The Differentiation Potentiality Of The Neonatal Rat Skin Epidermal Cells To Dental Epithelial Cells

The odontogenesis is initiated by the presumptive dental epithelium and then proceeded by the reciprocal interactions between mesenchyme and epithelium. Epithelial derived cells and mesenchymal derived cells are two kinds of cells which are necessary for bioengineering tooth regeneration. It has been reported that dentin/pulp complex can be generated by dental pulp stem cells (DPSCs) and adult bone marrow cells. These researches implied that embryonic dental mesenchymal cells could be replaced with adult cells during tooth regeneration. However, researches about the substitute for dental epithelium are lagging than dental mesenchyme. Fetal palatal epithelium has been proved to have the capability of forming enamel organ and secret matrices when recombined with fetal dental mesenchyme. Bone marrow cells can give rise to ameloblasts with the existence of both embryonic dental epithelium and dental mesenchyme. Nevertheless, the conversion of non-dental tissues/cells to dental epithelium in these studies was realized under the induction of embryonic tissues, which is not suitable for future clinical application due to ethical concerns. Therefore, to find alternatives from postnatal tissues will be preferable to those from embryonic tissues. According to auxanology, skin epithelium and dental epithelium are both derived from embryonic ectoderm. The same origin of both tissues promoted us to investigate the conversion potentiality of postnatal skin-epithelium-derived cells to dental epithelial cells. The following experiments were involved in this study:1. The differentiation of skin epithelial cells toward dental epithelial cells under the induction of dental papillae mesenchymal cells (DPMCs) in vitro. Skin epithelial cells were isolated and selected by extracellualr matrix before cultured and identification. The selected cells had behaved like stem cells and showed high activity of proliferation in vitro. The results of immunocytochemical staining showed that almost all of the cells had positive staining for CK19 and all of the cells had positive staining for CK14. Further FCM analysis showed most of the cells lied in the period of G0/G1. An indirect induction strategy was performed after successfully separated and cultured DPMCs. Tooth germ cells conditioned medium were added as supplementary induction medium. Gene expression for AMBN, AMGN and ALP were detected on the sixth day by RT-PCR analyses. Further immunochytochemical staining showed that the epithelial cells had positive staining for CK14. These results demonstrated that skin epithelial cells have the potential to convert to dental epitheial cells in our culture system.2. The differentiation of skin epithelial cells toward dental epithelial cells under the induction of dental papillae mesenchymal cells (DPMCs) in vivo. Firstly, the recombination patterns between dental mesenchyme and non-dental epithllial cells were selected. There are four kinds of recombination patterns in our study, which is cells pellets of DPMCs recombined with epidermal pieces, cell-scaffolds complex, epithelial cells pellets-mesenchymal cells pellets complex, cells pellets of epithelial cells and mesenchymal cells. The results showed that the cells pellets culture system of mixed cells is better than others and therefore is more suitable for tooth-like structure formation. Secondly, we recombined skin epithelial cells/ pieces with DPMCs as cells pellets and cultured in SD rat renal capsule for 14 days. The results showed that tooth like structure could be formed in both groups, which included enamel, ameloblasts, dentin, predentin and odontoblasts. Further immunohistochemical staining analysis and cell labeling tracing showed that enamel-forming cells are skin epithelial cells derived. These results showed that 1-dpn SD rat skin epithelia cells can convert to ameloblasts-like cells under the induction of 1-dpn SD rat DPMCs. Thirdly, we estimated the conversion possibility of older SD rat skin epithelium to dental epithelium and the results showed that there was no enamel-like structure formed.3. The differentiation of skin epithelial cells toward dental epithelial cells under the induction of dental pulp mesenchyme tissues or cells in vivo.Firstly, we cultured and compared the basic characteristics and odontogenic potentiality of dental pulp stem cells (DPSCs) and DPMCs. The results showed that DPSCs had similar denin-formation ability to DPMCs. Secondly, based on the conclusion we drawed from the above study, a special research was designed to estimate the induction ability of DPSCs or dental pulp tissues on skin epithelial cells. The results showed that there was no enamel-like structure formed when skin epithelial cells were recombined with the mesenchymal cells or tissues of dental pulp.In summary, skin epithelial cells of 1-dpn SD rat can convert to ameloblasts-like cells under the induction of DPMCs, but the conversion can not be found when skin epithelial cells recombined with dental pulp mesenchymal tissues or cells under the same condition. In this study, we demonstrated that skin epithelial cells could convert to ameloblasts when recombined with DPMCs under a cell pellet culture system and tooth-like structures could be formed after transplanted into renal capsule. The present study represents a brand new approach for tissue-engineered tooth by a successful application of non-dental-derived cells from postnatal rat skin.