Stem Cell Research: Applicability in Dentistry

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1 Stem Cell Research: Applicability in Dentistry Shivani Mathur, MDS 1 /Rahul Chopra, MDS 2 /I. K. Pandit, BDS, MDS 3 / Nikhil Srivastava, BDS, MDS 4 /Neeraj Gugnani, BDS, MDS 4 In the face of extraordinary advances in the prevention, diagnosis, and treatment of human diseases, the inability of most tissues and organs to repair and regenerate after damage is a problem that needs to be solved. Stem cell research is being pursued in the hope of achieving major medical breakthroughs. Scientists are striving to create therapies that rebuild or replace damaged cells with tissues grown from stem cells that will offer hope to people suffering from various ailments. Regeneration of damaged periodontal tissue, bone, pulp, and dentin is a problem that dentists face today. Stem cells present in dental pulp, periodontal ligament, and alveolar bone marrow have the potential to repair and regenerate teeth and periodontal structures. These stem cells can be harvested from dental pulp, periodontal ligament, and/or alveolar bone marrow; expanded; embedded in an appropriate scaffold; and transplanted back into a defect to regenerate bone and tooth structures. These cells have the potential to regenerate dentin, periodontal ligament, and cementum and can also be used to restore bone defects. The kind of scaffold, the source of cells, the type of in vitro culturing, and the type of surgical procedure to be used all require careful consideration. The endeavor is clearly multidisciplinary in nature, and the practicing dental surgeon has a critical role in it. Playing this role in the most effective way requires awareness of the huge potential associated with the use of stem cells in a clinical setting, as well as a proper understanding of the related problems. Oral Craniofac Tissue Eng 2012;2: doi: /octe.0052 Key words: dental pulp stem cells, dentistry, human cementum derived cells, periodontal ligament stem cells, stem cells from human exfoliated deciduous teeth, stem cells from root apical papilla Research on stem cells is based on the knowledge of how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. This promising area of science is also leading scientists to investigate the possibility of cell-based therapies for the treatment 1 Senior Resident, Department of Pedodontics and Preventive Dentistry, Government Dental College, Rohtak, Haryana, India. 2 Senior Resident, Department of Periodontics, Government Dental College, Rohtak, Haryana, India. 3 Professor and Head, Department of Pedodontics and Preventive Dentistry, D.A.V. (C) Dental College and Hospital, Yamuna Nagar, India. 4 Professor, Department of Pedodontics and Preventive Dentistry, D.A.V. (C) Dental College and Hospital, Yamuna Nagar, India. Correspondence to: Dr Shivani Mathur, Department of Pedodontics and Preventive Dentistry, Government Dental College, Rohtak, Haryana, India. shivani_bds@rediffmail.com 2012 by Quintessence Publishing Co Inc. of incurable diseases. This mode of treatment is often referred to as regenerative or reparative medicine. Stem cells are one of the most fascinating areas of biology today. Researchers have been investigating ways to use stem cells to replace damaged or diseased cells and tissues of the body. However, like many expanding fields of scientific inquiry, research on stem cells raises scientific questions about healing, ethics, and other issues as rapidly as it generates new discoveries. WHAT IS A STEM CELL? The cell is the basic unit of life. Life originates from a single cell the ovum which, when fused with the male pronucleus leads to the formation of a two-cell embryo 2 days after fertilization. The two cells then undergo a series of cell divisions by way of cleavage, ultimately proceeding to the formation of the morula. The morula becomes a blastocyst, which when develops 272 Volume 2, Number 4, 2012

2 further, giving rise not only to the tissues and organs of the embryo, but also to a number of structures that support the embryo and help it to acquire nutrition. At the very early stage, the embryo proper acquires the form of a three-layered disk, called the embryonic disk; this consists of endoderm, ectoderm, and mesoderm. These are the three germ layers. All the tissues of the body are derived from one or more of these layers 1 (Fig 1). Any damage to these tissues or organs, whether caused by aging or injury, may pose a threat to the whole system and thus has long been of concern. It has been observed that several tissues in the body (for example, blood, skin, and gastrointestinal tract) undergo rapid renewal and have regenerative abilities. This observation has led scientists to hypothesize that tissues with regenerative potential may contain cells that initiate their replacement. These cells have been termed stem cells. Stem cells are thus the pioneer of regenerative medicine. Exploring the possibility of using stem cells for cell-based therapies has become a very active area of investigation by researchers. There are three defining features of a stem cell 2,3 : A stem cell self-renews by a process called asymmetric division. A stem cell is multipotent; that is, it can form into multiple types of cells. A single stem cell completely recreates a particular kind of tissue when it is transplanted within the body. Different types of stem cells vary in their degree of plasticity, or developmental versatility. Stem cells are perhaps best understood in terms of how committed they are to become any particular type of cell. 4 TYPES OF STEM CELLS There are two main types of stem cells that are being investigated for their potential use in research and medicine: embryonic stem cells and adult stem cells (Table 1). They differ in their degree of differentiation and ability to self-renew and may be totipotent, pluripotent, or multipotent. 