Using engineering models to shorten cryoprotectant loading time for the vitrification of articular cartilage

Osteochondral allograft transplantation can treat full thickness cartilage and bone lesions in the knee and other joints, but the lack of widespread articular cartilage banking limits the quantity of cartilage available for size and contour matching. To address the limited availability of cartilage, vitrification can be used to store harvested joint tissues indefinitely. Our group's reported vitrification protocol [Biomaterials 33 (2012) 6061-6068] takes 9.5 h to load cryoprotectants into intact articular cartilage on bone and achieves high cell viability, but further optimization is needed to shorten this protocol for clinical use. Herein, we use engineering models to calculate the spatial and temporal distributions of cryoprotectant concentration, solution vitrifiability, and freezing point for each step of the 9.5-h protocol. We then incorporate the following major design choices for developing a new shorter protocol: (i) all cryoprotectant loading solution concentrations are reduced, (ii) glycerol is removed as a cryoprotectant, and (iii) an equilibration step is introduced to flatten the final cryoprotectant concentration profiles. We also use a new criterion-the spatially and temporally resolved prediction of solution vitrifiability-to assess whether a protocol will be successful instead of requiring that each cryoprotectant individually reaches a certain concentration. A total cryoprotectant loading time of 7 h is targeted, and our new 7-h protocol is predicted to achieve a level of vitrifiability comparable to the proven 9.5-h protocol throughout the cartilage thickness.