Application of the ferrocyanide-reduced osmium method for mineralizing cartilage: further evidence for the enhancement of intracellular glycogen and visualization of matrix components.

The ferrocyanide-reduced osmium (FRO) fixation method was applied to neonatal mouse mandibular condylar cartilage for its processing for electron microscopy. The results were compared to those obtained by the conventional glutaraldehyde-osmium tetroxide fixation method. Three different stages in the life cycle of condylar cartilage cells were examined. FRO enabled the visualization of delicate fibrillar mesh in the matrix of all three zones of the cartilage, resulting in a dense appearance of the intercellular matrix. The classical stellate shape of matric granules seen in cartilage fixed with glutaraldehyde-osmium tetroxide was not observed in FRO-processed tissues. Chondrocytes that were FRO-processed almost entirely filled their lacunar space. In their pericellular area, fibrillar material and electron-dense aggregates could be demonstrated by the FRO method. As a conclusion of this study, it is recommended to supplement a conventional protocol with the FRO fixation method for routine and research purposes.