CRYOPROTECTANT PENETRATION INTO INTACT PORCINE ARTICULAR CARTILAGE

Abstract

Cryopreserving agents (CPAs) can cryopreserve articular cartilage (AC) but their use is limited due to cellular toxicity. This study examined the time-dependent penetration of multiple CPAs into intact porcine AC. Porcine AC was immersed in CPAs for various amounts of time at three temperatures (4°C, 22°C, and 37°C). The results demonstrated an initial sharp rise in CPA concentration within the matrix for dimethyl sulfoxide and propylene glycol with maximum concentration after three to six hours. The trehalose and glucose concentration increased minimally even after twenty-four hours of exposure. The information from this study provides insight into the penetration kinetics of cryoprotectant agents into AC.

This study examined the time-dependent penetration of cryoprotectant agents (CPAs) [dimethyl sulfoxide (DMSO), propylene glycol (PG), trehalose and glucose] into intact porcine articular cartilage (AC).

Penetration of DMSO and PG into AC was rapid but time and temperature dependent while trehalose and glucose had poor penetration.

The information gathered from this study can determine concentrations of CPAs within the cartilage matrix to create cryopreservation/vitrification solutions while minimizing toxicity.

The results demonstrated there was a sharp rise in the CPA concentration within 15–30min exposure to DMSO and PG and the concentration peaked after three to six hours exposure at a concentration approximately 90% of the original concentration (6.5 molar). This was temperature dependent with slower penetration at lower temperatures. The trehalose and glucose had very poor penetration into the matrix at all temperatures, with a maximum penetration of 2% of the original concentration.

Dowels of porcine AC (10mm diameter) were immersed in high concentration of each CPA for various amounts of time (0min, 15min, 30min, 60min, 3hr, 6hr, and 24hr) at three temperatures (4°C, 22°C, and 37°C). The cartilage was excised and the amount of cryoprotectant within the matrix determined.

Successful cryopreservation of AC could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. However, successful cartilage cryopreservation is limited by chondrocyte death and matrix disruption due to inadequate CPA penetration.