ABSTRACT
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.
Subject(s)
Humans , Blotting, Western , Cartilage , Cell Proliferation , Collagen , Collagen Type II , DNA , Extracellular Matrix , Knee , Osteoarthritis , Proteoglycans , Sonication , UltrasonographyABSTRACT
In monolayer culture, articular chondrocytes are well known to proliferate and dedifferentiate by seum and transforming growth factor-beta(TGF-beta). These dedifferentiated cells regain the ability to express type II collagen in alginate bead culture. In this study, the effects of human serum and TGF-beta on the proliferation and phenotypical change of human chondrocytes were examined in both monolayer and alginate bead culture. Proliferation was measured by 3H-thymidine incorporation and cell counting, chondrocytic phenotype by Western blot analysis of type II collagen expression, and proteoglycan synthesis by dimethylmethylene blue assay. Both human serum and TGF-beta synergistically increased the proliferation of chondrocytes in monolayer culture. Human serum had effect to maintain the type II collagen expression, even with enhanced level, in monolayer culture and showed redifferentiation in alginate culture, similar to fetal bovine serum control. TGF-beta enhanced the production of proteoglycan in monolayer culture. In conclusion, the present study demonstrated that human serum and TGF-beta could be used as potent additives to increase chondrocyte proliferation and maintain its phenotype.
Subject(s)
Humans , Blotting, Western , Cell Count , Chondrocytes , Collagen Type II , Phenotype , Proteoglycans , Transforming Growth Factor betaABSTRACT
Articular cartilage is a unique tissue devoid of blood and nerve tissue and so its regeneration is very limited. Recently a clinical trial on transplantation using autologous chondrocyte with periosteal flap has drawn a great deal of attention. Chondrocytes cultured in a plastic flask in monolayer can rapidly dedifferentiate appearing fibroblastic, and exhibit a change in matrix gene expression characterized by a decrease in type II collagen synthesis. It is uncertain whether phenotypic change of dedifferentiated chondrocytes in vitro can be reversible to their original status alter long term culture. It is important to verify tile maintenance of the phenotype and determine the optimum period for culturing chondrocytes to be used in autologous chondrocyte transplantation. This study will be set up to confirm the reversibility of once-dedifferentiated chondrocytes with matrix-producing capability. The phenotype of cultured human chondrocyte is analysed by Northern blot and Western blot analysis for collagen type I and II. Chondrocytes appeared fibroblast right after adhering to the flask buttom at first week of culture. The proliferating rates of chondrocyte in a monolayer culture were maximum at 3rd and 4th week of culture. And thereafter, proliferation rate flowed down or stops as confluence rose. On Northern and Western blot analysis, collagen type II was well expressed by 3th to 4th week culture, thereafter progressively decreased its density with time. On the other hand, collagen type I m-RNA has not expressed until 3rd week of the culture, showing progressive increment of density thereafter. On Northern blot analysis in pellet culture, type II collagen m-RNA is apparantly reexpressed. This study indicates chat in the monolayor culture, the chondrocytic phenotype was lost with regards to morphology and mRNA expression and cartilage specific protein. However, these cells seemed to haute the potential to redifferentiate to well-differentiated chondrocytes in densely packed culture, such as pellet.