Reactive oxygen species (ROS) are implicated in both cartilage aging and the pathogenesis of osteoarthritis. We developed an in vitro model to study the role of chondrocyte-derived ROS in cartilage matrix protein degradation. Matrix proteins in cultured primary articular chondrocytes were labeled with [ 3 H]proline, and the washed cell matrix was returned to a serum-free balanced salt solution. Exposure to hydrogen peroxide resulted in oxidative damage to the cell matrix as established by monitoring the release of labeled material into the medium. Calcium ionophore treatment of chondrocytes, in a dose-dependent manner, significantly enhanced the release of labeled matrix, suggesting a chondrocyte-dependent mechanism of matrix degradation. Antioxidant enzymes such as catalase or superoxide dismutase did not influence matrix release by the calcium ionophore-activated chondrocytes. However, vitamin E, at physiological concentrations, significantly diminished the release of labeled matrix by activated chondrocytes. The fact that vitamin E is a chain-breaking antioxidant indicates that the mechanism of matrix degradation and release is mediated by the lipid peroxidation process. Lipid peroxidation was measured in chondrocytes loaded with cis-parinaric acid. Both resting and activated cells showed constitutive and enhanced levels of lipid peroxidation activity, which were significantly reduced in the presence of vitamin E. In an immunoblot analysis, malondialdehyde and hydroxynonenal adducts were observed in chondrocyte-matrix extracts, and the amount of adducts increased with calcium ionophore treatment. Furthermore, vitamin E diminished aldehyde-protein adduct formation in activated extracts, which suggests that vitamin E has an antioxidant role in preventing protein oxidation. This study provides in vitro evidence linking chondrocyte lipid peroxidation to cartilage matrix protein (collagen) oxidation and degradation and suggests that vitamin E has a preventive role. These observations indicate that chondrocyte lipid peroxidation may have a role in the pathogenesis of cartilage aging and osteoarthritis.Cartilage degeneration is a hallmark of cartilage aging and osteoarthritis (1). Degeneration of articular cartilage in osteoarthritis is accompanied by chronic pain and significant disability. In a series of reports (2-7), we and others have documented that chondrocytes produce reactive oxygen species (ROS).1 The production of ROS by chondrocytes can contribute to degradation of the cartilage matrix. For example, ROS can mediate intracellular signaling and gene activation of cytokine and growth factor-induced products in chondrocytes (8, 9). In activated neutrophils and monocytes/macrophages, the cellspecific gene products of "NADPH-oxidase complex" physically come together and initiate single electron reduction of oxygen and the release of ROS outside the cells. Phagocytes use the toxic properties of ROS to eliminate pathogens (10, 11); in contrast, the biological role of secreted ROS in cartilage is not known.The ob...

We fabricated "ligament analogs" in vitro by seeding high-strength resorbable collagen fiber scaffolds with intraarticular (anterior cruciate ligament, ACL) or extraarticular (patellar tendon, PT) rabbit fibroblasts. Fibroblasts attached, proliferated, and secreted new collagen on the ligament analogs in vitro. Fibroblast function depended on the tissue culture substrate (ligament analog vs. tissue culture plate) and the origin of the fibroblasts (ACL vs. PT) PT fibroblasts proliferated more rapidly than ACL fibroblasts when cultured on ligament analogs. Collagen synthesis by ACL and PT fibroblasts was approximately tenfold greater on ligament analogs than on tissue culture plates. The composition, structure, and geometry of the collagen fiber scaffolds may promote collagen synthesis within ligament analogs in vitro. Ligament analogs roughly approximate the structure and strength of native ligament tissue. Ongoing in vivo studies suggest that autogenous fibroblast-seeded ligament analogs remain viable after implantation into the knee joint. With further development, ligament analogs may be useful as implants for ACL reconstruction surgery.

We consider AR(q) models in time series with non-normal innovations represented by a member of a wide family of symmetric distributions (Student's t).Since the ML (maximum likelihood) estimators are intractable, we derive the MML (modi®ed maximum likelihood) estimators of the parameters and show that they are remarkably ef®cient. We use these estimators for hypothesis testing, and show that the resulting tests are robust and powerful.

No abstract

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.