Stem Cells in Dentistry and Medicine: The Dentist s Role

Transcription

1 Course Number: 133 Stem Cells in Dentistry and Medicine: The Dentist s Role Authored by Paul Krasner, DDS and Peter Verlander, PhD Upon successful completion of this CE activity 2 CE credit hours may be awarded A Peer-Reviewed CE Activity by Dentistry Today, Inc, is an ADA CERP Recognized Provider. ADA CERP is a service of the American Dental Association to assist dental professionals in indentifying 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 ada.org/goto/cerp. Approved PACE Program Provider FAGD/MAGD Credit Approval does not imply acceptance by a state or provincial board of dentistry or AGD endorsement. June 1, 2009 to May 31, 2012 AGD Pace approval number: Opinions expressed by CE authors are their own and may not reflect those of Dentistry Today. Mention of specific product names does not infer endorsement by Dentistry Today. Information contained in CE articles and courses is not a substitute for sound clinical judgment and accepted standards of care. Participants are urged to contact their state dental boards for continuing education requirements.

2 Stem Cells in Dentistry and Medicine: The Dentist s Role Effective Date: 01/1/2011 Expiration Date: 01/1/2014 LEARNING OBJECTIVES After reading this article, the individual will learn: Basic biology and sources of stem cells, and basic science and clinical applications of dental stem cells in particular. Emerging role of dental professionals in enabling medical and dental applications of dental stem cells, and indicators for quality control in dental stem cell banking and cryopreservation. ABOUT THE AUTHORS Dr. Krasner is a Diplomate of the American Board of Endodontics and is a scientific advisor and reviewer for the Journal of Endodontics. He is former director of postdoctoral endodontics and is presently a clinical professor at Temple University School of Dentistry. He is an international lecturer and author of more than 80 journal articles. He has been in the private practice of endodontics for more than 35 years and currently practices in Pottstown, Pa. He can be reached at via at [email protected]. Disclosure: Dr. Krasner invented the Save-A-Tooth emergency tooth preserving system and holds a patent on a device to transport teeth destined for cryopreservation. He is a founder and advisor for Provia Laboratories, LLC. Dr. Verlander is the former associate director for strategic development of the laboratory for molecular medicine within the Harvard Medical School Partners Healthcare Center for Genetics and Genomics. He led the team that developed the first clinical test for detection of EGFR Continuing Education Recommendations for Fluoride Varnish Use in Caries Management mutations predicting therapeutic response in lung adenocarcinoma. He is a molecular geneticist with 15 years of experience in various aspects of human genetics in both academia and industry, including research at The Rockefeller University in New York on the molecular genetics of Fanconi anemia, a DNA breakage disorder leading to bone marrow failure and increased risk of leukemia. He can be reached at [email protected]. Disclosure: Dr. Verlander is a founder and chief scientific officer of Provia Laboratories, LLC. INTRODUCTION New developments in stem cell research have created an environment in which dentists will be in the position to assume a leading role in the treatment chain of medical disease. Research at various institutions, including the National Institute of Dental and Craniofacial Research (NIDCR) at the NIH, has shown that mesenchymal stem cells (MSCs) can be obtained from a variety of odontogenic tissues including the dental pulp of third molars 1 and exfoliating deciduous teeth, 2 the periodontal ligament, 3 the apical papilla of extracted immature teeth, 4 and the dental follicle. 5 In the United States, the sheer volume of exfoliating deciduous teeth (80 million/year), extracted premolars (1.5 million/year), and extracted third molars (1.3 million/year) 6 presents a readily-available source of stem cells for the treatment of both dental and medical conditions. One of the barriers to the widespread use of stem cells on a routine basis has been the lack of easy accessibility to them, and the discovery of a source of easily-obtained postnatal stem cells in teeth provides one possible solution to this problem. Dental stem cells have demonstrated proof of concept in 2 human clinical studies, 7,8 and a broad range of clinical applications is emerging that could potentially benefit a majority of the population. Therefore, dentists may become a critical first link in the chain of collecting dental stem cells, to be used not just for the treatment of dental pathoses, but also for medical disease. In order for dentists to assume this new role in the healthcare chain, they must be knowledgeable about the biology of stem cells and the potential applications of dental stem cells in clinical practice, and be familiar with dental stem cell banking services. 1

