Objective: Oxidative stress occurs when the metabolic balance of a cell is disrupted through exposure to excess pro-oxidant. Whilst it is known that unregulated production or exposure to exogenous sources of pro-oxidants induces chondrocyte cell death and degrades matrix components in vitro, relatively little is known of the effects of pro-oxidants on articular cartilage in situ. The objective of this study was to determine if a single exposure to the pro-oxidant hydrogen peroxide (H(2)O(2)) induces a degenerative phenotype.
Methods: Articular cartilage explants were obtained from skeletally mature bovine steers and exposed to a single dose of hydrogen peroxide (0.1-1.0 mM) and cultured for up to 21 days. Cell death, and sulfated glycosaminoglycan loss into the medium and gene expression were quantitatively determined. Adoption of an abnormal chondrocyte phenotype was analyzed through the expression of 3B3(-), nitrotyrosine and procollagen type IIA epitopes in cartilage explants.
Results: Cell death occurred primarily at the surface zone of cartilage in a dose-dependent manner in H(2)O(2) treated explants, and supplementation of standard serum-free medium with insulin-selenium-transferrin significantly reduced cell death (>fourfold). Nitric oxide synthase-2 gene expression and proteoglycan loss increased in oxidant treated explants in a concentration-dependent manner. Antibody labeling to 3B3(-), procollagen type IIA and nitrotyrosine was present in all treated explants but absent in untreated explants.
Conclusions: This study demonstrates that a single exposure to high levels of pro-oxidant causes the expression of genes and antibody epitopes that are associated with early degenerative changes observed in experimental osteoarthritis.