5 Embryonic Stem Cells Embryonic stem cells are most often derived from embryos that develop from eggs that have been fertilized in vitro in a clinic and were then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman s body. The embryos from which human embryonic stem cells are derived are typically 4 or 5 days old and thus in the blastocyst stage. 5 Fertilized egg Morula Inner cell mass Blastocyst Stem cells Culture in incubator Fig 1 Stem cells from the inner cell mass of the blastocyst. Method of Growing Embryonic Stem Cells Growing of cells in the laboratory is known as cell culture (Fig 2). Human embryonic stem cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with mouse embryonic skin cells (these have been treated so that they will not divide) to give the inner cell mass cells a sticky surface to which they can attach. This coating layer of cells is called a feeder layer. The feeder cells release nutrients into the culture medium. Over the course of several days, the cells of the inner cell mass proliferate and begin to crowd the culture dish. When this occurs, they are re-plated and subcultured. Each cycle of subculturing the cells is referred to as a passage. Embryonic stem cells that have proliferated in cell culture for 6 or more months without differentiating are pluripotent and appear genetically normal. They are referred to as an embryonic stem cell line (Fig 2). After cell lines are established, or even before that stage, batches of them can be frozen and shipped to other laboratories for further culture and experimentation. 5 Differentiation of Embryonic Stem Cells As long as embryonic stem cells in culture are grown under certain conditions, they can remain undifferentiated (unspecialized). However, if cells are allowed to clump together to form embryoid bodies, they begin to differentiate spontaneously in the presence of certain growth factors (eg, activin, retinoic acid, sonic hedgehog, etc). They can form muscle, nerve, and many other cell types. Although spontaneous differentiation is a Oral & Craniofacial Tissue Engineering 273

3 Table 1 Categories of Stem Cells Cell type Totipotent Pluripotent Multipotent Defining features Capable of forming a completely new embryo that can develop into a new organism. A fertilized egg is totipotent. None of the stem cells used in research appear to have this capacity. Have the potential to develop into any of the cell types found in an adult organism. Embryonic stem cells are pluripotent. Have the potential to differentiate into only a few cell types in the body. Adult stem cells appear to be multipotent. Cleavage stage embryo Cells dissociated and replaced Cultured blastocyst Isolated inner cell mass Irradiated mouse fibroblast feeder cells New feeder cells Established embryonic stem cell cultures Fig 2 Techniques for generating embryonic stem cell cultures. good indication that a culture of embryonic stem cells is healthy, it is not an efficient way to produce cultures of specific cell types. Thus, scientists try to control the differentiation of embryonic stem cells. They can change the chemical composition of the culture medium, alter the surface of the culture dish, or modify the cells by inserting specific genes. If scientists can reliably direct the differentiation of embryonic stem cells into specific cell types, they may be able to use the resulting differentiated cells to treat certain diseases in the future. Adult Stem Cells The notion that stem cells exist during embryonic development has long been accepted, but the thought that stem cells remain in various tissues after birth (adult stem cells) is relatively new. Based on the observation that several tissues in the body undergo rapid renewal, scientists hypothesized that postnatal tissues must contain stem cells to initiate such replacement. The first definitive evidence came with the work of Till and McCulloch on blood-forming (hematopoietic) stem cells in the 1960s; they began analyzing bone marrow to determine which components were responsible for regenerating blood. It is now believed that virtually every tissue in the body contains some type of stem cells, potentially providing many avenues for tissue repair. An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ that can renew itself and can differentiate to yield the major specialized cell types of that particular tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. Some scientists now use the term somatic stem cell instead of adult stem cell. Unlike embryonic stem cells, which are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is unknown. Research on adult stem cells has recently generated a great deal