Experimental Study For The Treatment Of Osteonecrosis Of The Femoral Head With Bone Marrow Mesenchymal Stem Cells Modified By HCGRPαGene Combined With Autologous Bone Grafting

Objective(1) Construt recombinant retrovirus vector carrying human calcitonin gene-related peptideα(hCGRPα) gene.(2) To investigate the proliferation and osteogenic potential of hCGRPa-producing BMSCs(BMSCs/pLNCX2-hCGRPα) after virus infection.(3) The femoral-head osteonecrosis models on one side were established by liquid nitrogen. To investigate the treatment of osteonecrosis of the femoral head with BMSCs modified by hCGRPαgene combined with autologous bone graftingMethods1. Constrution of recombinant retrovirus carrying hCGRPαgene.(1) Synthetic hCGRPαgene:cDNA coding sequence in GenBank hCGRPα enzyme sites in the 5' end and 3' ends, respectively, together with HindⅢand EcoRⅤ, synthetic hCGRPa of DNA fragments.(2) Constrution of recombinant retrovirus vector carrying hCGRPa gene: pES-hCGRPa plasmidand pLNCX2 vector were double digested by EcoRⅠand HindⅢrestriction enzymes. hCGRPa and pLNCX2 cDNA fragments were collected by 1% agarous gel electrophoresis. After purification, hCGRPa and pLNCX2 fragments were mixed and T4 DNA Ligase was added into the mixture. The sequence of recombinant DNA was confirmed by DNA sequencing with universal primer pairs designed according to the multiple cloning sites of pLNCX2. The sequences were compared with GenBank database.(3) Virus packaging of pLNCX2-hCGRPa:The resuscitated PT67 cells were cultured in complete culture medium. Recombinant retroviral vector pLNCX2-hCGRPa transfected into PT67 cells packaging, collection of virus solution, centrifugation and filtration, the virus titer determination.2. Cytological experiments(1) Isolation and culture of BMSCs:Rabbits were sacrificed by air embolism. Then, rabbits were removed into clean bench and the long bones were isolated from animals under sterilized condition. Epiphyses were cut off, and the marrow was flushed out using serum-free DMEM. Ficoll lymphocyte separation medium gradient centrifugation for cell separation, culture, get from the body of the original generation of bone marrow stem cells.(2) Virus infection of BMSCs:The frozen retroviruses of pLNCX2-hCGRPa and pLNCX2 empty vector were thawed and were added onto BMSCs. In addition,8 mg/L Polybrene was applied. Forty-eight hours later, medium was removed and fresh culture medium supplemented with 1000mg/L G418 was added. After two weeks of continuous screening, G418-resistant clones were selected and cultured. Cells were divided into three groups:①Normal control group:normal culture of BMSCs, do not give special treatment.②Experimental group:infected by pLNCX2-hCGRPa BMSCs group.③Empty vector group:infected by pLNCX2. (3) Total RNA was extracted from passage 3 BMSCs/pLNCX2-hCGRPα, BMSCs/pLNCX2 or BMSCs by Trizol.β-actin was used as control. The mRNA expression of hCGRPαwas determined by RT-PCR; The protein expression of hCGRPαwas determined by western blotting.(4) Evaluation of cell proliferation by MTT; evaluating ALP,Collagen,LN and BGP by detection kit.3. Animal experiments(1) grouping experiment:take the New Zealand species of rabbit 74,2 to 3 months of age, body weight 2.7±0.5kg/only either male or female. The application of liquid nitrogen freezing randomly produced rabbit unilateral femoral head necrosis. 2 killed, radiological and histomorphological examination model after 8 weeks, were randomly selected. The remaining 72 were randomly divided into three groups of 24 experimental animals.①group A:Transgenic the BMSCs joint autologous support bone transplantation. Surgical exposure of femoral head, femoral trochanter chisel to take a small cortical bone, bone column, about 4mm long,2mm wide. Other small particles to the deep excavation of the number of group cancellous bone surgery standby. In the back of the neck femoral head cartilage edge of the bottom opening of a small bone window, the excavation of femoral internal necrotic bone. Preoperative alternate rabbit genetically modified BMSCs (containing small particles of pLNCX2-hCGRPαplasmid) and cancellous bone mixed planting at the bottom of the femoral head and the head surrounding. Followed by the central position of the femoral head support implanted in cortical bone column, the top and the cartilage surface of the top tight prison, remote card.