Ultrastructure of hyaline cartilage. I. A comparative study of cartilage from different species and locations, using cryofixation, freeze-substitution and low-temperature embedding techniques.

ABSTRACT

Important progress in the cryofixation of tissues has recently been made with the introduction of a new technique which permits a great reduction in the rates of ice-crystal growth and nucleation by rapid freezing under a pressure of 2 100 bar. Tissue pieces up to 0.5 mm in thickness can now be processed at a freezing rate sufficient to prevent the formation of detectable ice crystals at the ultrastructural level. In the present investigation this technique, in combination with freeze substitution and low temperature embedding was applied for ultrastructural and immunocytochemical studies of hyaline cartilage. No extraction of matrix proteoglycans was observed during the substitution procedure, and there are good reasons to believe that in preparations obtained by this technique the native state of the matrix components is preserved, since, for example, the collapse temperature of the macromolecules is not exceeded. Furthermore, no chemical fixatives or cryoprotectants are required. Ultrastructural differences in the hyaline cartilage of the growth-plate between normal rats and mice were observed, and also differences between cartilage at different locations, such as tracheal cartilage and growth-plate. Using this technique, further comparative ultrastructural studies enable us to obtain information about the macromolecular organisation of cartilage matrix under various normal and pathological conditions in vivo. In addition, using monoclonal antibodies to the main macromolecules of the matrix, it was found that this technique not only provides excellent tissue preservation but is also well suited for immunocytochemistry with colloidal gold as a marker.