3 The purpose of this paper is to: 1. Provide an understanding of the basic biology and sources of stem cells. 2. Review the basic science and clinical applications of dental stem cells in particular. 3. Discuss the emerging roles of dental professionals in enabling both medical and dental applications using stem cells from teeth. 4. Provide an understanding of the indicators for quality control in dental stem cell banking and cryopreservation services. stem cell cryopreservation: educating patients and/or their parents about the existence and use of dental stem cells, and helping them select an appropriate approach to collecting and preserving these stem cells. In addition, oral surgeons and pediatric dentists indeed, any dentist involved in extracting teeth may also be called on to assist with the collection of the teeth for cryopreservation. Patients now have a new choice for the use of these teeth to preserve their own stem cells in the anticipation that these cryopreserved stem cells may prove valuable for their dental or medical health in the future. THE CHANGING ROLE OF DENTISTS Medicine and dentistry are on the cusp of a new treatment modality. For both physicians and dentists to take advantage of this new modality, not only do the clinical applications need to be further developed, but sources of stem cells from teeth will be necessary. Similar to umbilical cord blood, teeth are one of the few tissues in the body that are naturally shed (or extracted during the normal course of dental care) that contain potent stem cells, creating an opportunity to save this tissue for when it is needed. Dentists and patients need to be aware that the option exists to preserve the stem cells from healthy extracted or exfoliating teeth as a resource for these future clinical applications (Figure 1). In 2008, the American Academy of Pediatric Dentistry published a policy on dental stem cells and dental stem cell banking. 9 This policy emphasizes the importance of the use of dental stem cells in dentistry, and demonstrates how dentists are poised to take a leadership position in this new emerging field. Patients increasingly want to know more information about dental stem cell banking and procedures using dental stem cells. The dentist will have a dual role to play in dental Figure 1. Teeth commonly removed in the course of normal dental care that contain stem cells. BIOLOGY OF STEM CELLS The term stem cell has roots as far back as Stem cells are generally defined as clonogenic cells with the capacity to both self-renew and give rise to differentiated cells Stem cells can be thought of as the building blocks of the body. Whereas most cells in the body, eg, muscle cells or red blood cells, are specialized cells that can form only the exact same type of cell and only perform narrow biological functions, stem cells are versatile and can develop into a number of different cell types. The layperson often associates the term stem cells 2

4 with embryonic stem cells, but there are also noncontroversial adult stem cells that can be collected from various tissue niches within the body and which retain important stem cell properties. In fact, adult stem cells, in particular from bone marrow or umbilical cord blood, have been used clinically for decades as discussed in the following section. SOURCES OF STEM CELLS As reviewed in this section, adult stem cells are typically characterized as either hematopoietic (capable of forming all types of white blood cells and red cells) or mesenchymal (capable of forming a wide variety of connective tissues such as bone, muscle, cartilage, fat, tendons but not internal organs or skin). Hematopoietic stem cells have been routinely used to treat blood cancers such as leukemia and other blood disorders. Bone marrow contains both types of stem cells and has been most extensively researched. Umbilical cord blood is another source of hematopoietic stem cells. Both deciduous teeth and wisdom teeth contain MSCs but not hematopoietic stem cells, as does adipose (fat) tissue. Bone Marrow Stem Cells The most commonly known procedure involving stem cells is the bone marrow transplant, first used by Dr. E. Donnall Thomas in the late 1950s to treat humans; early studies involved using bone marrow from one identical twin to attempt treatment of the other twin who had leukemia. 14 By the late 1960s, further research involving how to address graft versus host disease and other clinical advancements led to successful transplants between nontwins. 15 In 1990, Dr. Thomas was awarded the Nobel Prize in Medicine for his pioneering work in bone marrow transplantation. 16 Today, more than 50,000 bone marrow transplants are performed annually. 17 More than 1,500 clinical trials related to bone marrow are planned, ongoing, or have been completed, which study diseases ranging from leukemia to spinal cord injury. 18 Approximately 120 of these studies are focused on MSCs from bone marrow; the most advanced studies (Phase III trials) of MSCs from bone marrow are focused on the treatment of diabetes and associated conditions, Crohn s disease, graft versus host disease, bone defects, or cartilage injury. 18 A study specific to dentistry involved MSCs from bone marrow aspirate concentrate plus scaffold to regenerate hard tissueenabling dental implants. 19 Cord Blood Stem Cells In the mid 1980s, scientists proposed that hematopoietic stem cells in umbilical cord blood, resembling those in bone marrow, could be used when a bone marrow transplant was not available; in 1988 the first cord blood stem cell transplant was used to treat a young boy with Fanconi s anemia 20 (who remains alive and well to this date 21 ). The properties of cord blood stem cells to treat bloodrelated diseases and also to be readily collected and cryopreserved for future use formed the basis for the umbilical cord blood banking industry. Today, more than 9,300 patients have been treated with umbilical cord blood, and more than 200,000 units are publicly banked. 22 In addition to public banks, parents of more than 550,000 newborns have privately banked their child s umbilical cord blood at birth 23 for its proven use in treating leukemia and genetic blood disorders 24 and also for its potential to treat other diseases such as myocardial infarction, diabetes mellitus, and neurological disorders. 22 Adipose Tissue Stem Cells MSCs can be isolated from adipose tissue obtained from liposuction aspirates or abdominoplasty procedures, and are being studied for repairing tissue defects resulting from traumatic injury, tumor resection, and congenital defects, 25 calvarial defects following severe head injury, 26 and in dentistry for repair of jaw bone. 27 Embryonic Stem Cells In 1998, scientists derived cells from frozen human embryos to generate human embryonic stem cells for the first time. 28 Many scientists and clinicians were enthusiastic that embryonic stem cells would provide a way to cure 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 3

