| Articular cartilage injury is very common, which can be caused by various factors such as trauma,osteoarthritis et al. Articular cartilage, which is parimarily avascular and has an abnormally small cellular density, has limited capacity for self repair. Until recently, it was not generally thought that there was no effective clinical treatment that could restore a damaged cartilage and prevent the normal sequelae of degenerative joint disease. For the repair tissue typically was classified as fibrocartilage and not hyaline. Previous surgical treatments have not good long term effect while may have certain short term effect. At last, many patients can not restose fuction satisfactorily and have to accept joint arthroplastic procedures or joint replacement. Clinical workers have been trying to transplant chondrocyte and articular cartilage in order to restore damaged cartilages. Articular cartilage or chondrocytes transplantation was showing its promising future for the repair of demaged cartilage, along with the technique of autologous chondrocyte transplatation was approved by the Food and Drug Administration, and the rapid progress in the tissue engineering field. Because the transplantation using allogenic chondrocyte would lead to immunological rejection and transmission of infectious diseases, and the results of mes-enchymal stem cell transplantion were not definite positive, the autologous chondrocytes was considered to be a preferable choice. But the relatively rare source of the autologous chondrocytes extremely restrictthe use of this technique, how to expand rapidly chondrocytes within short term was practically proved to be crucial. The previous experimental work primarily has used immature or young animals. However most of the patients undergoing knee replacements are elder patients with osteoarthritis , because the stem cell population in the bone marrow and the number of articular chondrocytes decline with age , these protocols may be difficult to use with the elder population.MethodsRabbit articular chondrocytes were obtained from cartilage chips scraped from the tibial plateaus and femoral condyles of 24 to 28 ?month - old New Zealand White rabbits. The chips were washed with phosphate buffered saline. The cells were released by sequential digestion at 31 with 2mg/ml hyaluronidse,2mg/ml trypsin,and 4mg/ml collagenase ( class 2 ). Parimary culture were plated in DMEM culture medium with 20% FBS. ( 1 ) MTT assay:The primary chondrocytes of mature rabbits were incubated with bFGF at different concentrations, IGF - I at different concentrations and a combination of these two cy-tokines (50ng/ml) treatment. MTT assay was made at 24,48, 72 hours to determine the number of living chondrocytes. (2) Cell counting evaluate cell fold increase-. After setting control group, various groups incubated separately with 50ng/mlbFGF, 50ng/mlIGF - I and 50ng/ml combination of these two cytokines. All groups of cells were passaged at the same time when cells of any group reached near confluence. Compareing the final fold increase of cells among various groups after 3 passages. ( 3) Flowing cytometer evaluate proliferation index; After setting control, first passage chondrocytes of variousgroups were incubated with 50ng/mlIGF - I , 50ngml/bFGF andSOng. ml combination of these two cytokines. The proliferaton index of different groups were measured by flowing cytometer after 14 days.Result(1) MTT assay result shows: After IGF - I at different concentrations , bFGFat different concentrations and a combination of there two cytokines at 50ng/ml were applied, the number of living cells were obviously higher than that of control groups at 24,48,72 hours, the number of the group with a combination of these two cytokines was higher than that of any other group with any individual cytokine. The living cell number was highest when each cytokine was applied with concentration of 50ng/ml.(2) Cell counting shows;The final fold increase of any group incubated with cytokines was much higher than that of... |
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