②group B:normal BMSCs with autologous support the bone graft. The surgery as the same way as group A, the stem cells implanted were normal BMSCs (excluding pLNCX2-hCGRPαplasmid).③C Group:simply supported autologous bone graft. The surgery as the same way as group A, the bone implanted was not mixed with stem cells.(2) Evaluation of treatment:at the end of three months after bone graft surgery animals were sacrificed and the effectiveness of the therapy was evaluated by HE staining,Biomechanic.Results(1) pLNCX2-hCGRPαretrovirus suspension was analyzed by DNA sequencing. The sequence was in aggrement with that in GenBank database. The recombinant retrovirus was transfected into NIH3T3 cells and the titer was measured. The titer of recombinant retrovirus with hCGRPαgene was 1.7x106pfu/mL and the retrovirus was effectively transfected into NIH3T3 cells.(2) BMSCs/pLNCX2-hCGRPαcells could express hCGRPαmRNA Determinated by RT-PCR, and could express hCGRPαprotein Determinated by western blotting.(3) Compared with normal cultured BMSCs and BMSCs transfected with empty vector (BMSCs/pLNCX2 cells), hCGRPα-modified BMSCs (BMSCs/pLNCX2-hCGRPαcells) showed significantly increased proliferation (P<0.05). Three days after subculture, cells entered the logarithmic growth period and the proliferation rate of BMSCs/pLNCX2-hCGRPαcells was obviously increased. The morphology of BMSCs/pLNCX2-hCGRPαcells was not altered compared with that of control. Nevertheless, no significant difference was found in the proliferation rate between normal BMSCs and BMSCs/pLNCX2 cells (P>0.05). These results revealed that elevated hCGRPαexpression promoted the proliferation of BMSCs. Compared with that in BMSCs and BMSCs/pLNCX2 cells, the concentration of ALP,Collagen,LN and BGP was significantly elevated in BMSCs/pLNCX2-hCGRPαcells (P<0.05). However, no statistical difference was observed in the ALP,Collagen,LN and BGP value between BMSCs and BMSCs/pLNCX2 cells (P>0.05). Therefore, elevated hCGRPαexpression enhanced the osteogenic potential and differentiation of BMSCs.(4) Modeling freezing in liquid nitrogen for 8 weeks, histological observation to see the side of the femoral head within the organization of animal models, a large number of empty lacunae, and trabecular bone thinning, the organization sparse, disorganized. Part of the trabecular bone fracture, reduced bone marrow cavity of hematopoietic tissue, and bone cell degeneration and death of the performance of typical femoral head necrosis.3 months after treatment:group A new bone trabeculae basic rules, the trabecular bone of mature trabecular bone, and the formation of a large number of bone cells; Group B specimens of trabecular bone and irregular arrangement, the observed bonemajority of naive trabecular trabecular bone cells to produce; group C specimens of trabecular bone thinning, sparse, part of the fracture, the bone marrow cavity filled with necrotic tissue debris, degeneration and death of bone cells, hematopoietic necrosis, a large number of empty osteocytelacunae appear. Three months after surgery, measured in group A surgical side of the femoral head subchondral bone, the average elastic modulus and maximum compressive strength of cancellous bone than in group B, group C (p<0.05), with normalside compared to the difference was not statistically significant (p> 0.05). After six months the group A surgical side of the femoral head subchondral bone, cancellous bone of the average elastic modulus and maximum compressive strength compared with the normal side differences None was statistically significant (p> 0.05), while group C surgery side of the femoral head collapse.Conclusion(1) The recombinant retroviral vector pLNCX2-hCGRPαhad been constructed successfully, The titer of recombinant retrovirus with hCGRPαagene was 1.7x106 pfu/mL and the retrovirus was effectively transfected into NIH3T3 cells.(2) BMSCs/pLNCX2-hCGRPαcells could stably express hCGRPα.(3) BMSCs/pLNCX2-hCGRPαcells showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.(4) Genetically modified BMSCs by hCGRPαjoint autogenous bone graft can be increased in the animal body into a bone-inducing activity, and promote the repair of bone tissue, improve the success rate of treatment ONFH.