The tooth, which is an ectodermal organ whose development is regulated by reciprocal epithelial-mesenchymal interactions (Jussila et al., 2013), contributes to oral functions associated with mastication and enunciation, which are important aspects of general health and quality of life (Proffit et al., 2004). Teeth have a three-dimensional multicellular structure composed of characteristic hard tissues,
Recent approaches for tooth regenerative therapy have included tissue repair and whole tooth replacement. Tooth regenerative therapy and stem cell transplantation therapies are regarded as attractive approaches for repairing tissue that has been damaged by dental caries or periodontal disease. The transplantation of dental stem cells has been examined for the treatment of dental caries, pulp injury and periodontal disease.
2. The mechanisms of tooth development
Ectodermal organs, such as the teeth, hair and salivary glands, arise from their respective organ germs through reciprocal epithelial-mesenchymal interactions in the developing embryo. These interactions, which involve various signalling molecules and transcription factors, are the principal mechanism regulating organogenesis (Jussila et al., 2013). In tooth germ development, the dental lamina first thickens (lamina stage). This stage is followed by epithelial thickening (placode stage) at the future location of the tooth and subsequent epithelial budding to the underlying neural crest-derived ecto-mesenchyme. Tooth germ formation is initiated on embryonic days (EDs) 10-11 in mice by epithelial signals that include fibroblast growth factor (FGF) 8, bone morphogenetic protein (BMP) 4, sonic hedgehog (Shh), tumour necrosis factor (TNF) and Wnt10b. These signals induce the expression of several transcription factors in the dental mesenchyme that condense around the developing epithelial bud (bud stage) (Jussila et al., 2013; O’connell et al., 2013). At ED13.5-14.5, the first enamel knot, which acts as a signalling centre to orchestrate tooth development by controlling the gene expression of various signalling molecules and transcription factors, is formed in the dental epithelium (cap stage). At ED16-18, the epithelial and mesenchymal cells in the tooth germ terminally differentiate into the tooth-tissue progenitor cells, such as ameloblasts, odontoblasts, and dental follicle cells (bell stage). Ameloblasts and odontoblasts accumulate the enamel and dentin matrix, respectively, at the boundary surface between the epithelium and mesenchyme, while dental follicle cells differentiate into the periodontal tissues, which include the cementum, periodontal ligaments and alveolar bone (Avery, 2002).
3. A novel three-dimensional cell manipulation method for whole tooth regeneration
One current biological approach for the regeneration of three-dimensional organs is based on recapitulating organogenesis by mimicking the reciprocal epithelial-mesenchymal interactions that occur in the developing embryo, thereby developing fully functional bioengineered organs from a bioengineered organ germ generated from immature stem cells via three-dimensional cell manipulation
To realise whole tooth replacement, the first critical issue is to develop a three-dimensional cell manipulation method using completely dissociated epithelial and mesenchymal cells
In addition, the cell aggregation method, which aims to reconstitute a bioengineered organ germ, has been applied for the transplantation of cell aggregates constructed from dental epithelial and mesenchymal cells, and it has been reported that this approach can generate appropriate tooth formation (Hu et al., 2006). It has also been reported that mixed cell aggregates of tooth germ-derived epithelial and mesenchymal cells can develop into a tooth with the correct structure, following epithelial cell sorting and subsequent self-organisation of the epithelial and mesenchymal cells (Song et al., 2006). However, these approaches suffer from critical limitations, including a low frequency of tooth formation and irregularity of the resulting tooth tissue structures, for example with enamel-dentin complex formation and the arrangements of the ameloblast/odontoblast cell lineages.
To achieve precise replication of the processes in organogenesis, an
4. Functional tooth replacement therapy
Oral functions such as mastication, pronunciation, and facial aesthetics have an important influence on quality of life because they facilitate both oral communication and nutritional intake. These functions are achieved with the teeth, masticatory muscles and the temporomandibular joint, under control of the central nervous system. For the realisation of tooth replacement regenerative therapy, a regenerated tooth developing from bioengineered germ tissue or a transplanted bioengineered mature tooth unit must be capable of properly engrafting into the lost tooth region in an adult oral environment and acquiring full functionality, including sufficient masticatory performance, biochemical cooperation with periodontal tissues and afferent responsiveness to noxious stimulations in the maxillofacial region (Proffit et al., 2004).
4.1. Transplantation of bioengineered tooth germ or a bioengineered mature tooth unit as a tooth replacement therapy
The critical issue dictating the success of tooth regenerative therapy via the transplantation of bioengineered tooth germ tissue into the lost tooth region is whether the germ can erupt and occlude properly with the opposing tooth in an adult oral environment. It has previously been demonstrated that transplanted natural tooth germ erupts in a murine toothless diastema region (Ohazama et al., 2004). We have also reported that a bioengineered tooth germ can develop the correct tooth structure in an oral cavity and successfully erupt 37 days after transplantation (Ikeda et al., 2009). The bioengineered tooth subsequently reached the occlusal plane and achieved occlusion with the opposing tooth from 49 days onwards (Fig. 4A, B). In the case of a transplanted bioengineered mature tooth unit comprising mature tooth, periodontal ligament and alveolar bone, the most critical consideration is whether that unit can be engrafted into the tooth loss region through bone integration, which involves natural bone remodelling in the recipient. A bioengineered tooth unit transplanted at a position reaching the occlusal plane with the opposing upper first molar was successfully engrafted after 40 days and thereafter maintained the periodontal ligament originating from the bioengineered tooth unit through successful bone integration (Fig. 4C) (Oshima et al., 2011). The enamel and dentin hardness of the bioengineered tooth components were in the normal range when analysed by the Knoop hardness test (Ikeda et al., 2009; Oshima et al., 2011). These approaches demonstrate the potential to successfully recover masticatory performance and natural tooth tissue through state-of-the-art bioengineering technology.
