IADR Abstract Archives
Effect of Salivary MMPs and Cathepsins on Dentin Collagen Degradation
Objectives: Salivary enzymes may degrade dentin collagen and contribute to dentinal caries and erosion. We studied the effect of salivary MMPs and cysteine cathepsins (CCs) on demineralized dentin collagen with and without acid-activation.
Methods: Human whole saliva was centrifuged, supernatant was filtered to remove microbes and MMP-8, -2 and -9 activities were determined with Multiplex bead-based assay (Bio-Plex Pro™) and expressed as relative fluorescence units (RFU) compared to non-MMP controls. Saliva was then diveded into four groups: 1) no activation; 2) acid-activation with HCl, pH 4.5 for 30min and neutralized with NaOH; 3) acid-activated with MMP-inhibitor Galardin (GAL; 0.2mM); 4) acid-activated with CC-inhibitor E-64 (5µM). Demineralized human dentin beams (8/group) were dehydrated and weighed for initial mass, incubated in saliva for 96h and reweighed to calculate mass loss. To analyze MMP- and CC-activities, ICTP and CTX were measured from saliva using ELISA kits (UniQ ICTP EIA for ICTP and Serum Crosslaps Elisa for CTX). Salivary calcium was measured spectrophotometrically. Data was analyzed for normal distribution, and ANOVA/Tukey HSD or Kruskal-Wallis/Mann-Whitney tests were used for statistical significances.
Results: Saliva contained low MMP-2, intermediate MMP-8 and high MMP-9 (35.7+/-3.2, 2001.4+/-146.7 and 4679.9+/-153.5 RFU, respectively). Acid-activation, regardless of use of inhibitors, significantly increased mass loss and amount of calcium in saliva after 96h incubation of dentin beams. Inhibition of CC with E-64 resulted with significantly less available calcium in saliva than acid-activation alone. No significant differences were observed with ICTP and CTX release between the groups. The data is presented in Table.
Conclusions: Acid-activation of salivary collagen-degrading enzymes cause loss of dentin mass, which may relate to remineralization potential of saliva more than degradation of collagen itself. Enzyme inhibition was not able to eliminate the effect.
Methods: Human whole saliva was centrifuged, supernatant was filtered to remove microbes and MMP-8, -2 and -9 activities were determined with Multiplex bead-based assay (Bio-Plex Pro™) and expressed as relative fluorescence units (RFU) compared to non-MMP controls. Saliva was then diveded into four groups: 1) no activation; 2) acid-activation with HCl, pH 4.5 for 30min and neutralized with NaOH; 3) acid-activated with MMP-inhibitor Galardin (GAL; 0.2mM); 4) acid-activated with CC-inhibitor E-64 (5µM). Demineralized human dentin beams (8/group) were dehydrated and weighed for initial mass, incubated in saliva for 96h and reweighed to calculate mass loss. To analyze MMP- and CC-activities, ICTP and CTX were measured from saliva using ELISA kits (UniQ ICTP EIA for ICTP and Serum Crosslaps Elisa for CTX). Salivary calcium was measured spectrophotometrically. Data was analyzed for normal distribution, and ANOVA/Tukey HSD or Kruskal-Wallis/Mann-Whitney tests were used for statistical significances.
Results: Saliva contained low MMP-2, intermediate MMP-8 and high MMP-9 (35.7+/-3.2, 2001.4+/-146.7 and 4679.9+/-153.5 RFU, respectively). Acid-activation, regardless of use of inhibitors, significantly increased mass loss and amount of calcium in saliva after 96h incubation of dentin beams. Inhibition of CC with E-64 resulted with significantly less available calcium in saliva than acid-activation alone. No significant differences were observed with ICTP and CTX release between the groups. The data is presented in Table.
Conclusions: Acid-activation of salivary collagen-degrading enzymes cause loss of dentin mass, which may relate to remineralization potential of saliva more than degradation of collagen itself. Enzyme inhibition was not able to eliminate the effect.