Improved ultrastructural preservation of epiphyseal chondrocytes by the freeze-substitution method

The ultrastructure of epiphyseal chondrocytes was studied following quick-freezing and freeze-substitution, and was compared to that of cells fixed with aqueous aldehydes. The former approach provided an improved ultrastructural preservation whereby every type of chondrocyte exhibited a smoother cell contour. The plasma membrane as well as intracytoplasmic membranes revealed a trilaminar substructure. The intracytoplasmic ground substance was composed of flocculent materials which were in direct contact with the inner leaflet of the plasma membrane. Within the extracellular matrix the proteoglycan network adhered to the outer leaflet of the plasma membrane. Whenever cellular shrinkage took place, the flocculent matrix within the cytoplasm and the proteoglycan network in the pericellular matrix disappeared. The contents of the RER, the Golgi apparatus, and the intracellular vesicles and vacuoles were well retained. In the proliferative zone, the Golgi saccules of young cells contained a thread-like structure showing a clear periodicity. The cytoplasmic vesicles and vacuoles showed marked variation in their electron density. Intramitochondrial granules were sensitive to aqueous treatments, as evidenced by the observation that they disappeared after either floating on water or staining with aqueous solution. In the calcifying zone, mitochondrial granules were noted within hypertrophic chondrocytes, a feature that was not observed following conventional processing. Cytoskeletal elements were well preserved in all types of cells. A dense microfilamentous network occupied the pericellular cytoplasm. Bundles of microfilaments were seen in the cellular peripheral processes. Microtubules were distributed throughout the cytoplasm, and the Golgi complex was intimately associated with the microtubule network; it appears that the secretory processes are involved with the microtubules.