PRF applications in Endodontics [1 ed.] 0867158271, 9780867158274

Citation preview

Edited by

Mohammad Sabeti, dds, ma Edward S. Lee, dds Mahmoud Torabinejad, dmd, msd, phd

PRF Applications in Endodontics

PRF Applications in Endodontics

PRF Applications in Endodontics Edited by Mohammad Sabeti, dds, ma Professor and Endodontic Program Director Department of Preventive and Restorative Dental Sciences School of Dentistry University of California San Francisco San Francisco, California

Edward S. Lee, dds Clinical Instructor Department of Preventive and Restorative Dental Sciences School of Dentistry University of California San Francisco San Francisco, California

Mahmoud Torabinejad, dmd, msd, phd Adjunct Professor Department of Preventive and Restorative Dental Sciences School of Dentistry University of California San Francisco San Francisco, California

Library of Congress Cataloging-in-Publication Data Names: Sabeti, Mohammad (Mike) A., editor. | Lee, Edward S., editor. | Torabinejad, Mahmoud, editor. Title: PRF applications in endodontics / edited by Mohammad Sabeti, Edward S. Lee, Mahmoud Torabinejad. Description: Batavia, IL : Quintessence Publishing Co., Inc., [2020] | Includes bibliographical references and index. | Summary: “This handbook will help clinicians understand and use PRF in endodontics by discussing the science, clinical applications, and techniques for specialists and general practitioners”-- Provided by publisher. Identifiers: LCCN 2020002529 (print) | LCCN 2020002530 (ebook) | ISBN 9780867158274 (paperback) | ISBN 9781647240196 (epub) Subjects: MESH: Dental Materials--chemical synthesis | Platelet-Rich Fibrin | Regenerative Endodontics methods Classification: LCC RK652.5 (print) | LCC RK652.5 (ebook) | NLM WU 190 | DDC 617.6/95--dc23 LC record available at https://lccn.loc.gov/2020002529 LC ebook record available at https://lccn.loc.gov/2020002530

97% ©2020 Quintessence Publishing Co, Inc Quintessence Publishing Co, Inc 411 N Raddant Road Batavia, IL 60510 www.quintpub.com 5 4 3 2 1 All rights reserved. This book or any part thereof may not be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, or otherwise, without prior written permission of the publisher. Editor: Marieke Zaffron Editorial Assistant: Samantha Smith Design: Sue Zubek Production: Sue Robinson Printed in the United States

I dedicate this book to my wife, Parvin, and my children, Sara and Ali, with love. –MS I would like to thank my mentors, Dr Ronald J. Nicholson and Dr Chutima Mangkornkarn for inspiring me to become a better clinician and person, and to Dr Calvin Tae Nam for sharing his knowledge about PRF. –ESL

To the soul of my dear father whom we lost to cancer early in his and our lives. –MT

Contents

Foreword by Richard J. Miron   viii Preface  x Contributors  xi Introduction by Mahmoud Torabinejad   xii

1 2

Stem Cells in Regenerative Medicine   1

3

Nonsurgical Application: PRF for Regeneration Endodontics   1 7

4

Soft Tissue Applications   25

C. Cameron Taylor  |  Habib Torfi  |  Mohammad Sabeti

History, Science, Armamentarium, and How to Make PRF   9 Edward S. Lee

Mohammad Sabeti  |  Edward S. Lee

Kayvon Javid  |  Gregori M. Kurtzman  |  Carlos Fernando Mourão

5

Hard Tissue Applications  39

6

PRF in Endodontic Surgery  57

7

PRF for Socket Preservation  7 1

Kayvon Javid  |  Gregori M. Kurtzman  |  Carlos Fernando Mourão

Mohammad Sabeti  |  Gregori M. Kurtzman Edward S. Lee  |  Eric Wong

Yogalakshmi Rajendran  |  Yvonne Kapila

Index  77

Foreword

T

he use of platelet-rich fibrin (PRF) has seen a rapid increase over the past decade, owing to its ability to rapidly release autologous growth factors harvested quite easily from peripheral blood. While original case studies dating back nearly two decades focused primarily on its use in medicine for the treatment of hard-to-heal wound ulcers, it is now well known that its inclusion of high concentrations of platelets and leukocytes has served several benefits in dentistry. Specifically, platelets are largely responsible for the release of various regenerative growth factors favoring wound healing, whereas leukocytes (white blood cells) participate in host defense against incoming pathogens. The ability to concentrate both cell types found within PRF has shown pronounced benefits in the oral cavity, an area particularly concentrated with various oral bacteria. Over the years, several research articles focused on the use of PRF for multiple applications in regenerative dentistry; more recently, publications have begun to emerge dealing specifically with its use in endodontics. I have the great pleasure to announce the launch of this new book, PRF Applications in Endodontics, which addresses this topic in extensive detail. The book begins by providing background knowledge on various cell types found in regenerative medicine with particular focus on stem cells. Thereafter, the book rapidly enters into a variety of chapters dedicated to PRF with a brief history regarding its scientific background, including growth factors, armamentarium, and protocols utilized to fabricate PRF. The discussion then transitions to nonsurgical applications in regenerative endodontics, as well as its use in dentistry, particularly for the formation of a bone grafting material complex including bone grafting particles and autologous PRF (aka “sticky bone”). Its use as an alternative to bone grafts and other biomaterials is further discussed in later chapters dealing with endodontic surgery. These include various endodontic procedures indicated following common human “accidents” (accidental tooth loss and replantation, for instance), for improvements in furcation-involved teeth as a result of iatrogenic procedures, for root-end resection procedures, and for the management of surgical cysts. viii

This textbook is for both the beginner as well as the advanced endodontist and practicing dentist working in the field of endodontics wishing to further improve their practice by adopting some of the latest regenerative protocols. It is certainly a first of its kind and a must-read in the field of endodontics, highlighting the benefits of autologous blood concentrates specifically dedicated to endodontic procedures. Colleagues will certainly enjoy this read, and it will undoubtedly open many avenues of future research on the topic!



Richard J. Miron, dds, msc, phd Group Leader, The Miron Research Lab Lead Educator, Advanced PRF Education Venice, Florida

ix

Preface

A

s endodontists and periodontists, we are all familiar with the potential applications of platelet-rich plasma (PRP) in medicine and dentistry. But in the past few years, platelet-rich fibrin (PRF) has emerged as an alternative material in its own right. One of our first opportunities to observe the effects of PRF was in discussion with colleagues using it in oral surgery procedures. Their patients experienced remarkable hard and soft tissue healing with minimal postoperative discomfort. Intrigued, we dug further and discovered the widespread applications of PRF in dentistry and medicine. The appeal of PRF stems from the fact that it is made from a patient’s own blood. It is easy to prepare and can be used for many kinds of procedures, making it cost-effective. PRF has many potential applications in endodontics. It can be used in surgical endodontics and adjunctive surgical procedures such as root amputation and hemisection. In addition, it can be used for root perforation repair, vital pulp therapy, and regenerative endodontics. Furthermore, it can be used as a bone graft binder during socket preservation to create “sticky bone” for the closure of surgical sites. When the three of us first met, the idea of sharing these various applications of PRF was an immediate common ground. We were working with residents at the time and knew how much they could benefit from learning about PRF. After using PRF and observing successful outcomes in several cases, we decided to take things to the next level. We brought together some of the most forward-thinking endodontists, periodontists, oral surgeons, and general practitioners to share our thoughts regarding potential use of this material in endodontics and other fields of dentistry. This book, representing a collaboration of like-minded clinicians, is the first to introduce the idea of PRF and cord blood stem cells in endodontics. It contains an overview of PRF itself with up-to-date information on tissue regeneration, as well as step-by-step instructions on how to use PRF in a variety of endodontic and oral surgery procedures. We have been using this knowledge for years to improve tissue healing for our patients, and we hope this book will help you on your quest to improve healing for your patients. x

Contributors

Kayvon Javid, dds Private Practice San Pedro, California

Yvonne Kapila, dds, phd

Professor Department of Oral and Maxillofacial Surgery School of Dentistry University of California San Francisco San Francisco, California

Gregori M. Kurtzman, dds Private Practice Silver Spring, Maryland

Mohammad (Mike) Sabeti, dds, ma

Professor and Endodontic Program Director Department of Preventive and Restorative Dental Sciences School of Dentistry University of California San Francisco San Francisco, California

C. Cameron Taylor, phd

Research and Development Supervisor Invitrx Therapeutics Irvine, California

Edward S. Lee, dds

Mahmoud Torabinejad, dmd, msd, phd

Carlos Fernando Mourão, dds, msc, phd

Habib Torfi, mse

Private Practice San Pedro, California

CEO and President Invitrx Therapeutics Irvine, California

Yogalakshmi Rajendran, bds, ms

Eric Wong, dds

Clinical Instructor Department of Preventive and Restorative Dental Sciences School of Dentistry University of California San Francisco San Francisco, California

Assistant Clinical Professor, Health Sciences Director, Predoctoral Periodontics Department of Orofacial Sciences School of Dentistry University of California San Francisco San Francisco, California

Adjunct Professor Department of Preventive and Restorative Dental Sciences School of Dentistry University of California San Francisco San Francisco, California

Division Chair, Endodontics Department of Preventive and Restorative Dental Sciences School of Dentistry University of California San Francisco San Francisco, California

xi

Introduction

R

ecent studies using novel biomaterial scaffolds that contain host endogenous growth factors represent a departure from traditional clinical approaches and may result in better and more predictable regenerative solutions in medicine and dentistry. As early as 1966, Rule and Winter published a case report regarding continued root formation and apical closure in an immature human premolar tooth using pulp bleeding as a scaffold. Nygaard-Ostby et al, Nevins et al, Iwaya et al, Banchs and Trope, as well as others reported pulp revascularization in teeth with necrotic pulps and immature apices that showed continuous root maturation, dentinal wall thickening and, in some cases, a positive response to vitality tests. In 2011, we reported a case of pulp revascularization using platelet-rich plasma (PRP) in a second maxillary premolar with immature root that had been accidently extracted and then replanted. After removing the necrotic pulp, irrigating it with 5.25% sodium hypochlorite, and medicating it with a triple antibiotic paste for 3 weeks, we prepared PRP from the patient’s blood and injected it into the canal space. Mineral trioxide aggregate (MTA) was placed over the clotted PRP and double-sealed with Cavit (3M) and amalgam. Radiographic examination of this tooth 5.5 months later showed resolution of the periapical lesion, further root development, and continued apical closure. Vitality tests elicited positive responses like those found in the first premolar tooth. The shortcomings of PRP include the need to draw blood from the patient and the complexity of centrifugtion and purification in a clinical setting. Platelet-rich fibrin (PRF) is an autologous product that contains high concentrations of nonactivated, functional intact platelets within a fibrin matrix that release a relatively constant concentration of growth factors/cytokines over a few days. It is easier to produce but it has to be used immediately after blood drawing and centrifugation. PRF is a potential substitute for PRP in regenerative endodontics and other regenerative procedures involving reconstruction of hard tissues, such as surgical endodontics and adjunctive surgical procedures like root amputation, hemisection, and repair of root perforations. xii

The main purpose of PRF Applications in Endodontics is to stimulate research in regenerative procedures in endodontics and encourage clinicians to use PRF to improve healing of their patients and save natural dentition. The book has seven chapters and starts with the history of stem cells in regenerative medicine and its possible applications in endodontics, followed by PRF armamentarium and description of how to make PRF, use of PRF in nonsurgical endodontic procedures, its soft tissue applications, hard tissue applications, surgical endodontics, and finally socket preservation. It is assembled by well-known scientists and clinicians who are experts in their fields and interested in the use of innovative materials and techniques to improve human lives. Mahmoud Torabinejad, dmd, msd, phd

References Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: New treatment protocol? J Endod 2004;30:196–200. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part I: Technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e37–e44. Iwaya SI, Ikawa M, Kubota M. Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dent Traumatol 2001;17:185–187. Mao JJ, Kim SG, Zhou J, et al. Regenerative endodontics: Barriers and strategies for clinical translation. Dent Clin North Am 2012;56:639–649. Nevins A, Wrobel W, Valachovic R, Finkelstein F. Hard tissue induction into pulpless open-apex teeth using collagen-calcium phosphate gel. J Endod 1977;3:431–433. Nygaard-Ostby B, Hjortdal O. Tissue formation in the root canal following pulp removal. Scand J Dent Res 1971;79:333–349. Rule DC, Winter GB. Root growth and apical repair subsequent to pulpal necrosis in children. Br Dent J 1966;120:586–590. Torabinejad M, Faras H. A clinical and histological report of a tooth with an open apex treated with regenerative endodontics using platelet-rich plasma. J Endod 2011;38:864–868. Torabinejad M, Turman M. Revitalization of tooth with necrotic pulp and open apex by using platelet-­ rich plasma: A case report. J Endod 2011;37:265–268.

xiii

1

LEARNING OBJECTIVES • Gain a better understanding of stem cell biology and how it relates to regenerative medicine, specifically dentistry • Compare different sources of stem cells and the relative strengths and weaknesses associated with each source • Gain a better understanding of a mesenchymal stem cell secretome and why it is important therapeutically

Stem Cells in Regenerative Medicine C. CAMERON TAYLOR, PhD HABIB TORFI, MSE MOHAMMAD SABETI, DDS, MA

R

egenerative medicine, also commonly known as tissue engineering, is a discipline of medicine that is focused on restoring native tissue structure and functionality to an afflicted tissue. Dentistry has traditionally been at the forefront of regenerative medicine, commonly employing novel bioactive materials to stimulate bone growth and regeneration. Recently, stem cells and other cell-based therapies have attracted significant attention in this space due to their ability to not only treat patients’ symptoms but to improve physiologic activity and restore native tissue structure. Stem cells are characterized by a capacity for self-renewal while maintaining an undifferentiated state and, given the proper stimulus, the ability to differentiate into various types of specialized somatic cells. Stem cells are further classified by their relative differentiation potential. Stem cells that can differentiate into any cell type in the body are termed totipotent and have the widest differentiation potential. Mesenchymal stem cells (MSCs) are multipotent stem cells that are most closely associated with the mesodermal lineage and are known to differentiate into chondrogenic, osteogenic, myogenic, and adipogenic cell types.1 1

1

STEM CELLS IN REGENERATIVE MEDICINE

The discovery of stem cells and their multipotent potential has encouraged the development of the whole field of research, projected to have reached $170 billion by 2020. In particular, the multipotent MSCs, with their stem-like quality to differentiate into mesodermal cell types, have been a focus. Indeed, overall revenue for MSC products was projected to be $10.9 billion from 2010 to 2020. Alongside the possibilities of therapeutic successes (ranging from treating graft-versus-host disease, Crohn disease, spinal cord injury, and use in support of hematopoietic stem cell treatments) comes the inherent ethical and logistic dilemmas behind obtaining stem cells. This chapter focuses on MSCs due to their popularity for regenerative applications.

Mesenchymal Stem Cells Stem cell sources First isolated in bone marrow, bone marrow–derived MSCs were found to be precursors to multiple cell types and could be viably cultured while retaining their capacity for multilineage differentiation. Obtained from an invasive bone marrow harvesting procedure, bone marrow–derived MSCs avoid the ethical concerns as well as tumorigenicity of embryonic stem cells and have subsequently been used in a nearly exponential increase in research studies and trials.2 Unfortunately, bone marrow–derived MSCs are relatively low yield and limited to autologous use, requiring in vitro expansion that increases the risk of contamination. Additionally, harvesting the cells requires a surgical procedure with associated donor morbidity and risk, and the potency (ie, “stemness”) has been questioned when compared with more recently discovered sources of MSCs.3 One of these sources is umbilical cord blood (also known as cord blood), collected via venipuncture of the typically discarded umbilical cord. Painless and without morbidity, cord blood is considered superior to human bone marrow stem cells in its harvesting and yield. Cord blood is cryopreserved in two main methods using dimethyl sulfoxide (DMSO): (1) red cell reduction, which is less expensive to store and easier to defrost; and (2) plasma depletion, which is more economical to process. Public cord blood banks cost about $1,500 to $2,500 per unit stored, while private banks typically charge an initial processing fee of $1,400 to $2,300 plus annual storage costs of $115 to $150. However, MSCs only represent a small proportion—1,000 to 5,000 MSCs in one 100-mL unit of cord blood—of the cell types within cord blood, which includes hematopoietic cell types, endothelial and progenitor cells, as well as MSCs.4

2

Mesenchymal Stem Cells

There has also been recent attention toward Wharton’s jelly (WJ) within umbilical cords. WJ was found at the turn of the century to contain a multipotent, fibroblast-like MSC population with greater multipotent potency, faster proliferation, and longer life spans than adult bone marrow–derived MSCs.3 This is a result of reduced telomere length. Telomeres shorten with age, eventually resulting in cellular senescence. MSCs isolated from cord blood are much younger than adult MSCs and possess significantly longer telomeres.5 MSCs in WJ are an entity apart from cord blood MSCs and endothelial cells from the umbilical vein. The plentiful presence of MSCs in WJ is theorized to either be due to the trapping and retaining of fetal MSCs during the two waves of migration of fetal MSCs in early development or to the fact that the cells in WJ are actually primitive MSCs that originate from mesenchyme already present in the umbilical cord matrix. More research has been focused on not only the characterization and usage of these WJ-derived MSCs, but also on discrete differences of the stem cell populations depending on the anatomical region of the WJ.6 The most recent development is that what was once thought to be a single mass providing uniform MSCs is actually more anatomically distinct. There are six different zones of the cord with cells in various stages of differentiation: (1) the surface (amniotic) epithelium, (2) subamniotic stroma, (3) clefts, (4) intervascular stroma, (5) perivascular stroma, and (6) vessels. However, the descriptors separating these zones are not clear. It is thought that WJ is composed mainly of perivascular progenitors but may possibly include nonperivascular progenitors as they move away from the vasculature.6 In addition to the anatomical differences, there is concern that the MSCs may differ lengthwise and that the mother end of the umbilical cord may have different mesenchymal features than the fetus end of the umbilical cord.7 In addition to harvesting and potency advantages, cord blood and WJ-derived MSCs are also not limited to autologous use. Due to their excellent immunomodulatory properties and universally tolerated surface marker profiles, MSCs isolated from cord blood and WJ can be made available to patients as allografts.8,9 Using cells isolated from birth tissue as “off-the-shelf” allografts greatly simplifies the manufacturing process of MSCs for therapeutic use, providing a standardized, scalable method of producing cells that does not need to be personalized for each patient.

