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Journa C A L I F O R N I A

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November 2021 Dentofacial Orthodontic Treatment Oral and Maxillofacial Reconstruction Zirconia Abrasion

A S S O C I AT I O N

THE 20 th ANNIVERSARY

of Dental Stem Cells Rungnapa Yang Warotayanont, DDS, PhD

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Vol 49    Nº 11


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Nov. 2021

C D A J O U R N A L , V O L 4 9 , Nº 11

d e pa r t m e n t s

667 The Editor/Why My Staff Loves CDA 669

Impressions

715 RM Matters/Romantic Relationships With Patients: Your Obligation as the Employer

717 Regulatory Compliance/Regulated Waste Management 720 Tech Trends

f e at u r e s

673 The 20th Anniversary of Dental Stem Cells An introduction to the issue.

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Rungnapa Yang Warotayanont, DDS, PhD

677 Stem Cells and Dentofacial Orthodontic Treatment Potential This article is focused on the discovery of stem cells and their potential application in dentofacial orthodontic treatment. Phimon Atsawasuwan, DDS, MSc, MSc, MS, PhD C.E. Credit

685 Advances in Tissue Engineering and Implications for Oral and Maxillofacial Reconstruction This article is a review on tissue engineering strategies applicable to specialists who treat oral and maxillofacial defects. Caitlyn M. McGue, DDS; Victoria A. Mañón, DDS; and Chi T. Viet, DDS, MD, PhD

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Pre-Sintering Airborne Particle Abrasion Improves Surface and Biological Properties of Zirconia The present study aimed to investigate the effect of airborne-particle abrasion of pre-sintered zirconia on surface properties and biological performance of the resulting fully sintered zirconia. Tanjira Kueakulkangwanphol, DDS; Nichamon Chaianant, DDS, PhD; Yanee Tantilertanant, DDS, PhD; and Weerachai Singhatanadgit, DDS, PhD

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Journa C A L I F O R N I A

published by the California Dental Association 1201 K St., 14th Floor Sacramento, CA 95814 800.232.7645 cda.org

CDA Officers Judee Tippett-Whyte, DDS President president@cda.org Ariane R. Terlet, DDS President-Elect presidentelect@cda.org John L. Blake, DDS Vice President vicepresident@cda.org Carliza Marcos, DDS Secretary secretary@cda.org Steven J. Kend, DDS Treasurer treasurer@cda.org Debra S. Finney, MS, DDS Speaker of the House speaker@cda.org Richard J. Nagy, DDS Immediate Past President pastpresident@cda.org

D E N TA L

Management Peter A. DuBois Executive Director Carrie E. Gordon Chief Strategy Officer Alicia Malaby Communications Director

Editorial Kerry K. Carney, DDS, CDE Editor-in-Chief Kerry.Carney@cda.org Ruchi K. Sahota, DDS, CDE Associate Editor Brian K. Shue, DDS, CDE Associate Editor Gayle Mathe, RDH Senior Editor Rungnapa Yang Warotayanont, DDS, PhD Guest Editor

Volume 49 Number 11 November 2021

A S S O C I AT I O N

Jack F. Conley, DDS Editor Emeritus

Permission and Reprints

Journal of the California Dental Association Editorial Board

Robert E. Horseman, DDS Humorist Emeritus

Andrea LaMattina, CDE Publications Manager Andrea.LaMattina@cda.org 916.554.5950

Charles N. Bertolami, DDS, DMedSc, Herman Robert Fox dean, NYU College of Dentistry, New York

Production Shelly Peppel Senior Visual Designer

Manuscript Submissions

Upcoming Topics

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December/ Oral Health Literacy II January/ Dental Student Research February/ Post-Pandemic Assessment

Letters to the Editor www.editorialmanager. com/jcaldentassoc

Advertising Sue Gardner Advertising Sales Sue.Gardner@cda.org 916.554.4952

Andrea LaMattina, CDE Publications Manager Kristi Parker Johnson Communications Manager Blake Ellington Tech Trends Editor

The Journal of the California Dental Association (ISSN 1942-4396) is published monthly by the California Dental Association, 1201 K St., 14th Floor, Sacramento, CA 95814, 916.554.5950. The California Dental Association holds the copyright for all articles and artwork published herein.

Steven W. Friedrichsen, DDS, professor and dean, Western University of Health Sciences College of Dental Medicine, Pomona, Calif. Mina Habibian, DMD, MSc, PhD, associate professor of clinical dentistry, Herman Ostrow School of Dentistry of USC, Los Angeles Robert Handysides, DDS, dean and associate professor, department of endodontics, Loma Linda University School of Dentistry, Loma Linda, Calif. Bradley Henson, DDS, PhD , associate dean for research and biomedical sciences and associate professor, Western University of Health Sciences College of Dental Medicine, Pomona, Calif. Paul Krebsbach, DDS, PhD, dean and professor, section of periodontics, University of California, Los Angeles, School of Dentistry Jayanth Kumar, DDS, MPH, state dental director, Sacramento, Calif. Lucinda J. Lyon, BSDH, DDS, EdD, associate dean, oral health education, University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco Nader A. Nadershahi, DDS, MBA, EdD, dean, University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco Francisco Ramos-Gomez, DDS, MS, MPH, professor, section of pediatric dentistry and director, UCLA Center for Children’s Oral Health, University of California, Los Angeles, School of Dentistry Michael Reddy, DMD, DMSc, dean, University of California, San Francisco, School of Dentistry

The Journal of the California Dental Association is published under the supervision of CDA’s editorial staff. Neither the editorial staff, the editor, nor the association are responsible for any expression of opinion or statement of fact, all of which are published solely on the authority of the author whose name is indicated. The association reserves the right to illustrate, reduce, revise or reject any manuscript submitted. Articles are considered for publication on condition that they are contributed solely to the Journal of the California Dental Association. The association does not assume liability for the content of advertisements, nor do advertisements constitute endorsement or approval of advertised products or services.

Avishai Sadan, DMD, dean, Herman Ostrow School of Dentistry of USC, Los Angeles

Copyright 2021 by the California Dental Association. All rights reserved.

Brian J. Swann, DDS, MPH, chief, oral health services, Cambridge Health Alliance; assistant professor, oral health policy and epidemiology, Harvard School of Dental Medicine, Boston

Visit cda.org/journal for the Journal of the California Dental Association’s policies and procedures, author instructions and aims and scope statement.

Connect to the CDA community by following and sharing on social channels

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Harold Slavkin, DDS, dean and professor emeritus, division of biomedical sciences, Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry of USC, Los Angeles

Richard W. Valachovic, DMD, MPH, president emeritus, American Dental Education Association, Washington, D.C.


Editorial

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Why My Staff Loves CDA Kerry K. Carney, DDS, CDE

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he other day, I walked in to ask if there was anything the front desk staff members needed of me. There was a whirlwind of activity in the intake area. The office manager was trying to help a new patient register and her assistant was on the phone with another patient. The new patient preferred a health history questionnaire in Spanish. Because we do not keep much in the way of paper records anymore, my office manager did not have a hard copy at hand. She sat down at her computer and within two minutes she had logged on and signed in to the cda.org website, navigated to Practice Support and located and printed a Spanish version of our health history questionnaire. As the hardcopy was printing, she turned to me and said with a satisfied smile, “I love CDA.” I have related this sentiment to CDA staff members and they were overjoyed. During this pandemic, the Practice Support staff has taken an increased number of calls. The calls are not always pleasant or full of praise. There are the callers who complain that they sent in their CDA dues and are now getting a notice that their license has expired. (They confused the dental board with their professional organization.) The Practice Support staff has received calls that demanded to know why CDA closed down their offices. (They misunderstood and thought that CDA was the source of governmental directives regarding shelter-at-home and concomitant business restrictions). Agitated callers wanted to know why CDA decreed that their assistants, hygienists and dentists have to wear

When friends and family were panicking and fearful, my staff could rely on the information provided by CDA to become ambassadors of good science.

specific personal protective equipment and respirators that require annual fit tests. (They confused CDA with OSHA.) There have been callers whose feelings of anger and loss of control were so heated and misdirected that they threatened to sue CDA over the fact that they have to get vaccinated or comply with regular, frequent COVID-19 testing. (They conflate the state executive’s duties and directives with CDA’s Practice Support information and COVID-19 education.) These examples spring from the uncertainties of these times, but over the years, Practice Support has received calls about all sorts of issues that are out of their control. Members have called requesting documentation that would excuse them from jury duty. The Practice Support staff has been asked to give legal advice about specific aspects of practice sales. Frequently, the staff has been commanded to “force” dental benefit companies to increase their reimbursement levels. Even when they explain that forcing third parties to treat dentists better is beyond their scope, the callers still insist they should do something about the low reimbursements they are receiving. Members request legal, tax and financial advice about the advantages of incorporating or the purchase or sale of

real estate. The staff has even been asked to produce copies of a member’s fictitious name permit. These wide ranging and misdirected questions should be interpreted as a compliment. Our members think of their professional organization first when they need answers, any kind of answers. Practice Support has been helping California dentists in service to their patients since 2009. They provide consultative advice and guidance. They are experts in managing dental practices. As my staff can testify, Practice Support provides excellent materials to help our practices run smoothly. During this pandemic, the most frequently requested resources have been COVID-19-related regulatory compliance materials: ■  FAQs regarding State Public Health Orders on Required Vaccination or Testing of Health Care Workers ■  Religious Accommodation Request Form — COVID-19 Vaccination ■  COVID-19 Mandatory Policy Instructions for Employers and Sample Mandatory Vaccination Policy ■  Vaccine Confidence Toolkit ■  State Reopening Guide My staff found the “Back to Work” video extremely helpful. When friends and family were panicking and fearful, N OVEMBER 2 0 2 1

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my staff could rely on the information provided by CDA to become ambassadors of good science and educate others outside the dental field on logical and accepted safety precautions. Before the disruptive circumstances of the last two years, the most frequently requested materials were still related to regulatory compliance. (Anyone who has tried to parse their way through government-supplied regulatory guidelines understands why this is the case.) Here are just a few additional materials that can be handy for any practice: ■  Dental Benefits Issue Submission Form — the easiest way for members to get the help they need with dental plans. ■  New Employee Hiring and Onboarding Checklist ■  Starting a Dental Practice ■  Are You in Compliance? When Practice Support was established, the idea was that through Practice Support, CDA could act as

a trusted, supportive partner to help us all succeed in providing excellent care to our patients. It is no wonder that we look there first for all questions related to dentistry. And when those questions fall within the scope of practice management, the staff at Practice Support do everything they can to provide the appropriate information. So, the next time you get some helpful advice or materials from a Practice Support adviser, you might remember the wide range of inquiries they field each day. You might communicate a bit of kindness, a grateful acknowledgement of their excellent performance and let them know their work has made our work a little easier. n

The Journal welcomes letters We reserve the right to edit all communications. Letters should discuss an item published in the Journal within the last two months or matters of general interest to our readership. Letters must be no more than 500 words and cite no more than five references. No illustrations will be accepted. Letters should be submitted at editorialmanager.com/ jcaldentassoc. By sending the letter, the author certifies that neither the letter nor one with substantially similar content under the writer’s authorship has been published or is being considered for publication elsewhere, and the author acknowledges and agrees that the letter and all rights with regard to the letter become the property of CDA.

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Impressions

C D A J O U R N A L , V O L 4 9 , Nº 11

New Dental Implant Fights Bacteria

“The novel implant would implement two key technologies: a nanoparticleinfused material that resists bacterial colonization and an embedded light source to conduct phototherapy.” (Image: Courtesy of Albert Kim.)

Researchers at the University of Pennsylvania School of Dental Medicine have developed an innovative new dental implant that could help fight local inflammation and periodontitis, which often lead to implants needing to be replaced sooner than the implants were designed to last. The novel implant would implement two key technologies: a nanoparticle-infused material that resists bacterial colonization and an embedded light source to conduct phototherapy, powered by the natural motions of the mouth, such as chewing or toothbrushing. Geelsu Hwang, BS, PHD, an assistant professor in the University of Pennsylvania School of Dental Medicine, and colleagues presented their platform for the new implant in a paper in the journal ACS Applied Materials & Interfaces. The research could one day be integrated not only into dental implants but other technologies, such as joint replacements as well, according to the authors. “Phototherapy can address a diverse set of health issues,” Dr. Hwang said. “But once a biomaterial is implanted, it’s not practical to replace or recharge a battery. We are using a piezoelectric material, which can generate electrical power from natural oral motions to supply a light that can conduct phototherapy, and we find that it can successfully protect gingival tissue from bacterial challenge.” The material the researchers explored was barium titanate (BTO), which has piezoelectric properties that are leveraged in applications such as capacitators and transistors but has not yet been explored as a foundation for anti-infectious implantable biomaterials. To test its potential as a dental implant foundation, the team first used discs embedded with nanoparticles of BTO and exposed them to Streptococcus mutans. They found that the discs resisted biofilm formation in a dose-dependent manner. Discs with higher concentrations of BTO were better at preventing biofilms from binding. While earlier studies had suggested that BTO might kill bacteria outright using reactive oxygen species generated by light-catalyzed or electric polarization reactions, Dr. Hwang and colleagues did not find this to be the case due to the short-lived efficacy and off-target effects of these approaches. Instead, the material generates enhanced negative surface charge that repels the negatively charged cell walls of bacteria. The researchers say it’s likely this repulsion effect would be long-lasting. The power-generating property of the material was sustained and in tests over time the material did not leach. It also demonstrated a level of mechanical strength comparable to other materials used in dental applications. Finally, the material did not harm normal gingival tissue in the researchers’ experiments, supporting the idea that it could be used without ill effect in the mouth. Learn more about this research in ACS Applied Materials & Interfaces (2021); dx.doi.org/10.1021/acsami.1c11791. n

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Porphyromonas gingivalis bacteria, 3D illustration.

Identifying Genetic Risk Factors for Tooth Loss A group of researchers from Okayama University, Japan, have revealed insightful findings that could provide new directions to the treatment strategies for periodontitis. The study, published in the International Journal of Environment and Public Health Research, focuses on understanding the microbes associated with the presence of periodontitis and the host genetic factors that might facilitate the development of the conditions. Because the physiology of an individual directly affects the development of infection and genetic differences among hosts contribute to differences in susceptibility to specific pathogens and the chance of developing certain diseases, the researchers conducted a cross-sectional study in which they genotypically analyzed 14,539 participants and conducted saliva sampling of 385 individuals. They finally retained 22 individuals for statistical analysis, and based on their periodontal status, divided them into “periodontitis” and “control” groups. The team found that the “β-diversity” of the microbes, which refers to the ratio between regional and local species diversity, was significantly different between the periodontitis and control groups. Furthermore, they attributed the presence of the bacteria species, P. gingivalis, and the bacterial families, Lactobacillaceae and Desulfobulbaceae, to periodontitis. In contrast, they found no relation between genetic polymorphism and periodontitis. Taking these inferences into account, the team concluded that a person’s 670 N OVEMBER

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Research Finds Low Rates of Dental Fluoride Varnish Treatment for Children Fewer than 5% of well-child visits for privately insured young children included a recommended dental fluoride varnish application, despite mandatory insurance coverage for this service, according to a University of Massachusetts Amherst study. The new research, published Aug. 30 in the JAMA Network Open, was the first to assess delivery of this evidence-based service recommended by the U.S. Preventive Task Force and the American Academy of Pediatrics for privately insured children. Previous research showed that fewer than 8% of 1- to 5-year-olds covered by Medicaid receive fluoride varnish in medical settings. The study is part of a larger project in Massachusetts that will delve into more complex questions, such as why medical providers aren’t applying fluoride varnish during well-child visits. The preventive treatment is especially critical in light of the statistic that fewer than 1 in 3 children under age 5 have an annual dentist visit, where this service also could be provided. The research team examined data from 2016–2018 for privately insured young children in Connecticut, Maine, New Hampshire and Rhode Island. The sample included 328,661 well-child visits in the four states. Fluoride varnish application was more common among visits for younger children. A 2-year-old was nearly 8 percentage points more likely to receive fluoride varnish than a 5-year-old, an analysis of the data showed. Fluoride varnish applications were most common in Rhode Island, with a regression-adjusted probability of 8.7%. New Hampshire had the lowest rate, with a regression-adjusted probability of 2.2%. Co-author and pediatrician Sarah Goff, MD, associate professor of health policy and management at UMass Amherst, pointed to one “hopeful takeaway” from the study: The regression-adjusted probability of fluoride varnish application increased from 3.6% in 2016 to 5.8% in 2018. “That’s still really low, but it did go up over time,” she said. Learn more about this study in the JAMA Network Open (2021); dx.doi.org/10.1001/ jamanetworkopen.2021.22953.

oral microbiome affects the status of periodontitis more than their genes. The research team surmised that because the prevalence of periodontitis is associated with members of the microbiome rather than the genetic identity of an individual, clinicians will be motivated to pay more attention to

microbiome composition than to host factors in the routine work of periodontal examination and will design customized treatment strategies for periodontists. Read more of this study in the International Journal of Environment and Public Health Research (2021); dx.doi.org/10.3390/ijerph18126430.


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55-Million-Year-Old Fossils Reveal Oldest Known Mammal Caries A new University of Toronto study has discovered the oldest known caries ever found in a mammal, the likely result of a diet that included eating fruit. The caries were discovered in fossils of Microsyops latidens, a pointy-snouted animal no bigger than a racoon that

was part of a group of mammals known as stem primates. It walked the earth for about 500,000 years before going extinct around 54 million years ago. “These fossils were sitting around for 54 million years and a lot can happen in that time,” said Keegan Selig, PhD,

New Gel Whitens Teeth Without the Burn Researchers from Sichuan University, Chengdu, China, have developed a gel that, when exposed to near infrared (NIR) light, safely whitens teeth without the burn caused by high levels of hydrogen peroxide in dentists’ bleaching treatments. The study was published in the journal ACS Applied Materials & Interfaces. When a bleaching gel is applied to teeth, hydrogen peroxide and peroxidederived reactive oxygen species (mainly the hydroxyl radical) degrade pigments in stains. The hydroxyl radical is much better at doing this than hydrogen peroxide itself, so researchers have tried to improve the bleaching capacity of low-concentration hydrogen peroxide by boosting the generation of powerful hydroxyl radicals. The research team wanted to develop a safe, effective whitening gel containing a catalyst that when exposed to NIR light would convert low levels of hydrogen peroxide into abundant hydroxyl radicals. The researchers made oxygen-deficient titania nanoparticles that catalyzed hydroxyl radical production from hydrogen peroxide. Exposing the nanoparticles to NIR light increased their catalytic activity, allowing them to completely bleach tooth samples stained with orange dye, tea or red dye within two hours. Then, the researchers made a gel containing the nanoparticles, a carbomer gel and 12% hydrogen peroxide. They applied it to naturally stained tooth samples and treated them with NIR light for an hour. The gel bleached teeth just as well as a popular tooth whitening gel containing 40% hydrogen peroxide, with less damage to enamel. The nanoparticle system is highly promising for tooth bleaching and could also be extended to other biomedical applications, such as developing antibacterial materials, according to the researchers. Read more of this study in ACS Applied Materials & Interfaces (2021); dx.doi. org/10.1021/acsami.1c06774. Photographs of tea-stained teeth after bleaching for 0, 0.5, 1.5 and 3.5 h, respectively. HP + NIR group: 12% HP under 0.2 W/cm2 NIR; BT45/HP + NIR group: 2 mg/mL BT45 plus 12% HP under 0.2 W/cm2 NIR. These photographs are successive images of the same tooth. (Credit: Reprinted with permission from ACS Publications. Copyright 2021 American Chemical Society.)

Micro-CT reconstruction of (A) the cranium of the extant treeshrew Tupaia gracilis (AMNH 103620) and (B) the right upper jaw fragment (P3–M3) of M. latidens (USGS 17748) with carious lesions. (Credit: Selig K, et al. Licensed under Creative Commons CC BY-NC 4.0.)

lead author of the study. “I think most people assumed these holes were some kind of damage that happened over time, but they always occurred in the same part of the tooth and consistently had this smooth, rounded curve to them.” Very few fossils of M. latidens’ body have been found, but a large sample of fossilized teeth have been unearthed over the years in Wyoming’s Southern Bighorn Basin. While they were first dug up in the 1970s and have been studied extensively since, Dr. Selig is the first to identify the little holes in their teeth as being cavities. For the research, published in the journal Scientific Reports, Dr. Selig looked at the fossilized teeth of a thousand individuals under a microscope and was able to identify caries in 77 of them. To verify the results, he also did micro-CT scans on some of the fossils. The study, which received funding from the Natural Sciences and Engineering Research Council of Canada (NSERC), not only includes the largest and earliest known sample of caries in an extinct mammal, it also offers some clues into how the diet of M. Latidens changed over time. It also offers a framework to help researchers look for caries in the fossils of other extinct mammals. “It might be surprising to some that cavities are not a modern phenomenon, and they certainly are not unique to only humans,” Dr. Selig said. Read more of this study in Scientific Reports (2021); dx.doi.org/10.1038/ s41598-021-95330-x. N OVEMBER 2 0 2 1

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introduction C D A J O U R N A L , V O L 4 9 , Nº 11

The 20th Anniversary of Dental Stem Cells Rungnapa Yang Warotayanont, DDS, PhD

GUEST EDITOR Rungnapa Yang Warotayanont, DDS, PhD, received her dental degree from Chulalongkorn University in Bangkok and completed her doctoral degree at the Herman Ostrow School of Dentistry of USC with a focus on craniofacial molecular biology. Her interest in stem cell research began at USC, and she has worked with Dr. Songtao Shi, the pediatric dentist scientist who discovered dental pulp stem cells. Her research was in enamel matrix protein as well as the characterization of dental pulp stem cells. Dr. Yang continued her specialty training in pediatric dentistry at the University of California, San Francisco, and continued her research focusing on the clinical translation aspect of dental stem cells. Dr. Yang is a board-certified pediatric dentist and practices in Mountain View, Calif. Conflict of Interest Disclosure: None reported.

S

ince the outbreak of COVID-19, our dental offices have focused on new office safety protocols, having all personal protective equipment ready, installing new air purifier systems and buying extraoral high-velocity suction, while checking on availability of the vaccination and getting vaccinated. As scientists and clinicians work toward finding the effective treatment and cure for COVID-19, little did we, the dentists, know that the cells isolated from extracted teeth in our dental offices could be one of the most powerful therapeutic approaches to treat COVID-19 patients.1 Stem cells were discovered in tooth tissue over two decades ago. Dental pulp stem cells (DPSCs) were the first dental stem cells identified in human permanent teeth and primary teeth.2 These cells are characterized as stem cells because they possess the ability to self-renew and to differentiate into cells of various lineages. Stem cells from orofacial tissue have subsequently been identified in many other sites such as the periodontal ligament, gingival tissue, apical papilla and alveolar tissue. We can even find stem cells in an exfoliated deciduous tooth.3

Stem cells isolated from the oral cavity are derived from the cranial neural crest. These stem cells share many characteristics with mesenchymal stem cells (MSCs) isolated from other parts of the body, such as bone marrow and adipose tissue. Of particular interest, DPSCs have been extensively studied and characterized due to their advantages over other MSCs in many aspects. DPSCs are easily accessible in extracted teeth, have favorable regenerative property, possess unique angioneurogenic potential, have high proliferative ability and are anti-inflammatory. For medical applications, DPSCs are a prime candidate for cell-based therapy of many cardiovascular conditions including stroke, myocardial infarction and spinal cord injury.4 More recently, DPSCs have emerged as a potential treatment option for COVID-19. In recent clinical trials, the cells were injected intravenously in COVID-19 patients and showed promising outcomes, as they were shown to deregulate cytokine and promote regeneration of damaged lung tissue.1,5 There are increasing numbers of clinical trials for evaluation of the therapeutic property of various stem N OVEMBER 2 0 2 1

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cells for dentofacial restoration. Human deciduous pulp stem cells implanted into necrotic pulp showed continued root development and pulp regeneration with formation of blood and nerve tissue compared to traditional apexification treatment.6 Small blood stem cells as well as human adipose tissuederived MSCs were used to accelerate osteointegration in implant patients.7,8 As there are various categories of stem cells combining with a variety of clinical medical and dental applications, for this issue of the Journal, we invited practicing clinicians and scientists to give us a comprehensive review on their perspective of stem cells related to their scope of practice. The article from Dr. Phimon Atsawasuwan at the University of Illinois Chicago highlights the potential use of stem cells in orthodontics. The combination of stem cells and bone grafting was shown to increase biocompatibility for treatment of craniofacial anomalies. Stem cells injected into the rapid maxillary expansion site contributed to increased osteoclastic activity and accelerated rapid maxillary expansion. Transplantation of stem cells was shown to promote growth of the periodontal ligament after surgery. The article prepared by Dr. Chi T. Viet and colleagues at Loma Linda University provides a comprehensive review of the potential use of stem cell therapy to enhance oral and maxillofacial reconstruction. Research and strategies to enhance bone regeneration around implant tissue, a comparison of the autologous graft versus the stem cell-engineered graft, the regeneration of nerve tissue, the importance of vascular supply in the grafted tissue and the limitation of stem cell therapy in oral maxillofacial constructions are among the topics discussed. The response of stem cells to the modified zirconia surface of dental implants was assessed in the original 674 N OVEMBER

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research by Dr. Weerachai Singhatanadgit and colleagues at the Thammasat University in Thailand. The group studied approaches to modify the surface of a dental implant to optimize clinical outcome. Modification of a fully sintered zirconia dental implant surface can be problematic due to its high strength and bio-inert surface. The study demonstrated that well-optimized pre-sintering airborne-particle abrasion of a zirconia dental implant can render a highly roughened and hydrophilic surface of the fully sintered zirconia. The resulting zirconia showed an increased fibronectinto-albumin adsorption ratio and mesenchymal stem cell adhesion, therefore increasing a favorable clinical outcome. This technique may be suitable for modifying a zirconia surface to facilitate osseointegration of zirconiabased dental implants. The way we practice dentistry remains largely the same compared to many years, decades, if not a century ago. The treatment options offered to our patients remain focused on the use of synthetic materials to restore form and function of the lost craniofacial structures. Practical clinical application of the use of stem cells still faces many challenges. The major obstacles will be the cost of cell retrieval, expansion and maintenance. Given that our teeth do not regenerate as enamel-forming cells, the ameloblasts, undergo apoptosis at the completion of enamel formation, the concept of whole tooth tissue regeneration remains challenging. The questions that remain unanswered are the number of required stem cells, the length required for tooth development after stem cell implantation, how to achieve the correct size and shape of the tooth and how to prevent teratogenic formation, which is one of the major downsides of the use of stem cells.

