Comparative Study Of In Vivo And In Vitro On Osteogenic Effect Of BMSCs Transfected By Adenovirus-BMP-2-HIF-1α^mu And By BMP-2Single Gene

ObjectiveTo compare in vivo and in vitro osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) tansfected by asenovirus-bone morphogenetic protein2-internal ribsome entry site-hypoxia inducible factor1αmu (Ad-BMP-2-IRES-HIF-1αmu) and by Ad-cytomegalovirus(CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein1(hrGFP-1), respectively. optimize the source of osteoblasts.Methods1. BMSCs were separated and cultured from1-month-old New Zealand white rabbit. The BMSCs at passage3were transfected by virus.2. Experiment group In vitro: Group A: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu Group B: BMSCs were transfected by Ad-CMV-BMP-2-IRES-hrGFP-1Group C: BMSCs were transfected by Ad-CMV-IRES-hrGFP-1Group D: BMSCs were not transfected3. The optimum multiplicity of infection (MOI)(50,100,150, and200) was calculated and then the cells were transfected by the optimum MOI, respectively.4. Index detection of In vitro:the expressions of BMP-2protein and HIF-1α protein were detected by Western blot method. the expressions of BMP-2gene and HIF-1α gene were detected by RT-PCR. The osteogenic differentiation potential was detected by alkal ine phosphatase (ALP) activity5. Experiment group of In vivo:A-W-MGC/CS was utilized as support material in bone defect rabbit model. This experiment was divided into4groups:Group a:A-W-MGC/CS+Ad-BMP-2-IRES-HIF-1amuGroup b:A-W-MGC/CS+Ad-CMV-BMP-2-IRES-hrGFP-1Group c:A-W-MGC/CSGroup d:adopted rabbit bone6. Index detection of in vivo:After12weeks, The osteogenic differentiation potential in vivo were detected by histological examination, angiogenesis effection were detected by immunohistochemistry (IHC) of CD34protein.Results1. The optimum MOI of groups A, B, and C was200,150, and100, respectively.2. Both Western blot and RT-PCR showed that the expression level of group A, B was obviously higher than that of group C, D (P<0.05), and group A was higher than group B (P<0.05). The expression of HIF-1α protein in group A was significantly higher than those in the other3groups (P<0.05), no significant difference was found among the other3groups (P>0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P <0.05), group A was higher than group B (P<0.05).3. Histology displayed the degradation of carrier, the maturity of new bone and formation of bone trabecula in group a and d. Group b showed the connection of defected ends and incomplete degradation of support material. Group c revealed bad connection of defected end and padding of fibrillar connective tissue. Large number of CD34positive capillaries was observed in group a and d by IHC, and the microvessel density (MVD) value was obviously higher than that of group c and d (P<0.05). Conclusions1. The expression of BMP-2in vivo and in vitro were enhanced by H IF-1αmu, The formation of microvascular in defect parts of in vivo were en hanced by HIF-1αmu.2. BMSCs are good source of osteoblasts after transfected by Ad-BM P-2-IRES-HIF-1αmu; the tissue engieered bone constructed by Ad-BMP-2-IR ES-HIF-1αmu in combination with A-W-MGC/CS has favorable tissue bioacti vity that can be used to repaire large segmental bone defect and the effe ct of reparing is marked by comparison with the one only transfected by BMP-2single gene.