| Objective:Osteogenic differentiation of bone marrow mesenchymal stem cells?BMSCs?induced by the preparation of musk containing serum and to establish rat skull bone defect model,observation of the expression changes of bone morphogenetic protein 2?BMP-2?and fibroblast growth factor 2?FGF-2?in the process of bone defect healing,and explore the mechanism of musk promoting bone defect healing.Methods:In vitro culture of rat BMSCs by using adherent screening method,morphological observation,cell surface antigen detection and osteoinduction method identification BMSCs,musk containing serum interfered with the second-generation BMSCs,and the expression of BMP-2 and FGF-2 in the process of BMSCs osteogenic differentiation was detected by Western blotting on the 14th.The establishment of rat skull bone defect model,using random number table divided into model group and experimental group,each group of 150.The experimental group was given natural musk orally at a dose of 42 mg·kg-1.The model group was given 0.9%NaCl at the same volume once daily,all 1 times a day.The skull defects were harvested on the 7th,14th and 28th day after gavage respectively,and the expression of BMP-2 and FGF-2 in the bone defect was detected by Western blotting.Results:?1?Morphological observation of BMSCs:BMSCs just after inoculation can not be distinguished with blood cells or other cells.The culture solution is turbid.The cells are round,oval and irregular in size and suspended in the culture fluid;After 2 days of inoculation and cultivation,a small amount of cells began to adhere and the cell body was prolate or polygonal,the visual field was blurred and the growth was slow;On the 4th day of culture,a large number of cells adhered to the wall and began to divide.Some cells were spindle-shaped,and the hetero cells were obviously reduced and the visual field was clear.On the 8th day of culture,the cell division rate was significantly faster than that of the previous stage,and the cell volume increased,splits and proliferated vigorously,and arranged in a whirlpool arrangement.The adherent cells covered more than 80%of the bottom of the flask.The primary BMSCs was purified after passage to the first generation.A large number of cells began to adhere after 2 days of culture,The cell size was small,the shape was mainly short spindle shape,showing a few irregular shapes.When cultured on the 4th day,the growth rate of the cells was significantly increased and the entire bottle bottom was covered with multiple layers,the cells were fusiform,and there were cell colony groups,fused into pieces,and less impurities.After the first passage to the second passage,the cells were further purified,and cells adhered to the wall approximately 6 hours after passage,and the cell volume increased significantly.On the third day of culture,cell fusion is more than 70%,the arrangement was regular,the cell morphology was relatively uniform,a small number of cells showed an aging trend,and the proportion of nuclear plasma increased;On the 5th day of culture,cells arranged closely and swirled growth and covered the bottom of the bottle,almost no miscellaneous cells,but a small amount of aging cells.?2?Phenotype identification of BMSCs:The second generation BMSCs surface marker CD90 expression was 93.5%,was strongly positive,CD45 expression was 0.0%,was negative.?3?Osteogenic induction of BMSCs:10days after osteogenic induction of BMSCs,some cells changed from fusiform to triangular or irregular shape,and gradually appeared the trend of stacked and netted growth.After alizarin red staining,there appeared granular red deep calcified nodules in cells.After 14 days of osteogenic induction,cell density and calcium nodules gradually increased,aggregated into clumps,alizarin red staining of calcified nodules was dark red.?4?The expression of BMP-2and FGF-2 in the osteogenesis induced by BMSCs:The expression of BMP-2 musk group on the 7th and 14th day was higher than that of the blank group and the 28th day,the differences were statistically significant?all P<0.05?,the expression on day 14 was higher than that on osteogenic induction group,the difference was statistically significant?P<0.05?,the expression of 14th days in the osteogenic induction group was higher than that of the blank group and the expression of 28th in this group,the differences were statistically significant?all P<0.05?.FGF-2 musk group on the 14th day expression was higher than the blank group,the difference was statistically significant?P<0.05?,the expression of 7th days was higher than that of this group of 28th days,the difference was statistically significant?P<0.05?,the expression of osteogenic induction group on day 14th was higher than that of blank group and musk group,the differences were statistically significant?all P<0.05?.?5?Skull bone defect healing:After 7th days after modeling,it was found that the circular bone defect healed in different degrees,and the healing of the experimental group was better than that of the model group.On the 14th day,the circular defect was invisible in the experimental group,while the bone defect of the model group was more obvious than the normal bone tissue.On the 28th day,the experimental group almost completely healed,and the boundary between the round defect and the surrounding normal bone tissue was blurred.The circular defect of the model group was visible,but the healing degree was better than the 14th day.?6?The expression of BMP-2 and FGF-2 in the bone defect of the skull:The expression of BMP-2 in experimental group reached its peak on the 7th day,decreased on the 14th day,and lowest on the 28th day.FGF-2 peaked on the 14th day?P<0.05?,decreased on the 7th day,and lowest on the 28th day.Compared with the model group,the expression of BMP-2 in the experimental group on the7th day was significantly different?P<0.05?.The expression of FGF-2 on the 7th and 14th day was significantly different?all P<0.05?.The expression of FGF-2 on the 14th day in experimental group was higher than that on the 28th day?P<0.05?.Conclusions:Musk can promote the healing of the skull bone defect in rats,and its mechanism is related to the expression of BMP-2 and FGF-2. |
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