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Dental Tissue Engineering
75 Dental Tissue Engineering Yan Lin and Pamela C. Yelick INTRODUCTION It has long been appreciated that the oral cavity functions as an important barrier to microbial infections that can adversely affect the overall health and well-being of an individual. Diseased teeth and resulting compromised oral health present real threats to healthy lifestyles. This threat becomes more significant over time as teeth wear and/or become damaged after years of prolonged use and as the potential susceptibility to disease increases with advancing age. The need for improved dental tissue repair/regeneration methods has been recognized for many centuries, and a variety of approaches have been explored to restore teeth loss, including tooth autotransplantation, allotransplantation, and dental implant methods (Yelick and Vacanti, 2004; Yen and Sharpe, 2006). Although the current availability and use of prosthetic dental implants contributes to an improved quality of life for many individuals, certain limitations associated with those procedures, including limited tooth autograft available and the possibility of tooth allograft or foreign body rejection by the patient makes this method unsuitable for a number of potential recipients. Thus, the development of new methods to repair dental tissues or to replace whole teeth is in great demand. Recent advancements in stem cell biology and material science have highlighted tissue engineering as an emerging science with the potential, in the relatively near future, to facilitate the successful development of replacement tissues and organs. In ongoing preclinical studies, cell- and gene-based therapies are being developed for a variety of tissues and organs, including bone, heart, liver, and kidney. Methods to deliver stem/progenitor cells, biodegradable scaffolds, and growth factors and/or morphogen gradients to tissue injury sites are being used to accelerate and/or induce natural biological regeneration. With respect to dental applications, stem cell-based dental tissue engineering provides the opportunity to consider biologically based reparative and/or replacement tooth therapies, with the potential to regenerate dental tissues exhibiting physical and esthetic properties that are equal to, or better than, existing counterparts. Because of the rapid growth in this field, this chapter will present and discuss some of the most recent developments in various subfields of dental tissue engineering, which collectively highlight the powerful impact that biologically based dental tissue regeneration strategies will have on the field of dentistry in the foreseeable future. Natural Tooth Tissue Development and Repair Teeth consist of crown, neck, and root structures, and are composed of a variety of hard and soft dental tissues (Figure 75.1). Tooth crowns consist of an outer mineralized enamel layer, the subjacent mineralized dentin layer, and an inner dental pulp tissue. Tooth roots consist of a small root canal containing dental pulp and nerves, surrounded by mineralized dentin and cementum layers and integrated periodontal ligament (PDL) tissue, which secures the tooth to the underlying alveolar bone (Nanci, 2003). The enamel surface of the tooth is produced by specialized dental epithelial cells, called ameloblasts. Enamel is primarily mineral and contains enamel-specific proteins including amelogenin (Snead et al., 1983), ameloblastin, and enamelin (Krebsbach et al., 1996; Paine et al., 1998). Enamel is extremely hard and durable
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Enamel Crown
Dentin Pulp
Neck Cementum Root
Periodontal ligament Root canal
Figure 75.1 Erupted tooth structures.
but is not self-regenerative, since ameloblasts are no longer present in postnatal or adult teeth once they have erupted. With the exception of enamel, all of the other tooth tissues are derived from neural crest cell (NCC)derived dental mesenchyme. The tooth layer underlying the enamel is composed of dentin, which is approximately 75% mineralized and contains dentin-specific gene products, including dentin sialoprotein (DSP), dentin phosphoprotein (DPP), and dentin matrix protein-1 (DMP-1) (Begue-Kirn et al., 1998; Butler et al., 2002). Dentin is produced by NCC-derived dental mesenchymal cells called odontoblasts, which exhibit limited regenerative capacities in response to injury or disease. The dental pulp at the center of the tooth is composed of dental mesenchymal cells, including putative dental stem cells (DSCs), nerves, and blood vessels that thread through the root canal and support the tooth organ. The periodontium, which supports and attaches teeth to the jaw, primarily consists of four connective tissues: mineralized cementum, fibrous PDL, alveolar bone, and gingival tissue (Beertsen et al., 1997). Cementum is a bone-like mineralized tissue lining the dentin of the root that protects the root and also serves as an attachment surface to anchor the PDL to the tooth (Diekwisch, 2001). The PDL functions to secure the tooth to the underlying alveolar bone and is essential for the long-term survival of the tooth. Sub-functioning PDL tissue can result in tooth root damage and eventual ankylosis of the tooth. Cells of the PDL include fibroblasts, cementoblasts on the root surface, osteoblasts and osteoclasts present on the alveolar bone face, and undifferentiated mesenchymal tissues in the body of the ligament (Taba et al., 2005; Nanci and Bosshardt, 2006). The periodontium also possesses some regenerative capability, as tissue loss during the early phases of periodontal diseases can be restored to a certain degree. However, once periodontitis becomes established only therapeutic intervention has the potential to induce regeneration (Shi et al., 2005). When considering methods to regenerate teeth, it is first necessary to have a thorough understanding of de novo tooth development. Like many other organs, such as hair follicles, salivary glands, intestines, and kidneys, tooth development is the cumulative result of reciprocal signaling events between the ectoderm-derived dental epithelial and the NCC-derived mesenchyme (Jernvall and Thesleff, 2000). Molecular signals initiated in the dental epithelium induce gene expression in the subjacent dental mesenchyme, and the acquired odontogenic potential in the mesenchyme triggers further epithelial morphogenesis and cytodifferentiation. As tooth development progresses, dental mesenchyme differentiates into dentin, pulp, cementum, PDL, and alveolar bone, and the epithelial tissues produce dental enamel. The cellular and molecular natures of enamel,
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dentin, cementum, and root development have been extensively characterized (Linde and Goldberg, 1993; Zeichner-David et al., 1995; Bartlett and Simmer, 1999; Chai et al., 2000; Grzesik et al., 2000; Jernvall and Thesleff, 2000; Saygin et al., 2000; Diekwisch, 2001; Bosshardt, 2005; Luan et al., 2006). The morphological stages of natural tooth development consist of early bud and cap stages, and later bell stage where tooth histodifferentiation and morphodifferentiation occur (Thesleff et al., 2001). Tooth development begins as a localized thickening of the dental epithelium, which proliferates and invaginates into the underlying dental mesenchyme, appearing as a small epithelial bud. The primary “enamel knot,” a signaling center that forms at the tip of the epithelial tooth bud, and subsequently formed enamel knots, all of which mark the site of future cusp formation, are considered to be central regulators of tooth development, as they link dental cell differentiation to tooth morphogenesis (Thesleff et al., 2001). The primary enamel knot expresses at least 10 different signaling molecules belonging to the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (Hh), and Wnt (an amalgam of wingless- and int-related proteins) families, which are responsible for the transition from bud to cap stage (Thesleff and Sharpe, 1997; Jernvall and Thesleff, 2000). Epithelial signals emanating from the primary enamel knot induce the formation of the dental papilla, the mesenchymal cell layer underlying the dental epithelium, at the transition from bud to cap stage. Later on, cells of the dental papilla differentiate into odontoblasts, which subsequently differentiate and produce dentin (Thesleff et al., 2001). In bell stage teeth, so named for their characteristic bell-shaped appearance, odontoblasts induce ameloblast formation, and the ameloblasts differentiate and synthesize enamel. Also, at the bell stage the dental lamina connecting the tooth germ to the oral epithelium disappears, isolating the tooth from the oral cavity. Dental Tissue Engineering Realizing the full potential of tissue regenerative treatments for the oral complex will require the integration of three key elements: (1) dental stem/progenitor cells; (2)inductive morphogenetic signals (morphogens); and (3) scaffold material