Tooth bioengineering leads the next generation of dentistry.

BACKGROUND: As a result of numerous rapid and exciting developments in tissue engineering technology, scientists are able to regenerate a fully functional tooth in animal models, from a bioengineered tooth germ. Advances in technology, together with our understanding of the mechanisms of tooth development and studies dealing with dentally derived stem cells, have led to significant progress in the field of tooth regeneration. AIM AND DESIGN: This review focuses on some of the recent advances in tooth bioengineering technology, the signalling pathways in tooth development, and in dental stem cell biology. These factors are highlighted in respect of our current knowledge of tooth regeneration. RESULTS AND CONCLUSION: An understanding of these new approaches in tooth regeneration should help to prepare clinicians to use this new and somewhat revolutionary therapy while also enabling them to partake in future clinical trials. Tooth bioengineering promises to be at the forefront of the next generation of dental treatments.

[Dentification ability of inbred strain mice tooth germs homologically transplanted into oral submucosa].

OBJECTIVE: To establish a suitable environment for the bioengineered teeth in vivo by observing the dentification ability of BALB/C mice tooth germs homologically implanted into the oral submucosa. METHODS: The first molar tooth germs of BALB/C mice 4 days after birth were transplanted into the oral submucosa of BALB/C male mice, and then recycled for regular histological observation after 1, 2, 3, and 6 week transplantation. RESULTS: The tooth germs in the oral submucosa grew well with continuing developing enamelum and pulpodentinal complex, and the dentinal tubules were clear. CONCLUSION: The environment of the BALB/C male mice oral submucosa is favorable for the growth of tooth germs in inbred strain BALB/C mice, and it can provide a new environment for the development of bioengineered teeth in vivo.

Reconstructing mandibular defects using autologous tissue-engineered tooth and bone constructs.

PURPOSE: Current strategies for jaw reconstruction require multiple operations to replace bone and teeth. To improve on these methods, we investigated simultaneous mandibular and tooth reconstruction, using a Yucatan minipig model. MATERIALS AND METHODS: Tooth and bone constructs were prepared from third molar tooth tissue and iliac-crest bone marrow-derived osteoblasts isolated from, and implanted back into, the same pig as an autologous reconstruction. Implants were harvested after 12 and 20 weeks and evaluated by x-ray, ultrahigh-resolution volume computed tomographic (VCT), histological, and immunohistochemical analyses. RESULTS: Small tooth structures were identified, and consisted of organized dentin, enamel, pulp, and periodontal ligament tissues, surrounded by new bone. No dental tissues formed in implants without tooth-bud cells, and bone regeneration was observed to a limited extent. Immunohistochemical analyses using tooth-specific and bone-specific antibodies confirmed the identity of regenerated tissues. CONCLUSIONS: This pilot study supports the feasibility of tissue-engineering approaches for coordinated autologous tooth and mandible reconstruction, and provides a basis for future improvement of this technique for eventual clinical use in humans.

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.

Development and terminal differentiation of pulp and periodontal nerve elements in subcutaneous transplants of molar tooth germs and incisors of the rat.

Ectopic tooth transplants are known to receive rich innervation of local neurons, but the precise location and structural features of neurites in the pulp and periodontal ligament (PDL) of such transplants are unclear. In this experiment, the molar tooth germs of rat embryos and incisors of young rats were subcutaneously transplanted into the dorsal regions of rats and processed, at various time intervals, for immunohistochemical demonstration of neural elements. Teeth with periodontal tissue elements developed in most of the molar transplants in 6 or 8 wk and received rich innervation, including some autonomic fibres, in the pulp. Nerve elements were also confirmed to be present in the PDL of these transplants, including specialized nerve ending-like structures reminiscent of the periodontal Ruffini endings. Mechanoreceptor-like structures were also induced in the regenerated PDL of similarly transplanted incisors, although the success rate was low. We conclude that rich and highly ordered innervation of the pulp, and occasional development of mechanoreceptors in the regenerated PDL of ectopic dental transplants, imply a high probability of successful induction of teeth with both nociceptive and mechanical sensations in the ectopic tooth and/or tooth germ transplant systems, although differentiation of mechanoreceptor-like nerve endings occurred in only a few rare cases.

