Reconstruction Of Tissue Engineering Cartilage With Chondrocyte Transfected Human Vascular Endothelial Growth Factor 165 Gene.

Background Defection of auricle caused by injury or congenital microtia is a common disease which brings negative affection for patients' complexion and physiology in plastic surgery field.The major treatment method for these patients is auricular reconstruction surgery because bad regeneration of auricular cartilage.The surgery has many complications,such as defection deformity of donor site,infection,deformation,absorption of rib framework, even though autologous one has no rejection action.Artificial polymers exposed from skin easily because of rejection.Tissue engineering cartilage research brings the hope for patients.Although the use of autologous cartilage tissue engineering cartilage in clinical has been proved,but it only fit the defection less than 1mm.Cartilage is avascularity tissue,and chondrocyte metabolism depended on permeability of extracellular matrix(ECM).The more mature and thicker of extracellular matrix produced by external cells,the lower conduction efficacy induced dysbolism and lack of oxygen.Central chondrocytes of tissue were necrotic,while the circumjacent cells were survival,then,the "hollow" happened.Therefore,our experiments focus on retain the proliferation and survival of central chondrocytes,and keep them alive.Vascular endothelial growth factor(VEGF) is an important angiogenine. There is accumulating evidence shows that expression of VEGF in mature chondrocyte can promote cell redifferentiation and caryocinesia activity. VEGF plays a key role for chondrocyte to survival and proliferation during the chondrogenesis.We used gene engineering technical transfer VEGF into chondrocytes to construction gene -enhance engineering cartilage,which may benefit to the chondrocytes' survival,proliferation,differentiation and improve the internal construction in engineering cartilage.Objection Observation and analysis the biologic character of cultured chondrocyte from pediatric residual auricular cartilage of microtia patients.We constructed hVEGF₁₆₅ vector and transfected VEGF gene to chondrocyte in vitro,analyzed the expression of VEGF and evaluated the biologic character of chondrocytes transfected VEGF by ECM analysis.When chondrocytes transfected VEGF formed tissue engineering cartilage vivo,we evaluated its biology characters by evaluation of tissue structure,the expression of ECM proteins and related-genes.Method 1.Construction of recombinant adeno-associate virus vectors rAAV2-hVEGF₁₆₅-CMV-EGFP.2.Isolation and culture of chondrocytes from pediatric residual auricular cartilage.Test of double time. Immunohistochemistry,RT-PCR can be used to analyse extracellular matrix produced by chondrocyte.ECM includes Collagen typeâ…¡,â…©,â… and aggrecan. 3.When the transfection is completed,transfection efficiency has been quantities by flow cytometry.The expression pattern of VEGF and the chondrocyte extracellular matrix are examined by histology staining, immunochemistry,Real-time PCR,ELASA,Western Blot methods.4.Nude mouse has been implanted complex made up chondrocytes transfected rAAV2-hVEGF₁₆₅-CMV-EGFP and pluronic-F127,while chondrocyte transfected rAAV2-EGFP and pure chondrocvte also implanted respectively as control group. 10 weeks later,cartilage-like tissues were taken out,histology and immunohistochemical staining has been used to analyze the structure of cartilage.The differences among the 3 groups were compared by test of the tissue structure,ECM protein and the expression of gene about survival and proliferation of chondrocyte by Real-time PCR and Western Blot method.Result 1.We constructed VEGF vector rAAV2-hVEGF₁₆₅-CMV-EGFP successfully. 2.With proliferation of chondrocyte in vitro,the shape of it turned fiber-like shape from polygon.The expression of AG,COLâ…¡and COLâ…¹decreased gradually.The COhâ…¡,COLâ…¹disappeared,while COLâ… begin express in passage 5.3.rAAV2-hVEGF₁₆₅-CMV-EGFP tranfected chondrocyte successfully in vitro.The transfection efficiency reach 20.1±0.62%after 3 days and keep stable.Following extend of transfection phase,the expression of VEGF is up first,down later,and up again,as well as the expression of ECM. 4.All cell-materials complex groups produced cartilage with different size on nude mouse subcutaneously.Chondrocyte transfcted VEGF gene Pluronic F-127 complex produced homogeneous tissue,while the control tissues were "hollow".The result of Safranin O and Verhoeff stain showed GAG of the experimental groups was plentiful,chondrocyte and cartilage lacuna was well-distributed and elastic fiber distribution was reticulodromous.The result of immunohistochemistry and Western Blot suggested experimental group have more collagenâ…¡than control group.VEGF-positive cell were found in all group,but on boundary of experimental group tissue and all field of control group.â…§Factor could not be found in all groups.The result of Western Blot showed the expression of VEGF are more abundant than control group,and VEGFR-2 was just be found in experimental group.The result of real-time PCR showed expression of RunX2,Sox9,collagen typeâ…¡,â…©higher in experimental group,especially of GAG when compare to control group,but there has no significant change in PTH/PTHrR expression.There are not differences between the control groups.)Conclusion:VEGF gene can be transfer chondrocyte by rAAV2-hVEGF₁₆₅-CMV-EGFP vector successfully in vitro.They share the same pattern between VEGF expression and ECM protein.Cells become ageing when the major protein component of ECM decreased,VEGF expressed down.Moreover,senescence chondrocyte can redifferentiation and increase the expression of AG,COLâ…¡, COLâ…©and COL,while VEOF express up.The results from vivo showed chondrocytes transfected VEGF can form cartilage tissue.Compared with control group,more COLâ…¡,COLâ…©and AG and the expression of RunX2,Sox9 appeared in chondrocyte transfected VEGF experimental group.Elastic fibers were well-distributed,and vascular endothelial cell did not immigrate.We suggested that expression of ectogenic VEGF triggered paracrine and autocrine of VEGF of chondrocyte and co-acted with VEGF receptor 2 to enhance permeability of chondrocyte and improve internal construct of engineering cartilage,and prevent vascularize proceed.