| Objective:Study bone marrow mesenchymal stem cells with fibrin sealant for scaffolds based on combined with pravastatin common culture in vitro, observe the effect of pravastatin on bone marrow stromal cell growth, proliferation and osteoblast and vascular effects. Study the effect of pravastatin on the in vitro secretion of BMSCs/FS Bmp-2and VEGF in to determine the optimal concentration of the drug, for the subsequent directional differentiation of bone marrow stromal stem cells into osteoblasts and vascular differentiation and provide experimental basis.Method:Take the health of4New Zealand rabbits were only, male and female, using the whole bone marrow culture method, through changing the primary culture and passage of liquid, Growth of cells by flow cytometry in CD34, CD44and other surface markers, proved to be of high purity Take P3BMSCs, divided into four groups:Group A:P3generation of bone marrow stromal stem cells and fibrin sealant+osteogenic medium; group B:P3generation of bone marrow stromal stem cells and fibrin sealant+osteogenic medium+50u mol/L pravastatin; group C:P3generation of bone marrow stromal stem cells and fibrin sealant+osteogenic culture base+100u mol/L pravastatin; group D:P3generation of bone marrow stromal stem cells and fibrin sealant+osteogenic medium+150u mol/L pravastatin. Osteogenesis induced by1,2,4,7,14,21days, respectively under the light microscope, gross morphological changes of cells were observed; in the pravastatin under the action of growth kinetics determination of cell proliferation ability changes. The osteoblast differentiation of14d,21d groups of bone marrow mesenchymal stem cell line of alkaline phosphatase Gomori calcium cobalt method and modified Von-Kossa staining were observed under microscope, camera, cytoplasmic positive response showed black granular or lump precipitation. Osteogenesis induced by5,10,15,20days of each row of alkaline phosphatase and calcium quantitative detection, identification of osteogenic differentiation ability. In Fourteenth days,21by Elisa assay medium BMSCs secretion in Bmp-2and VEGF concentration. In fourteenth21days using real-time fluorescence quantitative PCR for detection of Bmp-2and VEGF mRNA expression quantity. Use SPSS17.0statistical software for data statistics and analysis.Result:In vitro osteogenesis induced by2D visible minority adherent cells were fibroblast-like cells.4days later, a small number of cells form elongated spindle, P3generation of adherent cells by flow cytometry, positive expression of CD44, greater than90%purity. Embedded in the FS stem cells containing pravastatin in culture medium,4days after the cells showed typical spindle cell morphology,7days after fibrin sealant edge portion begins to degrade, exfoliative cells in culture plates; in vitro culture for14days, the cells grow well, most of the FS degradation, exfoliated cells increased, three weeks after fibrin sealant completely degraded, the four groups were compared, growth of adherent cells in normal morphology, cell morphology was not affected. Growth kinetics of cells growth curve shows:four groups of passage cells were digested after undergoing1-2days of incubation, and then enter the6-9days of the logarithmic growth phase, then transition to the platform, in the MTT method for the detection of growth kinetics of OD, four groups of comparison between groups P>0.05, not statistically significant. The line GENMED cell alkaline phosphatase Gomori calcium cobalt method and Von-Kossa improved staining method, four groups of cells with culture time, staining positive cells and calcium nodule number gradually increased, in which C group simultaneously changes than the other three groups is more obvious. Alkaline phosphatase and calcium quantitative detection display, C group and A, B, D groups compared, for repeated measures analysis of variance, P<0.05, there is significance. Fourteenth21day was detected with Elisa BMSCs secretion of VEGF and Bmp-2concentrations, real-time fluorescent quantitative PCR for detection of VEGF and Bmp-2mRNA expression, four groups compare group C expression was significantly higher than that of A, B, D group, P<0.05, there is significance.Conclusion:1. BMSCs embedded in FS construction of injectable scaffold material has good biocompatibility and biodegradability, plasticity, and has a three-dimensional structure, is a kind of good engineering organization2. Pravastatin role following in vitro culture of engineering complex BMSCs/FS had no significant effect on cell morphology.100u mol/L concentration of pravastatin can promote the secretion of BMSCs Bmp-2and VEGF,150u mol/L concentration on stem cell secretion of Bmp-2and VEGF inhibition produced... |
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