| Bone morphogenetic protein-2 (BMP-2) has been demonstrated to be a potential growth factor in facilitating cell proliferation and differentiation into cells of the osteoblast lineage, and then accelerate bone formation both in vitro and in vivo. BMP-2 combined with many kinds of seed cells and scaffolds has been widely used in bone tissue engineering procedure, aiming to achive bone repair and reconstruction via osteoconduction and osteoinduction. Unfortunately it is hard to maintain BMP-2 to the necessary concentration in situ and revascularization of the transplanted bone remains to be an obstacle for clinical application of tissue engineering bone. According to the gene therapy technique, we transferred the human BMP-2 gene into the bone mesenchymal stem cells (BMSC) and then seeded the gene-modified cells into a free-antigen allogenetic bone scaffold in this study. As we expected, BMP-2 was carried special anatomic site using the MSC as delivery vehicle and stably was secreted. In both ectopic (subcutanously transplanted on the back of nude mice) and orthotopic (radial segmental defecit in rats) sites, the effects of BMSC expressing BMP-2 on bone formation and vascularization were evaluated. Furthermore we transplanted vascularized periosteum and vessel bundle into the BMP-2 gene modified engineered bone graft to repair bone defect to discuss the effect of an additional blood supply on bone hailing, in order to find the best method to bridge bone defect, and to look for an most applicable style of engineered bone repair.Part I: Adenovirus vector carrying hBMP-2 gene (Ad-BMP-2) infected human bone mysenchymal stem cell (hBMSC) and construction of gene therapeutic tissue engineering bone in vitro1. Construction, purification and titration of adenoviral vector Ad-BMP-2The recombinant adenoviral shuttle plasmid pAdCMV-BMP-2 was co-transfected into 293 cells with pJM17 using lipofectamine, and adenoviral vector Ad-BMP-2 was produced by in vivo homologous recombination. Identified by PCR and gene sequencing, construction of adenoviral vectors Ad-BMP-2 were successful achieved. After propagation of 293 cells transfected with the vector and purification of the virus, the titer of the adenovirus detected by CPE assay was 2×1010 pfu/ml.2. Ad-BMP-2 transfacted hBMSC in vitro and detection of BMP-2 expressionhBMSC was cultured in vitro and identified by it's potential to differentiate into multi-lineages like osteoblast and lipid cell et al. then Ad-BMP-2 was transferred into hBMSC and BMP-2 expression inside hBMSC was verified by immunocytochemistry and in situ hybrydization, while Western blot analysis of the cell culture medium of BMP-2 gene transferred hBMSC showed that BMP-2 was expressed in level of mRNA and protein and secreted into the medium by the gene modified cells. The recombinant adenovirus containing E. Coli lacZ gene [Adv- (beta) gal] was transduced by same method and used as control in the study. X-gal immunostaining was tested to assess the transfection efficiency that was higher than 90%. 3. Effects of BMP-2 gene transfaction on hBMSC proliferation, differentiation and VEGF expression in hBMSCFlow cytometer (FCM) examination showed the percentage of S phase cells in BMP-2 gene transferred hBMSC increased, while that of G1 phase cells decreased, which indicated that DNA synthesis and cell proliferation was promoted after gene transfaction. Osteocalcin immunofluorencence, type I collagen immunocytochemistry staining and ALP activity examination showed that BMP-2 gene transfection facilitate hBMSC differentiate into osteoblast. VEGF in situ hybridization, which was assessed by the mean staining gray value showed that BMP-2 gene transfaction up-regulated VEGF expression and then induce vessel regeneration indirectly.4. Construction of BMP-2 gene modified tissue-engineered bone in vitroFree-antigen bovine cancellous bone (BCB) was prepared according to the related references and transplanted into a muscle bag of the quadricept musle in rats, immunological examinatio...
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