of excitement. Scientists have found adult stem cells in many more tissues than they once thought possible, and this has given rise to speculation of whether adult stem cells could be used for transplants. Research on adult stem cells began about 40 years ago (Table 2). In the 1960s, researchers discovered that bone marrow contains at least two kinds of stem cells. One population, called hematopoietic stem cells, forms all the different types of blood cells in the body. A second population, called bone marrow stromal cells, was discovered a few years later. Stromal cells are a mixed cell population that generates bone, cartilage, fat, and fibrous connective tissue. It was not until the 1990s that scientists agreed that the adult brain contains stem cells that are able to generate the brain s three major cell types: astrocytes and oligodendrocytes, which are nonneuronal cells, and neurons, or nerve cells. 5 Identification and Functions of Adult Stem Cells. Adult stem cells have been identified in many organs and tissues. Stem cells are thought to reside in a specific area of each tissue, where they may remain quiescent (nondividing) for many years until they are 274 Volume 2, Number 4, 2012

4 Table 2 History of Stem Cell Discoveries and Research 1878 The first attempts are made to fertilize mammalian eggs outside the body The first animals are made by in vitro fertilization (IVF). 1960s Teratocarcinomas are determined to originate from embryonic germ cells in mice. Embryonal carcinoma (EC) cells are identified as a kind of stem cell The first human egg is fertilized in vitro. 1970s EC cells are injected into mouse blastocysts, resulting in chimeric mice. Cultured stem cells are explored as models of embryonic development in mice The first IVF baby is born in England; the hematopoietic stem cell is discovered in human cord blood Mouse embryonic stem (ES) cells are derived from the inner mass of blastocysts by scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Mouse ES cells are grown in vitro. ES cells are injected into mice from teratomas EC cells are developed as pluripotent clonal cells. When exposed to retinoic acid, these cells differentiate into neuronlike cells and other cell types A clonal line of human EC cells is derived that yields tissues from all three primary germ layers. They have limited replicative and differentiative capacity Neural stem cells are cultured in vitro Human blastocysts are generated, and the inner cell mass is maintained in culture. ES cell like cells form in the center and retain a stem cell like morphology Nonhuman primate ES cells are maintained in vitro from the inner cell mass of monkeys. These cells are pluripotent and differentiate normally into all three primary germ layers ES cells from the inner cell mass of normal human blastocysts are cultured and maintained normally for many passages. Embryonic germ cells are also derived and grown in vivo Scientists derive human ES cells from the inner cell mass of blastocysts. They proliferate in vitro for a long time and form all three germ layers and teratomas when injected into immunodeficient mice As human ES cell lines are shared and new lines are derived, more research groups are focusing attention on the differentiation of cells in vitro. Many methods focus on making human tissues for transplantation Dr Songtao Shi discovers a new source of adult stem cells in primary teeth Scientists in England create the first ever artificial liver cells using umbilical cord blood stem cells Scientists report the discovery of a new type of stem cell in amniotic fluid, which may be an alternative to ES cells for use in research and therapy. January 2008 Human ES cell lines are generated without destruction of the embryo. February 2008 Pluripotent stem cells are generated from adult mouse liver and stomach. activated by disease or tissue injury. The adult tissues that have been reported to contain stem cells include brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin and liver. Scientists in many laboratories are trying to find ways to grow adult stem cells in cell culture and manipulate them to generate specific cell types so that they can be used to treat injury or disease. Adult Stem Cell Differentiation. Scientists have reported that adult stem cells occur in many tissues and that they enter normal differentiation pathways to form the specialized cell types of the tissue in which they reside. Adult stem cells may also exhibit the ability to form specialized cell types of other tissues, which is known as transdifferentiation or plasticity. 5 Normal Differentiation Pathways of Adult Stem Cells. In a living animal, adult stem cells can divide for a long period and can give rise to mature cell types that have characteristic shapes and specialized structures and functions of a particular tissue, just as, for instance, hematopoietic stem cells give rise to all types of blood cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets; bone marrow stromal cells (mesenchymal stem cells [MSCs]) give rise to bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendons; neural stem cells in the brain give rise to its three major cell types: nerve cells (neurons), astrocytes, and oligodendrocytes; and so on. Adult Stem Cell Plasticity and Transdifferentiation. A number of experiments have suggested that certain adult stem cell types are pluripotent. The following are examples of adult stem cell plasticity (transdifferentiation) that have been reported during the past few years. Oral & Craniofacial Tissue Engineering 275