upon which progenitor cells attach and elaborate an extracellular matrix (Nakashima and Reddi, 2003). DSCs One way to regenerate whole teeth would be to mimic the process of natural tooth development, either in vitro or in vivo, using DSCs. DSCs are a somewhat elusive population of self-renewing cells that exhibit the potential to form biological replacement tooth tissues. A variety of DSC populations have been identified for potential use in tooth tissue engineering strategies, as recently reviewed (Krebsbach and Robey, 2002; Murray and Garcia-Godoy, 2004; Yelick and Vacanti, 2004; Risbud and Shapiro, 2005; Shi et al., 2005; Bartold et al., 2006; Yen and Sharpe, 2006). Because tooth development is dependent on epithelial and mesenchymal cell interactions, DSCs necessarily consist of two types: epithelial DSCs, which form ameloblasts, enamel, stellate reticulum, and stratum intermedium, and mesenchymal DSCs, which form the dental papilla, odontoblasts, predentin, dentin, cementum, PDL, and alveolar bone (Nanci, 2003). The exclusive environment in which stem cells reside, called the “stem cell niche,” is thought to support the maintenance and self-renewal of stem cells (Spradling et al., 2001). In the remaining paragraphs, we will focus on the applications of stem cells in dental tissue engineering. Significant efforts currently focus on the development of methods to control the differentiation and proliferation of DSCs, to facilitate the creation of stem cell-based therapies that can be used more effectively than current synthetic material-based dental treatment regimes (Yelick and Vacanti, 2006). For instance, Miura
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et al. (2003) isolated stem cells from human exfoliated deciduous teeth, which were found to be capable of differentiating into a variety of cell types, including neural cells, adipocytes, and odontoblasts. The reparative potential of these progenitor cells is now being carefully scrutinized. It has long been appreciated that the PDL harbors progenitor cells that can differentiate into fibroblasts, osteoblasts, and cementoblasts (McCulloch, 1995; Bartold et al., 2006). Many investigators, including Seo and coworkers (2004), have isolated multipotent postnatal human PDL stem cells and demonstrated their capacity to generate cementum/PDL-like tissue after in vivo transplantation. However, at the present time, limited knowledge exists about the manner in which the specialized periodontal tissues are organized and the identity of the cells that contribute to the formation of each type of periodontal tissue (Shi et al., 2005). What has become increasingly clear is the fact that as knowledge of stem cell biology continues to advance, including methods to identify, isolate, expand, and differentiate DSCs, biologically based dental repair and whole-tooth regeneration therapies will become realistic possibilities. Growth Factors and Morphogen-Based Cell Signaling Gradients A major focus of contemporary developmental biology has been to delineate the biological cues that drive stem cell proliferation and differentiation (Thesleff and Sharpe, 1997; Nakao et al., 2004). Knowledge of the responsiveness of DSCs to various morphogenetic signals is of potentially significant importance for improving dental regenerative therapies. Continued efforts to elucidate signaling pathways regulating natural tooth development focus on four families of growth factors, the BMP, FGF, Hh, and Wnt, which govern tissue-specific patterning and morphogenesis during odontogenesis (Yen and Sharpe, 2006). Growth factor ligands from each of these families are currently being evaluated for their utility in guided, stem cell-based dental tissue engineering efforts. It has been demonstrated that delivery of signaling molecules, including BMP (Ripamonti and Reddi, 1997; Nakashima and Reddi, 2003; Iohara et al., 2004) and platelet-derived growth factor (PDGF) (Rutherford et al., 1993; Giannobile et al., 2001; Jin et al., 2004) via gene therapy vectors, resulted in partial dental tissue regeneration, for example, dentin and periodontal tissues. The results suggest that such factors, alone or in combination with other agents, may eventually be used to promote dental tissue regeneration. Scaffolding Materials In the context of tissue engineering, scaffolds made of natural or synthetic materials play a central role in supporting cells during the formation of functional tissues. Therefore, a suitable three-dimensional carrier with tailored macroscopic properties, a well-tuned degradation profile, and specific biological cues are necessary to promote successful tissue growth (Kleinman et al., 2003; Abukawa et al., 2006; Ramseier et al., 2006). Polymers and ceramics are the two major families of biomaterials currently used in tissue engineering. To date, various synthetic biodegradable polymer scaffolds, such as polyglycolic acid (PGA) (Sumita et al., 2006) and poly-L-lactate-co-glycolate (PLGA) (Young et al., 2002, 2005) have been used in dental-related tissue regeneration. Jin et al. (2003) and Zhao et al. (2004) have also used PLGA polymer sponges to deliver cementoblasts, fibroblasts, and dental follicle cells to facilitate periodontal regeneration. These synthetic polymers can be fabricated into desirable sizes and shapes, with readily adjustable degradation profile and pore features. However, they exhibit poor biocompatibility due to release of acidic degradation products and lose mechanical properties very early during degradation (Gunatillake and Adhikari, 2003). Natural polymers, like collagen, have been explored as scaffolds in whole-tooth (Sumita et al., 2006) or periodontal (Rutherford et al.,
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Epithelium
Mesenchyme
Scaffold
Epithelium/mesenchyme interaction
Yellow oval: ameloblast Green oval: odontoblast Ameloblasts Enamel Dentin Odontoblasts Pulp
Figure 75.2 Scheme of whole-tooth tissue engineering.
1993) bioengineering. Properties that can compromise the efficiency of collagen-based scaffolds are rapid absorption and weak mechanical strength (Chevallay and Herbage, 2000). Ceramics, including hydroxyapatite (HA) and tricalcium phosphate (TCP) (Gronthos et al., 2000, Grzesik et al., 2000, Seo et al., 2004; Saito et al., 2005; Shi et al., 2005; Zhang et al., 2005), are also being examined for dental tissue regeneration applications, due to their biocompatibility, osteoconductivity, and structural similarity to the inorganic component of bone. However, the predictability of ceramic degradation is poor, as it is dependent on many factors, including material crystallinity, porosity, density, and host response (Theiss et al., 2005). Tissue regeneration is a highly coordinated process, involving stem cells, scaffolds, and regulatory signals. Scaffold chemistry, morphology, and structure directly impact cell–cell interactions and signaling cascades. For dental tissue regenerative purposes, studies of scaffolding materials are being conducted to identify compatible matches between materials and biological functions. In particular, since tooth development depends on interactions between dental epithelial and mesenchymal cells, to successfully bioengineer teeth of predetermined size and shape, biodegradable scaffolds are desired that can initially facilitate proper epithelial and mesenchymal cell orientation, to guide the subsequent formation of highly mineralized tooth tissues (Figure 75.2). Prior and Current Dental Tissue Regeneration Research The foundation of dental tissue bioengineering has ancient roots. Therapies to replace missing teeth can be traced back at least 2,500 years, when the Etruscans learned to substitute missing teeth with bridges made from
