennis F. Pawlak* Maya Prabhu*Δ Spencer C. Saint-Cyr*Δ Mark A. Schachman* Michele A. Scrime*Δ Asgeir SigurdssonΔ Marvin G. Weiss* Ira J. Zohn* Patron Sally Abdelkarim Thomas B. Allen* John J. Archible* Nicholas Addiego Keith Appelbaum* Yasmin Al-Zoubaidy Fabio G. Apolito Anthony Arena Benedict Bachstein* Mojitaba Bagheri

1537

DONOR ROLL, continued

Jaime Ball* Herbert D. Benkel* Sheldon S. Berkman* Melinda B. Blume* Sudharani Bodepudi* Oscar R. Bolanos Jeffrey L. Carroll* Jeffrey Chen* Daniel Choi Joseph ChikvashiliΔ Sheldon B. Cohen* J. Jeffrey Curry* Lauren C. D’Elia Donald A. DeRosaΔ Raney J. Deschenes* Duy Do Edwin F. Eisenberg*Δ Samuel R. Epley Carla Falcon Yuehong Fan Robert G. Farber* Anthony Fasciano* Alex C. Fitzhugh Sara M. Fonseca*Δ Arnold J. Goldberg* Joshua A. Goldfein* Robert L. Grove Jr.* Jose A. Guerra* Robert S. Gureasko* Kinga A. Haft Jack A. Hoffer* Aleksander Iofin Kevin D. Johnson Eunchong Ju Rattanjit Singh Kamboj Jean H. Kang* Peter Kapsimalis* Avery H. Kelner* Neha P. Khandhadiya Laurence W. Kielt* Alexander T. Kim Ana Kim Riki Kreitman Mervin Kroop* Paul M. Lafkowitz* Rajesh Lall* Ian J. Langer* Paul B. Langer* Lawrence R. Law* Stephen G. Lawson* Laurence C. Leff* Mara L. Leveson* Stephen M. Leveson* Jeffrey A. Levin* Wayne I. Levine* Timothy C. Lin Carmine J. LoMonaco* Mandy J. Louis Fredric A. Lubit* Bruce R. Lustbader* Steven Lustbader*Δ Tara Mahpour Dimple Malavia* Mansi Malavia Nikhil Mallick

1538

Carol E. Mann* Yandy Gonzalez Marrero Sarina K. Mercado* Hossein Shayei Moosavi Donald R. Morse* Marc E. Moskowitz* Samin F. Nawaz Francisco J. Nieves Achara O’Brien* Hyunjung Park Jin-Sir Park* Stephen D. Pascal* Nikeeta M. Patankar John W. Pawluk Christopher Pellicano Alejandro E. Pereda Thai V. Pham Rahi Rahnama Prathibha Rugmini Warren A. Radcliffe* Calvin Reeman* Arthur Royzner Ahsan Sadiq Arash Safaverdi Siddarth Sehgal Louis A. Seiden Gerald C. Selke* Robert J. Seltzer* Neha Shah* Bita Shakiba Lawrence J. Sheer* Emi Shimizu* Eric Y. Shon Anna Sidor Zack Siegler Priya Singh Ravinder Singh Arwa Siyam Stephen Smiley* Bruce E. Smith* Rosemichele SorvinoMacchia Raymond A. Sterling Jr.* Jeffrey T. Stewart Amy StoneΔ Douglas Szeto Fardad T. Tayebaty* Shalini Tewary Brian P. Trava* Zahabiyah Tsiamwala Harvey S. Waldman* Phianh Waldon Eric M. Weinberg Jay Weinberg* John E. Weise* Martin Weiselberg* Joel G. Weiss* Morton L. Wertheimer* Shayna Whiteman Joseph Zelig Winnie Zhang

New Mexico Diamond James. F. WolcottΔ* Topaz Charles J. Goodis*Δ Benefactor Kenneth E. Bray* Marcus R. MinerΔ* Brian R. Papworth* Patron Erick F. Carlgren* Shane T. Clark* Philip B. Edgerton* Bernard R. Gavron* Kenneth H. Kahn*Δ Edward S. Kaminsky* John W. King* Robert S. Lash* Marc A. Pacheco* Terryl A. Petropoulos*

New York Gold Michael J. FeldmanΔ* Randolph ToddΔ Diamond Stanley J. Einbender* Richard L. RubinΔ* Hank SchiffmanΔ* Barnet B. Shulman* Richard R. Weiss*Δ Topaz Robert S. GoldbergerΔ* Founder Albert L. GrangerΔ Michael D. Grassi*Δ Ming Shih Levine* Maria C. MarangaΔ Bruce H. Seidberg* Westchester-Putnam Endodontic Associates* (Marc Cohn, Gerry Hyman & Barry Rothenhaus) New York State Association of EndodontistsΔ Benefactor Benedict V. Alibrandi* Abe Ancselovics* B. Craig Anderson Robert W. BerlsΔ* George A. BruderΔ Daniel H. Flanders* David M. Gallin* Jamie S. Gartenberg* Fredric Goodman* Richard I. Herman*Δ

updated as of 7/26/2021

Arielle Chassen Jacobs* Jonathan M. Kamen* Eunah KohΔ* Kathryn M. Karpinski*Δ Matthew Malek* Richard A. Masucci* Les H. MuldorfΔ Barry L. Musikant* Elaine G. RogersΔ* Paul A. Rosenberg* Endo Association of Greater New York* AAE District II*, see New Jersey listing for New York State Assn. of EndodontistsΔ Patron Shira Ackerman H. Lee Adamo* Howard N. Aaronson* Casimir S. Ahamad* Sarah Alfadda Hashim Alhassany Amru Albeiruti* Adel S. Alobaid Riyadh Alroomy Abdulmajeed S. Alshahrani Denise A. Assogna* Talayeh Azarpajouh Peter J. Babick* Afshin Badii Albert Baharestani* Michael Baharestani* Bobak Bakhshoudeh Francisco A. Banchs* Seema Basati Patrick J. Battista* Phillip Bell Jhujhar S. Bhambra* Lynn S. Brinker* David P. Browdy* Nghia D. Bui Younghyup Byun* David G. Carter, Jr. Henry E. Chalfin Stuart E. Chassen* Yan Chen Brian Cheung Susan Chi Thomas Chillemi* Christine Choi Joseph H. Chu* Joshua M. Chubak Mark O. Coffiner* Chayne E. Costen Michael R. Cotter* Davide Cuocolo Mark D. Currell*Δ Jerome J. Cymerman* Timothy J. Daws John E. DeAngelo* Jason Deblinger* Harpreet Deol* Allan S. Deutsch* Kara Diamond*

Martin H. Diamond* Judy DiDonato Anthony J. Domenico* Wilson Duong Horst Dziura Harry J. Einbender* Kathryn E. Epkey Elliot J. Fidler* Howard B. Fine Cagatay Erakin* Yaela Faitlowicz-Gayer* Ehsan Farrokhmanesh Ali Forghani Alan C. Feldman* Mark J. Feldman* Mark J. Fishman* Denise Foran* Vincent J. Frazzetto* Lee E. Friedman* Atsushi Fujimura Anjani Gandhi* Ron Ganik* Roxene S. Gascoigne* Kamen I. Genov Larry M. Genser* Robert G. Giannuzzi* Jennifer L. Gibbs* Anna Glinianska Gary S. Goldstein* Stacey S. Goldstein David J. Goodcoff* Barry S. GoodmanΔ Deborah L. Gortler* Manisha Goswami Tod Reed Granger David Grassi* Frank L. Graziano Jr.* Brian D. Greenburg* Steven Grossman* Peter A. Guagliano* Rahul Gupta Jonathan M. Gutman* Jennifer Ha Thomas E. Hebert* Diane B. Heller* Susan P. Hoang* Robert Hochberg Jeffrey D. Hom Keith Hope Donald P. Horton* Sunna Huh Michael F. Hutton* Dean G. Hyde* Andrew Inagaki Timothy C. Ingrao* Aleksander S. Iofin* Edward M. Isaac* Yehuda Isseroff Gary P. Jablow* David P. Jacobson* Sid Poorya Jalali Nanthicha Jiratikal Christopher Joubert* Jae Ha Jung Frederick H. Kahn*

