I. I. . INTERDISCIPLINARY THERAPY: USING CONTEMPORARY APPROACHES FOR COMPLEX CASES This volume includes the proceedings of the Forty-Second Annual Moyers Symposium and the Fortieth Annual International Conference on Craniofacial Research March 6–8, 2015 Ann Arbor, Michigan Editors Sunil D. Kapila Mithran Goonewardene Assistant Editor Hera Kim-Berman Editorial Associate Kristin Y. De Koster Volume 52 Craniofacial Growth Series Department of Orthodontics and Pediatric Dentistry School of Dentistry; and Center for Human Growth and Development The University of Michigan Ann Arbor, Michigan ©2016 by the Department of Orthodontics and Pediatric Dentistry, School of Dentistry and Center for Human Growth and Development The University of Michigan, Ann Arbor, MI 48109 Publisher's Cataloguing in Publication Data Department of Orthodontics and Pediatric Dentistry and Center for Human Growth and Development Craniofacial Growth Series Interdisciplinary Therapy: Using Contemporary Approaches for Complex Cases Volume 52 ISSN 0162 7279 ISBN 0-929921-00-3 ISBN 0-929921-48–8 No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Editor-in- Chief of the Craniofacial Growth Series or designate. CONTRIBUTORS BRENT P. ALLAN, Oral and Maxillofacial Surgeon, Honorary Clinical Consultant, Orthodontics, The University of Western Australia, Perth, Australia; private practice, Leederville, Australia. ANACLAUDIAARAUJO-PIRES, Post-doctoral Research Fellow, Department of Periodontics and Oral Medicine, School of Dentistry, The University of Michigan, Ann Arbor, MI. SHARON ARONOVICH, Clinical Assistant Professor, Department of Oral and Maxillofacial Surgery, The University of Michigan, Ann Arbor, Ml. JOSH I. BECKER, Undergraduate Student, Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, MI. PETER H. BUSCHANG, Regents Professor and Director of Orthodontic Research, Peter H. Buschang Endowed Professor of Orthodontics, Department of Orthodontics, Baylor College of Dentistry, Texas A&M University, Dallas, TX. LUCIA H.S. CEVIDANES, Assistant Professor, Department of Orthodontics and Pediatric Dentistry, School of Dentistry, The University of Michigan, Ann Arbor, MI. PAULA L. CHEIB, Orthodontist, PhD Student, Graduate Program in Dentistry, Pontifical Catholic University of Minas Gerais, Belo Horizonte, Brazil. - ALEXANDRE F.M. DaSILVA, Assistant Professor, Biologic and Materials Sciences, School of Dentistry; Director and Founder, Headache and Orofacial Pain Effort (H.O.P.E.); Co-Director, fNIRS Lab, Center for Human Growth and Development, The University of Michigan, Ann Arbor, MI. ANN M. DECKER, Periodontics Resident and PhD Pre-candidate, Department of Periodontics and Oral Medicine, School of Dentistry, The University of Michigan, Ann Arbor, MI. LORENZO FRANCHI, Research Associate, Department of Surgery and Translational Medicine, The University of Florence, Italy; Thomas M. Graber Visiting Scholar, Department of Orthodontics and Pediatric Dentistry, School of Dentistry, The University of Michigan, Ann Arbor, MI. WILLIAM V. GIANNOBILE, Najjar Professor of Dentistry and Chair, Department of Periodontics and Oral Medicine, School of Dentistry; Professor, Department of Biomedical Engineering, College of Engineering, The University of Michigan, Ann Arbor, MI. MITHRAN S. GOONEWARDENE, Senior Lecturer and Program Director, Orthodontics, The University of Western Australia, Nedlands, Australia. CHRISTIAN GROTH, Orthodontist, private practice, Birmingham, MI. SUNITA P. HO, Associate Professor, Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, University of California-San Francisco, San Francisco, CA. JOHAN JANSMA, Consultant and Oral Maxillofacial Surgeon, Department of Oral Maxillofacial Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. DARNELL KAIGLER, Assistant Professor, Department of Periodontics and Oral Medicine, School of Dentistry; Department of Biomedical Engineering, College of Engineering, The University of Michigan, Ann Arbor, MI. SUNIL D. KAPILA, Thomas M. and Doris Graber Endowed Professor, Department of Orthodontics and Pediatric Dentistry, School of Dentistry, The University of Michigan, Ann Arbor, MI. SUJUNG KIM, Associate Professor, Department of Orthodontics, School of Dentistry, Kyung Hee University, Seoul, South Korea. LISA M. LARKIN, Associate Professor, Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, MI. Ji-HYUN LEE, Postdoctoral Fellow, Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, University of California-San Francisco, San Francisco, CA. JONAH D. LEE, Postdoctoral Fellow, Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, MI. JANE McCARTHY, Periodontist, private practice, Applecross, Australia. JAMES A. McNAMARA Jr., Thomas M. and Doris Graber Endowed Professor Emeritus, Department of Orthodontics and Pediatric Dentistry, School of Dentistry; Professor Emeritus of Cell and Development Biology, School of Medicine; Research Professor Emeritus, Center of Human Growth and Development, The University of Michigan, Ann Arbor, MI. BIRTE MELSEN, Certified Specialist in Orthodontics, Section of Ortho- dontics, Institute of Odontology, Aarhus University, Aarhus, Denmark. WAGNER MOYSES-BRAGA, Undergraduate Student, School of Dentistry, Pontifical Catholic University of Minas Gerais, Belo Horizonte, Brazil. DAVID NORMANDO, Associate Professor, Federal University of Pará, Belém-PA, Brazil. GIORGIO PAGNI, Periodontist, private practice, Florence, Italy; Visiting Professor, Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, Italy. YOUNGGUK PARK, Dean and Professor, Department of Orthodontics, School of Dentistry, Kyung Hee University, Seoul, South Korea. SOPHIA P. PILIPCHUK, PhD Candidate, Department of Biomedical Engineering, College of Engineering, The University of Michigan, Ann Arbor, MI. CATIA CARDOSO ABDO OUINTAO, Associate Professor, State University of Rio de Janeiro, Rio de Janeiro, Brazil. ARCHANA RAJAN, Orthodontist, private practice, Farmington Hills, MI. YIJIN REN, Chair, Professor and Program Director, Department of Orthodontics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. HECTOR RIOS, Assistant Professor, Department of Periodontics and Oral Medicine, School of Dentistry, The University of Michigan, Ann Arbor, MI. ANTONIO CARLOS RUELLAS, Associate Professor, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; Visiting Scholar, Department of Orthodontics and Pediatric Dentistry, School of Dentistry, The University of Michigan, Ann Arbor, MI. MARK I. RYDER, Professor, Division of Periodontology, Department of Orofacial Sciences, University of California-San Francisco, San Francisco, CA. BRADLEY G. SHEPHERD, Prosthodontist, Honorary Clinical Consultant, Orthodontics, The University of Western Australia, Perth, Australia; private practice, West Perth, Australia. BERNARDO O. SOUKl, Associate Professor, Graduate Program in Orthodontics, Pontifical Catholic University of Minas Gerais, Belo Horizonte, Brazil. HARRY STAMATAKIS, Orthodontic Resident and Oral Radiologist, Department of Orthodontics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. SUNJAY SURI, Associate Professor, Discipline Head of Orthodontics and Graduate Program Director, Faculty of Dentistry, University of Toronto, Toronto, Canada; Staff Orthodontist, Department of Dentistry, The Hospital for Sick Children, Toronto, Canada. vi PREFACE The management of patients with complex dental, periodontal, Orthodontic, skeletal and systemic conditions remains a substantial challenge in orthodontics and dentistry in general. Additionally, the aging population and consequently the changing patient demographics present further challenges and limitations inherent to this cohort. Achieving the best possible outcomes in such cases requires a Sound understanding of basic clinical principles across different disciplines, comprehensive diagnostic skills and optimal communication and coordination between various providers. This proceeding from the 42nd Annual Moyers Symposium and 40th Annual International Conference on Craniofacial Research (the Presymposium) contains clinical reports, original research and review articles written by international experts in interdisciplinary therapy with the goal of imparting current knowledge in this important and challenging area. The manuscripts describe the optimal management of complex interdisciplinary cases through coordinated teamwork and contemporary clinical methods, the caveats and management of periodontal defects, tissue engineering to replace lost and missing tissues, and expediting tooth movement. Besides discussing contemporary diagnostic and therapeutic approaches, the chapters present the uses of new technologies including 3D imaging in facilitating optimal and efficient outcomes for these patients. I believe that the readers will find the materials informative, timely and of practical value. AS in the past, the Symposium honored the late Dr. Robert E. Moyers, Professor Emeritus of Dentistry, and Fellow Emeritus and Founding Director of the Center for Human Growth and Development. The meeting was co-sponsored by the Department of Orthodontics and Pediatric Dentistry and Center for Human Growth and Development at the University of Michigan, for whose support we truly are grateful. We wish to thank Michelle Jones of the Office of Continuing Dental Education for Coordinating and efficiently running the Presymposium and Symposium, Kris De Koster for her invaluable help in editing this volume and Katherine Ribbens for her technical assistance in compiling the monograph. Vii We also extend out gratitude to the Friends of the Moyers Symposium, who make annual contributions to help offset the costs of the meeting. These include Dr. Robert J. Isaacson from West Long Beach, New Jersey, Dr. Neal C. Murphy from Agourz Hills, California, and Dr. Howard L. Tingling from Southfield, Michigan. Finally, we thank all the speakers and participants of the Symposium and Presymposium, as well as those who purchase the volumes of the Craniofacial Growth Series. All volumes that are still in available in print can be obtained through Needham Press either online (www.needhampress.com) or by phone (734.668.6666). Sunil D. Kapila Editor in Chief Ann Arbor, Michigan December 2015 viii TABLE OF CONTENTS Contributors Preface Rehabilitation of Complex Dentofacial Problems with the Interdisciplinary Team Mithran S. Goonewardene, Bradley G. Shepherd, Brent P. Allan, Jane McCarthy The University of Western Australia; private practice Dental Rehabilitation: Multidisciplinary or Interdisciplinary Treatment? Birte Melsen, Aarhus University, Denmark The Role of Orthodontics in the Interdisciplinary Preparation for Osseointegrated Dental Implants Sunjay Suri, University of Toronto Treatment of Class Ill Malocclusion with Anchored Maxillary Protraction in Cleft Children: A One-year Follow-up Study on 3D Surface Models Derived from CBCT Yijin Ren, Johan Jansma, Harry Stamatakis University Medical Center Groningen, The Netherlands Three-dimensional Changes in the Mandible and Articular Fossae Following Herbst Appliance Therapy: A Preliminary CBCT Study Bernardo O. Souki, Lucia H.S. Cevidanes, Paula L. Cheib, Wagner Moyses-Braga, Antonio Carlos Ruellas, Lorenzo Franchi, James A. McNamara Jr. Pontifical Catholic University of Minas Gerais; The University of Michigan Surgically Facilitated Orthodontics: What Does the Evidence Say? Peter H. Buschang, Texas A&M University Vii 43 85 99 113 135 Timing of Post-operative Orthodontic Intervention in Interdisciplinary Approaches for Overcoming Alveolar Defects Sujung Kim, Youngguk Park, Kyung Hee University Bioengineering of the Periodontal Ligament Ann M. Decker, Sophia P. Pilipchuk, Ana Claudia Arauj-Pires, William V. Giannobile, The University of Michigan Tissue Engineering Approaches in Today's Clinical Practice Hector Rios, The University of Michigan Tissue Engineering of Alveolar Bone Using Clinical Stem Cell Therapies se Giorgio Pagni, Archana Rajan, William V. Giannobile, Darnell Kaigler, Università degli Studi di Milano, private practice, The University of Michigan Fabrication and Morphological Characterization of a Three Dimensional, Multi-phasic, Scaffoldless, Tissue-engineered Temporomandibular Joint Disc-ligament Complex Construct Jonah D. Lee, Josh I. Becker, Lisa M. Larkin, Sunil D. Kapila The University of Michigan Single-stage Reconstruction of Acquired TMJ Ankylosis with Surgical Navigation and Prosthetic Total Joint Replacement Sharon Aronovich, The University of Michigan The Brain as a Therapeutic Target in Headache and Facial Pain: The Next Frontier in Pain Medicine and Health Technology Alexandre F.M. DaSilva, The University of Michigan Microanatomical Responses of Developing and Functionally Active Bone-periodontal Ligament-tooth Fibrous Joints Ji-Hyun Lee, Mark I. Ryder, Sunita P. Ho University of California-San Francisco 167 195 243 265 291 317 361 373 Retrospective Analysis of Efficiency and Efficacy of Computer- 389 Assisted Finishing Techology Christian Groth, Lorenzo Franchi, James A. McNamara Jr. The University of Michigan; private practice Malocclusion at the Navel of the Amazon River 401 David Normando, Cátia Cardoso Abdo Ouintão Federal University of Pará; State University of Rio de Janeiro Xi REHABILITATION OF COMPLEX DENTOFACIAL PROBLEMS WITH THE INTERDISCIPLINARY TEAM Mithran S. Goonewardene, Bradley G. Shepherd, Brent P Allan, Jane McCarthy ABSTRACT Interdisciplinary treatment plans are rewarding for both the treating clinicians and patients. For patients with these therapeutic needs, a rigorous systematic approach is necessary to develop a coordinated, patient-centered, goal-oriented treatment plan. It is critical to establish the needs of the individual patient within their own value system and institute a realistic plan. To achieve the best outcome, clinicians must establish a group of healthcare providers who will bring the necessary skills and knowledge to the interdisciplinary team. They must meet and communicate regularly and effectively through appropriate media. A sequence of activity with specific goals should be outlined, identifying the respective clinicians and their immediate goal(s), to which all clinicians and the patient may refer at any stage in treatment. The team leader has the responsibility of ensuring that the patient and clinicians all are cognizant of the stage that the patient has reached and what work needs to be performed next— in turn, facilitating communication. If these processes are followed, the patient and the team can anticipate a mutually rewarding experience with consistently excellent and reproducible outcomes. KEY WORDS: interdisciplinary treatment, communication, teamwork, treatment sequencing INTRODUCTION The contemporary clinician is fortunate to be able to draw on the significant advances in technology and material science relevant to dentistry. These developments enable the clinician to manage routine and more complex treatment problems with improved and predictable esthetic and functional outcomes. Unfortunately, a combination of the clinician's enthusiasm for these new and improved materials and technologies, combined with a lack of appreciation of the mechanisms by Complex Dentofacial Problems which complex malocclusions progress in pathological circumstances (e.g., periodontal tissue destruction or tooth surface loss) may result in overly ambitious treatment plans being offered to patients (Fig. 1). Moreover, Some clinicians overlook routine diagnostic measures as their enthusiasm for applying these new technologies overwhelms their decision-making processes (Fig.2). The purpose of this chapter is to provide the necessary background on optimized and realistic treatment goals in complex cases requiring interdisciplinary care. The chapter discusses the keys to Success in such cases, which requires a rigorous systematic approach to develop a coordinated, patient-centered and goal-oriented treatment plan. This approach emphasizes the importance of establishing a team of healthcare providers who will bring the necessary skills and knowledge to the interdisciplinary team to achieve the best possible outcomes. THE ADULT PATIENT Although two thirds of the population exhibit malocclusion, a few individuals are able to progress to later adulthood with minimal intervention from the restorative dentist (Proffit et al., 1998). Adult patients present significant challenges because they often have been affected by long-term multi-faceted progressive deterioration of restorative dentistry exacerbated by periodontal disease, attrition, abrasion and erosion (Musich, 1986; Musich and Crossetti, 1986; Abdullah et al., 1994; Spear et al., 2006). Teeth may have drifted secondary to tooth loss and/or periodontal destruction and further compensated from tooth surface loss. Moreover, a significant number of individuals may have had a pre-existing malocclusion with some degree of skeletal discrepancy and associated dental compensations; this can place unrealistic demands on the restorative dentist and the materials if attempts are made to restore the dentition in compensated positions (Fig. 1). Adult patients have many opportunities to become well informed of their possibilities and options through online searches; they often present with certain expectations and a general awareness, but usually lack a depth of knowledge of contemporary treatment options. Adults differ from the adolescent patient in many ways. The most significant difference is that they measure the costs and trade- offs of treatment carefully and make decisions for themselves, whereas Goonewardene et al. Restorative dentist - - COMPENSATES - crown form Figure 1. A-B: Intra-oral photos of patient who recently had the upper bridge placed in compensated positions, which resulted in edge-to-edge traumatic occlusion and abfraction of the ceramic margins due to overloading. C. The vertical dimension also was increased in an attempt to improve the incisor relationship. Figure 2. Veneers were placed in attempt to compensate for the Class || relationship and the irregularity without consideration for the periodontal issues. A: The upper left lateral incisor is over contoured. B-C: The lower right central and lower left lateral incisor have been built up into a crossbite relationship. decisions for adolescent patients usually are made by their parents. Adults generally are more fastidious and actively engaged in the treatment process, but have various degrees of psychological tolerance with the concept of treatment that must be considered carefully. The presence of appliances has an impact on physical appearance, comfort and function. It is important, therefore, to set realistic goals prior to treatment (Kokich and Spear, 1997) that will combine the objectives of both the clinician and the patient for a successful treatment outcome. Finally, from the Orthodontist's perspective, adults lack significant growth changes that influence orthodontic biomechanics and retention strategies, which places demands on the clinician to be more precise than in adolescents. Uni-disciplinary Treatment A restorative dentist may consider delivering treatment in- dependently and using restorative means alone for a patient with complex Complex Dentofacial Problems problems (Roblee, 1998). Treatment might be performed in this manner if the patient previously rejected intervention from other specialists and exerts significant pressure on the restorative clinician (Fig. 3). Some clinicians even attempt to deliver care without the necessary skills or knowledge in areas such as periodontics, endodontics and orthodontics. Regrettably, the clinical outcomes in these circumstances often are compromised and occasionally impose caveats or are irreversible when revision treatment is indicated or desired by the patient. Multidisciplinary Treatment Multidisciplinary treatment is a progression beyond uni- disciplinary treatment in which clinicians from multiple disciplines are integrated into the treatment in a haphazard fashion. In these cases, the clinician to whom the patient initially presents often takes the lead role in developing and initiating a treatment plan. This is problematic particularly in the adolescent patient with hypodontia who might require implants years later in adulthood. Decisions may be made unilaterally by the orthodontist regarding tooth position for optimal implant placement. The restorative dentist may confront these issues many years after fixed appliances are removed, only to discover that the patient refuses an additional course of orthodontics to idealize the tooth positions. Naturally the outcome may be a compromised restorative outcome. These shortcomings may not be isolated to restorative dentistry interactions, but may be equally important in individuals for whom oral surgery, periodontal and endodontic interactions are critical. As an example, a careful review of several case reports in published literature reveals undiagnosed periodontal disease in patients in whom orthodontic or restorative dentistry has been initiated. Multidisciplinary treatment often is characterized by poor communication between clinicians who may be excellent clinicians independently, but do not understand the considerations of all specialty areas (Fig. 4). It is necessary to appreciate the influence of discipline- specific implications on the overall management of the patient as a specific goal from one perspective may conflict with the ideal goal from another specialist area (Roblee, 1998). Goonewardene et al. Figure 3. The restorative dentist felt pressured against his better judgement into placing veneers to address the irregular teeth. The patient sought a second opinion when the veneered teeth exhibited spontaneous bleeding from the gingival tissues. A. Both upper lateral incisors have been overbuilt to improve the position of the labial surface. B: Marginal esthetic improvement from the frontal View is seen. Concepts in Interdisciplinary Treatment Planning In established offices, many orthodontic patients are referred from the restorative dentist with specific requests and clear comprehension of what the patient expects from treatment. However, many patients have little appreciation of their dental problems themselves and most do not seek treatment independently without referral for a specific complaint. The management of these patients is critical and sometimes challenging because of the disparity between the patient's and clinician's understanding of the problems. It is critical to establish the nature of the patient's primary complaint and his/her specific needs in order to determine what will define success or satisfactory outcome from the patient's perspective. A Common error in patient management is overwhelming the patient with Comprehensive and expensive diagnostic processes only to find that the patient will not consider any perceived complex treatment plans. It is essential, therefore, to establish the interest(s) of the patient. For this process to be effective, the clinician must possess the necessary wisdom to outline a program of treatment briefly along with the associated Complexities of treatment, which should include an approximation of time Complex Dentofacial Problems º - º º - - Figure 4. A: An adult patient presented to the office in appliances for completion of her orthodontic treatment, which included mandibular advancement surgery and preparation for prosthetic teeth. The referring orthodontist indicated that the patient was ready for the mandibular surgery and implant placement. B: The pre-treatment model showed a tipped molar. The treatment plan aimed to move the molar distally and open up the space. A progress model clearly demonstrates inadequate consideration by the orthodontist for the prosthetic replacement. There was too much space for a single implant and too little for two implants if an ideal occlusion was to be realized. The decision had been made Without a prosthodontist's input. C-D: The treatment plan was revised to place a single tooth implant in the premolar space to use as direct anchorage and protract the posterior teeth to close the excessive space. and cost implications (Fig. 5). A simple patient-specific set of records may be considered at this stage to satisfy the requirements of the initial evaluation and the information exchanged between clinicians (Fig. 6). While the orthodontist often assumes the role of facilitator, s/he must recognize that there are occupational biases between practitioners in various disciplines, which can influence the relative importance of specific aspects of the proposed treatment in achieving the mutually agreed goals (Kokich, 1990). Goonewardene et al. Introduce Complexity Preliminary Discussion Comprehension Biology Interest? Risk Benefit Cost $$$ Hygiene Response Therapeutic Diagnosis -> Control Phase Compliance Interdisciplinary Consultations Figure 5. Flowchart outlining the sequence of managing patients from the initial consultation to interdisciplinary consultations. It is important to ensure that this is performed in a progressive manner so that the patient understands the complexity and general goals of treatment. This may include a period of preliminary treatment, which may be considered as a therapeutic diagnostic process and may determine the suitability for more extensive treatment. In patients with complex long-standing periodontal pathology and/or compromised restorative dentistry, control of these problems is often a necessary prerequisite for future orthodontic treatment (Mathews and Kokich, 1997). Therefore, careful consideration must be given to planning complex treatment until all clinicians are satisfied that these issues can be resolved. The cost and significant time commitment of controlling disease and stabilizing the tissues will discriminate problematic patients in what often is called the “control” phase of treatment with the failure to comply with the demands placed on the patients by the practitioner. This obviously is critical before being committed to potentially risky tooth movement and placement of complex prostheses in the presence of uncontrolled disease (Fig. 7; Knocht et al., 1996; Mathews and Kokich, 1997; Axelsson and Lindhe, 1981). It also will allow the clinicians to identify the specific location and architecture of bony Complex Dentofacial Problems Figure 6. This patient has significant esthetic concerns with extensive dentistry performed in a piecemeal fashion over many years. A: Complex skeletal issues contribute to the inability to achieve an ideal occlusion with a narrow maxilla and significant dentofacial deformity. B: The panoramic radiograph reveals significant restorative dentistry and periodontal compromise associated with the restorations placed in compromised positions. These limitations will require careful and thorough evaluation and planning prior to implementing a comprehensive interdisciplinary treatment. Goonewardene et al. Figure 7. A: This patient presented with extensive dentistry that is failing and the periodontal condition also is guarded because of contribution from poorly Constructed prostheses. B: The panoramic radiograph revealed significant restorative dentistry and periodontal compromise associated with the restorations placed on teeth with compromised positions. and Soft tissue lesions (Becker and Becker, 1993), including crater defects, one-, two- and three-wall defects, furcation defects and localized horizontal bone loss (Schluger, 1949). This stage of treatment may be Considered a screening process. Complex Dentofacial Problems The adult patient often has pre-existing periodontal problems that need to be evaluated. These may include patients with: • Malocclusion that has resulted from tooth drift secondary to the periodontal tissue destruction. This is a significant challenge as these patients often have complex factors, including compliance with dental procedures, that need to be addressed. • Tooth drift and tipping secondary to loss of teeth that has predisposed the tissues to periodontal problems. • Recession-like defects and patients with thin gingival biotype, which may influence the clinician's ability to move teeth and maintain tissue integrity. The periodontist may consider soft tissue augmentation procedures either to prevent further breakdown of the gingival tissues because they are predisposed to recession by the previous history or because the tooth movement proposed by the interdisciplinary team may predispose the patient to further attachment loss (Fig. 8; Gartrell and Matthews, 1976; Dorfman, 1978; Steiner et al., 1981; Dorfman et al., 1982; Miller, 1982; Langer and Langer, 1985). Through careful consideration of the relative prognoses of various treatment procedures, the periodontist will determine if periodontal treatment alone is all that is indicated or whether the orthodontist may have to consider modifying the position of the teeth to effect changes in the bony architecture (Brown, 1973; Ingber, 1974). Consideration also may be given to bone regeneration procedures (Shallhorn and McClain, 1988; Kramer, 1992; Becker and Becker, 1993), but the orthodontist often is called upon to level teeth by selective vertical repositioning to establish a balanced and healthy bone level across affected teeth, particularly if they are associated intimately with restorative procedures (Brown, 1973; Ingber, 1974). These specific procedures ultimately may be incorporated into the biomechanical plan (Fig. 9). In some circumstances, patients are placed in a removable bite-plane or occlusal splint, in addition to the periodontal treatment. 10 Goonewardene et al. Figure 8. The lower incisor needs to be moved forward as part of decompensation prior to surgery. The thin gingival biotype requires special consideration as gingival augmentation may be required prior to orthodontics. Figure 9. A: The patient presented with tipped teeth due to previous extractions and periodontal breakdown resulting from the tipping of teeth and inadequate home care. After selective extractions and a period of periodontal treatment, pre-prosthetic orthodontics was performed to improve the angulations of the teeth. B: The panoramic radiograph exhibits excellent stability 17 years after definitive prosthodontics and regular periodontal maintenance. This enables the orthodontist to evaluate compliance with an appliance, as well as attention to appointments for orthodontic review (Fig. 10). COMPREHENSIVE INTERDISCIPLINARY TREATMENT PLANNING Once the relevant clinicians are satisfied that the patient is suitable for complex interdisciplinary treatment, they may acquire Complete diagnostic records and referral for interdisciplinary consultations with the appropriate clinicians. 11 Complex Dentofacial Problems Figure 10. In patients with a longstanding history of irregular dental attendance and neglect, it often is important to assess whether the patient is going to comply with the challenges of multiple appointments over the treatment time while control measures (e.g., periodontal treatment and provision restorative procedures) are performed. A removable appliance (e.g., bite plane) may be used with the dual purpose of relieving the direct gingival impingement on the palatal tissue margins and as a preliminary assessment for the patient's capacity to conform to the demands of treatment. Interdisciplinary treatment is characterized by a group of clinicians from various backgrounds, all of whom contribute to managing the patient in their area of expertise. It is important to establish a working team of specialists who can communicate effectively and meet regularly (Kokich and Spear, 1997; Roblee, 1998). The interdisciplinary team may include the orthodontist, restorative dentist or prosthodontist, periodontist, oral surgeon, endodontist, physicians, oral radiologist and computer scientist (Fig. 11; Kokich and Spear, 1997). Comprehensive commercial file-sharing programs are available to facilitate the transfer of information and reduce costs related to record duplication. In many Countries including the U.S., these programs have 12 Goonewardene et al. Oro Restorative Dentist - S. EGLET Cº. Leaderº ſ - ora Radiologist Computer Scientist Figure 11. The interdisciplinary team may include clinicians from many disciplines, an example of which is provided here. The key to success of interdisciplinary treatment lies in excellent communication in developing a patient-specific treatment plan with a detailed sequence of activities led by a delegated team leader. to be HIPAA compliant. For patients with more complex treatment needs, a consistently coordinated approach with a specific series of procedures must be considered and adhered to for successful management (Kokich and Spear, 1997; Roblee, 1998). These procedures include: 1. Establish goals to define success. 2. Establish a specific treatment plan. 3. Outline a sequence of events with responsible parties for treatment. 4. Clearly demarcate goals for each stage. 5. Facilitate exchange of information with all clinicians and the patient. 6. Measure outcomes against goals. Establish Treatment Goals As in routine diagnosis and treatment planning, a database will be outlined and a detailed, prioritized problem list will be developed 13 Complex Dentofacial Problems (Proffit et al., 2013). This problem list forms the basis for developing a list of ideal treatment goals (Fig. 12). It is important for the team to meet and discuss its patient(s) in either a roundtable discussion or using contemporary file-sharing programs. This provides an opportunity for valuable exchange of information, even if a specific case does not involve consideration of a specific specialist's skills (Kokich and Spear, 1997; Roblee, 1998). In formal training or treatment institutions, patients often are consulted in comprehensive multidisciplinary clinics, which facilitate interdisciplinary management with all clinicians present. In the private environment, it is more difficult to schedule meetings that will include all clinicians in the treatment team. Considerations in Goal Setting There are many factors to consider when establishing the goals for a patient's treatment, which includes the patient's primary complaint and dental history. Enhancement of dental and facial esthetics often is a primary motivation for seeking treatment. From a functional perspective, it often is difficult to reconcile changing the occlusal relationship if there is no obvious issue related directly to the occlusion. The best indication of need for intervention for an adult patient is the observation of any deteriorating status in the dentition related to that specific condition (e.g., an increased overbite). If the patient reveals signs of breakdown (e.g., tooth wear, soft tissue impingement and/or tooth migration related to their occlusal relationships), it is justified to consider addressing this aspect with their specific goals. It is a more difficult decision to address the deep overbite in the absence of adverse clinical findings. Various treatment plans with modified goals may be considered by the interdisciplinary team, each of whose members may suggest perfectly acceptable solutions not considered by the others in the group (Fig. 13; Roblee, 1998). Occupational bias can be observed when considering treatment options and this type of decision-making environment ensures a thorough evaluation of many possible scenarios. The decision to treat growing children is less complicated because they often are in optimal health, which helps achieve an ideal occlusal outcome. With adults, however, careful consideration must be focused on achieving realistic goals, which may not include the ideal goals (Kokich and Spear, 1997). Ultimately, it is important to outline how success will 14 Goonewardene et al. Pº Problem sººn Records List GOALS Figure 12. A routine process of developing treatment goals after comprehensive examination and record keeping will establish a set of ideal treatment goals, which must be assessed further to ensure that they are in keeping with the needs and desires of the patient. IDEAL TREATMENT GOALS … . . . Modified Goals 1 Modified Goals 2 Modified Goals 3 Modified Goals 4 Advantages *Disadvantages Clinicion Decision Patient FINAL TREATMENT PLAN Figure 13. The final treatment plan requires a systematic appraisal of the cost/ benefit analysis of a series of treatment options. A carefully considered treatment plan will be formulated which represents a joint decision between the patient and clinicians based on their relative value systems. be measured from both the clinicians' and patient's perspectives with appropriate consideration of quality-of-life issues (Phillips, 1999). Consideration of the cost/benefit analysis is important, particularly when complex surgery and/or prostheses are an integral part of the treatment. A more contemporary view of success includes esthetics 15 Complex Dentofacial Problems and periodontal health outcomes, as well as factors such as the longevity versus indexed cost, invasiveness and biological cost (Musich and Crossetti, 1986; Kokich, 1990, 2004, 2011; Knocht et al., 1996; Mathews and Kokich, 1997; Noack et al., 1999; Mayer et al., 2002; Weng et al., 2003; Proffit et al., 2013). If fixed prostheses are to be considered such as implants, biological and technical complications need to be fully explored. Issues such as peri-implantitis (occurring in 9.7% of cases), screw/ abutment loosening (12.7%), darkening of labial gingiva and resorption of the endosteally derived labial bone are the most commonly reported events even within the first five-year observation period (Esposito et al., 1993; Chang et al., 1999; Robertsson and Mohlin, 2000; Fürhauser et al., 2005; Belser et al., 2009). Moreover, the inability of implants to adapt to dynamic maturational changes experienced during adulthood may result in minor vertical and antero-posterior discrepancies even when the optimal time has been established to insert the implants following serial cephalometric review (Nordquist and McNeil, 1975; Iseri and Solow, 1996; Oesterle and Cronin, 2000; Thilander et al., 2001; Bernard et al., 2004; Jemt et al., 2006; Zachrisson, 2006; Fudalejet al., 2007). The team leader or appointed individual must complete the discussion carefully and systematically with the patient to make sure the final outcome aligns with the patient and clinicians' desires, acknow- ledged risks and sacrifices and associated financial considerations. DIAGNOSTIC MODEL SET-UP The occlusal outcome from routine orthodontic treatment is simpleforthe experienced clinicianto visualize; however, interdisciplinary plans involving complex tooth movements, restoration of tooth mass and replacement of teeth are more complex. These plans are analogous to the architectural drawings used in construction (Fig. 14). The application of digital study models and development of digitally assisted set-ups facilitate the assessment of numerous treatment options on a three- dimensional (3D) imaging program. Many clinicians, however, still prefer the plaster and wax set-up (Fig. 15). The diagnostic set-up is a key communication tool and reference point for all parties and facilitates appreciation of the: 16 Goonewardene et al. Figure 14. Contemporary digital imaging systems may be used to produce digital diagnostic set-ups such as the models displayed. This provides clinicians with the specific goals of the treatment outcome and allows more detailed information to be shared between all parties. Figure 15. A: Plaster models demonstrate a significant malocclusion and the treatment goals established for all parties by preparing a wax and plaster diagnostic set-up. B: The diagnostic work up incorporates upper incisor flaring and opening of Spaces to replace the missing incisors, canine and premolars; the anteroposterior occlusal relationships will be corrected with mandibular surgery. Proposed occlusal relationship. Anticipated space requirements for alignment and/or Compensatory tooth movements, which may neces- sitate extractions and/or interproximal reduction. 17 Complex Dentofacial Problems • Antero-posterior and vertical space requirements for restorative dentistry particularly relevant to tooth replacement. • Anchorage requirements for orthodontic tooth movement. Treatment Sequencing The final treatment plan reflects the combined decision of the treatment team and the patient. A treatment-sequencing document routinely is prepared principally by the team leader to facilitate communication and ensure that the goals for each stage are understood clearly (Kokich and Spear, 1997). This document ensures an efficient transition through treatment and allows the patient to review his/her progress. The orthodontist or restorative dentist frequently leads this process as s/he often spends significantly more time with the patient during the course of the treatment, although any team member may assume this role (Fig. 16). Pre-prosthetic Tooth Movement The restorative dentist routinely is challenged with restoring teeth that are not positioned ideally (Kokich and Spear, 1997). Minor modifications to ideal tooth preparation or occlusal reduction may compensate for a less-than-ideal position of teeth and excellent esthetic and functional results may be realized. This is considered to be — Figure 16. A: The patient presented with significant dental problems contributed to by tooth loss and a skeletal Class Il jaw base discrepancy. B: After refining a specific treatment plan, a sequencing document was developed that encompasses all details of the plan in a systemic way. Initials represent associated author/clinician. C. As outlined in this case, provisional prostheses are placed prior to orthodontics, jaw surgery and final prosthetics to realize the excellent occlusal outcome following treatment. 18 Goonewardene et al. SN: Sequencing Lab set-up and stent construction BA/BG Implants anterior (teeth 11, 22) BA Provisional bridge to ideal positions BG Pre-surgical orthodontics MG – align and prepare space on teeth 43,44, 36 Mandibular advancement EXO 36 BA - implant placement on teeth 36,43, 44 Finalize orthodontics MG Definitive prosthodontics – crown teeth 36, 43,44 and bridge teeth 11-22 B | Maintenance (splint) BG/MG -- 19 Complex Dentofacial Problems “conformative” restorative dentistry (Fig. 17). In many circumstances, however, tooth movement is required to ensure that the optimal form and position is achieved, including: 1. Teeth with atypical crown form a. Peg lateral incisors and other deviations in shape b. Teeth subjected to wear and/or erosion and subsequent compensatory tooth movements 2. Teeth that have drifted and tipped associated with tooth extraction or congenital absence Teeth with Atypical Crown Form. Absence of normal tooth form often results in compensatory tooth movements to maintain tooth contact and also may be a component of significant malocclusion traits. It is important to evaluate the spatial position of the crown and associated gingival anatomy before planning tooth movements in 3D. Ideally, the abnormally shaped tooth should be restored with a provisional restoration (usually a composite resin or heat-cured temporary crown), prior to tooth movement to an ideal position (Kokich, 1993b,c, 1996; Chiche et al., 1994; Kokich and Spear, 1997). Tooth movement often is required prior to build-up to create the necessary space in all three dimensions to achieve ideal form. This is significant particularly in cases with attrition with compensatory vertical eruption, antero- posterior uprighting and mesiodistal dimension reduction because of the converging crown form to the gingival (Fig. 18; Berry and Poole, 1976; Silness et al., 1994). It is necessary to intrude, procline and redistribute the teeth mesiodistally to facilitate restoration in all dimensions (Fig. 19; Dahl et al., 1975; Evans, 1997; Dyer et al., 2001; Kokich, 2001; Mizrahi, 2006). Following repositioning, the restorative material of choice in patients with previous attrition should match the wear properties with natural teeth and/or the dentition in the opposing arch (Fig. 20; Dahl et al., 1993). For those problems related to dentofacial deformities, space also may be created by vertically repositioning the mandible relative to the maxilla (Fig. 21). When planning tooth movements, the esthetic balance of the smile always must be considered, including 20 Goonewardene et al. Figure 17. A-C. This patient ideally requires orthodontics and restorative dentistry due to an increased overbite, tooth wear in the upper and lower incisors and a demand for an improved esthetic outcome. The patient did not want to consider orthodontics, so a conformative treatment plan was presented by the restorative dentist to increase the vertical dimension by adding composite to the palatal surface of the canines (D) and to create vertical space for final prosthetic rehabilitation (E-F). the mesio-distal and inciso-gingival proportions of the teeth (Fig. 22; Spear et al., 2006; Kokich, 1993b,c). Where significant deviations in form are found to influence esthetics substantially, several proposed guides (e.g., the mathematically derived golden proportions) can be utilized (Kokich et al., 1984; Kokich, 1993a). 21 Complex Dentofacial Problems 2. Decreased m-d dimension decrease arch length from decrease arch length Figure 18. Effects of tooth wear on inter- and intra-arch relationships. Attrition of teeth is a complex problem that results in tooth position compensations. The most significant effect is the loss of crown height and accompanying extrusion of the tooth/teeth that will affect the level of the gingival margin. The extent of the attrition often may be determined by the variation in level of the respective gingival margins. The secondary effect is a reduction in mesiodistal dimension of the teeth and subsequent uprighting of the teeth to assume a more vertical relationship. 22 Goonewardene et al. Figure 20. A-B: A female patient presents with a complaint of tooth wear and excessive gingival display. C. The incisors were intruded to create vertical space. D-E: When combined with minor gingival surgery, composite build-ups produced an excellent esthetic outcome. Figure 21. A vertical inter-occlusal gap for restoration of vertical space may be achieved by surgical vertical repositioning of the mandible relative to the maxilla as exhibited. A: The deep overbite and bruxing habit had reduced the bulk of the right central incisors such that the tooth was so worn that the darkened appearance that represents thinning from the palatal surface can be seen from the frontal view. B: Post-surgical position. C. The final outcome enabled the restorative dentist to restore the worn palatal surface and achieve an ideal vertical incisor relationship. 4– Figure 19. An adult patient presents with an esthetic complaint related to the incisor irregularity and tooth wear, which was impacted by placement of a ceramic crowns. A. Variation of the gingival margins represents a combination of recession and tooth extrusion secondary to the attrition. Orthodontics aims to align the teeth and gingival margins. B: The provisional outcome reveals improvement in gingival margins, provisional restoration of the central incisor mass with composite that requires further refinement as indicated by the reference lines. C. At deband, the gingival margins have been balanced. D: The final restorations in ceramics are most pleasing. 23 Complex Dentofacial Problems Figure 22. Ideal relationship of the dimensions and positions of the teeth to each other and the lip line have been recommended for optimal esthetics. This includes the tooth widths, gingival margin distribution, contact posits and the gingival papilla projection as well as the smile arc relative to the lower lip. ". Complex Tooth Movements in Partially Edentulous Patients. A partially edentulous patient may present with issues related to tooth drift (Fig. 23), tipping and eruption in addition to the need for tooth replacement (Fig. 24; Kokich and Spear, 1997). In this circumstance, there is a degree of maxillary vertical deficiency and facial asymmetry (Fig. 24). A problem list is developed (Fig. 25), which may involve a course of orthodontic therapy to idealize tooth position prior to prosthetic replacement. An interdisciplinary sequencing plan is outlined (Fig. 26) to achieve a pleasing final outcome (Fig. 27). The goals of treatment may include alignment of teeth to facilitate replacement of a prosthetic tooth of optimal size and form from an occlusal and periodontal perspective. Improved patterns of occlusal loading to improve prognosis of the restorative therapy and normalizing periodontal architecture hypothetically reduce the likelihood of plaque accumulation and facilitate home care. Moreover, it may be possible for orthodontic tooth movement to reduce the complexity of tooth replacement by reducing the number of prosthetic teeth, potentially even eliminating the need for prostheses. Historically, there have been biomechanical limitations on the clinician to manage severely tipped or extruded teeth. The introduction of osseointegrated dental implants and temporary anchor devices (TADs; screws and plates) has made a significant contribution to dental rehabilitation with what is now considered to be a relatively predictable prosthetic replacement (Noack et al., 1999; Mayer et al., 2002; Weng et al., 2003; ADA Council of Scientific Affairs, 2004; Kokich, 2004). Dental 24 Goonewardene et al. Figure 23. This patient exhibits problems with long-standing compromised restorative dentistry. Her previous clinicians attempted to restore her partial edentulism with associated tooth irregularity and a significant jaw base discrepancy. A-C: Facial evaluation reveals a significant asymmetry with an occlusal plane cant and insufficient tooth display at rest and when animated. D-H: Intra-oral views exhibit a heavily restored upper arch with two bridges replacing the upper right second premolar and upper left central incisor, as well as a crown on the upper left molar. The combination of poor periodontal morphology and compromised restorative dentistry eventually resulted in progressive failure of the treatment rendered. implants now may be considered for the majority of replacement options, but may be limited by anatomical factors (e.g., sufficient bone volume, space requirements and issues related to dentofacial growth). Osseointegrated implants may be placed strategically prior to or during orthodontic treatment to facilitate the magnitude and type oftooth movement (Fig. 28; Brânemark et al., 1969; Linkow, 1969; Roberts et al., 1984, 1990; Odman et al., 1994; Buser et al., 1997; Wehrbein and Merz, 1998; Umemori et al., 1999). The additional anchorage derived from 25 Complex Dentofacial Problems Figure 24. The panoramic and poster-anterior radiographs reveal the extent of the failing restorative dentistry from restorations placed on teeth in compromised positions (A) and the significant skeletal asymmetry (B). GS: Problem List Facial asymmetry - –Maxillary cant –Secondary mandibular cant Decreased tooth display –Maxillary vertical deficiency Heavily restored dentition - ? Prognosis for upper anterior teeth Figure 25. The problem list for the patient in Figure 23 is identified based on the diagnostic findings. GS: Sequencing Periodontal Stabilization GP FFAs align: redistribute space MG Oral sugery modified LeFort, mandibular osteotomy BA Orthodontic finishing MG Extraction of teeth 12, 21, 23, 24 BA Implant placement BA Provisional restorations BGS Retention appliances MG Final restorations BG Maintenance BG/MG 26 Goonewardene et al. Figure 27, A-C. The final outcome following periodontal maintenance, ortho- dontics and jaw surgery to address the skeletal asymmetry and targeted restor- ative dentistry reveals a symmetrical face and Smile. D-H: An implant-supported bridge now replaces the teeth from upper right lateral incisor to upper left molar and routine fixed bridges replace the missing lower teeth. 4–Figure 26. The sequencing plan is developed and circulated to all team members as well as the patient, to outline a specific set of activities. Initials represent the respective author/clinician. This process ensures that communication between all parties is enhanced and the document may be reviewed by any party through the course of treatment. The prognosis for the upper anterior teeth was considered guarded and the timing of their replacement was considered carefully as they would be replaced by an implant-supported prosthesis. 27 Complex Dentofacial Problems Figure28.A: This patient presented with missing upper lateral incisors and canines. Osseointegrated implants have been placed with provisional restorations in the lateral incisor positions prior to treatment. B: Implants were used as anchors to protract the posterior teeth. C. Space closure was relatively simple because of the stable anchorage provided by the implants. the implants have expanded the envelope of possible tooth movements and reduced significant side effects often encountered during extensive repositioning. Moreover, the implants have enabled significant intrusive movements and vertical control to be achieved with relative ease. Careful planning assisted by the diagnostic set-up helps the clinicians determine the final desired position of the implant when placed prior to or during orthodontic treatment. A range of TADs recently has been introduced including screws and bone plates (Kanomi, 1997; Nagasaka et al., 1999; Daimaruya et al., 2001; Freudenthaler et al., 2001). These devices may be placed outside the field of tooth movement and have been shown to be effective in facilitating significant tooth movements by reducing biomechanical side effects (Fig. 29). Retention Strategies Following the removal of fixed orthodontic appliances, retention strategies may vary depending on the types oftooth movements achieved. Vacuum-formed removable and fixed-wire retainers often are chosen to provide immediate stability following alignment. Bonded rigid retainers or provisional bridgework may be placed immediately in circumstances of edentulous spans; final refined prosthetic reconstruction may be delayed for three to six months following removal of appliances. It often is necessary to consider an occlusal Splint as part of the long-term retention regime and the interdisciplinary team must ensure that the delegated clinician takes the responsibility for the construction and maintenance of the appliance. 28 Goonewardene et al. Figure 29. A: The patient presented with a missing lower second premolar and an otherwise ideal occlusion. The deciduous second molar is resorbing. B: A skeletal anchorage plate, developed by Nagasaka and coworkers (1999), was used to close the space completely (C). The plate was placed with two fixation Screws between the lower premolar and canine. The exposed arm provides a series of possible attachment points for force application, which is dependent on the clinician’s desired line of action. CONCLUSION Interdisciplinary treatment plans can be rewarding for both the treating clinicians and patients. Regardless of the individual clinician's technical skill, however, significant regret can be encountered when a rigorous systematic approach is not pursued in developing a Coordinated, patient-centered, goal-oriented treatment plan (Figs. 30- 31). Subsequently, interdisciplinary treatment was undertaken to address the patient's concerns resulting in esthetically and functionally pleasing outcomes (Figs. 32–33). It is critical to establish the needs of the individual patient within his/her value system so that a realistic plan can be addressed regarding these needs. For the patient who presents with a degree of neglect, it is important to control disease initially and ensure that the patient is well informed of the complexities associated with such an interdisciplinary treatment. This disease control stage often is seen as a Screening tool for suitable patients. In many circumstances, the plan may approach an ideal, but alternate plans with various outcomes may be equally acceptable to the patient and clinician. To achieve the best outcome, clinicians must establish a group of healthcare providers who are willing to contribute to an interdisciplinary team. This will require regular meetings to discuss more complex problems and bring their respective expertise to share with other clinicians. They must be able to communicate regularly and effectively through appropriate media. A rigorous systematic case-specific database 29 Complex Dentofacial Problems Figure 30. A-C. The patient presented with a primary complaint of a poor smile arc following placement of two implant-supported prostheses in the maxillary central incisor region. She also expressed a desire to improve her chin projection. The restorative dentist placed the implants to conform to the current occlusal relationship without considering the overall esthetic and functional goals. D-H: The incisors are not projected sufficiently vertically. of clinical records must be established, a team leader for that specific treatment plan identified and an appropriate treatment goal established, which will include a diagnostic set-up in most cases. The patient with bilateral cleft lip and palate (CL/P) shown in Figures 34 to 37 illustrates the systematic and appropriate approach in a complex case that resulted in optimal results. 30 Goonewardene et al. Heavily resored mplant posſerior teeth 21 overeupted 37 33 47. 43 JM: Problem list Smile arc (implants 11, 21) Heavily restored teeth Increased overjet Mandibular retrognathism C Over-erupted lower posterior teeth JM: Sequencing Extraction of the lower third molars and placement of bone BA plates to facilitate intrusion of the second molars FFAs to align and coordinate the teeth within the arches— upper lateral incisors will be extruded to increase the tooth MG display—space opened to increase the mesiodistal size of the anteriors Mandibular orthognathic surgery (remove plates) BA Post-surgical detailing of the occlusion MG Implant crowns, elongated and widened BGS Final crowns BGS Modification of retainer MG D Splint as retainer longterm BGS Figure 31. A: The panoramic radiograph reveals extensive restorative dentistry and lower second and third molars that are unopposed and extruded. B: Surgical advancement of the mandible will be compromised by the vertical position of the molars, so skeletal anchorage plates were used to support intrusion of the Second molars prior to mandibular advancement. Consistent with the principles of interdisciplinary planning, a problem list is developed (C) and a sequence of activities established to which all clinicians can refer to (D). Initials represent the respective author/clinician. 31 Complex Dentofacial Problems Figure 32. The mandible is advanced after levelling the lower arch and aligning the upper teeth. A: Pre-surgery. B: Post-surgery. A sequence of activity with specific goals should be outlined, identifying the respective clinicians and their immediate goal, to which all clinicians and the patient may refer at any stage in treatment. The team leader takes the responsibility of ensuring that the patient and clinicians are all cognizant of the stage that the patient has reached and what is performed next, in turn, facilitating communication. If these processes are followed, the patient and team can anticipate a mutually rewarding experience with consistently reproducible outcomes. 32 Goonewardene et al. Figure 33. A-C. The final esthetic outcome satisfied the patient's primary complaint of increasing the tooth display, enlarging the mesiodistal dimensions and increasing chin projection. D-H: The occlusion was idealized and the tooth dimensions were increased to improve the balance of tooth size. 33 Complex Dentofacial Problems PROBLEM LIST Bilateral CL/P Oronasal fistula Missing incisors Maxillary vertical deficiency Maxillary retrognathism C Asymmetry (maxilla, mandible) RL: Sequencing - Placement of craniofacial implants BA Full fixed appliances MG Maxillary and mandibular advancement and leveling BA (downgraft) Complete fixed appliances MG Tongue graft to close fistula BA Bone graft to cleft BA Implant placement BA Definitive prosthodontics BG D Maintenance (splint) BG/MG Figure 34. A-B: A patient with a partially repaired bilateral CL/P presented with a primary complaint of a poor esthetics. C. A complex diagnostic and treatment planning process identified key issues related to the skeletal asymmetry and dentofacial deformity, the oro-nasal fistula and the missing teeth. D. 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Am J Orthod Dentofacial Orthop 1999;115(2):166-174. Wehrbein H, Merz BR. Aspects of the use of endosseous palatal implants in orthodontic therapy. J Esthet Dent 1998;10(6):315-324. Weng D, Jacobson Z, Tarnow D, Hürzeler MB, Faehn O, Sanavi F, Barkvoll P, Stach RM. A prospective multicenter clinical trial of 3i machined- surface implants: Results after 6 years of follow-up. IntJ Oral Maxillofac Implants 2003;18(3):417-423. Zachrisson BU. Single implant-supported crowns for the anterior maxilla: Potential esthetic long-term (>5 years) problems. World J Orthod 2006;7(3):306–312. 42 DENTAL REHABILITATION: MULTIDISCIPLINARY OR INTERDISCIPLINARY TREATMENTP Birte Melsen ABSTRACT Dentistry has developed into a discipline that takes care of the problems that the village doctor, blacksmith and barber handled when extraction was the only solution to toothache. The family dentist was considered capable of taking care of most tooth-related problems, but with increased comprehension and integration of biological mechanisms combined with development of treatment strategies and materials, a number of specialties have been defined, initially comprising only surgery and orthodontics. Orthodontics was considered a profession that focused on the treatment of children and took advantage of the growth that occurred during treatment. Today, however, with increased life expectancy and the associated focus of combating age-related changes, the demand for rehabilitation of a degenerated dentition has increased. It has been demonstrated clearly that the balance in the chewing organ will change with time, due to general age- related changes within the bone and local degeneration (e.g., loss of teeth due to caries or periodontal disease). As a consequence, Secondary malocclusions may develop or be aggravated. The re-establishment of an aesthetic and functionally satisfactory solution rarely can be achieved by replacing teeth by fixed prosthodontics and implants alone. Orthodontic treatment often is an indispensable part of rehabilitation of the degenerated dentition, though the patients and the different dental specialists may perceive the problem differently. A satisfactory and maintainable rehabilitation can be achieved only in an interdisciplinary collaboration in which all involved professionals have an understanding of each other's contribution and in which the patient agrees to invest the time and resources necessary for the chosen treatment approach. The maintenance of the treatment result likewise requires the understanding of age- related changes that also occur after rehabilitation. KEY WORDS: adult orthodontics, dental degeneration, interdisciplinary rehabilitation 43 Dental Rehabilitation INTRODUCTION Dentistry has developed into a discipline that takes care of the problems that previously were handled by the village doctor, blacksmith and barber when extraction was the preferred solution for most problems. The family dentist was considered capable of taking care of all tooth- related problems, but with increased comprehension and integration of biological mechanisms combined with development of treatment strategies and materials, a number of specialties have been defined. It is accepted, therefore, that the general dentist alone is unable to master and deliver all areas of dentistry to the highest level (Fig. 1). The patient, however, can benefit only from the advances within each specialty if the specialists communicate and collaborate actively. Surgery and orthodontics were among the first specialties within dentistry. Orthodontics initially was considered a profession that treated children and took advantage of the growth that occurred during treatment. Today, however, with increased life expectancy and the associated focus of combating age-related changes, the demand for rehabilitation of a degenerated dentition has increased. Removable dentures gradually are becoming unacceptable; therefore, the conse- quence is an increasing demand for fixed restorations. In order to pro- vide optimal care for these patients, it is important that the patient and dentist both recognize that the masticatory organ of the adult pa- tient undergoes changes with time, due to general age-related changes within the bone and due to local degeneration (e.g., loss of teeth due to caries or periodontal disease; Figs. 2-3). Consequently, secondary malocclusions may develop or be aggravated; re-establishment of an aesthetic and functionally satisfactory solution rarely can be achieved by substituting the lost teeth with fixed prosthodontics and implants alone; orthodontics, therefore, often is required as part of a rehabilita- tion (Behrents, 1985a,b; Melsen, 2012a). The aim of this chapter is three-fold: first, to demonstrate how different specialties perceive patient's problems differently; second, to explain how an interdisciplinary approach can make regeneration of even severely degenerated dentitions possible; and third, to show the importance of the general dentist in the maintenance of the results achieved since treatment should not aim for short-term results, but should focus on those that can be maintained long term. 44 Melsen Orthodontist Prosthodontist Periodontist Patient Evt. Gnathologist Figure 1. Survey of different specialties originating from dentistry. A Figure 2. Human trabecular obtained from the vertebrae of (A) an 18-year-old Woman and (B) a 60-year-old woman. PROBLEM LISTING: DIFFERENT SPECIALTIES, DIFFERENT PROBLEMS The influence of oral health on life quality, especially for the aging population, has been recognized and a number of indices have been developed to quantify the impact of individual factors. The validity of many of the indices has been assessed in several studies (Hebling and Pereira, 2007; Ozhayat et al., 2010; Kieffer et al., 2011; Sierwald et al., 45 Dental Rehabilitation Figure 3. Skulls from (A) a young adult and (B) an old adult that demonstrate age-related bone loss at the alveolar margins. 