Knowledge for Clinical Practice. A Guide for Dental Professionals

Transcription

1 DENTAL LEARNING Knowledge for Clinical Practice A PEER-REVIEWED PUBLICATION Dental Stem Cells: A Guide for Dental Professionals Jeffrey Gruneich, PhD, MS; and Fiona M. Collins, BDS, MBA, MA INSIDE Earn 2 CE Credits Written for dentists, hygienists and assistants Integrated Media Solutions Inc./DentalLearning.net is an ADA CERP Recognized Provider. ADA CERP is a service of the American Dental Association to assist dental professionals in identifying quality providers of continuing dental education. ADA CERP does not approve or endorse individual courses or instructors, nor does it imply acceptance of credit hours by boards of dentistry. Concerns or complaints about a CE provider may be directed to the provider or to ADA CERP at Integrated Media Solutions Inc./DentalLearning.net designates this activity for 2 continuing education credits. Approved PACE Program Provider FAGD/MAGD Credit Approval does not imply acceptance by a state or provincial board of dentistry or AGD endorsement. 2/1/2016-1/31/2020 Provider ID: # AGD Subject Code: 149 Dental Learning, LLC is a Dental Board of California CE Provider. The California Provider # is RP5062. All of the information contained on this certifi cate is truthful and accurate. Completion of this course does not constitute authorization for the attendee to perform any services that he or she is not legally authorized to perform based on his or her license or permit type. This course meets the Dental Board of California s requirements for 2 units of continuing education. CA course code is

2 DENTAL LEARNING ABOUT THE AUTHORS Dr. Jeffrey Gruneich was a worldwide biobanking specialist for IBM Healthcare and Life Sciences, providing IT solutions and consulting for leading pharmaceutical, biotechnology, academic, government, hospital, and consumer advocacy organizations as they created new processes, products, and services in translational research and regenerative medicine. Prior to IBM, Dr. Gruneich co-founded etechtransfer.com, an online exchange for biotech intellectual property, and Infoceutics Inc., a bioinformatics company that identified key infectious genes in microbiology. Jeff also developed immunoassays and reagents for food and environmental monitoring for Charm Sciences in Malden, MA. Dr. Gruneich received his Ph.D. in Bioengineering from the University of Pennsylvania with a focus in cell and gene therapy, an M.S. in chemistry at UC Berkeley as a National Science Foundation graduate fellow, and B.S. degrees in chemistry and mathematics at Southern Methodist University. He lives in Boston with his wife and two sons. AUTHOR DISCLOSURE: Dr. Gruneich is vice president and co-founder of Provia Laboratories. He can be reached at jgruneich@provialabs.com. Dr. Fiona M. Collins has authored and presented CE courses to dental professionals and students in the United States and internationally, and has been an active consultant in the dental industry for several years. She is a member of the American Dental Association and the Organization for Asepsis and Safety Procedures, and has been a member of the British Dental Association, Dutch Dental Association, the International Assocation for Dental Research and the Academy of General Dentistry Foundation Strategy Board. Fiona earned her dental degree from Glasgow University and holds an MBA and MA from Boston University. AUTHOR DISCLOSURE: Dr. Collins does not have a leadership position or a commercial interest with any products that are mentioned in this article, or with products and services discussed in this educational activity. Dr. Collins can be reached at fionacollins@comcast.net ABSTRACT The science of stem cells and their clinical use in regenerative medicine and dentistry have developed significantly over the last decade. Stem cells may be derived from various sources within the body: teeth and dental pulp, bone marrow, cord blood, and adipose tissue. Most importantly for the dental professional, teeth can provide a plentiful and potent source of stem cells which can be preserved rather than discarded as medical waste. Dental stem cells are a convenient, noncontroversial, and affordable source of stem cells for families that wish to preserve their stem cells for future use. Dental professionals are in a unique position to build awareness about the option of storing stem cells from teeth, as well as to assist patients with the collection of candidate teeth for subsequent lab processing and cryopreservation by the dental stem cell bank. In 2008, the American Academy of Pediatric Dentistry published a policy on stem cells that provides a useful framework for dental stem cells dental stem cell banking. This couse reviews the science behind dental stem cells and, specifically for dental professionals, the processes involved with collection of dental stem cells, their processing and preservation. EDUCATIONAL OBJECTIVES The overall objective of this article is to provide the participant with information on stem cells, particularly dental stem cells. Upon completing this course, the participant will be able to: 1. Delineate key elements of the AAPD policy on stem cells and how this translates into daily practice. 2. List the types, sources, and basic properties of stem cells. 3. List and describe the range of potential clinical uses for dental stem cells and the current status of these in type 1 diabetes and spinal cord injuries. 