Evaluation of chondrocyte cell-associated matrix metabolism by flow cytometry

Objective: To analyze human articular chondrocyte celf-associated matrix ag grecan, hyaluronan (HA) and type II collagen metabolism using flow cytometr y; and to compare the results obtained for aggrecan with classic (35)Sulfat e incorporation methods and an enzyme linked immunosorbent assay (ELISA). Design: Human articular chondrocytes obtained from five donors were culture d In gelled agarose and tested for their response to different concentratio ns of interleukin-1 beta (IL-1 beta). Synthesis and distribution of aggreca n in the cell-associated matrix (CAM), in the interterritorial matrix and i n the nutrient medium of the chondrocytes in culture were analyzed using (3 5)Sulfate incorporation. The results were expressed incorporated in aggreca n per 1 x 10(6) cells/h. Flow cytometry with FITC-conjugated monoclonal ant ibodies against aggrecan and as pg SO4 incorporated in aggrecan per 1 x 10( 6) cells/h. Flow cytometry with FITC-conjugated monoclonal antibodies again st aggrecan and type II collagen, and with the biotinylated hyaluronic acid binding protein (b-HABP), was used to investigate the synthesis and accumu lation of aggrecan, type I I collagen and HA in the CAM of the cultured cel ls. The packing of these macromolecules in the CAM of the chondrocytes, was assessed by measuring the mean fluorescence intensity (MFI) of the cell sa mple due to the binding of the specific monoclonal antibodies or b-HABP use d. ELISA was used in parallel to quantify CAM aggrecans after these macromo lecules were brought into solution with guanidinium chloride. Detection of aggrecan by flow cytometry was compared with S-35-incorporation in chondroc ytes from two subjects and with. ELISA in a further two donors. Results. IL-1 beta suppressed aggrecan synthesis by chondrocytes in agarose . An IL-1 beta dose-dependent suppression of S-35-aggrecan in the CAM refle cted the changes in the interterritorial matrix. IL-1 beta -induced aggreca n breakdown was followed by a rise in S-35-aggrecan metabolites In the incu bation media of the calls in culture. Flow cytometry and ELISA confirmed th is decreased accumulation of aggrecan in the CAM of the chondrocytes. The r esults obtained with flow cytometry were closely related to those obtained with ELISA. on the other hand, indirectly measures the glycosaminoglycan co ntent of the aggrecan and does not necessarily reflect the absolute amount of aggrecan molecules. Therefore, the effects of IL-1 beta on cell-associat ed aggrecan, where assessed with S-35-incorporation, did not correlate with the results of the flow cytometric assays. Flow cytometry enabled the dete ction of an impaired synthesis and accumulation of HA and of type II collag en in the CAM of the cultured chondrocytes, IL-1 beta -induced changes in C AM agrecan and hyaluronan closely agreed. Conclusions. Flow cytometry offers an efficient tool to study the metabolis m of the chondrocyte CAM. The MFI has been used as a parameter to quantify the ECM molecules in the CAM. (C) 2001 OsteoArthritis Research Society Inte rnational.