2011; Kenig and Nikolovska, 2012; Leon et al., 2014); the weaknesses of such methods also have been documented (Tsakos et al., 2012). A common finding due to the many confounders, nevertheless, is that independent of the method applied, life quality is related highly to the preservation of a functional and esthetically satisfactory dentition and that there is an ongoing decrease in oral health status with advancing age. As a consequence of the slow ongoing deterioration of the dental status, which often includes occlusal changes, patients may need major dental rehabilitation, but may be aware of and seek treatment only for small problems. In most cases, the patient presents with an acute problem—the tip of the iceberg—and the fact that this problem is the result of ongoing deterioration may not have been noted by the patient. The key difficulty when studying patients with a degenerated dentition, therefore, is that the patient's perception of the problem may diverge considerably from the true problem, which may lead to communication difficulties both between patient and dentists and between colleagues from different dental specialties (Melsen, 2012b). As an example, a 49-year-old woman was concerned with a gradual increase in labial displacement of her upper lateral incisor, which traumatized her lower lip (Fig. 4). She asked her family dentist for advice and received a referral to an Orthodontist for correction of the flared incisor; this appeared to be only a minor part of her problem. She had a deep bite, severe periodontal problems, and upper lateral teeth, a molar and a premolar, could not be maintained as they had almost 100% bone loss. The patient could have had more than one reaction to this situation: “Why didn't my dentist tell me about this before?" If the patient felt this way, she might not return to her dentist, who most likely would not refer another patient to this orthodontist. Another possible 46 Melsen Figure 4. A: Extra-oral view of a 49-year-old woman with complaints related to a progressively flaring upper lateral incisor traumatizing her lower lip. B-E: Intra- oral images, however, reveal other problems including crowding in both arches and a deep bite with impingement. F: Intra-oral radiographs demonstrate Severe periodontal problems to a degree that the upper right molar and second premolar needed to be extracted. reaction: “This specialist is only out to provide a complicated and expensive treatment; I would rather stay with my family dentist and accept his possible solution.” Regardless of the reaction, the patient ends up dissatisfied and frustrated. The flaring of the incisor did not occur overnight; it was related to the deepening of the bite, but the process 47 Dental Rehabilitation reached an unacceptable level because neither the dentist nor the patient was aware of the ongoing process. In this case, it was the patient that expressed a need. In other cases, as described below, it might be a new dentist seeing the patient. The clinician must have a comprehensive grasp of several aspects of dentistry in order to provide treatment or refer to the appropriate professional. When searching PubMed with the keywords “occlusion” and “aging,” only fifteen papers could be found, which perhaps represents a lack of understanding of the dynamics of the occlusion. The difference in perception of the problem by specialty domains also may act as a confounder, which contributes to the patient's mistrust and confusion with the dental profession. The divergence between problems registered by different specialties is illustrated in Figure 5. The patient's primary reason for seeking treatment was an increasing problem with a traumatic occlusion on a lower second molar that had tipped mesially following extraction of the first molar. The dentist suggested replacing the lost molar with an implant, but the implantologist's challenge was the lack of space for an implant and, therefore, recommended uprighting the second molar. The periodontologist's focus was gingival impingement, which led to periodontal problems on the lingual aspects of the upper molars. The gnathologist noted severe abrasion and signs of bruxism. The orthodontist also noted an over-erupted upper incisor, a midline discrepancy, a lower arch asymmetry and a distal canine and premolar relationship on the left side, as a result of the distal tipping of lower premolars and canine, secondary to the loss of the lower molar. Most, if not all of these problems could be ascribed to a failing replacement of the extracted molar; it also is likely that at the time of molar extraction, the patient was not counseled adequately regarding possible consequences of the extraction and, therefore, did not accept an immediate replacement. It is only through a coordinated approach when the specialists work together toward a common treatment plan and share this information consistently with the patient that a maintainable result can be achieved. STOPPING AND REVERSING THE DENTITION'S DEGENERATION: AN INTERDISCIPLINARY TASK The dentist's focus when treating young patients is mainly on caries prevention and monitoring occlusal development. The goal when 48 Melsen Figure 5. A-E: Intra-oral images of a patient who saw his family dentist due to traumatic occlusion relative to a mesially tipped lower third molar. The different problems perceived from the different dental specialists are described in the text. F: Intra-oral radiograph reveals the mesially tipped second molar. treating adult patients, on the other hand, is to prevent degeneration and, if possible, to regenerate or recreate an aesthetic and functionally satisfactory dentition that can be maintained for life. It generally is ac- cepted that dentists who treat children and adolescent patients should refer them to an orthodontist when occlusal development deviates from what is considered the norm. The general dentist who treats adult pa- tients also should be aware of age-related dynamics and the need for an interdisciplinary treatment approach that involves orthodontics if a main- tainable treatment result is needed. The benefit of referral to a specialist often is ignored, however, as the immediate solution to the patient's chief complaint usually is considered the main issue. The lack of interdisciplin- ary treatments also is reflected in the survival studies on implants and/ or single prosthetic reconstructions where such analyses of the occlusal changes have been overlooked. When a patient needs rehabilitation, the first step is to stop the ongoing degeneration with the necessary intervention (Table 1) and only after this determine which type of reconstruction should be rec- ommended. Through these interventions, not only is the progression of the pathological processes stopped, but the patient's response to treat- ment also can be assessed. This is important as the patient's willingness to invest resources—both money and time-will determine the final plan. It does not make sense to invest in an expensive reconstruction 49 Dental Rehabilitation Table 1. Essential procedures. • Oral prophylaxis • Provisional restorative treatment • Endodontics • Extractions if the patient is not willing or able to maintain the result; therefore, the choice of optional interventions depends on the reaction to the essential procedures (Table 1). When serving an aging population, the dentist's attention should be focused on maintenance. The risk of developing periodontitis as a Consequence of gingivitis and poor oral hygiene increases with age (Schei et al., 1959; Holm-Pedersen et al., 1975); nevertheless, the regeneration of the lost periodontal support has received limited and non-uniform influence on the treatment performed on patients with a degenerating dentition (Karring, 1993; Lindhe, 1997; Bartold, 2015). On the other hand, the introduction of dental implants has had an overwhelming impact on both research and clinical behavior, no doubt due to the influence of the marketing related to this approach (Brănemark et al., 1977; Albrektsson et al., 1986). When the keywords “rehabilitation” and “dental implants” were combined in a PubMed and Embase search, 4,290 publications were found, though it is striking that only 29 were randomized-controlled studies with long-term control and none focused on the advantage of an interdisciplinary approach in the treatment of the patients with a need for rehabilitation. In a Society where avoidance of degeneration and rehabilitation of the aging population is in focus, it seems odd that treatment of elderly patients with a degenerated dentition still is performed primarily via prosthodontics without any or only minor interaction with other disciplines (Zarb, 2003). If adult patients are to benefit from the existing knowledge and expertise within different disciplines, it is crucial that a total problem list is compiled, including why and how these problems can be resolved. Abstaining from collaboration with other disciplines regarding the patient as a “whole" can be regarded as a weakness and it is such abstinence that will prevent further development for the benefit of the patients. The prosthodontist's referral of patients to an orthodontic spe- cialist often is limited to the situation in which one or more teeth have 50 Melsen to be displaced before the prosthodontist can perform a replacement of a lost tooth. A median diastema of a magnitude that does not allow for the replacement of the incisor that cannot be maintained for caries risk or for periodontal reasons may cause the dentist to ask the orthodontist for a space reduction, which would allow him to replace the lost tooth with a bridge. This is an example of a multidisciplinary treatment where the treatment plan is already defined and alternatives may seem out of the question. The patient in Figures 6 and 7 was referred for space closure before the upper right central incisor, or possibly both central incisors, could be replaced by a bridge. Based on results described in both clinical studies and animals experiments (Melsen et al., 1988, 1989; Melsen and Kragskov 1992), the orthodontist suggested a conservative approach that involved an attempt to maintain the incisor planned for extraction by undertaking a modified Widman Flap surgery and intruding it while closing the space. This approach provided nothing to lose and everything to gain. At the end of treatment, the incisor to be replaced exhibited no pocket and the decision was made to refrain from extraction and stabilize the treatment result with a cast retainer. Retrospectively, the questions can be asked: Why was this diastema not treated before? Where was the neglect? The reason was the lack of information transfer. The patient had noted the increasing diastema on family photos, but was not aware that it could be prevented; only when the problem became acute with the loosening of the central incisor did he change to a dentist who focused on periodontal problems, from whom he received periodontal treatment and referral to an orthodontist. . MULTIDISCIPLINARY OR INTERDISCIPLINARYP Multidisciplinary collaboration is characterized by each of the involved dentists performing the same tasks as they traditionally have performed without interacting with their colleagues. In these circumstances, the treatment should be interdisciplinary, which indicates that treatment planning is an interactive process in which all disciplines involved express their opinion. The consensus from such a treatment plan would take the patient's general health, priority and expectations into consideration. The lack of an interdisciplinary approach to treatment of degenerated dentitions is reflected in the meager results when searching 51 Dental Rehabilitation Figure 6. A-B: Intra-oral images demonstrate a problem localized to the upper incisor region where flaring and an extrusion made prosthodontic reconstruction impossible. There is a need for a combined intrusion and retraction of the central incisors. C: Treatment began with an intrusion and mesial movement of the most protruded incisor with three-piece mechanics. The difference in point of force application resulted in a mesial tipping of the left incisor. D: Once the left incisor had reached the level of the right incisors, a group of three incisors was intruded and retracted as a block. The right lateral incisor was not included in the appliance until the finishing stage because it basically was placed in a desirable position. E-F: At the end of treatment, a normal sagittal and vertical incisor relationship had been reached and the clinical crowns were reduced significantly. PubMed, Embase and Cochrane with the keywords “dental regeneration,” “rehabilitation” and “interdisciplinary.” However, the lack of evidence- based literature on the topic not only may reflect a lack of interest, but also may indicate different facets of the topic. True lack of collaboration, or the difficulty related to research in order to establish the necessary evidence, would require randomized controlled trials, which are impossible to perform for various reasons that will be discussed later. The importance of dental rehabilitation for quality of life, however, is important and was stressed by Freud (Leibin, 2003) and Jung (2001), who analyzed the dreams of people and indicated that loss of teeth could be identified as loss of power and influence. This is corroborated by the aging edentulous face, which within art often is interpreted as a manifestation of weakness. In order to maximize the benefit of treatment, an interdisciplinary approach that takes advantage of advances within all disciplines is necessary. This has been stressed in relation to orthodontic treatment of adult patients (Ong et al., 1998; Turpin, 2001, 2010; Gkantidis et al., 2010; Aulakh and Melsen, 2011; Vinod, 2012). The orthodontist needs to recognize that without colleagues who 52 Melsen Figure 7. A. Before removal of the appliance, a cast retainer extending from canine to canine was placed. The teeth were prepared for the retainer as for a Maryland bridge and the retainer was equilibrated for occlusal contact and group contact on protrusion. B: Tracing demonstrates the combined intrusion and retraction that occurred. C-D: Ten-year follow-up revealed good stability. E-F: Twenty-five-year follow-up shows that the retainer was broken between the lateral incisors and the canines, and the four incisors had started migrating forward. However, the benefit of the intrusion still was present and it was decided to give the patient a night guard instead of using a fixed appliance again. can provide the healthy tissue necessary to avoid iatrogenic Consequences of orthodontics and those who can generate the optimal occlusion by restoring normal tooth anatomy and replacing lost teeth, the final result of an Orthodontic treatment for an adult patient with a degenerated dentition will not be satisfactory. PRESENTATION OF THE PROBLEM LIST: THE TIP OF THE ICEBERG The examples described above serve to illustrate that when treating adult patients, a major obstacle is communicating the problem list to the patient. It is a difficult task to explain that what the patient perceived as a simple problem is the Symptom of a highly complex situation. For example, having heard that a simple disharmony (e.g., the flaring of incisors) is a symptom of a more severe malocclusion, patients may react in different ways. They may reject the complexity of the 53 Dental Rehabilitation problem and opt for an immediate and easy prosthetic solution. In the case of crowding, this may consist of extraction or, in the case of a deep bite, reduction in the height of incisors by grinding or by adding lateral on-lays that will leave the incisors to erupt even further. Unfortunately, treatment of one arch often is demonstrated in many Internet advertise- ments and possible consequences are neglected. Sometimes acompromise has to be made when treating elderly adult patients (Zachrisson, 2004a,b). In those cases, the limitation in the treatment goal should be made clear to the patient, as should the influence on the prognosis for the dentition. Independent of the patient's chief complaint, a comprehensive problem list workup still is necessary, after which treatment options can be discussed. Figures 8-10 illustrate the controversy between the subjective and objective problems between the patient's chief complaint and the dentist's findings—in other words, treatment demand versus treatment need. A 48-year-old woman presented to a general dentist for the first time. She had just moved to the city and had experienced pain and developed an abscess buccal to the upper left first molar during a vacation. She had been given high dosages of antibiotics and was advised to see her dentist upon returning from vacation. She explained her acute problem and added to her story that she had a bridge which repeatedly was loose and asked, “By the way, now that I am here, could you fix a bridge on the other side since it has come loose several times?” Apart from these complaints, the patient generally was satisfied with her dentition. When asked about other complaints, she explained that she frequently suffered from headaches, which she ascribed to a stressful job situation. The result of the clinical examination and interdisciplinary approach in relation to this patient is described below. Extra-oral Examination The face appeared symmetrical in the frontal view with bi- maxillary protrusion and insufficient lip closure (Fig. 8A-C). The functional analysis revealed traumatic occlusion in the first premolar region bilaterally and sliding of the mandible to the left during clo- sure. This resulted in a slightly asymmetric occlusion and asymmetric con- dylar position, which later was confirmed by the large distance between 54 Melsen Figure 8. A-C: Extra-oral images of a 48-year-old patient who visited the dentist due to pain. The patient has a bimaxillary protrusion and insufficient lip closure. D: Periapical status demonstrates two granuloma on the upper right first molar and a deep caries on the upper left first premolar E-H: Intra-oral pictures reveal a neutral sagittal relationship with crowding in both arches and traumatic occlusion on the first lower premolars that are in Crossbite. the posterior contours of the mandibular ramus on the head film (Fig. 9A). Palpation of muscles revealed tender temporalis muscles on both sides. Before making any other analysis, it became obvious that the problems were more severe than the patient anticipated. The upper right molar, where she had experienced pain, had an abscess on two roots With insufficient root filling; this tooth could not be saved. Regarding the other chief complaint, the loose bridge, the radiographs showed 55 Dental Rehabilitation A Figure 9. A. Lateral tracing demonstrates an asymmetrical level of the mandible's posterior border as a possible sign of a forced bite. B: 3D virtual treatment object (VTO) made on a lateral tracing combined with an occlusogram was used to illustrate the treatment goal in 3D. C-E: Following extraction of the maxillary right first molar and maxillary left first premolar that could not be maintained, the second upper left molar was displaced mesially in combination with a distal rotation. Simultaneously, two lateral segments were used to give lingual root torque to the canines that had been in crossbite. In the lower jaw, the right and left premolars that had excessive buccal root torque were extracted and a levelling and space closure were performed with a continuous arch. a deep carious lesion beneath the crown on the maxillary left first premolar that involved part of the root and extended deep below the bone margin (Fig. 8D). Based on the findings from the clinical and functional examination, the periapical radiographic status supplemented with a lateral headfilm and a set of articulator mounted study casts, a problem list was created that detailed conditions of which the patient initially was not 56 Melsen Figure 10. Intra-oral and extra-oral photographs showing post-treatment aware or did not consider problematic, along with degenerations that Could not be reversed and had to be accepted. The problem list was the result of a standardized examination, the sequence of which is presented in Table 2. The problem list contains only the positive findings; because it is based on a standardized examination, items not mentioned are Considered normal. The patient's comments when presented with her problem list are shown in Table 3. Extra-oral Finding Insufficient lip closure. The patient had been suffering from insufficient lip closure related to her bi-alveolar protrusion. She was not Concerned by the state of her lip closure and liked her Smile (Fig. 8A-C). Function. Traumatic occlusion on premolars on both sides led to a severe loosening and bone dehiscence on the buccal aspect of the lower first premolars without loss of attachment. Whenever the patient 57 Dental Rehabilitation Table 2. Advisable sequence of clinical examination. EXTRA-ORAL • Facial Smile: lip line Profile • Lips Midlines (nose, maxillary dental, mandibular dental, chin) ORAL FUNCTION • Opening path • Maxillary opening • Lateral movements e TMJ • Muscles Tongue function Lip catch Mode of respiration Mode of swallowing INTRA-ORAL • Mucosa • Dental status based on periapical radiographs (missing teeth, morphological anomalies, restorations, attrition) • Periodontal status (pathological pockets, recessions) DENTAL CASTS • Positional anomalies—tipping—rotation • Occlusion (Sagittal, vertical, transverse) SPACE • Maxilla • Mandible ARCH SHAPE • Maxilla • Mandible CEPHALOMETRIC ANALYSIS 58 Melsen Table 3. Patient's chief complaint was a toothache in the upper right molar and a loose bridge on the upper left side. Note that the International Tooth Numbering System is utilized. PROBLEM LIST SOLUTION EXTRA-ORAL EXAMINATION Reduction of protrusion through • Bimaxillary protrusion, Space closure following extraction of insufficient lip closure teeth #16, 24, 34 and 44 FUNCTIONAL ANALYSIS • Extraction of teeth #34 and 44 • Traumatic occlusion on • Coordination between lower first premolars . structural position and • Laterally-forced bite Intercuspidation • Muscle tenderness INTRA-ORAL ANALYSIS • Missing tooth #25 • Space closure • Teeth #16 and 24 cannot • Tooth loss has to be be saved accepted • Gingival dehiscences • Bony recessions on teeth • Extraction of teeth #34 and 44 #34 and 44 • Bony fenestration apically • Lingual root torque of teeth on teeth #13 and 23 #13 and 23 • Irregular position and • Leveling following extraction crowding of upper and lower front chewed, these teeth were tipped slightly buccally, which led to a particular chewing pattern in order to avoid the traumatic occlusion and, in part, may explain her tension headache. She did not want to relate the headache to her dental situation, however, and did not consider it sensible to include this in the solution of her dental problems. The headfilm confirmed that the mandible was forced into an asymmetric position on closure into maximal intercuspation. The patient's reaction was that she had lived with this occlusion since she was a child and had managed to adapt her function when occluding, so should a problem arise, she would handle it in due time. 59 Dental Rehabilitation Dental Status. The dentition reflected a large caries experience, but no active caries apart from the one on the first left upper premolar (Fig. 8D). The second left upper premolar was missing and replaced by a bridge; the maxillary right first molar could not be saved. Apart from occlusal and Class Il restorations, many teeth had cervical composite fillings for defects resulting from aggressive brushing. The patient was not concerned about the irregular position of any teeth, but definitely wanted the missing teeth replaced (Fig. 8E-H). Periodontal Status. In general, the periodontium was healthy, though there was substantial gingival recession. The traumatic occlusion on the lower first premolar had led to loss of bone on the buccal aspect, which resulted in a loosening of these teeth. In the upper arch, the traumatic occlusion had led to lingual tipping of the canines with a fenestration of the bone in the apical region of the upper canines. This caused pain whenever she blew her nose since she basically was touching the apex of the canines. The patient's reaction was that she could simply avoid touching these areas. The problem with the gingival recession could not be treated because buccal fillings covered the exposed cementum. Therefore, they had to be accepted or disguised with replacement of the fillings or veneers on the incisors. Occlusion. The patient had neutral molar relationship, a tendency toward a distal canine relationship and bilateral crowding in the upper and lower arches, especially in the incisal region of the lower jaw (Fig. 8E-H). When the problem list was presented, the possible solutions were explained to the patient, including potential extraction of the first premolars in the lower jaw and a leveling that could solve the traumatic occlusion. This also would resolve the problem of the lower arch crowding. The patient felt that all of these problems had existed for so long and saw no reason to treat the crowding, particularly since the thought of wearing braces was unacceptable to her. Her only wish was to replace the missing teeth. A 3D VTO was worked out and used to illustrate to the patient that the space left when extracting the first upper right molar could be closed by a mesial displacement of the second molar, instead of being replaced with a bridge (Fig. 9B). A minor distal displacement of the premolar and canine would serve as anchorage and allow for correction 60 Melsen of the anterior crowding and lingual root torque of the canines (Fig. 9C). On the right side, the space left after extraction of the root of the first left premolar could alleviate the problem of anterior crowding; if the lower first premolars with severe loss of buccal bone were extracted, the patient would end up with neutral molar relationship bilaterally, no crowding and less protrusion. Treatment of the upper jaw would involve lingual root torque of the upper canines as well. Several matters were explained and discussed: the prognosis, both long- and short-term; the advantages and disadvantages of the prosthetic solution; the combined orthodontic prosthetic solution; and the timing and visibility of the appliance. The patient was told that the first part of the treatment could be performed with an appliance in the lateral segments only and that anterior brackets would be needed only for the last months (Fig. 9C-D). The patient accepted this treatment plan. Treatment Treatment began with extraction of the maxillary right first molar and maxillary left first premolar. The general practitioner separated the first premolar from the bridge, replacing the second premolar and extending to the first left molar. Following extensive cleaning and careful instruction, the first premolars in the lower jaw were slenderized until the patient felt pain. This not only allowed for the crowded lower incisors to start unravelling spontaneously before extraction, but also reduced the time for space closure (Fig. 9E). Space closure following extraction of the molar was performed lingually, since the force system generated between a power arm on the second molar and the anterior segment would lead to the desired combination of distal rotation and mesial movement. Lingual root torque was added to the upper canines by utilizing the posterior segment (Fig. 9C-D). On the left side, the space closure following extraction of upper left first premolar was performed with a type B anchorage (Burstone, 1982). Orthodontic treatment was completed with a neutral canine relationship without spacing or crowding. The patient was referred back to the prosthodontist with a fully equilibrated splint that was used to maintain the jaw position, which had changed following the extraction of the lower premolars that previously were in traumatic occlusion. The patient now no longer had headaches, but the occlusal surfaces that did not fit to the corrected occlusion needed to be restored. Following this, 61 Dental Rehabilitation the patient had achieved a dentition with no teeth at risk and without periodontal problems. The remaining problem of gingival recession needed to be accepted because mucogingival Surgery could not be performed due to the many composite fillings that were necessary to cover the defects generated by tooth-brushing habits (Fig. 10D-G). The treatment planning was interdisciplinary. The team included a dental hygienist, an orthodontist and a prosthodontist who also served as a gnathologist during the final establishment of the mandibular position before the final occlusal adjustment. Maintenance comprised two Essex splints and it was explained to the patient that stability could not be guaranteed; therefore, both biological maintenance—the regular periodontal control—and mechanical maintenance of the tooth position by means of a splint that should be used at night, is necessary. In terms of a cost–benefit analysis, the treatment compared positively to the one the patient requested. She had asked for restoration of the missing teeth, which would have involved insertion of two bridges or three implants. Due to the quality of the bone, it is likely that bone augmentation would have been needed if this treatment had been chosen. Following the interdisciplinary treatment—which included an eighteen-month orthodontic treatment and a subsequent six-month occlusal reconstruction—the patient spent less money and had no teeth at risk. The treatment focused on the cause of the problem, rather than just relieving the patient's Symptoms. - ETIOLOGY AND DEVELOPING A PROBLEM LIST It is important for the general dentist to be aware that the masticatory organ is not static in the adult population. Both the facial skeleton and the occlusion are subject to age-related changes, which results in both Class II and Ill malocclusions becoming more severe (Behrents, 1985b; Harris and Baker, 1990). When a patient requests dental rehabilitation, therefore, it is important to assess the dynamics in the situation. The etiology of the degenerating dentition can be found both in the general aging process and in the local environment (Fig. 2). Aging leads to a decrease in both the toughness and density of the bone and, thereby, in the resistance to spontaneous migration of teeth (Brockstedt et al., 1993; Wang and Puram, 2004). This phenomenon will be enhanced locally both by diseases of the periodontium and loss of teeth or tooth 62 Melsen substance, both of which will contribute to aggravation of already ex- isting malocclusions and development of secondary malocclusions that are not compatible with normal function. This generates a vicious cycle (Melsen and Kalia, 2012). When first meeting with a patient who is seeking rehabilitation of a degenerated dentition, it is important to let him/her express his/ her wishes freely in order to get an impression of the priorities and values the patient assigns to the dentition. After the patient has shared this information, the dentist should perform a general examination, not one related only to the patient's chief complaint(s). A suggested sequence for a clinical examination is found in Table 2 and was used in the cases described. When listing the results of the examination, only positive findings should be noted and should always appear in the same order, indicating that if no comments are found on a specific topic, the situation is considered normal. Based on this information, the dentist can explain to the patient that the problem(s) to be solved may differ from the chief complaint(s) of the patient and that what the patient perceived as a local problem actually may be a symptom of a somewhat more complicated problem that if solved satisfactorily, may require the collaboration of several colleagues. Because oral health closely reflects general health, it also is advisable to have the patient fill in a medical history form (Table 4). A reason for the inertia in the development of interdisciplinary treatment approach in treating complex cases may be the lack of evidence-based research. Patients in this category generally differ too much to be assigned to specific groups; randomly assigning patients with progressing degeneration into different treatment groups or observing the progressing degeneration does not seem acceptable ethically. Long-term observation of patients submitted to different treatment approaches seems the only way to move forward. It is, however, of utmost importance that colleagues from all specialties are informed on how progress within the other disciplines can be beneficial to the patient. This can be achieved best by personal contact, study groups at meetings and Skype or videoconferencing so that the database with registration material can be available to all colleagues involved. There are essential procedures that all can agree upon (Table 1), as well as optional procedures; the decision about which to use varies according to the knowledge and capacity of the dentist, along with the values and 63 Dental Rehabilitation Table 4. Suggested questionnaire regarding general health. MEDICAL HISTORY Patient name: Date of birth: Name of physician: Office phone: Office address: Date of last exam: 1. Do or did you have a general health problem? Yes [...] No […] lf yes, please explain: 2. Have you ever been hospitalized, had general anesthesia or emergency room visits? Yes [...] No [] lf yes, please explain: 3. Do you have allergies to any drugs, medical products (e.g., latex) or the environment (e.g., dust mites, pollen, mold)? Yes [...] No […] lf yes, please explain: 4. Do you take any medications regularly? Yes […] No […] lf yes, please list your daily medications: 5. Have you ever been treated by a physician for any of the following? Check all that apply. PROBLEM YES NO DON'T KNOW Problems at birth Heart murmur Heart disease Rheumatic fever Anemia Sickle cell anemia Bleeding/hemophilia 64 Melsen PROBLEM YES NO DON'T KNOW Blood transfusion Hepatitis A|DS Or HIV+ Tuberculosis Liver disease Kidney disease Diabetes Arthritis Cancer (state which kind) Cerebral palsy Seizures Asthma Osteoporosis Speech or hearing problems Eye problems/contact lenses Skin problems Tonsil/adenoid/sinus problems Sleep problems Emotional/behavior problems Radiation therapy Hormonal therapy Immune suppression therapy economy of the patient (Table 5). Unfortunately, the proposed treatment may be influenced by the dentist's attitude. It is not uncommon for a sub- optimal treatment to be offered to the uninformed patient because the dentist is reluctant to refer to a colleague due to fear that the patient may not come back. Before initiating treatment, creating a table that lists the chief complaints, problems and solutions to each problem is recommended (Table 3). Some problems must be accepted because there currently is no available cure and the patient must know what to expect from treatment. Discussing treatment also gives the patient an understanding of the necessity for maintenance, as age-related changes will continue after rehabilitation; the value of treatment is assessed not only by its immediate results, but also by its maintainability. 65 Dental Rehabilitation Table 5. Optional procedures. • Periodontal surgery • Gnathological treatment • Fixation of mandibular position • Implants • Orthodontic therapy (minor/major) • Orthognathic surgery The value that a patient assigns to his/her dentition has a major impact on the treatment plan aimed at rehabilitation, meaning restoring a condition of good health and the ability to function. The willingness of the patient to engage in a long-term investment, requiring both time and money, is the basis on which the individual treatment plan should be made (Melsen, 1991; Melsen and Agerbaek, 1994). If the patient is interested in a plan, but treatment seems too expensive, a multi-step outline can be generated and periods of maintenance with temporary restorations may be necessary. THE SEOUENCE OF TREATMENT If pain or symptoms indicating the presence of a temporoman- dibular disorder (TMD) are reported, it is important that no invasive and permanent measures are undertaken before change in the mandibular position as part of the treatment can be excluded. Because pain may or may not be related to occlusion, the treatment plan depends complete- ly on this aspect of the patient's problem (Pullinger et al., 1993; Oke- son, 2013). A final treatment plan for rehabilitation, therefore, can be achieved only when the optimal mandibular position is established. For patients with insufficient teeth to establish a fixed mandibular position, a bonded splint with sufficient interdigitation may be the first step in treat- ment. Once the mandibular position is established, a combination of the occlusogram and the lateral headfilm can be used to illustrate the tooth movement needed to obtain the treatment goal (Fig. 11). If dental implants are part of rehabilitation, they can be inserted at the beginning of treatment, providing the necessary space and bone are available. The insertion of an implant may be the first intervention and the implant may serve as part of the anchorage for orthodontic tooth movement. Another scenario is one in which missing teeth cannot 66 Melsen Figure 11. 3D VTO combine the lateral cephalogram and virtual models. be replaced by implants due to lack of space (e.g., Fig. 5). In that case, Orthodontic tooth movement can open up the necessary space and the implant insertion will follow orthodontic treatment. In the case of atro- phy of the alveolar process, tooth movement can contribute to the gen- eration of the alveolar bone necessary for an implant (Fig. 12). This pa- tient had a heavily restored permanent dentition with the presence of an implant, bridge and endodontically treated molars, all of which reflect an interest in maintenance. However, a recent extraction of the two right lower molars left the patient with reduced chewing capa- City on the right side, a problem that could not be solved with a fixed 67 Dental Rehabilitation Figure 12. A-B: Intra-oral images of a patient who lost the lower right molars and for whom the ridge atrophy of the alveolar process is so pronounced that an implant can not be inserted. C. Panoramic image confirms the atrophy of the alveolar process on the lower lateral region. D-E. With a temporary anchorage device (TAD) as anchorage, forces can be delivered at the level of the center of resistance in the second premolar. F-G: Radiographs demonstrate the bone formation generated by the distal displacement of the second premolar. H-l: Virtual models before and after treatment. Following treatment, an implant can be inserted between the first and second premolar. replacement because there was insufficient alveolar height for the in- sertion of an implant. The problem was solved by moving the second premolar distally toward a skeletal anchorage, an Aarhus mini-implant", that was placed on the anterior aspect of the ramus. This allowed for the generation of both buccal and lingual forces that facilitated translational tooth movement. When the orthodontic treatment was completed, the patient was ready for the final rehabilitation, which made normal func- tion possible. Orthodontic intervention itself should always be based on a defi- nition of the treatment goal including a clear understanding of the de- sired movement of individual teeth in three dimensions to be undertaken 68 Melsen (Figs. 9B and 11). Identification of the teeth to be moved facilitates determination of the active unit(s) requiring tooth movement and of those teeth belonging to the reactive or the anchorage unit. The sequence of the dental displacement may vary between patients; it is important for the orthodontist to recognize that growth and development—which are part of orthodontic treatment with growing patients—do not factor into the treatment of an adult patient. The treatment goals in these patients, therefore, can be obtained only by tooth movement and/or orthognathic surgery (Woodside et al., 1983, 1987; Ruf and Pancherz, 2000) or, in some cases, by mandibular repositioning (Fiorelli et al., 2016). When treating older patients, especially those with a reduced periodontal support, any force applied to teeth that are not in occlusion should include an intrusive component as the shearing of the root in relation to the alveolar wall will result in an extrusion that will become more pronounced with increasing marginal bone loss (Melsen and Fiorelli, 2000). Many adult patients present with an increasing overjet and anterior diastema developed by either extrusion due to periodontal disease or caused by collapse of the occlusion following loss of teeth, occlusal wear or wear of occlusal restorations. In the case of marginal bone loss and the development of pockets, treatment will require a combined retraction and intrusion of the incisors (Figs. 10-11). A pre-requisite for this procedure is a reduction of the periodontal pockets, which can be obtained by a reverse Widman Flap surgery with an apical displacement of the gingiva. The side effect of undesirable elongation of the clinical crown can be reversed by the combined retraction and intrusion, the result of which has proven to be maintainable over decades, although whether or not this treatment results in a long epithelial attachment or a real re-attachment still is not resolved (Melsen et al., 1988, 1989; Figs. 10-11). The correction of the vertical discrepancy generally is combined with or precedes the correction of the sagittal and the transverse pro- blems (Melsen and Fiorelli, 2000). Retroclined upper incisors may have to be proclined and intruded, allowing for a possible forward displacement of the mandible. Proclined and flared incisors have to be intruded and retracted in order to establish a normal lip closure. There may be individual problems, however, that may influence the sequence. Space 69 Dental Rehabilitation problems may require the transverse problems to be solved first or canines may prevent the movement of the incisors, for which reason the canine displacement may precede the repositioning of the incisors. The treatment of degenerated dentitions is goal oriented, meaning that there is clear differentiation between the teeth to be moved, the active unit and the teeth to serve as anchorage, the reactive unit. The appliance should be custom made, delivering the force system needed to generate the predefined tooth movement(s) (Melsen et al., 2012c, Fig. 11). This also indicates that a clear differentiation between the passive units (the teeth that should not be displaced) and the active units (the teeth to be moved) is needed. This can be performed only with segmented mechanics (Burstone, 1962, 1966; Burstone and Koening, 1976). Even major degeneration may be overlooked or considered a part of normal aging. A 55-year-old man brought two family portraits illustrating how the midline diastema developed over the years, even though he had seen his dentist every six months (Fig. 13). The patient reported that his molars were extracted as they became loose and were not replaced because he thought that losing teeth was part of the normal age-related process. It was not until he changed dentists that he heard the word “periodontitis.” When he presented to the orthodontist, his periodontium was healthy, but the occlusion was not compatible with normal function. The left central incisor, which exhibited grade Ill mobility combined with the midline diastema, could not be replaced by a bridge or an implant because the space was too large and there was insufficient alveolar bone. The patient's problems and possible solutions are listed in Table 6. The first step in treatment was to define the treatment goal. It was decided to maintain the occlusion relative to the right premolar and the left lateral segment and rotate, retract and intrude only the upper right incisors (Fig. 14). In the lower arch, there was a limitation due to the total lack of buccal bone relative to the right lower canine, for which a midline discrepancy and right canine relationship had to be accepted. Following treatment, a cast retainer was necessary in both jaws. In the upper arch, it was decided to accept a shortened arch. A bonded cast retainer combined with a bridge replacing a lower left incisor was fabri- 70 Melsen Figure 13, A-D: Family pictures illustrate the progressive degeneration of the dentition. E-I: Intra-oral images reveal that several posterior teeth were lost. In the anterior region, a large diastema and an openbite were the result of a migration of the upper right incisors. Cated for the lower arch (Fig. 15). This retainer was made by a prosthodontist with special expertise in gnathology and rendered occlusal Contacts to all teeth and anterior group function. The occlusal contacts were necessary to maintain normal swallowing. Long-term outcomes of this treatment and retention are shown in Figure 16. 71 Dental Rehabilitation Figure 14. A: Periapical radiographic status reveals a severe periodontal breakdown, especially in the anterior region. When these images were taken, the patient had just received lege artis periodontal treatment and there was no bleeding on probing and no pockets over 4 mm. B-E: First phase of treatment where the occlusal feedback from the lateral teeth was reinforced by a double transpalatal arch and occlusal build up with light-cured composite. The active unit (the teeth to be moved), comprised of the two right incisors, was connected with a piece of full-size rectangular stainless steel that extended toward the left canine region where it was connected to a 0.017" x 0.025" TMA" cantilever activated so that a clockwise rotation in the frontal and horizontal plane of space was generated. This brought the incisors into place and closed both the 72 Melsen Figure 15. A-D: Treatment was completed with cast retainers, combined with replacement of missing teeth, namely the upper first premolar and the lower left lateral incisor. Figure 16. A-C. Ten-year follow up showing the maintenance of the results. D-G. At the 15- year follow up, it appeared that the left lateral incisor had loosened from the cast retainer and the dentist had bonded it by holding the loosened incisor away from the retainer and pressed bonding material into the space. Thereby, an openbite was generated and, as a result, the tongue pressure was in the process of displacing the whole anterior segment. The patient had a renewed need for orthodontic treatment. (4– continued) openbite and diastema. F-J: Second phase of treatment, which was done With continuous arches and during which the lower arch was involved. The purpose of the phase of treatment was to establish an incisor relationship with group contact. Due to the total lack of buccal bone on the lower right canine, the asymmetry and midline deviation Could not be corrected. Following the completion, a connective tissue graft was placed on the right central incisor. 73 Dental Rehabilitation Table 6. Patient's chief complaint is concern about losing a central incisor and the progression of degeneration. The current dentist cannot replace the loose incisor due to a large diastema. Prognosis depends on periodontal health and maintenance of retainers. Note that the Universal Tooth Numbering system is utilized below. PROBLEMS SOLUTIONS EXTRA-ORAL • Smile influenced by the di- • Closure of the diastema astema and missing lower • Prosthetic replacement of incisor missing incisor(s) FUNCTION • Simple tongue pressure • Close the diastema and when Swallowing produce a replacement for • Lip catch of tooth #11 missing teeth |NTRA-ORAL • Accept a shortened arch from • Severely reduced dentition teeth #15 to 26 • Patient noted that previous • Upper arch (UJ): closure of dentist considered eXtraC- the medial diastema and tions necessary opening of space for a bridge • Healthy but significantly re- to replace tooth #14 duced periodontal support, especially relative to teeth #11 and 43 • Missing teeth: #18, 17, 16, 14, 27, 28, 32, 37, 38 • Tooth position: #15 tipped mesially; #12 and 11 ro- • Connective tissue graft rela- tive to tooth #11, if possible, to retain • Lower arch (LJ): proclincation and intrusion of the three lower incisors tated distally and flared; • Correction has to be main- #23 rotated mesially; #11 tained by cast retainers com- extruded bined with bonded bridges in • Occlusion overjet of 4 mm both arches • Overbite of 1 mm • Correction of positional and occlusal deviation consult VTO MAINTENANCE Preservation of the treatment result requires both biological maintenance of a healthy periodontium and mechanical retention of the 74 Melsen tooth position. Periodontal maintenance includes good hygiene and a regular dental hygiene program. Prosthodontic reconstruction is important to support normal function. The tendency toward relapse depends on the type of treatment performed and if the occlusion is not stabilized by equilibration and/ or restoration of occlusal surfaces that do not fit to the orthodontically established occlusion, the result cannot be maintained. Bonded retainer wires, routinely used in young patients, do not provide satisfactory retention in adult patients with bone loss. In patients with severely reduced periodontium (>30%) and consequently increased mobility, it is recommended strongly to fit the patient with a lingual cast retainer to the upper anterior teeth. This retainer has to be constructed by a prosthodontist or general dentist before the orthodontic appliance is removed (Melsen and Kalia, 2013). The reason for choosing this more complicated approach to retention is the need for generating a situation in which the remaining periodontium is loaded optimally. Consolidating the anterior teeth and canines brings the occlusal forces closer to the center of resistance. This can be achieved if the retainer is constructed with a build-up of the often-abraded lingual surface so that group function to the upper front teeth can be established. In lateral segments, cast on-lays or composite fillings may be necessary to maintain the mandibular position. This is relevant especially following the successful treatment of patients with TMD. Due to the reduced periodontium and as a result of the recently finished orthodontic movements, the teeth may be loosened; therefore, the retainer should be in place before debonding the patient. The orthodontic appliance that reduces the mobility of the teeth is left on in order to allow the dentist to perform the necessary preparation for the retainer. For patients who require a cast lingual retainer, the orthodontist should complete the treatment with a slight overjet allowing for the reconstruction of the lingual surface. As is the procedure for a bonded bridge, the lingual surface of such a retainershould be modelled for group contact in lateral and protrusive movement. This type of retainer can be connected with the replacements of single teeth, whereby retainer and reconstruction become one. Samama (1995) described a technique whereby the pontic can be replaced if gingival recession occurs during the post-treatment period. 75 Dental Rehabilitation Even a cast retainer is not a guarantee, however, as it can loosen and minor movements can occur and not be noticed by the patient. To help detect even small movements, both the patient and dentists involved can be given a copy of the virtual models that illustrate end- of-treatment status. This would allow the general dentist to note when minor changes occur before they become serious. If this process had been used, the relapse shown in Figures 7F and 16F-G could have been prevented. The patient in Figure 6 had been provided with a cast retainer. It was fractured and not replaced. When the patient was seen by the orthodontist as part of a long-term follow-up, it was obvious that the anterior segment had migrated forward, leaving a space between the posterior and anterior segments. Despite this occurrence, improvement of the periodontium and shortening of the clinical crown have been maintained for 25 years. The patient in Figure 16 maintained the orthodontic results for the first fifteen years after treatment, but the patient then noticed that the right lateral incisor was loose in relation to the cast splint. The general dentist solved the problem and rebonded the tooth to the splint. This was achieved with the tooth being mobile due to the lost marginal bone being displaced and bonding material squeezed between the splint and the tooth. A minor open bite was generated, normal swallowing was no longer possible and simple tongue pressure caused the anterior segment to displace. Consequently, the patient developed an anterior open bite and required a second course of orthodontic treatment as a consequence of failing to understand the dynamics of the occlusion; even a small open bite may lead to tongue pressure. The routine use of a tooth pajama, a fully balanced splint also can be an effective maintainer. The patient in Figures 17 and 18 pre- sented in 1975 with complaints of an increasing median diastema. She had suffered from extrusion and flaring of the upper incisors and intra- oral x-rays revealed horizontal bone loss. Before seeing the orthodon- tist, she had received periodontal treatment with a modified Widman Flap surgery, which replaced the marginal gingiva apically to reduce the pocket depth. Combined retraction and intrusion were performed with a 0.018" titanium-molybdenum alloy (TMA) base archwire. The side seg- ments that served as anchorage were maintained with heavy stainless 76 Melsen Figure 17, A-B: A 30-year-old woman presented with an increasing median diastema. Note the marginal bone loss and the absence of lamina dura of the marginal bone. C-D: Following eighteen months of combined retraction and intrusion with the line of action almost parallel to the long axis of the central incisors, the overjet and overbite are normalized, and the marginal bone level and density have improved significantly. Steel segments and atranspalatal arch. The result was a combined intrusion and retraction that led to closure of the diastema and reduction of both Overbite and overjet, in addition to shortening the clinical crowns. In the lower arch, the lower left lateral incisor was extracted and the incisors were aligned. At the end of orthodontic treatment, the intra-oral radiograph demonstrated that the bone level relative to the cemento-enamel border improved significantly and no pathological pockets could be detected. Before orthodontic treatment, 5.8 mm cementum was visible; after treatment only 1.5 mm was visible. The follow-up almost forty years later revealed that the periodontal gain and optimal incisal relationship could be maintained, providing the periodontium was kept healthy by effective tooth brushing and regular periodontal control, as well as maintenance over the tooth position by a bonded retainer and a fully balanced splint—a tooth pajama—used at night. Endodontic treatment of the left upper incisor due to caries was detected in relation to a lingual groove, a dens in dente had resulted in discoloration of the tooth and the patient had a Veneer on the incisor. 77 Dental Rehabilitation Figure 18. A:Ten-year follow-up. B: Twenty-year follow-up. C. An endodontictreat- ment of upper right central incisor led to discoloration and the patient received a veneer. D: The periodontium has been maintained. E-F: Forty-year follow-up. The gingival adaptation was far from satisfactory, but intra-oral radiographs revealed that the improved bone level was maintained. The tissue reaction in relation to such treatment was analyzed later in monkey and dog experiments, and it was demonstrated that lost attachment could be regained by a combined Widman flap surgery and intrusion performed with light forces 5 to 10 CN per tooth (Melsen et al., 1988, 1989; Melsen and Kragskov, 1992; Diedrich, 1996; Diedrich et al., 2003; Reet al., 2004). The patient described above went to her dentist regularly; her upper right first molar was treated with a crown; the lower first molar was extracted and not replaced, which allowed the upper molar to extrude. Long-term observation showed that periodontal improvement could be maintained with a night guard. Whether improvement of the bone and gingival level reflects a long epithelium or regain of lost attachment still is a matter of discussion (Bartold, 2015), however maintenance has been demonstrated clearly (Melsen, 2012d). 78 Melsen CONCLUSION This chapter has discussed the reason for the increasing need for oral rehabilitation and the importance of performing it in an interdisciplinary approach. The necessity for the general dentist to note changes that occur in the dentition and the severe consequence of fail- ureto do so, both before and after rehabilitation, has been demonstrated. To prevent degeneration from progressing, it is recommended that the family dentist take intra-oral photos or generate virtual models as part of a “dental health card,” which also should include information on the patient's dental, periodontal and general health status. This would resemble the general health card kept by family doctors in many countries and would allow the dentist to detect changes when patients have their routine check-up. Through case reports, it was demonstrated that the results of rehabilitation can be preserved and that maintenance is crucial to Success. REFERENCES Albrektsson T, Zarb G, Worthington P, Eriksson AR. The long-term efficacy of currently used dental implants: A review and proposed criteria of success. Int J Oral Maxillofac Implants 1986;1(1):11-25. Aulakh RS, Melsen B. When should orthodontics be part of recon- struction of a degenerating dentition? A case report. Prog Orthod 2011;12(2):161-168. Bartold PM. Periodontal regeneration: Fact or fiction? 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Int J Prosthodont 2003(16 Suppl):7–10. 83 THE ROLE OF ORTHODONTICS IN THE INTERDISCIPLINARY PREPARATION FOR OSSEOINTEGRATED DENTAL IMPLANTS Sunjay Suri ABSTRACT Interdisciplinary treatment broadens the scope of treatment solutions for complex situations beyond what traditional approaches can provide in isolation. Participating in collaborative interdisciplinary treatment is a clinically and intellectually rewarding experienceforthe orthodontistand when performed with diligence, it contributes to optimal treatment results. The use of osseointegrated dental implants as a predictable and reliable method of tooth replacement has stood the test of time in clinical practice. Orthodontic treatment often is necessary to prepare the recipient sites in the dental arch to facilitate such treatment. This chapter uses clinical vignettes to illustrate and discuss the role of orthodontics in the interdisciplinary preparation for osseointegrated dental implants, the clinical mechanics used and precautions exercised during orthodontic space distribution and implant site development to optimize treatment results. KEY WORDS: osteointegrated dental implants, implant site development, interdisciplinary treatment, esthetic zone INTRODUCTION Orthodontics is a beautiful branch of dentistry. Its beauty stems from several aspects, some foundational and others evolutionary. Facial beauty and optimal dentofacial esthetics are prominent foundational goals of orthodontic treatment. Treating malocclusion and achieving Such goals in the growing face—which is a dynamically changing sys- tem—as well as in the adult face or in the face with a failing dentition, offers unique challenges to the orthodontist. With the ever-increasing de- mand from patients seeking solutions to complex dentofacial problems and malocclusions, orthodontists today are collaborating with clinicians 85 Interdisciplinary Role of Orthodontics in other branches of dentistry like never before. This has resulted from the great strides that different disciplines of dentistry and medicine have made as they have evolved in the last fifty years, as well as a willingness of professionals to work across disciplinary boundaries and collaborate in an open forum of holistic treatment. Such collaborations provide tremendous opportunities not only for continuous learning and intellectual growth for the orthodontist, but also are likely to contribute to optimized outcomes for patients with complex challenges. Among the complexities that challenge the orthodontist and complicate the treatment of malocclusions are: 1. Ectopically erupting teeth and impacted teeth; 2. Dentomaxillofacial trauma; 3. Craniofacial imbalance such as that seen in cleft lip and palate (CL/P) and other congenital or acquired craniofacial anomalies; 4. Multiple missing teeth, especially when located in the same quadrant in the esthetic zone; 5. Treatment necessitating atypical extractions; 6. Overjets greater than 9 mm or negative overjets exceeding -3.5 mm; 7. High mandibular plane angles, such as when the Frankfurt mandibular plane angle is greater than 35°, with an anterior open bite; 8. Low mandibular plane angles, such as when the Frankfurt mandibular plane angle is smaller than 10°, with a severe deep bite; and 9. Posterior missing teeth or loss of posterior tooth Structure. This chapter does not aim to present interdisciplinary treatment in all these types of situations. Instead, its purpose is to highlight the role of interdisciplinary orthodontics in preparation for dental implants in the esthetic zone—a clinical scenario not discussed frequently in textbooks and monographs—by using two case reports as examples. 86 Suri CASE REPORTS Case 1 Malocclusion exists in a large proportion of humans. However, since it does not contribute directly to health problems, seeking treat- ment for malocclusion is an elective choice for most individuals. How- ever, a sudden unplanned event such as a motor vehicle accident can inflict maxillofacial injury that brings orthodontic treatment into the management plan, even though the affected individual may have had a malocclusion before the accident and lacked any prior desire to seek Such treatment. p A 22-year-old woman was the victim of a motor vehicle accident and sustained a parasymphyseal fracture of the mandible along with avulsive loss of the maxillary left lateral incisor and canine (Fig. 1). Loss of multiple teeth in the same quadrant, especially in the esthetic zone of the highly visible anterior part of the arch, increased the complexity of her problem. Surgical management had involved fracture reduction and stabilization with bone plates; implant replacement of the lost teeth was a part of the long-term treatment plan that was determined during the interdisciplinary assessment. Unrelated to the trauma, she had an untreated Class || malocclusion, which was more severe on the left side. The examination also revealed that the maxillary dental midline was shifted to the left and the space between the maxillary left central incisor and first premolar was inadequate for placing appropriately sized lateral incisor and canine implant supported prostheses. The patient described that this part of the arch had been crowded prior to the accident. A moderate deep bite was present. Overall, the patient had pleasing facial esthetics. Orthodontic treatment was necessary in order to create adequate space for the dental implants. Although the patient may not have considered orthodontic treatment prior to the maxillofacial injuries from the accident, she now desired correction of her Class || malocclusion, along with the preparation of space for the implants. The goals of orthodontic treatment included space distribution for receiving 87 Interdisciplinary Role of Orthodontics Figure 1. Pre-treatment malocclusion with the Class || molar relation, which is more severe on the left side, moderate overbite, midline discordance and avulsed loss of the maxillary left lateral incisor and canine. osseointegrated dental implants, midline correction and resolution of the deep bite and Class || malocclusion. The orthodontic treatment plan involved derotation and distalization of the maxillary left first molar, followed by the distalization of the premolars of that quadrant, correction of maxillary dental midline and use of Class || mechanics using intermaxillary elastics to resolve the malocclusion and create space for the missing teeth. This would be followed by surgical placement of two dental implants in the maxillary left lateral incisor and canine sites, which would be restored with ceramic crowns after implant osseointegration. Treatment was initiated by bonding brackets on the maxillary and mandibular arches and using a flipper-type interim removable partial denture (Fig. 2). Low-profile brackets were bonded to the maxillary left lateral incisor and canine denture teeth by removing the surface of the acrylic denture tooth with a bur, coating the pontic with a plastic conditioner and bonding the bracket with a HEMA bonding resin. Decreasing the facial-lingual dimension of the acrylic teeth with the use of low-profile brackets allowed easy denture removal and reinsertion despite the presence of the archwire, which otherwise would have engaged the bracket slots. After initial alignment, the interim partial denture was replaced by pontics with bonded brackets, which were tied securely to rectangular archwires (riding pontics; Suri et al., 1999; Suri and Utreja, 2003). An open vertical loop was used to distalize and de- rotate the maxillary left first molar. A sliding jig was used to reinforce anchorage and Counter the reactionary force of the open loop and prevent the undesirable effect of protruding or proclining the maxillary anterior teeth (Fig. 3). This jig also allowed engaging the Class II intermaxillary elastics from the mandibular first molars to the maxillary canines symmetrically on both sides, despite the absence of the maxillary left 88 Suri Figure 2. Interim removable partial denture with brackets bonded to the denture teeth allowed restoration of esthetics and function in the early phase of treatment. Figure 3. Intra- and inter-arch mechanics employed in achieving molar correction. Riding pontics were used to restore the dental arch in the region of tooth loss and maintain good esthetics during treatment. Canine. The smile esthetics thus were restored and maintained through- out the orthodontic treatment. After nine months of orthodontic preparation, two dental im- plants were placed following a flapless surgical protocol (Fig. 4). The rid- ing pontics for the maxillary left lateral incisor and canine were modified by grinding the lingual surface and cingulum area to allow clearance for the implant heads. The archwire, jig and pontics were replaced immedi- ately after the implant surgery. Following completion of the orthodontic treatment and successful osseointegration, the implants were restored by ceramic crowns (Fig. 5). Osseointegrated dental implants have stood the test of time as a reliable and predictable method for replacing teeth, with impressive long-term survival rates in both prospective and retrospective studies (Jung et al., 2012). Several factors contribute to success with this treat- ment modality and special considerations are needed when planning 89 Interdisciplinary Role of Orthodontics Figure 4. Implants placed after nine months of orthodontic treatment. Pontics and archwire were replaced immediately following implant placement. The lingual surfaces of the pontics were adjusted to allow clearance for implant heads. Figure 5. Final implant restorations with ceramic crowns. treatment with dental implants in esthetically important zones of the arch (Spear et al., 1997; Kokich, 2004). Inadequate distance between neighboring implant heads where two or more implants are placed and between an implant head and the neighboring tooth leads to several unfavorable outcomes, including crestal bone loss and loss of papilla (Tarnow et al., 2000; Buser et al., 2004). Knowledge of the dimensions and geometry of the implant to be used is helpful to ensure that the space distribution is adequate when managing sites for one or more implants; this is particularly important for dental implant sites in the esthetic zone. The orthodontist can use the dimensions of the teeth on the contralateral side-if they are of normal shape and size—as a 90 Suri reference for selecting pontics for use during treatment, which then can be used as an index for space distribution. The dimensions of the arch in the edentulous area must be measured during the pre-implant orthodontic preparation to determine and achieve adequacy of the spaces for implant placement. Many implantology clinicians consider that a minimum of 1.5 mm space should exist between the implant head and the cementoenamel junction of a natural tooth and two neighboring implant heads should be spaced 3 mm apart. The space distribution should be planned in collaboration with the restorative dentist and implant surgeon. During space distribution, the orthodontist must ensure that the roots are positioned optimally and reflect the measurements made at the level of the crest of the alveolar ridge by undertaking mechanics that prevent tipping of the roots into bone where the implants will be placed (Kokich, 2004). It also should be recognized that the basal bone in the apical region is narrower than the surface of the alveolar ridge. For patients who have suffered tooth loss, orthodontic preparation should be managed efficiently since alveolar bone is lost rapidly in the first year following extraction (Scropp et al., 2003). In the treatment of the patient in Case 1 presented above, collaborating with the implant surgeon and restorative dentist allowed the orthodontist to distribute and manage the space optimally to allow adequate space between the natural teeth and the implants while simultaneously correcting the midline, moderate deep bite and Class Il malocclusion in a timely manner. Case 2 A 20-year-old man with a previously repaired CL/P needed interdisciplinary treatment to restore maxillomandibular relationship and the occlusion for a failing maxillary left central incisor. This tooth was proximal to a complete unilateral cleft that had alveolar bone grafting during the mixed dentition period. The patient had a missing maxillary left lateral incisor and it was decided to substitute it with the left maxillary canine. There was an anterior crossbite due to maxillary retrusion, which was corrected by surgical orthodontic treatment and maxillary advancement (Fig. 6). Four months following orthognathic surgery, attention was directed toward the restoration of the occlusion related to the maxillary left central incisor, which was breaking down structurally. This tooth previously had received endodontic treatment and also had undergone internal and external apical and lateral root resorption. The 91 Interdisciplinary Role of Orthodontics Figure 6. Steps involved in orthodontic forced eruption process. Top: Before orthognathic surgery. Bottom: After implant restoration. The photos between the top and bottom panels represent Intermediate stages of treatment. marginal gingiva had a bluish tinge and was inflamed. Pocket depths in excess of 5 mm were noted. The crown of this tooth had received 92 Suri Several prior restorations to preserve it until a definitive implant support- ed prosthetic replacement could be placed. Radiographic examination revealed horizontal bone loss with the alveolar crest margins located at the mid-root level. A slightly overextended root canal filling also was evident. While it was a failing tooth with poor prognosis for survival, its periodontal ligament was viable. Taking advantage of the viable liga- ment, orthodontic site development of the alveolar bone using forced eruption of the tooth was included in the interdisciplinary treatment Strategy. The eruptive mechanics were conducted over a nine-month duration (Fig. 6). Tooth extrusion was conducted by altering bracket height and placing step bends in the archwire. Care was taken to place adequate torque in the stepped archwire in order to avoid a lingual tipping of the tooth as it extruded and the incisal edge of the tooth was reduced progressively to keep it out of occlusion. The patient was advised to avoid biting with his incisors and preferably to use a medium soft diet to avoid impacting the eruptive mechanics. Halfway through the forced eruption treatment, the gutta percha in the maxillary left central incisor was removed and replaced with calcium hydroxide and a screw post was placed to support an interim restoration of the badly broken- down crown. The marginal gingiva spontaneously migrated toward the occlusal plane as the tooth progressively moved in that direction. After achieving 4 mm of extrusive movement, the patient was evaluated and the alveolar bone height gain was considered sufficient for the implant placement phase of the interdisciplinary treatment (Fig. 7). The dentition was stabilized, debonded and mild occlusal recontouring of the crown of the maxillary left canine was performed. The patient was referred for the extraction of the maxillary left central incisor followed by immediate dental implant placement. The patient was made aware that following extraction and visual inspection of the implant site, if bone quality and quantity were determined to be inadequate, bone grafting and a two-stage implant placement procedure might be required instead of an immediate implant. Following orthodontic forced eruption, a significant improvement was noted in the crestal bone height. As desired, healthy keratinized gingiva was present at the vertical position of the gingival zenith of the adjoining maxillary right central incisor. During the surgical phase of treatment, the maxillary left central incisor was extracted carefully and 93 Interdisciplinary Role of Orthodontics Figure 7. Gain in alveolar bone height from the orthodontic forced eruption as visualized in a series of periapical radiographs. atraumatically. The bone quantity and character were determined to be acceptable for immediate implant placement. A bone-level immediate implant was placed using a flapless surgical technique, along with a mixture of Cortical and cancellous bone chips and autologous bone between the implant and the labial cortical plate. An interim denture was provided to maintain esthetics. After four months of healing, osseointegration was verified and the restorative phase of the treatment plan was completed. Orthodontic implant site development was described by Salama and Salama (1993) and involves an adjunctive role of Orthodontics in the extrusion of hopeless teeth to enhance soft- and hard-tissue dimensions of future implant sites. Such orthodontic forced eruption was described in several publications in the 1970s (Brown, 1973; Heithersay, 1973; 94 Suri Ingber, 1974, 1976) and refers to the controlled movement of a tooth root in a coronal direction under the effects of a sustained physiologic orthodontic force that maintains the existing periodontal attachment apparatus (modified from Korayem et al., 2008). A predictable biologic response follows the sustained orthodontic traction on the periodontal ligament fibers and the supracrestal gingival fibers (Mantzikos and Shamus, 1997, 1998, 1999). Cellular changes within the ligament lead to new bone formation along the bone surface nearest the stretched ligament and the existing alveolar crest. Hochman and associates (2014) described the manipulation of the periodontium by controlled tooth extrusion mechanics as providing the only desirable and non-surgical technique of producing new bone and the most predictable means of correcting the loss of the interdental papilla. Amato and colleagues (2012) described that the orthodontic efficacy of such procedures to generate new bone was about 70% and orthodontic efficacy to generate new gingiva was about 65% in a series of thirteen patients who underwent orthodontic forced eruption. There are certain precautions clinicians should follow while conducting orthodontic forced eruption. If the goal of the treatment is to develop an implant site along the long axis of the affected tooth root, then the force should be kept light and sustained, and along the long axis of the root, in order to have a uniform pattern of traction loading the ligament. Current mechanics employ step bends, which lead to an interrupted continuous pattern of force delivery. In the author's opinion, rectangular titanium molybdenum alloy archwires allow a longer range of force activation, lower forces and the ability to torque the archwire in order to counter the moment of the extrusive force applied from the labial side of the tooth, which tends to tip the crown lingually. In other instances, buccal root torque may be placed if the intent is to develop the labiolingual thickness of alveolar bone. The extrusive force should be reactivated every four weeks to achieve a rate of eruption of about 1 mm per month, although this recommendation is more empiric than supported by definitive research data. The rate of movement may need to be altered depending upon the state of the periodontium. Occlusal reduction is needed at almost every activation appointment to avoid occlusal interference and traumatic impact. The technique is confronted by additional challenges that should be considered prior to starting treatment. The slow nature of the process can be challenging 95 Interdisciplinary Role of Orthodontics for the patient to tolerate, particularly when it involves the esthetic zone. Thus, patient education prior to commencing treatment is critical to ensure that the patient is cognizant of and accepts that this is a te- dious and exacting process that will be accompanied by significant dis- crepancies in gingival height during the course of treatment. A stabiliza- tion period should follow the active eruption phase to give the tissues time to mature through mineralization and modeling of the alveolar process. The duration of such stabilization may need to be determined on an individual basis depending upon the state of the tooth undergo- ing forced extrusion and the status of its periodontium. The extraction of teeth or tooth roots should be done atraumatically and flaplessly, while preserving as much of the newly developed bone height as pos- sible. Bone grafts may be needed during the extraction and immediate implant placement procedure. CONCLUSION Attaining a normal, stable and harmonious relationship of the teeth with their surrounding structures and providing an esthetically balanced face are essential goals of orthodontic treatment. Creative thinking is required when working as a part of an interdisciplinary clinical team to correct unusual or complex situations. Two cases were presented to illustrate how the scope of orthodontic treatment was extended to address interdisciplinary problems in the esthetic zone. Stepping out of the traditional box of orthodontic mechanics helps to evolve treatment planning into an open process in which a spectrum of viable choices that are not restricted by mechanistic disciplinary boundaries become available. While such present-day approaches facilitate creative treatment options, the future holds even greater promise for interdisciplinary collaborations, treatment outcomes and range of therapies. Such novel big steps in interdisciplinary therapy include the integration of molecular technology to deliver superior and more optimal treatment outcomes in complex situations as presented in several other chapters of this monograph. Such technologies, that surely will revolutionize the conduct of treatment, may not just be knocking at the orthodontist's door for use in the future—they already may be here. 96 Suri ACKNOWLEDGEMENTS The orthodontic treatments illustrated in this chapter were conducted by the author. The participation of Amit Brar, Ashok Utreja, Christopher Forrest, Harpinder Chawla, Henry Choi, Justin Garbedian, Robert Carmichael, Satinder Pal Singh and Vidya Rattan in the interdisciplinary management of the patients described is acknowledged with appreciation. REFERENCES Amato F, Mirabella AD, Macca U, Tarnow DP. Implant site development by orthodontic forced extraction: A preliminary study. IntJ Oral Maxillofac Implants 2012;27(2):411-420. Brown IS. The effect of orthodontictherapy on certain types of periodontal defects: I. Clinical findings. J Periodontol 1973;44(12):742-756. Buser D, Martin W, Belser UC. Optimizing esthetics for implant restorations in the anterior maxilla: Anatomic and surgical considerations. Int J Oral Maxillofac Implants 2004;(19 Suppl):43-61. Heithersay GS. Combined endodontic-orthodontic treatment of transverse root fractures in the region of the alveolar crest. Oral Surg Oral Med Oral Pathol 1973;36(3):404–415. Hochman MN, Chu SJ, Tarnow DP. Orthodontic extrusion for implant site development revisited: A new classification determined by anatomy and clinical outcomes. Semin Orthod 2014;20(3):208-227. Ingber JS. Forced eruption: I. A method of treating isolated one and two wall infrabony osseous defects: Rationale and case report. J Periodontol 1974;45(4):199-206. Ingber JS. Forced eruption: Part II. A method of treating nonrestorable teeth: Periodontal and restorative considerations. J Periodontol 1976;47(4):203-216. Jung RE, Zembic A, Pjetursson BE, Zwahlen M, Thoma DS. Systematic review of the survival rate and the incidence of biological, technical, and aesthetic complications of single crowns on implants reported in longitudinal studies with a mean follow-up of 5 years. Clin Oral Implants Res 2012;23(Suppl 6):2-21. 