Tooth Bioengineering and Regenerative Dentistry.

Over the past 100 y, tremendous progress has been made in the fields of dental tissue engineering and regenerative dental medicine, collectively known as translational dentistry. Translational dentistry has benefited from the more mature field of tissue engineering and regenerative medicine (TERM), established on the belief that biocompatible scaffolds, cells, and growth factors could be used to create functional, living replacement tissues and organs. TERM, created and pioneered by an interdisciplinary group of clinicians, biomedical engineers, and basic research scientists, works to create bioengineered replacement tissues that provide at least enough function for patients to survive until donor organs are available and, at best, fully functional replacement organs. Ultimately, the goal of both TERM and regenerative dentistry is to bring new and more effective therapies to the clinic to treat those in need. Very recently, the National Institutes of Health/National Institute of Dental and Craniofacial Research invested $24 million over a 3-y period to create dental oral and craniofacial translational resource centers to facilitate the development of more effective therapies to treat edentulism and other dental-related diseases over the next decade. This exciting era in regenerative dentistry, particularly for whole-tooth tissue engineering, builds on many key successes over the past 100 y that have contributed toward our current knowledge and understanding of signaling pathways directing natural tooth and dental tissue development-the foundation for current strategies to engineer functional, living replacement dental tissues and whole teeth. Here we use a historical perspective to present key findings and pivotal advances made in the field of translational dentistry over the past 100 y. We will first describe how this process has evolved over the past 100 y and then hypothesize on what to expect over the next century.

Bioengineered Tooth Buds Exhibit Features of Natural Tooth Buds.

Tooth loss is a significant health issue currently affecting millions of people worldwide. Artificial dental implants, the current gold standard tooth replacement therapy, do not exhibit many properties of natural teeth and can be associated with complications leading to implant failure. Here we propose bioengineered tooth buds as a superior alternative tooth replacement therapy. We describe improved methods to create highly cellularized bioengineered tooth bud constructs that formed hallmark features that resemble natural tooth buds such as the dental epithelial stem cell niche, enamel knot signaling centers, transient amplifying cells, and mineralized dental tissue formation. These constructs were composed of postnatal dental cells encapsulated within a hydrogel material that were implanted subcutaneously into immunocompromised rats. To our knowledge, this is the first report describing the use of postnatal dental cells to create bioengineered tooth buds that exhibit evidence of these features of natural tooth development. We propose future bioengineered tooth buds as a promising, clinically relevant tooth replacement therapy.

Decellularized Tooth Bud Scaffolds for Tooth Regeneration.

Whole tooth regeneration approaches currently are limited by our inability to bioengineer full-sized, living replacement teeth. Recently, decellularized organ scaffolds have shown promise for applications in regenerative medicine by providing a natural extracellular matrix environment that promotes cell attachment and tissue-specific differentiation leading to full-sized organ regeneration. We hypothesize that decellularized tooth buds (dTBs) created from unerupted porcine tooth buds (TBs) can be used to guide reseeded dental cell differentiation to form whole bioengineered teeth, thereby providing a potential off-the-shelf scaffold for whole tooth regeneration. Porcine TBs were harvested from discarded 6-mo-old pig jaws, and decellularized by successive sodium dodecyl sulfate/Triton-X cycles. Four types of replicate implants were used in this study: 1) acellular dTBs; 2) recellularized dTBs seeded with porcine dental epithelial cells, human dental pulp cells, and human umbilical vein endothelial cells (recell-dTBs); 3) dTBs seeded with bone morphogenetic protein (BMP)-2 (dTB-BMPs); and 4) freshly isolated nondecellularized natural TBs (nTBs). Replicate samples were implanted into the mandibles of host Yucatan mini-pigs and grown for 3 or 6 mo. Harvested mandibles with implanted TB constructs were fixed in formalin, decalcified, embedded in paraffin, sectioned, and analyzed via histological methods. Micro-computed tomography (CT) analysis was performed on harvested 6-mo samples prior to decalcification. All harvested constructs exhibited a high degree of cellularity. Significant production of organized dentin and enamel-like tissues was observed in dTB-recell and nTB implants, but not in dTB or dTB-BMP implants. Micro-CT analyses of 6-mo implants showed the formation of organized, bioengineered teeth of comparable size to natural teeth. To our knowledge, these results are the first to describe the potential use of dTBs for functional whole tooth regeneration.

