High concentrations of cryoprotective agents (CPA) are required during articular cartilage cryopreservation but these CPAs can be toxic to chondrocytes. Reactive oxygen species have been linked to cell death due to oxidative stress. Addition of antioxidants has shown beneficial effects on chondrocyte survival and functions after cryopreservation. The objectives of this study were to investigate (1) oxidative stress experienced by chondrocytes and (2) the effect of antioxidants on cellular reactive oxygen species production during articular cartilage exposure to high concentrations of CPAs. Porcine cartilage dowels were exposed to a multi-CPA solution supplemented with either 0.1 mg/mL chondroitin sulfate or 2000 μM ascorbic acid, at 4 °C for 180 min (N = 7). Reactive oxygen species production was measured with 5 μM dihydroethidium, a fluorescent probe that targets reactive oxygen species. The cell viability was quantified with a dual cell membrane integrity stain containing 6.25 μM Syto13 + 9 μM propidium iodide using confocal microscopy. Supplementation of CPA solutions with chondroitin sulfate or ascorbic acid resulted in significantly lower dihydroethidium counts (p < 0.01), and a lower decrease in the percentage of viable cells (p < 0.01) compared to the CPA-treated group without additives. These results indicated that reactive oxygen species production is induced when articular cartilage is exposed to high CPA concentrations, and correlated with the amount of dead cells. Both chondroitin sulfate and ascorbic acid treatments significantly reduced reactive oxygen species production and improved chondrocyte viability when articular cartilage was exposed to high concentrations of CPAs.

中文翻译：

---

抗氧化添加剂可减少暴露于冷冻保护剂期间关节软骨中活性氧的产生

关节软骨冷冻保存期间需要高浓度的冷冻保护剂 (CPA)，但这些 CPA 可能对软骨细胞有毒。由于氧化应激，活性氧与细胞死亡有关。添加抗氧化剂已显示出对冷冻保存后软骨细胞存活和功能的有益影响。本研究的目的是调查 (1) 软骨细胞经历的氧化应激和 (2) 关节软骨暴露于高浓度 CPA 期间抗氧化剂对细胞活性氧产生的影响。将猪软骨销暴露于补充有 0.1 mg/mL 硫酸软骨素或 2000 μM 抗坏血酸的多 CPA 溶液中，4 °C 下 180 分钟（N = 7）。用 5 μM 二氢乙锭测量活性氧的产生，一种靶向活性氧的荧光探针。使用共聚焦显微镜，使用含有 6.25 μM Syto13 + 9 μM 碘化丙啶的双细胞膜完整性染色剂对细胞活力进行定量。与不含添加剂的 CPA 处理组相比，用硫酸软骨素或抗坏血酸补充 CPA 溶液导致二氢乙锭计数显着降低（p < 0.01），活细胞百分比降低（p < 0.01）更低。这些结果表明，当关节软骨暴露于高浓度的 CPA 时，会诱导活性氧的产生，并与死细胞的数量相关。