Morphological investigation of epiphyseal cartilage after glutaraldehyde-malachite green fixation

The glutaraldehyde-malachite green (GlMG) fixation has been reported to allow preservation of tissue lipids, mainly acidic phospholipids, that in the absence of malachite green are lost in the glutaraldehyde solution. This method has been used for the morphological study of calcifying epiphyseal cartilage, with particular reference to the presence of lipids in chondrocytes, cartilage matrix and calcification nodules. Both light and electron microscopy show that after GlMG-osmium fixation the chondrocytes contain cytoplasmic dense bodies. These have the same morphology as those found with the same method in other cells and tissues and considered to be lipid globules. However, they are not modified by treatment of GlMG-fixed specimens with chloroform-methanol. Formic acid decalcification induces partial solubilization of these bodies, whereas EDTA decalcification leaves them unchanged. They are found also in the pericellular space and in calcifying matrix. GlMG fixation improves the preservation of the crystal-associated organic structures (crystal ghosts) when the cartilage is decalcified before embedding. When these structures are demonstrated by the post-embedding decalcification and staining method, their morphology is similar to that found in controls. Moreover, GlMG fixation demonstrates the presence of typical rod-like, beaded structures, probably corresponding to proteoglycans, on the outer surface of the chondrocyte membrane. Although composition and role in calcification of the dense bodies remain uncertain, the important physiological implications that can be drawn from their presence in chondrocytes and cartilage matrix require further investigation.