GelMA-Encapsulated hDPSCs and HUVECs for Dental Pulp Regeneration.

Pulpal revascularization is commonly used in the dental clinic to obtain apical closure of immature permanent teeth with thin dentinal walls. Although sometimes successful, stimulating bleeding from the periapical area of the tooth can be challenging and in turn may deleteriously affect tooth root maturation. Our objective here was to define reliable methods to regenerate pulp-like tissues in tooth root segments (RSs). G1 RSs were injected with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) encapsulated in 5% gelatin methacrylate (GelMA) hydrogel. G2 RSs injected with acellular GelMA alone, and G3 empty RSs were used as controls. White mineral trioxide aggregate was used to seal one end of the tooth root segment, while the other was left open. Samples were cultured in vitro in osteogenic media (OM) for 13 d and then implanted subcutaneously in nude rats for 4 and 8 wk. At least 5 sample replicates were used for each experimental group. Analyses of harvested samples found that robust pulp-like tissues formed in G1, GelMA encapsulated hDPSC/HUVEC-filled RSs, and less cellularized host cell-derived pulp-like tissue was observed in the G2 acellular GelMA and G3 empty RS groups. Of importance, only the G1, hDPSC/HUVEC-encapsulated GelMA constructs formed pulp cells that attached to the inner dentin surface of the RS and infiltrated into the dentin tubules. Immunofluorescent (IF) histochemical analysis showed that GelMA supported hDPSC/HUVEC cell attachment and proliferation and also provided attachment for infiltrating host cells. Human cell-seeded GelMA hydrogels promoted the establishment of well-organized neovasculature formation. In contrast, acellular GelMA and empty RS constructs supported the formation of less organized host-derived vasculature formation. Together, these results identify GelMA hydrogel combined with hDPSC/HUVECs as a promising new clinically relevant pulpal revascularization treatment to regenerate human dental pulp tissues.

Bioengineered post-natal recombinant tooth bud models.

The long-term goal of this study is to devise reliable methods to regenerate full-sized and fully functional biological teeth in humans. In this study, three-dimensional (3D) tissue engineering methods were used to characterize intact postnatal dental tissue recombinant constructs, and dental cell suspension recombinant constructs, as models for bioengineered tooth development. In contrast to studies using mouse embryonic dental tissues and cells, here the odontogenic potential of intact dental tissues and dental cell suspensions harvested from post natal porcine teeth and human third molar wisdom tooth dental pulp were examined. The recombinant 3D tooth constructs were cultured in osteogenic media in vitro for 1 week before subcutaneous transplantation in athymic nude rat hosts for 1 month or 3 months. Subsequent analyses using X-ray, histological and immunohistochemical methods showed that the majority of the recombinant tooth structures formed calcified tissues, including osteodentin, dentin cementum, enamel and morphologically typical tooth crowns composed of dentin and enamel. The demonstrated formation of mineralized dental tissues and tooth crown structures from easily obtained post-natal dental tissues is an important step toward reaching the long-term goal of establishing robust and reliable models for human tooth regeneration. Copyright © 2014 John Wiley & Sons, Ltd.

The Contributions of the Ribosome Biogenesis Protein Utp5/WDR43 to Craniofacial Development.

Fairly recently, it was recognized that human ribosomopathies-developmental defects caused by mutations in ribosome biogenesis proteins-can exhibit tissue-specific defects rather than the expected global defects. This apparent anomaly-that seemingly ubiquitously expressed and required ribosomal proteins can have distinct functions in cell and tissue differentiation-has spurred new areas of research focused on better understanding translational mechanisms, biogenesis, and function in diverse cell types. This renewed appreciation for, and need to better understand, roles for ribosomal proteins in human development and disease has identified surprising similarities and differences in a variety of human ribosomopathies. Here, we discuss ribosomal protein functions in health and disease, focusing on the ribosome biogenesis protein Utp5/WDR43. New and exciting research in this field is anticipated to provide insight into a variety of previously understudied craniofacial dysostoses and result in significantly improved knowledge and understanding of roles for translational machinery in human craniofacial development and disease.