JOE • Volume 47, Number 9, September 2021

Yasuhiko Kamura Howard Kang Stanley M. Kaplan* Brian Kardon* Mitchell A. Kellert Anya K. Kent* Eiman Khalili-Araghiz Niloofar Khosravian Pedram Khosravian Jimin Kim Melanie Y. Kim* Sean Kim Abhishek Kirpal Hae Jung Ko Nassim Kohly Justin R. Kolnick* John A. Korkosz* Andrew W. Krieger Ladam Kuang Neena Lakhanpal Michael A. Landau* Jeffrey Levine Christina L. Boyd Stephen Langan* Naghmeh Latifi Andrew Lee Claire J. Lee Myong Lee* Nicholas Leon-Guerrero Kenneth Liao* Bernard S. Lichtenstein*Δ Jane Lieberman Cheng I. Lin Louis M. Lin* Edward J. Lipke*Δ Jeffrey Lo Kimberly Long Ivan Lukachynets William W. Lyons* Joceyln P. Magahis Alison Mancia Victor Kwame Marfo Firas Marsheh* Samuel Masyr* Obianuju R. Mbamalu Joseph P. McMenamin* Andrew S. Melinger* Bruce A. Merriam* Stanley Michel Richard M. Moodnik* Rick C. Moser* Gurpreet S. Narula William H. Nesbitt* Alan J. Nevins* Michael E. Newman* Michele Nguyen Vinh-Thy H. Nguyen Katsushi Okazaki Tatiana Oliveira Wael Oweity Lawrence F. Pace* Kaveh Pajouhan Carole T. Pantera* Sanghyun Park Wohlgeruth Pierre

Yumna Piracha Jeffrey A. Pobiner* Lawrence A. Popkin* Victoria Ra Jaclyn Rabadi Ramin Rahmani Pardis Rajabi Veena Ramesh Austin Ramsey Ariyan S. Ravangard Bart M. Rizzuto* Wendy B. Rosen* Olivia Rotondi Jack S. Roth* Amir Saadati Kamyar Sadeghein Anthony Salierno John L. Santopolo* Jerrold Schapiro* Danielle L. Schulman Hilton Z. Segal* Jeffrey S. Senzer* Hoon Sagong John Shamul* Dara A. Siegel Randy N. Simon Lisa Siu Rachel Snyder Charles S. Solomon* Kamolthip Songtrakul Sara Soumeeh Joseph K. Spector* Jadwia Stec Laura C. Steff Jeffrey H. Stein* Joseph C. Stern* Steven M. Stern* Kent D. StrickmanΔ* Prashant Sukhani Steven D. Svetcov* Hess M. Tagouri* Doreen F. Toskos Victoria E. Tountas Lisa Tran* John B. Turco* Nikita Vakil Callie M. Vasilakis* Justin Vice Christopher B. Vivona Navid Vosoughi Leiza Walia Paul D. Weseley* Chris Wood Noboru Yamaki* Joshua Yanover Rabia Yilan Jay Ziffer* Dianna L. Zosche Ante Zovko

North Carolina Silver Sandra Madison*Δ∞ Mary T. PettietteΔ

JOE • Volume 47, Number 9, September 2021

Robert L. Sherman*Δ Diamond D. Gregory Chadwick*Δ Stuart B. Fountain* Linda G. LevinΔ John S. Olmsted* Emerald Hal W. Mohorn*Δ Topaz Ashraf FouadΔ Leslie A. Malueg*Δ William K. Morgan Jr.*Δ George B. ShupingΔ Founder Matthew W. BairdΔ Jessica L. BarrΔ* Curt W. BeachΔ* Richard A. BeaversΔ Jason E. BergmanΔ Thomas K. Buttler* Gregory C. GellΔ* Henry H. Hanco*ck IIIΔ John D. Hartness* Prenard R. MickensΔ Stuart O. Miller*Δ Steven G. Mohorn*Δ Charles G. New*Δ Alessandra R. RitterΔ Gary Russell Sugg*Δ Jennifer TedderΔ Benefactor Gary D. Behrend*Δ Daniel L.Bondra* Kathleen S. BoydΔ Nona I. Breeland*∞ Thomas M. Buttke Joe H. Camp* Steven J. CardΔ* Luis A. Chamorro*Δ Harold I. Coe, Jr.Δ William F. Freccia* Thomas M. Gilbert* Henry H. Hanco*ck*Δ Robert E. Jepko* Seung S. Jung* Glen Ajith Karunanayake Tom J. Kleitches* William M. Kopp* Maria Cristina MarescaΔ Samuel V. Mesaros* Evan N. Miller* James Y. Morris* James M. Musselwhite*∞ Richard J. Pockat* Cameron B. Ritter Christopher L. RoweΔ* Zachary P. Schnoor*Δ Robert SopkoΔ Lisiane Ferreira Susin Jennifer Tedder*Δ

Thank you for your generosity to the Foundation for Endodontics! The Honor Roll represents cumulative giving, not necessarily the amounts of individual donations. Donors move up recognition levels with additional individual gifts and/or pledge commitments. Optional contributions made during the AAE membership process are tallied separately. The Honor Roll goes to print two months prior to arrival of the JOE by USPS mail. We sincerely apologize for any errors or omissions—please email [emailprotected] or call 312-517-2159 about any discrepancies. Eric B. Van Huss* W. Christopher Ward* John D. Wells* Henry M. Wright Jr.*Δ Patron Christopher Ammons Krista Andersen Elizabeth A. Applebaum* Elisa Arnarsdottir Homa Azargoon* Charles M. Beavers* Geraldine R. Bills* Daniel L. Bondra* Erich T. Brewer*Δ Steven A. Brofsky* Carrie L. Burger* Ryan W. Burleson* Thomas M. Buttke*Δ Terry Callison* Linda Chan* Deborah A. Conner* Mark Conrad Harold I. Coe, Jr.* James R. Corcoran Eric Cottle* John P. Crisp* Daniel Crossen Marcus C. Curry Iryna Hryvenko Daline Luke K. Dalzell* Derek J, Duggan Romi M. Farber* J.B. Freedland* James M. Gambill* Gregory C. Gell* James Goglia Alvin S. Goodman* Worth B. Gregory* Robert R. Haglund, Jr. Ethan Hamer E. Flynn Harris*Δ Robert M. Herman* Anthony L. Horalek* Feifei Huang James G. Hupp*Δ Alabbas Hussain Jeffrey D. Hutcheson* Katherine Kuntz Jakuc Keenon L. Johnson Mary Ann Johnson* Thomas L. Jones*Δ Kevin A. Keating* Jennifer E. Key*Δ Asma A. Khan*Δ James W. King II Daniel J. Kolzet* Jason E. Lambert I. Joel Leeb* Eric J. Lensgraf*

David R. Lukosik* William D. Luper*Δ A.K. Bobby Mallik Roger A. McDougal* Robin E. McGurkin-Smith* Michael N. McKee* Prenard R. Mickens* Timothy H. Mihle* E. James Mistak* Michael Mittelsteadt Zackyry Tyler Mohorn Josh C. Morrow William C. Myers* Keith Napolitano* Richard G. Neal* James M. Nelson Achara O’Brien* Nancy Osborne* Jeffrey M. Parker Lesleigh A. Payne Nicholas Pettit John L. Phillips* Eric M. Rivera*Δ J. Christian Sheaffer* Guy Shipper* Dara A. Siegel Asgeir Sigurdsson*Δ Robert Sopko* David L. Spencer* Alison St. Paul Robert J. Stancill* William Stanley* William C. Stiefel* Ramesh K. Sunar* Mark Tadrissi* Tanjit Taggar Peter Z. Tawil* Arya M. Tehrany Aaron J. Thompson My Trinh Tran Ung Aurelia N. Vanderburg Robert C. Verhelle* Stephen C. Wheeler Robert E. Widis*Δ Benjamin J Williams* John M. Williams Andrew Vo Shizuko Yamauchi William Yeung

