Abstract
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.
Highlights
During the fetal period, most bones are derived from endochondral bone formation, in which mesenchymal stem cells (MSCs) differentiate into chondroblasts, forming a cartilage mold that is gradually replaced with bone (Adams et al, 2007)
At 7 days, only cultures of chondrocytes treated with caffeine at a concentration of 0.5mM exhibited MTT conversion in the formazan crystals significantly lower compared to the control and significantly lower compared to the other groups treated with caffeine
At 14 and 21 days of culture, all groups treated with caffeine at concentrations of 0.5, 1.0 and 2.0mM showed less cell viability, which was characterized by a significant reduction in the conversion of MTT in the formazan crystals
Summary
Most bones are derived from endochondral bone formation, in which mesenchymal stem cells (MSCs) differentiate into chondroblasts, forming a cartilage mold that is gradually replaced with bone (Adams et al, 2007). Chondroblasts secrete a matrix rich in collagen type II and aggrecan during the chondrocyte maturation phase, which is characterized by cellular hypertrophy and the expression of collagen type X and alkaline phosphatase (Garimella et al, 2004). These matrix constituents, in turn, are regulated by the transcription factors Sox-9 and Runx-2, which exert their effects during the early and late stages of the chondrogenic differentiation (Komori, 2003; Hino et al, 2014). To elucidate the genesis of the changes caused by caffeine on the cartilage, further studies on the cellular and molecular mechanisms by which caffeine acts on chondrocytes are needed
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