Osteoarthritis

P137 - Progress in Cartilage Cryopreservation Using Nanowarming and Ice-free Vitrification

Abstract

Purpose

Resurfacing of articular cartilage with cold stored osteochondral allografts is employed clinically for repair of trauma and osteoarthritis-induced articular cartilage surface damage. Chondrocyte viability of transplanted articular cartilage is accepted as one of the determinants of outcome following osteochondral allograft transplantation. My team has previously developed an ice-free vitrification method of cryopreservation that maintains excellent chondrocyte viability in small articular cartilage samples. However, it had not previously been possible to rewarm larger samples due to ice nucleation during rewarming that results in loss of chondrocyte viability. The innovation in this presentation relates to a new rewarming method that does not have the limitations of boundary convection warming called nanowarming using magnetic nanoparticles (Figure).nanowarming slide-2.jpg

Methods and Materials

Pig knees were procured post-mortem from Yorkshire cross-farm pigs (~25Kg) at a slaughterhouse. Vitrification was initiated after gradual infiltration with a formulation consisting of 12.6 M of cryoprotectants (4.65M DMSO, 4.65M formamide, and 3.31M 1,2-propanediol in EuroCollins solution) at 4°C. Precooled dilute vitrification solutions were added in sequential steps of increasing concentration on ice. Two mgs/mL Fe magnetic nanoparticles were added in a final step at -10°C. Samples were cooled to -135°C and and stored in a mechanical freezer. Fresh, convection warmed and nanowarmed samples were evaluated by metabolic assay with alternative assays to confirm metabolic activity outcomes. Biomaterial assys were also performed.

Results

Nanowarming by inductive heating system at 500 Amps and 234kHz warmed from -135°C to -30°C in 80 seconds. Longer times and higher settings resulted in overheating and decreased chondrocyte viability. Statistically significant viability (p<0.05) was obtained using nanowarming compared with convection warming using the 12.4M cryoprotectant formulation. Lower cryoprotectant concentrations resulted in dose dependent reductions in viability.

Conclusion

The combination of ice-free cryopreservation by vitrifcation and nanowarming results in the possibility of banking osteocartilage grafts. In vivo porcine studies are in progress.

Collapse