The Study Of BMSCs Transfected By HVEGF165 And HBMP2 Double Gene Adenovirus Combined With Multihole N-HA/PA66 To Repair The Radius Defect Of Rabbit

Background Bone marrow mesenchymal stem cells?BMSCs?with multi-directional differentiation potential,thus recognized as good seed cells.Bone morphogenetic protein 2?BMP2? and Vascular endothelial growth factor 165?VEGF165?are currently accepted osteogenesis induced factor and angiogenesis factor.Multihole nano hydroxyapatite/polyamide 66?n-HA/PA66?because of its good biological and mechanical properties,which are widely used.HVEGF165 and hBMP2 gene are adopted in this experiment,therefore,double gene adenovirus vector transfect BMSCs composite multihole n-HA/PA66 to construct tissue engineering bone implanted and observation,aiming at providing reliable technical support and theoretical basis for clinical application of tissue engineering bone to treat bone defect.Objective In this experiment by in vitro to construct the tissue engineering of bone which is hBMP2 and hVEGF165 gene adenovirus transfected BMSCs combine with porous n-HA/PA66 and to access its osteogenesis ability of differentiation,and then implanted to assess its in the model of rabbit radial defect repair ability.Methods 1.Puncture pumping using iliac bone marrow fluid,density gradient centrifugation BMSCs,grown in 25 cm³ culture bottle,after 48 h in liquid,observe the general form of the BMSCs,more than 80% of the cells in the alignment of subculture,take the third generation of BMSCs to identify the cell phenotype.2.Take the third generation grew well BMSCs as experimental object,the calculation of cells,using different plural BMSCs infection,infection after 48 h in liquid,2 d cells were observed under inverted phase contrast microscope after stateand use the inverted fluorescence microscope transfection situation,find out the best MOI values,and ELISA to detect the expression of cell hBMP2 and hVEGF165;Will be infected cells inoculated to n-HA/PA66 biological materials,in turn and then 1 w,2 w,3 w,4 w for Alkaline phosphatase,Alkaline phosphatase,ALP)activity determination and in 3 w,4 w and alizarin red staining observation and electron microscopy scanning.3.The use of 1% pentobarbital sodium anesthesia?3.5 ml/kg? of New Zealand white rabbit,surgery to make 15 mm long interruption of radial bone defect,the tissue engineering bone defects of bone graft is local,closing a wound.4.At postoperative 2 w,4 w and 8 w,12 w,X-ray,CT three dimensional reconstruction bone defect healing,and remove the tissue engineering bone and HE,toluidine blue,Masson staining to observe osteogenesis and angiogenesis.Results1.After 48 h in liquid,were observed under inverted microscope,a small stick wall cells,a polygon or fusiform,with the extension of time,cell colony growth and increased,shape or form vortex herringbone shape cell arrangement,primary cell 5-7d can reach more than 80% alignment,as the batches number increase in spindle cells change,cell morphology more than aligned,about 3 generation cells purity can reach more than 95%.Three generations of cells by flow cytometry instrument to detect the phenotype: CD29?99.82%?and CD44?94.14%?,low expression of CD14?3.11%?and CD34?0.34%?.2.Ad-BMP2,Ad-VEGF165,Ad-BMP2-VEGF165 recombinant adenovirus vector,by the Shanghai sangon build,virus drops 1×10¹⁰ pfu/ml.Transfection respectively after BMSCs,ELISA to detect hBMP2 and hVEGF165 protein expression level,BMP2,VEGF165 in 3-28 d were expressed,reaches its peak when the 3-5 d,5 d after began to reduce.Growth in good condition observed under inverted phase contrast microscope,inverted fluorescence microscope to observe the GFP expression,normal transfection efficiency is above 90%.In 3 w,4 w alizarin red staining observation groups have been formed calcium nodules and mineralization zone,but the double genome is superior to other groups,BMP2,VEGF165 times,BMSCs is the worst.Calcified nodules can be seen cells closely observed under scanning electron microscopy?sem?adhesion on the scaffold,and 4 w week in double genome is dense calcium nodule formation,other groups have different amounts of calcium nodule formation.Alkaline phosphatase activity tests showed double genome in each period were higher than other groups,each group differences were statistically significant?P < 0.05?.3.The interruption of rabbit radial bone defect model was constructed successfully,about 15 mm long,ulna intact,no damage,each implant implantation and the displacement of intraoperative postoperatively.Postoperative observation local operation without infection,inflammation and other anomalies.4.At postoperative 2 w,4 w and 8 w,12 w,observe X-ray,CT three dimensional reconstruction observations show that implant position is good,and closely with the surrounding tissue growth,while without phase can be observed clearly have different degrees of bone defect repair,visible when 12 w double composite porous n-HA gene/PA66 group of scaffold materials have been bone cortex complete package,and remove the tissue engineering bone and HE,toluidine blue,Masson staining to observe osteogenesis and angiogenesis.C2 group each phase points when the observation indexes were better than the other groups?P < 0.05?;B1 group blood vessel number is optimal in the B2 group?P < 0.05?,blood count and C1 group was obviously better than A2 group?P < 0.05?;When groups of each phase observations were superior to A1?P < 0.05?.Conclusion1.BMSCs both in vivo and in vitro have their potential to differentiate into osteoblasts,can be used as seed cells and used in bone tissue engineering.2.BMSCs were Ad-BMP2-VEGF165 gene adenovirus after transfection can safe,healthy growth,and can be sustained high quantity of protein expression purpose,at the same time,after the transfection BMSCs can on porous n-HA/PA66 mesh good growth,adhesion and mineralization.3.Studies have shown that hBMP2 hVEGF165 and gene adenovirus transfection BMSCs composite porous n-HA/PA66 to construct tissue engineering bone caneffectively promote the bone defect repair.