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

Abstract

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 A(2) (PLA(2))-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 PLA(2), 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.

Biological Calcification

“…Histochemical and transmission electron microscope investigations confirmed the presence of lipids in areas of early bone calcification and at the calcification front (Ponlot 1958;Ngoma and Haumont 1974;Bonucci and Silvestrini 1995;Zini et al 1996), so supporting the view that acidic lipids may be involved in calcification (discussed by Vogel and Boyan-Salyers 1976;Dziak 1992). Studies carried out with the antibody MC22-33F, which displays a specific reaction with phosphatidylcholine, sphingomyelin and dimethylphosphatidylethanolamine, showed a strong immunoreaction along the osteoblast membrane, in the interfibrillary spaces of the osteoid border, in matrix vesicles and in calcification nodules and their peripheral zone (Fig.…”

“…Zini N, Sabatelli P, Silvestrini G, Bonucci E, Maraldi NM (1996) Influence of specimen preparation on the identification of phospholipids by the phospholipase A 2 -gold method in mineralizing cartilage and bone. Histochem Cell Biol 105:283-296 8 Calcifying Matrices: Dentin and Cementum…”

Calcifying Matrices: Cartilage

“…Lipid accumulation in calcified cartilage areas is also shown by staining with sudan black after pyridine extraction (Bonucci et al 1978), and using the MC22-33F antibody, which displays a specific reaction with phosphatidylcholine, sphingomyelin and dimethylphosphatidylethanolamine: the immunoreaction was found in the cytoplasm of maturing, hypertrophic and, to a lesser extent, proliferating and degenerating chondrocytes, at the periphery of calcification nodules, and at the periphery of the calcified matrix (Bonucci et al 1997). The presence of phospholipids in epiphyseal cartilage was confirmed by combining malachite green fixation with phospholipase A 2 -gold complex, a method which localizes phospholipids: the labeling intensity was higher at the periphery of the calcification nodules than in the central, fully calcified matrix (Silvestrini et al 1996;Zini et al 1996). The proteolipid/dry weight and proteolipid/total lipid ratios were greater in the lower than in the upper (resting) part of the epiphyseal cartilage (Boyan and Ritter 1984).…”