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artificial teeth carved from the bones of oxen (Ring, 1995). More recent efforts to regenerate teeth from ectopically grafted embryonic tooth buds have been previously reviewed (Yelick and Vacanti, 2004; Yen and Sharpe, 2006), demonstrating the feasibility of growing teeth in an appropriate environment. State-of-the-art dental tissue engineering methodologies, which employ in vitro expanded presumptive DSCs that are then seeded onto three-dimensional biodegradable scaffolds and implanted back into individuals, are rapidly advancing. Reparative Pulp and Dentin Dentin, a highly calcified connective tissue, is similar to, but distinct from bone (Linde and Goldberg, 1993). Dental pulp tissue exhibits the potential to regenerate dentin in response to noxious stimuli, such as caries, and it was hypothesized that the stem/progenitor cells present in the dental pulp differentiate into odontoblasts in response to BMP signaling (Nakashima and Akamine, 2005). BMP-driven stem cell therapies have shown considerable promise in dentin–pulp complex regeneration (Shi et al., 2005; Murray and GarciaGodoy, 2006). Iohara et al. (2004) discovered that when stimulated by the morphogenetic signal BMP2 porcine pulp cells differentiate into odontoblasts, as indicated by the expression of dentin sialophosphoprotein (DSPP) and enamelysin/matrix metalloproteinase 20 (MMP20). The autogenous transplantation of the BMP2-treated pulp cells into an amputated pulp cavity resulted in reparative dentin formation in dogs. Zhang et al. (2005) isolated dental pulp cells from maxillary incisors of rats and found that these cells, cultured on either titanium or TCP scaffolds, differentiated into odontoblast-like cells and produced calcified nodules similar to dentin. Furthermore, Gronthos and collaborators (2000) have isolated highly proliferative progenitor cells from adult human dental pulp, which produced densely calcified nodules in culture. After in vivo transplantation of cultured cells into immune compromised mice, a dentin/pulp-like complex was generated, again demonstrating the potential use of this approach for successful dental tissue regeneration. Periodontal Tissue Regeneration The wide prevalence of periodontal disease, the limited regenerative capability exhibited by the PDL, and the critical role of the PDL in maintaining tooth health and function have made periodontal tissue engineering an extremely active area of research. As mentioned above, the PDL complex contains putative stem cells that can commit to a variety of cell fates, such as cementum, ligament, and bone. Periodontal tissue engineering offers a powerful approach to supplement existing treatment regimens for periodontal disease, as recently reviewed (Taba et al., 2005; Bartold et al., 2006; Ramseier et al., 2006). A considerable amount of research has explored periodontal regeneration capability of progenitor cells isolated from the PDL of humans (Grzesik et al., 2000; Seo et al., 2004; Shi et al., 2005), bovine (Saito et al., 2005), and mice (Jin et al., 2003; Zhao et al., 2004). According to the findings of Grzesik and colleagues (2000), human cementum-derived cells (HCDCs), expanded in vitro, formed a mineralized matrix when attached to a ceramic carrier and transplanted subcutaneously into immunodeficient mice. The mineralized matrix exhibited features identical to cementum, with typical organized bundles of collagen fibers (Sodek et al. 1977). Saito and coworkers (2005) transplanted bovine cementoblast progenitor cells subcutaneously into nude mice on an HA/TCP scaffold, also resulting in the formation of cementum-like tissue. In addition, Zhao et al. (2004) reported that rat cementoblasts, delivered via PLGA polymer sponges, have a marked ability to promote periodontal regeneration by inducing mineralization and PDL formation in periodontal wounds. Also in this study, the demonstrated healing of alveolar bone defects was found to be limited only by the size of the transplanted PLGA carrier.
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Interestingly, Kawaguchi et al. (2004) used autologous bone marrow stem cells (BMSCs), in combination with atelocollagen (2% type I collagen), to regenerate periodontal tissues in beagle dogs. The repaired experimental Class III defects consisted of cementum, PDL, and alveolar bone, suggesting that autotransplantation of BMSCs, embedded in the appropriate environment niche, is a novel option for the regeneration of complex tissues, including periodontium. Growth factors or morphogens modulate the cellular activities of, and induce cell differentiation and extracellular matrix production in, developing tissues. The effects of several growth factors, including BMPs (Ripamonti and Reddi, 1997; Giannobile and Somerman, 2003; Wikesjo et al., 2004; Xu et al., 2004; Dunn et al., 2005; Miranda et al., 2005), insulin-like growth factor-I (IGF-I) (Saygin et al., 2000), PDGF (Giannobile et al., 2001; Jin et al., 2004; Nevins et al., 2005; Sarment et al., 2006), and transforming growth factor-β (TGF-β) (Tatakis et al., 2000), on periodontal regeneration of various animal models, including baboons (Miranda et al., 2005), dogs (Tatakis et al., 2000; Wikesjo et al., 2004), rats (Jin et al., 2004; Dunn et al., 2005), and humans (Xu et al., 2004; Nevins et al., 2005; Sarment et al., 2006), have been evaluated. For instance, Saygin et al. (2000) reported that several growth factors, including IGF-I, PDGF-BB, and TGF-β, influence the mitogenetic potential, phenotypic gene expression profile, and biomineralization potential of cementoblasts. Even though preclinical and early clinical data for these growth factors appear promising in stimulating periodontal regeneration, they are not sufficient for definitive conclusions at this time (Ramseier et al., 2006). Furthermore, the methods to deliver growth factors, and the kinetics of release must also be considered to optimize the dosage and exposure time to the cells, and to eventually make periodontal tissue engineering a widely practiced, clinically available therapy. Based on Melcher’s (1976) proposition that cells that repopulate the periodontal wound would determine the type of new attachment onto the root surface, a surgical procedure called guided tissue regeneration (GTR) has demonstrated tremendous potential for periodontal tissue regeneration, by using a barrier membrane to selectively encourage progenitor cell populations to remain at the wound site (Quinones et al., 1996; Laurell and Gottlow, 1998). For example, Aukhil et al. (1986) reported the use of a cell-occlusive barrier to prevent gingival epithelium and connective tissue from growing into the periodontal space and to provide a favorable niche to promote maximal functional PDL regeneration in beagle dogs. More recently, Wikesjo et al. (2003) have shown that the combined use of BMP-2 with a physical barrier, such as a bioabsorbable, space-providing polymer membrane, significantly enhanced periodontal bone and soft tissue regeneration in dogs. However, clinical results using this method to achieve complete restoration of periodontal defects are often unpredictable and vary significantly, possibly due to variations in defect morphology among individuals. Whole-Tooth Tissue Engineering Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continue to adversely affect most individuals at some time in their lives. Recent achievements in tooth tissue engineering promise to provide viable alternatives to currently available human tooth replacement therapies. Young et al. (2002) and Duailibi et al. (2004) have demonstrated the potential to bioengineer complex tooth structures from pig or rat tooth bud tissues, respectively. Using a tissue engineering approach, cells from tooth buds of either 6-month-old porcine third molar or 4-day postnatal (dpn) rats were seeded onto biodegradable polymers. After a period of growth in the omenta of adult rat hosts, a conducive environment to promote vascularization and tissue growth, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig’s root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Both of these studies demonstrated that continuous,
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de novo tooth development had occured in the implants, suggesting the potential utility of this method for the regeneration of mammalian dental tissues. As mentioned above, tooth development originates from interactions between dental epithelial and mesenchymal cells, with the epithelium providing the instructive signals for tooth initiation and shape determination. Based on these properties, Ohazama et al. (2004) examined the odontogenic potential of embryonic stem cells, neural stem cells, or adult bone marrow-derived cells, combined with oral epithelium. The oral epithelium was found to stimulate odontogenic responses in each of these three non-dental mesenchymal cell types. Transfer of the tissue recombinants into adult mice renal capsules resulted in the formation of tooth structures and associated bone. In addition, transfer of embryonic tooth primordia into the adult jaw led to the development of tooth structures, indicating that an embryonic primordium can develop in an adult environment. The ability for heterogeneous postnatal and adult cell populations obtained from rodents to form bone and teeth in tissue-engineered rudiments may have important implications for the further development of these procedures on humans. The ability to bioengineer whole teeth inspires additional important therapeutic strategies. For instance, a logical therapeutic approach for treatment of edentulism and accompanying alveolar bone loss would be to combine whole-tooth bioengineering with bone tissue engineering approaches (Young et al., 2005; Yelick and Vacanti, 2006). The combined usage of these methods would result in the generation of both jaw and tooth root structures, to provide teeth and supporting periodontal tissues for individuals born without, or to replace structures lost to disease or injury. The widespread availability of such coordinated therapeutic treatments would provide potentially powerful tools for dental tissue therapeutic strategies and dramatically alter the current landscape of treatments for a variety of craniofacial anomalies.
