Effects Of Different Oxygen Concentrations On Proliferation,Survival,Migration,and Osteogenic Differentiation Of MC3T3-E1 Cells

Background:Osteoblasts,which are cells of mesenchymal origin,are the most important cells in the process of osteogenesis.They eventually become quiescent cells lining the bone surface or are transforamed to osteocytes embedded in the bone matrix synthesized by themselves.In addition,more and more emerging evidence demonstrate that the osteoblasts also have some endocrine functions.In a word,the osteoblasts can not only regulate osteogenesis but also influence the whole body's homeostasis.The oxygen concentration of physiological environment osteoblasts residing in,much lower than that in atmospheric air and the normal incubator,range from 3%to 9%.Taking pathological conditions such as fractures into account,the oxygen concentration osteoblasts experience is about 0.8%-9%.Since the range of oxygen concentrations osteoblasts contact is so wide,how they affect the proliferation,survival,migration,and osteogenic differentiation of osteoblasts,and whether there is an optimal oxygen concentration have important significance for the intervention of some clinically diseases such as fracture,osteoporosis and some vascular diseases.However,No one had systematically studied the above issue.Methods:we cultured the osteoblast precursor cell line MC3T3-E1 cells in small individual chambers with oxygen concentrations of 1%,3%,6%,9%,and 21%respectively.The cell proliferation was evaluated by the proliferation index test and EdU staining.To test the cell survival,Live/Dead assay was performed.Tablet scratch assay was performed to detect the migratory ability of the cells.Immunofluorescence and Western blotting were performed to assess the osteogenic differentiation of them.Results:We found that proliferation and osteogenic differentiation ability of MC3T3-E1 cells against oxygen level were both approximate bell-shaped curve,and the optimal oxygen concentration was about 6%and 9%respectively.Live/Dead assay showed that the survival of MC3T3-E1 cells in different oxygen concentrations was affected by the amount of serum.In the 10%FBS group,there was no statistical difference in survival rate among the different oxygen concentration tests.In the 1%FBS group,the oxygen concentration of 6%resulted in worse cell survival rate than the other oxygen concentrations.In the 0%FBS group,the cells survived better in the oxygen concentration of 1%than in the other oxygen concentrations.The tablet scratch experiment showed that the oxygen concentration of 1%,3%,and 21%resulted in greater cell migration than 6%and 9%.Conclusions:for the first time we systematically explored the effects of different oxygen concentrations on proliferation,survival,migration,and osteogenic differentiation of the MC3T3-E1 cells and determined the approximate ranges of the optimum oxygen concentrations of them.The results have significant reference value for the intervention of the pathological processes involving osteoblasts,such as fracture,osteoporosis,and some vascular diseases.They also have an important guiding role for the new scientific idea that osteoblasts as a treatment cell repair bone defects.However,we have not conducted any further research on the specific mechanism,which needs to explore the related gene transcription,protein expression,cytokine release,and response to various stimuli of osteoblasts at different oxygen concentrations more comprehensively and thoroughly.