Importance of the secretome The therapeutic effectiveness of MSCs is well documented, especially as it pertains to wound healing. However, the mechanisms of action are not well understood. Stem cells are partially defined as cells that are capable of differentiation into a 3

1

STEM CELLS IN REGENERATIVE MEDICINE

variety of specialized somatic cells, and this knowledge has fueled speculation that cell differentiation upon engraftment is responsible for the observed therapeutic effects. On further investigation, it would appear that this is not the case. Recent research has shown that MSCs introduced therapeutically primarily function through trophic and immunomodulatory signaling pathways, and the stem and progenitor cells of the host actually do most of the work.10 This is why the secretome, or the collection of bioactive molecules secreted from the cells, has been receiving more attention from researchers. Rich in growth factors and cytokines that are associated with modulating inflammation and promoting angiogenesis, the MSC secretome seems perfectly suited to enhance wound healing. This is evidenced in human physiology by the ability of MSCs to zero in on areas of inflammation and injury and secrete bioactive factors.11 The regenerative effects of growth factors and cytokines have been well documented in dentistry. Peptides in the transforming growth factor β (TGF-β), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and interleukin (IL) families are crucial components driving regeneration, especially as it relates to bone growth.12 These factors stimulate host cells in regenerative pathways, but it can be challenging to maintain dosing and ensure efficient cell uptake of these factors therapeutically. The MSC secretome is rich in many of these peptides, suggesting that the secretome could be responsible for some of the observed therapeutic effects.13

Stem Cells in Regenerative Endodontics The potential utility of cord blood MSCs has not yet been fully realized in the relatively new field of regenerative endodontics. Since its development in 2004 by Banchs and Trope, regenerative endodontics has been employed as a root-preserving alternative to a root canal, utilized to eradicate pulp infections in immature permanent teeth, thus permitting further root development and preservation of teeth in patients who are still growing.14 Liao et al15 revealed the presence of osteogenic MSCs in both inflamed pulp tissue and inflamed periapical tissues, and further investigation by Chrepa et al16 revealed that the evoked bleeding step demonstrated an increase in local accumulation of undifferentiated MSCs even in a mature tooth. Because of these revelations, the recommended American Association of Endodontists protocol was revised to use irrigants that are less toxic to stem cells and propose the use of platelet-rich plasma, platelet-rich fibrin, and autologous fibrin matrix in place of the simple blood clot. However, there is no evidence in the literature of regeneration of the dentin pulp complex. The effect of noninflamed apical residual tissue in regeneration 4

Stem Cells in Regenerative Endodontics

of pulp has been investigated recently in an animal model. Torabinejad et al17 conducted a study providing evidence that noninflamed apical residual pulp has the capability to regenerate the normal pulp. Given the discovery of the presence of stem cells involved in the restoration of the root, studies are now underway to assess if implantation of stem cells may in fact accelerate pulp tissue regeneration and healing and perhaps shorten the wait time. Tissue engineering permitted two strategies: (1) the direct implantation of freshly isolated stem cells with or without biodegradable scaffolds, and (2) implantation of preassembled tissue constructs containing in vitro cultured cells in the scaffold.18 Studies showed that the implantation of stem/progenitor cells isolated from a human root in a mouse model resulted in formation of a pulp-like tissue with a layer of dentin-like tissue along the canal wall.19 Unfortunately, retrieval of autologous dental stem cells is difficult, especially in the common case where all other teeth are healthy. It is even more difficult to obtain one of the five subtypes of dental stem cells: (1) dental pulp stem cells (DPSCs), (2) stem cells from human exfoliated deciduous teeth (SHEDs), (3) stem cells from apical papilla (SCAPs), (4) periodontal ligament stem cells (PDLSCs), and (5) tooth germ progenitor cells (TGPCs).20 As such, recent efforts have investigated the use of MSCs derived from other origins, such as human bone marrow–derived MSCs, which have potential for osteogenesis as a more multipotent stem cell than the differentiated dental stem cells. However, bone marrow–derived MSCs are associated with risks, mortality, and high cost of bone marrow harvesting and processing. Induced pluripotent stem cells have also been investigated,21,22 which studies show contribute to mesenchymal progenitors to create early cells in the osteogenic lineage. Unfortunately, the study by Sueyama et al23 using a rat model found that implanting MSCs alone showed incomplete dentin bridges, while coimplantation of MSCs with endothelial cells resulted in pulp healing with complete dentin bridge formation. As such, a viable strategy to allow osteogenic regeneration involves the use of cord blood MSCs. This avoids the ethical concerns of embryonic stem cells and the morbidity of bone marrow acquisition while retaining the multipotency required for the regeneration of complex endodontic tissue. In 2018, Chen et al24 showed that MSCs derived from cord blood were able to achieve successful osteogenic and angiogenic properties, in addition to density, when cocultured with human umbilical vein endothelial cells. These cord blood MSCs had similar capabilities and performance as human bone marrow–derived MSCs and human embryonic stem cells.24 Currently, the literature does not indicate any effect of cord blood stem cells used in combination with residual dental pulp tissue on the ability of a tooth to regenerate pulpal tissues. The authors propose that cord blood stem cells will enhance the ability of residual pulp tissue to regenerate.  5

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STEM CELLS IN REGENERATIVE MEDICINE

a

b

c

FIG 1-1  The different stages of cord blood processing. (a) Blood bag as it was received being processed under aseptic conditions. (b) Conical tube after centrifugation. Note plasma layer (top), buffy coat layer (middle), and red blood cells (bottom). (c) PBMCs suspended in Stem-Cellbanker ready for storage in a cryovial.

MSC Isolation Methods Cord blood–derived and WJ-derived MSCs are an excellent option for therapeutic use because they are easily collected, readily available, and highly proliferative. They can also be used as allografts. Procedures used to isolate MSCs from these sources are described in the following sections.

Cord blood Cord blood is collected from eligible donors at the time of delivery and transported to the processing facility on ice (2°C to 8°C) in a blood bag. Upon arrival, the blood is processed immediately under aseptic conditions (Fig 1-1a). The blood bag is first drained into conical tubes and centrifuged at 1500 × g to separate components. This results in distinct layers in the conical tube, with plasma rising to the top and red blood cells forming a pellet at the bottom of the tube. There is also a distinct buffy coat layer below the plasma that contains the cells of interest. The buffy coat layer is then collected and diluted with a phosphate-­ buffered saline (PBS) solution before undergoing density gradient centrifugation. The buffy coat and PBS mixture is added to Ficoll-Paque (GE Healthcare) density gradient media and then centrifuged at 409g. This results in an isolation of peripheral blood mononucleated cells (PBMCs) in the resulting buffy coat (Fig 1-1b). 6

References

The PBMCs are then collected and diluted once again with PBS before being centrifuged at 409 × g. This wash step is repeated until the resulting supernatant is no longer cloudy or hazy, and the resulting pellet is then resuspended in Stem-Cellbanker (Amsbio). Cell count, viability, and surface marker profile are then tested using flow cytometry. This information is used to aliquot the appropriate cell number into cryovials, and the cells are immediately stored at –80°C (Fig 1-1c).

Wharton’s jelly Umbilical cords are collected from eligible donors at the time of delivery and transported to the processing facility on ice (2°C to 8°C) in Dulbecco’s Modified Eagle Media (DMEM). Cords are processed immediately under aseptic conditions, and MSCs are collected for culture according to the procedure described below. To start a primary explant culture, a roughly 1 × 1–cm segment of cord is obtained using sterile forceps and scalpel. This segment is dissected to remove blood vessels and isolate the WJ. The resulting segments of WJ are then added to 10 mL of a collagenase-DMEM solution and incubated at 37°C for 4 hours. The collagenase-­ DMEM solution is prepared to a strength of 300 collagenase degrading units (CDU) per mL. Digested pieces of tissue are then collected using sterile forceps and transferred to a T-25 flask containing 10 mL of MSC-Brew Xeno-Free Media (Miltenyi Biotec). Cultures are then incubated for 72 hours at 37°C, at which point the media is replaced and the pieces of tissue are removed from the flask. Once the primary culture has been established, regular media changes occur every 2 to 3 days, and cells are allowed to grow to 80% to 90% confluency. At this point, cells are passaged using trypsin-EDTA solution (0.25%) and reseeded into a T-75 flask. The culture is maintained this way until the target number of cells has been reached, at which point passaged cells are suspended in Stem-Cellbanker and frozen at –80°C.

References 1. Rodriguez Y, Baena R, Rizzo S, Graziano A, Lupi SM. Bone regeneration in implant dentistry: Role of mesenchymal stem cells. In: Kalantar Motamedi MH (ed). A Textbook of Advanced Oral and Maxillofacial Surgery, vol 3. Rijeka, Croatia: IntechOpen, 2013. 2. Samsonraj RM, Raghunath M, Nurcombe V, Hui JH, van Wijnen AJ, Cool SM. Concise review: Multifaceted characterization of human mesenchymal stem cells for use in regenerative medicine. Stem Cells Transl Med 2017;6:2173–2185. 3. Kim DW, Staples M, Shinozuka K, Pantcheva P, Kang SD, Borlongan CV. Wharton’s jelly-derived mesenchymal stem cells: Phenotypic characterization and optimizing their therapeutic potential for clinical applications. Int J Mol Sci 2013;14:11692–11712. 4. Roura S, Pujal JM, Gálvez-Montón C, Bayes-Genis A. The role and potential of umbilical cord blood in an era of new therapies: A review. Stem Cell Res Ther 2015;6:123.

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STEM CELLS IN REGENERATIVE MEDICINE

5. Trivanović D, Jauković A, Popović B, et al. Mesenchymal stem cells of different origin: Comparative evaluation of proliferative capacity, telomere length and pluripotency marker expression. Life Sci 2015;141:61–73. 6. Davies JE, Walker JT, Keating A. Concise review: Wharton’s jelly: The rich, but enigmatic, source of mesenchymal stromal cells. Stem Cells Transl Med 2017;6:1620–1630. 7. Bharti D, Shivakumar SB, Park JK, et al. Comparative analysis of human Wharton’s jelly mesenchymal stem cells derived from different parts of the same umbilical cord. Cell Tissue Res 2018; 372:51–65. 8. Gharibi T, Ahmadi M, Seyfizadeh N, Jadidi-Niaragh F, Yousefi M. Immunomodulatory characteristics of mesenchymal stem cells and their role in the treatment of multiple sclerosis. Cell Immunol 2015;293:113–121. 9. Brunstein CG, Petersdorf EW, DeFor TE, et al. Impact of allele-level HLA mismatch on outcomes in recipients of double umbilical cord blood transplantation. Biol Blood Marrow Transplant 2016;22:487–492. 10. Mancuso P, Raman S, Glynn A, Barry F, Murphy JM. Mesenchymal stem cell therapy for osteoarthritis: The critical role of the cell secretome. Front Bioeng Biotechnol 2019;7:9. 11. Kim JY, Xin X, Moioli EK, et al. Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing. Tissue Eng Part A 2010;16:3023–3031. 12. Hughes FJ, Turner W, Belibasakis G, Martuscelli G. Effects of growth factors and cytokines on osteoblast differentiation. Periodontology 2000 2006;41:48–72. 13. Huang CC, Narayanan R, Alapati S, Ravindran S. Exosomes as biomimetic tools for stem cell differentiation: Applications in dental pulp tissue regeneration. Biomaterials 2016;111:103–115. 14. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: New treatment protocol? J Endod 2004;30:196–200. 15. Liao J, Al Shahrani M, Al-Habib M, Tanaka T, Huang GT. Cells isolated from inflamed periapical tissue express mesenchymal stem cell markers and are highly osteogenic. J Endod 2011;37:1217– 1224. 16. Chrepa V, Henry MA, Daniel BJ, Diogenes A. Delivery of apical mesenchymal stem cells into root canals of mature teeth. J Dent Res 2015;94:1653–1659. 17. Torabinejad M, Alexander A, Vahdati SA, Grandhi A, Baylink D, Shabahang S. Effect of residual dental pulp tissue on regeneration of dentin-pulp complex: An in vivo investigation. J Endod 2018;44:1796–1801. 18. Gong T, Heng BC, Lo EC, Zhang C. Current advance and future prospects of tissue engineering approach to dentin/pulp regenerative therapy. Stem Cells Int 2016;2016:9204574. 19. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 2010;16:605–615. 20. Bansal R, Jain A, Mittal S. Current overview on challenges in regenerative endodontics. J Conserv Dent 2015;18:1–6. 21. Duan X, Tu Q, Zhang J, et al. Application of induced pluripotent stem (iPS) cells in periodontal tissue regeneration. J Cell Physiol 2011;226:150–157. 22. Hynes K, Menicanin D, Han J, et al. Mesenchymal stem cells from iPS cells facilitate periodontal regeneration. J Dent Res 2013;92:833–839. 23. Sueyama Y, Kaneko T, Ito T, Kaneko R, Okiji T. Implantation of endothelial cells with mesenchymal stem cells accelerates dental pulp tissue regeneration/healing in pulpotomized rat molars. J Endod 2017;43:943–948. 24. Chen W, Liu X, Chen Q, et al. Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co-culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs, human induced pluripotent stem cell-derived MSCs and human embryonic stem cell-derived MSCs. J Tissue Eng Regen Med 2018;12:191–203.

8

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LEARNING OBJECTIVES • Discuss the benefits of using platelet-rich fibrin as a biomaterial for surgical and nonsurgical endodontic applications • Gain a better understanding about the history of PRF and its advantages over other platelet concentration techniques • Describe the armamentarium involved with making PRF • Gain a better understanding of how to make leukocyte- and platelet-rich fibrin

History, Science, Armamentarium, and How to Make PRF EDWARD S. LEE, DDS

D

entoalveolar soft and hard tissue healing is a self-regulated dynamic process. The initial stage of healing involves locally mediated events followed by a systemic response. Mediators such as growth factors and cytokines play an important role. These mediators are found within blood and, without blood, healing cannot occur. Inflammation is also a necessary part of this process. As a result, the path to healing can be slow and painful. This chapter discusses how it is possible to modulate the inflammatory response and accelerate healing by creating a natural, custom-tailored biomaterial made from the body’s own resources.

The Science of Platelet-Rich Fibrin Whole blood contains many of the molecular and cellular components needed for healing. Whole blood is comprised of plasma and three main types of cells: (1) red blood cells, (2) white blood cells, and (3) platelets. The type of cells of particular interest to us are platelets.1,2 Platelets are produced in the bone marrow 9

2

HISTORY, SCIENCE, ARMAMENTARIUM, AND HOW TO MAKE PRF

daily and are found in a normal range of 150 to 400 × 109 per liter of blood.3 The average life span of circulating platelets is 8 to 9 days. Old platelets are removed by the spleen and liver. Platelets play a significant role in innate immunity, either by recruiting leukocytes through platelet adhesion receptors attaching to leukocytes or by releasing cytokines that affect leukocyte function.1,4,5 These activities help promote tissue healing and regeneration.6 Table 2-1 describes the growth factors and cytokines identified in platelet-rich fibrin (PRF) and their role in the inflammatory and proliferative stages of wound healing.7,8

TABLE 2-1  Growth factors and cytokines in PRF and their roles in wound

healing

Growth factor/cytokine

Action

Interleukin-1 (IL-1)

Stimulates T helper cells, mediates inflammatory response

Interleukin-4 (IL-4)

Proliferates and differentiates activated B cells, moderates inflammation, increases fibroblast synthesis of fibrillary collagen

Interleukin-6 (IL-6 )

Promotes B cell differentiation, activates T cells, secretes antibody stimulation, mediates inflammatory response and remodeling

Tumor necrosis factor α (TNF-α)

Activates monocytes, stimulates fibroblast remodeling, increases phagocytosis and neutrophil cytotoxicity, modulates IL-1 and IL-6 expression

Vascular endothelial growth factor (VEGF)

Increases vascular permeability, initiates angiogenesis, stimulates endothelial cell proliferation

Transforming growth factor β1 (TGF-β1)

Controls collagen and fibronectin synthesis, increases chemotaxis of neutrophils and monocytes, expresses autocrine, generates additional cytokines (TNF-α, IL-1β, PDGF, and chemokines)

Platelet-derived growth factor (PDGF)

Regulates migration, proliferates survival of mesenchymal cell lineages, increases chemotaxis of neutrophils and monocytes, promotes angiogenesis, factors potent fibroblast recruitment

Insulinlike growth factors (IGF) 1 and 2

Mediates cell multiplication in apoptosis, produces chemotactic effects toward human osteoblasts

10

Comparing PRP and PRF

Platelets contain three main populations of granules: (1) alpha granules, (2) dense granules, and (3) lysosomes. When platelets are activated, the granules release their contents and cause a cascade of events regulating hemostasis and thrombosis. The granules are also known to secrete substances that play an integral role in intercellular communication and help mediate inflammatory and immunomodulatory functions during the healing process. Alpha granules are the most abundant of the three granule types and release growth factors that have an impact on angiogenesis, stem cell migration, and cell proliferation. Platelet concentrates are biomaterials with a higher concentration of platelets compared with whole blood. Platelet concentrates are made by centrifugation of whole blood and can be classified into a four-family system based on the presence of leukocytes and fibrin architecture9,10 (Table 2-2). TABLE 2-2  The four-family system of platelet concentrates Type of platelet concentrates

Leukocytes

Fibrin network

Application state

Pure PRP

No

Low-density

Gel/liquid

Leukocyte- and platelet-rich plasma (L-PRP)

Yes

Low-density

Gel/liquid

Pure PRF

No

High-density

Solid

Leukocyte- and platelet-rich fibrin (L-PRF)

Yes

High-density

Solid

Our focus is on leukocyte- and platelet-rich fibrin (L-PRF) because of the additional benefit of having leukocytes in this biomaterial. Leukocytes help fight infection, and leukocytes in PRF can promote the in vivo recruitment of new leukocytes.11,12 Leukocytes also produce large amounts of vascular endothelial growth factor (VEGF), which fosters healing and stimulates angiogenesis.13,14

Comparing PRP and PRF Platelet-rich plasma (PRP) is a first-generation platelet concentration technique developed in the 1970s.15 L-PRF is a second-generation platelet concentration technique developed in 2001 by Choukroun et al.16 Unlike PRP, PRF forms a flexible, high-density organized fibrin network that entraps growth factors. Studies have demonstrated that this network structure allows for angiogenesis, cell migration, 11

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HISTORY, SCIENCE, ARMAMENTARIUM, AND HOW TO MAKE PRF

and proliferation.17–19 PRP involves multiple centrifugation cycles, multiple steps in handling the blood product, and addition of coagulant additives for growth factor release. Within the first hour of PRP preparation, 95% of the growth factors are released.20 In contrast to PRP, PRF involves one centrifugation cycle and no blood additives. PRF studies have shown that this biomaterial will produce a continuous slow release of growth factors during and for more than 7 days, allowing PRF to stimulate the environment for a significant time during wound healing.11,17,19,21,22 PRF is 100% compatible with the patient’s body because it is derived from the patient’s own blood.

