| Objective:To investigate the biological characteristics and osteogenic potential of rAAV2-hBMP-2 Gene Modified Marrow Stromal Stem Cells. To investigate the effect of treatment for osteonecrosis of femoral head by rAAV2-hBMP-2 gene modified marrow stromal stem cells.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 6-month old weighing 2.0-2.5Kg. Rabbit BMSCs were isolated by density the cluture media. The cellular histological characteristics and growth aspects were observed under phase contrast microscope, dyeing of AKP and AKP in cell detection ability of bone formation on BMSCs。the osteogenic potential of BMSCs was investigated by ultrastructural observation under transmission electron microscope. rAAV2-hBMP-2 plasmids were extracted and were transfected into rabbit marrow stromal stem cells. hBMP-2 mediated by adeno-associated virus (AAV) transfected rBMSCs. Virus transfection to stay overnight after 80% cell converged. MOI was 105v. g./cell,1:2 dilutus virus about 4ml was added at bottle, continue to culture after changing liquid.The transfection supernatant was harvestd 24h to 6d after transfection, The transcription and expression of hBMP-2 protein expression were investigated by Western blotting.The animal models of femoral head necrosis established in 36 rabbits by delivery of liquid nitrogen into femoral head. Each group had 12 rabbits. A group was control group;B group was BMSCs group; C group was rAAV2-hBMP-2/BMSCs group. When animal models of femoral head necrosis confirmed, the BMSCs were transplanted back to the rabbit under C-arm, The femoral head was inspected by histology postoperative 4-12weeks.Results:In primary culture, fibroblastic cells appeared on the bottom of the flasks when first changing the culture medium on day 3, with a few colonies forming, which were the Colony-forming unit-fibroblasts. During day 5 to day 10, the colonies increased in number and size gradually, forming the larger clonies with the diameter ranging 5mm-8mm and small clonies with the diameter less than 5mm., cytotostatic and increment. Strong reaction for AP and calcified nodules were observed. The expression of hBMP-2 gene could be found distinctly in the transfected rabbit BMSCs and hBMP-2 protein in the supernatants of transfected cell cultures possesses highly biological activity, which could promote rabbit osteoblast increased and augment cytoactive effective. The concentration of BMP-2 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 bone was 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 12 weeks after treatment.Conclusions:Rabbit BMSCs cultured in vitro are fibroblastic cells with characteristics of colonyforming, self-renewal, and double potential of differentiating into adipocyte and osteoblasts. hBMP-2 gene transfected rabbit BMSCs could express hBMP-2 with highly biological activity, which provided the foundation on learning hBMP-2 gene and BMSCs based gene therapy for ONFH repairing and regeneration. Implantation of human BMP-2 gene transfected BMSCs could repair early- stage experimental femoral head necrosis. |
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