The Construction Of Nets N-HA/PA66/ Osteoblast/vegf Tissue Engineering Bone

Background: The repair of large bone defect is a tough question, given the aging of the population structure in our society appeared, it will face a greater challenge. Vascularization is recognized to be one of the key factors in the bone repair process , and Vascular endothelial growth factor (VEGF) in the fracture areas is the most important bioactive molecules. Gene therapy has arisen as a novel and effective method of delivering therapeutic proteins at a site of desired bone regeneration. Gene therapy has shown tremendous potential in preclinical studies of fracture healing, but to date, no clinical trials have occurred. The delivery of therapeutic proteins VEGF to the local tissues or cells has great application prospect in the healing of bone defects caused by trauma and delayed or nonunion of fractures.Objective: Isolation,culture and identification of rabbit osteoblasts. The optimization of transfection conditions of recombinant plasmid pEGFP/hVEGF165 into rabbit osteoblast in vitro . The construction of nets n - HA/PA66 / osteoblast/VEGF tissue engineering bone .Materials and methods: Using tissue mass law culture method , rabbit osteoblasts was separated, extracted, cultivated . Using calcium cobalt dyeing method, rabbit osteoblasts was appraised. Using liposome law, exogenous gene VEGF was transfected into osteoblasts.Applying different amounts and ratio of plasmid and liposome, in the 24 well plate, recombinant plasmid pEGFP/hVEGF165 was transfected into rabbit osteoblasts by liposome .After transfection for 48h,transfention ratio was detected by fluorecent microscopy.Through the body precipitation planting technology , the seed cell was planted in the nets n - HA/PA66 scaffold materials . Using the cell count method seed cells in scaffold materials was observeed about the proliferation, the expression level and express time was detected by ELISA.Results: The rabbit osteoblast was extracted successfully,and alkaline phosphatase dyeing positive; It is confirmed that liposomes and plasmid can affect transfecting efficiency, and that the interaction between them also has a significant impact on transfecting rate. and under the conditions of plasmid liposome 2.5ul and plasmid 1.2ug,there had a highest transfection efficiency. A new tissue engineering bone was successfully constructed. It has been discovered that the tissue engineering bone can continuously express for nine days, and that the express peak was on the third day .Conclusions: A new tissue engineering bone was successfully constructed, the net n - HA/PA66 / the osteoblast/VEGF bone tissue engineering has a further research prospect.