PRF and Regenerative Endodontics Regenerative endodontics uses the concept of tissue engineering to restore the root canals to a healthy state, allowing for continued development of the root and surrounding tissue. There are three key elements for tissue engineering: stem cells, scaffold, and growth factors.23 Since PRF forms a high-density organized fibrin network, it has been recognized as a scaffold material for regenerative endodontics.24 PRF has also been enriched with growth factors, and the presence of leukocytes in the fibrin network helps with the self-regulation of the inflammatory and infectious processes in areas of regeneration.1,25

Surgical and nonsurgical PRF applications • Surgical

—— Root resection procedures —— Root-end resection procedures —— Socket preservation procedures

• Nonsurgical

—— Regenerative endodontics —— Soft tissue barrier

12

L-PRF Open-Source Protocol

L-PRF Open-Source Protocol Armamentarium • Tourniquet and bandage • 21-gauge safety butterfly needle set • 10-mL sterile glass vacuum-sealed blood collection tubes (eg, Vacutainer,

Becton Dickinson) with no additives • Table centrifuges:

—— IntraSpin by Intra-Lock International —— PRF DUO Quattro by PRF Process Choukroun —— Medifuge MF200 by Silfradent —— Blood Spin Centrifuge BSC 10 by Boca Dental Regenerative

• PRF press box:

—— Xpression Fabrication Kit by Intra-Lock International —— PRF Box by PRF Process Choukroun —— PRF Box by Boca Dental Regenerative

Technique Venous blood is drawn using a 10-mL sterile glass tube (eg, BD Vacutainer) with no additives. The blood needs to be centrifuged as soon as possible because fibrin polymerization begins immediately after the blood enters the tube. The recommended speed is 2,700 rpm (400g force) for 12 minutes in a table centrifuge.26,27 Depending on the clinical situation, multiple tubes of blood can be drawn and balanced in the centrifuge. The blood will separate into three layers: red blood cells (RBCs) at the bottom, platelet-poor plasma (PPP) on the top, and a fibrin clot of PRF in the middle (Fig 2-1a). Remove the middle PRF clot layer with tweezers. Cut out the bottom RBC layer if it is still attached to the PRF clot (Fig 2-1b). The PRF clot can be pressed into a membrane and used whole or cut into pieces (Figs 2-1c to 2-1e). The PRF membrane is made by squeezing out the fluid using a membrane press box. The membrane press box can also make cylinder plugs (Fig 2-1f) for socket preservation procedures (see chapter 7). The membranes and plugs can be sutured.

13

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HISTORY, SCIENCE, ARMAMENTARIUM, AND HOW TO MAKE PRF

a

b

c

d

e

f

FIG 2-1  (a) Centrifuged blood. RBCs at the bottom, PPP on the top, and a fibrin clot of PRF in the middle. (b) The middle PRF clot layer is removed with tweezers. (c) PRF in the membrane press box before being pressed. (d) PRF after being pressed. (e) PRF cut into pieces. (f ) PRF pressed in cylinder and made into plugs.

14

References

References 1. Jenne CN, Urrutia R, Kubes P. Platelets: Bridging hemostasis, inflammation, and immunity. Int J Lab Hematol 2013;35:254–261. 2. Parrish W, Roides B. Physiology of blood components in wound healing: An appreciation of cellular co-operativity in platelet rich plasma action. J Exerc Sports Orthop 2017;4(2):1–14. 3. Yun SH, Sim EH, Goh RY, Park JI, Han JY. Platelet activation: The mechanisms and potential biomarkers. Biomed Res Int 2016;2016:9060143. 4. Thomas MR, Storey RF. The role of platelets in inflammation. Thromb Haemost 2015;114:449– 458. 5. Herter JM, Rossaint J, Zarbock A. Platelets in inflammation and immunity. J Thromb Haemost 2014;12:1764–1775. 6. Anitua E, Andia I, Ardanza B, Nurden P, Nurden AT. Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb Haemost. 2004;91:4–15. 7. Hotwani K, Sharma K. Platelet rich fibrin—A novel acumen into regenerative endodontic therapy. Restor Dent Endod 2014;39:1–6. 8. Kaigler D, Cirelli JA, Giannobile WV. Growth factor delivery for oral and periodontal tissue engineering. Expert Opin Drug Deliv 2006;3:647–662. 9. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: From pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol 2009;27:158–167. 10. Dohan Ehrenfest DM, Sammartino G, Shibli JA, Wang HL, Zou DR, Bernard JP. Guidelines for the publication of articles related to platelet concentrates (Platelet-Rich Plasma - PRP, or Platelet-Rich Fibrin - PRF): The international classification of the POSEIDO. POSEIDO 2013;1:17–27. 11. Zumstein MA, Berger S, Schober M, et al. Leukocyte- and platelet-rich fibrin (L-PRF) for longterm delivery of growth factor in rotator cuff repair: Review, preliminary results and future directions. Curr Pharm Biotechnol 2012;13:1196–1206. 12. Bielecki TM, Gazdzik TS, Arendt J, Szczepanski T, Król W, Wielkoszynski T. Antibacterial effect of autologous platelet gel enriched with growth factors and other active substances: An in vitro study. J Bone Joint Surg Br 2007;89:417–420. 13. Nielsen HJ, Werther K, Mynster T, et al. Bacteria-induced release of white cell- and platelet-­ derived vascular endothelial growth factor in vitro. Vox Sang 2001;80:170–178. 14. Werther K, Christensen IJ, Nielsen HJ. Determination of vascular endothelial growth factor (VEGF) in circulating blood: Significance of VEGF in various leukocytes and platelets. Scand J Clin Lab Invest 2002;62:343–350. 15. Soomekh DJ. Current concepts for the use of platelet-rich plasma in the foot and ankle. Clin Podiatr Med Surg 2011;28:155–170. 16. Choukroun J, Adda F, Schoeffler C, Vervelle A. The opportunity in perio-implantology: The PRF [in French]. Implantodontie 2001;42:55–62. 17. Dohan Ehrenfest DM, de Peppo GM, Doglioli P, Sammartino G. Slow release of growth factors and thrombospondin-1 in Choukroun’s platelet-rich fibrin (PRF): A gold standard to achieve for all surgical platelet concentrates technologies. Growth Factors 2009;27:63–69. 18. Khiste SV, Tari RN. Platelet-rich fibrin as a biofuel for tissue regeneration. ISRN Biomaterials 2013;2013:627367. 19. Schär MO, Diaz-Romero J, Kohl S, Zumstein MA, Nesic D. Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro. Clin Orthop Relat Res 2015;473: 1635–1643. 20. Marx RE. Platelet-rich plasma (PRP): What is PRP and what is not PRP? Implant Dent 2001;10: 225–228.

15

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HISTORY, SCIENCE, ARMAMENTARIUM, AND HOW TO MAKE PRF

21. Dohan Ehrenfest DM, Bielecki T, Jimbo R, et al. Do the fibrin architecture and leukocyte content influence the growth factor release of platelet concentrates? An evidence-based answer comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte- and platelet-rich fibrin (L-PRF). Curr Pharm Biotechnol 2012;13:1145–1152. 22. Zumstein MA, Bielecki T, Dohan Ehrenfest DM. The future of platelet concentrates in sports medicine: Platelet-rich plasma, platelet-rich fibrin, and the impact of scaffolds and cells on the long-term delivery of growth factors. Oper Techn Sports Med 2011;19:190–197. 23. Sharma S, Sikri V, Sharma NK, Sharma VM. Regeneration of tooth pulp and dentin: Trends and advances. Ann Neurosci 2010;17:31–43. 24. American Association of Endodontists. AAE Clinical Considerations for a Regenerative Procedure Revised 4/1/2018. https://www.aae.org/specialty/wp-content/uploads/sites/2/2018/06/ ConsiderationsForRegEndo_AsOfApril2018.pdf. Accessed 2 October 2019. 25. Simonpieri A, Del Corso M, Sammartino G, Dohan Ehrenfest DM. The relevance of Choukroun’s platelet-rich fibrin and metronidazole during complex maxillary rehabilitations using bone allograft. Part I: A new grafting protocol. Implant Dent 2009;18:102–111. 26. Dohan Ehrenfest DM, Pinto NR, Pereda A, et al. The impact of the centrifuge characteristics and centrifugation protocols on the cells, growth factors, and fibrin architecture of a leukocyte- and platelet-rich fibrin (L-PRF) clot and membrane. Platelets 2018;29:171–184. 27. Eren G, Gürkan A, Atmaca H, Dönmez A, Atilla G. Effect of centrifugation time on growth factor and MMP release of an experimental platelet-rich fibrin-type product. Platelets 2016;27:427–432.

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LEARNING OBJECTIVES • Understand the benefits of using autologous blood for regenerative endodontics • Understand the use of autologous blood concentrates as a scaffold material • Understand the benefit of autologous blood concentrates for improved healing and continued developing of root and surrounding tissues

Nonsurgical Application: PRF for Regeneration Endodontics MOHAMMAD SABETI, DDS, MA EDWARD S. LEE, DDS

A

s discussed in chapter 2, regenerative endodontics uses the concept of tissue engineering to restore the root canals to a healthy state, allowing for continued development of the root and surrounding tissue. There are three key elements for tissue engineering: stem cells, scaffold, and growth factors.1–4 Platelet-rich fibrin (PRF) has been recognized as a scaffold material for regenerative endodontics5 because it has all the properties required for regenerative endodontic therapy. Because PRF is derived from the patient’s own blood, it is 100% compatible with the patient’s body. PRF has been recognized for use as a scaffold material in revascularization of immature permanent teeth with necrotic pulps (Box 3-1). Stem cells and growth factors found in PRF promote cellular proliferation and differentiation and allow for continued development of the root and surrounding tissue.6 PRF has also been considered a stabilizing and nourishment sheath.7 It releases growth factors and stem cells that enhance cellular migration for neoangiogenesis and vascularization and promote healing.7

17

3

NONSURGICAL APPLICATION: PRF FOR REGENERATION ENDODONTICS

BOX 3-1  PRF as a scaffold for regenerative endodontics Inclusion criteria • Restorable permanent necrotic tooth • Maxillary or mandibular single-rooted immature permanent tooth with open apex • At least 5 mm of root development • Patient is cooperative • Patient is available and commits to recall appointments Exclusion criteria • Nonrestorable teeth • Patient is unable to give consent or unwilling to participate • Patient is not available for follow-up visits • Immunocompromised patients

Using PRF as a Scaffold First appointment During the first appointment, it is important to review the patient’s medical and dental records and baseline radiograph to verify the eligibility for the PRF procedure. Anesthesia with 2% lidocaine with epinephrine 1:100,000 will be needed for this procedure. Once verified, complete the following steps: 1. Place rubber dam to isolate the tooth. 2. Disinfect the field with 5.2% sodium hypochlorite (NaOCl) followed by 5% sodium thiosulfate. 3. Once the tooth has been accessed and work length is determined, touch the canal wall lightly with a hand file to disrupt the bacterial biofilm. 4. Irrigate the root canal system first with 1.5% NaOCl (20 mL/canal for 5 minutes) and then with saline (20 mL/canal for 5 minutes). 5. Irrigate the canal with 5% sodium thiosulfate and dry with sterile paper points. 6. Slowly inject calcium hydroxide into the canal space using a 3-mL syringe fitted with a capillary tip inserted to working length. 7. Place micro sponges and a temporary restoration consisting of a GC Fuji TRIAGE (GC America Inc) in the access.

Second appointment (1 to 4 weeks later) Perform a clinical exam to ensure that there are no signs or symptoms (ie, moderate to severe pain to palpation, percussion, sinus tract, or swelling). If the patient 18

Using PRF as a Scaffold

presents with any symptoms or signs, repeat the treatment provided at the first visit. If not, complete the following steps: 1. Determine tooth shade by using the VITA classical A1–D4 shade guide (VITA North America). 2. Provide anesthesia with 3% mepivacaine, and place rubber dam to isolate the tooth. 3. Disinfect the field first with 5.2% NaOCl followed by 5% sodium thiosulfate. 4. Once the root canal system is accessed, remove calcium hydroxide by irrigating with saline (20 mL/canal for 5 minutes) followed by a final flush of 17% ethylenediaminetetraacetic acid (EDTA) (20 mL/canal for 5 minutes). 5. Perform a final flush with saline (20 mL/canal for 1 minute), and dry the canal with sterile paper points. 6. Draw blood using a 10-mL glass Vacutainer (Becton Dickinson) without any additives and perform the following steps: a. Centrifuge at 2,700 rpm for 12 minutes (Fig 3-1a). The blood will separate into platelet-poor plasma, a PRF fibrin clot, and red blood cells (RBCs) (Figs 3-1b and 3-1c). b. Separate the PRF fibrin clot by pulling it with tweezers, and cut as necessary (Fig 3-1d).

a

Acellular plasma supernatant (PPP)

Fibrin clot exudate (PRF)

Red blood cells

PPP

PRF clot

RBCs

b

FIG 3-1  (a) The blood is centrifuged at 2,700 rpm for 12 minutes. (b and c) A fibrin clot will form in the middle of the tube. The top contains acellular plasma (ie, platelet­-­poor plasma or PPP), and the bottom contains RBCs. (d) Pull out the fibrin clot with tweezers.

c

d

19

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NONSURGICAL APPLICATION: PRF FOR REGENERATION ENDODONTICS

FIG 3-2  (a and b) The PRF clot is compressed in a PRF box. (c and d) After this, the PRF is cut into pieces as needed.

a

c

b

d

c. Use a PRF box to compress the PRF clot and cut into pieces as needed (Fig 3-2). 7. Irrigate the root canal with the liquid that is obtained by compression of the PRF clot. 8. Use a file to induce bleeding to fill the apical portion of the root canal (Fig 3-3). The blood should fill one-fourth to one-half of the apical portion of the root canal. 9. Insert the PRF membrane plug into the root canal to the apex of the tooth using a premeasured gutta-percha or endodontic hand plugger (Fig 3-4). 20

Using PRF as a Scaffold

Stem cells of the apical papilla (SCAP) invading PRF

MTA seal

Composite coronal seal

a

b

FIG 3-3  (a) Bleeding is induced by using a pre-bent k-file size 25 at 2 mm past the foramen. (b) The blood should fill the apical one-fourth to one-half of the root canal. FIG 3-4  A premeasured gutta-percha or endodontic hand plugger is used to insert the PRF.

a

b

FIG 3-5  (a) When there is excess moisture in root canal over PRF membrane plug, a CollaPlug (b) can be trimmed to size and placed over the PRF membrane plug to absorb the moisture.

10. Place mineral trioxide aggregate (MTA) or a bioceramic (eg, BC putty, bio­dentine) over the PRF membrane plug. If there is excessive moisture in the root canal, a small piece of CollaPlug (Zimmer Biomet) can be placed over the PRF membrane plug before MTA or bioceramic placement (Fig 3-5). 11. Flow a 1- to 2-mm layer of glass ionomer gently over bioceramics layer and light cure for 40 seconds. 12. Place a composite restoration over the glass ionomer. 21

3

NONSURGICAL APPLICATION: PRF FOR REGENERATION ENDODONTICS

Using an Apical Plug An apical plug can be used as a barrier outside the root. Then, the whole root is filled with MTA or a bioceramic material.

First appointment Review the patient’s medical and dental data and baseline radiograph to verify their eligibility (Box 3-2). Once assessed, complete the following: 1. 2. 3. 4.

Obtain anesthesia, and place rubber dam to isolate the tooth. Determine working length once the tooth is accessible. After access, clean and shape the canal. Irrigate the root canal system first with 1.5% NaOCl (20 mL/canal for 5 minutes), and then irrigate with saline (20 mL/canal for 5 minutes). 5. Slowly inject calcium hydroxide into the canal space using a 3-mL syringe fitted with a capillary tip inserted to working length. 6. Place micro sponges and a temporary restoration consisting of GC Fuji TRIAGE in the access. BOX 3-2  Apical plug as a barrier Inclusion criteria • Restorable permanent necrotic tooth • Maxillary or mandibular single-rooted immature permanent tooth with closed apex • Patient is cooperative Exclusion criteria • Nonrestorable teeth • Patient is unable to give consent or not willing to participate • Patient is not available for follow-up visits • Immunocompromised patients

Second appointment (after 4 weeks) Perform a clinical exam to ensure that there are no signs or symptoms (ie, moderate to severe pain on palpation, percussion, sinus tract, or swelling). If the patient presents with any symptoms or signs, repeat the treatment provided at the first visit. If not, complete the following steps: 22

Using an Apical Plug

PRF

MTA seal

Composite coronal seal

FIG 3-6  MTA condensed into the root canal to the cementoenamel junction.

1. 2. 3. 4.

5. 6.

7. 8. 9. 10. 11.

FIG 3-7  Composite restoration over the MTA.

Determine tooth shade by using the VITA classical A1–D4 shade guide. Obtain anesthesia with 2% lidocaine with 1:100,000 epinephrine. Place rubber dam to isolate the tooth. Once the root canal system is accessed, remove the calcium hydroxide by irrigating with saline (20 mL/canal for 5 minutes) followed by a final flush of 17% EDTA (20 mL/canal for 5 minutes). Perform a final flush with saline (20 mL/canal for 1 minute), and dry the canal with sterile paper points. Draw blood using a 10-mL Vacutainer without any additives and perform the following steps (see Figs 3-1 and 3-2): a. Centrifuge at 2,700 rpm for 12 minutes. The blood will separate into platelet-poor plasma, a PRF fibrin clot, and RBCs. b. Separate the fibrin clot by pulling it with tweezers and cut as necessary. c. Use a PRF box to compress the PRF clot. Cut the compressed PRF membrane to size and insert into the root canal (see Figs 3-2c and 3-2d). Insert the PRF membrane plug into the root canal to the apex of the tooth with a premeasured gutta-percha or endodontic hand plugger (see Fig 3-4). Place multiple pieces until a firm apical barrier is formed. Fill the canal with MTA to the cementoenamel junction (Fig 3-6). Place a composite restoration over the MTA (Fig 3-7). 23

3

NONSURGICAL APPLICATION: PRF FOR REGENERATION ENDODONTICS

References 1. Sharma S, Sikri V, Sharma NK, Sharma VM. Regeneration of tooth pulp and dentin: Trends and advances. Ann Neurosci 2010;17:31–43. 2. Howard D, Buttery LD, Shakesheff KM, Roberts SJ. Tissue engineering: Strategies, stem cells and scaffolds. J Anat 2008;213:66–72. 3. Mikos AG, Herring SW, Ochareon P, et al. Engineering complex tissues. Tissue Eng 2006;12: 3307–3339. 4. Forghani M, Parisay I, Maghsoudlou A. Apexogenesis and revascularization treatment procedures for two traumatized immature permanent maxillary incisors: A case report. Restor Dent Endod 2013;38:178–181. 5. American Association of Endodontists. AAE Clinical Considerations for a Regenerative Procedure Revised 4/1/2018. https://www.aae.org/specialty/wp-content/uploads/sites/2/2018/06/ ConsiderationsForRegEndo_AsOfApril2018.pdf. Accessed 2 October 2019. 6. Keswani D, Pandey RK. Revascularization of an immature tooth with a necrotic pulp using platelet-rich fibrin: A case report. Int Endod J 2013;46:1096–1104. 7. Simonpieri A, Del Corso M, Sammartino G, Dohan Ehrenfest DM. The relevance of Choukroun’s platelet-rich fibrin and metronidazole during complex maxillary rehabilitations using bone allograft. Part II: Implant surgery, prosthodontics, and survival. Implant Dent 2009;18:220–229.