The tremendous advancement in stem cell research gives us hope that stem cell-based therapy will become a viable treatment option and provide promising benefits compared to traditional dentistry. n RE F E RE N C E S 1. Zayed M, Iohara K. Immunomodulation and regeneration properties of dental pulp stem cells: A potential therapy to treat coronavirus disease 2019. Cell Transplant 2020 Jan–Dec;29:963689720952089. doi: 10.1177/0963689720952089. PMID: 32830527; PMCID: PMCPMC7443577. 2. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000 Dec 5;97(25):13625– 30. doi: 10.1073/pnas.240309797. PMID: 11087820; PMCID: PMCPMC17626. 3. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: Stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 2003 May 13;100(10):5807–12. doi: 10.1073/pnas.0937635100. Epub 2003 Apr 25. PMID: 12716973; PMCID: PMCPMC156282. 4. Yamada Y, Nakamura-Yamada S, Kusano K, Baba S. Clinical potential and current progress of dental pulp stem cells for various systemic diseases in regenerative medicine: A concise review. Int J Mol Sci 2019 Mar 6;20(5). doi: 10.3390/ijms20051132. PMID: 30845639; PMCID: PMCPMC6429131. 5. Ye Q, Wang H, Xia X, Zhou C, Liu Z, Xia ZE, et al. Safety and efficacy assessment of allogeneic human dental pulp stem cells to treat patients with severe COVID-19: Structured summary of a study protocol for a randomized controlled trial (Phase I/II). Trials 2020 Jun;21(1):520. doi: 10.1186/ s13063-020-04380-5. PMID: 32532356; PMCID: PMCPMC7290137. 6. Xuan K, Li B, Guo H, Sun W, Kou X, He X, et al. Deciduous autologous tooth stem cells regenerate dental pulp after implantation into injured teeth. Sci Transl Med 2018 Aug 22;10(455)eaaf3227. doi: 10.1126/scitranslmed.aaf3227. PMID: 30135248. 7. Feng SW, Su YH, Lin YK, Wu YC, Huang YH, Yang FH, et al. Small blood stem cells for enhancing early osseointegration formation on dental implants: A human phase I safety study. Stem Cell Res Ther 2021 Jul 2;12(1):380. doi: 10.1186/ s13287-021-02461-z. PMID: 34215319; PMCID: PMCPMC8254299. 8. Tzur E, Ben-David D, Gur Barzilai M, Rozen N, Meretzki S. Safety and efficacy results of BonoFill first-in-human, phase I/IIa clinical trial for the maxillofacial indication of sinus augmentation and mandibular bone void filling. J Oral Maxillofac Surg 2021 Apr;79(4):787–98 e2. doi: 10.1016/j. joms.2020.12.010. Epub 202 Dec 17. PMID: 33434518.


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dentofacial treatment C D A J O U R N A L , V O L 4 9 , Nº 11

Stem Cells and Dentofacial Orthodontic Treatment Potential Phimon Atsawasuwan, DDS, MSc, MSc, MS, PhD

abstract Background: In recent years, stem cell therapy has become a very promising and advanced scientific research topic. With advanced technology in tissue engineering, the development of this approach has evolved with great expectations. Methods: This article is focused on the discovery of stem cells and their potential application in dentofacial orthodontic treatment. For craniofacial deformities, stem cell-based therapy has been applied as a part of a tissue engineering approach to regenerate bone and tissues to reconstruct the deformities. With a limitation of tissue defects, several stem cells could be great candidates for the treatment. Conclusions: Stem cell-based therapy could be applied for TMJ regeneration with great potential for the development of scaffold regenerative materials. Current evidence demonstrates the potential of stem cellbased therapy for regenerate cementum, collagen and alveolar bone for the benefit of accelerated tooth movement, increased envelope of discrepancy and improved posttreatment stability. Practical implications: However, there is a need for more in vivo and clinical trials for stem cell-based therapy to be used as a standard treatment. Keywords: Stem cells, dentofacial, orthodontic, tissue engineering

AUTHOR Phimon Atsawasuwan, DDS, MSc, MSc, MS, PhD, is a tenured associate professor in the department of orthodontics at the University of Illinois Chicago and a diplomate of the American Board of Orthodontics. His research interest is focused on accelerated tooth movement, craniofacial/ bone biology, microRNA and advances in

orthodontic treatment. Dr. Atsawasuwan’s research involves clinical research, bench-top molecular biology research, cell culture and animal research. Conflict of Interest Disclosure: None reported.

D

entofacial orthodontic treatment involves treatment of dental malocclusion, dentofacial deformities and developmental anomaly. Orthodontic problems can affect several oral functions such as chewing, biting, swallowing and speaking and create abnormal habits such as tongue thrust and mouth breathing.1 On several occasions, the problems are coincident with several developmental deformities and impact dentofacial aesthetics,

psychosocial self-confidence and quality of life.2–5 The worldwide prevalence of malocclusion is 56% without difference in sex, with varying severity in different parts of the world.6 The prevalence of malocclusion was found to be higher and more severe in individuals with intellectual disabilities.7 The prevalence of craniofacial anomalies and jaw deformities is less frequent compared to dental malocclusion, but the treatment is more challenging and needs multi- and N OVEMBER 2 0 2 1

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Stem cells

Fetal stem cells (FSCs)

Embryonic stem cells (ESCs)

Adult stem cells (ASCs)

Totipotent

Pluripotent

Multipotent

Oligopotent

Unipotent

Whole organism

All tissues except whole organism

Several tissues

Certain tissues

One tissue

FIGURE 1. Classification of stem cells.

interdisciplinary approaches.8 Many approaches to these conditions involve regenerative medicine and stem cell therapy. This review article describes the applications of stem cells in the treatment of dentofacial orthodontic treatment. Stem cells are undifferentiated cells of a multicellular organism that can differentiate into various types of cells under suitable conditions and have the ability of self-renewal.9,10 Stem cells exist in embryonic, fetal and adult (somatic) stem cells. Developmental potency is very high in embryonic stem cells and is reduced with each step during the development. Stem cells can be classified in the following ways.11 Totipotent stem cells are able to proliferate and differentiate into cells of the whole organism. They have the highest differentiation potential for the cells to form both embryo and extraembryonic structures. Pluripotent stem cells (PSCs) form cells of all germ layers but not embryonic structures. The pluripotency starts from completely pluripotent cells and ends on representatives with less potency 678 N OVEMBER

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as multi-, oligo- or unipotent cells. PSCs can be derived from embryonic or fetal stem cells; however, in certain conditions, it could be derived from adult cells via the introduction of specific genes.12 Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can be driven to differentiate into specific cell lineages. These cells can become oligopotent cells and later differentiate into unipotent cells. Oligopotent stem cells can differentiate to several cell types while unipotent stem cells have limited differentiation capabilities and a unique capability to divide themselves continuously (FIGURE 1 ). Somatic or adult stem cells are undifferentiated and are located among differentiated cells in the whole organism during development. These cells have limited ranges of differentiation and their functions are to replace the dying cells and promote regeneration, healing and growth of tissues. The stem cells derived from somatic stem cells have limited

differentiation potential and tend to be unipotent stem cells and differentiate to a specific type of tissue such as skin, nerve or bone marrow (FIGURE 2 ). Induced pluripotent stem cells (iPSCs) are the somatic cells that achieve reverse pluripotency by forcing the expression of octamer-binding transcription factor (Oct4), sex-determining region Y (SOX2), Krüppel-like factor 4 (KLF4) and Myc genes encoding transcriptional factor. These processes convert somatic cells into pluripotent stem cells.12 The application of stem cells for dentofacial orthodontic treatment emphasized the regenerative potential aspect of stem cells. Several stem cells such as mesenchymal stem cells have been used in combination with grafting material for regenerative tissue engineering in several treatments such as cleft lip/palate augmentation, distraction osteogenesis, rapid palatal expansion and temporomandibular disorders.13 Many procedures involve bone grafting to enhance the posttreatment stability and provide a scaffold for regenerative


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Umbilical cord blood Hematopoietic stem cells (HSCs) Bone marrow Mesenchymal stem cells (HSCs) Adult/somatic stem cells (ASCs)

Skin stem cells

Fat tissue

Dental/periodontal tissue

Keratinocytes Neural stem cells Oligodendrocytes/ astrocytes Induced pluripotent stem cells (iPSCs)

+Oct4, Sox2, Klf4, Myc

FIGURE 2 . Differentiation potentials of somatic/adult stem cells.

cells and new vascularity in the surgical or treatment area. Until now, adult stem cells from postnatal tissues have great potentials for clinical application due to several restrictions on embryonic stem cells including legal issues. Adult stem cells can be isolated from several tissues including the gastrointestinal tract,14 skeletal muscle,15 central nervous system,16 adipose tissue17 and neural crest derived tissues such as dental/periodontal tissues.18,19 The adult bone marrow shelters various stem cells including hematopoietic (HSCs)20 and mesenchymal stem cells (MSCs).21 The process of stem cells-based therapy includes isolation, selection, testing and delivery of the stem cells to the surgical sites with a bioresorbable carrier or injection as shown in FIGURE 3 . Currently in the United States, the only stem cell-based products approved by the U.S. Food and Drug Administration (FDA) are hematopoietic progenitor

cells (HPC) derived from cord blood.22 However, several cell-based therapies have been approved by the FDA such as autologous CAR-positive viable T cells (Breyanzi, Kymriah, Tecartus and Yescarta) for treatment of B-cell lymphoma, autologous fibroblast intradermal injection (laViv) for treatment of nasolabial fold wrinkles, autologous cultured chondrocytes on porcine collagen membrane (Maci) for regeneration of knee cartilage defects and autologous CD54+ cells activated with PAP­GM-CSF (Provenge) for treatment of certain types of prostate cancers. The cell-based therapy for intraoral use is Gintuit, the allogeneic cultured human keratinocytes and dermal fibroblasts in bovine Type I collagen for the treatment of mucogingival conditions such as gaining gingival keratinized tissue.22 These cell-based therapies will provide a navigation for the development of stem cells-based therapy in the future.

Application in Treatment of Dentofacial/Craniofacial Deformities

These conditions encompass many different defective developmental conditions that cause the malformation of complete structures of the face and head. The most common conditions include cleft lip/palate (CLP) and craniosynostosis syndromes. These conditions require multi- and interdisciplinary treatment approaches that involve pediatricians, genetic physicians, geneticists/genetic counselors, plastic/craniofacial surgeons, orthodontists, pediatric dentists, prosthodontists, speech therapists, nurses, psychologists and social workers. CLP is one of the most prevalent congenital craniofacial deformities. CLP occurs in approximately 1 per 940 births,23 and 80% of the orofacial cleft are nonsyndromic and of multifactorial genetic and environmental origin. Craniosynostosis syndromes occur when N OVEMBER 2 0 2 1

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Delivery of stem cells with scaffolds

Selection for niche of stem cells

Extraction of stem cells from skeletons, muscles, adipose tissues, teeth or periodontal ligament

FIGURE 3 . Process of stem cells-based therapy.

one or more of the sutures in a baby’s skull closes too early. Children who have this condition might have an abnormal skull shape, forehead shape and asymmetrical eyes and/or ears. This condition occurs in approximately 1 per 2,000 births. Many cases of these conditions are of syndromic involvement while some cases are nonsyndromic. The treatment of these craniofacial deformities involves reconstruction of the craniofacial defects including autogenous bone grafts, allogeneic material and prosthetic compounds such as metals and polymers.24–26 Distraction osteogenesis 680 N OVEMBER

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has been widely applied in orthopedic surgery for correction in the treatment of several craniofacial deformities.27 While increasingly utilized as endogenous bone tissue engineering, distraction osteogenesis could involve postoperative complications such as infection, scarring device failure and nonunion healing in up to 35% of cases.28,29 The combination of stem cells with bone grafting for craniofacial reconstruction may alleviate these limitations due to its potential for regeneration for the tissues. The need for biocompatible scaffolds for the cells is under investigation due to the size

of deformities such as palatal fissures.30 Another concern is the number of stem cells for a large defect such as alveolar or palatal cleft. The bone marrow MSCs have been used in animal models, such as the dog model, and have demonstrated promising outcomes.31,32 Bone marrow MSCs have been evaluated in a case report using a patient-specific, 3D printed bioresorbable polycaprolactone scaffold with autogenous bone marrow stromal cells collected from the iliac crest. The report showed 45% defect regeneration six months after transplantation, with a 75% bone mineral density compared to


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the surrounding bone.33 Adipose-derived MSCs are another good candidate for stem cell-based therapy for CLP reconstruction due to their availability and easy handling. Stem cells from human exfoliated deciduous teeth are also good candidates for CLP reconstruction because they are less invasive to obtain and have higher accessibility; however, the number of stem cells could be a concern. Animal studies showed that these stem cells have the ability to repair the cleft site effectively and could be a good alternative for bone regeneration.34–36 A recent elegant study from the University of Southern California reported the approach using a combination of biodegradable grafting material with MSCs to reverse craniosynostosis phenotypes and sustain calvarial bones and suture homeostasis of Twist1+/– mice.37 A human-based report discussed how stem cell-based therapy using stem cells and blood from an umbilical cord and placenta injection into the surgical sites was used during a rhinocheiloplasty procedure. All CLP patients were followed up for two years, and the outcome showed a smaller alveolar cleft and improved maxillary alignment; however, there was no evidence of osseous development.38 A clinical trial evaluated mandibular regeneration using bone-derived MSCs for a patient with severe posterior mandibular ridge atrophy. The MSCs and biphasic calcium phosphate granules were used as scaffolds and were inserted subperiosteally onto the resorbed alveolar ridge. The results were assessed 12 months after the procedure; the bone augmentation of the posterior mandibular was successful for all patients.39 A randomized controlled feasibility trial was performed to investigate the treatment of jaw bone defects using a mixture of CD90+ MSCs and CD14+ monocytes/macrophages and mononuclear cells from bone marrow. The

treatment with MSCs accelerated alveolar bone regeneration and reduced the need for secondary bone grafting compared to conventional treatment.40 The same group of authors further studied the potential of the mixture of CD90+ MSC cells and CD14+ monocyte/macrophages in maxillary sinus bone regeneration and found greater regenerative effects in these cells.41 A study reported how autologous MSCs that were expanded in the laboratory were injected back into the patients’ mandibular surgical sites during the consolidation phase

Rapid maxillary expansion is an approach used to correct the constricted maxilla in certain patients.

after distraction osteogenesis surgery that demonstrated favorable treatment outcomes.42 Another study showed that grafting a combination of autologous deciduous dental pulp stem cells (DDPSC) and a hydroxyapatite-collagen sponge in alveolar bone defects in a group of CLP patients revealed satisfactory bone healing without significant complication after a six-month follow-up.43

Application in Treatment of Temporomandibular Disorders

Temporomandibular disorders (TMD) manifest their symptoms as pain, myalgia, headaches and structural destruction, collectively known as degenerative joint disease.44 The prevalence of TMD in the U.S. is about 5%.45 The temporomandibular joint (TMJ)

comprises both osseous and cartilaginous structures and, like other synovial joints, is also prone to rheumatoid arthritis, injuries and congenital anomalies.44 The severe form of TMD necessitates surgical replacement of the TMJ.46 The complications of a surgical replacement of the TMJ include infection, implant wear, dislocation, donor site limitation and morbidity.47 The application of stem cells for the treatment of TMJ replacement, including the delivery of stem cells in the existing defective TMJ structure, is used to promote tissue regeneration,48–53 to introduce stem cells growth factors and biomolecules into the degenerative sites54–57 and to incorporate stem cells with injectable polymers to form a scaffold with the cells.58–60 The first human case reported was performed by injecting in vitro expanded autologous MSC cells from nasal septum into the patient’s degenerative TMJ by arthrocentesis. After six months, computed tomography images showed new cortical bone formation and partial repair of condylar and temporal bones.61

Application in Orthodontic Treatment Rapid Maxillary Expansion

Rapid maxillary expansion is an approach used to correct the constricted maxilla in certain patients. The action on the maxilla is similar to distraction osteogenesis either with or without surgical interventions. A preclinical study in rats showed that local injection of MSCs labeled with green fluorescent dye into an intermaxillary suture after activation of an expansion screw resulted in increased new bone formation in the suture by increasing the number of osteoclasts and new blood vessels compared to the control group.62 In turn, the cells isolated from midpalatal sutures of mice could exhibit MSC markers, CD73, CD90, CD105 and N OVEMBER 2 0 2 1

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Sca-1 after exposure to cyclic tensile force for two hours.63 This implicates that the stem cell-based therapeutic approach could potentially benefit the rapid maxillary expansion and may help the posttreatment stability for bone regeneration after the expansion.

Surgically Facilitated Orthodontic Therapy

In 1994, Proffit and Ackerman first highlighted the importance of “the envelope of discrepancy” and how it portrays the range limitations of the maxillary and mandibular teeth during orthodontic treatment (inner envelope), orthodontic treatment combined with growth modification (middle envelope) and orthognathic surgery (outer envelope).64 If the movement violates beyond the envelope of discrepancy, the dental and periodontal health of the patient may be compromised. With a shallow osteotomy, with shallow perforations or cuts made on the cortical alveolar bone only, the trabecular bone is left intact; with bone grafting, the envelope of discrepancy could be expanded for the orthodontic treatment and the rate of tooth movement could be accelerated.65 As stated previously, MSCs could be used in combination with grafting materials to enhance bone regeneration as well as trophic mediator secretion to promote tissue regeneration around the tooth after movement. In addition to the rate of tooth movement being accelerated, this implicates the increased stability after surgically facilitated orthodontic therapy as well.66

Regeneration of Periodontal Tissue

Gingival recession or bony dehiscence could be unwanted consequences after orthodontic treatment. Interdental papilla recession after alignment of the crowded teeth is a major posttreatment 682 N OVEMBER

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concern. The solution to improve the “black triangle” is to remove the enamel thickness to squeeze the soft tissue between the teeth. This approach has limitations due to the thickness of enamel on the tooth, and it could harm the intact structure of enamel if the interdental papilla loss is excessive or the thickness of the enamel is limited. If periodontal ligament derived stem cells (PDSCs) could be used to regenerate cementum or periodontal bone, the alternative approach to gain the interdental papilla could be used. Several studies evaluated

If the movement violates beyond the envelope of discrepancy, the dental and periodontal health of the patient may be compromised.

the potential of human PDSCs to induce cementum regeneration by transplanting PDSCs into athymic rat models. Human PDSCs demonstrated their capability to regenerate cementum,67 bone and collagen fibers.68,69

Conclusion

The current literature revealed potential applications of stem cells in tissue engineering therapy for dentofacial orthodontic treatment. These stem cellbased therapies could be stem cells alone as cells and trophic factors for regeneration or in combination with regenerative scaffolds in the defect of the deformities. In addition, stem cell-based therapy could improve the treatment outcomes and duration as well as posttreatment stability. However, most of the evidence

has shown in vitro and stem cell-based therapy to be expensive. In addition, the stem cell niche will be an important factor to consider when delivering stem cellbased therapies. More in vivo or clinical trials are required to assess the possibility of these innovative interventions. n AC KN OW L E DG M E N T This work was supported by the University of Illinois Chicago, Brodie Craniofacial Endowment fund and Biomedical Research Awards from the American Association of Orthodontists Foundation and DE024531, the National Institute of Dental and Craniofacial Research, National Institute of Health. RE F E RE N C E S 1. Grippaudo C, Paolantonio EG, Antonini G, et al. Association between oral habits, mouth breathing and malocclusion. Acta Otorhinolaryngol Ital 2016 Oct;36(5):386–394. doi: 10.14639/0392-100X-770. 2. Dos Santos PR, Meneghim MC, Ambrosano GM, Filho MV, Vedovello SA. Influence of quality of life, self-perception and self-esteem on orthodontic treatment need. Am J Orthod Dentofacial Orthop 2017 Jan;151(1):143–147. doi: 10.1016/j.ajodo.2016.06.028. 3. Kiyak HA. Does orthodontic treatment affect patients’ quality of life? J Dent Educ 2008 Aug;72(8):886–94. 4. Silvola AS, Varimo M, Tolvanen M, et al. Dental esthetics and quality of life in adults with severe malocclusion before and after treatment. Angle Orthod 2014 Jul;84(4):594–9. doi: 10.2319/060213-417.1. Epub 2013 Dec 5. 5. Dalle H, Vedovello SAS, Degan VV, et al. Malocclusion, facial and psychological predictors of quality of life in adolescents. Community Dent Health 2019 Nov 28;36(4):298–302. doi: 10.1922/CDH_4633Dalle05. 6. Lombardo G, Vena F, Negri P, et al. Worldwide prevalence of malocclusion in the different stages of dentition: A systematic review and meta-analysis. Eur J Paediatr Dent 2020 Jun;21(2):115–122. doi: 10.23804/ejpd.2020.21.02.05. 7. Cabrita JP, Bizarra MF, Graca SR. Prevalence of malocclusion in individuals with and without intellectual disability: A comparative study. Spec Care Dentist 2017 Jul;37(4):181–186. doi: 10.1111/scd.12224. Epub 2017 Jun 9. 8. Salzmann JA. Editorial: Seriously handicapping orthodontic conditions. Am J Orthod 1976 Sep;70(3):329–30. doi: 10.1016/0002-9416(76)90340-7. 9. Atala AL, et al. Handbook of Stem Cells. 2nd ed. San Diego: Academic Press; 2013. 10. Parker GC, Anastassova-Kristeva M, Broxmeyer HE, et al. Stem cells: Shibboleths of development. Stem Cells Dev 2004 Dec;13(6):579–84. doi: 10.1089/scd.2004.13.579. 11. Zakrzewski W, Dobrzynski M, Szymonowicz M, Rybak Z. Stem cells: Past, present and future. Stem Cell Res Ther 2019 Feb 26;10(1):68. doi: 10.1186/s13287-019-1165-5. 12. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006 Aug 25;126(4):663–76. doi: 10.1016/j.cell.2006.07.024. Epub 2006 Aug 10. 13. Safari S, Mahdian A, Motamedian SR. Applications of