4. Describe the key elements involved in discussing dental stem cell banking services with patients and in providing these services. SPONSOR/PROVIDER: This is a Dental Learning, LLC continuing education activity. COMMERCIAL SUPPORTER: This course has been made possible through an unrestricted educational grant from Store-A-Tooth. DESIGNA- TION STATEMENTS: Dental Learning, LLC is an ADA CERP recognized provider. ADA CERP is a service of the American Dental Association to assist dental professionals in identifying quality providers of continuing dental education. ADA CERP does not approve or endorse individual courses or instructors, nor does it imply acceptance of credit hours by boards of dentistry. Dental Learning, LLC designates this activity for 2 CE credits. Dental Learning, LLC is also designated as an Approved PACE Program Provider by the Academy of General Dentistry. The formal continuing education programs of this program provider are accepted by AGD for Fellowship, Mastership, and membership maintenance credit. Approval does not imply acceptance by a state or provincial board of dentistry or AGD endorsement. The current term of approval extends from 2/1/2016-1/31/2020. Provider ID: # EDUCATIONAL METHODS: This course is a self-instructional journal and web activity. Information shared in this course is based on current information and evidence. REGISTRATION: The cost of this CE course is $29.00 for 2 CE credits. ORIGINAL RELEASE DATE: February REVIEW DATE: January EXPIRATION DATE: December REQUIREMENTS FOR SUCCESSFUL COMPLETION: To obtain 2 CE credits for this educational activity, participants must pay the required fee, review the material, complete the course evaluation and obtain a score of at least 70%. AUTHENTICITY STATEMENT: The images in this course have not been altered. SCIENTIFIC INTEGRITY STATEMENT: Information shared in this continuing education activity is developed from clinical research and represents the most current information available from evidence-based dentistry. KNOWN BENEFITS AND LIMITATIONS: Information in this continuing education activity is derived from data and information obtained from the reference section. EDUCATIONAL DISCLAIMER: Completing a single continuing education course does not provide enough information to result in the participant being an expert in the field related to the course topic. It is a combination of many educational courses and clinical experience that allows the participant to develop skills and expertise. PROVIDER DISCLOSURE: Dental Learning does not have a leadership position or a commercial interest in any products that are mentioned in this article. No manufacturer or third party has had any input into the development of course content. CE PLANNER DISCLOSURE: The planner of this course, Joe Riley, does not have a leadership or commercial interest in any products or services discussed in this educational activity. He can be reached at jriley@ims.co. TARGET AUDIENCE: This course was written for dentists, dental hygienists, and assistants, from novice to skilled. CANCELLATION/REFUND POLICY: Any participant who is not 100% satisfied with this course can request a full refund by contacting Dental Learning, LLC, in writing. Please direct all questions pertaining to Dental Learning, LLC or the administration of this course to jriley@ims.co. Go Green, Go Online to take your course Copyright 2015 by Dental Learning, LLC. No part of this publication may be reproduced or transmitted in any form without prewritten permission from the publisher. DENTAL LEARNING 500 Craig Road, First Floor, Manalapan, NJ Director of Content JULIE CULLEN Creative Director MICHAEL HUBERT Art Director MICHAEL MOLFETTO 2

3 Dental Stem Cells: A Guide for Dental Professionals Introduction The term stem cell dates back more than a century. 1 Since then, clinicians and scientists have come to define stem cells as undifferentiated cells that possess both the capacity for self-renewal and the ability to give rise to differentiated cells. Due to these properties, stem cells will have increasingly transformational uses in medicine and dentistry. The layperson often associates stem cells with embryonic stem cells that come from embryos. However, while the collection of embryonic stem cells is controversial, adult stem cells can be collected in noncontroversial ways from various tissue niches within the body. These stem cells retain the properties that are important for clinical use. In fact, adult stem cells, such as those extracted from bone marrow transplants, have been used in medicine for over 50 years to treat hundreds of thousands of people, primarily patients with leukemia or genetic blood disorders. The use of bone marrow in medicine is an example of regenerative medicine. The U.S. Department of Health and Human Services has identified regenerative medicine as an emerging field that will revolutionize health care treatment, potentially treat heretofore incurable diseases, combat rising healthcare costs, and serve as an important new area of U.S. competitiveness. 2 The interdisciplinary field of regenerative medicine is rapidly evolving, with a wide array of approaches to repairing, replacing, or enhancing biological function lost to congenital abnormalities, injury, disease, or aging. Historically, identifying a potent, noncontroversial, accessible, relatively inexpensive, and convenient source of stem cells has been challenging one solution is dental stem cells. Since scientists at the National Institutes of Health discovered these cells in 2000, dental professionals and dental patients have become increasingly aware of the emerging uses of both dental stem cells and dental stem cell banking services. Dental stem cells can be readily sourced from various dental tissues for potential autologous use at a future date. Advances in stem cell research and interest in dental stem cells and banking services are increasing such that there are now policies relating to dental stem cells. In 2008, the AAPD Council on Clinical Affairs published a Policy on Stem Cells, which was revised in It provides a useful framework for dental professionals as the dental stem cell field emerges. AAPD Policy on Stem Cells The AAPD policy acknowledges that the public is expressing increasing interest in dental stem cell banking and encourages dentists to follow evidence-based literature in order to educate patients about the collection, storage, viability, and use of dental stem cells with respect to autologous regenerative therapies. The policy also highlights that while sources of dental stem cells are readily accessible, those cells must be secured and stored properly to maintain the potential to proliferate and differentiate. Finally, the policy gives an overview of the properties of dental stem cells and highlights a number of conditions for which stem cell therapy in general is currently being clinically tested as a treatment for conditions including diabetes, Parkinson s disease, and spinal cord injuries. Suggested regenerative dentistry applications mentioned include the repair of craniofacial defects as well as dental and periodontal tissues. Stem Cell Basics A simple way to conceptualize stem cells is to envision the paths leading from embryogenesis to the development of each cell in a fully developed organism. Cells start with January

