Effects Of Gene Transfection On The Expression Of Celluer Growth Factor In Mandible Distraction Area

Background: Distraction osteogenes, which is an important endogenous bone tissue engineering, provide an important method for correction the congenital, acquired deformities and defects. Meanwhile,distraction osteogensis have relatively long time, probably lead to some complications and social-mental problems, which remain to be further resolved. On the other hand, the mechanisms of how the distraction mechanical tension convert to biological signal is still unclear to us. Therefore, how to promote new bone formation and regulate the cellular factors during distraction became one of hot topics at present. In the perious study, we have established electroporation-mediated gene therapy animal models of rabbit mandibular distraction osteogenesis successfully, and the studies suggest that the electroporation-mediated gene therapy can promote angiogenesis and calcium deposition during early stage of mandible DO, strengthen the bone mineral density and siffness of new formed bone. Based on those studies, the present study is to further investigate the effect on cytokine expression in the distraction area after electroporation-mediated gene therapy. The aim of this study is to explore the mechanism of it promote new bone formation.Objective: To observe the changes of several of the bone growth factors local expression in the distraction area, explore the mechanism of gene therapy promote new bone formation in mandibular distraction erea, provide experimental data and theoretical basis for solving the complications of mandibular distraction osteogenesis clinically.Methods: Forty-five New-Zeland rabbits were employed, submandible extraoral incisions to osteotomy and place the distaction devices. The osteotomy is completed by inserting and rotation an osteotome to demonstrate separation of the bony segments. The arms of distractors are placed percutaneously outside the oral. The wound closed with interrupted suture respectively. The latency period was 3 days. Activation of the device was commenced after the latency period and proceeds at the rate of 0.8mm per day for 7days. After the completion of activation, the device was maintained in position, and then the rabbits were randomly divided into 5 groups: group A: recombinant plasmid 2μg(0.1μg/μl)pIRES-hVEGF165-hBMP2,was injected into the distraction area, after the completion of activation; group B: recombinant plasmid pIRES-hBMP2 was injected into the distraction area ; group C: recombinant plasmid pIRES-hVEGF165 was injected into the distraction area; group D: pIRES was injected into the distraction area, and group E: normal saline (NS) group NS was injected into the distraction area. After injection every group employed electroporation. Each group will be killed by air embolism at 7th, 14th and 28th of consolidation stage, the tissue of the distraction area were harvested for histological examination and immunohistochemical staining detection of BMP, VEGF, FGF, TGF-β, CyclinsA, D1, E expression respectively. The data were analyzed by image analysis system.Results: It was shown that BMP, VEGF, FGF, TGF-β, CyclinsA, D1. E mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues arrounding the new bone. These growth factor reach to the peak at 7th day, and weakened at 14th day of of consolidation stage,at 28th day, some of those growth factors expressed weakly or negatively. It was shown by image analysis that the expression of growth factor in group A, B and C, which subjected to gene therapy, were remarkable higher than those in the control group D and E at the same time.Conclusion: The electroporation-mediated gene therapy can promote multiple cytokines expression effectively, which plays an important role in angiogenesis, cell differentiation and proliferation during distraction osteogenesis. Theses growth factors stimulate extracellular matrix synthesis, induce the proliferation and differentiation of fibroblasts, osteoblasts, vascular endothelial cells and osteocyte, promote angiogenesis effectively during early distraction stage, which promote the new bone formation and repair . This is the probably molecular mechanism of the gene therapy promoting new bone formation in distraction gap.