| Bone defect is one of the most common diseases in osteology clinic.Autogeneic bone transplantation, xenogeneic bone transplantation as wellas vascular anatomizes bone transplantation are the traditional strategiesused today. All these proposals undergo some disadvantages such as scarcesource, immunologic rejection and disease transmission. In recent years,rapidly developed gene engineering and tissue engineering technology givea promising method to deal with bone defect. It has become a hot researchspot which makes tissue engineering bone used in clinic practice. To construct a tissue engineering bone, seed cells, scaffolds as well ascell factors are the three critical elements. Tissue engineering boneconstructed in vitro is a compound of cells and scaffolds, it has to be nourished by blood and intracellular fluid after implantation. Poor bloodsupply of planting areas or long revascularization would result in nutritiondeficiency of the seed cells grow on the scaffold, which leads to disordersin cell metabolism, proliferation, differentiation and secretion, at last,devitalizing of the cells. Then the transplanted bone would just act as apure bone transduction material and lost the osteogenesis ability. Therefore,fast revascularization after tissue engineering bone transplantation is thepremise for keeping the activity of seed cells. At present, it is in a period ofinitiative exploring. In this study, VEGF 121 gene modified tissue engineering bone wasconstructed to transplant into rabbit models of large segment bone defect,the capabilities of the revascularization and bone defect repairing wasobserved and detected in vivo. This study was composed of five portions described as follows:1. Construction of the scaffold with deproteinated bone and detectionof its biological characters. Metaphysis cancellous were collected from adult New Zealand whiterabbit, the biological derivative scaffold, which was called deproteinatedbone (DPB), was prepared with a method of hydrogen peroxide-diethylether digestion. DPB was undergone a serials of physical and chemicaltests 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 theDPB scaffold maintained a proper dimensional structure that met thecriteria of a tissue engineering bone scaffold.2. Isolation, culture and identification of rabbit osteoblasts. The cranium was isolated from one-week prenatal fetus rabbit byenzyme-digestion combined with tissue culture. The isolated osteoblastswere undergone a primary and passage culture. The viability of theosteoblasts was determined with morphological, cytochemical and enzymetests. The cell morphological character and proliferation ability was noobvious difference between passaged and primary cultured osteoblasts,which were good seed cells for construction of tissue engineering bone.3. Construction of pEGFP/VEGF 121 recombinant plasmid and itsexpression in osteoblasts. VEGF 121 gene was amplified with PCR from human fetus brainlibrary, then, the PCR product was ligated with pEGFP-C1 to construct therecombinant plasmid pEGFP/VEGF121. After confirmed with PCR,enzyme redigestion and DNA sequencing, pEGFP/VEGF121 wastransfected into osteoblasts with lipofectamine. Fluorescent microscopeand ELISA were engaged to detect the expression of EGFP and VEGF,respectively. Thus, the recombinant plasmid was confirmed to express VEGF protein in osteoblasts.4. Construction of the gene modified tis...
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