4 DENTAL LEARNING Stem Cell Potency Figure 1. Stem Cell Characteristics Nails/Lens/Enamel CNS/PNS Spinal Cord/Brain Skeleton Muscles/Heart Connective/Tissues Blood Tissues Red Cells Kidney Ovary/Uterus/Testes Lung Liver Digestive System Pancreas Totipotent Pluripotent Multipotent Unipotent high potential in a fertilized egg and, in fact, are considered at this point to be totipotent. This means that a single cell (for example, from a blastocyst) can give rise to a complete organism. In practice, embryonic stem cells are obtained at this point in development from embryos or blastocysts. As development proceeds, the cells divide and become increasingly specialized, developing into the four germ layers: ectoderm, mesoderm, endoderm, and germ layers (Figure 1). At this point the cells are multipotent, meaning that they still have the potential to differentiate into more limited arrays of cell types. Ultimately in a developed organism nearing or following birth, the vast majority of cells are oligopotent or unipotent that is, they have terminally differentiated into the over 200 specialized cell types making up the trillion cells that do the work of the body (e.g., producing blood, bone, muscle, organs, and neurons). Some stem cells remain in tissues after birth and into adulthood; they are by convention termed adult stem cells. These cells reside in specialized stem cell niches in various tissues. The niches preserve the capacity of stem cells to proliferate and differentiate into many cell types. 4 In disease or injury, the body naturally relies in part on stem cell niches to help repair and regenerate tissues over one s lifetime. Clinicians are increasingly using this knowledge, for example by transplanting or mobilizing stem cells from their stem cell niche, to improve clinical outcomes. Practically speaking, the most accessible stem cell niches are bone marrow, the umbilical cord, adipose tissue, and dental pulp. 5 Importantly for dental professionals, the dental stem cell niche is proving to be easy to access and contains a range of cell types that may be useful for regenerative dentistry and medicine. Adult mesenchymal stem cells, of which dental stem cells are a subset, are highly proliferative and have the ability to proliferate and differentiate into many cell lines. AAPD policy Sources of Stem Cells Stem cells can be organized into two main sources: embryonic stem cells sourced from embryos, and adult stem cells sourced from an adult stem cell niche (e.g., bone marrow, umbilical cord blood, dental pulp, adipose tissue). If it is necessary to have a discussion with a patient about stem cells before collecting them, it is important to realize that laypersons often oversimplify the field and associate all stem cells with embryonic stem cells. For completeness, this article addresses embryonic stem cells and then focuses on adult stem cells particularly dental stem cells. A. Embryonic Stem Cells and Induced Pluripotent Stem Cells When human embryonic stem cells were first isolated in 1998, 6 scientists and clinicians were very hopeful that this technology would provide a ready source of stem cells useful for the cure of most, if not all, diseases due to their pluripotent nature (the ability to form most if not all cell types). However, this has proven difficult in practice for many reasons. First, there are ethical, legal, moral, and social issues associated with embryonic stem cells for example, determining the rights of the embryo, manifold issues around therapeutic cloning, and whether U.S. federal funding should be used to support this field. Second, there are significant practical clinical and engineering challenges to using pluripotent stem cells, including inadequate cell numbers, immunorejection (caused by a mismatch of the immune systems of the donor and recipient), and tumori- 4

5 Dental Stem Cells: A Guide for Dental Professionals genesis (the pluripotent nature of embryonic stem cells that frequently leads to teratomas tumors containing one or more of the three embryonic germ tissue layers). 7 The first human clinical trials of embryonic stem cells in the United States were initiated in 2009 after the federal ban on embryonic stem cell therapy was lifted. Compared to adult stem cell approaches, which have been used safely and effectively for decades, the enormous challenges to using embryonic stem cells are clearly illustrated by the leading embryonic stem cell therapeutics company Geron. After investing millions of dollars in R&D for over 10 years, Geron not only terminated its embryonic stem cell clinical trial for spinal cord injury but also completely exited the business of embryonic stem cell therapeutic development. 8 Partially addressing the ethical, legal, moral, and social issues associated with embryonic stem cells, genetic engineers in 2006 induced the first pluripotent states in unipotent cells by a technique known as induced pluripotent stem cell or ips technology. Basically, genetic engineering could reset cells back to an embryonic-like state. 9 While this technology avoids the ethical issues of having to extract embryonic stem cells and uses a patient s own tissues (thus reducing immunologic incompatibility), ips cells have also been shown to cause teratomas and may, in practice, be prohibitively expensive to generate on a personalized basis. 10 B. Adult Stem Cells 1. Bone Marrow Stem Cells In contrast to embryonic stem cells or ips cells, adult stem cells from bone marrow have been used clinically for over 50 years. In the late 1950s, bone marrow from one identical twin was used to treat the other twin s leukemia. 