Root development of rat tooth germs implanted in the tooth socket and in the subcutaneous tissue.

OBJECTIVE: This study was designed to investigate root development of a rat tooth germ implanted in a tooth socket or in a subcutaneous region. MATERIALS AND METHODS: Tooth germs of the upper left first molars in 2-week-old rats were extracted and implanted in the original tooth socket or in the subcutaneous region of the back. The upper right first molar was used as a control. The rats were fixed in weeks 1, 2, 4, 8 and 12. The root development was examined quantitatively with X-ray radiographic morphometry. The cellular activity of producing matrix proteins was assessed using in situ hybridization for type I collagen. RESULTS: Root development was observed in the implanted teeth in the tooth socket as also in the control teeth. In contrast, roots hardly developed in subcutaneously implanted teeth. Histology showed that periodontal ligaments were arranged around roots of implanted teeth in the tooth socket as around control teeth, but few periodontal ligaments were identified in the subcutaneous implantation. Dentin and cementum formed in both the implanted teeth as also in the control teeth and odontoblasts, cementoblasts and cementocytes expressed type I collagen. CONCLUSION: Tooth sockets may possess specific environments that allow root development of a tooth germ.

Whole-tooth regeneration: it takes a village of scientists, clinicians, and patients.

A team of senior scientists was formed in 2006 to create a blueprint for the regeneration of whole human teeth along with all of the supporting structure of the dentition. The team included experts from diverse fields, each with a reputation for stellar accomplishment. Participants attacked the scientific issues of tooth regeneration but, more importantly, each agreed to work collaboratively with experts from other disciplines to form a learning organization. A commitment to learn from one another produced a unique interdisciplinary and multidisciplinary team. Inspired by the Kennedy space program to send a man to the moon, with its myriad of problems and solutions that no one discipline could solve, this tooth regeneration team devised an ambitious plan that sought to use stem cell biology, engineering, and computational biology to replicate the developmental program for odontogenesis. In this manner, team members envisioned a solution that consisted of known or knowable fundamentals. They proposed a laboratory-grown tooth rudiment that would be capable of executing the complete program for odontogenesis when transplanted to a suitable host, recreating all of the dental tissues, periodontal ligament, cementum, and alveolar bone associated with the canonical tooth. This plan was designed to bring regenerative medicine fully into the dental surgery suite, although a lack of funding has so far prevented the plan from being carried out.

[Experimental study on the development of tissue-engineered tooth germ with heterotopic allotransplantation].

OBJECTIVE: To compare the growth and development of tissue engineered tooth germ implanted into different tissues, and explore a suitable growing environment for the tissue engineered teeth in vivo. METHODS: SD rat/porcine tooth germ cells from postnatal 4 days were used as seeding cells, which combined various scaffolding biomaterials to construct the compound with tissue engineered teeth. The allografts were implanted into renal subcapsule, the mesenteries and subcutaneous tissues. Then, the implants were retrieved at special time points for histological analysis. RESULTS: Further developments were not observed in the graft implanted into mesenteries and subcutaneous tissues. Partial grafts were fallen off and lost from the subcutaneous tissues after implanted, and there were obvious lymphocyte infiltrations in the mesenteries. Moreover, the enamel and pulp-dentin complex were observed within the graft implanted in the subrenal capsule, which indicated there to be good condition. CONCLUSION: The subrenal capsule can provide a promising implantation environment for the further growth of allogeneic tissue engineered tooth germ, and the subrenal capsule implantation can be used as a new alternative method for tissue-engineering tooth in vivo.

The cell re-association-based whole-tooth regeneration strategies in large animal, Sus scrofa.

