| Background Osteoarthrosis is commonly seen in orthopaedics clinical practice, and articular cartilage is usually involved. It was known that articular cartilage has little capacity to self-repair 200 years ago. Damage to the articular cartilage may therefore progress to osteoarthritis by mechanic wear and enzymatic degradation, which often results in a painful and dysfunction joint. During several decades, many strategies including periosteal and perichondrial tissue grafting, osteochondral autografting etc have been investigated to regenerate it, but the best method is not found yet. Currently, prosthetic joint replacement is the most common approach for treating severe and extended degeneration of the cartilage, but it has many complications including infection, long-term loosening and it may be needed to repaired repeatedly. Instead of replacement, plerosis and reconstruction have been a tendency in the research field. Tissue engineered cartilage cultivated in vitro is tried to repair articular cartilage lesion.With the development of genetic engineering, molecular tissue engineering has been a subject that combines molecular biology and tissue engineering, to study cells, tissue-inducing factors, biomaterials and the subtlerelationships between them at molecular level, and finally develops better biological substitutes which would restore, maintain, or improve tissue function.New thought would be provided for obtaining cytokine in tissue engineering after occurrence of gene transfer technology based on virus vector. As a transfer vector, adenovirus has many special advantages: infection and expression of genes in replicating and non-replicating cells; high expression level of functional proteins; no insertional mutagenesis and free from inactivation by complement. Adenovirus vector has been regarded as one of up and coming tools in gene therapy. Combination of molecular biology and tissue engineer brings new expectation for the repair of articular cartilage lesions.Objective To obtain high titers level recombinant adenovirus taking chondroplasia factors genes using replication defective recombinant adenovirus Adeno-X?as a carrier, and infect rabbit marrow mesenchymal stem cells and induce expression of cartilage cell phenotype by influence of growth factors coding by exogenous genes. Then the tissue-engineered cartilage is constructed by combination of infected MSCs and bone matrix gelatin, and its capacity to repair articular cartilage was tested in rabbit knee bone-cartilage defect model.Methods (1) To obtain TGF-β1 gene from several tissue and cells by RT-PCR method, sequence and analyze it after cloning it. (2) Subcloned TGF-β1 and BMP-7 genes separately into the p-Shuttle vector. The resultant plasmid was cut by restriction endonuclease PI-Sce I and I-Ceu I , and subsequently linked linearized Adenovirus . Then the recombinant plasmid was transfected into HEK 293 cells by Lipofectamine 2000, and high titer virus is obtained through HEK 293 cell package. (3) Bone marrow was drawn from human posterior superior iliac spine and mononuclear cell layer was collected by using density centrifugation. Marrow-derived MSCs were incubated in complete medium(10% fetal bovine serum, penicillin 100U/ml,streptomycin 100ug/ml) at 37C in humidified atmosphere containing 95% air and 5%CO2. Primary cultured and passage cultured MSCs were observed to evaluate the feasibility of being seed cell in cartilage tissue-engineering. (4) Adenovirus vector mediated by chondroplasia factors gene were applied into culture medium(30pfu/cell) to induce bone marrow mesenchymal stem cells to express chondrocytic phenotype. Infected cell form and cell cycle were observed to compare with non-infected cell. Immunohistochemistric staining in situ hybridization and Western blot of chondroplasia factors and its genes were detected to show the expression of exogenous genes and its coding proteins. Procollagen II mRNA in cells was detected by RT-PCR, hexuronic acid in culture medium was assayed by carbazole-sulfuric acid method to...
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