ABSTRACT
For the successful autologous chondrocyte transplantation, it is important to maximize the number of chondrocyte and maintain its original morphology and phenotypic change of the chondrocyte in the culture. In this study, the effect of ascorbic acid and human serum which are known to promote cell proliferation and collagen synthesis was observed in the culture of human chondrocyte. Media were prepared with the conditions of fetal bovine Serum(FBS) treated group, FBS +ascorbic acid(asc) treated group, human serum(HuS) treated group, and HuS+asc treated group, respectively. Proliferation was measured by cell counting using trypan-blue staining method. We used to determine the degree of expression of aggrecan of mRNA and type II collagen using RT-PCR. Type II collagen in cultured cell and medium was measured by western blot analysis and proteoglycan synthesis by DMB (Dimethylene Blue) assay. Under all conditions, aggrecan on mRNA level was well expressed. On the other hand, expression of type II collagen was reduced on HuS treated group than FBS treated group, and ascorbic acid treated groups showed decreased expression of type II collagen. Western blot analysis showed increased expression of type II collagen on HuS treated group than FBS treated group, and ascorbic acid treated groups showed increased level. HuS+asc treated group showed the most significant effect than the other groups. The increased effects of ascorbic acid on the proliferation and collagen synthesis were more prominent in the culture with human serum. It might be due to the synergic effect with some growth factors which were present in human serum.
Subject(s)
Humans , Aggrecans , Ascorbic Acid , Blotting, Western , Cell Count , Cell Proliferation , Cells, Cultured , Chondrocytes , Collagen , Collagen Type II , Hand , Intercellular Signaling Peptides and Proteins , Phenotype , Proteoglycans , RNA, MessengerABSTRACT
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.