Osteogenic Character Of Autologous Bone Marrow Stromal Cells Cocultured Under Hypoxia With Endothelial Cells Induced By Bone Marrow Stromal Cells

OBJECTIVE:To investigate the influence of hypoxia and co-culture of endothelial cells(EC) induced by BMSC and autologous bone marrow stromal cells(BMSC) on proliferation and osteogenetic ability of BMSC.METHODS:1.The rabbit BMSC were isolated by centrifugation in Percoll solution(1.077g/ml) followed by gradient centrifuge method.Then they were cultured in basic culture medium in culture flasks.2.The BMSC were induced into endothelial cells using endothelial cell medium. Then induced endothelial cells were identified by CD34 immunocytochemistry.3.The BMSC and induced endothelial cells were divided into four groups.(1) BMSC group cultured under normoxia;(2) BMSC group cultured under hypoxia; (3) BMSC+EC group cocultured under normoxia;(4) BMSC+EC group cocultured under hypoxia.Four groups were cultured for 7 days in vitro and the proliferation was detected by MTT assay once a day.Then the growth curves were drawn according to OD values.4.The BMSC and induced endothelial cells were divided into four groups.(1) BMSC group cultured in osteogenic medium under normoxia;(2) BMSC group cultured in osteogenic medium under hypoxia;(3) BMSC+EC group cocultured in osteogenic medium under normoxia;(4) BMSC+EC group cocultured in osteogenic medium under hypoxia.Four groups were cultured for 6 days in vitro and alkaline phosphatase(ALP) activity was detected by PNPP assay once a day. Then the ALP activity curves were drawn according to OD values.Mineralization nodules of four groups were dyed by alizarin monosulfonate after 7 days' cultivation.RESULTS:1.A small percentage of isolated cells adhered to the flasks and grew as fusiform and polygon shape in 3 days;Then BMSC proliferated rapidly and cell colonies were formed in about 5 to 6 days.The shape of cells was more unity after passage.2.Endothelial cells induced by BMSC adhered to the flasks in 3 days;Then cells grew as fusiform shape and cell colony were formed in about 5 to 6 days.The cells grew larger in polygon shape after passage and showed positive staining in the CD34 immunocytochemistry test.3.The study of cells' proliferation among four groups:No significant proliferation difference was observed among these groups in the 1 st and 2nd day(P＞0.05).The cells of each goup proliferated rapidly in 3 days.The proliferative ability of groups cultured under hypoxia were stronger than that of groups cultured under normoxia(P＜0.01).But there was no significant proliferation difference between BMSC groups and BMSC+EC group under the same cultured condition(P＞0.05).4.The study of ALP activity among four groups:The ALP activity of BMSC+EC group cocultured in osteogenic medium under hypoxia was the strongest in four groups(P＜0.05).The ALP activity of BMSC group cultured in osteogenic medium under hypoxia was stronger than that of BMSC group cultured in osteogenic medium under normoxia(P＜0.05).The ALP activity of BMSC+EC group cocultured in osteogenic medium under normoxia was stronger than that of BMSC group cultured in osteogenic medium under normoxia(P＜0.05).The mineralization nodules were formed only in BMSC+EC group cocultured in osteogenic medium under hypoxia and their dyeings were positive.There were no mineralization nodules formed in other groups.CONCLUSION: 1.The rabbit BMSC can be isolated by gradient centrifuge method and proliferates rapidly when cultured in vitro.The BMSC can be induced into endothelial cells in endothelial cell medium.2.The hypoxia can increase the proliferated ability of BMSC.The endothelial cells induced by BMSC can not increase the proliferated ability of BMSC.3.Both hypoxia and endothelial cells induced by BMSC can increase the osteogenetic ability of BMSC cutured under osteogenic medium.Autologous BMSC cocultured under hypoxia with endothelial cells induced by BMSC can significantly increase the proliferated ability of BMSC.