5 of the embryo, or therapeutic cloning, and whether federal funding should be available to support this field. Second, there are significant medical and engineering challenges to using pluriopotent stem cells including inadequate cell numbers, immunorejection (caused by a mismatch of the immune systems of the donor and recipient), and tumorigenesis (the pluripotent nature of embryonic stem cells leads to teratomas tumors containing one or more of the 3 embryonic germ tissue layers). 29 Induced Pluripotent Stem Cells In 2006, it was discovered that by using genetic engineering techniques, unipotent cells could be reprogrammed back into cells that resemble pluripotent embryonic stem cells. 30 While this technology avoids the ethical issues of embryonic stem cells and uses the patient s own tissues, thus reducing immunologic incompatibility, induced pluripotent stem cells cells have also been shown to cause teratomas and are expensive to generate. 31 Dental Stem Cells A new source of readily available MSCs teeth was discovered in 2000 by scientists at the NIH. 1 Stem cells can be collected from deciduous teeth when they naturally exfoliate from approximately age 6 to 11 years, and also from teeth that are surgically removed, such as premolars for orthodontia and third molars during wisdom teeth extraction. Dental stem cells allow for autologous use, meaning the adult stem cells can be collected from and used on the same person, so there are no issues of immunological incompatibility. It may seem remarkable that stem cells can be found in such an everyday source as exfoliating or extracted teeth, but as early as 1965, scientists studying pulpal tissues from children s premolars had noticed interesting properties of the pulp. 32 In fact, scientists have studied dental pulp in vitro and in vivo for more than 70 years. 33 Many of the nowmature tools and techniques developed during the past half century to study adult and embryonic stem cells apply directly to dental stem cells. As a result, the field has advanced quickly. Continuing Education Dental Follicle Stem Cells Stem Cells from Apical Papilla Figure 2. Dental stem cell niches. REVIEW OF BASIC RESEARCH OF DENTAL STEM CELLS Niches of Dental Stem Cells Dental stem cells reside in multiple niches 34 of deciduous and Dental Pulp Stem Cells Periodontal Ligament Stem Cells permanent teeth (Figure 2). The first conclusive study demonstrating the presence of stem cells in teeth was published in 2000 by dental scientists at the NIDCR. 1 Suspecting that a stem cell population was responsible for the similarities between dental pulp and bone marrow, they identified MSCs in the pulp of extracted third molars. Termed dental pulp stem cells (DPSC), these stem cells are more prolific than bone marrow MSCs, and were shown to generate calcified nodules and neuron-like cells. 1,35 Stem cells from human exfoliated deciduous teeth (SHED), 2 periodontal ligament stem cells (PDLSC), 3 Stem cells from apical papilla (SCAP), 4 and dental follicle stem cells (DFSC) 5 were subsequently identified. Collectively, these dental stem cells are currently being investigated for numerous scientific and clinical uses summarized in the next section. Characteristics of Dental Stem Cells All of these sources of dental stem cells display typical mesenchymal stem cell characteristics Overall, dental stem cells have the capacity to generate a broad range of mesenchymal tissue or cell types in cell culture or in animal studies including dentin-producing odontoblasts, adipocytes, osteoblasts, bone, cartilage, and smooth and skeletal muscle. 1,35,44-46 Additionally, dental stem cells either contain or can switch lineage to form ectodermal tissues such as neurons, 47 or epithelial-like stem cells. 48 There are also 4

