| Background: Bone marrow stromal stem cells ( bone marrow stromal cell,BMSC ) is a class derived from bone marrow, have the ability of self-renewal and multiple differentiation potential of pluripotent stem cells. Under the specific culture conditions, can differentiate into osteoblasts, chondrocytes, endothelial cells and mesenchymal cells, and has a wide range of sources, Easy isolation and culture characteristiced. As tissue engineering the most ideal seed cells. In recent years onischemic necrosis of femoral head, bone tissue engineering research shows, bonemarrow mesenchymal stem cells not only acts as a bone cell production sources, but also can be used as gene therapy technology suitable cell carriered.Avascular necrosis of the femoral head restoration and reconstruction is a common orthopaedic problems, In retained the treatment of femoral head, one must complete removal the necrotic bone and provide condition for femoral head restoration.While the lesions following removal of how to more effectively so that the area of bone defect bone regeneration and revascularization clinical need, can effectively promote the new repair method of the femoral head. Vascular endothelial growth factor ( vascular endothelial growth factor,VEGF ) is one of the key angiogenic factor, and occurs in bone tissue development and regeneration, plays an important regulatory role. However, direct application of the VEGF-165 in avascular necrosis of the femoral head is easy to be in the tissues surrounding the enzymes to degrade, short action time. Gene therapy can overcome this shortcome. In recent years, the application of VEGF-165in gene therapy of bone defect and other orthopedic diseases become a hotspot at home and abroad, and has started clinical trialed. Therefore, application of VEGF enhances the osteogenic capacity of avascular necrosis of the femoral head in clinical therapy is expected to become the new way.This study is intended to rabbit BMSCs cells in vitro model, in vitro culture,constructing adeno-associated virus ( Recombinant Adeno-Associated Virus,rAAV hVEGF-165) mediated transfection of rabbit BMSCs complexes, arthroscopic inferior explants, discusses the repair in rabbits with avascular necrosis of the femoral head avascular necrosis of the femoral head, for gene therapy for experimental foundation.Avascular necrosis of the femoral head cartilage defects has been preserved head in treatment of delayed femoral head collapse is a serious problem, Bio-gide collagen membranes loaded with bone marrow mesenchymal stem cells in repairing cartilage defect test study for this problem and provide theoretical supportObjective: The experimental study on rabbit bone marrow mesenchymal stem cell isolation, culture and identification method and to discuss the bone marrow stromal stem cells into osteoblasts and chondrocytes transforming characteristics,detection of bone marrow stromal stem cells into osteoblasts and chondrogenesis,detection of rAAV-2-hVEGF-165 transfection of bone marrow mesenchymal stem cells after transfection of gene transfection efficiency and expression,study on the avascular necrosis of the femoral head bone and cartilage injury and repair of seed cells in tissue engineering feasibility and bone marrow stromal cells and Bio-gide composite culture,detection of bone marrow stromal cells in Bio-gide biofilm growth,discusses the Bio-gide collagen membrane as carrier of bone marrow stromal cell growth advantage,to study of core decompression with arthroscopic hVEGF gene transfected bone marrow mesenchymal stem cells in treatment of ischemic necrosis of femoral head in the role and valued.Metheds: Under aseptic conditions,bone marrow was obtained by double external condyle of femur and proximate of tibia aspiration from an adult male New Zealand white rabbit 4-month old weighing 2.5-3.OKg under chloral hydrate anesthesia (1.5ml/Kg). Using density gradient centrifugation method for the isolation of rabbit bone marrow mesenchymal stem cells and a large number of amplification,joined the 8ml DMEM - low sugar medium ( containing 10% fetal bovine serum )were cultured cells were observed by inverted microscope, growth and morphology of hematoxylin - eosin, cell specimens (HE) staining in the cell,80% tol.: 3generations,MTT mapping,2,3,4,first,5 generation of cell growth, cell immunofluorescence identification of rabbit bone marrow stromal stem cells, In vitro induction of bone marrow stromal stem cells into osteoblasts and chondrocytes differentiation, and detection of osteogenesis and chondrogenesis.The cellular histological characteristics and growth aspects were observed under phase contrast microscope.The osteogenic potential of MSCs was investigated by ultrastructural observation under transmission electron microscope. rAAV-2-hVEGF-165 plasmids were extracted and weretransfected into rabbit marrow stromal stem cells. hVEGF-165 mediated by adeno-associated virus(AAV) transfected rMSCs. To investigate transfection efficiency