Introduction: Osteoarhthritis is a degenerative disease affecting a large proportion of the population. Recently, there has been renewed interest in the use of neutraceuticals (such as glucosamine) for the treatment of symptomatic pain and pathology in arthritic joints. However, little research has been carried out to assess the biochemical mechanisms by which glucosamine imparts its effects on the disease process. Biochemically, an early change in the cartilage metabolism is a loss of the large aggregating proteoglycan, aggrecan. Functionally, this loss results in a decreased capacity for the tissue to sustain mechanical loading that leads to cartilage destruction and a painful joint. The enzymes responsible for the loss of aggrecan from the tissue are commonly referred to as the aggrecanases and are members of the ADAMTS family of enzymes. Degradation of aggrecan by the aggrecanases can be detected using a specific neoepitope monoclonal antibody BC-3 (1). Model systems using cartilage explant cultures that mimic the degradative processes seen in osteoarthritis have been developed in which cytokine such as IL-1 are used to initiate the catabolic processes leading to cartilage degradation.
Methods: Cartilage explant cultures (bovine) were established using published methodologies (1). Explants were then incubated in either DMEM, DMEM supplemented with a chemically modified glucosamine (0.5–15mM) or DMEM supplemented with glucosamine hydrochloride (0.5–15mM) for 1 hour. IL-1 (10ng/ml) was then added to half of the explant cultures in each experimental group. Cultures were maintained for 4 days in the experimental media after which media and explants were harvested for analysis. Glycosaminoglycan (GAG) concentrations of media samples and cartilage extracts were determined using the DMMB assay. RNA was extracted from cartilage explants and RT-PCR was performed using primers to cartilage matrix molecules, ADAMTS and MMPs. Western blot analysis was performed on the experimental media using MAb BC-3 to determine the presence of aggrecanase-generated aggrecan catabolites.
Results: Experiments show that glucosamine hydrochloride (0.5–15mM) was unable to inhibit the release of GAG from explant cultures induced by treatment with IL-1. However, explant cultures preincubated with 10–15mM chemically-modified glucosamine were able to inhibit the release of GAG induced by IL-1 to that of control culture levels. The decreased release of GAG corresponded to a decrease in the detection of aggrecanase-generated aggrecan catabolites as assessed by Western blotting with MAb BC-3.
Discussion: This data questions the effectiveness of glucosamine hydrochloride in the inhibition of biochemical mechanisms involved in the IL-1 induced degradation of aggrecan in articular cartilage. However, the data suggests a role for a chemically modified glucosamine in the IL-1 induced degradative pathways involved in the loss of aggrecan from cartilage. The use of glucosamine in the treatment of arthritic diseases is controversial, however, the modified form of glucosamine used in this study helps to support the potential use of the dietary ingestion of glucosamine and its beneficial effects in arthritis patients. 1. Hughes, C.E., et al. (1995). Biochem. Journal. 305, 799–80
