The role of matrix metalloproteinases and cysteine-cathepsins on the progression of dentine erosion.

OBJECTIVE: To evaluate in vitro the effect of the inhibition of endogenous dentinal enzymes (matrix metalloproteinases-MMPs and cysteine cathepsins-CCs) on dentine erosion. DESIGN: Dentine blocks (4mm×4mm×2mm) from sound human teeth were randomly divided into 7 groups (n=17) according to the treatment: MMP- and CC-inhibitor chlorhexidine digluconate (CHX, 10mM); MMP-inhibitor galardin (G, 0.2mM); specific cathepsin B inhibitor (CCB, 0.2mM); non-specific CC inhibitor (CCE-64, 0.5µM); fluoride (F, 1.23% NaF); placebo (P) and untreated (UT). Inhibitors were applied as gels once for 1min. Specimens were submitted to 5 days of pH cycling including the erosive challenge (Coke, pH 2.64, 90s/day) and remineralisation (artificial saliva). Demineralised organic surface loss was determined profilometrically. Demineralised organic matrix (DOM) was removed with collagenase and the profile was re-evaluated in the absence of collagen fibrils. The differences in profilometric results and DOM thickness among the groups were analysed with ANOVA and Tukey's test (p<0.05). RESULTS: Loss of demineralised tissue (µm, mean±SD) was: CHX 8.4±1.7 b, G 8.6±1.9 b, CCB 9.6±1.4 a, CCE-64 9.9±1.3 a, F 9.9±1.7 a, P 10.9±2.2 a, UT 11.0±1.5 a. Loss of mineralised tissue was: CHX 15.4±2.2 b, G 16.0±1.8 b, CCB 17.6±2.4 a, CCE-64 17.6±2.0 a, F 17.3±2.8 a, P 19.1±2.1 a, UT 18.9±2.4 a. MMP-inhibitors significantly reduced organic matrix and mineral loss in comparison to all the other groups (p<0.05). No statistically significant differences were found in the thickness of the remaining DOM (p=0.845). CONCLUSION: Dentine endogenous MMPs seem to be the main enzymes responsible for DOM loss and erosion.

Sodium fluoride inhibits MMP-2 and MMP-9.

The importance of fluoride (F) in preventing dental caries by favorably interfering in the demineralization-remineralization processes is well-established, but its ability to inhibit matrix metalloproteinases (MMPs), which could also help to prevent dentin caries, has not been investigated. This study assessed the ability of F to inhibit salivary and purified human gelatinases MMPs-2 and -9. Saliva was collected from 10 healthy individuals. Pooled saliva was centrifuged, and supernatants were incubated for 1 hr at 37°C and subjected to zymography. Sodium fluoride (50-275 ppm F) was added to the incubation buffer. The reversibility of the inhibition of MMPs-2 and -9 by NaF was tested by the addition of NaF (250-5,000 ppm F) to the incubation buffer, after which an additional incubation was performed in the absence of F. F decreased the activities of pro- and active forms of salivary and purified human MMPs in a dose-response manner. Purified gelatinases were completely inhibited by 200 ppm F (IC50 = 100 and 75 ppm F for MMPs-2 and -9, respectively), and salivary MMP-9 by 275 ppm F (IC50 = 200 ppm F). Inhibition was partially reversible at 250-1,500 ppm F, but was irreversible at 5,000 ppm F. This is the first study to describe the ability of NaF to inhibit MMPs completely.

Activity of matrix metalloproteinases in bovine versus human dentine.

Metalloproteinases (MMPs) have been implicated with metabolism of collagen in physiological and pathological processes in human dentine. As bovine teeth have been used as a substitute for human teeth in laboratory analysis, this study evaluated the activity of MMP-2 and -9 in bovine versus human dentine. Bovine and human dentine fragments, from crowns and roots, were powderized. Protein extraction was performed by two protocols: a neutral extraction with guanidine-HCl/EDTA (pH 7.4) and an acidic extraction with citric acid (pH 2.3). Gelatinolytic activities of extracts were revealed by zymography. MMP-2 and -9 were detected in crown and root dentine from bovine and human teeth. Total activities of MMP-2 were 11.4 ± 2.2, 14.6 ± 2.0, 9.7 ± 1.2 and 12.4 ± 0.9 ng/ml for bovine root, human root, bovine crown and human crown dentine, respectively. Corresponding activities for MMP-9 were 14.9 ± 2.0, 15.3 ± 1.3, 15.4 ± 1.3 and 15.5 ± 1.3 ng/ml, respectively. Bovine dentine was found to be a reliable substrate for studies involving the activity of MMP-2 and -9.