OBJECTIVES: Whole-tooth regeneration for tooth loss has long been a goal of dentistry. There is also an increasing demand to carry out pre-clinical in vitro and in vivo research methods in large animal model similar to human. The miniature pig has proven to be an alternative as a large mammal model owing to its many similarities to human. However, whole-tooth regeneration in large animal remains a challenge. Here, we investigated the feasibility of cell re-association-based whole-tooth regeneration in miniature pigs. MATERIALS AND METHODS: Single cells from the forth deciduous molar germs (p4) of pig were reconstituted to bioengineered tooth bud using different treatment for in vitro culture and in vivo transplantation in mouse subrenal capsules and jawbones. RESULTS: The bioengineered tooth bud from re-aggregated epithelial to mesenchymal single cells with and without compartmentalization restored the morphogenesis, interactions or self-sorting between 2 cells in vitro culture. The pig bioengineered tooth bud transplanted in mouse subrenal capsules and jawbones restored odontogenesis and developed into large size tooth. CONCLUSIONS: We characterized the morphogenesis and interaction of single-tooth germ cells in vitro, and first addressed efficient long-term survival and growth through transplantation of pig bioengineered tooth bud under mouse subrenal capsules or in mouse jawbones, where it can develop into large size tooth. Our study extends the feasibility of whole-tooth regeneration in large animal.

Practical whole-tooth restoration utilizing autologous bioengineered tooth germ transplantation in a postnatal canine model.

Whole-organ regeneration has great potential for the replacement of dysfunctional organs through the reconstruction of a fully functional bioengineered organ using three-dimensional cell manipulation in vitro. Recently, many basic studies of whole-tooth replacement using three-dimensional cell manipulation have been conducted in a mouse model. Further evidence of the practical application to human medicine is required to demonstrate tooth restoration by reconstructing bioengineered tooth germ using a postnatal large-animal model. Herein, we demonstrate functional tooth restoration through the autologous transplantation of bioengineered tooth germ in a postnatal canine model. The bioengineered tooth, which was reconstructed using permanent tooth germ cells, erupted into the jawbone after autologous transplantation and achieved physiological function equivalent to that of a natural tooth. This study represents a substantial advancement in whole-organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future clinical regenerative medicine.

Inhibition of apoptosis in early tooth development alters tooth shape and size.

Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.

Bioengineered teeth from tooth bud cells.

Advances in tissue engineering and materials science have led to significant progress in hard and soft tissue repair and regeneration. Studies demonstrate the successful application of tissue engineering for bioengineering dental tissues. The ability to apply tissue engineering to repair or regenerate dental tissues and even whole teeth is becoming a reality. Current efforts focus on directing the formation of bioengineered dental tissues and whole teeth of predetermined size and shape. Advances in dental progenitor cell characterizations, combined with improved methods of fabricating biodegradable scaffold materials, bring closer the goal of making tooth tissue engineering a clinically relevant practice.

Tissue-engineered hybrid tooth and bone.

Tooth loss accompanied by alveolar bone resorption presents a significant clinical problem. We have investigated the utility of a tissue-engineering approach to provide corrective therapies for tooth-bone loss. Hybrid tooth-bone tissues were bioengineered as follows. Tooth implants were generated from pig third molar tooth bud cells seeded onto polyglycolide (PGA) and polyglycolide-colactide (PLGA) scaffolds, and grown for 4 weeks in the omenta of adult rat hosts. Bone implants were generated from osteoblasts induced from bone marrow progenitor cells obtained from the same pig, seeded onto PLGA fused wafer scaffolds, and grown for 10 days in a rotational oxygen-permeable bioreactor system. The tooth and bone implants were harvested, sutured together, reimplanted, and grown in the omenta for an additional 8 weeks. Histological and immunohistochemical analyses of the excised hybrid tooth-bone constructs revealed the presence of tooth tissues, including primary and reparative dentin and enamel in the tooth portion of hybrid tooth-bone implants, and osteocalcin and bone sialoprotein-positive bone in the bone portion of hybrid tooth-bone constructs. Collagen type III-positive connective tissue resembling periodontal ligament and tooth root structures were present at the interface of bioengineered tooth and bone tissues. These results demonstrate the utility of a hybrid tooth-bone tissue-engineering approach for the eventual clinical treatment of tooth loss accompanied by alveolar bone resorption.

Contribution of donor and host mesenchyme to the transplanted tooth germs.