97 Interdisciplinary Role of Orthodontics Kokich VG. Maxillary lateral incisor implants: Planning with the aid of orthodontics. J Oral Maxillofac Surg 2004;62(9 Suppl 2):48–56. Korayem M, Flores-Mir C, Nassar U, Olfert K. Implant site development by orthodontic extrusion: A systematic review. Angle Orthod 2008;78 (4):752–760. Mantzikos T, Shamus I. Case report: Forced eruption and implant site development. Angle Orthod 1998;68(2):179-186. Mantzikos T, Shamus I. Forced eruption and implant site development: An osteophysiologic response. Am J Orthod Dentofacial Orthop 1999;115(5):583-591. Mantzikos T, Shamus I. Forced eruption and implant site development: Soft tissue response. Am J Orthod Dentofacial Orthop 1997;112(6): 596–606. Salama H, Salama M. The role of orthodontic extrusive remodeling in the enhancement of soft and hard tissue profiles prior to implant placement: A systematic approach to the management of extraction site defects. Int J Periodontics Restorative Dent 1993;13(4):312-333. Schropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing and Soft tissue contour changes following single-tooth extraction: A clinical and radiographic 12-month prospective study. Int J Periodontics Restorative Dent 2003;23(4):313-323. Spear FM, Mathews DM, Kokich VG. Interdisciplinary management of single-tooth implants. Semin Orthod 1997;3(1):45-72. Suri S, Utreja A. Management of a hyperdivergent Class Ill malocclusion, maxillary midline diastema, and infected mandibular incisors in a young adult. Am J Orthod Dentofacial Orthop 2003;124(6):725-734. Suri S, Utreja A, Singh SP. Riding pontics: Useful adjuncts with twin bracket appliances. J Ind Orthod Soc 1999;32:127-129. Tarnow DP, Cho SC, Wallace SS. The effect of inter-implant distance on the height of inter-implant bone crest. J Periodontol 2000;71(4):546-549. 98 TREATMENT OF CLASS III MALOCCLUSION WITH ANCHORED MAXILLARY PROTRACTION IN CLEFT CHILDREN: A ONE-YEAR FOLLOW-UP STUDY ON 3D SURFACE MODELS DERIVED FROM CBCT Yijin Ren, Johan Jansma, Harry Stamatakis ABSTRACT OBJECTIVE: To evaluate the treatment outcome of mild to moderate Class || malocclusion with anchored maxillary protraction in young cleft patients with a one-year follow-up on 3D surface models derived from a cone-beam computed tomography (CBCT). SUBJECTS AND METHODS: In this prospective case series study, patients with unilateral complete cleft lip and palate (CL/P) below the age of twelve with a sagittal overjet between 0 and -5 mm were included. Four Bollard bone plates were placed at eleven years of age. Maxillary protraction with intermaxillary elastics was started three weeks after the placement with a force of 200 g per side. The CBCT scans for each patient were performed before and twelve months after active protraction. RESULTS: In total, eleven patients were included (age = 10.9 to 11.6; mean overjet = -2.1 mm). Nearly all subjects showed improved lip projections and/or a fuller midface projection. The most significant skeletal changes are at the zygomatic arches (1.82 mm forward and downward displacement), at the maxillary complex (1.28 mm forward and 1.08 mm downward displacement of the A point) and at the mandible (1.27 mm backward and 2.07 mm downward displacement of the B point; 2.55 mm backward and downward displacement of the Pogonion [Pg] point). Three patients with a substantial displacement of the A-point, the B-point and transverse palatine suture opening, respectively, were demonstrated with 3D illustrations. CONCLUSIONS: For the first time, successful treatment outcome of Class Ill malocclusion with maxillary protraction in cleft children was shown during one year of therapy with favorable skeletal changes and improved facial profiles. KEY WORDS: orthodontics, cleft lip and palate (CL/P), skeletal anchorage, CBCT, maxillofacial protraction 99 Anchored Maxillary Protraction in Cleft Children INTRODUCTION Class Ill malocclusion as a consequence of maxillary deficiency and/or mandibular prognathism conventionally is treated with a facemask with a heavy anterior traction applied to the maxilla to stimulate forward and downward movement and to restrain and redirect mandibular growth. However, the optimal treatment timing and duration for facemask therapy remains controversial. Facemask wear usually is for a short and limitedly effective treatment time and, therefore, often is associated with undesirable treatment outcomes. These include dental compensations as a consequence of the application of forces on the teeth and an increased vertical dimension of the face as a result of posterior rotation of the mandible (Baik et al., 2000; Toffol et al., 2008; Watkinson et al., 2013). In recent years, the use of titanium miniplates for anchorage has been advocated as an alternative treatment modality to apply pure bone-borne orthopedic forces between the maxilla and the mandible for 24 hours per day, thereby minimizing dentoalveolar compensations. Though this treatment approach has showed favorable results in healthy growing subjects (De Clerck and Proffit, 2015), no previous study has investigated the treatment effect with anchored maxillary protraction on Class Ill malocclussion in cleft patients. To date, an early age maxillary protraction with facemasks with or without a combination with rapid maxillary expansion remains the most common treatment modality in growing cleft lip and palate (CL/P) patients with Class Ill malocclusions (Liou et al., 2005; Borzabadi-Farahani et al., 2012). The goal of this study was to evaluate the treatment outcome of mild to moderate Class III malocclusion with anchored maxillary protraction in young CL/P patients with a one-year follow-up on three- dimensional (3D) surface models derived from cone-beam computed tomography (CBCT). SUBJECTS AND METHODS Subjects This is a prospective case series study. All patients with unilateral complete CL/P younger than twelve years of age were included. All 100 Ren et al. patients had been under treatment by one orthodontist at the Department of Orthodontics at the University Medical Center Groningen (The Netherlands) and had undergone a series of interdisciplinary treatments coordinated by the Cleft Team at the same medical center. The inclusion Criteria were: 1. All patients had undergone a secondary bone transplantation procedure by the same surgeon. 2. None of the patients has started comprehensive orthodontic treatment with full fixed appliances. 3. Both lower permanent canines had erupted. 4. Sagittal overjet was between 0 and -5.0 mm. Bone Plates Four Bollard bone plates were placed at eleven years of age under general anesthesia (Cornelis and De Clerck, 2007). Maxillary protraction with intermaxillary elastics was started three weeks after placement with a force of approximately 200 g per side. CBCT Imaging Acquisition The CBCT scans were performed using the Kavo 3D eXam CBCT unit (Kavo Dental GmbH, Bismarckring, Germany) using a 17 x 23 cm field of view (FoV), the default 8.5 s acquisition time resulting at an average of 24 mAs at 120kV/5 mA and an isotropic voxel size of 0.3 mm. The patients were placed in the scanner with the Frankfurt Horizontal Plane (FHP) parallel to the ground and positioned centrally in the FoV with the aid of the laser alignment lights of the unit. - The CBCT scans for each patient were performed immediately before the placement of the bone anchorage (TO) following the De Clerck technique and after a period of approximately twelve months (range: eleven to fourteen months) of active protraction of the maxilla with Class Ill elastics (T1). The scan data for each patient were exported from the unit's dedicated software in DICOM format and imported to a specialized software (DeVIDE, Delft Technical University, Delft, The Netherlands) for 3D model construction following the segmentation of the hard tissues. The segmentation technique was based on pixel intensity differentiation thresholding and active contour tracing. Following this technique, the 101 Anchored Maxillary Protraction in Cleft Children segmented hard tissue data were transformed to a polygon 3D surface model comprising around five million surface polygons per skull. Superimposition of the 3D Models The 3D models for each patient subsequently were imported in Geomagic Studio v.2012 (Geomagic Solutions", Rock Hill, SC, USA) for 3D comparison before (T0 model) and after (T1 model) maxillary protraction. First, the registration procedure was performed, based on initial manual registration based on the anterior cranial fossa structures, followed by automatic best-fit match for optimal superimposition of the T1 model over the T0 model (Cevidanes et al., 2009). In addition to visualization of the surface discrepancies by means of color mapping, different regions of interest (ROI) were defined on all models by the same examiner who is experienced in 3D imaging in order to quantify the skeletal differences at Nasion (N), right and left zygomatic processes (Zyg), A-point and B-point. For each ROI, an area of approximately 100 polygons was defined arbitrarily. The software provided the mean positive or negative difference in mm of all individual surface polygons within the defined ROIs, which translate into their total displacement in space. Such a translation comprises both a horizontal and vertical component. By using transparency layers, each ROI could be visualized simultaneously on both the superimposed pre- and post- treatment 3D models, making it possible to measure the horizontal and vertical components separately, while maintaining the FHP as a reference. Finally, axial and sagittal reconstructions were used in order to measure any opening of the transversal palatal suture as a response to the bone-borne Class Ill traction. The schematic representation of Figure 1 outlines the measurement method for the displacement of A-point. Using as reference the FHP, the total displacement of A-point comprises a horizontal and a vertical component. The same principle was applied for B-point. Again, its total displacement comprises a horizontal and a vertical component. As both the zygomatic-maxillary complex and mandible exhibited significant downward displacement, it is important to make an analysis of the horizontal and vertical components of A- and B-point displacement measurements. Cornelis and De Clerck's study (2007) referred a total displacement in mm as the horizontal displacement of 102 Ren et al. A-point pre-ty horizontal —-ſ, š displacement A-point post-ty B-point pre-ty horizontal K- Figure 1. An illustration of the analysis of the horizontal and vertical components of A- and B-point displacement measurements. The schematic representation outlines the measurement method for the displacement of A-point. A: Using as reference the Frankfurt Horizontal plane (FHP), the total displacement of A-point 99mprises a horizontal and a vertical component. B: The same principle applied for B-point with its total displacement comprised of a horizontal and a vertical COmponent. A-point resulted in an overestimation of the treatment effect at the Sagittal plane. 103 Anchored Maxillary Protraction in Cleft Children RESULTS Twelve patients were included in this case series study. Increased mobility and local inflammation at the maxillary bone plates occurred in one patient four months after the protraction was initiated. These bone plates had to be removed and consequently, the patient was excluded from the study. The mean age of the remaining eleven subjects was 11.2 years (range: 10.9 to 11.6 years). The mean overjet of the subjects at the start of maxillary protraction was -2.1 mm (range: 0 to -5.0 mm). Lip Projection and Facial Profile Changes Lip projection and facial profile changes of the included patients one year after maxillary protraction are illustrated in Figure 2. Variations in individual treatment outcomes were observed in both genders, with more than half of the patients showing improved lip projections from a Class III concave profile toward a more straight or Class Il convex profile (Fig. 2A-C, G-I). The remaining subjects, however, did not show improvement in lip projection and presented a fuller midface projection (Fig. 2D-E, J-K). Only one subject had worse lip projection after one year of treatment (Fig. 2F); this patient also had the most severe Class || malocclusion (overjet -5.0 mm) among all included subjects at the start of the protraction. . Skeletal Changes on 3D Surface Models Superimposition of pre- and post-treatment 3D models from an 11.2-year-old male is illustrated in Figure 3. The overall changes took place mainly at the zygomatic-maxillary complex (forward and downward movement) and the mandible (downward and clockwise rotation). These findings are similar to previous reports on non-cleft patients with similar ages treated with maxillary protraction (Nguyen et al., 2011; De Clercket al., 2012). A patient with a favorable response of the maxilla to the bone- borne Class Ill protraction showed a significant forward displacement of the A-point (Fig. 4). The total displacement is 3.75 mm, indicating positive/ forward movement of 3.33 mm and downward displacement of 1.7 mm. 104 Ren et al. Figure 2. Treatment changes with maxillary protraction in the eleven patients before (extra-oral left panel; intra-oral top panel) and one year (extra-oral right panel; intra-oral bottom panel) after Class III maxillary protraction. A patient with a downward rotation of the mandible after the bone-borne class II protraction showed a significant downward/ backward displacement of the B-point (Fig. 5). The total displacement 105 Anchored Maxillary Protraction in Cleft Children Figure 3. Superimposition of pre- and post-treatment 3D models. The 3D hard-tissue models derived from the pre- and post-treatment CBCT data were registered and aligned on the anterior cranial base structures using the best-fit matching method. Green = pre-treatment model; mesh = post-treatment model. A-point pre-tz. +3.33 mm º A-point post-tz. Figure 4. Superimposition of pre- and post-treatment 3D models of a patient with substantial A-point forward displacement. Light blue = pre-treatment CBCT model; yellow = post-treatment CBCT model; dashed red circle = area analyzed. of the mandible was -5.53 mm, which indicates a downward displace- ment of 5.11 mm and a backward displacement of 2.09 mm as a result of mandibular backward rotation. The transverse palatine suture has been demonstrated to have the largest separation of all sutures in response to forward extra- oral forces (Kambara, 1977). Experimentally, the transverse palatine, zygomaticotemporal and pterygopalatine sutures exhibited the greatest 106 Ren et al. B-point pre-bº ( B-point pre-bº Figure 5. Superimposition of pre- and post-treatment 3D models of a patient With significant B-point downward/backward displacement. Light blue = pre- treatment CBCT model; yellow = post-treatment CBCT model; dashed red circle = area analyzed. ſeSponse to extra-oral forces with active osteogenesis and dramatically Stretched fibers (Jackson et al., 1979; Zhao et al., 2008). Here we refer to the present study on a CBCT model with a significant opening of a transversal palatal suture of nearly 2.5 mm after one year of bone-borne Class || maxillary traction (pre-treatment: ANS-PNS = 48.81 mm; suture Opening = 0.90mm; post-treatment: ANS-PNS=51.79 mm; suture opening 3.31 mm; Fig. 6). Although such an opening typically was not found in *Very patient treated with the same protocol, it clearly demonstrated the Potential of suture opening at the transversal palatal region at a later age than previously stated in the literature (Watkinson et al., 2013). Although the sample size was relatively small, we demonstrated Clearly that after one year of treatment with bone-borne maxillary Protraction, the most significant skeletal changes took place at the */80matic arches (1.82 mm forward and downward displacement), at the maxillary complex (1.28 mm forward and 1.08 mm downward displacement of A-point) and at the mandible (1.27 mm backward and 2.07 mm downward displacement of the B-point; 2.55 mm backward and downward displacement of Pogonion point (Pg). This means the Sagittal skeletal profile changes in terms of the net difference between A- and B-point one year after treatment is 2.55 mm (Table 1: Fig. 7). These 107 Anchored Maxillary Protraction in Cleft Children Figure 6. A CBCT illustration of a patient with an opening of the transversal palatal suture. A: Axial view. B: Sagittal view. Arrows indicate opening of the transversal palatal suture. Table 1. Descriptive table with measurements of the N-, Zyg-, A- and B-point displacement. The values are the mean for the total analyzed CBCT models measured in mm; forward and downward vectors are denoted positive; backward vectors are denoted negative. Point mm. SD N-point 0.35 0.23 Zyg-point 1.82 0.40 A-point 1.45 1.23 A-point horizontal 1.28 1.13 A-point vertical 0.58 0.62 B-point –2.29 2.31 B-point horizontal -1.47 1.39 B-point vertical 1.63 1.97 results are similar to the unpublished data from a poster presentation at the 2015 Moyer's Symposium (Yatabe et al., 2015), the only study known to the authors that used a similar treatment protocol on growing cleft patients. 108 Ren et al. + 1.82mm W º + 1.45mm | Nº hori. - 1.28 mm N Ş + 0.35mm 9 vent. -- 1.08 mm Q %) // - 2.29mm B hori, -1.27 mm vent. 4-2.07mm 2/* - 2.55mm Figure 7. An illustration of the overall changes taking place at the zygomatic arch, maxillary complex and the mandible. In this schematic representation. The mean displacement after treatment of N-, Zyg-, A- and B-points in our sample are shown (red and blue font) together with their horizontal and vertical vectors (gray font). DISCUSSION AND CONCLUSIONS Treatment of Class III malocclusion with anchored maxillary Protraction in growing non-cleft subjects previously showed favorable results (De Clerck and Proffit, 2015); however, to date, no study has been published on the treatment effect using the same method on growing cleft patients. Our study provided treatment outcomes for 3D CBCT Surface models for the first time that showed favorable skeletal changes at the zygomatic arches and the maxillary complex, both of which showed forward and downward displacement, and the mandible that had a backward rotation and downward displacement. This was accompanied by improved facial profile with a fuller mid-face and lip projections more .." a Straight or a Class || convex profile. The complication rate was OW. 109 Anchored Maxillary Protraction in Cleft Children A number of limitations in our study need to be acknowledged. First, there was no CBCT data available on non-treated subjects that can serve as a control to evaluate the skeletal changes attributed to the treatment itself instead of a combined effect from treatment and growth. Alternatively, comparisons could be made with non-cleft patients treated with bone-borne maxillofacial protraction based on 3D CBCT models, though publications on this subject are scarce. Another alternative to overcome this drawback is to compare analyses on linear and/or angular measurements with non-cleft patients treated with conventional facemasks or untreated Class Ill malocclusions subjects based on 2D cephalometric (Cevidanes et al., 2010; Baccetti et al., 2011). Accuracy and reliability of 3D measurements based on CBCT data may differ, however, when compared to 2D techniques (Oh et al., 2014; Pittayapat et al., 2015). Our study focused on skeletal changes based on 3D surface models, therefore dentofacial effects were not analyzed. Only subjects with mild and moderate Class Ill malocclusion were included in our study. Future studies should be directed to define treatment indications and identify subjects that could benefit most from this treatment modality. On the other hand, a unique outcome from our results is forward and downward displacement of the zygomatic arches. Such displacement was demonstrated consistently by the significantly smaller variability (standard deviation) in Table 1 compared with those of the A- and B-points. This is an important advantage that a later Le Fort | jaw surgery cannot offer. Therefore, an argument could be made to include more severe Class III malocclusions for this treatment modality— not with the goal to avoid a jaw surgery at a later age, but to provide better mid-face support to facilitate and complement the treatment outcome of a jaw surgery that already is indicated. In addition, longer follow-ups are needed to demonstrate long-term treatment effects and possible skeletal relapse. ACKNOWLEDGEMENTS The authors would like to thank the clinical support staff at the Department of Orthodontics, University Medical Center Groningen, The Netherlands for their contribution to the treatment and care to the cleft patients included in this study. 110 Ren et al. REFERENCES Baccetti T, De Clerck HJ, Cevidanes LH, Franchi L. Morphometricanalysis of treatment effects of bone-anchored maxillary protraction in growing Class III patients. Eur J Orthod 2011;33(2):121-125. Baik HS, Han HK, Kim DJ, Proffit WR. 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Cochrane Database Syst Rev 2013;9:CD003451. Zhao N, Xu Y, Chen Y, Xu Y, Han X, Wang L. Effects of Class III magnetic orthopedic forces on the craniofacial sutures of rhesus monkeys. Am J Orthod Dentofacial Orthop 2008;133(3):401–409. 112 THREE-DIMENSIONAL CHANGES IN THE MANDIBLE AND ARTICULAR FOSSAE FOLLOWING HERBST APPLIANCE THERAPY: A PRELIMINARY CBCT STUDY Bernardo O. Souki, Lucia H.S. Cevidanes, Paula L. Cheib, Wagner Moyses-Braga, Antonio Carlos Ruellas, Lorenzo Franchi, James A. McNamard Jr. ABSTRACT OBJECTIVE: The purpose of this study was to compare three-dimensional (3D) skeletal changes in the mandible and the articular fossae of a group of Class || malocclusion subjects treated with the Herbst appliance (HAG) and a Class || Comparison group (CG) treated using conventional fixed orthodontic appliances. PATIENTS AND METHODS: The preliminary findings of the study were evaluated using 3D virtual models generated from cone-beam computed tomographies (CBCTs) taken at two time points (TO = baseline and T1 = eight months following treatment) of sixteen HAG and five CG subjects. Voxel-based registration on the anterior Cranial fossa was used to assess mandibular displacement/articular fossa remodeling, while regional registration on the mandibular symphysis was performed to evaluate mandibular growth. RESULTS: At the end of Herbst therapy (T1), the condyles of HAG subjects were in similar orientation within the temporomandibular joint (TMJ) region as at T0. Downward displacement of the mandible was observed in both HAG and CG, but forward displacement of the mandible was measured only in the Herbst group. An increase in mandibular length (approximately 2 mm), associated with additional growth of the posterior surface of the condyles and rami, was observed in the HAG patients at T1; however, no change in mandibular length was found in CG. Vertical growth of the mandible was greater slightly in HAG than in CG. No bone remodeling was found in the articular fossae in both groups after eight months of treatment or observation. CONCLUSIONS: After Herbst appliance therapy, mandibular forward displacement was achieved due to increased bone deposition and remodeling in the mandibular posterior rami and condylar surfaces of HAG patients. No changes in the articular fossae were found. KEY WORDS: Herbst appliance, malocclusion, Angle Class II, orthodontics, computer-generated 3D imaging 113 Herbst Appliance Therapy INTRODUCTION Class || malocclusion is highly prevalent worldwide (Massler and Frankel, 1951; Emrich et al., 1965; Proffit et al., 1998; Almeida, 2011) and is a major reason for orthodontic treatment to be initiated (Laganà et al., 2013; Kandasamy and Goonewardene, 2014). This type of malocclusion can be restricted to the dentoalveolar region, but the majority of Class Il patients exhibit a strong skeletal component to the malocclusion, including most frequently mandibular skeletal retrusion and an increased lower anterior facial height (McNamara, 1981; Buschang et al., 1988; Buschang and Martins, 1998). With increased severity of the mandibular deficiency, the orthodontist is challenged to treat the underlying skeletal problem appropriately. For growing patients, the use of fixed and removable mandibular advancement appliances has been advocated for many decades (Ruf et al., 2002; Kinzinger et al., 2007; Li et al., 2014). A variety of fixed Herbst appliance designs have achieved worldwide acceptance in that they eliminate most major patient compliance factors (Cope et al., 1994; Karacay et al., 2006; Kinzinger et al., 2011). The Herbst appliance (HA) is by far the most frequently used mandibular jumping device in the United States (Pancherz, 2013), with more HA being fabricated than all other functional appliances combined. The HA originally was introduced by Emil Herbst in the early 20th century (Herbst, 1910), but it did not achieve worldwide clinical acceptance until its re-introduction by Hans Pancherz 70 years later (Pancherz, 1979). Since then, a significant number of clinical and scientific studies have been conducted about HA (Pancherz and Anehus-Pancherz, 1980; Pancherz, 1981, 1982a,b, 1985, 1991; Pancherz and Hägg, 1985; Pancherz and Hansen, 1986, 1988; Hägg and Pancherz, 1988; Pancherz et al., 1989; Pancherz and Fackel, 1990). Two-dimensional (2D) cephalometric studies have reported increases in the length of the mandible and in forward displacement of the mandible following HA when compared with matched untreated controls (Pancherz et al., 1998; Hägg et al., 2008; Serbesis-Tsarudis and Pancherz, 2008). On average, 2 mm of mandibular length gain (measured from Gonion to Pogonion) and 1.5° increase in the SNB angle can be observed following HA therapy (Pancherz, 1982a). 114 Souki et al. Investigations in experimental animals have provided the histological evidence of the changes in the condyles, rami and articular fossae when the mandible is advanced in a laboratory setting (Chayanupatkul et al., 2003; McNamara et al., 2003; Peterson and McNamara, 2003; Rabie et al., 2003; Voudouris et al., 2003a,b). Peterson and McNamara (2003) found an increased proliferation of the condylar Cartilage in monkeys that had their mandible advanced with the HA. These adaptations occurred primarily in the posterior and posterosuperior regions of the condyle. They also reported significant bone deposition along the posterior border of the mandibular ramus during the early part of the experimental period, as well as significant deposition of new bone on the anterior surface of the post-glenoid spine at the articular fossae. It should be noted, however, that the post-glenoid spine is not defined well in humans. Clinical investigations using MRI and lateral cephalograms have shown that changes in the mandibular condyles and articular fossae may occur with Herbst therapy. Ruf and Pancherz (1999) demonstrated that Some articular fossa remodeling is observed at the anterior surface of the post-glenoid spine following Herbst therapy, which causes a relocation of the articular fossae in a downward and forward direction. Such fossa adaptation in Herbst patients is less extensive than in experimental animals, however, with many variations in individual responses. Although most studies with HA are consistent with the extent of Skeletal and dentoalveolar adaptations, some questions still remain: • How much mandibular growth can be achieved with therapeutic mandibular advancement using HAP • How much mandibular rotation and skeletal bite opening occurs following HA therapy? • How much of the original mandibular advancement is maintained long term? • Are there adaptive changes in the articular fossa after mandibular advancement, with bone remodeling occurring in the fossa? In the last decade, a new methodology using 3D virtual modeling has been developed allowing a change in the paradigm of assessing the skeletal changes associated with growth and treatment (Cevidanes et al., 2009); this technology has opened new horizons in scientific 115 Herbst Appliance Therapy investigations of dentofacial orthopedics. Le Cornu and associates (2013) recently presented the first 3D report on Herbst treatment, a pilot study in which seven Class II subjects treated with the HA were compared with seven Class II subjects treated with fixed appliances and intermaxillary elastics. They found that Class Il patients undergoing Herbst treatment demonstrated anterior displacement of the condyles and articular fossae along with maxillary restraint when compared with the Class Il patients using Conventional fixed appliance therapy. The aim of this prospective-controlled clinical trial was to investigate the skeletal changes in the mandible and articular fossae in Class Il patients undergoing HA therapy, using 3D virtual models in comparison to a control group comprising Class || Subjects treated with conventional fixed appliances. This chapter presents preliminary data from the first sixteen included in the Herbst Appliance Group (HAG) and from the first five subjects in the comparison group (CG). PATIENTS AND METHODS Approval for this clinical trial was granted by the Institutional Review Board of the Pontifical Catholic University of Minas Gerais at Belo Horizonte, Brazil (CAAE: 21534013.8.5137). The trial design included 60 skeletal Class || pubertal subjects. Thirty individuals who had an HA inserted at the beginning of the treatment with a one- step full activation were included in the HAG, while 30 skeletal Class || malocclusion individuals—with indication for HA therapy, but with other clinical conditions that required prior treatment before HA insertion— were allocated to the CG. Mandibular teeth were not bonded and no intermaxillary elastics were used. Figure 1 shows a patient from the HAG, where a one-step full activation into a Class I canine relationship was achieved immediately after T0. Figure 2 shows a patient from the CG. The need for restorative dentistry, as well as orthodontic decompensation of maxillary incisors, was necessary prior to the insertion of the HA. The two groups were matched chronologically (between twelve and sixteen years of age), by stage of skeletal maturation (patients in the peak of pubertal growth, i.e., CS3 and CS4) and by the stage of dental development (permanent dentition). 116 Souki et al. Figure 1. Patient from Herbst Appliance Group (HAG) with a one-step full activation. A: Pre-treatment. B. Immediately after HA insertion. HA Design The HA design included bilateral telescoping arms (3M Abzil, São José do Rio Preto, Brazil) articulated with pivots that were positioned in both the maxillary and mandibular arches (Fig. 3). The pivots were Welded to a rigid cantilever wire (0.040” stainless steel) extending from the lower first permanent molar bands (TP Orthodontics, La Porte, |N) to the canine regions of the mandible. In the maxillary arch, the pivots were welded to bands on the permanent first molars. A Hyrax *Pander (Morelli Ortodontia, Sorocaba, Brazil) and a 0.040” stainless Steel lower lingual arch were added to the HA structure to improve Herbst Appliance Therapy Figure 2. Patient from the Comparison Group (CG). The malocclusion needed dental decompensation, leveling and alignment prior to HA placement. Figure 3. HA design used in this study. appliance stability and transverse dimensional control. Two 0.028” stain- less steel wires were used as occlusal rests in the permanent second molars 118 Souki et al. to avoid their extrusion after activation. The occlusal rests were removed if they interfered with the occlusion to avoid bite opening. HA Treatment Protocol Depending on the patient's inter-arch transverse discrepancy, rapid maxillary expansion was necessary one week prior to the installation of the mandibular component of the HA. One-step full activation, into a Class I relationship in the canines region, was performed on the day of the complete HA installation. On average, the mandibular advancement was 5.3 mm (ranging from 3 to 10 mm). Method of Registration Patients from the HAG presented two time-point records: T0, at baseline, before any orthodontic treatment; and T1, eight months after HA insertion, at the end of HA treatment. For the CG, two time-point records were available: T0, at baseline; and T1, at the end of alignment and leveling, eight months after T0. Data derived from cone-beam computed tomography (CBCT) included in the patient's orthodontic records were used for the construction of virtual three-dimensional (3D) surface models. All patients were instructed to bite into maximum occlusal contacts during the scan capture. Method of Measurement Analysis of serial CBCT images to evaluate changes between T0 and T1 included 3D analysis methodology using ITK-SNAP (open-source software, www.itksnap.org; Yushkevich et al., 2006); SLICER (open-source Software, www.slicer.org; Simmross-Wattenberg et al., 2005); and Vectra Analysis Model (VAM) software version 3.7.6 (Canfield Scientific Inc., Fairfield, NJ). The 3D image analysis procedures included: Approximation of T0 and T1 scans; Construction of 3D label models; Voxel-based image registration; and Ouantitative and qualitative assessments using 3D mesh surface models. : Ouantitative assessment of the positional changes between the 3D surface models of the mandible was performed using point-to-point landmarks measurements (VAM software) and virtual analytics (SLICER 119 Herbst Appliance Therapy software). Two landmarks were identified: right Condylar Point (CoP) and Pogonion Point (PogP). CoP was defined as the mid-most superior aspect of the right and left condyles, while PogP was defined as the most anterior midsagittal point along the anterior border of the mandible. Changes in the Cartesian coordinates of CoP and PogP between T0 and T1 were used to assess the 2D-projected linear condylar displacement. The posterior-anterior (AY) and inferior-superior (AZ) 2D-project- ed linear displacements of CoP and PogP from T0 to T1 were measured in the sagittal plane (YZ). The medial-lateral (AX) 2D-projected linear displacement was measured in the axial plane (XY). The Mann-Whitney non-parametric test was used to assess the differences between HAG and CG in the CoP displacements. Interactive visual analytics included graphic displays with quali- tative assessments using semi-transparent overlays of the 3D surface models at T0 and T1 and quantitative assessments of the condylar and chin surface displacements using color-coded surface distance maps. All visual analytics assessments were performed using two modules (Model- to-Model Distance and Shape Population Viewer) in the SLICER software. For the assessment of mandibular displacement and articular fossa remodeling, a total superimposition of the virtual 3D surface models with a volumetric voxel based registration at the cranial base was undertaken (Fig. 4A). To investigate the amount of mandibular growth, a regional superimposition at the mandibular symphysis was performed (Fig. 4B). RESULTS Preliminary results are presented according to three of the possible skeletal adaptive processes associated with HA therapy: 1. Mandibular growth; 2. Mandibular displacement; and 3. Articular fossae bone remodeling. To illustrate the findings, representative composite images of four individuals from each group are presented in Figures 5, 6, 8 and 9. Semi-transparent overlays and color mappings are shown for those 120 Souki et al. Figure 4. Voxel-based registration. A: Cranial base. B: Mandibular symphysis. Cases. Overlays give the direction of the changes, while the color-mapping Scale provides the quantification of such changes. Mandibular Growth Figure 5 shows the semi-transparent overlay of four patients treated with the HA and four patients from the CG. The regional super- imposition was registered at the mandibular symphysis. In the CG, mandibular growth occurred mainly along the superior Surface of the condyle, with a minimal amount of growth observed along the posterior surface of the rami. In the HAG, however, mandibular growth Was observed not only along the superior surface of the condyles, but also along the posterior surface of the condyles and along the posterior Surface of the rami. Figure 6 shows that following eight months of observation, verti- Cal growth of the condyle in the CG individuals was approximately 2 mm (indicated by purple). In the HAG, greater vertical growth (approximately 3 mm, as indicated by yellow) and approximately 2 mm of growth along the posterior surface of the rami (indicated by purple) was observed. Fig- uſe 7 presents a vectorization of mandibular growth in the two investi- §ated groups. The CG patients exhibited an upward growth of the rami and condyles, while the HA patients presented an upward and backward Śrowth vectorization, both in the rami and condyles. 121 Herbst Appliance Therapy - - Comparison group Figure 5. Mandibular growth evaluation with semi-transparent overlay from two time-point mandibular virtual models: T0 (red) and T1 (black mesh). Registration at the mandibular symphysis. Comparison group Figure 6. Mandibular growth evaluation with color mapping (model-to-model distance) from two time-point mandibular virtual models: T0 and T1. Registration at the mandibular symphysis. Mandibular Displacement Figure 8 shows the semi-transparent overlay of four patients from the HAG and four patients from the CG, based on cranial base registration. In the CG, the mandible was displaced mainly downward (2 mm, indicated by the purple color in Fig. 9). However, in the HAG, a downward (2 to 3 mm) and forward (3 to 4 mm) displacement of the mandible was noted. 122 Souki et al. Comparison group Herbst group Figure 7. Illustration of the mandibular growth with vectorization of the growth direction. Registration at the mandibular symphysis. Comparison group Figure 8. Mandibular displacement evaluation with semi-transparent overlay from two time-point mandibular virtual models: TO (red) and T1 (black mesh). Registration at the cranial base. Table 1 gives the point-to-point measurements of the changes between T1 and To of Pogonion of the two groups. The HAG showed a 3.4 mm forward mandibular displacement and 2.8 mm downward mandibular displacement; the CG showed only a 0.1 mm forward and a 2.4 mm downward mandibular displacement. The difference in vertical mandibular displacement was not significant statistically between the two 123 Herbst Appliance Therapy Comparison group Figure 9. Mandibular displacement evaluation with color mapping (model-to- model distance) from two time-point mandibular virtual models: T0 and T1. Registration at the cranial base. groups. The condyles appeared to maintain or resume their original position within the glenoid fossa in both groups. Only minor positional changes were observed and were not significant statistically. Articular Fossae Bone Remodeling To assess the articular fossa changes, the color-coding scale that was used was narrower than for the assessment of mandibular changes. The range was set between -1.5 and +1.5 mm. The scale was adjusted to identify changes greater than 0.75 mm. No significant bone remodeling within the articular fossa was observed in either group (Fig. 10). DISCUSSION The evaluation of dentofacial orthopedics outcomes using CBCT and 3D virtual modeling has the potential to improve the understand- ing of modifications in the size, shape and change in relative positions of the skeletal facial components that underpin the response to treatment (Cevidanes et al., 2011). 3D volumetric superimpositions are effective in revealing areas of bone displacement and remodeling after dentofacial Orthopedic intervention (Cevidanes et al., 2009). Moreover, the differ- entiation between dentofacial changes caused by treatment from those resulting from normal growth can be made with a matched comparison 124 Table 1. Evaluation of displacement changes (point-to-point measurements) of the Pogonion Point (PogP) and Condylar Point (CoP) in the HAG and in the CG with registration at the cranial base. NS = not significant. * = P × 0.05. Herbst Appliance Group Comparison Group º, Minimum Maximum Mean SD Minimum Maximum Mean SD Pogonion Lateral 0.008 1,713 0.579 0.497 0.012 0.113 0.038 0.049 0.071 NS Point Forward 0.050 5,788 3.437 1,430 0.011 0.185 0.078 0.070 0.022* Downward 0.458 5.143 2.841 1,483 1.459 3.241 2,351 0.727 0.317 NS Condylar Lateral 0.000 0.040 0.008 0.018 0.000 0.050 0.055 0.017 0.682 NS Point Forward 0.001 0.500 0.357 0.182 0.002 0.050 0.035 0.017 0.122 NS Downward 0.003 0.900 0.550 0.197 0.002 0.050 0.037 0.017 0.094 NS § Herbst Appliance Therapy Comparison Figure 10. Articular fossae remodeling evaluation with color mapping (model- to-model distance) from two time-point cranial base virtual models: T0 and T1. Registration at the cranial base. group. Therefore, the contribution of this prospective trial to the HA literature is a way of validating previous evidence provided by 2D investigations using the new 3D virtual modeling technology. Since the HA was re-introduced to the orthodontic specialty (Pancherz, 1979), many investigative efforts have been undertaken to provide clinical and experimental evidence concerning the mechanism of action of such devices (Pancherz and Anehus-Pancherz, 1980; Pancherz, 1981, 1982a,b, 1985, 1991; Pancherz and Hägg, 1985; Pancherz and Hansen, 1986, 1988; Hägg and Pancherz, 1988; Pancherz et al., 1989; Pancherz and Fackel, 1990). Most of the previous 2D cephalometric data, as well as the histological findings concerning mandibular displacement and growth, have been confirmed by 3D preliminary data from the current study. Although previous studies mentioned the 126 Souki et al. possibility of significant remodeling of the articular fossae following treatment with HA (Woodside et al., 1987; Pancherz and Michailidou, 2004; LeCornu et al., 2013), our preliminary 3D results indicate no such remodeling in the articular fossae region. The preliminary findings extracted from the initial 21 subjects of this clinical trial (sixteen patients in the HAG and five in the CG) do not allow for definitive conclusions about the nature of the adaptations produced by the HA, especially because significant individual biological variation in treatment responses have been observed. Analysis of the entire sample may provide answers as to why some patients experienced greater skeletal changes than others. Due to the limited sample size, linear regression analysis—which potentially could explain the correlation between the magnitude of the Herbst activations and skeletal outcome—was not performed. Therefore, the direct relationship between the amount of bite advancement at the Start of the treatment and the treatment effects could not be determined. The present findings, however, corroborate previous cephalometric data that have shown that HA stimulates condylar growth in a posterior direction; as a consequence, mandibular length is increased (Pancherz and Ruf, 2008). The results of this preliminary study showed that for a mean Herbst activation of 5.3 mm (ranging between 3 and 10 mm; SD = 1.9 mm), there was a marked growth of the condyles and rami (approximately 3 mm in the superior-posterior surfaces of the condyles and 2 mm in the posterior surface of the rami). The increase in mandibular length in the present study, considering the additional bone remodeling in the posterior areas of the condyles and rami in comparison with the CG, varied between 2 and 4 mm. Such numbers are similar to those reported by Pancherz (1982a) in his 2D cephalometric study where an average of 2.5 mm of mandibular length growth after six months of HA treatment was noted. Similarly, the results of this study indicate that the forward dis- placement of the mandible after eight months of HA therapy ranged be- tween 0.1 and 5.8 mm measured at the chin point (Pogonion). Taking into account that the initial one-step HA activation ranged from 3 to 10 mm in this sample, we can infer that a significant amount of the origi- nal activation was not maintained during the therapeutic period. The 127 Herbst Appliance Therapy rebound of the original amount of bite jumping has been described previ- ously (Pancherz, 1982a) with an estimated 54% loss of original activation. The preliminary data from our results indicate a rebound rate of 50 to 75%. This loss of activation most probably occurs because of the muscle activity that pulls the mandible back to its original position, even against the mechanical component of the Herbst bite-jumping mechanism. The forward displacement of the mandible at the end of HA wear (the ultimate therapeutic aim of such treatment), however, was observed in all HAG patients. The mean forward displacement was 3.4 mm. In contrast, the CG patients did not show significant forward repositioning of the mandible, with a mean forward displacement of 0.1 mm. The anterior 3D displacement of the mandible observed in HAG subjects and the resulting improvement in the facial profile is in accordance with previous 2D studies (Ruf and Pancherz, 2006). In our investigation, the condyle-fossa relationship was unaf- fected by HA therapy. In all sixteen HA individuals analyzed thus far, the spatial positions of the condyles maintained their original position; this observation deserves emphasis. One of the concerns about HA insertion is the alteration of a previously balanced TMJ relationship into a patho- logical condition (Woodside et al., 1987). The “return” of the condyles into their original positions has been described previously (Ruf and Pan- cherz, 1998). It has been hypothesized that the translation of the articular fossae might be one of the adaptive processes associated with HA therapy (Woodside et al., 1987; Pancherz and Fischer, 2003). Le Cornu and associates (2013) recently reported with 3D data that the HA alters the growth pattern of the articular fossa, resulting in a more anteriorly positioned fossa (greater than 1 mm) and, therefore, a more anteriorly positioned mandible. In the analysis of our initial 16 subjects from the HAG, however, no significant bone remodeling could be measured in the articular fossae region. This study methodology used a 1.5 mm color-coding scale that ranged between minimum (coded dark blue) and maximum (coded red) values, with a range of detection of difference of 0.75 mm shown as cyan (-0.75 mm) and yellow (+0.75 mm) for assessment. Previous clinical 2D studies using MRI and lateral headfilms described only small changes in the articular fossae. Pancherz and Fischer (2003) 128 Souki et al. reported 0.4 mm forward and 1.3 mm downward displacement following HA therapy. Such numbers probably are within or close to the measurement margin of error and probably are insignificant clinically. Therefore, further analysis of the contribution of the articular fossae in the anterior displacement of the mandible during Herbst treatment must be revisited with larger samples. As in many clinical investigations, the variability of the changes in mandibular position and growth among different individuals in this study was large; however, a clear, uniform trend of mandibular position and growth was observed within each group. As a preliminary report, the sample size is an inherent limitation and caution must be taken in the interpretation of the findings. A more robust data on this topic may be expected following analysis of the entire sample. CONCLUSIONS The preliminary results of this study showed: 1. Changes in the direction and magnitude of growth of the mandibular condyles and rami were observed after eight months of Herbst therapy. 2. A greater forward displacement of the mandible was observed in patients treated with HA than in comparison individuals. 3. No mandibular rotation was observed following Herbst therapy. 4. Bone remodeling in the articular fossae was not ob- served in either group. REFERENCES Almeida MR, Pereira ALP, Almeida RR, Almeida-Pedrin RR, Silva Filho OG. Prevalence of malocclusion in children aged 7 to 12 years. Dental Press J Orthod 2011;16(4):123-131. Buschang PH, Martins J. Childhood and adolescent changes of skeletal relationships. Angle Orthod 1998;68(3):199-206. Buschang PH, Tanguay R, Demerjian A, LaPalme L, Turkewicz J. 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Condyle-fossa modifications and muscle interactions during Herbst treatment: Part 1. New technological methods. Am J Orthod Dentofacial Orthop 2003b;123(6):604-613. Woodside DG, Metaxas A, Altuna G. The influence of functional appliance therapy on glenoid fossa remodeling. Am J Orthod Dentofacial Orthop 1987;92(3):181-198. Yushkevich PA, Piven J, Hazlett HC, Smith RG, Ho S, Gee JC, Gerig G. User-guided 3D active contour segmentation of anatomical struc- tures: Significantly improved efficiency and reliability. Neuroimage 2006;31(3):1116-1128. 134 SURGICALLY FACILITATED ORTHODONTICS: WHAT DOES THE EVIDENCE SAYP Peter H. Buschang ABSTRACT Faster tooth movement is the orthodontist's holy grail. Shorter treatment times would reduce costs and decrease the risks of gingival inflammation, decalcifica- tion, dental caries and root resorption. The idea of shortening treatment duration through surgical intervention was introduced first in the late 1800s; it resurfaced temporarily in the early to mid 1900s and reemerged in 2001. Interest increased dramatically after faster movements were linked to the biological process, the regional acceleratory phenomenon (RAP), which explained the response of bone to injury. Since then, a great deal of evidence has been accumulated showing that corticotomies—the most commonly used surgical intervention—increase rates of tooth movement approximately two-fold; however, the duration of the effect is limited. For humans, the orthodontist can expect the effect to last two to three months, during which the teeth can be moved 4 to 6 mm. There also is good evidence that the greater the injury, the greater the rate of tooth move- ment, but this does not affect the duration of the effect. While the evidence pertaining to less invasive, flapless, corticision procedures is controversial, more rapid tooth movements may be possible when the surgical cuts penetrate deep- er into medullary bone. There is weak evidence supporting the efficacy of the least invasive flapless procedures involving limited numbers of 2 to 3 mm deep perforations into bone. Finally, there is limited evidence supporting the most in- vasive approaches that move teeth up to 1 mm/day by means of periodontal or dentoalveolar distraction osteogenesis. Although the evidence for some ap- proaches may be limited, their potential is great. Surgical intervention presently provides the best means for orthodontists to accelerate tooth movement sub- stantially and decrease treatment time. More experimental studies are needed to understand and eventually be able to control the biological events that occur with surgical intervention. KEY WORDS: tooth movement, corticotomies, regional accelatory phenomenon (RAP), piezocision 135 Surgically Facilitated Orthodontics INTRODUCTION Orthodontic treatment typically takes two or more years to complete. The average treatment duration for patients who have had extractions is approximately 28.4 months, although some studies have reported times of up to 35 months (Table 1). Non-extraction treatments require approximately 25 months. Longer treatment times result in patient “burn out,” increased travel and time costs to the patient, and increased chair time for the orthodontists. Long treatment durations are problematic particularly for working adults, who are seeking treatment in increasing numbers. Most importantly, longer treatment times increase the risk of gingival inflammation, decalcification, dental caries and especially root resorption (Artun and Brobakken, 1986; Kurolet al., 1996; Segal et al., 2004; Ristic et al., 2007; Lopatiene and Dumbravaite, 2008). The best way to shorten orthodontic treatment time is to accelerate tooth movement. Teeth move approximately 0.8 to 1.2 mm/month when continuous forces are applied (Samuels et al., 1993; Daskalogiannakis and McLachlan, 1996; Dixon et al., 2002; Barlow and Kula, 2008). Experimentally, movements have been accelerated by local injections of osteocalcin, prostaglandins and vitamin D3, as well as by the application of pulsed electromagnetic fields and direct electric currents (Davidovitch et al., 1980; Yamasaki et al., 1982; Stark and Sinclair, 1987; Collins and Sinclair, 1988; Kobayashi et al., 1998). However, none of these approaches have been applied clinically, due to potential detrimental inflammatory responses, pain, discomfort, adverse metabolic effects and tissue damage. Surgery remains the most thoroughly evaluated and clinically applicable means of rapidly moving teeth. Bryan (1882) introduced the notion and Cunningham (1894) more fully described the procedures at the World's Columbian Dental Congress in Chicago. He noted that teeth could be moved rapidly—up to a quarter of an inch (Fig. 1)—by cutting “the alveolar bone with a thin circular saw” and using forceps, elevators or other instruments “for pushing, pulling or rotating the tooth sections into place,” which often required “great strength carefully applied.” While the goal was “to move each tooth with its socket entire as far as may be possible,” Cunningham had “no hesitation in preparing a way for that tooth by removing the bone behind it.” 136 Buschang Table 1. Treatment duration (months) associated with extraction and non- extraction treatments. Extraction Non-extraction O'Brien et al., 1995 30.6 24.8 Alger, 1988 26.6 22.0 Fink and Smith, 2005 26.2 22.0 Popowich et al., 2005 25.7 25.0 Skidmore et al., 2006 24.6 21.3 Vu et al., 2008 35.2 27.4 Vig et al., 1990 31.2 31.3 Callaway, 1999 , 26.7 22.4 Buckman, 1999 28.4 25.7 Mean 28.4 24.7 Figure 1. Left: Upper right first molar, which was decayed extensively, was extracted with forceps. Right: The second premolar was “forcibly rotated on its axis." With considerable application of force in an outward direction, the alveolar yielded and the tooth was drawn into the position recently occupied by the first molar. Patient treated by Cunningham (1894). While this approach was never accepted widely, the idea of cutting Cortical bone to accelerate tooth movement reemerged during the early 1900s. Starting in the 1920s, various Europeans (Cohnstock, Bichlmayr, Skogsborg, Ascher) advocated removing the cortical plates to accelerate tooth movement. This treatment approach was based on the belief that the cortical plates served as the major resistance to tooth movement. By the late 1940s, the procedures involved less invasive vertical interdental OSteotomies. In his classic paper, Kole (1959) introduced Corticotomies to achieve faster tooth movement. Convinced that the cortex provided the main resistance to tooth movement, he advocated performing buccal and lingual vertical corticotomies, leaving the medullary bone intact, and through-and-through horizontal osteotomies, approximately 1 cm 137 Surgically Facilitated Orthodontics apical to the roots of the teeth being moved. Suya (1991) reported having treated 395 cases with corticotomies and being able to achieve space closure in an average of 107 it 20 days. Unlike Kole, Suya recommended making shallow—just through the cortical bone–horizontal cuts 2 to 3 mm apical to the root tips that connected the vertical cuts. While this generated additional studies evaluating revascularization and bone healing (Bell and Levy, 1972) and the effects of corticotomies on the periodontium (Gantes et al., 1990), orthodontists largely lost interest in corticotomy procedures until 2001, when Wilcko and coworkers (2001) introduced Accelerated Osteogentic Orthodontics (AOO)—a clinical procedure that combined alveolar corticotomies—particulate bone grafting and orthodontic forces. Unlike previous investigators who thought that the faster tooth movement was a mechanical process due to bony block movements, Wilcko and coworkers were the first to suggest that the teeth moved faster due to a biological process, the regional acceleratory phenomenon (RAP). The RAP is a well-established, normal, localized reaction of soft and hard tissues to noxious stimuli (Frost, 1983). Corticotomies injure the bone and the disrupt circulation, increase bone turnover, decrease the amount of bone and decrease bone density, which in turn increases orthodontic tooth movement rates (Fig.2). EVIDENCE FOR THE EFFICACY OF SURGICALLY FACILITATED ORTHODONTICS Four types of questions or procedures and their relative levels of evidence for efficacy in tooth movement are summarized below: 1. There is strong evidence that corticotomies produce faster tooth movement, but the duration of the ef- fect is limited. 2. There is a moderate amount of evidence showing that the amount of surgical insult matters. 3. There is moderate evidence supporting the less inva- sive corticision/piezocision procedures, but contro- versy remains. 4. There is weak and contradictory evidence for the least invasive approaches. These concepts and findings are discussed in greater detail below. 138 Buschang Corticotomies njure Bone/Disrupt Circulation TBone Turnover J Bone Amt J Bone Density Increased Tooth Movement Figure 2. The process by which corticotomies produce the regional acceleratory phenonmenon (RAP) and faster tooth movements. EFFICACY OF CORTICOTOMIES IN ENHANCING TOOTH MOVEMENT RATES Case Reports Numerous case reports support the use of Corticotomies for accelerating tooth movement (Table 2). Such reports demonstrate the ability rotate the teeth within six to eight weeks, distalize premolars Within eight to twelve weeks, procline incisors within eight to twelve Weeks, extrude molars within four to eight weeks, intrude molars in four to Sixteen weeks and decompensate anterior teeth within twenty weeks. Others have reported cases in which corticotomies shorten full treatment times, with most patients completing treatment within eighteen months. While case reports provide an excellent way to describe the treatments of individual patients, they are not a valid for describing expected treatment effects (i.e., the average change expected for a Sample of patients). Since they do not include controls, case reports are subject to bias. Most importantly, case reports do not control inter- individual variation. Due to genetic, environmental and epigenetic differences between patients, the same treatment approach should be expected to produce a wide variety of outcomes. This explains why treatment outcomes (e.g., duration) exhibit a normal distribution of Variation, with some patients completing treatment earlier than others. 139 Surgically Facilitated Orthodontics Table 2. Case reports of patients treated with corticotomy assisted orthodontics. Single Treatment Procedures Patient Treatment Reference Problem Age Duration Rotated teeth N/A Six to eight weeks Kole, 1959 Premolar requiring distalization N/A #:0 twelve Upper incisor proclination N/A Eight to ten weeks * g. * ~ * Ten to twelve Lower incisor proclination N/A weeks Hwang and Lee Intrusion of maxillary first molars 21 y/o ºf Four weeks 2001 †ed mandibular second 20 y/o P Four weeks Moon et al., 2007 Extruded first and second molars 26 y/o Q Two months • §º decompensation for 25 y/o P Five months Kim et al., 2011 Anterior d ion f §º ecompensation for 26 y/o P Five months gº º Intrusion of first and second T d a half Oliveira et al., molars 36 y/o º #. d Extruded maxillary molars 39 y/o (3 Four months Bilateral maxillary posterior Kanno et al., 2007 intrusion 24 y/o P One month º maxillary posterior 21 y/o & Two months Ahn et al., 2014 Unilateral maxillary posterior intrusion 15 y/o Q Four months Full Treatment - - Patient Treatment Reference Problem Age Duration Anholm et al., Narrow arches, unilateral Class || 1986 malocclusion 23 y/o 3 Eleven months Owen, 2001 Mild anterior crowding Adult cº Eight weeks Anterior crowding and posterior Six and a half Wilcko et al., 2001 crossbite g and p 24 y/o (; months • ? * Six and a half Moderate to severe crowding 17 y/o P months §ar et al., Class ll, division 2 with crowding 41 y/o & Eight months jeº et al., Class Ill with crowding 22 y/o P Sixteen months lino et al., 2006 ºcclusion bimaxillary 24 y/o (P Twelve months Aljhani et al., 2011 ºne flared and 22 y/o Q Five months Spena et al., 2007 §ºwth proclined maxillary 18 y/o 9 Eleven months Posterior crossbite; deep bite 21 y/o P Nineteen months Hassan et al., 2010 e • i4 - - - & §ºnd posterior crossbites; 24 y/o Q Eighteen months 140 Buschang For example, Vig and associates (1990) reported that the average treat- ment duration of 236 extraction cases was 31.2 months (Fig. 3). Based on their standard deviations, approximately 38 (16%) of their patients com- pleted treatment in less than 18 months and 6 (2.5%) required less than 5.3 months. These findings demonstrate that a case report based on any one of the subjects in this study certainly would not be representative of the larger sample. Experimental Evidence Because experiments can be controlled well, they make it pos- sible to establish causal relationships and, most importantly, allow the biological effects to be identified. They provide the best way of evaluat- ing whether there are treatment effects and why they occur. It also must be understood that findings from animal models used in experimental Studies evaluating tooth movements may not be translatable to humans. Thus, while rats are excellent models for studying whether effects (e.g., RAP) occur or whether some experimental approach increases tooth movements, due to their size and physiological differences from humans, they are not well suited for translating the basis for orthodontic tooth movements to humans. The best models for studying tooth movements should have bone composition, density, quality and turnover rates that are similar to human bone. The composition (ash weight, hydroxyproline, extractable proteins, IGF-1 and density of dog bone) more closely approximates hu- man bone than does sheep, pig, cow or chicken bone (Aerssens et al., 1998). Cortical and cancellous bone of dogs and humans also has been shown to be similar in terms of water fraction, organic fraction and vola- tile inorganic fraction (Gong et al., 1964). Nevertheless, there are important differences between dogs and humans. Dogs have faster bone turnover rates than humans; the forma- tion rate of trabecular iliac bone of beagles is approximately twice that of humans (Melsen and Mosekilde, 1978). Skeletal remodeling rates of dogs are approximately 42% faster than those of humans (Parfitt et al., 1987). Dog bone also has significantly higher mineral density than human bone (Wang et al., 1998). Despite these differences, the simi- larities in these parameters between dogs and humans have led to the conclusion that with the exception of other primates, dogs have bone that most closely approximates human bone (Aerssens et al., 1998; 141 Surgically Facilitated Orthodontics 2.5% (6) 5.3 18.0 31.2 44.4 57.1 Months Figure 3. The distribution of treatment times of 236 normally treated extraction cases, with 38 and 6 patients completing treatment in less than 18 and 5.3 months, respectively. Based on data reported by Vig et al., 1990. Pearce et al., 2007). Larger animal models (e.g., dogs) also are better than smaller animals for studying the microstructure of cancellous bone (Egermann et al., 2005). The size of the teeth and dentoalveolar structures of dogs more closely approximate those of humans, making the surgical insults produced, as well as the forces and appliances used to move teeth, more comparable. Establishing That Corticotomies Produce RAP Experiments have shown that corticotomies initiate the RAP increase modeling rates, reduce mineral density and create a transient osteopenia (Frost, 1983; Lee et al., 2008; Sebaoun et al., 2008). Bone turnover has been reported to be up to five times higher in long bones adjacent to corticotomy sites than at unoperated control sites (Bogoch et al., 1993). Although the precise relations remain to be established, it is thought that the alveolar response to decortication is a function of time and proximity to the injury site (Sebaoun et al., 2008). Compared to untreated control bone, perforations of the maxillary buccal and lingual cortical plates produced three times as many osteoclasts, three times faster bone apposition, two times less calcified spongiosa and greateſ PDL surface area around the roots of teeth (presumably associated 142 Buschang with decreases in calcified spongiosa; Teixeira et al., 2010). These effects were reported to be localized to the area immediately adjacent to the site of injury. Rats that had orthodontic forces, tissue flaps and perforations of the cortical plates exhibited a greater elevation of cytokines, greater number of osteoclasts and greater bone remodeling than control rats that had orthodontic forces and soft tissue flaps (Teixeira et al., 2010). Experiments also have shown that corticotomies are safe. As one example, Düker (1975) performed vertical buccal and horizontal bicortical osteotomies 5 mm above the maxillary root apices in six beagle dogs and showed that the pulp was vital and the periodontium was healthy after 4 mm of tooth movement. Studies Evaluating Effects of Corticotomies on Tooth Movement The first split-mouth design evaluating the effects of corticotomies on rates of tooth movement was performed on two beagle dogs. Four weeks after the second premolars were extracted, a mucoperiosteal flap was retracted and twelve perforations were made with a round bur into the buccal and lingual cortical plates of the right maxillary and mandibular quadrants. The third premolars were protracted for eight weeks with 150 g nickel titanium (NiTi) coil springs (Cho et al., 2007). Corticotomies produced tooth movement in the maxilla and mandible that were approximately four and two times faster, respectively, than movements of control teeth. While differences between the mandibular experimental and control teeth increased over the entire eight-week observation period, mandibular tooth movement showed no rate differences after six weeks. In another study, the second premolars of twelve adult beagle dogs were extracted and corticotomies were performed around the left third premolars sixteen weeks later. The third premolars then were moved mesially with a 0.5 N NiTi coil spring for four weeks (lino et al., 2007). Overall, the corticotomy side showed approximately twice as much tooth movement as the control side. Tooth movement was significantly faster on the experimental side compared to the sham control side over the first two weeks only; there were no differences in tooth movement rates between weeks two and four. The corticotomy side showed hyalinization of the periodontal ligament only during the first week, while the control side showed evidence of hyalinization throughout the four-week experi- mental period. 143 Surgically Facilitated Orthodontics To appreciate how long the treatment effects might be expected to last, it is important to understand that the RAP is simply an acceleration of normal biological processes. In other words, corticotomies accelerate normal bone healing, which includes three phases (Frost, 1989; Brighton and Hunt, 1991, 1997). The first inflammatory or reactive phase starts with immediate constriction of blood vessels to mitigate bleeding, followed by blood clot formation within a few hours. Whether or not hematomas develop depends on the extent of the injury. During this phase, there also is resorption of bone and the formation of granulation tissue. During the transition from the first to the second reparative phase, cells within the granulation tissue proliferate and differentiate into fibroblasts and chondroblasts. During the reparative phase, there is deposition of woven bone by osteoblasts; the formation of immature lamellar bone begins as soon as the tissues become mineralized. During the last phase, bone is modeled and remodeled into functionally competent mature lamellar bone. The actual rates at which these phases occur depend on the extent of the injury, the stability of the segments and the blood supply. Corticotomies, which are stable, and undisplaced fractures that injure the periosteum and dentoalveolar bone might be expected to heal rapidly. Berglundh and colleagues (2003), who provided some of the best estimates of healing time for dentoalveolar bone, showed that the first two phases should be completed within twelve weeks (Fig. 4). Prospective Human Trials Prospective clinical trials are necessary to determine how much faster human tooth movement is with corticotomies. Fischer (2007) performed a split-mouth design using six consecutively treated patients with partially impacted canines and showed that the corticotomy side required 28 to 33% less treatment time than the side where conventional surgical techniques (11.5 months versus 16.6 months) were used, with no side differences in the final periodontal conditions. Lee and associates (2007) compared 29 adult female patients who had conventional orthodontic treatment to 20 individuals who had corticotomy-assisted orthodontics in the maxilla. The corticotomy-assisted group completed treatment eight months faster than the conventional orthodontic group (19 + 6 months versus 27 it 7 months). However, the sample size and pre-treatment morphological differences (50% of the measures 144 Buschang • 7-14 days for bone resorption and "...” the formation of granulation tissue • 12 weeks for woven bone and Reparative - - Phase immature lamellar bone formation • Months to years for the formation Remodeling Phase of mature lamellar bone Figure 4. The three phases of normal bone healing. Adapted from Berglundh et al., 2003. Were not comparable) suggest that the groups initially were not equivalent. Four years after the Fischer study, Aboul-Ela and coworkers (2011) performed a split-mouth clinical study evaluating the effects of Corticotomies in thirteen adult Class II, division 1 malocclusion extraction Cases. After leveling and alignment, 8 mm-long miniscrew implants were placed, the first maxillary premolars were extracted, a full-thickness mucoperiosteal flap was raised and perforations were made extending from the lateral incisors to the first premolar area. On the side with perforations, rates of canine retraction increased for approximately 1.3 months and then decreased (Fig. 5). On the control side, rates increased rapidly, albeitless rapidly than on the experimental side, for approximately 1.2 months and then much more slowly for the next three months. The Corticotomy side exhibited significantly faster rates of tooth movement during the first three months only. MAGNITUDE OF SURGICAL INSULT AFFECTS RATES OF TOOTH MOVEMENT As stated above, currently there is a moderate amount of evidence showing that the amount of surgical insult determines the rates of orthodontic tooth movement. It is important to understand that the RAP effect on tooth movement depends on the extent of the injury; the 145 Surgically Facilitated Orthodontics 2.5 –Control —Experimental 1. 