| Objective: To explore efficient induction system of inducing Human bone marrow mesenchymal stem cells into osteoblasts.Methods: Mesenchymal stem cells were isolated and cultured by density gradient centrifugation. The growth curves of passage 1 and 2 cells were drawn by MTT method. The phenotype of passage 2 mesenchymal stem cells was analyzed by the following conjugated monoclonal antibody:CD14-APC, CD34-PE, CD44-FITC, CD45-FITC, CD90-FITC and CD105-PE. Data were acquired using a FACScalibur flow cytometer. Different concentrations of dexamethasone,β-Glycerophosphate and vitamin C were divided into 4 groups, and the control group was set, mesenchymal stem cells were induced into osteoblasts. After 10 days, the character of osteoblasts was detected by gomori method for alkaline phosphatase (ALP).Results: The cell number of passage 1 cells went down in the first three days after the MSCs were planted, then increased rapidly in the log phase during the 4th to 10th day, at last, the growth curve went to the stationary phase after the 10th day. While the cell count of passage 2 cells went down in the first 2 days after planted, then increased rapidly in the log phase during the 3rd to 10th day, at last, the growth curve went to the stationary phase after the 10th day. The data of flow cytometric analysis showed that CD44,CD90and CD105 were positive , while CD14, CD34 and CD45 were negative. Integral optical density was analyzed by the Imagine-Pro Plus 6.0. The mean value of 4 experimental groups was 634500.14, 716254.91, 808213.05, 577479.49 respectively, while the mean of control group was 177411.99. There were differences compared the experimental groups to the control group by nonparametric rank sum test using SPSS software program (SPSS 13.0). Among the experimental groups, there were differences compared the experimental group 3 to the others by nonparametric rank sum test using SPSS software program (SPSS 13.0).Conclusion: By density gradient centrifuge culture, relatively high purity of mesenchymal stem cells can be obtained. Under the concentrations of dexamethasone was 1×10-8 mol/L,β-glycerophosphate was 1×10-2 mol/L and vitamin C was 3×10-4 mol/L, mesenchymal stem cells induced into osteoblasts was the most efficient. Objective: Under the osteogenic culture conditions, the tumorigenicity and the Chromosome karyotype of the mesenchymal stem cells were detect which can be used to analyze the biological security.Method: Different concentrations of dexamethasone,β-Glycerophosphate and vitamin C were divided into 4 groups, and mesenchymal stem cells were osteogenically-induced for 10 days. Ten days later, the induced cells were injected into nude mice, and the positive control group and negative control group were set. Then we can observe whether there was suspicious tumor or nodule in the inoculation site for the evaluation of tumorigenicity during 10 weeks. At the same time, Chromosome G banding of the induced mesenchymal stem cells was used to analyze the karyotype which was used to observe whether there were chromosomal inversion, translocation, deletion, duplication or fusion.Result: Any abnormal changes in the nude mice could not be found and no abnormal changes of the chromosome G-banding karyotype of the induced mesenchymal stem cells were detected in vitro osteogenic culture by different concentrations of dexamethasone,β-glycerophosphate and vitamin C in the experiment groups.Conclusion: Under the osteogenic culture according to the different concentrations of dexamethasone,β-glycerophosphate and vitamin C for Human bone marrow mesenchymal stem cells, the suspicious tumor or nodule in the inoculation site were not detected and chromosome G-banding karyotype was normal. Mesenchymal stem cells induced into osteoblasts were biological security acted as the ideal material for tissue engineering.
|
PDF Full Text Request