CONCLUSIONS Dental tissue engineering is a newly emerging field, with just over a decade of creation and development. Although still largely experimental in nature, recent progress in this field promises that tissue engineering approaches will be used to bioengineer replacement teeth in the foreseeable future. The successful development of methodologies for dental tissue repair/regeneration from autologous adult tissues will change the face of modern dentistry and reduce the need for synthetic materials in clinical dental practice. The pressing challenge confronting dental tissue engineering is how to perfect the current techniques, such that bioengineered dental tissues or whole teeth are integrated physically and functionally with preexisting dental tissues (Yelick and Vacanti, 2006). Ideally, the bioengineered teeth would be fabricated to occlude properly with opposing and adjacent teeth and be anchored to the underlying alveolar bone via the PDL to transmit mechanical signals when necessary. Biological teeth would also exhibit proper proprioception, facilitating the life of the substitute itself, as well as surrounding teeth. To accomplish these goals, some important hurdles need to be overcome, including: the identification and maintenance of multipotential DSCs in vitro; the establishment of conditions that foster DSC growth and differentiation; and the development of suitable scaffolds and inductive factors that help DSC-scaffold constructs integrate into the surrounding environment for the reconstruction of dental tissues. With parallel and synergistic advancements in stem cell biology and material science, it is possible to envision that the day may soon come when a patient can visit his or her dentist to obtain a biological, living tooth, grown from the patient’s own cells. ACKNOWLEDGMENTS The authors would like to thank Dan McCloskey and Susan Orlando for library science expertise.
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REFERENCES Abukawa, H., Papadaki, M., Abulikemu, M., Leaf, J., Vacanti, J.P., Kaban, L.B. and Troulis, M.J. (2006). The engineering of craniofacial tissues in the laboratory: a review of biomaterials for scaffolds and implant coatings. Dent. Clin. N. Am. 50: 205–216. Aukhil, I., Pettersson, E. and Suggs, C. (1986). Guided tissue regeneration. An experimental procedure in beagle dogs. J. Periodontol. 57: 727–734. Bartlett, J.D. and Simmer, J.P. (1999). Proteinases in developing dental enamel. Crit. Rev. Oral. Biol. Med. 10: 425–441. Bartold, P.M., Shi, S. and Gronthos, S. (2006). Stem cells and periodontal regeneration. Periodontol. 2000 40: 164–172. Beertsen, W., McCulloch, C.A. and Sodek, J. (1997). The periodontal ligament: a unique, multifunctional connective tissue. Periodontol. 2000 13: 20–40. Begue-Kirn, C., Ruch, J.V., Ridall, A.L. and Butler, W.T. (1998). Comparative analysis of mouse DSP and DPP expression in odontoblasts, preameloblasts, and experimentally induced odontoblast-like cells. Eur. J. Oral Sci. 106(Suppl 1): 254–259. Bosshardt, D.D. (2005). Are cementoblasts a subpopulation of osteoblasts or a unique phenotype? J. Dent. Res. 84: 390–406. Butler, W.T., Brunn, J.C., Qin, C. and McKee, M.D. (2002). Extracellular matrix proteins and the dynamics of dentin formation. Connect. Tissue Res. 43: 301–307. Chai, Y., Jiang, X., Ito, Y., Bringas Jr., P., Han, J., Rowitch, D.H., Soriano, P., McMahon, A.P. and Sucov, H.M. (2000). Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127: 1671–1679. Chevallay, B. and Herbage, D. (2000). Collagen-based biomaterials as 3D scaffold for cell cultures: applications for tissue engineering and gene therapy. Med. Biol. Eng. Comput. 38: 211–218. Diekwisch, T.G. (2001). The developmental biology of cementum. Int. J. Dev. Biol. 45: 695–706. Duailibi, M.T., Duailibi, S.E., Young, C.S., Bartlett, J.D., Vacanti, J.P. and Yelick, P.C. (2004). Bioengineered teeth from cultured rat tooth bud cells. J. Dent. Res. 83: 523–528. Dunn, C.A., Jin, Q., Taba Jr., M., Franceschi, R.T., Bruce, R.R. and Giannobile, W.V. (2005). BMP gene delivery for alveolar bone engineering at dental implant defects. Mol. Ther. 11: 294–299. Giannobile, W.V. and Somerman, M.J. (2003). Growth and amelogenin-like factors in periodontal wound healing. A systematic review. Ann. Periodontol. 8: 193–204. Giannobile, W.V., Lee, C.S., Tomala, M.P., Tejeda, K.M. and Zhu, Z. (2001). Platelet-derived growth factor (PDGF) gene delivery for application in periodontal tissue engineering. J. Periodontol. 72: 815–823. Gronthos, S., Mankani, M., Brahim, J., Robey, P.G. and Shi, S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl. Acad. Sci. USA 97: 13625–13630. Grzesik, W.J., Cheng, H., Oh, J.S., Kuznetsov, S.A., Mankani, M.H., Uzawa, K., Robey, P.G. and Yamauchi, M. (2000). Cementum-forming cells are phenotypically distinct from bone-forming cells. J. Bone Miner. Res. 15: 52–59. Gunatillake, P.A. and Adhikari, R. (2003). Biodegradable synthetic polymers for tissue engineering. Eur. Cell Mater. 5: 1–16. Iohara, K., Nakashima, M., Ito, M., Ishikawa, M., Nakasima, A. and Akamine, A. (2004). Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. J. Dent. Res. 83: 590–595. Jernvall, J. and Thesleff, I. (2000). Reiterative signaling and patterning during mammalian tooth morphogenesis. Mech. Dev. 92: 19–29. Jin, Q.M., Zhao, M., Webb, S.A., Berry, J.E., Somerman, M.J. and Giannobile, W.V. (2003). Cementum engineering with three-dimensional polymer scaffolds. J. Biomed. Mater. Res. A 67: 54–60. Jin, Q.M., Anusaksathien, O., Webb, S.A., Printz, M.A. and Giannobile, W.V. (2004). Engineering of tooth-supporting structures by delivery of PDGF gene therapy vectors. Mol. Ther. 9: 519–526. Kawaguchi, H., Hirachi, A., Hasegawa, N., Iwata, T., Hamaguchi, H., Shiba, H., Takata, T., Kato, Y. and Kurihara, H. (2004). Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells. J Periodontol. 75: 1281–1287.
