Effects of IGF (Insulin-like growth factor) on sub-populated articular chondrocytes by Percoll density gradient / 대한정형외과연구학회지

PURPOSE: Articular chondrocytes have been known to have heterogeneity in articular cartilage. The different responses of chondrocytes to various cytokines and growth factors have been reported. These variations are likely a result of metabolic differences among the cell populations. We used the Percoll density gradient method to separate chondrocytes from articular cartilage into distinct subpopulations. Several growth factors are known to enhance the synthesis of cartilage matrix. In particular, IGF has specific anabolic effects. Addition of IGF to chondrocytes increased the synthesis of proteoglycans and collagen type-II while inhibiting the degradation and release of proteoglycans. MATERIALS AND METHODS: Chondrocytes were isolated from rabbit knee articular cartilage by collagenase digestion. In brief, male rabbits weighing 250g were euthanized by injecting an overdose of Nembutal, and nonfibrillated articular cartilage of the knee was removed by sterile dissection. Isotonic Percoll was mixed with 10x PBS to give a 60% stock solution. This was further diluted with PBS to give Percoll concentrations of 10, 20, 30, 40, 50, and 60%. RT-PCR, western blot analysis, immunocytochemistry, and immunohistochemistry were done for examination of collagen type II and aggrecan as the specific marker of extracellular matrix and proteoglycan synthesis on cultured chondrocytes. RESULTS: The sub-populated cells were proliferated variously. On the other hand, the addition of IGF to the sub-populated cells increased the proliferation in all fractions. Also the expression of collagen type-II and TIMP-2 was increased by IGF treatment. After alginate culture, collagen type-II expression was not significantly different between the IGF treated and the control groups in high density fractions. However, the addition of IGF to chondrocytes increased the expression of collagen type-II in low density fractions. The expression of collagen type-II after IGF addition was decreased in monolayer culture while it was increased in alginate culture. CONCLUSION: The effects of IGF are various among the subpopulated chondrocytes. These results will provide useful information for the separation of articular chondrocytes with an active metabolic activity and extracellular matrix for the investigation of the pathogenesis of articular cartilage.

The effects of sonication on human osteoarthritis cartilage in ex vivo tissue culture / 대한정형외과연구학회지

This study was initiated to investigate the effect of ultrasound(US) stimulation on therapeutic effects on human osteoarthritic cartilage repair. Cartilage explants from human osteoarthritic knee were sonicated for 10 minutes every day using continuous wave at frequency 1 MHz US signals with spatial and temporal average intensities of 0, 40, 200, 500 and 700mW/cm2. One group of explants was exposed to sham ultrasound as a control. After 1 week of culture, the intensity-dependent effects of US on DNA, proteoglycan (PG) and collagen synthesis were measured by 3H-thymidine, 35S-sulfate, 3H-proline incorporation, respectively. The expression of PG and type II collagen released into medium were measured by DMB (dimethylmethylene blue) method and western blot analysis. Safranin O/fast green and immunohistochemical staining with anti-collagen type II antibody were performed using the serial sections of cartilage explants. The histochemical examination showed that the expression of PG at the pericellular area in the deep layer increased continuously up to 700mW/cm2. In contrast, the depth of the superficial layer significantly decreased after treatment of sonication at 500 and 700mW/cm2. The expression of PG and type II collagen assessed by the isotope incorporation was significantly enhanced to the level up to 140%, 120% respectively, although US had no stimulatory effect on cell proliferation. These results suggest that optimum intensity of US for the effective expression of extracellular matrix in osteoarthritic cartilage may be around 200mW/cm2. In conclusion, our study suggests the possibilities that sonication may be therapeutically utilized for the repair of human osteoarthritic cartilage.