Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats

Abstract

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.