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Kleinman, H.K., Philp, D. and Hoffman, M.P. (2003). Role of the extracellular matrix in morphogenesis. Curr. Opin. Biotechnol. 14: 526–532. Krebsbach, P.H. and Robey, P.G. (2002). Dental and skeletal stem cells: potential cellular therapeutics for craniofacial regeneration. J. Dent. Educ. 66: 766–773. Krebsbach, P.H., Lee, S.K., Matsuki, Y., Kozak, C.A., Yamada, K.M. and Yamada, Y. (1996). Full-length sequence, localization, and chromosomal mapping of ameloblastin. A novel tooth-specific gene. J. Biol. Chem. 271: 4431–4435. Laurell, L. and Gottlow, J. (1998). Guided tissue regeneration update. Int. Dent. J. 48: 386–398. Linde, A. and Goldberg, M. (1993). Dentinogenesis. Crit. Rev. Oral Biol. Med. 4: 679–728. Luan, X., Ito, Y. and Diekwisch, T.G. (2006). Evolution and development of Hertwig’s epithelial root sheath. Dev. Dynam. [Epub ahead of print]. McCulloch, C.A. (1995). Origins and functions of cells essential for periodontal repair: the role of fibroblasts in tissue homeostasis. Oral Dis. 1: 271–278. Melcher, A.H. (1976). On the repair potential of periodontal tissues. J. Periodontol. 47: 256–260. Miranda, D.A., Blumenthal, N.M., Sorensen, R.G., Wozney, J.M. and Wikesjo, U.M. (2005). Evaluation of recombinant human bone morphogenetic protein-2 on the repair of alveolar ridge defects in baboons. J. Periodontol. 76: 210–220. Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, L.W., Robey, P.G. and Shi, S. (2003). SHED: stem cells from human exfoliated deciduous teeth. Proc. Natl. Acad. Sci. USA 100: 5807–5812. Murray, P.E. and Garcia-Godoy, F. (2004). Stem cell responses in tooth regeneration. Stem Cell Dev. 13: 255–262. Murray, P.E. and Garcia-Godoy, F. (2006). The outlook for implants and endodontics: a review of the tissue engineering strategies to create replacement teeth for patients. Dent. Clin. N. Am. 50: 299–315. Nakao, K., Itoh, M., Tomita, Y., Tomooka, Y. and Tsuji, T. (2004). FGF-2 potently induces both proliferation and DSP expression in collagen type I gel cultures of adult incisor immature pulp cells. Biochem. Biophys. Res. Commun. 325: 1052–1059. Nakashima, M. and Akamine, A. (2005). The application of tissue engineering to regeneration of pulp and dentin in endodontics. J. Endod. 31: 711–718. Nakashima, M. and Reddi, A.H. (2003). The application of bone morphogenetic proteins to dental tissue engineering. Nat. Biotechnol. 21: 1025–1032. Nanci, A. (2003). Ten Cate’s Oral Histology, Development, Structure, and Function, 6th edn. St. Louis: Mosby[A1]. Nanci, A. and Bosshardt, D.D. (2006). Structure of periodontal tissues in health and disease. Periodontol. 2000 40: 11–28. Nevins, M., Giannobile, W.V., McGuire, M.K., Kao, R.T., Mellonig, J.T., Hinrichs, J.E., McAllister, B.S., Murphy, K.S., McClain, P.K., Nevins, M.L., Paquette, D.W., Han, T.J., Reddy, M.S., Lavin, P.T., Genco, R.J. and Lynch, S.E. (2005). Platelet-derived growth factor stimulates bone fill and rate of attachment level gain: results of a large multicenter randomized controlled trial. J. Periodontol. 76: 2205–2215. Ohazama, A., Modino, S.A., Miletich, I. and Sharpe, P.T. (2004). Stem-cell-based tissue engineering of murine teeth. J. Dent. Res. 83: 518–522. Paine, C.T., Paine, M.L. and Snead, M.L. (1998). Identification of tuftelin- and amelogenin-interacting proteins using the yeast two-hybrid system. Connect. Tissue Res. 38: 257–267. Quinones, C.R., Casellas, J.C. and Caffesse, R.G. (1996). Guided periodontal tissue regeneration (GPTR): an update. Pract. Periodontics Aesthet. Dent. 8: 169–180. Ramseier, C.A., Abramson, Z.R., Jin, Q. and Giannobile, W.V. (2006). Gene therapeutics for periodontal regenerative medicine. Dent. Clin. N. Am. 50: 245–263. Ring, M.E. (1995). A thousand years of dental implants: a definitive history. Comp. Cont. Educ. Dent. 16: 1060–1069. Ripamonti, U. and Reddi, A.H. (1997). Tissue engineering, morphogenesis, and regeneration of the periodontal tissues by bone morphogenetic proteins. Crit. Rev. Oral Biol. Med. 8: 154–163. Risbud, M.V. and Shapiro, I.M. (2005). Stem cells in craniofacial and dental tissue engineering. Orthod. Craniofac. Res. 8: 54–59. Rutherford, R.B., Ryan, M.E., Kennedy, J.E., Tucker, M.M. and Charette, M.F. (1993). Platelet-derived growth factor and dexamethasone combined with a collagen matrix induce regeneration of the periodontium in monkeys. J. Clin. Periodontol. 20: 537–544.
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Saito, M., Handa, K., Kiyono, T., Hattori, S., Yokoi, T., Tsubakimoto, T., Harada, H., Noguchi, T., Toyoda, M., Sato, S. and Teranaka, T. (2005). Immortalization of cementoblast progenitor cells with Bmi-1 and TERT. J. Bone Miner. Res. 20: 50–57. Sarment, D.P., Cooke, J.W., Miller, S.E., Jin, Q., McGuire, M.K., Kao, R.T., McClain, P.K., McAllister, B.S., Lynch, S.E. and Giannobile, W.V. (2006). Effect of rhPDGF-BB on bone turnover during periodontal repair. J. Clin. Periodontol. 33: 135–140. Saygin, N.E., Giannobile, W.V. and Somerman, M.J. (2000). Molecular and cell biology of cementum. Periodontol. 2000 24: 73–98. Seo, B.M., Miura, M., Gronthos, S., Bartold, P.M., Batouli, S., Brahim, J., Young, M., Robey, P.G., Wang, C.Y. and Shi, S. (2004). Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364: 149–155. Shi, S., Bartold, P.M., Miura, M., Seo, B.M., Robey, P.G. and Gronthos, S. (2005) The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod. Craniofac. Res. 8: 191–199. Snead, M.L., Zeichner-David, M., Chandra, T., Robson, K.J., Woo, S.L. and Slavkin, H.C. (1983). Construction and identification of mouse amelogenin cDNA clones. Proc. Natl. Acad. Sci. USA 80: 7254–7258. Sodek, J., Brunette, D.M., Feng, J., Heersche, J.N., Limeback, H.F., Melcher, A.H. and Ng, B. (1977). Collagen synthesis is a major component of protein synthesis in the periodontal ligament in various species. Arch. Oral Biol. 22: 647–653. Spradling, A., Drummond-Barbosa, D. and Kai, T. (2001). Stem cells find their niche. Nature 414: 98–104. Sumita, Y., Honda, M.J., Ohara, T., Tsuchiya, S., Sagara, H., Kagami, H. and Ueda, M. (2006). Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering. Biomaterials 27: 3238–3248. Taba Jr., M., Jin, Q., Sugai, J.V. and Giannobile, W.V. (2005). Current concepts in periodontal bioengineering. Orthod. Craniofac. Res. 8: 292–302. Tatakis, D.N., Wikesjo, U.M., Razi, S.S., Sigurdsson, T.J., Lee, M.B., Nguyen, T., Ongpipattanakul, B. and Hardwick, R. (2000). Periodontal repair in dogs: effect of transforming growth factor-beta 1 on alveolar bone and cementum regeneration. J. Clin. Periodontol. 27: 698–704. Theiss, F., Apelt, D., Brand, B., Kutter, A., Zlinszky, K., Bohner, M., Matter, S., Frei, C., Auer, J.A. and von, R.B. (2005). Biocompatibility and resorption of a brushite calcium phosphate cement. Biomaterials 26: 4383–4394. Thesleff, I. and Sharpe, P. (1997). Signalling networks regulating dental development. Mech. Dev. 67: 111–123. Thesleff, I., Keranen, S. and Jernvall, J. (2001). Enamel knots as signaling centers linking tooth morphogenesis and odontoblast differentiation. Adv. Dent. Res. 15: 14–18. Wikesjo, U.M., Lim, W.H., Thomson, R.C., Cook, A.D., Wozney, J.M. and Hardwick, W.R. (2003). Periodontal repair in dogs: evaluation of a bioabsorbable space-providing macroporous membrane with recombinant human bone morphogenetic protein-2. J. Periodontol. 74: 635–647. Wikesjo, U.M., Sorensen, R.G., Kinoshita, A., Jian, L. and Wozney, J.M. (2004). Periodontal repair in dogs: effect of recombinant human bone morphogenetic protein-12 (rhBMP-12) on regeneration of alveolar bone and periodontal attachment. J. Clin. Periodontol. 31: 662–670. Xu, W.P., Shiba, H., Mizuno, N., Uchida, Y., Mouri, Y., Kawaguchi, H. and Kurihara, H. (2004). Effect of bone morphogenetic proteins-4, -5 and -6 on DNA synthesis and expression of bone-related proteins in cultured human periodontal ligament cells. Cell Biol. Int. 28: 675–682. Yelick, P.C. and Vacanti, J.P. (2004). Handbook of Stem Cells, Vol. 2. Academic Press, pp. 279–292. Yelick, P.C. and Vacanti, J.P. (2006). Bioengineered teeth from tooth bud cells. Dent. Clin. N. Am. 50: 191–203. Yen, A.H. and Sharpe, P.T. (2006). Regeneration of teeth using stem cell-based tissue engineering. Expert Opin. Biol. Ther. 6: 9–16. Young, C.S., Terada, S., Vacanti, J.P., Honda, M., Bartlett, J.D. and Yelick, P.C. (2002). Tissue engineering of complex tooth structures on biodegradable polymer scaffolds. J. Dent. Res. 81: 695–700. Young, C.S., Abukawa, H., Asrican, R., Ravens, M., Troulis, M.J., Kaban, L.B., Vacanti, J.P. and Yelick, P.C. (2005). Tissueengineered hybrid tooth and bone. Tissue Eng. 11: 1599–1610.