North Dakota Diamond Jay K. TaylorΔ* Founder Jerry M. CookΔ* Kelli Swenson*Δ

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DONOR ROLL, continued

Benefactor Robert B. Panther*Δ Patron Kent A. Spriggs* Marvin A. Zerr*

Ohio Gold Steven J. Katz*Δ John M. NussteinΔ* Silver Richard A. Menke* Diamond Steven M. Klayman*Δ Andre K. MickelΔ Topaz Rubin GutartsΔ* Phillip L. Michaelson*Δ∞ Founder Anita AminoshariaeΔ* F. Charles Arens*Δ Kent W. Cunningham* Vicki M. Houck*Δ James G. Kotapish, Jr.Δ* James C. Kulild* Harris B. Levine* Alan B. MikesellΔ* Stephen H. MooreΔ Leigh A. R. Pickenpaugh William Robertson* Joseph W. Shiveley*Δ Kerry R. SteinΔ Howard J. Synenberg*Δ - In honor of Dr. Samuel and Dr. Rossman Naija Usman Benefactor J. Jason BigbyΔ David E. Claffey IVΔ* Elizabeth M. Claffey*Δ James A. DeVengencie* Mark E. Dinkins*Δ Melissa M. Drum*Δ Christopher A. Ettrich William E. FosterΔ* John J. Haidet*Δ Lawrence J. HannanΔ Vicki M. Houck* A.L. Christopher Kayafas Navid Khalighinejad George R. LawleyΔ* Philip B. MikesellΔ Thomas A. Montagnese* Kerry D. MooreΔ Matthew S. NiemiecΔ* Isaac Pratt Al W. Reader*Δ Shelley M. Ridenour*Δ

1540

Louis SusiΔ* David B. Swartz* Chandra D. Sykes SmithΔ Robert A. Uhle*Δ David J. Wesley* Michael E. Whitcomb Jr.*Δ Patron Mona Adams Vaishali Agarwala* Tasneem Ahmad Moeen Al Weshah Oday H. Alhalasa Waleed Almutairi Iman Attar Caroline Ghattas Ayub Matthew Balasco* Seth Barnett John Barrett* Jennifer L. Barrord Gerhard A. Berberich*Δ Hannah Beus Stephen A. Billy* Shaletha M. Bolden Eric M. Bramy* Vikram Brar George T. Brown Saadia Bukhari Thomas Burke Aaron Burleson Kenneth B. Chance* Andrew S. Chandler Joseph C. Charnas Lo Shen Chin Stephen J. Clark* Vivian Click Allen D. Colic* Olivia Cook Harvey E. Cooper* Chase Crowley Brian Crump Donald B. Davies* Ryan C. Duval Giselle C. Eitter Nivine El Refai Kerolos Elsayed Grace S. Evans* Sonja Evans Jose J. Ferrari Thomas J. Fogarty* Sara M. Fowler Sanford A. Fox* Michael D. Fuller Jenna Gaw Galen Phil Geraets Brandon N. Glenn Mark C. Gorman* John R. Grady* Anna Maria GuglielmoΔ Joseph A. Haase* A. Todd Halcomb* Andrew J. Haase* Alexis Joy Herring Alven L. Herstig* Stewart A. Hinkley*

Gregory J. Hutchison Jason C. Hyde Zachary Imperial* Marcus D. Johnson Joseph J. Jurcak* Dania Kafri* Arshia Kasravi Russell Kiser II* Matthew Kotapish Timothy Kreimer* Ben Kushnir Sarah Kwang Emily Lammers Gary L. Laukhuf* Joseph T. Lee Lisa Leone Raymond Lev* Matthew V. Lindemann Herbert M. Litton*Δ Ellen MacDonald Daniel Magness Carl L. Marion* Amy Martinkus* Rachel E. Matthews Mayes A. McEntire* Eric R. Menke* Alex Mihailoff* Stephen H. Moore* Jussara M. Morsani James R. Murrin* Gregory S. MyersΔ John W. Myers* William J. Meyers* Ellen G. Nist*Δ Robert A. Nist* Long Nguyen Frederick M. Nuzum David F. O’Connor*Δ John N. Odai* Mark L. Oleson* Dawn M. Ondrik Lindsay A. Pabst Jeffrey B. Pafford Rodney P. Paladino* Morgan Palya Jayesh B. Patel Victor A. Peritore Nelson M. Petrov John J. Plant* Sumesh Potluri Deron J. Reisman Karan J. Replogle* Mina Rizk Douglas N. Robertson Thomas P. Russin* Khalid N. Sahly Maria D. Santos Steffan J. SchererΔ J. Richard Schleder* Scott B. Shellhammer* Michael G. Simpson Richard L. Slate* Steven M. Smith* William E. Smith James Smithson II

updated as of 7/26/2021

Anna E. Smothers Poonam D. Solanki B. Christopher Space* Paul M. Stabile* Alex W. Stamos William D. Stanley Daniel Stentz Christopher A. Thompson* Craig J. Tyler* Michael Uhrich* Peyman Vaziri* Vinson Vig* Patricia S. Walter* Larry K. Wells Jennifer W. Willett* Toby S. Wilson* Dustin M. Wirig Ronald M. Wolf Leigh Yarborough Thomas A. Yonchak* Kathryn J. Younkin* Kevin Younkin*

Oklahoma $500,000 Wm. Ben Johnson*Δ Founder William Lee Beasley*Δ David C. Bird*Δ Myron S. Hilton*Δ Denny W. Southard*Δ Benefactor Victoria BallΔ* David L. Maddox*∞ Michael J. MindiolaΔ James B. Roane* Laurie L. Southard* Jackson L. SullivanΔ Patron Ronald W. Anderson* Richard G. Beatty* Fred W. Benenati*Δ L. Joe Bradley* Nicole B. Chung* David J. Clement* Darrell W. Daugherty* Andrew P. Goldbeck* Edward W. Grimes*Δ Leslie B. Hardy* Spencer P. Hinckley Jeffrey R. Johnson* James E. Leonard* Roger S. Nishimura Jr.* Steven E. Powell David B. Shadid* Amy E. StoneΔ* J. Michael Strand J. Scott Sullins* David R. Woodard*

Oregon Diamond Donna J. Mattscheck* Founder Scott L. Barry Jeffrey A. Dryden*Δ Bill R. Scharwatt* William F. Warren Jr.* Kenneth B. WiltbankΔ Benefactor Cary J. Cunningham*Δ William J. Girsch Jr.* Dennis E. Holt* Michael J. OndoΔ Christine M. SedgleyΔ H. Clayton Stearns* Patron Michael J. Allen* Amir Arad Matthew R. Baumgarth* William J. Beeler* Chris J. Bowman* Paul D. Brent* Wells Brockbank Jarid A. Burley* Richard L. Calhoun* Kevin R. Christiansen* Kelly A. Conlon* Michael T. Dryden Douglas J. Duncan* Daniel J. Erickson* Robert K. Frisk Glen S. Gerdes* Michael C. Hall* Julia A Javarone* Walter R. List* Paul M. Madden* F. James Marshall* Chester V. Mayo Jr. Todd A. Miller Leslie A. Morgan Steven M. Murata* Gregory J. Reams* Dennis E. Reed* Lynn Ross Jim E. Ruckman* Brian J. Strand* Leila Tarsa* Tai Truong* James P. Walker Brian H. Whitten* John Wiens Kathryn C. Wright* Russell S. Yamada* Jon D. Yatsushiro* Tracie M. Zielinski*

