Oxidant conditioning protects cartilage from mechanically induced damage

Abstract

Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the antioxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that antioxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant preconditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 h with tert-butyl hydrogen peroxide (tBHP) at nonlethal concentrations (25, 100, 250, and 500 microM) for a varying number of times (one, two, or four) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post-compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 microM tBHP applied four times. RNA analysis revealed significant effects of 100 microM tBHP on gene expression. Catalase, hypoxia-inducible factor-1alpha (HIF-1alpha), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated four times with 100 microM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase-3 (MMP-3) expression. These findings demonstrate that repeated exposure of cartilage to sublethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide-induced changes in gene expression that could render chondrocytes more resistant to oxidative damage.

Figure 1. Fluorescent Viability Stains

The micrographs show representative sagittal sections of cartilage stained with calcein AM and ethidium homodimer (A,B), or DAPI (C,D) 24 hours after compression. Cartilage was either untreated (A,C) or treated 4 times with 100 µM tBHP (B,D). The bar in D represents 500 µm.

Figure 2. Effects of Oxidant Pre-conditioning on Chondrocyte Viability

Viability was measured in compressed explants (A) and uncompressed explants (B) after treatment with the indicated doses of tBHP. Explants were treated with tBHP once (white columns), twice (grey columns), or 4 times (black columns) prior to compression. Columns and bars show means and standard deviations based on 6 explants. Symbols above the columns indicate significant differences versus 0 tBHP control for 1 day treatment (*), 2 days of treatment (^), and 4 days of treatment (#).

Figure 3. Effects of Oxidant Pre-conditioning on Lactate Production

Lactate was measured in the culture medium from compressed explants (A) and non-compressed explants (B) treated once (white columns), twice (grey columns), or 4 times (black columns) with the indicated doses of tBHP. Symbols above the columns indicate significant differences versus 0 tBHP control for 1 day treatment (*), 2 days of treatment (^), and 4 days of treatment (#). The columns and bars represent means and standard deviations based on 9 explants.

Figure 4. Effects of Oxidant Pre-conditioning on Glycosaminoglycan Content and Release in Compressed Explants

The glycosaminoglycan (GAG) content of explants (A) and proteoglycan released to the culture medium (B) was measured after compression in explants that had been treated once (white columns), twice (grey columns), or 4 times (black columns) with the indicated doses of tBHP. Columns and error bars show means and standard deviations based on analysis of 6 explants. Symbols above the columns indicate significant differences versus 0 tBHP control for 1 day treatment (*), 2 days of treatment (^), and 4 days of treatment (#).

Figure 5. Effects of Oxidant Pre-conditioning on Gene Expression

Real time PCR analysis of gene expression is shown for explants treated with 100 µM tBHP once, twice, or 4 times. Expression levels for the indicated genes were normalized to untreated controls (Relative Expression). Columns and error bars show means and standard deviations based on 4 explants.