Characterization of the functional stages of osteoclasts by enzyme histochemistry and electron microscopy

Abstract

To investigate the functional stages of osteoclasts, the ultrastructural histochemical distribution of the lysosomal enzymes [acid phosphatase (tartrate-sensitive) and neutral phosphatase], the plasma membrane enzymes [alkaline phosphatase, Ca(++)-ATPase, and alkaline ouabain-insensitive p-nitrophenylphosphatase (alkaline p-NPPase)], and the mitochondrial enzyme (cytochrome C oxidase) was evaluated in the chicken tibial metaphysis. Both active-appearing and detached (resting) osteoclasts were studied. Serial sectioning was used to identify detached osteoclasts which were present in the perivascular space. The ultrastructure of detached osteoclasts was similar to that of active osteoclasts, except for the lack of a ruffled border and clear zone, and an altered distribution pattern of small vesicles. Small vesicles were uniformly distributed in the cytoplasm of resting osteoclasts, whereas they were concentrated beneath the ruffled border of active osteoclasts. Alkaline p-NPPase, a marker enzyme for the basal ruffled border, was also apparent on the membrane of small vesicles. However, the vesicles did not possess Ca(++)-ATPase, a marker enzyme for the apical plasma membrane. These findings support the concept that small vesicles serve as a membrane reservoir for the ruffled border membrane. Pre-osteoclasts contained abundant mitochondria and lysosomes, prominent Golgi complexes, moderately developed endoplasmic reticulum, and lacked small vesicles. Pre-osteoclasts appear to fuse with osteoclasts which are attached to the bone surface, but not with detached osteoclasts. The small vesicles, from which the ruffled border arises, are absent from pre-osteoclasts, suggesting that they develop after fusion with pre-existing osteoclasts or after attachment to the bone surface. Alkaline p-NPPase appears to be a marker for differentiation of pre-osteoclasts to mature osteoclasts.

Cytodifferentiation of the odontoclast prior to the shedding of human deciduous teeth: An ultrastructural and cytochemical study

“…The mononuclear cells fused with mononuclear cells or multinucleate odontoclasts attached to the resorbed surface, but no multinucleation phenomenon could be found between mononuclear precursor cells away from the resorbed surface. These results well agree with those of osteoclast multinucleation reported by Baron et al (1986) and Fukushima et al (1991b). Furthermore, we found that cell fusion also can take place between multinucleate odontoclasts that had ruffled border and clear zone against the resorption surface.…”

Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption

“…One of the critical steps in osteoclastic bone resorption is the attachment of osteoclasts to bone and the subsequent formation of a sealing zone, which can be visualized as a clear zone by electron microscopy (5)(6)(7)(8). This attachment is a prerequisite for bone resorption because it creates a sequestered microenvironment into which osteoclasts secrete protons, creating an acidic milieu suitable for the dissolution of bone mineral.…”

Aminopeptidases and Dipeptidyl Peptidases Activities in Chicken Bone Tissue.

“…One of the reasons seems to be that the natural substrates of these peptidases in the bone tissues are unknown. To investigate the roles of these peptidases, the present study attempted to demonstrate their localiza- AP-M (EC 3.4.11.2, also called aminopeptidase-N, microsomal aminopeptidase or CD 13) The present study indicated that AP-M was positive in active osteoclasts which were attached to the bone matrix, not in detached osteoclasts [4] or pre-osteoclasts.…”