11 This work was inspired by research into the survival of irradiated dogs following nuclear radiation poisoning. The dogs survived because their spleens contained cells that repopulated their otherwise radiationablated blood systems. When applied to twins where one twin had leukemia and the other was healthy, initial bone marrow transplants restored the diseased twin s health for a few months, but many of the first patients died from relapse of leukemia or graft-versus-host disease. Years of research and clinical advancements led to successful transplants between non-twins in the late 1960s. 12 In 1990, Dr. E. Donnall Thomas was awarded the Nobel Prize in Medicine for his pioneering work in bone marrow transplantation. 13 Today, clinical bone marrow stem cell treatments are commonplace; over 50,000 bone marrow transplants are performed each year, 14 and more than 1,500 clinical trials related to bone marrow are planned, ongoing, or have been completed, treating conditions ranging from leukemia to spinal cord injury Cord Blood Stem Cells In the mid-1980s, a lack of matched donors available for bone marrow transplants in children with leukemia motivated the alternative use of hematopoietic stem cells from umbilical cord blood. By 1988, the first cord blood stem cell transplant was used to treat a young boy with Fanconi s anemia. 16 That patient remains alive and well to this date. 17 Today dozens of diseases are treatable with fresh or cryopreserved umbilical cord blood. The properties of cord blood stem cells to treat leukemia or blood-related genetic diseases and to be readily collected and cryopreserved for future use formed the basis for the umbilical cord blood banking industry. To date, more than 9,300 patients have been treated with stem cells from umbilical cord blood, and over 200,000 units are publicly banked. 18 In addition to public banks, parents of over 550,000 newborns have privately banked their children s umbilical cord blood at birth 19 due to its proven use in treating leukemia and genetic blood disorders, as well as for its potential to treat other diseases such as myocardial infarction, diabetes mellitus, and neurological disorders. 20 The American College of Obstetricians and Gynecologists 21 and the American Pediatric Association 22 each have official policies or position statements on umbilical cord blood banking. 3. Adipose Tissue Stem Cells Adipose tissue obtained from liposuction aspirates or abdominoplasty procedures can provide a source of mesenchymal stem cells. Stem cells from adipose tissue January

6 DENTAL LEARNING are being investigated for repairing tissue defects resulting from traumatic injury, tumor resection, and congenital defects, 23 as well as from calvarial defects following severe head injury, 24 and in dentistry for maxillary and mandibular repair Dental Stem Cells Pulpal tissue of primary teeth and surgically removed third molars may serve as a source of mesenchymal stem cells. AAPD policy In 2000, scientists at the National Institute of Dental and Craniofacial Research discovered that mesenchymal stem cells can be readily accessed from the dental pulp. 26 Suspecting that a stem cell population was responsible for the similarities between dental pulp and bone marrow, they identified mesenchymal stem cells in the pulp of extracted third molars. Termed dental pulp stem cells (DPSCs), these stem cells are more prolific than bone marrow mesenchymal stem cells, and were shown to generate calcified nodules and neuron-like cells. 27 Stem cells from human exfoliated deciduous teeth (SHED), 28 periodontal ligament stem cells (PDLSCs), 29 stem cells from apical papilla (SCAPs), 30 and dental follicle stem cells (DFSCs) 31 were subsequently identified. The Science of Dental Stem Cells Hundreds of peer-reviewed scientific studies have since examined dental stem cells in laboratory, animal, and human studies, showing that dental stems display typical mesenchymal stem cell characteristics These include generating dentin-producing odontoblasts, adipocytes, osteoblasts, bone, cartilage, and smooth and skeletal muscle. 27, 40, 41,42 Additionally, dental stem cells either contain or can switch lineage to form ectodermal tissues such as neurons 43 and epithelial-like stem cells, 44 as well as cells associated with the endodermal lineage, including endothelial cells, 40 hepatocytes, 45 and insulinproducing cells. 46 In addition, populations of dental stem cells have been identified that express embryonic Figure 2. Milestones in the Adult Stem Cell Field 6

7 Dental Stem Cells: A Guide for Dental Professionals stem cell markers In other words, stem cells found associated with teeth may contain many of the cell types required for regenerative medicine. The biology of odontogenesis provides some clues as to the existence of dental stem cells. Early in fetal development, teeth arise from the neural crest through a series of interactions between neural, mesenchymal, and epithelial tissues. 50,51 The developed tooth can be thought of as a source of an encapsulated population of quiescent stem cells. It has also been shown that the pluripotency of dental stem cells 52 may be a function of the age of the tooth or the age of its donor. 53 In other words, primary teeth, molars, and wisdom teeth of young adults all contain potent sources of dental stem cells. Current and Emerging Clinical Uses of Dental Stem Cells In human clinical studies, dental stem cells have demonstrated proof of concept to regenerate alveolar bone 54 and for periodontal disease. 55 In animal studies, human dental stem cells have shown the potential to treat a wide variety of conditions including the ability to regenerate damaged pulp; 56 reconstruct craniofacial defects; 57 generate lamellar bone; 40 engineer new teeth; 58 repair damaged corneas; 59 treat liver disease; 60 repair myocardial infarction; 61 and treat muscular dystrophy, 62 stroke, 63 and spinal cord injury. 