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4

LEARNING OBJECTIVES • Understand the benefits of patient-derived blood concentrates and its healing and cell regeneration components • Understand the usage of autologous blood concentrates for various applications that are related to hard tissue grafts • Understand the benefit of autologous blood concentrates for improved soft tissue grafting • Understand that all forms of autologous blood concentrates behave the same clinically and are used in similar methods to achieve the desired clinical benefits

Soft Tissue Applications KAYVON JAVID, DDS GREGORI M. KURTZMAN, DDS CARLOS FERNANDO MOURÃO, DDS, MSc, PHD

T

he chapter reviews how autologous blood concentrates are fabricated and how they are used in various techniques for soft tissue applications. Discussions include how to use these patient-derived blood products and fabricate membranes with the benefits of using fibrin, stem cells, and other blood products to enhance soft tissue healing and accelerate the desired clinical outcome.

Blood-Derived Biologics as Membranes Blood-derived biologics (also referred to as autologous blood concentrates) have been steadily growing in dental treatment over the past 20 years, and the growth factors present in the blood-derived products have proven very beneficial. Frequently, when dental procedures require flap elevation, a membrane is needed to prevent soft tissue ingrowth into the underlying osseous graft, which allows the hard tissue graft to establish before the gingiva can invade that space. Soft tissue grows more rapidly than a hard tissue graft can establish, so forgoing the use of a membrane may result in loss in volume of the hard tissue graft. Hence, guided tissue regeneration allows hard tissue grafts to establish and angiogenesis 25

4

SOFT TISSUE APPLICATIONS

to begin, which limits ingrowth of the overlaying gingival tissue and maximizes the volume of the graft and desired clinical results.1,2 Resorbable and nonresorbable membranes are commercially available. Nonresorbable membranes require a second surgery at a later date for removal, and when primary closure cannot be achieved, membrane exposure increases over the short term. This may necessitate early removal of the membrane or may cause some soft tissue inflammation at the flap’s margins due to accumulating plaque, and the associated bacteria may limit the volume of the underlying graft. Resorbable membranes eliminate the issue of membrane removal with a second surgery and are better tolerated by the overlaying soft tissues. Typically, when exposed at surgical placement due to an inability to achieve primary closure, the gingival tissue attaches to the membrane and grows over the exposure, sealing the area over a few days or a week. Yet, these nonresorbable and resorbable membranes share several things in common: Their use increases treatment cost due to the cost of the materials, and cost increases when larger areas need coverage. An alternative is to fabricate membranes from patient-drawn blood at the time of surgery. These membranes are a sufficient size to cover any surgical need that may be encountered in the dental practice. The cost of blood-derived membranes is minimal per patient use, and the only products needed at each treatment are Vacutainer tubes (Becton Dickinson), making them practical for routine usage.3,4 Several different blood-derived products have been discussed in the literature, including platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concentrated growth factor (CGF).5 These all involve preoperatively centrifuging blood drawn from the patient and involve different speeds and times to separate the components of the blood. Each product has identical uses, and opinions vary as to which product provides superior effects over the other, but the literature indicates similar results with each formulation.1 Although the centrifugal conditions are different for PRF and CGF, they are prepared by essentially identical mechanisms; therefore, it is conceivable that membranes fabricated from either product have similar mechanical and chemical properties.5 Both PRF and CGF preparations contain high amounts of growth factors capable of stimulating periosteal cell proliferation. This suggests that PRF and CGF function not only as a scaffold, but also as a reservoir to deliver growth factors at the site of application.6 This chapter and the following will focus on CGF and its uses in endodontic and related surgeries. CGF fabrication is initiated by phlebotomy using two different Vacutainer tubes, one with a red top and the other with a white top (Fig 4-1). The red-top Vacutainer tubes are used to create a solid-phase product used for membranes, resulting in a fibrin clot on completion of centrifuging. The tube is coated with silicon to aid in fibrin clot formation (see Fig 4-1a). Upon centrifugation, the blood in the Vacutainer tube separates into four phases or layers. The layer at the top of the 26

Blood-Derived Biologics as Membranes

FIG 4-1  Upon completion of centrifuging blood, layers develop in the tube. (a) Red-top Vacutainer tube. The tube is coated with silicon to aid in the formation of a fibrin clot. (b) White-top Vacutainer tube with no coating or additional substances.

Serum Serum

Platelet-poor plasma

Platelet-poor plasma

Clot

a

Platelet-rich plasma

Platelet-rich plasma

White layer

White layer

Red blood cells

Red blood cells

Fragments

b

Fragments

tube is serum, and an interim layer below that consists of the fibrin buffy coat (clot). A liquid layer lies around the clot containing growth factors, and at the bottom of the tube, the lower layer consists of the red blood cells (RBCs). CGF is a fibrin-rich organic matrix that contains growth factors, platelets, leukocytes, and CD34+ stem cells that aid in the process of regeneration. Additionally, immunologic cells that are effective in regulating inflammation and minimizing the risk of infection are present.7 The fibrin clot is the PRF or CGF that consists of five representative growth factors in platelets: platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β1 (TGF-β1), insulinlike growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).8,9 A quantity of growth factors are located on the interface between the fibrin clot and the RBC layer. Therefore, a certain amount of RBCs should be retained when the fibrin clot is removed from the tube to ensure a concentration of the growth factors. The resulting fibrin clot is used as the membrane when compressed and as a plug when left uncompressed. The liquid portion of the buffy coat, referred to as CGF glue, is a fibrin tissue adhesive with hemostatic and tissue sealing properties. When injected over the soft tissue or graft material, it promotes wound healing and accelerates osteogenesis. 27

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CGF glue improves wound stability and new connective tissue attachment to the root surface by promoting epithelial, endothelial, and epidermal regeneration, while decreasing scarring. Additionally, it has antimicrobial properties due to high concentrations of leukocytes, which act as an anti-antigenic agent on chronic nonhealing wounds, while providing a scaffold to support cellular migration. The liquid phase of CGF (LPCGF) is the rich portion of the plasma near the buffy coat zone. It is extremely different from PRP because it can be produced without anticoagulants and clot activators. The white-top Vacutainer tube contains no coating in the tube or additional substances. On centrifugation, a liquid phase results with no clot formation in the tube (see Fig 4-1b). The liquid exhibits glue-like properties when placed into osseous graft material and is used for infiltration over the soft tissue wound (flap closure). It needs to be used within 20 to 30 minutes of centrifuging. Delay in use beyond that time results in the liquid becoming jelly-like, so it cannot be injected or flowed over the site. The liquid has also been used in esthetic and cosmetic applications such as facial rejuvenation, facial volumization, elimination of fine lines and wrinkles, and other uses.

Fabrication of CGF Membranes Membrane fabrication begins with drawing the patient’s blood at the time of treatment into Vacutainer tubes with red and white tops and centrifuging those tubes at the appropriate speed to cause separation in the tubes. Fibrin clots will be formed during separation in the tubes with red tops and are removed from the tube with cotton pliers (Figs 4-2a and 4-2b). The RBC layer at the bottom of the tube is separated from the fibrin clot, leaving approximately 10 mm attached to the fibrin clot (Figs 4-2c and 4-2d). The resulting fibrin clot may be compressed or left uncompressed. When flattened, it may be used as a membrane over a graft to cover a soft tissue defect, used to bulk out soft tissue (ie, Chao Pinhole or gum drop techniques), or used to repair perforations to the maxillary sinus membrane when sinus augmentation is being performed. Uncompressed fibrin clots may be used without bone for extraction socket grafting, maxillary sinus augmentation, or to fill crestal defects at implant placement. The fibrin clot is placed on a sterile tray ready to be used as is (uncompressed) (Fig 4-2e) or may be compressed using the weighted tray cover to press liquid out of the fibrin clot (Fig 4-3a), allowing for it to be used as a membrane (Fig 4-3b). Therefore, two types of fibrin clots, either compressed or uncompressed, may be fabricated depending on what they will be used for (Fig 4-3c). The yellow buffy

28

Fabrication of CGF Membranes

a

b

c

d

FIG 4-2   (a) Following centrifugation of the patient’s blood, grab the fibrin clot formed in the red-top Vacutainer tube with cotton pliers. (b) Remove the fibrin clot from the red-top Vacutainer tube. (c) The coagulated RBC portion may be separated from the fibrin clot, and only the segment attached to the fibrin clot is left attached, as this contains a high concentration of growth factors. (d) Approximately 10 mm of the RBCs are left attached to the fibrin clot, as this portion contains the highest concentration of growth factors. (e) Fibrin clots are placed on a sterile tray and may be used as is (uncompressed) or compressed.

e

layer is withdrawn from the white-top Vacutainer tube with a syringe (Fig 4-3d). This liquid has concentrated fibroblasts and growth factors and may be used as a “tissue glue” over the fibrin clot membrane or to seal the flap following suture placement (Fig 4-3e). Additionally, it may be mixed with the liquid from the red-top Vacutainer tube when creating “sticky bone.”

29

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a

b

c

d

e

FIG 4-3  (a) When compressed membranes are desired, place the weighted tray cover over the fibrin clots in the sterile tray to press the liquid out of the clots. (b) Compressed fibrin clots ready to be used as membranes. (c) CGF membranes shown uncompressed (left) and compressed (right). (d) Draw the buffy yellow liquid layer out of the white-top Vacutainer tube with a syringe. (e) Either use the liquid from the white-top Vacutainer tube as a “tissue glue” or mix with liquid from the red-top Vacutainer tube to aid in creation of sticky bone.

30

Clinical Applications

Clinical Applications Blood-derived membranes have multiple clinical uses. They act as (1) a barrier to oral bacteria when primary closure may not be possible, (2) a source of stem cells, and (3) a scaffold when placed alone in a defect (eg, an apical surgical site or extraction socket and a host of other applications).

Sinus augmentation without osseous graft material Insufficient available height may present in the posterior maxilla related to enlargement of the maxillary sinus, crestal resorption, periodontal disease associated with an affected tooth, endodontic apical pathology, or a combination of these factors that may hamper implant placement. Lack of available height will require an increase in crestal height to permit sufficient osseous volume to house implants. Apical endodontic surgery in the posterior maxilla follows similar procedures, as access into the sinus area is required to access the apical area of the tooth. The following case illustrates the steps involved that can be applied to either apical endodontic surgery in the posterior maxilla or implant-related sinus augmentation using CGF membranes alone without osseous graft materials added to the surgical area (Fig 4-4). Similar to endodontic apical surgery, after the sinus membrane has been elevated, the next actions are achieving access to the apical area and performing apical surgery. CGF membranes are created following blood draw from the patient and centrifugation. The uncompressed membranes are then placed into the elevated sinus area between the sinus membrane and gently compacted to fill the site to the level of the buccal lateral wall of the sinus. The added benefit of using the CGF membranes is that they can help resolve the common complication of sinus membrane perforation. If a tear occurs through the sinus membrane, the CGF membrane can seal the tear and prevent any related complications.

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membranes. 2. Apply local anesthetic. 3. For a lateral sinus augmentation: a. Make an incision and raise a full-thickness flap to expose the lateral aspect of the maxillary bone. b. Create a lateral osseous window with burs or piezoelectric instruments and either rotate the window into the superior position or remove the window and reserve. c. Elevate the sinus membrane. 31

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a

b

c

d

e

FIG 4-4  (a) Place noncompressed CGF mem­branes around the recently placed implant after sinus membrane elevation without osseous grafting to augment the sinus. (b) Place noncompressed CGF over the lateral sinus window. (c) Place a compressed CGF membrane over the lateral bony window under the flap. (d) Suture the flap to achieve primary closure. (e) Radiograph of the area 6 months after sinus augmentation with CGF membranes alone and simultaneous implant placement. Bone is evident around the apical areas where augmentation was performed.

d. Place CGF membranes into the area created by membrane elevation, making sure to fill the medial aspect and elevated sinus area. e. If the osseous window was removed, place it over the CGF-augmented sinus. f. Reposition the flap and secure with sutures. 4. For a crestal sinus augmentation: a. Create a crestal osteotomy using appropriate crestal sinus elevation burs. b. Place CGF membranes into the crestal sinus elevation osteotomy and gently push apically into the elevated sinus area. c. Place the implant into the osteotomy to the appropriate depth, further elevating the previously placed CGF membranes. 32

Clinical Applications

Soft tissue thickening for periodontal or prosthetic considerations Soft tissue may be thin in areas near teeth or implants, and these areas may benefit from increasing the thickness of that soft tissue. CGF membranes are well suited for this application; due to the stem cells and other blood factors that they contain, they can provide better results than commercially available membranes. Additionally, use of CGF membranes eliminates the need to procure connective tissue from elsewhere intraorally, avoiding potential issues with donor sites. The following case illustrates how tissue thickening may be performed with CGF membranes (Fig 4-5).

a

b

FIG 4-5  (a) Flap closure without osseous grafting at an implant placement site where tissue thickening over the implant is desired. (b) A CGF membrane has been placed over the implant and crest under the flap. (c) The flap has been sutured to achieve primary closure over the implant site.

c

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membranes. 2. Following local anesthetic application, make an incision and elevate the soft tissue to create a void between the soft tissue and underlying bone. 3. Place CGF membranes into the created void or tunnel to thicken the tissue. 4. Place sutures to close the initial incision. 33

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SOFT TISSUE APPLICATIONS

a

b

c

d

e

f

FIG 4-6  (a and b) Generalized recession in the absence of periodontal inflammation with associated thin gingival tissue. (c) The interproximal contacts have been bonded to act as anchorage points for sutures to be placed, and holes have been created just above the posterior mucogingival junction (bilaterally) and midline at the mucogingival junction with subsequent internal elevation of the gingival tissue across the arch. (d) CGF membranes were introduced through the gingival holes that had been previously corrected to bulk the tissue out and move the gingival margins coronally to cover the recession that had been present. Sutures were placed looping over the composite, anchoring the soft tissue in the new desired position to allow healing. (e and f) Soft tissue healing after 30 days, demonstrating elimination of the recession and thickening of the gingival tissue.

34

Clinical Applications

Gingival recession and soft tissue thickening with CGF membranes Gingival recession is a common finding and occurs most frequently in patients with thin gingival tissue. This procedure has been advocated under various names for the technique using commercial collagen membranes and membranes derived from different blood products (ie, PRP, PRF, and CGF). Once placed, the fibrin in blood product membranes act like tissue glue to help tack the tissue in the new position and provide more rapid healing than collagen membranes. The following case illustrates use of CGF membranes for treatment of recession and a need to provide tissue thickening, but membranes derived from other blood products may also be used (Fig 4-6).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membranes. 2. Following local anesthetic application, make an incision in the attached gingiva, sparing the papilla. 3. Create a tunnel extending past the teeth with recession and mobilize the soft tissue without tension. 4. Contour the cervical area of tooth at the recession with a finishing bur or diamond bur. 5. Treat the areas of recession with citric acid gel for 60 seconds, and then rinse thoroughly. 6. Place CGF membranes into the tunnel to reposition the gingival margin to cover the areas of recession. 7. Place sutures to hold the gingival margin in the new desired position and loop over composite at the contact spots. 8. Place sutures to close the initial vertical incision. 9. See patient 2 weeks postoperatively to remove the sutures and composite at the contact spots.

Immediate implant placement at extraction to seal the site without an osseous graft Frequently, when immediate implant placement is to be performed at the time of extraction, especially in molar sites, the implant being placed does not fill the entire extraction socket and leaves gaps. When those gaps are not filled at the time of surgery, as healing progresses, gingival tissue may grow down into the space and fill it, and food and oral bacteria may hamper healing and potentially affect the 35

4

SOFT TISSUE APPLICATIONS

a

c

b

d

e

FIG 4-7  (a) Implant placed at time of extraction at a maxillary molar site performed without flap elevation, demonstrating exposed portions of the socket that were occupied by the tooth’s roots. (b) These CGF membranes have had a hole placed in the center that will allow the healing abutment to pass through the membrane. (c) Healing abutments have been inserted through the holes in the CGF membranes, creating a poncho. (d) A CGF membrane has been used with the poncho technique to seal the socket with a healing abutment inserted through the membrane. (e) Sutures are placed to hold down the edges of the membrane that were tucked under the gingiva on the buccal and lingual aspects.

long-term success of the implant. Occluding the space and blocking entry of oral bacteria or food allows bone development to proceed from the periphery with the clot that forms at surgery. Clinicians recommend the “poncho” technique, which uses a membrane fabricated from blood products to cover the area using the implant healing abutment to retain it in place. The following case illustrates the use of a CGF membrane without additional osseous graft material in a poncho 36

References

technique to treat immediate implant placement in extraction sockets when large gaps are present due to a mismatch of the implant diameter and socket cross section (Fig 4-7).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membranes. 2. Following local anesthetic application, extract the tooth and curette the socket to remove any residual tissue or pathology. 3. Create the implant osteotomy in the extraction socket, and place the implant. 4. Place CGF membranes into any voids between the implant and extraction socket walls. 5. Place a piece of membrane over the implant with a hole in the center, and place a healing abutment to secure the membrane in place. 6. Tuck the membrane under the buccal and lingual soft tissue margins. Place sutures if desired to ensure the membrane is secure.

Conclusion Blood-derived membranes have many applications in dental treatment either alone or in combination with sticky bone created from those blood products. The clinical examples are an illustration of how these may be used, and additional uses may be considered by the practitioner in endodontic, oral, and periodontal surgery.

References 1. Thoma DS, Buranawat B, Hämmerle CHF, Held U, Jung RE. Efficacy of soft tissue augmentation around dental implants and in partially edentulous areas: A systematic review. J Clin Periodontol 2014;41(suppl 15):S77–S91. 2. Dawson DR 3rd, El-Ghannam A, Van Sickels JE, Naung NY. Tissue engineering: What is new? Dent Clin North Am 2019;63:433–445. 3. Mamajiwala AS, Sethi KS, Raut CP, Karde PA, Mangle NM. Impact of different platelet-rich fibrin (PRF) procurement methods on the platelet count, antimicrobial efficacy, and fibrin network pattern in different age groups: An in vitro study. [epub ahead of print 25 July 2019] Clin Oral Investig doi:10.1007/s00784-019-03022-8. 4. Miron RJ, Dham A, Dham U, Zhang Y, Pikos MA, Sculean A. The effect of age, gender, and time between blood draw and start of centrifugation on the size outcomes of platelet-rich fibrin (PRF) membranes. Clin Oral Investig 2019;23:2179–2185. 5. Isobe K, Watanebe T, Kawabata H, et al. Mechanical and degradation properties of advanced platelet-rich fibrin (A-PRF), concentrated growth factors (CGF), and platelet-poor plasma-­ derived fibrin (PPTF). Int J Implant Dent 2017;3:17.