1. 0.5 Months Figure 5. Comparisons of maxillary canine movements rates on the experimental corticotomy and control sides of humans. Data from Aboul-Ela et al., 2011. greater the injury, the greater the tooth movement. However, the amount of surgical insult does not extend the duration of the RAP probably because cortictomies produce only limited amounts of injury. Mostafa and colleagues (2009) were among the first to demonstrate this relationship by showing that corticotomies performed along with extractions produced greater tooth movements than extractions alone. They extracted the maxillary second premolars of six dogs, immediately raised a full-thickness mucoperiosteal flap and then performed eight to ten buccal perforations on the right side only. The first premolars then were distalized using 400 g of force using miniscrews for skeletal anchorage. During the four-week experimental period, the teeth on the corticotomy/extraction side moved approximately twice as far (2.3 versus 4.7 mm) as the premolars on the extraction side. Differences in rates of tooth movements between the experimental and control teeth, which were statistically significant after only one week, decreased during the first four weeks, such that there were no differences in rates of tooth movements after the fourth week. Their histomorphometric analyses showed that bone remodeling was more active and extensive on the corticotomy side than on the sham control side. Using five foxhounds, Sanjideh and colleagues (2009) performed a split-mouth study to evaluate more closely the effects of extractions versus extractions plus corticotomies on rates of tooth movements. The mandibular second premolars and mandibular first premolars were ex- tracted, followed immediately by flap surgeries and corticotomies on the 146 Buschang buccal and lingual surfaces around the mandibular second premolars on one randomly chosen side. A 200 g Niſi coil spring was used to provide a constant mesial force on the teeth for eight weeks. There was approximately twice as much tooth movement on the experimental side (2.4 mm) compared with the sham control side (1.3 mm). Rates of tooth movement accelerated until days 22 and 25 on the experimental and control sides, respectively, and then decelerated with no significant rate differences between sides after seven weeks (Fig. 6). Sanjideh and associates (2009) also showed that performing a second maxillary corticotomy four weeks after the first corticotomy increased tooth movements only by 15%. Interestingly, mandibular tooth movement was greater than maxillary tooth movement, which was unexpected given the differences in bone density. They explained the difference by noting that the maxilla only had buccal side corticotomies, while the mandible received buccal and lingual corticotomies, which probably produced a greater RAP effect. Cohen and coworkers (2010) performed an experiment purposefully designed to evaluate the effects of increased surgical trauma on tooth movement. On both sides, the maxillary first premolar was elevated and extracted. On the RAP side, the interseptal bone mesial to the second premolar was undermined with a fissue bur by cutting vertical grooves inside the extraction socket along the buccal and lingual sides and by cutting oblique grooves toward the base of the interseptal bone. On the RAP+ side, a full thickness gingival flap was raised, the buccal plate between the second premolars and canines was removed and a vertical osteotomy extending to, but not through the lingual cortex was performed. The osteotomy was connected to the extraction site by a horizontal corticotomy 1 to 2 mm deep, performed 3 to 5 mm apical to the Second premolar. The second premolars were protracted 0.5 mm/day for fifteen days using a custom jack-screw distractor. After 28 days, the Second premolars on the RAP+ side showed significantly more movement than those on the RAP side (2.9 versus 1.8 mm). A recent follow-up study by McBride and colleagues (2014) evaluated the dentoalvolear bone on the RAP and RAP+ sides after seven weeks of consolidation. Micro-CT analyses showed significantly less bone on the RAP+ side than on the RAP side (Fig. 7). The bone on the RAP+ side also was less dense and less mature than the bone on the 147 Surgically Facilitated Orthodontics —Control —Experimental Weeks Figure 6. Comparisons oftooth movements rates on the experimental corticotomy and control sides for the mandibular second premolars. Data reported by Sanjideh et al., 2010. RAP RAP+ Mesal Distal Buccal Lingual Mesial Radicular | Distal Radicult - A 700 750 800 850 900 B Figure 7. A. Histogram of material densities (mg HA/cm3) of bone surrounding the maxillary second premolar. B: Sites and probabilities. Data from McBride et al., 2014. RAP side. Histology showed greater numbers of osteoclasts and more osteoclasts per unit area on the RAP+ side. As detailed in the findings below, currently there is moderate evidence supporting the less invasive corticision/piezocision procedures on the rates of tooth movement; nevertheless, some controversy remains 148 Buschang about these findings. Once investigators realized that corticotomies produced faster tooth movement, they tried to produce the RAP without flaps, which often are not accepted well by the patients, increase post- operative discomfort and elevate the risk of crestal bone loss. Kim and associates (2009) introduced corticision as a minimally invasive dentoalveolar surgical procedure to accelerate tooth movement. Using Cats as the animal model, they either applied orthodontic forces in the sham control group or orthodontic forces plus corticision in the experimental group. Corticision was performed on the mesio-buccal, disto-buccal and disto-palatal aspects of the maxillary canines. A reinforced surgical blade was positioned in the interradicular-attached gingiva and malleted into the bone marrow (depth was not specified); Corticision was repeated every three days throughout the experimental period. The canines were retracted with 100 g Sentalloy coil springs for 28 days. Bone apposition mesial to the retracted canines (e.g., on the tension side) was 3.5 times greater in the corticision group than in the Sham Control group. The same investigators also evaluated the effects of Corticision in beagle dogs (Kim et al., 2009) in which the corticision was performed by matting the surgical blade up to 10 mm into bone. Compared to the sham control group, the corticision group showed 3.75 times more maxillary second premolar movement. Bony apposition behind the trailing root was almost three times greater in the corticision than in the sham control group. The same group evaluated a less invasive procedure, which used a piezosurgical instrument to puncture the bone to a depth of 3 mm (Kim et al., 2013). They allocated ten beagles into either a sham Control or piezopuncture group. A total of sixteen piezopunctures were performed on the mesio-buccal, disto-buccal and mesio-lingual and disto-lingual aspects of the second premolars. The second premolars in both arches were moved using an orthodontic force of 100 g. The results showed significantly more tooth movements (> twice as much) in the piezopuncture group after one week in the maxilla and after two weeks in the mandible. However, group differences in rates of tooth movement did not extend beyond the third week. Dibart and colleagues (2014) introduced piezocision, which combined incisions to the buccal gingiva and piezoelectric knife cuts to decorticate the alveolar bone. Using rats as their experimental model, they inserted a Piezotome 0.5 mm deep mesial and distal to the maxillary first molars. The first molars 149 Surgically Facilitated Orthodontics then were moved mesially with 25 g coil spring. Their results indicated that decreases in bone content were initiated earlier in the piezocision group than in the control group. Not all Studies have found faster tooth movements with Corti- cision/piezocision. Murphy and associates (2014) evaluated the effects of corticison on tooth movements in rats. They delivered both light (10 g) and heavy (100 g) forces between the maxillary first molars and inci- sors. Corticision was performed at appliance placement and one week later. They reported that corticision had no effect on tooth movements. Ruso and coworkers (2014) evaluated buccal tooth movements with and without flapless alveolar decortication in dogs using a split-mouth design. They performed 5 mm deep piezosurgery on the mesial and distal aspects as well as in the furcation of the second premolar root. The second pre- molars were moved buccally for seven weeks. They showed significantly (20%) more premolar movement on the experimental side than control side. The experimental teeth also tipped more than the control teeth. De- hiscences were evident on both the experimental and control sides (Fig. 8). Micro-CT analyses showed less mature bone, but significantly more. new bone formation around the experimental premolars than the control premolars. Current findings show weak or contradictory evidence of the effects of the least invasive approaches on rates of tooth movement. This conclusion is based on the findings of several studies that have sought to determine whether tooth movement rates increase using flapless procedures that rely on a limited number of perforations into bone, often penetrating only 2 to 3 mm. These procedures are based on an experimental study performed by Teixeira and coworkers (2010) who divided 48 adult rats into four groups: control, tooth movement only and two flap surgery groups. One of the two flap surgery groups that had soft tissue flaps raised around the first molars also received three perforations with a round bur, 0.25 mm deep and 0.25 mm in diameter, approximately 5 mm mesial to the left first molars. They reported significantly faster tooth movements, greater osteopenia and the greater cytokine levels in the group that received the perforations than in the other three groups. A subsequent study by a different group used a split-mouth design in a canine model, in which 25 holes were drilled, 2 mm deep, 150 Buschang Control Experimental Figure 8. Buccal view of 3D micro-CT reconstructions showing dehiscences after eleven weeks of buccal tooth movements on the control side (orthodontics only) and experimental (orthodontics plus flapless piezosurgery). From Ruso et al., 2014. Without flaps on one randomly chosen side of the mouth (Safavi et al., 2012). The second premolars were moved with 150 g of force on both Sides. The results showed significantly faster tooth movements during the first month on the side with osteoperforations, no difference during the Second month and significantly slower movement during the third month. Overall, there were no significant differences in tooth movement between sides. Swapp and colleagues (2015) performed a split-mouth design * Seven foxhounds. On one randomly chosen side, they produced 60 151 Surgically Facilitated Orthodontics awl injuries (without flaps) into the buccal and lingual cortices around the tooth being moved, often penetrating 2 to 3 mm. The awl alone produced approximately 21 mm” of injury and approximately 93 mm” of injury if microfractured bone was included. The extent of cortical bone injury was similar to that produced in previous corticotomy studies showing twice as much tooth movement on the experimental side compared with the control side (Cho et al., 2007; lino et al., 2007; Sanjideh et al., 2009). Despite the fact that the control bone had been damaged substantially, there were no significant differences in tooth movement between sides because the effect of the RAP did not extend down to the bone through which the roots that were moved. The limited extent of the RAP emphasizes the important role of the periosteal blood supply; the periosteum is the primary supplier of blood to the cortical bone. In long bones, it supplies 70 to 80% of the arterial blood, 100% of the venous return and 20 to 30% of the medullary blood supply (Chanavez, 1995). While percentages of blood supply are not available for facial bones, the greater number of periosteal than centromedullary vessels indicates that the periosteum is also the primary supplier of blood to the mandible (Saka et al., 2002). Yaffe and associates (1994) showed dramatic bone resorption after mucoperiosteal flap surgery alone on both mandibular cortical and medullary bone. A recent split-mouth canine model study showed that flap surgery alone increases tooth movement approximately 30%, due to a reduction in the amount of medullary bone mesial to the tooth being moved (Owens, 2014). The implication is that elevation of the flap decreases circulation to the medullary bone, which in turn decreases bone and increases tooth movementS. Only one study has attempted to verify the clinical application of osteoperforations (Alikhani et al., 2013). Twenty adult Class II, divi- sion 1 patients were allocated randomly to either an osteoperforation or control group. The arches were leveled and aligned and then the first premolars were extracted. Six months after the extractions, the canines were retracted with 100 g NiTi springs. In the osteoperforation group, three perforations (1.5 mm wide and 2 to 3 mm deep) were made distal to the canines using a Propel (Ossining, NY) device. Tooth movement, which was measured from casts before and after 28 days of canine re- traction, were significantly greater in the group that received osteoper- forations than in the control group. Unexpectedly, the rates of canine 152 Buschang retraction reported for the osteoperforation group closely compares with rates of canine retraction previously reported for patients treated with conventional orthodontics, while the rates of the control group were substantially less than previously reported (Table 3). Finally, there is limited evidence for the effects of the most invasive approaches on rates of tooth movement as summarized here. Several clinicians also have tried to move teeth at much faster rates than can be obtained with corticortomies. Instead of moving teeth at 2 mm/month, they have sought to move teeth at rates approximating 1 mm/day. Liou and coworkers (1998) introduced periosteal distraction, based on the notion that the periodontal ligament serves as a growth site between the alveolar bone and the teeth. After first premolar extractions, the interseptal bone distal to the canines was removed with vertical and oblique undermining grooves, which was thought to "weaken its resistance.” A distraction device then was used to move the canine at rates of 0.5 to 1 mm/day. Using this procedure, they retracted the canines of fifteen patients 6.5 mm within three weeks and reported no adverse periodontal defects and minimal root resorption. Realizing the need for experimental validation, Ai and coworkers (2008) evaluated periosteal distraction in dogs using the same procedures. They activated the appliance 7 mm, but obtained only 3.7 mm of canine retraction, which was less than expected, but more than the 1.2 mm of retraction obtained on the control side using power chains (100 g). Again, no evidence of root resorption was reported. Kişnisci and associates (2002) introduced a more invasive procedure called dentoalveolar distraction osteogenesis to retract maxillary canines. Using eleven patients, they performed vertical and horizontal osteotomies around the canines, then extracted the premolars and removed the buccal bone over the extraction site, as well as any other bone that could interfere with canine retraction. After the transport segment, which included the buccal cortex and underlying medullary bone surrounding the canine had been mobilized, dentoalveolar distraction was initiated immediately and continued at a rate of 0.8 mm/day until the canines made contact with the second premolar. Contact occurred after eight to twelve days, with no evidence of tooth discoloration or radiographic evidence of vitality loss. Three years later, the same investigators used the same procedures, this time with a three- day latency period between appliance activations, to retract the canines of 153 Surgically Facilitated Orthodontics Table 3. Published rates of canine retraction (mm/mo.) with description of how these evaluations were performed. REFERENCES N RATE EVALUATION Huffman and Way, 1983 25 1.2-1.4 Clinical Yamasaki et al., 1984 8 1.3 Clinical Ziegler and Ingervall, 1989 21 1.3 Clinical Daskalogiannakis and McLachlan, 1996 6 1.2 Models Lotzof et al., 1996 12 2.3 Models Iwasaki et al., 2000 7 0.9–1.3 Models Hayashi et al., 2004 4 1.8 Models Herman et al., 2006 14 1.3 Models Boester and Johnston, 1974 10 1.0 Radiographs Tanne et al., 1995 10 2.4 n/a Lee, 1995 7 2.2 Clinical Hasler et al., 1997 22 0.9 Models Martins et al., 2009 10 1.1-2.1 Radiographs Average • 1.5 subjects 13 to 26 years of age fully. Retraction was completed in 10.4 + 1.9 days, with no changes in the width of attached gingiva during their twelve-month follow-up (Gürgan et al., 2005; Iseri et al., 2005). Sukurica and associates (2007) used the procedures to retract the canines of eight patients 3 to 8 mm in 12 to 28 days. While the periodontal evaluations were within normal limits and there were no clinical signs of discoloration, only seven of the twenty teeth showed electrical vitality six months after distraction. Finally, Kharkar and Kostrashetti (2010) used these procedures to retract twenty canines 6.5 mm in 12.5 + 0.5 days, with approximately 11° of tipping, minimal anchorage loss and no evidence of root resorption. Unfortunately, there are limited numbers of experimental studies evaluating these procedures. A pair of studies has evaluated the effects of latency and rates of tooth movement on the quality and quantity of bone produced by dentoalvolar transport osteogenesis (Moore et al., 2011; Spencer et al., 2011). Moore and coworkers' study showed that with the exception of slight and expected differences in maturation, 154 Buschang a five-day latency had no effect on the regenerate bone produced. Spencer and colleagues' study showed that the quantity or quality bone produced was not different significantly when comparing teeth distracted at rates of 1 mm/day versus 2 mm/day. Both studies showed that dentoalveolar transport osteogenesis produced bone of adequate quantify and quality for dental implant restorations (Fig. 9). SUMMARY AND CONCLUSIONS Surgical intervention presently provides the greatest hope for Substantially shortening treatment times; however, more experimental Studies are needed to understand and eventually be able to control the biological events that occur following these procedures. Experiments provide the biological principles upon which meaningful clinical studies can be designed. It is important particularly to consider both the least and most invasive procedures for these purposes. Much remains to be known about the minimally invasive flapless procedures before they can be considered efficient and effective for accelerating tooth movements. For example, it remains unknown how many perforations are needed or how deep they should extend into medullary bone for maximum effect. It also is unclear how far the effects of the RAP extend from the perforation sites. Furthermore, it presently is unclear whether accelerated tooth movements can be maintained if additional perforations are performed at some later time and how often they should be performed. The most invasive procedures clearly hold the greatest potential for faster tooth movements, but they also hold the greatest risks. More experimental studies are needed to assess and understand the risks and limitations of these procedures. For example, it remains unknown how fast the extraction space fills with tissues that could limit movement of the transport segment, or how much bone needs to be removed. It also is unclear how optimally to create the interface between the transport Segment and bone toward which it is being moved. 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Am J Orthod Dentofacial Orthop 1989;95(2):99-106. 165 TIMING OF POST-OPERATIVE ORTHODONTIC INTERVENTION IN INTERDISCIPLINARY APPROACHES FOR OVERCOMING ALVEOLAR DEFECTS Sujung Kim and Youngguk Park ABSTRACT Differential healing states of the surgical alveolar defect have been assumed to affect the timing of post-operative orthodontic intervention. The in vivo studies were designed to: 1) elucidate the effect of timing of force application on the rates of tooth movement and defect regeneration; and 2) investigate differential gene expression pattern in the tissue regenerated in the defect over time with or Without Orthodontic tooth movement. The beagle model used involved a critical-sized defect including the extracted socket of a maxillary first premolar and protraction of the second premolar into the defect. The animals were allocated into a control group, a defect-only group, a bone-grafted defect group and a laser-irradiated grafted group. Force application time points were established at immediate, two weeks and twelve weeks after surgery. For the intergroup comparison, the weekly rate of tooth movement, micro-CT quantitative analysis, triple-fluorochrome staining and microarray gene screening were performed. The findings suggest that tooth movement into the surgical gap in this animal model can be favorably undertaken at the two-week time period. We also show that immediate traction could be permissible following bone grafting; however, a longer duration of effective tooth movement was observed even with delayed force initiation after surgery. In contrast, low-level laser therapy (LLLT), inhibited tooth movement possibly due to accelerated defect healing. Based on the differential gene expression pattern between the natural and tooth movement-accompanied healing, we concluded that optimal timing of post- Surgical force application is dependent on sustaining the woven bone period. This study provides a basis to elucidate biological modulators controlling the formation of woven bone state to achieve optimal tooth movement. KEY WORDS: orthodontic tooth movement, surgical alveolar defect, healing progress, woven bone 167 Overcoming Alveolar Defects INTRODUCTION Surgically assisted orthodontic treatment has been studied and applied as an adjunctive procedure in accelerating the rate of orthodontic tooth movement (OTM) and reducing total treatment time (Long et al., 2013; Gkantidis et al., 2014; Hoogeveen et al., 2014; Liem et al., 2015). Because of concerns over the invasiveness and associated morbidity of these surgical procedures has led to the consideration of non-invasive biostimulation methods for this purpose in recent years (Andrade et al., 2014; Jawad et al., 2014; Ge et al., 2015; Kalemaj et al., 2015). Nevertheless, surgically assisted orthodontics remains an indispensable procedure in overcoming the physical limitations of periodontal Supporting tissue such as a thin buccolingual Cortex, highly resistant bone with poor cancellous/cortical ratio and atrophic alveolar ridges (Mimura, 2013; Uribe et al., 2013). It is not uncommon for unexpected cessation or retardation of tooth movement to occur during orthodontic treatment, when an interdisciplinary approach with strategic intervention between the surgeon and orthodontist may be considered a viable option. When such a surgical intervention is undertaken to improve the quality and quantity of the bone matrix, and where appropriate based on the surgical procedure, the post-operative orthodontic protocol should be optimized to take advantage of this surgical intervention to enhance OTM. Among various surgical modalities including osteotomy, corticotomy and minimally invasive cortical activation (Fig. 1), segmental alveolar osteotomy may be considered to correct an ankylosed tooth (Rodrigues et al., 2014), retract anterior teeth en masse with poor periodontium (Kim et al., 2015) or close a severely constricted extraction space (Öncü et al., 2015). An example of this is shown in the case of an elderly patient with high bone density and low cellular activity (Fig. 2), for whom anterior segmental osteotomy (ASO) could be a good option for immediate correction of anterior dentoalveolus, to prevent possible problems such as root or alveolar bone resorption secondary to extended conventional tooth movement. The surgical defect created by segmental alveolar osteotomy is reported to have a different healing pattern biologically and physically from the defect by corticotomy or cortical activation (Wang et al., 2009). The surgical gap with complete loss of integrity from cortex to cancellous and marrow created with this 168 Kim and Park • Alveolar / PDL distraction osteogenesis • Individual tooth osteotomy - Block osteotomy Corticotomy-facilitated orthodontics - Individual / Bock corticotomy - Selective alveolar decortication - Periodontally accelerated osteogenic orthodontics | Minimally-invasive cortical activation | _/ • Corticision • Piezocision - Piezopuncture Figure 1. Categorization of alveolar surgery assisting orthodontic treatment, according to surgical insult. procedure might undergo a fracture-like wound healing process in addition to a regular regional acceleratory phenomenon (RAP; Teng et al., 2014). Considering that the regenerated tissue would provide a matrix for subsequent movement of the adjacent teeth, comprehension of the Specific healing mechanism occurring in the osteotomy gap is required for establishing an orthodontic protocol. To determine the timing of force initiation after ASO, biological evidence related to the time course healing progress of the surgical gap is required. Nevertheless, the decision of timing depends on clinical criteria due to the lack of biological evidence. When the distance from the Osteotomy gap to the adjacent root surface is approximately more than 1.5 to 2.0 mm radiographically or the direction of root movement is not directly into the defect, immediate force application might be acceptable regardless of surgical injury (Tuncer et al., 2008). This is based on the Premise that mechanically induced PDL and alveolar modeling activity ſequires a time of approximately two to three weeks to recover from tissue hyalinization (von Böhl and Kuijpers-Jagtman, 2009). Moreover, Surgically induced local alveolar osteopenia may occur, leading to a 169 Overcoming Alveolar Defects Figure 2. A 51-year-old female patient received a lower anterior segmental osteotomy (ASO) prior to orthodontic tooth movement. A and C: Initial records. B and E. Two weeks after surgery. D: At the time of surgery, revealing the sectioned bone surface with high density. She had a skeletal Class III malocclusion, but desired camouflage treatment only. Due to her poor cancellous/cortical ratio and thin Symphysis (indicated by arrow in D), ASO was performed to close the lower extraction space and correct the anterior crossbite. synergistic environment for accelerating tooth movement around the defect (Huang et al., 2014). In addition, soft tissue wound healing may indicate whether the passive segmental wire could be changed to an active continuous wire after surgery. Given that soft tissue wound healing is crucial for underlying hard tissue healing (Williams et al., 2014) and healing can be disturbed by immediate force toward an open wound (Nemcovsky et al., 2007), force application around two weeks after surgery is recommended in most circumstances (Fig. 3); howeveſ, prolonged intervention by one to two weeks may be considered if necessary. On the other hand, a delay in force application for more than three months after surgery may inhibit OTM into the surgical defect in 170 Kim and Park Figure 3. Intra-oral photographs showing a surgical defect area taken immediately (A), one week (B) and two weeks (C) after an ASO. A. The osteotomy gap between the canine and second premolar is seen before soft tissue suturing. B; Incompletely closed soft tissue wound right after suture removal at one-week post-operative. Sectional passive wire still is maintained. C. Complete soft tissue healing is seen at two weeks post-operative, when a continuous active wire was inserted to initiate OTM toward the defect. - º º º E. F. ºº - - - - - - Figure 4. Micro-computed tomography (micro-CT; A-D) and histologic images (E- H) demonstrating the time-course of healing of the surgical alveolar defect in beagles. A and E: Immediately after surgery with coagulum in the fresh wound. B and F. Two weeks after surgery with a high fraction of woven bone under the Completely recovered crestal bone bridge with soft tissue integrity. C and G: Twelve weeks after surgery showing non-union of the bony defect, D and H. Twelve weeks after surgery with hyper-matured lamellar bone. Reprinted with Permission from Ahn et al., 2014. Cases of poor healing (e.g., epithelial or cortical invagination, non-union of the gap or hyper-maturation; Fig. 4). In circumstances when unfavorable defect healing seems to in- terrupt tooth movement, a bone graft may be considered to promote healing (Fig. 5). Although studies have suggested novel graft materials and techniques from the periodontal perspective (Zhang et al., 2010; 171 Overcoming Alveolar Defects Figure 5. Alveolar decortication (A) accompanied by bone grafting (B) and Bio- OSs collagen (TM, Geistlich, German) and collagen membrane (C) for enhancing tooth movement into the atrophic alveolar ridge. The protraction of the lower left first molar into the grafted area was initiated three weeks after surgery (D-E) but the timing of intervention was not based on biologic evidence. Horowitz et al., 2012), there is little data reporting the effect of an alveolar graft on the rate of tooth movement. Early tooth movement into a grafted site may cause root resorption or graft failure by collision between the particles and the root, whereas late force application may inhibit the movement associated with graft maturation or healing residues. Nevertheless, the post-graft orthodontic time-points have not been evaluated specifically in terms of the timing for initiating tooth movement to achieve optimal rates of tooth movement while minimizing adverse consequences (Reichert et al., 2010). Amit and colleagues (2012) suggested immediate force application toward bone-grafted buccal cortices in periodontally accelerated osteogenic orthodontics; however, there was lack of biologic evidence. Araújo and coworkers (2001) showed that there was no significant difference in the rate of tooth movement and the amount of root resorption between the bone- grafted and non-grafted alveolar defects; however, the tooth movement was initiated at three months post surgery, when maturation of the 172 Kim and Park defect is relatively advanced. Zhang and associates (2011) inferred that tooth movement earlier than four weeks after grafting is reasonable, based on their experiment which showed the fastest tooth movement in the most poorly regenerated defect and the slowest in the favorably regenerated defect when the force was initiated at eight weeks post surgery. To enhance orthodontic movement rate simultaneously with successful defect regeneration after graft implantation, the healing-based time points need to be investigated. In addition to surgical intervention to regenerate periodontal tissues, biostimulation may be a good tool for controlling the healing rate. Thus, for example, low-level laser therapy (LLLT) has been applied to enhance the healing potential of a surgical wound (Pinheiro and Gerbi, 2006) or grafted defects (Gergi et al., 2005; Weber et al., 2006; de Almeida et al., 2014). The effects include the stimulation of angiogenesis, earlier onset of inflammation, increased turnover of collagen, greater Synthesis of growth factors, osteosynthesis by activated bone cells and improvement in bone strength in relation to hyper-mineralization (Nicola et al., 2003; Pinheiro et al., 2003; Kim et al., 2009a). Some of the previous Studies, however, investigated these acceleratory effects on long bones (Weber et al., 2006) or calvaria (de Almeida et al., 2014), which have different biologic properties from alveolar bone surrounding the teeth. Considering that several studies have also reported that LLLT affects the rate of OTM itself regardless of alveolar surgery (Altan et al., 2012; Torri et al., 2013; Jawad et al., 2014), it currently is not known whether LLLT would accelerate the rate of OTM into the grafted defect by stimulating paradental remodeling activities or decelerate it by primarily stimulating defect maturation and mineralization. On the assumption that differential healing states of the surgical alveolar defect might be a key determinant for the optimal timing of post-operative OTM, we undertook two in vivo studies with the goals of: 1) elucidating the effect of timing of force application on the rates of tooth movement and defect regeneration in relation to different healing States; and 2) determining differential gene expression patterns in the regenerate tissue of the surgical defect with and without OTM using microarray analysis. 173 Overcoming Alveolar Defects PART I: TIMING OF TOOTH MOVEMENT INTO SURGICAL DEFECTS |N DIFFERENT HEALING STATES Objective The objective was to compare the rates of tooth movement between different post-surgical healing conditions with the goal of identifying time-points that offer optimal tooth movement rates combined with favorable defect regeneration. The variables for intergroup comparisons included bone graft, laser biostimulation and timing of post- Surgical force application. Materials and Methods Sixteen beagles were allocated randomly into three groups: defect only, O group; defect with graft, OG group; and defect with graft and laser, OGL group (Fig. 6). Each group was subdivided into immediate traction group, two-week group and twelve-week traction group according to the timing of force application following surgery. The twelve-week time point was excluded in the OGL group because excessive bone maturation was found with laser irradiation just two weeks into the preliminary study. Critical-sized defects of 5x5x7 mm (mesiodistal, buccolingual, vertical) were prepared including the extracted socket of the maxillary first premolar approximately 1.5 to 2.0 mm apart from the mesial root surface of the second premolar (Fig. 6A). The defect was filled with a 1:1 mixture of deproteinized bovine bone matrix (Bio-Oss"; Geistlich Sons Ltd., Wolhusen, Switzerland) and demineralized bone matrix (OrthoBlastll; Isoſis, Irvine, CA; Fig. 6B). In the OGL group, a GaAIAS diode laser (Laser HandTM; MM Optics Ltda, São Carlos, Brazil) with a wavelength of 780 nm was applied at six points around the defect every other day for two weeks after surgery (Fig. 60). The power intensity was 1.75 W/cm”, energy dose was 5J/cm” and the total daily dose was 30J/ cm”. The maxillary second premolar was protracted into the defect for six weeks using a 0.019" x 0.025" stainless steel sectional wire and nickel titanium (NiTi) coil spring activated by 100 gm of force in all groups (Fig. 6D). The magnitude of OTM was measured on stone models every week and calculated as the difference of the distances measured from the mesial cervical margin of the second premolar to the mesial cervical 174 Kim and Park Figure 6. Photographs of experimental procedures. A: Preparation of four-wall, Cuboidal-shaped, critical-sized defect mesial to the maxillary second premolar. B: Bone graft into the defect with a mixture of Bio-Oss" and DBM. C. Application of LLLſ around the grafted defect. D: Orthodontic protraction of a second premolar into the defect using a canine as an anchorage. Reprinted with permission from Kim et al., 2015. margin of the first molar before and after tooth movement. Micro-CT images were obtained to determine the type of tooth movement into the defects and analyze quantitatively maturational status of the defect using SkyScan 1173 (Skyscan N.V., Kontich, Belgium). Triple-fluorochrome Staining was performed to determine the anabolic-modeling rate on the tension side of the moved tooth. Results The weekly rate of tooth movement was the greatest in two §ſoups: the non-grafted group in which the traction was initiated two Weeks post surgery (O-2) and the grafted group in which the traction was initiated immediately following surgery (OG-0; Fig. 7). Initiation of traction twelve weeks after surgery resulted in the slowest tooth movement in the non-grafted group (O-12), while the rate was slightly greater in the twelve-week grafted group (OG-12), but which still was lower 175 Overcoming Alveolar Defects 4.00 £ 3.50 5. # E 3.00 º -> 3. = 2.50 --O-0 3 -----O-2 - ---O-12 § 2.00 --OG-0 # -----OG-2 º ---OG-12 3 1.50 --OGL-0 # -----OGL-2 # E 1.00 O º º 5 § 0.50 º 0.00 1 2 3 4. 5 6 (week) Figure 7. Mean accumulated distance of tooth movement over six weeks. Red lines = non-grafted groups; blue lines = grafted groups; green lines = laser- irradiated grafted groups. Reprinted with permission from Kim et al., 2015. than the groups with earlier traction. Micro-CT images confirmed that the teeth moved mostly by translation in O-2 and OG-0 groups consistent with favorable defect healing, while the tooth was rotated in place in the O-12 group showing a distorted tooth image with bony discontinuity (Fig. 8). Interestingly, in the laser-irradiated groups, the rates of tooth movement were significantly reduced both in the immediate (OGL-0) or early traction (OGL-2) groups relative to their non-grafted (O-0 and O-2) and grafted (OG-0 and OG-2) counterparts and were comparable to the level of O-12 group. Triple-fluorochrome staining to visualize the rate of alveolar modeling on the tension side of the moved tooth demonstrated the greatest apposition rate in O-2 and OG-0 groups, which paralleled the findings on the rate of tooth movement (Fig. 9). The O-12 group showed no such gaps between the color bands, which corresponded to the IoWest 176 Kim and Park Figure 8. Micro-CT images representing the type of tooth movement associated With defect maturation. O = non-grafted groups; OG = grafted groups; OGL = laser-irradiated groups. Reprinted with permission from Kim et al., 2015. ſate of tooth movement. The laser-irradiated groups showed thicker and more diffuse color bands with little spacing between the dyes, which Correspond to limited tooth movement in this group. Interestingly, active intra-cancellous osteonal remodeling rather than surface cortical modeling was seen in this group. In the histological findings, the O-2 and OG-0 groups revealed the defect still was filled with a high fraction of woven bone lined by active bone forming cells under an intact crestal bone bridge (Fig. 10). In Contrast, poorly repaired defects with low quantity and quality of matured bone were observed in the O-12 group, whereas the vertical bone ſegeneration to the marginal level was found in the OG-12 group. In the 177 Overcoming Alveolar Defects Group O - Group OG Group OGL O-0 O-2 O-12 OG-0 OG-2 OG-12. OGL-0 OGL-12 Figure 9. Microphotographs of triple-fluorochrome staining on the tension side of the moved teeth. The yellow dye represents mineralizing front one day prior to initiating tooth movement, while the red and green dyes represent the same parameter at three and six weeks, respectively, after initiating tooth movement. Reprinted with permission from Ahn et al., 2014. Kim and Park irradiated groups, the defects were full of highly dense lamellar boneconsistent with the slow tooth movement. Also notably in the OGL- 2 group, root resorption was detected on the mesial surface of tipped tooth, supposedly in relation to resistance offered by accelerated bone maturation by laser biostimulation. These descriptive findings on the tissue composition and maturity in the defect area were confirmed quantitatively by micro-CT morphometric analysis (Fig. 11). DiSCuSSion The present study elucidated that the timing of post-surgical orthodontic intervention depended on the healing states of alveolar defect. Based on our experimental protocols, two-week post-operative traction was recommended when tooth movement was planned into the Surgical gap. On the other hand, immediate traction could be a viable option when the defect is implanted with resorbable allograft and/ or xenograft. It is an interesting finding that LLLT inhibited OTM into the defect, even with immediate traction, presubmably by stimulating healing and maturation of the defect with an increase of bone density evident within two weeks post surgery. As a modulator of defect healing, a bone graft appears to extend the duration of woven bone—a favorable matrix for OTM-during, while laser irradiation curtailed this phase of regenerative bone by earlier transition into lamellar bone. As a key intrinsic modulator of bone healing, woven bone formation is of great importance for both tissue regeneration and Subsequent OTM, especially in the case of a critical sized defect (CSD; Cardaropoli et al., 2005). Woven bone is formed from osteoid tissue by mineralization, which is known to occur in a dual mode: appositional formation from the existing defect walls mediated by osteoclast- osteoblast coupling process; and de novo formation from the center of the defect regardless of initial osteoclast function (Williams et al., 2014). In this context, the main causative factor of non-union in the O-12 group— where the defect was left undisturbed for twelve weeks—might be a failure of de novo bone formation in the CSD. In contrast, the activated {- Figure 10. Descriptive histologic findings on the defect healing pattern in each group. O = non-grafted groups; OG = grafted groups; OGL = laser-irradiated groups. Arrow indicates the resorbed root surface. H&E staining, original magnification of x20. Reprinted with permission from Kim et al., 2015. 179 Overcoming Alveolar Defects | Immediate raciongroup |Two-week traction group Bone mineral density (g/cm3) --- --- *… - I --~ - 1:… - - … - --~~ tº c oz os-2 ost-2 ºc--- B - Percent bonevolume (%) | & - I I I - I f I I 5- - -- 2: . - - - - - olo co-o cºlo c. o.2 oc-2 ocu-2 Control (C) D Defect (O) - Defectºgraft (OG) --- - Defectraraftºulſ (OGL) - --- --- -- º- OG-0 - - oc--0 F c º-2 Cº-2 Cºl.-- Trabecular number(mm) I T - - - - | T 2 . - - - - - - - - - --- Cº-º ºc--0 H c O-2 oG-2 OcL-2 Figure 11. Diagrams on the intergroup comparisons of four bone morphometric parameters from micro-CT analysis. Converted values of bone mineral density (A–B), percent bone volume (C-D), trabecular thickness (E-F) and trabecular number (G-H) relative to that of a control group (C) with normal sound alveolar bone tissue were compared among groups subjected to traction immediately following surgical procedures (left column) and among groups subjected to traction two weeks following surgical intervention (right column). PDL and bone cells around the moving tooth (Henneman et al., 2008) or osteoconduction by the bone graft, enhanced woven bone formation by aiding de novo bone formation, which could account for the clinical 180 Kim and Park importance of early orthodontic traction and graft implantation into the defect site, respectively. Woven bone maturation into lamellar bone, in addition to for- mation from osteoid, is another aspect to consider for estimating an active period of OTM after surgery (Gorski, 1998). We found that early OTM could act not only as a stimulator of woven bone formation, but also as a retardant of lamellar bone maturation. This inversely affected OTM favorably for extended movement into the defect, which corroborates previous reports (Vardimon et al., 2001; Nemcovsky et al., 2004). On the contrary, biostimulation by LLLT proved to be an accelerator of bone maturation. This suggests that LLLT is not recommended for sustaining fast movement into injured bone (Kim et al., 2009b), although it might be used for accelerating or decelerating tooth movement when applied to the moving tooth surrounded by normal sound bone (Seifi and Vahid- Dastjerdi, 2015). With a focus on controlling the period of woven bone as a tran- Sient osteopenic state, further experimentation is required to explore major regulators of woven bone formation and/or maturation using a genetics-based approach. The ultimate goal is to develop a non-invasive procedure comprising a local delivery system of woven bone-specific regulators instead of an aggressive surgical insult. Basically, OTM is known to involve a cytokine-mediated bone adaptive response, similar to the wound healing process including an inflammatory osteopenic state and subsequent stimulation of bone apposition (Melsen, 1999; Milne et al., 2009; Hassan et al., 2015). As such, it might be postulated that each biologic potential of OTM and defect regeneration would contribute to each other Synergistically as long as woven bone healing could progress well (Fig. 12). Several studies have been performed regarding differential gene expression in woven bone as compared to lamellar bone (McKenzie and Silva, 2011) or enhanced single gene expression in the periodontium during OTM (Kanzaki et al., 2004, 2006; Takahashi et al., 2006). However, the mechanisms for woven bone formation, maintenance and maturation in the injured alveolar bone subjected to active tooth movement remains uncertain. To pursue specific functional genes for favorable woven bone formation (Fig. 12), another experiment was performed to screen the genetic events as a whole between natural healing and OTM-induced healing using the same surgical model in beagles as described above. 181 Overcoming Alveolar Defects Figure 12. Photomicrographs of the moved tooth and the regenerated defect in the O-2 group, which showed the fastest tooth movement. The defect is filled with woven bone up to the crestal level, in spite of bone resorptive activity facing the moving tooth by compression. Arrow indicates the direction of tooth movement and the dotted line demarcates the original defect walls. Masson's trichrome staining, original magnification of x12.5. PART II: DIFFERENTIAL GENE EXPRESSION IN THE SURGICAL DEFECT WITH AND WITHOUT OTM Objective As described above, we have found that OTM could play an es: sential role in forming and sustaining woven bone on its own or in Syn- ergistic combination with grafts used for healing bone defects. Current knowledge on the biologic mechanism of OTM is that it involves an in- flammatory response in which chemokines, cytokines and growth fac tors involved in bone metabolism modulate cell recruitment, activation, proliferation, differentiation and survival (Teixeira et al., 2010; Andrade et al., 2014). This raises the possibility that local delivery of biomolecules of gene therapy could be used to regulate OTM; however, there still is little information on the specific genes or proteins involved in OTM, especial- ly in the woven bone states and during the earlier inflammatory States. It is uncertain how different the whole gene expression pattern would be according to the elapsed time after surgery when combined with 182 Kim and Park OTM. This rationale was the basis for selecting specific genes or biomol- ecules for further study. The present study aimed to investigate the impact of OTM on the gene expression pattern in the regenerate tissue during the healing process of a surgical alveolar defect in beagles. Specifically, we wanted to determine whether the mRNA profile in regenerate tissue in proximity of a tooth moving for six weeks would be similar to that in the potential woven bone at two weeks of natural healing, or to that in the maturing bone at six weeks of healing without tooth movement. Materials and Methods One each of four male beagles were assigned as follows: normal alveolar bone as a baseline control, C; defect with two-week natural healing, D2; defect with six-week natural healing, D6; and defect with six- week healing accompanied by OTM, DT6 (Fig. 13). A critical-sized defect was prepared including the extracted Socket of the maxillary first premolar in all experimental animals. In the DT6 animal, the second premolar was protracted into the defect starting immediately after surgery for a duration of six weeks. Two tissue samples (left and right) from the regenerate tissue in the defect area in each animal were retrieved, and mRNA extracted and analyzed separately. After a quality check on RNA purity and integrity, Affymetrix whole transcript expression array process was executed according to the manufacturer's protocol (GeneChip Whole Transcript PLUS Reagent Kit). Raw data were extracted automatically in Affymetrix data extraction protocols using the software provided by Affymetrix GeneChip” Command Console° Software (AGCC) and analyzed bioinformatically. To compare the expression levels of mRNAs between the groups, the median of probe intensities in the same gene was calculated and used for further analysis. Principal component analysis was performed using “proomp" function in R Statistical language v3.1.2. (www.r-project.org) and hier- archical cluster analysis using complete linkage and Euclidean distance as a measure of similarity. Differentially expressed gene (DEG) analysis between the test and control sample was carried out using fold change and a Local Pooled Error (LPE) test in which the null hypothesis was that no difference exists among groups. False discovery rate (FDR) was 183 Overcoming Alveolar Defects Defect only TM into defect Natural healing Healing with TM 2 Wieks 6 Weeks 6 Wies N Figure 13. Diagram of experimental procedures and animal assignment. controlled by adjusting the p value using a Benjamini-Hochberg algorithm. Differentially expressed genes were defined as those with changes of at least 1.5-fold between a pair of samples at a false FDR of 5%. Results In the hierarchical clustering analysis (Fig. 14A), the DT6 sample showed similar clustering to the D2 sample, rather than to the D6 sample. The D6 had similar clustering as the C sample. The D2 and D6 groups were identified with different clustering as expected. This corresponded to the result of principal component analysis representing distributional proximity among groups (Fig. 14B). The baseline C sample was in the farthest position from all the tested samples. Among all samples, the DT6 group was distributed closer to the D2 group than to the D6 group. Microarray analysis identified functionally upregulated genes in six pairs among the four animals, designated by the number of DEGs (Fig. 15). The DT6 sample showed the lowest level of DEGs in pair of the D2 sample (DT6-D2), while it had a larger number of DEGs in pair of the C Sample (DT6-C) or of the D6 sample (DT6-D6). The D2 sample represented a large number of upregulated genes in pairs of the C (D2-C) as well as of the D6 group (D2-D6). 184 Kim and Park § Z-score . c. c. § 5. ; - D 3. 1. s g - : - dist-tº- -10000 -5000 A n-lust tºº..., B Pct Figure 14. A: Diagram of Sig hierarchical clustering by cluster analysis where grouping of samples was explored. B: Distributional proximity among groups represented by principal components analysis. Two samples (left and right represented by _1 and 2) from each animal were used for these analyses. 0. 5 do o Discussion This pilot study elucidated differential gene expression between different healing states of the regenerate tissues, which was affected by OTM, The gene expression pattern showed significant differences between the two-week and six-week time course of defect healing. At the Six-week time point, the regenerate tissue in close proximity of a moving tooth represented no significant similarity to the naturally healed tissue Without OTM, rather, it showed great similarity to that in the woven bone-like tissue at two weeks of natural healing. This confirmed at the gene level that activated bone remodeling activities induced by OTM could be synchronized with active bone healing potentials, sustaining a Woven bone state as an optimal matrix for efficient tooth movement and favorable defect regeneration. With further analyses of gene enrichment and functional annotation, some candidate genes specifically functioning In WOVen bone formation and maturation could be selected for future therapeutic application. Recently, gene-based therapeutics have been tested as a novel non-invasive approach for modulating the rate of OTM by modifying the individual biological milieu. Local genetransfer has been used to overcome the drawback such short duration of action and/or rapid degradation 185 Overcoming Alveolar Defects 603 247 152 Dº DTG (UP) (UP) 67 57 C. C. C (DOWN) (DOWN) (DOWN) 230 D2–C D6-C DT6-C D6 DTG 124 DTS 64 (UP) (UP) (UP) 21 32 44. D2 D2 Dº (DOWN) (DOWN) (DOWN) 192 DT6-D2 Figure 15. The number of up-/down-regulated DEGs in six pairs of inter-sample comparisons C, D2, D6 and DT6. Pairwise comparison; cut-offs, fold change 2 1.5; LPE p-value < 0.05; FDR 10%. of traditional protein therapy as a pharmacologic approach (Kanzaki et al., 2006; Zhao et al., 2012). The study by Iglesias-Llnares and colleague: (2011) compared the effect of RANKL gene transfer with corticotomy and suggested that the local gene transfer method is promising to make up for the gradual decrease of the acceleratory effect on OTM Oveſ time by Surgical methods. Nonetheless, no studies have investigated gene therapy aimed at regulating OTM and the reconstruction of 186 Kim and Park poor underlying tissue in combination, which was the focus of our study. In addition, the previous studies used an already known single gene without background on the whole gene expression and interaction seen in the best environmental condition for OTM, which might be a risky factor for clinical application to patients. The primary purpose of our study was to establish gene-based biological evidence for an optimal time period of post-surgical OTM and, in sequence, to set up a basis for the development of more specific gene-based therapeutic procedures. This may alleviate possible problems such as an immune response and local or Systemic imbalance. Experimental limitations of our pilot study included a lack of adequate sample and specificity in microarray measurements. The sample size of one animal in each group is inadequate for reliable statistical validity and the beagle has only limited gene ontology information available (Higgins et al., 2003; Wayne and Ostrander, 2007). However, this model has been shown to be advantageous in obtaining a critical- sized alveolar defect where spontaneous healing does not occur and to extract sufficient amount of mRNAs from each single object. Despite the possibility of still-omitted functional annotations on whole genes, microarray or further RNA sequencing analysis holds promise not only for integrating and reinterpreting already known biologic pathways, but also for discovering unknown hidden mechanisms by providing extensive genetic resources and a high-quality genome sequence position (Draghici et al., 2006; Koltai and Weingarten-Baror, 2008). CONCLUSION Optimal timing of orthodontic force application into a surgical alveolar defect relies on its healing state, which was influenced by bone graft and laser biostimulation. Bone grafting not only could allow immediate force application into the defect, but it also enables a longer duration of effective tooth movement in case of delayed force application after surgery. Although LLLT would be a beneficial tool for enhancing periodontal healing after alveolar reconstruction procedures, it is not recommended when OTM toward the surgical site is planned Subsequently. The woven bone state was verified as a desirable potential biological environment for fast tooth movement with favorable defect regeneration. This phase during periodontal bone regeneration lasted 187 Overcoming Alveolar Defects longer when combined with immediate OTM. 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Zhang ZY, Teoh SH, Chong MS, Lee ES, Tan LG, Mattar CN, Fisk NM, Choolani M, Chan J. Neo-vascularization and bone formation mediated by fetal mesenchymal stem cell tissue-engineered bone grafts in critical-size femoral defects. Biomaterials 2010;31(4):608–620. Zhao N, Lin J, Kanzaki H, Ni J, Chen Z, Liang W, Liu Y. Local osteoprotegerin gene transfer inhibits relapse of orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2012;141(1):30-40. 193 BIOENGINEERING OF THE PERIODONTAL LIGAMENT Ann M. Decker, Sophia P. Pilipchuk, Ana Claudia Araujo-Pires, William V. Giannobile ABSTRACT The periodontal ligament (PDL) is a complex and integral Supporting component of the periodontal complex. Proper healing of defects requires the repair of the PDL for normal form and function. Strategies such as the delivery of biologics, cell therapies and biomaterial scaffolds for bioengineering of the PDL all are rooted in the fundamental understanding of the steps that are involved during development, disease and repair of this tissue. This chapter reviews the essential Components of PDL regeneration and formation during development and dis- cusses how the ligament is affected by disease and biomechanical tooth move- ment. Currently available strategies for bioengineering the PDL are reviewed in both a fundamental and functional context. KEY WORDS: periodontal ligament (PDL), bioengineering, cell therapy, biologics, biomaterials INTRODUCTION The periodontal ligament (PDL) is an important structure within the greater periodontal complex. The periodontal complex is comprised of gingiva, cementum, alveolar bone and PDL. Each of these four ana- tomic Components individually has a unique structure and function. The interface that connects each component together is essential to overall dental function. Disruption of any one of these anatomical components, through disease or trauma, results in apical migration of the periodontal tissues, bone resorption and tooth loss if the etiology is not addressed ap- propriately in a timely manner (Wang et al., 2005; Nanci and Bosshardt, 2006). Complete periodontal tissue regeneration is the ideal therapeu- tic goal in most cases, but is not seen predictably in many clinical situa- tions with existing conventional therapeutic techniques (Bosshardt and 195 Bioengineering of the Periodontal Ligament Sculean, 2009). Furthermore, complete tissue regeneration of the peri- odontium presents a significant challenge because it requires the simul- taneous re-establishment of structure and function to PDL, cementum, bone and gingival fibroblasts. In addition, these periodontal structures must be developed as both independent and interfacing components to prevent bacterial invasion and future attachment loss (Taba et al., 2005). This chapter will focus on the current progress of PDL bioengineering in the context of development, health, disease and biomechanical tooth movement. DEVELOPMENT Development of the PDL is entwined with the development of both the alveolar process and cementum (Lindhe et al., 2008). Under- standing both the structural process and gene expression involved in embryologic development is essential to the regeneration of these struc- tures and their interfaces. PDL formation coincides with root cementum and alveolar bone formation following the establishment of the cervical loop during the bell stage of tooth-bone complex development (Fig. 1; Ten Cate, 1997). The cervical loop is comprised of an epithelial bilayer called the Hertwig's epi- thelial root sheath (HERS) that initiates root dentin formation. Following root development, the HERS cells lose their linear integrity and become remnants known in the mature PDL space as epithelial cell rests of Mal- assez (ERM; Tummers and Thesleff, 2003; Rincon et al., 2006). Currently, there is controversy pertaining to the role of the ERM in the adult PDL; however, Xiong and colleagues (2013) reported stem cell characteristics in these cells and suggest that ERM might have a role in maintaining the PDL through prevention of tooth resorption or ankylosis. As such, the ERM potentially could be a therapeutic target for periodontal structure regeneration in the future. Fragmentation of the HERS element allows the mesenchymal cells from the neural crest cell-derived dental follicle to contact the newly formed root dentin (Bosshardt et al., 2015). HERS cell secretion of bone morphogenetic protein 2 (BMP2) and BMP7 along with direct contact of the root dentin initiates differentiation of mesenchymal cells into cement- oblasts (Yang et al., 2013). In addition to hydroxyapatite crystalline matrix, cementoblasts also secrete a dense bundle of collagen fibers into the pre- 196 Decker et al. Development Bud Stage Cap Stage BellStage Tooth Eruption Healthy Tooth osteoblast osteoclast osteocyte fibroblast - Cell Types Figure 1. PDL in the context of development and health. Proper development leads to periodontal structures, which include cementum, PDL, alveolar bone and epithelium. These structures are supported by a network of vasculature and homeostatic cell types. collagen fibers - - dentinstructure prior to mineralization. Bundles of collagen are presentin the mature root surface as Sharpey's fibers (Bosshardt and Nanci, 2004). On the opposite side of the forming PDL space, alveolar bone develops directly from dental mesenchyme by intramembranous ossi- fication through both molecular signaling and mechanical loading (So- ſherman et al., 1999; Zhang et al., 2003; Diep et al., 2009). Collagenous Sharpey's fibers of the bone also are inserted into the developing alveo- lar bone proper to span the width of the PDL space (Bosshardt et al., 2015). Together, these features mature into the healthy periodontium (Fig. 1). HEALTH In the healthy adult, the PDL maintains a consistent width of between 0.15 to 0.38 mm (Nanci and Bosshardt, 2006). The PDL is 197 Bioengineering of the Periodontal Ligament comprised of an extracellular matrix (ECM), cellular component, as well as vascular and neuronal networks (Figs. 1-2). The ECM of the PDL is made up of collagen and elastic system fibers within the context of a proteo- glycan matrix (Fig. 2). The majority of fibers in the PDL are collagen type | and Ill, with smaller quantities of collagen type V, VI and XII (Lukinmaa and Waltimo, 1992; Berkovitz, 2004). These fibers are arranged in bun- dles of five distinct orientations (the alveolar crest group, the horizontal group, the oblique group, the apical group and the interradicular group), which are continuous coronally with the gingival collagen fibers. Individ- ual strands in each bundle are remodeled continuously, while the overal fiber bundle maintains its structure, function and orientation (Nanci and Bosshardt, 2006). Elastic system fibers (including the oxytalan, elaunin and elastic fibers) constitute a minority of the fiber network within the PDL (Beer- tsen et al., 1997; Strydom et al., 2012). The elastic system fibers are an important complement to the collagen fiber system because they con- tribute properties of elasticity and resilience to the connective tissue (Kielty et al., 2002). Maintenance of these fibers is correlated directly with mechanical loading, age-related factors and systemic conditions (Ren et al., 2008; Tsuruga et al., 2009; Zheng et al., 2009). In addition, human and pre-clinical in vivo studies suggest that biomechanical tooth movement results in increased number, size and length of elastic system fibers (Edwards, 1968; Boese, 1969; Sims, 1976; Jonas and Riede, 1980; Kalajzic et al., 2013). There are many cells and molecular factors involved in the main- tenance and repair of the PDL on a daily basis. The cellular components of the PDL include primarily ectomesenchyme-derived fibroblastic cells and macrophages within the PDL space (Ten Cate, 1997). At the alveo- lar bone interface, osteoblasts and osteoclasts reside. At the cementum interface there are ERMs, cementoblasts and cementoclasts (Nanci and Bosshardt, 2006; Rodrigues et al., 2007). Fibroblastic cells in the PDL include both PDL stem cells and fi- broblasts (Seo et al., 2004). Fibroblasts of the PDL uniquely express both osteoblastic and cementoblastic properties through bone-associated markers, regulate osteoclastogenesis and have the capability of secreting cementum-like ECM (Nohutcu et al., 1997; Flores et al., 2008; Paula-Silva et al., 2010). 198 Decker et al. | Figure 2. Anatomy of the PDL. The PDL is a complex structure containing fibro- blasts, immune cells, collagen fibers, blood vessels and stem cells, as seen in the schematic (middle) and histological and electron microscopy images (right). Adapted from Marchesan et al., 2011. Due to the commonalities of phenotypic expression between PDL fibroblasts with cementoblasts and osteoblasts, PDL fibroblast iden- tification is essential for bioengineering applications and therapeutic use. solation of these cells through in vitro culture protocols have identified many cytoskeletal and secreted proteins that are present in the popu- lation that provide specific function to the PDL fibroblast phenotype (Table 1). In addition to the ECM fiber network present in the PDL space, extensive vasculature and innervation also is present (Fig. 2). The vas- cular network of the PDL not only supplies the nutrients required to Sustain cellular and acellular components of functional PDL, but also Contributes to force dissipation through viscoelastic properties of the fluid within the vessels (Bien, 1966; Strydom et al., 2012). An extensive Vascular plexus is formed throughout the PDL space from vessels emerg- ing from Volkman's Canals of the alveolar bone anastomosing with ves- Sels from both the apex of the tooth and coronally to the gingival tissue (Matsuo and Takahashi, 2002). Nerve supply in the PDL is an important sensory input for mas- tication, providing information regarding pressure, touch, tooth position and tissue injury (Taylor, 1990; Maeda et al., 1999). Histological sections demonstrate that the nerve supply is associated anatomically with the blood vessels in the PDL space (Byers and Holland, 1977; Heyeraas et al., 199 Bioengineering of the Periodontal Ligament Table 1. Markers of the PDL fibroblast. Adapted from Marchesan et al., 2011. Transcription factors mechanical loading CATEGORY FACTOR FUNCTION REFERENCES * ~ *~ . Saito et al., 2002; PerioStin Bone formation; Rios et al., 2008; Iwata et al., 2010 Runt-related transcription factor 2 Essential for osteoblast dif- ferentiation Saito et al., 2002 Twist Negative regulator of osteoblast differentiation Komaki et al., 2007 Extracellular N-Cadherin Mineral nodule formation Lin et al., 1999 Neural cell adhesion marker Function unknown Iwata et al., 2010 Tissue factor pathway inhibitor-2 Extracellular matrix regulation Fujita et al., 2007 Mineralization; Pitaru et al., 1995, Fibril assembly matrix Cementum collagen-like protein; 2002: proteins attachment protein partial homology to y collagen I and XI Bar-kana et al., 1998 Cementum protein- Involved in cementum for- Alvarez-Perez et al., 23 mation; related to 2006 collagen type X & . * ingival epi- e Laminin-2/4 and 8/9 Chemotaxis of gingival epi Ohshima et al., 2006 thelial cells e g * * * Jäger et al., 2010; Sclerostin Mineralization Lehnen et al., 2012 & - * & Yamada et al., 2001 & Positi lat g y Asporin/PLAP-1 º tive/ . regulator | 2007. Of mineralization Li et al., 2012 & Duarte et al., 1998; º: calcium Inhibitor of mineralization Park et al., 2001; inding protein Iwata et al., 2010 Others Fibromodulin Lallier et al., 2005 Periodontal ligament specific-17 Function unknown Park et al., 2001 Major histocompat- ibility complex-D-B Receptor molecules Ohyama et al., 2002; Fujita et al., 2007 Uncoordinated-like protein PDL fibroblast differentiation; mechanical stress Kim et al., 2007 200 Decker et al. 1993). These structural associations have functional consequences in that Sympathetic fibers and their secreted neuropeptides regulate blood vessel diameter and local blood flow (Karita et al., 1988). Innervation of the PDL tissues also may play a role in homeostasis of the PDL, as well as the remodeling process in orthodontic tooth movement, through the release of neurotransmitters, including Substance P (SP) and calcitonin gene-related peptide (CGRP; Wakisaka et al., 1985; Nicolay et al., 1990; Heyeraas et al., 1993). These components of the PDL in health provide tooth attachment and dissipation of occlusal forces to the alveolar bone during function. The collagen fibers, elastic system fibers and proteoglycans represent the primary ECM components of PDL, but their maintenance by nerve sup- ply, blood Supply and fibroblastic cells within the space are a necessary Continued function in health, with dysfunction leading to disease. BIOMECHANICAL TOOTH MOVEMENT Biomechanical tooth movement is possible due to mechanically induced remodeling of the PDL and alveolar bone in an aseptic environ- ment. Signaling from immune, nerve and fibroblastic cells mediate these remodeling events (Krishnan and Davidovitch, 2006). Following force ap- plication to the tooth, there is a mechanical strain on the ECM of the PDL and alveolar bone (Fig. 3). This mechanical strain induces deformational changes of the cells connected intimately through integrins with the PDL ECM (Meeran, 2012). This cellular deformation induces cytoskeletal re- modeling, secretion of cytokines and changes in gene expression includ- ing upregulation of prostaglandins in as little as fifteen minutes following force application (Ngan et al., 1990). In sites of PDL compression, focal necrotic areas develop. Ne- Crotic areas become free of cells and hyalinized, requiring influx of mac- rophages to clear debris. In later stages following compression necrosis, multi-nucleated osteoclasts are recruited from the adjacent alveolar bone through an increase in receptor activator of NF-kB ligand (RANKL) at the site of compression, seen as early as three hours after applica- tion of force in a murine model (Brooks et al., 2009). RANKL expres- sion is mediated by fibroblasts, osteoblasts and stromal cells following 201 Bioengineering of the Periodontal Ligament Biomechanical Movement Force Tension - Compression II osteoblast osteoclast osteocyte fibroblast Cell Types - - Figure 3. PDL plays an essential role in tooth movement. As teeth move when a force is applied, cells respond to the tension and compression of the PDL. This force results in the apposition or resorption of alveolar bone, respectively. collagen fibers RANKL - - E º application of forces, resulting in resorption of alveolar bone at the sites of PDL compression (Nishijima et al., 2006). In sites of PDL tension, mechanical strain on PDL fibroblasts and osteoblasts induces expression of runt-related transcription factor 2 (RUNX2), a transcription factor that is linked closely with osteoblast differentiation (Franceschi and Xiao, 2003). In addition, following applica: tion of mechanical force, osteoblastic precursors begin to activate geneº necessary for osteoblast differentiation and mineralization (Brooks et al., 2009). New osteoid is deposited on the site of tension within 24 houſ: following osteoblastic differentiation (Pavlin et al., 2001). PDL fibroblasts are involved circumferentially in ECM remodel- ing during biomechanical tooth movement, both on the side of Comº pression and tension (Cantarella et al., 2006). PDL fibroblasts also ini- tiate an inflammatory cascade through secretion of interleukins and tumor necrosis factors (TNFs). The inflammatory cascade then recruits and promotes osteoclastogenesis and resorption of the alveolar bonº (Krishnan and Davidovitch, 2006). This inflammatory response parallels 202 Decker et al. the events seen in periodontal disease; however, no lymphocytes or granulocytes are seen to amplify the signal in a positive feedback mecha- nism, which indicates that the environment is aseptic in biomechanical tooth movement. DISEASE Periodontal disease remains a public health issue that affects in- dividuals on a global scale (Albandar and Tinoco, 2002; Eke et al., 2012; Kassebaum et al., 2014). It is characterized as an inflammatory lesion in response to subgingival biofilm formation resulting in alveolar bone loss, Subsequent loss of the PDL and apical migration of the long junctional ep- ithelium (Fig. 4; Terheyden et al., 2014). Currently, successful periodontal therapy is achieved by the standard of bacterial removal and prevention of additional tissue damage. However, loss of periodontal apparatus of ten results in tooth sensitivity, poor esthetics and increased risk of tooth loss (Badersten et al., 1984; Claffey and Egelberg, 1995). - Biofilm Removo/ Disease Regeneration osteoblast osteoclast osteocyte fibroblast T-cell B-cell macrophage RANKL collagen fibers Cell Types y oC = - - Q = Figure 4. Goals of periodontal regeneration. Damage to the PDL can occur either through disease or tooth movement. Regeneration is required to restore health, Which can be promoted clinically through the use of biomaterial scaffolds, cell therapy or delivered biologics to promote wound healing. 