64 Dental stem cells have also proven capable of generating islet-like aggregates that secrete insulin in a glucose-responsive manner 65 and of modulating immune responses, 66 both of which may be important strategies for treating diabetes (Table 1). A complete review of all the progress in this field is beyond the scope of this article. 67 Therefore, this section focuses on two potentially groundbreaking uses of dental stem cells that illustrate the transformative role that dental professionals may play in this emerging field. A. Diabetes Today, there is no cure for type 1 diabetes (insulindependent or juvenile-onset diabetes). In the United States alone, nearly 15,000 children and teenagers are newly Table 1. Potential Applications for Dental Stem Cells Dental Applications Pulpal regeneration Craniofacial reconstruction Engineering of new teeth Medical Applications Corneal repair Lamellar bone generation Treatment of liver disease Cardiac repair following myocardial infarction Treatment of muscular dystrophy Treatment following a stroke Spinal cord regeneration Treatment of diabetes Mixed Dentition Dental Pulp Stem Cells Stem Cells from Human Exfoliating Deciduous Teeth Permanent Dentition Dental Follicle Stem Cells Stem Cells from Apical Papilla Periodontal Ligament Stem Cells Figure 3. Dental Stem Cell Niches January

8 DENTAL LEARNING diagnosed each year. 68 Standard treatment for type 1 diabetes includes lifestyle management, frequent blood glucose monitoring, and daily insulin injections or the use of insulin pumps. Although the discovery and use of insulin by Frederick Banting and Charles Best 69 is one of the major breakthroughs in medical history, Banting himself recognized in his Nobel Lecture delivered in 1925 that: Insulin is not a cure for diabetes; it is a treatment. The expected total lifetime medical and indirect costs for those newly diagnosed is estimated at about $10 billion, and over $400 billion in costs would be avoided if the disease was eliminated. 70 Stem cells are being aggressively investigated as part of a cure for type 1 diabetes. This research is focused on two fronts: inducing tolerance to pancreatic antigens, 71 and developing islet-like cells that secrete insulin in a glucose-responsive manner for cell replacement therapy. Sourcing cells that will work on either or both fronts has historically remained a challenge. However, cells sourced from exfoliating or extracted teeth have been reported to possess potentially useful properties pertaining to both of these strategies: immunomodulation 66,72,73 and islet generation. 65 As a result, dental stem cells may represent an easily available source of stem cells for potentially both of these therapeutic approaches to type 1 diabetes. In the future, we may see patients own exfoliating or extracted teeth (heretofore discarded) serving as the cellular starting materials for treating their own diabetes. B. Spinal Cord Injury The dental pulp originates from the neural crest, 74 a structure in the developing animal that gives rise to the autonomic nervous system including the spinal cord. 75 It is therefore not a huge leap that neurons have been generated from dental stem cells, 76 including from PDLSCs, 77 DPSCs, 43 DFSCs, 78 and SHEDs. 79 More than 10 years ago, an animal model of spinal cord injury showed that dental stem cells increased the survival rates of motoneurons following injury. 64 When implanted into animals, dental pulp promotes proliferation, cell recruitment, and maturation of endogenous stem/progenitor cells by modulating the local microenvironment, 80 and also secretes factors that induce the axon guidance of endogenous neurons. 81 Continuing work since then has led to increased understanding of the techniques for inducing dental pulp into functional neural cells. 82,83 Recently, dental pulp from human exfoliating deciduous teeth and from extracted third molars were studied in an animal model of a spinal cord injury and demonstrated multifaceted abilities to mediate the inflammatory process, direct axon growth, and even provide a source of neurons for cell therapy, resulting in the gain of significant hind leg motor function. 84 Although significant hurdles remain before applying dental pulp becomes a routine clinical practice, dental stem cells may represent an easily available Figure 4. A Typical Dental Stem Cell Banking Process 8

9 Dental Stem Cells: A Guide for Dental Professionals source of stem cells for therapeutic treatments for spinal cord injuries. Compared to the earlier technology that uses embryonic stem cells, collecting dental stem cells is simpler, avoids the use of embryos, and is also less expensive. The Basics of Stem Cell Cryopreservation The ready availability, simplicity, convenience, low cost, and high potency of dental stem cells means that, as with stem cells derived from bone marrow, they may become just another logical and routine source for regenerative medicine. Given that the clinical need for stem cells and the loss of teeth typically do not happen simultaneously, there is a need to store these tissues until they are needed. This can be accomplished through cell cryopreservation, a well-established practice with, for example, bone marrow, 85 cord blood, 86 and fertilized embryos. 87 Dental stem cells from the periodontal ligament, 88 apical papilla, 89 and dental pulp 90,91 have been successfully cryopreserved while retaining their ability to differentiate into other tissue types. Dental stem cell banking works in the same manner as cord blood banking, and these are complimentary. However, there is only one opportunity for an individual s umbilical cord blood to be banked at birth. The stem cells from cord blood are used primarily for hematopoietic uses, while stem cells from dental pulp are primarily mesenchymal stem cells and neuroprogenitors thereby providing a complimentary cell type for patients. The cryopreservation of dental stem cells simply represents the application of existing technology to a new source of stem cells. After the dentist collects a tooth, it is transported to the laboratory in a sterile, isotonic solution. This package is shipped chilled to reduce the growth of contaminating microbes, and is delivered to the laboratory as quickly as possible. Although stem cells can successfully be recovered from teeth several days post-extraction, the yield declines significantly with time. 92 The laboratory must have validated processes with appropriate quality control metrics in place, verifying its ability to remove contaminating oral flora from the tooth and to recover viable stem cells. Ideally, the laboratory should have the ability to grow these cells in culture and to verify that the cultured cells exhibit the baseline set of cell surface markers expected for mesenchymal stem cells. 