Experimental Study Of Rabbit Bone Marrow Stem Cells Transfected By Adenovirus Mediated Bone Morphogenetic Protein-2 Gene Plus Nano-hydroxyapatite To Construction Tissue Engineered Bone In Vitro

【Objective】Bone defect, due to trauma,tumor,congenital malformations,infection,pathology and other factors, is one of the challenges facing in the clinic. Bone graft is the main method to solve this problem. There were some disadvantages or limitations at the current methods, such as lackage of donor bone, damage of transplant bone area and transplant rejection reactions complications after bone transplant. In recent years, combined the scaffold material, seed cells and growth factors to build bone tissue engineering can improve the above drawbacks for the clinical treatment of bone defects bring about a better future. It brings a good prospects for the clinical treatment of bone defect repair. To explore the target gene expression and osteoblastic differentiation of human bone morphogenetic protein-2 recombinant adenovirus expression vector transfection of rabbit bone marrow-derived mesenchymal stem cells (rBMSCs). On this basis, to observation the adhesion,proliferation of adenovirus-mediated human bone morphogenetic protein-2 expression vector transfected with rabbit bone marrow mesenchymal stem cells after composite nano-hydroxyapatite (Nano-HA) to construction biomimetic artificial bone in vitro. Using transgenic technology and the adenovirus vector with the purpose gene transfection target cells then compound of Nano-HA, cell carrier complex stable and long-term secrete growth factor, constructed artificial bionic in vitro. For the further application of the tissue-engineered bone to repair bone defect. 【Methods】1. Take a healthy, 2～3 months age, New Zealand white rabbits of under sterile conditions, with 18 size marrow puncture needle to extract 3～4ml bone marrow from the lateral tibial tubercle.Using Percoll (1.073g/ml) liquid separation BMSCs. To cultured bone marrow cells to prepare to the further experiment. In which use a small amount of cells to observe cell morphology and growth curve and use the conditioned media induced BMSCs osteoblast differentiation. Calcium cobalt method to detect alkaline phosphatase expression, alizarin red staining to detect calcium nodule-forming ability.2.Taken part of the subculture rBMSCs, with adenovirus (Ad-BMP-2/GFP)carrying BMP-2 gene transfection for next step. Taken portion of transfected cells to identify as follows: detection of Ad-GFP transfection efficiency,immunohistochemistry,RT-PCR and western blot detection of Ad-BMP-2 gene expression in rBMSCs after transfection.3.Trypsin digestion and collected third generation rBMSCs of the untransfected and transfected 48h cells, made them into single cell suspension, then evenly inoculate the suspension cell on pre-wet Nano-HA material, taken the two groups of materials by scanning electron microscopy detection the surface cell attachment, distribution and growth at 3d,5d. Western Blot detect expression of BMP-2 protein.【Results】1.Primary and subculture rBMSCs showed long fusiform ortex-like, radial alignment . BMSCs grew well after continuous subculture, using the conditioned medium for 3 weeks culture, it can induced cell transformation into osteoblast, alizarin red staining is positive and formation of mineralized nodules after induction. ALP test detection is positive ,the cell were colonies growth formed calcium nodules. 2. The cells morphology is rule and vigorous growth after transfected, the cells became larger with the time and in the trend to polygonal chang. BMP-2 immunohistochemistry showed that the cytoplasm was positive expression in the transfected cells. RT-PCR can detect a positive bands about 338bp size, BMP-2 gene mRNA high level expression in different periods in the transfected cells. Western-blot can detect a positive bands about 30kD size. After transfection, Ad-BMP-2 transfected with BMP-2 positive bands, and bands were strongly positive over time, without transfection was visible weaker bands, the virus transfected cells at the protein level also highly expressed.3.Cells and scaffold co-cultured for 3 days, scanning electron microscopy can see the surface of scaffold materials is irregular, scaffold materials have many micropores covered cells,at Nano-HA material lacuna and voids block transfected cells grows well and well adhesion ,cells attach and stretch well. Composite 5 days,cells completely spreading and outstretched pseudopodium, Cells and materials fusion closely, cell edge can visible calcium secretion and deposition. Collection composited cells,western-blot to show BMP-2 protein high expression of transfection group at third,fifth days,cell-Nano-HA compound may continue secretion BMP-2.【Conclusion】1. BMSCs is convenient to get through density gradient centrifugation method can be get a large number of BMSCs. BMSCs have stable performance and easy to amplification. In certain conditions, it can be induced into osteoblasts. BMSCs is suitable as seed cells for bone tissue engineering.2. Using GatewayTM technology to construct hBMP-2 adenovirus vector. Ad-BMP-2 can efficiently transfect bone marrow mesenchymal stem cells, after transfection, BMP-2 can sustained efficiently secreted expression。3. Successful constrction BMP-2 gene enhanced tissue engineering biomimetic artificial bone in vitro.