The Experiment Research About The Promoting Effect Of PIRES-hBMP2-hVEGF165 Transfected BMSCs On The Osteogenesis And Vascularization Of Tissue Engineering Bone

Cranio-maxillo-facial bone defect is common both in daily life and during the war. So many cinical bone defect cases also needed to be reconstructed . Bone Morphogenetic Proteins (BMPs) play a critical role in the differentiation of osteoprogenitor cells and the bone formation process. Vascular Endothelial Growth Factor (VEGF) is the recently discovered cellular growth factor related to the angiogenesis which is important in the wound healing process. VEGF and BMP-2 have synergistic interaction in ossification of the cranium bone defect in different healing stages.In this study, an rabbit canium defect model was established , by the prophase research of vector containing combinations of condensed plasmid DNA encoding for hBMP2 and hVEGF165. The cultured rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with cation liposome-mediated transfection method to express the exogenous VEGF and BMP-2. The transfected BMSCs could improve the bone regeneration. Then we put the bone marrow mesenchymal stem cells (BMSCs) into the absorbtable polylatic acid, all of them were implantded into the bone defect area together,so we can observe the regeneration of the defect bone. The reconstruction of cranium bone defect was detected in the transfected rBMSCs by gross observation , X-ray examination , histological test and immunohistochemistry examination.Results1. The gross morphological alternation The cranium bone defect in the transfected group was replaced with neogenetic bone tissue with the generation of os integumentable 4 weeks later. No obvious osteotylus was found in the control group, however the defect area was encapsuled with granulation tissue. A large amount of lipid bony callus formed in the cranium bone defect in the transfected group 8 weeks later. Partial bone callus could be found merely encircled the cranium bone defect in the control group. The cranium bone defect in the transfected group was completely replaced with neogenetic rigid bone 12 weeks later. The callus encircled the cranium bone defect proliferated in bigger number without the cental converase of the bone in the control group.2. X-ray examinationThe undefined bone callus appeared in the transfected group 2 weeks later, with scattered spot-like density enhancement in the center. The defected margin exposed evidently without the formation of osteotylus in the control group. The callus emerged in cranium bone defect 4 weeks later in the transfected group , lamellar density enhancement could be found in the center. While in the control group the marginal callus increased . The cranium bone defect area in the transfected group vanished basically still with higher bone density than perimeter 12 weeks later. The cranium bone defect area declined without central bone formation in the control group.3. histological alterationConsiderable fibrous osteotylus could be found at cranium bone defect area 2 weeks after the occurence of the bone defect .Mesenchymal stem cells in the medial of the periosteum around the cranium bone defect area proliferated, converting into massive osteoblasts and chondrocytes. Come out with the cartilaginous insula. The chondral tissue regenerated rapidly to fill the interspace of the cranium bone defect area. Fibrous osteotylus , chondral osteotylus and osseous osteotylus mixed together at the bone defect area 4 weeks later, by the majority of fibrous osteotylus and chondral osteotylus together with more neogenetic vascellum. With the appearance of the of intral-chondral ossification, partial chondral ossification formed bridge-grafting could be seen. The osteoblasts , fibroblasts declined . The fibrous osteotylus disappreared replaced by the chondral osteotylus and osseous osteotylus 8 weeks later. The bone construction proned to be perfect at the 12th week and the medullary canal was formed basically.4. The expression of VEGF165In the transfected group, we found that VEGF was positively expressed in the chondrocytes , osteoblasts and matrix at the 2nd week in the bone defect area. The chondrocytes proliferated and arranged in the clusters. The expression of VEGF reached peak value and was hadro-positive 4 weeks later. While the expression of VEGF was postive in the neogenetic chondrocytes and osteoblasts 2 weeks later , only partial reserved positive 4 weeks later after the occurence of the defect in the control group.5.The expression of BMP2In the transfected group, BMP2 was expressed in the osteoblasts, osteocytes and regenerated cartilage matrix, granular matrix in the primary callus at the bone defect area 2 weeks later after the bone defect. Strong expression of BMP-2 was observed in the regenerated chondrocytes and cartilage matrix, junior chondral cells and osteoblasts at the surface of the callus 4 weeks later. While the expression of BMP2 was postive in the neogenetic chondrocytes and osteoblasts 2 weeks later , only partial reserved positive 4 weeks later after the occurence of the defectin the control group.Conclusions1.The constructed pIRES-hBMP2-rVEGF165 can be successfully transfected into BMSCs of rabbits by bangosome. So this way can faciliate the bone ossification in tissue engineering.we can also advance our reserch from vitro study to vivo study primarily with this cation liposome-mediated transfection method. VEGF and BMP-2 have synergistic interaction in ossification. BMP2-VEGF165 can be steadily expressed in the transfected BMSCs both immediately and 4 weeks later.2. The differentiation abilities of BMSCs is enhanced, so to promote the reconstruction of canium bone defect.3. This study can provide an experimental base for rectification enormous bone defect in tissue engineering way.