93 While sources of dental stem cells are readily accessible, those cells must be secured and stored properly to maintain the potential to proliferate and differentiate. AAPD policy Cryopreservation typically involves equilibrating the cells with a cryoprotectant solution, a solvent that protects the cells from the formation of ice crystals and helps preserve the integrity of cell membranes upon thawing. The temperature is typically slowly brought down to freezing using programmable controlled-rate freezers. The frozen Step 1. Decide n Decide if your practice will offer dental stem cell banking services. n Would your patients want to know? n Will you do education or tooth collection or both? Step 2. Select a Vendor Checklist: n Professional support n Patient education & support n Tooth collection & transport process n Stem cell processing, storage, reporting Step 3. Get Started n Delegate point person n Internal/external practical plan n Patient education n Tooth collection Figure 5. A Simple Guide to Help You Get Started with Dental Stem Cell Banking January

10 DENTAL LEARNING cells are then transferred to vapor-phase liquid nitrogen freezers for long-term storage at ultra-low temperatures, typically at about 150 C. Freezing drastically slows down all biochemical processes, allowing the frozen cells to remain stable indefinitely. The clinical use of cryopreserved tissues is regulated at both state and federal levels. Laboratories storing or expanding human cells for future clinical use must be registered with the FDA, 94 licensed by the health department of the state where the laboratory operates, and should be accredited by the American Association of Blood Banks and/or the American Association of Tissue Banks. Incorporation of Dental Stem Cell Services into Your Practice The American Academy of Pediatric Dentistry recognizes the emerging field of regenerative medicine and encourages dentists to follow future evidence-based literature in order to educate parents about the collection, storage, viability, and use of dental stem cells with respect to autologous regenerative therapies. AAPD policy Incorporating dental stem cell banking into any dental practice is surprisingly simple and boils down to two things: 1) informing patients that this is an option; and 2) helping patients with tooth collection. Since every practice is unique, this section is intended to provide a general guide to help you develop your own approach that works for your practice. Step 1: Decide if you will incorporate dental stem cell banking into your practice. Collectively, 20 to 30 million patients in the United States lose teeth every year many of which are intact, vital teeth that can be suitable sources from which to obtain dental stem cells. This includes exfoliating or atraumatically extracted teeth (e.g., extracted third molars, teeth pulled in relation to orthodontic procedures). The enormous number of patients eligible for this new service means that dental professionals are in a unique position to be at the forefront of this field by building awareness about the option to store stem cells from teeth and collecting teeth for stem cell banking. All dental professionals can provide information to patients and the public about this important new field. General dentists and some specialists dealing with exfoliating or extracted teeth will also be called on by their patients to assist with high-quality tooth collection. Cost considerations Different vendors provide different options for providers and patients. The costs to a dental practice may range from $0 to $90 or more per year. The cost to patients for privately banking their dental stem cells is typically around $600 for the first year plus an annual charge of roughly Table 2. Dental Professionals Play a Number of Roles from Education to Providing High-quality Service Patient/Parent Tooth Collection Specialty Education Exfoliating Ortho 3rd molars Pediatric/General/Family Dentist Orthodontist Oral Maxillofacial Surgeon Hygienist Periodontist Endodontist Dental Assistant/ Office Staff 10

11 Dental Stem Cells: A Guide for Dental Professionals $100 for cryopreservation. Some companies provide a range of pricing and financing plans that provide families with more options. For reference, dental stem cell banking is substantially less expensive than private umbilical cord blood banking which is typically $1,000 2,000 for the first year and $ per year after that. Step 2: Considerations when selecting a vendor Once you decide to offer patients the opportunity to bank their dental stem cells, the next step is to select a vendor. The AAPD s Policy on Stem Cells may help provide a useful framework during your vendor selection and evaluation process. Below are a few factors to consider: Professional support. Look for the availability of dedicated professional support to help your practice and to coordinate with your point person. This can make the difference between success and failure. Training, continuing education courses, webinars, and progress updates in the field are important for keeping current. Patient education. Look for the availability of simple and accurate tools for patient education from vendors such as informational brochures, videos, online Web tools, and newsletters. These can greatly simplify awareness building in your patient base. Consider the level of customer support you want from a vendor to assist those patients who want more information. Tooth collection and transport. The AAPD policy highlights the need for appropriate informed consent and patient privacy. Ask about how the company transports your