Intramatrix events during cryopreservation of porcine articular cartilage using rapid cooling

Abstract

Cryopreservation of articular cartilage may improve long-term transplantation results if cell and matrix integrity can be maintained. This study examined intramatrix events in intact porcine articular cartilage that occurred during a rapid-cooling technique with various concentrations of dimethyl sulfoxide (DMSO) (1, 3, 5, 6 and 7 M). Thermocouples were inserted into the solution and in the cartilage matrix to record the temperature during rapid cooling. In addition, scanning electron microscopy of freeze-substituted samples was performed and quantitatively evaluated for the areas representing ice in the matrix. The results of this study showed that low concentrations of DMSO resulted in the largest temperature gradient between the matrix and the surrounding solution, which occurred near the freezing point of the cryoprotectant solution. At higher concentrations of DMSO, the peak temperature gradient occurred near the glass transition temperature. The temperature measurements suggested that a significant amount of ice formed within the matrix at lower DMSO concentrations. At higher DMSO concentrations that resulted in vitrification of the external solution, there was evidence of some ice in the matrix. The scanning electron micrographs demonstrated significantly more matrix disruption (likely due to ice formation) ( P<0.02) in the lower DMSO concentrations (1 and 5 M) while the 6 M DMSO concentration demonstrated minimal matrix disruption. Cryopreservation of articular cartilage with a rapid-cooling technique and high concentrations of DMSO resulted in partial vitrification of the matrix and significantly less matrix disruption. It appears that successful cryopreservation of viability and function in articular cartilage will require high concentrations of cryoprotectants and rapid cooling.