203 Bioengineering of the Periodontal Ligament In particular, loss of the PDL allows expedited progression of mi- crobes toward the apical and anaerobic portion of the tooth. The event of PDL breakdown is due to a combination of proteolytic enzymes secreted by virulent bacteria within the periodontal pocket and the inflammatory host response (Ximénez-Fyvie et al., 2000; Sorsa et al., 2006). An exam- ple of bacterial proteolysis is gingipain, secreted from Porphyromonas gingivalis, which subsequently degrades fibronectin, a crucial fibroblastic ECM protein (Potempa et al., 1995; Travis et al., 1997). ECM degrada- tion by bacterial proteolysis results in host cell apoptosis and tissue de- struction (Ruggiero et al., 2013). Pathogen associated molecular proteins (PAMPs), such as bacterial membrane protein lipopolysaccharide, also in- duce the release of additional host-mediated pro-inflammatory signaling molecules including TNF-O, interleukin-1 and interleukin-8 (IL-1 and IL-8; Agarwal et al., 1995; Yoshimura et al., 1997; Harris et al., 2002). These pro-inflammatory signals induce fibroblasts, macrophages and epithelial cells to release matrix metalloproteases (MMP), primarily MMP-1, MMP- 3, MMP-8, MMP-9 and MMP-13, which are enzymes responsible for PDL and soft tissue breakdown (Sorsa et al., 2006). The breakdown of bone follows PDL and soft tissue destruction through an imbalance in bone remodeling between osteoblasts and oS- teoclasts through the RANK/RANKL/osteoprotegerin (OPG) signaling sys- tem. RANKL binding to the RANK receptor located on monocytes induces differentiation and formation of multi-nucleated osteoclasts, which di- rectly resorbs bone (Khosla, 2001). The release of pro-inflammatory signals from the host response to bacterial invasion in the periodontal pocket results in activation of adaptive immune cells and subsequent Se- cretion of RANKL. Secretion of RANKL from both T and B cells promotes osteoclast differentiation, survival and alveolar bone resorption in the context of periodontal disease (Kawai et al., 2006). Interestingly, popula- tions of cells within the PDL also express RANKL and contribute to bone remodeling in both disease and orthodontic tooth movement (Kanzaki et al., 2002, 2006). REGENERATION Regeneration of damaged or diseased periodontium long has been the goal of clinical periodontology with many challenges due to 204 Decker et al. the Complexity of the wound healing process (Fig. 4; Giannobile, 2014). Wound healing is comprised of four phases including hemostasis, inflam- mation, proliferation and tissue remodeling (Guo and DiPietro, 2010). Of these phases, hemostasis often is the most important because stabiliza- tion of the clot, along with platelet secreted cytokines and growth fac- tors, initiate the healing process (Wikesjö and Selvig, 1999). Following non-regenerative periodontal therapy, epithelial cell growth permeates throughout the periodontal pocket and along the surface of the root, forming a long junctional epithelium. The biological presence of the long junctional epithelium is fundamental to expediting wound closure, which innately prevents bacterial invasion of susceptible tissues (Polimeni et al., 2006). PDL fibers are inhibited from formation in this case because there is no mechanism for attachment into the cementum surface. Guided-tissue regeneration techniques (e.g., the use of barrier membranes) provide a mechanism to stabilize the clot, which prevents apical migration of epithelial cells (Wikesjö and Nilvéus, 1990). Provided adequate space and time, PDL progenitors and stem cells can regenerate PDL insertion into cementum and alveolar bone, resulting in tissue re- generation of true periodontal apparatus structure and function (Haney et al., 1993). There are many limitations to the current techniques both in ex- ecution and consistent tissue regeneration and, specifically, regeneration of the PDL. The PDL is a particularly challenging structure to regenerate due to its complex developmental origin, heterogeneous composition and its dynamic function in the context of health and tooth movement. Progenitor cells are present in the PDL; however, their ability to function in a regenerative capacity is diminished severely in wound healing if the clot is disrupted or invaded by more proliferative cell types (Karring et al., 1993). As such, much effort has been applied toward the development of technologies for precise, functional bioengineering of the PDL. GOALS OF PDL BIOENGINEERING Restoration of the periodontal complex requires: 1. Elimination/control of infection and uncontrolled in- flammation; - 2. Regeneration of cementum on the root surface; 205 Bioengineering of the Periodontal Ligament Restoration of alveolar bone height; Regeneration of the PDL; Regeneration of PDL insertion into cementum; and Re-establishment of connective tissue formation and epithelial seal circumferentially (Aukhil, 1991; Gar- rett, 1996; Grzesik and Narayanan, 2002). : The goal for periodontal engineering is expanding from guided tissue regeneration alone to cell-based tissue engineering and therapies that promote regeneration of tissues through growth of cells resident in the PDL (Bosshardt et al., 2015). These cell-based targeted strategies are multi-faceted. Following disease and/or biomechanical tooth movement, tissue regeneration requires a biomaterial scaffold, biological cells that can re-populate the areas and biological agents that promote the clinical outcomes to regulate the regenerative process (Fig. 4). These three ele- ments are discussed in detail in the remainder of this chapter. BIOLOGICS Biologics, such as growth factors, are important to guide the PDL regenerative process. Specifically growth factors, chemokines and cyto- kines are important molecular components of cell-to-cell communication and regulate migration, progenitor cell differentiation and cellular pro- liferation (Bosshardt et al., 2015). Growth factors that have been under particular investigation regarding PDL regeneration include BMP, plate- let-derived growth factor (PDGF), fibroblast growth factor (FGF), enamel matrix derivative (EMD) and growth and differentiation factor-5 (GDF-5; Table 2). Bone Morphogenetic Proteins (BMPs) BMPs are related to the transforming growth factor-3 superfam- ily of proteins and most notably regulate bone formation. Additionally, BMPs have been implicated in other tissue formation including carti- lage, brain, kidney and nerves, and are an essential regulator of em- bryonic patterning. More than twenty BMPs have been identified; of those, several have been engineered into human recombinant proteins and studied in the context of PDL regeneration including BMP2, BMP7 (osteogenic protein 1) and BMP14 (growth differentiation factor 5. 206 Decker et al. Table 2. Biologics used for PDL regeneration. TGF-31 = tissue growth factor-31; EMD = enamel matrix derivative; BMP2 = bone morphogenetic protein 2; BMP7/ OP-1 = bone morphogenetic protein 7/osteogenic protein-1; BMP14/GDF-5 = bone morphogenetic protein 14/growth differentiation factor-5; PDGF = platelet- derived growth factor; FGF-2 = fibroblast growth factor-2; PDLSCs = PDL stem cells. GENERAL BIOLOGIC BIOLOGIC IMPACT EFFECT ON PDL REFERENCES Stimulates • * g. proliferation Fujii, 2010 increases perpstin ex- Rios, 2008 pression Wound healing through angiogenesis, inhibition of Induces differentiation of • * * F- TGF-31 inflammation and PDLSCs into PDL cells Fujii, 2010 deposition of ECM Induces secretion of ECM - - e Fujita, 2004 (e.g., osteonectin) Promotes PDL Nishimura and migration Terranova, 1996 Cellproliferation, angio- genesis, osteogenesis and Enhanced proliferation Gestrelius, 1997 * EMD cementogenesis Enhanced ECM LyngstadaaS production et al., 2001 Bone metabolism, Induces differentiation of Kobayashi, 1999; BMP2 formation maintenance PDLSCs into b Skodie, 2014 and repair S IntC) OOne Odje, º bone gene ex- Dereka, 2009 BMP7/ Osteogenic differentiation p OP-1 of mesenchymal stem cells increase PDL precursor Rajshankar, 1998 cell proliferation BMP14/ Bone, cartilage, tendon and GDF-5 ligament Enhances proliferation Nakamura, 2003 development Stimulates Matsuda, 1992; Cell proliferation, proliferation yºpoulou. PDGF angiogenesis and migration Promotes migration Matsuda, 1992; g Mumford, 2001 Stimulates Takayama, 1997 proliferation FGF-2 Cell proliferation Promotes migration Terranova, 1989 inhibits osteogenic dif- Lee, 2012 ferentiation 207 Bioengineering of the Periodontal Ligament GDF-5). BMP2 generally is involved in bone metabolism and has been shown to induce osteogenic differentiation of PDL stem cells (PDLSCs) (Kobayashi et al., 1999; Skodje et al., 2014). Additionally, BMP2 can induce ectopic bone formation and ankylosis with no functional PDL formation (Selvig et al., 2002). BMP7 promoted osteogenic gene expression, includ- ing alkaline phosphatase (ALP) and osteocalcin in PDL cells. Additionally, following BMP7 application, PDL precursor cells demonstrated increased proliferation rates, shown through Bromodeoxyuridine (BrdU) incorpora- tion assays (Rajshankar et al., 1998). In vivo, BMP7 application has been shown to improve periodontal regeneration—including new PDL attach- ment—of Class III furcation defects (Giannobile et al., 1998). BMP14 is expressed in the dental follicle during tooth development and is of inter- est in the regeneration of periodontal tissues. In vitro, BMP14 has been shown to increase the proliferation of human PDL cells (Nakamura et al., 2003). Additionally in vivo, GDF-5 generated a two-fold increase in PDL during periodontal regeneration of twenty chronic periodontitis patients (Stavropoulos et al., 2011). Transforming Growth Factor Beta1 (TGF-31) TGF-31 is one of three isoforms in the Transforming Growth Fac- tor family (TGF-31, -32, -33). Specifically, TGF-31 stimulates angiogenesis, inhibits inflammation and promotes synthesis and deposition of ECM during the wound healing process. Application of TGF-31 on PDL cells in vitro significantly increased proliferation rates, both compared to PDLS not administered TGF-31 and gingival fibroblasts that also were admin- istered TGF-31 (Dennison et al., 1994). Additionally, delivery of TGF-31 through the use of a chitosan/collagen scaffold in vivo demonstrated similar results (Zhang et al., 2006). Though TGF-31 has been shown to exert a proliferative effect on terminal PDL cells, it appears to inhibit proliferation of stem cells and progenitor cells in many tissues including the PDL (Cochran and Wozney, 1999). More specifically, TGF-31 was shown to increase proliferation in primary PDL fibroblasts and inhibited proliferation of PDL stem cells from the cell line 1-11 (Fujii et al., 2010). Thus, TGF-31 has dual proliferative effects on cells with different differentiation States. 208 Decker et al. Enamel Matrix Derivative (EMD) EMD is a porcine-derived combination of peptides, mainly com- prised of amelogenins and is the first Food and Drug Administration (FDA)-approved biologic for periodontal regeneration. EMD can aid in the regeneration of intrabony defects and, as such, may prove a good supple- ment to directed PDL bioengineering (Esposito et al., 2009). EMD signifi- cantly increased cell proliferation and total cell number when applied to PDL fibroblasts (Gestrelius et al., 1997; Cattaneo et al., 2003). In addition, EMD increased PDL fibroblast synthesis of total ECM protein production (including increased ALP activity and in vitro mineralized nodule forma- tion), TGF-31, IL-6 and PDGF (Gestrelius et al., 1997; Lyngstadaas et al., 2001; Rodrigues et al., 2007). However, there was no significant effect on migration of PDL fibroblasts following EMD application (Gestrelius et al., 1997; Palioto et al., 2004). Platelet Derived Growth Factor (PDGF) ~. PDGF is a homodimer that can present in five different isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. PDGF is released by platelets and involved extensively in wound healing of both hard and Soft tissues by promoting cell migration, proliferation and angiogenesis following injury (Kaigler et al., 2011). More specific to periodontal re- generation, PDGF-BB was shown to promote regeneration of bone, ce- mentum and the PDL (Lynch et al., 1989; Khoshkam et al., 2015). PDGF also promotes both migration and proliferation of PDL fibroblasts, among other cell types (Matsuda et al., 1992; Mumford et al., 2001; Marcopou- lou et al., 2003). Histological analysis of intrabony defects associated with advanced periodontitis revealed new bone, cementum and PDL formation following application of the recombinant human (rh) PDGF-BB (Camelo et al., 2003). Furthermore, in a prospective, randomized clini- cal trial, rhpDGF-BB stimulated significant increase in the rate of clini- cal attachment level gain and bone defect fill (Nevins et al., 2005, 2013). Similar results were found in a trial of combination rhpDGF-BB + B-TCP treatment (Jayakumar et al., 2011). A subsequent systematic review of available literature on the use PDGF-BB or FGF-2 (see below) in treat- ment of periodontal defects supported the conclusion that these factors 209 Bioengineering of the Periodontal Ligament promote linear defect fill and increased clinical attachment gain (Khosh- kam et al., 2015). Fibroblast Growth Factor-2 (FGF-2) FGFs are signaling molecules that are involved in tissue repair mediated through tyrosine kinase signaling. FGF-2 specifically promotes proliferation of mesodermal cells and angiogenesis, essential elements for the wound healing process (Ledoux et al., 1992). In PDL cells, FGF-2 increased cellular proliferation, chemotaxis and the production of ECM proteins (Terranova et al., 1989; Takayama et al., 1997). However, ALP expression and osteogenic differentiation was inhibited (Lee et al., 2012). In beagle dogs, topical application of FGF-2 initiated PDL regeneration without ankylosis or root resorption in Class Il furcation defects (Muraka- mi et al., 2003). In a multi-center clinical trial, periodontal regeneration and bone fill was improved significantly for patients with two- and three- walled intrabony defects who received FGF-2 therapy (Kitamura et al., 2011). Cell Therapy Cell therapy represents administration of living cells in a patient to restore, maintain or enhance the function of tissue and organs. Cells play a key role in the regenerative process, providing the machinery for new tissue neogenesis and growth (Mao et al., 2006; Rios et al., 2011; Pagni et al., 2012). For recovery of normal PDL, cell therapy emerges as a strategy to improve this highly specialized tissue regeneration process. In this re- gard, stem cells could be used directly on the defect or by loading onto a biomaterial. Different pre-clinical and clinical studies demonstrate the efficacy of cell therapy to regenerate periodontal defects using post-natal stem cells (e.g., mesenchymal stem cells or MSCs; Kawaguchi et al., 2004; Yamada et al., 2006; Chen et al., 2012; Yoshida et al., 2012; Yamada et al., 2013). MSCs have the multi-potent capacity to differentiate into mes- enchymal-derived tissues and have been isolated from bone marrow and other sites (e.g., adipose tissue, muscle, liver, pancreas, cartilage and PDL; Caplan, 1991, 2005; Pittenger et al., 1999; Seo et al., 2004; Mao et al., 2006; Ward et al., 2010). MSCs function in the post-natal en- 210 Decker et al. vironment to supply replacement units for terminal cells that naturally expire due to injury and disease (Caplan, 2005; Dong and Caplan, 2012). Thus, MSCs have been used widely in regenerative medicine (Caplan, 2005). In addition, MSCs secrete growth factors and have immunoregu- latory properties that favor the tissue regeneration (Caplan, 2005; Rios et al., 2011; Knaån-Shanzer, 2014). In fact, when activated by an inflam- matory milieu, MSCs provide subsequent immunosuppressive feedback for restoring tissue homeostasis and triggering the tissue reparative pro- cesses (Mooney and Vandenburgh, 2008; Shi et al., 2012; Araujo-Pires et al., 2014). As such, MSCs provide a good strategy for periodontal tissue regeneration. Both bone marrow-derived mesenchymal stem cells (BM- MSCs) and PDL-MSCs provide viable options for cell therapy for periodon- tal regeneration. BM-MSCs are the most widely studied MSCs for periodontal re- generation because they are accessible in quantities appropriate for clini- cal application (Chen et al., 2012; Kimura et al., 2014). Moreover, PDL- MSCS represent a promising cell therapy in periodontal regeneration due to their optimal location within the context of the normal periodontum (Seo et al., 2004; Feng et al., 2010; Maeda et al., 2011). Thus, the remain- der of this section will focus on the use of both BM-MSCs and PDL-MSCs for the purpose of PDL bioengineering. Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs) BM-MSCs have been used to regenerate the periodontal tissues in both pre-clinical animal models (Yamada et al., 2004, 2013; Hasegawa et al., 2006; Li et al., 2009; Wei et al., 2010; Yang et al., 2010; Simsek et al., 2012) and human clinical trials (Yamada et al., 2004, 2013). Re- cently, it was shown that BM-MSCs communicate with dental tissues and become tissue-specific mesenchymal progenitor cells to maintain tissue homeostasis (Zhou et al., 2011). Translational studies conducted in both Class II and Class Ill furcation defects in dogs used BM-MSCs to achieve Complete periodontal regeneration with formation of new bone, cemen- tum and PDL (Kawaguchi et al., 2004; Hasegawa et al., 2006; Simsek et al., 2012; Yamada et al., 2013). In patients, BM-MSCs have been used to fill bony defects, an adjunct for alveolar ridge preservation, and also as a graft in sinus-lift 211 Bioengineering of the Periodontal Ligament surgeries (Yamada et al., 2004, 2006, 2013; McAllister et al., 2009; Gon- shor et al., 2010; Kaigler et al., 2010, 2013, 2015). In these clinical stud- ies, BM-MSC transplantation provided a safe and effective mechanism for PDL and periodontal regeneration. Although use of BM-MSCs has proved a promising periodontal regeneration strategy, the harvest mechanism is an extremely invasive procedure. As such, many clinical investigators are keen to use cells that are more accessible for cell sources in the dental clinic (e.g., PDL-MSCs), which can be obtained from the root surface of extracted teeth. Periodontal Ligament Stem Cells (PDLSCs or PDL-MSCs) PDL-MSCs are a small population of stem cells found within the PDL space and are responsible for maintaining and regenerating peri- odontal tissue structures (Chen et al., 2012). These cells first were iso- lated from the PDL of third molars (Seo et al., 2004). Similar to other MSCs, PDL-MSCs are multi-potent; however, PDL-MSCs exhibit a unique potential to form cementum and PDL-like tissues—including Sharpey's fiber-like structures—in vivo (Seo et al., 2004). Few studies have demonstrated complete periodontal regenera- tion using PDL-MSCs. In a porcine model, PDL-MSCs were used success- fully for treatment of severe periodontitis in conjunction with a standard surgical procedure (Liu et al., 2008). In a human case report of three patients, PDL-MSCs were capable of regenerating cementum, collagen fibers including Sharpey's fibers cells (Feng et al., 2010). Interestingly, PDL-MSCs also may be isolated from inflamed PDL tissues, retaining the potential to differentiate into cementum-, bone- and PDL-forming cells (Park et al., 2011). To this end, cell therapy is a field that requires further investiga- tion, but is a promising strategy for future use in the clinic due to the multi-potency and immunoregulatory properties of MSCs. Both BM- MSCs and PDL-MSCs have been shown to regenerate cementum, PDL and alveolar bone formation when properly stimulated. However, PDL-MSCs provide a more accessible cell type specific to periodontal regeneration for clinical application. 212 Decker et al. BIOMATERIALS AS SCAFFOLDS FOR PERIODONTAL LIGAMENT REGENERATION A biomaterial scaffold ideally is intended to provide a cell-adhe- sive, three-dimensional (3D) microstructural framework for the guidance and support of physiologically functional tissue formation—reminiscent of the ECM that provides native “scaffolding” in healthy tissue. Several important scaffold development criteria include: 1. Biological functionality capable of supporting cell infil- tration, proliferation and differentiation; 2. ECM-mimicking permeable microstructure with po- rosity to facilitate nutrient and oxygen delivery and exchange; 3. Rate of degradation consistent with rate of tissue re- generation and remodeling; 4. Mechanical properties to maintain tissue defect archi- tecture and support applied loads (e.g., mastication- induced compressive forces on the periodontal liga- ment); 5. Neovascularization for tissue homeostasis and reper- fusion; and 6. Targeted cell, growth factor and/or gene delivery for enhanced regenerative capacity. While scaffolds formed from naturally derived materials includ- ing proteins (i.e., collagen, fibrin, gelatin, hyaluronic acid) and polysac- Charides (i.e., chitosan, alginate) have innate biological functionality, their mechanical properties and rates of degradation cannot be tailored as precisely as is possible with synthetically derived materials. Synthetic polymers are used widely in the clinic, with members of the polyester family most prevalent due to their proven safety profile, biodegradable properties and biocompatibility. Among these, polylactic acid (PLA), poly- glycolic acid (PGA), their co-polymer polylactic-co-glycolic acid (PLGA) and polycaprolactone (PCL) have been investigated extensively in pre-clini- cal studies for PDL regeneration. Additionally, ceramic-based materials 213 Bioengineering of the Periodontal Ligament that are used most commonly as bone grafts in periodontal regeneration studies include calcium phosphate (CaR)-derived matrices (i.e., tricalci- um phoshate [TCP) and hydroxyapatite [HA]) and bioactive glass. Instead of serving as scaffolds for directed growth of PDL, these are employed mostly as barrier membranes for guided bone regeneration (GBR) and can assist in maintaining the space needed for connective tissue growth that may have PDL-like formation (Hayashi et al., 2006). The method of delivering a scaffold to the site of defect varies depending on material properties, with naturally based protein scaffolds frequently seen in the form of highly hydrated gels or particulates, whereas synthetic materi- als are more likely to be delivered in solid form (Fig. 5, Table 3; Hollister, 2009; Scheller et al., 2009; Atala et al., 2012). Natural Polymers Among the various candidate scaffolds for PDL regeneration (Ta- ble 1), biological polymers are advantageous given their low cytotoxic- ity and immunogenicity, including their resemblance to an endogenous ECM. Most commonly formed as hydrogels or sponges, these polymers and polysaccharides can be used to fill irregular defects and provide a delivery vehicle with high cell-seeding efficiency. Hydrogels typically are prepared by chemically or physically crosslinking the polymer chains, us- ing chemical crosslinkers or pH-based reactions, respectively (Van Vlier- berghe et al., 2011). Cell-encapsulation within hydrogels prior to trans- plantation can be used for cell and gene delivery into periodontal defects. Collagen scaffolds alone still can facilitate improved periodontal tissue formation as compared to a vehicle control (Kosen et al., 2012), but not to the same extent as with cell-based and growth factor-based delivery. Jin and associates (2004) used a collagen matrix to deliver adenoviral PDGF-B to the periodontium, stimulating PDL formation and cemento- genesis with fiber insertion, including vascularization and newly formed periodontium not observed with collagen matrix alone. Interestingly, the majority of the collagen matrix delivered in combination with PDGF-B was resorbed by two weeks, with more scaffold remnants present at le- sions sites with collagen matrix alone (Jin et al., 2004). It is likely that the addition of growth factors increases the rate of tissue regrowth and inva- sion into the scaffold, thereby increasing its rate of degradation. 214 Decker et al. - Natural Polymers Forms of Delivery • Polysaccharides (chitosan, alginate) * Injectable * Proteins (collagen, fibrin) (PEG-PLGA, alginate) Synthetic Polymers • Polyesters (PLA, PGA, PCL) • Polyethers (PEG) Q • Particulate Bioceramics/Glass (collagen, B-TCP) * CaFOA ceramics (HA, B-TCP) • Bioactive glass Composites * Copolymers (PLGA, PEG-PLGA) • Solid * Polymer blends (PLA-chitosan) (PCL, PLGA) * Polymer-ceramics (PLGA-HA) _ Figure 5. Overview of biomaterials used for PDL regeneration. Polymers and ce- Famics can be combined into composite materials, which then are delivered as an injectable gel, in particulate form, or as a solid biomaterial scaffold. Adapted from Pilipchuket al., 2015. The presence of anti-microbial properties and anti-inflammatory effects of naturally occurring materials (e.g., chitosan) also make them applicable matrices and PDL cell-carriers for periodontal tissue regen- ºration (Yan et al., 2014). Composite collagen/chitosan scaffolds can be formed for mechanical reinforcement and have shown increased human PDL cell adhesion and growth compared to collagen or chitosan alone. Composite scaffolds increased the retention of Water, contributing to an increase in pore size and, thereby, the total internal surface area available for PDL cell invasion (Peng et al., 2006). To prolong the residence time of Scaffolds in vivo for complete tissue growth and remodeling, increased Stiffness and a decreased rate of degradation of natural matrices is pref- erable and can be accomplished through their integration with synthetic polymers. 215 Bioengineering of the Periodontal Ligament Table 3. Summary of PDL regenerative outcomes using scaffold design ap- proaches. SCAFFOLD TYPE BIOMATERIAL AND BIOLOGIC REGENERATIVE OUTCOME (WITH TIMEPOINT) REFERENCE Natural polymers Collagen Class || furcation defects in beagle dogs; increased PDL formation (4 weeks) Kosen et al., 2012 Chitosan Rat primary PDL cells Denuded root of rat intrabony periodontal defect; > 35% of surface covered by functional ligament, collagen fibers (4 weeks) Yan et al., 2015 Synthetic polymers PCL hPDL, BMP7 Rat periodontal fenestration defect; showed oriented ligament tissues with high periostin immunoreactivity; athymic rats (6 weeks) Park et al., 2012 PLGA Adipose-derived stromal cells Periodontal fenestration defects in Sprague Dawley rats; increased thickness of PDL tissue formation in the ASC/PLGA groups compared to PLGA alone at tooth crown and central areas (5 weeks) Akita et al., 2014 Bioceramics 3-TCP FGF-2 Beagle dog one-wall periodontal defect; increased length of PDL for- mation with Sharpey's fibers connecting regenerated cementum to bone (6 weeks) Anzai et al., 2010 Cap Experimental periodontitis induced in healthy beagle dogs; healing of periodontal tissues and PDL-like tissue formation observed between Cap and root surface (12 weeks) Hayashi et al., 2006 Composites PLGA/ Cap bilayered COnStruct Class || furcation defect in dogs; PDL with parallel and perpendicular collagen fibers, blood vessels and Sharpey fiber insertions (60 days) Carlo Reis et al., 2011 216 Decker et al. Table 3. (continued) SCAFFOLD BIOMATERIAL REGENERATIVE OUTCOME REFERENCE TYPE AND BIOLOGIC (WITH TIMEPOINT) Acute-type buccal dehiscence periodontal Bioglass/silk fibrin defects in beagle dogs; PDL glass/ region approaching 90% of Zhang et al., original height; similarity of 2015 BMP7, PDGF-B tissue to native PDL structure observed * (8 weeks) Composites e Histological characteristics PLGA/gelatin of PDL-like tissues observed using root-shaped Chen et al Dentin matrix, jaw bone implant socket 2015 • ? dental follicle in adult miniature swines at tooth extraction sites stem cells (DFSCs) (12 weeks) Athymic rat mesial Human-derived dehiscence model; Hasegawa PDL cell sheet immature thin PDL fibers et al., 2005 (4 weeks) Hvaluronic acid- Dehiscence defects in & tº º cell beagle dogs on molars; new Akizuki et al., PDL tissue formation in 3/5 2005 sheets of defects (8 weeks) Cell sheet e e Canine 3-wall infrabony de- engineering Woven PGA- fect with removed Iwata et al supported, three- cementum; well-oriented 2009 • y layered cell sheets collagen fiber formation (6 weeks) Three-layered Canine denuded root with cell sheets with one-wall intrabony defect; TSumanuma oriented PDL fibers, nerve et al., 2011 B-TCP/collagen fibers (8 weeks) Synthetic Polymers Synthetic biodegradable polymers (e.g., PLGA and PCL) are used Clinically in drug delivery systems, surgical sutures and orthopedic fixa- tion devices. These hydrolytically degradable materials undergo cleav- age of polymer chains to oligomers and monomers, producing lower molecular weight molecules in the process (Yildinimer and Seifalian, 2014). As with naturally-derived matrices, these polymers are most ap- plicable for periodontal regeneration when used in combination with cell 217 Bioengineering of the Periodontal Ligament and growth factor delivery (Park et al., 2012; Akita et al., 2014), as the material itself has little biological functionality, although it can be im- proved with the application of protein coatings. However, one of the key advantages of synthetic polymers includes their ability to be formed and processed in a variety of ways to yield highly aligned structures that can be achieved using electrospinning—a technology that allows for the for- mation of long, thin fibers on the nano and micron scale. This technique offers potential for controlled fiber orientation that can be used to influ- ence cell behavior through structural and physical cues that mimic ECM architecture, and allows control over a variety of parameters that deter- mine fiber dimension, density and porosity (Agarwal et al., 2008). Chen and coworkers (2015) used electrospun PLGA/gelatin sheets that were applied at tooth extraction sites in combination with dentin matrix, re- sulting in the formation of PDL-like tissues. Typically, poly-O-hydroxy acids (e.g., PLGA) result in an inflamma- tory reaction involving multi-nucleated cells. Rates of degradation vary depending on the molecular weight and copolymer ratios of PLGA, allow- ing it to be tailored more easily to the expected rate of tissue re-growth at the defect site. After treatment of critical-size supra-alveolar periodon- tal defects in dogs with rhGDF-5-coated B-TCP/PLGA scaffolds, Kwon and coworkers (2010) observed limited residual rhGDF-5/B-TCP/PLGA in two of five site at eight weeks, with higher residual amounts present in four of five sites with B-TCP/PLGA only. Given that residual material may obstruct periodontal tissue regeneration, it has been noted with synthetic materi- als as much as with natural materials that incorporation of growth factors increases the rate of matrix remodeling. BioCeramics A similar phenomenon of increased material resorption rates also has been noted with bioceramics. Koo and colleagues (2007) used a ceramic-based calcium carbonate carrier with TGF-3 in a critical-size, supra-alveolar periodontal defect, finding that the growth factor acceler- ated the degradation of the carrier relative to calcium carbonate with- out TGF-3. While growth of PDL-like tissues has been observed against Cap-based materials (Anzai et al., 2010), including TGF-3 with FGF-2 (Hayashi et al., 2006), these are not ideal for soft tissue regeneration. Instead, bioceramics are applied widely for periodontal therapy to treat 218 Decker et al. bone defects due to their biocompatibility, biodegradation and osteoin- ductive activity. Their use as guided-tissue regeneration (GTR) and GBR membranes prevents the down-growth of epithelial tissue into areas of the defect that require the regeneration of PDL, cementum and bone. Cap materials have been blended with polymers for this purpose in or- der to decrease their rate of resorption (Sowmya et al., 2013). Likewise, the incorporation of Cap can be advantageous for composite materials that serve as matrices for interphase tissue engineering of ligament-bone structures. Incorporation of HA onto electrospun collagen/PCL fibers re- Sulted in increased roughness due to calcium phosphate deposition and increased scaffold mineralization, and showed increased osteoinductive properties that caused osteogenic differentiation of scaffold-seeded PDL cells (Wu et al., 2014). Therefore, partial HA coating of hybrid scaffolds with potential for PDL regeneration may be used as a technique to pro- mote osteogenic differentiation of cells in specific regions of the scaffold that incorporate Cap-based materials. Cell Sheet Engineering Given the importance of biological factor delivery and retention of a viable cell population at the site of the defect, cell sheet engineering has emerged as a novel approach for targeted cell delivery without the use of biodegradable scaffolds. Cell sheet monolayers with intact cell-to- cell contacts and ECM can be harvested non-enzymatically using thermo- responsive polymeric surface cell culture plates that enable controlled cell adhesion (Matsuura et al., 2014). In vitro cultures of human-derived hPDL cell sheets show periostin expression and high alkaline phosphatase activity (Washio et al., 2010) and several studies confirm their potential for PDL regeneration using small and large animal models (Hasegawa et al., 2005; Iwata et al., 2009). Despite this scaffold-free approach, the in- Corporation of supporting synthetic (Iwata et al., 2009) or natural mem- branes (Akizuki et al., 2005) can be useful for increased cell sheet stability and ease of transplantation. The most promising approaches for the re- generation of PDL, therefore, lie in the design and fabrication of scaffolds that can be suited appropriately to the delivery of cells and growth fac- tors that will stimulate the regenerative process and allow for the remod- eling and growth of tissue that is similar structurally and functionally to native ligament. 219 Bioengineering of the Periodontal Ligament COMBINATORIAL SCAFFOLD APPROACHES Customized scaffold constructs are being developed to fit a pa- tient's periodontal defect better using novel technologies and unique combinations of formulated scaffolds. These include the use of 3D print- ing technology to generate material, which conform to defect site param- eters obtained via computed tomography (CT) or cone-beam tomogra- phy (CBT) scans. Multi-phasic designs incorporating several biomaterial layers that serve as regenerating platforms for each region of the peri- odontum (i.e., PDL, alveolar bone) increasingly are being investigated in combination with the delivery of cells and other biologic factors. These include the layer-by-layer deposition of a polymer that allows for con- trolled pore-size formation depending on printer resolution or the print- ing of a mold that can be cast to produce the final scaffold shape. Lee and associates (2014) demonstrated the use of PCL-HA layer deposition with microchannels ranging from 100-600 pum for the PDL, bone and ce- mentum/dentin regions of a hierarchical scaffold, which incorporated the spatiotemporal delivery of amelogenin, connective tissue growth factor and BMP-2 within each of the phases, respectively. Seeding of the constructs with dental pulp stem/progenitor cells and subcutane- ous implantation in immunodeficient mice resulted in aligned PDL-like collagen fiber formation connected to bone tissue that was sialoprotein- positive (Lee et al., 2014). Biphasic scaffold designs also have been ex- plored combining fabrication technologies (e.g., 3D printing and elec- trospinning with cell sheet engineering). Vaquette and coworkers (2012) developed a construct of PCL with B-TCP for the bone region and an electrospun PCL membrane for the PDL region with placement of sev- eral PDL cell sheets, which improved attachment to the dentin surface relative to constructs that did not incorporate the sheets. Hybrid PCL/ PLGA and fiber-guiding PCL scaffolds with perpendicularly oriented mi- crochannels were developed by Park and colleagues (2010, 2012) utiliz- ing computational design and solid-free form fabrication for the direct guidance of PDL fibers in vivo, resulting in more predictable formation of organized periostin-positive ligamentous structures. Findings from these studies indicate that bone-ligament complex regeneration in the clinic has the potential to be guided via 3D printed, hybrid scaffolds with a multi-compartmental architecture designed based on patient defect CT scans (Park et al., 2013). This system recently was demonstrated suc- 220 Decker et al. cessfully in a patient over the span of one year (Rasperini et al., 2015). The anatomical complexity of PDL tissue makes combinatorial approach- es and advanced multi-phasic scaffold designs appealing for improving its Structural and functional regeneration. Ligaplants An additional, exciting and clinically relevant combinatorial ap- proach is the use of PDL fibroblast-seeded scaffolds. This technology is motivated by findings that implants placed near retained root tips devel- oped a periodontal ligament (Buser et al., 1990). Use of these “ligaplants” in Vivo has provided a novel engineering Strategy to create physiologically and anatomically correct PDL (Gault et al., 2010). In a human case series of nine patients, autologous cells were isolated from the PDL, cultured on titanium implants in a bioreactor system and subsequently implanted. Implantation of the PDL-coated implant fixtures demonstrated regen- eration of alveolar defects both radiographically and histologically. His- tological sections in this study depicted transplanted PDL cells oriented obliquely in relation to the bone and implant surfaces. Lin and associates (2011) demonstrated similar results using PDL-derived progenitor cells in a rat model. In the future, such hybrid, bio-inspired implants using a combination of proper biomaterials and cellular platforms could allow for movement of an implant using standard orthodontic treatments (Gian- nobile, 2010). FUTURE DIRECTIONS Bioengineering the PDL is one component of the larger goal of Complete periodontal regeneration. Many clinical techniques are avail- able to aid in achieving this goal; however, none are predictable Com- pletely. Future research is needed to understand fully the mechanisms involved in PDL development in the context of interfacing structures, cell migration in the periodontal apparatus, the effects of inflammation me- diators on regeneration and also the individualized patient wound heal- ing response. Tissue engineering has provided tools to aid in tissue regenera- tion including biologics, autologous stem cells and biomaterials. More research is needed to optimize these components in a regenerative con- text. Currently, pharmaceutical strategies to bring the endogenous cells 221 Bioengineering of the Periodontal Ligament to the injury site need to be investigated further. Specifically, temporal and spatial resolution of biologics is critically important to direct cell mi- gration, proliferation and ECM secretion, particularly in PDL regenera- tion. Furthermore, the use of biomaterials as a cell carrier provides mor- phological boundaries, multi-agent release characteristics and dynamic structural changes to facilitate the proper mechanical and structural properties of the new tissue. Further optimization of biomaterials that facilitate tissue growth and simultaneous biologically compatible degra- dation is important to ensure long-term success and prevent microbial infection (or reinfection) in the periodontal microenvironment. Understanding periodontal and specifically PDL regeneration in the context of personalized medicine will be an important consideration moving forward in this field. Providing treatment therapies for specific patients, as opposed to a generalized treatment modality for all defects, is paramount for successful and predictable regenerative outcomes. In this regard, a specific patient may be more likely to have an exacerbated inflammatory response that would require a more biologically sensi- tive biomaterial, while another patient with a similar periodontal defect might require a longer chemotherapeutic release to achieve the same regenerative response. Epigenetics is a field that is beginning to provide this essential patient-specific information. However, more extensive re- search in this area is needed to make epigenetic tools accessible in a clini- cal setting that is realistic for therapeutic use. ACKNOWLEDGEMENTS This work was supported by NIH/NIDCR DE13397 to WVG, NSF Graduate Research Fellowship DGE1256260 to SPP and The University of Michigan School of Dentistry Dean's Scholarship to AMD. 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This chapter highlights the developing science and technologies in tissue engineering for potential application for oral hard and soft tissue reconstructions. KEY WORDS: regeneration, wound healing, growth factors, scaffolds, cell therapy INTRODUCTION Tissue engineering approaches to restore an adequate periodon- taland/or alveolar ridge anatomy substantially have advanced the field of periodontal regenerative medicine and promise to bring greater predict- ability to common clinical challenging scenarios (Fig. 1). Most such thera- pies require one or more factors to succeed including bioactive agent(s), Cells and scaffolds. This chapter presents current and emerging tissue en- gineering strategies and approaches for the successful regeneration of oral hard and soft tissues. BIOLOGICAL EFFECTS OF GROWTH FACTORS A substantial effort in periodontal research has been geared toward understanding the impact of tissue growth factor applications on bone and tissue regeneration (Giannobile, 1996; Anusaksathien and Giannobile, 2002; Nakashima and Reddi, 2003; Raja et al., 2009). Advances in molecular cloning have made unlimited quantities of recombinant 243 Tissue Engineering Approaches Periodontal Defects - Figure 1. Today's clinical practice demands treatment alternatives to overcome challenging situations that address compromised patient's esthetics and achieve a functionally adequate alveolar anatomy and long-term tissue stability. growth factors available for applications in tissue engineering. Recombi- nant growth factors known to promote skin and bone wound healing, such as platelet-derived growth factors (PDGFs; Rutherford et al., 1992, Giannobile et al., 1994; Camelo et al., 2003; Ojima et al., 2003; Nevins et al., 2005; Judith et al., 2010), insulin-like growth factors (IGFs; Lynch et al., 1991; Giannobile et al., 1994, 1996; Howell et al., 1997), fibroblast growth factors (FGFs; Terranova et al., 1989; Sigurdsson et al., 1995, Giannobile et al., 1998; Takayama et al., 2001; Murakami et al., 2003) and bone morphogenetic proteins (BMPs; Gao et al., 1995; Wikesjö et al., 2004; Huang et al., 2005) have been used in pre-clinical and clinical trials for the treatment of large ridge and alveolar deficiencies (Jung et al., 2003; Fiorellini et al., 2005; Nevins et al., 2005). Currently, there are two recombinant proteins approved to enhance and promote alveolar ridge regeneration: Bone Morphogenetic Protein-2 (BMP-2) and Platelet Derived Growth Factor-BB (PDGF-BB). Platelet Derived Growth Factor (PDGF) PDGF is a member of a multi-functional polypeptide family that binds to two-cell membrane tyrosine kinase receptors (PDGF-R and PDGF-R) and subsequently exerts its biological effects on cell prolifera tion, migration, extracellular matrix synthesis and anti-apoptosis (Kaplan et al., 1979; Seppä et al., 1982; Heldin et al., 1989; Rosenkranz and Ka- zlauskas, 1999). PDGF-o and -3 receptors are expressed in regenerating 244 Rios periodontal soft and hard tissues (Parkar et al., 2001). In addition, PDGF initiates cell chemotaxis (Nishimura and Terranova, 1996), mitogenesis (Oates et al., 1993), matrix synthesis (Haase et al., 1998) and attachment (Zaman et al., 1999). More importantly, in vivo application of PDGF alone or in combination with IGF-1 enhances mineralized tissue repair (Lynch et al., 1991; Rutherford et al., 1992; Giannobile et al., 1994, 1996; Haase et al., 1998). PDGF has been shown to have a significant regenerative impact on PDL cells as well as on osteoblasts (Figs. 2-3; Matsuda et al., 1992; Oates et al., 1993; Marcopoulou et al., 2003; Ojima et al., 2003). Bone Morphogenetic Proteins (BMP) BMPs are multi-functional polypeptides that belong to the TGF-3 superfamily of proteins (Wozney et al., 1988). The human genome encodes at least twenty BMPs (Reddi, 1998). BMPs bind to type I and II receptors that function as serine-threonine kinases. The type I receptor protein kinase phosphorylates intracellular signaling substrates are Cailed Smads (Sma gene in C. elegans and Mad gene in Drosophila). The phosphorylated BMP-signaling Smads enter the nucleus and initiate the production of bone matrix proteins leading to bone morphogenesis. The most remarkable feature of BMPs is the ability to induce ectopic bone formation (Urist, 1965). BMPs not only are powerful regulators of cartilage and bone formation during embryonic development and regeneration in postnatal life, they also participate in the development and repair of other organs such as the brain, kidney and nerves (Reddi, 2001). Studies have demonstrated the expression of BMPs during tooth development and periodontal repair including that of alveolar bone (Aberg et al., 1997; Amar et al., 1997). Investigations in animal models have shown the potential repair of alveolar bony defects using recombinant human bone morphogenetic protein-12 (rhBMP-12) or rhBMP-2 (Lutolf et al., 2003; Wikesjö et al., 2003). In a clinical trial by Fiorellini and colleagues (2005), rhEMP-2 delivered by a bioabsorbable Collagen Sponge revealed significant bone formation in a human buccal wall defect model following tooth extraction when compared to collagen Sponge alone. Furthermore, BMP-7, also known as osteogenic protein-1, Stimulates bone regeneration around teeth, endosseous dental implants and in maxillary sinus floor augmentation procedures (Rutherford et al., 1992; Giannobile et al., 1998; van den Bergh et al., 2000). 245 Tissue Engineering Approaches Clinical Presentation Etiology Treatment DxMocalized chronic moderate Primary bacterial plaque in a jº host GTR + biologic USal trauma periodontiis Secondary ename pear 4 Figure 2. The use of PDGF-BB for periodontal regeneration is currently an existing treatment modality that favors early healing events geared to promote the recruitment of periodontal ligament cells to the wound site. Nonetheless, incorporating a growth factor to enhance a regenerative outcome requires a proper diagnosis, recognition of the primary and known secondary etiologic factors, and the use of sound surgical principles to optimize an adequate wound healing process. CELL THERAPY Cells are the center of new tissue growth and differentiation. In cell-based regenerative medicine, cells are delivered to a defect Site with the goal of improving the regeneration process (Mao et al., 2006). Cell delivery approaches are used to accelerate alveolar ridge regeneſ ation through two primary mechanisms: 1) to use cells as carriers tº deliver growth or cellular signals; and 2) to provide cells that are able to differentiate into multiple cell types to promote regeneration. The use of cells as vehicles to deliver growth factors can stimulate an eſ' dogenous regeneration process (Discher et al., 2009). This strategy has been investigated intensively in both soft and hard periodontal tissuº 246 Figure 3. The successful use of PDGF has been reported in combination therapy in the context of implant site development. Autograft, alloplast, allograft or Kenograft material is hydrated using the soluble growth factor to correct existing alveolar defects. In the highly esthetic area, the treatment often is staged over a period of four to six months to allow the alveolar bone maturation prior to implant placement. ſºgeneration. Stem cell research has gained substantial momentum in the past few years and its effects on healing and regenerative potential have been studied extensively. Mesenchymal stem cells (MSCs) are self-renewing cells that ºn differentiate into a variety of cell types that form mesenchymal and ºnnective tissues (Pittenger et al., 1999; Mao et al., 2006). Bone marrow Stromal cells are the most widely investigated MSCs because they ** accessible easily. Bone marrow stromal cells initially were isolated and described nearly 50 years ago based on their ability to adhere to plastic substrates of cell culture plates (Becker et al., 1963). Since then, 247 Tissue Engineering Approaches this simple protocol has been used widely to isolate MSCs from many tis- sues such as adipose tissue, muscle, liver, pancreas and Cartilage (Ward et al., 2010). MSCs have a tremendous potential in regenerative medicine owing to their multi-potency and capability to form a variety of tissues. Regarding periodontal tissue engineering, both extra-oral and intra-oral stem cells can be harvested and then subjected to enrichment and expan- sion techniques. Within this context, multiple sources of stem cells have been evaluated for the treatment and regeneration of the alveolar ridge (Huang et al., 2009). There is strong potential for the use of MSC from sources out- side the oral cavity for regeneration of the oral and craniofacial complex (Noth et al., 2010; Ward et al., 2010). Bone marrow stromal cells also have been shown to promote bone healing and dental implant osseoin- tegration (Bueno and Glowacki, 2009). In a series of studies, Yamada and associates (2004) used a combination of Platelet Rich Plasma (PRP) as an autologous scaffold with in vitro-expanded bone marrow stromal cells to increase osteogenesis in dental implant surgery. This “autogenous in- jectable bone treatment” resulted in higher marginal bone levels, better bone-implant contact and increased bone density compared to controls. Recently, cells harvested from the bone marrow have been driven down MSC pathways via a single-pass perfusion process to promote bone re- generation in tooth extraction socket and sinus floor augmentation pro- cedures (Kaigler et al., 2010). Just as PDL is essential for the osteogenesis and cementogenesis during development and remodeling, cells derived from this tissue are necessary for the appropriate healing response to injury (Shimono et al., 2003). Transplantation of PDL cells has shown the potential to regenerate alveolar bone in vivo (Nakahara et al., 2004). Besides stem cell delivery, other strategies have been developed based on the concept that transplanted cells will promote regeneration by secreting growth factors via autocrine and paracrine pathways. Allo- genic foreskin fibroblasts recently have been shown to be safe and are able to promote keratinized gingiva formation at gingival recession de- fects (Nevins et al., 2005). A tissue-engineered living cellular construct composed of viable neonatal keratinocytes and fibroblasts was report- ed to achieve comparable clinical outcome as gingival graft (McGuire 248 Rios et al., 2008) with strong potential to promote tissue neogenesis through the stimulation of angiogenic signals (Morelli et al., 2011). The significant potential of cell-based therapy to form a variety of periodontal tissues is documented well in the references mentioned above. This approach delivers important cues that would drive the regenerative process (Fig. 4). Scaffolding Matrices Used to Deliver Cells and Genes Scaffolding matrices are used in tissue engineering to provide an environment in which space is created and maintained over a period of time for cellular growth and tissue in-growth. These matrices serve as BEFORE Figure 4. The use of allogenic-cultured keratinocytes and fibroblasts in bovine Collagen has been used successfully for intra- and extra-oral applications. This therapy brings together existing tissue engineering principles and acts as a biologically active dressing to promote soft tissue regeneration. Intra-orally, its therapeutic effect supports the long-term sustainable increase of the attached Singiva and a highly esthetic outcome. 249 Tissue Engineering Approaches three-dimensional (3D) template structures to support and facilitate peri- odontal tissue regeneration physically when combined with cell- or gene- based tissue engineering. Over the past two decades, scaffolds have been developed, studied and utilized extensively. Several fundamental require- ments have been proposed for scaffold design in their applications to tis- sue engineering (Murphy and Mooney, 1999). These include: 1. Provide a 3D architecture that supports a desired volume, shape and mechanical strength; 2. Consist of a high porosity and surface-to-volume ratio with a well-interconnected open pore structure to promote high seeding density and embracing of bioactive molecules; 3. Be biocompatible; and 4. Degrade at a controlled rate and pattern that allows sufficient support until tissue defects are re-grown fully. Scaffolds also can be engineered to serve as supportive carriers that conduct a sustained release of bioactive factors, thereby inducing stimuli for tissue formation. Cells can be transplanted via tissue engi- neered scaffolds (Murphy and Mooney, 1999) that provide adhesion and anchorage for interacting stem cells in order to control the presenta- tion of adhesion sites, thereby improving cell survival and participation (Alsberg et al., 2003; Davis et al., 2005). By furthering the pattern of tissue structure formed by stem cells, a new mandible was formed in a patient by using a metal and polymer scaffold seeded with stem cells and BMPs (Warnke et al., 2004). Bioactive molecules, such as growth factors, also may be encapsulated into nano/micro particles that are embedded into the matrices to aid in their sustained release from scaf- folds. Other approaches in using scaffolds include mimicking stem cell niches to regulate daughter cell proliferation, differentiation and disper- sion into surrounding tissue or attracting useful cells to a desired ana- tomic site (Discher et al., 2009). Several scaffold fabrication technologies as applied to periodontal tissue engineering will be discussed including conventional pre-fabricated scaffolds (e.g., particulated, solid form and injectable scaffolds that are adapted or administered into a periodontal 250 Rios defect) and novel image-based designs that result in a 3D-printed scaffold that is custom fit to a defect. PRE-FABRICATED SCAFFOLDING MATRICES Conventional scaffolds used to regenerate tissue in vivo are pre- fabricated and many techniques have been described that produce both natural and synthetic polymeric scaffolds. Naturally derived scaffolds include autografts, allografts and xenografts. Alloplasts and other polymers are synthetically engineered materials that consist of bioactive molecules serving a purpose similar to that of natural scaffolds. Naturally Derived Scaffolds There are many naturally derived scaffolds used for tissue engineering applications. Freeze dried bone allograft (FDBA) is a mineralized bone graft that has been suggested to promote osteo- inductive and osteo-conductive bone regeneration, although reports of its regenerative effectiveness have been mixed (Altiere et al., 1979; Dragoo and Kaldahl, 1983; Goldberg and Stevenson, 1987). Variability in preparations of the allograft, its regenerative potential and the osteoinductive ability exists in different bone banks (Shigeyama et al., 1995; Schwartz et al., 1996). Nonetheless, FDBA appears to be a practical material of choice for regeneration of periodontal attachment apparatus. Xenogenic grafts' physical and chemical similarities to human bone matrix have allowed them to demonstrate success in various periodontal and implant-related bone repair cell delivery applications (Nevins et al., 2006). Deproteinized bovine bone mineral have osteo-conductive properties (Figs. 5-6; Hämmerle et al., 1998). Synthetic Biomimetic Polymer Scaffolds Synthetic polymers have been studied extensively as gene therapy delivery systems since they provide greater freedom for property modification (e.g., control of macrostructure and degradation time), Compared to naturally derived scaffolds (Jang et al., 2004). Furthermore, the release mechanism and exposure duration of bioactive molecules (e.g., growth factors) can be controlled (Ramseier et al., 2006). By acting as a localized gene depot, Synthetic polymer scaffolds have the ability to maintain the therapeutic level of encoded proteins that limit 251 Tissue Engineering Approaches CBCT EVALUATION OFIMPLANT SITE 6 m AFTER GBR Figure 5. Naturally derived scaffolds for soft and hard tissue regeneration are used successfully for implant site development. The success of such therapies can be determined by volumetric assessment of the regenerated tissues using cone-beam computed tomography (CBCT). This can help the clinician further in customizing and optimizing the treatment outcomes and the quality of the healed site. unwanted immune response and potential side effects (Ghali et al., 2008). Polymers such as the poly-lactic-co-glycolic acid (PLGA) have drawn much attention for their excellent properties for encapsulation of genes (Mundargi et al., 2008). PLGA microspheres have been used pre- viously to deliver antibiotics, as an occlusive membrane for guided tissue regeneration, as a growth factor carrier for periodontal regeneration and for cementum and complex tooth structure engineering (Williams et al., 2001; Kurtis et al., 2002; Young et al., 2002; Jin et al., 2003; Cetiner et al., 2004; Moioli et al., 2006). Microsphere systems have demonstrated promising results in the past; however, more novel approaches to micro- technologies today are focusing on nano-sized particles (Agarwal and Mallapragada, 2008). Nanotechnology has attracted much attention for use as a therapeutic agent and for gene delivery with several studies and reviews having delineated its contribution and capability to meet challenges of current regeneration therapy (Agarwal and Mallapragada, 2008; Mundargi et al., 2008; Sanvicens and Marco, 2008). 252 Rios Figure 6. The concept of bone graft osteo-conductivity and bio-functionality of the regenerated tissue often is inferred from clinical observations based on tissue volume stability over time. Histologically, this property is reflected on the native bone growing over the scaffold and supports an increased cellularity that assists the host in the remodeling processes that are necessary to sustain the COnStant mechanical demands. In the dental field, hyaluronic acid (HA) has been demonstrated to festore periodontal defects and to carry and deliver growth factors such as BMPs and FGF-2 (Wikesić et al., 2003). Inorganic calcium phosphate- based materials also have been used as delivery systems. Materials such as B-tricalcium phosphate (B-TCP) are synthetic scaffolds that can be used to repair osseous defects around teeth or dental implants by acting as a bone substitute or as a carrier for growth factor delivery (Gille et al., 2002). Hydrogels, formed by the crosslinking or self-assembly of a Variety of natural or synthetic hydrophilic polymers to produce structures 253 Tissue Engineering Approaches that contain over 90% water, are obtained from natural materials (e.g., collagen chitosan, dextran, alginate or fibrin). They are favorable for tissue engineering due to their innate ability to interact with and mediate degradation by cells (De Laporte and Shea, 2007; Moioli et al., 2007; Agarwal and Mallapragada, 2008). Vector release from hydrogels is dependent upon the physical structure and degradation of the hydrogel, and its interactions with the vector (De Laporte and Shea, 2007). COMPUTER-BASED APPLICATIONS IN SCAFFOLD DESIGN AND FABRICATION Computer-based applications in tissue engineering are Some of the more recent developments in scaffold design and fabrication for cell and gene delivery (He et al., 2010). This type of technology—image-based design—has been used in recent years to define virtual 3D models for surgical planning by utilizing data from computed tomography (CT) and magnetic resonance imaging (MRI). Specifically, in tissue engineering, CT or MRI data is used to define the 3D anatomical geometry of a defect and can be used to create a template for a scaffold on a global anatomical level. This 3D-printed scaffold, since it is produced from the 3D model, would fill the defect space precisely (Fig. 2). Furthermore, the architecture of the scaffold can be defined to design the heterogeneous internal structure in a way to create region-specific variations in porous microstructures and scaffold surface topography, thereby altering material and biological properties in specific regions of the scaffold (e.g., modulus, permeability and cell orientation; Hollister et al., 2002). Various novel delivery scaffolding systems are being studied and fabricated extensively and are demonstrating capabilities to meet the challenges of current regeneration therapy. There are several techniques and technologies that have been developed and applied to fabrication of scaffold matrices. Only through further research and development in this area, along with cell-based and gene therapy, can tissue engineering continue to advance. FUTURE PERSPECTIVE Tissue engineering is making an important impact on bone regenerative therapy. The use of cell and gene therapy to enhance and 254 Rios direct periodontal wound healing into a more predictable regenerative path is being exploited in bioengineering efforts that aim to develop a therapeutic system to promote bone repair; however, numerous challenges remain. Today, there are a number of developing systems that have the potential to optimize tissue-healing biology. A major obstacle that remains is how to maximize the utility of cells/gene delivered to a passive or permissive environment where there is context for the type of Cell needed, but in which few biological signals are provided to encourage normal cell function. The tissue-engineering field still needs to confront other hurdles, such as identifying cell sources and clinically relevant cell numbers, the integration of new cells into existing tissue matrices and the achievement of functional properties of tissue equivalents using an expanded repertoire of biomaterials. Major constraints to the cell- and gene-transfer fields remain in the practical and regulatory requirements to apply these technologies to the clinical arena. Collectively, the cell-based, scaffold and gene therapy methods interface and complement each other to enhance the potential to restore tissue function and structure in a predictable manner. It is expected that the increased use of bioactive molecules (e.g., BMPs and PDGF) to accelerate and enhance the healing potential of the defects will bring about faster, easier and predictable treatment outcomes in the future. The success and future of periodontal regenerative therapy thereby will be supported by our understanding and ability to recognize clinical situations that will benefit from any of these new emerging technologies. CONCLUSIONS Much research has been done in the field of advanced bone grafting for implant dentistry. 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Effect of recombinant human platelet- derived growth factor-BB and bone morphogenetic protein-2 application to demineralized dentin on early periodontal ligament cell response. J Periodontal Res 1999;34(5):244-250. 264 TISSUE ENGINEERING OF ALVEOLAR BONE USING CLINICAL STEM CELL THERAPIES Giorgio Pagni, Archana Rajan, William V. Giannobile, Darnell Kaigler ABSTRACT It has been established that the loss or absence of teeth leads to alveolar ridge resorption. To replace missing teeth with dental implants at sites with atrophied ridges, there is a need for adequate bone quality and volume. The use of innovative ridge augmentation techniques utilizing stem cells can enhance treatment outcomes as part of dental implant therapy. Our group has initiated and completed clinical trials investigating stem cell therapies for the regeneration and engineering of alveolar bone. These trials have been regulated by the U.S. Food and Drug Administration (FDA) and conducted under investigational new drug (IND) approvals. These first-in-human studies have served as an important foundation upon which to develop widespread clinical protocols using oral- derived stem cells for bone and tooth regeneration. This chapter outlines promising results of these early-stage clinical studies and continued efforts toward these translational projects are ongoing. KEY WORDS: stem cells, alveolar bone, FDA, clinical trial, bone regeneration ALVEOLAR BONE DEFICIENCIES AND CURRENT THERAPIES Oral and craniofacial bone defects secondary to congenital deformities, disease and injury are common and highly variable. The Clinical management of these conditions represents a significant healthcare burden (Abbott, 2014). When these conditions are associated with tooth loss or the congenital absence of teeth, the alveolar bone of the jaw does not receive the functional stimulus innately generated by the teeth and their supporting structures resulting in further bone resorption (Cardaropoliet al., 2005). The consequence of these processes is severe horizontal and vertical bone deficiencies and inadequate bone volume to restore these areas with functional and esthetic tooth 265 Tissue Engineering of Alveolar Bone replacements. In such cases, advanced alveolar bone reconstruction, followed by dental prosthetic rehabilitation, is needed to re-establish form, function and esthetics to these regions of the oral cavity (Greenberg et al., 2012). With the appropriate clinical conditions, dental implant therapy serves as the most functional and esthetic therapeutic treatment option for the replacement of missing teeth (Fugazzotto, 2005). A key determinant that underlies the success of dental implant therapy, however, is the qualitative and quantitative nature of the bone support into which implants are placed (Stanford, 2003). It is established well that a significant number of patients who seek dental implant therapy lack sufficient bone and require reconstructive bone grafting procedures to enable implant placement and improve the function, esthetics and longevity of dental implants (Esposito et al., 2008). Regardless of the extent or severity of the bone defects, the success of bone regenerative and reconstructive procedures used to correct them depends on the presence of osteogenic and vascular precursor cells resident in the surrounding tissues (McAllister and Haghighat, 2007; Meijer et al., 2008). As such, the wound healing and regenerative processes make small, localized defects more manageable and predictable to treat, while larger reconstructions of severe deficiencies are significantly more challenging. Despite recent advances in tissue engineering and regenerative medicine, reconstruction of large defects still relies primarily on treatments involving large autogenous grafts, allografts, xenografts and synthetic alloplastic materials (Cochran, 1996; Coulthard et al., 2003; Peleg et al., 2010). Current treatment modalities in the rehabilitation of oral and craniofacial tissues provide functional and structural restoration of the compromised or lost tissue, though many of these approaches do not meet the need for more biologic and physiologic treatment outcomes. Newer, more targeted cell and tissue- based therapies are needed to overcome the problematic limitations of traditional treatments (Ohazama et al., 2004; Mao et al., 2006; Caplan, 2007; Zaky and Cancedda, 2009). STEM CELL THERAPIES Stem cell therapy is a promising tissue engineering strategy to enhance tissue regeneration and promote de novo formation of both 266 Pagni et al. hard and soft tissues (Ouarto et al., 2001; Macchiarini et al., 2008; Rayment and Williams, 2010). The emergence of cell therapy science has been a gateway to new paradigms of treatment for tissue regeneration; however, a major challenge in this new arena is the identification of the most appropriate cell source for use in these regenerative approaches. Recent evidence cites different cell types of different origins that have "stem-like” properties and the capacity to regenerate a variety of Craniofacial tissues including cartilage, bone, blood vessels, salivary gland, gingiva and tooth tissues (Krebsbach, and Robey, 2002; Yan et al., 2006; Macchiarini et al., 2008; Delaere et al., 2010). Due to their bone-forming capacity in pre-clinical model systems, bone marrow- derived mesenchymal stem cells have gained much attention for use in Cell-based tissue regenerative approaches (Krebsbach et al., 1997; Gao et al., 2001; Holtorf et al., 2005; Kaigler et al., 2006). Recently, clinical reports have emerged that investigate cell therapy for craniofacial applications (Gimbel et al., 2007; Meijer et al., 2008; Shayesteh et al., 2008; McAllister et al., 2009; Mendonça and Juiz-Lopez, 2011; Rickert et al., 2011; Behnia et al., 2012; Yamada et al., 2013; Zamiri et al., 2013; Janssen et al., 2014; Sándor et al., 2014). These early reports have had modest and mixed results, but a major limitation they share is the limited characterization of the cell populations used for therapy. Limited knowledge regarding the cell population used as part of the cell therapy makes it difficult to understand the basis for and mechanisms underlying the study outcomes (Gimbel et al., 2007; Marcacci et al., 2007; Pelegrine et al., 2010; Soltan et al., 2010). Tissue Repair Cells (TRC; developed by Aastrom Biosciences Inc., Ann Arbor, MI) represent an autologous, bone-marrow-derived mixed-cell population containing mesenchymal stem cells. TRC are produced by an automated cell manufacturing process that uses a single-pass perfusion (SPP) system. In SPP, culture medium is replaced continuously by fresh medium at a slow, controlled rate without the disturbance, removal or passaging of cells; this enables a clinical-scale expansion of TRC to numbers not achievable through conventional culturing techniques. Through this process, cell populations are produced, which are enriched for CD90+ mesenchymal stem cells and CD14+ monocytes. TRC cell populations have been shown to have strong regenerative and bone differentiation potential in vitro and in vivo (Dennis et al., 2007). In this chapter, we describe our more recent work that has utilized TRC as part 267 Tissue Engineering of Alveolar Bone of autologous clinical cell therapies to regenerate bone defects of the alveolar ridge and enable dental implant therapy (Fig. 1). CLINICAL REPORTS OF ALVEOLAR BONE ENGINEERING USING CD90+ ENRICHED STEM CELLS Extraction Defects In a randomized controlled clinical trial (RCT) involving 24 pa- tients, we evaluated TRC therapy for the treatment of localized alveolar ridge defects created following tooth extraction (www.clinicaltrials.gov; CT00755911). There were four experimental groups with six patients in each group, as follows: 1. Six-week control group (guided bone regeneration [GBR] therapy); 2. Six-week TRC treatment group; 3. Twelve-week control group; and 4. Twelve-week TRC treatment group. Figure 2A shows the packaged TRC cell product; Figure 2B shows collection of cells into a 5 cc syringe just prior to use. Two co of 1.5 x 10' cells/cc cell suspension are shown being loaded onto a gelatin sponge scaffold (Fig. 2C) to the point of saturation of the sponge (Fig. 2D). Cells were allowed to adhere to the gelatin sponge for fifteen minutes prior to their placement into the osseous defect site. Scanning electron microscopy (SEM) shows the distribution of cells within the internal aspect of the Sponge just prior to transplantation and confirms that cells were distributed throughout the sponge and tended to adhere in large clusters (Fig. 2E). Figure 3A shows standardized digital radiographic (SDR) images of GBR and TRC treated sites at the time of tooth extraction (baseline) and six weeks after therapy. At six weeks, there was greater radiographic bone height achieved in the TRC group than in the GBR group, as measured by the percentage of the radiographic bone fill within the extraction defect (Fig. 3B; p = 0.01). At twelve weeks, the GBR group displayed 74.6 + 3.3% bone fill while the TRC groups showed 80.1 + 2.0% bone fill (p = 0.28; Table 1). Figure 4A shows photographic images of TRC and GBR treatment sites at baseline, six weeks after treatment and one year after intial 268 Pagni et al. ne ſnarrow Harvest bo Prepare stem cells Via SPP for 12d Load stem cells onto Cells repair scaffold bone defect Figure 1. Cell therapy approach. Cell therapy approach for the treatment of jawbone defects. Reproduced with permission from Mary Ann Liebert, Inc.; Kaigler et al., 2010. Surgery, fully restored. Clinically, in the GBR-treated sites, the regen- erated tissues at six weeks appeared highly vascular and fibrous and most Specimens were notably soft during biopsy harvest. Overall, the ſegenerated tissues in the TRC sites exhibited a bone-like appearance Clinically, were denser and demonstrated high vascularity during biopsy harvest. In all four groups, dental implants were placed in a stable man- ſleſ. Due to a greater number of residual bone defects present in the §ſoups initially treated with GBR (Fig. 4B), however, there was a greater need for the GBR groups (six and twelve weeks) to receive secondary bone grafting procedures at the time of implant placement, relative to the need within the TRC treated groups (Table 2). Additionally, in the GBR groups at both six and twelve weeks, there was an approximate six-fold greater percentage implant exposure that necessitated more ex- tensive Secondary grafting, compared to this need in the TRC-treated 269 Tissue Engineering of Alveolar Bone Figure 2. Cell preparation for cell delivery. A Cell packaging following production of TRC. B. Collection of TRC into syringe for loading of cell scaffold. C. Loading of gelatin sponge scaffold with TRC cell suspension. D: Saturation of gelatin Spongº with 2 ml of 1.5 x 107 cells/ml suspension. E. Scanning electron micrograph of cells within the interior aspect of the gelatin sponge fifteen minutes following loading. groups (p & 0.04; Fig. 4B, Table 2). For each of the treatment group: following the regenerative procedures, all implants achieved clinical evidence of integration into the restored bone and were able to sustain biomechanical loading when restored with an implant restoration sº months following placement. Implant stability was followed for one yeaſ and all implants remained integrated functionally at study completion. 