Long-term storage of articular cartilage (AC) remains challenging due to poor post-thaw viability. An initial step towards addressing this issue is characterizing cryoprotectant (CPA) transport, since ensuring adequate CPA equilibration throughout the tissue offers protection during cooling. This study takes a systematic approach in determining CPA transport rates through bovine AC and uses that information in mathematical models to determine CPA equilibration times.
Diffusion of high concentration single (6.9 M dimethyl sulfoxide (DMSO)) and multi-component CPA solutions (VS55, 3.1 M DMSO+2.2 M 1,2-propanediol (PD)+3.1 M formamide (FM)) was measured through AC using (1)H nuclear magnetic resonance (NMR) imaging and localized spectroscopy, respectively. Using experimentally calculated effective diffusivities, diffusion models describing CPA transport through the tissue matrix and across chondrocyte membranes were combined to design a CPA addition and removal scheme for a cartilage plug of clinically relevant dimensions.
(1)H NMR imaging and localized spectroscopy experiments suggested that the permeation of CPAs through AC (5 mm diameter, 5-10 mm in thickness) took on the order of 4 h for full equilibration at 22 degrees C. Imaging clearly showed the permeation of DMSO into cartilage over time and localized spectroscopy was able to distinguish the permeation rates of the individual VS55 components and water. Experimentally measured diffusivity values were used in CPA addition/removal simulations with a cartilage plug of clinically relevant dimensions (5 mm diameter, 2 mm in thickness). Results suggested a multi-step approach for adding and removing high concentration CPAs, with the addition and removal each taking approximately 2 h to complete.
This study provides a foundation for designing CPA addition and removal protocols for effective long-term storage of cartilage tissue using a novel approach to measure CPA permeation.