Experiment Study On Osteoblast Differentiation Of Rabbit Adipose Mesenchymal Stem Cells By Gene Bone Morphogenetic Protein-7 Transfaction In Vitro

PurposeSpinal fusion has been one of the most popular procedures performed in spine surgery,however,some clinical investigations have shown a considerable rate of failure to achieve a solid bony union.The most common clinical approach for preventing nonunions has been the use of internal fixation,which provides the initial stability and mechanical environment for bony fusion.Despite the improved mechanical strength of the recent spinal instrumentations,spinal bony fusion is the objective.Traditional spinal fusion procedures use autogenous bone as a graft to provide the osteoinductive and osteoconductive components necessary for the formation of new bone at the operative site.Although autogenous bone graft is though to be a gold standard in the achievement of solid spinal fusion,there is frequently an inadequate supply of autogenous bone graft for performing multilevel spinal arthrodesis.In addition,the morbidity of autogenous bone graft harvest is reported to be as high as 30%,with the most frequent complications including chronic donor site pain,nerve injury,infection,fracture,hematoma,and increased operative time.The healing of a spine fusion is a multifactorial process,the donor site morbidity,limited supply,and imperfect success rate of autogenous bone graft has alerted researchers to search for new suitable alternatives.The ideal autograft alternative is a material that may be used entirely in place of autogenous bone graft to achieve the same or a better fusion success rate.The rationale underlying the use of a matrix/stem cells/growth factor combination as an alternative to autogenous bone is that the osteoconductive and osteoinductive components of such a combination will attract,support,and simulate host osteogenic cells to form new bone tissue.Currently,there has been a considerable clinical interest in the use of matrix/stem cells/growth factor combination for bone regeneration and osseous graft supplementation therapy.Materials and methods1.Study of the isolation culture and identification of rabbit Adipose Tissue-derived Stem Cells.(1)Fat tissue aspirate of New Zealand white rabbits was digested by typeâ… collagenase and cultured in plastic culture bottles.(2)CD₄₄,CD_49d,CD₁₀₆detect the cells' immunophenotype;osteoblastdifferentiation of rabbit adipose mesenchymal stem cells in vitro by Vc,DEX andβ-sodium glycerophosphate.(3)The cells were examined by invert microscope,the growth curve was drawn and flow cytometry.2.Experimental study on osteoblast -differentiation of rabbit adipose mesenchymal stem cells by gene bone morphogenetic protein-7 transfection of in vitro.(1)The Reconstitution,amplification and appraisement of eukaryotic expression plasmid(2)Amplification and purification of plasmid DNA of pcDNA3.1-BMP-7.BMP-7 were detected by PT-PCR.(3)The cells were examined by invert microscope and Collagen I were analysied by Immunofluorescence.Results1.Success of the isolation culture of rabbit Adipose Tissue-derived Stem Cells.(1)After the digestion of collagenase I,the obtained cell mostly is the circular mononuclear cell.ADSCs had a similar long-spindle morphology and tightly arrayed to grow. (2)The results of cell's immunophenotype;The expression CD₄₄,CD_49dis masculine,but CD₁₀₆is negative.The extracellular ALP staining,alizarin Bordeaux staining of induction ADSCs were masculine.(3)Growth curve showed the proliferation abilitys' velocity of ADSCs are quickly.2.The effect of bone morphogenetic protein-7 in order transfer on the proliferation of rabbit Adipose Tissue-derived Stem Cells and its osteoblast differentiation potential.(1)Amplification and purification of plasmid DNA of pcDNA3.1-BMP-7 were success.(2)stable transfection BMP-7 to ADSCs was success.(3)the ADSCs still have a high reproductive activity after gene transfection.(4)ADSCs transfected with BMP-7 gene showed stable expression of collagen I.Conclusion1.The combination of digestion of collagenase I and adherent culture is an effective method to isolate ADSCs from fat tissue aspirate.ADSCs has higher proliferation ability and can be used in tissue engineering.2.ADSCs transfected with BMP-7 gene showed stable expression of collagen I.