Chemerin-ChemR23 signaling in tooth development.

Our long-term goal is to identify and characterize molecular mechanisms regulating tooth development, including those mediating the critical dental epithelial-dental mesenchymal (DE-DM) cell interactions required for normal tooth development. The goal of this study was to investigate Chemerin (Rarres2)/ChemR23(Cmklr1) signaling in DE-DM cell interactions in normal tooth development. Here we present, for the first time, tissue-specific expression patterns of Chemerin and ChemR23 in mouse tooth development. We show that Chemerin is expressed in cultured DE progenitor cells, while ChemR23 is expressed in cultured DM cells. Moreover, we demonstrate that ribosomal protein S6 (rS6) and Akt, downstream targets of Chemerin/ChemR23 signaling, are phosphorylated in response to Chemerin/ChemR23 signaling in vitro and are expressed in mouse tooth development. Together, these results suggest roles for Chemerin/ChemR23-mediated DE-DM cell signaling during tooth morphogenesis.

Dental tissue regeneration - a mini-review.

BACKGROUND: with today's 21st century technological advancements, it is expected that individuals will either retain their natural teeth or obtain functional tooth replacements throughout their entire life. Modern dental therapies for the replacement of missing teeth largely utilize partial or complete dentures and titanium implants capped with prosthetic crowns. Although these prostheses serve a purpose, they are not equivalent, neither in function nor aesthetics, to natural teeth. Recent progress in dental tissue engineering has lent significant credibility to the concept that biological replacement teeth therapies may soon be available to replace missing teeth. OBJECTIVE: in this review, we summarize the emerging concepts of whole-tooth replacement strategies, using postnatal dental stem cells (DSCs) and dental tissue engineering approaches. METHODS: we provide a thorough and extensive review of the literature. RESULTS: current approaches to achieve clinically relevant biological replacement tooth therapies rely on the cultivation of DSCs capable of relaying odontogenic induction signals, through dental epithelial-mesenchymal cell interactions. DSC expansion and differentiation can be achieved by programming progenitor stem cells to adopt dental lineages, using instructive, bioengineered scaffold materials. Periodontal ligament regeneration in particular has demonstrated significant progress recently, despite the somewhat unpredictable clinical outcomes, with regard to its capacity to augment conventional metallic dental implants and as an important component for whole-tooth tissue engineering. Following recent advances made in DSC and tissue engineering research, various research groups are in the midst of performing 'proof of principle' experiments for whole-tooth regeneration, with associated functional periodontal tissues. This mini-review focuses on recent and promising developments in the fields of pulp and periodontal tissue DSCs that are of particular relevance for dental tissue and whole-tooth regeneration. CONCLUSION: continued advances in the derivation of useable DSC populations and optimally designed scaffold materials unequivocally support the feasibility of dental tissue and whole-tooth tissue engineering.

Bioengineered periodontal tissue formed on titanium dental implants.

The ability to use autologous dental progenitor cells (DPCs) to form organized periodontal tissues on titanium implants would be a significant improvement over current implant therapies. Based on prior experimental results, we hypothesized that rat periodontal ligament (PDL)-derived DPCs can be used to bioengineer PDL tissues on titanium implants in a novel, in vivo rat maxillary molar implant model. Analyses of recovered implants revealed organized PDL tissues surrounding titanium implant surfaces in PDL-cell-seeded, and not in unseeded control, implants. Rat PDL DPCs also exhibited differentiative potential characteristic of stem cells. These proof-of-principle findings suggest that PDL DPCs can organize periodontal tissues in the jaw, at the site of previously lost teeth, indicating that this method holds potential as an alternative approach to osseointegrated dental implants. Further refinement of this approach will facilitate the development of clinically relevant methods for autologous PDL regeneration on titanium implants in humans.

Alternative splicing, phylogenetic analysis, and craniofacial expression of zebrafish tbx22.