patient s tooth to the lab while maintaining high cell viabilities. Stem cell harvesting and storage. Ultimately the cells collected may be used clinically. Ask how the company meets industry standards for human tissue banking for clinical use, and how they will provide you and or your patient with evidence that the stem cells were properly stored. Step 3: Incorporate dental stem cell services into your everyday practice. Now you are ready to incorporate dental stem cell services into your everyday practice workflows and deal with patient education and tooth collection. Patient education: Patients appreciate being informed about this option by their dental provider in time to make an informed decision about banking their own stem cells. Like any other service you provide, patients will expect the dental team to be current and provide them with information on dental stem cell banking. Accurate information becomes especially important if a patient s family has the types of diseases discussed above, such as diabetes, where dental stem cells may play an important role in their future health. These patients and their families will be particularly grateful to your team for providing them with this information. Your vendor can provide you with support and materials to help you streamline patient education. In turn, providing this information to patients is not only potentially valuable for their health, it may also help retain patients and attract new ones. The layperson already has a general idea of what stem cells are; therefore, patient education can be streamlined. For practices that see children, new patient orientation, newsletters, or hygiene visits prior to a tooth exfoliating can all be good opportunities for sharing information. When teeth need to be extracted as part of a treatment plan, the initial treatment planning visit and the extraction consultation are both examples of good times when information fits naturally into the process. This gives patients time to consider their options between when they first learn they are eligible to when they need to bring their tooth collection kit to your office. Tooth collection. Instead of being discarded, stem cells collected during the normal course of dental care can be readily cryopreserved for future clinical application. 42,49,95,96,97 As a result, dental stem cells may present the simplest, least expensive way for families and medical professionals to collect potent stem cells from each person. When it comes to tooth collection, pulpal viability should be evaluated to decide if each tooth is a good candidate for banking. Different dental stem cell banking vendors approach this issue differently. Consult with your vendor about which teeth provide high-quality banked samples. January

12 DENTAL LEARNING Evidence suggests that dental stem cells may be more proliferative the younger the tooth/patient is, although patients with teeth extracted in their 30s and beyond can still be eligible. Typically third molars yield a higher total number of cells than primary teeth about to exfoliate, especially when multiple teeth are extracted as part of the treatment plan. Getting started. As with other services in your practice, having a designated point person in the practice who can work with patients, your team, your network of referring providers, and vendors can make it easier to get started. This person can be a dentist, hygienist, office manager, patient coordinator, or experienced dental assistant anyone on the team who is enthusiastic about this service can be a good point person. Depending on your practice philosophy, this is also an opportunity to develop a participatory process where the dentists and hygienists may develop or co-develop a plan with the extended team for dental stem cell banking. Practical considerations include determining how you will let your patients, your referral network, and the outside world know that you offer this service. The public is increasingly aware of this emerging science, and more parents are expressing interest in harvesting/banking dental stem cells. AAPD policy Conclusions Interest in dental stem cells for regenerative dentistry and medicine continues to increase as more research demonstrates their viability for the potential treatment of a range of diseases and conditions. The technology to collect and store tissues that contain these cells for future use is proven and available today. Millions of people will become eligible for tooth banking this year. Dental professionals are ideally positioned to help patients by providing both education and dental stem cell banking services, thereby giving patients the opportunity to store stem cells from viable teeth for future use. Terminology Allogeneic Use: This use requires a human leukocyte antigen (HLA) match; the donor and the recipient are two different people. Autologous Use: The donor is the same person as the recipient (biological definition) or is a close blood relative (FDA definition); the implantation, transplantation, infusion, or transfer of human cells or tissue back into the individual from whom the cells or tissue were recovered ensures an HLA match. Biomarker: A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Frequently biomarkers are measurable proteins such as DNA or RNA. Cryopreservation: The process of using very low temperatures, typically around -300 F (-150 C), to keep cells preserved for future use. Differentiation: The process by which a less specialized cell becomes a more specialized cell. Flow Cytometry: A technique for measuring the presence of various biomarkers associated with cells. Hematopoietic: Blood forming. Homologous Use: The repair, reconstruction, replacement, or supplementation of a recipient s cells or tissues with those that perform the same basic function(s) in the recipient as in the donor. Human Leukocyte Antigen: The major histocompatibility complex in humans. HLAs on the surface of cells inform the immune system as to the contents of the cell (class I) and its environment (class II), and are therefore critical to the success of cell and organ therapies. Mesenchymal: The type of tissues arising from the mesoderm germ layer (e.g., myogenic or muscle forming, osteogenic or bone forming, adipogenic or fat forming, chondrogenic or cartilage forming). Multipotent: Capable of developing into many cell or tissue types. Pluripotent: A type of stem cell that has the potential to differentiate into endoderm, mesoderm, or ectoderm. Pluripotent stem cells can give rise to any fetal or adult cell type, but cannot develop into a fetal or adult animal because they lack the potential to contribute to extra-embryonic tissue, such as the placenta. 12

