Effectiveness of Clinical-Grade Chondroitin Sulfate and Ascorbic Acid in Mitigating Cryoprotectant Toxicity in Porcine Articular Cartilage

High concentrations of cryoprotective agents (CPAs) are required to achieve successful vitrification of articular cartilage; however, CPA cytotoxicity causes chondrocyte death. To reduce CPA toxicity, supplementation with research-grade additives, in particular chondroitin sulfate (CS) and ascorbic acid (AA), have previously been shown to improve chondrocyte recovery and metabolic function after exposure to CPAs at hypothermic conditions. However, it is necessary to evaluate the pharmaceutical equivalent clinical grade of these additives to facilitate the supplementation of additives into future vitrification protocols, which will be designed for vitrifying human articular cartilage in tissue banks. We sought to investigate the effectiveness of clinical-grade CS, AA, and N-acetylcysteine (NAC) in mitigating toxicity to chondrocytes during CPA exposure and removal, and determine whether a combination of two additives would further improve chondrocyte viability. We hypothesized that clinical-grade additives would exert chondroprotective effects comparable to those of research-grade additives, and that this protective effect would be enhanced if two additives were combined when compared with a single additive. The results indicated that both clinical-grade and research-grade additives significantly improved cell viability (p < 0.10) compared with the negative control (CPA with no additives). CS, AA, and NAC+AA increased cell viability significantly (p < 0.10) compared with the negative control. However, NAC, NAC+CS, and CS+AA did not improve cell viability when compared with the negative control (p > 0.10). We demonstrated that supplementation with clinical-grade CS or AA significantly improved chondrocyte viability in porcine cartilage subjected to high CPA concentrations, whereas supplementation with clinical-grade NAC did not benefit chondrocyte viability. Supplementation with clinical-grade additives in CPA solutions can mitigate CPA toxicity, which will be important in translating previously developed effective protocols for the vitrification of articular cartilage to human tissue banks.

Automated summary

Both clinical-grade and research-grade additives significantly improved cell viability compared with the negative control. CS, AA, and NAC+AA also increased cell viability significantly compared with the negative control. However, NAC, NAC+CS, and CS+AA did not show improvement in cell viability compared with the negative control. Supplementation with clinical-grade CS or AA significantly improved chondrocyte viability in porcine cartilage at high CPA concentrations, while supplementation with clinical-grade NAC did not have a beneficial effect. Supplementing with clinical-grade additives in CPA solutions can mitigate CPA toxicity and is crucial for developing effective vitrification protocols for human tissue banks.