The Experimental Study On Repairing The Knee Cartilage Defect Of Rabbit Using The Allogenic Tissue Engineering Cartilage Which Gene Enhanced

Arthrodial cartilage injure is the very common problem in clinic. Arthrodial cartilage has very limited self-repairing capability, because it is a kind of avascular tissue and well-differentiated chondrocyte loss fission ability in the main. The rapid development of cartilage tissue engineering brings cartilage repairing a new hope, but many researches discovered that engineering cartilage using normal chondrocyte usually unable achieve integral repairing, furthermore, calcification and degeneration often appear some time later. However, gene transfer techniques offer a new way to solve above-mentioned problems. The modified target cells are utilized to construct engineering cartilage, and then the cells not only act as seeds cells, but also secrete growth factor which make important role in the regenerative process of cartilaginous tissue.Being one of the most important growth factor-1 in cartilage development and homeostasis accommodation, insulin-like growth factor-1 can stimulate chondrocyte division growth, moreover, it enhance chondrocyte to secrete II type collagen and proteoglycan that are essential to maintain the phenotype of chondrocyte. In addition, some research indicated that IGF-I can promote cartilage recovery in vivo. Therefore, transferring IGF-I to chondrocyte is helpful for the engineering cartilage to produce a marked effect. Various methods have been developed to deliver the genes into cells. These methods involve using physical approaches to delivery such as electroporation, chemical approaches such as encapsulation of DNA into liposomes and biological approaches such as viruses-mediated transfection. Advantages and disadvantages are concomitant in all these methods, and none achieved the Ideal request of gene transfer and release system. Relatively speaking, viruses-mediated transfection possesses longer exogenous gene expression, lower cytotoxicity and is more efficient at delivering genes. Furthermore, the prepared virus liquid can be preserved for long-term and added into culture fluid directly with no other reagent participation. Therefore, viruses-mediated transfection is especially suitable to the requirement of the seeds cell proliferated by large scale. Among the viruses vector, retroviral vector is the most frequently used vector, which possesses longer exogenous gene expression and is more efficient at delivering genes, and have the predominance to construct the engineering cartilage. In our previous study, we have transferred hIGF-I to engineering cartilage using Lipofectin(a kind of cathodolyte liposome), but the transferred gene could not express for long time. In our present study, we have constructed retroviral vector pLNC-IGF-GFP which contains IGF and GFP coexpression box by the technique of gene engineering. Then, the retroviral vector pLNC-IGF-GFP was transferred to rabbit arthrodial cartilage cells and the influence of gene transfer was investigated. Following, transgenic tissue engineering cartilages were constructed using transferred cells to repair rabbit articular cartilage defects.1.Construction and verification of retroviral vector containing hIGF-I gene and marker gene GFPpCG plasmid containing GFP sequence, pIRES-neo plasmid containing CMV promoter and endogenous ribosome binding site IRES, pC-IGF plasmid containing IGF sequence and retroviral vector pLNCL were primitive plasmids. Retroviral vector pLNC-IGF-GFP which contains IGF and GFP coexpression box and was controlled by CMV promoter was constructed after series enzyme incising and conjunction. Enzyme incising showed that the retroviral vector was constructed successfully.2.Packaging and verification of retroviral vector pLNC-IGF-GFPUsing hangosome method, etroviral vector pLNC-IGF-GFP was packaged by helper cell line PA317 and then was transferred to fibroblast NIH3T3. G418 screen transferred NIH3T3. Observed by fluorescence microscope, the transferred NIH3T3 expresses GFP. Virus titer was 4×106. These studies established foundation for the next research of chondrocyte.3.The effluence of hIGF-I gene transfection-mediated by retroviral vector on biological behaviour of chondrocyteDetection showed that the transferred chondrocytes had higher reproductive activityas well as the secretion of II type collagen and proteoglycan than non- transferred chondrocytes within 6 weeks after transferred. The results indicated that hIGF-I gene transfection could promote chondrocytes proliferation and maintain chondrocytes'phenotype for a comparatively long time (at least 6 week). In addition, RT-PCR demonstrated that the transferred chondrocytes still remained high hIGF-ImRNA expression after transferred for 6 weeks, which indicated the retroviral vector could express for a long time in host chondrocyte. This parts of results established good foundation for the following research.4.Tissue engineering cartilaginous constructed by the transferred chondrocytes forming cartilaginous tissue inin vivoThe transferred chondrocytes were cultivated on three diamensions condition in vitro for three days, and then the tissue engineering cartilaginous were transplanted subcutaneously to rabbit to develop for 6 weeks. After 6 weeks, the tissue lump had many Chondrocyte lacune and deep blue cartilage matrix showed by alcian blue staining, without blood vessel structure, which showed typical cartilaginous tissue formming. The results demonstrated the transplanted engineering cartilaginous can survive well, without serious rejection, and develop to cartilaginous tissue. In addition, RT-PCR demonstrated that the transferred group had hIGF-ImRNA expression and non-transferred groups dad not, which indicated the engineering cartilaginous transferred by retroviral vector could express IGF-Ⅰfor a long time in vivo. Furthermore, the transferred group had relatively higher II type precollagen expression and lower I type precollagen expression than those of non-transferred groups, which indicated the transferred engineering cartilage was more close to normal cartilaginous tissue than non-transferred engineering cartilage.5.Tissue engineering cartilaginous repairing articular cartilage defectsEstablish patella side cartilage defects model of rabbit articulatio genus. Animals were divided randomly into 5 groups: model group, bracket group, non-transferred group, transferred group, auto-cartilage grafting group. The results showed neogenetic cartilaginous tissue in transferred group had more cartilage matrix secretion and healed better than those in non-transferred group at all time point(4w. 8 w. 16 w. 2 w). In order to appreciate the repairing roles of the tissue engineering cartilaginous overall, Wakitani method was used to score cartilage defect repair. The results showed that there was significant difference between transferred group and non-transferred group in Wakitani indexes except cell morphous and surface evenness. Furthermore, except the difference of the whole score and cell morphous, transferred group had no difference with auto-cartilage grafting group at surface evenness, matrix staining, the thickness of neogenetic cartilaginous, and recipient site integration, which indicated transferred group got close to auto-cartilage grafting group on some facts.Conclusions:1. Retroviral vector containing hIGF-I gene and marker gene GFP was constructed successfully.2. Transferred chondrocytes had higher reproductive activityas well as the secretion of II type collagen and proteoglycan than non- transferred chondrocytes.3. hIGF-I-transplanted tissue engineering cartilaginous could survive well, and develop to cartilaginous tissue in vivo.4. To rabbit articular cartilage defects, transferred tissue engineering cartilage had the analogous effect as auto-cartilage.In short, rabbit arthrodial cartilage cells were transferred by retroviral vector containing hIGF-I gene and marker gene GFP in this study, which solved the problem existed in other transferred methods such as low transfection efficiency, and short expression time. Meanwhile, tissue engineering cartilaginous constructed by the transferred chondrocytes could repair rabbit full-thickness articular cartilage defects well.