Quantitative analysis of gene expression in human articular chondrocytes in monolayer culture

Abstract

Human articular chondrocytes (HACs) were isolated from cartilage samples of normal joints and cultivated in monolayer for 56 days. Collagen type I, II, IX, aggrecan, and versican expression was quantified by real-time polymerase chain reaction (PCR). The expression of the genes was highly time-dependent and changed over the culture time. Collagen type I was not present at the beginning of the culture and increased 100-fold during the culture time. Collagen type II and IX expression was found during the entire culture period and decreased more than 100-fold. Aggrecan expression was downregulated 100-fold whereas versican expression increased 10 times. Indices of cell differentiation, defined as ratios of collagen type II to I (CII/I), collagen type IX to I (CIX/I), and aggrecan to versican (Agg/Ver), were significantly higher at the beginning of the culture and decreased over the culture period. The CII/I and CIX/I ratios formed three phases, with a plateau at the beginning, followed by a transition phase and a steady state at the end of the culture time, whereas the Agg/Ver ratio declined constantly. The accurate quantitative assessment of extracellular matrix gene expression in samples of chondrocytes taken from monolayer culture can be used to monitor chondrocyte metabolism as well as changes in the cell differentiation status.