Study On Bone Formation Of Rabbit Mesenchymal Stem Cells Modified By Bone Morphogenetic Protein 2 Gene

Objective To induce expression of osteogenic phenotype by hBMP-2 gene the rabbit marrow mesenchymal stem cells were infected by recombinant adenovirus. Then the tissue-engineered bone was constructed by combination of infected MSCs and PLGA (75:25 polylactic acid-polyglycolic acid) and its feasibility and ability to enhance bone formation was explored.Methods Bone marrow was obtained from dual femoral greater trochanter of rabbit and mononuclear cell layer was isolated by density centrifugation. Marrow-derived MSCs were incubated in complete medium(10% fetal bovine serum, penicillin 100U/ml, streptomycin 100μg/ml) at 37℃in humidified atmosphere containing 95% air and 5%CO2. Primary cultured and passage cultured MSCs were observed to evaluate the feasibility of being seeding cell in bone tissue-engineering. The proliferation and cell cyle analysis of gene-modified MSCs were assessed by MTT method and FCM after transfected with Ad-BMP-2. The methods of RT-PCR and immunohistochemical staining were used to test the expression of BMP-2 gene. ALP testing kit were used to measure ALP activity and immumomradiatory testing kit were used to measure OCN contents. PLGA(with diameter of 150~300μm and porosity of 90%) was prepared and modified MSCs were seeded onto it. Scanning electronic microscoping were used to observe cell-matrix interaction. The ectopic bone formation was observed with histological method.Results Mononuclear cells collected by density centrifugation were cultured and passaged and morphological obsevation suggested these cells were rabbit MSCs. No significant changes in morphology of infected MSCs were observed. There was no significant difference in proliferation and cell cycle between infected and non-infected MSCs. Results showed that MSCs transfected with Ad-BMP-2 could expressed BMP-2 protein effectively. After being transfected the MSCs gained the structural and morphological characters which were similar to those of osteoblasts. The ALP activity and OCN contents in transfected MSCs were increased significantly(p<0.05). PLGA prepared was a grid-like porous structure, with definite ductility and strength, and could be trimmed into different models. Adenovirus-infected MSCs seeded onto PLGA showed high level of proliferation and mass synthesis of cell matrix was observed with electronic microscoping . New bone formation were observed in 8 weeks after implanted into the muscle pouches of rabbit.Conclusions Density centrifugation is a feasible method to isolate MSCs and these can be cultured and passaged in vitro . All results shows that 1-5 era passaged MSCs are suitable for seeding cells in tissue engineering. PLGA can promote cell adhesion, proliferation and contribute to new bone formation. It should be a good scaffold for tissue-engineered bone. Autologous marrow mesenchymal stem cells infected by BMP-2 gene can differentiate into osteoblast cells. The composite of transgeneic MSCs and PLGA can be used to construct tissue-engineered bone.