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6. Kim TH, Kim SH, Sándor GK, Kim YD. Comparison of platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concentrated growth factor (CGF) in rabbit-skull defect healing. Arch Oral Biol 2014;59:550–558. 7. Masuki H, Okudera T, Watanebe T, et al. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF). Int J Implant Dent 2016;2:19. 8. Nityasri AS, Pradeep KY, Kalaivani V, Rajapandian K. Role of CGF (concentrated growth factor) in periodontal regeneration. J Dent Health Oral Disord Ther 2018;9:350–352. 9. Qiao J, An N, Ouyang X. Quantification of growth factors in different platelet concentrates. Platelets 2017;28:774–778.

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5

LEARNING OBJECTIVES • Understand the usage of autologous blood concentrates for various applications that are related to hard tissue grafts • Understand the benefit of autologous blood concentrates for improved hard tissue grafting • Understand how to fabricate “sticky bone” using hard tissue graft materials combined with autologous blood concentrates to improve handling of the graft material and prevent separation during the healing phase

Hard Tissue Applications KAYVON JAVID, DDS GREGORI M. KURTZMAN, DDS CARLOS FERNANDO MOURÃO, DDS, MSc, PHD

T

his chapter reviews the fabrication of autologous blood concentrates and their application in various techniques for hard tissue grafting. Benefits of autologous blood concentrates are also discussed, such as using the stem cells and other blood products to enhance hard tissue healing and accelerate the desired clinical outcome.

Blood-Derived Biologics as Osseous Graft Enhancers Osseous grafting is used to fill a void in the bone, created either by infection or as a result of surgery, to access an area such as the apical region of a tooth undergoing endodontic surgery. This graft provides a scaffold for the surrounding bone to fill the defect with what will become native bone following healing. An important component of the healing is angiogenesis, as the bone being maintained needs vascularization. In the absence of a graft, it may be difficult to achieve the desired volume of bone to fill the defect, or the defect will be slower to fill as the area fills with a blood clot that must be broken down peripherally to be replaced by new host bone over time. 39

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HARD TISSUE APPLICATIONS

FIG 5-1  “Sticky bone” providing a moldable osseous graft material fabricated with cells and growth factors from the patient’s blood.

As outlined in chapter 4, patient-derived blood products have applications in the regeneration of oral soft tissues. These benefits also extend to hard tissue applications, which improves both the management of the osseous graft materials and results of osseous grafting by incorporating the patient’s own stem cells, growth factors, and other beneficial components found in the blood. Some studies have indicated that concentrated growth factor (CGF) provides better results than platelet-rich fibrin (PRF) in early bone formation.1 However, other studies indicate that use of any of the blood-derived products improved bone formation at the sixth week, and all were comparable clinically.2 These preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application. Results are improved when compared with situations when blood-derived products are not used with the osseous graft material.3 The PRF/CGF in the centrifuged blood contains growth factors that include platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β 1 (TGF-β1), insulinlike growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).4 These growth factors help induce cell migration from the periphery that uses the osseous graft material as a scaffold to accelerate conversion to host bone over time.5 Soluble factors and fibrin in CGF were found to promote initial cell stretching, proliferation, and osteoblastic differentiation of rat bone marrow cells in vitro with resulting bone regeneration.6 Particulate graft materials—whether placed dry and then wet at the site or wetted with saline prior to placement—present with handling challenges. These materials tend not to stay confined to the defect during placement and may migrate after placement, limiting the volume of defect fill that was desired. Incorporating CGF or other blood product fibrin-containing liquids yields a moldable product referred to as “sticky bone,” as the fibrin content congeals the mass (Fig 5-1). The sticky bone can be adapted to the defect, filling the void without dispersing of the graft particles either during placement or during the early healing phase, which is an important feature when filling a surgical defect in the mandibular premolar area to prevent migration of particles into the mental foramen that may result in 40

Fabrication of CGF Sticky Bone

a

b

c

FIG 5-2 (a) An appropriate osseous graft material is dispensed into a sterile dish. (b) Blood from the patient has been drawn into white-top Vacutainer tubes and centrifuged to separate the blood into layers. (c) The CGF liquid layer is withdrawn from the white-top tube with an 18-gauge needle on a syringe approximately 10 mm above the RBC portion of the tube contents.

a paresthesia. Due to the moldable nature of the sticky bone, the practitioner also has the ability to build up the area contour-wise should that be desired.

Fabrication of CGF Sticky Bone To fabricate sticky bone, follow these clinical steps: 1. Following phlebotomy, draw blood into both white- and red-top Vacutainer tubes (Becton Dickinson) and centrifuge to separate the blood into layers. The white tube separates the blood into (1) plasma, (2) buffy coat, and (3) red blood cells (RBCs). The red tube separates the blood into (1) serum, (2) CGF clot including the buffy coat, and (3) RBCs. 2. Dispense an appropriate osseous graft material into a sterile dish (preferably glass) to begin creation of the sticky bone (Fig 5-2a). 3. Withdraw a centrifuged white-top Vacutainer tube of the patient’s blood (Fig 5-2b) with the liquid CGF from the tube with an 18-gauge needle syringe (Fig 5-2c). 4. Extend the tip of the needle into the tube to about 10 mm from the RBC layer where the highest concentration of growth factors resides in the centrifuged blood. Withdraw all the liquid above that point into the syringe (volume of about 2 mL). 5. Dispense this liquid into the dish containing the osseous graft material previously dispensed (Fig 5-2d). 41

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HARD TISSUE APPLICATIONS

e

d

f

g

FIG 5-2 (cont) (d) The liquid PRF withdrawn from the white-top Vacutainer tube is expressed into the dish containing the osseous graft material (e) The CGF liquid is mixed with the osseous graft material to make a paste. (f) The patient’s blood that was drawn into white-top Vacutainer tubes has been centrifuged with the red-top Vacutainer tubes. (g) The liquid PRF is withdrawn from the tube with a syringe. The fibrin clot is left in the tube or is withdrawn and placed on a tray.

6. Lightly mix the material to incorporate the liquid with the osseous graft particles, wetting the material (Fig 5-2e). 7. With another syringe, withdraw the top layer from the red-top Vacutainer tube (Fig 5-2f). It will have formed a liquid serum, not a fibrin clot, and its volume is about 1 mL (Fig 5-2g). 8. Add the liquid in the syringe to the previously mixed osseous graft material in the dish (Fig 5-2h), and allow it to rest. After 1 to 2 minutes, the material has congealed into a mass referred to as “sticky bone” (Fig 5-2i). The sticky bone is ready for placement intraorally (Fig 5-2j). 42

Fabrication of CGF Sticky Bone

h FIG 5-2  (cont) (h) The liquid from the red-top Vacutainer tube that was drawn into the syringe is added to the dish containing the osseous graft material mixed with the liquid from the white-top Vacutainer tube that was previously mixed. (i) The mixed osseous graft is combined with the liquid from the red- and white-top Vacutainer tubes and allowed to congeal into what will become sticky bone after 1 to 2 minutes. ( j) After 2 minutes, the graft material has become a moldable gelatinous mass referred to as “sticky bone” and is ready for placement intraorally.

i

j

The CGF fibrin clot from the white tube is PRF consisting of five representative growth factors in platelets. Those PDGF-BB, TGF-β1, IGF-1, VEGF, and bFGF.5 Additionally, a quantity of growth factors are located at the interface between the CGF fibrin clot and the RBC layer. Therefore, a certain amount of RBCs should be retained to the CGF fibrin clot when it is removed from the tube. This will ensure a concentration of the growth factors from the patient’s centrifuged blood. The resulting CGF fibrin clot may be used as a membrane when compressed or as a plug when left uncompressed. The liquid portion dispensed from the white tube, referred to as platelet-poor plasma (PPP) and also known as CGF glue, is a fibrin tissue adhesive with hemostatic and tissue-sealing properties. When injected over a soft tissue incision or graft material, it promotes wound healing and accelerates osteogenesis, thus improving wound stability and new connective tissue attachment to the root surface by promoting epithelial, endothelial, and epidermal regeneration while decreasing scarring. Additionally, it has antimicrobial properties due to high concentrations of leukocytes, acting as an anti-antigenic agent on chronic nonhealing wounds while providing a scaffold supporting cytokine attachment and cellular migration. 43

5

HARD TISSUE APPLICATIONS

a

b

c

d

FIG 5-3  (a) Palatally positioned erupted supernumerary tooth requiring extraction. (b) Extraction socket following removal of the tooth. (c) CGF sticky bone is carried to the site and placed into the extraction socket. (d) The socket has been filled with CGF sticky bone.

Clinical Applications Osseous socket grafting Socket preservation at the time of extraction aids in preservation of the remaining osseous structure and prevents crestal resorption observed due to resorption that normally accompanies extraction sockets when site grafting is not performed. Various products are available commercially that range between allografts (human source), xenografts (other species sourced), synthetics, and bone derived from the patient being treated (autogenous). Combining these with blood-derived products such as PRP, PRF, or CGF allows better handling for the socket grating material and the contained stem cells, which accelerates conversion to host bone. This combination may be applied when the entire tooth is extracted or extended to treatment of the empty socket when hemisection is performed to stabilize the bone at the furca, preventing bone loss on the remaining root. The following case illustrates use of CGF sticky bone and a CGF membrane when primary closure is not achievable (Fig 5-3). 44

Clinical Applications

e

f

g

h

FIG 5-3  (cont) (e) A CGF membrane is placed into the socket over the previously placed CGF sticky bone. (f) A condenser is used to compress the CGF membrane over underlying CGF sticky bone placed into the extraction socket. (g) Sterile gauze is used to compress the CGF membrane over the site and remove any residual blood from the site. (h) CGF membrane has been compressed into the site over the CGF sticky bone socket graft. (i) One week postsurgery, the patient returned for suture removal. Though he presented with some gingival inflammation, he did not experience postoperative sensitivity.

i

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membranes and sticky bone. 2. Combine the CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 3. Extract the tooth following local anesthetic application. 4. Curette the extraction socket to remove residual tissue and any pathology. 5. Fill the extraction socket with sticky bone. 6. Place a CGF membrane over the sticky bone, and tuck the edges under the buccal and lingual soft tissue margins. 7. Place a figure-of-eight suture over the site to contain the membrane and underlying sticky bone. 8. Remove the sutures 2 weeks postsurgery. 45

5

HARD TISSUE APPLICATIONS

a

c

b

d

FIG 5-4  (a) Extensive dehiscence of the buccal maxillary plate at time of extraction. (b) The socket has been filled with sticky bone and the buccal plate around the dehiscence overfilled to restore the lost arch contour. (c) A CGF membrane has been placed over the sticky bone and over the extraction socket. Primary closure will not be achieved at the top of the crest because the extraction was performed in conjunction with the buccal plate repair. (d) As primary closure of the site was not possible at the crest, a membrane was necessary to limit the underlying sticky bone in the socket from being lost intraorally and to prevent oral contamination of the graft material.

Dehiscence correction at extraction Dehiscence or fenestration may present either following extraction or on teeth that are to be maintained. The techniques used for this procedure also apply when endodontic apical surgery is being performed to fill the osseous defect created either by the apical lesion or surgical removal of bone to access the apical portion of the root (see chapter 7). The following case illustrates management of these type osseous defects (Fig 5-4).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membranes. 2. Combine CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 46

Clinical Applications

3. Following local anesthetic application, extract the tooth and explore the socket to identify any dehiscence or fenestration that may be present. 4. When a dehiscence or fenestration is identified, elevate a full-thickness flap to expose the osseous defect and at least one tooth distance mesial and distal to the site. 5. Curette the extraction socket to remove residual tissue and any pathology. 6. Fill the extraction socket with sticky bone. 7. Place a CGF membrane over the sticky bone, filling the defect on the buccal aspect and covering the crestal aspect of the extraction socket. This may require more than one membrane. 8. Tuck the edges under the buccal and lingual soft tissue margins. 9. Reposition the flap and place sutures to close the vertical incisions. 10. Place a figure-of-eight suture over the site to contain the membrane and underlying sticky bone. 11. Remove the sutures 2 weeks postsurgery.

Lateral ridge augmentation with a membrane Loss of the width of the ridge may occur either related to periodontal disease associated with failing teeth that had been extracted, resorption of the ridge vertically over time, or a combination of these factors. When implants are planned for the area, augmentation may be required to accommodate the implants being placed if insufficient width is present. Should removable prosthetics be planned, and a narrow ridge is creating or will create patient comfort issues under that prosthesis, augmentation may be necessary. The following case illustrates management of a narrow ridge with treatment using CGF sticky bone to increase ridge width and a CGF membrane to allow flap closure that cannot be achieved by primary intention (Fig 5-5).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membranes and CGF sticky bone. 2. Combine CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 3. Following local anesthetic application, make a crestal incision and elevate a full-thickness flap. It may be required to create a mesial vertical releasing incision to achieve adequate soft tissue elevation. 4. Create bleeding points in the lateral aspect of the ridge with a bur. 5. Place CGF sticky bone over the lateral aspect of the ridge where the site will be widened. 47

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a

b

c

d

e

f

g

48

FIG 5-5  (a) A crestal incision with a distal vertical releasing incision was made with a scalpel. (b) Following full-thickness flap elevation, the deficient width of the posterior mandible is evident. (c) Decortication is performed over the lateral aspect of the ridge to be grafted using a small round carbide to allow an avenue for angiogenesis from the medullary bone of the mandible. (d) The lateral wall of the mandible shows decortication points created to improve integration of the graft being placed (arrows). (e) CGF sticky bone has been adapted to the lateral aspect of the deficient posterior mandible being grafted. (f) The CGF membrane is placed under the flap and overlaid on the CGF sticky bone graft. (g) The flap has been reapproximated over the underlying CGF sticky bone and CGF membrane and secured with sutures.

Clinical Applications

a FIG 5-6   (a) The lateral wall of the ridge is prepared by creating bleeding points through the cortical plate with a bur after flap elevation. (b) Sticky bone is placed over the lateral aspect of the maxilla to augment the width of the ridge. (c) The flap has been scored on the periosteum at the mucogingival line internally to allow stretching of the tissue to achieve primary closure and secured with sutures.

b

c

6. Place CGF membranes over the sticky bone. 7. Reposition the flap to get crestal closure when possible without tension on the flap. Some exposure of the CGF membranes is permissible. 8. Place sutures to secure the soft tissue. 9. Remove the sutures 2 weeks postsurgery.

Lateral ridge augmentation without a membrane When primary closure of the flap can be achieved and there is no need for a membrane, ridge width augmentation may be necessary. The following case illustrates how augmentation of the lateral ridge to increase width with primary intention can be achieved due to reapproximation of the flap margins over the placed osseous graft material (Fig 5-6).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the CGF sticky bone. 2. Combine CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 3. Following local anesthetic application, make a crestal incision and elevate a full-thickness flap. It may be required to create a mesial vertical releasing incision to achieve adequate soft tissue elevation. 49

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4. Create bleeding points in the lateral aspect of the ridge with a bur. 5. Place CGF sticky bone over the lateral aspect of the ridge where the site will be widened. 6. Reposition the flap to get crestal closure without tension on the flap. 7. Place sutures to secure the soft tissue. 8. Remove the sutures 2 weeks postsurgery.

Lateral ridge defect augmentation Defects in the lateral wall of the crest may result following past extraction of a tooth that previously presented with endodontic issues. These defects may affect the fit of both removable and fixed prosthetics, leading to a food trap in the vestibule that can be an ongoing irritant for the patient. The following case illustrates augmentation of a defect in the lateral wall of the ridge to provide a better ridge contour either to prevent a vestibular food trap or prepare the ridge for subsequent implant placement (Fig 5-7).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the CGF sticky bone. 2. Combine CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 3. Following local anesthetic application, make a crestal incision and elevate a full-thickness flap. It may be required to create a mesial vertical releasing incision to achieve adequate soft tissue elevation. 4. Create bleeding points in the lateral aspect of the ridge with a bur. 5. Place CGF sticky bone over the lateral aspect of the ridge where the site will be widened. 6. Reposition the flap to get crestal closure without tension on the flap. 7. Place sutures to secure the soft tissue. 8. Remove the sutures 2 weeks postsurgery.

50

Clinical Applications

a

b

c

d

FIG 5-7  (a) A defect is present at the pontic site on the buccal wall related to resorption following long-term healing of an extraction. (b) After the flap is raised, the deep buccal osseous defect can clearly be seen on the lateral aspect of the ridge. (c) The CGF membrane is placed under the buccal flap, and CGF sticky bone is placed between the membrane and the buccal lateral bone to fill the osseous defect. (d) The CGF membrane is overlaid on the CGF sticky bone placed into the lateral defect. (e) The flap is closed over the graft for primary intention healing and secured with sutures.

e

Immediate implant placement at extraction to seal site with osseous graft As outlined in the previous chapter, frequent placement of an implant in an immediate extraction site may result in large gaps between the implant and socket walls. These large gaps may require some grafting to fill the void and ensure that bone is connecting with the implant circumferentially after healing. When the gap is very wide, placement of osseous graft material prevents potential soft tissue ingrowth into the gap, and following healing, bone obliterates the gaps that were present at implant placement. The following case illustrates use of CGF sticky bone to fill 51

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a

b

c

d

f

e

g

FIG 5-8  (a) Failing premolar with vertical root fracture requiring extraction. (b) The premolar has been extracted without elevating a flap. (c) A flap has been elevated and an implant has been placed into the extraction site, noting a gap between the socket wall and implant on the buccal and lingual aspects. (d) CGF sticky bone has been packed into the gaps on the buccal and lingual aspects between the implant and socket wall. (e) The CGF membrane has been perforated by the implant healing abutment to act as a poncho at the site covering the CGF sticky bone placed. (f) Implant healing abutment with CGF membrane is inserted into the implant to act as a poncho over the CGF sticky bone. (g) Sutures are placed to close the previously elevated flap. The CGF membrane poncho protects the underlying CGF sticky bone during the initial healing phase, allowing soft tissue closure where it would not have been easily achievable without the poncho placement.

52

Clinical Applications

gaps in the socket that were occupied by roots not filled by the implant. A CGF membrane is used with the poncho technique to seal the area and promote better healing when primary intention is not achievable (Fig 5-8).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the membrane. 2. Combine CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 3. Following local anesthetic application, make a crestal incision and elevate a full-thickness flap. It is required to place a mesial vertical releasing incision to achieve adequate soft tissue elevation. 4. Create bleeding points in the lateral aspect of the ridge with a bur. 5. Place CGF sticky bone over the lateral aspect of the ridge where the site will be widened. 6. Reposition the flap to get crestal closure without tension on the flap. 7. Place sutures to secure the soft tissue. 8. Remove the sutures 2 weeks postsurgery.

