Initial Investigation In Vitro Of The Effection Of Hypoxia On Osteo-differentiation Of Human Mesenchymal Stem Cells

Part One Proliferation and identification of human bone marrow mesenchymal stem cells in vitroObjective To supply seed cells for tissue engineering, the human bone marrow- derived mesenchymal stem cells (hMSCs) were isolated from adult human whose bone marrow is normal and cultured in vitro.Methods Bone marrow was obtained by aspirated in the posterior superior iliac spine from three adult patients whose marrow is normal. Cells were isolated by gradient centrifugation,.the cell morphologic features and growth status were observed by lightmicroscope. The cell surface antigen and osteo-differntiation were used to identify hMSCs.Results Isolated cells grow adherence,and the cells exhibited dominant growth of spiral type. Immunocytochemistry analysis demonstrated hMSCs were uniformly positive for CD44 and CD71,while negative for CD34 and CD45,which indicated no contamination of hematopoietic cells,the cell surface marker is consistent with character of mesenchymal stem cells.Isolated cells were induced by bone formation condition medium,2 weeks later cells alkali phosphatase were stained positively,4 weeks later calcium nodule could be found,all of these data showed that isolated cells were human mesenchymal stem cells.Conclusion Cells were isolated by human lymphocyte separating medium,could harvest more purity of hMSCs,which supply stable separation method for hMSCs. Part Two Effection of hypoxia on biological behavior of human bone marrow mesenchymal stem cellsObjective To establish a model of hypoxia culture and osteoblast differentiation in vitro, and investigate the biological characteristics of cultured adult mesenchymal stem cells(MSCs) in hypoxia. That will be experimental basis of MSCs used as the functional cells in bone tissue engineering.Methods Bone marrow was obtained by aspirated in the posterior superior iliac spine from three adult patients whose marrow is normal. Cells were isolated by gradient centrifugation and grown to confluence.The second passages According to the oxygen concentrations and nutrient mediums, was categorized into four groups: normoxic group ( 20 % O2, DMEM-LG), hypoxic group(1%O2, DMEM-LG), normoxic osteoblast induction group(20%O2 , conditioned medium), hypoxic osteoblast induction group(1%O2, conditioned medium).Proliferations and apoptosis of all the cultured cells were observed by an inverted phase contrast microscope,growth curve,detection of PCNA,apoptosis proteins and Colony forming unit-fibroblast (CFU-F); effection of osteo-differentiation of hMSCs were observed by real-time RT-PCR,activities of alkaline phosphatase and Alizarin red staining;Detected expression of Fibronectin,Laminin and CollagenⅠproteins,was used to view extracellular matrix secretary in low oxygen.Results the cells cultured in hypoxic group proliferated continuously throughout the culture period, while maintaining significantly higher colony-forming unit capabilities than hMSC cultured at normoxic oxygen(P<0.05);In low oxygen groups,apoptosis cells decreased,and anti-apoptosis protein bcl-1 increased(P<0.05). Upon induction, hMSCs of hypoxic group expressed less levels of osteoblast than normoxic osteoblast induction (P<0.01); Immunocytochemistry and Western blot detected,expression of FN and LA proteins increased(P<0.05),no signify -cant difference in expression of CollagenⅠ(P>0.05)Conclusion hMSCs maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSCs niche.chronic hypoxia is a key parameter that infuences the in vitro biological behavior of hMSCs.Part Three Effects of chronic hypoxia on the expression of HIF-1α,SDF-1 and VEGF165 in human bone marrow mesenchymal stem cellsObjective To establish a model of chronic hypoxia in vitro culture , and observe the epression of HIF-1α,SDF-1 and VEGF165 in cultured adult human bone marrow stromal cells(hMSCs) ,That will be experimental basis of MSCs used as the functional cells for hypoxic disease therapy;by comparision of expression of hypoxia-inducible factor-1α(HIF-1α),stomal cell- derived factor (SDF-1) and Vascular Endothelial Growth Factor 165(VEGF165 ) proteins,to document mechanism of hMSCs osteo-differentiation.Methods Bone marrow was obtained by aspirated in the posterior superior iliac spine from three adult patients whose marrow is normal. Cells were isolated by gradient centrifugation and grown to confluence.The second passages According to the oxygen concentrations and nutrient mediums, was categorized into four groups: normoxic group ( 20 % O2, DMEM-LG), hypoxic group(1%O2, DMEM-LG), normoxic osteoblast induction group(20%O2 , conditioned medium), hypoxic osteoblast induction group(1%O2, conditioned medium ) .HIF-1α,SDF-1 and VEGF proteins were measured by immuocytochemistry,Western-blot was used to detect HIF-1αand SDF-1 protein. HIF-1α,SDF-1 and VEGF165 mRNA were detected by real-time RT-PCR.Results cytoplasm of hMSCs of n group and nos group were stained by HIF-1αpositive,and strong positive staining were found in cytoplasm and cell nuclei of hMSCs in h group and hos group. Under chronic hypoxic condition,hMSC expressed mRNA of HIF-1α,SDF-1 and VEGF165 more strongly than that under normal oxygen condition(P<0.05),expression of HIF-1αmRNA in n group and nos group were in low level(P<0.05), Quantity of SDF-1 mRNA and VEGF165 mRNA increased with time,reached the peak maximium value at fourteenth days, then decreaed quickly(P<0.05).Western blot detected that,expression of HIF-1αand SDF-1 proteins in h and hos group were more than proteins in n and nos group(P<0.05),SDF-1 proteins in nos group increased greatly 7 days later(P<0.05),then decresed after 14 days.Conclusions in normoxia condition,SDF-1 and VEGF play a role in osteogenic capacity of hMSCs,although HIF-1 stimulate SDF-1 and VEGF secretion, inhibit function of SDF-1 and VEGF in osteo-differentiatio of hMSCs, perhaps it has another pathway for regulation of SDF-1 and VEGF secretion.