Effect of polysulfated glycosaminoglycan on osteoarthritic equine articular cartilage in explant culture

J. P. Caron From the Departments of Large Animal Clinical Sciences (Caron, Toppin), Michigan State University, East Lansing, MI 48824, and the Departments of Medicine (Rheumatology) and Biochemistry, Rush Presbyterian-St Lukes Medical Center, Chicago, IL (Block).

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D. S. Toppin From the Departments of Large Animal Clinical Sciences (Caron, Toppin), Michigan State University, East Lansing, MI 48824, and the Departments of Medicine (Rheumatology) and Biochemistry, Rush Presbyterian-St Lukes Medical Center, Chicago, IL (Block).

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J. A. Block From the Departments of Large Animal Clinical Sciences (Caron, Toppin), Michigan State University, East Lansing, MI 48824, and the Departments of Medicine (Rheumatology) and Biochemistry, Rush Presbyterian-St Lukes Medical Center, Chicago, IL (Block).

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Summary

Middle carpal cartilage explants from 4 horses with mild osteoarthritis involving that joint were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan (psgag) on proteoglycan synthesis and degradation. Cultures were exposed to 0.025 or 25 mg of psgag/ml for 48 hours, after which the medium was replaced with medium containing similar doses of psgag and 35S. Subsequently, the sulfated proteoglycan content of the medium and extracts of the explants was measured. Gel filtration chromatography was used to estimate the size and to purify the principal, large proteoglycan monomer, which was further characterized by digestion, using glycosidic enzymes. In a second experiment, explants were incubated with 35S for 48 hours, and were subsequently exposed to the same concentrations of the psgag for an additional 48 hours. The amount of remaining labeled proteoglycan was determined for culture medium and cartilage extracts. Gel filtration chromatography was used to assess the hydrodynamic size of the large proteoglycan monomer. Aliquots of proteoglycans from the second experiment were incubated in high-molecular weight hyaluronate and chromatographed to assess reaggregation.

Polysulfated glycosaminoglycan caused a significant (P ≤ 0.04) decrease in sulfated proteoglycan synthesis by cartilage explants. Radioactive proteoglycan content in explants labeled prior to exposure to psgag were similar. Large proteoglycan monomer size was similar in both experiments (median partition coefficient [KAV] = 0.40), and was not influenced by psgag treatment. Prelabeled explants exposed to hyaluronate and chromatographed under associative conditions had similar proportions of the radiolabel eluting as proteoglycan aggregate. Enzymatic digestion of newly synthesized large monomer revealed a mild dose-dependent increase in the proportion of keratan sulfate substitution on core protein. It was concluded that psgag in vitro, at the dosages evaluated, caused a decrease in proteoglycan synthesis, had little effect on labeled proteoglycan degradation, did not influence the size of large monomer, and caused a modest increase in the concentration of keratan sulfate in proteoglycans synthesized by osteoarthritic equine chondrocytes.

Summary

Middle carpal cartilage explants from 4 horses with mild osteoarthritis involving that joint were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan (psgag) on proteoglycan synthesis and degradation. Cultures were exposed to 0.025 or 25 mg of psgag/ml for 48 hours, after which the medium was replaced with medium containing similar doses of psgag and 35S. Subsequently, the sulfated proteoglycan content of the medium and extracts of the explants was measured. Gel filtration chromatography was used to estimate the size and to purify the principal, large proteoglycan monomer, which was further characterized by digestion, using glycosidic enzymes. In a second experiment, explants were incubated with 35S for 48 hours, and were subsequently exposed to the same concentrations of the psgag for an additional 48 hours. The amount of remaining labeled proteoglycan was determined for culture medium and cartilage extracts. Gel filtration chromatography was used to assess the hydrodynamic size of the large proteoglycan monomer. Aliquots of proteoglycans from the second experiment were incubated in high-molecular weight hyaluronate and chromatographed to assess reaggregation.

Polysulfated glycosaminoglycan caused a significant (P ≤ 0.04) decrease in sulfated proteoglycan synthesis by cartilage explants. Radioactive proteoglycan content in explants labeled prior to exposure to psgag were similar. Large proteoglycan monomer size was similar in both experiments (median partition coefficient [KAV] = 0.40), and was not influenced by psgag treatment. Prelabeled explants exposed to hyaluronate and chromatographed under associative conditions had similar proportions of the radiolabel eluting as proteoglycan aggregate. Enzymatic digestion of newly synthesized large monomer revealed a mild dose-dependent increase in the proportion of keratan sulfate substitution on core protein. It was concluded that psgag in vitro, at the dosages evaluated, caused a decrease in proteoglycan synthesis, had little effect on labeled proteoglycan degradation, did not influence the size of large monomer, and caused a modest increase in the concentration of keratan sulfate in proteoglycans synthesized by osteoarthritic equine chondrocytes.

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