270 Pagni et al. Baseline defect 6 Weeks GBR A Linear Radiographic Bone Height GBR 100 >k TRC º º º 3. º 5 E 40 g 3 sº 20 0 B 6 weeks 12 weeks figure 3. Tissue repair cells (TRC) promote regeneration of alveolar bone de- ſects. A Digital radiographic images of linear bone height density measures for guided bone regeneration (GBR, control treatment) and TRC groups at the time of tooth extraction (baseline) and six weeks after treatment. Standardized digi- tal radiography (SDR) was used to assess linear changes in radiographic bone height from baseline to six and twelve-week time points. In the baseline im- *ées, the orange lines show the full extent (height) of the original defect, created following extraction of the tooth. In the six-week images, the green lines show the extent to which there was radiographic bone fill. B: Heights were calculated 271 Tissue Engineering of Alveolar Bone (Fig. 3 continued) and the linear length of the bone fill was determined by calculating the percentage of the original defect, which was filled with radiographic evidence of bone. Reprinted with permission from Cognizant Communication Corporation; Kaigler et al., 2013. Baseline defect 6 weeks Implant reconstruction --- A GBR TRC Residual Defects - 45 LGBR º º TRC # as ; : # 25 # 20 15 º 10 º: º: - - - - º 5 B - - o 6 weeks 12 weeks Figure 4. A: Clinical photographs of the defect area created immediately following removal of the tooth, at re-entry into the site six weeks following treatment and twelve months following treatment after full restoration of the site with an oral implant supported crown. B: In some patients, residual bony defects were noted at the time of re-entry into the defect sites; in other patients, remaining bone deficiencies were identified during implant placement. There was significantly greater implant exposure in those patients receiving GBR versus those patients receiving TRCs (p & 0.04). Reprinted with permission from Cognizant Communication Corporation; Kaigler et al., 2013. Table 1. Linear radiographic bone height changes. LINEAR BONE HEIGHT (%) Time point 6 weeks 12 weeks Group GBR TRC GBR TRC Mean 55.3 78.9 74.6 30.1 Mean difference (GBR & TRC) 23.6 5.4 95% CI 6.0.2, 41.09 -12.11, 22.95 p-value 0.01 0.28 272 Pagni et al. Table 2. Residual bone defects and secondary grafting. 6 weeks 12 weeks GBR TRC GBR TRC Mean 9% linear implant exposure 29.2 5.1 25.3 3.8 95% CI (-1.2, 60) (-8.5, 18.7) (-0.9, 51.5) (-6.1, 14) p-value 0.04 0.03 CaseS requiring Secondary bone 5 2 3 2 grafting Time point Mean amount ºftona graft used 0.23 0.09 | 0.08 0.05 CC 95% CI (0.02, 0.44) (-0.01, 0.2) (-0.02, 0.2) (-0.05, 0.16) p-value 0.08 0.31 Atrophic Posterior Maxilla Defects Our next application for cell therapy with TRC involved reconstruction of the atrophic posterior maxillae as part of sinus augmentation procedures. In contrast to the gelatin sponges used as a Cell Carrier in the extraction defects, beta-tricalcium phosphate (B-TCP) particles were used as a scaffold for delivery of cells into the bone defects. This study also was a RCT and involved 30 patients in need of sinus bone- grafting procedures for dental implant placement (www.clinicaltrials. goväCT00980278). There were two treatment groups, one receiving the Cell Scaffold alone and the other receiving TRC on the scaffold. Figure 5 Shows representative cone-beam computed tomographic (CBCT) images Oftreated sites before and after treatment. Four months following delivery of the cells or the scaffold alone, bone biopsies from the regenerated tissue were retrieved and evaluated with three-dimensional (3D) micro- Computed tomography (ucT). These analyses enabled the determination of the extent to which regenerated tissue was comprised of residual B-TCP graft particles or bone tissue (Fig. 6). In the stem cell therapy 3roup, the percentage of CD90+ cells in different cell populations yielded an enhancement in the regenerated bone quality, showing a significant positive correlation between percent CD90+ cells and bone volume fraction (BVF, r = 0.56; p = 0.05; Fig. 7). This relationship was consistent With the clinical observation that clinical bone density was highest in the Patients treated with the stem cell therapy. 273 Tissue Engineering of Alveolar Bone LOCalized reconstruction Multiple site reconstruction - Control Stem cell therapy Control Stem cell therapy Figure 5. Radiographic evaluation of sinus grafts and implant stability six months following functional loading. Cross-sectional images from CBCT scans showing initial bone height in localized reconstructions (single site/tooth) of the (A) control and (B) stem cell therapy groups and bone height four months following treatment (C,D) in both groups. E-F: Periapical radiographs show bone consolidation around the implants six months following functional restoration of the restored areas with a tooth. Cross-sectional images from CBCT SCans showing initial bone height in multiple site reconstructions (two to four teeth) of the (G) control and (H) stem cell therapy groups and bone height four months following treatment (I,J) in both groups. K-L: Periapical radiographs show bone consolidation around the implants six months following functional restoration of the restored areas with teeth. Reprinted with permission from John Wiley & Sons, Inc.; Kaigler et al., 2015. CBCT was used to evaluate 3D changes in the bone volume within the treated areas of the sinus cavity. Overall, there was a significant increase in bone volume in both treatment groups, but no difference was observed between groups in the ratio of regenerated bone volume to initial grafted volume (Fig. 8A). Using uCT and histological analyses to evaluate the qualitative nature of the de novo bone, it appeared that the BVF of the regenerated bone was higher in biopsies retrieved 274 Pagni et al. Control (scaffold alone) Stem cells + scaffold .. - regenerated bone residual scaffold Figure 6. Cell populations with higher percentages of CD90+ cells yield better quality of regenerated bone, uCT images of representative bone biopsies from the control (scaffold only) and stem cell therapy groups clearly show residual grafted scaffold (B-TCP) particles in the grafted zone four months following grafting. The zone of regenerated bone is delineated from the native bone (yellow hashed line) and in the stem cell group is comprised of more bone relative to residual graft compared to the control group. Reprinted with Permission from John Wiley & Sons, Inc.; Kaigler et al., 2015. from patients who received the stem cell therapy. Ouantitatively, the BWF for biopsies from the stem cell therapy group (0.49) was higher than that for the control group (0.43). Additionally, the bone quality of ſºgenerated bone was enhanced significantly in patients receiving the Stem cell therapy in the most severe deficiencies (> 50% deficiency in bone height). In these cases, the regenerated bone biopsies from patients ſeceiving the stem cell therapy had a significantly greater BVF than those "Ores harvested from the control group (0.5 versus 0.4, respectively; p = 004: Fig. 8B). 275 Tissue Engineering of Alveolar Bone % CD90+ cells vs. BVF r = 0.56 45 (p = 0.04) 40 35 30 25 © 20 4) O 15 © 10 50 | O O 30 40 50 60 70 Bone volume fraction (BVF) Figure 7. For all patients in both the control and stem cell therapy groups, there was a significant inverse relationship between the initial graft volume and the bone volume fraction (BVF), which is the proportion of regenerated bone comprised of mineralized bone tissue of the bone biopsy. For only those patients who received the stem cell therapy, there was a significant positive correlation between the percentage of the CD90+ cells (within the Cell populations delivered) and the BVF, Reprinted with permission from John Wiley & Sons, Inc.; Kaigler et al., 2015. Trauma Defect Our next indication for cell therapy with TRC was in the treatment of trauma defects. Traumatic injuries involving the face are common and half of these injuries result in a loss of teeth and the supporting bone (Gassner et al., 2003; Thoren et al., 2010). The resultant loss of bone support compromises replacement of the lost teeth and leaves the afflicted individual functionally and emotionally debilitated. This study highlighted the first clinical report of a stem cell therapy for craniofacial trauma reconstruction. The study involved aſ individual who lost seven teeth and 75% of the supporting bone due to a traumatic injury to the face. Following the injury, the deficient bone precluded her from receiving dental implant therapy to replace the missing teeth. To restore the deficient bone, the patient received stem 276 Pagni et al. Control º sº º:" - region Stem cell therapy -- Regenerated ºf bone : region CBCT: Bone volume/graft volume HCT: Bonevolumefraction 1.00 0.90 60.00 >k 0.80 0.70 50.00 0.50 40.00 9% 0.50 0.40 0.30 0.20 30.00 20.00 10.00 0.10 0.00 0.00 B control cell therapy Figure 8. A: Representative images of 3D reconstructions of occlusal and lateral Open views into the maxillary sinus cavity of the skull show the bone volume that Was grafted (blue) in the control and stem cell therapy groups in severe bone defects. Histological and corresponding uCT images of bone biopsies harvested from the grafted regions of the two groups show a greater degree of mineralized bone tissue in the stem cell therapy group. B: CBCT analysis of the bone volume to graft volume ratio (bone volume/graft volume) was no different between the Control and stem cell therapy groups in treating severe defects, uCT analyses of the bone biopsies revealed that compared to the control, BVF was significantly higher in the stem cell therapy group in treating severe defects. Reprinted with Permission from John Wiley & Sons, Inc.; Kaigler et al., 2015. Cell therapy utilizing a B-TCP as a scaffold to deliver TRC. The 75% hori-zontal bone deficiency clearly was evident radiographically and using volumetric evaluation of 3D reconstructed CBCT images prior to treatment (Fig. 9A-B). Immediately after grafting, a second CBCT was performed and showed a 10 to 12 mm increase in horizontal width of the jawbone (Fig. 9C-D). Four months after grafting and immediately prior to implant placement, a third CBCT was performed and showed that there was an overall 25% reduction of the initial grafted width compared 277 Tissue Engineering of Alveolar Bone Figure 9. Cone-beam computed tomographic (CBCT) scan of cell therapy treated area. CBCT scans were used to render 3D reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points: A-B: Initial clinical presentation shows 75% jawbone width deficiency. C-D: Immediately following cell therapy grafting, jawbone width is restored fully. E-F: 25% resorption of graft at four months and overall net 80% regeneration of original ridge width deficiency. Reprinted with permission from Alpha Med Press; Rajan et al., 2014. to four months prior, immediately following grafting (Fig. 9E-F). Relative to the original jawbone deficiency, however, there was a net 5 to 6 mſſ horizontal gain in width of the jawbone, resulting in 80% regeneration of the original jawbone deficiency. Four months following healing, oral implants were placed in the grafted site and there was clinical evidence of bone regeneration with 278 Pagni et al. new horizontal ridge width of 8 to 9 mm (Fig. 10A-B). Oral implants then were placed stably in the previously grafted sites and torqued biomechanically to standard-of-care guidelines of 35 Ncm (Fig. 10C- D). Implants were left submerged under the gingival tissue (Fig. 10E-F) for six months of healing. The bone biopsy core retrieved at the time of implant placement revealed that the engineered bone was viable histologically (Fig. 10G-H). The regenerated bone also was dense enough biomechanically to enable the stable placement of dental implants for subsequent restoration with prosthesis to re-establish the patient's full function and smile in the previously edentulous area (Fig. 11). DISCUSSION The field of regenerative medicine aims to use tissue engineering and biomimetic strategies to restore and replace damaged and lost tissue functionally (Langer and Vacanti, 1993). The prospect of stem cell therapies offers significant advantages over traditional approaches for Oral and craniofacial reconstruction and has led to the development of an immense body of work characterizing different stem cell populations and their regenerative potential. Despite these efforts, there has been limited translation of this work toward clinical applications. Through the studies described in this chapter, we have initiated early investigations of stem Cell therapies for alveolar bone deficiencies. The extraction socket created following tooth removal serves as a good model of human bone regeneration in that it is highly reproduc- ible and yet has a limited capacity to regenerate without intervention. Our tooth extraction defect study was the first randomized, controlled, human trial employing stem cell therapy for the regeneration of cranio- facial bone. Most clinical protocols that employ grafting procedures for reconstruction of alveolar bone allow healing periods minimally of three to six months. The rationale for choosing the six-week time point was to determine if wound repair could be accelerated by delivering cells to the defect at the time of tooth extraction. Clinical and laboratory anal- ySes of treatment sites demonstrated that the cell therapy accelerated the regenerative response as determined clinically, radiographically and histologically. Further, there was a significantly reduced need for second- ary bone-grafting procedures in the group that originally received the cell therapy. Additional studies need to be performed to determine the 279 Tissue Engineering of Alveolar Bone Figure 10. Surgical re-entry of grafted site and implant placement. Following elevation of a full thickness gingival flap, (A) frontal and (B) occlusal views of the treated site reveals regenerated tissue and a reconstructed alveolar ridge with a clinically measured width of 8 to 10 mm. C. Frontal and (D) occlusal views of the placement of dental implants in the regenerated sites and (EF primary closure of the site. A bone core biopsy was retrieved from one of the 280 Pagni et al. (Fig. 10 continued) regenerated sites (G) to determine the presence of mineral- ized tissue with L-CT analysis and (H) to confirm the histomorphometric appear- ance of bone tissue histologically with H&E staining. Green arrows = residual B-TCP; yellow arrows = bone tissue; 40x and 100x magnification. Reprinted with permission from Alpha Med Press; Rajan et al., 2014. Figure 11. Clinical presentation of the patient before and after oral reconstruction With cell therapy. mechanism of this accelerated regenerative response, yet two possible mechanisms for these observations exist: 1. Theregenerated tissue is derived from the transplanted cells (Jensen and Terheyden, 2009); and 2. The transplanted cells act in a trophic fashion and provide signals to the host cells, thereby "jump- Starting” the regenerative process (Caplan, 2007). These results provided the first published evidence that TRC, eXpanded from a small amount of bone marrow, have the regenerative Capacity to produce highly vascular bone tissue in a human craniofacial bone defect. In our study involving maxillary sinus augmentation, we evaluated TRC therapy to engineer bone tissue in severe bone deficiencies of the maxilla. Autogenous, allogeneic and alloplast bone “void fillers" typically are grafted in the sinus cavity. All these modalities have been 281 Tissue Engineering of Alveolar Bone shown to generate sufficient height and bone volume for stable placement of oral implants to support functional tooth replacements (Esposito et al., 2014). Nevertheless, the study by Esposito and associates (2014) showed that while both control and stem cell therapies yielded sufficient bone height and volume for the placement of oral implants, that regenerated bone in the stem cell group was of higher quality as defined by the BVF. This was apparent, particularly in the larger reconstructions where the most severe deficiencies were present. In these cases, the bone tissue formed in the control group was comprised of a higher proportion of residual alloplast B-TCP carrier, whereas the stem cell therapy yielded a greater proportion of regenerated bone being comprised of viable, highly vascular, mineralized bone tissue. De novo bone formed in sinus reconstructive procedures is dependent primarily upon osteogenic and vascular cells from the sinus cavity to infiltrate and remodel the graft material, ultimately to form bone. In our study, the provision of a cellularized graft to the defect appeared to accelerate the process of remodeling, demonstrated by less residual graft particles being present four months following treatment in biopsy samples obtained in the experimental group. This concept of accelerated remodeling is supported by the findings from the extraction socket studies. De novo bone regeneration was sufficient to place oral implants stably, which ultimately were used to support dental prostheses functionally. A key finding was that better quality bone was formed in patients who received the stem cell therapy compared to the control group and bone quality correlated significantly with the percentage of autologous CD90+ cells transplanted. This study was the first to evaluate how cell phenotype correlates to clinical regenerative outcomes, showing that irrespective of the number of cells delivered, the best bone quality was achieved in patients whose cell populations had the highest percentages of CD90+ cells. This specific relationship needs to be evaluated further in a larger number of patients, but the finding could be important to optimize and personalize clinical cell therapy protocols to meet specific needs of different patients. Like most regenerative studies that apply novel therapies, our clinical trial was designed to evaluate safety and efficacy; unlike most studies, however, we also aimed to acquire information relative to the treatment protocol from the patient perspective. This “quality-of-life" assessment often is overlooked or not reported when trying to determine 282 Pagni et al. the initial feasibility of emerging therapies; yet, if the therapy is deemed effective, these factors could underscore the acceptance and widespread use of these procedures. Traditional treatments for large oral and cra- niofacial defects routinely utilize large autogenous grafts, which often require significant recovery time and, due to the associated post-oper- ative pain and distress, most patients would not elect to undergo them again, if they are deemed necessary (Myeroff and Archdeacon, 2011). In contrast, our study found that at the completion of treatment for pa- tients who received the cell therapy, all participants reported that the treatment regimen and procedures involved did not impact their daily life activities significantly and that, if necessary, they would undergo them again. Despite the promising results identified in the extraction socket defects and sinus augmentation Studies, the most appropriate application for this cell therapy is in more complex and severe craniofacial defects, which often occurs following oral-facial trauma. A severe defect result- ing from a traumatic injury does not resolve naturally without significant intervention and also results in significant functional and esthetic defi- Ciencies. As such, these defects typically require advanced bone grafting procedures with autogenous blocks of bone or GBR procedures (Melcher, 1976). The GBR approach uses a protective barrier membrane to cover the allogeneic or alloplastic graft material during healing. Following GBR for large reconstructions of alveolar bone, however, most protocols re- quire a minimal healing period of six to eight months prior to re-entry for oral implant placement (McAllister and Haghighat, 2007). In our final case presentation of this cell therapy approach, we described the oral reconstruction of a patient who lost teeth and support- ing jawbone tissue as a result of a traumatic injury to the face. Through delivery of 100 million cells using a tissue engineering cell therapy ap- proach, we were able to regenerate 80% of the patient's original jaw- bone deficiency in only four months, sufficient to place oral implants Stably to support a dental prosthesis biomechanically. Additionally, opti- mized parameters for cell attachment and survival were defined for the Cell transplantation protocol used in this approach. To date, this study represents the most advanced craniofacial trauma reconstruction us- ing a stem cell-based therapy for oral rehabilitation involving oral im- plants. Despite the promising results of this case report, it is important to note that these results were obtained in a single patient, which limits 283 Tissue Engineering of Alveolar Bone any general conclusions that can be drawn. This case presentation is part of a larger, ongoing U.S. FDA-regulated, randomized, controlled phase I/ II trial where a larger number of patients have been treated with TRC in similar types of defects. CONCLUSIONS AND FUTURE DIRECTIONS There is a growing interest in cell therapy strategies to regenerate craniofacial tissues, particularly bone; however, critical questions to be considered in using these strategies are: 1. What is the source of cells used in these approaches? 2. How will the cells be processed and expanded to reach clinical-scale numbers for application? 3. What are the phenotypes and regenerative capacities of the cells produced? Though the studies presented herein do not provide the answers to the aforementioned questions, they do provide insight toward the clinical regenerative potential of craniofacial cell therapy. Additionally, it should be noted that throughout all the studies, there were no serious adverse events reported which were related specifically to the stem cell therapy. To this end, it should be recognized that cell-based therapies require nav- igation through a rigorous regulatory process before they can be Studied clinically and certainly before they can be practiced widely (Caunday et al., 2009; Zaky and Cancedda, 2009). Nonetheless, additional clinical in- vestigations certainly are warranted and currently underway as the ther- apeutic potential for these therapies is promising. These studies provide evidence that stem cell therapy could be considered for treatment of other challenging oral and craniofacial bone defects including segmental/continuous defects secondary to disease or congenital malformations (i.e., cleft palate). Additionally, these applications potentially could be combined with other treatment modalities where accelerated bone healing and highly viable bone is desired. ACKNOWLEDGEMENTS The authors would like to acknowledge Gustavo Avila, Ronnda Bartel, Judy Douville, Andrew Eisenberg, Andrei Taut and Suncica Travan 284 Pagni et al. for their technical and clinical assistance with these studies. These studies were supported generously by the Burroughs Wellcome Fund (CAMS), the ITI Foundation, the National Institute of Dental and Craniofacial Research/NIH (R56DE23095-01A1), the National Center for Advancing Translational Sciences/NIH (UL1TR000433) and the Oral-Maxillofacial Surgery Foundation. REFERENCES Abbott MA. Cleft lip and palate. 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Occurrence and types of dental injuries among patients with maxillofacial fractures. Int J Oral Maxillofac Surg 2010;39(8):774-778. Yamada Y, Nakamura S, Ito K, Umemura E, Hara K, Nagasaka T, Abe A, Baba S, Furuichi Y, Izumi Y, Klein OD, Wakabayashi T. Injectable bone tissue engineering using expanded mesenchymal stem cells. Stem Cells 2013;31(3):572-580. Yan Q, Zhang Y, Li W, DenBesten PK. Differentiation of human ameloblast- lineage cells in vitro. Eur J Oral Sci 2006;114(Suppl 1):154-158. Zaky SH, Cancedda R. Engineering craniofacial structures: Facing the challenge. J Dent Res 2009;88(12):1077–1091. Zamiri B, Shahidi S, Eslaminejad MB, Khoshzaban A, Gholami M., Bahr- amnejad E, Moghadasali R, Mardpour S, Aghdami N. Reconstruc- tion of human mandibular continuity defects with allogenic scaffold and autologous marrow mesenchymal stem cells. J Craniofac Surg 2013;24(4):1292-1297. 289 FABRICATION AND MORPHOLOGICAL CHARACTERIZATION OF A THREE DIMENSIONAL, MULTI-PHASIC, SCAFFOLDLESS, TISSUE-ENGINEERED TEMPOROMANDIBULAR JOINT DISC-LIGAMENT COMPLEX CONSTRUCT Jonah D. Lee, Josh I. Becker, Lisa M. Larkin, Sunil D. Kapila ABSTRACT BACKGROUND: The temporomandibular joint (TMJ) disc-ligament complex is an important structural component of the TMJ because of its role in normal joint movements and because its degeneration leads to compromised function. Tissue- engineering strategies that are customized to regenerating and integrating this Complicated structure could be highly beneficial in restoring tissue integrity and function in diseased joints. The aim of this study was to utilize a novel method to design and construct a multi-phasic, tissue-engineered TMJ disc-ligament- bone Complex and to evaluate the morphology and histology of this construct relative to that of the native counterpart from a large animal model. MATERIALS AND METHODS: Following initial differentiation of sheep bone marrow stromal Cells into osteoblastic, fibroblastic and chondrocytic lineages, we assembled three-dimensional multi-phasic bone-, ligament- and disc-like, scaffoldless tissue-engineered constructs. These constructs were compared with native TMJ complexes from skeletally mature sheep for morphological dimensions and for type I collagen, elastin and glycosaminoglycans (GAGs). RESULTS: We were able to assemble a multi-phasic, 3D disc-ligament-bone construct successfully whose structural dimensions compared favorably to tissues from the sheep TMJ. Histologically the tissue-engineered construct mimicked the native tissue phases with similar regional distribution of type I collagen and elastin. The constructs, however, did not demonstrate presence of GAGs found in the native discal apparatus. CONCLUSIONS: These data demonstrate the feasibility of tissue engineering a multi-phasic TMJ disc and attachment complex similar in size and partially analogous in matrix content to that of native sheep tissues. Our data provide the framework and methodologies for future work aimed at achieving optimal functional properties and organization for engineering the TMJ disc- ligament-bone complex toward translational applications. KEY WORDS: temporomandibular joint (TMJ), tissue engineering, sheep, SCaffoldless, disc-ligament 291 TMJ Disc-Ligament Complex Construct INTRODUCTION The temporomandibular joint (TMJ) is classified as a ginglymo-ar- throdial joint due to its ability to rotate and translate uniquely (Wadhwa and Kapila, 2008). These joint movements are facilitated by the presence of a multi-phasic disc interposed between the joint's articulating surfaces that divide the joint compartment into inferior and superior cavities and enable rotational and translational movements, respectively. Specifical- ly, during jaw movement, the disc first is stabilized between the articu- lar fossa and condylar head as the condyle rotates during initial mouth opening, followed by its coordinated movement with the condyle as they translate together down the articular eminence. Such complex and co- ordinated movements are facilitated by the ligamentous attachments comprised of varied regional composition and elasticity that attach the periphery of the disc to the condyle inferiorly and the temporal bone superiorly. The disc is attached at its antero-medial surface to the lateral pterygoid muscle that plays a critical role in the coordinated movement of the disc with the condyle during translational movements. The disc also serves to transmit and dissipate loads acting on the joint and adapts its shape to changing geometry of the articular surfaces, which helps to minimize small contact areas and local peak forces (Sperber, 2001; Ben- jamin and Ralphs, 2004). Because the disc is subject to tensile, compres- sive and shear forces, it is configured for anisotropic mechanical behavior and has a matrix composition and organization designed to withstand these complex mechanical challenges (Nakano and Scott, 1989; Milam et al., 1991; Landesberg et al., 1996; Scapino et al., 1996; Detamore and Athanasiou, 2003b). These complex mechanical demands result in a het- erogeneous tissue with differences in the regional distribution of extra- cellular matrices and cell phenotypes. Thus, the mature disc has a fibrous tendinous structure with fibroblastic cells at sites of tension—a primarily cartilaginous matrix and chondrocyte-like cells at sites of compression— and mixed tissue composition and intermediate fibrochondrocytic cells at sites where it is subjected to diverse tensile, compressive and shear forces (Rees, 1954; Nagy and Daniel, 1992; Mills et al., 1994; Scapino et al., 1996; Ahmad et al., 2012; Leonardi et al., 2012). 292 Lee et al. The importance of the TMJ disc also is evident from the find- ings that its degeneration contributes to compromised joint function and symptoms (Sperber, 2001; Tanaka et al., 2006; Carini et al., 2007; Santos et al., 2013). Many such TMJ disorders involve progressive degen- eration of the disc and associated tissues that require replacement or regenerative approaches at advanced stages of the disorder (Detamore and Athanasiou, 2003b; Tanaka et al., 2008; Wadhwa and Kapila, 2008; Athanasiou, 2009; Ingawalé and Goswami, 2009; Murphy et al., 2013b). The functional, organizational and compositional complexity of the TMJ, Combined with a poor understanding of TMJ disorders, necessitates de- vising customized therapeutic and tissue regenerative strategies for TMJ degenerative conditions that may differ from those required for other leSS Complex joints (Wadhwa and Kapila, 2008; Ingawalé and Goswami, 2009). Although substantial work has been performed toward engineer- ing a TMJ disc (Detamore and Athanasiou, 2003a,b; Almarza and Atha- nasiou, 2005; Johns and Athanasiou, 2007a,b; Athanasiou, 2009; Zhang et al., 2009; Almarza et al., 2011), it is apparent that the fabrication of the disc in isolation is not an adequate solution for its integration into joint structures and restoration of complex movements during normal joint function (Willard et al., 2012; Murphy et al., 2013a). Such advances necessitate the design and engineering of a complex multi-phasic struc- ture that would be composed of the fibrocartilaginous disc, ligament and bone. Thus, as opposed to an isolated engineered TMJ disc, a construct Composed of these diverse tissue-types would facilitate its surgical im- plantation into the condyle head and temporal bone that could be inte- grated more readily into the joint and be compatible with an expedited return to normal joint function. The aim of this study was to use a novel method to develop Such a multi-phasic tissue that integrates a fibrocartilaginous central tissue with a ligamentous tissue that, in turn, is attached to bone. The Outcome of this approach was assessed by comparing the morphology and histology of this in vitro-engineered construct with that of its native Counterpart from a large-animal model. The successful fabrication of the engineered disc and discal attachments with its desirable structural 293 TMJ Disc-Ligament Complex Construct organization and promising biochemical content validates the application of our three-dimensional (3D), multi-phasic, scaffoldless approach to engineering whole TMJ disc-ligament-bone complex constructs and holds promise for its applications to TMJ replacement and regenerative therapies. MATERIALS AND METHODS Animal Care Bone marrow stromal cells (BMSCs) were harvested from marrow aspiration using a previously established approach in adult Black Suffolk sheep (Ma et al., 2012). The sheep were given access to food and water ad libitum. All animal care and surgical procedures were performed in accordance with The Guide of Care and Use of Laboratory Animals and the experimental protocol was approved by The University of Michigan's Committee for the Use and Care of Animals. Preparation of Cell Culture Supplies The media used in this experiment have been described previ- ously (Ma et al., 2012) as follows. The baseline growth medium (GM) consisted of 78% Dulbecco's Modified Eagle Medium (DMEM: Gibco, Grand Island, NY, USA) with 20% fetal bovine serum (FBS; Gibco), 2% antibiotic antimycotic (Grand Island, NY, USA), 6ng/mL basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ, USA), 0.13 mg/mL ascor- bic acid-2-phosphatase (Sigma-Aldrich, St. Louis, MO, USA) and 0.05mg/ mL L-proline. Differentiation medium (DM) for ligamentous/fibrous tis- sue consisted of 91% DMEM, 7% horse serum albumin (HS; Gibco, Grand Island, NY, USA), 2% antibiotic-antimycotic, 0.13mg/mL L-Proline and 2ng/mL transforming growth factor-beta (TGF-3; Peprotech, Rocky Hill, NJ, USA). For culture of cells in osteoblastic conditions, 10°M dexametha- sone (DEX; Sigma-Aldrich) was added to GM and DM while 107M DEX, 10ng/mL of TGF-B and 50mg/mL ascorbic acid-2-phos-phatase was add- ed to both media for cells subjected to chondrogenic conditions. Construct dishes were prepared as previously described to form and maintain 3D constructs (Ma et al., 2012). Briefly, 100 mm diameter cell culture plates were coated with 12mL Sylgard (Dow Chemical Corp., Midland, MI, USA; type 184 silicon elastomer) and allowed to cure for 294 Lee et al. three weeks at room temperature. Prior to culturing cells, the plates were decontaminated with UV light (wavelength 253.7nm) for 60 minutes and rinsed with 70% ethanol and Dulbecco's phosphate-buffered saline (DPBS). Isolation and Expansion of BMSCs BMSCs were obtained from marrow aspirations from the iliac Crest of a sheep with the animal under general anesthesia induced by intravenous Propofol and sustained with inhalation of isoflurane in oxygen. Marrow was collected using heparinized Monoject Illinois needles (Sherwood Medical Company, St. Louis, MO, USA) and dispensed into EDTA blood collection tubes (Becton Dickinson, San Jose, CA, USA) for processing. The marrow aspirate was filtered and isolated using methods previously described (Mahalingam et al., 2015). Briefly, a 15ml Ficoll-Paque Premium (MNC; GE Healthcare, Munich, Germany) was added on top of the aspirate solution and centrifuged. The upper layer of plasma was removed and the mesenchymal cells contained in the mononuclear cell layers were transferred. The isolate was centrifuged again, Supernatant removed and an equivalent ammonium-chloride- potassium lysing buffer (Gibco) was added. The isolate was washed, Centrifuged and supernatant removed in preparation for a cell count. Cells were plated at 40,000-60,000 cells/cm3 in cell culture dishes in GM Specific for each of the desired cell lineage. Fabrication of TMJ Constructs The BMSCs were expanded into ligament cell-, osteoblast- and Chondrocyte-like lineages in their respective GM as described previously (Ma et al., 2009, 2012; Syed-Picard et al., 2009; Mahalingam et al., 2015; Step 2 in Fig. 1). Following expansion, passage-3 cells in the ligament pathway and passage-4 cells in the bone and cartilage pathways were Seeded at density of ~21,000 cells/cm3 and switched to DM eight days after plating (Step 3 in Fig. 1). After two more days of culture in appropriate DM for each lineage, the monolayers from the osteoblast and chondrocyte differentiation lineages were rolled with sterile tweezers into rectangular and square-shaped 3D 'blocks,' respectively, then transferred and pinned to Sylgard plates (Step 4 in Fig. 1). The ligament was maintained as a monolayer construct. After two additional days in DM, the bone constructs and fibrocartilage constructs were pinned 295 TMJ Disc-Ligament Complex Construct ~ 1 7 º º Bone Cartilage Ligament Pathway Pathway Pathway ! ... ( ) || <- s Figure 1. Summary schematic for fabrication of a 3D, scaffoldless, fused assembly, multi-phasic TMJ disc-ligament-bone complex. Step 1: Bone marrow stroma cells first were isolated from sheep by bone marrow aspiration. Step 2: Cells were proliferated and differentiated into osteoblast-, chondrocyte- and ligament- like cells using appropriate growth media and growth factors. Step 3: Cells were seeded on 150 mm cell culture plates and grown to confluence forming a monolayer of each cell type. Step 4: Formed monolayers were transferred from cell culture dishes to Sylgard-coated dishes with minutien pins placed on each monolayer to guide formation of TMJ disc and attachment tissue types in appropriate DM. Step 5: Formation of 3D structure was initiated with further differentiation and delamination of the respective cell-type monolayers that carefully were transferred sequentially so that the bone- and cartilage-like tissues were pinned in series and on top of a delaminating ligament monolayer. Step 6: The different components of constructs fuse together with continual media feeding over the course of one to two weeks. Step 7: The constructs containing bone-like ends (for implantation anchors), ligament-like interfaces, with a discº like central region, yielding a complete multi-phasic construct approximately 5 to 6 cm in length are designed to achieve a fully functional tissue-engineered construct for the purposes of surgical implantation as a viable option for TM repair in a sheep model. 296 Lee et al. Sequentially onto a series of ligament monolayers and held together with pins (Step 5 in Fig. 1). Confluent ligament monolayers then were rolled to create the 3D shape that incorporated bone construct ends and a putative cartilaginous center in a ligament envelope. The multi-phasic tissues then were transferred carefully to a new Sylgard plate and pinned back into a single complex construct with appropriate dimension adjustments (Step 6 in Fig. 1). The baseline DM was changed every two days for up to two weeks for proper construct fusion. Completely formed disc- ligament-bone constructs (Step 7 in Fig. 1) were removed from the plate and samples (n = 5) pinned to popsicle sticks, coated in tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC, USA), frozen in isopentane cooled by dry ice and stored at -80°C for future analyses. Native Sample Preparation TMJs were excised en bloc from five skeletally mature sheep (Suffolk or Dorset; Valley View Farms, Cockeysville, MD or Cornell Farms, Dryden, NY, USA). Segments of the zygomatic process, squama tempo- ralis, mastoid and condyle were dissected to maintain the integrity of the joint capsule and the TMJ removed en bloc. The joints were washed with phosphate-buffered saline (PBS: Sigma-Aldrich, St. Louis, MO, USA), wrapped in gauze soaked with PBS containing protease inhibitors (1 mM N-ethylmaleimide and 1 mM phenylmethylsulfonyl fluoride, Sigma-Al- drich) and frozen at -20°C until subsequent sample analyses. For histolog- ical analyses, the TMJ was thawed, the disc-attachment complex isolated and released from the joint capsule, temporal and condylar attachments, and verified to be grossly normal with an absence of perforation or de- formity. Histochemical and Immunohistochemical Analyses Frozen samples of TMJ construct and native TMJ disc and discal attachments were cryo-sectioned at 12 pum thickness from the midline in the antero-posterior direction, mounted on Superfrost Plus micro- Scopy Slides and stained for histological analysis. Disc-ligament sections were analyzed in entirety in a region-specific manner including: the fibro- cartilage only in the disc region; the ligament/fibrocartilage interface 297 TMJ Disc-Ligament Complex Construct at the immediate posterior and anterior periphery to the disc regions; and the posterior and anterior ligament only. Sections were immuno- stained with collagen type I and elastin antibody, and histochemically for Safranin-O for determining the regional content and/or alignment of collagen, elastin and glycosaminoglycans (GAGS), respectively. Sections were fixed with ice-Cold methanol for ten minutes and rinsed with DPBS. The sections were submerged for fifteen minutes in PBS with 0.05% Triton X-100 (PBST, Sigma-Aldrich) and blocked with 3% Bovine Serum Albumin in PBS (PBST-S; Sigma-Aldrich, A2153-10g). The sections then were incubated overnight at 4°C with primary antibodies to collagen type | (Abcam, Cambridge, MA, USA, #AB292) or elastin (Millipore, Billerica, MA, USA, #AB2039) diluted in PBST-S. Following PBST washes, sections were incubated in 1:500 dilutions of Alexa-fluor anti-mouse or anti-rabbit antibodies (Life Technologies, Carlsbad, CA, USA) for three hours. Sections were fixed in Prolong Gold with DAPI (Sigma-Aldrich) and covered. To evaluate GAG content and distribution, the samples were stained with fast green/Safranin-O solutions, rinsed and then mounted for analyses. Sections were imaged with equivalent setting on an Olympus BX-51 microscope and analyzed using Image J Software package. Image analysis quantification consisted of selecting and averaging at minimum six independent non-overlapping fields of view for each Selected region of interest of the TMJ disc-ligament complex for both the native sheep TMJ (n = 5) as well as the engineered TMJ constructs (n = 5). The percent area staining positive for each of the assayed matrix molecules within the field of view for each region of interest was calculated from equivalent sized fields of view for all determinations. Statistical Analysis The percent area staining positive for collagen and elastin was determined at three sites: the periphery of the ligament where it would attach to bone; the ligament-disc interface; and the fibrocartilaginous disc of the native tissue and disc-ligament construct. Since the native tissues had variable matrix content in the anterior and posterior regions of each of these sites, these sites were plotted and analyzed separately. In contrast, because of the symmetric disc-ligament-bone construct and, therefore, its lack of anterior and posterior tissue definition, this data for both locations of the construct were combined for plotting and analyses. 298 Lee et al. The data was plotted as means #: S.E. and Statistical analyses performed using JMP statistical analysis software (JMP, Cary, NC, USA) by a one- way analysis of variance with Tukey post-hoc analysis when indicated, or paired Student's t-test analysis. Differences were considered significant at p < 0.05. RESULTS Size and Morphology By using BMSCs that were subjected separately to differentiation along ligament cell, osteoblast and chondrocyte differentiation lineages followed by assembly of resultant cells and matrices into a tissue Complex, we generated a tissue with the size and shape characteristics similar to the TMJ disc-ligament-bone complex from sheep. These Constructs measuring approximately 1.0 to 1.5 cm in width, 5.0 to 6.0 cm antero-posteriorly and approximately 1.0 to 1.5 mm in thickness at the Center representing the putative fibrocartilage (Figs. 2 and 3) had similar dimensions to that of the native sheep TMJ discal complex. However, as opposed to the irregular configuration both in width and thickness of the native tissues resulting from the functional and anatomic demands placed on it in vivo, the TMJ disc-ligament-bone construct was symmetric in shape. Histochemical and Immunohistochemical Analysis Histological methods and analyses of bone-ligament interfaces utilizing equivalent sample preparation and Construct fabrication have been described previously (Ma et al., 2009, 2012; Mahalingam et al., 2015) and will not be discussed in this manuscript. Immunohistochemistry for type I collagen—a primary extracellular matrix of the ligament and fibrocartilaginous disc complex—demonstrated a well-defined, region- Specific orientation throughout the tissue-engineered TMJ disc and attachment construct, as well as the native TMJ disc-ligament complex and interfaces (Fig. 4). The tissue-engineered TMJ construct demonstrates an obvious antero-posterior alignment of collagen (left to right) in the intermediate fibrocartilage region, while the immediate periphery of this region that encircles the disc and represents the ligament shows less clear alignment of the fibers. The native TMJ disc is attached along its entire periphery to both the condyle and temporal bone, as well as the superior head of the lateral pterygoid muscle through a complex 299 TMJ Disc-Ligament Complex Construct - |- ºn. * TTT Ligament Putative Cartilaginous Disc Bone Figure 2. Representative images of multi-phasic TMJ disc-ligament-bone complex fabricated from BMSC monolayers differentiated under appropriate conditions and manipulated into a 3D complex. The fabrication of this complex involves pinning the monolayers to Sylgard plates to encourage appropriate dimension development. Monolayers then are assembled sequentially to facilitate fusion into a complex construct. A: Confluent ligament monolayers rolled to create the 3D shape. B: The ligamentenvelope is assembled sequentially with bone construct ends and a disc-like center and allowed to fuse. The construct is pinned to retain dimension and shape during complex development. C. TMJ disc-ligament-bone complex construct after two weeks of maturation following construct assembly and fusion. Note the antero-posterior symmetry of the construct. network of fibrous connective tissues that form a synovial capsule enveloping the joint (Athanasiou, 2009). Nonetheless, in the native primarily fibrocartilaginous TMJ disc, the characteristic crimped fibeſ pattern of the attachment connective tissue is replaced by fibers that have a greater antero-posterior orientation that transitions obliquely once again toward the periphery to establish a circumferential organization of the capsular attachments. This arrangement of collagen fibers is similar to that of the tissue-engineered TMJ disc-ligament construct The posterior of the native complex is comprised of a blended network 300 Lee et al. - º - A ſº |- milliºn. - 1...." ºnly, º ----- Bone Ligament Fibrocartilage Disc Ligament Figure 3. Region-specific dimensions of (A) native TMJ disc-attachment complex of the sheep, compared to the (B) in vitro tissue-engineered TMJ disc-ligament. bone Complex. Regions consist of multi-phasic connective tissue including bone, ligament and putative cartilaginous disc-like structure. offibro-elastictissues and vasculature, which transitions into thick bundled dense Collagen arrangements in the periphery. Through quantification (Fig. 4C), we demonstrate that collagen I staining in the tissue-engineered ligament is greater than in both the anterior and posterior regions of the native TMJ ligament and greater at the disc-ligament interface of the construct than in its counterpart region of the native tissue. No significant differences in collagen I staining were detected in the putative fibrocartilage regions of the construct versus that in the native disc. 301 TMJ Disc-Ligament Complex Construct E. º º O O A Ligament (posterior) Fibrocartilage disc Ligament (anterior) B 80 -- ºr 60 § § 40 Hr- [] Posterior §. [] Anterior 20 Construct O - - C Ligament Ligament/disc Fibrocartilage disc Figure 4. Immunohistochemistry for type I collagen of the (A) in vitro TMJ disc- ligament-bone construct and (B) native TMJ disc-attachment complex. The native and engineered tissues demonstrate similar organization and antero-posterior collagen I fiber alignment. C. A lower percent area in the immediate posterior ligament/disc region of the native TMJ stains positive for collagen I compared to that in the tissue-engineered TMJ construct. The TMJ construct also has a greater percent area for collagen I staining than the posterior and anterior ligament regions of the native sheep TMJ discal attachment. The native and tissue-engineered TMJ disc have similar areas staining for collagen I. Due to the antero-posterior symmetry of the construct, there is no distinction between anterior and posterior regions and, therefore, the data is combined for anterior and posterior regions. Significant difference between * in vitro versus in vivo, p * 0.05. Scale bar = 100 um. Elastin staining was performed to compare its distribution and organization between the tissue-engineered and native disc-ligament complex. Elastin fibers were found throughout the disc and discal regions that complement the collagen distribution, but were a much smaller proportion of connective tissue than collagen throughout the complex. In general, the elastin fibers are less organized than collageſ, with a higher degree of branching and multi-directional orientation (Fig. 5). Within the native tissues, the anterior disc-ligament attachment had significantly lower percentage area staining for elastin than the posterior 302 Lee et al. # O O Ligament (posterior) Fibrocartilage disc Ligament (anterior) B 50 - º: 40 º: º § 30 | ; 20 ITI D. Posterior Sº # |-- D. Anterior 10 Construct 0 + C Ligament Ligament/disc Fibrocartilage disc Figure 5. Immunohistochemistry for elastin of the (A) in vitro TMJ disc- ligament-bone construct and (B) native disc-attachment complex demonstrates region-specific organization in both tissues. Staining for elastin of the tissue- engineered construct is more generalized, particularly in the ligament regions. C. Ouantification of percent area staining for elastin demonstrates a larger area of the ligament and ligament-disc of the in vitro TMJ stains positive for elastin than either the posterior or anterior discal attachments of the native sheep TMJ. Elastin content is similar in the disc region between native tissues and the engineered constructs. Due to the antero-posterior symmetry of the construct, there is no distinction between anterior and posterior regions and, therefore, the data is combined for anterior and posterior regions. Significant difference between * in vitro versus in vivo and # between in vivo regions, p < 0.05. Scale bar = 100 um. region (Fig.5C). Both the anterior and posterior regions of the ligament, as Well as the disc-ligament region in the native tissues, also had significantly lower elastin staining than the engineered construct. Finally, the native tissues and the construct have equivalent elastin content in the disc fibrocartilage region. Despite the relatively greater percentage of tissue Staining for elastin in the ligament and disc-ligament interface regions of the construct relative to the native tissues, this staining is diffuse in the construct, while it has an organized focal distribution in the native tissues. This indicates a lack of organizational structure for elastin in the tissue construct relative to the native sheep tissues. 303 TMJ Disc-Ligament Complex Construct Safranin-O staining to identify the presence and location of GAGS demonstrated that no region of the tissue-engineered disc-ligament complex construct stained positive for GAG (Fig. 6A). In contrast, GAG was present within the disc and immediate posterior disc periphery regions, with light staining also present in the anterior disc-ligament interface in the native sheep (Fig. 6B-C). The native tissues also showed large cartilage-like cells Surrounded by lacunae in areas staining for GAGS, which were absent in the tissue construct. This reflects a lack of adequate differentiation of the cells in the tissue construct relative to the native tissue. DISCUSSION The development and morphological characterization of tissue- engineered TMJ disc-ligament-bone complex constructs is essential to establish an efficacious therapeutic strategy for replacing irreversibly damaged and functionally vital tissues of the TMJ. We have adapted a previously defined method used to construct bone-ligament constructs (Ma et al., 2009, 2012; Mahalingam et al., 2015) toward a novel ap- proach for fabricating a disc-ligament-bone construct for the TMJ. This approach advances current TMJ tissue-engineering studies that have fo- cused on fabrication of the TMJ disc in isolation (Detamore and Athana- Siou, 2003a,c, 2004; Almarza and Athanasiou, 2005; Athanasiou, 2009; Mäenpää et al., 2010; Hagandora et al., 2013; Shu et al., 2015). Macro- scopically, we were able to fabricate a multi-phasic disc-ligament-bone construct that is comparable in dimensions to the native sheep tissue counterpart. The regional variation in this multi-phasic connective tis- Sue construct was achieved by using a strategic tissue-engineering pro- cess designed to maintain the phenotype of individual tissue types via initial divergent differentiation phases for ligament, putative cartilage and bone followed sequentially by an integration and fusion of these phases (Figs. 1 and 2). In addition to characterizing the ligament and putative cartilage phenotypes, due to the crucial importance of tissue interfaces in the discal complex (Willard et al., 2012), we phenotyped the immediate disc periphery and discal attachments to determine whether appropriate connective tissue interfaces were achieved suc- cessfully while concurrently maintaining the multi-phasic nature of the TMJ disc-ligament complex. Our unique fabrication process yielded a construct with robust collagen and elastin content and similar collagen 304 Lee et al. Figure 6. Staining for glycosaminoglycan (GAG) with Safranin-O demonstrates (A) lack of staining in the TMJ disc-ligament complex construct and (B) region- Specific GAG staining in native sheep TMJ disc-attachment complex. C. GAG Staining in sheep disc visualized in lower magnification composite image. Scale bar = 100 um. Orientation to that of native TMJ disc complex, but it was not successful in establishing an adequate GAG biochemical profile. Despite the lack of GAG staining in the engineered disc, our methodologies and the characterization of TMJ disc-ligament constructs offer valuable benchmarks and advances in developing and improving therapeutic Strategies to engineering this complex musculoskeletal joint structure. While the findings on generating bone and characterizing the bone- ligament interface by this approach are reported elsewhere (Ma et al., 2009, 2012; Mahalingam et al., 2015), the findings reported here reveal a multi-phasic tissue construct that successfully integrates various tissue types to create a disc-ligament complex. Much of the research on tissue engineering of TMJ tissues has focused on fabricating the condyle or the disc in isolation (Alhadlaq and Mao, 2003; Detamore and Athanasiou, 2003a; Schek et al., 2005; Mao et al., 2006; Athanasiou, 2009). With regard to the TMJ disc engineering, Studies to date primarily have concentrated efforts on characterizing native tissue composition, organization and mechanical properties, identifying appropriate scaffolds and testing cell types and densities (Scapino et al., 1996, Zaucke et al., 2001, Detamore and Athanasiou. 305 TMJ Disc-Ligament Complex Construct 2003c, 2004; Almarza and Athanasiou, 2004, 2005, 2006; Allen and Athanasiou, 2007, 2008; Wang and Detamore, 2009; Kalpakci et al., 2011a,b; Segu et al., 2011). For example, in identifying optimal cell types, it has been shown that following stimulation with insulin- like growth factor-1, cells from hyaline cartilage generate greater amounts of GAGs and collagen than those from the condylar cartilage, which results in fibrocartilaginous versus fibrous tissues, respectively (Kalpakci et al., 2011a). Others have shown that a mixture of cartilage and fibrocartilage cells treated with transforming growth factor-31 results in a construct with favorable mechanical properties (Detamore and Athanasiou, 2004). While these previous findings together provide relatively promising outcomes toward fabricating a TMJ disc, this strategy fails to address how this tissue would be integrated into and establish a normally functional unit within the joint. Our approach to develop a disc-ligament-bone complex through parallel differentiation of BMSCs into three cell lineages, their initial independent expression of relevant matrices and subsequent assembly into a 3D construct of putative cartilage, ligament and bone provides a unique and promising approach to this challenge. Toward this end, we recently have immortalized, cloned and characterized TMJ disc cells that retain stem cell-like characteristics (Park et al., 2015) and could serve in developing discal and attachment tissues as done in the present study using BMSCs. The TMJ disc is highly fibrous and shows a ring-like alignment of collagen fibers throughout the periphery and their antero-posterior alignment through the central region. This anisotropy contributes to optimizing the structure-function relationship of the disc, with antero- posterior alignment supporting the tensile forces imposed on the disc during functional movements, while the circumferential orientation of peripheral fibers serves to resist and displace compressive loads radially away from the locus of the applied hoop stress pressures from the con- dyle contact (Scapino et al., 2006; Athanasiou, 2009). Despite the rela- tively small differences in the collagen and elastin content between the ligamentous attachments and the disc, these together with the region- ally distinct distribution of cartilage specific matrices likely influence the variability in the functional properties between these two types of tissues (Tanaka et al., 1999, 2002, 2003). The regional content of colla- gen and elastin in the TMJ construct are comparable to those previously 306 Lee et al. published in pig (Willard et al., 2012) where the presence of collagen and elastin was demonstrated throughout all regions of the disc and discal attachment, a finding also corroborated in sheep (Figs. 4 and 5). The lowest relative collagen content in pig is in the anterior ligament attachment, although the content did not vary greatly amongst the remaining regional attachments (Willard et al., 2012). We demonstrate a similar trend in sheep in that the lowest relative collagen content also is found in the anterior ligament attachment (Fig. 4C); however, the engineered construct has significantly, greater collagen content than the posterior and anterior ligament, as well as posterior disc- ligament interface of the native tissues (Fig. 4). It is not surprising that collagen composition is uniformly high throughout the construct Since the fabrication was performed to maximize robustness through Sequential monolayer manipulation, differentiation, time for fusion and a Symmetrical engineering configuration. Future studies in which some of these variables are modulated may offer approaches to refine and improve further on the organization and properties of the TMJ disc- attachment construct to mimic that of the native tissues more closely. Cross-linked elastin fibers are relatively small and comprise only 1 to 2% of the tissue mass distributed throughout the disc and discal at- tachments. Because elastin fibers have a compliant characteristic, they serve the important function of restoring the original shape of the tis- Sue complex following loading (Christensen, 1975; Carvalho et al., 1993; Gross et al., 1999). Here we show equal distribution of elastin content in the posterior and anterior of the fibrocartilage disc region in native sheep, as well as in the immediate posterior and anterior ligament-disc attachments; however, there was a lower proportional area of stain- ing in the anterior compared to the posterior ligament region in na- tive sheep tissues (Fig. 5C). With respect to the disc itself, it has been found that the anterior portion of the fibrocartilage disc in humans has the greatest elastin content (Gross et al., 1999), whereas the pig dem- onstrates the greatest elastin content in the posterior portion of the disc (Christensen, 1975; Detamore et al., 2005). The other known elas- tin content comparison of TMJ ligament-disc attachments is for the pig, which demonstrates a relatively low elastin content in the posterior at- tachment; the highest levels are found in the anterior attachment (Wil- lard et al., 2012). These data suggest that the distribution of elastin in the disc and attachment tissues is specific to region and species. The 307 TMJ Disc-Ligament Complex Construct dearth of information on matrix transitions between tissue phases highlights the need to characterize the composition and organization of these interfaces further. Such information will enable the determination of the functional role of these transitional tissues to the TMJ complex and provide useful information for future studies like ours aimed at developing viable tissue-engineered constructs. GAG confers properties required to resist compressive loading and its presence indicates that the TMJ disc is placed under such functional demands. GAG staining was observed in the native sheep disc-ligament complex localized to the fibrocartilaginous disc (Fig. 6), which corresponds to findings in pigs, rabbits and humans (Detamore and Athanasiou, 2003b; Kalpakci et al., 2011b; Willard et al., 2012). However, others have shown absence of GAG staining in the pig disc, which may be attributable to species, age and regional specificity of these molecules (Kalpakci et al., 2011b). We demonstrate an absence of GAG staining in tissue engineered disc-ligament complex constructs (Fig. 6), which we anticipate would limit the potential for the construct to resist compressive loading. Since compressive biomechanical cues rather than biochemical stimulation alone may be necessary to promote GAG expression, the absence of GAG staining in the constructs may have resulted from a lack of compressive mechanical stimulation of the disc-ligament constructs, which were subjected only to tensile loading via Sylgard pinning. Beside the possible lack of mechanical stimulation that may be needed for deriving appropriately mature tissue content, the lack of GAG staining in our disc-ligament construct also may have resulted from inadequate duration and conditions required for robust chondrogenic differentiation and cartilage formation. This is validated by previous studies that show that chondrocytic differentiation requires high-density cultures under chondrogenic conditions over several weeks for robust GAG expression (Xu et al., 2008; Park et al., 2015). Thus, the chondrogenic differentiation performed in monolayer cultures for twelve days followed by differentiation in more high-density conditions for two weeks in our studies may not have been optimal for this purpose. A proposed modification to this approach likely would entail culturing the cells in high-density chondrogenic DM for a few weeks prior to assembly with ligament and bone. From a practical standpoint, this would require a carefully choreographed timing for initiating differen- tiation of the respective cell lineages including an early chondrocytic 308 Lee et al. differentiation in high cell density environments to provide the time and Conditions for achieving the optimal tissue types in the assembled tissue Complex. CONCLUSIONS Our studies demonstrate a scaffoldless method utilizing BMSCs for fabricating a multi-phasic TMJ disc-ligament-bone complex containing Several key matrix molecules and with an organizational configuration that has several similarities to that of its counterpart native tissues. Further work with temporal and density modifications during the differentiation process and assembly protocols—including possibly the integration of a bilaminar asymmetric ligamentous construct–should help achieve a more valid construct. This, together with more detailed evaluation of biochemical composition, organizational structure, cellular phenotyping and biomechanical properties of the multi-phasic disc-attachment complex constructs both in vitro and in vivo, will help complement our findings and advance this novel approach toward clinical applications. Future clinical applications will require intermediate in vivo translational Studies in a large animal model, which offers the biochemical and mechanical loading milieu needed to improve the efficacy of the tissue- engineered TMJ complex construct. Our approach and data provide an important foundation to advance this innovative therapeutic strategy to fabricate and replace critically important degenerated tissues of the TMJ. ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health/ National Institutes of Dental and Craniofacial Research #K12DE023574 awarded as The University of Michigan's TMJD and Orofacial Pain Interdisciplinary Consortium (UM-TOPICS) Training Award. REFERENCES Ahmad N, Wang W, Nair R, Kapila S. Relaxin induces matrix- metalloproteinases-9 and -13 via RXFP1: Induction of MMP-9 involves the PI3K, ERK, Akt and PKC-C pathways. Mol Cell Endocrinol 2012;363(1-2):46–61. 309 TMJ Disc-Ligament Complex Construct Alhadlaq A, Mao J.J. 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Zaucke F, Dinser R, Maurer P, Paulsson M. Cartilage oligomeric ma- trix protein (COMP) and collagen IX are sensitive markers for the 314 Lee et al. differentiation state of articular primary chondrocytes. Biochem J 2001;358(Pt 1):17-24. Zhang L, Hu J, Athanasiou KA. The role of tissue engineering in articular cartilage repair and regeneration. Crit Rev Biomed Eng 2009;37(1-2):1- 57. 315 SINGLE-STAGE RECONSTRUCTION OF ACOUIRED TMJ ANKYLOSIS WITH SURGICAL NAVIGATION AND PROSTHETIC TOTAL JOINT REPLACEMENT Sharon Aronovich ABSTRACT Temporomandibular joint (TMJ) ankylosis is a rare but challenging clinical problem with various treatment options. Alloplastic total joint replacement (TJR) is recognized as the treatment of choice for ankylosis that is refractory to conventional surgical treatments. This chapter explores the use of current technological advances that simplify and enhance these reconstructive efforts. KEY WORDS: ankylosis, navigation, alloplastic, TMJ, surgical simulation INTRODUCTION Temporomandibular joint (TMJ) ankylosis is a rare but Challenging clinical problem with various treatment options. Alloplastic total joint replacement (TJR) is recognized as the treatment of choice for ankylosis that is refractory to conventional surgical treatments. This chapter explores the evolution of alloplastic TJR of the TMJ, indications for use and contemporary technological advances that simplify and enhance these reconstructive efforts. Current uses and techniques will be illustrated through clinical cases. Finally, a case report illustrating the Work-up and management of a patient with refractory TMJ ankylosis will be demonstrated. Prosthetictotal joint components are biocompatible implants that offer a mechanical solution to end-stage arthritis, as well as congenital and acquired disorders of the TMJ. Christensen (1960) described a cast Vitallium hemi-joint replacement with a fossa-eminence prosthesis. The resulting degenerative changes to the native condylar head were a significant disadvantage. In 1980, the Kent-Vitek prosthesis was introduced containing a Teflon-coated Proplast fossa. Despite early en- 317 Acquired TMJ Ankylosis thusiasm and positive short-term outcomes, this fell into disfavor as reports emerged of proliferative giant cell reactions, condylar degeneration, pain and malocclusion. Given the extensive use of alloplastic materials in orthopedic surgery, the experience with total hip and knee replacement surgery would serve as a valuable model for prosthetic TMJ components. In 1960, Sir John Charnley reported on a well-tolerated and biocompatible option for total hip replacement using a stainless steel femoral head on a titanium shaft and a titanium-backed high molecular weight polyethylene acetabular cup (Brand, 2010). Between 1985-1990, surgeons from Rostock, Germany described the evolution from a condylar prosthesis to a total TMJ endoprosthesis made of titanium alloy with a high density polyethylene fossa (Sonnenburg and Sonnenburg, 1985, 1990). Soon after, results of Technedica's CADCAM alloplasts (Henry et al., 1993; Wolford et al., 1994, 1997; Mercury et al., 1995), which currently are known as TMJ Concepts, and Biomet stock prosthesis have helped establish these producers as leaders in the TMJ TJR market. The current materials used have become the gold standard for low friction orthopedic TJR. TMJ Concepts incorporates a condylar head made of cobalt-chromium-molybdenum alloy (ASTM F1537) with a ra- mus shaft made of medical grade titanium alloy containing 90% titanium, 6% aluminum and 4% vanadium (ASTM F136). The fossa is made of Com- mercially pure titanium mesh (ASTM F67) with an articular surface of ultra-high molecular weight (UHMW) polyethylene (ASTM F648). Each al- loplastic component is designed and manufactured to be patient specific, meaning that it will conform to the patient's unique skeletal anatomy in the temporal and mandibular areas and provide maximal surface Con- tact (Fig. 1). This precise mating between the host bone and prosthetic components is considered to be critical for long-term function under the loads exerted by mastication. In some cases such as congenital hypopla- sias or dysplasias of the mandible, or ablative defects as a result of tumor resection or osteomyelitis, a longer span can be used to replace a signifi- cant portion of the mandible or the entire mandible (Fig. 2). To achieve fabrication and TMJ TJR with these customized CAD- CAM components, a two-stage process traditionally is employed. During the first stage of surgery, pre-auricular and submandibular dissections are 318 Aronovich Figure 1. Patient-fitted alloplastic TJR components. A long-span body-ramus- Condyle unit is shown articulating with an ultra-high molecular weight fossa. Figure 2. A: CBCT 3D reconstruction of a patient with Hajdu–Cheney Syndrome and resulting excessive mandibular resorption leading to an acquired loss of the condyle, ramus and posterior body. B: Pre-operative retrognathic profile With unsupported mandible. C. Placement of bilateral long-span alloplasts for replacement of the TMJ, as well as ramus and posterior body of the mandible. D-E. Post-operative CBCT with custom prosthetic components implanted. F-G: Post-operative frontal and profile views with good mandibular projection. 