5 Hematopoietic stem cells may differentiate into the three major types of brain cells (neurons, oligodendrocytes, and astrocytes); skeletal muscle cells; cardiac muscle cells; and liver cells. Bone marrow stromal cells may differentiate into cardiac muscle cells and skeletal muscle cells. Brain stem cells may differentiate into blood cells and skeletal muscle cells. Current research is aimed at determining the mechanisms that underlie adult stem cell plasticity. If these mechanisms can be identified and controlled, existing stem cells from healthy tissue might be induced to repopulate and repair diseased tissue. 5 Comparison of Adult and Embryonic Stem Cells Advantages of Adult Stem Cells. Both lines of stem cells have an enormous therapeutic potential. Whereas embryonic stem cells offer the potential for wider therapeutic applications, adult stem cells avoid the ethical issues associated with embryonic stem cell research. Therefore, many stem cell therapies are currently being tested using adult stem cells. Additionally, adult stem cells offer the potential for autologous stem cell donation, which may help to prevent issues of immune rejection. Advantages of Embryonic Stem Cells. A major advantage of embryonic stem cells is that they offer one cell source for multiple indications. They provide the potential for a wider variety of applications than do adult stem cells. Additionally, they theoretically have the possibility of being immunoprivileged because of their highly undifferentiated state. A privileged immune status would remove one of the main barriers of stem cell therapies, as self-rejection is one of the most common complications of stem cell therapy. The idea that embryonic stem cells can be immunoprivileged must be viewed skeptically, however, as this theory has not yet been proven. Another advantage of embryonic stem cells is that they appear to be immortal in vitro, whereas adult and differentiated stem cells cannot be cultured indefinitely in the laboratory. Once they have differentiated, embryonic stem cells seem to die off like typical tissue cells. Embryonic stem cells can become all cell types of the body because they are pluripotent. Adult stem cells are merely multipotent and are therefore generally limited to differentiating into different cell types of their tissue of origin only. However, some evidence suggests that adult stem cell plasticity may exist, increasing the number of cell types that a given adult stem cell can become. POSSIBLE SOURCES OF STEM CELLS Embryonic Stem Cell Sources Embryonic stem cells can be obtained from embryos created via in vitro fertilization (whether for infertility treatment or for research purposes), embryos or fetuses obtained through elective abortion, and embryos created via somatic cell nuclear transfer, or cloning. Adult Stem Cell Sources Adult stem cells can be obtained from bone marrow, peripheral blood, neurons, muscles, liver, pancreas, cornea and retina, mammary glands, salivary glands, skin, tendon, synovial membrane, heart, cartilage, thymic progenitors, adipose tissue, umbilical cord blood, amniotic stem cells, and blood vessels. Dental sources of adult stem cells include dental pulp (dental pulp stem cells [DPSCs]), primary teeth (stem cells from human exfoliated deciduous teeth [SHED]), cementum (human cementum derived cells [HCDCs]), papillae (stem cells from root apical papilla [SCAP], and periodontal ligament stem cells (periodontal ligament stem cells [PDLSCs]). CLINICAL THERAPIES Clinical Applications of Stem Cells Stem cell therapies have the potential to make a tremendous impact on the general well-being of people physically, economically, and psychologically. About 128 million people in India suffer from chronic, degenerative, and acute diseases, and stem cell therapies hold great promise in the treatment of many of these diseases. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases. To be useful for transplant purposes and to bring the stem cells for cell-based treatments into the clinic, scientists must precisely control stem cells so that they can: Proliferate extensively and generate sufficient quantities of tissue Differentiate into the desired cell type(s) Survive in the recipient after transplantation Integrate into the surrounding tissue after transplantation Function appropriately for the duration of the recipient s life Avoid harming the recipient in any way 276 Volume 2, Number 4, 2012