6 reports of dental stem cells giving rise to cells typically of the endodermal lineage, such as endothelial cells, 44 hepatocytes, 49 and insulin-producing cells. 50 In addition, populations of dental stem cells have been identified which express embryonic stem cell markers. 46,51,52 Taken together, this research suggests that the stem cells found associated with teeth contain a hierarchy of highly potent pluripotent stem cells, multipotent mesenchymal, endodermal, and ectodermal stem cells, and more committed progenitors amongst fully differentiated cells. In simpler terms, teeth have now been proven to contain stem cells with similar capabilities to, but without many of the challenges of, embryonic stem cells and this has important implications for tissue banking and clinical use. Dental Stem Cells Normal Biology The existence of stem cells in teeth is a robust phenomenon and required for odontogenesis. Early in fetal development, teeth arise from the neural crest through a series of interactions between neural, mesenchymal, and epithelial tissues. 53,54 The developed tooth can be thought of as an encapsulated population of quiescent stem cells. 52,55 The finding of stem cells in natal teeth, 56 supernumerary teeth, 57 and odontoma 58 reinforces the concept that stem cells play a key role in organogenesis of every tooth. It has also been shown that the pluripotency of dental stem cells may be a function of the age of the tooth or the age of the donor. 59 In other words, primary teeth, molars, and wisdom teeth of young adults all contain potent sources of dental stem cells. teeth can be readily cryopreserved for future clinical application. Relevant for the everyday practice of dentistry, stem cells have been identified in diseased or injured teeth such as primary incisors extracted because of disease, 62 crownfractured adult teeth resulting from trauma, 63 and inflamed dental pulp. 64 An exploratory study demonstrated that even in the permanent teeth of 6 to 40 years old which were diagnosed with irreversible pulpitis necessitating root canal treatment or pulpotomy, there are endogenous stem cells (albeit at lower numbers and proliferative capacity due to disease) that could potentially be utilized in pulp therapy. 65 In subsequent years, dentists will not only be using the knowledge of stem cells to develop new treatment methodologies, implants, and restorative materials, but also will be helping patients save their own dental stem cells for medical and dental applications. REVIEW OF CLINICAL APPLICATIONS FOR DENTAL STEM CELLS Dental stem cells have already been used in the clinic to treat humans. These and additional clinical applications will continue to emerge in the near term and longer term. This section is intended to provide a brief overview of the tremendous potential of dental stem cells in clinical dentistry and medicine (Figure 3). Current Uses of Dental Stem Cells Alveolar Bone Regeneration Only 9 years after the first Practical Aspects of Dental Stem Cells In addition to the above sources of dental stem cells in extracted third molars and exfoliating deciduous teeth, every year millions of healthy teeth that possess stem cells are routinely discarded as medical waste in the course of normal dental care, eg, from extracted deciduous teeth and permanent teeth 46,60,61 and the developing tooth germ of impacted third molars. 52 Instead of discarding these sources, stem cells from these patients Figure 3. Clinical applications of dental stem cells. 5

7 published literature involving dental pulp stem cells, dental stem cells were used in humans to regenerate dental bone in human clinical studies. 7 Defects of at least 1.5 cm in the alveolar ridge of 17 human volunteers were filled with a construct of stem cells collected from third molars and seeded onto a collagen matrix. One year later in many cases, the gap was filled with bone. Periodontal Ligament Following research showing that human periodontal ligament stem cells contribute to periodontal tissue repair in both rat 3 and swine, 66 Feng, et al 8 demonstrated clinical and experimental evidence supporting the safe and efficacious use of autologous PDL cells to treat periodontitis in 3 humans in a multiyear study. Regenerative Dentistry Regenerative dentistry is based in part on the concept that stem cells will become increasingly useful in dentistry; strategies include using the patient s stem cells in situ to stimulate healing, or the use of laboratory-cultured stem cells with or without tissue engineering scaffolds. 67 A recent survey of practicing endodontists found that more than half of those surveyed thought that stem cell banking would be useful to regenerate dental tissues, and almost 90% would be willing to save teeth and dental tissues for banking. 68 Promising examples of using stem cells in dentistry include the conservative endodontic treatments of immature teeth, 69 pulp regeneration, 70 evaluation of bone formation on dental implants, 71 evaluation of cytotoxicity of resinbased sealers, 72 and for drug screening. 73 Clearly, the phenomenon of dental stem cells is already starting to affect the practice of dentistry. Near Future Pulp Regeneration The use of stem cells to regenerate dental pulp tissues is being studied as an alternative method to conventional root canal treatment. 74,75 Cranial Defects Human dental stem cells isolated from deciduous teeth were useful for correcting large cranial defects in rats, providing a promising model for reconstruction of large cranial defects in craniofacial surgery. 76 Lamellar Bone Stem cells from the dental pulp have been used to produce bone chips in cell culture with implantation of this construct into an animal model resulting in vascularized lamellar bone. 44 This demonstrates how the use of dental stem cells may impact both dentistry and medicine. Long-Term Third dentition (bioengineered teeth) A method has been developed to regenerate tooth buds in a single procedure by combining dental pulp and bone marrow on a scaffold and implanting this into surgically created defects. After a number of months, the construct led to organized dentin, enamel, pulp, cementum, and periodontal ligament surrounded by regenerated alveolar bone, suggesting a method that could translate directly to humans. 77 Since periodontal disease is the primary cause of tooth loss in adults aged 35 years and older 78 and approximately 25% of those more than age 65 years have lost all their teeth, 79 dental applications alone may justify the banking and use of dental stem cells for future uses by millions of people. Cornea Based upon similarities of human dental stem cells with limbal cells in the eye, 80 human dental stem cells were used to successfully treat an animal model for cornea damage by chemical burn. 81 Liver Disease Stem cells from third molars were differentiated into hepatocytes in cell culture, and in an animal model of liver disease, they prevented liver fibrosis and increased levels of albumin and billirubin. 82 Myocardial Infarction (heart attacks) Human dental stem cells injected intramyocardially into a rat model of acute myocardial infarction showed an increase in angiogenesis, improvement in cardiac function, and a reduction in infarct size. 83 More recent work studying the application of induced pluripotent stem cell techniques to dental pulpal cells demonstrated the possibility of creating functional (beating) cardiomyocytes. 84 Muscular Dystrophy The best animal model for muscular dystrophy is the golden retriever muscular dystrophy (GRMD) dogs. Four GRMD dogs were treated with human dental stem cells via intraarterial or intramuscular injection. No signs of cross species immune rejection were detected, and at least one dog remained clinically stable for at least 25 months longer than expected. 85 Diabetes The discovery and use of insulin by Banting, et al 86 is one of the major breakthroughs in medical history. However, as Fredrick Banting himself recognized in his Nobel Lecture delivered in 1925, Insulin is not a cure for 6

