CRYOPROTECTANT PERMEATION INTO INTACT PORCINE ARTICULAR CARTILAGE

Abstract

Effective cryopreservation of articular cartilage (AC) could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. Vitrification (a form of cryopreservation) incorporates high concentrations of cryoprotectant agents (CPAs) and rapid cooling rates to preserve cells in suspended animation without detrimental ice formation. Effective vitrification requires high concentrations of CPAs within the cartilage matrix but the time-dependent toxicity of CPAs hinders their usefulness. The objective of this experiment was determine the CPA permeation parameters for four commonly used CPAs. This data will enable the use of mathematical models to develop novel vitrification procedures to preserve AC. We hypothesised that the time dependency of CPA permeation into intact AC can be determined by exposing AC to CPAs for specific times and then allowing the CPA to be removed into a known volume of PBS, the osmolarity of which is then measured.

Full thickness 10mm diameter osteochondral dowels were harvested from the medial femoral condyles of sexually mature pigs. The dowels were randomly immersed in one of four CPAs (DMSO, propylene glycol, ethylene glycol, and glycerol) for various lengths of time (1–15 min). The cartilage was then immersed in 4ml of 1X PBS in a sealed container for twenty-four hours. The equilibrated solution was measured for osmolarity. The cartilage was weighed before and after treatment and this data was used to calculate the CPA concentration within the AC. This will be repeated in triplicate.

Preliminary results (minimum n=2) indicated a marked difference in permeation for the four CPAs. Ethylene glycol had the most rapid permeation with almost complete permeation (84%) within 15 min. Conversely, glycerol had the least permeation (29%) after 15 min most of which occurred within the first minute. DMSO (63%) and propylene glycol (40%) had intermediate rates of permeation that gradually increased over time.

Cryoprotectant agent permeation into intact porcine AC can be calculated using the method described in this study. This will allow us to successfully document the permeation kinetics of four commonly used CPAs within intact AC. This valuable data will markedly improve our ability to create novel vitrification solutions using mathematical models to add and remove CPAs to limit their toxic effects at high concentrations.