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stem cells in orthodontics and dentofacial orthopedics: Current trends and future perspectives. World J Stem Cells 2018 Jun 26;10(6):66–77. doi: 10.4252/wjsc.v10.i6.66. 14. Slack JM. Stem cells in epithelial tissues. Science 2000 Feb 25;287(5457):1431–3. doi: 10.1126/ science.287.5457.1431. 15. Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004 Jan;84(1):209–38. doi: 10.1152/physrev.00019.2003. 16. Daniela F, Vescovi AL, Bottai D. The stem cells as a potential treatment for neurodegeneration. Methods Mol Biol 2007;399:199–213. doi: 10.1007/978-1-59745-504-6_14. 17. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002 Dec;13(12):4279–95. doi: 10.1091/mbc.e02-02-0105. 18. Mitsiadis TA, Feki A, Papaccio G, Caton J. Dental pulp stem cells, niches and notch signaling in tooth injury. Adv Dent Res 2011 Jul;23(3):275–9. doi: 10.1177/0022034511405386. 19. Tomokiyo A, Wada N, Maeda H. Periodontal ligament stem cells: Regenerative potency in periodontium. Stem Cells Dev 2019 Aug 1;28(15):974–985. doi: 10.1089/ scd.2019.0031. 20. Spangrude GJ, Smith L, Uchida N, et al. Mouse hematopoietic stem cells. Blood 1991 Sep 15;78(6):1395– 402. 21. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997 Apr 4;276(5309):71–4. doi: 10.1126/science.276.5309.71. 22. U.S. Food and Drug Administration. Approved cellular and gene therapy products. March 02, 2021 ed. www.fda. gov/vaccines-blood-biologics/cellular-gene-therapy-products. Accessed March 12, 2021. 23. Parker SE, Mai CT, Canfield MA, et al. Updated National Birth Prevalence estimates for selected birth defects in the United States, 2004–2006. Birth Defects Res A Clin Mol Teratol 2010 Dec;88(12):1008–16. doi: 10.1002/ bdra.20735. Epub 2010 Sep 28. 24. Rah DK. Art of replacing craniofacial bone defects. Yonsei Med J 2000 Dec;41(6):756–65. doi: 10.3349/ ymj.2000.41.6.756. 25. Bruens ML, Pieterman H, de Wijn JR, Vaandrager JM. Porous polymethylmethacrylate as bone substitute in the craniofacial area. J Craniofac Surg 2003 Jan;14(1):63–8. doi: 10.1097/00001665-200301000-00011. 26. Shenaq SM. Reconstruction of complex cranial and craniofacial defects utilizing iliac crest-internal oblique microsurgical free flap. Microsurgery 1988;9(2):154–8. doi: 10.1002/micr.1920090218. 27. McCarthy JG, Stelnicki EJ, Mehrara BJ, Longaker MT. Distraction osteogenesis of the craniofacial skeleton. Plast Reconstr Surg 2001 Jun;107(7):1812–27. doi: 10.1097/00006534-200106000-00029. 28. McCarthy JG, Schreiber J, Karp N, Thorne CH, Grayson BH. Lengthening the human mandible by gradual distraction. Plast Reconstr Surg 1992 Jan;89(1):1–8; discussion 9–10. 29. Mofid MM, Manson PN, Robertson BC, et al. Craniofacial distraction osteogenesis: A review of 3278 cases. Plast Reconstr Surg 2001 Oct;108(5):1103–14; discussion 11157. doi: 10.1097/00006534-200110000-00001. 30. Nyberg EL, Farris AL, Hung BP, et al. 3D-printing technologies for craniofacial rehabilitation, reconstruction and regeneration. Ann Biomed Eng 2017 Jan;45(1):45–57. doi:

10.1007/s10439-016-1668-5. Epub 2016 Jun 13. 31. Yoshioka M, Tanimoto K, Tanne Y, et al. Bone regeneration in artificial jaw cleft by use of carbonated hydroxyapatite particles and mesenchymal stem cells derived from iliac bone. Int J Dent 2012;2012:352510. doi: 10.1155/2012/352510. Epub 2012 Mar 26. 32. Tanimoto K, Sumi K, Yoshioka M, et al. Experimental tooth movement into new bone area regenerated by use of bone marrow-derived mesenchymal stem cells. Cleft Palate Craniofac J 2015 Jul;52(4):386–94. doi: 10.1597/12-232. Epub 2013 Jun 19. 33. Ahn G, Lee JS, Yun WS, Shim JH, Lee UL. Cleft alveolus reconstruction using a three-dimensional printed bioresorbable scaffold with human bone marrow cells. J Craniofac Surg 2018 Oct;29(7):1880–1883. doi: 10.1097/ SCS.0000000000004747. 34. Pourebrahim N, Hashemibeni B, Shahnaseri S, et al. A comparison of tissue-engineered bone from adipose-derived stem cell with autogenous bone repair in maxillary alveolar cleft model in dogs. Int J Oral Maxillofac Surg 2013 May;42(5):562–8. doi: 10.1016/j.ijom.2012.10.012. Epub 2012 Dec 7. 35. Lee JM, Kim HY, Park JS, et al. Developing palatal bone using human mesenchymal stem cell and stem cells from exfoliated deciduous teeth cell sheets. J Tissue Eng Regen Med 2019 Feb;13(2):319–327. doi: 10.1002/term.2811. Epub 2019 Jan 30. 36. Nakajima K, Kunimatsu R, Ando K, et al. Comparison of the bone regeneration ability between stem cells from human exfoliated deciduous teeth, human dental pulp stem cells and human bone marrow mesenchymal stem cells. Biochem Biophys Res Commun 2018 Mar 11;497(3):876–882. doi: 10.1016/j.bbrc.2018.02.156. 37. Yu M, Ma L, Yuan Y, et al. Cranial suture regeneration mitigates skull and neurocognitive defects in craniosynostosis. Cell 2021 Jan 7;184(1):243–256.e18. doi: 10.1016/j. cell.2020.11.037. 38. Mazzetti MPV, Alonso N, Brock RS, et al. Importance of stem cell transplantation in cleft lip and palate surgical treatment protocol. J Craniofac Surg 2018 Sep;29(6):1445– 1451. doi: 10.1097/SCS.0000000000004766. 39. Gjerde C, Mustafa K, Hellem S, et al. Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial. Stem Cell Res Ther 2018;9(1):213. doi. org/10.1186/s13287-018-0951-9. 40. Kaigler D, Pagni G, Park CH, et al. Stem cell therapy for craniofacial bone regeneration: A randomized, controlled feasibility trial. Cell Transplant 2013;22(5):767–77. doi: 10.3727/096368912X652968. 41. Kaigler D, Avila-Ortiz G, Travan S, et al. Bone engineering of maxillary sinus bone deficiencies using enriched CD90+ stem cell therapy: A randomized clinical trial. J Bone Miner Res 2015 Jul;30(7):1206–16. doi: 10.1002/jbmr.2464. 42. Lim HJ, Lee EM, Kim WK, et al. Application of autologous human bone marrow-derived mesenchymal stem cells in distraction osteogenesis for the treatment of bilateral mandibular hypoplasia. J Craniofac Surg 2018 Sep;29(6):1629–1632. doi: 10.1097/ SCS.0000000000004614. 43. Tanikawa DYS, Pinheiro CCG, Almeida MCA, et al. Deciduous dental pulp stem cells for maxillary alveolar reconstruction in cleft lip and palate patients. Stem Cells Int 2020 Mar 12;2020:6234167. doi:

10.1155/2020/6234167. eCollection 2020. 44. Okeson JP. Evolution of occlusion and temporomandibular disorder in orthodontics: Past, present and future. Am J Orthod Dentofacial Orthop 2015 May;147(5 Suppl):S216–23. doi: 10.1016/j.ajodo.2015.02.007. 45. Lipton JA, Ship JA, Larach-Robinson D. Estimated prevalence and distribution of reported orofacial pain in the United States. J Am Dent Assoc 1993 Oct;124(10):115–21. doi: 10.14219/jada.archive.1993.0200. 46. Laskin DM, Renapurkar SK. Current Controversies in the Management of Temporomandibular Disorders. Oral Maxillofac Surg Clin North Am 2018 Aug;30(3):xiii. doi: 10.1016/j.coms.2018.05.005. Epub 2018 Jul 5. 47. Jacobs JJ, Gilbert JL, Urban RM. Corrosion of metal orthopaedic implants. J Bone Joint Surg Am 1998 Feb;80(2):268–82. doi: 10.2106/00004623-19980200000015. 48. Brady MA, Sivananthan S, Mudera V, et al. The primordium of a biological joint replacement: Coupling of two stem cell pathways in biphasic ultrarapid compressed gel niches. J Craniomaxillofac Surg 2011 Jul;39(5):380–6. doi: 10.1016/j.jcms.2010.07.002. Epub 2010 Aug 31. 49. Chen K, Man C, Zhang B, Hu J, Zhu SS. Effect of in vitro chondrogenic differentiation of autologous mesenchymal stem cells on cartilage and subchondral cancellous bone repair in osteoarthritis of temporomandibular joint. Int J Oral Maxillofac Surg 2013 Feb;42(2):240–8. doi: 10.1016/j. ijom.2012.05.030. Epub 2012 Jul 2. 50. Yoshimura H, Muneta T, Nimura A, et al. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue and muscle. Cell Tissue Res 2007 Mar;327(3):449–62. doi: 10.1007/s00441-006-0308-z. Epub 2006 Oct 13. 51. Koga H, Muneta T, Nagase T, et al. Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: Suitable conditions for cell therapy of cartilage defects in rabbit. Cell Tissue Res 2008 Aug;333(2):207–15. doi: 10.1007/s00441-008-0633-5. Epub 2008 Jun 17. 52. Wu Y, Gong Z, Li J, et al. The pilot study of fibrin with temporomandibular joint derived synovial stem cells in repairing TMJ disc perforation. Biomed Res Int 2014;2014:454021. doi: 10.1155/2014/454021. Epub 2014 Apr 15. 53. Okano T, Yamada N, Sakai H, Sakurai Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J Biomed Mater Res 1993 Oct;27(10):1243–51. doi: 10.1002/ jbm.820271005. 54. MacFarlane RJ, Graham SM, Davies PS, et al. Antiinflammatory role and immunomodulation of mesenchymal stem cells in systemic joint diseases: Potential for treatment. Expert Opin Ther Targets 2013 Mar;17(3):243–54. doi: 10.1517/14728222.2013.746954. Epub 2013 Jan 8. 55. Zhang J, Guo F, Mi J, Zhang Z. Periodontal ligament mesenchymal stromal cells increase proliferation and glycosaminoglycans formation of temporomandibular joint derived fibrochondrocytes. Biomed Res Int 2014;2014:410167. doi: 10.1155/2014/410167. Epub 2014 Nov 10. 56. Kato T, Miyaki S, Ishitobi H, et al. Exosomes from IL-1beta stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes. Arthritis Res Ther 2014 Aug 4;16(4):R163. doi: 10.1186/ar4679. 57. Wang Y, Yu D, Liu Z, et al. Exosomes from embryonic N OVEMBER 2 0 2 1

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mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix. Stem Cell Res Ther 2017 Aug 14;8(1):189. doi: 10.1186/s13287-017-0632-0. 58. Lim EH, Sardinha JP, Myers S. Nanotechnology biomimetic cartilage regenerative scaffolds. Arch Plast Surg 2014 May;41(3):231–40. doi: 10.5999/aps.2014.41.3.231. Epub 2014 May 12. 59. Liu M, Zeng X, Ma C, et al. Injectable hydrogels for cartilage and bone tissue engineering. Bone Res 2017 May 30;5:17014. doi: 10.1038/boneres.2017.14. eCollection 2017. 60. Wang LS, Du C, Toh WS, et al. Modulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymatically crosslinked hydrogel with tunable mechanical properties. Biomaterials 2014 Feb;35(7):2207– 17. doi: 10.1016/j.biomaterials.2013.11.070. Epub 2013 Dec 12. 61. de Souza Tesch R, Takamori ER, Menezes K, et al. Temporomandibular joint regeneration: Proposal of a novel treatment for condylar resorption after orthognathic surgery using transplantation of autologous nasal septum chondrocytes and the first human case report. Stem Cell Res Ther

2018;9(1):94. doi: 10.1186/s13287-018-0806-4. 62. Ekizer A, Yalvac ME, Uysal T, Sonmez MF, Sahin F. Bone marrow mesenchymal stem cells enhance bone formation in orthodontically expanded maxillae in rats. Angle Orthod 2015 May;85(3):394–9. doi: 10.2319/031114-177.1. Epub 2014 Jul 23. 63. Mojarrad S. Effect of Expansive Force on Mesenchymal Stem Cells Isolated From the Mid-Palatal Suture of Mice. University of Pennsylvania Scholarly Commons: University of Pennsylvania; 2018. 64. Proffit W, et al. Diagnosis and Treatment Planning in Orthodontics. In: Graber LW, Vig KW, Vanarsdall RL, Huang GJ, eds. Orthodontics: Current Principles and Techniques. St. Louis: Mosby; 1994:3–95. 65. Hoogeveen EJ, Jansma J, Ren Y. Surgically facilitated orthodontic treatment: A systematic review. Am J Orthod Dentofacial Orthop 2014 Apr;145(4 Suppl):S51–64. doi: 10.1016/j.ajodo.2013.11.019. 66. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006 Aug 1;98(5):1076–84. doi: 10.1002/jcb.20886. 67. Yamato M, Okano T. Cell sheet engineering. Mater Today 2004 7(5)42–7. doi.org/10.1016/S1369-

7021(04)00234-2. 68. Grimm WD, Dannan A, Becher S, et al. The ability of human periodontium-derived stem cells to regenerate periodontal tissues: A preliminary in vivo investigation. Int J Periodontics Restorative Dent Nov–Dec 2011;31(6):e94–e101. 69. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004 Jul 10–16;364(9429):149–55. doi: 10.1016/S0140-6736(04)16627-0.

THE AU THOR , Phimon Atsawasuwan, DDS, MSc, MSc, MS, PhD, can be reached at patsawas@uic.edu.

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reconstructive surgery C D A J O U R N A L , V O L 4 9 , Nº 11

C.E. Credit

Advances in Tissue Engineering and Implications for Oral and Maxillofacial Reconstruction Caitlyn M. McGue, DDS; Victoria A. Mañón, DDS; and Chi T. Viet, DDS, MD, PhD

abstract Background: Reconstructive surgery in the oral and maxillofacial region poses many challenges due to the complexity of the facial skeleton and the presence of composite defects involving soft tissue, bone and nerve defects. Methods: Current methods of reconstruction include autologous grafting techniques with local or regional rotational flaps or microvascular free flaps, allografts, xenografts and prosthetic devices. Results: Tissue engineering therapies utilizing stem cells provide promise for enhancing the current reconstructive options. Conclusions: This article is a review on tissue engineering strategies applicable to specialists who treat oral and maxillofacial defects. Practical implications: We review advancements in hard tissue regeneration for dental rehabilitation, soft tissue engineering, nerve regeneration and innovative strategies for reconstruction of major defects. Keywords: Stem cells, oral and maxillofacial surgery, tissue engineering, nerve regeneration, organoids, reconstructive surgery

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AUTHORS Caitlyn M. McGue, DDS, is a resident in the department of oral and maxillofacial surgery at the Loma Linda University School of Dentistry. Conflict of Interest Disclosure: None reported. Victoria A. Mañón, DDS, MBA, is a resident in the department of oral and maxillofacial surgery at the University of Texas Health Science Center at Houston School of Dentistry. Conflict of Interest Disclosure: None reported.

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Chi T. Viet, DDS, MD, PhD, is an assistant professor in the department of oral and maxillofacial surgery at the Loma Linda University School of Dentistry. Conflict of Interest Disclosure: None reported.

T

he goal for reconstructive surgery is the restoration of form, function and aesthetics. Dental and medical specialties are faced with many challenges when considering reconstruction of maxillofacial defects due to congenital deformities, trauma and benign or malignant pathology. The maxillofacial area plays a significant role in how patients define themselves and how they relate to others. Their facial appearance is an integral part of their identity, and their ability to display emotion, converse and eat are all controlled by the complex anatomic features in the facial skeleton. This article highlights current practices in soft and hard tissue reconstruction in the maxillofacial region and discusses advances in tissue regeneration research that have significant implications for the future of reconstructive surgery. Many factors must be considered when deciding on the reconstructive method, including the size of the defect, the types of tissue missing, the vascular pattern present, the availability of tissue for transfer and patient and surgeon preference.1 The current gold standard for small hard tissue defects involves nonvascularized autologous tissue transfers. Autologous block bone grafts can be harvested from the iliac crest or from intraoral sites including the mandibular ramus or symphysis.2 Small soft tissue defects can be reconstructed with local rotational flaps. For large soft tissue or composite (both soft and hard tissue) defects, vascularized grafts (i.e., free tissue transfer) are utilized with predictable success. A large study on reconstruction with microvascular free flaps demonstrated a 95% success rate.3 As for nerve tissue reconstruction for motor or sensory defects, autologous nerve grafts have traditionally been considered the standard of care. However, advancements in microneurosurgery

have created additional surgical options including allografts, xenografts or a combination of multiple grafting modalities. Finally, there are nonsurgical restorative options for major soft and hard tissue defects, such as prosthetic devices. FIGURE 1 provides an overview of the different reconstructive options currently available for hard and soft tissue defects. Autologous tissue transfer is unfortunately associated with multiple disadvantages. Vascularized free flap transfer is often complicated by scarring, poor color and size matching and longer surgical time.4 A second surgical site leads to donor site morbidity such as pain and neurosensory disturbances as well as longer surgical procedures and recovery time.2 Free flap transfers are also restricted due to limited availability of competent donor sites.5 Because most grafts lack adequate innervation, there is often loss of motor function and sensation.6 Tissue engineering is defined as “an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain or improve tissue function.”7 Tissue engineering is an attractive alternative to the current surgical options discussed previously and relies on stem cell research. Stem cells are capable of self-renewal and differentiation to a more specialized cell type. They can be classified into three different groups based on this differentiation potential. Totipotent stem cells are able to form an entire embryo, including the extraembryonic tissues. Pluripotent stem cells can differentiate into any of the three germ cell layers (endoderm, mesoderm, ectoderm). A special type of pluripotent stem cell is “induced” pluripotent stem cells (iPSC) that can be generated directly from adult cells. Unipotent or progenitor stem cells are limited to one defined cell type.8


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Possible applications of tissue engineering in oral and maxillofacial surgery include hard tissue regeneration for dental rehabilitation, soft tissue engineering, nerve regeneration and the reconstruction of major defects, with the possibility of eventual organoid fabrication and utilization (FIGURE 2 ). We will discuss some of the challenges and advances in tissue engineering for each of these categories. The purpose of this article is to provide a succinct overview of several recent advances in tissue engineering in oral and maxillofacial surgery for dentists across multiple specialties.

Hard Tissue Engineering

The restoration of bony defects continues to remain a challenge in dental rehabilitation. While autogenous grafts have been shown to be successful in repairing some of these defects, the associated donor site morbidity has encouraged research into other more innovative options. Some studies have demonstrated improved clinical outcomes with the use of stem cells and tissue engineering. Bone marrow derived mesenchymal stem cells (BMDSCs) and adipose derived mesenchymal stem cells (ADSCs) have been shown to induce improved bone formation in animal and human models when compared to no treatment or acellular management strategies.5 Rickert et al. utilized a splitmouth design to compare implant stability in maxillary sinuses augmented with Bio-Oss treated with mesenchymal stem cells to Bio-Oss treated with autogenous bone. The authors found mesenchymal stem cells induced bone comparable to that of autogenous bone.9 Osteocel, which contains mesenchymal stem cells seeded on demineralized freeze-dried bone allograft, has been used for sinus grafting and implant site development. A systematic review by Al-Moraissi et al.

Autologous block bone grafts Local/regional rotational flaps

Prosthetic devices Current reconstructive options

(Vascularized) free flaps

Allografts/xenografts

Autologous nerve grafts

FIGURE 1. Schematic summarizing current options for reconstructive treatment modalities.

reported no significant increase in bone formation between tissue-engineered bone using mesenchymal stem cells and conventional bone grafts at three to four months, but a statistically significant increase in bone in the tissue-engineered bone group at six months. Additionally, there was no difference found in residual graft particles, connective tissue, bone gained or implant failure rate.10 Stem cells also hold promise for implant therapies and peri-implant defects. A study on the efficacy of adiposederived stem cell-impregnated scaffolds in dogs demonstrated a significant increase in bone regeneration in peri-implant marginal gaps when used at the time of implant placement.11 In a clinical trial of 11 patients using BMDSCs combined with biphasic calcium phosphate granules for horizontal ridge augmentation, there were significant increases in alveolar width and volume sufficient for the placement of implants in all patients.12 However, studies

by Rickert et al. reported decreased implant survival compared to autologous grafting techniques with survival rates of 91% and 100%, respectively, within the first 12 months.13 Furthermore, a systematic review of the literature of stem cell use in maxillary sinus augmentation by Niño-Sandoval et al. showed that stem cells, when compared to other graft types, did not lead to a significant difference in multiple outcome measures including implant survival rate, bone height, marginal bone loss following implant placement or new bone formation.14 Alveolar clefts are another type of hard tissue defect that could benefit from stem cell therapy. Alveolar clefts are formed when there is improper fusion of the maxillary prominences during the fifth and sixth weeks of gestation.15 Tissue engineering may eventually offer an alternative to autologous bone grafts to limit morbidity associated with the donor surgical site. Preclinical studies in N OVEMBER 2 0 2 1

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Tissue engineered reconstruction

Hard tissue

Nerve regeneration

Soft tissue

Major defects

Sinus lift

Oral mucosa

Skin

Axonal regeneration

Ridge augmentation

Schwann cell differentiation

Peri-implant defects Gingiva

Alveolar cleft

Myelination

Musculocutaneous grafts

Neurotrophic In vitro growth constructs factors

In vivo bioreactor constructs

Organoids

Myelin protein synthesis

Periodontal ligament

Mucocutaneous grafts

Vascularized tissue engineered grafts

FIGURE 2 . This diagram demonstrates potential areas for utilization of stem cell therapies including hard and soft tissue engineering, nerve regeneration and reconstruction of major defects using bioreactors and eventual organoids.

animal models provided promising results regarding the use of stem cells to augment bone formation across alveolar clefts.16 Zhang et al. showed that mesenchymal stem cells combined with a beta-tricalcium phosphate scaffold were as effective in bone generation as autologous bone in a dog model and allowed for adequate bony support for orthodontic movement.17 While clinical trials are lacking, many case reports have demonstrated efficacy of stem cells in bone formation in alveolar clefts in humans.16 The incorporation of mesenchymal stem cells not only improved bone formation 688 N OVEMBER

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but also allowed for tooth eruption in multiple case studies.18,19 Further studies confirmed the usefulness of stem cells in improving alveolar cleft defects to allow for orthodontic tooth movement.20,21 While most studies have not provided long-term follow-up, Chai et al. showed that bone formed utilizing BMDSCs and demineralized bone matrix was maintained for up to three years.22 Although many of the studies using stem cells for the regeneration of bone in alveolar clefts have been promising, one randomized control trial in patients with horizontal alveolar bone deficiencies

demonstrated that stem cells had limited efficacy in larger alveolar defects.2 While artificial transplant materials such as hydroxyapatite or beta-tricalcium phosphate are another alternative to autografts, their use has been limited due to mixed clinical outcomes, especially in studies involving orthodontic movement.23 A study in dog models demonstrated significantly improved bone formation when stem cells were added to carbonated hydroxyapatite (CAP) versus CAP alone with improved radiopacity in the experimental sites. They also demonstrated significantly


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greater numbers of capillary vessels on the experimental side, implying that stem cells may also improve vascularity in the newly formed bone, posing a promising adjunct to artificial transplant materials.24 While the current literature shows much promise for the use of stem cells as an alternative therapy for bone augmentation and improvement of hard tissue defects, there are major variations in reports of their success rates. Additionally, autologous grafts still demonstrate superiority compared to stem cellengineered grafts, although this finding varies from study to study.25 There is a need for further studies and optimization of stem cell protocols and therapies before they become a widely used treatment in dental rehabilitation and alveolar clefts.

Soft Tissue Engineering

Innovations in tissue engineering have produced the ability to create different tissue types such as skin, mucosa, bone and cartilage.26 Autologous-engineered skin substitute grafts have been used widely in burn victims, especially in those patients who have limited healthy skin sites for autologous grafting.27 However, skin is a complex structure composed of epidermis, dermis, vascular plexus, melanocytes and hair follicles. Currently, no engineered substrates can truly replicate this complexity.28 Comparatively, oral mucosal equivalents have also been fabricated using tissue engineering. Izumi et al. reported enhanced maturation of the submucosal layer and vascular ingrowth using a tissue-engineered oral mucosa construct in patients with premalignant or cancerous lesions when compared to AlloDerm alone.29 Soft tissue constructs composed of different tissue types have remained more elusive. Mucocutaneous human tissue constructs have been fabricated to

replicate tissues with a mucocutaneous junction, such as the vermilion of the lip.30 Kim et al. further developed a mucocutaneous construct in vitro that was then grafted over the latissimus dorsi muscle in rats in an attempt to create a prelaminated musculocutaneous flap for lip reconstruction. This served to develop a mature trilaminar flap that could then be harvested and placed into the defect site.26 These constructs hold promise for the ability to restore complex soft tissue defects with better function and aesthetics than current options allow.

gingiva remained nonsignificant between the treatment groups. The only significant finding was improved root coverage in the stem cell impregnated membrane group.34 Although soft tissue engineering modalities hold potential for regeneration and reconstruction of soft tissue defects in the oral and maxillofacial region, clinical studies focused on soft tissue engineering are limited when compared to the literature on hard tissue engineering. Well-designed clinical trials are needed to develop efficacious and viable treatment options that utilize stem cell therapies.

Nerve Regeneration

Innovations in tissue engineering have produced the ability to create different tissue types such as skin, mucosa, bone and cartilage.

