| Avascular necrosis of femoral head (AVNFH) is a kittle disease that usually leads to destruction of the hip joint and increasing musculoskeletal morbidity in young and middle aged patients in whom total hip replacement is best avoided because of an increased risk of revision and more troubles after revision. Thus, it's significant to delay or decrease the need for hip replacement by treating this condition with a method that preserves the femoral head before collapse. Recently, many references were involved in VEGF gene transfer to promote bone healing or facilitate the repair of bone defect and most of the results were positive. No matter in the case of bone defect or fracture, however, the blood supply around them is almost normal. Experiment indicated there was obviously increasing blood supply in the VEGF transfected ischemic muscles and the transfection efficiency was sixfold higher in ischemic muscles than in nonischemic control muscles. So elicited form it, we want to know how could be the effect of VEGF gene transfer therapying osteonecrosis? Till now there is seldom pubished article that reported the effect of VEGF on enhancing the repair of osteonecrosis. DPB possesses natural network pore system that is advantageous for capillary vessels and mesenchymal cells immigration which induces osteogenesis. Deproteinized bone (DPB), as it has stronger mechanical intensity compared with soft implant, is relatively ideal bone repair material. In present, there is no any report involved in using DPB as vector combining with VEGF plasmid to therapy AVNFH. We implanted DPB-VEGF compound in the necrotic femoral head hoping to induce angiogenesis and promote the repair process of the necrotic femoral head. This study was composed of four portions described as follows: 1. Construction of the scaffold with deproteinized bone and detection of its physical and chemical characters Metaphysis cancellous were collected from adult New Zealand white rabbit, the biological derivative scaffold, which was called deproteinated bone (DPB), was prepared with a method of hydrogen peroxide-diethyl ether digestion. DPB was undergone a serials of physical and chemical tests to explore its porus diameter, porosity, transconnection as well as the pattern of the internal surface of the pori. The protein and lipid were almostremoved, which eliminated immunogenicity. It was confirmed that the DPB scaffold maintained a proper dimensional structure that met the criteria of a tissue engineering bone scaffold. 2. Construction of the model of early AVNFH of rabbit New Zealand rabbits were distributed into three groups randomly. Horse serum and dexamethasone sodium phosphate were used to induce osteonecrosis models in group A, dexamethasone sodium phosphate only in group B and saline in group C. Test and analyze the values of serum lipid, AST and ALT of all rabbits. Rabbits from all groups were observed by MRI to prove osteonecrosis, sacrificed to observe the structure of femoral head through HE staining. The Results showed that typical avascular necrosis of femoral head happened in the first group. The values of serum lipid, AST and ALT are significantly different from other groups of which rabbits did not develop osteonecrosis. Horse serum combining dexamethasone sodium phosphate can induce rabbit osteonecrosis model successfully. Vasculitis, abnormal serum lipid metabolism and liver functional lesion result in avascular necrosis of femoral head together. High dose steroid can not induce osteonecrosis in short time. 3. The Reconstruction of pcDNA3.1/VEGF165 and biological activity identification of it Obtained the target gene segment after restriction endonuclease digesting the pUC18/VEGF165 containing the whole length of humanVEGF165 cDNA. Then cloned the segment into pcDNA3.1(+) to turn into reconstructed plasmid-pcDNA3.1/VEGF165 which was identified by restriction endonuclease, PCR and sequencing analysis. ELISA detected the biological activity of hVEGF165 protein secreted by ECV304 72h after transfection of reconstructed plasmid... |
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