319 Acquired TMJ Ankylosis carried out for access and a condylectomy is performed with resection of associated tissues, including the articular disc and lateral capsule. Any necessary osteoplasty of the glenoid fossa, eminence or ramus is performed. The desired occlusion is set using archbars or fixed orthodontic appliances and maxillomandibular fixation (MMF) is achieved. Finally, a spacer (e.g., silicone block or tobramycin-impregnated polymethyl methacrylate [PMMA]) is placed to fill the space between the glenoid fossa and the ramus. As soon as is feasible after surgery, a CT scan is obtained using a product-specific protocol and the Digital Imaging and Communications in Medicine (DICOM) data is submitted to the company. Fabrication of the prosthetic joint components requires an eight- to- twelve-week process. During this time, the patient remains in MMF while awaiting the Second stage of Surgery. Placement of a spacer eliminates dead space, thus discouraging hematoma formation and preventing fibrous tissue in-growth from fill- ing the space created by the gap arthroplasty. A thin but well-defined fibrous capsule forms around the spacer, which facilitates re-entry and implantation of the prosthesis. Maintenance of MMF until the second stage of surgery is essential for several reasons. First, any function of the mandible will promote movement of the spacer to encourage a hema- toma, or worse, to cause resorptive remodeling of the adjacent bones and thus, an inaccurate prosthetic fit. Second, movement may lead to dislocation of the spacer into the infra-temporal fossa, which is difficult to retrieve and results in fibrous tissue in-growth that complicaties future implantation. Finally, allowing mandibular function during this period carries the risk of erosion of the spacer into the external auditory canal (EAC), thus contaminating the TMJ Space with ear flora. In such cases, the risk of prosthetic infection and failure of the alloplastic components is high and requires an additional surgical intervention to repair the EAC perforation prior to TJR. How are the prosthetic joint components fabricated? DICOM data from the CT scan is used for the fabrication of a 3D-printed stereolithographic (SLA) model. These anatomical SLA models have a high degree of dimensional accuracy, approaching 98% (Mercuri, 2012a). The shape of the mandibular implants is designed to fit the 3D model using CAD software and then waxed up onto the ramus and fossa region. A laser scanner is used to assess the wax-up and allows for necessary refinements to improve the mating geometry of the components to the 320 Aronovich host bone. The surgeon is asked to verify and approve the proposed design. Then the titanium mesh is customized to fit the patient's glenoid fossa and that shape is transferred to the UHMW polyethylene. The Surface is scanned and the bone-side surface of the polyethylene is milled to specification. The UHMW polyethylene then is bonded to the formed mesh with a thermal bond under pressure. An interference fit is created between the condylar head (Co-Cr-Mdb) and the titanium alloy block (Ti- 6Al-4V) via a cylindrical trunion. Once this is assembled, the mandibular implant is milled to specification on a CNC mill. During the second stage of surgery, the same incisions and dissections must be repeated to access the TMJ and mandibular ramus. The spacer is removed and the alloplastic joint components are implanted beginning with the fossa component. Once initial fixation is achieved, the MMF may be released and the mandible mobilized to confirm a reproducible occlusion and determine the patient's risk of dislocation. Fixation is completed once satisfactory adaptation is confirmed and the incisions are closed. The obvious disadvantages associated with such a prolonged pro- Cess and the possible morbidity associated with two surgical procedures has led clinicians to consider if comparable component fit is possible with a one-stage TJR. If feasible, the advantages of a one-stage TJR include: • Avoidance of prolonged MMF period; • Avoidance of the cost and morbidity associated with two operations and two anesthetics; • A decreased risk of cranial nerve 7 injury; • Decreased overall recovery and time away from work or school; • Reduced scarring of the peri-mandibular soft tissue envelope; • Immediate post-operative physiotherapy (PT); and • The creation of stable joint for combined orthognathic TJR Cases. The one-stage approach also has possible disadvantages including a risk of a less-than-ideal fit between the prosthetic components and the host bone, especially when the surgeon must perform extensive Osseous reshaping. This may compromise the distribution of chewing 321 Acquired TMJ Ankylosis loads and lead to loosening or mechanical failure of the prosthetic components. To justify a one-stage approach, we must be confident in our ability to incorporate CAD-CAM and navigation technology into the operating room. Since the inception of patient-specific alloplastic TMJ TJR, the value of customized prosthetic components has been highlighted in the management of end-stage osteoarthritis, as well as in patients with grossly abnormal TMJ anatomy secondary to multiple operations, or as a result of foreign body giant cell reactions to previously implanted Teflon- Proplast alloplasts. The gross inflammatory reactions and abnormal anatomy associated with the latter clearly constitute a need for a staged approach where gross debridement and osteoplasty—the extent of which was unpredictable—was essential prior to obtaining a CT scan and printing a 3D model. While success rates are inversely proportional to the number of prior operations, an early retrospective study with 215 patients found significant reductions in mean pain levels by 58%, 51% increase in mandibular function, a 55% improvement in diet consistency tolerated and a 27% increase in maximal interincisal opening (MIO; Mercuri et al., 1995). Moreover, a mailed questionnaire and exam kit allowing a mean follow-up of 11.4 years in 193 patients continued to support a sustained improvement in MIO by 6.4 mm and an improved quality of life (Mercuri et al., 2007). In a separate patient cohort, a prospective study based on the registry in the United Kingdom found a significant improvement in MIO from 22.4 to 33.7 mm, a significant increase in diet on visual analog scale (VAS) from 38 to 92, and a high level of patient satisfaction with the treatment results after a mean follow-up of twelve months (Sidebottom et al., 2013). Aside from severe degenerative changes associated with osteo- arthritis and multiple prior operations, the indications for alloplastic TMJ TJR include condylar degeneration of varying etiologies (e.g., rheumatoid arthritis, juvenile idiopathic arthritis, idiopathic/progressive condylar resorption and avascular necrosis), congenital condylar malformations (e.g., hypoplasias or dysplasias associated with hemifacial microsomia) as a reconstructive option in ablative defects resulting from condylar-ramus pathology (e.g., benign tumors) and in cases of primary or refractory TMJ ankylosis (Westermark et al., 2011). 322 Aronovich APPLICATIONS OF TMJ TJR IN A ONE-STAGE APPROACH Case 1 A 50-year-old female presented with a two-year history of progressive condylar resorption, a receding chin, Class II anterior openbite malocclusion and severe retrognathia with a high mandibular plane angle (Fig. 3). A polysomnogram demonstrated moderate obstructive sleep apnea (OSA) with an AHI of 21. This was associated with significant Symptoms including daytime hypersomnolence, lethargy and morning headaches. On exam, she had a 4 mm openbite and an overjet of 9 mm. An elongated and tapered lower facial third was evident along with a convex facial profile and poorly defined gonial angle. She denied pain and had unhindered mouth opening. A rheumatological survey was negative and Serial cephalograms with clinical correlation confirmed the progressive nature of this process. Panoramic radiography and CBCT demonstrated Severely resorbed condyles with loss of normal morphology. Articulation of the maxillary and mandibular dental casts produced the patient's prior Occlusion with good intercuspation. - - - - - Figure 3. A patient with progressive condylar resorption exhibited the features asSociated with loss of ramus height; receding chin, excessive anterior face height, short posterior face height, high mandibular plane angle, poorly defined 30nial angle and Class || malocclusion with apertognathia. 323 Acquired TMJ Ankylosis The goals of treatment included stable occlusal correction, nor- malization of the facial skeleton, increased upper airway dimensions, treatment of the OSA and normalized oral function. Given her Symp- tomatology associated with sleep-disordered breathing, the patient was motivated for a surgical correction at the earliest convenience. Surgical options included conventional orthognathic surgery, costochondral rib grafts (CCRG) and alloplastic TJR. Given the progressive condylar resorp- tion and large mandibular advancement needed, orthognathic Surgery carried significant risk of occlusal instability and relapse. Autogenous grafts (e.g., CCRG), along with condylectomies, also are susceptible to remodeling changes or possible post-surgical resorption with associated occlusal instability. Moreover, there may be significant donor site mor- bidity associated with CCRG harvest (Dimitroulis, 2014). While alloplastic TJR of the TMJ carry a 1.51% risk of infection (Mercuri et al., 2011) and the unlikely possibility of mechanical failure, prosthetic components pro- vide unparalleled stability in the skeletal and occlusal correction without the risk of resorption and relapse (Mercuri, 2007). This was chosen as the most predicable treatment option in this case. Correction of skeletal symmetry, occlusion and a steep occlu- sal plane angle was planned using computer-aided Surgical Simulation, which commonly is used for virtual surgical planning of orthognathic cases, due to its high degree of accuracy (Xia et al., 2007; Sheng-Pin et al., 2013). The virtual plan included bilateral condylectomies, mandib- ular advancement with counterclockwise (CCW) rotation and a Le Fort 1 maxillary osteotomy (Fig. 4). Cutting guides were provided for accu- rate placement of condylectomy osteotomies and leveling guides were provided for lateral ramus osteoplasty (Fig. 5A). No modifications were made to the glenoid fossa or eminence. The plan also included coronoid process osteotomies to allow complete mobilization and advancement of the mandible. The final occlusion was set with dental casts and the — Figure 5. A: 3D printed custom condylectomy cutting guides and ramus resurfacing guides for accurate reproduction of the simulated plan. B: This 3D anatomical SLA model, based on the desired final skeletal and occlusal position, is used for the fabrication of patient-fitted alloplastic joint components. C: Post-operative lateral cephalograms after bilateral alloplastic TJR and Le Fort 1 maxillary osteotomy. 324 Aronovich Pre-operative Position Figure 4. Computer-assisted surgical simulation used to planned con- dylectomy osteotomies, mandibular advancement with CCW rotation, maxillary advancement and lateral ramus osteoplasty. 325 Acquired TMJ Ankylosis virtual plan data was used to generate a 3D SLA model (Fig. 5B). This was reviewed and submitted for fabrication of prosthetic joint Com- ponents. Once fabrication was complete, the patient was taken to the operating room for one-stage correction. Surgical access was obtained via a pre-auricular incision for access to the TMJ and a submandibu- lar incision provided access to the angle and ramus. After completion of condylectomies and coronoidectomies, the mandible was mobilized with simultaneous CCW rotation, advanced to the intermediate occlu- sion and the TMJs were reconstructed with prosthetic joint compo- nents. Since no fossa reshaping was planned, removal of residual in- tra-articular soft tissues was sufficient to obtain ideal adaptation of the fossa component. The cutting guides and accuracy of the intermediate occlusal splint were used to ensure the ideal fit of the ramus-condyle component. Once the transfacial incisions were closed, the maxillary operation could be accomplished with stable mandible and a reliable centric relation (CR; Fig. 5C). The patient's recovery was uneventful. Her occlusion and skeletal correction remained stable at her two-year follow-up (Fig. 6). A substantial increase in the airway dimensions was achieved and a post- operative polysomnogram demonstrated complete resolution of OSA. Normal mouth opening was rehabilitated with passive range of motion appliance (Fig. 7). Case 2 A 33-year-old female presented following iatrogenic subcondy- lar fractures during attempted treatment of TMJ internal derangement with modified condylotomies. Her exam revealed a loss of posterior vertical dimension, a large anterior openbite measuring 17 mm, Class || malocclusion with a high mandibular plane angle, lip incompetence with mentalis muscle strain and a long lower facial third (Figs. 8-9). A CBCT survey indicated significant condylar dislocation and resorption with nar- rowing of the airway. An overnight polysomnogram revealed OSA. The lack of temporomandibular articulation resulted in mandibular malpo- sition and soft tissue contracture, which represented a contraindica- tion to conventional orthognathic techniques for mandibular advance- ment. While autogenous grafting with CCRGs represented a viable op- tion, the planned advancement of 27.9 mm at pogonion was associated with significant lengthening of suprahyoid muscular attachments, which 326 Aronovich Figure 6. Pre- (left) and two-year post-operative (right) pho- tos with notable facial changes including improved mandibular projection, well-defined gonial angles and inferior border, and adequately supported facial soft tissue envelope. 327 Acquired TMJ Ankylosis occlusion with positive overbite and overjet (middle) and normal mouth opening (bottom). Figure 8. The patient sustained an iatrogenic condylectomy with 17 mm anterior openbite and facial changes associated with loss of ramus height. 328 Aronovich Figure 9. Pre-operative situation on lateral cephalograms and panoramic radio- graph. Could produce excessive soft tissue tension and relapse tendency. This may favor resorptive remodeling of CCRGs or even loss of fixation. For these reasons, a reliable biomechanical solution with alloplastic TJR was deemed most appropriate. Dental cast analysis indicated the need for pre-Surgical orthodontics. After several months of orthodontic preparation and Confirma- tion of the desired final occlusion with model Surgery, the approach described in Case 1 was used for planning and execution of the Surgical treatment for this patient. Specifically, a large mandibular advancement, ramus lengthening and CCW rotation was simulated (Fig. 10). This usu- ally creates a significant clearance for the prosthetic components, so the Condylectomy osteotomies were placed close to the site of prior Subcon- dylar fractures. In addition, an osteoplasty of the ramus was planned to reduce the lateral mid-ramal prominences in this case (Figs. 11-12). This decreases the lateral width of the face, which would be an undesirable masculinizing feature in this case. After releasing the pterygomasseteric attachments and achiev- ing the desired condylectomies, coronoidectomies and osteoplasties, the mandible was advanced with a bone hook engaging the lingual cortex of the Symphysis transcutaneously (Fig. 13A). The intermediate occlusion Was established with the intermediate surgical splint and stabilized with 24-gauge wire loops. Once the prosthetic components were implant- ed and stabilized (Fig. 13B), the transfacial incisions were closed 329 Acquired TMJ Ankylosis Figure 10. Virtual surgical planning shows pre-operative, intermediate occlu- sion and final positions. Mandible first surgery with implantation of Custom alloplastic components restores a functional articulation in centric relation. 1.3 mm 1.8 mm Figure 11. Custom condylectomy guide and plan for lateral ramus osteoplast). 330 Aronovich Figure 12. Wax-up of the proposed prosthetic joint components on the post- Simulation SLA model. Figure 13. A Typical sterile prep and drape for pre-auricular and submandibular approaches to the TMJ and ramus. B: Intra-operative position of fossa and ramus- Condyle units. 331 Acquired TMJ Ankylosis in layers with care to re-approximate the medial pterygoid and masseter muscles around the inferior border; this guides muscular reattachment of the Sling, which may prevent condylar sag and malocclusion. With stable condyles established, a two-piece segmental Le Fort 1 maxillary osteotomy was completed to correct a transverse discrepancy of the occlusion (Fig. 14). The patient was maintained in MMF for five days to allow initial muscular reattachment and help prevent the risk of early mandibular dislocation with mouth opening. Guiding elastics were used for several weeks to support the mandible further and guide the teeth into occlusion. Mandibular functional rehabilitation must start early in the process and typically includes active and passive Figure 14. A-B: Post-operative panoramic and frontal films reveal a well-seated fossa mesh and occlusion in the final splint. C: Pre- and post-operative lateral cephalograms with an increase in posterior airway space after mandibular advancement and a two-piece segmental Le Fort 1 osteotomy. 332 Aronovich range of motion appliances. The latter may include the use of stacked tongue blades for static stretch and dynamic passive range of motion devices that are activated manually by the patient. With an accurate simulation of the ramus osteoplasty required, the use of cutting guides and excluding the need to modify the shape of the glenoid fossa-eminence complex, this case illustrates the ease of incorporating a one-stage approach with prosthetic TJR and conventional maxillary osteotomy (Fig. 15). - - - - Figure 15, Pre- (top) and eight-week post-operative (middle and bottom) facial and occlusal photographs. 333 Acquired TMJ Ankylosis Case 3 This is the case of an 18-year-old female with fetal alcohol Syn- drome (FAS), refractory bilateral TMJ ankylosis, severe microretrognathia and OSA (Fig. 16). She was born at 38 weeks gestation to a mother who had a history of alcohol abuse. At birth, her exam was notable for Con- genital micrognathia, TMJ ankylosis, high palatal vault, bilateral clubbed feet and oculomotor apraxia. As an infant and toddler, she suffered from a failure to thrive (at or below 5th percentile for height and weight), re- current otitis media and was found to have developmental delays in both motor skills and language. The patient's relevant surgical history included bilateral TMJ gap arthroplasties with fat graft, bilateral coronoidectomies and masseter myotomies eight years prior to presentation. Despite the recommended PT and adjuvant care from members of the craniofacial team, she developed recurrent ankylosis of the bilateral TMJs. Three years later, she underwent repeat bilateral TMJ gap arthroplasty with temporalis muscle flaps and buccal fat grafts. In addition to attempts at home and professional PT, several occasions of TMJ manipulation were performed under anesthesia. She presented with multiply recurrent TMJ ankylosis that was re- fractory to prior treatments. On exam, dysmorphic facial features were apparent and included severe microretrognathia, shortened posterior vertical dimension, vertical maxillary excess, long anterior face height, severe transverse maxillary hypoplasia, apertognathia, high arched pal- ate, lip incompetence and mentalis strain, immobile mandible, deep la- biomental fold, obtuse cervico-mental angle and probable disuse atro- phy of the masseter and medial pterygoid muscles (Fig. 17). Imaging with panoramic radiography demonstrated a dysmorphic mandible with aper- tognathic, prominent antegonial notches, high mandibular plane angle, coronoid hyperplasia and possible re-ankylosis of the TMJs with evident heterotopic bone islands in the right TMJ area. — Figure 17. Note the severe maxillary narrowing with apertognathia and poste- rior impingement of the maxillary arch by the ankylosed mandible. 334 Aronovich Figure 16. A patient with fetal alcohol syndrome (FAS), ankylosis, microretrogna- thia, lip incompetence and apertognathia. - 335 Acquired TMJ Ankylosis Given her hypoplastic craniofacial anatomy, there was high clini- cal suspicion for upper airway constriction and possible sleep-disordered breathing. An overnight polysomnogram revealed moderate OSA with an apnea-hyponea index of 23.2 respiratory events/hour and a minimal oxygen saturation of 90%. Of note, her sleep architecture was distorted significantly with no recorded REM sleep on EEG monitoring. This cor- related with non-restorative sleep clinically. Based on initial clinical, radiographic and polysomnographic data, a treatment plan was created to address the patient's specific problem list (Table 1). A CT angiogram (CTA) is obtained to visualize the extent of the TMJ ankylosis and associated bony dysmorphology in three dimen- sions (3D). It also allows the surgeon to assess the relationship of the external carotid and internal maxillary artery branches to the ankylotic bone mass. In some cases, especially in the presence of exuberant ex- tra-articular ankylosis and more commonly in recurrent ankylosis cases, blood vessels may be found traveling directly adjacent to or through the bony ankylotic mass. In the latter case, severe bleeding may be en- countered during gap arthroplasty if proper precautions are not taken. Therefore, consultation with an interventional radiologist to accomplish selective embolization of the involved blood vessels must be carried out within a day or two of the operation. A small case series provides sup- port for this treatment protocol (Susarla et al., 2014). As an additional precaution, a type and cross is performed and blood products should be made available during the operation. The patient's maxillofacial CTA demonstrated a left TMJ bony an- kylosis and a right TMJ fibrous ankylosis with heterotopic bone islands, bilateral coronoid elongation and an intimate association between the ankylotic TMJ mass and the adjacent medial maxillary artery and ptery- goid plexus; however, there was no evidence of vessels coursing through the bony ankylosis. Craniofacial dysmorphology of the maxilla and man- dible also was demonstrated, as noted previously (Figs. 18–19). – Table 1. Itemized problem list and treatment strategy to normalize form and function in a patient with FAS, ankylosis and microretrognathia. 336 Aronovich PROBLEM LIST TREATMENT GOALS Restore normal mouth opening: 1. Resection of bilateral bony TMJ ankylosis and heterotopic bone 2. Coronoidectomies 3. Release of scarred soft tissue envelope 4. Separation of mandibular and temporal bone with alloplastic total joint components and abdominal fat grafts 5. Immediate and aggressive post- operative PT with passive ROM appliance, profession PT visits and TMJ manipulation under anesthesia Bony ankylosis with coronoid elongation and post-Surgical Scar tissue Large mandibular advancement with CCW rotation and reconstruction with alloplastic total joint components: 1. Lengthen posterior facial height 2. Shorten anterior face height and advance the mandible to a mild Class Ill angle relationship; facilitates improved lip competence 3. Alleviate impingement on the maxillary posterior dentition 4. Normalize mandibular plane angle 5. Facilitate oral hygiene, dental prophylaxis, restoration of caries and access for orthodontics 6. Enlarge the upper airway 1. Severe micrognathia with high occlusal plane angle 2. Upper airway obstruction (OSA) 3. Inadequate access for orthodontics — blocked out maxillary arch by impinging mandibular arch (Fig. 17) 4. Inability to perform effective oral hygiene on posterior dentition 5. Lip incompetence Strengthening exercises for jaw closing muscles to start post-operatively: 1. Includes gum chewing 2. Assisted jaw closure exercises Ankylosis mediated disuse atrophy of masseter and medial pterygoid muscles Restore normal occlusion via Residual post-operative Class Ill orthodontic treatment: malocclusion with anterior open | 1. SARPE bite and transverse 2. Orthodontic appliances and Le Fort maxillary hypoplasia 1 maxillary osteotomy 337 Acquired TMJ Ankylosis Figure 18. CTA reveals a dysmorphic and retrognathic mandible, bony ankylosis of the left TMJ, fibrous ankylosis of the right TMJ with heterotopic bone islands and coronoid enlargement with ankylosis. Figure 19. Reconstructed views of CTA revealing the relationship of vascular structures medial to the ankylosis. 338 Aronovich The CT image files in DICOM format then were converted to STL files for 3D segmentation and computer-assisted surgical simulation. This was combined with scanned data of the occlusal detail and merged to reflect the patient's pre-surgical occlusion. The planned ankylosis resec- tion, condylectomies, coronoidectomies and mandibular advancement with CCW rotation then were simulated virtually. Familiar cephalometric measurements and clinical information were used to guide the degree of skeletal movement desired. Treating her to cephalometric norms pro- duced a 31.6 mm advancement at pogonion and a 15.4 mm advancement at the mandibular incisal edge (Fig. 20). To reproduce this surgically, addi- tive manufacturing technology was used to create a 3D-printed occlusal Splint, condylectomy cutting guides and fossa reshaping templates (Fig. 21). The final desired skeletal position was established and a 3D anatomic SLA model was printed and submitted for custom alloplast fabrication (Fig. 22). The use of a fossa-reshaping template constitutes one of several techniques to reproduce the planned glenoid fossa/eminence anatomy for an accurate fit between the host bone and the prosthetic joint fossa Post-operative Position Figure 20. Virtual surgical planning for mandibular advancement and CCW rotation, 31 mm advancement at pogonion, simulated fossa reshaping and Condylectomy osteotomies. 339 Acquired TMJ Ankylosis Figure 22. Wax-up of patient-fitted alloplastic components with care taken to avoid the inferior alveolar nerve displayed as red dashed line. component. This ideally is supplemented with the use of surgical navigation technology to improve safety and accuracy. The navigation system is composed of a computer, monitor, detector and a series of emitters. DICOM files of the pre- and post-operative skeletal situations are transferred to the navigation system. The surgeon picks landmarks on the CT reconstruction images to register the patient during surgery. A link is 340 Aronovich made between the patient and his/her 3D CT images, which enables the Surgeon to track the instruments (e.g., pointer or hand piece) against the backdrop of the CT images. The use of a facemask or surface matching allows further refinement of this registration. The position of the patient's head is tracked by the patient tracker, which typically is mounted on a fixed skull post in these cases. After the working tip is calibrated using the calibration station, the position of any surgical instrument and its cutting tip may be followed by the instrument tracker (Fig. 23). Thus, a hand piece may be navigated during osteotomy to identify its proximity to vital structures and to ensure reproducibility of pre-surgical plan. Specifically, after safe resection of the ankylotic mass, data of the desired final margins of glenoid fossa-eminence complex (visible on the post- operative dataset) are used to navigate a precise fossa reshaping. Based on the preference of the clinician, there are several ways to establish the bony margins of the fossa-eminence complex: 1. When the fossa reshaping is simulated virtually, DICOM files of the post-operative situation may be uploaded directly to the navigation system. 2. A 3D SLA model is provided. Fossa-eminence reshap- ing then is achieved with standard rotary instruments until deemed satisfactory and reproducible clinically. The SLA model, with new temporal resection margins, is stabilized and imaged using a CBCT scanner. The Dl- COM data is uploaded to the navigation system (Fig. 24). 3. A 3D SLA model is provided. Fossa-eminence reshap- ing then is achieved with navigated rotary instruments to track the bony reshaping. To accomplish this, the desired CT data is imported into the surgical naviga- tion system, the SLA model is registered with a fixed post and reshaping of the model is achieved as noted above. The new margins are stored for use during sur- gery (Malis et al., 2007). Surgical preparation requires a strict sterile protocol to disinfect the patient's hair and ear canals (Mercuri, 2012b). A second oral table is needed on which to place oral appliances and adapt occlusal splints with MMF wires. Surgical access is obtained via a pre-auricular incision 341 Acquired TMJ Ankylosis Figure 23. Surgical navigation. A: Skull-post/patient tracker. B: Navigation tower with IR receiver. C. Calibrated hand piece ready for navigated rotary instrumen- tation. Figure 24. SLA model that has been reshaped and scanned with a CBCT to generate a data set with the desired final temporal bone margins. for access to the TMJs and a submandibular incision to access the angle and ramus. The operator must be oriented properly during the approach to the ankylotic mass (Fig. 25). Local landmarks may be distorted and thus, using a CT-based navigation probe is advantageous. Gap arthro- plasty may be created with rotary instruments, saws or piezoelectric in struments. In the author's experience, the use of the piezoelectric 342 Aronovich Figure 25. Intra-operative view after right TMJ ankylosis release and demon- Stration of left TMJ ankylosis. bone scalpel is a safe and efficient method of performing an osteotomy through the ankylotic mass with minimal risk of vascular or soft tissue injury medially. This tool uses piezo crystals to convert and amply electric Signals into mechanical vibrations that allow the ultrasonic cutting of bone with a blunt round tip at 22,500 Hz. The ultrasonic tip provides tactile feedback throughout the ostectomy and simultaneous irrigation to minimize thermal damage to bone that will be supporting prosthetic Components. This ultrasonic tip is calibrated for surgical navigation, Which allows the operator to avoid perforation into the middle cranial fossa or middle ear while monitoring the gap arthroplasty as it progresses medially. After completion of condylectomies and coronoidectomies, the fossa is reshaped using one of the methods described above (Fig. 26). Mobilizing the mandible a great distance can be a challenge. First, the pterygomasseteric sling is released subperioteally, the temporalistendon is detached during coronoidectomy and the suprahyoid musculature 343 Acquired TMJ Ankylosis Figure 26. A: Exposure of ramus. B: Placement and fixation of custom cutting guide. C. Condylectomy. D: Removal of heterotopic bone islands and elongated coronoid process (bottom right of panel). remains. After isolating the sterile dissections bilaterally, the oral cavity is approached. A bone hook is used to puncture the submental skin and engage the lingual aspect of the mandible. This is used to advance the mandible while digital distraction forces are applied at the molars to lengthen the ramus vertically. Occasionally, a three-pronged Smith spreader or Turvey spreader with soft rubber covers may be used to distract the molars and loosen perimandibular scar tissue. Active back and forth forces are most effective to advance the mandible adequately. While a suprahyoid myotomy may be an option if the desired 344 Aronovich advancement cannot be achieved, the benefits of upper airway enlarge- ment may not be realized if this is employed. The mandible is stabilized with the surgical stent and maxillo- mandibular wire fixation (Fig. 27). Good hemostasis must be achieved and may require the use of local hemostatic agents medial to the glenoid fossa. The surgical sites are irrigated with antibiotic-impregnated saline Solution and the prosthetic components also are soaked in this solution. The fossa and ramus-condyle components are implanted sequentially and fixed in position with pre-measured screws based on the patient's anatomic 3D model. Accurate seating of the prosthetic components is paramount to long-term biomechanical stability and to prevent dislo- Cation. The condyle is seated in the most posterior and superior aspect of the UHMW polyethylene fossa to maximize range of motion, provide Condylar stability and reduce the risk of anterior dislocation. Harvested abdominal adipose tissue graft is placed medial and lateral to the pros- thesis. Finally, the pterygomasseteric sling is reconstructed around the inferior border by identifying and suturing the medial pterygoid and mas- Seter muscles together with longer lasting resorbable sutures. This guides muscular reattachment to the ramus, supports the condyle vertically to prevent condylar sag and help maintain the desired occlusion. Elastic guidance with heavy rubber bands provides adequate support while this muscular reattachment takes place. After prosthetic implantation is achieved, the MMF wires are released to confirm the desired occlusion is obtained with stable hinge Figure 27. Advancement of the mandible to the proposed occlusion and reten- tion with surgical stent and heavy SS wires. 345 Acquired TMJ Ankylosis motion at the condyles. The incisions are closed in a layered fashion (Fig. 28). Post-operative lateral cephalometric film demonstrates the new mandibular position, alloplastic components and a significant increase in upper airway dimension (Fig. 29). A six-month post-operative polysomnogram revealed normalized sleep architecture with mild snoring, but no OSA. Photographic documentation two months after surgery reveals a substantial increase in mandibular projection and lower anterior face height; however, there is persistent evidence of poor muscle tone with associated incomplete closure, lip incompetence and drooling (Fig. 30). Leveling of the mandibular occlusal plane has eliminated previously noted impingement of the maxillary arch to facilitate oral hygiene, restorative dental treatment and future surgically assisted rapid palatal expansion and maxillary advancement (Fig. 31). After surgery, a drastic improvement in mouth opening to 35 mm is noted while undergoing mandibular manipulation under anesthesia (Fig. 32). Achieving such a significant improvement clinically depends on the patient's unwavering commitment to vigorous daily PT that includes static mandibular stretches, active and passive range of motion (ROM) exercises, and closing exercises to strengthen elevator muscles. This expectation clearly is communicated to patients prior to surgery and a passive ROM appliance is obtained to facilitate immediate post-operative exercises. DISCUSSION While TMJ ankylosis is a rare condition, there are several risk factors that may predispose a patient to this complication. Etiologies described in the literature include trauma, otitis media, septic arthritis, congenital (e.g., idiopathic, gene mutation, Syndrome), iatrogenic (e.g., distraction osteogenesis, arthroplasty and discectomy, MMF) and high inflammatory states (e.g., juvenile idiopathic arthritis or rheumatoid arthritis). Facial trauma is the most common cause of ankylosis. Mandibular fractures that may lead to ankylosis include intra-capsular condylar fractures treated with prolonged periods of MMF and laterally displaced subcondylar fractures with a symphyseal fracture that lead to mandibular widening. In the latter situation, inadequate reduction of the fractured segments, or open approaches that do not preserve the articular disc, may lead to TMJ ankylosis. Given their higher number of 346 Aronovich Figure 28. Pre- and immediately post-operative change in facial projection after ankylosis release, mandibular advancement and TJR. Figure 29. Post-operative lateral cephalograms and frontal view with adequate fossa mesh-to-temporal bone adaptation. 347 Acquired TMJ Ankylosis Figure 30. Notable post-operative facial changes with resolution of OSA. progenitor stem cell, rapid cytokine-mediated reparative fibroplasia and predisposition to rapid bone regeneration, this risk is more pronounced in children compared to adults. Given the rare occurrence of ankylosis, even in the setting of trauma, some authors hypothesize that a genetic predisposition may play a greater role (Hall, 1994; Gu et al., 2008; Braeſſ and Lories, 2012). While ShoX2-deficiency and ANKH gene mutation lead to fibrous ankylosis in mice, the role of genes has yet to be elucidated clearly in animal and human studies (Hall, 1994; Gu et al., 2008; Braeſſ 348 Aronovich Figure 31. Occlusal change to a Class III position without any impingement of the maxillary arch post-operatively. - - Figure 32. Mandibular manipulations under anesthesia as an adjunct to home and professional PT after surgery. and Lories, 2012). Further molecular research on the role of osteoin- ductive and inhibitory proteins that regulate bone regeneration in dis- eased States may pave the way for adjunctive gene therapies or medical treatments. Rittenberg and associates' study (2005) that focused on the mechanisms regulating BMP-induced heterotopic bone formation found that alpha2-HS-glycoprotein (Fetuin) inhibits osteocyte cell signaling by binding to TGF-beta/BMP receptors. In vivo experiments comparing wild 349 Acquired TMJ Ankylosis heterozygous and knockout mice revealed a significantly higher incidence Of ectopic ossification in the latter group (Rittenberg et al., 2005). TMJ hemarthrosis in the setting of articular surface damage—a potential outcome of facial trauma–also may lead to heterotopic bone formation and ankylosis (Yan et al., 2014). Early mobilization with range of motion exercises is a recommended preventative strategy. In the de- veloping world, where otitis media may go untreated in children, the local spread of infection to the TMJ may be a source of septic TMJ ar- thritis (Kumar et al., 2013). This may damage the synovium and erode the osteochondral surfaces, which may be followed by reparative fibrosis and periostitis that are predisposed to the risk of a fibrous or bony TMJ ankylosis. A similar mechanism may be responsible for ankylosis in pa- tients with high inflammatory states (e.g., juvenile idiopathic arthritis). The true etiology of longstanding ankylosis in the last patient presented (Case #3) cannot be determined with certainty, but she had a history of frequent otitis media infections as a child. While there is no known asso- ciation of TMJ ankylosis with FAS, syndromes that have been associated with ankylosis include ankylosing spondylitis, arthrogryposis, SAPHO Syn- drome (Müller-Richter et al., 2009), acrocephalosyndactyly syndromes (e.g., Pfeiffer and Apert) and Treacher Collins Syndrome (TCS). A report on congenital ankylosis of the TMJ identified difficult forceps delivery and hydramnios as possible etiological factors (Burket, 1936). Mandibular hy- pomobility, facial asymmetry or unilateral hemiatrophy, difficulty feed- ing, microretrognathia and upper airway obstruction with cyanosis may be early signs and symptoms that should raise clinical suspicion for TMJ ankylosis. Radiographic studies, ideally CT, are required for a definitive diagnosis. Treatment options for ankylosis generally include gap arthro- plasty (GA) alone, GA with interpositional tissue (GAIPT) or GA with au- togenous or alloplastic (prosthetic TJR) reconstruction of the articulation. Coronoidectomy or coronoidotomy are adjunctive procedures used to gain adequate mouth opening in the operating room. Aggressive post- surgical PT, including active and passive range-of-motion exercises, is a critical element of any protocol and may be supplemented by chemode- nervation of masticatory muscles with botulinum toxin. Gap arthroplasty alone involves an ostectomy to separate the temporomandibular bones and allow immediate mobility of the mandi- 350 Aronovich ble. This usually is followed by a loss of posterior vertical ramus height as the condyle settles back into the fossa to achieve a superior stop and thus, brings the separated bones into close proximity once again. This also may be accompanied by facial asymmetry and openbite malocclusions. Topazian (1966) found a 53% re-ankylosis rate after GA alone compared to no recurrence after GA with autogenous interpositional tissue placement. He and colleagues (2011) found a 36.4% re-ankylosis rate after lateral gap arthroplasty. There is debate over the extent of gap arthroplasty needed to prevent recurrence. Minimal gap arthroplasty (GA), at least 5 to 10 mm GA, or as much as 2 cm GA all have been described (Nitzan et al., 2012). Bhatt and coworkers (2014) compared GA to GAIPT with temporalis myofascial flap in 262 patients with either a first-time ankylosis or previously treated patients with a re-ankylosis. Patients with first- time ankylosis had a 14.6% re-ankylosis rate with GA compared to 4.8% with GAIPT using the temporalis myofascial flap. However, among the 42 recurrence cases, re-ankylosis occurred in 34.5% with GA and 30.8% with GAIPT. With a mean follow-up beyond three years, an openbite was identified in 9.2% with GA and 34.5% with GAIPT (Bhatt et al., 2014). These results highlight the value of interpositional tissue in first-time Cases, but an excessively high re-ankylosis rate for recurrent cases in both groups. Moreover, vertical changes in the length of the ramus tend to produce openbite malocclusions. While GA leads to anterior openbite tendency, insertion of a bulky temporalis myofascial flap produces a posterior openbite with eventual reduction of flap volume and occlusal uncertainty. To maintain vertical ramus height while preserving the occlu- Sion and facial symmetry, the use of autogenous bone grafts have been advocated. Examples include the costochondral rib graft (CCRG), sterno- Clavicular graft, metatarsal graft, iliac crest graft, coronoid process graft (pedicled or free), posterior ramus sliding osteotomy and transport dis- traction osteogenesis (DO) of the ramus. Kaban (2009) described a pro- tocol for managing TMJ ankylosis that incorporated an extensive GA, Coronoidectomies, lining the joint with temporalis fascia or native disc, CCRG or DO, and aggressive post-operative PT. Outcomes on 14 patients (18 joints) treated with his protocol were reported. With a one-year follow-up, mean mouth opening improved from 16.5 to 37.5 mm; how- ever, occlusal outcomes were not reported and the pre-operative mouth opening indicates that not all patients had bony ankylosis. In addition, 351 Acquired TMJ Ankylosis to allow revascularization of the CCRG, ten days of MMF were employed. There are several possible disadvantages to this approach. First, signifi- cant donor site morbidity and complications necessitating return to the operating room have been reported in association with costochondral rib graft harvest (Dimitroulis, 2014). Moreover, both the placement of an au- togenous bone graft and immobilization the mandible after resection of a bony ankylosis seems counterintuitive where there is a substantial risk of re-ankylosis and heterotopic bone formation (Mercuri, 2000). Placement of an autogenous bone graft after GA provides an osteoconductive and inductive scaffold for bone formation while shortening the distance be- tween articulating surfaces (Cheung et al., 2007). Finally, the CCRG may undergo lateral overgrowth or resorptive remodeling that lead to signifi- cant malocclusions and skeletal asymmetries. An alternative CT-guided protocol was advocated (Nitzan et al., 2012) using minimal and selected gap arthroplasty to release the ankylosis, preserve the displaced condyle, use the articular disc as interposition tissue when available and guided post-operative PT. A series of thirteen patients treated with this proto- col had a mean mouth opening improvement from 18.4 to 41.2 mm and all patients preserved a stable occlusion as well as good facial symmetry over a five-year follow-up period (Nitzan et al., 2012). There is a lack of controlled clinical studies to develop evidence- based recommendation for the treatment of a rare condition such as TMJ ankylosis. The majority of publications on TMJ ankylosis and its management include expert opinions, surgical techniques, case series and retrospective studies with small sample sizes. Among the published studies, there is significant heterogeneity within the patient sample, pre-operative characteristics and surgical techniques; however, there are many challenges, both ethical and logistical, that preclude a pro- spective randomized clinical trial for this condition. A review and meta- analysis that compared GAIPT to GA-CCRG found a significantly greater maximal mouth opening in patients who had GAIPT without a rib graft. Occlusal stability and skeletal symmetry were not assessed (Katsnelson et al., 2012). A comparative retrospective study (Loveless et al., 2010) reviewed patients treated with GAIPT or GA-prosthetic TJR with ab- dominal fat grafts at two different institutions. After performing a re- gression analysis adjusting for age, etiology, number of previous proce- dures, laterality and institution, they found no significant difference in 352 Aronovich maximal mouth opening or pain levels with these two treatment modali- ties. Most patients in the prosthetic TJR group had multiple previously failed operations, significant pain and chronic opioid use, making true comparison to the GAIPT group difficult. This study did not report out- comes such as diet consistency, occlusal stability or facial symmetry that have significant quality of life implications. In a well-matched retrospective study (Saeed et al., 2002), 49 pa- tients with CCRG reconstruction were compared to 50 patients treated with alloplastic TJR. While each group had several pre-operative diagno- ses, ankylosis was present in 35 patients in the CCRG group and in 15 patients undergoing alloplastic TJR. They found greater improvements in maximal mouth opening and diet in the alloplastic TJR group. In the latter group, mean mouth opening improved from 17.1 to 25.2 mm, whereas the CCRG group has an insignificant change from 23.2 to 24.6 mm. A strik- ing finding was the high rate of complications requiring re-operation in the autogenous CCRG group. The latter group had 27 complications in 23 patients including overgrowth of the rib in three patients and anky- losis in 18 patients. Moreover, 26 re-operations were required in 18 pa- tients including repeat arthroplasty in 16 subjects, graft removal in five, osteotomy in two and alloplastic TJR in three patients. In contrast, the alloplastic TJR group required six additional re-operations for dislocation and infection of prosthetic components, with the majority experiencing temporary complications involving motor nerve paresis and neurosenso- ry alterations (Saeed et al., 2002). Careful review of the limited evidence indicates that alloplastic TJR is a safe and effective treatment for TMJ ankylosis or re-ankylosis, with better outcomes than autogenous CCRG in terms of mouth opening and improved likelihood of occlusal stability compared to both GAIPT and CCRG. To date, the use of alloplastic TJR is not accepted well in children. Despite the advantages and disadvantages of the treatment modalities described, post-operative PT is likely to be the most impor- tant determinant of success in terms of mouth opening and preven- tion of re-ankylosis. It is a difficult variable to measure since patient Compliance and anamnestic reports of frequency are unreliable. Set- ting up effective PT requires significant patient counseling and attain- ment of a passive mandibular rehabilitation appliance prior to surgery. A structured regimen with calendar reminders is best. Pain associated with range of motion exercises may create a significant hindrance to 353 Acquired TMJ Ankylosis adequate PT for children. Rewards and incentives can be beneficial when used by parents to help motivate growing patients. A consultation with a child psychiatrist or behavioral specialist may be warranted in select situations. The use of NSAIDs, opioids and muscle relaxers prior to range of motion exercises may help increase the frequency and efficacy of stretching exercises. Mobility is a key inhibitor of bone regeneration at a fracture or ankylosis site. Clinicians must monitor the maximal interinci- sal opening frequently and consider mandibular manipulation under an- esthesia when appropriate. Future research may employ memory chips in mandibular rehabilitation appliances to measure the frequency of PT performed by patients and assess its role in treatment outcome. As highlighted in Case 3, when advancing the mandible simulta- neously with a GA, there is significant posterior soft tissue pull that arises from the suprahyoid muscles that must be overcome with rigid fixation in order to maintain the desired occlusion. The thinness and flexibility of the CCRG may not be suited ideally to overcome these soft tissue forces. In the author's opinion, alloplastic TJR is the ideal choice for superior oc- clusal and skeletal stability when combining TMJ replacement with man- dibular advancement and occlusal plane rotations. Furthermore, in the setting of TMJ ankylosis, a large bone gap is created intra-operatively to minimize the risk of re-ankylosis and the patient may initiate immediate post-surgical PT. To limit peri-articular heterotopic bone and fibrous tissue in- growth further, abdominal fat grafts were harvested and placed around the alloplastic TMJ. Grafted adipose tissue may reduce the extent of post-operative peri-articular fibrosis, reduce the chance of heterotopic bone formation and provide hemostasis by filling dead space. Although the volume of fat decreases over the first year, MRI and histological data from patients who underwent re-operation has confirmed the vi- ability and persistence of free adipose grafts (Dimitroulis et al., 2008). A review of 20 patients (33 TMJs) diagnosed with ankylosis or re-anky- losis demonstrated significant objective and subjective quality of life improvements after treatment with alloplastic TJR and autogenous fat grafts (Mercuri et al., 2008). Harvested dermal-fat grafts also have been used as interpositional material after gap arthroplasty and after discec- tomy. A post-operative MRI analysis to determine the fate of dermal- fat grafts revealed maintenance of peri-articular adipose tissue volume posterolateral to the joint space (in 71% of TMJs) with more than two 354 Aronovich years of follow up, but a predominance of grey signal intensity, consistent with fibrous tissue, in most joints (Dimitroulis et al., 2008). Donor site morbidity, however, is a known disadvantage. The opportunity to use a local or regional interpositional fat tissue would eliminate the additional morbidity and time associated with distant fat harvest. The presence of the locally available buccal fat pads with their extensions can be used readily for a variety of applications (e.g., reconstruction of ablative de- fects, closure of oro-antral fistulas and palatoplasty). A technique for its use as interpositional tissue for the treatment of TMJ ankylosis had been described (Singh et al., 2011) where the pterygoid extension of the buc- cal fat pad is accessed after completion of coronoidectomy and incision of the periosteum anterio-medial to the coronoid process. Ten patients with a mean follow-up of 14.8 months were treated with a 5 to 10 mm gap arthroplasty and pedicled interpositional buccal fat achieving a mean mouth opening of 35.1 mm (range: 32 to 41 mm). Notably, a satisfactory occlusion and facial symmetry were maintained with no reported cases of facial nerve paresis (Singh et al., 2011). The advantages of Surgical navigation also have been discussed. Surgical navigation is a valuable tool for precise execution during opera- tive treatment of intra-cranial or skull base tumors, functional endoscopic Sinus surgery, in the anatomic reduction of facial fractures and for man- agement of TMJ ankylosis (Klimek et al., 1993; Watzinger et al., 1998; Schmelzeisen et al., 2002, 2004; Voss et al., 2009; Yu et al., 2009). To- gether with the use of computer simulation, surgical navigation provides the operator with a road map and real-time information about the loca- tion of a navigation pointer, as well as rotary or ultrasonic instruments used to excise the ankylotic mass. A recent series of six patients were managed with virtual simulation for the planned resection and naviga- tion-guided lateral gap arthroplasty with interpositional tissue (Gui et al., 2014). A comparison of post-operative CT images with the virtual sur- gical plan confirmed the accuracy of simulation based planning with a discrepancy smaller than 0.8 mm. The accuracy of CT-guided navigation already has been described elsewhere (Xia et al., 2007) with precision ranging from 0.2 to 1 mm. Patients in this study had an improvement in mouth opening from a mean of 10 mm to a post-operative range of 35 to 40 mm in all patients. Moreover, the ramus height was preserved. The added safety and accuracy provided by navigation includes preserva- tion of adjacent vital structures and the ability to remove the ankylotic 355 Acquired TMJ Ankylosis bone mass precisely while preserving the displaced condyle and articular disc (Guiet al., 2014). CONCLUSION This chapter described three cases to illustrate the feasibility of single-stage reconstruction of TMJ and mandibular structures using patient-fitted alloplastic TJR. Along with surgical navigation, the use of computer-aided surgical simulation, 3D-printed custom cutting guides and resurfacing guides allow precise surgical execution, preservation of vital structures and accurate mating between the custom fossa compo- nent and the underlying temporal bone. The efficacy of alloplastic TMJ TJR has been illustrated in Wolford and coworkers' study (2015) with up to 20 years of follow-up. However, the long-term success of a single-stage protocol with prosthetic TMJ replacement for ankylosis requires further research. Finally, the increasing use of this treatment modality for grow- ing patients with TMJ ankylosis will require careful follow-up to deter- mine effects on facial growth, mandibular function and revision rates. REFERENCES Bhatt K, Roychoudhury A, Bhutia O, Pandey RM. Functional outcomes of gap and interpositional arthroplasty in the treatment of temporoman- dibular joint ankylosis. J Oral Maxillofac Surg 2014;72(12):2434–2439. 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Navigation-guided gap arthroplasty in the treatment of temporomandibular joint ankylosis. Int J Oral Maxillofac Surg 2009;38(10):1030-1035. 360 THE BRAIN AS A THERAPEUTIC TARGET IN HEADACHE AND FACIAL PAIN: THE NEXT FRONTIER IN PAIN MEDICINE AND HEALTH TECHNOLOGY Alexandre F.M. DaSilva ABSTRACT There is limited in vivo information available on the dysfunctional brain systems in headache and orofacial pain patients and how they directly respond to precise and non-invasive modulation. Currently an extensive array of technologies is available to investigate mechanisms in chronic pain, thereby immensely expanding our understanding of its pathophysiology. Herein, we aim to explain Central molecular pain regulatory mechanisms such as in migraineurs and temporomandibular disorder patients. In addition, while understanding that Central mechanisms in these patients using advanced neuroimaging is important, developing novel concepts for future clinical application is equally important. The neurotechnologies and studies described in this chapter represent a change of paradigm in pain medicine as we now directly can investigate and modulate in vivo the major central mechanisms associated with pain pathophysiology and relief. KEY WORDS: temporomandibular disorders (TMD), migraine, neuroimaging, neuromodulation, 3D visualization INTRODUCTION Pain is the main reason for seeking medical care as it highly impairs our quality of life. Chronic pain alone affects 116 million Americans and Costs an estimated $635 billion per year. Lipton and colleagues (1993) reported that almost 22% of the American population suffered from at least one type of orofacial pain with temporomandibular disorders (TMDs) being the most prevalent condition in which many subjects experience severe chronic pain and dysfunction (Salonen et al., 1990). Despite the impact of TMD on our society, few treatment advances have been made in this field. The condition represents a clinical problem 361 Pain Medicine and Health Technology in which empirical treatment options offer uncertain relief for a large number of patients. Most therapies for chronic TMD that focus on mechanical and peripheral factors are ineffectual, which leads to persistent treatment failure and/or poor iatrogenic-induced results, including multiple surgeries (Okeson, 2013). Chronic TMD also is associated with increased sensitivity to sensory stimulation and jaw function/movement, and the increased incidence of the Comorbidities of depression, fibromyalgia, anxiety and migraine among patients with chronic TMD is documented well (Plesh et al., 1996; Madland et al., 2000; Yap et al., 2003). Migraine—a primary headache disorder that affects nearly 16% of women and 6% of men worldwide (Lipton et al., 1994; Stewart et al., 1994; Lipton and Bigal, 2005)—also has marked increased sensitivity to noxious (hyperalgesia) and non-noxious stimulus (allodynia; Burstein et al., 2000). More specifically, studies show that migraine patients, both adults and children (Liljeström et al., 2001, 2005; Dando et al., 2006), more frequently report severe symptoms of facial pain, including tenderness upon palpation and higher sensitivity to pain on the masticatory muscles and temporomandibular joint (TMJ). However, the pathophysiology of primary headaches and facial pain are not understood fully. Although magnetic resonance techniques have provided insights into some brain mechanisms of headache and facial pain in humans, many questions regarding their chronification and treatments still are unanswered. Recent advances in molecular neuroimaging and neuromodulation have provided important new information including the molecular mechanisms in the brain that are affected during the course of the patients' suffering in vivo and how these mechanisms can be modulated safely for therapeutic and research purposes. In this chapter we discuss the new paradigm in headache and facial pain therapy as understood by in vivo investigations and modulation of the brains of these patients. MOLECULAR NEUROIMAGING OF HEADACHE AND OROFACIAL PAIN IN VIVO One of the hypotheses for primary headaches and facial pain pathophysiology, in addition to sensitization due to abnormal trigeminal 362 DaSilva afferent inputs (Waeber and Moskowitz, 2005; Younger et al., 2010), is the dysfunction of the descending modulatory mechanisms, whose understanding could help decipher the chronic pain in migraine and TMD by disinhibitory sensitization. For instance, projections to the periaqueductal gray (PAG), where there is an elevated expression of p-opioid receptors (LOR; Bausch et al., 1995; Kruit et al., 2009), would produce their modulatory effect on trigeminovascular neurons in migraine inefficiently (Goadsby, 2005). Not surprisingly, many migraineurs routinely use opiates; 72% of admitted patients in tertiary care pain clinics report using them (Nijjar et al., 2010). However, the use of opiates poses a high risk of side effects in primary headaches and facial pain patients where the recurrent nature of the attacks and consequently, frequent rescue opiate intake, can increase hyperalgesia/allodynia severely (De Felice and Porreca, 2009), medication-overuse headaches and other Comorbidities (Lipton and Bigal, 2004). Independent of complications associated with opiate use, endogenous opioid release and LOR concentrations are critical elements for the understanding of general pathophysiology and alleviation of Suffering in migraineurs and orofacial pain patients. The L-opioid system is the main target of opiate analgesia and highly regulates pain nociceptive Signals. Our group published the first direct evidence of dysfunctional endogenous pu-opioid activations during spontaneous migraine and allodynia in vivo, by measuring puCR non-displaceable binding potential (BP) in vivo with [C]carfentanil (DaSilva et al., 2014b). We noticed a reduction in LOR BP, during a spontaneous migraine attack compared to baseline in pain-modulatory regions of the opioid system, mostly the thalamus, as well as the anterior cingular cortex, PAG and insula (Fig. 1). This effect was consistent with the activation of endogenous pu-opioid neurotransmission in trigeminal neuropathic pain (TNP) patients who Were non-opiate users (Dossantos et al., 2012). When compared with age- and gender-matched healthy controls who also were scanned with positron tomography, the TNP patients showed significant decrease in LOR BP, in the nucleus accumbens, an area associated with pain modulation and reward/aversive behaviors. Furthermore, the LOR BP, in the NAc was correlated negatively with pain ratings in the TNP patients. 363 Pain Medicine and Health Technology Figure 1. Full virtual reality 3D data navigation in a migrainous brain. We investigated for the first time actual migraine neuroimaging data in a full immersive virtual 3D reality. This included unrestricted navigation through the data regarding availability of p-opioid receptors (u0R) non-displaceable binding potential (BP) in the brain during the migraine attack in vivo. It has been used for data analysis and as a novel educational tool (DaSilva et al., 2014c). NEUROMODULATION OF HEADACHE AND OROFACIAL PAIN IN VIVO Conventional therapies are unable to target selectively many of the neuronal structures discussed above and there is a paucity of data on how to reverse neuroplastic molecular mechanisms when available medications fail. Interestingly, studies with motor cortex stimulation (MCS) have shown that epidural electrodes in the primary motor Cortex (M1) are effective in providing analgesia in patients with central pain (Lima and Fregni, 2008) resulting from their indirect modulation of thalamic activity (García-Larrea et al., 1999). Unfortunately, the invasive nature of such a procedure limits its indication to highly severe pain disorders. New non-invasive neuromodulatory methods for M1 (e.g., transcrania direct current stimulation ſtDCSI), now safely can modulate the LOR system (DoSSantos et al., 2012, 2014), providing relatively longlasting 364 DaSilva relief in pain patients (DaSilva et al., 2011; http://www.jove.com/de- tails.php?id=2744). Transcranial direct current stimulation (tDCS) is based on the application of a weak direct current to the scalp that flows between two relatively large electrodes—anode and cathode (Fig. 2). Its effects depend on polarity of stimulation: cathodal stimulation tends to induce a decrease in cortical excitability; anodal stimulation induces an increase in cortical excitability that may last beyond the duration of the stimulation. Recently, we were able to decrease pain significant- ly in chronic migraine patients following ten non-invasive Sessions of M1-SO tBCS (DaSilva et al., 2011c, 2012a). In our investigation with the pu-opioid-specific radiotracer, [*C]carfentanil, the immediate effect of M1-tDCS application induced significant pu-opioid system activation in the descending inhibitory system, including the PAG (Dossantos et al., 2014). However, a more concentrated area of stimulation allows for better targeting and modulation, and could emulate the successful effects of implanted MCS. The term for this new montage is high-definition tPCS (HD-tDCS). It increases the accuracy of current delivery to the brain by using arrays of smaller electrodes, instead of the larger pad electrodes of conventional t0CS (Fig. 2). In order to evaluate the analgesic effect of focally targeted M1 modulation, we have developed a novel M1 HD- tDCS montage with 2x2 electrode design, which was tested on a cohort of patients with TMD, a chronic trigeminal pain illness. Our computational model simulated our montage's currentflow through tissues captured with 3D imaging, accounting for the tissue type, tissue shape, tissue resistance, electrode positioning and strength of the current. Greatest density was focused on the lower region of the precentral gyrus/sulcus, targeting the putative homuncular craniofacial M1 region and immediately within the HD-tDCS 2x2 electrodes. This is the region where invasive motor cortex Stimulation produces maximal analgesic effect for head and facial pain (Nguyen et al., 1999). We screened 78 patients for this study and of those, 24 (30.8%) were randomized, with twelve per control and experimental group. All patients completed the five daily sessions of active and placebo M1 HD-tDCS according to the protocol. There were statistically significant differences between groups for pain and motor measurements in the active HD-tDCS group: responders with pain relief above 50% in the visual analogue scale (VAS) at one-month follow-up (p = 0.04); pain-free mouth 365 Pain Medicine and Health Technology tuae aelºoºººº：： | __| |_| |_| plºu º aeºnº tuwa sae'n tweed Zºº1.^º SOC]]-CIH MOļOW tuae aeroºººº：0 | TTT . tuae aero，ae SOCI} ioļOW 366 DaSilva 4– Figure 2. Computational model comparison between conventional tBCS (A) and 4x1-ring high-definition (HD)-tDCS (B) using a standard bipolar sponge montage (Villamar et al., 2013). Skin, skull and CSF masks are suppressed to reveal the underlying gray matter mask. Induced cortical surface electric-field magnitude plot for 2 mA stimulation. HD-tDCS resulted in more precise brain Current flow that is restricted to within the ring perimeter with dominant inward current on gyri. Conventional t0CS resulted in comparatively diffuse current flow with clusters of peaks and bidirectional stimulation on opposing walls between the electrodes. opening at one week follow-up (p < 0.01); and most importantly, improvement of contralateral but not ipsilateral sensory-discriminative pain measures during the treatment week (p<0.01; Fig. 3; Donnell et al., 2015). The sensory pain information was collected using PainTrek, a free and interactive mobile application developed by our laboratory and the 3DLab at The University of Michigan. It allowed our researchers to track, display and analyze the headache and facial pain information that was acquired from patients during our migraine research protocol (Fig. 4). Additionally, we found no significant evidence that mood changes differed overall between the groups, as evaluated by the Positive and Negative Affect Schedule, indicating that our focused M1 HD-tDCS montage selectively modulated sensorimotor outcomes. CONCLUSIONS The most recent neuroimaging and neuroimaging protocols have demonstrated that dysfunctional endogenous pu-opioid activation in the brains of primary headache and facial pain patients are related to their Symptomatology and to the molecular neuroplasticity in humans. Such a crucial neurotransmitter system can be targeted (in)directly and neuromodulated for pain relief. CONFLICT OF INTEREST STATEMENT: Dr. Alexandre DaSilva co-owns Health- Trek Solutions, L.L.C., which is seeking an option to license the PainTrek technology from the Office of Technology Transfer at The University of Michigan. 367 Pain Medicine and Health Technology Less Pain - No Change More Pain F3 C. +3 Figure 3. Sample of pain relief in three patients in the active HD- tDCS group. Colors represent change in the pain area and intensity from baseline to final session on the side of the head contralateral to stimulation. REFERENCES Bausch SB, Patterson TA, Ehrengruber MU, Lester HA, Davidson N, Chavkin C. 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Neurology 2005;64(10 Suppl 2):S9-S15. 371 Pain Medicine and Health Technology Yap AU, Dworkin SF, Chua EK, List T, Tan KB, Tan HH. Prevalence of temporomandibular disorder subtypes, psychologic distress, and psychosocial dysfunction in Asian patients. J Orofac Pain 2003;17(1): 21–28. Younger JW, Shen YF, Goddard G, Mackey SC. Chronic myofascial temporomandibular pain is associated with neural abnormalities in the trigeminal and limbic systems. Pain 2010;149(2):222-228. 