Tissue engineering has also been evaluated for its possible therapeutic effects in gingival defects. The utilization of stem cell therapy may help overcome limitations of free gingival and connective tissue grafts, such as donor site morbidity and limited tissue for grafting.31 One study of five patients with missing keratinized mucosa or mucogingival defects showed a gain of keratinized gingival width, but no significant change in probing depths.32 A systematic review by Gaubys et al. reported that stem cell therapy had the ability to enhance periodontal ligament and cementum regeneration.33 A more recent study evaluated the ability of a stem cell impregnated membrane to improve gingival recession when compared to membrane alone; however, the differences in gingival recession and keratinized

Dysfunction of the trigeminal or facial nerve following injury or disease of the maxillofacial region is significantly distressing and debilitating for patients. It can lead to paresthesia or dysesthesia, dysgeusia, paralysis of the muscles of facial expression, inability to chew and maintain lip and cheek competence and altered speech patterns. In cases of trigeminal nerve injury, the inferior alveolar nerve is most frequently affected, followed by the lingual and infraorbital nerves.35 Initial treatment options for trigeminal nerve injury without indications for immediate surgical intervention are often pharmacological, using medications such as NSAIDs or antiepileptic drugs like gabapentin or carbamazepine. Other options such as local and regional anesthesia have also been used. Low-level laser therapy has also demonstrated efficacy, but the effect is decreased with time from injury.36,37 Microneurosurgical repair with end-to-end anastomosis or grafting procedures is explored in circumstances where nonsurgical options are ineffective. However, neurorrhaphy can be challenging in cases of inferior alveolar nerve injury due to a limited ability to advance the nerve across a gap without N OVEMBER 2 0 2 1

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tension. Grafting procedures include the use of both allogenic or autologous nerve grafts.38 One commercial decellularized allogenic nerve graft is available, which is heavily marketed among surgical specialists who perform sensory or motor nerve repair. Studies using allogenic nerve graft show some success in the repair of nerve defects and reinnervation of distal targets with the allograft comparable to that seen with autografts. But the effect is diminished in longer gaps when utilization of an allograft would be most beneficial.39,40 Cell-based therapies pose a promising alternative treatment option that would minimize some of the disadvantages associated with autologous nerve grafts, such as donor site morbidity, neuroma formation and limited length of available grafts.39 Bone marrow-derived mesenchymal stem cells can differentiate into myelinating cells and support nerve fiber regeneration.41 ADSCs have also been shown to physically engraft and myelinate regenerating axons and are comparable to BMDSC in in vivo studies.42 BMDSCs can be induced to express neural stem cell markers. Studies utilizing pre-differentiated stem cell transplantation showed they accelerated regeneration of transected axons and achieved improved myelination that was comparable to the results observed after Schwann cell transplantation.43,44 However, contrasting studies showed primary Schwann cells were significantly better than BMDSCs and ADSC-loaded conduits at promoting distal stump sprouting.42 De Carvalho Raimundo et al. demonstrated improved whisker movement and eyelid closure in rats following nerve injury with a 5 mm gap when stem cells were injected into the polyethylene conduit connecting the two segments. The study also demonstrated improved nerve fiber area and myelin sheath thickness in the stem cell groups.45 Another study by Choi 690 N OVEMBER

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et al. compared the effectiveness of nerve repair in a 15 mm defect in rabbits between a vein conduit with BMDSCs to a vein conduit alone. The vein conduit filled with BMDSCs demonstrated superiority in axon formation, the number of nerve fibers generated and the diameter of the nerve fibers.46 Stem cells also play a role through other supporting measures for nerve regeneration. They can secrete a variety of growth factors, such as nerve growth factor, brain-derived neurotrophic factor, vascular endothelial growth factor and

Cell-based therapies pose a promising alternative treatment option that would minimize some of the disadvantages associated with autologous nerve grafts ... glial cell-derived neurotrophic factors, that act as neurotrophic molecules to help provide a beneficial microenvironment for neural cell survival and neurogenesis. Additionally, they synthesize myelin proteins that serve to enhance myelination and function of the regenerated nerves.47 While many of the current in vitro and in vivo studies provide promising results for the use of tissue engineering in nerve regeneration, few clinical trials have been conducted.

Reconstruction of Major Defects

Microvascular reconstruction of large defects in the oral and maxillofacial regions is the current standard of care for restoration of form and function, as it provides the most predictable results. It allows for the regeneration of

both hard and soft tissue and carries its own blood supply, which is crucial in defects where a sufficiently vascularized tissue envelope may not be feasible due to lack of adequate healthy tissue, such as in traumatic or oncologic defects. Despite the improvements osteocutaneous flaps provide, donor site morbidity, limited tissue availability and compromised aesthetics due to mismatch in tissue color and dimension prove to be challenging (FIGURE 3 ). Tissue engineering may provide an alternative for the repair of these large defects. However, there are significant challenges that need to be addressed before this becomes a viable treatment option. Large tissue-engineered constructs created in vitro have a limited vascular supply that is unable to support the constructs and prevents their utilization in clinical settings.48 One method to overcome this problem is the use of in vivo bioreactors composed of nondegradable custom-shaped chambers filled with either osteoconductive or osteoinductive materials. Allowing the graft to mature in vivo generates a tissue-engineered vascularized graft that can then be harvested and transferred with a vascular pedicle for reconstruction (FIGURE 2 ).49 Kasper et al. summarized the case reports of five different prefabricated vascularized free flap approaches in patients. While all the reports demonstrated bone formation within the in vivo bioreactor chambers, two out of five of the constructs failed or required significant revisions.49 Cheng et al. designed a prefabricated bone graft that was transferred to a mandible that had deficient bony dimensions for implant placement following fibula free flap. The transferred tissue was able to maintain dental implants at 16 months, although the patient eventually died of hepatocellular carcinoma before the implants could be restored.50


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Two other case reports described restoration of large mandibular defects (angle-to-angle and parasymphysis-toretromolar region) that were restored using tissue-engineered constructs utilizing in vivo bioreactors. The first study by Orringer et al. in 1990 created a mandibular-shaped polyurethane tray packed with autograft from the iliac crest combined with human bone morphogenetic protein (BMP). The prefabricated graft was used to restore the mandible and lower lip.51 In the second case report, Warnke et al. used a titanium mesh scaffold filled with mineral bone blocks coated with BMP and augmented with bone marrow aspirate from the iliac crest. The graft was implanted into the latissimus dorsi muscle for seven weeks, then harvested with a vascular pedicle containing the thoracodorsal artery and vein and transplanted to the area of defect. The case report only described up until postoperative week four at which time the patient recovered some masticatory ability, even though he remained edentulous at that time.52 These studies lead to questions about the possible fabrication of organoids for the maxillofacial region. Organoids are “self-organizing 3D structures grown from stem cells that mimic the in vivo architecture and multilineage differentiation of the original tissue in mammals.”53 Various studies have demonstrated that organoids can be produced from multiple different types of stem cells including embryonic, adult and patient-derived pluripotent stem cells. It is theorized that these organoids could be utilized for cancer research, drug screening and eventually reconstruction.54 However, at this time none have been transplanted into patients. Furthermore, while there are lingual and salivary gland organoids, there are no mandibular or bony organoid constructs.55,56

A

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F FIGURE S 3 . The panoramic radiograph is of a patient with ameloblastoma of the left posterior mandible (3A). The patient is treated with the “Jaw in a Day” technique, in which the patient undergoes a mandibulectomy (here, with osteotomies through tooth No. 19 anteriorly and the sigmoid notch posteriorly to achieve clear tumor margins), with fibula microvascular free flap reconstruction, where dental implants are also placed in the fibula and a prosthesis is cemented to the dental implants (3B). This entire fibula, implant and prosthesis construct (shown in this picture, while still connected by the vascular pedicle in the leg) is transferred to the mandible defect, and microvascular surgery is performed to connect the fibula pedicle with an artery and vein in the patient’s neck. This technique allows for immediate reconstruction of hard and soft tissue defects and missing teeth in one surgery, efficiently restoring the patient’s form and function after tumor ablation. The picture shows the immediate postoperative occlusion after microvascular reconstruction (3C), the final implantsupported restorations (3D), final periapical radiograph (3E) and final frontal occlusion (3F). This case was performed by Dr. Chi T. Viet (microvascular surgery), Dr. Alan Herford (tumor resection) and Dr. Jui Min Su (prosthodontics) at Loma Linda University.

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While the development of prefabricated flaps and organoids presents exciting possibilities for the future of reconstructive and regenerative medicine, there is still significant preclinical research to be performed before we could even begin to plan clinical trials. Organoids could represent an alternative to the in vivo bioreactor approach, but there are still few applications for the oral and maxillofacial surgeon. Ultimately, the goal is to produce viable constructs for large multitissue maxillofacial defects that would abolish the need for vascularized free flaps altogether.

Conclusions

Despite the promise that stem cell therapy holds, it is not without limitations. Tissue engineering still requires the harvesting of autologous bone cells, which has associated donor site morbidity; however, the collection procedures are less invasive and traumatic than autograft harvesting.14,47 Obtaining stem cells may or may not require general anesthesia or sedation, depending on the selected site for cell collection. Prefabricated flaps also still require multiple-staged procedures for the implantation of the construct and eventual transfer. Additionally, because the grafts utilizing an in vivo bioreactor are buried beneath the skin as they mature, they are not visible for observation and rely on alternative modalities such as Doppler ultrasound to monitor their maturation.26 Currently, stem cell therapies are inefficient, as they require culturing and expansion, especially in grafts requiring vascularity and perfusion.6 This extended treatment timeline is impractical in patients who have large oncologic defects, as they will need to be reconstructed with a reliable vascularized graft prior to adjuvant radiation and chemotherapy. 692 N OVEMBER

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There is also concern for tumorigenic potential of stem cells because they share many characteristics with cancer cells.17 Both have long life spans with abilities to self-renew and replicate for long periods of time.57 For example, in one study of a rat model with sciatic nerve injury transplanted with neural stem cells, 25% developed large neuroblastoma-like tumors.58 The degree of differentiation of the stem cell may play an important role in determining the true risk of malignant transformation.57 Tissue engineering using stem cell therapies represents an innovative step forward in regenerative medicine. As we continue to search for ideal methods to restore form, function and aesthetics in the maxillofacial region, there is much hope that these treatment modalities will provide viable alternatives to the current restorative options. Currently, there are limited publications regarding the utilization of tissue engineering in patients, especially for complex soft tissue constructs. Furthermore, there are many variations in protocols used with no consensus on the optimal harvesting and isolation techniques.14 There are also few studies evaluating long-term stability of tissue-engineered constructs.16 These limitations highlight the importance of focused and thoughtful research in tissue engineering to optimize and standardize protocols before stem cells can be used routinely in clinical practice. n RE FE RE N CE S 1. Young S, Kasper FK, Melville J, et al. Tissue engineering in oral and maxillofacial surgery. In: Lanza R, Langer R, Vacanti JP, Atala A, eds. Principles of Tissue Engineering. 5th ed. Cambridge, Mass.: Academic Press; 2020:1201–1220. 2. Bajestan MN, Rajan A, Edwards SP, et al. Stem cell therapy for reconstruction of alveolar cleft and trauma defects in adults: A randomized controlled, clinical trial. Clin Implant Dent Relat Res 2017 Oct;19(5):793–801. doi: 10.1111/cid.12506. Epub 2017 Jun 28. 3. Wei FC, Jain V, Celik N, Chen HC, Chuang DC, Lin CH. Have we found an ideal soft-tissue flap? An experience with 672 anterolateral thigh flaps. Plast Reconstr Surg

2002 Jun;109(7):2219–26; discussion 2227–30. doi: 10.1097/00006534-200206000-00007. 4. Lubek JE, Ord RA. Lip reconstruction. Oral Maxillofac Surg Clin North Am 2013 May;25(2):203–14. doi: 10.1016/j. coms.2013.01.001. Epub 2013 Mar 17. 5. Khojasteh A, Behnia H, Dashti SG, Stevens M. Current trends in mesenchymal stem cell application in bone augmentation: A review of the literature. J Oral Maxillofac Surg 2012 Apr;70(4):972–82. doi: 10.1016/j.joms.2011.02.133. Epub 2011 Jul 16. 6. Kim RY, Bae SS, Feinberg SE. Soft tissue engineering. Oral Maxillofac Surg Clin North Am 2017 Feb;29(1):89–104. doi: 10.1016/j.coms.2016.08.007. 7. Langer R, Vacanti JP. Tissue engineering. Science 1993 May 14;260(5110):920–6. doi: 10.1126/science.8493529. 8. Lakshmipathy U, Verfaillie C. Stem cell plasticity. Blood Rev 2005 Jan;19(1):29–38. doi: 10.1016/j.blre.2004.03.001. 9. Rickert D, Sauerbier S, Nagursky H, Menne D, Vissink A, Raghoebar GM. Maxillary sinus floor elevation with bovine bone mineral combined with either autogenous bone or autogenous stem cells: A prospective randomized clinical trial. Clin Oral Implants Res 2011 Mar;22(3):251–8. doi: 10.1111/j.1600-0501.2010.01981.x. Epub 2010 Sep 10. 10. Al-Moraissi EA, Oginni FO, Mahyoub Holkom MA, Mohamed AAS, Al-Sharani HM. Tissue-engineered bone using mesenchymal stem cells versus conventional bone grafts in the regeneration of maxillary alveolar bone: A systematic review and meta-analysis. Int J Oral Maxillofac Implants Jan/Feb 2020;35(1):79–90. doi: 10.11607/jomi.7682. Epub 2019 Sep 18. 11. Bressan E, Botticelli D, Sivolella S, et al. Adipose-derived stem cells as a tool for dental implant osseointegration: An experimental study in the dog. Int J Mol Cell Med Fall 2015;4(4):197–208. 12. Gjerde C, Mustafa K, Hellem S, et al. Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial. Stem Cell Res Ther 2018;9(1):213. doi. org/10.1186/s13287-018-0951-9. 13. Rickert D, Vissink A, Slot WJ, Sauerbier S, Meijer HJ, Raghoebar GM. Maxillary sinus floor elevation surgery with BioOss mixed with a bone marrow concentrate or autogenous bone: Test of principle on implant survival and clinical performance. Int J Oral Maxillofac Surg 2014 Feb;43(2):243–7. doi: 10.1016/j.ijom.2013.09.006. Epub 2013 Oct 30. 14. Nino-Sandoval TC, Vasconcelos BC, SL DM, CA AL, Pellizzer EP. Efficacy of stem cells in maxillary sinus floor augmentation: Systematic review and meta-analysis. Int J Oral Maxillofac Surg 2019 Oct;48(10):1355–1366. doi: 10.1016/j.ijom.2018.04.022. Epub 2019 Apr 11. 15. Coots BK. Alveolar bone grafting: Past, present and new horizons. Semin Plast Surg 2012 Nov;26(4):178–83. doi: 10.1055/s-0033-1333887. 16. Gladysz D, Hozyasz KK. Stem cell regenerative therapy in alveolar cleft reconstruction. Arch Oral Biol 2015 Oct;60(10):1517–32. doi: 10.1016/j. archoralbio.2015.07.003. Epub 2015 Jul 13. 17. Zhang D, Chu F, Yang Y, et al. Orthodontic tooth movement in alveolar cleft repaired with a tissue engineering bone: An experimental study in dogs. Tissue Eng Part A 2011 May;17(9–10):1313–25. doi: 10.1089/ten. TEA.2010.0490. Epub 2011 Mar 17. 18. Hibi H, Yamada Y, Ueda M, Endo Y. Alveolar cleft


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C .E. CREDIT QUESTIONS

November 2021 Continuing Education Worksheet This worksheet provides readers an opportunity to review C.E. questions for the article “Advances in Tissue Engineering and Implications for Oral and Maxillofacial Reconstruction” before taking the C.E. test online. You must first be registered at cdapresents360.com. To take the test online, please click here. This activity counts as 1.0 of Core C.E. 1. Which of the following is not associated with autologous tissue transfer? a. Multiple options for competent donor sites b. Poor color and size matching of vascularized free flap c. Pain and neurosensory disturbances at donor site d. Longer recovery time 2. Which of the following stem cells classifications is able to form an entire embryo? a. Pluripotent b. Totipotent c. Unipotent or progenitor d. An induced pluripotent (iPSC) 3. A literature review of stem cell use in maxillary sinus augmentation, when compared to other graft types, showed that stem cell grafts were comparable in which of the following areas? a. Implant survival rate b. Bone height c. New bone formation d. All of the above 4. Stem cells show promise for supporting nerve regeneration because of their ability to do which of the following (mark all that apply)? a. Secrete nerve growth factor b. Secrete vascular endothelial growth factor c. Regenerate transected axons d. Synthesize myelin proteins 5. Organoids are “self-organizing 3D structures grown from stem cells that mimic the in vivo architecture and multi-lineage differentiation of the original tissue in mammals.” Which of the following statements about organoids is incorrect? a. They can be produced from multiple different types of stem cells. b. They are currently being utilized for cancer research. c. There are salivary gland organoids. d. There are not yet bony organoids.

6. True or False: A promising method for overcoming the lack of vascularity of large, in vitro-created tissue-engineered constructs is to instead allow the tissue to mature in vivo bioreactors filled with bone conductive or inductive materials. 7. Preliminary results from stem cells studies used for alveolar cleft repair show promise for which of the following (mark all that apply)? a. Augmenting bone formation across alveolar clefts b. Improving the vascularity of newly formed bone c. Superiority to autologous grafts d. All of the above 8. Which stem cells have been shown to physically engraft and myelinate regenerating axons? a. Adipose-derived mesenchymal stem cells b. Bone-derived mesenchymal stem cells c. Nerve-derived mesenchymal stem cells 9. Cell therapies pose a promising alternative treatment option that may minimize which of the disadvantages associated with autologous nerve grafts (mark all that apply)? a. Donor site morbidity b. Neuroma formation c. Limited length of available grafts d. None of the above 10. True or False: Though reviews are mixed, autologous grafts still demonstrate superiority compared to stem cell-engineered grafts. Hence, there is a need for further studies and optimization of stem cell protocols and therapies before they become a widely used treatment in dental rehabilitation and alveolar clefts.

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osteoplasty using tissue-engineered osteogenic material. Int J Oral Maxillofac Surg 2006 Jun;35(6):551–5. doi: 10.1016/j.ijom.2005.12.007. Epub 2006 Apr 11. 19. Pradel W, Tausche E, Gollogly J, Lauer G. Spontaneous tooth eruption after alveolar cleft osteoplasty using tissueengineered bone: A case report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008 Apr;105(4):440–4. doi: 10.1016/j.tripleo.2007.07.042. Epub 2008 Feb 21. 20. Behnia H, Khojasteh A, Soleimani M, Tehranchi A, Atashi A. Repair of alveolar cleft defect with mesenchymal stem cells and platelet derived growth factors: A preliminary report. J Craniomaxillofac Surg 2012 Jan;40(1):2–7. doi: 10.1016/j. jcms.2011.02.003. Epub 2011 Mar 21. 21. Behnia H, Khojasteh A, Soleimani M, et al. Secondary repair of alveolar clefts using human mesenchymal stem cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009 Aug;108(2):e1–6. doi: 10.1016/j.tripleo.2009.03.040. 22. Chai G, Zhang Y, Hu XJ, et al. [Repair alveolar cleft bone defects with bone marrow stromal cells]. Zhonghua Zheng Xing Wai Ke Za Zhi 2006 Nov;22(6):409–411. 23. Hossain MZ, Kyomen S, Tanne K. Biologic responses of autogenous bone and beta-tricalcium phosphate ceramics transplanted into bone defects to orthodontic forces. Cleft Palate Craniofac J 1996 Jul;33(4):277–83. doi: 10.1597/1545-1569_1996_033_0277_broaba_2.3.co_2. 24. Yoshioka M, Tanimoto K, Tanne Y, et al. Bone regeneration in artificial jaw cleft by use of carbonated hydroxyapatite particles and mesenchymal stem cells derived from iliac bone. Int J Dent 2012;2012:352510. doi: 10.1155/2012/352510. Epub 2012 Mar 26. 25. Yuan J, Cui L, Zhang WJ, Liu W, Cao Y. Repair of canine mandibular bone defects with bone marrow stromal cells and porous beta-tricalcium phosphate. Biomaterials 2007 Feb;28(6):1005–13. doi: 10.1016/j. biomaterials.2006.10.015. Epub 2006 Nov 7. 26. Kim RY, Fasi AC, Feinberg SE. Soft tissue engineering in craniomaxillofacial surgery. Ann Maxillofac Surg 2014 Jan– Jun;4(1):4–8. doi: 10.4103/2231-0746.133064. 27. Payne KF, Balasundaram I, Deb S, Di Silvio L, Fan KF. Tissue engineering technology and its possible applications in oral and maxillofacial surgery. Br J Oral Maxillofac Surg 2014 Jan;52(1):7–15. doi: 10.1016/j.bjoms.2013.03.005. Epub 2013 Apr 16. 28. Supp DM, Boyce ST. Engineered skin substitutes: Practices and potentials. Clin Dermatol Jul–Aug 2005;23(4):403–12. doi: 10.1016/j.clindermatol.2004.07.023. 29. Izumi K, Feinberg SE, Iida A, Yoshizawa M. Intraoral grafting of an ex vivo produced oral mucosa equivalent: A preliminary report. Int J Oral Maxillofac Surg 2003 Apr;32(2):188–97. doi: 10.1054/ijom.2002.0365. 30. Izumi K, Song J, Feinberg SE. Development of a tissueengineered human oral mucosa: From the bench to the bed side. Cells Tissues Organs 2004;176(1–3):134–52. doi: 10.1159/000075034. 31. McGuire MK, Scheyer ET, Nunn ME, Lavin PT. A pilot study to evaluate a tissue-engineered bilayered cell therapy as an alternative to tissue from the palate. J Periodontol 2008 Oct;79(10):1847–56. doi: 10.1902/jop.2008.080017. 32. Izumi K, Neiva RF, Feinberg SE. Intraoral grafting of tissueengineered human oral mucosa. Int J Oral Maxillofac Implants Sep–Oct 2013;28(5):e295–303. doi: 10.11607/jomi.te11. 33. Gaubys A, Papeckys V, Pranskunas M. Use of autologous stem cells for the regeneration of periodontal defects in

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animal studies: A systematic review and meta-analysis. J Oral Maxillofac Res 2018 Jun 29;9(2):e3. doi: 10.5037/ jomr.2018.9203. eCollection Apr-Jun 2018. 34. Zanwar K, Kumar Ganji K, Bhongade ML. Efficacy of human umbilical stem cells cultured on polylactic/polyglycolic acid membrane in the treatment of multiple gingival recession defects: A randomized controlled clinical study. J Dent (Shiraz) 2017 Jun;18(2):95–103. 35. Schultze-Mosgau S, Reich RH. Assessment of inferior alveolar and lingual nerve disturbances after dentoalveolar surgery and of recovery of sensitivity. Int J Oral Maxillofac Surg 1993 Aug;22(4):214–7. doi: 10.1016/s09015027(05)80638-1. 36. Khullar SM, Brodin P, Barkvoll P, Haanaes HR. Preliminary study of low-level laser for treatment of long-standing sensory aberrations in the inferior alveolar nerve. J Oral Maxillofac Surg 1996 Jan;54(1):2–7; discussion 7–8. doi: 10.1016/ s0278-2391(96)90290-6. 37. Miloro M, Repasky M. Low-level laser effect on neurosensory recovery after sagittal ramus osteotomy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000 Jan;89(1):12–8. doi: 10.1016/s1079-2104(00)80006-2. 38. Bagheri SC, Meyer RA. Management of mandibular nerve injuries from dental implants. Atlas Oral Maxillofac Surg Clin North Am 2011 Mar;19(1):47–61. doi: 10.1016/j. cxom.2010.11.004. 39. Moore AM, MacEwan M, Santosa KB, et al. Acellular nerve allografts in peripheral nerve regeneration: A comparative study. Muscle Nerve 2011 Aug;44(2):221–34. doi: 10.1002/mus.22033. Epub 2011 Jun 9. 40. Porzionato A, Stocco E, Barbon S, Grandi F, Macchi V, De Caro R. Tissue-engineered grafts from human decellularized extracellular matrices: A systematic review and future perspectives. Int J Mol Sci 2018 Dec 18;19(12):4117. doi: 10.3390/ijms19124117. 41. Dezawa M, Takahashi I, Esaki M, Takano M, Sawada H. Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur J Neurosci 2001 Dec;14(11):1771–6. doi: 10.1046/j.0953816x.2001.01814.x. 42. di Summa PG, Kingham PJ, Campisi CC, Raffoul W, Kalbermatten DF. Collagen (NeuraGen) nerve conduits and stem cells for peripheral nerve gap repair. Neurosci Lett 2014 Jun 20;572:26–31. doi: 10.1016/j.neulet.2014.04.029. Epub 2014 May 2. 43. Tomita K, Madura T, Mantovani C, Terenghi G. Differentiated adipose-derived stem cells promote myelination and enhance functional recovery in a rat model of chronic denervation. J Neurosci Res 2012 Jul;90(7):1392–402. doi: 10.1002/jnr.23002. Epub 2012 Mar 15. 44. Wang X, Luo E, Li Y, Hu J. Schwann-like mesenchymal stem cells within vein graft facilitate facial nerve regeneration and remyelination. Brain Res 2011 Apr 6;1383:71–80. doi: 10.1016/j.brainres.2011.01.098. Epub 2011 Feb 3. 45. de Carvalho Raimundo R, Landim FS, Gomes ACA, Castro C, Silva Junior VA, Vasconcelos B. Morphofunctional effect of stem cells on the regeneration of the facial nerve in a rat model. J Oral Maxillofac Surg 2019 Oct;77(10):2168.e1–2168. e12. doi: 10.1016/j.joms.2019.06.008. Epub 2019 Jun 21. 46. Choi BH, Zhu SJ, Kim BY, Huh JY, Lee SH, Jung JH. Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration. Int J Oral Maxillofac Surg 2005 Jul;34(5):537–42. doi: 10.1016/j.