Pennsylvania Titanium Louis E. Rossman*

JOE • Volume 47, Number 9, September 2021

Silver Clara M. Spatafore*Δ∞ Diamond Matt E. Hoshino Henry J. Rankow* Samuel Seltzer* Emerald Western Pennsylvania Academy of Endodontics Drs. Ronald Ferrari, Richard Leffel, Andrew Michanowicz, John Michanowicz and Ronald Lindaburg* Topaz Patrick E. DahlkemperΔ Ronald G. Linaburg*Δ Margot T. KusienskiΔ Founder Frederic BarnettΔ Gregory H. Kadel*Δ Frank C. Setzer Martin TropeΔ* Pennsylvania Association of Endodontists Benefactor William A. AdamsΔ* Alan M. Barnett* Patrick J. Carrigan* Jay M. Goldberg*Δ Andrew D. Greenstein* Heidi Ray Monteleone Stephen NiemczykΔ* Robert Saylor and Mark Ritchey* Herbert L. Ray Bruce R. TerryΔ Carl L. Tinkelman* Joshua B. Wolgin Patron Allyson A. Abbott* Edward S. Abrams* M. Reza Akhavan-Sharif Fouad Al-Maki Alaa A. Babeer Julianna Bair Michael J. Barkan* Brian T. Barker Justin W. Batz George E. Biron* Raquel Braga Peter Brothman* Lorel E. Burns Luke Cantamessa Robert A. Caruso Jr.* Prasad Challagulla John J. Charletta*Δ Shilpa S. Chaudhry Hari Priya Chebrolu Zan Chen

Jason C. Cho Brett Cole Serena M. Colletti* Shane Curtis Francine T. Cwyk* Kenneth C. De Nardo* Patrick A. DeRose Nima Dejbod Gary C. Dennis Jr. Jason P. Devey* Matthew D. DiAndreth* Alan M. Ehrenreich* Amanda B. Eidelson Reid S. Elattrache*∞ Ameir Eltour Paulina Erdle* Anas M. Fatayer Adam S. Feuer Robert M. Fleisher*Δ Alexander E. Fuller Adam J. Gatan Nikolaos G. Gavrilis Laura Gecina Saju George Ryan Germann Thomas J. Gillen* Joel M. Glickman* Bruce H. Godick* Michael E. Gonsky* James R. Granite* Terry G. Han Ronald E. Hand* Bryan Hays* Anthony R. Harlacher* Martin D. Hickey*∞ Nathan J. Hinckley* Richard J. Horwat Yu Kai Hsu Craig A. Hurtt* James D. Isett*Δ Austin Jang Joshua R. Jeppson Marc Jiorle* Michelle. C. Jordan Yi-Tai Jou* Maria Jun George M. Just* Renee R. Kalp* Bekir Karabucak* Adnan A. Kazim Shahryar N. Khaliq Mir Khan Daniel D. Kim* Hayoung Kim Jessica S. Kim* Myoung Kim Saehee Kim Sahng Gyoon Kim* Syngcuk Kim* Alan Kirsch* Meetu R. Kohli* Michael Korch* Larry Koren* Paul R. Krasner* Samuel I. Kratchman*

JOE • Volume 47, Number 9, September 2021

Robert M. Krauss* David G. Kuntz* Larisa Kushnir Lyudmila Kuznetsova Haris Lakisic Jason E. Lambert* Kelly R. Lanning Eun Young Lee Kang Lee* Kenneth Lee* Mindo Lee Sodam Lee Richard Leffel* Mark D. Lentz Robert E. Lesniak* Daniel Leung Fred J. Levin* Scott E. Levy Timothy C. Lin* Michael Lisien Jessica J. Liu Swati Malhotra Paul R. Mancia* Janel Marcelino Michael S. Marmo* Antonio J. Marques Salome K. Masrani Lisa M. Matisko* Celia G. McLean* John R. Mellett* Paula A. Mendez Rachel L. Mitrani David D. Moore Craig E. Nixon* Marcus L. Palermo Rinku Parmar Patrick J. Petley* Tanya Reiter Xiomara Y. Rivera Ross Rosenblatt Bruce Sailor* Aaron Salimnia Salar Sanjari Alan R. Schatz Jennifer Schlesinger Gary B. Schultz Gregory M. Semashko* Karla J. Sermeno De Castillo Kevin B. Seto Shaveta Settie Sweta B. Shah Sumei Z. Sharma Kedy Shen Yenshuo Shen Rajesh V. Shenoy Jane Shin Lauren Shin Joseph A. Silvaggio III* Rebecca J. Silver* Jenelle Silvers Gregory M. Smee* John S. Snee Craig B. Soffin Joshua J. Steffen Steven Stein

Puerto Rico Benefactor Patricio J. Sumaza Patron Gabriel Fuentes-Arroyo Raymond Halais* Jose A. Medina* Ronaldo Ramirez Ramos* Luz D. Visbal

Rhode Island Benefactor Edwin S. Mehlman* Patron J. Patrick Morganti* Janet S. Tanzi*

South Carolina Diamond Fred L. SykesΔ* Founder Thomas E. Harvey* Shawn R. Nally* Benefactor Charles E. FriedmanΔ* Bruce T. Howell*Δ Kenneth T. King* Marc E. LevitanΔ Walter R. Long*Δ Michael W. Nimmich*Δ

Geoffery Steinkruger*Δ Robert B. Whitten Gregory B. Wiggins*Δ Patron Omar Abusteit Craig A. Atchison Victoria Ball Nathan E. Beam* Todd R. Barrett* Harry G. Bobotis* Timothy E. Bodey Sr. Aaron Burleson* Chalbourne R. Brasington Christopher T. Carter Keith C. Carver* Jordan M. Christensen Alyana Corden James A. Coward* B. Clark Dalton* Pranav D. Desai* William M. Edwards* E. Stephen Fragale William T. Gillespie* Robert J. Gohean* William N. Gressette Jr.* Wallace L. Guilford* Stephen B. Haas* Mark J. Hauser*Δ Thomas J. Heeren Arash Jahanbakhsh Anthony P. Joyce* Mahmoud Karimipour* Kevin A. Kerley George G. Kitchens Jr.* Jeffrey C. Kotz* Lauren E. Kuhn Scott Loomis Chris M. Maltezos* Rachel Matthews* Justin R. McAbee Katherine M. McKitrick Peter M. Murphy* J. Wade Nichols* Christopher S. Noel* George S. Parsons* Mary B. Ringler* Brad Sleeth John D. Snowden* April Kemp Spitz* Rodney G. Southern Kyan Salehi South Carolina Association of Endodontists*

South Dakota Founder Richard D. Allen*Δ

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DONOR ROLL, continued

Patron Lyde J. Adams Marshall T. Lavin* Nathan W. Schwandt* Andrew Wiswall*

Tennessee Gold Terryl A. PropperΔ* Diamond William D. Powell*∞ Founder B. Keith Elliott* Erik E. JansenΔ William G. Linebarger* F. Graham LockeΔ* John T. McSpadden* Charles A. Scott Jr.* Tennessee Association of EndodontistsΔ Benefactor G. Matthew Brock Carol T. Coffey*Δ Arthur L. Cole* Vince E. HicksΔ A.V. Hill Jr.*Δ Karen R. Kamer* Ben F. Locke Jr.* Edward M. MackΔ* Wallin H. Myers*Δ Sidney C. Roberts* Benjamin D. Scott*Δ James F. Woods*Δ Patron Frank S. Balaban*Δ Jeffrey Bell* Maria W. Bryan* Kevin P. Bryant*Δ Yvette Burns* Christopher W. Cain* Clifton B. Chunn* Robert J. Clayton* Christian L. Culbreath* John E. Davis* Larry Durand*Δ William S. Emery* Mark Freeland* Heather L. Gnau* Tamara C. Gravely Jordan J. Hansen Bradley M. Harris*Δ Elliot Haybarger* Phillip W. Head*Δ Thomas Heerin* John P. Hoover Jr.*Δ Richard Horwat* George T.J. Huang* D. Wayne Hughart* Christopher M. James Margaret Ann Jones*

1542

Bryce F. McCreary* Amir H. Mehrabi*Δ Joseph B. Milholm* Richard E. Moore* Mark E. Morison* Geraldine Navarrete Casey L. Noble* Daniel K. Price* Philip Sherman Jr.* Manuel Sir* Bindu A. Soni John E. Sullivan Jr. Stephanie A. Tran Austin VanDusen Ben Wheeler* Dr. & Mrs. James M. Wilson* John Workman Ronald S. Wright Jr.* Tenn. Acad. of Endodontists*

