Mixtures of glucosamine and chondroitin sulfate reverse fibronectin fragment mediated damage to cartilage more effectively than either agent alone.
OBJECTIVE
To test the effectiveness of glucosamine (GluNH(2))-HCl, chondroitin sulfate (CS) and mixtures in protecting cartilage exposed to fibronectin fragments (Fn-fs), an exposure known to enhance catabolic cytokines and matrix metalloproteinases (MMPs).


METHODS
Pharmacologic formulations of GluNH(2) (FCHG49) and CS (TRH122) (Nutramax Laboratories, Inc.) were added at 1, 10 or 100 microg/ml singly or in mixtures to bovine cartilage cultures in serum or serum-free conditions with or without Fn-f. Proteoglycan (PG) release into media and remaining cartilage PG content were measured by dye binding analysis and effects on PG synthesis by assays of 35-sulfate incorporation. Effects on MMP-3 and -13 expression were measured by Western blotting of conditioned media.


RESULTS
In serum-free conditions, the agents singly or as mixtures did not block Fn-f mediated matrix degradation. In serum, single agents were weakly effective at 100 microg/ml, while the mixture of each agent at 0.1 microg/ml decreased PG loss by about 50% by day 7 and at 1 microg/ml restored nearly 50% of the PG after 7 days in Fn-f pretreated cartilage. However, both agents singly and as mixtures at 0.1-100 microg/ml decreased MMP release. In serum, the single agents at 1-10 microg/ml weakly reversed Fn-f mediated PG synthesis suppression, while the mixtures were 100% effective at 1 microg/ml.


CONCLUSIONS
GluNH(2) and CS act synergistically in reversing damage and promoting repair at concentrations found in plasma after oral ingestion of these agents. Reversal of PG synthesis suppression correlates more with these activities than suppression of MMP-3 or -13 expression.
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA. ghomandberg@medicine.nodak.edu
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D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002358:Cartilage, Articular / Q000293:injuries* / Q000378:metabolism; D002417:Cattle; D002809:Chondroitin Sulfates / Q000627:therapeutic use*; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004359:Drug Therapy, Combination; D005353:Fibronectins / Q000378:metabolism*; D005944:Glucosamine / Q000627:therapeutic use*; D008163:Luminescent Measurements; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D046509:Tissue Culture Techniques; D014945:Wound Healing
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D010446:Peptide Fragments; D011509:Proteoglycans; D002809:Chondroitin Sulfates; D053509:Matrix Metalloproteinase 13; D019278:Matrix Metalloproteinase 3; D005944:Glucosamine