6 Immune Rejection To avoid the problem of immune rejection, scientists are experimenting with different research strategies to generate tissues that will not be rejected. Human leukocyte antigen (HLA) typing is a testing process that is used to match patients and donors for cord blood or bone marrow transplants. HLA antigens are proteins found in most cells in the human body. The immune system uses these proteins or markers to recognize which cells belong in the body and which do not. If the immune system determines that a cell does not belong to the body, the cell is attacked. Thus, HLA typing is done to reduce the risk of the transplanted stem cell being attacked by the immune system of the recipient. A close match between the patient s HLA antigens and those of the donor can reduce the risk of the patient s immune cells attacking the donor s cells or vice versa. HLA typing is usually done for all allogeneic transplants using a blood sample. A well-matched donor is important to the success of a stem cell transplant. Six HLA markers are matched; for a transplant to be successful, at least four of the six markers must match. Because everyone inherits half of their HLA antigens from their mother and half from their father (ie, 50%), a sibling has a 25% chance of matching another. During a transplant, the patient s parents and/or children are also tested to confirm the HLA typing and to make sure no possible donors are overlooked 4 (Table 3). HLA typing improves the chances of a successful transplant. Matching HLA tissue traits before a transplant is mandatory because it promotes engraftment and reduces the risk of graft-versus-host disease. Engraftment occurs when the donated cells that were transplanted begin to grow and make new cells. For the transplant to succeed, the donated cells need to engraft quickly, as the patient is at a high risk of infection. Graft-versus-host disease is a posttransplant complication that occurs when the immune cells from the graft attack the patient s own immune cells. Potential Clinical Applications in the Orofacial Complex Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all the craniofacial structures are derivatives of MSCs. Cells with characteristics of adult stem cells have been isolated from the dental pulp, primary teeth, and the periodontium. Several craniofacial structures, such as the mandibular condyle, calvarial bone, the cranial suture, and subcutaneous adipose tissue, have been engineered from MSCs. Transplanted skeletal or dental stem cells may one day be used to repair craniofacial bone or even repair Table 3 HLA Typing and Relationships Relationship Chances of matching Sibling sibling 25% Parent child 50% Cousins 6.25% or regenerate teeth. While most often caused by ablative surgery to remove cancerous tissue, craniofacial osseous deficiencies can also arise from infection, trauma, congenital malformations, and progressive deforming skeletal diseases. Techniques used to repair craniofacial skeletal defects are similar to the accepted surgical therapies for bone loss elsewhere in the skeleton. These include the use of autogenous bone and alloplastic materials. 6 However, despite the usefulness of these reparative strategies, each method has inherent limitations that restrict their universal application. Transplantation of a bone marrow stromal cell population that contains skeletal stem cells may provide a promising alternative approach for reconstruction of craniofacial defects by circumventing many of the limitations of autografting and allografting methods. 7,8 Identification of Stem Cells in Other Mineralized Tissues in the Oral Cavity. Caries, pulpitis, apical periodontitis, and other craniofacial diseases increase health costs and lead to loss of economic productivity. They ultimately result in premature tooth loss and therefore diminish the quality of life. Tissue engineering is a new concept that might solve the problem of craniofacial regeneration. Tissue engineering is the science of the design and manufacture of new tissues to replace tissues that have been damaged by disease or trauma. The three key elements of tissue engineering are: a signal for morphogenesis, stem cells for responding to morphogens, and a scaffold of extracellular matrix. 9 In light of recent discoveries of adult stem cells in tissues that were not thought to contain stem cells, it was hypothesized that other hard tissues in the oral cavity may also contain these unique entities. Using techniques previously established to isolate progenitors from bone and marrow, it is now apparent that these tissues do in fact contain clonogenic cells with extensive proliferative capacity. 6 Furthermore, in vivo transplantation has further characterized their ability to regenerate hard tissue. Isolation and Characterization of Cementoblastlike Cells. Although there are differences in the organization of bone and cementum, it is not clear whether they are formed by distinct cell types or by Oral & Craniofacial Tissue Engineering 277

7 a bone-forming cell that has different environmental cues. Distinguishing between these two possibilities has been difficult because, to date, no specific marker for cementum or cementocytes has been identified. Cultures of murine or primary HCDCs have been established from healthy teeth using a collagenase pretreatment, as had been established previously for the culture of trabecular bone cells. With primary HCDCs, discrete colonies that included cells exhibiting fibroblastlike morphology are formed, and when the colonies became sufficiently large, cells from individual colonies were isolated and subcultured. HCDC matrix was found to contain unorganized collagen bundles, as is seen in cementum. Cells in the HCDC matrix were positive for fibromodulin and lumican and were also devoid of hematopoietic