Study of Expression Level of Cartilage Genes in Rat Articular Chondrocyte Monolayer and 3D Cultures using Real Time PCR

Purpose: to compare the expression level of certain genes related to cartilage and non-cartilage tissues at monolayer and alginate cultures derived from rat articular cartilage. Materials and Methods: Articular cartilage was harvested from knee joints of 10 male rats and was digested using enzymatic solution consisting of 0.2% collagenase I and 0.1% pronase. Released chondrocyte were then plated in 25-cm2 culture flasks and expanded. For alginate culture, about 5´106 passaged-5 cells were mixed with 1 ml alginate solution and cultivated as small beads for a period of two months. During the culture, the expression of Sox9, collagen II, I and aggrecan genes were quantified by real time PCR and compared with the gene expressions at monolayer cultures. Furthermore, the cell morphology, in this study, was observed using either conventional or inverted light microscopy. Results: The cells at monolayer cultures were observed as spindly shaped cells, while within alginate, chondrocytes tended to be morphologically spherical cells. Real time PCR analysis indicated that at monolayer cultures, the expression of Sox9, collagen II and aggrecan were significantly down-regulated while the expression of collagen I was largely up-regulated. In contrast to monolayer cultures, the expression levels of sox 9, collagen II and aggrecan, at alginate cultures, tended to be statistically high while collagen I was observed to be expressed at negligible level. All these differences were statistically significant (p<0.05). Conclusions: While at chondrocyte monolayer cultures, the cartilage-specific gene expressions appeared to be significantly down-regulated, alginate culture tended to stimulate the cells to express the genes in statistically high levels.