Crestal sinus augmentation As discussed previously, various causes may result in insufficient height for implant placement, requiring augmentation to increase the height to house the implant. When that height increase is 5 mm or less, a crestal approach is an alternative to the traditional lateral window approach. The following case illustrates a crestal sinus augmentation with use of CGF sticky bone to tent the elevated sinus membrane and encase the apical portion of the implant that sits superior to the native crestal bone (Fig 5-9).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the CGF sticky bone. 2. Combine CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 3. Following local anesthetic application, create a crestal osteotomy using appropriate crestal sinus elevation burs. 4. Place CGF sticky bone into the crestal sinus elevation osteotomy and gently push apically into the elevated sinus area. 5. Place the implant into the osteotomy to the appropriate depth. This further elevates the previously placed CGF sticky bone. 53

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b

a

c

d

e

FIG 5-9  (a) An osteotomy has been created at the site to accommodate implant placement, and the sinus membrane has been elevated with a crestal approach. (b) CGF sticky bone is compacted into the osteotomy to elevate the sinus using the crestal approach. (c) CGF sticky bone has been placed into the osteotomy to complete the crestal sinus augmentation. (d) Implant at placement with crestal sinus augmentation performed to increase available height to house the implant. (e) The flap was reapproximated and secured with sutures.

54

Clinical Applications

a

b

c

d

FIG 5-10 (a) Following flap elevation, lesions are apparent where the infection has not healed from the prior endodontic treatment on the central incisors. (b) CGF sticky bone is pressed into the apical surgery sites to fill the defects created by endodontic surgery and the causative lesions. (c) Additional CGF sticky bone is adapted to the entire facial plate of the ridge to improve the osseous thickness over the anterior teeth. (d) Once primary flap closure is achieved, the area is secured with sutures.

Grafting apical surgical defects Endodontic surgery typically requires enlargement of apical defects created by a lesion related to endodontic failure of prior endodontic treatment or when a large lesion is present, necessitating surgical removal of the apical pathology. Traditionally, these defects following surgical intervention were left to fill on their own over time. Placement of commercially available graft material has been advocated to provide better osseous fill in those defects, and use of CGF sticky bone or only the CGF membrane accelerates this osseous fill. This also decreases potential for the defect to not fill when left without filling the surgical void prior to flap closure (Fig 5-10).

Clinical steps 1. Draw blood from the patient at the start of the appointment and place into the centrifuge to fabricate the CGF sticky bone. 2. Combine CGF liquid with the chosen type of osseous graft particles to create CGF sticky bone, and then set aside. 55

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3. Following local anesthetic application, elevate a full-thickness flap to expose the surgical site to access the tooth requiring endodontic surgery. 4. Expose the apical area of the tooth by removing bone. 5. Resect the apical aspect of the tooth with a bur, and place a retrograde filling into the remaining tooth to seal the canal system. 6. Curette the apical lesion to remove any pathology. 7. Place CGF sticky bone into the osseous defect. 8. Reposition the flap and secure with sutures. 9. Remove the sutures 2 weeks postsurgery.

Conclusion Incorporation of patient-derived blood products when performing osseous grafting adds stems cells and other factors that can accelerate graft healing and improve the clinical result desired. Additionally, the fibrin component congeals the osseous graft into a moldable mass that improves handling during placement and prevents particle separation during the initial healing phase. When combined with blood product–derived membranes, healing avoids potential issues reported with commercial membranes while stimulating overall healing.

References 1. Park HC, Kim SG, Oh JS, et al. Early bone formation at a femur defect using CGF and PRF grafts in adult dogs: A comparative study. Implant Dent 2016;25:387–393. 2. Kim TH, Kim SH, Sándor GK, Kim YD. Comparison of platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concentrated growth factor (CGF) in rabbit-skull defect healing. Arch Oral Biol 2014;59:550–558. 3. Masuki H, Okudera T, Watanebe T, et al. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF). Int J Implant Dent 2016;2:19. 4. Qiao J, An N, Ouyang X. Quantification of growth factors in different platelet concentrates. Platelets 2017;28:774–778. 5. Schär MO, Diaz-Romero J, Kohl S, Zumstein MA, Nesic D. Platelet-rich concentrates differentially re­lease growth factors and induce cell migration in vitro. Clin Orthop Relat Res 2015;473:1635–1643. 6. Takeda Y, Katsutoshi K, Matsuzaka K, Inoue T. The effect of concentrated growth factor on rat bone marrow cells in vitro and on calvarial bone healing in vivo. Int J Oral Maxillofac Implants 2015;30:1187–1196.

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LEARNING OBJECTIVES • Discuss the benefits of platelet-rich fibrin as an alternative to autogenous bone grafting material • Understand the benefit of autogenous blood concentrates for various applications in procedures to treat accidents. • Understand the benefit of autogenous blood concentrates for improving furcation involvement as a result of iatrogenic procedures • Understand the benefit of autogenous blood concentrates for various applications in root-end resection procedures • Understand the benefit of autogenous blood concentrates to improve the surgical crypt after root-end resection procedures

PRF in Endodontic Surgery MOHAMMAD SABETI, DDS, MA GREGORI M. KURTZMAN, DDS EDWARD S. LEE, DDS ERIC WONG, DDS

E

ndodontic surgery related to either apical lesion removal, the need to amputate the apical aspect of the root, or a combination results in an osseous defect or void. There has been a long-standing debate regarding filling these defects with graft material or allowing them to fill on their own peripherally. Periodontal issues may also have an endodontic component related to perforations during endodontic treatment, leading to furcation bone loss in posterior teeth.1 Depending on their location in relation to the crestal bone, these furcal lesions may expand and communicate with the oral environment and can be identified by periodontal probing. Endo-perio lesions are a clinical challenge because they require removal of the apical lesion, repair of the perforation, and regeneration of the lost osseous tissue.2,3 Studies suggest that the use of guided tissue regeneration (GTR) of furcation lesions produced by endodontic perforations has resulted in significant new bone and connective tissue attachment.4 Systematic reviews have reported that GTR was more effective than curettage without graft placement into the void in reducing furcation depth, with improvement in horizontal and vertical attachment levels and reduction of pocket depths.5,6 GTR fills the osseous defect and has resulted 57

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in improved clinical outcomes when compared with those defects not filled by graft material.7 This is also true with regard to endodontic GTR, specifically with molar furcation repairs.8

Guided Tissue Regeneration Various graft materials have been advocated for GTR of furcation and apical surgeries, including autogenous bone, allografts, xenografts, and synthetic materials.9–11 Grafting materials essentially act as space fillers and scaffolds for peripheral migration of osteoblast cells and other host components that are able to create bone at the needed site. Depending on the graft material placed, it may take varying amounts of time for the host tissue to replace the graft. Some materials, specifically xenografts (eg, bovine bone), may present with a significant percentage of residual particles after 6 months of healing.12 Reports indicate that some particles may remain beyond 12 months and may not ever fully resorb.13 Allograft products have also been widely used. These materials fully resorb over time and may be cortical or cancellous in nature. This will determine how long they last before the host tissue removes them. Cancellous bone is less dense than cortical bone and has a shorter resorption time. This means that in larger voids, it may not remain long enough to be replaced by host bone in sufficient volume. Yet, cortical particles may slow down the replacement process due to their longer resorption time related to their higher density. The alternative with this has been the use of a corticocancellous mixture to provide a graft material that remains long enough to allow replacement with cortical bone without loss of volume of the graft placed following healing. Yet, like xenografts, this type of graft has no osteogenic cells within it that may stimulate bone formation in the grafted site. Autologous blood concentrates, platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concentrated growth factor (CGF) have been used to greater degrees with respect to endodontic treatment replacing commercial products. As discussed in other chapters, autogenous bone provides stem cells and other components from the patient to speed healing and regeneration without the potential of possible allogeneic reactions that may accompany the use of commercial graft products. When they are used without autologous blood concentrates, commercial grafting products do not provide components that stimulate host hard tissue regeneration via stem cells and other blood-derived factors. As these components are derived from that individual patient, potential allogeneic reactions may occur when commercial products are used. Additionally, inflammatory reactions are also minimized when more rapid healing potential is possible. Regarding their use in endodontic surgery, it has been reported that patients treated with these 58

Root Resection and Root-End Resection

products presented with no pain or signs of reinfection with radiographic fill of the apically grafted area over time. PRP was reported to be better than PRF and the induced bleeding technique with respect to periapical wound healing when used in regenerative endodontic procedures.14 An added benefit of these autologous blood concentrates is the elimination of a commercial membrane. The blood products are centrifuged to create a natural membrane that contains stem cells and fibroblasts and limits overlaying soft tissue invasion into the grafted material. Endo-perio lesions share the same anatomical origin, with the exact etiology difficult to determine at times. PRF may be used to enhance osseous augmentation when treating periapical defects as a potential treatment addition to stimulate faster healing, and it may also enhance osseous fill compared with commercial graft materials.15

Root Resection and Root-End Resection Furcation involvement in the molars presents a challenge with a multitude of treatment options. Specific periodontal, endodontic, and restorative factors must be taken into consideration when deciding on the appropriate treatment.16 Root amputation can be a valuable procedure when the tooth in question has a very high strategic value or when there are specific problems that cannot be solved by other therapeutic approaches and the tooth is restoratively sound.17,18 Teeth in proximity to anatomical landmarks, such as the maxillary sinus, can be safely treated by a root resection procedure. Root-end resection may also be performed to save a tooth. Osseous grafting is used to fill voids in the bone created either by infection or as a result of surgical access, such as the apical region of a tooth undergoing endodontic surgery. Grafting material provides a scaffold for the surrounding bone to fill the defect with what will become native bone after healing. An important component of wound healing is angiogenesis, which is the ability to develop new blood vessels. Improved angiogenesis allows growing bone to maintain its need for vascularization. The advantages of PRF as a grafting material are the presence of growth factors that improve angiogenesis and the ease of managing the material. This preparation speeds up both management of osseous graft materials and healing of the osseous graft by incorporating the patient’s own stem cells, growth factors, and other beneficial components found in the blood. Root amputation and hemisection have been successfully performed as a means to preserve dentition for over 100 years.19,20 Variable success and survival rates have been demonstrated in root resection studies.21,22 In retrospective evaluation on the outcome of 90 root-resected molars, the overall survival rate was 90.6% 59

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after 10 years. There was a median survival rate of 20 years, and mandibular molars had a lower risk of loss than those in the maxilla, showing a survival rate of almost 80% after 20 years.23 Prolonging the life of a tooth, rather than early extraction and replacement with an implant, may best serve the patient. Regarding the resection-related factors, one study reported that molars resected due to periodontal problems had a higher survival rate than those resected because of nonperiodontal problems (eg, tooth fracture, dental caries, endodontic problems). The authors reported that roots that had more than 50% bone support had a better survival rate. This evidence supports the grafting of these endodontic surgical sites to fill the voids present while increasing bone support.24 Implant treatment, on the other hand, may require multiple procedures with significant associated costs, complications, and risks. A recent systematic review based on a European consensus conference revealed that the prevalence of peri-implant mucositis and peri-implantitis ranges from 19% to 65%. Implant survival rates do not far exceed those of compromised but adequately treated and maintained teeth, supporting the notion that the decision to extract a tooth and place an implant should be made cautiously.25 The restorability of the tooth needs to be taken into consideration when making these treatment planning decisions. When the tooth is structurally sound, endodontic treatment that may include a surgical component should be considered. When the prognosis is poor, extraction should then be considered. Cumulative success rates when comparing teeth undergoing endodontic surgery and replacement with implants in molars reported 96.8% for root-resected molars and 97.0% for molar implants.26  In light of this information, there is a strong interest in preserving the natural dentition.27 A recent systematic review and meta-analysis of the literature concerning outcome rates for crown resection and root resection procedures reported an overall cumulative survival rate of 85.6%.28 Clinicians agree that bone formation can be achieved by using any of the blood-derived products discussed in this book, and there is no notable clinically significant difference in bone formation after the sixth week.29 PRF and sticky bone not only act as a scaffolding material, but also as a reservoir to deliver certain growth factors. Compared with situations when blood-derived products are not used with the osseous graft material, results are improved when PRF assists in preparation sites.30 PRF contains growth factors which include platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β1 (TGF-β1), insulinlike growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).31 These growth factors help induce cell migration from the periphery that uses the osseous graft material as a scaffold to accelerate conversion to host bone over time.32

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FIG 6-1  Allograft bone mixed with PRF liquid. The PRF liquid is a fibrin adhesive binder and prevents the bone graft material from migrating.

Due to the nature of growth factors and their ability to improve angiogenesis in PRF, there is strong evidence that using PRF in an osseous grafting material can be effective in areas of bone dehiscence and fenestrations caused by surgical access in root-end resection procedures. These techniques also apply when dehiscence or fenestration may present on teeth that are to be maintained following rootend resection. Many common types of bone grafting material are particulate in nature, and because the material in particulate grafts does not tend to clump and cling together, the material often tends to migrate out of the surgical crypt. Prior to placement in osseous defects, particulate graft material is generally used moistened with sterile saline to create a material that can be difficult to manipulate and handle. PRF and other fibrin-containing blood products have properties that allow them to be moldable and sticky in nature and tend to congeal in a mass. By incorporating PRF with graft material such as bone allograft, it can create a more malleable, congealed sticky mass that tends to stay within the surgical crypt and have a lesser tendency to migrate or wash out. After root-end resection, particulate graft materials wetted with saline prior to placement in the osseous defect present with management challenges. When placed with saline, these materials tend not to stay confined to the defect and may migrate, thereby limiting the desired volume of defect fill. Incorporating PRF or other blood product fibrin-containing liquids yields a moldable product referred to as “sticky bone” as the fibrin content congeals the mass (Fig 6-1). The sticky bone can be adapted to the defect, filling the void without dispersing the graft particles either during placement or during the early healing phase.

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Root-end resection technique clinical steps 1. Assess the necessity for apical surgery using various imaging sources and clinical evaluation (Fig 6-2a). 2. Review the patient’s medical history, medications, and potential contraindications to treatment. 3. Discuss treatment options with the patient, including risks, benefits, and alternatives, and establish a written informed consent for treatment. 4. Design the surgical flap after taking into consideration intraoral anatomy, dentition, restorations, and esthetics. 5. Topically sterilize the surgical flap is with antiseptic (17% chlorhexidine). 6. Choose a local anesthetic with vasoconstrictors to obtain proper hemostasis (ie, 2% lidocaine hydrochloride with 1:50,000 epinephrine). 7. Incise the surgical flap with a microsurgical or 15C surgical blade. 8. Elevate the full-thickness flap with periosteal elevators. 9. If the root end is not exposed by bony dehiscence, initiate the osteotomy by using sterile burs on a surgical handpiece, performed with copious continuous irrigation with sterile saline until the root end is located and the extent of the osseous lesion is observed. 10. Stain the root end with 2% methylene blue to check the anatomy and expose any fractures (Fig 6-2b). 11. Resect approximately 3 mm of the root end at 90 degrees to the long axis of the root or, if necessary, to the superior extension of the osseous lesion (see Fig 6-2b). 12. Establish or maintain hemostasis with the use of hemostatic agents (ie, epinephrine pellets). 13. Prepare the root end approximately 3 mm in depth to remove gutta-percha with a piezoelectric handpiece and retrograde surgical tips. 14. Evaluate the prepared root end with microsurgical mirrors, and pack residual gutta-percha with retrograde pluggers. 15. Irrigate the root end with sterile saline, and dry with paper points. 16. Complete the retrograde filling by condensing mixed mineral trioxide aggregate (MTA) or bioceramic cement and burnishing smooth and flush to the root surface. 17. Take a radiograph to verify the depth and angulation of the root end preparation and the density of the retrograde filling. 18. Cleanse the surgical crypt with gentle irrigation with sterile saline to remove the hemostatic agents and debris. 19. Reinduce hemorrhaging with gentle manipulation of the bony crypt with instruments such as a surgical curette. 62

Root Resection and Root-End Resection

a

b

c

FIG 6-2  (a) An appropriate osseous graft material is dispensed into a sterile dish. (b) View of rootend resection after the root end has been prepared using an ultrasonic device. (c) PRF was placed to fill the surgical crypt. FIG 6-3  Immediate postoperative sutures of the surgical site.

20. Place grafting material such as PRF and sticky bone to fill the surgical crypt (Fig 6-2c). 21. Reapproximate and suture the surgical flap into place with interrupted or mattress sutures. Sutures with resorbable and monofilamented features are recommended for this procedure (Fig 6-3). 22. Prescribe and discuss appropriate postoperative instructions, medications, and rinses. An ice pack can be applied to reduce swelling. 23. Schedule a postoperative appointment in 4 to 7 days to remove sutures and assess primary healing of the surgical flap. 24. Make follow-up appointments at 1 month, 6 months, and 1 year after surgery. (The follow-up appointments at 6 months and 1 year should include radiographs to assess healing of the osseous lesion.) 63

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Case study 1: Root-end resection and PRF site grafting This case illustrates the management of the surgical crypt after root-end resection, apical preparation, and the retrograde filling (see Fig 6-2). A horizontal papillary incision was made, and a full-thickness mucoperiosteal flap was reflected without any incident. An osteotomy to expose the root tip was completed with copious irrigation with sterile saline, granulation tissue was removed, and the root end was stained with methylene blue. Staining did not reveal any signs of isthmus or cracks. Using an ultrasonic device, retropreparations at a depth of 3 mm in the buccal and lingual canals were performed. ProRoot MTA (Dentsply) root repair material was used for retrograde fills. The surgical crypt was irrigated with sterile saline, PRF was placed to fill the surgical crypt, and the mucoperiosteal flap was reapproximated and sutured.

Case study 2: Root-end resection and PRF site grafting A male patient presented with the complaint of pain associated with a mandibular left first molar that had undergone prior endodontic treatment. A periapical radiograph was taken to evaluate the quality of the endodontic treatment and any possible associated pathology (Fig 6-4a). Evaluation of the radiograph noted a large apical lesion associated with the mesial root with some external resorption present related to a perforation from the previous endodontic treatment. A clinical examination noted deep periodontal probing in the furcation with a Naber probe (Fig 6-4b). Clinical findings were discussed with the patient, and it was recommended to re-treat the endodontics, followed by apical surgery to resect the affected root apex. Local anesthetic was administered, and the tooth was isolated with rubber dam. Access was achieved through the porcelain-fused-to-metal crown, and the obturation material removed from the mesiobuccal and mesiolingual canals. Obturation was then completed using MTA as the sole obturation material to provide an apical seal for the canals following the planned apical surgery. The access through the crown was sealed with a resin restoration, rubber dam removed, and the patient dismissed. Allowing the MTA material within the canals to set, the patient returned a week later for apical surgery, where local anesthetic was administered. Blood was drawn from the patient and centrifuged to create a PRF membrane. A sulcular incision was made alone with a vertical releasing incision mesial to the planned surgical site, and a full-thickness flap was elevated. Bone was noted to be missing in the furcation, and examination with a probe noted a strip perforation on the distal aspect of the mesial root that was not evident in the radiograph (Fig 6-4c). An 64

Root Resection and Root-End Resection

b

a

c FIG 6-4  (a) Preoperative periapical radiograph demonstrating a lesion apical to the mesial root with associated root resorption. (b) A deep pocket with a Class II furcation was identified with a Naber probe. (c) Following flap elevation, a strip perforation was identified on the distal aspect of the mesial root that led to the furcation involvement and associated bone loss. (d) An osseous window was created over the apical aspect of the mesial root to be resected, and the apical portion of the root was resected and removed. (e) Periapical radiograph following root resection to verify that the apical portion of the root had been completely extracted.

d

e

osseous window was created with a carbide bur at a high speed over the apical area of the mesial root. The apical portion of the mesial root was resected and removed from the site, followed by curettage and enucleation of the granulation tissue (Fig 6-4d). Methylene blue dye was applied to the area of the exposed root to rule out presence of a fracture. A periapical radiograph was taken to confirm that the entire apical portion of the root had been removed and no residual root pieces remained (Fig 6-4e). The PRF membrane created at the start of the appointment was placed into the vacancy created by the osseous defect and packed to fill the void and furcation area to the level of the adjacent unaffected bone (Fig 6-4f). The flap was repositioned to cover the surgical site, with the crestal margin located at 65

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f

g FIG 6-4  (cont) (f ) PRF was prepared and placed to fill the void at the mesial root, filling the defect to the buccal contour of the adjacent bone. (g) The flap was repositioned and secured with sutures. (h) Periapical radiograph at 3 months postsurgery demonstrating that the PRF placed into the void has converted to bone and continues to become denser than the adjacent native bone.

h

a higher position in relation to the crown to eliminate the recession noted before treatment. Sutures were placed to secure the flap (Fig 6-4g). The patient returned 10 days later for suture removal, and he indicated that the prior discomfort had resolved with some minor irritation at the sutures. A recall was scheduled to check the graft healing and organization 3 months postsurgically. A periapical radiograph was taken at that appointment. Evaluation of the radiograph demonstrated that the PRF placed into the void had converted to bone and continued to become denser compared with the adjacent native bone (Fig 6-4h).