Texas Gold Kirk A. CouryΔ Samuel O. Dorn*Δ James C. Douthitt*Δ James L. Gutmann*Δ Diamond Gerald N. GlickmanΔ Kenneth M. Hargreaves*Δ John W. Myers* Kurt W. Myers Robert L. Reames*∞ Richard S. Schwartz* Topaz Ron C. HillΔ Founder Robert A. AugsburgerΔ*∞ Stephen O. Cheff* Deborah Creel LothΔ Anibal Diogenes* Michael W. Ford*Δ Julio Gaitan*Δ Robert S. HamiltonΔ* Jason Koh Scott R. MakinsΔ* Katherine E. Olson Triska*Δ Hedley Rakusin*Δ John D. ReganΔ Nikita B. RuparelΔ Karl WoodmanseyΔ Benefactor Basil Al Shaikhly John D. Andrews* Justin E. Aurbach* Karla Ham Bishop* Jose M. Bisquerra Susana M. Bruce* Paul Buxt*Δ Clinio C. CerrudΔ Stephanie Chan

Lindsey Chang Tracy M. ClarkΔ* Christopher L. Coleman* Craig M. Curd*Δ Joanna Davis*Δ Mercedes S. Dominguez*Δ Joy W. FieldΔ* Lee T. Fox*Δ Thomas H. GarrettΔ Shawn D. Gilbert*Δ Brittany Gillard David Gruber* Brad Hajdik* Jenny HeΔ Jeffrey Hoover* Helen L. Joiner*Δ Albert JowidΔ Karl KeiserΔ* Carolyn A. Koenig*Δ John G. Kostohryz* Richardson L. McGuireΔ* Anthony G. McNaught Mahnaz Messkoub* Rita F. Ne Kian Nikdel Katherine E. Olson Triska Terry W. Ott*∞ Jill Peterson*Δ John Reynolds Charles S. Sanford* Paul G. SatchellΔ* Allen P. SchusterΔ Stephen F. Schwartz* Jordan L. SchweitzerΔ* Alan G. Selbst* Kevin Selden* E. Steve Senia* Sandra Shambarger* Renato M. Silva*Δ Joel C. Small* Peter M. Spradling* Fabricio B. Teixeira Jeremy Martin Thompson*Δ Ramona M. Torgerson* Frank B. Trice* William A. Walker III* Sarah Jo Welch* Jeffrey S. Woodson*Δ Carl R. Wright Patron Abdulmajeed Al Shujaa Ahmed A. Alelyani Louis W. Adams III* Chad R. Allen Jason S. Allen Michael J. Allen Riyadh I. Althumairy Dina Ahme AlSharif Samuel J. Angulo* Donald R. Anthony* Sayeed Attar* Obadah N. Austah Murat Ayik* Homa Azargoon

updated as of 7/26/2021

Kendall L. Baginski Cole W. Barnett Christopher F. Bates* Thomas J. Beeson* Antonio Berto Charles D. Bishop Jose M. Bisquerra Bill Bledsoe*Δ William M. Bolak* Percy G. Bolen III Jordan A. Bolles John L. Bond* Raison S. Bose David J. Bowers Robert E. Boynton* Dina Bramipour David R. Bright* Tod T. Bruchmiller* Kirk D. Brown* Nadia Budhani* Angela M. Bullock Shiwei Cai* Johnny G. Cailleteau* Steven D. Calkins* Mark A. Camp* David L. Carnes Jr.* Eric K. Cato* Peter A. Cecic* David A. Cervantes*Δ Robert Chavez Thomas W. Choate* Evan Chugerman Elizabeth A. Chybowski Gavin J. Convey David J. Coon Callee E. Cosby Taylor P. Cotton* Michael E. Crabtree* Kathleen R. Craig* Darrell M. Curtis W. Murray Cutbirth Jr.* Lori Anna Dees* Gary Dennis* Parvin Dinyarian* Kavita Doddamane* John T. Dominici* Jason M. Duggan* J. Brian Duncan* Julius W. Eickenhorst* James A. Elliott* Richard A. Ellis* Michael Eskander Sara Fayazi* Kim Freeman* Ross C. Fruithandler* Jonathan Fu Robert R. Galvan Jr.* Deepika L. Ganne Manish Garala* Alexander Garcia-Godoy Todd M. Geisler Lilley Gharavi James A. Gilles* Ray C. Gillespie* Steven Chad Goodman

Ryan L. Gordon* Terence M. Gordon* Fang Gu* John W. Ham* Jason R. Hansen* John W. Harrison* John R. Hayco*ck William H. Heggen III* William D. Henderson* William T. Henson* Jason L. Hicks Vi C. Ho Paul G. Hobeich* Kimberly G. Holt* Fabian G. Hosein Michael D. Hosking* Xuan V. Huynh* Ryan L. Gordon* Terence M. Gordon* Fang Gu* Brad Hajdik John W. Ham* Jason R. Hansen* John W. Harrison* John R. Hayco*ck Jianing He* William H. Heggen III* William D. Henderson* William T. Henson* Jason L. Hicks Vi C. Ho Paul G. Hobeich* Kimberly G. Holt* Fabian G. Hosein Michael D. Hosking* Xuan V. Huynh* Olga M. Iglesias Michael A. Koch* Paul Kogan George D. Kohout Shab R. Krish* Kevin R. Kunz* Michael C. Larsen* Debora F. Levine* John Loeffelholz* John R. Ludington Jr.* Victor Luikham* Stephen M. Magers* Thomas G. Marino* Warren H. Marrow* David E. Martin* Edwin J. Martin Jr.* Margaret J. Martin* Matthew J. Martin* Jack T. Mayhew* Joseph McFarland Ryan P. McNamara Liliana M. Meeker* Miguel E. Mego* Maria F. Messing Danny K. Miller* Brad Mize Brandi L. Molina Edward M. Moore III* Saeed Bayat Movahed

JOE • Volume 47, Number 9, September 2021

Tuomas Niemi Mbachan Collins Okwen* Le O’Leary* Yoshie Onoe* Christopher Brett Owatz William Pack Panchali Patel Tushar B. Patel* Yogesh T. Patel* Kavita Patil-Doddamane Jimmy Ray Patten* Michael A. Peck* Elineida Perez* Brandon Pitcher Micah L. Porter W. Paul Radman* Kelly A. Ramey* Ahmad Rayyan Walter J. Redmond* Todd W. Remmers* Adrian Reyes Alejandro Rios Ivan E. Rodriguez* John B. Ross* Parastou Rouhani* Kade A. Roundy* Richard E. Rutledge* Sukhpreet Kaur Sandhu Chad M. Sargent* Norman M. Sawyers* William G. Schindler* Scott V. Schlofman* Scott A. Schwartz* Michael J. Schwarze* Dontra B. Scott*Δ Jelana C. Seibold Paulomi R. Shah Jeff T. Shell* Michou T. Shell* Stephen H. Simon Ravisher Singh* Lance J. Skidmore* Jeremy J. Smith* W. Randy Snyder* Eva Stanley* Torsten H. Steinig* Michael H. Stern* Robert P. Stites* Charles H. Stuart* Orsure W. Stokes* Brett M. Strong* John A. Suchina* Michael R. Suhler* Jenny Y. Sun Timothy A. Svec* Koyo Takimoto Esther C. Tam* Matthew Tonioli Ernesto G. Trevino* Carlene Tsai* Mary J. Um Rebeca Weisleder Urow* B. Aaron Vaughn* Adali Velez* Ryan M. Walsh

Glenn R. Walters* Pei Wang Jelani T. Washington Blake E. Wayman* Rebeca Weisleder* Robert A. Welch David J. Weyh Sterling J. Whipple* Robert K. White* Harry V. Whitehill* Brent Winward David E. Witherspoon* K. Paul Wong* Andrew Y. Xu Peter P. Yancich* James H. Yao