ijom.2004.10.017. Epub 2005 Jan 26. 47. Jiang L, Jones S, Jia X. Stem cell transplantation for peripheral nerve regeneration: Current options and opportunities. Int J Mol Sci 2017 Jan 5;18(1):94. doi: 10.3390/ijms18010094. 48. Liu Y, Chan JK, Teoh SH. Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems. J Tissue Eng Regen Med 2015 Feb;9(2):85–105. doi: 10.1002/term.1617. Epub 2012 Nov 19. 49. Kasper FK, Melville J, Shum J, Wong M, Young S. Tissue engineered prevascularized bone and soft tissue flaps. Oral Maxillofac Surg Clin North Am 2017 Feb;29(1):63–73. doi: 10.1016/j.coms.2016.08.005. 50. Cheng MH, Brey EM, Ulusal BG, Wei FC. Mandible augmentation for osseointegrated implants using tissue engineering strategies. Plast Reconstr Surg 2006 Jul;118(1):1e–4e. doi: 10.1097/01. prs.0000221120.11128.1a. 51. Orringer JS, Shaw WW, Borud LJ, Freymiller EG, Wang SA, Markowitz BL. Total mandibular and lower lip reconstruction with a prefabricated osteocutaneous free flap. Plast Reconstr Surg 1999 Sep;104(3):793–7. doi: 10.1097/00006534199909030-00028. 52. Warnke PH, Springer IN, Wiltfang J, et al. Growth and transplantation of a custom vascularised bone graft in a man. Lancet 2004 Aug 28–Sep 3;364(9436):766–70. doi: 10.1016/S0140-6736(04)16935-3. 53. Dutta D, Heo I, Clevers H. Disease modeling in stem cell-derived 3D organoid systems. Trends Mol Med 2017 May;23(5):393–410. doi: 10.1016/j.molmed.2017.02.007. Epub 2017 Mar 21. 54. Ashok A, Choudhury D, Fang Y, Hunziker W. Towards manufacturing of human organoids. Biotechnol Adv Mar–Apr 2020;39:107460. doi: 10.1016/j.biotechadv.2019.107460. Epub 2019 Oct 15. 55. Ferreira JN, Hasan R, Urkasemsin G, et al. A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids. J Tissue Eng Regen Med 2019 Mar;13(3):495–508. doi: 10.1002/ term.2809. Epub 2019 Mar 6. 56. Hisha H, Tanaka T, Kanno S, et al. Establishment of a novel lingual organoid culture system: Generation of organoids having mature keratinized epithelium from adult epithelial stem cells. Sci Rep 2013;3:3224. doi.org/10.1038/srep03224. 57. Herberts CA, Kwa MS, Hermsen HP. Risk factors in the development of stem cell therapy. J Transl Med 2011 Mar 22;9:29. doi: 10.1186/1479-5876-9-29. 58. Johnson TS, O’Neill AC, Motarjem PM, Nazzal J, Randolph M, Winograd JM. Tumor formation following murine neural precursor cell transplantation in a rat peripheral nerve injury model. J Reconstr Microsurg 2008 Nov;24(8):545–50. doi: 10.1055/s-0028-1088228. Epub 2008 Sep 25. T HE CORRE S P ON DIN G AU T HOR , Caitlyn M. McGue, DDS, can be reached at caitlyn.mcgue@gmail.com.


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zirconia implants C D A J O U R N A L , V O L 4 9 , Nº 11

Pre-Sintering Airborne Particle Abrasion Improves Surface and Biological Properties of Zirconia Tanjira Kueakulkangwanphol, DDS; Nichamon Chaianant, DDS, PhD; Yanee Tantilertanant, DDS, PhD; and Weerachai Singhatanadgit, DDS, PhD

abstract Background: Surface topography of a zirconia dental implant has a major impact on osseointegration and clinical success. Modification of a fully sintered zirconia surface can be problematic due to its high strength and bio-inert surface. Methods: The present study therefore aimed to investigate the effect of airborne-particle abrasion of presintered zirconia on surface properties and biological performance of the resulting fully sintered zirconia. Results: The results showed that surface topographies of fully sintered zirconia were influenced by abrasive particle size and blasting time used in repeated pre-sintering airborne particle abrasion. Well-optimized pre-sintering airborne-particle abrasion of zirconia surface could render a highly roughened and hydrophilic surface with increased fibronectin-to-albumin adsorption ratio and mesenchymal stem cell adhesion. Practical implications: This technique may be suitable for modifying zirconia surface to facilitate osseointegration of zirconia-based dental implants. Keyword: Pre-sintering airborne-particle abrasion, zirconia, surface roughness, surface hydrophilic, mesenchymal stem cells

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zirconia implants C D A J O U R N A L , V O L 4 9 , Nº 11

AUTHORS Tanjira Kueakulkangwanphol, DDS, is a postgraduate student in the Master of Science program in dentistry (prosthodontics) at Faculty of Dentistry, Thammasat University in Pathumthani, Thailand. Conflict of Interest Disclosure: None reported. Nichamon Chaianant, DDS, PhD, is a lecturer in oral epidemiology and statistics at Faculty of Dentistry, Thammasat University in Pathumthani, Thailand. Conflict of Interest Disclosure: None reported.

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Yanee Tantilertanant, DDS, PhD, is a clinical lecturer in the department of operative dentistry at Faculty of Dentistry, Chulalongkorn University in Bangkok. Conflict of Interest Disclosure: None reported. Weerachai Singhatanadgit, DDS, PhD, is an associate professor in oral biology at Faculty of Dentistry, Thammasat University in Bangkok. Conflict of Interest Disclosure: None reported.

D

ental implants provide aesthetic and functional tooth replacement due to their high survival rate and success rate.1 The demand for use of implants has increased. A titanium dental implant is reliable for tooth replacement supported by numerous studies with long-term clinical success.2–4 However, its natural gray color is an important concern in the aesthetic zone where the titanium color can show through thin peri-implant tissues.5 Furthermore, titanium may cause metal allergy and metal toxicity produced by corrosion of titanium as reported in multiple titanium implant cases.6–12 To solve the aesthetic and metal particle problems, new materials should be tooth colored and nonmetallic materials such as ceramics. Zirconia, namely 3-mol% yttrium oxide-stabilized zirconia (3Y-TZP), has widely been introduced for dental implants.13 In addition to good biocompatibility, zirconia has very high flexural strength and fracture toughness. Compared to a titanium implant, the zirconia implant demonstrated good performance in osseointegration in several studies that reported no significant differences in bone-to-implant contact and removal torque value or even better than a titanium implant.14–20 Moreover, zirconia was superior to the titanium material in lower bacterial adhesion.21–24 The conventional surface of zirconia implants is usually a smooth surface. Surface topographies and surface roughness significantly affect osseointegration by increasing biological responses, enhancing osseointegration and increasing torque resistance.25–31 It is then undoubtedly necessary to modify the surface of a zirconia implant. There are several ways to modify the zirconia implant’s surface roughness, including

airborne-particle abrasion, acid etching, plasma spraying, bioactive material coating and UV-light treatment.32 Airborne abrasion was most commonly used to increase the surface roughness of the zirconia because it is simple and inexpensive to use. Airborne abrasion also yielded a microroughness surface of the zirconia when compared to other techniques.27,28,32–35 Due to its high strength and bioinert property, increased surface roughness of a fully sintered zirconia surface can be problematic, requiring high blasting pressure for airborne-particle abrasion and strong acid with high temperature for acid etching.28,36,37 Moreover, an airborne-particle abrasion of a fully sintered zirconia is associated with phase transformation from a tetragonal to a monoclinic phase that induced a negative effect on long-term clinical performance by increasing the rate of aging and zirconia fracture.38–42 Additionally, airborne-particle abrasion of fully sintered zirconia can cause flaws, microcracks, plastic deformation or even embedding of abrasive particles on the zirconia surface.38,43 These unfavorable results have also been reported to be associated with nonoptimized abrasive particle size and shape, blasting distance, blasting duration and blasting pressure.38,43 A number of airborne-particle abrasion methods to modify the zirconia surface before the final sintering step have recently been introduced. It is possible that airborne-particle abrasion of pre-sintered zirconia produces higher surface roughness compared with that of fully sintered zirconia.38,39,44–51 Moreover, the modification of pre-sintered zirconia may contribute to greater durability of the implant because zirconia presents no monoclinic phase from reverse transformation after the sintering process.49,50 However, the optimized


C D A J O U R N A L , V O L 4 9 , Nº 11

25 µm airborne-particle abrasion protocol in pre-sintered zirconia has not yet been established, and nonoptimized abrasive particle size and shape, blasting distance, blasting duration and blasting pressure could induce a negative effect to the zirconia. Longer blasting duration and higher blasting pressure could produce volume loss and height loss of the zirconia surface.43,47,48,51,52 The purpose of the present study was to investigate the use of airborne abrasion and its effect on zirconia surfaces. Different sizes of abrasive particles and various blasting times were used under controlled blasting pressure on fully sintered zirconia. The parameters measured included zirconia surface topographical characteristics, zirconia surface hydrophilicity and in vitro biological response of mesenchymal stem cells (MSCs).

Materials and Methods Specimen Preparation

All specimens in this study were prepared from pre-sintered yttriastabilized zirconia blocks (Prettau zirconia, Zirkonzahn, Gais, Italy). The blocks were cut into 5 x 5 x 2 mm3 rectangular pieces using a precision high-speed lathe machine (CL4070, YAM, Taiwan) under copious water by setting the rotational speed at 1200 rpm. To standardize the initial surface roughness and create a finishing smooth surface, all specimens were polished with silicon-carbide grit papers for 10 vertical strokes in each grit value from #600, #800, #1000 and #2000 manually. The surfaces of the specimens were then cleaned to eliminate remnants of the powder produced through the cutting and polishing process using a strong stream of air and dried under room temperature. The specimens were randomly allocated into nine groups as shown in FI GURE 1 .

Control

110 µm

1 second

1 second

2 seconds

2 seconds

3 seconds

3 seconds

4 seconds

4 seconds

FIGURE 1. Summarized experimental groups used in the present study. The pre-sintered yttria-stabilized zirconia specimens were randomly allocated into one control group and eight tested groups receiving different airborne-particle abrasion protocols. Two aluminium oxide (Al2O3) particle sizes (25 and 110 µm) and four blasting durations (one to four seconds) were used. All nine groups include a control group (control), 25 µm Al2O3 blasting for one second group (25 µm-1s), 25 µm-2s, 25 µm-3s, 25 µm-4s, 110 µm Al2O3 blasting for one second group (110 µm-1s), 110 µm-2s, 110 µm-3s and 110 µm-4s.

An airborne-particle abrasion was performed using a sandblasting machine (Basic eco, Renfert, Hilzingen, Germany). Fine (25 µm) and coarse (110 µm) aluminum oxide particles were used to create submicron-scale and micronscale surface roughness, respectively, under the conditions used in the present study. These roughness scales have been shown to have a positive impact on osseointegration.25–31 Airborne abrasion was used 25 µm and 110 µm aluminum oxide particles (Cobra, Renfert) with varying blasting times under control 1 bar pressure and 20 mm in perpendicular distance from nozzle tip to specimen surface. One-second blasting specimens were blasted in one direction with a single motion and the blasting was repeated two, three and four times in the same manner in two-, three-, and four-

second blasting specimens respectively. Our preliminary study suggested that for most of the roughness parameters studied, longer blasting duration (e.g., five to eight seconds) was found to produce similar roughness values observed on the samples abraded for one to four seconds. Thus, a maximum blasting duration of four seconds was used. After blasting, all specimens were cleaned ultrasonically in 99% isopropanol for three minutes to remove residual Al2O3 particles then sintered to the final temperature at 1600 degrees C in a furnace (Zirkonofen 700 Ultra-Vakuum, Zirkonzahn) as recommended by the manufacturer.50,51 Specimens were cleaned again in an ultrasonic bath of acetone for one minute and absolute ethanol for three minutes then stored in a desiccator for 24 hours before further tests.53 N OVEMBER 2 0 2 1

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Surface Roughness and Topography

Surface roughness was characterized under a confocal laser scanning microscope (OLS4500, Shimadzu, Kyoto, Japan). Four different areas of each of the three specimens were used to measure surface roughness parameters (n = 12). The evaluating parameters include vertical roughness profiles (Ra, Sa, Rq, Rz, Rt), horizontal roughness profiles (Sm, S) and surface area as previously defined by Gadelmawla.54 Ra is defined as the average absolute deviation of the roughness irregularities from the mean line over one sampling length. Sa is defined as the average absolute deviation of the roughness irregularities compared to the arithmetical mean of the surface. Rq, root mean square roughness (RMS), represents the standard deviation of the distribution of surface height. Rq is more sensitive than the arithmetic average height (Ra) to large deviation from the mean line. Rz, 10-point height, is defined as the difference in height between the average of the five highest peaks and the five lowest valleys along the assessment length of the profile. Rmax or Rt is defined as the vertical distance between the highest peak and the lowest valley along the assessment length of the profile. Sm is defined as the mean spacing between profile peaks at the mean line. S is defined as the average spacing of adjacent local peaks of the profile measured along the assessment length. Surface topography and elemental compositions were characterized by scanning electron microscope (SEM) (JCM-6000, Jeol, Tokyo) and energydispersive X-ray spectroscopy (SU5000 FE-SEM, Hitachi, EDS HORIBA, Kyoto, Japan). In this test, three representative areas were randomly selected for each group and SEM surface topography was also analyzed. 700 N OVEMBER

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Surface Hydrophilicity

Surface hydrophilicity was used in the present study to demonstrate the importance of zirconia surface topographical changes by different airborne-particle abrasion protocols. Hydrophilicity has been shown to significantly influence cell adhesion and subsequent osseointegration of the implanted biomaterials because water molecules are rapidly adsorbed within seconds on the implant surface following implant placement.53,54 Surface hydrophilicity was determined

Surface hydrophilicity was used in the present study to demonstrate the importance of zirconia surface topographical changes by different airborneparticle abrasion protocols. by static contact angles using the sessile water drop method under atmospheric conditions at room temperature.55 A drop (0.5 µl) of water was gently deposited on the specimen surface using a droplet dispenser and computer-controlled stage by dispensed 0.5-µl of water so a drop freely hung at the dispenser tip, then moved the stage upward until the drop contacted with the specimen surface and moved the stage downward to detach the drop from the dispenser tip. The water contact angle was measured by a video-based optical contact angle measuring system (OCA 40 Micro, Data Physics Instruments GmbH, Filderstadt, Germany). Four drops were obtained per group. Water contact angles were analyzed 60 seconds after the droplet contacted the surface of specimens.56

Measurements of Fibronectin and Albumin Adsorption

Solutions of human plasma fibronectin (Fn, Harbor Bio-Products, Norwood, Mass.) and bovine serum albumin (Sigma-Aldrich, St. Louis) were prepared at a concentration of 0.5 mg/ml (pH 7.4), and the fully sintered zirconia samples (control, 110 µm-2s and 110 µm-4s) were incubated in the protein solutions at 37 degrees C for 60 minutes. The samples were then washed with PBS to eliminate nonadsorbed protein molecules and then air dried at 37 degrees C. The adsorbed Fn and Alb were extracted and measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, Mass.) according to the manufacturer’s instructions. The amounts of protein adsorbed were calculated as ng/cm2 and the ratio of adsorbed Fn/Alb was calculated accordingly, with the four replicates being performed for two independent experiments with similar results.

Cell Culture

In the present study, human MSCs (Lonza Biologics plc, Cambridge, U.K.) were maintained in a standard medium consisting of the α-minimum essential medium (α-MEM, Gibco Life Technologies Ltd, Paisley, U.K.) containing 15% heat-inactivated fetal calf serum (FCS, PAA Laboratories, Yeovil, U.K.) supplemented with 200 U/ml penicillin, 200 μg/ml streptomycin and 2 mM L-glutamine (all from Gibco) at 37 degrees C in a humidified atmosphere of 5% CO2 in air. The media were changed every two to three days. Cells between passages 5–6 were used.

Cell Adhesion

MSCs were seeded on tested zirconia surfaces at a density of 1.5 x 104 cells/ cm2, and after six hours, the samples were fixed with 4% paraformaldehyde for 10 minutes and stained with rhodamine


C D A J O U R N A L , V O L 4 9 , Nº 11

phalloidin (Thermo Fisher Scientific) for actin and the nucleus counterstained with DAPI (Sigma-Aldrich). The morphology and number of adhered MSCs were examined under a confocal fluorescence microscope (Nikon Ti Eclipse, Nikon Instruments Inc., Melville, N.Y.). The results were expressed as the mean number of attached MSCs ± SD per mm2 derived from three experiments.

Cell Proliferation Index

Flow cytometric analysis was performed using the CytoFLEX Flow Cytometer and the CytExpert software (both from Beckman Coulter, Brea, Calif.) for data acquisition and analysis, respectively. Cells were collected and the cell pellets were resuspended in ice cold 70% ethanol for 30 minutes. Cells were then rinsed twice in PBS and resuspended in 20 μg/ ml propidium iodide (PI) in PBS with 50 μg/ml RNase A (both from Sigma) at 4 degrees C for 30 minutes and subjected to flow cytometry. The distribution of cells in three major phases of the cycle (G0/G1 versus S versus G2/M) was analyzed and the cell proliferation was calculated by the following equation: Proliferation index (%) = (S + G2/M) / (G0/G1 + S + G2/M) x 100% Proliferative index is expressed as mean percentage and the data are presented as the mean percentage ± SD from three independent experiments.

Determination of Osteogenic Differentiation and Mineralization by MSCs

For osteogenic differentiation induction, MSCs were seeded onto the tested surfaces at a density of 1.5 x 104 cells/cm2 and allowed to grow with the standard medium for the first 48 hours until the cells reached

80% confluence. Then, the cells were incubated for 14 to 21 days with an osteogenic medium (OM) (standard medium with 100 nM dexamethasone, 50 μM ascorbate-phosphate and 10 mM β-glycerolphosphate, all from SigmaAldrich). The expressions of runt-related transcription factor 2 (Runx2), type-I collagen (COL-I), alkaline phosphatase (ALP) and osteocalcin (OC) mRNAs were determined on day 14 using a quantitative real-time PCR, and the formation of mineralization on day 21 was examined using alizarin red S staining.

Alizarin red S-stained mineralized matrices observed as bright-red deposits were photographed under a Nikon digital camera ... To determine the expression of osteogenic genes, total RNA was isolated and first strand cDNA was synthesized from 1 μg RNA. The first strand cDNA was subjected to Q-PCR using SYBR Green I dye performed in an iQ5 iCycler (Bio­Rad, Bradford, U.K.), with specific primers for the RUNX2, COL-I, ALP, OCN and GAPDH mRNA. GAPDH was used as an endogenous control. SYBR Green PCR reaction mixtures using SYBR Green I Master kit (Roche Diagnostic Co., Basel, Switzerland) were set up as suggested by the manufacturer. The amplification conditions consisted of 40 cycles at 95 degrees C for 15 seconds, followed by 60 degrees C for 30 seconds and subsequently 72 degrees C for 30 seconds. The specificity of the PCR products was verified by melting

curve analysis. The PCR reactions were performed in six replicates, and each of the gene signal was normalized to the GAPDH signal in the same reaction. The mRNA expression is expressed as mean fold-change of control (1.0). Data are presented as the mean fold-change ± SD from three independent experiments. Primer sequences were as follows: RUNX2 F 5′‐TGGTTACTGTCATGGCGGGTA-3′, R 5′‐TCTCAGATCGTTGAACCTTGCTA-3′; COL-I F 5′‐GAGGGCCAAGACGAAGACATC-3′, R 5′‐CAGATCACGTCATCGCACAAC-3′; ALP F 5′‐ACTGGTACTCAGACAACGAGAT-3′, R 5′‐ACGTCAATGTCCCTGATGTTATG-3′; OC F 5′‐ CACTCCTCGCCCTATTGGC-3′, R 5′‐ CCCTCCTGCTTGGACACAAAG-3′; GAPDH F 5′‐ CTGGGCTACACTGAGCACC-3′, R 5′‐ AAGTGGTCGTTGAGGGCAATG-3′.57,58 For the alizarin red S staining, the samples were fixed with cold methanol for 30 minutes at 4 degrees C and washed with distilled water. The 1% alizarin red S solution (pH 4.2, Sigma) was added and incubated for 10 minutes at room temperature, and the samples were rinsed twice with methanol to remove unbound alizarin red S. Alizarin red S-stained mineralized matrices observed as bright-red deposits were photographed under a Nikon digital camera, and the amount of stained dye was eluted by 10% cetylpyridinium chloride and measured spectrophotometrically at 560 nm. The mineralization is expressed as mean foldchange of the absorbance of control (1.0).

Statistical Analyses

To compare means of surface roughness and water contact angles between experimental groups and the control N OVEMBER 2 0 2 1

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(25 µm Al2O3 blasting group versus control and 110 µm Al2O3 blasting group versus control), the one-way analysis of variance (ANOVA) was used followed by Tukey’s post-hoc test for multiple comparisons. Pearson’s correlation coefficient was used to evaluate the relationship between surface roughness and water contact angle. In addition, the linear regression model was performed to assess the association between particle size, blasting duration and water contact angle after adjusting for covariates (SPSS software, version 23, IBM, Armonk, N.Y). A value of p < 0.05 was considered statistically significant for all analyses.

A

B

Results Surface topographies of fully sintered zirconia were influenced by abrasive particle size and blasting time used in repeated airborne-particle abrasion in the pre-sintering stage.

C

D FIGURE S 2 . Scanning electron micrographs of Al2O3 abrasive particles, pre-sintered zirconia surface following presintered zirconia airborne-particle abrasion and fully sintered zirconia surface following pre-sintered zirconia airborneparticle abrasion. Al2O3 particle sized 25 µm and 110 µm showing polygonal shape which average sizes of 25.54 µm and 102.93 µm, respectively (x100 magnification) (2A). Pre-sintered zirconia surface after airborne-particle abrasion of control (nonblasting group), (i) 25 µm Al2O3 blasting group at different blasting times, (ii) 110 µm Al2O3 blasting group at different blasting time (x500 magnification) (2B). Fully sintered zirconia surface treated by airborne-particle abrasion of control and blasting groups as described in 2B (x500 magnification) ( 2C). EDS spectra of fully sintered zirconia surface after pre-sintered zirconia airborne-particle abrasion (2D). Note that a very low Al signal (0.1 wt%) was detected.

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We first examined the shape and size of the commercial Al2O3 particles, and the SEM results showed that both 25 µm and 110 µm Al2O3 particles appeared to be polygonal and vary in size with average sizes of approximately 25.5 ± 5.7 µm and 102.9 ± 17.7 µm, respectively (FIGURE 2A ). In FIGURE 2B , SEM micrographs showed different topographical appearances of pre-sintered zirconia after airborne-particle abrasion. The control surface was relatively smooth with characteristics of polished surface with silicon-carbide grit papers, which could also be observed only in the 25 µm-1s group (FIGURE 2B ). In general, the surface of all pre-sintered samples became roughened with increasing blasting time in both 25 µm and 110 µm groups, except the 110 µm-4s group, the surface of which appeared smoother than the 110 µm-3s group (FIGURE 2B ). The results also demonstrated that the samples in 110 µm groups were rougher


C D A J O U R N A L , V O L 4 9 , Nº 11

than the corresponding samples in the 25 µm groups. All the surface topography of fully sintered zirconia previously receiving pre-sintered zirconia airborneparticle abrasion (FI GURE 2C ) seemed to have similar patterns that were observed in the corresponding presintered specimens. This suggests that the surface characteristics of the fully sintered zirconia surface were influenced by particle size and blasting time that were used in the pre-sintered zirconia airborne-particle abrasion protocol. To determine whether ultrasonic cleansing before the sintering process could eliminate any possible Al2O3 particle remnants, ultrasonic cleansing as described in the materials and methods was performed. The SEM-EDS was used to examine the presence of any possible Al element remaining on the fully sintered zirconia. The results showed that the main composition of both control and tested surfaces consisted of Zr and O (approximately 95 wt%) and very little (0.1 wt%) of Al residues was detected (FIGURE 2D ). This indicated that ultrasonic cleansing was an effective procedure to clean the surface before the sintering process.

