Osteogenic Ability Of BMP-2 And VEGF Gene Co-modified Bone Marrow Stromal Cells Under Hypoxia

OBJECTIVE:BMP2 has the strongest osteogenetic ability. VEGF can promote the angiogenesis, simultaneously can increase the expression of BMP2. This study is aimed to investigate the effects of Ad-hBMP-2 and VEGF transfection on the osteogenetic ability of BMSC in vitro under hypoxia.METHODS:1.The rat BMSCs 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.Examine the biological characteristics of BMSC after hBMP-2 and VEGF were transfected according to cell morphological survey and cell growth curve. Determine the expression of BMP-2 and VEGF by Elisa. After the Ad-GFP was transfected into BMSC, the adenovirus infection rate was determined under fluorescent microscopy, and the green fluorescent protein was examined in the subcultured BMSC.3.The BMSCs were divided into four groups.(1)BMSC group;(2) Ad-hBMP-2+BMSCgroup;(3)VEGF+BMSCgroup;(4)Ad-hBMP-2+VEGF+BMSC group. Four groups were cultured under hypoxia 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 BMSCs were divided into four groups.(1)BMSC group;(2) Ad-hBMP-2+BMSCgroup;(3)VEGF+BMSCgroup;(4)Ad-hBMP-2+VEGF+BMSC group. Four groups were cultured under hypoxia 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 day' 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 proliferate rapidly and cell colonies were formed in about 5 to 6 days.The shape of cells became unity subculture.2.hBMP-2 and VEGF were transfected into BMSC in vitro. The green fluorescent protein was detected at 12 hours after the transfection and the average transfection rate was about 90%. We also detected the green fluorescent protein in the fifth subcultured cells.3.The result of the proliferation of the four groups of BMSC:No significant proliferation difference was observed among these groups in the first 2 days (P>0.05). The cells of each group proliferation rapidly after 3 days. The Ad-hBMP-2 transfected groups have stronger proliferation ability than other groups(P<0.01);there was no significant proliferation difference between Ad-VEGF groups than no transfected group (P>0.05).4. The osteogenetic ability of hBMP-2 and VEGF transfected BMSC in the hypoxia control system.1)The ALP activity among four groups:The ALP activity of Ad-hBMP-2+VEGF+BMSCgroup was stronger than other groups (P<0.05). The Ad-hBMP-2+BMSC group was stronger than BMSC group and VEGF+BMSC group (P<0.05). The BMSC group and VEGF+BMSC group have no significant difference. (P>0.05)2) The mineralization nodules:the Ad-hBMP-2+VEGF+BMSC group has the most mineralization nodules. The Ad-hBMP-2+BMSC group and VEGF+BMSC group are almost the same, more than the BMSC group. The BMSC group has the least mineralization nodules. CONCLUSION:BMP-2 can promote the osteogenetic ability in 7 days' observation.VEGF has no significant effects on the osteogenetic ability of BMSC.The osteogenetic ability can be obviously increased due to the combined action of BMP-2 and VEGF.