Abstract
The growing need for improved methods of viable tissue cryopreservation has stimulated debate regarding the relative merits of traditional freezing methods versus ice-free vitrification methods. Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, full-thickness rabbit femoral head articular cartilage was used to compare a freezing method of cryopreservation and an ice-free, vitrification method of cryopreservation with fresh controls in vitro. Chondrocyte viability was determined in vitro using vital fluorescent staining with calcein-acetoxymethyl ester and an oxidation-reduction assay with alamarBlue™ (Accumed International, Westlake, Ohio). Cryosubstitution with 1% osmium tetroxide in 100% methanol was used to detect ice during cryopreserved storage. Cryosubstitution studies of frozen and vitrified articular cartilage pieces revealed negligible ice in the vitrified specimens and extensive ice formation in frozen specimens with the exception of the articular surface. Frozen specimens retained <13% viability. In contrast, postvitrification tissue had >80% of control viability. The distribution of ice observed in cartilage cryopreserved by freezing suggests that ice formation was the cause of poor cryopreserved cartilage viability reported in the literature. The results demonstrate the feasibility of vitrification as a storage method for cartilaginous tissues.
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