Effects of increasing concentrations of dimethyl sulfoxide during cryopreservation of porcine articular cartilage

Cryopreservation of intact articular cartilage (AC) may provide a source of tissues that will enhance long-term results of osteochondral transplantation for large, unconfined joint defects. This study investigated the effects of increasing dimethyl sulfoxide (Me₂SO) concentrations on chondrocyte recovery in intact porcine AC after a rapid-cooling cryopreservation technique. Osteochondral dowels 10 mm in diameter were harvested, immersed in various concentrations of Me₂SO (1 M, 3 M, 5 M, 6 M, and 7 M), and cryopreserved using a rapid-cooling technique by plunging into liquid nitrogen after initial cooling in an ice-water bath. Fresh controls, toxicity controls, and an experimental group were compared. Slices 70 μm thick (divided into three layers - superficial, middle, and deep) were evaluated after membrane integrity dye staining by fluorescence microscopy. High concentrations of Me₂SO (6 M and 7 M) demonstrated significant protection from cryoinjury, likely due to vitrification, but this was compromised by significant cellular toxicity. Low concentrations of Me₂SO (1 M and 3 M) exhibited significantly less cellular toxicity but did not protect against cryoinjury. The superficial layer demonstrated significantly increased cellular toxicity compared with the deeper layers at all Me₂SO concentrations. Me₂SO at 6 M demonstrated the highest recovery of chondrocytes after the cryopreservation procedure, with 42% recovery compared with fresh controls (59% recovery compared with toxicity controls), with the middle layer of the sample demonstrating the optimal balance between cellular toxicity and cryoprotection with an intact cell recovery of 56% (compared with fresh controls). This study demonstrated that concentrations of Me₂SO sufficient to result in vitrification have significant potential to successfully cryopreserve intact AC. Further investigation is required to limit potential causes cell loss, including cryoprotectant toxicity, devitrification, and heat transfer.