| Chondral injuries involving the knee are common. Chondral or osteochondral defects are observed in many occasions such as trauma, osteochondritis dissecans, and osteoarthritis disease. Chondrocytes are highly specialized cells. Articular cartilage is an avascular, aneural tissue with limited capacity for self-repair after injury. Although many varieties of treatment options for cartilage injuries, for instance joint lavage, tissue debridement, bone marrow stimulating procedure (i.e., subchondral drilling and microfracture technique), or transplantation of autologous or allogeneic osteochondral grafts, have been used to repair such defects. Unfortunately, these treatments do not result in complete regeneration of the original hyaline architecture. A valuable technique to restore a defect in the articular surface is still non-existent. To date, the repair of articular cartilage lesions remains a significant clinical and groundwork challenge.Tissue engineering which has been grew rapidly in recent decade introduce new therapeutic options in the repair or replacement of diseased and/or damaged cartilage tissues. The construction of tissue engineered cartilage is influenced by three key factors including seeding cells, scaffolds and constructing methods. The seeding cell play a key role in tissue engineered constructs. Autologous chondrocyte is still the only one permitted in clinical usage. Autogenous chondrocytes-transplantation has proved as a promising method of cartilage repair, but it had a few problems such as restricted availability of donor sites for chondrocyte harvest, the limit capacity of proliferation of mature chondrocytes, and senescence during cell culture in vitro. Therefore, shortage of sufficient seeding cell hinder the technique widely application in clinical bedside .Mesenchymal stem cells (MSC) as alternative cell source have recently received widespread attention because of their potential utility in tissue engineering. MSC obtained from adipose tissue have been termed adipose-derived stem cells(ADSC) which is a kind of adult stem cells and has. been recently characterized from adipose tissue. ADSC has strong proliferation capacity and multi-lineage differentiation potential similar to bone mesenchymal stem cell (BMSC), but ADSC are available in large quantities with minimal possibility of morbidity and discomfort clinically. In view of these practical advantages, ADSC become an alternative for chondrocytes or BMSCs used in research of engineered cartilage.Many materials have been used as the scaffolds for tissue engineered cartilage. It is also essential to select a suitable scaffolds for cartilage engineering. The ideal scaffolds material should have good biodegradability, biocompatibility and 3-D space structure similar to extracellular matrix (ECM) .The study was divided into three phases. The first phase was aimed at developing a means of separating and culturing stem cells from subcutaneous adipose tissues of rabbit and subsequently characterizing the cells in terms of morphological and functional criteria. The second phase was aimed at constructing tissue engineered cartilage by ADSC in vitro for further use in in vivo manipulations for the treatment of cartilage defects. The third phase was aimed at implantation of tissue engineered cartilage composite and reconstituting full-thickness Chondral defects in rabbits, and assessing the reconstituted defects grossly and histologically.Part I Isolation and characterization of adipose-derived stem cells from rabbitADSC were isolated from rabbit subcutaneous adipose tissues by enzymatic digestion and were expanded in monolayer with serial passages at confluence. The morphologic and growth of cell were observed by convert microscope. The proliferation of cell was evaluated by 3(4,5dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and flow cytometry. The CD44 phenotypes expression of these cells in Undifferentiated states was examined by immunofluorescence technique. To demonstrate the multilineage differentiation potential, P3 cell were cultured in specific differentiation medium to be induced to osteogenesis, chondrogenesis and adipogenesis. This results showed that, Fibroblast-like cells were obtained from rabbit subcutaneous adipose tissues. Large numbers of CD44-positive cells could be observed by immunofluorescence technique. MTT colorimetric assay and flow cytometry demonstrated these cells had the remarkable potential of division and proliferation. Under appropriate culture conditions, these cells were induced to differentiate to the adipocyte, osteoblast, and chondrocyte.Present results indicate that we succeed in isolating ADSC from rabbit subcutaneous fat. These cells possess the potential of multilineage differentiation and stable proliferation over extended periods.Part II Construction and characterization of cartilage engineered from ADSCs and BMG scaffolds"Two-phase" bone matrix gelatin(BMG) scaffold (one side of cancellous bone and the other side of cortical bone) was prepared from iliac bone of rabbits by sequentially chemical method. The P3 ADSC were collected and incubated within the porous cancellous bone side of BMG-scaffold in a high density and three-dimensional culture with control medium or chondrogenic medium. The proliferation of ADSC within the BMG was investigated by scanning electron microscope (SEM) .The production of extracellular matrix (ECM) was detected by safranine 0 staining and reverse transcriptase polymerase chain reaction (RT-PCR) .The results showed that the "two-phase" BMG consisted of the dense cortical part and the loose porous cancellous part which had good biocompatibility. Results of safranine 0 staining and RT-PCR confirmed the expression of cartilage-specific collagen type II and proteoglycan aggrecan after chondrogenic differentiation for 2 weeks.These results revealed that BMG could work as scaffold of ADSC. After induced, the ADSC-BMG composite possess characteristics histologically similar to native hyaline cartilage which could be for further used in in vivo manipulations for the treatment of cartilage defects.Part III Transplantation of ADSC in the BMG-ScaffoldThe P3 ADSC were collected and then seeded at a high density onto the round BMG-scaffold (diameter, 4 mm; thickness, 2 mm) that ADSC and BMG come from the same donator. The scaffold containing ADSC was cultured in chondrogenic medium or control medium for 2 weeks before autologous transplantation which served as experimental groups I and II. Defects that were left empty or treated with cell-free scaffold served as controls. Gross Evaluations and Histological examination were carried out to measure the repair of defects at 4, 12, 20 weeks after operation. The results revealed that , the defects in the both experimental groups were filled with smooth and white reparative tissue when compared with the two control groups , the reparative tissue showed a smooth surface being connected with adjacent normal cartilage. In contrast, the surface of control group was irregular and the thickness of the tissue was smaller than that of the cell-containing scaffold experimental groups. Furthermore, the means in Wakitani' s score of the experimental groups exhibited significantly better than those of the other two groups at every time point. But the difference between both experimental groups was not obviouslyThe present study demonstrated that tissue engineered cartilage constructed in vitro repaired the Chondral defect successfully and the ADSC-BMG composite need not induction before transplantation.In conclusion, our research suggested that adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage, it is feasible to construct a tissue engineered cartilage composite using ADSC as seed cells while BMG as scaffold, and the ADSC-BMG composite need not induction before transplantation.
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