13 Dental Stem Cells: A Guide for Dental Professionals Regenerative Medicine: The process of creating living, functional tissues to repair or replace tissue or organ function lost due to age, disease, damage, or congenital defects. Stem Cell: An unspecialized cell that has the potential to develop into many different cell types and that, under certain conditions, can be induced to become a tissue- or organ-specific cell. Totipotent: A type of stem cell that is capable of differentiating into all cell types. Totipotent stem cells can even form a complete organism. Unipotent: Capable of developing into only one type of cell or tissue. Vitrification Agent: This is added to the cryopreservation solution prior to freezing In order to prevent cell death due to the freezing process. It reduces the formation of ice crystals that can damage cells in the freeze-to-thaw process. This improves the post-thaw viability of cells. References 1. Ramalho-Santos M, Willenbring H. On the origin of the term stem cell. Stem Cell Jun 7;1(1): Department of Health and Human Services. Regenerative Medicine. August American Academy of Pediatric Dentistry Council on Clinical Affairs. Policy on Stem Cells. Ped Dent ;30(7 Suppl):84. Revised Available at: media/policies_guidelines/p_stemcells.pdf 4. Scadden DT. The stem-cell niche as an entity of action. Nature Jun 29;441(7097): Li L, Xie T. Stem cell niche: structure and function. Annu Rev Cell Dev Biol. 2005;21: Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391): Fong CY, Gauthaman K, Bongso A. Teratomas from pluripotent stem cells: A clinical hurdle. J Cell Biochem Nov 1;111(4): Accessed January 31, Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell Aug 25;126(4): Sun N, Longaker MT, Wu JC. Human ips cell-based therapy: considerations before clinical applications. Cell Cycle Mar 1;9(5): Thomas ED, Lochte HL, Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med. 1957;257: Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet Dec 28;2(7583): Appelbaum FR. Hematopoietic-cell transplantation at 50. N Engl J Med Oct 11;357(15): Accessed September 24, Gluckman E, Broxmeyer HA, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med Oct 26;321(17): Accessed September 24, Accessed September 23, and Accessed September 23, Reimann V, Creutzig U, Kögler G. Stem cells derived from cord blood in transplantation and regenerative medicine. Dtsch Arztebl Int Dec;106(50): Committee on Obstetric Practice; Committee on Genetics. ACOG committee opinion number 399, February 2008: umbilical cord blood banking. Obstet Gynecol Feb;111(2 Pt 1): Lubin BH, Shearer WT. Cord blood banking for potential future transplantation. Pediatrics Jan;119(1): Gomillion CT, Burg KJ. Stem cells and adipose tissue engineering. Biomaterials Dec;27(36): Lendeckel S, Jödicke A, Christophis P, et al. Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: case report. J Craniomaxillofac Surg Dec;32(6): Mesimäki K, Lindroos B, Törnwall J, et al. Novel maxillary reconstruction with ectopic bone formation by GMP adipose stem cells. Int J Oral Maxillofac Surg Mar;38(3): Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA Dec 5;97(25): Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stem cells. J Dent Res Aug;81(8): Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA May 13;100(10): Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet Jul 10-16;364(9429): Sonoyama W, Liu Y, Yamaza T, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod Feb;34(2): Morsczeck C, Götz W, Schierholz J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol Apr;24(2): Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res Sep;88(9): Tziafas D, Kodonas K. Differentiation potential of dental papilla, dental pulp, and apical papilla progenitor cells. J Endod May;36(5): Yamada Y, Fujimoto A, Ito A, Yoshimi R, Ueda M. Cluster analysis and gene expression profiles: a cdna microarray system-based comparison between human dental pulp stem cells (hdpscs) and human mesenchymal stem cells (hmscs) for tissue engineering cell therapy. Biomaterials Jul;27(20): Zhang W, Walboomers XF, Van Kuppevelt TH, et al. In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages. J Tissue Eng Regen Med Mar-Apr;2(2-3): Lindroos B, Mäenpää K, Ylikomi T, Oja H, Suuronen R, Miettinen S. Characterisation of human dental stem cells and buccal mucosa fibroblasts. Biochem Biophys Res Commun Apr 4;368(2): Koyama N, Okubo Y, Nakao K, Bessho K. Evaluation of pluripotency in human dental pulp cells. J Oral Maxillofac Surg Mar;67(3): Schoenebeck B, Hartschen HJ, Schindel M, et al. Molecular characterization of human impacted third molars: diversification of compartments. Cells Tissues Organs. 2009;189(5): Song J, Park B, Byun J, et al. Isolation and characterization of human dental tissuederived stem cells in the impacted wisdom teeth: comparison of dental follicle, dental pulp, and root apical papilla-derived cells. J Korean Assoc. Oral Maxillofac Surg. 2010;36: d Aquino R, Graziano A, Sampaolesi M, et al. Human postnatal dental pulp cells codifferentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ Jun;14(6): Jo YY, Lee HJ, Kook SY, et al. Isolation and characterization of postnatal stem cells from human dental tissues. Tissue Eng Apr;13(4): Kerkis I, Kerkis A, Dozortsev D, et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs. 2006;184(3-4): Arthur A, Rychkov G, Shi S, Koblar SA, Gronthos S. Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells Jul;26(7): Nam H, Lee G. Identification of novel epithelial stem cell-like cells in human decidu- January