Localization of freezing injury in articular cartilage

In order to improve techniques for cryopreservation of articular cartilage, a study has been carried out to assess localization of cryoinjury in intact articular cartilage. Osteochondral dowels taken from the femoral condyles of sheep were subjected to graded freezing in the presence and absence of a cryoprotectant (10% DMSO). The graded freezing technique involves slow cooling (1 degree C/min) to various subzero temperatures before either rapid warming or rapid cooling by plunging in liquid nitrogen. This protocol allows assessment of the separate effects of rapid and slow freezing which damage cells in different ways, and the effects of cryoprotectants on the different types of damage. To assay damage, thin slices of cartilage were cut with a vibratome, which allows viable cells within the matrix to be observed microscopically. Injury was assessed by staining with fluorescent dyes to indicate damage to the plasma membrane. In general, tissue response was similar to that of cell suspensions, showing at least two mechanisms of injury acting on the cells: one at slow cooling rates and another at rapid cooling rates. The primary effect of DMSO was to reduce injury due to slow cooling. When the location of injury within the tissue was examined, it was found that chondrocytes of the intermediate layer were injured more extensively than those of either the deep or superficial layers.