N-acetylcysteine inhibits post-impact chondrocyte death in osteochondral explants

Abstract

Background: Chondrocyte death has been linked to injury-induced oxidative damage, suggesting that antioxidants could substantially improve viability. However, since reactive oxygen species play roles in normal physiology, there are concerns that antioxidants may have deleterious side effects. To address these issues, we studied the effects of N-acetylcysteine, a potent free radical scavenger, on chondrocyte viability and cartilage proteoglycan content in an in vitro cartilage injury model. We hypothesized that treatment with N-acetylcysteine soon after an impact injury would have significant chondrocyte-sparing effects and would prevent injury-induced proteoglycan losses.

Methods: Bovine osteochondral explants were subjected to a single impact load with use of a drop-tower device. Chondrocyte viability was measured at multiple time points post-impact with use of fluorescent probes and confocal microscopy. Forty-eight hours after impact, the effects on viability of immediate post-impact treatment with N-acetylcysteine were compared with the effects of the caspase inhibitor N-CBZ-Val-Ala-Asp(O-Me) fluoromethyl ketone and those of the cell-membrane-stabilizing surfactant poloxamer 188. The effect of N-acetylcysteine on proteoglycan content was determined at seven and fourteen days post-impact.

Results: Chondrocyte viability declined sharply within an hour and reached a steady state within six to twelve hours after impact. Immediate treatment with N-acetylcysteine doubled the number of viable chondrocytes assayed forty-eight hours after impact, and this effect was significantly greater than that of N-CBZ-Val-Ala-Asp(O-Me) fluoromethyl ketone. Even when N-acetylcysteine treatment was delayed for up to four hours after injury, it still had significant positive effects on cell viability at forty-eight hours. Moreover, N-acetylcysteine treatment significantly improved proteoglycan content at the impact sites at both seven and fourteen days after injury.

Conclusions: Treatment with N-acetylcysteine soon after a blunt impact injury can reduce chondrocyte death and proteoglycan loss measured seven to fourteen days after injury.

Fig. 1

Structural damage from impact injuries. Micrographs show sections from a nonimpacted control cartilage explant (A), a 7-J/cm²-impact site (B), and a 14-J/cm²-impact site (C) (safranin-O and fast green). The bar in A is 0.5 mm long.

Fig. 2

Laser confocal imaging of viability stains. A nonimpacted control cartilage explant (A), an explant subjected to a 7-J/cm² impact (B), and an explant subjected to a 7-J/cm² impact and treated immediately with N-acetylcysteine were stained with calcein AM (green) and ethidium homodimer (red) to label live and dead cells, respectively. The micrographs are z-axis reconstructions of images made at the surface and to 200 μm beneath the surface at intervals of 40 μm The bar in C represents 200 μm.

Fig. 3

Time course of chondrocyte death after impact injury. Cell viability was evaluated in nonimpacted control explants (open triangles) and explants subjected to an impact of 7 J/cm² (open circles) or 14 J/cm² (filled squares). Each symbol represents the average of the results for three explants. The error bars indicate standard deviations.

Fig. 4

Effects of drug treatment on post-impact chondrocyte viability. Cell viability was evaluated in nonimpacted control explants (Con), explants subjected to an impact of 7 J/cm² without drug treatment (No Tx), and explants subjected to an impact of 7 J/cm² and treated immediately with N-acetylcysteine (NAC), tocopherol (Toc), poloxamer 188 (P188), or z-VAD-fmk (Z-Vad). Columns and error bars represent the means and standard deviations based on six explants.

Fig. 5

Effect of delaying N-acetylcysteine treatment. Explants were subjected to an impact of 7 J/cm² and were left untreated (No Tx) or were treated with N-acetylcysteine immediately (0) or after a delay of one, two, three, four, or twelve hours. Columns and error bars indicate means and standard deviations based on three explants.

Fig. 6

Effects of N-acetylcysteine on glycosaminoglycan (GAG) content seven and fourteen days after injury. Glycosaminoglycan content within each impact site was normalized to the glycosaminoglycan content of an adjacent, nonimpacted control site (percent of the control value). Results are shown for explants that were treated with N-acetylcysteine (NAC) and for untreated controls (Con). The columns and error bars indicate means and standard deviations based on six explants. The numbers above the bars are p values, which indicate significant differences between the N-acetylcysteine-treated and untreated specimens.