Dimensional Changes of Articular Cartilage during Immersion-freezing and Freeze-substitution for Scanning Electron Microscopy

In this study, the dimensional changes of articular cartilage during cryofixation (immersion-freezing) followed by freeze-substitution and critical-point-drying or tertiary-butyl alcohol drying were compared with conventional fixation techniques. The cryotechnique resulted in 11% shrinkage of the articular cartilage. During conventional chemical fixation, the sample shrank by 28%. No difference in shrinkage could be observed between the two drying methods. Morphologic examination by scanning electron microscopy (SEM) showed no difference between the rabbit tibial plateau cartilage of small excised samples and whole knee joints fixed by the cryotechnique. The collagen structure was unaffected, but the chondrocytes showed formation of segregation artifacts (ice crystal damage) compared to conventional fixation. For the study of articular cartilage with SEM, cryofixation followed by freeze-substitution is superior to conventional fixation methods with respect to specimen shrinkage, but ice crystal artifacts will be introduced into the ultrastructure. It allows immediate preservation of large samples with now damage to collagen structure.