Vitrification of porcine intact femoral condyle through different protocol lengths and loading strategies

Cryopreservation of articular cartilage (AC) allografts is a potential candidate to solve the shortage of fresh osteochondral allografts to treat large AC defects. This technique requires optimization to address issues related to the actual process of cryopreservation and the translation of the technology to tissue banks. In the present study, we compared different vitrification protocols consisting of multiple cryoprotective agent (CPA) loading timings and loading strategies to achieve successful vitrification-rewarming protocols for porcine intact femoral condyles. Porcine medial femoral condyles (n = 6) were used to test 5 vitrification protocols differing in lengths of time and the method of CPA loading. The effects of these protocols on post-warming normalized chondrocyte viability, metabolic activity, and tissue cracking frequency were documented. We demonstrated that all 5 CPA loading-vitrification protocols resulted in high normalized chondrocyte viability (>88 % of Fresh control) post vitrification in porcine intact femoral condyles. Moreover, the Room Temperature Gel (RG) protocol showed high viability and metabolic activity of chondrocytes post-warming, and also decreased cracking frequency in AC after rewarming of vitrified intact femoral condyles. To facilitate translation of this technology to tissue banks, we showed that by shortening (total time of 15 min in Protocol RG) or combining our established Protocol 8 (430 min) with a hydrogel, including use of cryobags in liquid nitrogen vapour as the conventional storage method in tissue banks, we could introduce two reproducible protocols for clinical applications involving vitrified osteochondral tissue.