| Objective:1. To observe the osteoblastic proliferation, differentiation and osteogenetic phenotype of rat bone marrow stromal cells (MSCs) in culture medium.2. To construct recombinant adenovirus vector carrying the vascular endothelial growth factor 165 (VEGF165),and to observe the change of growth of MSCs transfected by Ad-VEGF165 and to assay expression of VEGF165 of MSCs.3.To investigate the expression of VEGF165 and the proliferation of MSCs into type I collagen.4.To investigate the effect of compound pattern of ceramic bovine bone (CBB) and collagen on the attachment and proliferation of cells.5.To investigate using CBB / collagen as extracellular matrix for the MSCs to repair bone defects.6.To investigate the osteogenic and angiogenic characteristic in vivo of rat MSCs with Ad-VEGF165 combined onto CBB and collagen. Methods:1.Rat MSCs were cultured in Dulbecco's modified eagle medium (DMEM) with supplement. The cells proliferation capability and osteogenic property were examined by phase-contrast microscope, transmission electron microscope, histochemistry staining and ALP and Von kossa. 2. pAdTrack-VEGF165 was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.pAdEasy-VEGF165 transfected into 293T cells to package the adenovirus, followed by identification by means of EGFP. Ad-VEGF165 transfected into MSCs, VEGF165 was determined by EGFP, RT-PCR and ELISA.3.MSCs transfected by Ad-VEGF165 were mixed with type I collagen.4.Rat MSCs were cultured in DMEM with supplement. Thesurface of CBB was covered with type I collagen. Rat osteoblasts were compounded to CBB/collagen, the attachment and morphology of osteoblasts were observed by SEM, ALP and histochemistry. 5. Cranial defects with 8mm in diameter were painted with CBB/collagen and MSCs, new bone regeneration in the defects was investigated by histological analysis.6.MSCs transfected by Ad-VEGF165 and obsteoblasts were mixed and loaded onto CBB /collagen, and cells-scaffold compound was implanted into the corresponding rat. Osteogenic and angiogenic characteristic in vivo were investigated by histochemistry. Results:1 .The formation of mineralized nodules, synthesis of collagen and expression of osteocalcin and ALP activities of cells in medium with supplement were superior to those of in medium without additive.2.Ad-VEGF165 constructed successfully, which was detected by restriction enzyme digestion, gene sequence andEGFP. MSCs transfected by Ad-VEGF165, the expression of VEGF165 was confirmed by RT-PCR, immunohistochemical staining and ELISA.3.MSCs could be extended into type I collagen. MSCs in transfection group could express VEGF 165.4.The cells could grow on the surface of CBB, collagen fiber bundles and mineralized nodules were observed in the experimental group.5.The skull defects were covered with regenerated new bones and fibrous tissues.6.Histological study demonstrated that cell-scaffold compound of experimental group had an ability of heterotopic osteogenesis. There were blood vessel around the osseous island. Conclusion:1. Rat MSCs had a strong activity of proliferation capability and osteogenic property. MSCs could be used as seed cells in the bone tissue engineering.2.Ad-VEGF165 successfully constructed, VEGF 165 could be expressed in MSCs, which may offer the possibility of a novel approach to local gene therapy of bone tissue engineering. 3. Type I collagen was a good scaffold of tissue engineering, which could offer a novel approach to observethe expression of exogenous gene.4. CBB had a good biocompatibility with the osteoblasts. The collagen surface coating was beneficial to osteoblasts attachment and its osteogenic phenotype.5.Transplantation of MSCs with CBB/collagen could treat skeletal defects and would be a potential technique to regenerate bone defects.6.VEGF165 could improve vessels growth and accelerate osteogenic of tissue engineering bone. The experiment was an interesting research of vascular tissue engineering bone.
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