Influence of specimen preparation on the identification of phospholipids by the phospholipase A2-gold method in mineralizing cartilage and bone

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A₂ (PLA₂)-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resin-embedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA₂, which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mineralization suggests some hypotheses on the possible involvement of these molecules in the mineral-phase deposition process.