Comparative Analysis Of In Vitro Biological Charactersitics Of Dental Pulp Stem Cells On Rat Tail Collagen And Titanium

Objective:Dental pulp stem cells(DPSCs) is a kind of adult stem cells derived from dental pulp and possess self-renewal capability, high proliferation rate and multiple differentiation capability.VThe study of DPSCs presents important significance in dental tissue engineering, such as dental restoration Implant and dental pulp regeneration. Gronthos et al. And can express bone Sialophosphoprotein(BSP, bone metabolic markers, guided bone mineralization, promoting the formation of bone and cementum), dentin phosphoprotein(DSPP, dentin cell-specific marker proteins, can be used to detect biochemical indexes dentin cell activity), osteocalcin(OC, a synthetic composed of mature osteoblasts non-vitamin K dependent bone collagen enzyme-modified). In the comparative study of dental pulp stem cells in bone marrow stromal cells, they found the two have a similar phenotype.Current research DPSCs biological characteristics of multi-directional differentiation of dry cells. Experimental method:Complete removal of root yet fully developed orthodontic teeth or third molars never had dental periodontal disease, with hair tissue primary culture DPSCs, after grouping. Divided into three groups, the control group, normal culture(10% FBS + α-MEM culture medium); experimental group A: Join rat tail collagen and 10% FBS + α-MEM culture medium; the experimental group B: added titanium metal and 10% FBS + α-MEM culture liquid. Three groups were observed under inverted microscope DPSCs primary cells and the growth and passaging features passaged cells. After three groups of P2 cells were seeded on coverslips, using immunofluorescence techniques to detect DPSCs showed expression of markers STRO-1. Then after the logarithmic growth of the three groups of cells into cell suspension, inoculated into 6-well plates and cultured 7D, added crystal violet dye staining, colony formation. Continuous 10 D by MTT OD values were measured in three groups DPSCs P3 cells, growth curves of the three groups DPSCs. Take the three groups in P2 cells were cultured in adipogenic was observed after the use of lipid droplets-O oil red staining. Three groups of cells were treated with osteogenic 4W be west after red staining, two parallel semi-quantitative determination of calcium content Stem Cells to compare rat tail collagen and titanium ability to form mineralized nodules on DPSCs impact. Mineralization induced 4W three groups DPSCs using immunofluorescence osteogenic markers alkaline phosphatase(alkaline phosphatase, ALP), bone Sialophosphoprotein(bone sialophosphoprotein, BSP), dentin sialoprotein(dentin sialoprotein, DSP) osteocalcin(osteocalcin, OC) expression. Application of PCR System 7900 respectively detect m RNA expression levels of the three groups DPSCs osteogenic 3D, 14 D, 21 D of the ALP, BSP, DSP, OC genes, quantitative comparison of rat tail collagen and titanium stem cells into bone / a tooth ability to influence bone. Results:After continuous culture 7D, inverted microscope three groups were seen with adherent cells in tissue blocks centered radially growth. The three most cells of fibroblast-like growth, the shuttle was loaded, clean cut, basically the same size. Three groups of P0 DPSCs climbing film anti STRO-1 immunofluorescence, endoscopic third group DPSCs nuclei showed blue fluorescence, green fluorescence cytoplasm, is positive. 7D were cultured dye crystal violet staining, microscope were seen significant cell clusters are formed, wherein A genomic clone formation rate of 8.5% in group B was 5%. After statistical analysis, A group formed higher than group B, the difference was statistically significant(p <0.05). DPSCs the growth curve was "S" type, 1-2D is the incubation period, 3-8D logarithmic growth phase, cell growth accelerated after 8D into the plateau, cell growth slowed down, in line with the characteristics of stem cells slow cycle, P4 start A group A and group B compared statistically significant differences(p <0.05). Performed three sets DPSCs vitro adipogenesis 3W-O after oil red staining were positive, inverted microscope to observe significant red lipid droplets. After mineralization induced 4W, three groups of cells using Alizarin Red staining, showing a orange mineralized nodule formation. Alizarin Red semi-quantitative detection of calcium content in three groups of cells, the calcium content of which A group was 5.89 ± 0.41, significantly higher than group B 3.83 ± 0.27, the difference was statistically significant(p <0.05). Mineralization of induced 4W DPSCs after anti ALP, BSP, DSP, immunofluorescence OC, the result is positive for group A fluorescence intensity was significantly higher than the other two groups. Quantitative PCR results showed that with the increase of induction time, BSP, DSP and OC gene expression levels increased, while the level of gene expression of ALP decreased with increasing induced increase in time, and the A group of m RNA for each gene expression levels were higher than the other two groups, the difference was statistically significant(p <0.05). ConclusionThe successful experiment of human permanent dental pulp stem cells Tao culture, and to identify in vitro respectively rat tail collagen and titanium for permanent dental pulp stem cells into bone / tooth-borne and related osteogenic differentiation markers and compared and confirmed the influence and role in promoting rat tail collagen DPSCs on more than a review of each indicator.