Case study 3: Root resection and PRF site grafting A 67-year-old woman with a noncontributory medical history presented with mild discomfort and swelling around the maxillary left first molar. Clinical and radiographic findings confirmed a vertical root fracture of the mesial buccal root (Fig 6-5a). Treatment options were discussed, including extraction and replacement with implants or a removable partial denture. Implant treatment would require bone grafting, sinus augmentation, and multiple implants with a new fixed prosthesis. The patient decided to do a mesial buccal root amputation procedure and save the existing tooth and fixed partial denture (Fig 6-5b). Venous blood was drawn from the patient’s median cubital vein and collected in three 10-mL Vacutainer (Becton Dickinson) tubes without anticoagulants. 66

Root Resection and Root-End Resection

a

b

c

d

FIG 6-5  (a) A J-shaped lesion associated with the mesial buccal root of the maxillary first molar indicative of a vertical root fracture. (b) The mesial buccal root was resected with a bur, and a composite retrofill was placed. (c) Allograft bone has been mixed with PRF to create a putty-like bone mi­xture. (d) The PRF graft particle mixture was placed into the site and molded to fill the surgically created osseous defect. (e) PRF membranes were then placed over the graft and surgical site.

e

The blood was immediately centrifuged at 2,700 rpm for 12 minutes. The portion containing the PRF clot was cut and removed from the RBC layer. The PRF clot was compressed to form a membrane using a membrane-processing box. The liquid from the compressed membrane was collected and mixed with allograft bone (Fig 6-5c). A triangular flap incision was made, and the mesial buccal root was exposed, sectioned, and removed. The exposed gutta-percha was retroprepared with ultrasonic instrumentation and retrofilled with composite. The allograft bone mixture was placed into the bony defect followed by coverage with PRF membranes (Figs 6-5d and 6-5e). Figure 6-5f shows the immediate postoperative radiograph. 67

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PRF IN ENDODONTIC SURGERY

f

g

h

i FIG 6-5  (cont) (f ) Immediate postoperative radiograph following endodontic surgery. (g) At 1 month postsurgery, minimal recession was noted around the amputated mesial buccal root area. (h) The radiograph at 7 months postsurgery demonstrates good osseous fill of the grafted site. (i) The 7-month recall demonstrates good gingival architecture with minimal recession noted. ( j) The 14-month recall demonstrates tissue stability at the site without inflammation in the soft tissue.

j

The gingival tissues were repositioned and closed with 5.0 monofilament sutures. The sutures were removed after 1 week. At 1 month postsurgery, there was minimal recession around the amputated mesial buccal root area (Fig 6-5g). Figures 6-5h and 6-5i show the healing after 6 months. The bone has filled in, and the gingival architecture is excellent with no inflammation. Long-term follow up at 14 months demonstrated minimal recession and tissue stability at the site without inflammation (Fig 6-5j). In this case, immediate bone grafting with PRF after mesial buccal root amputation helped maintain bone height and prevent ridge deficiency. 68

References

References 1. Tang PM, Chan CP, Huang SK, Huang CC. Intentional replantation for iatrogenic perforation of the furcation: A case report. Quintessence Int 1996;27:691–696. 2. Mills ML. Perio/endo lesions: A brief review. J N Z Soc Periodontol 1988;(65):14–16. 3. Christie WH, Holthuis AF. The endo-perio problem in dental practice: Diagnosis and prognosis. J Can Dent Assoc 1990;56:1005–1011. 4. Leder AJ, Simon BI, Deasy M, Fenesy KE, Dunn S. Histological, clinical, and digital subtraction radiographic evaluation of repair of periodontal defects resulting from mechanical perforation of the chamber floor using ePTFE membranes. Periodontal Clin Investig 1997;19(2):9–15. 5. Eickholz P, Hausmann E. Evidence for healing of class II and III furcations after GTR therapy: Digital subtraction and clinical measurements. J Periodontol 1997;68:636–644. 6. Jepsen S, Eberhard J, Herrera D, Needleman I. A systematic review of guided tissue regeneration for periodontal furcation defects. What is the effect of guided tissue regeneration compared with surgical debridement in the treatment of furcation defects? J Clin Periodontol 2002;29(suppl 3):103–116. 7. Tsesis I, Rosen E, Tamse A, Taschieri S, Del Fabbro M. Effect of guided tissue regeneration on the outcome of surgical endodontic treatment: A systematic review and meta-analysis. J Endod 2011;37:1039–1045. 8. Duggins LD, Clay JR, Himel VT, Dean JW. A combined endodontic retrofill and periodontal guided tissue regeneration technique for the repair of molar endodontic furcation perforations: Report of a case. Quintessence Int 1994;25:109–114. 9. Gagnon K, Morand MA. Guided tissue regeneration in endodontics. Part 1 [in French]. J Can Dent Assoc 1999;65:394–398. 10. Gagnon K, Morand MA. Guided tissue regeneration in endodontics (2) [in French]. J Can Dent Assoc 1999;65:440–443. 11. Taschieri S, Testori T, Azzola F, Del Fabbro M, Valentini P. Guided-tissue regeneration in endodontic surgery [in French]. Rev Stomatol Chir Maxillofac 2008;109:213–217. 12. Molly L, Vandromme H, Quirynen M, Schepers E, Adams JL, van Steenberghe D. Bone formation following implantation of bone biomaterials into extraction sites. J Periodontol 2008;79:1108–1115. 13. Artzi Z, Kozlovsky A, Nemcovsky CE, Weinreb M. The amount of newly formed bone in sinus grafting procedures depends on tissue depth as well as the type and residual amount of the grafted material. J Clin Periodontol 2005;32:193–199. 14. Shivashankar VY, Johns DA, Maroli RK, et al. Comparison of the effect of PRP, PRF and induced bleeding in the revascularization of teeth with necrotic pulp and open apex: A triple blind randomized clinical trial. J Clin Diagn Res 2017;11(6):ZC34–ZC39. 15. Patel GK , Deepika PC, Sisodia N, Manjunath MK. Platelet rich fibrin in management of complex endoperio cases. Kathmandu Univ Med J (KUMJ) 2017;15(57):102–105. 16. Hülsmann M. Retreatment decision making by a group of general dental practitioners in Germany. Int Endod J 1994;27:125–132. 17. Langer B, Stein SD, Wagenberg B. An evaluation of root resections. A ten-year study. J Periodontol 1981;52:719–722. 18. Kim Y. Furcation involvements: Therapeutic considerations. Compend Contin Educ Dent 1998; 19:1236–1240. 19. Green EN. Hemisection and root amputation. J Am Dent Assoc 1986;112:511–518. 20. Guldener PH. Hemisection, tooth separation and root amputation [in German]. SSO Schweiz Monatsschr Zahnheilkd 1976;86:795–811. 21. Bühler H. Survival rates of hemisected teeth: An attempt to compare them with survival rates of alloplastic implants. Int J Periodontics Restorative Dent 1994;14:536–543. 22. Fugazzotto PA. A comparison of the success of root resected molars and molar position implants in function in a private practice: Results of up to 15-plus years. J Periodontol 2001;72: 1113–1123.

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23. Derks H, Westheide D, Pfefferle T, Eickholz P, Dannewitz B. Retention of molars after root-­ resective therapy: A retrospective evaluation of up to 30 years. Clin Oral Investig 2018;22: 1327–1335. 24. Park SY, Shin SY, Yang SM, Kye SB. Factors influencing the outcome of root-resection therapy in molars: A 10-year retrospective study. J Periodontol 2009;80:32–40. 25. Lang NP, Berglundh T; Working Group 4 of Seventh European Workshop on Periodontology. Periimplant diseases: Where are we now?—Consensus of the Seventh European Workshop on Periodontology. J Clin Periodontol 2011;38(suppl 11):178–181. 26. Carnevale G, Pontoriero R, di Febo G. Long-term effects of root-resective therapy in furcation-­ involved molars. A 10-year longitudinal study. J Clin Periodontol 1998;25:209–214. 27. Setzer FC, Kim S. Comparison of long-term survival of implants and endodontically treated teeth. J Dent Res 2014;93:19–26. 28. Setzer FC, Shou H, Kulwattanaporn P, Kohli MR, Karabucak B. Outcome of crown and root resection: A systematic review and meta-analysis of the literature. J Endod 2019;45:6–19. 29. Kim TH, Kim SH, Sándor GK, Kim YD. Comparison of platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concentrated growth factor (CGF) in rabbit-skull defect healing. Arch Oral Biol 2014;59:550–558. 30. Masuki H, Okudera T, Watanebe T, et al. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF). Int J Implant Dent 2016;2:19. 31. Qiao J, An N, Ouyang X. Quantification of growth factors in different platelet concentrates. Platelets 2017;28:774–778. 32. Schär MO, Diaz-Romero J, Kohl S, Zumstein MA, Nesic D. Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro. Clin Orthop Relat Res 2015;473:1635– 1643.

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LEARNING OBJECTIVES • Describe the characteristics and biologic properties of platelet-rich fibrin and its various applications in medicine • Understand the preparation protocol for PRF for socket preservation • Understand the clinical and histological data supporting PRF use for socket preservation

PRF for Socket Preservation YOGALAKSHMI RAJENDRAN, BDS, MS YVONNE KAPILA, DDS, PhD

P

latelet-rich fibrin (PRF) is an autologous blood derivative prepared without the use of any anticoagulants like bovine thrombin. Platelet concentrates such as PRF have been used in regenerative procedures in various fields of medicine, including dentistry, reconstructive surgery, vascular surgery, cardiothoracic surgery, plastic surgery, and dermatology.1–5 In vascular surgery, it is used to seal microvascular bleeding, and in plastic surgery, it is used to seal facial defects. The regenerative potential of the PRF membrane has led to its use in oral and maxillofacial applications, including sinus floor augmentation, ridge augmentation, socket preservation, root coverage, and intrabony and furcation defects.6–9 The PRF protocol involves drawing venous blood and centrifuging it in a sterile glass tube. After centrifugation, three layers are formed, including a base of red blood cells (RBCs) at the bottom, a supernatant layer of acellular plasma on the top, and a layer of PRF in the middle. The PRF is a fibrin-rich matrix that consists of platelets (98%), cytokines, leukocytes, and growth factors.10–13 PRF is a safe, cost-effective autologous blood product that accelerates tissue healing in part by release of growth factors and cytokines. Growth factors released by PRF are a chemoattractant to various cell types, including monocytes, fibroblasts, and endothelial cells. Macrophages are also released from PRF, and they have important roles in tissue regeneration, wound healing, and prevention of infection. A study 71

7

PRF FOR SOCKET PRESERVATION

reported that the infection rate after third molar removal was significantly reduced to 1% of cases when PRF was used in the sockets.6 Platelets contain alpha granules that degranulate to release cytokines. The cytokines in turn can stimulate cell migration and initiate cellular events, which expedite wound healing.10–13 A study has revealed that the PRF matrix mediates a very slow release of potential growth factors, including transforming growth factor β1 (TGF-β1), platelet-derived growth factor AB (PDGF-AB), vascular endothelial growth factor (VEGF), and matrix proteins (ie, fibronectin, vitronectin, and thrombospondin-1) for a period of 1 week.14 These growth factors are essential for hard and soft tissue regeneration.15,16 The gel-like consistency of the PRF membrane enables easy manipulation and suturing. The membrane can be obtained by compressing the PRF plug between metal plates using a commercially available box. There are studies discussing the improved properties of PRF as a result of changing the centrifugation parameters.

Socket Preservation PRF has been used in ridge preservation techniques to minimize the dimensional changes and preserve the alveolar bone following extraction of teeth. Araújo and Lindhe17 have shown in their study that there is substantial change in the alveolar ridge dimension after extraction. The dimensional changes are more pronounced in the buccal bone than in the palatal bone. Extraction of more than one tooth can cause further significant alterations in the height and width of the alveolar ridge.18 A systematic review19 concluded that the horizontal bone loss at 6 months was approximately 2.46 to 4.56 mm, and the vertical bone loss was approximately 0.8 to 1.5 mm.19 There is a rapid resorption pattern during the first 6 months after extraction, followed by a slow gradual reduction throughout life. These postextraction alveolar width dimensional changes compromise the esthetic and prosthodontic rehabilitation for a patient. Ridge preservation is used to preserve the alveolar ridge dimensions during the healing of an extraction site. To minimize the resorption of the alveolar ridge, various types of bone graft materials and growth factors are used in the preserve the ridge postexraction.20,21 A systematic review21 on this topic revealed that the clinical gains after socket preservation with a bone graft is up to 1.89 mm in a buccolingual dimension and up to 2.07 mm of midbuccal height. Several growth factors,5 such as platelet-derived growth factors (PDGFs), insulinlike growth factor (IGF), and bone morphogenetic proteins (BMPs), have been used to promote bone regeneration when treating sockets to preserve alveolar ridge dimension.22,23

72

PRF Preparation for Ridge Preservation

PRF Preparation for Ridge Preservation PRF can be used to promote bone regeneration for postextraction healing. PRF is first prepared by collecting venous blood in a 10-mL sterile glass tube via venipuncture of the antecubital vein. The blood is then centrifuged immediately at 1,300 rpm for 8 minutes. After centrifugation, the PRF clot is removed from the test tube. It can be directly placed as a plug in the extraction sockets, or it can be cut into small pieces. Bone graft material can be mixed with PRF and placed in the extraction socket. Many studies have evaluated the beneficial use of PRF in preserving the alveolar ridge postextraction.24–29 One study found a significant difference at 1 month and 3 months postextraction in the horizontal and vertical alveolar ridge dimensions by using PRF to treat extraction sockets compared with using a blood clot alone.25 Studies have noted decreased postoperative pain when PRF is used for alveolar ridge preservation and accelerated changes in soft tissue healing.25,26 The socket orifice is reported to close and mature within 4 weeks when PRF is used.26 A recent study conducted by the authors’ group evaluated the use of PRF in extraction sockets.24 The study focused on the histologic and micro-CT analysis of extraction sockets treated with different materials, including (1) a blood clot only, (2) PRF only, (3) PRF plus freeze-dried bone allograft (FDBA), and (4) FDBA only. This study found that the application of PRF in extraction sockets led to statistically significant new bone formation compared with using FDBA alone.24 Histologic analysis of bone cores taken at 3 months after grafting showed that sockets treated with PRF only had more trabecular bone formation than those treated with only a blood clot (Fig 7-1). Sockets treated with FDBA plus PRF showed more bone formation and less graft material remaining compared to sockets treated only with FDBA. Micro-CT analysis of the bone cores showed more trabecular bone formation in sockets treated with PRF only and PRF mixed with FDBA. However, the sockets treated with the FDBA showed less trabecular bone formation and had bone graft particles still present. The presence and release of growth factors and the potential to accelerate wound healing are important characteristics of PRF. These PRF properties are key to regenerative applications, especially in hard tissue regeneration, such as socket preservation. PRF, when used alone or in combination with bone grafting materials for site preservation, shows improved bone remodeling, accelerated healing, and improved clinical dimensional changes. Thus, PRF is a useful adjunct to socket preservation procedures.

73

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PRF FOR SOCKET PRESERVATION

a

b

c

d FIG 7-1  Bone cores harvested 3 months after alveolar ridge preservation and at the time of implant placement. (a) Blood clot only group. (b) PRF only group. (c) PRF + FDBA group. (d) FDBA only group.

References 1. Desai CB, Mahindra UR, Kini YK, Bakshi MK. Use of platelet-rich fibrin over skin wounds: Modified secondary intention healing. J Cutan Aesthet Surg 2013;6:35–37. 2. Danielsen PL, Agren MS, Jorgensen LN. Platelet-rich fibrin versus albumin in surgical wound repair: A randomized trial with paired design. Ann Surg 2010;251:825–831. 3. Sclafani AP. Safety, efficacy, and utility of platelet-rich fibrin matrix in facial plastic surgery. Arch Facial Plast Surg 2011;13:247–251. 4. Sclafani AP, McCormick SA. Induction of dermal collagenesis, angiogenesis, and adipogenesis in human skin by injection of platelet-rich fibrin matrix. Arch Facial Plast Surg 2012;14:132–136. 5. Gorlero F, Glorio M, Lorenzi P, Bruno-Franco M, Mazzei C. New approach in vaginal prolapse repair: Mini-invasive surgery associated with application of platelet-rich fibrin. Int Urogynecol J 2012;23:715–722.

