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

Objective:To study the effects of extreme hypoxia on the culture and osteogenic differentiation of mouse BMSCs in vitro.Methods:(1)Isolation,purification and culture of mouse BMSCs in vitro:the cells were isolated using whole bone marrow adherent method,cultured and purified under hypoxia(1%)and normoxia(21%)respectively,and the cell morphology and proliferation were viewed under microscope.(2)Hypoxia and normoxia mouse BMSCs were induced to differentiate into osteogenic,adipogenic and neurogenic cells respectively:the third generation of mouse BMSCs were cultured with osteogenic inducer,adipogenic inducer and neurogenic inducer for 14,21 and 3 days,respectively.Cell morphology and proliferation were observed under inverted microscope.Osteoblasts,adipoblasts and neuroblasts were identified by alizarin red,oil red O and Nissl staining.(3)Flow cytometry of bone marrow mesenchymal stem cells under hypoxia and normoxia:When the cells grew to 80%-90%fusion,the third generation of BMSCs from under hypoxia and normoxia were suspended with a cell scraper,inoculated in the flow tube according to the cell concentration of 1 x 10~6/m L,washed with D-Hanks twice,and detected by adding fluorescent labeled monoclonal antibodies Sca-1,CD29,CD117 and CD45.(4)Idexes of BMSCs under hypoxia and normoxia:the proliferation of BMSCs in each group was detected by CCK-8 assay from the 1st to the7th day,and the proliferation curve was performed.The scratch test of cells was performed to show the cell migration efficiency at 0,6,12,24 and 48h.Mito Tracker staining was performed to observe the structure and morphology of mitochondria,and the fluorescence intensity was performed by flow cytometry.Cells were induced in osteogenic inducer for 3,5,7,10 and 14 days,then the activity of ALP was detected.The bone formation efficiency was aslo observed with alizarin red staining under microscope after induction with osteoblast inducer.Meanwhile,the calcium nodule was dissolved by cetylpyridine chloride for semi quantitative detection.The oxygen consumption(OCR)of cells in each group was measured by Seahorse cell metabolic energy analyzer.What’s more,the expression of osteogenic gene Runx2 and OPN was detected by and Western Blot.Results:(1)The mouse BMSCs extracted by whole bone marrow adherent method grew both like long fusiform and polygonal clones under both normoxia and hypoxia conditions.(2)Under the induction of osteogenic inducer,adipogenic inducer and nerve inducer,the results of alizarin red,oil red O and Nissl’s staining were all positive in mouse BMSCs,orange-red mineralized nodules,red lipid droplets and dark purple Nissl bodies could be observed.(3)After flow cytometry analysis,it was found that Sca-1 and CD29 of BMSCs of normoxia and hypoxia cells were positive,while CD117 and CD45 were negative,indicating that the normoxia and hypoxia BMSCs have stem cell characteristics and are a group of uniform undifferentiated stem cells.(4)There was no significant difference between the two groups on the first and second day of culture,however,the cell proliferation of mouse BMSCs was promoted obviously from the 3rd day to the 7th day.Compared with normoxia group,hypoxia could significantly promote cell migration,and Mito Tracker staining showed that the structure of mitochondria in hypoxia group was more mature than that in normoxia group,showing a short rod shape.Flow cytometry showed that the fluorescence intensity of Mito Tracker staining in hypoxia group was significantly stronger than that in normoxia group.Alizarin red staining showed that calcium nodules in hypoxia group were significantly more than that in normoxia group,and ALP activity of hypoxia was also higher than that of normoxia.Seahorse test results showed that the OCR of hypoxia was higher than that of normoxia group,too.Futhermore,Western Blot analysis indicated that the osteogenic gene expression level of BMSCs in hypoxia group was significantly higher than that in normoxia group(p<0.05).Conclusion:Both normoxia and hypoxia conditions are suitable for the culture of mouse BMSCs.Hypoxia can not only improve the proliferation and migration efficiency of mouse BMSCs,but also promote the osteogenic differentiation of mouse BMSCs in vitro.