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Influence of polysulfated polysaccharides on aggrecans synthesized by differentiated human articular chondrocytes
G Verbruggen1, M Cornelissen, D Elewaut
1Department of Rheumatology, Ghent University Hospital, University of Ghent, Belgium.
Objective:
Synthesis rates of aggrecans by phenotypically stable human articular chondrocytes and the immobilization of these aggrecans in large aggregates have been used as variables reflecting the capability of these cells to restore the extracellular matrix of articular cartilage in vitro. The effects of heparin, chondroitin polysulfate, and xylosan polysulfate on these variables were investigated.
Methods:
Human articular chondrocytes were maintained in culture in gelled agarose. After 2 weeks of culture, the media were supplemented for one week with 10 microg/ml each of heparin, chondroitin polysulfate, and xylosan polysulfate. Synthesis of aggrecans was investigated using Na2(35SO4) as a radioactive precursor during the last 24 h of exposure to polysaccharides. Size exclusion chromatography was used to assay the proportions of aggrecans immobilized in aggregates. Native aggrecan aggregates accumulated during culture were liberated in associating conditions by agarase digestion of the artificial agarose matrix and studied by electron microscopy. Hyaluronan synthesis was studied in monolayer cultured chondrocyte derived fibroblasts. The cells were exposed to 3H glucosamine for 24 h after the nutrient media were supplemented with 3 polysaccharides for one week. Size exclusion chromatography was used to assess the length of the hyaluronan filaments.
Results:
Xylosan polysulfate and chondroitin polysulfate but not heparin significantly increased total 35S incorporation rates in aggrecan. Electron microscopy study of aggrecan aggregates showed that xylosan and chondroitin polysulfate but not heparin significantly increased aggrecan aggregate sizes. The same polysulfated polysaccharides increased the synthesis of high molecular weight hyaluronan by chondrocyte derived fibroblast-like cells.
Conclusion:
Polysulfated polysaccharides significantly increased the synthesis rates and the accumulation of aggrecan in aggregates in the extracellular environment in this in vitro system.