8 diabetes; it is a treatment. The use of stem cells to treat diabetes is focused on 2 fronts: developing cells that secrete insulin in a glucose-responsive manner, and using MSCs to regulate the immune response by inducing tolerance to pancreatic antigens. 87 Dental stem cells have been shown to produce insulin 50 and to modulate the immune system by suppressing T-cell response in laboratory and animal testing Dental stem cells therefore represent an easily available source of stem cells for potentially both of these therapeutic approaches to diabetes. Stroke Neuronal stem cells from human third molars were used to treat a rat model of middle cerebral artery exclusion (induced stroke). The treatment group showed decreased neurologic dysfunction relative to those who were not treated. 91 Spinal Cord Injury and Other Neurological Diseases/ Disorders The dental pulp originates from the neural crest, a structure in the developing animal that gives rise to the autonomic nervous system including the spinal cord. Neurons have been generated from dental stem cells, 92 including from PDLSC, 93 DPSC, 94 DFSC, 95 and SHED. 95 In animal models, dental stem cells have been found to secrete factors that attract axon growth 96 and lead to increased survival of motorneurons in spinal cord injury. 97 Altogether, the ability of the dental pulp to mediate the inflammatory process, direct axon growth, and even provide a source of neurons for cell therapy suggest that dental stem cells could play an important role in treating human spinal cord injury, as well as neurodegenerative diseases such as Parkinson s disease. THE POTENTIAL OF DENTAL STEM CELLS It is clear from this brief overview that there is tremendous clinical potential for the use of dental stem cells in treatments involving not just dentistry, but many medical applications. More than 30 million people in the United States this year will have teeth removed or exfoliated most of them children many of whom will be affected by one of the diseases or injuries listed above in their lifetime. Patients need to know the facts so they can make an educated decision about what they should do with the stem cells found in their teeth, which today most dentists simply discard as medical waste. A clear challenge in the field of dental stem cells is the need to have more dentists understand the value of these cells and the important role dentists can play in educating their patients. As the field grows, dental stem cells may become a key enabler of regenerative medicine, thereby expanding and enhancing the role of dentists in overall healthcare. THE SCIENCE OF STEM CELL BANKING Stem Cell Cryopreservation The practice of cryopreserving and later using cells for clinical use is well established. Bone marrow, cord blood, and fertilized embryos have been routinely preserved for decades. For example, cord blood began being preserved for clinical use immediately after its potential for therapeutic use was reported 98 and has successfully been recovered from longterm storage. 99 Therefore, the cryopreservation of dental stem cells simply represents the application of existing technology to a new source of stem cells. In fact, commercial dental stem cell banking services are now available. Figure 4. The process of dental stem cell banking. 7