Mutations in human TBX22 cause X-linked cleft palate with ankyloglossia syndrome (CPX; OMIM 303400). Since the secondary palate was an adaptation to breathing on land, we characterized zebrafish tbx22 to study molecular mechanisms regulating early vertebrate craniofacial patterning. Rapid Amplification of cDNA Ends (RACE) analyses revealed two zebrafish tbx22 splice isoforms, tbx22-1 and tbx22-2, encoding proteins of 444 and 400 amino acids, respectively. tbx22-1 resembles canonical Tbx22 orthologs, while tbx22-2 lacks conserved N-terminal sequence. Developmental RT-PCR revealed that tbx22-1 is maternally and zygotically expressed, while tbx22-2 is expressed zygotically. WISH analyses revealed strong tbx22 mRNA expression in ectomesenchyme underlying the stomodeum, a bilaminar epithelial structure demarcating early mouth formation, and in early presumptive jaw joints. Zebrafish tbx22 expression mirrored some aspects of mammalian Tbx22, consistent with roles in early vertebrate face patterning. These studies identify an early transcription factor governing vertebrate facial development, which may underlie common craniofacial birth disorders. Developmental Dynamics 238:1605-1612, 2009. (c) 2009 Wiley-Liss, Inc.

Bioengineered dental tissues grown in the rat jaw.

Our long-term objective is to develop methods to form, in the jaw, bioengineered replacement teeth that exhibit physical properties and functions similar to those of natural teeth. Our results show that cultured rat tooth bud cells, seeded onto biodegradable scaffolds, implanted into the jaws of adult rat hosts and grown for 12 weeks, formed small, organized, bioengineered tooth crowns, containing dentin, enamel, pulp, and periodontal ligament tissues, similar to identical cell-seeded scaffolds implanted and grown in the omentum. Radiographic, histological, and immunohistochemical analyses showed that bioengineered teeth consisted of organized dentin, enamel, and pulp tissues. This study advances practical applications for dental tissue engineering by demonstrating that bioengineered tooth tissues can be regenerated at the site of previously lost teeth, and supports the use of tissue engineering strategies in humans, to regenerate previously lost and/or missing teeth. The results presented in this report support the feasibility of bioengineered replacement tooth formation in the jaw.

Developmental analysis and computer modelling of bioengineered teeth.

Here we present the developmental progression of bioengineered pig teeth from 1 to 25 weeks of development. We demonstrate that 2-25 week implants contained embryonic tooth bud- and cap-stage tooth structures consisting of dental epithelium expressing the sonic hedgehog gene and condensed dental mesenchyme. Implants harvested at 18-25 weeks also contained tooth bud-like structures, as well as mature tooth structures containing enamel, dentin and pulp tissues. Immunohistochemical analyses confirmed the expression of dentin- and enamel-specific proteins in differentiated bioengineered tooth tissues. Three-dimensional computer modelling further demonstrated a spatial organization of enamel, dentin and pulp tissues resembling that of natural teeth. We conclude that bioengineered teeth commonly exhibit morphological stages characteristic of naturally forming teeth. Furthermore, the presence of immature tooth buds at all times assayed and increased numbers of bioengineered tooth structures over time suggests that porcine dental progenitor cells maintain the ability to form teeth for at least 25 weeks.

Bioengineered teeth from cultured rat tooth bud cells.

The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.

Alk8 is required for neural crest cell formation and development of pharyngeal arch cartilages.

The type I TGFbeta family member receptor alk8 acts in bone morphogenetic protein (BMP) signaling pathways to establish dorsoventral patterning in the early zebrafish embryo. Here, we present evidence that alk8 is required for neural crest cell (NCC) formation and that alk8 signaling gradients direct the proper patterning of premigratory NCCs. We extend our previous functional studies of alk8 to demonstrate that ectopic expression of constitutively active and dominant negative Alk8, consistently results in more medially or laterally positioned premigratory NCCs, respectively. We also demonstrate that patterning defects in premigratory NCCs, induced by alk8 misexpression, correlate with subsequent defects in NCC-derived pharyngeal arch cartilages. Furthermore, an anteroposterior effect is revealed, where overexpression of Alk8 more severely affects anterior arch cartilages and decreased Alk8 activity more severely affects posterior arch cartilage formation. Ectopic expression studies of alk8 are supported by analyses of zygotic and maternal-zygotic laf/alk8 mutants and of several BMP pathway mutants. Pharyngeal mesodermal and endodermal defects in laf/alk8 mutants suggest additional roles for alk8 in patterning of these tissues. Our results provide insight into alk8-mediated BMP signaling gradients and the establishment of premigratory NCC mediolateral positioning, and extend the model for BMP patterning of the neural crest to include that of NCC-derived pharyngeal arch cartilages.

Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, 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. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Zebrafish E-cadherin: expression during early embryogenesis and regulation during brain development.

Zebrafish E-cadherin (cdh1) cell adhesion molecule cDNAs were cloned. We investigated spatial and temporal expression of cdh1 during early embryogenesis. Expression was observed in blastomeres, the anterior mesoderm during gastrulation, and developing epithelial structures. In the developing nervous system, cdh1 was detected at the pharyngula stage (24 hpf) in the midbrain-hindbrain boundary (MHB). Developmental regulation of MHB formation involves wnt1 and pax2.1. wnt1 expression preceded cdh1 expression during MHB formation, and cdh1 expression in the MHB was dependent on normal development of this structure.

Functional characterization and genetic mapping of alk8.

The novel type I TGFbeta family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.

Regulation of tooth development by the novel type I TGFbeta family member receptor Alk8.

We have recently identified, in zebrafish, a novel type I receptor of the TGFbeta family, alk8, that participates in Bmp signaling pathways to mediate early dorsoventral patterning of neurectodermal and mesendodermal tissues. Since Bmps play significant roles in tooth specification, initiation, and differentiation, we hypothesized that alk8 may play a role in directing the Bmp-mediated epithelial mesenchymal cell interactions regulating tooth development. Immunohistochemical analysis demonstrates that Alk8 is expressed in developing zebrafish and mouse teeth. Examination of tooth development in zebrafish with disrupted alk8 signaling revealed specific defects in tooth development. Ectopic expression of constitutively active Alk8 results in the formation of elongated tooth structures, while expression of dominant-negative Alk8 results in arrested tooth development at the bud stage. These results are consistent with the established requirements for Bmp signaling in tooth development and demonstrate that Alk8 is a key regulator of tooth development.

zALK-8, a novel type I serine/threonine kinase receptor, is expressed throughout early zebrafish development.

Here, we report the isolation and characterization of zebrafish activin receptor-like kinase-8 (zALK-8), a novel type I serine/threonine (ser/thr) kinase receptor of the transforming growth factor beta (TGF-beta) family. zALK-8 is novel, in that it contains an extracellular domain that is quite distinct from that of previously identified ALK receptors 1 through 7. Analysis of the predicted amino acid sequence of the 506 amino acid zALK-8 receptor reveals an ser/thr kinase domain characteristic of type I TGF-beta family member receptors. zALK-8, therefore, is a traditional type I ser/thr kinase receptor of the TGF-beta family, but it may exhibit novel ligand-binding activities. The developmental expression of zALK-8 mRNA was examined by wholemount in situ hybridization analysis using a probe from the 3'-untranslated sequence of zALK-8, which does not cross react with other members of the highly conserved TGF-beta receptor family. zALK-8 mRNA is present as a maternal message that is expressed ubiquitously before the start of zygotic transcription. By 16 hr postfertilization (hpf), zALK-8 mRNA is still expressed fairly evenly throughout the embryo. In 24-hpf embryos, zALK-8 mRNA is expressed predominantly in the developing eye and neural structures. By 48 hpf, zALK-8 mRNA is faintly detectable as a diffuse signal throughout the head. zALK-8 mRNA is not detectable by this method in 72-hpf or 96-hpf embryos. Northern analysis of zALK-8 mRNA in poly(A+) mRNA isolated from 6-9 hpf embryos detects a major transcript of 3.6 kb and a minor transcript of 4.3 kb. zALK-8 mRNA expression correlates well with known functions of TGF-beta family members as early axial patterning and mesoderm-inducing growth factors and as potent growth and differentiation factors in craniofacial development.

Vertebrate genome evolution and the zebrafish gene map.

In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.