AN ULTRASTRUCTURAL EVALUATION OF THE EFFECTS OF CYSTEINE-PROTEINASE INHIBITORS ON OSTEOCLASTIC RESORPTIVE FUNCTIONS

This study was designed to evaluate the effects of specific and potent cathepsin inhibitors on osteoclastic resorptive functions in vitro by means of a novel ultrastructural assay system. Mouse bone marrow cell -derived osteoclasts were suspended on dentine slices and cultured for 48 hours in the presence of either E-64 (a generalized cysteine prote inase inhibitor) or Z-Phe-Phe-CHN2 (a selective cathepsin L inhibitor) . After the removal of cultured osteoclasts, co-cultured dentine slice s were examined using electron microscopy: backscattered (BSEM), scann ing (SEM), and atomic force (AFM). In morphometric analyses of BSEM im ages, there were no significant differences in the areas of deminerali zed dentine surfaces between control and inhibitor-treated groups, sug gesting that cathepsin inhibitors had no effect on dentine demineraliz ation by cultured osteoclasts. However, in SEM and AFM observations, b oth inhibitors remarkably reduced to the same extent, the formation of deep resorption lacunae on dentine slices that had resulted from degr adation of matrix collagen. In addition, Z-Phe-Phe-CHN2 treatment prod uced deeper, ring-like grooves with little collagen exposure in shallo w resorption lacunae. These results strongly suggest that (1) cathepsi ns released by osteoclasts are involved in the formation of deep resor ption lacunae, and (2) cathepsin L plays a key role in bone resorption .