Aggrecan degradation in chondrocytes is mediated by reactive oxygen species and protected by antioxidants

Reactive oxygen species (ROS) are implicated in aging of cartilage and in the pathogenesis of osteoarthritis. However, the biological role of chondrocytes-derived ROS has not been elucidated. An in-vitro model was developed to study the role of chondrocyte-derived ROS in cartilage matrix degradation. The primary articular chondrocytes were cultured and the aggrecan matrix was radiolabeled with 35-sulfate. The labeled aggrecan matrix was washed to remove unincorporated label and chondrocytes were returned to serum free balanced salt solution. The cell-monolayer-matrix sensitivity to oxidative damage due to either hydrogen peroxide or glucose oxidase was established by monitoring the release of labeled aggrecan into the medium. Lipopolysaccharide (LPS) treatment of chondrocyte-monolayer enhanced the release of labeled aggrecan. Catalase significantly prevented the release of labeled aggrecan in LPS-chondrocyte cultures, suggesting a role for chondrocyte-derived hydrogen peroxide in aggrecan degradation. Superoxide dismutase or boiled catalase had no such inhibitory effect. The effect of several antioxidants on LPS-chondrocyte-dependent aggrecan degradation was examined. Hydroxyl radical scavengers (mannitol and thiourea) significantly decreased aggrecan degradation. A spin trapping agent N-tert-butyl-phenylnitrone (but not its inactive analog tert-butyl-phenylcarbonate) significantly decreased aggrecan degradation. Butylated hydroxytoluene also inhibited aggrecan degradation, whereas the other lipophilic antioxidant tested, propyl gallate, had a marked dose-dependent inhibitory effect. These data indicate that general antioxidants, hydroxyl radical scavengers, antioxidant vitamins, iron chelating agents, lipophilic antioxidants, and spin trapping agents can influence chondrocyte-dependent aggrecan degradation. These studies support the role of a chondrocyte-dependent oxidative mechanism in aggrecan degradation and indicate that antioxidants can prevent matrix degradation and therefore may have a preventive or therapeutic value in arthritis. The enhancement of oxidative activity in chondrocytes and its damaging effect on matrix may be an important mechanism of matrix degradation in osteoarthritis.