Autologous tooth germ transplantation of immature teeth is an alternative method of tooth replacement that could be used instead of dental implants in younger patients. However, it is paramount that the dental pulp remain vital and that root formation continue in the transplanted location. The goal of this study is to characterize the healing of allogenic tooth grafts in an animal model using GFP-labeled donor or host postnatal mice. In addition, the putative stem cells were labeled before transplantation with a pulse-chase paradigm. Transplanted molars formed cusps and roots and erupted into occlusion by 2 wk postoperatively. Host label-retaining cells (LRCs) were maintained in the center of pulp tissue associating with blood vessels. Dual labeling showed that a proportion of LRCs were incorporated into the odontoblast layer. Host cells, including putative dendritic cells and the endothelium, also immigrated into the pulp tissue but did not contribute to the odontoblast layer. Therefore, LRCs or putative mesenchymal stem cells are retained in the transplanted pulps. Hertwig's epithelial root sheath remains vital, and epithelial LRCs are present in the donor cervical loops. Thus, the dynamic donor-host interaction occurred in the developing transplant, suggesting that these changes affect the characteristics of the dental pulp.

Transplantation of bovine odontogenic tissues and dissociated odontogenic cells to hypothymic mice.

It was demonstrated in this study that bovine odontogenic tissues and the dissociated cell preparations of such tissues, when transplated to the subcapsular kidney site of hypothymic mice, retained their phenotypic expression and continued function to produce their recognisable products, dentine and enamel matrices. It was also shown that in the grafts of dissociated odontogenic tissues, cell sorting occurred so that histotypical tissues were formed and that their function produced readily recognisable dentine and enamel matrices. Attention has been drawn to the usefulness of this model for studying a variety of events that occur during odontogenesis.

Nerve growth to tooth buds after homotopic or heterotopic autotransplantation.

Feline permanent incisor tooth buds (bell stage) were autotransplanted to mandibular alveolar sockets (homotopic site) or to the submandibular subcutis or the leg (heterotopic sites). This was done in 34 kittens aged 1-2 months. After survival times of 3-8 months the animals were fixed by glutaraldehyde perfusion. A total of 56 mineralized teeth, which had developed at the recipient sites, were removed, demineralized and processed for light microscopic (LM) general evaluation. Fourty-four teeth, which were judged to be grossly normal in the LM, were selected for electron microscopic (EM) analysis with respect to the occurrence of pulpal nerve fibres. The highest proportion of normal teeth (16 of 16) was obtained from the alveolar site, followed by the submandibular (11 of 14) and hindlimb (17 of 26) sites. Most of the grossly normal grafts possessed pulpal axons (37 of 44). The alveolar grafts were all innervated and exhibited a largely normal appearance qualitatively and in terms of percentage of myelinated fibres. The proportion of innervated pulps was lower among the heterotopic mandibular (10 of 11) and hindlimb (11 of 17) grafts. In addition, signs of nerve fibre degeneration appeared more frequently at the heterotopic sites. On the basis of these findings, and in view of the results of other workers, we conclude that tooth germs are attractive targets for all divisions of the trigeminal nerve and for cutaneous nerves outside the trigeminal system. However, the morphological picture tends to become increasingly abnormal with increasing distance from the normal locus.

Murine tooth organ transplantation after in vitro culture.

Molar tooth organs were transplanted from fetal and neonatal mouse donors as well as from cap-stage molar tooth organs following ten days of in vitro culture. Tooth organs from neonatal donors appeared to develop better than those from fetal donors in syngeneic recipients. Cultured allogeneic molar tooth organs appeared to survive longer than uncultured controls.

Tooth allografts fail to stimulate immunologic memory in mice.

Tooth transplants should not preclude future transplants in the recipient. In this study teeth failed to stimulate immunologic memory in weakly disparate mice. The teeth did carry transplantation antigens and were rejected after second-set skin grafts. The teeth probably enhanced their own survival through some immunoregulatory mechanism.

Influence of serum on cultured murine tooth organ allotransplants.

Previous investigations indicated that in vitro culture of fetal tooth germs before allotransplantation prolonged graft survival. This study assessed the influence of different sources of serum in culture medium on post-transplant development of neonatal tooth organs. The results indicate that recipient-strain serum, but not donor-strain serum or calf serum, increased tooth survival compared with that of uncultured controls, which were rejected by the fourteenth post-transplant day. The survival curve for cultured tooth organs was shifted to the right by two days. No tooth transplant survived beyond three wk.