4.2. Biological response of bioengineered teeth to mechanical stress
Biological oral functions require cooperation between teeth and the maxillofacial region through the connection of periodontal ligaments (Dawson, 2006). Tooth loss and periodontal disease cause fundamental problems for oral function, including mastication, as well as associated health issues. The periodontal ligament plays an essential role in the pathogenic and physiological tooth response to extreme mechanical forces from bone remodelling accompanied by orthodontic tooth movement (Proffit et al., 2004). Studies on autologous tooth transplantation have indicated that healthy periodontal tissue remaining on the tooth root can successfully restore physiological tooth function, including bone remodelling, and effectively prevent ankylosis. In contrast, the absence of a periodontal ligament in osseo-integrated dental implants is associated with deficiencies in essential tooth functions and in the natural structural relationship between the tooth root and alveolar bone (Dawson, 2006). The periodontal ligament of bioengineered teeth that erupted following the transplantation of bioengineered tooth germ and mature tooth units achieved functional tooth movement comparable with that of natural teeth. Bioengineered teeth also successfully underwent bone remodelling in response to mechanical stress via the proper localisation of osteoclasts and osteoblasts, indicating that a bioengineered tooth can reproduce critical tooth functions by restoring and re-establishing cooperation with the surrounding jawbone (Ikeda et al., 2009; Oshima et al., 2011).
4.3. Perceptive neuronal potential of bioengineered teeth
The peripheral nervous system is established by the growth of axons that navigate and establish connections with developing target organs during embryogenesis (Guyton & Hall, 2000). The perceptive potential for noxious stimulation, including mechanical stress and pain, is important for proper organ function (Guyton & Hall, 2000). Additionally, it is believed that the recovery of the nervous system, which requires the re-entry of nerve fibres following organ transplantation, is critical for reconstituting organ function. Teeth are a peripheral organ for sensory and sympathetic nerves, both of which play important roles in tooth function and protection (Dawson, 2006). It is anticipated that tooth regenerative therapies will be able to recover the neuronal ability related to the perception of mechanical forces that are lacking in implant patients. Importantly, sensory and sympathetic nerve fibres innervate both the pulp and periodontal ligament of a bioengineered tooth following its eruption (Ikeda et al., 2009). Thus, these bioengineered teeth possess appropriate perceptive potential for nociceptive pain stimulations, such as pulp injury and orthodontic treatment, and can properly transduce these events to the central nervous system through c-Fos immunoreactive neurons (Ikeda et al., 2009; Oshima et al., 2011). In this way, bioengineered teeth can indeed restore the perceptive potential for noxious stimuli in cooperation with the maxillofacial region.
5. Future directions for tooth regeneration
To realise the use of tooth regenerative therapy in future clinical applications, one of the major research hurdles remaining is the identification of appropriate cell sources. The cell source may be optimised by using the patient’s own cells for regenerative therapy to avoid immunological rejection. Tooth tissue-derived stem cells found in pulp and periodontal ligaments can differentiate into dental cell lineages and contribute to the supply of various progenitor cells (Egusa et al., 2012, 2013). While these tissues are good candidate cell sources for stem cell transplantation therapy for tooth tissue repair, epithelial-mesenchymal interaction driven tooth inductive potential has not been reported for these stem cells. Other candidate cell sources for whole tooth regeneration include embryonic stem (ES) cells and iPS cells, which are capable of differentiating into endoderm, ectoderm and mesoderm (Takahashi et al., 2006). Recently, iPS cells have been established from various oral tissues, and reprogramming procedures for dental epithelial and mesenchymal fates have been established (Arakaki et al., 2013; Otsu et al., 2013). Another important direction for future research on tooth regenerative therapies is the identification of key factors for reprogramming non-dental cells into dental epithelium and mesenchyme. Notably, the self-organisation of various tissues such as the optic cup and adenohypophysis using uniform pluripotent stem cells in three-dimensional culture has been reported (Eiraku et al., 2011; Suga et al., 2011). A three-dimensional
The technology of regenerative medicine has progressed remarkably, and many patients and clinicians are anticipating the realisation of whole tooth regenerative therapy. Tooth regenerative therapy is now regarded as a crucial model for future organ replacement regenerative therapies for severe diseases and will contribute substantially to the understanding of tissue regeneration for more complex organs.
This work was partially supported by Health and Labour Sciences Research Grants from the Ministry of Health, Labour, and Welfare (No. 21040101) to Akira Yamaguchi (Tokyo Medical and Dental University), a Grant-in-Aid for Scientific Research (A) (No. 20249078) to T. Tsuji (2008–2010) and a Grant-in-Aid for young Scientists (B) to M. Oshima from the Ministry of Education, Culture, Sports and Technology, Japan.