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.

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.

Failure of Tooth Formation Mediated by miR-135a Overexpression via BMP Signaling.

MicroRNAs (miRNAs) are known to regulate a variety of gene functions in many tissues and organs, but their expression and function in tooth development are not well-understood. A specific miRNA, miR-135a, was determined to be highly expressed at the bud stage. Interestingly, after the cap stage, miR-135a was expressed in the epithelium and mesenchyme but not in the inner enamel epithelium. To identify the relationship between miR-135a and its putative target genes, Bmpr-Ia and Bmpr-Ib, in early tooth development, miR-135a was ectopically overexpressed with a lentivirus. This overexpression resulted in the repression of Bmpr-Ia and Bmpr-Ib. Furthermore, miR-135a inhibited both Bmpr-Ia and Bmpr-Ib transcription. BMP2 proteins were expressed ectopically in tooth germs during the cap stage to determine the relationship between miR-135a and BMP signaling in early tooth development. When miR-135a was ectopically expressed, no tooth formation was observed after 4 wk of incubation in the kidney capsule. This study suggested that Bmp signaling, specifically Bmpr-Ia and Bmpr-Ib, regulates tooth formation via miR-135a.

Application of induced pluripotent stem cells in generation of a tissue-engineered tooth-like structure.

Stem cells, such as adult stem cells or embryonic stem cells, are the most important seed cells employed in tooth tissue engineering. Even though dental-derived stem cells are a good source of seed cells for such procedures, they are not often used in clinical applications because of the limited supply. Induced pluripotent stem (iPS) cells, with their high proliferation and differentiation ability, are now considered a promising alternative. The objectives of this study were to assess the role of iPS cells in tooth tissue engineering. We used real-time polymerase chain reaction to confirm that mouse iPS (miPS) cells can be induced to express both odontogenic and osteogenic gene profiles. We then established a tooth germ model and transplanted the recombinant tooth germ into a mouse subrenal capsule for 4 weeks to reproduce early-tooth organogenesis. After 4 weeks, hematoxylin and eosin staining results showed newly formed bone-like and dental pulp-like areas. Further immunohistochemical staining confirmed that osteopontin was present in the apical part of the tooth-like structure. These results demonstrate that miPS cells have the potential to differentiate into odontogenic cells, confirming that they could be a new source of seed cells for use in tooth tissue engineering.

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.

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.

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.

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.

[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.

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.

Mouse embryonic diastema region is an ideal site for the development of ectopically transplanted tooth germ.

The anterior eye chamber and the kidney capsule of the mouse have been traditionally used for long-term culture of tooth germ grafts. However, although these sites provide an excellent growth environment, they do not represent real in situ sites for the development of a grafted tooth germ. Here, we describe a protocol to transplant a tooth germ into the mandibular diastema region of mouse embryos using exo utero surgery. Our results demonstrate that the mouse embryonic diastema region represents a normal physiological environment for the development of transplanted tooth germs. Transplanted tooth germs developed synchronically with and became indistinguishable from the endogenous ones. These ectopic teeth were vascularized and surrounded with nerve fibers, and were able to erupt normally. Thus, the exo utero transplantation approach will provide a new avenue to study tooth development and regeneration.

Regulating the role of bone morphogenetic protein 4 in tooth bioengineering.

PURPOSE: Culture of the whole organ and regulation of its development using biologic and engineering principles can be used to produce structures and organs for reconstructing defects. The application of these bioengineering approaches in artificial tooth development may be the alternative way to replace missing dentition. MATERIALS AND METHODS: For the artificial bioengineering of a mouse tooth, tooth buds were dissected and transplanted into the diastema of the developing mandible. The mandiblular primordia containing transplanted tooth buds were culture in vitro and in vivo using a bioengineering method. In addition, to regulate the development of tooth germs, bone morphogenetic protein 4 (BMP4) or its antagonist, Noggin was administered. RESULTS: After the period of in vitro and in vivo culture, the transplanted tooth germ in the diastema showed tooth development with supportive structure formation. In the BMP-treated group, the bioengineered tooth was observed with increased maturation of cusp and enamel matrix. However, in the Noggin-treated tooth germs, the developing molar had a crater-like appearance with the immature development of the cusp and suppressed formation of the enamel matrix. CONCLUSIONS: This study confirmed that tooth germ transplantation in the diastema and culture with administration of BMP4 could lead to the mature development of the dental structures. In addition, these results suggest the possibility of bioengineering the tooth in morphogenesis and differentiation even in the toothless area.

[A histological innovative study of Balb/c mouse tooth germ transplanted to nude mouse in vivo].

PURPOSE: To observed the changes of the developed mouse's dental germs after transplanting into nude mouse and find some theoretical foundation for establishing an innovative experimental model. METHODS: Eight tooth germs from four 5th day postnatal Balb/c mice were transplanted to the back muscles of the adult nude mice. At seventh and fourteenth day after grafting, the germs were collected, fixed, demineralized, dehydrated, and embedded in wax. Serial sections of 5 microm thick were made following the routine methods, stained with haematoxylin-eosin dying solution, and observed under a light microscope. The mandibular first molars were taken out from the 12th and 19th day postnatal mouse. Serial sections of 5 microm thick were made following the routine methods, then compared with the germs after graft. RESULTS: All implantations were located in the superficial muscles with abundant capillary vessels. The dental germs could further developed after grafting under the microscope, but slower than dental germs self-development. The layer of dentin was thin, plenty of dentin with disorganized dentin tubule formed after grafting. Although the location of Hertwig's epithelial root sheath hardly moved, the roots developed further. The floor of pulp chamber could form and the pulp chambers were shrinking. The degree of calcification in the area of root increased very clearly. The inflammatory reaction was found at 7th day while hardly noted at 14th day after grafting. CONCLUSIONS: It suggests that late development of mouse tooth germs could further develop after heterotopic transplantation within the superficial muscles of the nude mouse. This model is useful for study of tooth root development in short time.

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.