Abrasive particle size and blasting duration in repeated pre-sintering airborne-particle abrasion affected surface roughness cycle.

Surface topographical characteristics of the fully sintered zirconia following repeated pre-sintering airborne-particle abrasion were analyzed in terms of vertical roughness parameters (Ra, Sa, Rq, Rz and Rt) and horizontal roughness parameters (S and Sm) and surface area. Almost all the tested groups possessed higher values of all the roughness parameters and surface area compared with the control group (p < 0.05) (FIGURE 3A ). In the 25 µm-1s

group, however, only the Ra, Rq and Rz were statistically higher than those of the control group (Ra and Rq p = 0.003; Rz p = 0.002) whereas the Sa and Rt showed a nonsignificant difference (p > 0.05). Peak values of vertical roughness parameters (FIGURE 3A ) in the 25 µm and 110 µm groups were at two seconds and three seconds repeated blasting duration, respectively, whereas the peak values of horizontal roughness parameter (FIGURE 3B ) in the 25 µm and 110 µm groups were at three to four seconds and two seconds repeated blasting

The SEM-EDS was used to examine the presence of any possible Al element remaining on the fully sintered zirconia.

duration, respectively. In FIGURE 3C , repeated pre-sintering airborne-particle abrasion using 25 µm abrasive particles did not increase the surface area of the fully sintered samples regardless of the blasting duration (p < 0.05), but 110 µm abrasive particles statistically significantly increased the surface area at all blasting times used, with the peak surface area being observed with three seconds repeated blasting duration (p < 0.05). Taken together, using 110 µm abrasive particles in pre-sintering airborne-particle abrasion of zirconia was more effective in creating increased surface roughness and surface area, and optimal, but not the longest, duration was required to obtain fully sintered zirconia with maximal values of these parameters, suggesting the presence

of surface roughness cycles following repeated pre-sintering airborne-particle abrasion. One cycle of surface roughness profiles thus included mild roughness at one second of blasting duration, maximum roughness during repeated blasting for two to three seconds and subsequently lesser roughness in the last phase of the cycle (i.e., repeated blasting for four seconds).

Increased surface hydrophilicity of a fully sintered zirconia was obtained by an optimal pre-sintering airborne-particle abrasion protocol with maximum surface roughness.

In the present study, surface hydrophilicity was determined by water contact angle and under the conditions used here, only the 110 µm-2s group was found to have increased surface hydrophilicity by statistically significantly decreased water contact angle from approximately 70 degrees in the control group to 60 degrees (p = 0.005) (FIGURE 4 ). Statistical analyses further suggested that surface hydrophilicity was correlated with blasting time and horizontal, but not vertical, roughness profiles (Ra, Sa, Rq, Rz and Rt, p > 0.05). Pearson’s correlation coefficient revealed that water contact angle was moderately negatively correlated with horizontal roughness profiles, S (r = – 0.36; p = 0.039) and Sm (r = – 0.30; p=0.085). However, only S value, indicating average spacing of adjacent local peaks of the profile measured along the assessment length, was statistically significantly correlated with water contact angle (TA BLE 1 ). A linear regression model concurrently assessed associations between water contact angle, particle size and blasting time after being adjusted for horizontal roughness profile. The results showed that only blasting time and S value were statistically significant predictors for N OVEMBER 2 0 2 1

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25 µm

Ra

Ra (µm) 1.2

Sa

Sa (µm) 1.2

0.8 C 0.4

C

C

0.4

A 0.0

control

1s

1.2

0.8

B

2s

3s

4s

Time (sec.)

0.0

A

A,B

control

1s

C

2s

C

3s

4s

B

0.4 Time (sec.)

0.0

C

C

C

2s

3s

4s

A control

1s

Time (sec.)

Rt

Rt (µm) 8 6

4 A

2 0

0.8

C

Rz

Rz (µm) 6

Rq

Rq (µm) 1.6

B

B

1s

A

2

A control

B

4

B

2s

3s

4s

Time (sec.)

0

B

B

3s

4s

A control

1s

2s

Time (sec.)

110 µm

Ra

Ra (µm) 1.2 B

B,C

B,C

B

1.2

0.8

B

B

0.4

A control

1s

2s

3s

4s

Time (sec.)

0.0

Rz

Rz (µm) 6 B

B

C

1.2

B

B,C

1s

2s

B

0.8 A

0.4

control

1s

2s

3s

4s

Time (sec.)

8 B

B

B

1s

2s

6

4

0.0

Rt

Rt (µm)

C

B

Rq

Rq (µm) 1.6

0.8

0.4 0.0

Sa

Sa (µm) C

A control

3s

4s

Time (sec.)

B B

4 2 0

2

A control

1s

2s

3s

4s

Time (sec.)

0

A control

3s

4s

Time (sec.)

FIGURE S 3A

FIGURE S 3 . Surface roughness of fully sintered zirconia surfaces following different pre-sintered zirconia airborne-particle abrasion protocols. Vertical roughness profiles (Ra, Sa, Rq, Rz, Rt) (3A). Horizontal roughness profiles (S, Sm) (3B). Surface area (3C). The results from one-way ANOVA and Tukey’s post hoc test explain as characters above bar charts. Different character indicates a statistically significant difference (p < 0.05). A proposed diagram demonstrating the representative profile of a surface roughness cycle consisting of up and down surface roughness changes and the surface roughness trendline during repeated blasting for one to four seconds using abrasive particles sized 110 µm under 1 bar of blasting pressure (3D). This suggests that for obtaining maximum surface roughness, optimal blasting time is pivotal.

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25 µm

S

S (µm) 10 8

B,C

6

A

water contact angle. A blasting time at four seconds increased water contact angle (β = 15.21; p < 0.001) while an increasing of S value decreased water contact angle (β = –2.47; p < 0.001) (TA BLE 2 ).

4

In vitro biological responses of 110 µm-2s and 110 µm-4s surfaces.

110 µm

To test whether the most hydrophilic surface, which showed strong correlation with the S roughness value, possessed good in vitro biological responses, the effects of 110 µm-2s (higher hydrophilic and S value) and 110 µm-4s (lesser hydrophilic and S value) surfaces on protein adsorption and adhesion, proliferation, osteogenic differentiation and mineralization of MSCs were examined. The results showed that compared with the control machined surface, only the 110 µm-2s, but not 110 µm-4s, surface showed a significantly higher Fn/Alb adsorption ratio (FIGURE 5 A ). A significantly higher MSC adhesion to the 110 µm-2s surface was observed when compared with that in the control surface (FIGURE 5 A ), whereas only less than a 20% increase in MSC adhesion was evident on 110 µm-4s (FIGURE 5B ). In addition, only limited cell cytoplasmic spreading was seen in the control surface; both 110 µm-2s and 110 µm-4s surfaces appeared to be better substrates for MSC spreading (FIGURE 5B ). In contrast to their stimulatory effect on cell adhesion, 110 µm-2s and 110 µm-4s surfaces showed only a comparable effect to the control surface regarding MSC proliferation, osteogenic differentiation of MSCs and formation of mineralization (FIGURES 5C–5E , respectively).

0

control

B,C

A,C

A

20

1s

2s

3s

4s

Time (sec.)

S C

8

0

control

Sm (µm) 40 B,C

B

A,B

A

1s

2s

3s

4s

Time (sec.)

Sm B

B

30

B

B

A

20

4

10

2 0

A

10

S (µm) 10

6

B

30

A,B

2

control

1s

2s

3s

4s

Time (sec.)

0

control

1s

2s

3s

4s

Time (sec.)

FIGURE S 3B 25 µm

110 µm

Surface area (x103 µm2)

Surface area

Surface area (x103 µm2) 81

77 73 69 65

Surface area

77 B

73 A control

A 1s

A

2s

A

3s

A

4s

69 Time (sec.)

65

B,C

C B,C

A control

1s

2s

3s

4s

Time (sec.)

FIGURE S 3C

Surface roughness

Discussion

In the present study, the surface roughness of zirconia treated by pre-sintered airborne-particle abrasion was improved significantly compared with the control

A

Sm

Sm (µm) 40

C

1 2 3 4

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TABLE 1

TABLE 2

Correlations Between Water Contact Angle and Roughness Profile

Results of Linear Regression Model Predicting Contact Angle, Adjusted for S, Sm

Roughness profile

r

Df

p-value

Particle size

Ra

–0.09

33

0.615

Sa

–0.08

23

0.734

Rq

–0.11

33

0.563

Rz

–0.14

33

0.454

Rt

–0.21

33

0.239

S

–0.36

33

0.039*

Sm

–0.30

33

0.085

Surface area

–0.08

23

0.734

β -Coefficient

Variables 25 µm

(ref.)

110 µm

–0.54

1s 2s

3.61

(–1.33, 8.56)

0.145

2.60

(–1.88, 7.08)

0.244

4s

15.21

(9.94, 20.47)

< 0.001*

S

–2.47

(–3.57, –1.36)

< 0.001*

Sm

–0.08

(–0.38, 0.21)

0.557

Constant

81.13

(75.21, 87.05)

< 0.001*

*: Indicates a statistically significant association.

2s

3s

4s

1s

2s

3s

4s

25 µm

110 µm FIGURE 4A 25 µm

110 µm

Water contact angle

Contact angle (0)

100 75

100 A

A

A

75

50

50

25

25

0

control

1s

2s

3s

4s

Time (sec.)

0

Water contact angle

A,D

control

A,B

1s

B,C

2s

A,C

3s

A,D

4s

Time (sec.)

FIGURE S 4B FIGURE S 4 . Surface hydrophilicity of the fully sintered zirconia samples following pre-sintered zirconia airborne-particle abrasion. Water droplets on zirconia surfaces of control (nonblasting group), 25 µm Al2O3 blasting group at different blasting times, and 110 µm Al2O3 blasting group at different blasting times (4A). Mean water contact angles (degree) of control group and experimental groups (25 µm and 110 µm Al2O3 blasting group at different blasting times) (4B). The results from one-way ANOVA and Tukey’s post-hoc test explain as characters above bar charts. Different character indicates a statistically significant difference (p < 0.05).

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0.763

3s

1s

A

(–4.17, 3.09)

(ref.)

Control

A

p-value

Blasting time

r: Pearson’s correlation coefficient *: Indicates a statistically significant correlation

Contact angle (0)

(95% CI)

surface. This increase in roughness is within the range reported by several previous studies that demonstrated that zirconia treated by pre-sintered airborneparticle abrasion increased the surface roughness (Ra) by three to 17 times compared to the unmodified control surface.38,39,44,46,51,59 Due to the lack of standardized methods, surface roughness values reported in the literature generally vary tremendously. It has been suggested that a moderate rough surface with Ra or Sa of 1-2 µm is optimal for dental implants and possess higher bone responses over a smoother or rougher surface.25,30 However, a rougher implant surface might have a greater risk to develop peri-implantitis due to bacterial accumulation.60,61 In the present study, the roughness obtained from presintering airborne-particle abrasion with 110 µm Al2O3 showed surface roughness with Sa = 0.94-1.28 µm and Ra = 0.73-0.99 µm while 25 µm Al2O3 blasting resulted in surface roughness with Sa = 0.38-0.51 µm and Ra = 0.23-0.40 µm. Therefore, the method carried out in the present study provided surface roughness within the suitable range for enhancing performance of a dental implant. A number of considerations should be weighed to obtain optimal conditions for pre-sintered


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Adsorbed amount (ng/cm2) 5 4 3 2 1 0

Protein adsorption

B

Control

110 µm-2s

110 µm-4s

45 ± 10A

80 ± 12B

55 ± 10 A

A,B

A

control

110 µm-2s

110 µm-4s

FIGURE 5A

FIGURE 5B

Proliferation index (%)

5

100

4

80 60 40

A

A

NS _________

NS _________

Control 110 µm-2s

NS _________

3

A

NS _________

110 µm-4s

2

20 0

Osteogenic gene expression

mRNA expression

Cell proliferation

1 control

110 µm-2s

0

110 µm-4s

FIGURE 5C

airborne-particle abrasion. These considerations are discussed below. Extensive surface damage in presintered Y-TZP produced in the CNC milling process has been reported.62 It has been suggested that this machininginduced damage cannot be naturally removed during the subsequent sintering process, and thus can cause stress concentrations under mechanical loads.63 This damage causes weakly interconnected porous structures in the pre-sintered state, thus resulting in intragranular and transgranular fractures.63 The currently studied method of pre-sintering airborneparticle abrasion is therefore suitable for eliminating such damage after the milling process. Although some scratches from the polishing process remained on the surface of the 25 µm-1s group as shown in FIGURE 2C , the other conditions showed no evidence of those scratches, suggesting that optimized pre-sintering airborne-particle abrasion conditions could effectively remove the

Runx2

COL-I

ALP

OC

FIGURE 5D

Control

110 µm-2s

110 µm-4s

1.0 A

0.9A

1.1A

FIGURE 5E FIGURE S 5 . Biological responses of 110 µm-2s and 110 µm-4s surfaces. Fn-to-Alb adsorption ratio was measured using a BCS protein assay kit (5A). Representative immunofluorescence images showing cell attachment on different surfaces for six hours (5B). The number of attached MSCs per mm2 were shown below the images. Cell proliferation was determined by the Proliferation Index of the cells cultured on the different surface for 48 hours (5C). Expression of osteogenic genes in MSCs cultured under osteogenic medium for 14 days (5D). Representative images of alizarin red S staining of cells cultured under osteogenic induction for 21 days (5E). Amounts of the extracted staining were determined and compared to the control surface (defined as 1.0). The results are expressed as means ± SD derived from three experiments. Different character indicates a statistically significant difference (p < 0.05).

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milling process-induced damage of the pre-sintered surface of the zirconia. Under the conditions tested, the repeated blasting for four seconds using 110 µm Al2O3 particles constituted one cycle, which demonstrated mild roughness at one second, maximum roughness during repeated blasting for two to three seconds and subsequently less roughness in the last phase of the cycle (i.e., repeated blasting for four seconds). It is possible that increasing blasting time to five to six seconds could add a more complete profile of one cycle and that a longer blasting duration may produce another cycle of surface roughness profile with reduced thickness of the zirconia due to the loss of material from the surface. It has been shown that a high number of grits hitting the substrate modified the uppermost roughen surface, thus resulting in a flattened surface.64 Compared with 110 µm abrasive particles, the use of abrasive particles sized 25 µm also demonstrated a similar profile of surface roughness but possibly with a longer blasting duration constituting of one complete cycle and lower values of all of the roughness parameters. These present results, therefore, indicated that the surface roughness of fully sintered zirconia was markedly dependent on abrasive particle size and blasting duration used in presintering airborne-particle abrasion. Moreover, our unpublished data showed that initial surface roughness (i.e., created by machining) influenced the roughness cycle profile. It is possible that different manufacturing systems and different types of zirconia-based material may produce pre-sintered zirconia implants with different surface roughness, suggesting the need for optimization of the pre-sintering airborne-particle abrasion in order to achieve the optimal surface roughness 708 N OVEMBER

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and hydrophilicity with minimum loss of abraded material for each of the systems used. A representative profile of a surface roughness cycle consisting of up and down surface roughness changes and the surface roughness trendline during the repeated blasting for one to four seconds using abrasive particles sized 110 µm under 1 bar of blasting pressure is proposed in FIGURE 3D . This shows the changes of surface roughness with an increasing abrasion time under indicated conditions, suggesting that for obtaining maximum surface roughness,

Volume loss of zirconia during the pre-sintering abrasion may affect the mechanical properties of the resulting sintered zirconia.

optimal blasting time is pivotal and depends on conditions used in the pre-sintering airborne-particle abrasion protocol. It is important to note that the pre-sintering airborne-particle abrasion protocol was well defined and controlled to ensure precise effect of increasing times for repeated blasting on the surface roughness. To the best of our knowledge, the present study is the first to demonstrate the profile of the surface roughness cycle of fully sintered zirconia following repeated pre-sintering airborne-particle abrasion. Undefined blasting times reported in previous studies makes comparison with our results impossible.38,39,44–47,49–51,59,65–67 Volume loss of zirconia during the pre-sintering abrasion may affect the mechanical properties of the resulting

sintered zirconia. A small volume loss of zirconia in the present study may be estimated from the surface roughness, as previously described.68 Previous studies suggest that air-abrasion pressure and blasting duration have positive correlation to the amount of surface loss.51,52 Using low air-abrasion pressure (1 bar) and short blasting durations (one to four seconds), the protocols used in the present study are thus unlikely to cause a significant amount of zirconia loss during the abrasion process. It is noteworthy that air abrasion may initiate some flaws or microcracks in zirconia structure, although we did not investigate this aspect in the current study. Abi-Rached and colleagues reported in 2015 that the flexural strength of abraded and nonabraded sintered materials showed no difference.49 The study also confirmed by XRD analysis that abraded and nonabraded sintered materials contained no monoclinic phase (0.0%wt) because of the sintering process. This can imply that the grain of sintered materials may rearrange after air abrasion from phase toughening and reverse to a tetragonal phase as reported by Monaco and colleagues in 2013.38 We assumed that with low pressure of air abrasion, a very small amount of microcrack can occur, but it may not be significant when the air abrasion was performed before the sintering process. However, the aging degradation of the material and different sintering moments should be further investigated. Previous studies suggest that residual aluminum oxide particles on a dental implant surface following airborneparticle abrasion could negatively affect osseointegration and diminish the clinical success.69,70 In the present study, elemental composition analysis showed that no residual alumina particles on


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a fully sintered surface receiving presintering airborne-particle abrasion were found. This is consistent with previous studies that reported that no byproduct or contamination of residual alumina particles were found on a modified surface after receiving pre-sintering airborne-particle abrasion and ultrasonically cleaned with 99% isopropanol for 10 minutes.45,65 In contrast, Monaco suggested that blasting zirconia in a pre-sintered state induced surface contamination from alumina embedding after airborneparticle abrasion.38 Martins also found alumina residue remained on a fully sintered surface after blasting presintered zirconia with Al2O3 under 0.5 bar pressure and a working distance of 10 mm.50 The explanation of this discrepancy may be the differences in air pressure and the working distance used. We again emphasize the importance of the optimization of each parameter used in pre-sintering airborne-particle abrasion. It is also noteworthy that the cleansing method used in our study was ultrasonic cleansing with isopropanol to remove any possible remaining Al2O3 particles. This also shows that the cleansing agent is one of the important factors. Among several surface roughness parameters tested in the present study, only the S value, which is defined as the average spacing of adjacent local peaks of the profile measured along the assessment length, was found to be statistically correlated with the surface hydrophilicity of fully sintered zirconia from the linear regression model (p < 0.001). The strong correlation of these two surface properties can be explained by the Wenzel theory, which stated that adding surface roughness can enhance hydrophilicity based on the assumption that the liquid penetrates

into the roughness grooves.71 Although several previous studies reported the use of Ra and Sa to exhibit surface roughness that represents only vertical roughness profiles,38,39,46,47,50,51 we propose that other surface roughness values, especially the horizontal parameter, e.g., S values, must be included when performing analysis of surface roughness and hydrophilicity. Following implant placement, it is generally accepted that MSCs play a key role in bone healing and osseointegration of dental implants. Certain serum proteins adsorbed on the implant

Zirconia dental implants have been shown to be a successful alternative to titanium dental implants in the aesthetic zone …

surface may regulate MSC adhesion, proliferation and differentiation toward an osteoblast lineage.72 Responses of MSCs to the modified zirconia surfaces were thus investigated to assess the biological significance of the zirconia surface modification technique. The present results also showed that the most hydrophilic 110 µm-2s surface, which correlates well with the S roughness value, had enhanced in vitro biological responses by promoting Fn-to-Alb adsorption ratio and initial MSC adhesion compared with either the control machined surface or 110 µm-4s surface. The levels of Fn-to-Alb adsorption ratio, initial MSC adhesion, surface hydrophilicity and S value of the three surfaces appeared in the following order (from high to low): 110 µm-2s

> 110 µm-4s > control, adding more evidence to support the importance of the S value. Although, MSCs grown on the 110 µm-2s surface showed comparable ability to proliferate, differentiate and form mineralization compared to those grown on the 110 µm-4s and control surfaces, the 110 µm-2s surface-enhanced Fnto-Alb adsorption ratio and initial MSC adhesion could hasten the osseointegration process. The adhesive protein fibronectin enhances osteoblast adhesion whereas the nonadhesive proteins, such as albumin, appear to be an inhibitor for osteoblast adhesion,72 consistent with the finding in this study that high Fn-to-Alb adsorption ratio of the 110 µm-2s surface is associated with increased MSC adhesion. This is supported by previous studies.59,73 However, a lack of effect on osteoblast differentiation and mineralization by MSCs shown in the present study is inconsistent with a previous report.74 This could be due to a difference in the resulting surface roughness, cell type used and type and chemistry of the zirconia used. Further studies to test these hypotheses would help gain more knowledge about cellular responses to zirconia. These data suggest that well-optimized pre-sintering airborneparticle abrasion of zirconia, such as the 110 µm-2s surface tested in the present study, may improve osseointegration of a zirconia dental implant by increasing MSC adhesion through increased surface roughness and hydrophilicity. Zirconia dental implants have been shown to be a successful alternative to titanium dental implants in the aesthetic zone, where a deficiency in alveolar bone volume is common. Adequate peri-implant tissues in the aesthetic zone is thus important for long-term maintenance of bone levels N OVEMBER 2 0 2 1

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and aesthetic outcomes. An immediate restoration protocol to replace a natural tooth in the aesthetic zone may require simultaneous MSC-based bone tissue engineering for reconstruction of alveolar bone deficiency.75 The improved surface properties of modified zirconia demonstrated in the present in vitro study might help maintain the number of MSCs (simultaneously loaded while placing dental implants) that are sufficient to regenerate bone in vivo. Further studies on the optimal surface characteristics of zirconia that can also stimulate MSC proliferation and osteogenic potency will enable the advancement and better understanding of stem cell research and implant dentistry. A key strength of the present study is the well-defined and well-controlled pre-sintering airborne-particle abrasion protocol. The present study is the first to demonstrate the profile of the surface roughness cycle of fully sintered zirconia following repeated pre-sintering airborne-particle abrasion. The key findings of the study suggest some potential clinical implications. First, over-repeating airborne-particle abrasion leads to lower surface roughness. Maximum surface roughness could be achieved only by optimal blasting duration. Second, hydrophilicity could be predicted by certain surface roughness parameters. There is a significant association between the surface roughness parameters, especially horizontal roughness profiles (S and Sm) and the zirconia surface hydrophilicity. Thus, it might be possible to evaluate the surface roughness and approximate for biological responses of the dental implant surface. The present study has some limitations, as only one type of zirconia was used. Optimization of this method for other types of zirconiabased dental implants remains challenging. In addition, this study lacks 710 N OVEMBER

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the evaluation of key mechanical properties of the modified zirconia samples. This has a significant impact on the success of a dental implant.

Conclusion

In conclusion, the present study has shown, for the first time, that repeated pre-sintered zirconia airborne-particle abrasion with increasing blasting times resulted in a range of surface roughness with up and down changes of roughness, constituting one roughness cycle. The abrasive particle size and blasting duration significantly influenced surface roughness, hydrophilicity and certain in vitro biological responses of fully sintered zirconia. Well-optimized presintering airborne-particle abrasion of the zirconia surface could accomplish the highly roughened and hydrophilic surface of a fully sintered zirconia dental implant with improved Fn-to-Alb adsorption ratio and MSC adhesion. This inexpensive and simple airborne-particle abrasion technique may be suitable for modifying the surface of zirconia in order to facilitate osseointegration and enhance the performance of zirconia-based dental implants. n AC KN OW LE DGM E N T S This study was financially supported by the National Metal and Materials Technology Center (Grant number P-18-50741 (MT-B-61-BMD-13-231-G)), Thailand, and Research Unit in Mineralized Tissue Reconstruction, Thammasat University, Thailand. We thank Terawat Tosiriwatanapong, Anucha Wannagon, Pattarawan Choeycharoen and Kannaporn Pooput for their technical assistance. RE FE RE N CE S 1. Moraschini V, Poubel LAC, Ferreira VF, Barboza ESP. Evaluation of survival and success rates of dental implants reported in longitudinal studies with a follow-up period of at least 10 years: A systematic review. Int J Oral Maxillofac Surg 2015 Mar;44(3):377–88. doi: 10.1016/j. ijom.2014.10.023. Epub 2014 Nov 20. 2. Leonhardt Å, Gröndahl K, Bergström C, Lekholm U. Long-term follow-up of osseointegrated titanium implants using clinical, radiographic and microbiological parameters. Clin Oral Implants Res 2002 Apr;13(2):127–32. doi: 10.1034/j.1600-0501.2002.130202.x.