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6. Hoaglin DR, Lines GK. Prevention of localized osteitis in mandibular third-molar sites using platelet-rich fibrin. Int J Dent 2013;2013:875380. 7. Simon BI, Zatcoff AL, Kong JJ, O’Connell SM. Clinical and histological comparison of extraction socket healing following the use of autologous platelet-rich fibrin matrix (PRFM) to ridge preservation procedures employing demineralized freeze dried bone allograft material and membrane. Open Dent J 2009;3:92–99. 8. Mazor Z, Horowitz RA, Del Corso M, Prasad HS, Rohrer MD, Dohan Ehrenfest DM. Sinus floor augmentation with simultaneous implant placement using Choukroun’s platelet-rich fibrin as the sole grafting material: A radiologic and histologic study at 6 months. J Periodontol 2009;80:2056–2064. 9. Miron RJ, Zucchelli G, Pikos MA, et al. Use of platelet-rich fibrin in regenerative dentistry: A systematic review. Clin Oral Investig 2017;21:1913–1927. 10. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part I: Technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101(3):e37–e44. 11. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part II: Platelet-related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101(3):e45–e50. 12. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part III: Leucocyte activation: A new feature for platelet concentrates? Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101(3):e51–e55. 13. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part IV: Clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101(3):e56–e60. 14. Dohan Ehrenfest DM, de Peppo GM, Doglioli P, Sammartino G. Slow release of growth factors and thrombospondin-1 in Choukroun’s platelet-rich fibrin (PRF): A gold standard to achieve for all surgical platelet concentrates technologies. Growth Factors 2009;27:63–69. 15. Choukron J, Ghanaati S. Reduction of relative centrifugation force within injectable platelet-­ rich-fibrin (PRF) concentrates advances patients’ own inflammatory cells, platelets and growth factors: The first introduction to the low speed centrifugation concept. Eur J Trauma Emerg Surg 2018;44:87–95. 16. Fujioka-Kobayashi M, Miron RJ, Hernandez M, Kandalam U, Zhang Y, Choukroun J. Optimized platelet-rich fibrin with the low speed concept: Growth factor release, biocompatibility, and cellular response. J Periodontol 2017;88:112–121. 17. Araújo MG, Lindhe J. Ridge alterations following tooth extraction with and without flap elevation: An experimental study in the dog. Clin Oral Implants Res 2009;20:545–549. 18. 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;23:313–323. 19. Tan WL, Wong TL, Wong MC, Lang NP. A systematic review of post-extractional alveolar hard and soft tissue dimensional changes in humans. Clin Oral Implants Res 2012;23(suppl 5):1–21. 20. Wang HL, Kiyonobu K, Neiva RF. Socket augmentation: Rationale and technique. Implant Dent 2004;13:286–296. 21. Avila-Ortiz G, Elangovan S, Kramer KW, Blanchette D, Dawson DV. Effect of alveolar ridge preservation after tooth extraction: A systematic review and meta-analysis. J Dent Res 2014; 93:950–958. 22. Fiorellini JP, Howell TH, Cochran D, et al. Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. J Periodontol 2005; 76:605–613. 23. Becker W, Lynch SE, Lekholm U, et al. A comparison of ePTFE membranes alone or in combination with platelet-derived growth factors and insulin-like growth factor-I or demineralized freeze-dried bone in promoting bone formation around immediate extraction socket implants. J Periodontol 1992;63:929–940.

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24. Clark D, Rajendran Y, Paydar S, et al. Advanced platelet-rich fibrin and freeze-dried bone allo­ graft for ridge preservation: A randomized controlled clinical trial. J Periodontol 2018;89: 379–387. 25. Temmerman A, Vandessel J, Castro A, et al. The use of leucocyte and platelet-rich fibrin in socket management and ridge preservation: A split-mouth, randomized, controlled clinical trial. J Clin Periodontol 2016;43:990–999. 26. Anwandter A, Bohmann S, Nally M, Castro AB, Quirynen M, Pinto N. Dimensional changes of the post extraction alveolar ridge, preserved with leukocyte- and platelet rich fibrin: A clinical pilot study. J Dent 2016;52:23–29. 27. Castro AB, Meschi N, Temmerman A, et al. Regenerative potential of leucocyte- and platelet-­ rich fibrin. Part B: Sinus floor elevation, alveolar ridge preservation and implant therapy. A systematic review. J Clin Periodontol 2017;44:225–234. 28. Strauss FJ, Stähli A, Gruber R. The use of platelet-rich fibrin to enhance the outcomes of implant therapy: A systematic review. Clin Oral Implants Res 2018;29(suppl 18):6–19. 29. Alzahrani AA, Murriky A, Shafik S. Influence of platelet rich fibrin on post-extraction socket healing: A clinical and radiographic study. Saudi Dent J 2017;29:149–155.

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Index

Page references followed by “f” denote figures, “t” denote tables, and “b” denote boxes.

A Allografts description of, 44 freeze-dried bone, 73, 74f guided tissue regeneration uses of, 58 mesenchymal stem cells as, 3 platelet-rich fibrin with, 61, 61f, 67f Alpha granules, in platelets, 11, 72 Alveolar ridge preservation, 72–73, 74f American Association of Endodontists, 4 Angiogenesis, 39, 59 Apical plug, 22–23, 23f Apical surgical defects, 55f, 55–56 Autogenous bone, 58 Autologous blood, 17 Autologous blood concentrates. See also Platelet-rich fibrin; Platelet-rich plasma. as membranes, 25–28 as osseous graft enhancers, 39–41 soft tissue applications of. See Soft tissue.

B Basic fibroblast growth factor, 40, 43, 60 bFGF. See Basic fibroblast growth factor. Blood-derived biologics concentrated growth factor. See Concentrated growth factor; Concentrated growth factor membranes.

gingival recession treated with, 34f, 35 immediate implant placement at extraction using, 35–37, 36f, 51–53, 52f as membranes, 25–28 as osseous graft enhancers, 39–41 platelet-rich fibrin. See Platelet-rich fibrin. platelet-rich plasma. See Platelet-rich plasma. sinus augmentation without osseous graft material using, 31–32, 32f soft tissue thickening using, 33, 35, 33f–34f BMP. See Bone morphogenetic protein. Bone formation, 40, 60 Bone marrow mesenchymal stem cells derived from, 2, 5 platelets from, 9–10 Bone morphogenetic protein, 4, 72 Bone regeneration. See also Guided tissue regeneration. growth factors for, 72 platelet-rich fibrin for, 73 Buffy coat, 6, 6f

C Cancellous bone, 58 CGF. See Concentrated growth factor. CGF membranes. See Concentrated growth factor membranes. Collagenase degrading units, 7 CollaPlug, 21f Concentrated growth factor antimicrobial properties of, 43 bone formation uses of, 40 fabrication of, 26–28, 27f fibrin clot, 27–28, 43

77

INDEX

liquid phase of, 28 platelet-rich fibrin versus, 40 Concentrated growth factor glue, 27–28, 43 Concentrated growth factor membranes fabrication of, 28–30, 29f–30f fibrin clot, 28–29, 29f gingival recession treated with, 34f, 35 growth factors in, 26 immediate implant placement at extraction using, 36, 36f osseous socket grafting uses of, 44, 45f sinus augmentation without osseous graft material using, 31–32, 32f soft tissue thickening using, 33, 35, 33f–34f Concentrated growth factor sticky bone apical surgical defects grafted using, 55f, 55–56 crestal sinus augmentation uses of, 53, 54f definition of, 40 dehiscence correction at extraction using, 46f, 46–47 fabrication of, 41–43, 42f–43f illustration of, 40f immediate implant placement at extraction to seal site with osseous graft using, 51–53, 52f lateral ridge augmentation uses of, 47–50, 48f–49f, 51f osseous socket grafting uses of, 44–45, 44f–45f root-end resection and, 61 as scaffold, 60 Cord blood cell types in, 2 costs associated with, 2 cryopreservation of, 2 processing of, 6f Cord blood mesenchymal stem cells description of, 2 isolation methods for, 6–7 osteogenic regeneration uses of, 5 processing of, 6f pulp tissue and, 5 Cortical bone, 58 Crestal sinus augmentation concentrated growth factor membrane for, 32 concentrated growth factor sticky bone for, 53, 54f Cytokines, 4, 10, 10t, 72

78

D Decortication, 48f Dehiscence correction at tooth extraction, 46f, 46–47 Dental pulp stem cells, 5 Dental stem cells, 5 Dentin pulp complex, 4 Dentoalveolar soft tissue healing, 9 Dimethyl sulfoxide, 2 DMEM. See Dulbecco’s Modified Eagle Media. DMSO. See Dimethyl sulfoxide. DPSCs. See Dental pulp stem cells. Dulbecco’s Modified Eagle Media, 7

E Embryonic stem cells, 2, 5 Endodontic surgery guided tissue regeneration, 57–59 osseous defects created by, 57 root resection. See Root resection. root-end resection. See Root-end resection. Endo-perio lesions, 57, 59 Exfoliated deciduous teeth, stem cells from, 5 Extraction, tooth alveolar ridge changes after, 72 dehiscence correction at, 46f, 46–47 immediate implant placement at, 35–37, 36f, 51–53, 52f

F FDBA. See Freeze-dried bone allograft. FGF. See Fibroblast growth factor. Fibrin clot concentrated growth factor, 27–29, 29f, 43 growth factors in, 27 platelet-rich, 13, 14f, 19f–20f, 27 Fibroblast growth factor, 4 Fibronectin, 72 Flap elevation, 25 Freeze-dried bone allograft, 73, 74f Furcation lesions, 57–59

G Gingival recession, 35 Graft(s) osseous. See Osseous graft/grafting. particulate, 61

INDEX

Granules, in platelets, 11, 72 Growth factors. See also specific growth factor. alpha granule release of, 11 bone regeneration uses of, 72 concentrated. See Concentrated growth factor. in fibrin clot, 27 in platelet-rich fibrin, 4, 10t, 12, 17, 40, 60, 71 Guided tissue regeneration. See also Bone regeneration. description of, 25 furcation lesions treated with, 57–59

H Hard tissue applications alveolar ridge preservation, 72–73, 74f grafting. See Osseous graft/grafting. socket preservation, 72–73 tissue regeneration, 73

I IL-1. See Interleukin-1. IL-4. See Interleukin-4. IL-6. See Interleukin-6. Immediate implant placement concentrated growth factor membrane for, 36, 36f concentrated growth factor sticky bone uses in, 51–53, 52f Implant(s) disadvantages of, 60 immediate placement of. See Immediate implant placement. Innate immunity, platelets’ role in, 10 Insulinlike growth factors 1 and 2, 10t, 40, 43, 60, 72 Interleukins -1, 10t -4, 10t -6, 10t description of, 4

L Lateral ridge augmentation concentrated growth factor sticky bone for, 47–50, 48f–49f, 51f

lateral wall defect treated with, 50, 51f with membrane, 47–49, 48f–49f without membrane, 49f, 49–50 Leukocyte- and platelet-rich fibrin characteristics of, 11t development of, 11 open-source protocol for, 13, 14f Leukocyte- and platelet-rich plasma, 11t Leukocytes, 10 L-PRF. See Leukocyte- and platelet-rich fibrin. L-PRP. See Leukocyte- and platelet-rich plasma.

M Macrophages, 71 Membranes blood-derived biologics as, 25–28 nonresorbable, 26 platelet-rich fibrin, 13, 14f, 20, 21f, 72 resorbable, 26 Mesenchymal stem cells allograft uses of, 3 applications for, 2 bone marrow–derived, 2, 5 cell types derived from, 1 from cord blood. See Cord blood mesenchymal stem cells. definition of, 1 mechanism of action, 3–4 osteogenic, 4 pulp tissue regeneration uses of, 5 regenerative endodontics use of, 4–5 revenue from, 2 secretome from, 3–4 signaling pathways of, 4 sources of, 2–3 from Wharton’s jelly, 3, 7 wound healing applications of, 3–4 Methylene blue, 65 Mineral trioxide aggregate, 20, 22–23, 23f, 64 Molar root resection, 60 MSCs. See Mesenchymal stem cells. MTA. See Mineral trioxide aggregate.

N Nonresorbable membranes, 26

79

INDEX

O Osseous graft/grafting apical surgical defects filled using, 55f, 55–56 blood-derived biologics as enhancers of, 39–41 concentrated growth factor sticky bone use as, 44–45, 51, 52f, 53, 44f–45f dispensing of, 41f immediate implant placement uses of, 51, 52f, 53 platelet-rich fibrin added to, 61, 63f, 66f as scaffold, 40, 59 Osteogenic mesenchymal stem cells, 4 Osteogenic regeneration, 5 Osteotomy, 54f

P PBMCs. See Peripheral blood mononucleated cells. PBS. See Phosphate-buffered saline. PDGF. See Platelet-derived growth factor. PDLSCs. See Periodontal ligament stem cells. Peri-implant mucositis, 60 Peri-implantitis, 60 Periodontal ligament stem cells, 5 Peripheral blood mononucleated cells, 6f, 6–7 Phosphate-buffered saline, 6 Plasma depletion, 2 Platelet(s) bone marrow production of, 9–10 granules in, 11, 72 innate immunity role of, 10 Platelet concentrates, 11, 11t Platelet-derived growth factor, 10t, 72 Platelet-derived growth factor-AB, 72 Platelet-derived growth factor-BB, 40, 43, 60 Platelet-poor plasma, 43 Platelet-rich fibrin allografts with, 61, 61f, 67f autologous blood for, 17 centrifugation of, 12, 19f, 26, 71 composition of, 71 concentrated growth factor versus, 40 definition of, 71 endodontic surgery uses of, 58–59 fibrin network created by, 11–12 freeze-dried bone allograft with, 73, 74f as grafting material, 59, 61 growth factors in, 10t, 12, 17, 26, 40, 60, 71 leukocyte and, 11, 11t, 13, 14f

80

liquid plasma, 42 as membrane, 13, 14f, 20, 21f, 72 nonsurgical applications of, 12 in osseous defect, 64–66, 65f–66f osseous graft material with, 63f platelet-rich plasma and, comparisons between, 11–12 pure, 11t regenerative endodontics applications of, 12, 71 ridge preservation uses of, 72–73, 74f root resection and, 66–68, 67f root-end resection and, 64–66, 65f–66f as scaffold, 17–21, 18b, 19f–21f, 60 scientific characteristics of, 9–11 socket preservation uses of, 72–73 stem cells in, 17 surgical applications of, 12 Platelet-rich fibrin clot, 13, 14f, 19f–20f, 27 Platelet-rich plasma centrifugation cycle for, 12 endodontic surgery uses of, 58–59 leukocyte, 11t platelet-rich fibrin and, comparisons between, 11–12 “Poncho” technique, 36–37, 36f, 53 Posterior maxilla, 31 PPP. See Platelet-poor plasma. PRF. See Platelet-rich fibrin. PRP. See Platelet-rich plasma. Pulp tissue regeneration, 5

R RBCs. See Red blood cells. Red blood cells, 27 Red cell reduction, 2 Regenerative endodontics apical plug, 22–23, 23f applications of, 4 definition of, 12, 17 mesenchymal stem cells use in, 4–5 platelet-rich fibrin applications in, 12, 71 Regenerative medicine, 1–8 Resorbable membranes, 26 Ridge preservation, 72–73, 74f Root resection indications for, 59 molars, 60 platelet-rich fibrin site grafting with, 66–68, 67f survival rates for, 59–60

INDEX

Root resorption, 65f Root-end resection concentrated growth factor sticky bone with, 61 indications for, 59 platelet-rich fibrin site grafting with, 64–66, 65f–66f technique for, 62–63, 63f

S Scaffold osseous graft material as, 40, 59 platelet-rich fibrin as, 17–21, 18b, 19f–21f, 60 SCAPs. See Stem cells from apical papilla. Secretome, 3–4 SHEDs. See Stem cells from exfoliated deciduous teeth. Sinus augmentation without osseous graft material, 31–32, 32f Sinus membrane perforation, 31 Socket preservation, 72–73 Soft tissue, blood-derived biologics applications in concentrated growth factor. See Concentrated growth factor. immediate implant placement, 36, 36f as membranes, 25–28 thickening of, 33, 35, 33f–34f Stem cells autogenous bone as source of, 58 characteristics of, 1 definition of, 3–4 dental, 5 embryonic, 2, 5 mesenchymal. See Mesenchymal stem cells. multipotent potential of, 2 periodontal ligament, 5 pulp tissue regeneration uses of, 5 regenerative endodontics use of, 4–5 in regenerative medicine, 1–8 totipotent, 1 Stem cells from apical papilla, 5 Stem cells from exfoliated deciduous teeth, 5 Sticky bone. See Concentrated growth factor sticky bone.

T

TGF-b1. See Transforming growth factor b1. TGPCs. See Tooth germ progenitor cells. Thrombospondin-1, 72 Tissue engineering. See also Regenerative medicine. key elements for, 12, 17 strategies for, 5 TNF-α. See Tumor necrosis factor α. Tooth germ progenitor cells, 5 Totipotent stem cells, 1 Transforming growth factor b, 4 Transforming growth factor b1, 10t, 40, 43, 60, 72 Tumor necrosis factor α, 10t

U Umbilical cord blood from. See Cord blood. Wharton’s jelly from, 2–3 zones of, 3

V Vascular endothelial growth factor, 10t, 40, 43, 60, 72 VEGF. See Vascular endothelial growth factor. Vertical root fracture, 67f Vitronectin, 72

W Wharton’s jelly, 3, 7 White-top Vacutainer tube, 28–29, 30f, 41 Whole blood, 9–10 WJ. See Wharton’s jelly. Wound healing angiogenesis in, 59 cytokines in, 10t growth factors in, 10t mesenchymal stem cells in, 3–4 whole blood for, 9

X Xenografts, 44, 58

Telomeres, 3 TGF-b. See Transforming growth factor b.

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Mohammad (Mike) Sabeti, DDS, MA, currently serves as Director of Postgraduate Endodontics at University of California, San Francisco (UCSF). He is a Diplomate of the American Board of Endodontics and has been invited to present at many meetings. In addition, he has received a Certificate in Recognition of Outstanding Services as a Faculty Member to the enhancement of education and clinical excellence, presented by the Advance Endodontics Herman Ostrow School of Dentistry of USC, and Certificate of Appreciation for the Service and Significant Contribution to Periodontal Division, University of Texas, Dental Branch. He was recognized by the UCSF’s the Haile T Academy of Medical Educators with an Excellence in Teaching Award for 2019. Edward S. Lee, DDS, is currently teach-

ing at the UCSF Postgraduate Endodontic Clinic. Dr Lee is a recognized innovator in the field of endodontics. He invented and patented the MTA Pellet Forming Block over 20 years ago. The MTA Block is still in production and sold worldwide. He has lectured nationally and internationally about the MTA Pellet Forming Block technique and has published articles in peer-reviewed journals. He also participates in Dental Humanitarian Projects in Suqian and Chengdu, China.

Mahmoud Torabinejad, DMD, MSD, PhD, is currently the President and CEO of the Torabinejad Institute of Surgical Education and Research Venues in Irvine, California. He has teaching positions in endodontics at multiple schools and is the former professor of endodontics and Director of Advanced Program in endodontics at Loma Linda University School of Dentistry. He lectures internationally and has authored six textbooks and more than 350 publications. He is the #1 cited author in classic literature and regenerative endodontics in the field of endodontics. He is a past president of both the California Association of Endodontics and the American Association of Endodontists and has received numerous awards and accolades in the field of endodontics.

About the book Platelet-rich fibrin (PRF) is a second-­generation autologous platelet aggregation biomaterial with a wide range of applications in medicine and dentistry. PRF is already frequently used for procedures such as bone grafting and sinus augmentation, but its application can extend to surgical and nonsurgical endodontic procedures as well. This hand­ book, the first of its kind, is designed to help the clinician understand and use PRF in endodontics. It discusses the science, clinical applications, and techniques for specialists and general practitioners in a concise, easy-to-read format. The applications for PRF for each endodontic procedure are laid out step-by-step with accompanying photographs and case studies.

ISBN 978-0-86715-827-4

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9 780867 158274