ab2005

Influence of polysulphated polysaccharides and hydrocortisone on the extracellular matrix metabolism of human articular chondrocytes in vitro

L. Wang, J. Wang, K.F. Almqvist, E.M. Veys, G. Verbruggen

Department of Rheumatology, Ghent University Hospital, Ghent University, Ghent, Belgium

ABSTRACT
Objective
To evaluate the influence of hydrocortisone and two polysulphated polysaccharides (xylosan polysulphate and chondroitin polysulphate) on the extracellular matrix metabolism of chondrocytes cultured in gelled agarose.

Methods
Isolated chondrocytes from normal femoral cartilage of the knee joints of 7 donors were cultured in gelled agarose to maintain their differentiated phenotype. After two weeks of culture, hydrocortisone (0.2 µg/ml), xylosan polysulphate (10 µg/ml) and chondroitin polysulphate (10 µg/ml) were added to the culture media supplemented with or without interleukin (IL)-1b. After one week of incubation, the cells were liberated from the agarose with agarase. Isolated cells were labelled with antibodies against aggrecan and type II collagen, as well as biotinylated hyaluronic acid binding protein to analyse the extracellular matrix (ECM) molecules in the cell-associated matrix (CAM). The levels of matrix metalloproteinase (MMP)-1, -3, and -13, as well as tissue inhibitor of metalloproteinase (TIMP)-1 and -3 were determined after the cells had been permeabilised and stained with the appropriate antibodies. Triplicate samples were analysed with flow cytometry.

Results
IL-1b decreased the accumulation of aggrecan, hyaluronan and type II collagen in the CAM and increased intracellular MMP-1, -3 and -13 at a concentration of 100 pg/ml. Xylosan polysulphate and chondroitin polysulphate restored the expression of these CAM molecules in these IL-1b-treated cultures. Hydrocortisone stimulated the accumulation of CAM aggrecan and hyaluronan whether or not under the exposure to IL-1b. Intracellular MMP-1, -3, -13 and TIMP-1 and -3 of IL-1b-treated cells was downregulated after treatment with hydrocortisone.

Conclusion
Both hydrocortisone and the two polysulphated polysaccharides could stimulate the accumulation of CAM macromolecules of IL-1b-treated chondrocytes. This effect probably resulted in part from the downregulation of MMPs. These agents showed cartilage structure modifying effects in vitro.

Key words
Chondrocytes, extracellular matrix, polysulphated polysaccharide, hydrocortisone, IL-1b, flow cytometry.

Abbreviations: CAM: cell-associated matrix; CPS: chondroitin polysulphate; ECM: extracellular matrix; HA: hyaluronan; IL: interleukin; MFI: mean fluorescence intensity; MMP: matrix metalloproteinase; OA: osteoarthritis; TIMP: tissue inhibitor of metalloproteinase; XPS: xylosan polysulphate.

Dr. Lai Wang and Dr. Jun Wang contributed equally to this study.
This work was supported by a FWO-grant 3G013201 and in collaboration with the Institut de Recherches SERVIER.
Please address correspondence and reprint requests to: Gust Verbruggen, MD, Polikliniek Reumatologie - 0K12 - Universitair Hospitaal, De Pintelaan no. 185, B-9000 Ghent, Belgium.
E-mail: gust.verbruggen@rug.ac.be