Studies On Repair Of Bone Defect By Rabbit Mesenchymal Stem Cells Transfected By Adenovirus-mediated Bone Morphogenetic Protein 7 Gene

Objective To clone human bone morphogenetic protein 7 (hBMP-7)gene cDNA full length and generate high titers level recombinant adenoviruses containing hBMP-7 gene the replication defective recombinant adenoviruses AdEasy system was used. To induce expression of osteogenic phenotype by hBMP-7 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 segmental bone defect healing of rabbit radius was explored.Methods (1) hBMP-7 gene was cloned from human placent tissue by RT-PCR method, then sequenced and analyzed in Genbank. (2) hBMP-7 gene was cloned into Shuttle vector to generate pAdTrack-CMV-BMP-7,which was linearized by PmeI and co-transformed into adenoviral backbone plasmid competent cells-AdEasier-1 Cells to obtain recombinant adenoviral plasmid- pAdBMP-7 by homologous recombination. Recombinants were selected and confirmed by restriction endonuclease analysis. After packed in 293 cells, the recombinant adenoviruses AdBMP-7 were generated. (3) 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 seed cell in bone tissue-engineering. (4) The proliferation and cell cyle analysis of gene-modified MSCs were assessed by MTT method and FCM after transfected with Ad-BMP-7. The methods of RT-PCR, immunohistochemical staining and western blot were used to test the expression of BMP-2 protein. Gomori stain and ALP testing kit were used to measure ALP activity and immumomradiatory testing kit were used to measure OCN contents. In addition, the collagen type I were tested with the methods of RT-PCR and immunohistochemical staining. (5) PLGA(with diameter of 150³00um and porosity of 90%) was prepared and infected MSCs were seeded onto it. Scanning electronic microscoping were used to observe cell-matrix interaction. The ectopic bone formation was observed with histological method. (6) Segmental bone defect model of rabbit radius was made and infected MSCs-PLGA composites were transplanted into defects(Group A).At the same time, non-infected MSCs seeded onto PLGA repair group(Group B), PLGA repair group(Group C) and defect model group(Group D) were employed as control. The defect repaired capabilities for each of the reconstructive modalities were assessed by gross observation, radiograph and histological analysis.Results (1) A 1296bp cDNA was amplified by RT-PCR from human placent tissue and its sequence analysis was documented as was expected. (2) The recombinant plasmid pAdBMP-7 was established by homologous recombination and confirmed by restriction endonuclease digestion. GFP expression could be observed on the third day after packing of the linearized pAdBMP-7 in 293 cells and 3 × 10⁹ efu/L titer of Ad-BMP-7 was obtained by CsCl gradient purification. (3) Mononuclear cells collected by density centrifugation were cultured and passaged and morphological obsevationsuggested these cells were rabbit MSCs. (4) 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-7 could expressed BMP-7 protein effectively. After the gene transfection 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). Analysis revealed that collagen type I expression remarkably increased after transfection(1 .32 ±0.16 in the experimental group versus 3.65 ± 0.31 in the control group) (p<0.01). (5) 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 4-8 weeks after implanted into the muscle pouches of rabbit. (6) The defects treated in the Group A were repaired and regenerated much more new bone, bridged earlier and stronger than those in the Group B. The defects treated in the Group C and D could not attain osseous tissue healing.Conclusions (1) Human BMP-7 gene is successfully cloned and its recombinant adenoviruses are constructed. This may offer the possibility of a novel approach to local gene therapy of bone regeneration. (2) AdEasy system is a simplified and efficient system for rapid generation of recombinant adenoviruses, which ensure the target gene can be expressed in MSCs efficiently. (3) 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 seed cells in tissue engineering. (4) PLGA can promote cell adhesion, proliferation and contribute to new bone formation. It should be a good scaffold for tissue-engineered bone. (5) Autologous marrow mesenchymal stem cells infected by hBMP-7 gene can...