Recent advances in the ultrastructural assessment of osteoclastic resorptive functions

Ultrastructural enzyme and immunocytochemical studies have made great contributions to clarifying intriguing questions as to the actual role of osteoclastic ruffled borders in bone resorption. In the present study, vacuolar-type H(+)-ATPase and cysteine-proteinase (cathepsin) were localized in osteoclasts by means of light and electron microscopic immunocytochemistry. The specific immunoreactivity of vacuolar-type H(+)-ATPase was detected along the ruffled border membranes, associated pale vacuoles, and cisterns of the rough-surfaced endoplasmic reticulum of osteoclasts. Anti-cathepsin B immunoreaction occurred in Golgi vesicles, lysosomes, pale vesicles and vacuoles, and the extracellular canals of ruffled borders of osteoclasts. The resorbing bone surfaces were also immunoreactive for anti-cathepsin B. In a coculture system of osteoclasts with devitalized dentine slices, a specific H(+)-ATPase inhibitor (bafilomycin A1) markedly reduced both demineralized areas and resorption lacuna formation on the dentine slices. On the other hand, the cathepsin inhibitor, E-64, inhibited only resorption lacuna formation but had no effect on demineralization of the dentine slices. These results suggest that H(+)-ATPase and cathepsins in osteoclasts are involved, respectively, in the extracellular solubilization of apatite crystals and subsequent degradation of bone matrix and that the ruffled border-clear zone complex of osteoclasts is the main site of cell-matrix interactions during bone resorption processes.