Cysteine cathepsins activity in sound and carious dentin

Objective: Host proteases (matrix metalloproteinases, MMPs) have been indicated to be important in caries pathogenesis (Tjäderhane et al. 1998, Sulkala et al. 2001). The aim of this study was to examine the role of other host enzymes, cysteine cathepsins activity in sound and carious dentin related to lesion depth. Method: Carious teeth were assigned to following groups: sound dentin (control n=15); superficial (2-3 mm, n=6); deep (3-5 mm, n=6); and pulp exposure (n=5). Sound dentin was removed using carbide bur and carious dentin with sterile spoon excavator, then immersed in 1,0 ml of Tris-HCl 50 mM buffer, pH 7.4, NaCl 100 mM. Proteolytic activities were determined spectrofluorometrically with fluorescently labeled cysteine-specific substrate, and the rate of substrate hydrolysis was determined by measuring the rate of fluorescence increase at 460 nm. To verify the specificity of the cysteine proteinases the assays were carried out with and without protease inhibitors: E-64 (cysteine cathepsin inhibitor), PMSF (serine protease inhibitor), pepstatin A (aspartyl protease inhibitor), 1,10 phenanthroline, EDTA and phosphoramidon (metalloprotease inhibitors). The amount of protein was determined by the Bio-Rad dye-binding assay with BSA as standard, and specific activities were calculated with reference to protein concentration. Data of cysteine cathepsin B specific activities (unit/µg) was analyzed by ANOVA and Tukey post-hoc test (p<0.05). Results: All intact dentin samples demonstrated cysteine cathepsin-specific activities. Statistically significant increase in activity was observed with the increasing depth of the dentinal caries lesions, especially in lesions with pulp exposure. Conclusion: It can be concluded that along with previously demonstrated role of MMPs in caries pathogenesis, also cysteine proteinases are important. This may be especially true in deep and active caries, possibly due to the pulpal origin of cysteine cathepsins in caries lesions. (Supported by CAPES, CNPq FAPESP and the Academy of Finland).