Utah Topaz Brent C. Sonnenberg*Δ Founder Robert L. Tayler* Benefactor Wallace B. Brown* Kent Christiansen* John M. Coats* Brad L. Holmes* Randell Madsen Robert S. Richards* Mitchell G. Rudd*Δ Steven G. Starr*Δ Justin D. ThorntonΔ* Patron Val S. Cox* Richard W. Elggren* Sean D. Fessenden* Robert K. Flath* Stuart G. Gibby* Boyd L. JacobsonΔ David G. Johnson* David P. Koelliker*Δ Steven S. Larsen* Steve Montgomery* R. Blake Nielsen Craig C. Smith* Keith D. Sonntag*

Vermont Titanium Cheryl Lang UllmanΔ Benefactor John A. Pane* Victor L. Ratkus* Patron Brooke Blicher* John D. Dresser* Arthur W. Harris* Karen K. Parolin*

JOE • Volume 47, Number 9, September 2021

Ramesh Thondapu

Virgin Islands Patron E. Olutayo Delano*

Virginia Diamond David M. KeneeΔ Bruce W. Overton Emerald Garry L. MyersΔ* Topaz Gary R. Hartwell*Δ Founder Lawrence M. Kotler*Δ Richard D. ArcherΔ Heidi L. Moos*Δ Colleen C. Shull*Δ Benefactor Robert CheronΔ Edward Chun* John S. Ehreth*Δ Tawana D. FeimsterΔ Paige Holbert*Δ Husain A. Karashi R. Denby Lewis* C. Vaughn MayoΔ* Jayesh S. Patel* Sudha P. Patil* Patron Nitin Agrawal Hussameldin M. Ahmed Fawaz Al-Foraih Sarah Alkanderi Lisa Anderson* Matthew T. Ankrum* Eshwar Arasu Jeffrey C. Bailey* Preeti Batra* William E. Bernier Joseph A. Bernier-Rodriguez* Sanjay Bhagchandani Rashin T. Bidgoli Samuel A. Black, Jr.* John D. Bramwell* William F. Bussey* B. Ellen Byrne Frederick L. Canby* James Y.M. Chau* Albert Citron*Δ Paul D. Clark David J. Coon* David M. Craig*Δ Leo V. Crowley* Richard J. Dellork* Matthew Detar William Smith Dodson Jr.*

Thank you for your generosity to the Foundation for Endodontics! The Honor Roll represents cumulative giving, not necessarily the amounts of individual donations. Donors move up recognition levels with additional individual gifts and/or pledge commitments. Optional contributions made during the AAE membership process are tallied separately. The Honor Roll goes to print two months prior to arrival of the JOE by USPS mail. We sincerely apologize for any errors or omissions—please email [emailprotected] or call 312-517-2159 about any discrepancies. Wayne J. Dollard* Jeffrey D. Domark* Virginia N. East* Michael A. Fabio* Erik Foisy Steven G. Forte*Δ Madelyn G. Gambrel* Laura T. Garden Scott E. Gerard* Timothy J. Golian*Δ Richard J. Gray* Megan E. Green Robert E. Grover* Chin-Lo Hahn Jesse B. Harris* Carolyn C. Herring* Robin E. Hinrichs* Michael G. Hunt*Δ Dr. & Mrs. James R. Lance* Leander Lanier Sr.* Sean O. Lawson* Katherine Lee* Gary S. Leff* Richard J. Lieb* Frederick R. Liewehr* Matthew Lloyd Harold J. Martinez* W. Anthony Meares* Kenneth J. Mello* Robert L. Merian* Michael Scott Monts* Margaret M. Mossler* Edward M. O’Keefe* Robert David Pagan* Raymond Pandez Louis C. Peron* Michael V. Piccinino* Frank R. Portell* Dustin S. Reynolds* Sherma R. Saif Adam Sarnowski* Madison W. Saunders Kyle J. Schmidt* Scott Seago* Richard W. Sedwick* Mariah A. Shojaei* Michael S. Smith James L. Stanley*Δ David L. Stepp* Edward J. Strittmatter*Δ Katherine Thomas* Jeffrey R. Thorpe* Michelle K. Toms Ellison P. Turner B. Aaron Vaughn Thomas L. Walker* Lesley A. West* Evan Whitbeck David S. Wozniak* Todd E. Wynkoop*

Washington Platinum Albert C. Goerig*Δ Gold John D. WestΔ* Silver Patrick E. TaylorΔ*∞ Diamond Maureen L. Swift Sean L. WittmerΔ* Emerald Heidi K. KanningΔ Topaz James C. McGraw* Founder Timothy A. BachmanΔ Marshall L. Batchelor*Δ Neil W. Bryant* Billy Don Card Jr.* Shahin Etemadi* Roy N. Kaldestad* I. Blake McKinley Jr.Δ Kathleen Mulligan*Δ Stephen R. Ottosen*Δ Kai Reynolds Jeffrey A. Short*Δ Benefactor Kenji Beppu* Bradley P. Brown*Δ R. John Bull* Allen D. Colic*Δ Thomas G. Davidson* Ryan Duval Lisa A. Ellingsen* Michelle A. Ellingsen* Natasha FlakeΔ Mark A. Freeman*Δ John B. Goessman, Jr.* Frederick L. Gonzales* Neil A. Gray* Michael P. HarwoodΔ Michael W. HueyΔ James D. Johnson* Louis F. Kramp*Δ Edmund H. Kwan*Δ Paul E. Lovdahl* Suzanne R. Megenity* Marcus R. Miller Ardon L. Overby* Don H. Pratten* James W. ReidΔ Shahrzad Sarram*Δ David R. Steiner*

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DONOR ROLL, continued

Timothy L. Sweatman Roderick W. Tataryn* I. Emily Wang*Δ Steve W. WidmanΔ* Scott A.D. Williams*Δ Patron Sami Ali Karim Z. Alibhai* Wallis E. Andelin* Gary A. Backlund* Charles A. Backman* Steven D. Baerg* Steven E. Baker* Linda K. Bascom* Neil Begley*Δ Michael D. Benner* John A. Berude* Diane M. Brighton* Raymond Bogaert Jeanette L. Brandal* Myron L. Brown*Δ F. Chester Burrell* Randall Rex Calvert* Varvara Chrepa* Wayland Chu Matthew S. Clegg* Nestor Cohenca* Nima Dejbod* Hayley Denison Daryl J. Detwiler Kristi R. Detwileri Fergus Duddy Ryan Duval* Paul A. Erben Theron Eichenberger* Daniel Estrada Ali Etemad* Willis P. Gabel* Dustin L. Gatten Scott W. George* Saman R. Gharai* David Goerig* H. George Golden* Michael E. Gonzales Susan Hagel-Bradway* Gerald W. Harrington* Jeremiah J. Hawkins* Norbert L. Hertl* Thomas W. Hooks* Monica Jethwani A. Edward Kim* Timothy S. Kim* Charles L. Kimberly* Dennis H. Kobata* David A. Kosa* Ronald H. Kuritani* Andrew M. JohnsonΔ Eric C.K. Law Adelheid Nerisa Limansubroto Mandy J. Louis* Karen F. Lovato* David M. Macdonald* Matthew A. Mandel*Δ Michael Marcello

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Gerald F. McCann* I. Blake McKinley* Ryan P. McNamara* David S. Mehlhaff* Dennis L. Meidinger* Michael C. Moran Boyd F. Munson* Amanda Y. Ngan Vicky Nguyen Brett J. Nydegger Robert J. Oswald* Tiina Oviir* Avina K. Paranjpe Mary T. Parlee* John M. Patierno* Sidney R. Patten* Joseph J. Pawlusiak*Δ Theodore F. Pilot* Loubna C. Pla* James Reinmiller* Joy A. Rivero* Bart F. Robison* Rajnish Rohila Roman Rossmeisl* Thomas J. Rude* Ronald A. Sabins Jeffrey A. Samyn* Brandon G. Seto Rupinderpal Singh Andrew J. Slisco* Jerald W. Smith* Robert H. Smith* Scott J. Starley* James R. Stephens* Mary L. Stewart*Δ Richard H. Tangen* Bart C. TirrellΔ Orapin Veeraqutthwilai Richard. A. Volwiler* John B. HarwoodΔ Derrick I. Wang* Nancy S.Y. Wang* Jordan C. West Don B. Wilson* Anne E. Wiseman Walter Wittgow* Grace Hsiao Wu* Ryan W. Wynne Jerome K. Yamada* Brandon T. Yamamura* Peter Yamamura Bernard J. Zeldow* Ruvim Zhuk David R. Zielke*