372 MICROANATOMICAL RESPONSES OF DEVELOPING AND FUNCTIONALLY ACTIVE BONE-PERIODONTAL LIGAMENT-TOOTH FIBROUS JOINTS Ji-Hyun Lee, Mark I. Ryder, Sunita P Ho ABSTRACT In this qualitative study, the presence of various types of molecules prompting cell behavior at the periodontal ligament (PDL)-bone and PDL-cementum attachment sites (AS) has been documented using three-week- and three-month-old Scleraxis-green fluorescent protein (GFP) transgenic mouse line. Strain-dominant regions were identified by overlapping both angular distributions of GFP- expressing PDL cells and phalloidin-stained actin filaments of the cytoskeleton along the direction of collagen fibers of the coronal and apical extracellular matrix (ECM) in both age groups, except in the apical region of three-week-old animals. Differences in biomolecular expression with age were indicative of a dominance of passive pullout stretch in a developing molar of a three-week-old, Compared to an active pullout stretch in a fully functional, cyclically loaded three- month-old dentoalveolar complex. In general, presence of CD146+ cells around capillaries were CD31+ and were detected in the PDL, dental pulp, endosteal Space and bone marrow 50 microns (pum) from the ligament-bone AS. In three- week-old mice, NG2+ cells were along the PDL-cementum AS and concentrated at the root apex, an active site for developing root through growth of secondary cementum. The three-month-old mice showed increased NG2 expression in their apical region and alveolar crest, which are active biomechanical sites during tooth function. The NG2+ cells were not associated with blood vessels. OSX- positive cells—a marker for precursors to osteoblasts and cementoblasts—were identified directly at the respective AS. Bone sialoprotein was distributed evenly in coronal regions with a rich expression in cement lines of apical bone in three- week-old mice, which contrasted with that of three-month-old animals—a result that indicates active remodeling during tooth eruption. Results of this study Show overlapping but unique location of putative progenitor cells, which may be related to their regenerative capacity necessary for tooth eruption and function. Conceivably, the cells and matrix molecules can form respective interfaces from the original PDL-bone and PDL-cementum AS in a load-bearing dentoalveolar Complex. KEY WORDS: periodontal ligament, interfaces, entheses, PDL-bone, PDL-cementum 373 Microanatomical Responses INTRODUCTION From materials science and applied mechanics perspectives, function-mediated pullout forces at the site of attachment of disparate materials (e.g., ligament-bone, tendon-muscle tissue attachment sites [AS] and interfaces) are dampened by the presence of various types of interacting adhesive molecules. In addition, the presence of these molecules can aid in providing shape memory or recoverable charac- teristics for the AS. From a mechanobiology perspective, given these innate characteristics, it is hypothesized that the AS play a role as semi- autonomous differentiating zones. It also is the differentiating cells that interact in a milieu of adhesive globular and fibrillar proteins (e.g., a highly viscous medium to maintain strain-adaptive nature of biologi- cal interfaces that subsequently transform to osteoid from the origi- nal PDL-alveolar bone and cementoid from the original PDL-cementum AS). Within this context, various types of molecules that are sensitive to stem cell identification, differentiation, mechanical forces and blastic lineage will be discussed along the ligament-bone and ligament-cemen- tum biological interfaces. In this study, we chose the ligament-bone and ligament- cementum AS in a bone-ligament-tooth complex as our biological interface to explain events that then would lead to mineral forming osteoid and cementoid matrices. The bone-periodontal ligament (PDL)-cementum complex functions through an orchestrated action of multiple types of tissue. PDL is a soft compliant tissue attached to stiff hard tissues (e.g., the alveolar bone of the jaw and cementum of the tooth) and provides a natural docking of the tooth in the alveolar socket. Based on previous studies, it is possible that docking can be affected as a result of bony adaptations at the PDL-bone (PDL-B) and PDL-cementum (PDL-C) entheses (Hurng et al., 2011; Ho et al., 2013). Although tissue- related transformations have been interrogated in the musculoskeletal system (Rufai et al., 1995; Benjamin and Ralphs, 2001; Kumai et al., 2002; Moriggi et al., 2003; Benjamin et al., 2004; Ho et al., 2007; Liu et al., 2012), few studies have been performed at the enthesial sites of the bone-PDL-tooth fibrous joint. This most likely is due to the challenge in accessibility of these 1 to 5 microns (pum) wide regions in a 70 to 150 pum wide functional space, specifically in rat and mice models. Despite the 374 Lee et al. common denominators between various entheses, fundamental differ- ences in mineralization exist between the oral and craniofacial complex when compared to that of the musculoskeletal system. Thus, the novel aspect of this study is to investigate changes in levels of key biomolecules in development and growth of functional interfaces and biomechanically ac- tive AS within the bone-PDL-cementum complex. Biophysical cues play a critical role in the development of liga- ment and bone, not only in musculoskeletal system, but also in the load- bearing dentoalveolar complex (Wang et al., 1993; Thomopoulos et al., 2003; Luo et al., 2008; Boyle et al., 2014). Mechanical strains created by mastication and through tooth contact influence the complex in various ways. PDL, which is a vascularized ligament unlike that the in musculo- skeletal system, gets blood supplied through the physical continuum it maintains with alveolar bone (Ho et al., 2010). Therefore, blood flow under function can be an effective means of delivering chemokines for cell migration, differentiation and proliferation (Park et al., 2004). Strain gradients in a blood vessel and interacting extracellular matrix (ECM) also can act as cues for a synchronized cell differentiation and migration to occur. Orchestrated events including innate stretch within cytoplasm and tissue, protein stabilization and conformation and protein docking are necessary to increase the bio-affinity for inorganic crystal forma- tion on an organic macromolecular substrate and subsequent forma- tions of mineralized fronts at the AS. Interestingly, due to the interplay of passive and active forces, the continuous modeling and remodeling in alveolar bone prompts a reactive response in cementum to maintain functional space and congruency of the joint throughout the life of an organism. Within this context, we will discuss the localization of matrix molecules in the bone-ligament-tooth complex at the differentiating zone adjacent to the ligament-bone and ligament-cementum attach- ment sites. This will be accomplished by highlighting a few molecules that are deemed necessary to form mineralized tissue. These molecules include bone sialoprotein (BSP) as a putative mechanosensor at the PDL-B and PDL-C AS and osterix (OSX) as a cell differentiation marker to detect blastic lineage. In addition, it is postulated that the force driven ligament-specific marker, scleraxis (SCX), can be used to identify tension- dominated regions and correlate these spatial regions to expressions of molecules specific to genesis of inorganic fronts. Therefore, detection of 375 Microanatomical Responses these organic molecules at the concatenated sites and proposed differentiation zone was found by using transgenic Scx-reporter mice in the context of cell ontogeny. MATERIALS AND METHODS Specimen Preparation Mouse mandibles from a generation of Scx-GFP transgenic reporter mice (Pryce et al., 2007) were harvested at three weeks and three months postnatally and fixed in 1% paraformaldehyde, demineralized in 0.5 M ethylene diaminetetraacetic acid (EDTA, pH 8.0) for two weeks. Following a thorough rinse, with phosphate-buffered saline (PBS), the specimens were equilibrated in 15 and 30% sucrose sequentially at 4°C and then embedded in Optimum Cutting Temperature compound (OCT; Feng et al., 2003). The mandibles were sectioned serially in sagittal plane at 10 Jm with a cryostat. Tissue sections were placed onto Superfrost glass slides (Fisher Scientific, Pittsburgh, PA). Three consecutive sections of left mandibles were processed with the purpose of staining the first with NG2 and CD31, the second with CD146 and CD31, and the third with OSX. Similarly, three consecutive sections of right mandibles were stained with bonesialoprotein (BSP), phalloidin and picrosirius red (PSR). Following antigen retrieval, the specimens were washed twice in PBS containing 0.1% Tween-20 (PBST) for two minutes, then incubated in blocking solution consisting of normal donkey serum (Sigma-Aldrich, St Louis, MO), bovine serum albumin in PBS for one hour before overnight incubation in primary antibodies at 4° C. Primary antibodies included rabbit anti-mouse NG2 (1:100, AB5320, Chemicon, Temulca, CA), rabbit anti-mouse CD146 (1:250, AB75769, Abcam, Cambridge, MA), rat anti- mouse CD31 (1:20, DIA-310, Dianova, Hamburg, Germany), rabbit anti- mouse OSX (1:200, AB22252, Abcam, Cambridge, MA) and rabbit anti- mouse BSP (1:100, sc-292394, Santa Cruz Biotechnology, Santa Cruz, CA). Slides were rinsed in PBST and then incubated in Alexa Fluor 594 donkey anti-rabbit or Alexa Fluor 647 goat anti-rat secondary antibody (Molecular Probes, Eugene, OR) at a 1:200 dilution. Primary and secondary antibodies both were diluted in blocking solution. Slides were rinsed and incubated in Hoechst 33342. For detection of actin cytoskeleton, sections were incubated for 30 minutes in rhodamine-conjugated 376 Lee et al. phalloidin (5ml/ml, Cytoskeleton Inc., Denver, CO) at room temperature. Controls were prepared by replacement of primary antibodies with non- immune isotype immunoglobulin obtained from the primary antibody's host species (isotype control) and by omitting primary antibodies (blank control). Microscopy and Image Analysis Light microscopic analysis was performed with a BX51 Olympus microscope (Olympus America Inc., San Diego, CA) with x4 and X20 objectives. Images were obtained using camera controlled by Image Pro Plus v6.0 software (Media Cybernetics, Inc., Silver Spring, MD). For PSR Staining, polarized light was used to enhance the birefringence of collagen to illustrate changes in collagen fiber orientation and birefringence intensity throughout the complex. Immunoreactivity was visualized with a wide-field fluorescence microscope (Nikon Ti-E Microscope; Nikon Imaging Center at the University of California San Francisco [UCSF), CA) with a x20 objective and a confocal microscope (Nikon FN-1 Microscope; Nikon Imaging Center at UCSF, CA) with a x40 plain lens objective. Images were collected using EZ- C1 and Nikon-element software (v4.20). RESULTS Orientations of Actin in Scx+ PDL Cells and Collagen Fibers of PDL Matrix Collagen fiber orientation highlighted by birefringence was utilized to identify the location and shape of PDL fibroblasts in Scx-GFP mice. At three weeks, the angular distribution of collagen fibers, fibroblasts and the PDL-cell cytoskeleton coincided within the PDL complex in the coronal region, but not in the apical region; collagen fibers were perpendicular to secondary cementum with less birefringence, while the orientation of actin filaments and fibroblasts was observed to be oblique toward the root apex (Fig. 1A). At three months, the directionality of collagen fibers, PDL fibroblasts and the PDL-cell cytoskeleton were similar in both coronal and apical regions, and less aligned actin filaments in fibroblasts (Fig. 1B). In comparison to three weeks, collagen fibers and PDL fibroblasts manifested in stronger and more uniform alignment at three months. 377 Microanatomical Responses º º º º C - F. º º | * : * s = - c c c. c. c. Iºnºsuºuſ DIO SM89/A 391 || NG2+ Cells, Not in Spatial Concordance with CD146+ Cells The NG2 molecule is a chondroitin sulfated proteoglycan and Was observed in proximity of endothelial cell in microvasculature, in addition 378 Lee et al. 4– Figure 1. Strain-dominant regions were identified by overlapping angular distributions of SCX, PSR and phalloidin stains. Left panel: NG2+ cells were not always with blood vessels, but were co-localized with OSX-positive. NG2+ were identified directly at PDL-C and PDL-BAS. Middle panel: BSP was identified in coronal regions with a rich expression in cement lines of apical bone in three- week-old (a) mice contrasting three-month-old (b) animals, indicating active remodeling during tooth eruption. Right panel: CD146+ cells were detected adjacent to CD31+ cells and were observed in the PDL, dental pulp, endosteal Space and bone marrow. B = bone; C = cementum; D = dentin. to CD146. However, NG2+ cells not only overlapped with CD146+ cells, they also were observed along PDL-B and PDL-C interfaces (Fig. 1), al- though the pattern of distribution between the two was different. At PDL- B interface, more NG2+ cells were located coronally than apically, but the three-months-old group showed increased NG2 expression in the apical region, as well as on the alveolar crest. On the other hand, at the PDL-C interface of the three-weeks-old group, NG2+ cells were concentrated in actively developing secondary cementum of the apical portion of the root. This pattern also was observed in the three-months-old group, but not as obviously as in the three-weeks-old group (Fig. 1). OSX and BSP Expressions at Interfaces and in Periodontal Tissue At three weeks, OSX was expressed in alveolar bone, cellular cementum-lining cells, odontoblasts lining dental pulp chamber and root canals, bone marrow stromal cells and dental follicle cells (data not shown as it parallels with positive expression of NG2). At three months, however, OSX expression was limited only to bone lining cells and no OSX positive cells on cellular cementum were observed. Immunoreactivity of OSX at three months was lower than that observed in three-week-old mice (data not shown as it parallels with positive expression of NG2). Anti-BSP antibody stained all the mineralized structures within the complex. To be specific, immunoreactivity for BSP was observed in the primary and secondary cementum, as well as alveolar bone, while only weak staining was observed in the predentin layer. Within alveolar bone, BSP expression was more intense in alveolar crest or the coronal region of interradicular bone than in the bottom of the alveolar process. 379 Microanatomical Responses Distribution and Co-localization of CD146 and CD31 CD146 (Melanoma Cell Adhesion Molecule, or MCAM) was co- localized with CD31 and was observed in the vicinity of PDL-B interface. Expression of CD146 was detected around capillaries with CD31+ cells, through PDL-space, dental pulp, endosteal Space and bone marrow. Three-week-old mice had more CD31 and CD146 expressions, and CD146 expression was significantly higher in the three-week-old mice compared with three-month-old mice (Fig. 1) with co-localized cells in channels that are continuous from bone marrow to the PDL. The fluorescent intensity of CD31 and CD146 was higher at the respective AS. ". DISCUSSION The PDL of the bone-tooth complex is highly vascularized and innervated (Freezer and Sims, 1987). A dense capillary network is a unique feature at the bone remodeling site (Kristensen et al., 2013) and cells adjacent to blood vessels have been proven to be enriched with tissue-resident mesenchymal stem cells (MSCs; Chen et al., 2006; Crisan et al., 2008; Iwasaki et al., 2013). It is hypothesized, therefore, that the perivascular niche is located preferentially along the AS and is a resource for development and movement of mineralizing fronts identified as osteoid and cementoid interfaces within bone-PDL-cementum complex. The CD146/CD31 co-localization and prevalence of CD146+ cells in the PDL-space parallel the distribution of vasculature along the PDL-B attachment site. This finding is congruent with previous studies on vessel distribution at the alveolar socket surface (Freezer et al., 1987; Prisby et al., 2011; Kristensen et al., 2013). Interestingly, the distribution of vasculature indicated by CD31+ cells is limited to PDL-B interface, although both PDL-B and PDL-C interfaces are strained mechanically and functionally are active soft-hard tissue interfaces as indicated by PDL birefringence and Scx expressions (Fig. 1). The difference in blood vessel density and prevalence of putative stem cells identified as CD146+ within the PDL-space implies that the vessels are a distinct and rich resource for development and maintenance of each functional attachment site and PDL-B interface. In contrast, cementum does not undergo remodeling under normal conditions and seems to function without such a complex cell system, albeit signals from the adjacent PDL likely influence cementoblast function (Bosshardt, 2005). Results of this study 380 Lee et al. provide insights into the potential of PDL-matrix and its proteins partially delivered through blood vessels could irrigate the PDL with needed matrix molecules to form functional PDL-C AS from which cenentoid layers are formed (Grzesik and Narayanan, 2002). Bone remodels through a conserved regulation of osteoblasts, osteocytes and osteoclasts (Miller, 2012). Additionally, vascular invasion is essential for regeneration and repair that predominantly involves migration of osteoblastic and osteoclastic progenitors (Karsenty and Wagner, 2002). Furthermore, osteoclasts are of hematopoietic lineage and their differentiation involves multiple steps associated with cytokines such as RANKL and OPG (Liet al., 2000). Overall, it can be postulated that bone requires tight-knit assistance of hematopoietic environment, although bone per se originated from mesenchymal lineage, whereas it could be the local environment of the cementum matrix that is thought to maintain cementum homeostasis (Grzesik and Narayanan, 2002). Pericyte marker NG2 was observed immediately adjacent to endothelial cells of pulptissue rather than in PDL, at PDL-Band PDL-CAS. As pericytes have been named after their location rather than their function and extinct phenotype, NG2+ cells surrounded the CD31+ endothelium (Fig. 1) in the pulp and CD146+ cells at the endosteal spaces in alveolar bone. Despite the observation, the reasons for co-localization of NG2+ cells with vasculature (CD31+) in the pulp, rather than PDL-space, needs further investigation in the lines of lineage tracing and potential relevance to the size and type of the vessel. NG2+ cells also were populated along the PDL-B and PDL-C AS (Fig. 1). Cells specific to the PDL-C attachment site cannot be considered as perivascular mesenchymal stem cells due to the absence of CD31. It is not possible to track the origin of the particular cell population using the present model. However, NG2+ cells had a typical phenotype of OSX positive polarized and cuboidal shaped cells adjacent to newly formed bone/cementum-like tissue. Therefore, it is plausible that NG2+ cells at the AS could differentiate into osteoblast/cementoblast-like cells and synthesize osteoid/cementoid tissue. Anatomically, the NG2+/OSX+ cells were populated on biomechanically active sites such as the alveolar bone crests, interradicular bone and secondary cementum. At three weeks, the tooth is not under functional load, but the eruption and root formation of the second mandibular molar is mostly complete (Shibata et al., 1995). Both NG2 and OSX were negative at the apical region 381 Microanatomical Responses of the bone surface and intense at the secondary cementum surface (Fig. 1). The OSX positive cells can be speculated to be progenitors committed to be in charge of the development and homeostasis of the tissue interface, whether or not they are of pericyte origin. Specifically those at the PDL-C site need further investigation and it is likely that the NG2+ cells at the PDL-C attachment site are progenitors from the PDL- matrix. Evidence that corroborates with the hypothesis is the direction of the cementocytes canaliculi in the direction of the PDL. Hence, it is possible that the development of the functional interfaces from the respective attachment site could be due to synchronized events between macromolecules in the ECM of the PDL prompting progenitors and their communication with osteocyte and cementocytes at the respective AS. Although tooth eruption initially depends on osteoclast activity to generate an eruptive path through the bone developmentally (Wise et al., 2002), the stage of the eruption prior to active function (chewing forces) seems to be driven by cementum apposition rather than bone remodeling. However, the functionally active three-month-old mice molars showed NG2+ cell population on both secondary cementum and the surface of apical bone, which indicates that PDL space is maintained during function via cementum apposition and bone formation/resorp- tion events in apical region. A similar pattern was observed in the biomechanically active site of the interradicular complex. This finding implies that the driving force for active eruption in the three-week-old mice lies in the apical and interradicular bone-PDL-cementum complex undergoing differentiation. In the three-months-old molars, NG2+ apical bone surface indicates that following active function, remodeling sites for tissue homeostasis also include alveolar crest, interradicular and apical regions. BSP was chosen to document the lineage end product of mesenchymal origin with a rich expression on the apical alveolar bone surface noted at three weeks, but not at three months. The lack of BSP immunolabeling on the apical bone surface of three-months-old mice was counterintuitive because BSP is known to be mechanosensitive as well as osteogenic and cementogenic (MacNeil et al., 1995; Carvalho et al., 2002; Foster et al., 2013). However, previous in vitro studies showed that BSP also is involved in regulation of osteoclast differentiation and activity (Raynal et al., 1996; Malaval et al., 2008). Little or no BSP expression, despite constant stimulation by occlusal mechanical force, 382 Lee et al. may indicate low bone resorption activity and dominance in a developing matrix of a three-week-old than in a remodeling matrix of a three- month-old mouse. By contrast, increased level of BSP at three weeks may facilitate osteoclast activity, which is required for tooth eruption (Sundquist and Marks, 1994; Cielinski et al., 1995). Osteoclasts and their precursors appear in dental follicle (Marks et al., 1983; Volejnikova et al., 1997) and they adhere to BSP through ovſ;3 integrin; this interaction plays a regulatory role in their differentiation and activity (Nakamura et al., 2007) is limited to passive stretch of a developing matrix. In all the three components, the organization of collagen fibers (PSR staining to enhance collagen birefringence), fibroblasts (SCX) and cytoskeleton (phalloidin stained actin filaments) within PDL representing ECM, cells and intracellular structures respectively showed Coinciding directionality within the PDL, except at the apical region of three-weeks-old mice. Although the three-weeks-old molar did not reach the occlusion, the coronal part of its PDL already showed a preferential orientation of collagen birefringence, PDL fibroblasts and their cytoskeletons, indicating that internal erupting force generated by developing and organized tissues is dominant in the coronal regions of the periodontal complex. In the apical region of a three-week-old molar, the directionality of collagen birefringence was not parallel to that of PDL fibroblasts and their cytoskeleton. From these results, it can be ascertained that physical alterations within intracellular machinery or changes in cell shape are regulated by the innate mechanical integrity of the ECM. The cell and matrix directionality with the spatial distribution of the biomolecules investigated in this study—when presented within the context of organ function—provides new insights to the presence of progenitors at the mechanical strained alveolar crests, interradicular and apical regions, where robust tissue generation was observed in the present study. Hence, it can be postulated the erupting molar at three weeks undergoes mechanical stimuli generated by volumetric expansion due to internal tissue growth and its manifestation, though location Specific, it is not different necessarily from the co-localization of type of molecules observed in a periodontal complex under functional load. Function-induced strains promote PDL turnover (Beertsen et al., 1997; McCulloch et al., 2000), perpetuate bone remodeling (Rubin and Lanyon, 1984) and control cementum growth (Niver et al., 2011). In the present study, either still erupting (three weeks old) or under functional load 383 Microanatomical Responses (three months old), the cellular and molecular responses for continuous physiologic remodeling and homeostasis were concentrated at the alveolar crest, interradicular and apical region. Whether the source of mechanical strain is intrinsic (within tissues or cells) or extrinsic (from external environment of a given organ), it plays a role in Sculpting and maintaining the organ shape by coordinating the spatial arrangement of cells and controlling the cell proliferation and its directionality (Ingber, 2006; Nelson and Gleghorn, 2012). ACKNOWLEDGEMENTS The authors thank Nikon Imaging Center (NIC) at UCSF for their facility and assistance in fluorescence microscopy. 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Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site. J Orthop Res 2003;21(3):413-419. Volejnikova S, Laskari M, Marks SC Jr, Graves DT. Monocyte recruitment and expression of monocyte chemoattractant protein-1 are developmentally regulated in remodeling bone in the mouse. Am J Pathol 1997;150(5):1711-1721. Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton. Science 1993;260(5111):1124-1127. Wise GE, Frazier-Bowers S, D'Souza RN. Cellular, molecular, and genetic determinants of tooth eruption. Crit Rev Oral Biol Med 2002;13(4): 323-335. 388 RETROSPECTIVE ANALYSIS OF EFFICIENCY AND EFFICACY OF COMPUTER-ASSISTED FINISHING TECHNOLOGY Christian Groth, Lorenzo Franchi, James A. McNamara Jr. ABSTRACT OBJECTIVE: The aim of this study was to evaluate the efficiency and efficacy of computer-assisted orthodontic treatment technology (SureSmile” or SS) com- pared to a comparison group (CG) of patients treated with conventional fixed appliances. PATIENTS AND METHODS: Records for two treatment groups (SS group = SSG and CG) of patients treated without extractions were collected from five orthodontists. Patients in the SSG were matched to patients in the CG based primarily on a modified version of the Discrepancy Index (DI) developed by the American Board of Orthodontics (ABO) and secondarily on Angle classification. The final sample contained 89 patients in each group. The ABO Cast and Radio- graph Evaluation (CRE) was used to assess final treatment outcome. Treatment duration and the number of adjustment appointments also were calculated. Two- Sample t tests and Mann-Whitney tests were used for statistical comparisons. RESULTS: The matched samples had no statistical differences in any of the modi- fied DI measures or total score. Both groups showed mild malocclusions at T1. Treatment duration was significantly shorter and the number of appointments was significantly fewer (p < 0.001) in the SSG versus the CG (13.4 months and 9.2 appointments, compared to 22.4 months and 17.2 appointments, respectively). No clinically significant differences were found in the CRE scores between the two groups. CONCLUSIONS: In patients with a mild malocclusion treated without extractions, the use of the SS system results in shorter treatment durations and fewer appointments with no loss in the quality of outcome when compared to patients treated with conventional appliances. KEY WORDS: orthodontic finishing, computer-aided orthodontics, SureSmile”, treatment quality, treatment duration 389 Computer-assisted Finishing Technology INTRODUCTION Over the last decade, a technological revolution has occurred in orthodontics, giving rise to appliances that are intended to streamline treatment and give more predictable and consistent treatment outcomes in less time than with conventional fixed appliance therapy. The utiliza- tion of computer-aided design and computer-aided manufacturing (CAD/ CAM) technology has allowed custom orthodontic appliances to become a reality. Orametrix, Inc. (Richardson, TX) developed the SureSmile” (SS) system in the early 2000s as a novel way to finish an orthodontic case. The cornerstone of the SS system is the ability to design the finish of a case digitally, utilize a Software program to design a series of archwires and utilize a robot to bend the finishing archwires (Sachdeva, 2001). This system allows orthodontists to use their individual choice of brackets and initial archwires. It is not until after the leveling and aligning stage of treat- ment that the patient's dentition is scanned, either with an optical intra- oral scanner or a CBCT scan, that the digital treatment plan is established and executed. The software and robot have the ability to compensate for both bracket position and built-in prescription so that a full-sized arch- wire is not necessary to express the desired tooth movements (Sachdeva, 2001). Previous investigations (Saxe et al., 2010; Alford et al., 2011) have shown that the SS system results in shorter treatment times when com- pared to comparable cases treated with conventional appliances, along with high quality finishes. By using the Objective Grading System (OGS) score developed by the American Board of Orthodontics (ABO), Saxe and colleagues (2010) reported that SS patients showed an OGS score that was 14% better than for patients treated with conventional fixed appli- ances. Alford and coworkers (2011) found that SS patients were treated to better ABO Cast and Radiograph Evaluation (CRE) scores for first-order rotation and interproximal space closure compared to similar cases treat- ed with conventional methods. On average, however, malocclusions in the SS cohort were less complex and second order root alignment was inferior compared with patients treated with a conventional approach. It should be noted, however, that both of these studies had limited numbers of providers and patients in the sample. 390 Groth et al. The aim of this retrospective clinical study, therefore, was to quantify the quality of finish based on both the efficiency (treatment du- ration and number of adjustment appointments) and the efficacy (occlu- sal outcome) of the SS system when compared to conventional orthodon- tic treatment utilizing large samples of subjects. PATIENTS AND METHODS This study was performed using de-identified records following exemption approval from The University of Michigan's human research institutional review board. Records for two treatment groups (SS group = SSG and a comparison, conventionally treated group = CG) of patients treated without extractions were collected from five orthodontists, all of whom were aware that they were participating in a study on the finishing quality and efficiency of the treated cases. The SSG received computer- designed and robot-fabricated finishing archwires after the leveling and aligning phase of treatment. The CG was finished using traditional meth- ods including manual wire bending, bracket repositioning and vertical elastics. Consecutively treated patients who met the inclusion criteria from five different private practices of ABO-certified orthodontists were selected for this study; one practice contributed both SSG and CG. The three practices that contributed to the SSG had at least five years of expe- rience using the SS system. The three practices that contributed to the CG were matched to the SSG practices based on bracket slot size and the use of pre-adjusted appliances with conventional elastomeric and stainless Steel ligation. Two practices utilized 0.022” bracket slots and one prac- tice used 0.018” bracket slots in each group. All of the practitioners had at least fifteen years of clinical experience. Author CG traveled to each of the five private practices included in the study to verify the quality and consistency of the records, as well as to ensure the integrity of the Sample. Inclusion criteria for this study required that: • Patients did not need orthognathic surgery; • Patients were at least twelve years old and younger than eighteen at the start of treatment; 391 Computer-assisted Finishing Technology • Patients had a full, natural permanent dentition with no prosthetic replacement or congenitally missing teeth; and • Patients had second molars in occlusion at the time of final records. Patients were excluded from the study when records were incom- plete or of insufficient quality for analysis. Patients who were debonded early for any reason, including poor Compliance or hygiene, also were excluded. Treatment outcome was not an exclusion criterion. Records from 244 patients were collected initially. The SSG con- tained 126 patients; the CG had 118 patients. Due to incomplete records or early appliance removal, a total of 45 patients were excluded from the study, resulting in a sample size of 93 in the SSG and 106 in the CG. In order to control for any differences in case complexity be- tween groups and among practices, a control-matching procedure was performed. Patients in the CG were matched to patients in the SSG, based primarily on a modified version of the Discrepancy Index (DI) developed by the ABO (Casko et al., 1998) and secondarily on Angle Classification (Table 1). The modified Dl score was determined by grading the digital pre-treatment dental casts only; it must be noted that no cephalometric variables were considered, thus reducing the overall Dl score substantial- ly. Following the control-matching procedure, the final sample contained 89 patients in each group (Table 2). The records collected included initial and final panoramic and lat- eral cephalometric radiographs, digital or stone initial models (T1), stone final models (T2) and treatment notes for each patient. All records were transferred to The University of Michigan and assigned randomized identifiers by an independent third party to blind the investigators as to the treatment groups. All stone models were con- verted to digital format using the 3Shape R700 model scanner (3Shape, Copenhagen, Denmark). 3Shape OrthoAnalyzer" v.2010-2 was used for all digital model analyses. The modified version of Dl score (without cephalometrics) was scored on the digital models according to ABO con- vention. As the ABO has not validated the CRE scoring for use on digital models, the CRE was scored entirely on stone models (Casko et al., 1998). 392 Groth et al. Table 1. Prevalence rate of Angle classification in the SureSmile” group (SSG) and the conventional group (CG). SSG CG Angle Classification Number % Number % Class | 69 77.5 61 68.5 Class | End-On 8 9.0 15 16.9 Class || 12 13.5 13 14.6 Class || O 0.0 O 0.0 Total 89 100.0 89 100.0 Table 2. Demographics of patients from individual practices. M=male; F=female. T1 T2 T2-T1 Age (years) Age (years) º gºton Practice Group Mean SD Mean SD Mean SD Practice 1 (19 M, 15 F) SSG 13.3 1.0 14.4 1.1 12.7 4.2 Practice 2 (11M, 21 F) SSG 13.6 1.4 14.18 1.4 13.8 3.5 Practice 3 (11 M, 12 F) SSG 13.4 1.4 14.5 1.4 13.9 4.5 Practice 1 (10 M, 17 F) CG 13.3 1.5 14.8 1.5 17.5 5.4 Practice 4 (11 M, 19 F) CG 13.3 1.6 15.4 1.6 24.8 4.3 Practice 5 (12 M20 F) CG 13.1 1.1 15.1 1.1 24.4 5.9 SSG (41 M, 48 F) 13.4 1.3 14.6 1.3 13.4 4.1 CG (33 M, 56 F) 13.2 1.4 15.1 1.4 22.4 6.2 An ABO examiner with training and experience in DI and CRE scoring (DP) aided in the scoring calibration and performed random checks to ensure consistency. All models were scored randomly to prevent bias. In order to verify intra-rater agreement, a random set of twenty models was select- ed four weeks after initial scoring. In addition, a second investigator (JMJ) scored 35 models using the CRE criteria to evaluate inter-grader agree- ment. Both intra- and inter-grader agreement for the CRE total were as- sessed by means of intra-class correlation coefficient (ICC) with two-way random effect model. Age at T1, age at T2, treatment duration and number of adjust- ment appointments were determined from subject treatment notes. Final models of patients who used a tooth positioner at the end of treatment were handled no differently than the other models with 393 Computer-assisted Finishing Technology regard to grading. Emergency appointments were not counted as an ad- justment appointment. STATISTICAL ANALYSIS Chi-square tests were performed to check male/female distribu- tion and distribution of Angle classifications at T1 between the SSG and CG. Descriptive statistics including means and standard deviations were calculated for age, treatment duration, number of appointments, modi- fied Dl and CRE scores. Independent sample t-tests were used to com- pare the treatment duration and the number of appointments. Due to the ordinal nature of both the DI and CRE indices, non-parametric statis- tics (Mann-Whitney U test) were used to compare DI and CRE categories between the two groups (SPSS, Version 12.0, SPSS Inc., Chicago, IL). The power of the study for independent t-test was calculated on the basis of minimum expected difference for CRE score of 2.8 with a standard deviation of 6.5 as derived from a previous study (Alford et al., 2011). The power was 0.81 at an alpha level of 0.05 (SigmaStat 3.5, Systat Software, Point Richmond, CA). An ICC score of 0.989 was calculated on twenty sets of models randomly chosen and re-scored four weeks after initial scoring, indicating a high degree of intra-rater reliability. An ICC score of 0.961 was calcu- lated on 35 sets of models score independently by the two independent observers, indicating a high degree of inter-rater reliability. RESULTS Demographics of the treatment sample are shown in Table 2. Chi-square analysis showed that there were no significant differences in male/female distribution or in the distribution of Angle classifications (Table 1) between the SSG and CG (p = 0.287 and p = 0.264, respectively). There were no significant differences in the individual Dl categories or the modified total Dl score between the treatment groups at T1 (Table 3). The CG had a significantly longer treatment duration (22.4 + 6.2 months) than the SSG (13.4 + 4.1 months; p < 0.001). The number of adjustment appointments also was significantly greater in the CG with 17.2 + 2.6 appointments versus the SSG with 9.2 + 3.5 appointments (p < 0.001). 394 Groth et al. Table 3. Comparison of starting forms for the Discrepancy Index (DI). SSG CG N = 89 N = 89 SSG vs. CG ABO DI measures Mean SD Mean SD oº::ce Smg Overjet 1.5 0.9 1.4 1.1 0.1 NS Overbite 1.7 1.6 1.3 1.6 0.4 NS Anterior openbite 0.6 1.6 0.3 1.1 0.3 NS Lateral openbite 0.1 0.9 0.0 0.1 0.1 NS Crowding 1.3 0.9 1.4 1.0 –0.1 NS Occlusion 1.8 2.2 2.2 2.5 -0.4 NS Lingual crossbite 0.2 0.7 0.4 , 1.3 –0.2 NS Buccal crossbite 0.2 O.7 0.3 1.1 –0.1 NS Other complexities 0.4 0.8 0.5 1.1 –0.1 NS Total DI score 7.7 4.3 7.8 4.3 -0.1 NS As for the ABO CRE scores for treatment outcome, the only cat- egories that showed significant differences were buccolingual inclination and root angulation (Table 4). For buccolingual inclination, the SSG was 0.7 points more than the CG; for root angulation, the SSG was 0.4 points lower than the CG. There was no significant difference between the two groups for the total CRE score. Table 4. Comparison of CRE scores in SSG vs. CG. * = P × 0.05. SSG CG SSG vs. CG CRE Measures N = 89 N = 89 Mean SD Mean SD Diºce Sig Alignment and rotations 4.1 1.8 3.9 1.8 0.2 NS Marginal ridges 2.8 1.6 3.4 2.0 -0.6 NS Buccolingual inclination 2.9 1.9 2.2 1.6 0.7 >k Overjet 2.8 1.9 2.8 2.1 –0.1 NS Occlusal contacts 4.7 2.7 4.3 3.2 0.4 NS Occlusal relationships 5.5 2.9 4.8 3.1 0.7 NS Interproximal contacts 0.3 0.8 0.5 1.0 -0.2 NS Root angulation 1.1 0.9 1.5 1.1 -0.4 >k Total CRE 24.1 6.6 23.4 6.9 0.7 NS 395 Computer-assisted Finishing Technology DISCUSSION The average modified D] score for the respective groups was low. Any score under 10, according to the ABO, is considered a mild maloc- clusion; however, the Dl scores were calculated without the cephalomet- ric component of the Dl, a potentially significant contributor to the total DI. The cephalometric component was eliminated in order to keep this a study based only on dental cast analysis. Had the cephalometric compo- nent been included, the DI scores for many of the patients undoubtedly would have increased to above 10, which is the threshold for board case eligibility. While this study recognizes the generally mild nature of the cases treated, the investigators had no control over this aspect of the study. The cases were collected based solely on the inclusion criteria, not on initial discrepancy. One of the most interesting results of this study was the marked difference in treatment duration between the SSG and CG of about 9.0 months with the SSG completing treatment faster. The treatment dura- tion was 40% shorter, which is faster than what was reported by Alford and associates (2011). The average treatment time within the CG was 22.4 months, which has been shown in the literature to be a typical non- extraction average treatment time (Alger, 1988; Vig et al., 1990; Fink and Smith, 1992). The practice submitting to both treatment groups showed a similar result, with the SSG taking 4.9 fewer months to complete treat- ment. It has been reported that the decrease in unintentional tooth movements with the SS system reduces the treatment duration (Sachde- va, 2001). Thus, decreased “round tripping” during tooth movement may be a contributing factor related to the decrease in treatment duration ob- served with SS. When using SS, a tooth is moved the shortest distance to its final position, thus decreasing unintentional tooth movements (Mah and Sachdeva, 2001). Larson and colleagues (2013) evaluated the effec- tiveness of computer-assisted orthodontic treatment technology to pro- duce the tooth position prescribed by the virtual treatment plan (Larson et al., 2013). They reported that the effectiveness of orthodontic treat- ment delivered using SS technology to achieve predicted tooth position varies with tooth type and dimension of movement. 396 Groth et al. One of the major factors that can increase treatment time is the accurate positioning of brackets by the orthodontic provider at the initial bracket-bonding visit (Skidmore et al., 2006). With the SS system, there is a limited need to reposition brackets as the robotically bent archwires have the ability to compensate for inaccuracies in bracket position at ini- tial placement. A major operating cost within an orthodontic office is doctor, staff and patient chair time. Treatment time has been shown to be sig- nificantly shorter with the SSG, which potentially can lead to decreased operating cost for the clinician. Faster treatment time may not translate to lower operating costs, however, due to substantial fees associated with the SS system for each patient. The decreased number of adjustments to complete treatment in- dicates that not only are these patients finishing treatment faster, but also that they are using less chair time when compared to the patients treated conventionally. On average, the SSG needed eight fewer adjustment ap- pointments to complete treatment. Similarly, the practice that submit- ted to both treatment groups required 4.5 fewer appointments when us- ing SS. The reason for the difference may be due to the SS wires, which contain most, if not all, of the bends required in orthodontic finishing. The overall CRE scores between the SSG and CG were similar, 24.1 and 23.4, respectively. There were no significant differences be- tween the two groups for nearly all CRE categories with the exception of buccolingual inclination and root angulation. The SSG was less than one point lower than the CG for root angulation and the CG was ap- proximately one point lower than the SSG for buccolingual inclination. While there is no clinical difference between the groups for these two CRE measures, the results raise two interesting points. Many of the pro- viders using the SS system finish their cases in either copper nickel tita- nium (CuINiTi) or titanium molybdenum alloy (TMA) wires, both of which are not used routinely to engage the bracket slot fully. Considering the short treatment duration and the undersized wires being used, it is pos- sible sible that adequate posterior torque control may be difficult to at- tain in such instances. Along the same lines, Alford and coworkers (2011) recommended that the SS wires used for finishing do not possess the 397 Computer-assisted Finishing Technology strength necessary to upright the roots effectively. In their study, the root angulation score of the SSG was higher than that of the CG. Lin and Getto (2010) suggested that using the SS system to place the roots ideally within the bone may impact relapse rates; however, studies have shown that stability is unpredictable and no investigation has demonstrated that minor aberrations in root positioning has a dele- terious effect on stability (Linklater and Fox, 2002; Nett and Huang, 2005; de Freitas et al., 2007) or periodontal health (Årtun et al., 1987). CONCLUSIONS This study compared SS system versus conventional fixed appli- ance therapy in patients with a mild malocclusion treated without extrac- tions. The study has shown that in the selected sample of subjects and practices: 1. The treatment duration using the SS system was on average 40% shorter than using conventional orth- Odontic treatment. 2. The number of adjustment appointments needed to complete treatment was on average 47% less when using the SS system. 3. There were no clinically significant differences in case finish between the SSG and CG when assessed by the CRE Criteria. ACKNOWLEDGEMENTS The authors would like to thank all of the clinicians involved in this investigation (Drs. Scott Tyler, John Dumas, Brian Reyes, Ronald Snyder, Randy Moles, James McNamara, Jr., and Josephine Weeden). In addition, the authors appreciate Drs. Debbie Priestap and Joel Johnson for their assistance and expertise with the ABO and Peer Assessment Rating. 398 Groth et al. REFERENCES Alford TJ, Roberts WE, Hartsfield JK Jr, Eckert GJ, Snyder R.J. Clinical out- comes for patients finished with the SureSmile” method compared with conventional fixed orthodontic therapy. Angle Orthod 2011; 81(3):383-388. Alger DW. Appointment frequency versus treatment time. Am J Orthod Dentofacial Orthop 1988;94(5):436-439. Ártun J, Kokich VG, Osterberg SK. Long-term effect of root proximity on periodontal health after orthodontic treatment. 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Linklater RA, Fox NA. The long-term benefits of orthodontic treatment. Br Dent J 2002;192(10):583–587. Mah J, Sachdeva R. Computer-assisted orthodontic treatment: The Sure- Smile” process. Am J Orthod Dentofacial Orthop 2001;120(1):85-87. 399 Computer-assisted Finishing Technology Nett BC, Huang GJ. Long-term posttreatment changes measured by the American Board of Orthodontics objective grading system. Am J Or- thod Dentofacial Orthop 2005;127(4):444-450. Sachdeva RC. SureSmile” technology in a patient-centered orthodontic practice. J Clin Orthod 2001;35(4):245-253. Saxe AK, Louie LJ, Mah J. Efficiency and effectiveness of SureSmile”. World J Orthod 2010;11(1):16-22. Skidmore KJ, Brook KJ, Thomson WM, Harding WJ. Factors influencing treatment time in orthodontic patients. Am J Orthod Dentofacial Or- thop 2006;129(2):230-238. Vig PS, Weintraub JA, Brown Q, Kowalski CJ. The duration of orthodontic treatment with and without extractions: A pilot study of five selected practices. Am J Orthod Dentofacial Orthop 1990;97(1):45-51. 400 MALOCCLUSION AT THE NAVEL OF THE AMAZON RIVER David Normando, Cdtia Cardoso Abdo Ouintão ABSTRACT Studies of human and nonhuman primates have shown that the increased occur- rence of malocclusion can be attributed to diet. In a prior study, four indigenous populations were examined, which showed low intra-tribal genetic variation and large inter-tribal variation with traditional dietary habits. These isolated indig- enous populations provide an interesting opportunity to investigate factors re- lated to the development of dental malocclusion and face morphology, which might be camouflaged in modern human populations. Indigenous people of the Xingu River present similar tooth wear pattern, practice breastfeeding ex- clusively, do not use pacifiers and have a large inter-tribal genetic distance. Oc- clusion and facial features of five semi-isolated Amazon indigenous populations were evaluated and compared to data from urban Amazon people. Malocclusion prevalence in the indigenous group ranged from 33.8 to 66.7%, which is lower overall when compared to urban people. A high inter-tribal diversity was found regarding dental malocclusion, facial morphology, dental arch dimension and dental crowding, while low diversity within groups was observed. The results indicate that genetic factors may contribute substantially to the morphology of occlusal and facial features in the indigenous groups studied. This is supported by previous genetic studies and current findings that show similar patterns of tooth wear among the inhabitants of the Xingu River and high inter-tribal diversity of occlusal and facial features. KEY WORDS: dental malocclusion, dental crowding, arch dimensions, tooth size, face morphology INTRODUCTION Malocclusion is a public health problem that affects 39 to 93% of the world's population (Thilander et al., 2001) for which millions of people around the world undergo treatment every year. Despite the high prevalence, the etiology of this problem remains controversial (Mew, 2009) due to complex human dento-facial variations where ge- netic, epigenetic and environmental influences all interact to produce 401 Malocclusion at the Amazon River the final observable phenotypes. Models commonly used to evaluate the role of environment, epigenetic and genetics in malocclusion are based on animal experiments (Ulgen et al., 1997; Katsaros et al., 2002; He and Kiliaridis, 2003; Larsson et al., 2005; Burn et al., 2010), analysis of skull remains from ancient populations (Varrela, 1990; Harper, 1994; Lindsten et al., 2002; Mockers et al., 2004; Evensen and Øgaard, 2007; Defraia et al., 2008), research on living twins (Corruccini et al., 1990b; Hughes et al., 2001; Eguchi et al., 2004; Kawala et al., 2007; Townsend et al., 2009) and investigations of primitive human populations (Niswander, 1967; Corruc- cini, 1984, 1990; Corruccini et al., 1990a). Most scientific studies indicate a large influence of the environment on dental malocclusion, sustained by changes in the dietary consistency (Beecher and Corruccini, 1981; Cor- ruccini and Beecher, 1982, 1984; Beecher et al., 1983; Corruccini et al., 1985; Ciochon et al., 1997) to more softened foods, which leads to a re- duction of dental attrition. Wear of enamel produced by dental attrition was defined by Begg (1954) and later by Corruccini (1990) as an impor- tant mechanism for reducing mesiodistal tooth size, providing space in the dental arch. The available evidence suggests that the human dentition is “de- signed” on the premise that extensive wear will occur, described as “ad- aptation with passive structural changes” (Kaifu et al., 2003). Neverthe- less, the issue is not as simple as it may seem and, therefore, requires further deliberation and investigation. The alternative working assump- tion is that different malocclusions likely result from a combination of different etiological factors and occlusal alterations. A critical analysis of models used in previous studies to evalu- ate the etiology of malocclusions should take into consideration some important methodological issues. Reports examining skulls of ancient populations have advocated that a primary factor in the occurrence of dental malocclusion, mainly dental crowding, is the environment. How- ever, the lower frequency of malocclusion in such isolated populations may be a primary consequence of a homogenic genetic background ex- pressed in a small population. This hypothesis is supported by studies examining ancient populations with dental crowding, even in the pres- ence of severe tooth wear (Harper, 1994; Lindsten et al., 2002; Mock- ers et al., 2004). Furthermore, it seems difficult to determine the exact age in such skulls/individuals and its influence on dental wear. Thus, the 402 Normando and Ouintão observation of tooth wear in human skulls is limited and may be associ- ated not only with diet, but also with aging (Vieira et al., 2015). Animal studies have provided the most widely-used research model to define the primary role of dietary consistency on malocclusion. Animals raised on a hard diet showed more rapid tooth wear than those raised on a soft diet (Teaford and Oyen, 1989). The extrapolation of this result to craniofacial and occlusal development in humans is critical since electromyography data demonstrate that jaw-closing muscle recruitment patterns for macaques and baboons differ from those of humans (Hy- lander and Johnson, 1994). Furthermore, changes arising from manipu- lation of food consistency in animals are small in magnitude and mainly related to transverse maxillary growth. Data derived from twins and their families have been applied to study genetic and environmental influences on human dental variation (Corruccini et al., 1990b; Hughes et al., 2001; Eguchi et al., 2004). Tradi- tional models have advantages and limitations, and special features of the twinning process are important to consider (Townsend et al., 2009). A major issue of concern has been the accuracy of zygosity determination by comparison of physical appearance. However, recent analysis based on twin data—with accurate zygosity determination including the use of highly polymorphic regions of DNA-has proven to be more accurate and reliable (Nyholt, 2006). The epidemiologic study of primitive human populations pro- vides an interesting opportunity to determine the influence of genetics, epigenetics and environment in the occurrence of dental malocclusion. However, these studies mostly are inconclusive, since it is not possible to define the genetic background and ethno-historic factors for popula- tions under analysis. In this chapter, we discuss the possible contributions of diet as an environmental factor and genetics to malocclusion through findings of a series of studies on five semi-isolated Amazon indigenous populations. CULTURAL AND GENETICS BACKGROUND OF INDIGENOUS PEOPLE FROM XINGU RIVER Some Brazilian indigenous groups experienced a major process of acculturation throughout history and part of this indigenous population 403 Malocclusion at the Amazon River now live in urban centers. The Amazon, however, is a region where sev- eral different indigenous ethnic groups still can be found living in partial or total isolation. The middle valley of Xingu River, in the state of Pará-Brazil, is home to indigenous groups who are isolated geographically and Still maintain traditional habits. This indigenous population exhibit low intra- tribal diversity. The eating habits of these people in this region are tra- ditional and based on cassava, nuts, fish, meat of wild animals, Sweet potato and wild fruits (Carvalho et al., 1989). Processed foods rarely are consumed due to large geographical distance and lack of transportation to urban areas. Previous investigations (Normando et al., 2011, 2013; de Souza et al., 2015) have reported that tooth wear, which is direct evi- dence of what an individual ate in the past, is similar among the popula- tion living in these isolated villages. All children are breastfed until the birth of the next child, which usually occurs after one and a half to two years. The use of pacifiers or bottles is not observed. In contrast, the Amerindian population exhibits genetic diversity even though they live in a similar environment as the indigenous group. Genetic studies of the indigenous village populations report a large inter-tribal genetic distance (i.e., genetic divergence between popula- tions or tribes) associated with a low intra-tribal diversity (Zago et al., 1996; Vallinoto et al., 1998; Ribeiro-dos-Santos et al., 2001). Thus, the large variation between tribes compensates for the low variation within tribes, a feature attributed to the genetic drift acting on small, isolated populations. However, a small founder population (i.e., the first small population to compose a group of people living together) with a low genetic diversity is another factor that may contribute to the low intra- tribal diversity. Small founder populations are frequent events among the Amerindians, but typically are not recorded well. Therefore, study- ing semi-isolated indigenous people with similar dietary habits (Carval- ho et al., 1998) confirmed by similar tooth wear pattern (Normando et al., 2011, 2013; de Souza et al., 2015), large genetic distance and low intra-tribal diversity (Zago et al., 1996; Vallinoto et al., 1998; Ribeiro- dos-Santos et al., 2001) may contribute to elucidate the role of genet- ics and environment on the etiology of dental malocclusion and facial features characteristics. A comparative analysis with data obtained from Amazon urban populations (Brandão et al., 1996, 1997; Normando et 404 Normando and Ouintão al., 1999) may provide an insight into the role of genetics and environ- ment on the etiology of dentofacial disturbances. THE INDIGENOUS VILLAGES Our data was collected between 2009-2011 during three expedi- tions to the Middle Valley of Xingu River (Fig. 1); some of the data were published previously (Normando et al., 2011, 2013; Barbosa et al., 2015; de Souza et al., 2015; Vieira et al., 2015) and some are unpublished (Nor- mando et al., 2015). Clinical epidemiology, dental cast measurements and facial photogrammetric records were obtained from 611 individu- als. Five indigenous populations were evaluated: Arara-Laranjal (n = 239) and Arara-Iriri villages (n = 80), from Arara's ethinicity; Koatinemo from Assurini's ethnicity (n = 140); Pat-Krô (n = 83) and Pikayaká (n = 69), both of Xicrin-Kayapó ethnicity. For more clarity, the villages will be abbre- viated as follows: Arara-Laranjal (A-1), Arara-Iriri (A-2), Pat-Krô (XK-1), Pikayaká (XK-2) and Koatinemo (AS). Arara-Laranjal and Arara-Iriri tribes share the same ethnicity, the Arara. The Arara-Iriri village was formed by the descendants of a - - - - --- º Assurini --- º **Antºna -- ºne anºtha- - - - § canao º “”º stantºna ºº º º º cachoemasºca 0. - --- - º - - - - *-º- º -- º - º - - - - - - -- * … - - - º - wºº. ' - º º - Arara-iriri iº º º - - - - - * lº-ºº ºl tº a º – º º --- © tº . ~~ scale 1-2-250,000 - - º --- -º- --------- - - ºiseasºn. P------- -5 Figure 1. Map of South America with location of indigenous groups investigated in the present study. The magnified box highlights the Xingu River region and its tributaries, the Iriri River, where the Arara-Laranjal (A-1, green) and Arara-Iriri (A2; red) villages are located and the Bacajá River where the Xicrin-Kaiapó (XK-1, XK-2) villages are located (black). The Assurini Village (AS) is located in the main river (blue). Reprinted from Barbosa et al., 2015. 405 Malocclusion at the Amazon River single couple expelled from the Arara-Laranjal village, a phenomenon known as linear fission and characterized by large genetic variation be- tween the villages (Ribeiro-dos-Santos et al., 2001), justifying the need to evaluate the villages of this ethnic group separately. While the Arara- Laranjal, Xicrin-Kayapá and Assurinis villages were expanded by noncon- sanguineous relations, the initial expansion of the Arara-Iriri group oc- curred through the mating of closely-related people (e.g., parents and offspring, siblings) and later by marriages between more distant relatives such as uncle-niece, aunt-nephew and first cousins (Ribeiro-dos-Santos et al., 2001). The arrival of the Xikrin-Kayapó indigenous in the Bacajá River, a tributary of Xingu River, has been determined to have occurred some- time in 1926 or 1927. When they arrived at the Bacajá region, they heav- ily explored both banks of the river, building a number of villages. Contact between the explorers and the Xikrin took place during the 1960s. After this period, several epidemics caused many deaths and the indigenous headed back into the forests. Sometime later, they were transferred to the site of the current villages. The first reports of the Assurini date from the end of the 19th century. In the 1960s, the hunting of wildcats and extraction of rubber led the regional population to move further up the tributaries of the right bank of the Xingu, provoking hostile encounters with the indigenous pop- ulation. After contact with Brazilian society in 1971, the Assurini of the Xingu-so called by the attraction expeditions—suffered a drastic popu- lation loss. DENTAL MALOCCLUSION EPIDEMIOLOGY AND FACIAL FEATURES The findings presented here are summarized from two recent manuscripts (Normando et al., 2011; de Souza et al., 2015). The popula- tion of village A-1 has a normal facial morphology (98%) for this ethnic- ity (see Fig. 2), associated with high rate (66.2%) of normal occlusion. In contrast, village A-2, which has the same ethnicity and same ancestral decent, a high prevalence of malocclusion and the highest occurrence of Class III malocclusion (32.6%) and long face (34.8%) are found (Fig. 3). Whereas Class Ill malocclusion was a common characteristic among indigenous people in village A-1, Class Il malocclusion, associated with a 406 Normando and Ouintão - - - - - - - Figure 2. A-B: Adult male from village A-1, normal facial pattern. C. Class I maloc- clusion. D: Severe dental crowding. Convex pattern, was prevalent in the XK-1 population (43.9%), who are of Xicrin-Kayapá ethnicity (Fig. 4). Another important feature of XK-1 popula- tion was the low frequency of anterior openbite, with no observed case of anterior crossbite. On the other hand, the population of village XK-2, who share the same ethnicity as XK-1, show a high frequency of anterior openbite and anterior crossbite. The difference in the overall prevalence of malocclusion among the villages also is observed in the prevalence of each subtype of mal- occlusion. Whereas more than three quarters of the A-1, AS and XK-2 populations showed a harmonious Sagittal intermaxillary relation, about 50% of the population in villages A-2 and XK-1 had disharmonic inter- arch relations as described in our previous publications. Comparatively, Amazon urban populations are more similar to groups with the highest 407 Malocclusion at the Amazon River Figure 3. A-B: Adult male from village A-2, long face and concave profile C. No crowding. D: Class III malocclusion. prevalence of harmonious relations between the dental arches (Brandão et al., 1996, 1997; Normando et al., 1999; Fig. 5). The prevalence of malocclusion in indigenous villages overall is moderately lower than that in urban populations (Brandão et al., 1996, 1997; Normando et al., 1999). Previous reports using the same examiner showed an occurrence of dental malocclusion of approximately 48% in primary, 86% in mixed and 85% in permanent dentitions for people living in cities of the Amazon. Among the investigated indigenous villages, the prevalence of malocclusion ranged between 10% to 46% in the primary dentition, 44% to 68% in the mixed dentition and 34% to 86% in the per- manent dentition. The lowest prevalence of dental malocclusion in remote indige: nous populations might be associated with environmental etiologic fact 408 Normando and Ouintão Figure 4. A-B: Adult male from village XK-1, convex profile. C-D: Class Il maloc- clusion. tors (e.g., the absence of pacifiers in all villages). Furthermore, all chil- dren are breastfed until the birth of the next child, which usually occurs after one and a half to two years. Several studies in urban populations (Kobayashi et al., 2010; Bueno et al., 2013; Corrêa-Faria et al., 2013) have associated dental malocclusion with the practice of deleterious oral hab- its. The low prevalence of deep overbite in villages A-1, A-2, AS and XK-2 indicate that this malocclusion type is less frequent when com- pared with Amazon urban populations (Brandão et al., 1996, 1997; Normando et al., 1999). In contrast, the population of village XK-1 has a high prevalence of deep overbite, similar to the urban populations. The high incidence of deep overbite could be associated with increased 409 Malocclusion at the Amazon River 100 90 80 70 -Urban [37-39) -Pat-krº 60 - Assurini 50 -Pykaikā -º-Arara-iriri (30) 40 --Arara-Laranjal (30) 30 20 10 Primary Dentition Mixed Dentition Permanent Dentition Figure 5. Comparative analysis of the prevalence of malocclusion represented as a percentage of the sample on the Y axis obtained from urban populations. Reprinted from de Souza et al., 2015. frequency of Class || malocclusion present in this village, which is compa- rable to urban populations. Anteroposterior incisor discrepancies, or overjet, is uncommon in villages A-1, AS and XK-2. However, these malocclusions have a higher prevalence in village A-2, predominantly Class III, and in village XK-1, pri- marily Class || malocclusions. With regard to the prevalence of anterior crossbite, our findings (de Souza et al., 2015) show similarity among the populations of villages A-1, AS and XK-2 and urban populations of the Amazon region. The indigenous populations that were investigated showed a low rate of posterior crossbite compared with those of urban popula- tions. This may be related to the absence of pacifiers and the exclusive breastfeeding practice of indigenous populations. Kobayashi and col- leagues (2010) reported that children who were breastfed for a longer duration have a lower risk of prevalence of posterior crossbite. Recent studies (Normando et al., 2011, 2013; de Souza et al., 2015) evaluating tooth wear in these villages showed no differences among them, Sup- 410 Normando and Ouintão porting the observation that feeding habits and diet are similar among indigenous people of the Xingu River (Carvalho et al., 1989). Although the populations of villages A-1, AS, XK-1 and XK-2 exhibit similar levels of crowding, the absence of this malocclusion in A-2 individuals was a meaningful finding. These data do not give support to Begg's theory (1954), which associates dental attrition with a lower prevalence of den- tal crowding in Australian Aboriginals. When comparing Amazon urban populations to Amazon indigenous people, dental crowding frequency was relatively similar to most villages (de Souza et al., 2015). Dental crowding has been described as a common dental malocclusion, even in ancient populations (Harper, 1994; Vodanović et al., 2012). Further- more, differences in prevalence of dental crowding have been reported among ancient populations (Vodanović et al., 2012). The highest frequency of biprotrusion (89.1%) and the absence of dental crowding were observed in the A-2 population; the highest rate of dental crowding (37.3%) and lowest prevalence of biprotrusion (37.9%) were observed in the AS population. These data suggest that dental crowding appears to be associated with the absence of dental bi- protrusion and decreased arch dimensions (de Souza et al., 2015), rather than severity of tooth wear. Facial typology was another feature that proved to be distinct among the villages. Dolichofacial typology observed in village A-2 is asso- ciated with increased frequency of anterior openbite seen in this village. No cases of increased overbite were observed in village A-2. Moreover, in villages A-1, AS and XK-2, predominantly brachyfacial and mesofacial typology, low frequency of increased overbite and a moderate rate of anterior openbite was observed. From these results, it could be inferred that only a small part of the incisor vertical relationship appears to be related to facial patterns among remote indigenous populations. DENTAL ARCH DIMENSIONS, TOOTH SIZE AND DENTAL CROWDING The assessment of ancient populations (Evensen and Øgaard, 2007; Defraia et al., 2008) including indigenous Amazon people (Niswander, 1967) have revealed the occurrence of dental crowding, even in the presence of severe tooth wear. Most of these studies are 411 Malocclusion at the Amazon River inconclusive, however, given the lack of genetic information and the in- adequate ethnic and historical accounts on these populations. The bite force of incisors among the indigenous populations in- habiting the Brazilian Xingu region has been reported to be twice that of urban populations (Regalo et al., 2008). Among these individuals, eating habits remain predominantly traditional due to their geographic isolation and lack of regular transportation between villages and urban centers. Vieira and colleagues (2015) reported that dental wear among the indig- enous inhabitants of the middle Xingu Valley is associated strongly with age, whereas no significant association could be found in the Amazon urban population. The intergroup variation in dental arch dimensions in particular, as well as the size of the teeth of the upper arch, was shown to be greater significantly than the intragroup variation (Normando et al., 2015). This finding can be attributed to genetic drift, an ecostatic process that more often affects small, isolated populations, inducing loss of genetic varia- tion and thus altering the prevalence of certain traits in a given popula- tion (Ribeiro-dos-Santos et al., 2001). Low intragroup genetic diversity is related to high inter-tribal genetic variation. Our findings fully support the manifestation of genetic drift on occlusal features in these populations. The five indigenous populations can be divided into three discriminant groups, which support the genetic drift theory (de Souza et al., 2015). The first includes the XK-1, XK-2 and A-1 groups, which showed no significant differences in most dimensions. The second consists of AS subjects, who displayed larger tooth size and dental arch dimensions than the first group. Irregularities in the posi- tion of anterior teeth were similar between first and second groups. The third discriminant group can be differentiated from previous groups. It includes individuals from village A-2, whose tooth dimensions were simi- lar to those in the first group (A-2, XK-1 and XK-2), but whose dental arch dimensions were closer to those of the second group (AS). The scenario of having smaller tooth size and decreased arch length results in a group with the lowest irregularity index. Thus, Little's Irregularity Index values in the A-2 group close to zero, confirming the low prevalence of crowding in this population (Normando et al., 2011, 2013). 412 Normando and Ouintão Vela and associates (2011) reported that tooth size may differ among individuals of diverse ethnic backgrounds, a finding supported by the results of our investigations in the Xingu region (Normando et al., 2013, 2015). Individuals of the AS group were found to have larger up- per teeth compared with individuals from the other villages; however, no significant difference was observed in the lower arch dimensions. These different morphological patterns strengthen the hypothesis that the ge- netic or epigenetic mechanism behind tooth size can be different for each dental arch or group of teeth. Furthermore, the data do not substantiate the hypothesis that human teeth are larger in modern populations due to a softer diet. Another debate in the literature concerns the role of tooth size in the occurrence of dental crowding. Whereas Puri and colleagues (2007) and Normando and coworkers (2013) have stated that tooth size is a deci- sive factor in the development of crowding, Howe and coworkers (1983) argued that the variability of dental arch dimensions would play the most important role. Bernabé and Flores-Mir's study (2006) using multivariate statistics reported that individuals with larger teeth are more susceptible to developing crowding. Normando and associates (2015) have indicated that groups of individuals with larger upper teeth (e.g., AS) may exhibit just as much crowding as individuals with smaller teeth. In this case, the increased tooth size was offset by similarly increased dental arch dimen- sions. On the other hand, the group with the lowest irregularity index values in terms of tooth position (e.g., A-2) had tooth sizes that were similar to those of groups with larger irregularity values (e.g., XK-1, XK-2 and A-1). Thus, the discriminant factor in this group seemed to be the dental arch dimensions. FACE BIOMETRY The discriminant facial analysis of female individuals revealed that there are homogeneous features of females from within the same village, while a high inter-tribal heterogeneity was found. This finding allows for association of females subjects to their specific village using facial measurements. For males, the distinct facial measurement char- acteristics associated with their village was not as evident as in females. The male natives of the A-1, XK-1 and XK-2 villages showed an overall facial 413 Malocclusion at the Amazon River similarity tendency; however, a clear difference in facial features could be observed between the AS and A-2 males and with A-1, XK-1 and XK-2 villages. These findings do not support the concept that sex differences in genetic determination were significantly higher for boys than for girls (Carels et al., 2001). A comparative analysis of each variable used to evaluate facial morphology revealed striking differences among the villages. The A-1 indigenous, both males and females, presented a less prominent nose and A’ point (maxilla) and the lowest values for facial heights, showing a marked tendency toward a brachyfacial pattern. Therefore, these indig- enous can be characterized as brachyfacials associated with a less promi- nent maxilla and nose. * The people of all tribes showed a more acute nasolabial angle when compared to non-indigenous Brazilians, characterizing a greater lip protrusion for the indigenous people. For Caucasian Brazilian adults, the mean for nasolabial angle is reported as 108° (Reis et al., 2006), while the highest nasolabial angle mean among the indigenous people was 101°, observed in A-1 males. The most protruded upper lip was found in male and female from the XK-1 and XK-2 tribes, while the less prominent up- per lip was found in AS female and A-1 male; the same pattern was found for the lower lip in the female group. These findings confirm the highest prevalence of biprotrusion in the A-2, XK-1 and XK-2 populations and the lowest prevalence of biprotrusion in the AS and A-2 groups (de Souza et al., 2015). Knowledge of the degree to which various subsets of morpho- logical data reflect molecular relationships is crucial for studies attempt- ing to estimate genetic relationships from patterns of morphological variation (Smith and Heather, 2009). The important influence of heredity on craniofacial dimensions and facial biometrics (Kohn, 1991; Häjek et al., 2008; Smith and Heather, 2009; Djordjevic et al., 2013), supported by the large inter-tribal heterogeneity and a low intra-tribal variation, may explain the great homogeneity in facial morphology of individu- als from the same village and the marked difference among villages. Our findings from evaluating indigenous populations (Normando et al., 2011, 2013, 2015; Barbosa et al., 2015; de Souza et al., 2015; Vieira et al., 2015) lend support to the concept that variation in facial morphol- ogy is linked with genetics determination (Smith and Heather, 2009; 414 Normando and Ouintão Djordjevic et al., 2013; Hughes et al., 2013) and that we should expect a highly polygenic basis for complex traits such as human craniofacial and dentition morphology and development (Carrol, 2003). However, we should consider that the relative contribution of genetic and environ- mental factors is different for different areas that compose craniofacial morphology (Smith and Heather, 2009; Djordjevic et al., 2013). CONCLUSIONS Our findings reveal a high diversity of occlusal patterns and fa- cial morphology among the investigated populations; the challenge was to identify similar patterns in these dental and facial features within at least two groups. The morphologic diversity between groups was large, even when comparing those from the same ethnicity. A low diversity for some facial or occlusal characteristics within groups could be observed in some villages, but not in all of them. These findings demonstrate that prevalence of malocclusion in indigenous populations cannot be deter- mined from examining a single village or even an entire ethnicity. Simi- larly, subjects from different villages should not be examined within the Same group. These results suggest that a highly polygenetic base for the craniofacial morphology characteristics and dental human development should be expected (Carrol, 2003). Therefore, our results from isolated indigenous population do not support the theory, widely reported in the literature, that diet consistency plays the most important role in contrib- uting to the increase of malocclusion, which is mainly the dental crowd- ing, in modern human populations. 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