9 The concept of banking dental stem cells for future clinical uses was considered shortly after their discovery. Independent groups have reported the ability to successfully cryopreserve stem cells from both periodontal ligament 100 and dental pulp, 101,102 while retaining their ability to differentiate into other tissue types. This has recently been extended to isolated SCAP, 89 and multiple groups have reported the ability to recover viable stem cells from the pulp and periodontal ligament of intact frozen teeth Continuing Education The Process of Banking Dental Stem Cells After the tooth or teeth sample is collected by the dentist, the sample is transported from the dentist office to the laboratory (Figure 4). Cells should be transported in a sterile, isotonic solution, shipped chilled to reduce the growth of contaminating microbes, and 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. 103 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 MSCs 106 (Figure 5). Cryopreservation of cells typically involves equilibrating the cells with a cryoprotectant solution a solvent that protects the cells from the formation of ice crystals and that helps preserve the integrity of cell membranes upon thawing. The temperature is typically slowly brought down to freezing using programmable controlled-rate freezers. Frozen cells are then transferred to vapor-phase liquid nitrogen freezers for long-term storage at ultra-low temperatures, typically at about -150 C. The clinical use of cryopre- Figure 5. Biomarker Validation. served tissues is regulated at the state and federal level. Laboratories storing or expanding human cells for future clinical use must be registered with the US Food and Drug Administration, 107 licensed by the department of health 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. SUMMARY Research has shown that teeth are a source of high quality stem cells that may be used for the treatment of medical and dental disease. The discovery that odontogenic tissues are a source of adult stem cells has opened up a new role for dentists in the field of medicine. Dentists are positioned to become one of the key providers of stem cells, and as a result, their linkage with the medical field will become very intimate. Dental stem cells have the potential to be used in the treatment of a full range of oral pathoses. Dentists can be involved in the extraction, collection, and storage of the stem cells from their patients teeth. Ongoing research suggests that these stem cells will soon be used for dental purposes such as to replace lost bone around teeth, periodontal ligament or dental pulp; treat periodontal disease; and someday even produce new teeth, as well as for medical applications. In order for dentists to fully participate in this new role, they should become aware of the applications, clinical use, and banking of dental stem cells. 8

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Molecular characterization of human impacted third molars: diversification of compartments. Cells Tissues Organs. 2009;189: Song J, Park B, Byun J, et al. Isolation and characterization of human dental tissue-derived stem cells in the impacted wisdom teeth: comparison of dental follicle, dental pulp, and root apical papilla-derived cells. J Korean Assoc Maxillofac Plast Reconstr Surg. 2010;36: d Aquino R, Graziano A, Sampaolesi M, et al. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ. 2007;14: Jo YY, Lee HJ, Kook SY, et al. Isolation and characterization of postnatal stem cells from human dental tissues. Tissue Eng. 2007;13: 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, et al. Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells. 2008;26: Nam H, Lee G. Identification of novel epithelial stem celllike cells in human deciduous dental pulp. Biochem Biophys Res Commun. 2009;386: Ishkitiev N, Yaegaki K, Calenic B, et al. Deciduous and permanent dental pulp mesenchymal cells acquire hepatic morphologic and functional features in vitro. J Endod. 2010;36: Huang CY, Pelaez D, Dominguez-Bendala J, et al. Plasticity of stem cells derived from adult periodontal ligament. Regen Med. 2009;4: Siqueira da Fonseca SA, Abdelmassih S, de Mello Cintra Lavagnolli T, et al. Human immature dental pulp stem cells contribution to developing mouse embryos: production of human/mouse preterm chimaeras. Cell Prolif. 2009;42: Yalvac ME, Ramazanoglu M, Rizvanov AA, et al. Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis. Pharmacogenomics J. 2010;10: Sharpe PT. Neural crest and tooth morphogenesis. Adv Dent Res. 2001;15: Goldberg M, Smith AJ. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med. 2004;15: Graziano A, d Aquino R, Laino G, et al. Dental pulp stem cells: a promising tool for bone regeneration. Stem Cell Rev. 2008;4: Karaöz E, Doğan BN, Aksoy A, et al. Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol. 2010;133: Huang AH, Chen YK, Lin LM, et al. Isolation and characterization of dental pulp stem cells from a supernumerary tooth. J Oral Pathol Med. 2008;37: Song JS, Stefanik D, Damek-Poprawa M, et al. Differentiation and regenerative capacities of human odontoma-derived mesenchymal cells. Differentiation. 2009;77: Takeda T, Tezuka Y, Horiuchi M, et al. Characterization of dental pulp stem cells of human tooth germs. J Dent Res. 2008;87: Nakamura S, Yamada Y, Katagiri W, et al. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod. 2009;35: Govindasamy V, Abdullah AN, Ronald VS, et al. Inherent differential propensity of dental pulp stem cells derived from human deciduous and permanent teeth. J Endod. 2010;36: Coppe C, Zhang Y, Den Besten PK. Characterization of primary dental pulp cells in vitro. Pediatr Dent. 2009;31: Huang AH, Chen YK, Chan AW, et al. Isolation and characterization of human dental pulp stem/stromal cells from nonextracted crown-fractured teeth requiring root canal therapy. J Endod. 2009;35: Alongi DJ, Yamaza T, Song Y, et al. Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential. Regen Med. 2010;5: Wang Z, Pan J, Wright JT, et al. Putative stem cells in human dental pulp with irreversible pulpitis: an exploratory study. J Endod. 2010;36: Liu Y, Zheng Y, Ding G, et al. Periodontal ligament stem cellmediated treatment for periodontitis in miniature swine. Stem Cells. 2008;26: Rimondini L, Mele S. Stem cell technologies for tissue regeneration in dentistry. Minerva Stomatol. 2009;58:

12 68. Epelman I, Murray PE, Garcia-Godoy F, et al. A practitioner survey of opinions toward regenerative endodontics. J Endod. 2009;35: Huang GT. A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration. J Dent. 2008;36: ClinicalTrials.gov. Tissue characterization in teeth treated with a regeneration protocol. NCT Accessed November 1, Mangano C, De Rosa A, Desiderio V, et al. The osteoblastic differentiation of dental pulp stem cells and bone formation on different titanium surface textures. Biomaterials. 2010;31: Trubiani O, Caputi S, Di Iorio D, et al. The cytotoxic effects of resin-based sealers on dental pulp stem cells. Int Endod J. 2010;43: Okamoto Y, Sonoyama W, Ono M, et al. Simvastatin induces the odontogenic differentiation of human dental pulp stem cells in vitro and in vivo. J Endod. 2009;35: Gotlieb EL, Murray PE, Namerow KN, et al. An ultrastructural investigation of tissue-engineered pulp constructs implanted within endodontically treated teeth. J Am Dent Assoc. 2008;139: ClinicalTrials.gov. Regenerative endodontic techniques. clinicaltrials.gov/ct2/show/nct ?term=regenera tive+endodontic+techniques&rank =1. Accessed November 1, de Mendonça Costa A, Bueno DF, Martins MT, et al. Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells. J Craniofac Surg. 2008;19: Zhang W, Abukawa H, Troulis MJ, et al. Tissue engineered hybrid tooth-bone constructs. Methods. 2009;47: American Academy of Periodontology. Fallacies about gum disease. perio.org/consumer/f1.html. Accessed November 1, Centers for Disease Control and Prevention, Health Data Interactive. Edentulism (loss of all natural teeth), ages 18+: US, (Source: NHIS). ReportFolders/reportFolders.aspx. (Search: edentulism. ) Accessed November 1, Monteiro BG, Serafim RC, Melo GB, et al. Human immature dental pulp stem cells share key characteristic features with limbal stem cells. Cell Prolif. 2009;42: Gomes JA, Geraldes Monteiro B, Melo GB, et al. Corneal reconstruction with tissue-engineered cell sheets composed of human immature dental pulp stem cells. Invest Ophthalmol Vis Sci. 2010;51: Ikeda E, Yagi K, Kojima M, et al. Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease. Differentiation. 2008;76: Gandia C, Armiñan A, García-Verdugo JM, et al. Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction. Stem Cells. 2008;26: Oda Y, Yoshimura Y, Ohnishi H, et al. Induction of pluripotent stem cells from human third molar mesenchymal stromal cells. J Biol Chem. 2010;285: Kerkis I, Ambrosio CE, Kerkis A, et al. Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: local or systemic? J Transl Med. 2008;6: Banting FG, Best CH, Collip JB, et al. Pancreatic extracts in the treatment of diabetes mellitus. Can Med Assoc J. 1922;12: Furth ME, Atala A. Stem cell sources to treat diabetes. J Cell Biochem. 2009;106: Pierdomenico L, Bonsi L, Calvitti M, et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation. 2005;80: Ding G, Wang W, Liu Y, et al. Effect of cryopreservation on biological and immunological properties of stem cells from apical papilla. J Cell Physiol. 2010;223: Yamaza T, Kentaro A, Chen C, et al. Immunomodulatory properties of stem cells from human exfoliated deciduous teeth. Stem Cell Res Ther. 2010;1: Yang KL, Chen MF, Liao CH, et al. A simple and efficient method for generating Nurr1-positive neuronal stem cells from human wisdom teeth (tnsc) and the potential of tnsc for stroke therapy. Cytotherapy. 2009;11: Nosrat IV, Smith CA, Mullally P, et al. Dental pulp cells provide neurotrophic support for dopaminergic neurons and differentiate into neurons in vitro; implications for tissue engineering and repair in the nervous system. Eur J Neurosci. 2004;19: Widera D, Grimm WD, Moebius JM, et al. Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery. Stem Cells Dev. 2007;16: Arthur A, Rychkov G, Shi S, et al. Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells. 2008;26: Morsczeck C, Völlner F, Saugspier M, et al. Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro. Clin Oral Investig. 2010;14: Arthur A, Shi S, Zannettino AC, et al. Implanted adult human dental pulp stem cells induce endogenous axon guidance. Stem Cells. 2009;27: Nosrat IV, Widenfalk J, Olson L, et al. Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury. Dev Biol. 2001;238: Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA. 1989;86: Broxmeyer HE, Srour EF, Hangoc G, et al. High-efficiency 11

