| Background and Objective: Bone tissue defects caused by various causes are common clinical diseases,with the rise of bone tissue engineering technology,to the traditional treatment of difficult bone defects brought some dawn,tissue engineering using autologous cell biological activity,given external factors specific Interfere with the induction of differentiation into the required cells,so as to achieve the ability to repair tissue.Traditional mesenchymal stem cells and embryonic stem cells have been successfully induced in vitro,but some ethical and material limitations have been involved in the study.Therefore,they have been restricted in histology as a seed.Induced pluripotent stem cells,which are self-renewing and have pluripotent differentiation potential,which can be obtained by using several specific transcription factors to induce somatic reprogramming,can be used as tissue.The results showed that the i PSCs could be differentiated into i PSCs-MSC by induction factor,and the optimized seed was used as bone seed tissue.Cells were transfected into i PSCs-MSC,and the feasibility of vector-mediated transfection of seed cells was investigated.At the same time,Under the effects of i PSCs-MSC,which on the expression of BMP-2,observe its ability to proliferate and differentiate orthogenesis in vitro.Methods: 1.IPSCs-MSC was preincubated in vitro and cultured in vitro to the third generation of i PSCs-MSC,which was propagated in vitro according to the culture medium of BMP-2 and adenovirus-BMP-2.The rats were randomly divided into third groups: control group,negative control group and experimental group.Then,osteogenic induction fluid was added to each group.The culture medium was cultured in vitro.The cells were cultured in vitro.After 1,2,3,4,5,6,7days,detecting the survival and proliferation of i PSCs-MSC;2.Three groups of treated cells were transfected and the culture was stopped after 3,7 and 14 days respectively.The expression of alkaline phosphatase was detected by alkaline phosphatase kit.3.after three days of cell culture and proliferation,the alizarin red dye was used.Then,the formation of mineralized nodules was observed under light microscope at 1 week and 2 weeks after staining.4.The expression of BMP-2 was detected by Western Blotting.Results: 1.After the detection of CCK-8,the visible light absorption OD of the experimental group was higher than that of the other two control groups,which indicated that the activity of cell proliferation was stronger than that of the other two groups.2.The ALP activity of the three groups of cells transfected with adenovirus was positive,and the intracellular ALP activity increased with the prolongation of time.However,the ALP content in the experimental group was significantly higher than that in the other two groups.Meanwhile,with the prolongation of time,the negative control group Compared with the blank control group also showed strong ALP expression ability.3.The expression of mineralized nodules in the negative control group was slightly higher than that in the blank control group,and there was no obvious red nodule in the blank control group.4.The expression of BMP-2 in the experimental group was significantly higher than that in the negative control group,and the expression of BMP-2 in the negative control group was not obvious.Conclusions: 1.Confirmed the feasibility of i PSC-MSCs in vitro osteogenesis;2.The results showed that BMP-2 could significantly promote the differentiation and function of i PSC-MSCs into osteoblasts.3.Adenovirus can significantly accelerate the differentiation rate of BMP-2 to i PSC-MSCs in osteoblasts in vitro.4.BMP-2 gene transfection induced pluripotent mesenchymal stem cells in vitro can significantly promote its differentiation into osteoblasts,as the bone tissue engineering to optimize the seed cells laid the experimental basis. |
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