Improved structural preservation of high-pressure frozen cartilage

Abstract

Adult cartilage is a unique tissue in that it is avascular and lacks innervation. The chondrocytes are embedded in an extracellular matrix which in most cases occupies 60 to 90% of the tissue volume. The ultrastructure and composition of rat growth plate cartilage has been investigated (1). It was found that during conventional chemical fixation of me tissue (2), about 60% of the extracellular matrix proteoglycans were lost (3). This artefact can be prevented if proteoglycans are precipitated by cationic dyes (eg. ruthenium hexaamine trichloride) during fixation (4) which forms relatively large precipitates by rendering high resolution imaging impossible. High pressure frozen extracellular matrix revealed a fine meshwork which was attributed to proteoglycan distribution (5). A meshwork due to segregation during freezing was not seen in recent experiments where adaequately frozen cartilage was investigated.200μm thick sections of rat growth plate cartilage were excised in a bath of hexadecene (6). The sections were again processed under hexadecene, firstly punched (diameter 1.7mm) and then transferred to an aluminum sandwhich (corresponding in size to the sample).