marrow. These results show that cells from normal human cementum can be isolated and expanded in vitro. Furthermore, these cells are capable of differentiating and forming a cementumlike tissue when transplanted into immunocompromised mice. 6 Regrowing Dental Enamel from Cultured Cell. Dental enamel is the hardest tissue produced by the body. It cannot regenerate itself because it is formed by a layer of cells that is lost by the time the tooth appears in the mouth. The enamel spends the remainder of its lifetime vulnerable to wear, damage, and decay. Although researchers have experienced some success in producing enamellike and toothlike tissues, problems remain to be solved before the technology can be tested in humans. One of the issues has been how to produce, in culture, a sufficient number of enamel-forming cells. There are reports that a new technique is being developed for culturing cells that have the capacity to produce enamel The authors group has recently shown that epithelial cells extracted from the developing teeth of 6-month-old pigs continue to proliferate when they are cultured on top of a special feeder layer of cells (the feederlayer cells are known as the 3T3-J2 cell line). 13 This crucial step increases the number of dental epithelial cells that are available for enamel production. In a recent study, researchers seeded the cultured dental epithelial cells onto collagen sponge scaffolds, along with cells from the middle of the tooth (dental mesenchymal cells). The scaffolds were then transferred into the abdominal cavities of rats, where conditions were favorable for the cells in the scaffolds to interact and develop. When removed after 4 weeks, the remnants of the scaffolds were found to contain enamellike tissue. The key finding of this study was that, even after the multiple divisions that occurred during propagation of the cells in culture, the dental epithelial cells retained the ability to produce enamel, as long as they were provided with an appropriate environment. 14 Adult Human DPSCs. Another mineralized tissue that has a great deal of similarity to bone is dentin. Although dentin does not turn over throughout life, as does bone, limited dentinal repair in the postnatal organism does occur. It was postulated that the ability for limited repair is maintained by a precursor population, associated with pulp tissue, that has the ability to mature into odontoblasts. 15 Clonogenic and highly proliferative cells have been derived from enzymatically disaggregated adult human dental pulp (ie, DPSCs) that form sporadic but densely calcified nodules in vitro. When DPSCs were transplanted with hydroxyapatite/ tricalcium phosphate (HA/TCP) into immunocompromised mice, they generated a dentinlike structure, with collagen fibers running perpendicular to the mineralizing surface, as is found in vivo, and contained dentin sialophosphoprotein, a dentin-enriched protein. The newly formed dentin was lined with human odontoblastlike cells that extended long cellular processes into the mineralized matrix and surrounded an interstitial tissue; that is, it resembled pulp in vivo with respect to the organization of the vasculature and connective tissue. 16 Regenerating Human PDL. Periodontal diseases can destroy the PDL, bone, and cementum. Destruction of this tissue is a cause of tooth loss. Recently, Seo et al discovered stem cells in human PDL. These stem cells have the potential to generate PDL and cementum. 17,18 In another study, MSCs were used to treat periodontal defects. 19 The authors transplanted bone marrow derived MSCs into experimental Class III periodontal defects. Four weeks after transplantation, the periodontal defects were almost regenerated with periodontal tissue. In the regenerated periodontal tissues, cementoblasts, osteoblasts, osteocytes, and fibroblasts were detected. 20 Stem Cell Mediated Root Regeneration. Dental implant therapy has achieved long-term success in the clinic for the recovery of tooth function, but preexisting high-quality bone structures are required to support implants. Reconstruction of teeth in patients without adequate bone support would be a major advancement. Stem cell mediated root regeneration offers opportunities to regenerate a biologic root and its associated periodontal tissues, which are necessary for maintaining the physiologic function of teeth. A biologic root/periodontal complex can be built up by postnatal stem cells, including SCAP and PDLSCs, to which an artificial porcelain crown can be affixed. This hybrid strategy of autologous dental stem cell engineering may be applicable to human tooth regeneration. In a study using a minipig model, both human SCAP and PDLSCs were transplanted to generate a root/pdl complex that was capable of supporting a porcelain crown. 21 Since most human tissue at the developing stage is not clinically available for stem 278 Volume 2, Number 4, 2012

