Donor organ transplantation is currently an essential therapeutic approach to the replacement of a dysfunctional organ as a result of disease, injury or aging in vivo. Recent progress in the area of regenerative therapy has the potential to lead to bioengineered mature organ replacement in the future. In this proof of concept study, we here report a further development in this regard in which a bioengineered tooth unit comprising mature tooth, periodontal ligament and alveolar bone, was successfully transplanted into a properly-sized bony hole in the alveolar bone through bone integration by recipient bone remodeling in a murine transplantation model system. The bioengineered tooth unit restored enough the alveolar bone in a vertical direction into an extensive bone defect of murine lower jaw. Engrafted bioengineered tooth displayed physiological tooth functions such as mastication, periodontal ligament function for bone remodeling and responsiveness to noxious stimulations. This study thus represents a substantial advance and demonstrates the real potential for bioengineered mature organ replacement as a next generation regenerative therapy.

The arrangement of cells within a tissue plays an essential role in organogenesis, including tooth development. Organ morphogenesis and physiological functions induced by three-dimensional tissue organization are well known to be regulated by the proper spatiotemporal organization of various signaling molecules, including cytokines, extracellular matrix proteins, and adhesion molecules. Development of a three-dimensional cell manipulation technology to create a bioengineered organ germ, designated as the organ germ method, enabled the generation of a structurally correct and fully functional bioengineered tooth in vivo. This method is expected to be utilized as a valuable technique for analyzing gene and protein functions during organogenesis. Here, we describe protocols for tooth germ reconstitution using the organ germ method and methods for analyzing tooth development in vitro and in vivo.

Ectodermal organs, such as teeth, hair follicles, and mammary glands, arise from their respective germs through epithelial-mesenchymal interactions during organogenesis. Growth arrest and DNA damage-inducible gene gamma (Gadd45g) have been shown to play important roles in various biological processes, such as stress responses, cell differentiation, and tumor suppression, through the regulation of cell proliferation and gene expression. We found that Gadd45g was expressed in enamel knots, which orchestrate tooth germ development as epithelial signaling centers. Gadd45g induced the expression of p21 and inhibited the proliferation of dental epithelial cells. The up-regulation of p21 expression was regulated by Gadd45g-mediated activation of the p38 MAPK pathway. Thus, our results suggest that Gadd45g is involved in the regulation of p21-mediated epithelial cell proliferation through the p38 MAPK pathway during tooth organ development.

Dihydropyrimidinase-related protein 4 (Dpysl4) is a known regulator of hippocampal neuron development. Here, we report that Dpysl4 is involved in growth regulation, polarization and differentiation of dental epithelial cells during tooth germ morphogenesis. A reduction in Dpysl4 gene expression in the tooth germ produced a loss of ameloblasts, resulting in the decrease of synthesis and secretion of enamel. The inhibition of Dpysl4 gene expression led to promotion of cell proliferation of inner enamel epithelial cells and inhibition of the differentiation of these cells into pre-ameloblasts, which was confirmed by analyzing cell polarization, columnar cell structure formation and the expression of ameloblast marker genes. By contrast, overexpression of Dpysl4 in dental epithelial cells induces inhibition of growth and increases the expression of the inner enamel epithelial cell marker gene, Msx2. These findings suggest that Dpysl4 plays essential roles in tooth germ morphogenesis through the regulation of dental epithelial cell proliferation, cell polarization and differentiation.