Antioxidants block cyclic loading induced chondrocyte death

Abstract

Articular cartilage in congruous joints benefits from the moderate stresses and strains associated with normal cyclic loading. However, loading of joints with surface incongruities can lead to local stress and strain elevation at "step-off' sites where cartilage is not fully buttressed b ysurrounding matrix. Excessive stresses and strains predicted to occur at such sites may induce apoptosis, a process thought to promote cartilage degeneration and osteoarthritis (OA) through chondrocyte attrition. We hypothesized that the induction of apoptosis is mediated by oxidants, and that antioxidants can reduce elevated stress-induced chondrocyte attrition. To test this we exposed cylindrical cartilage explants from human articular cartilage to radially unconfined cyclic axial compression (3600 cycles, 1 Hz, 50% duty cycle) using two different physiologic loads (2MPa and 5 MPa). We found that 30% of chondrocytes in the superficial zone died within 24 hours of exposure to loading with 5 MPa axial compression, whereas mortality was limited to less than 15% with 2 MPa axial compression. Similarly, lactate accumulation in the medium was suppressed by compression with 5 MPa, but not 2 MPa. Approximately 80% of cell death induced by 5 MPa compression was blocked by pre-incubation of the explants in a variety of anti-oxidants including vitamin E, n-acetyl cysteine (NAC), and a superoxide dismutase mimetic (SOD). SOD and NAC also prevented the suppression of lactate secretion after 5 MPa compression. These observations support the hypothesis that the harmful effects of abnormal cyclic loading are mediated by oxidants and suggest that treatments to prevent OA may include methods of minimizing oxidative damage to chondrocytes.

Figure 1

Explant Sstrains During Axial Ccompression. Accumulated strains at maximum compression (MAX) and at minimum compression (MIN), and peak-to-peak strains (the difference between the maximum and minimum stains for each cycle) are plotted. (A) 2 MPa compression. (Bb) 5 MPa compression. Each panel shows strains over a 3600 cycle run for a typical explant.

Figure 2

Effects of Axial Ccompression on Cchondrocyte Viability. Rrepresentative photomicrographs show living chondrocytes (green) or dead chondrocytes (red) in explants that were either unloaded (A), or exposed to 2 MPa axial compression (Bb), or 5 MPa axial compression (Cc). Tthe surface of the explant faces upward in each panel. bar in panel Cc represents a length of 500 µm. (D) columns show average viabilities (mean and standard error based on 6 explants) in the superficial (Ss), middle (M) and deep (D) zones.

Figure 3

Cchondrocyte Apoptosis Induced by 5 MPa Axial Ccompression. Ccompressed (5 MPa, 5 MPa+NACc) and uncompressed controls (control) were stained using the TUtuNELl reaction. Tthe number of positive cells in the superficial zone (Ss), middle (M) or deep zone (D) were counted and normalized to the total number of cells in the zone (% Positive). Ccolumns show the mean and standard errors based on analysis of 6 explants per group. Asterisks indicate significant differences between the 5 MPa group and the control or 5MPa+NACc group.

Figure 4

Effects of Antioxidants and Ll-NAME on Chondrocyte Viability. (A) 2 MPa axial compression. (B) 5 MPa axial compression. Columns represent means and error bars show standard errors for 6 explants.

Figure 5

Effects of Ccompression and Antioxidants and on Llactate Production. Tthe concentration of lactate in culture medium (mM) was measured in unloaded controls (white columns) or 24 hours after compression with 2 MPa (hatched columns), or 5 MPa (black columns). Explants were not pre-treated (Ccon) or were pre-treated with SsOD, NACc, or catalase (CcATt). Ccolumns and bars show means and Standard errors based on 6 explants.