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Zeichner-David, M., Diekwisch, T., Fincham, A., Lau, E., MacDougall, M., Moradian-Oldak, J., Simmer, J., Snead, M. and Slavkin, H.C. (1995). Control of ameloblast differentiation. Int. J. Dev. Biol. 39: 69–92. Zhang, W., Walboomers, X.F., Wolke, J.G., Bian, Z., Fan, M.W. and Jansen, J.A. (2005). Differentiation ability of rat postnatal dental pulp cells in vitro. Tissue Eng. 11: 357–368. Zhao, M., Jin, Q., Berry, J.E., Nociti Jr., F.H., Giannobile, W.V. and Somerman, M.J. (2004). Cementoblast delivery for periodontal tissue engineering. J. Periodontol. 75: 154–161.
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Enamel Crown
Dentin Pulp
Neck Cementum Root
Periodontal ligament Root canal
Figure 75.1 Erupted tooth structures.
but is not self-regenerative, since ameloblasts are no longer present in postnatal or adult teeth once they have erupted. With the exception of enamel, all of the other tooth tissues are derived from neural crest cell (NCC)derived dental mesenchyme. The tooth layer underlying the enamel is composed of dentin, which is approximately 75% mineralized and contains dentin-specific gene products, including dentin sialoprotein (DSP), dentin phosphoprotein (DPP), and dentin matrix protein-1 (DMP-1) (Begue-Kirn et al., 1998; Butler et al., 2002). Dentin is produced by NCC-derived dental mesenchymal cells called odontoblasts, which exhibit limited regenerative capacities in response to injury or disease. The dental pulp at the center of the tooth is composed of dental mesenchymal cells, including putative dental stem cells (DSCs), nerves, and blood vessels that thread through the root canal and support the tooth organ. The periodontium, which supports and attaches teeth to the jaw, primarily consists of four connective tissues: mineralized cementum, fibrous PDL, alveolar bone, and gingival tissue (Beertsen et al., 1997). Cementum is a bone-like mineralized tissue lining the dentin of the root that protects the root and also serves as an attachment surface to anchor the PDL to the tooth (Diekwisch, 2001). The PDL functions to secure the tooth to the underlying alveolar bone and is essential for the long-term survival of the tooth. Sub-functioning PDL tissue can result in tooth root damage and eventual ankylosis of the tooth. Cells of the PDL include fibroblasts, cementoblasts on the root surface, osteoblasts and osteoclasts present on the alveolar bone face, and undifferentiated mesenchymal tissues in the body of the ligament (Taba et al., 2005; Nanci and Bosshardt, 2006). The periodontium also possesses some regenerative capability, as tissue loss during the early phases of periodontal diseases can be restored to a certain degree. However, once periodontitis becomes established only therapeutic intervention has the potential to induce regeneration (Shi et al., 2005). When considering methods to regenerate teeth, it is first necessary to have a thorough understanding of de novo tooth development. Like many other organs, such as hair follicles, salivary glands, intestines, and kidneys, tooth development is the cumulative result of reciprocal signaling events between the ectoderm-derived dental epithelial and the NCC-derived mesenchyme (Jernvall and Thesleff, 2000). Molecular signals initiated in the dental epithelium induce gene expression in the subjacent dental mesenchyme, and the acquired odontogenic potential in the mesenchyme triggers further epithelial morphogenesis and cytodifferentiation. As tooth development progresses, dental mesenchyme differentiates into dentin, pulp, cementum, PDL, and alveolar bone, and the epithelial tissues produce dental enamel. The cellular and molecular natures of enamel,
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dentin, cementum, and root development have been extensively characterized (Linde and Goldberg, 1993; Zeichner-David et al., 1995; Bartlett and Simmer, 1999; Chai et al., 2000; Grzesik et al., 2000; Jernvall and Thesleff, 2000; Saygin et al., 2000; Diekwisch, 2001; Bosshardt, 2005; Luan et al., 2006). The morphological stages of natural tooth development consist of early bud and cap stages, and later bell stage where tooth histodifferentiation and morphodifferentiation occur (Thesleff et al., 2001). Tooth development begins as a localized thickening of the dental epithelium, which proliferates and invaginates into the underlying dental mesenchyme, appearing as a small epithelial bud. The primary “enamel knot,” a signaling center that forms at the tip of the epithelial tooth bud, and subsequently formed enamel knots, all of which mark the site of future cusp formation, are considered to be central regulators of tooth development, as they link dental cell differentiation to tooth morphogenesis (Thesleff et al., 2001). The primary enamel knot expresses at least 10 different signaling molecules belonging to the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (Hh), and Wnt (an amalgam of wingless- and int-related proteins) families, which are responsible for the transition from bud to cap stage (Thesleff and Sharpe, 1997; Jernvall and Thesleff, 2000). Epithelial signals emanating from the primary enamel knot induce the formation of the dental papilla, the mesenchymal cell layer underlying the dental epithelium, at the transition from bud to cap stage. Later on, cells of the dental papilla differentiate into odontoblasts, which subsequently differentiate and produce dentin (Thesleff et al., 2001). In bell stage teeth, so named for their characteristic bell-shaped appearance, odontoblasts induce ameloblast formation, and the ameloblasts differentiate and synthesize enamel. Also, at the bell stage the dental lamina connecting the tooth germ to the oral epithelium disappears, isolating the tooth from the oral cavity. Dental Tissue Engineering Realizing the full potential of tissue regenerative treatments for the oral complex will require the integration of three key elements: (1) dental stem/progenitor cells; (2)inductive morphogenetic signals (morphogens); and (3) scaffold material upon which progenitor cells attach and elaborate an extracellular matrix (Nakashima and Reddi, 2003). DSCs One way to regenerate whole teeth would be to mimic the process of natural tooth development, either in vitro or in vivo, using DSCs. DSCs are a somewhat elusive population of self-renewing cells that exhibit the potential to form biological replacement tooth tissues. A variety of DSC populations have been identified for potential use in tooth tissue engineering strategies, as recently reviewed (Krebsbach and Robey, 2002; Murray and Garcia-Godoy, 2004; Yelick and Vacanti, 2004; Risbud and Shapiro, 2005; Shi et al., 2005; Bartold et al., 2006; Yen and Sharpe, 2006). Because tooth development is dependent on epithelial and mesenchymal cell interactions, DSCs necessarily consist of two types: epithelial DSCs, which form ameloblasts, enamel, stellate reticulum, and stratum intermedium, and mesenchymal DSCs, which form the dental papilla, odontoblasts, predentin, dentin, cementum, PDL, and alveolar bone (Nanci, 2003). The exclusive environment in which stem cells reside, called the “stem cell niche,” is thought to support the maintenance and self-renewal of stem cells (Spradling et al., 2001). In the remaining paragraphs, we will focus on the applications of stem cells in dental tissue engineering. Significant efforts currently focus on the development of methods to control the differentiation and proliferation of DSCs, to facilitate the creation of stem cell-based therapies that can be used more effectively than current synthetic material-based dental treatment regimes (Yelick and Vacanti, 2006). For instance, Miura
Dental Tissue Engineering
