Objective To validate flow cytometry as an experimental technique for the study of the homeostasis of the extracellular matrix (ECM) of human articular cartilage.
Methods Given the established insights in the relation between the transforming growth factor (TGF)-β type II Receptor (TGF-βRII)/TGF-β auto/paracrine pathway, the intracellular levels of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), and the accumulation of ECM molecules in the ECM of articular cartilage, this metabolic pathway was used as a reference model to fulfill the objective. Chondrocytes were liberated from visually intact femoral condyle cartilage and cultured in gelled agarose to maintain their differentiated phenotype. After 2 weeks of culture, the chondrocytes were isolated from the agarose and flow cytometry was used to analyse the expression of TGF-βRII on the plasmamembrane, the expression of TGFβ1, MMP-1, MMP-3, TIMP-1 and TIMP-3 inside the cells, as well as the amounts of aggrecan, type II collagen and hyaluronan in the cell-associated matrix (CAM). The expression of the different substances was analysed with flow cytometry and reported as mean fluorescence intensity (MFI), which is due to the binding of FITC-labeled antibodies to their specific antigens. In addition, the effects of exogenous TGFβ1 on the expression of these proteins was investigated on chondrocytes cultured in serum-free media. Enzyme Linked Immunosorbent Assay (ELISA) was performed to evaluate the MMP-1, MMP-3, TIMP-1 and MMP-1/TIMP-1 complex in the culture medium collected after the last 3 days of the culture period. The correlations between the data were analysed with the Spearman's test.
Results Exogenous TGF-β1 increased the accumulation of aggrecan and hyaluronan in the CAM of chondrocytes and down-regulated the intracellular levels of MMP-1 and -3. TIMP-1 and -3 were increased after exposure to TGF-β1. Baseline expression of TGF-βRII on the plasmamembrane of normal human articular chondrocytes significantly correlated with the intracellular levels of TGFβ1, TIMP-1 and TIMP-3. TGFβ1 was correlated with TIMP-1, TIMP-3 and MMP-1. Aggrecan in the CAM was inversely correlated with the ratio of MMP-1 to TIMPs. In addition, there were correlations between TIMP-1 and TIMP-3, aggrecan and hyaluronan. ELISA also revealed the correlation between MMP-1 and TIMP-1 secreted by the chondrocytes into the nutrient medium. MMP-1/TIMP-1 complex was hardly found in the medium.
Conclusions Some aspects of ECM metabolism of normal cartilage were evaluated by flow cytometry. Chondrocytes from normal human cartilage, when cultured in gelled agarose, showed correlations between the expression of TGF-βRII/TGF-β1 and the intracellular levels of TIMPs, indicating that TGF-β autocrine pathway may contribute to homeostasis of the ECM in the normal cartilage. The relations between MMPs, TIMPs and the ECM molecules support that a physiological balance between MMPs and TIMPs results in a well-controlled matrix turnover in normal cartilage.