West Virginia Diamond A. Eddy Skidmore* Founder Anthony T. Borgia* L. Keith Hildebrand*Δ Christopher L. Hugh*Δ John K. Kuyk*Δ

Jeffrey G. Minchau*Δ William Douglas RobertsonΔ Benefactor Mark A. Byron* Catherine E. Connor* Jamie M. Day*Δ Lora B. FordΔ Troy L. McGrew*Δ James W. Vargo*Δ Patron Behrouz Beheshtin Daniel Brawley Amanda Boustany* Ryan W. Burleson Gavin L. Criser* Stephen Fragale* Lori L. GochenourΔ* Ryan J. Greene Pamela P. Harrington Sean M. Horan C. Russell Jackson* Sloan G. Lanctot* Mehran Malakpour Kent A. McBride* Travis R. Moore Brian Nalls Lauren P. Namsupak Nathaniel Nicholson Pejman Parsa Tuyen T. Pham* James Poulos Mark D. Richey Douglas N. Robertson* Michelle Romeo Rodney G. Southern* Asmi Shah Marvin L. Speer*Δ Spencer R. Stiles* Michael A. Tran Albert L. Tomsic Jr.*

Wisconsin Diamond Michael Miles* Topaz David Landwehr Founder Eva C. Dahl* Frederick L. KatzΔ David Landwehr* John T. Streiff* Benefactor David W. Belardi* Kendra K. BodaΔ Joseph L. Gaffney* Gregory Haasch* Blair T. Johnson*Δ Thomas G. Langkammer* Michael Mindiola*

updated as of 7/26/2021

John G. Newman*Δ Derek B. Nordeen*Δ Kyle P. Schroeder*Δ Patron B. Peter Austin* James K. Bahcall*Δ Benjamin W. Baker Bruce A. Begotka* Megan Bollman* Joy C. Borszowski* Jacob Burry Mary Ann E. Campbell* Craig R. Carr* S. William Clark* Joseph D. deGuzman* Anne H. Fergus* Douglas Ferris* Benjamin Fravel* Timothy Gainey David J. Gamm* Thomas L. Goddard* David Goerig Chad Hansen Hunter R. Housley Gregory T. Isermann* Andris Jaunberzins*Δ James J. Jesperson* Kenneth J. Jozwiak*Δ Nabeel A. Khan* Kevin P. King*Δ Terry L. Kippa*Δ Richard V. Knoff* Gordon D. Korthals* Robert Lee* Wonhee Lee Allen M. Lepinski* Neill H. Luebke* Andrew R. Lulloff* Scott A. MacDonald George R. Machian*Δ Sushant Mahajan Ryan Margel Maksim Montatskiy David S. Moresi* Thomas O. Mork* Paul W. Moyer* Daniel J. Nencka* F. Kris Olsen* Gary R. Ries* Michael Smoljan Sheila Stover* Igor Sulim Patrick W. White* Thomas J. Witek* Andrew P. Wright* Reid Wycoff Abby E. Yavorek Kandace M. Yee Kenneth L. Zakariasen

Wyoming Topaz R. Cary MeadΔ*

Founder Paul J. Steele*Δ Benefactor Bryan W. Stowe* Ted J. Stowe* Patron Lisa M. Anderson Bryant W. Stowe*

International Brazil Benefactor Quintiliano De Deus, in memoriam by Ben B. Maze Patron Luiz Magalhães, in memoriam by Ben B. Maze

Canada Founder D. Wayne Acheson*∞ Douglas W. Conn*Δ Hugh Maguire*Δ Benefactor Howard K. Bittner* Rene ChuΔ Shimon FriedmanΔ Gary Glassman* Carl E. Hawrish* Manjinder S. Lalh* Julie LeducΔ* Mark A. Olesen*Δ Marshall D. Peikoff* Calvin G. PikeΔ W.C. Fred Weinstein* Patron Nawfal Al-Hashimi Emanuel Alvaro* Glenn L. Anderson* Kourosh Asgari Normand Aubre* Deborah E. Battrum* Craig Bellamy Alexander M. Brown* Rebecca Chan Jason T. Conn* Dominic Cote* Theodore Damas* Luis A. DaSilva* Shaul M. Dwosh* Howard M. Fogel* John G. Fraser* Manfred Friedman* Raphael Garofalo* Gerald J. Gass* Michael Gossack* Marie Gosselin*

JOE • Volume 47, Number 9, September 2021

Jacqueline Lopez Gross Dan-Linh Ha* David C. Hall David Harris Richard F. Hunter* Brian D. Jafine Fadi Kano* Christian B. Kecht Abhisheic Kirpal Gillian Landzberg Wing Man Wendy Lai Eric C. Law* Alice Li Helena Sooyeon Lim Gevik Malkhassian* Muna Marashdeh Patrick J. McCarthy* Alex G.R. McLean* Timothy J. McManus*Δ Brent Moore Robert D.S. Munce* Bobby Nadeau Georgia Nikoloudaki Babak Nurbakhsh* Christopher Owatz* Hiran Perinpanayagam* Aldo del Carpio Perocherna Simona Pesun*Δ Alysen Phee* Myrto Piperidou Keith Plain* Jonathan Rapp* G.R. Rastegar* Robyn H. Reiter* Alexander Sevo* Annie Shrestha Deborah M. Szabo Christine Teed Michael Tiedemann Calvin D. Torneck* Charles Tra Stephen L. Walker Maria E. Waslen* Caithlin Williams-Beecher

Australia William A. Kahler

Colombia Patron Sociedad de Endodoncia de Bogota

Denmark Patron Jens Ove Andreasen

England Patron B. David Cohen* Alessandro Falanga

Anthony E. Hoskinson*

Germany Patron Oliver Pontius

Greece Patron Spyridon Floratos*

Hong Kong Patron David Wong

Japan Patron Tetsuro & Kazuko Kawaguchi*

Kuwait Patron Faisal A. Amir Hadi Faras* Mohammed N. Mohammed

Mexico Patron Jose Hernandez-Mejia*

Saudi Arabia Patron Abdulaziz AbuMelha* Hussein R. Mokhlis

Spain Benefactor Asociacion Española de Endodoncias*

Ukraine Patron Andriy Polovinshchykov

Gifts Alaska Association of Endodontists Alliance of the American Association of Endodontists Associates in Endodontics Augusta Endodontic Study Club Michael D. CarterΔ Lynn B. Crookston Allan S. Deutsch Matthew D. Evans Scott Fehrs

JOE • Volume 47, Number 9, September 2021

Ian B. Glick Herbert N. GutentagΔ Alyson Hall Edon Y. Hirt* Illinois Association of Endodontists Iowa Association of Endodontists Matthew L. Kjar Brian S. Kunz Chris W Lee Jack Levi Brian J. Licari Jeffrey W. Linden Fay Mansouri Richard Mautner & Steven Oppenheimer Richard E. Mounce New York State Association of Endodontists Allison G. Ritch-Glick Steven H. Sanders J. Richard Schleder Michele A. Scrime Troy S. Thomson U.S. Navy Association of Endodontists Clinton E. Weaver Jack A. Weichman Eric Weinstock West Virginia Association of Endodontists Kenneth J. WidelkaΔ* D. Wayne Acheson Jessica L. Barr G. Matthew Brock Brian P. Chuang Douglas Conn Mark Desrosiers Mark Doherty John Dolbec Christopher D. Dorr Cami E. Ferris-Wong Fredric Goodman John Thomas Hanco*ck Yanliang Jiang Akbar Khayat Alvin A. Krakow Roger R. Lacoste Harold Levin Ben F. Locke Jr. F. Graham Locke Mahnaz Messkoub Peter A. Morgan Thomas Ollerhead Terrell F. Pannkuk James Penney Robert J. Rosenkranz & Judith E. Rosenkranz Natalie Shlosman Douglas W. Stewart Paul B. Talkov Cheryl L. Ullman Ian Watson John D. West

