The effects of inhibitors of cysteine-proteinases and collagenase on the resorptive activity of isolated osteoclasts

Abstract

The effects of specific inhibitors of cysteine-proteinases [Z-Phe-Ala-CHN 2: benzyloxycarbonyl-phenylalanyl alanyl diazomethane and E-64: trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane) and collagenase [Cl-1: N-(3- N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide) have been tested on the osteoclastic resorption of dentine. Chick osteoclasts were cultured in the presence or absence of 12.5 μM Z-Phe-Ala-CHN 2, 40 or 60 μM E-64, or 40 or 100 μM Cl-1 for 1 or 2 days. In addition, osteoclasts were cultured on oyster shell calcitostracum with or without 12.5 μM Z-Phe-Ala-CHN 2. Specimens were studied by light microscopy to count cells and resorption features and by scanning electron microscopy (SEM) stereophotogrammetry for the measurement of the depths, plan-areas and volumes of resorption pits. The numbers, depths and volumes (but not the plan-areas) of the resorption pits in dentine were significantly reduced by Z-Phe-Ala-CHN 2 and E-64. Thus, for a given plan-area, the volumes and the depths of resorption pits were smaller in these experimental groups compared with control dentine specimens. The overall inhibition of resorption was at least 75%. Cl-1 did not have this inhibitory effect on the numbers or sizes of resorption pits in dentine. When the oyster calcitostracum was used as a substrate for the osteoclasts, Z-Phe-Ala-CHN 2 did not reduce the numbers or volumes of pits, but increased the planareas and prevented the formation of deeper pits. These results indicate that cysteine-proteinases participate in the resorption of adult calcified connective tissues by osteoclasts, but do not provide evidence for an obligatory role for collagenase in this process.