8 cell isolation, this apical papilla stem cell population is inaccessible in clinical practice. However, SCAP can be isolated from extracted third molars. Therefore, it may be possible to bank these high-quality dental stem cells for future autologous use. Direct Orthotopic Transplantation into Segmental Defects. Investigators have developed a number of animal models of segmental defects in mice, 7 dogs, 22 and sheep. 23 In sheep, ceramic blocks loaded with bone marrow stem cells (BMSCs) led to complete healing of long-bone defects. In mice and dogs, investigators created critical-size defects in the cranium and filled them with ex vivo expanded BMSCs, either in collagen sponges or in association with HA/TCP. In both cases, the defects healed completely, and the newly formed bone integrated into the margin. 3 Researchers have succeeded in using porous silk fibroin scaffolds as a support for growth and differentiation of BMSCs in a bioreactor before transplantation into a cranial critical-size defect in mice. 24 Alveolar Ridge Augmentation. Restoration of alveolar ridge height is of utmost concern to practicing dentists in trying to prevent the loss of a tooth as a result of bone destruction induced by periodontal disease and in maintaining the ability of edentulous patients to wear dentures. 3 Appropriate ridge height is also essential for the placement and long-term retention of dental implants. Standard practice involves the use of autologous or allogeneic bone grafts or ceramics, both with and without growth factors, but the outcomes are variable. In animal models, BMSCs are used in conjunction with HA/TCP. They have been successful in building alveolar bone, 25 and a number of small studies in human patients have used BMSCs along with allogeneic bone fragments, 26 or together with platelet-rich plasma 27 and another ceramic scaffold such as TCP, to name just a few possibilities. 28 With further refinement, these types of procedures would mark a major advancement in dental reconstruction. Vascularized Bone Grafts. Recently, clinical researchers reconstructed part of a mandible in a patient who had undergone extensive tumor resection. 29 In this case, researchers used computed tomographic scanning to model the jaw, fabricated a custommade titanium mesh cage to match the dimensions of the defect, filled it with bovine bone powder, and infiltrated it with the patient s own bone marrow (rather than ex vivo expanded BMSCs) and bone morphogenetic protein. They then placed the cage in the highly vascularized latissimus dorsi muscle. Seven weeks later, they removed the cage, along with part of the muscle containing the thoracodorsal artery and vein, and moved it into the mandible. Here, they attached the blood vessels to the external carotid artery and cephalic vein, which they relocated from the patient s upper arm into the neck. Using bone scintigraphy, the researchers found that the graft was biologically active. 30 Long-term results for this patient have yet to be reported. In this case, the researchers used bone marrow, and one can envision that the use of ex vivo expanded BMSCs could hasten the development of bone, thereby shortening the period during which the transplant needs to be grown at the donor site. 3 Tissue Engineering of the Temporomandibular Joint from Stem Cells. Temporomandibular disorders (TMD) manifest as pain, myalgia, headaches, and structural destruction collectively known as degenerative joint disease. 31 The temporomandibular joint (TMJ), like other synovial joints, is also prone to rheumatoid arthritis, injuries, and congenital anomalies. 32 The severe form of TMD necessitates surgical replacement of the mandibular condyle. 33 Recently, researchers reported the tissue engineering of a mandibular condyle exhibiting the shape and dimensions of a human cadaver TMJ. The engineered mandibular condyle had stratified layers of cartilage and bone from a single population of bone marrow derived MSCs and was molded into the shape and dimensions of a human cadaver mandibular condyle. MSCs were isolated from femoral and tibial bone marrow of adult rats and exposed separately to either chondrogenic or osteogenic supplemented culture medium. 34,35 De novo formation of a structure with the same shape and dimensions as the cadaver human mandibular condyle was observed after 4 weeks of in vivo implantation. The tissue-engineered mandibular joint condyles retained the macroscopic shape and dimensions of the cadaver mandibular condyle. However, there are certain challenges with tissue engineering of the TMJ: To promote matrix synthesis and tissue maturation of stem cell derived chondrogenic and osteogenic cells encapsulated in biocompatible and bioactive scaffolds, it may be necessary to incorporate an array of growth factors and/or transcription factors separately for tissue-engineered chondrogenesis and osteogenesis. The mechanical properties of a tissue-engineered mandibular condyle must be enhanced for ultimate in situ implantation into the human TMJ. The remodeling potential of a tissue-engineered mandibular condyle has yet to be fully elucidated. Stem Cells from Human Exfoliated Deciduous Teeth. Recently, researchers found a population of high-quality human stem cells in exfoliated human primary teeth. 36 Remnant dental pulp derived from exfoliated primary teeth contains a multipotent stem cell population. These stem cells can be isolated and Oral & Craniofacial Tissue Engineering 279

