Morphogenetic Protein-2 Gene Transfection Of Rabbit Bone Marrow Mesenchymal Stem Cells Combined With Allogeneic Demineralized Bone Matrix Graft Material In Vitro The Study Of Biological Safety Of Adenovirus Mediated Bone

Objective: To observe the biocompatible of rabbit bone marrow mesenchymal stem cells(BMSCs) on allogeneic demineralized bone matrix graft material after transfection recombinant adenoviral human bone morphogenetic protein-2 expression vector. Methods: According to Ursit, the method is to get the rabbit limbs metaphysis and the cancellous bone tissue. The bone tissue was cryopreservated for 72 hours, which follows the removal of the soft tissue and marrow. The decalcification of bone tissue took for 72 hours, and was delipidated with both ethanol and ether for 24 hours. They soaked in distilled water for 24 hours after the process of removing acid treatment. After purification, the bone tissues were becoming 4mm * 4mm * 3mm demineralized bone matrix, ethylene oxide sterilization, and storage of 4 degrees for backup. Thawing the bone marrow mesenchymal stem cells(BMSCs) stores in liquid nitrogen tank. The recombinant adenovirus of human bone morphogenetic protein-2(Ad-rh BMP-2) transfected the same generation of the BMSCs. Immunohistochemical observation of transfected bone marrow mesenchymal stem cells of bone morphogenetic protein-2(BMP-2) expression after transfection. After transfected for 48 hours, cells were planted on the allograft demineralized bone matrix, and scanning electron microscopy was used to observe the cell growth and the conditions of adhesion on material. MTT was used to assay the proliferation case of BMSCs. Results: After the transfection for 48 hours, the fluorescence microscope showed the BMSCs grow well after the transfection. The green fluorescence were able to see under the fluorescence microscope, which infers the transfection efficiency can reach above 95%. The BMSCs showed cytoplasmic brown after transfection immunohistochemical staining, while normal cytoplasm showed unstained. The inverted microscope showed the decalcified bone matrix material with good porosity and three-dimensional structure. After transfected for 48 hours, the BMSCs were planted on the allograft, and the demineralized bone matrix showed the green fluorescence under the fluorescence microscope. The adherence of cells in DBM were(70±3.7)% after planted for 6 hours,(77±4.6)% after planted for 12 hours, and(80±3.8)% after for 24 hours. The scanning electron microscopy showed that the DBM has porous structure with different pore sizes, irregular shape and mutual traffic. The pore diameter of DBM was 215±86μm, and the porosity was 76±3.51%. The scanning electron microscopy showed that the cells grow well around/ in pores and proliferate on allogeneic DBM graft material. MTT assay showed: after transfection, the proliferation of the cells that planted on demineralized bone matrix was normal, and this phenomenon indicates there is no significant difference when compared with the control group(P>0.05). Conclusion: Allogenic DBM has a good three-dimensional structure. The result of(Ad-rh BMP-2) transfected BMSCs was successful and efficient. The Ad-BMP-2 transfected BMCSs are biocompatible to allogeneic DBM graft material.