with enhanced green fluorescent protein under fluorescence microscope.hVEGF-165 mediated by adeno-associated virus(AAV) transfected rMSCs. Virustransfection to stay overnight after 90% cell converged. MOI was 105, 1:3 dilutusvirus about 4ml was added at bottle, continue to culture after changing liquid . Thetransfection supernatant was harvestd 24h to 6d after transfection. The transcription and expression of hVEGF-165 protein expression were investigated by RT-PCR and Western blotting. Methods Established the animal models of femoral head necrosis in 144 rabbits by delivery of liquid nitrogen into femoral head. Each group had 36 rabbits.A group was control group;B group was decompression with MSCs group; C group was decompression with rAAV-2-hVEGF-165/MSCs group. necrosis bone was cleared out,then MSCs was implanted on arthroscope; D group was decompression with rAAV-2-hVEGF-165/MSCs/Bio-gide collagen composited roup was inspected by histology postoperative 2, 4,8 weeks.Results: Using density gradient centrifugation combined with adherence screening method can obtain high purity and high activity of bone marrow stromal stem cells, in the MTT testing found that the third generation of cell proliferation rate was first,2,4,5 generation slightly faster, logarithmic phase duration is relatively long,Immunofluorescence staining showed that high expression of CD44, CD90 positive,CD34 negative, the obtained cells are fibroblasts, hematopoietic cells and endothelial cells; Bone marrow stromal stem cells under certain conditions can to convert osteoblasts and chondrocyteed, Intracellular alkaline phosphatase content determination, calcium nodule staining, alkaline phosphatase staining, type I collagen immunohistochemical staining showed osteoblast biological characteristics, toluidine blue staining was found to see each generation of cartilage cells after passage of coloring, dyeing becomes shallow gradually; Type II collagen immunohistochemical staining seen within 5 generations of chondrocytes within brown granules of precipitationed.The expression of hVEGF-165 gene could be found distinctly in the transfected rabbit MSCs and hVEGF-165 protein in the supernatants of transfected cell cultures possesses highly biological activity,which could promote rabbit osteoblast increased and augment cytoactive effective. The transfection efficiency of adeno-associated virus(AAV) transfected rMSCs was 70%. And rAAV-2-hVEGF-165 transfected rMSCs can be achieved effective expression by RT-PCR and Western bloting. hVEGF-165 can be fined after transfected 48 hours and achieve hingest after 10 days. The concentration of hVEGF-165 in femoral head of C group was higher than that of A group at different time point. Empty lacunae and fibrotic marrow were demonstrated before implantation. New bonewas evident in the implantation field of C group while fibrous tissues were evident in that of B group at 4th week after treatment. Articular surface collapse were observed in the X ray photograph of untreated group. Regular shape and normal density of femoral head in C group compared with the irregular shape and low density of femoral head in B group could be demonstrated in the X- ray photograph 8 weeks after treatment. The compressive strength of the hVEGF-165 gene group higher than that of A group significantly.Conclusions: BMSCs can be used as avascular necrosis of the femoral head cartilage defects repair with tissue engineering seed cell source. In vitro under certain conditions of bone marrow stromal stem cells into chondrocytes transforming potential with. HVEGF-165 gene transfection of rabbit bone marrow mesenchymal stem cells can be effective expression of biologically active hVEGF-165 protein,For the hVEGF-165gene and bone marrow mesenchymal stem cell-based gene therapy in the treatment of avascular necrosis of the femoral head restoration and reconstruction in the application to provide experimental basised. BMSCs and Bio-gide collagen membranes in vitro co-culture, line morphology visible on BMSCs in collagen membrane surface compound layer growth, higher density, and com., growth in good condition obvious matrix secretioned, showed Bio-gide collagen membrane as a cell carrier for BMSCs scaffold for cartilage tissue engineering research, Through the arthroscope can complete removal of dead bone, so that hVEGF-165 gene modified bone marrow mesenchymal stem cells transplanted accurate into back bone necrosis.Can be for avascular necrosis of the femoral head restoration reinforcing, avascularnecrosis of the femoral head in the treatment of great significanced. Implanted into rabbit femoral head hVEGF-165 gene can effectively express. HVEGF-165 gene therapy in a rabbit model of avascular necrosis of femoral head after gene therapy group on compressive strength was stronger than the pure BMSCs treatment groupand blank control group, normal control group and close femoral compressive strength. |
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