13 recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years. Proc Natl Acad Sci U S A. 2003;100: Seo BM, Miura M, Sonoyama W, et al. Recovery of stem cells from cryopreserved periodontal ligament. J Dent Res. 2005;84: Papaccio G, Graziano A, d Aquino R, et al. Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol. 2006;208: Zhang W, Walboomers XF, Shi S, et al. Multilineage differentiation potential of stem cells derived from human dental pulp after cryopreservation. Tissue Eng. 2006;12: Woods EJ, Perry BC, Hockema JJ, et al. Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology. 2009;59: Lee SY, Chiang PC, Tsai YH, et al. Effects of cryopreservation of intact teeth on the isolated dental pulp stem cells. J Endod. 2010;36: Kaku M, Kamada H, Kawata T, et al. Cryopreservation of periodontal ligament cells with magnetic field for tooth banking. Cryobiology. 2010;61: Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8: Code of Federal Regulations. Title 21: Food and Drugs. Part 1271 Human Cells, Tissues, and Cellular and Tissue-Based Products. access.gpo.gov/nara/cfr/waisidx_10/21cfr1271_10.html. Accessed November 1,

14 POST EXAMINATION INFORMATION To receive continuing education credit for participation in this educational activity you must complete the program post examination and receive a score of 70% or better. Traditional Completion Option: You may fax or mail your answers with payment to Dentistry Today (see Traditional Completion Information on following page). All information requested must be provided in order to process the program for credit. Be sure to complete your Payment, Personal Certification Information, Answers, and Evaluation forms. Your exam will be graded within 72 hours of receipt. Upon successful completion of the postexam (70% or higher), a letter of completion will be mailed to the address provided. Online Completion Option: Use this page to review the questions and mark your answers. Return to dentalcetoday.com and sign in. If you have not previously purchased the program, select it from the Online Courses listing and complete the online purchase process. Once purchased the program will be added to your User History page where a Take Exam link will be provided directly across from the program title. Select the Take Exam link, complete all the program questions and Submit your answers. An immediate grade report will be provided. Upon receiving a passing grade, complete the online evaluation form. Upon submitting the form your Letter Of Completion will be provided immediately for printing. General Program Information: Online users may log in to dentalcetoday.com any time in the future to access previously purchased programs and view or print letters of completion and results. POST EXAMINATION QUESTIONS 1. The defining characteristics of stem cells are their ability to: a. Self-renew and give rise to differentiated cells. b. Regenerate cell membranes. c. Produce limited numbers of new cells. d. Only produce healthy cells. 2. Mesenchymal stem cells (MSCs) can be obtained from which of the following odontogenic tissues? a. Deciduous teeth. b. Apical papilla of third molars. c. Dental pulp of third molars. 3. Which tissue, besides teeth, contains stem cells that are naturally shed? a. Corneal. b. Epithelial skin. c. Umbilical cord blood. d. Nasal mucosa. 4. The most commonly performed stem cell procedure is: a. Corneal reconstruction. b. Coronary artery bypass graft. c. Kidney transplant. d. Bone marrow transplant. 5. The presence of stem cells in teeth was first conclusively demonstrated in: a b c d Which of the following are dental stem cells? a. Dental pulp stem cells. b. Stem cells from human exfoliated deciduous teeth. c. Periodontal ligament stem cells. 7. Dental stem cells can be collected from: a. Extracted third molars. b. Exfoliating deciduous teeth. c. Healthy extracted deciduous teeth. 8. Dental stem cells have the capacity to generate which of the following? a. Odontoblasts. b. Adipocytes. c. Bone. 13

15 9. Dental pulp contains which of the following subtypes of progenitor cells? a. MSCs. b. Embryonic-like stem cells. c. Neurogenic progenitor cells. 10. Dental stem cells have been used clinically in humans for: a. Tooth enamel replacement. b. Alveolar bone regeneration. c. Palate defect repair. d. Maxillary sinus repair. 11. Dental stem cells have been shown to produce: a. Insulin. b. Honadotropins. c. Acetylcholine. d. Dopamine. 12. Dental uses of dental stem cells may include: a. Treating periodontal disease. b. Pulp regeneration. c. Engineering new teeth. 14. Which of the following has/have been successfully cryopreserved? a. Umbilical cord blood. b. Bone marrow. c. Dental pulp. 15. During cryopreservation of stem cells, a cryoprotectant is used to prevent: a. Crushing of the cells. b. The formation of large ice crystals which can damage cell membranes. c. Accumulation of surface oils from the cells. d. Cell adhesion. 16. The laboratory that performs tooth cryopreservation for consumer tissue banking should have which of the following capabilities? a. Ability to remove microorganisms from the tooth prior to banking. b. Ability to grow cells in culture in a US Food and Drug Administration-compliant facility. c. Ability to evaluate cell biomarkers for the presence of stem cells. 13. Human dental stem cells have been used in animals to treat: a. Corneal damage. b. Muscular dystrophy. c. Myocardial infarction. 14

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