9 expanded ex vivo, thereby providing a unique and accessible population of stem cells from an unexpected tissue resource. Previous experiments have shown that dental pulp tissue of adult teeth contains a population of DPSCs that are capable of differentiating into odontoblasts and adipocytes, as well as expressing nestin and glial fibrillary acidic protein, and form a dentin/pulp like complex after in vivo transplantation. 37 Primary teeth are significantly different from permanent teeth with regard to their developmental processes, tissue structure, and function. Therefore, SHED are distinct from DPSCs with respect to their higher proliferation rate, increased cell population doublings, spherelike cell-cluster formation, and osteoinductive capacity in vivo. However, they fail to reconstitute a dentin/pulp like complex, perhaps as a result of their more immature characteristics versus other postnatal stem cell populations. The mechanisms controlling the growth and replacement of teeth are largely unknown, particularly with respect to how craniofacial components, including bone and the soft tissues surrounding teeth, participate in the process of tooth development. SHED demonstrated a strong capacity to induce recipient cell mediated bone formation in vivo. SHED do not differentiate directly into osteoblasts, but they do induce new bone formation by forming an osteoinductive template to recruit murine host osteogenic cells. These data imply that primary teeth may not only provide guidance for the eruption of permanent teeth, as is generally assumed, but they may also be involved in inducing bone formation during the eruption of permanent teeth. It is notable that SHED express neuronal and glial cell markers, which may be related to the neural crest cell origin of the dental pulp. Neural crest cells play a pivotal role in embryonic development, giving rise to a variety of cell types, such as neural cells, pigment cells, smooth muscle, craniofacial cartilage, and bone. 37 Recent studies 36,38 have provided evidence that SHED represent a population of postnatal stem cells that is capable of extensive proliferation and multi potential differentiation. Primary teeth therefore may be an ideal source of stem cells to repair damaged tooth structures, induce bone regeneration, and possibly to treat neural tissue injury or degenerative diseases. However, the biologic significance of the existence of SHED remains to be determined. CHALLENGES Stem cell research has undergone huge advancements in the past few years. It has been particularly challenging for scientists to ensure the long-term proliferative ability and pluripotency of embryonic stem and germ cells. These are important characteristics to maintain, as accurate models are necessary to understand the unique genetic and molecular basis by which these cells are able to replicate indefinitely. In addition to providing accurate models, culturing of stem cells in vitro is also necessary to ensure that sufficient quantities of stem cells are available to treat specific diseases. Teratoma formation has also produced a hurdle that needs to be overcome. Formation of these tumorlike masses of cells at injection sites significantly limits the potential therapeutic applications of embryonic stem cells. Immune challenges also prove a significant barrier to the application of stem cell therapies. If the stem cells are recognized as foreign, they are rejected and destroyed. Two potential solutions to this problem have been proposed. One is the creation of universal donor stem cells through genetic engineering techniques. Theoretically, stem cells could be created that lack outer surface labels. The absence of these labels, which normally identify cells as foreign, would eliminate the problem of immune rejection. Another solution to immune rejection would be to engineer stem cells identical to the recipient s cells using the patient s own DNA. However, the former solution of universal donor stem cells would prove less labor intensive and more cost effective than the latter solution. Adult stem cells have also presented unique problems of their own for researchers. Not only are they difficult to maintain in culture, similar to embryonic stem cells, but they are also very rare in adult tissues. Thus, adult stem cells are very difficult to isolate and identify. 39,40 PROSPECTS Clearly, advances in adult stem cell biology have provided a great deal of impetus for the biomedical community to translate these findings into clinical application. Given the fact that researchers possess populations of stem cells that can reproducibly re-form bone and its marrow, cementum, dentin, and perhaps even PDL, it is possible to envision complete restoration of the hard tissues in the oral cavity using the patient s own cells, thereby avoiding issues of histocompatibility. Furthermore, advances in techniques to modify the gene activity of stem cells during their ex vivo expansion offers the unique possibility to improve a patient s own stem cells. However, the replacement of dental tissues with either cell- or gene-based therapy may be complicated in areas of unresolved inflammation, thus highlighting the need for more research to understand potential complicating factors. While the technical hurdles to achieve these goals should not be underestimated, the recent recognition of stem 280 Volume 2, Number 4, 2012

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