Ultrastructural changes of articular cartilage chondrocytes associated with freeze-thawing

In an attempt to justify the use of cryopreserved versus fresh articular cartilage (AC) allografts, we used transmission electron microscopy (TEM) to study the ultrastructure of fresh versus frozen-thawed AC with or without a dimethyl sulfoxide (DMSO) treatment. AC explants were cut aseptically from the femoral condyles of healthy, mature rabbits when they were killed. Half of all explants were incubated in Ham's F-12 medium, supplemented with antibiotics and with or without 7.5% DMSO, frozen to -80 degrees C, stored for 24 h, and thawed rapidly. These, and the control explants, were fixed with glutaraldehyde, paraformaldehyde, and acrolein in cacodylate buffer. Sections were stained for acid phosphatase (APase), postfixed with osmium, embedded, and examined under TEM. The typical organization of the matrix and the cells was noted in control sections. The chondrocytes contained intact nuclei, organelles, and discrete plasma membrane. Although some endoplasmic reticula and nuclear membrane appeared intact, distinct ultrastructural changes were observed in frozen-thawed samples treated with DMSO. These changes included condensation of chromatin, large lipid droplets, partly disrupted plasma membrane, and pericellular precipitation of APase-positive crystalites. In sections not treated with DMSO, the cytoplasm was extensively vacuolated and no distinct organelles could be detected in the chondrocytes. Little difference was noted between the matrix organization of fresh or frozen-thawed samples. Our results suggest that distinct ultrastructural changes occur in the chondrocytes following freeze-thawing of intact AC and that DMSO pretreatment may contribute to improvement in the cryopreservation of AC.