Comparative Study On Immunophenotype And Ability Of Differentiation Between Dental Pulp Stem Cell And Ecto-mesenchymal Stem Cells

Primitive stem subgroup-cells deposit in some tissue and develop into adult stem cells when they gradually lost the part of differentiation ability after the embryo becomes mature. During the oral maxillofacial region developing-period, cephalic neural crest stem cells (CNCSCs) immigrate and develop into ectomesenchymal stem cells (EMSCs), which take part in the development of the most part of oral maxillofacial region tissue. It is unknown whether EMSCs still exist in the corresponding part of maxillofacial region tissue and work as stem cells and whether cell phenotype and biological behavior has changed. Embryo dental pulp is derived from ectomesenchyme. It is confirmed that dental pulp stem cells (DPSCs) existed in the adult dental pulp. But it is still unknown whether there is relationship and distinction of the cell phenotype and biological behavior between EMSCs and DPSCs. DPSCs and EMSCs are derived from the mesenchyme. Bone mesenchymal stem cells (BMSCs) have been detailedly studied now. In this study, the biological behaviors of DPSCs and EMSCs including growth proliferation characteristics, cell phenotype, differentiation abilities in vivoand in vitro and injury repair abilities were studied and analyzed with BMSCs as the standard. The main results are as follows:1 Observation of separation culture and growth proliferation characteristics of both DPSCs and EMSCsThe culture method of rat DPSCs in vitro was established successfully with enzyme digestion and clone screening. The morphological characteristics and growth proliferation activities were observed and analysed with BMSCs as standard. The results showed that the morphological characteristics of DPSCs and EMSCs were similar to them of BMSCs. Both of them looked like the mesenchymal cells. But the shape of DPSCs was uniform, and appears shortly fusiform. The shape of EMSCs appears various. The clone of EMSCs contained various kinds of cells. So DPSCs maybe the special stem cells in the tissue. The composition of cell is relatively single. EMSCs coming from embryo tissue maybe contain many kinds of cells. The proliferation ability of EMSCs is higher than it of DPSCs by determining the clone formation ability, cell proliferation and cell period, which shows that EMSCs as the mesenchymal stem cells in the maxillofacial region has the higher proliferation ability. The influence of cytokine on the growth of DPSCs and EMSCs was evaluated with MTT method. The results showed that the proliferation of DPSCs was inhibited by the TGF- β, but not EMSCs. The proliferation of DPSCs and EMSCs was not influenced by BMP-2. The bFGF can facilitate significantly the proliferation of DPSCs and EMSCs. So the reaction of both the two kinds of cells to the cytokine is similar.2 Study of the cell phenotype of DPSCs and EMSCsSome special differentiation marker expressions on the DPSCs, EMSCs and BMSCs were determined with immunohistochemistry method. The results showed: cell phenotypes of rat DPSCs appeared diverse. But the expression amount of some antigen on the DPSC had difference. The expression spectrum of DPSCs and EMSCs was similar. Both of them expressed the neuro-marker such as Nestin, NSE, NF and HNK-1. 5% STRO-1 positive cells were screened from the DPSCs with immunomagneticbeads, which expressed the EMSCs marker including the HNK-1 and Nestin with immunofluorescence double marking method. The cells co-expressing STRO-1 and HNK-1 were distributed around the vessel in the dental pulp tissue, which scattered in the dental pulp tissue like undifferentiated mesenhymal cells. It was suggested that DPSCs maybe the residual component of EMSCs which are mature. But the cell phenotype changed partly during the cell migration. The dentine special protein molecular-DSPP was not expressed on the DPSCs and EMSCs, but little expressed on the BMSCs of rats. It was suggested that DSPP were not only the mature dentine marker but also the bone marker. 3 Study of differentiation abilities of DPSCs and EMSCsIt is showed that STRO-1 positive DPSCs has the mineralization and differentiation potency which can be accelerated after adding mineralization inducing fluid by cell morphology method, mineralization stain and Hoechst33342 stain. DPSCs were differentiated into the odontoblast with the mineralization inducing fluid. But EMSCs differentiated into the osteoblasts. The differentiation procedure was regulated by the odontoblasts and osteoblasts special phenotype expression or expression upregulaion. DPSCs and EMSCs were implanted in the nude mouse subcutaneous part with the ceramics bone as the carrier. DPSCs can continue to differentiate into dentin-pulp complex, but EMSCs were differentiated into various structure. 4 days old tooth germ cells were chosen as the inducing tooth growth microenvironment. The germ cells containing the epithelial component were co-cultured with BrdU-marked DPSCs and EMSCs. It is observed that dentine special protein DSP was expressed by the DPSCs and EMSCs in the co-culture system. The inducing transforming rate of EMSCs is higher than that of DPSCs. The alkaline phosphoase activity of co-culture cells was significantly increased. Both DPSCs and EMSCs have the potency of differentiating into the fat and nerves, but DPSCs don't differentiate into the muscle cells. This suggests that the differentiation abilities in vivo and in vitro of both DPSCs and EMSCs are continuous. The multi-differentiation ability...