Effects Of Hypoxia On Bone Marrow Mesenchymal Stem Cells Culture And Osteogenic Differentiation In Vitro In Rabbits

Objective:Rabbit bone marrow mesenchymal stem cells(BMSCs)were isolated and cultured in vitro.Bone mesenchymal stem cells(BMSCs)were cultured in vitro using low oxygen microenvironment to investigate the effect of low oxygen microenvironment on BMSCs culture,proliferation and osteogenic differentiation.Methods;The bone marrow of healthy New Zealand white rabbits was extracted,and the bone marrow mesenchymal stem cells were isolated and purified with the method of "wall dressing".According to the characteristics of the bone marrow mesenchymal stem cells that can stick to the wall in the culture bottle,we purified the bone marrow mesenchymal stem cells by changing the fluid regularly.The third-generation cells were taken and immunohistochemistry was used to detect the surface marker CD44 of the adherent cells,which preliminarily confirmed that the isolated cells were BMSCs The morphological characteristics and growth status of primary cells and subcultured cells were observed.BMSCs were obtained by whole bone marrow adherent separation,fluid exchange and passage.Let's take generation 3,According to the cultivation of oxygen concentration and medium type is divided into four groups normal oxygen routine training group(20%02/F12)plus Dulbecco modified eagle medium,low oxygen routine training group(1%02/F12)plus Dulbecco modified eagle medium,normal oxygen osteogenesis induced group(20%02 and osteogenesis)induction medium and low oxygen induced osteogenesis group(1%02 and osteogenesis induction medium),through the cell count and the measurement of cell proliferation,pcna detection,to observe the effects of hypoxia on BMSCs proliferation apoptosis;Alkaline phosphatase activity detection and Gomori calcium cobalt staining were used to study the effect of low oxygen environment on osteoblastic differentiation of BMSCs.Results:isolated primary cells showed a small amount of adherent growth after 48h,and about 96h later,the isolated cells were radially arranged and proliferated like colonies.Immunohistochemistry detected CD44 of adherent cells,and we found that the expression of cell surface markers was consistent with the characteristics of mesenchymal stem cells,proving that the cultured cells were bone marrow mesenchymal stem cells.After the bone marrow mesenchymal stem cells were cultured in osteogenic induction medium,it was observed that the growth of the cells would have a long growth plateau of one to two days.Although the growth of the cells at this time was slow,most of the cell morphology and volume would change during this growth plateau.After trypsin digestion and continuous subculture,we found that the cells went through an incubation period of about 1 to 2 days by the third generation.At about 7 to 8 days,the cell growth began to show logarithmic growth,and we found that the cell growth still transited to the plateau.After the isolated cells were cultured in DMEM/F12 standard medium,the number of BMSCs cells in the hypoxic group was significantly increased compared with that in the normal oxygen group.PCNA staining cells increased and the proliferation rate was higher.TUNEL positive rate was significantly lower than that of normal oxygen group,which inhibited BMSCs apoptosis.The growth difference was significant(P<0.05).Continuous culture in four groups of cells respectively,the results found that the cells cultured in low oxygen osteogenesis medium of alkaline phosphatase activity,and in the normal oxygen osteogenesis compared the cells cultured in the induction medium,its alkaline phosphatase activity increased obviously,after statistics analysis,the results show that both have obvious difference(P<0.01);The isolated,purified and subcultured cells were cultured in osteogenic induction medium for 14 days,and the results showed that alkaline phosphatase staining was positive.After 4 weeks of continuous culture,the results showed that the alkaline phosphatase staining in the normal oxygen osteogenic induction group showed more calcium salt deposition and brown-black calcium nodules,while the other three groups showed no significant difference.Conclusion:In the experiment,the cells obtained after isolation and purification were bone marrow mesenchymal stem cells.Hypoxia increased the proliferation rate of BMSCs cultured in vitro,inhibited osteoblast differentiation,and maintained stem cell characteristics.