3. Buser D, Janner SF, Wittneben JG, et al. 10-year survival and success rates of 511 titanium implants with a sandblasted and acid-etched surface: A retrospective study in 303 partially edentulous patients. Clin Implant Dent Relat Res 2012 Dec;14(6):839–51. doi: 10.1111/j.17088208.2012.00456.x. 4. Chappuis V, Buser R, Brägger U, et al. Long-term outcomes of dental implants with a titanium plasma-sprayed surface: A 20-year prospective case series study in partially edentulous patients. Clin Implant Dent Relat Res 2013 Dec;15(6):780– 90. doi: 10.1111/cid.12056. Epub 2013 Mar 18. 5. Lops D, Stellini E, Sbricoli L, et al. Influence of abutment material on peri‐implant soft tissues in anterior areas with thin gingival biotype: A multicentric prospective study. Clin Oral Implants Res 2017 Oct;28(10):1263–1268. doi: 10.1111/ clr.12952. Epub 2016 Oct 3. 6. Bianco P, Ducheyne P, Cuckler J. Local accumulation of titanium released from a titanium implant in the absence of wear. J Biomed Mater Res 1996 Jun;31(2):227–34. doi: 10.1002/(SICI)1097-4636(199606)31:2<227::AIDJBM9>3.0.CO;2-P. 7. Weingart D, Steinemann S, Schilli W, et al. Titanium deposition in regional lymph nodes after insertion of titanium screw implants in maxillofacial region. Int J Oral Maxillofac Surg 1994 Dec;23(6 Pt 2):450–2. doi: 10.1016/s09015027(05)80045-1. 8. Fage SW, Muris J, Jakobsen SS, Thyssen JP. Titanium: A review on exposure, release, penetration, allergy, epidemiology and clinical reactivity. Contact Dermatitis 2016 Jun;74(6):323–45. doi: 10.1111/cod.12565. Epub 2016 Mar 29. 9. Hosoki M, Nishigawa K, Miyamoto Y, Ohe G, Matsuka Y. Allergic contact dermatitis caused by titanium screws and dental implants. J Prosthodont Res 2016 Jul;60(3):213–9. doi: 10.1016/j.jpor.2015.12.004. Epub 2016 Jan 8. 10. Hosoki M, Nishigawa K, Tajima T, Ueda M, Matsuka Y. Cross-sectional observational study exploring clinical risk of titanium allergy caused by dental implants. J Prosthodont Res 2018 Oct;62(4):426–431. doi: 10.1016/j. jpor.2018.03.003. Epub 2018 Apr 16. 11. Yan H, Afroz S, Dalanon J, et al. Metal allergy patient treated by titanium implant denture: A case report with at least four-year follow-up. Clin Case Rep 2018 Aug 28;6(10):1972–1977. doi: 10.1002/ccr3.1753. eCollection 2018 Oct. 12. Siddiqi A, Payne AG, De Silva RK, Duncan WJ. Titanium allergy: Could it affect dental implant integration? Clin Oral Implants Res 2011 Jul;22(7):673–680. doi: 10.1111/j.16000501.2010.02081.x. Epub 2011 Jan 20. 13. Denry I, Kelly JR. State of the art of zirconia for dental applications. Dent Mater 2008 Mar;24(3):299–307. doi: 10.1016/j.dental.2007.05.007. Epub 2007 Jul 19. 14. Wenz HJ, Bartsch J, Wolfart S, Kern M. Osseointegration and clinical success of zirconia dental implants: A systematic review. Int J Prosthodont Jan–Feb 2008;21(1):27–36. 15. Depprich R, Zipprich H, Ommerborn M, et al. Osseointegration of zirconia implants compared with titanium: An in vivo study. Head Face Med 2008 Dec 11;4:30. doi: 10.1186/1746-160X-4-30. 16. Aboushelib MN, Salem NA, Taleb ALA, El Moniem NMA. Influence of surface nano-roughness on osseointegration of zirconia implants in rabbit femur heads using selective infiltration etching technique. J Oral Implantol 2013


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Oct;39(5):583–90. doi: 10.1563/AAID-JOI-D-11-00075. Epub 2011 Sep 9. 17. Hoffmann O, Angelov N, Zafiropoulos GG, Andreana S. Osseointegration of zirconia implants with different surface characteristics: An evaluation in rabbits. Int J Oral Maxillofac Implants Mar–Apr 2012;27(2):352–8. 18. Gahlert M, Röhling S, Wieland M, et al. Osseointegration of zirconia and titanium dental implants: A histological and histomorphometrical study in the maxilla of pigs. Clin Oral Implants Res 2009 Nov;20(11):1247–53. doi: 10.1111/j.1600-0501.2009.01734.x. Epub 2009 Jun 15. 19. Gahlert M, Roehling S, Sprecher C, et al. In vivo performance of zirconia and titanium implants: A histomorphometric study in mini pig maxillae. Clin Oral Implants Res 2012 Mar;23(3):281–6. doi: 10.1111/j.16000501.2011.02157.x. Epub 2011 Aug 2. 20. Rocchietta I, Fontana F, Addis A, Schupbach P, Simion M. Surface-modified zirconia implants: Tissue response in rabbits. Clin Oral Implants Res 2009 Aug;20(8):844–50. doi: 10.1111/j.1600-0501.2009.01727.x. 21. Al-Radha ASD, Dymock D, Younes C, O’Sullivan D. Surface properties of titanium and zirconia dental implant materials and their effect on bacterial adhesion. J Dent 2012 Feb;40(2):146–53. doi: 10.1016/j.jdent.2011.12.006. Epub 2011 Dec 9. 22. Scarano A, Piattelli M, Caputi S, Favero GA, Piattelli A. Bacterial adhesion on commercially pure titanium and zirconium oxide disks: An in vivo human study. J Periodontol 2004 Feb;75(2):292–6. doi: 10.1902/jop.2004.75.2.292. 23. Maltagliati A, Angiero F, Zaky S, Blasi S, Ottonello A. Reduction of bacterial proliferation by zirconium collar in dental implants. Annu Res Rev Biol 2018;23(1):1–8. doi: 10.9734/ ARRB/2018/38270. 24. Rimondini L, Cerroni L, Carrassi A, Torriceni P. Bacterial colonization of zirconia ceramic surfaces: An in vitro and in vivo study. Int J Oral Maxillofac Implants Nov–Dec 2002;17(6):793–8. 25. Shalabi M, Gortemaker A, Van’t Hof M, Jansen J, Creugers N. Implant surface roughness and bone healing: A systematic review. J Dent Res 2006 Jun;85(6):496–500. doi: 10.1177/154405910608500603. 26. Sennerby L, Dasmah A, Larsson B, Iverhed M. Bone tissue responses to surface-modified zirconia implants: A histomorphometric and removal torque study in the rabbit. Clin Implant Dent Relat Res 2005;7 Suppl 1:S13–20. doi: 10.1111/j.1708-8208.2005.tb00070.x. 27. Kohal RJ, Wolkewitz M, Hinze M, et al. Biomechanical and histological behavior of zirconia implants: An experiment in the rat. Clin Oral Implants Res 2009 Apr;20(4):333–9. doi: 10.1111/j.1600-0501.2008.01656.x. 28. Gahlert M, Gudehus T, Eichhorn S, et al. Biomechanical and histomorphometric comparison between zirconia implants with varying surface textures and a titanium implant in the maxilla of miniature pigs. Clin Oral Implants Res 2007 Oct;18(5):662–8. doi: 10.1111/j.16000501.2007.01401.x. Epub 2007 Jun 30. 29. Albrektsson T, Wennerberg A. Oral implant surfaces: Part 1 — review focusing on topographic and chemical properties of different surfaces and in vivo responses to them. Int J Prosthodont Sep–Oct 2004;17(5):536–43. 30. Wennerberg A, Albrektsson T. Effects of titanium surface topography on bone integration: A systematic review. Clin Oral Implants Res 2009 Sep;20 Suppl 4:172–84. doi:

10.1111/j.1600-0501.2009.01775.x. 31. Hempel U, Hefti T, Kalbacova M, et al. Response of osteoblast-like SAOS-2 cells to zirconia ceramics with different surface topographies. Clin Oral Implants Res 2010 Feb;21(2):174–81. doi: 10.1111/j.16000501.2009.01797.x. Epub 2009 Aug 25. 32. Schünemann FH, Galárraga-Vinueza ME, Magini R, et al. Zirconia surface modifications for implant dentistry. Mater Sci Eng C Mater Biol Appl 2019 May;98:1294–1305. doi: 10.1016/j.msec.2019.01.062. Epub 2019 Jan 16. 33. Aboushelib MN, Kleverlaan CJ, Feilzer AJ. Selective infiltration-etching technique for a strong and durable bond of resin cements to zirconia-based materials. J Prosthet Dent 2007 Nov;98(5):379–88. doi: 10.1016/S00223913(07)60123-1. 34. Bacchelli B, Giavaresi G, Franchi M, et al. Influence of a zirconia sandblasting treated surface on peri-implant bone healing: An experimental study in sheep. Acta Biomater 2009 Jul;5(6):2246–57. doi: 10.1016/j.actbio.2009.01.024. Epub 2009 Jan 31. 35. Abi-Rached FO, Martins SB, Campos JA, Fonseca RG. Evaluation of roughness, wettability and morphology of an yttria-stabilized tetragonal zirconia polycrystal ceramic after different airborne-particle abrasion protocols. J Prosthet Dent 2014 Dec;112(6):1385–91. doi.org/10.1016/j. prosdent.2014.07.005. 36. Aboushelib MN, Osman E, Jansen I, Everts V, Feilzer AJ. Influence of a nanoporous zirconia implant surface of on cell viability of human osteoblasts. J Prosthodont 2013 Apr;22(3):190–5. doi: 10.1111/j.1532849X.2012.00920.x. Epub 2013 Feb 22. 37. Fischer J, Schott A, Märtin S. Surface micro-structuring of zirconia dental implants. Clin Oral Implants Res 2016 Feb;27(2):162–6. doi: 10.1111/clr.12553. Epub 2015 Jan 30. 38. Monaco C, Tucci A, Esposito L, Scotti R. Microstructural changes produced by abrading Y-TZP in pre-sintered and sintered conditions. J Dent 2013 Feb;41(2):121–6. doi: 10.1016/j.jdent.2012.06.009. Epub 2012 Jul 13. 39. Ramos-Tonello CM, Trevizo BF, Rodrigues RF, et al. Presintered Y-TZP sandblasting: Effect on surface roughness, phase transformation and Y-TZP/veneer bond strength. J Appl Oral Sci 2017 Nov–Dec;25(6):666–73. doi: 10.1590/16787757-2017-0131. 40. Kosmac T, Oblak C, Jevnikar P, Funduk N, Marion L. The effect of surface grinding and sandblasting on flexural strength and reliability of Y-TZP zirconia ceramic. Dent Mater 1999 Nov;15(6):426–33. doi: 10.1016/s0109-5641(99)00070-6. 41. Turp V, Sen D, Tuncelli B, Goller G, Özcan M. Evaluation of air-particle abrasion of Y-TZP with different particles using microstructural analysis. Aust Dent J 2013 Jun;58(2):183–91. doi: 10.1111/adj.12065. 42. Zhang Y, Lawn BR, Rekow ED, Thompson VP. Effect of sandblasting on the long-term performance of dental ceramics. J Biomed Mater Res B Appl Biomater 2004 Nov 15;71(2):381–6. doi: 10.1002/jbm.b.30097. 43. Hallmann L, Ulmer P, Reusser E, Hämmerle CH. Effect of blasting pressure, abrasive particle size and grade on phase transformation and morphological change of dental zirconia surface. Surf Coat Technol 2012;206(19–20):4293–302. doi.org/10.1016/j.surfcoat.2012.04.043. 44. He M, Zhang Z, Zheng D, Ding N, Liu Y. Effect of sandblasting on surface roughness of zirconia-based ceramics

and shear bond strength of veneering porcelain. Dent Mater J 2014;33(6):778–85. doi: 10.4012/dmj.2014-002. Epub 2014 Oct 11. 45. Skienhe H, Habchi R, Ounsi H, Ferrari M, Salameh Z. Structural and morphological evaluation of pre-sintered zirconia following different surface treatments. J Contemp Dent Pract 2018 Feb 1;19(2):156–165. doi: 10.5005/jpjournals-10024-2230. 46. Okutan Y, Yucel MT, Gezer T, Donmez MB. Effect of airborne particle abrasion and sintering order on the surface roughness and shear bond strength between Y-TZP ceramic and resin cement. Dent Mater J 2019 Mar 31;38(2):241– 249. doi: 10.4012/dmj.2018-051. Epub 2018 Dec 11. 47. Passos SP, Linke B, Major PW, Nychka JA. The effect of airabrasion and heat treatment on the fracture behavior of Y-TZP. Dent Mater 2015 Sep;31(9):1011–21. doi: 10.1016/j. dental.2015.05.008. Epub 2015 Jun 24. 48. Kurtulmus-Yilmaz S, Aktore H. Effect of the application of surface treatments before and after sintering on the flexural strength, phase transformation and surface topography of zirconia. J Dent 2018 May;72:29–38. doi: 10.1016/j. jdent.2018.02.006. Epub 2018 Mar 1. 49. Abi-Rached FO, Martins SB, Almeida-Júnior AA, et al. Air abrasion before and/or after zirconia sintering: Surface characterization, flexural strength and resin cement bond strength. Oper Dent Mar–Apr 2015;40(2):E66–75. doi: 10.2341/14-013-LR1. Epub 2014 Dec 23. 50. Martins SB, Abi-Rached FO, Adabo GL, Baldissara P, Fonseca RG. Influence of particle and air-abrasion moment on Y-TZP surface characterization and bond strength. J Prosthodont 2019 Jan;28(1):e271–e278. doi: 10.1111/ jopr.12718. Epub 2017 Dec 13. 51. Ebeid K, Wille S, Salah T, et al. Evaluation of surface treatments of monolithic zirconia in different sintering stages. J Prosthodont Res 2018 Apr;62(2):210–17. doi. org/10.1016/j.jpor.2017.09.001. 52. Su N, Yue L, Liao Y, et al. The effect of various sandblasting conditions on surface changes of dental zirconia and shear bond strength between zirconia core and indirect composite resin. J Adv Prosthodont 2015 Jun;7(3):214–23. doi: 10.4047/jap.2015.7.3.214. Epub 2015 Jun 23. 53. Lorente MC, Scherrer SS, Richard J, et al. Surface roughness and EDS characterization of a Y-TZP dental ceramic treated with the CoJet Sand. Dent Mater 2010 Nov;26(11):1035–42. doi: 10.1016/j.dental.2010.06.005. Epub 2010 Sep 15. 54. Gadelmawla E, Koura M, Maksoud T, Elewa I, Soliman H. Roughness parameters. J Mater Process Technol 2002 Apr;123(1):133–45. doi.org/10.1016/S09240136(02)00060-2. 55. Gittens RA, Olivares-Navarrete R, Cheng A, et al. The roles of titanium surface micro/nanotopography and wettability on the differential response of human osteoblast lineage cells. Acta Biomater 2013 Apr;9(4):6268–77. doi: 10.1016/j. actbio.2012.12.002. Epub 2012 Dec 8. 56. Drelich J. Guidelines to measurements of reproducible contact angles using a sessile-drop technique. Surf Innov 2013;1(4):248–54. doi.org/10.1680/si.13.00010. 57. Wang X, Spandidos A, Wang H, Seed B. PrimerBank: A PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Res 2012 Jan;40(database issue):D1144–9. doi: 10.1093/nar/gkr1013. Epub 2011 Nov 15. N OVEMBER 2 0 2 1

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58. Wang X, Seed B. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res 2003 Dec 15;31(24):e154. doi: 10.1093/nar/gng154. 59. Al Qahtani WM, Schille C, Spintzyk S, et al. Effect of surface modification of zirconia on cell adhesion, metabolic activity and proliferation of human osteoblasts. Biomed Tech (Berl) 2017 Feb 1;62(1):75–87. doi: 10.1515/bmt-20150139. 60. Becker W, Becker BE, Ricci A, et al. A prospective multicenter clinical trial comparing one- and two-stage titanium screw-shaped fixtures with one-stage plasma-sprayed solidscrew fixtures. Clin Implant Dent Relat Res 2000;2(3):159– 65. doi: 10.1111/j.1708-8208.2000.tb00007.x. 61. Åstrand P, Anzén B, Karlsson U, et al. Nonsubmerged implants in the treatment of the edentulous upper jaw: A prospective clinical and radiographic study of ITI implants — results after one year. Clin Implant Dent Relat Res 2000;2(3):166–74. doi: 10.1111/j.1708-8208.2000. tb00008.x. 62. Alao AR, Stoll R, Song XF, et al. Surface quality of yttriastabilized tetragonal zirconia polycrystal in CAD/CAM milling, sintering, polishing and sandblasting processes. J Mech Behav Biomed Mater 2017 Jan;65:102–116. doi: 10.1016/j. jmbbm.2016.08.021. Epub 2016 Aug 20. 63. Yin L, Nakanishi Y, Alao AR, et al. A review of engineered zirconia surfaces in biomedical applications. Procedia CIRP 2017;65:284–90. doi: 10.1016/j.procir.2017.04.057. 64. Asl SK, Sohi MH. Effect of grit-blasting parameters on the surface roughness and adhesion strength of sprayed coating. Surf Interface Anal 2010;42(6–7):551–54. doi. org/10.1002/sia.3184. 65. Skienhe H, Habchi R, Ounsi H, Ferrari M, Salameh Z. Evaluation of the effect of different types of abrasive surface treatment before and after zirconia sintering on its structural composition and bond strength with resin cement. Biomed Res Int 2018 May 27;2018:1803425. doi: 10.1155/2018/1803425. eCollection 2018. 66. Moon JE, Kim SH, Lee JB, Ha SR, Choi YS. The effect of preparation order on the crystal structure of yttria-stabilized tetragonal zirconia polycrystal and the shear bond strength of dental resin cements. Dent Mater 2011 Jul;27(7):651–63. doi: 10.1016/j.dental.2011.03.005. Epub 2011 Apr 29. 67. Zhang M, Zhang Z, Ding N, Zheng D. Effect of airborne-particle abrasion of pre-sintered zirconia on surface roughness and bacterial adhesion. J Prosthet Dent 2015 May;113(5):448–52. doi: 10.1016/j. prosdent.2014.12.012. Epub 2015 Mar 5. 68. Suh AY, Polycarpou AA, Conry TF. Detailed surface roughness characterization of engineering surfaces undergoing tribological testing leading to scuffing. Wear 2003;255(1– 6):556–68. doi:10.1016/S0043-1648(03)00224-2. 69. Zinelis S, Thomas A, Syres K, Silikas N, Eliades G. Surface characterization of zirconia dental implants. Dent Mater 2010 Apr;26(4):295–305. doi: 10.1016/j.dental.2009.11.079. Epub 2009 Dec 16. 70. Piattelli A, Degidi M, Paolantonio M, Mangano C, Scarano A. Residual aluminum oxide on the surface of titanium implants has no effect on osseointegration. Biomaterials 2003 Oct;24(22):4081–9. doi: 10.1016/s0142-9612(03)00300-4. 71. Wenzel RN. Resistance of solid surfaces to wetting by water. Ind Eng Chem 1936;28(8):988–94. doi. org/10.1021/ie50320a024. 72. Puleo D, Bizios R. Mechanisms of fibronectin-mediated

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attachment of osteoblasts to substrates in vitro. Bone Miner 1992 Sep;18(3):215–26. doi: 10.1016/01696009(92)90808-q. 73. Nassif W RM. Surface characterization and cell adhesion of different zirconia treatments: An in vitro study. J Contemp Dent Pract 2018 Feb 1;19(2):181–188. doi: 10.5005/jpjournals-10024-2234. 74. Rezaei NM, Hasegawa M, Ishijima M, et al. Biological and osseointegration capabilities of hierarchically (meso-/ micro-/nano-scale) roughened zirconia. Int J Nanomedicine 2018 Jun 8;13:3381–3395. doi: 10.2147/IJN.S159955. eCollection 2018. 75. Irandoust S, Müftü S. The interplay between bone healing and remodeling around dental implants. Sci Rep 2020;10(1):1–10. doi.org/10.1038/s41598-020-60735-7.

THE CORRE SP ONDIN G AU THORS , Weerachai Singhatanadgit, DDS, PhD, and Yanee Tantilertanant, DDS, PhD, can be reached at s-wrch@tu.ac.th and littlengi@hotmail.com.


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LIC #01418359 LIC #01886221 (916) 812-0500 (916) 812-3255 (619) 694-7077 (925) 330-2207 (949) 300-0312 (707) 391-7048 (714) 318-4911 (951) 314-5542 (408) 687-5001 (619) 889-6492 (949) 675-5578 46 Years in Business 39 Years in Business 47 Years in Business 38 Years in Business 36 Years in Business 31 Years in Business 31 Years in Business 27 Years in Business 18 Years in Business 17 Years in Business 12 Years in Business

PRACTICE SALES • VALUATIONS/APPRAISALS • TRANSITION PLANNING • PARTNERSHIPS • MERGERS • ASSOCIATESHIPS NORTHERN CALIFORNIA ALAMEDA COUNTY SOUTH-ORTHO: New Listing! 3 Chairs, below-market rent w/ over 50% Net, 2020 GR $505K. Orthowave and Digital Pan/Ceph. Great satellite office or for first-time buyer. #CA2785 EAST BAY AREA PEDO: Well-established with 8 Ops, Digital, plumbed for Nitrous, and high NP count. Associate-driven with Delta PPO. 2019 GR $832K on 3-4 days/wk., 2020 Production $560K. #CA2523 FAIRFIELD AREA: High traffic area, 7 Ops Digital, Pano/CB, 23+ NP/mo. with 8+ Hyg. days/wk. Room to grow with specialties. 2019 GR $1.7M and 2021 on track to exceed 2019. #CA1824 FAIR OAKS/CITRUS HEIGHTS AREA: Successful practice w/ 38 yrs. Goodwill. Nice décor, Digital, 6 hyg days/wk. Growth potential with Ortho/Implants. 4 Ops in 1,100 sf. 2019 GR $970K+ on 32 hrs/wk. #CA656 FREMONT ORAL SURGERY: New Listing! 34 yr history, diverse high-tech community. 4 Ops Digital, 7-10 y/o equipment, Pano. 2019 GR $548K on 3.5 days/wk. #CA2754 GREATER SONORA AREA: Rural lifestyle GP/Real Estate, 5 Ops, Dentrix, Strong hyg prog in stable community. 2019 GR $698K. #CA1713 HAYWARD: New Listing! Great neighborhood practice +RE opportunity. 4 Ops, digital, updated. 2019 GR $730K. #CA2771 LAKE TAHOE AREA: 4 Ops, 37+ yrs Goodwill. Rural lifestyle GP in growing resort community. 2019 GR $760K. #CA1715 LAKE TAHOE AREA: 5 Ops w/ 6th Open, Operatory views of Lake Tahoe, only 34 Delta Premier patients, 2,100 sf. 2019 GR $579K on 22 avg. Dr. hrs/wk. #CA608 MILLBRAE: New Listing! Great practice in the heart of the peninsula with 60 yrs goodwill. 5 Ops. 2019 GR $1M+ on 4 days/ wk. and 6 Hygiene days. Owner will work back for a short time for transition. Digital, Pano, Waterlase & Periolase. #CA1139 NORTHERN SACRAMENTO: Busy location, Paperless, 3 Ops+4th shared, CEREC, Digital Pano. 2019 GR $671K on 24-32 hrs/wk. #CA1745 OAKLAND: New Listing! Pill Hill area, walk to BART, 2019 GR $473K + postCOVID recovery $595K in 12 months since reopening. 3 Ops, Digi X-rays and Pano. #CA2839 REDDING AREA: Price reduced by $100K under valuation price! Modern office with 5 Ops, 4 Eq., Digital, Newer CEREC, 23 NP/ mo with no marketing. Strong Hygiene, specialties referred. 2019 GR $558K. #CA1742 ROCKLIN/GRANITE BAY: New Listing! High-end 4 Op GP/Cosmetic practice in affluent area. Paperless, digital, iTero scanner, 8+ hyg. Days/wk. 2019 GR $1.6M+, 2021 Prod projected at $2M+. RE for sale with practice. #CA2793 ROSEVILLE/CITRUS HTS: New Listing! 6 Ops, high traffic area, 13 yrs goodwill, Digital, lasers, 26 NP/mo, 5 days Hygiene, specialties referred. Seller will work back. #CA2749