Thank you for your generosity to the Foundation for Endodontics! The Honor Roll represents cumulative giving, not necessarily the amounts of individual donations. Donors move up recognition levels with additional individual gifts and/or pledge commitments. Optional contributions made during the AAE membership process are tallied separately. The Honor Roll goes to print two months prior to arrival of the JOE by USPS mail. We sincerely apologize for any errors or omissions—please email [emailprotected] or call 312-517-2159 about any discrepancies. Susan Wood In honor of Mentor Dr. John W. Harrison Kathryn Jurosky In honor of Dr. Linda Levin Terryl A. Propper In honor of Dr. Sandra Madison William Powell Kenneth Sunshine In honor of Dr. Peter Morgan Harold J. Levin Terryl A. Propper Dr. and Mrs. Louis and Val Rossman Yuri Shamritsky Fiza Singh In honor of Dr. John Olmsted, the 2021 Edgar D. Coolidge Award winner Sandra Madison In honor of Dr. Mary Pettiette Dr. and Mrs. Louis and Val Rossman In honor of Dr. William Powell’s 2013 President’s Award David C. and Rebecca Funderburk In honor of Dr. Terryl A. Propper David C. and Rebecca Funderburk John S. Olmsted In honor of AAE Foundation President Dr. Louis E. Rossman Mary T. Pettiette Terryl A. Propper In honor of Drs. Samuel Rossman and Louis Rossman Howard J. Synenberg In honor of AAE Foundation President Dr. A. Eddy Skidmore Frank S. Balaban Mark A. Byron Kimberly A. Dettori Kenneth M. Hargreaves Gary R. Hartwell L. Keith Hildebrand James G. Kotapish Jr. James C. Kulild Ralph W. Niemann William D. Powell Martha E. Proctor Clara M. Spatafore Kenneth P. Sunshine West Virginia Association of Endodontists

In honor of Dr. A. Eddy Skidmore’s 2014 Edgard D. Coolidge Award David C. and Rebecca Funderburk Gary R. Hartwell Sandra Madison Harvey Matheny Kenneth Sunshine In honor of Dr. Clara Spatafore Weld County Dental Society Sandra Madison In honor of Dr. Patrick E. Taylor, Peter A. Morgan Boston University graduating class of 1999 in honor of Dr. & Mrs. Herbert Schilder Allan & Janet Jacobs in honor of 2005 Edgar D. Coolidge Award Winner Philip W. Cohen Emily Rothenberg in honor of friend Dr. Marc Balson Massachusetts Association of Endodontists in tribute to Dr. Stephen Niemczyk Jessica Barr & Sandra Madison in honor of Ms. GorgAnna Randolph E.K. Pomerantz-Miller in honor of Dr. Louis E. Rossman Sandra Madison in honor of Dr. Jessica Barr’s Board Certification James F. Wolcott in honor of Dr. Susan L. Wood’s Board Certification Sandra Madison in honor of Dr. William T. Johnson, Dr. Louis E. Rossman, Dr. Keith Krell and Dr. A. Eddy Skidmore In honor of Phase Two Associates, LLC Sandra Madison

Memorials In memory of Wayne Alley Larry R. Faraskian Judith Steinberg, David Laks & Family Frank J. Woolman In memory of Don Arena Sandra Madison In memory of Joseph and Mary Bartling Alyssa Bartling Daniel Bartling

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DONOR ROLL, continued

Justina Bartling Matthew Bartling Sheryl L. Dando Kimberly A. Dettori Clara Spatafore and Phil Bartling In memory of I.B. Bender Edward M. O’Keefe In memory of Richard C. Burns L. Stephen Buchanan Noah Chivian Michael & Barbara Heuer Irma Kudo Sandra Madison North Texas Endodontic Associates Glenn& Beverly Paulk Jerome V. Pisano Cherilyn G. Sheets Denis E. Simon III* Joel C. Small Robert A. Uchin Van Strom & Towne, Inc. Franklin S. Weine David Witherspoon In memory of Jack Cochran Sandra Madison In memory of Philip W. Cohen Canadian Academy of Endodontics Noah Chivian Dr. & Mrs. Joel Dunsky Allan & Janet Jacobs Edwin S. Mehlman Mark A Oliver In memory of Alice J. Cooper American Association of Endodontists AAE Board of Directors AAE Foundation Board of Trustees JOE Editorial Board In memory of Mrs. Anita Cunningham, wife of Dr. Charles Cunningham Sandra Madison In memory of Marietta Dermody Louis and Val Rossman In memory of Robert L. Ellison Rodrick L. Barden Fred L. Sykes In memory of Alfred L. Frank Samuel O. Dorn John S. Olmsted In memory of Jacob B. Freedland William F. Freccia In memory of Dudley Glick John S. Olmsted Charles L. Siroky In memory of Carole Goldstein

1546

Dr. & Mrs. Louis & Val Rossman Dr. Clara Spatafore Dr. Kenneth and Ms. Shelley Zucker In memory of Fred Gossack Arnold H. Gartner In memory of Carl S. Haga Joseph Wells In memory of Robert Hamilton Department of Endodontics at Texas A&M University Baylor College of Dentistry In memory of Rosalind Hirsh Louis and Val Rossman In memory of Col. Michael J. Hutter Jr. Jessica L. Barr Samuel O. Dorn Kenneth M. Hargreaves Sandra Madison John S. Olmsted Denis E. Simon III In memory of Wilma M. Hutter Jessica L. Barr Sandra Madison Denis E. Simon III In memory of Mian K. Iqbal American Association of Endodontists In memory of Dr. Ray Kauffman Dr. and Mrs. Louis & Val Rossman In memory of Tom Kennedy’s Son, Steven Kennedy Mary T. Pettiette Terryl A. Propper Louis E. Rossman In memory of John Madonia Alan T. Azar Gary W. Brankin In memory of Tony S. Madison Kenneth Hargreaves Louis Rossman Clara Spatafore In memory of Jay Marlin Massachusetts Association of Endodontists Peter Morgan Deborah G. Roher In memory of Seymour Oliet Noah Chivian Louis and Val Rossman In memory of the mother of Dr. John S. Olmsted Louis E. and Val Rossman In memory of Edward M. Osetek Martha Proctor In memory of Josie Propper Bruce Cha David Funderburk

Kenneth Hargreaves Gary R. Hartwell Craig Hirschberg Steven Katz Keith Krell Mark Laramore Linda Levin Sandra Madison Maria Maranga Peter Morgan John Olmsted Mary Pettiette Louis & Val Rossman Richard Rubin Hank Schiffman Patrick Taylor Stefan Zweig In memory of Drs. Herbert Schilder & Robert Matusow Kevin L. Peterson Raina A. Trilokekar Lisa M. Wendell In memory of Charles A. Scott Jr. Kevin P. Bryant Stokely Doster Jr. Louise & David Lane Joe L. Mosier In memory of Charles L. Siroky Noah Chivian In memory of Joe Robert Spatafore Alyssa Bartling Daniel Bartling Justina Bartling Matthew Bartling Sheryl L. Dando Kimberly A. Dettori John S. Olmsted Denis E. Simon III Clara Spatafore and Phil Bartling In memory of Dr. Barbara Steinberg’s mother Louis and Val Rossman In memory of Besharat Torabinejad Kenneth Hargreaves Clara Spatafore John Olmsted Denis Simon In memory of Arnold Zuroff Joel L. Dunsky

Friends From the Industry Individuals Diamond Mark Oliver* Tulsa Dental Products Michael StoneΔ* Schick Technologies Founder William E. Newell* Tulsa Dental Products Russell Vanderslice* Tulsa Dental Products John Voskuil DENTSPLY Tulsa Dental Specialties Benefactor Mark LaramoreΔ Pacific Dental Services, Inc. Patron Jack Burlison* Brasseler USA Tom Kennedy* PBS Endo Arnaldo & Jennifer Castellucci* Quality Dent

Founder Yvonne Simon, in memoriam* Benefactor Edward M. Osetek, in memoriam*

updated as of 7/26/2021

JOE • Volume 47, Number 9, September 2021