et al. (2003) isolated stem cells from human exfoliated deciduous teeth, which were found to be capable of differentiating into a variety of cell types, including neural cells, adipocytes, and odontoblasts. The reparative potential of these progenitor cells is now being carefully scrutinized. It has long been appreciated that the PDL harbors progenitor cells that can differentiate into fibroblasts, osteoblasts, and cementoblasts (McCulloch, 1995; Bartold et al., 2006). Many investigators, including Seo and coworkers (2004), have isolated multipotent postnatal human PDL stem cells and demonstrated their capacity to generate cementum/PDL-like tissue after in vivo transplantation. However, at the present time, limited knowledge exists about the manner in which the specialized periodontal tissues are organized and the identity of the cells that contribute to the formation of each type of periodontal tissue (Shi et al., 2005). What has become increasingly clear is the fact that as knowledge of stem cell biology continues to advance, including methods to identify, isolate, expand, and differentiate DSCs, biologically based dental repair and whole-tooth regeneration therapies will become realistic possibilities. Growth Factors and Morphogen-Based Cell Signaling Gradients A major focus of contemporary developmental biology has been to delineate the biological cues that drive stem cell proliferation and differentiation (Thesleff and Sharpe, 1997; Nakao et al., 2004). Knowledge of the responsiveness of DSCs to various morphogenetic signals is of potentially significant importance for improving dental regenerative therapies. Continued efforts to elucidate signaling pathways regulating natural tooth development focus on four families of growth factors, the BMP, FGF, Hh, and Wnt, which govern tissue-specific patterning and morphogenesis during odontogenesis (Yen and Sharpe, 2006). Growth factor ligands from each of these families are currently being evaluated for their utility in guided, stem cell-based dental tissue engineering efforts. It has been demonstrated that delivery of signaling molecules, including BMP (Ripamonti and Reddi, 1997; Nakashima and Reddi, 2003; Iohara et al., 2004) and platelet-derived growth factor (PDGF) (Rutherford et al., 1993; Giannobile et al., 2001; Jin et al., 2004) via gene therapy vectors, resulted in partial dental tissue regeneration, for example, dentin and periodontal tissues. The results suggest that such factors, alone or in combination with other agents, may eventually be used to promote dental tissue regeneration. Scaffolding Materials In the context of tissue engineering, scaffolds made of natural or synthetic materials play a central role in supporting cells during the formation of functional tissues. Therefore, a suitable three-dimensional carrier with tailored macroscopic properties, a well-tuned degradation profile, and specific biological cues are necessary to promote successful tissue growth (Kleinman et al., 2003; Abukawa et al., 2006; Ramseier et al., 2006). Polymers and ceramics are the two major families of biomaterials currently used in tissue engineering. To date, various synthetic biodegradable polymer scaffolds, such as polyglycolic acid (PGA) (Sumita et al., 2006) and poly-L-lactate-co-glycolate (PLGA) (Young et al., 2002, 2005) have been used in dental-related tissue regeneration. Jin et al. (2003) and Zhao et al. (2004) have also used PLGA polymer sponges to deliver cementoblasts, fibroblasts, and dental follicle cells to facilitate periodontal regeneration. These synthetic polymers can be fabricated into desirable sizes and shapes, with readily adjustable degradation profile and pore features. However, they exhibit poor biocompatibility due to release of acidic degradation products and lose mechanical properties very early during degradation (Gunatillake and Adhikari, 2003). Natural polymers, like collagen, have been explored as scaffolds in whole-tooth (Sumita et al., 2006) or periodontal (Rutherford et al.,
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Epithelium
Mesenchyme
Scaffold
Epithelium/mesenchyme interaction
Yellow oval: ameloblast Green oval: odontoblast Ameloblasts Enamel Dentin Odontoblasts Pulp
Figure 75.2 Scheme of whole-tooth tissue engineering.
1993) bioengineering. Properties that can compromise the efficiency of collagen-based scaffolds are rapid absorption and weak mechanical strength (Chevallay and Herbage, 2000). Ceramics, including hydroxyapatite (HA) and tricalcium phosphate (TCP) (Gronthos et al., 2000, Grzesik et al., 2000, Seo et al., 2004; Saito et al., 2005; Shi et al., 2005; Zhang et al., 2005), are also being examined for dental tissue regeneration applications, due to their biocompatibility, osteoconductivity, and structural similarity to the inorganic component of bone. However, the predictability of ceramic degradation is poor, as it is dependent on many factors, including material crystallinity, porosity, density, and host response (Theiss et al., 2005). Tissue regeneration is a highly coordinated process, involving stem cells, scaffolds, and regulatory signals. Scaffold chemistry, morphology, and structure directly impact cell–cell interactions and signaling cascades. For dental tissue regenerative purposes, studies of scaffolding materials are being conducted to identify compatible matches between materials and biological functions. In particular, since tooth development depends on interactions between dental epithelial and mesenchymal cells, to successfully bioengineer teeth of predetermined size and shape, biodegradable scaffolds are desired that can initially facilitate proper epithelial and mesenchymal cell orientation, to guide the subsequent formation of highly mineralized tooth tissues (Figure 75.2). Prior and Current Dental Tissue Regeneration Research The foundation of dental tissue bioengineering has ancient roots. Therapies to replace missing teeth can be traced back at least 2,500 years, when the Etruscans learned to substitute missing teeth with bridges made from
Dental Tissue Engineering
artificial teeth carved from the bones of oxen (Ring, 1995). More recent efforts to regenerate teeth from ectopically grafted embryonic tooth buds have been previously reviewed (Yelick and Vacanti, 2004; Yen and Sharpe, 2006), demonstrating the feasibility of growing teeth in an appropriate environment. State-of-the-art dental tissue engineering methodologies, which employ in vitro expanded presumptive DSCs that are then seeded onto three-dimensional biodegradable scaffolds and implanted back into individuals, are rapidly advancing. Reparative Pulp and Dentin Dentin, a highly calcified connective tissue, is similar to, but distinct from bone (Linde and Goldberg, 1993). Dental pulp tissue exhibits the potential to regenerate dentin in response to noxious stimuli, such as caries, and it was hypothesized that the stem/progenitor cells present in the dental pulp differentiate into odontoblasts in response to BMP signaling (Nakashima and Akamine, 2005). BMP-driven stem cell therapies have shown considerable promise in dentin–pulp complex regeneration (Shi et al., 2005; Murray and GarciaGodoy, 2006). Iohara et al. (2004) discovered that when stimulated by the morphogenetic signal BMP2 porcine pulp cells differentiate into odontoblasts, as indicated by the expression of dentin sialophosphoprotein (DSPP) and enamelysin/matrix metalloproteinase 20 (MMP20). The autogenous transplantation of the BMP2-treated pulp cells into an amputated pulp cavity resulted in reparative dentin formation in dogs. Zhang et al. (2005) isolated dental pulp cells from maxillary incisors of rats and found that these cells, cultured on either titanium or TCP scaffolds, differentiated into odontoblast-like cells and produced calcified nodules similar to dentin. Furthermore, Gronthos and collaborators (2000) have isolated highly proliferative progenitor cells from adult human dental pulp, which produced densely calcified nodules in culture. After in vivo transplantation of cultured cells into immune compromised mice, a dentin/pulp-like complex was generated, again demonstrating the potential use of this approach for successful dental tissue regeneration. Periodontal Tissue Regeneration The wide prevalence of periodontal disease, the limited regenerative capability exhibited by the PDL, and the critical role of the PDL in maintaining tooth health and function have made periodontal tissue engineering an extremely active area of research. As mentioned above, the PDL complex contains putative stem cells that can commit to a variety of cell fates, such as cementum, ligament, and bone. Periodontal tissue engineering offers a powerful approach to supplement existing treatment regimens for periodontal disease, as recently reviewed (Taba et al., 2005; Bartold et al., 2006; Ramseier et al., 2006). A considerable amount of research has explored periodontal regeneration capability of progenitor cells isolated from the PDL of humans (Grzesik et al., 2000; Seo et al., 2004; Shi et al., 2005), bovine (Saito et al., 2005), and mice (Jin et al., 2003; Zhao et al., 2004). According to the findings of Grzesik and colleagues (2000), human cementum-derived cells (HCDCs), expanded in vitro, formed a mineralized matrix when attached to a ceramic carrier and transplanted subcutaneously into immunodeficient mice. The mineralized matrix exhibited features identical to cementum, with typical organized bundles of collagen fibers (Sodek et al. 1977). Saito and coworkers (2005) transplanted bovine cementoblast progenitor cells subcutaneously into nude mice on an HA/TCP scaffold, also resulting in the formation of cementum-like tissue. In addition, Zhao et al. (2004) reported that rat cementoblasts, delivered via PLGA polymer sponges, have a marked ability to promote periodontal regeneration by inducing mineralization and PDL formation in periodontal wounds. Also in this study, the demonstrated healing of alveolar bone defects was found to be limited only by the size of the transplanted PLGA carrier.