ROSEVILLE/ROCKLIN: New Listing! 7 Ops, high end practice in desirable area. Digital, CAD/CAM, lasers, Pano. 10+ hyg. Days/wk, 2019 GR $2.3M, 2021 projected $2.5M Production. Lease with purchase option. #CA2770 SACRAMENTO: HUGE PRICE REDUCTION! 5 Ops+RE in a busy medical/ dental/retail area. Digital, 50 years Goodwill, 6 days hygiene/wk. and 3.5 Dr. days/wk. 2019 GR of $697K with specialties referred. #CA2620 SAN JOSE: Est for 35 yrs, 2019 GR of $1.3M with Adj. Net of 38%. 6 Ops, Digital X-rays and Pan, CAD/CAM, Laser. Upscale building near shopping. Seller can stay on P/T.#CA1140 SAN MATEO: 5 Ops, Digital, iTero Scan, CEREC, Laser, Paperless, Microscope. Sellerowned stand-alone building to lease. $1.4M GR on 4 days/wk. #CA2596 SONOMA COUNTY: New Listing! 4 Ops with room to expand into suite next door. GR over $1M for last 3 yrs. Est. 30+ years. Strong hygiene, digital, space available to lease or buy. #CA2790 SONOMA COUNTY: 4 Ops in spacious layout in heart of the area off main highway. Est 22 yrs with 5 star Google reviews, Paperless with CEREC, Scope, Laser, Strong Hyg. Retiring seller. 2019 GR $782K with good post-COVID recovery. #CA2594 SONOMA COUNTY: Stand-alone 3,000 sf, 72 NP/mo. & 10 hyg days. 6 Ops, Pano, Dexis, Cameras, Laser, Dentrix. Business & RE for sale or Lease. Doctor Retiring. 2019 GR $2.3M+. #CA544 VACAVILLE AREA: Price Reduced over $35K! Seller will work back for up to 6 mo. Centrally-located & hi-traffic location with 25 + yrs goodwill. 5 Ops in 1,700 sf. 2019 GR $556K on 32 hrs/wk. #CA645 VACAVILLE AREA: 4 Ops, 3 equipped, 45 years goodwill, Digital, paperless, most specialties referred. 2019 GR $723K on 30 hour week. #CA2748

CENTRAL CALIFORNIA CENTRAL COAST: 5 Ops, digital, 25+ yrs Goodwill. Newly renovated, practice sees 30 NP/mo. Strong hyg prog. 2019 GR $1.1M+. #CA1218 CENTRAL VALLEY/MODESTO: New Listing! 8 Ops, high visibility retail, Open 20+ yr, Digital, soft/hard tissue lasers, 3,300+ active pts., 24+ NP/mo., 4 hyg days/ wk., 18.5 hour Dr. work week. 2019 GR $852K, 2020 84% of 2019. #CA2721 FRESNO AREA: New Listing! 6 Op Valley gem, great staff in desirable area. Paperless, Trios Scanner, Digital Pan/Ceph, Lasers and 12 days of hyg/wk. 2019 GR $1.4M, 2021 projected at $1.4M again. Seller may consider option to purchase RE. #CA2004 MODESTO AREA: Est. area with 60+ yrs. goodwill. 5 Ops, 2019 GR $1.1M+ on 3 days/ wk. Dental Condo also available for purchase or lease, Seller may consider financing. #CA635 MONTEREY: New Listing! 4 Ops, Paperless, Digital, Pano. 2019 GR $1.1M with Adj. Net over $450K. Post-COVID revenue has grown even more! RE for sale, non-Delta Premier office, FFS, some PPOs. #CA2614

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PALM DESERT: 4 Ops 27 yrs Goodwill. Strong hyg prog w/ hi-end patient base of locals/ snowbirds. 2019 GR $809K on only 16 days/ mo. with low overhead. Call today! #CA691 PALMDALE/LANCASTER: New Listing! SANTA CRUZ/APTOS PERIO: New Listing! 7 Op office in fast-growing community. Paperless with Dentrix, digital x-rays, 8 days of 4 Ops+RE, Paperless, Digital, CBCT, 27 years hyg./week and dedicated staff. Room to grow goodwill. Seller will help with smooth transition with specialties! #CA2612 of strong referral base. #CA2725 SOUTH BAY LOS ANGELES: Ready to STOCKTON: Practice+RE available, 5 Ops, 5 retire! 7 Ops, real estate for sale also. 50% DentiHyg. Days/wk. 2019 GR $812K on 32 hr. week. Cal, some HMO and PPO. 2019 GR $568K. #CA1050 High level of Ortho, seller can work back. #CA2006 SAN BERNARDINO: New Listing! 6 Ops, established 33 years, cash, HMO, Denti-Cal in a busy area with parking. Estimated GR for 2021 SOUTHERN CALIFORNIA at $960K+. Seller offering RE for sale with 2 BAKERSFIELD: 6 Ops, 40 yrs Goodwill, lease tenants adjacent to practice. Room to great reputation in the area. 6 hyg ds/wk and expand with spec. #CA2843 most specialty work referred. Digital pano, SANTA BARBARA: 4 Ops in beautiful digital X-rays. 2019 GR $600K. RE also for setting. Digital, FFS, strong hygiene, and room sale. #CA1274 to grow with specialties. Consistently collects BAKERSFIELD: New Listing! 6 Ops, 5 $1M+/yr. with manageable overhead. #CA2531 Equipped, Digital, 2020 Collections $1M+ TORRANCE: New Listing! 3 Ops, room for a with 6 days hygiene and 2 P/T associates. 4th. Dentrix, digital, refers most specialties with #CA2587 low overhead and high net. GR $600K. BURBANK: Big opportunity for large #CA2815 practice merger, 6 Ops, Digital, seller retiring. TORRANCE: New Listing! 3 Ops, retiring 6 days of hygiene, specialties referred. Seller seller with 34 yrs goodwill. Ready to take to the will transition, open to financing options. 2019 next level with technology of your choosing. GR $918K. #CA2632 Amazing location in desired area. 2019 GR of COASTAL ORANGE COUNTY: $300K with low expenses, a wonderful New Listing! 5 Ops, 4 Equipped, Digital Pano opportunity to grow. #CA2807 and X-rays, well-established neighborhood, SAN DIEGO very desired area. 2019 GR over $1M. #CA2787 DEL MAR: New Listing! 4 Ops, Digital, HUNTINGTON BEACH: PRICE Open Dental, Conservative Practitioner who REDUCED FOR QUICK SALE! 5 Ops, refers out specialties. 4 days of hygiene per desirable loc, Digital, Strong hyg prog. 2019 week. Seller is eager for a quick sale. GR $604K. #CA685 Excellent opportunity in a very desirable INDIO: 4 Ops, single-story medical/retail location. #CA2724 center. Digital, CEREC w/milling unit and oven. GR $764K in 2019 and $535K in 2020. SAN DIEGO: New Listing! Rare opportunity, seller retiring, 4 Ops in desirable location with 7 Hyg days/wk. Great Opportunity. good cash flow. High quality work. Digital, #CA2619 Dentrix. #CA2851 LONG BEACH: RE Ownership an option! Upper middle-class residential practice est. in OUT OF CALIFORNIA 1950. Existing 4 Ops, 3 Equip, Digital, Easy expansion next door to add 3 Ops, 2 are equip. BIG ISLAND, HAWAII: 3 Ops, nonMost Specialty referred. Strong post-COVID digital, excellent location plus rare option to production. 2019 GR $696K. #CA671 purchase office space. Room to grow! LOS ANGELES: Cash/PPO office in great #HI1929 DTLA Location. 3 Ops with low rent. Digital with scanner and lasers. 2020 GR $299K on 2 PORTLAND, OR: New Listing! Great location. 5 Ops, 4 equipped, Digital, Pano, days/wk. #CA2493 50% Medicaid. Turn-key practice on main MONTEBELLO: New Listing! 3 Ops in busy road. 2019 GR $646K. #OR2757 strip center location with 2 Associates, Digital SOUTHERN OREGON: 5 Ops, Paperless, x-rays, and all specialty work referred out. CEREC, Laser, and much more. Doctor is #CA2786 available to stay on for transition, if desired. ORANGE COUNTY: Price Reduced! 5 Turn-key office. 2020 GR $1.5M. Ops, Digital, Retiring seller. Excellent #OR2688 reputation, affluent area, high quality care. SOUTHERN OREGON: Quaint GP in ideal Modern, welcoming office with strong hyg location in desirable town. 4 Ops with room prog. Room to grow specialties. 2019 GR to grow adding days and specialties. Open 31 $642K. #CA1676 yrs. Digital with EagleSoft. $276K GR in ORANGE COUNTY: 4 Ops in sought-after 2020. #OR2574 area. 34 yrs Goodwill, many hi-end procedures TRI-CITIES, WASHINGTON: Small done in-house but room to grow other modified start up, fully equipped! Access to specialties. Digital. FFS/Cash. #CA2704 1500 patient records, Open Dental software, OXNARD: 4 Ops, Digital X-rays, Est. 35+ yrs laser, x-ray sensors. Desirable location, ago. Seller owned it for 3 yrs and has a primary affordable rent. #WA2629 office in LA. 2019 GR $662K. #CA1164 SANTA CRUZ COUNTY: New Listing! 4 Ops Close to beach in strip center. Digital Pano and x-rays, CEREC, 40 years goodwill. 2019 GR $392K on 3.5 days. #CA2822

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RM Matters

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Romantic Relationships With Patients: Your Obligation as the Employer TDIC Risk Management Staff

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s it ever okay to date patients? The unqualified answer is simply no. The ethical considerations of personal relationships with patients are addressed in the ADA Principles of Ethics and Code of Professional Conduct. Under the Principle of Nonmaleficence (“do no harm”), the ADA states, “Dentists should avoid interpersonal relationships that could impair their professional judgment or risk the possibility of exploiting the confidence placed in them by a patient.” When it comes to matters of the heart, however, objectivity can be compromised. Romantic chemistry happens, and there are risks beyond the ethics that can have profound impacts on your practice, whether or not the relationship works out. If an attraction develops, emotions and stakes are heightened. Consider beforehand how personal relationships could evolve (or devolve) into troublesome situations in the future. If you or any member of your dental team intends to pursue a personal relationship with a patient, the patient must be referred to another practice for care before the relationship begins. The Dentist Insurance Company provides a no-cost Risk Management Advice Line to help CDA members and TDIC policyholders navigate challenging situations. As calls to the Advice Line illustrate, romances that evolve in business and health care settings often have an imbalance-of-power aspect that creates even more tension and risk — even when the relationship is between a staff member and a patient.

A case study in dating a patient

In a recent Advice Line call, a dentist shared how the complications of a romantic relationship between a staff member and patient were fueling drama and discontent in his practice. The office’s receptionist, who was married, had been dating an elderly patient who was professionally successful. The patient

sent gifts to her and took her on lavish dates. The receptionist shared the details of her dating life with other staff members and admitted that flowers sent to her at the practice were from the patient. The receptionist’s husband discovered the relationship and had “lost control” — getting into an argument with her in the office parking lot, which escalated into

answers

From one-on-one risk management advice by phone to informed consent forms to expert-led seminars, we’re here to help you practice with confidence. We are The Dentists Insurance Company. Learn more at tdicinsurance.com/rm

Protecting dentists. It’s all we do.

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800.733.0633 | tdicinsurance.com | Insurance Lic. #0652783

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him throwing an item at his wife. The police had to be called to put an end to this disruptive and embarrassing scene. In this case, the dentist did not wish to dismiss the patient due to the patient’s high standing in the community. To complicate the issue, while the dentist’s employee manual did include a policy that specifically referenced not dating patients, not everyone on his staff had provided a signed acknowledgment that they had received the manual. The analyst urged the dentist to meet with the employee and remind her of the office policy and clearly communicate their expectation that she adhere to these guidelines. In addition, given the disruption that occurred when the patient’s husband confronted his wife at the practice, the dentist may consider obtaining a restraining order against the husband to prevent further occurrences. While employee termination may also be an option, the dentist was advised to consult an employment attorney prior to taking any definitive action. Regardless whether the potential relationship with a patient is to be with the dentist or a staff member, the patient must seek dental care from another office. This can prevent potential financial and privacy concerns. What if the patient’s balance was forgiven or an unauthorized credit was placed on their account? If the relationship doesn’t work out, the patient could voice concerns about unauthorized access to private health information. Patients should be able to trust their health care providers and have the expectation that any confidential information revealed will be used only in their best interest. This dynamic must not be exploited, regardless of the relationship status. If a romance ends, hurt feelings can even lead to retaliatory action taken by a patient, such as a complaint to the dental board or filing a malpractice claim. 716 N OVEMBER

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What can you do? ■

Lead by example. As practice owners and employers, dentists must model the behavior they want to see in others. They should neither initiate relationships with patients nor encourage romantic interest. If staff members observe a practice leader setting clear boundaries with a patient and referring a patient to another dental practice when needed, they are more likely to make prudent decisions about their own behavior. Proceed with caution. If you have weighed the potential consequences of dating a patient and are serious about pursuing a relationship, you must refer the patient to another provider. Include staff in your reasoning about these types of decisions, as it will demonstrate accountability and encourage discussions around similar situations among the team. Put your policy in writing. The Dentists Insurance Company recommends a written office policy that is applied universally, regardless of the staff role. In addition to having a policy in place, you should also communicate what behavior is unacceptable in the workplace, such as certain displays of affection and discussing relationship issues. For any employment policy, the consequences of violation should be equitable and clear. It can be helpful to establish an anonymous reporting process to allow employees to feel more comfortable to share when they’ve witnessed violations or concerning situations. Add a layer of protection. If you haven’t already done so, consider adding employment practices liability (EPLI) coverage as an endorsement to your

professional liability policy. EPLI can provide protection if you or one of your employees is sued for harassment, discrimination, wrongful termination, failure to promote or other employmentrelated issues. Talk to your trusted insurance advisor about the right coverage for your practice’s needs. In any workplace, navigating relationships is complicated. But the potential for unforeseen and possibly high-risk issues increases when a dentist — or anyone else on the practice team — chooses to date a patient. Protect yourself, your staff and your practice by keeping relationships professional. n The Dentists Insurance Company’s Risk Management Advice Line is a benefit available at no cost to CDA members, as well as to policyholders protected by TDIC. To schedule a consultation, visit tdicinsurance. com/RMconsult or call 800.733.0633.


Regulatory Compliance

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Regulated Waste Management CDA Practice Support

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ental practices have two types of regulated waste: hazardous and medical. Each type is regulated by separate laws and, in many communities, different agencies. This article summarizes what comprises each waste type, rules on storage and disposal and registration and documentation requirements.

of toxic hydrogen sulfide gas as the material decomposes. Recycling is the preferred option for disposing of this material. If closing a dental practice and in possession of a lot of models, contact the local landfill office for information on disposal options. Other hazardous wastes generated by dental practices have the option of being managed as universal waste. Universal

waste is a category of hazardous waste widely produced by households and different types of businesses. Universal wastes cannot be disposed in landfills and should be recycled. Hazardous and universal wastes may not be stored for longer than one year. Each type of waste should be collected in their own nonbreakable and closeable container and labeled with the contents and the

Hazardous Waste

Dental wastes in this category include glutaraldehyde, amalgam, photographic fixer and lead. State and federal hazardous waste laws regulate waste characterization. California imposes rules on storage and disposal of hazardous waste. Waste generators must register with their local agency, which works in conjunction with the state Department of Toxic Substances Control. A hazardous waste management plan and California EPA ID number may be required. Glutaraldehyde waste must be managed as a hazardous waste unless it is treated to render it nonhazardous. Treatment of hazardous waste in California typically requires a permit. However, a permit is not necessary if glutaraldehyde waste is treated with a solution containing glycine as its only active ingredient. A dental practice should check with its local sanitation agency to verify that the treated solution may be disposed safely down the drain. Although not categorized as a hazardous waste, large amounts of plaster disposed at a landfill can potentially lead to the formation N OVEMBER 2 0 2 1 LDM_CDA_Journal_1.3_Square_LindaBrown_05_23_17.indd 1

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date waste collection in the container was started. If using a mail-back service for disposal, the service will provide the appropriate container. Another disposal option is local household hazardous waste programs. Several programs in the state accept waste from very small-quantity generators. Contact information for these programs is available in “Household Hazardous Waste and Small-Quantity Generator Programs by County” on cda.org/practicesupport.

Medical Waste

Dental wastes in this category are contaminated sharps, pharmaceutical waste (not including controlled substances), used anesthetic carpules with aspirated blood and absorbent materials that are dripping or flaking blood. The California Medical Waste Management Act (MWMA) dictates the rules for medical waste management and disposal. Medical waste generators are required to register with either the California Department of Public Health (CDPH) or local enforcement agency and pay a regular fee. In some circumstances, a medical waste hauler will collect the fee. The MWMA applies different rules to small-quantity generators, such as dental practices, than to larger generators. A smallquantity generator generates less than 200 pounds of medical waste per month. Generators must have a written medical waste management plan. Sharps are the most common medical waste generated by dental practices. It includes but is not limited to hypodermic needles, hypodermic needles with syringes, blades, needles with attached tubing, acupuncture needles, root canal files, broken glass items used in health care such as Pasteur pipettes and blood vials contaminated with biohazardous waste and any item 718 N OVEMBER

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capable of cutting or piercing from trauma scene waste. Contaminated sharps and anesthetic carpules with aspirated blood should be collected in FDA-cleared sharps containers that are closeable and difficult to reopen after sealing shut, puncture resistant and leak proof on all sides. Sharps containers can be any color and should be labeled “Biohazardous Waste” or “Sharps Waste” with fluorescent orange or orange-red labels with letters and the hazard symbol in a contrasting color. The container should be maintained upright, easily

The California Medical Waste Management Act (MWMA) dictates the rules for medical waste management and disposal.

accessible to the immediate area of sharps use and not filled past the fill line. The sharps container should be disposed of within 30 days of the container being three-fourths full or filled or within 90 days if the sharps container is stored at less than 32 F. If the sharps container is combined with biohazardous waste in another container, the combined waste must be disposed within the shorter disposal schedule for biohazardous waste. Pharmaceutical waste includes expired drugs and unused drugs, but it does not include controlled substances. Expired or unwanted controlled substances must be disposed of through a DEA-registered service, sometimes the same ones that will accept other pharmaceutical waste. Pharmaceutical

waste should be placed in a leak-proof container that is closeable and has a tight-fitting lid. The container should be labeled “High Heat,” “Incineration Only” or have other wording approved by the CDPH on the lid and on the sides. The pharmaceutical waste must be disposed of within one year that waste accumulation starts if the dental practice generates less than 10 pounds of waste per year or within 90 days if more than 10 pounds per year is generated. Biohazardous waste includes containers, equipment or disposables (e.g., gauze and cotton rolls) that drip blood or saliva when compressed or that flake dried blood when shaken. Pathology waste includes human body parts, with the exception of teeth, removed at surgery and surgery specimens or tissues removed at surgery or autopsy that are suspected by the health care professional of being contaminated with infectious agents known to be contagious to humans or that have been fixed in formaldehyde or another fixative. Biohazardous waste must be placed in a red biohazardous bag and pathology waste placed in a white biohazardous bag. The biohazardous bags should be placed in rigid, leakproof containers of any color that are closeable and have a tight-fitting lid. A container with biohazardous waste must be labeled “Biohazardous Waste” or “Biohazard” on the lid and sides. A container with pathology waste must be labeled “Pathology Waste,” “PATH” or other label approved by the CDPH on the lid and sides. Before disposal, bags must be tied to prevent leakage or expulsion of contents during storage, handling or transport. Biohazardous and pathology waste, stored at room temperature, must be disposed of within 30 days of the date


C D A J O U R N A L , V O L 4 9 , Nº 11

waste accumulation starts if the dental practice generates less than 20 pounds of such waste per month or within seven days if more than 20 pounds of waste is generated per month. Dental practices that generate less than 20 pounds of biohazardous waste per month may store the waste at 32 F or below for up to 90 days before disposal. Mail-back programs are available.

What Is Not Medical Waste

Medical waste does not include disposal items, such as gauze or cotton, soiled with nonfluid blood or saliva; teeth not meeting the definition of biohazardous waste; or urine, feces, saliva, sputum, nasal secretions, sweat, tears or vomitus unless it contains visible or recognizable fluid blood. These items are not considered “regulated medical waste” and may be disposed of as regular solid waste. With respect to extracted teeth, neither Cal/OSHA nor the MWMA prohibit dentists from giving patients back their own extracted, nonbiohazardous teeth. Teeth containing amalgam or other heavy metal should be managed either as universal waste or hazardous waste and should never be discarded as regulated medical waste or as solid waste. n Regulatory Compliance appears monthly and features resources about laws that impact dental practices. Visit  cda.org/ practicesupport  for more than 600 practice support resources, including practice management, employment practices, dental benefit plans and regulatory compliance.

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Tech Trends

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A look into the latest dental and general technology on the market

Linq (Free, offers in-app purchases, Linq, LLC) As COVID-19 restrictions ease leading up to more in-person professional events, networking takes on a whole new meaning in this pandemic era. Physical business cards, long a mainstay of networking tools for in-person meetings, may not provide enough relevant information or ease of use in this digital age. Linq brings modern networking through digital business cards and products aimed at sharing customized information quickly and easily. Linq is a networking platform that starts with a customizable user profile page and requires signing up for a free account using an email address or a mobile phone number. This page can include a profile picture, description, contact card information, social media profile icons, custom links, photo galleries, files and text. More advanced features, such as embedded videos and music, forms, calendar integrations and additional profile pages, are available with a paid subscription to Linq Pro. Users can share their profile page to others using a simple QR code that can be sent through email or text, copied to the clipboard or added to their mobile phone digital wallet for easy access. The QR code contains a link that points to the user profile page, where connections can be established and contact information can be downloaded. The platform can be accessed entirely through the web browser. For a more integrated experience, users can download the mobile app for iOS or Android. A wide array of business products is available for purchase, such as bracelets, cards, Apple Watch bands and more, to help users connect their profile with other people while networking. These products either use a QR code or are NFC-enabled to link recipients to a user profile on the platform. The core features of the platform with a free account offer many options to share information and network, but users who want to further customize their profiles and experience may want to consider supplementing their accounts with products or a subscription to Linq Pro. Returning to fully in-person networking is sure to happen. For nearly the past two years, the entire world has grown accustomed to virtual meetings. Linq offers a modern way to network by taking the advancements learned virtually and providing an in-person connection experience that is both robust and versatile. — Hubert Chan, DDS

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Qlone

(Free, offers in-app purchases, EyeCue Vision Technologies) Every day, new scanners, printers and display devices are coming on the market aiming to push the envelope on what can be accomplished in the 3D technology space. One such innovation is the mobile app Qlone, “an all-in-one tool for 3D scanning.” The app claims that scanning is “super fast,” “super easy” and allows for exporting of files in popular formats like STL, OBJ and PLY. A notable characteristic is that Qlone is optimized to scan smaller objects like figurines, candy bars and sunglasses as opposed to larger objects and locations that are the focus of other competing scanning applications. Qlone has free and paid versions, and both were evaluated in this review. For those interested in the free Qlone product, the app is limited to a primitive scanning experience. Before scanning, a printed paper mat must be placed under the object of interest. With this mat in place, Qlone creates a virtual dome over the object and users are prompted to circumnavigate the object to complete the scan. After scanning in the free version, models can be viewed and edited. Matless scanning, file exporting and automatically animating the objects are available for the premium version only. The matless scanning is almost worth the price of the upgrade by itself, and combined with file exporting, makes it the clearly superior option. Both versions can wrap photos around scanned objects to create visually appealing models. File sharing in the premium version is easy to navigate, though the user interface is cluttered. Despite the visual appeals, scanning apps are best judged by scan quality, and unfortunately, Qlone scans are not detailed enough to be used clinically. Scans of casts do not contain clear finish lines or occlusal anatomy. Ultimately, Qlone is a convenient, easy-to-use scanning app for the tinkerer, but is of limited use for a clinician looking to enhance their digital workflows. — Alexander Lee, DMD


Time is running out The Mandate for Electronic Prescriptions Starts on January 1, 2022

Soon, California will require all dental practices to issue prescriptions electronically. Act fast to avoid penalties — and choose a solution that offers more than compliance: ePrescribe. • Gain seamless workflows by integrating with practice management systems. • Get access to the California Prescription Drug Monitoring Program. • Clarify communication by eliminating handwritten notes and transmitting instantly to pharmacies.

• Improve safe practices by automatically checking for drug allergies and interactions, dosage errors and duplicate therapies. • Help save patients’ money by using insurance and pharmacy information to estimate drug costs.

Don’t Get Caught Unprepared — Get ePrescribe Today Call 833.907.1747 for more info or visit: HenryScheinOne.com/ePrescribe/CA

©2021 Henry Schein One. Henry Schein One makes no representations or warranties with respect to the contents or use of this documentation, and specifically disclaims any express or implied warranties of title, merchantability, or fitness for any particular use. All contents are subject to change. Third-party products are trademarks or registered trademarks of their respective owners.


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