| The genesis and development of tooth are completed by interaction between dental epithelium which forms dental enamel and dental mesenchyme which forms dentin-pulp complex. Therefore there are at least two kinds of cells involved in the regeneration of tooth, that is, epithelium cells and mesenchyme cells. The key of tissue engineering of tooth is that to obtain rich seeded cells which have the potential to form a tooth and can develop and differentiate in a favorable local environment. Due to the limited resource of dental seeded cells, more researches are focused on looking for available, stable and easy-expansion adult stem cells. Human gingival fibroblasts are the major cells in the gingival tissue of oral mucosa which play an important role in the physiology and pathology of gingival and periodontal tissues. The cells are rich in recourses and have strong growth and self-reproductive ability. On the basis of the progress of dermal stem cells, there should be similar adult stem cells in the oral mucosa because it is analogous to skin. Owning to its plasticity, if we can separate mesenchyme stem cells from lamina propria of gingival tissue and induce nonodontogenic adult stem cells to differentiate into odontogenic stem cells, we will provide new seeded cells for the construction of tissue engineering of tooth. Human gingival mesenchyme stem cells, periodontal ligament stem cells and dermal stem cells were prepared by tissue explant culture technique and pancreatic enzyme digestion method and the biological characterization of gingival mesenchyme stem cells were identified. Then we made use of the signal molecule which secreted by the dental germ cells of early development of SD rats to provide suitable microenvironment and induced gingival mesenchyme stem cells to differentiate into the specific direction. We discussed the differentiation ability of gingival mesenchyme stem cells into the odontogenic mesenchyme stem cells and consequently, provided references for screening and inducing seeded cells of tooth tissue engineering.1. separation, culture, identification and biological characterization of gingival mesenchyme stem cells.The gingival tissues were cut from the extraction of the impacted third molars or artificial eruption after the patients gave their informed consent. Gingival mesenchyme stem cells were prepared by tissue explant culture technique and pancreatic enzyme digestion method. Primary culture cells were screened and purified by magnetic cell sorting and limited dilution. The morphology and growth of the clusters of cells clones were observed under Inverted Microscope and the proliferation ability, phenotype and cell cycle of screening cells were analyzed. Results showed that cultured gingival mesenchyme stem cells were long-spindle, which is the typical appearance of fibroblast. Flow cytometry showed that GMSC were positive for CD29 and CD90, which are makers of mesenchyme stem cells, and the positive rates were 99.8% and 99.4% respectively. The low expression of CD34 and CD45 indicated that cultured fibroblast-derived mesenchyme stem cells were not hematopoietic-derived cells. GMSC were capable of forming colonies and osteogenetic and lipogenetic differentiation under induction. Flow cytometry analysis of cell cycle demonstrated that cultured cells were G0/G2. Immunochemical assay demonstrated that GMSC were positive for CD105,STOR-1 and CD146, which are makers of mesenchyme stem cells, and most of the GMSC were positive for proliferating cell nuclear antigen Ki67. From above we concluded that separated cells were mesenchyme stem cells.2. Compare biological features among the gingival mesenchymal stem cells, periodontal ligament stem cells and dermal stem cellsTo investigate the different biological features among the gingival mesenchymal stem cells, periodontal ligament stem cells and dermal stem cells, three types cells were cultured by the orthodox method of tissue adhering. Morphological appearance, cell cycle analysis, MTT assays, alkaline phosphatase(ALP) activity, gene expression and differentiation capacities of three types cells were evaluated. The results showed that three types cells have a similar morphological appearances under light microscope. The proliferation activity of gingival mesenchymal stem cells was significantly higher than periodontal ligament stem cells. Flow cytometry assay showed that the expression of stem cell surface molecules CD90 and CD105 were highest in the gingival lamina mesenchymal stem cells. PCR analysis revealed that the expression of type I collagen in periodontal ligament stem cells and gingival mesenchymal stem cells was stronger than in the dermal stem cells, the expression of type III collagen was strongest in periodontal ligament stem cells, followed by dermal stem cells and gingival mesenchymal stem cells. ALP activity of gingival mesenchymal stem cells was higher than dermal stem cells, but less than periodontal ligament stem cells. The osteogenic and adipogenic differentiation capacity of periodontal ligament stem cells were highest than gingival mesenchymal stem cells and the dermal stem cells. In conclusion, periodontal ligament stem cells is a ideal seed cells for tissue engineering of tooth. Gingival mesenchymal stem cells had some features, such as easy to culture, strong porliferation ability, therefor it also could be considered as seed cells for tissue engineering of tooth.3. The study on the tooth differentiation potential of gingival mesenchymal stem cells induced by apical tooth germ cell-conditioned medium in vitroTo investigate the tooth differentiation potential, the gingival mesenchymal stem cells were induced by conditioned medium from developing apical tooth germ cells. The results showed that the induced gingival mesenchymal stem cells exhibited increased proliferation, calcified nodule formation and the expression of osteogenic-related genes, for example DMP-1,BSP,OPN,ALP in vitro。The study showed that the non-dental mesenchymal cells could substitude the dental mesenchymal cells for tooth differentiation and apical tooth germ cell-conditioned medium could promote gingival mesenchymal stem cells to dental mesenchymal cell differentiation in vitro.
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