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Interestingly, Kawaguchi et al. (2004) used autologous bone marrow stem cells (BMSCs), in combination with atelocollagen (2% type I collagen), to regenerate periodontal tissues in beagle dogs. The repaired experimental Class III defects consisted of cementum, PDL, and alveolar bone, suggesting that autotransplantation of BMSCs, embedded in the appropriate environment niche, is a novel option for the regeneration of complex tissues, including periodontium. Growth factors or morphogens modulate the cellular activities of, and induce cell differentiation and extracellular matrix production in, developing tissues. The effects of several growth factors, including BMPs (Ripamonti and Reddi, 1997; Giannobile and Somerman, 2003; Wikesjo et al., 2004; Xu et al., 2004; Dunn et al., 2005; Miranda et al., 2005), insulin-like growth factor-I (IGF-I) (Saygin et al., 2000), PDGF (Giannobile et al., 2001; Jin et al., 2004; Nevins et al., 2005; Sarment et al., 2006), and transforming growth factor-β (TGF-β) (Tatakis et al., 2000), on periodontal regeneration of various animal models, including baboons (Miranda et al., 2005), dogs (Tatakis et al., 2000; Wikesjo et al., 2004), rats (Jin et al., 2004; Dunn et al., 2005), and humans (Xu et al., 2004; Nevins et al., 2005; Sarment et al., 2006), have been evaluated. For instance, Saygin et al. (2000) reported that several growth factors, including IGF-I, PDGF-BB, and TGF-β, influence the mitogenetic potential, phenotypic gene expression profile, and biomineralization potential of cementoblasts. Even though preclinical and early clinical data for these growth factors appear promising in stimulating periodontal regeneration, they are not sufficient for definitive conclusions at this time (Ramseier et al., 2006). Furthermore, the methods to deliver growth factors, and the kinetics of release must also be considered to optimize the dosage and exposure time to the cells, and to eventually make periodontal tissue engineering a widely practiced, clinically available therapy. Based on Melcher’s (1976) proposition that cells that repopulate the periodontal wound would determine the type of new attachment onto the root surface, a surgical procedure called guided tissue regeneration (GTR) has demonstrated tremendous potential for periodontal tissue regeneration, by using a barrier membrane to selectively encourage progenitor cell populations to remain at the wound site (Quinones et al., 1996; Laurell and Gottlow, 1998). For example, Aukhil et al. (1986) reported the use of a cell-occlusive barrier to prevent gingival epithelium and connective tissue from growing into the periodontal space and to provide a favorable niche to promote maximal functional PDL regeneration in beagle dogs. More recently, Wikesjo et al. (2003) have shown that the combined use of BMP-2 with a physical barrier, such as a bioabsorbable, space-providing polymer membrane, significantly enhanced periodontal bone and soft tissue regeneration in dogs. However, clinical results using this method to achieve complete restoration of periodontal defects are often unpredictable and vary significantly, possibly due to variations in defect morphology among individuals. Whole-Tooth Tissue Engineering Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continue to adversely affect most individuals at some time in their lives. Recent achievements in tooth tissue engineering promise to provide viable alternatives to currently available human tooth replacement therapies. Young et al. (2002) and Duailibi et al. (2004) have demonstrated the potential to bioengineer complex tooth structures from pig or rat tooth bud tissues, respectively. Using a tissue engineering approach, cells from tooth buds of either 6-month-old porcine third molar or 4-day postnatal (dpn) rats were seeded onto biodegradable polymers. After a period of growth in the omenta of adult rat hosts, a conducive environment to promote vascularization and tissue growth, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig’s root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Both of these studies demonstrated that continuous,
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de novo tooth development had occured in the implants, suggesting the potential utility of this method for the regeneration of mammalian dental tissues. As mentioned above, tooth development originates from interactions between dental epithelial and mesenchymal cells, with the epithelium providing the instructive signals for tooth initiation and shape determination. Based on these properties, Ohazama et al. (2004) examined the odontogenic potential of embryonic stem cells, neural stem cells, or adult bone marrow-derived cells, combined with oral epithelium. The oral epithelium was found to stimulate odontogenic responses in each of these three non-dental mesenchymal cell types. Transfer of the tissue recombinants into adult mice renal capsules resulted in the formation of tooth structures and associated bone. In addition, transfer of embryonic tooth primordia into the adult jaw led to the development of tooth structures, indicating that an embryonic primordium can develop in an adult environment. The ability for heterogeneous postnatal and adult cell populations obtained from rodents to form bone and teeth in tissue-engineered rudiments may have important implications for the further development of these procedures on humans. The ability to bioengineer whole teeth inspires additional important therapeutic strategies. For instance, a logical therapeutic approach for treatment of edentulism and accompanying alveolar bone loss would be to combine whole-tooth bioengineering with bone tissue engineering approaches (Young et al., 2005; Yelick and Vacanti, 2006). The combined usage of these methods would result in the generation of both jaw and tooth root structures, to provide teeth and supporting periodontal tissues for individuals born without, or to replace structures lost to disease or injury. The widespread availability of such coordinated therapeutic treatments would provide potentially powerful tools for dental tissue therapeutic strategies and dramatically alter the current landscape of treatments for a variety of craniofacial anomalies.
CONCLUSIONS Dental tissue engineering is a newly emerging field, with just over a decade of creation and development. Although still largely experimental in nature, recent progress in this field promises that tissue engineering approaches will be used to bioengineer replacement teeth in the foreseeable future. The successful development of methodologies for dental tissue repair/regeneration from autologous adult tissues will change the face of modern dentistry and reduce the need for synthetic materials in clinical dental practice. The pressing challenge confronting dental tissue engineering is how to perfect the current techniques, such that bioengineered dental tissues or whole teeth are integrated physically and functionally with preexisting dental tissues (Yelick and Vacanti, 2006). Ideally, the bioengineered teeth would be fabricated to occlude properly with opposing and adjacent teeth and be anchored to the underlying alveolar bone via the PDL to transmit mechanical signals when necessary. Biological teeth would also exhibit proper proprioception, facilitating the life of the substitute itself, as well as surrounding teeth. To accomplish these goals, some important hurdles need to be overcome, including: the identification and maintenance of multipotential DSCs in vitro; the establishment of conditions that foster DSC growth and differentiation; and the development of suitable scaffolds and inductive factors that help DSC-scaffold constructs integrate into the surrounding environment for the reconstruction of dental tissues. With parallel and synergistic advancements in stem cell biology and material science, it is possible to envision that the day may soon come when a patient can visit his or her dentist to obtain a biological, living tooth, grown from the patient’s own cells. ACKNOWLEDGMENTS The authors would like to thank Dan McCloskey and Susan Orlando for library science expertise.
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