The Experimental Study Of Repairing Larynx Catilage Defect By CDMP1Transfection On In Vitro Chondrogenicand Differentiation Of Rabbit Bone Marrow Mesenchymal Stem Cells

ObjectiveTo evaluate the feasibility of repairing larynx catilage defect by CDMP1transfection on in vitro chondrogenicand differentiation of rabbit bone marrowmesenchymal stem cells and PLGA porous foam scaffolds by tissueengineering techniques.This is a new ways to provide for the repair of cartilagedefects and functional reconstruction by the Department of OtolaryngologyHead and Neck Surgery and clinical departments.MethodTaking the BMSCs of the one month rabbit in aseptic conditions.To isolationthe BMSCs through the combination of density gradient centrifugation andadhere wall culture.Take the third generation of rabbit BMSCs divided into thefollowing three groups,A group (the transfection group) was transfected byAd-CMV-hCDMP1-IRES-eGFP, B group (the untransfected group) wastransfected by Ad-CMV-eGFP. C group (the control group) was not transfected.Then hCDMP1expression at mRNA and protein level was measured byRT-PCRand western blot method． Expression of Type Ⅱ collagen(ColⅡ)，Glycosaminogl-ycans and growth of the cells were all measured by biologicalmethods to evaluate the effects of gene transfer on the differentiation of rabbitBMSCs.Making the mode of thyroid cartilage defects in the general anesthesia state. Divided the rabbits into the following three groups:Ⅰ group is implantedthe transfection group cells and PLGA compounds; Ⅱgroup is implantedBMSCs and PLGA compounds;Ⅲ group is implanted stents only.Drawingmaterials after4and8weeks,Then sacrificed the recovery result for gross andhistological observation.ResultsSubculture of BMSCs with active proliferative capacity, adenovirus infectedBMSCs with hCDMP1gene did not cause excessive proliferation or inhibit，theexpression of colⅡ and glycosaminogl-ycans in BMSCs transfected withCDMP-1gene increased significantly than control cells.Ⅰ group defect areashave chondrocytes generation,surfaces smooth.Toluidine blue and type Ⅱimmunohistochemical showed that newborn cells of Ⅰgroup secrete matrixGAG and cartilage collagen Ⅱ increase significantly than control cells;ⅡandⅢ groups defect only a small fiber tissue repair,surfaces rough.Conclusion1.Combination of adhere wall culture and density gradient centrifugation isa simple and pragamic method for cell dissociation and culture.By this method,rabbit bone marrow mesenchymal stem can be obtained with higher purity.2.With adenovirus,exogenou human CDMP-1gene can be transfected intoBMSCs in vitro and express hCDMP1mRNA and protein stably in the cell.3.The BMSCs transfected with hCDMP1gene present stronger capability tosecret cartilage-specific matrix such as type Ⅱ collagen andglycosaminogl-ycans, so these cells are possibily differentiated to chondrogeniccells.4.Combined with biological mounting, the BMSCs transfected withrecombinant hCDMP1gene can be transplanted in vivo and promote therestoration of cartilage defect.