HVEGF-165 And HBMP-2 Eukaryotic Co-expression Vector And Its Transfection Osteogenic Potential Of Human Bone Marrow Mesenchymal Stem Cells

Cranio-maxillo-facial bone defect is common both in daily life and during the war. 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.In this study, an animal cranium defect model was estabolished to monitor the levels of BMP and VEGF in the wound by immunohistochemistry and in situ hybridization technique. Vector containing combinations of condensed plasmid DNA encoding for hVEGF and hBMP-2, pIRES-hBMP2-hVEGF165, were constructed by molecular cloning and gene transfection technique. The cultured human bone marrow mesenchymal stem cells (hBMSCs) were transfected with cation liposome- mediated transfection method to express the exogenous VEGF and BMP-2. The transfected hBMSCs could improve the bone regeneration and bone healing. It also provided modified seed cells for bone tissue engineering.The expression of VEGF 165, BMP-2, and osteocalcin mRNA were detected in the transfected BMSCs by immunohistochemistry, immunofluorescence and reverse transcription polymerase chain reaction (RT-PRC). The production of type I collagen and the activity of alkaline phosphatase (ALP) were also observed in the transfected BMSCs.We found that VEGF was expressed in the chondrocytes and osteoblasts at the bone defect area 3 days after the occurrence of cranium bone defect, but slightly expressed in the matrix. The expression of VEGF decreased within the first week, increased at the second week, and reached the peak value at the third week. The proliferated chondrocytes arranged in clusters. The expression of VEGF was still expressed during the fourth week, and was positive in individual cells during the fifth week. BMP-2 was firstly expressed in several undifferentiated mesenchymal cells and in the intercellular matrix at the bone defect area 3 days after the occurrence of bone defect. One week later, the osteoblasts, osteocytes and regenerated cartilage matrix, granular matrix in the primary callus expressed BMP-2. The lower expression of BMP-2 appeared 2 weeks later. 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 3 weeks later. The expression of BMP-2 declined 4 weeks later and was only observed in individual cells 5 weeks later. The expression of BMP-4 mRNA was positive in mesenchymal cells at the defect site 1 week later, increased markedly at the third week and...