A.R. Hannas

The role of matrix metalloproteinases in the oral environment

This review focuses specifically on matrix metalloproteinases (MMPs) and their role in physiological and pathological extracellular matrix (ECM) remodeling and degradation processes in the oral environment. A group of enzymes capable of degrading almost all ECM proteins, MMPs contribute to both normal and pathological tissue remodeling. The expression of different MMPs may be upregulated in pathological conditions such as inflammation and tumor invasion. The balance between activated MMPs and tissue inhibitors of metalloproteinases (TIMPs) controls the extent of ECM remodeling. Prior to mineralization, MMPs may participate in the organization of enamel and dentin organic matrix, or they may regulate mineralization by controlling the proteoglycan turnover. There is evidence indicating that MMPs could be involved in the etiology of enamel fluorosis and amelogenesis imperfecta. They seem to play a part in dentinal caries progression, since they have a crucial role in dentin collagen breakdown in caries lesions. MMPs have been identified in pulpal and periapical inflammation and are strongly correlated with periodontal diseases, since they are the major players in collagen breakdown during periodontal tissue destruction. The use of MMP inhibitors could help the prevention and treatment of many MMP-related oral diseases.

Chlorhexidine and green tea extract reduce dentin erosion and abrasion in situ

OBJECTIVES This in situ/ex vivo study aimed to analyse the impact of possible MMP-inhibitors (chlorhexidine and green tea extract) on dentin wear induced by erosion or erosion plus abrasion. METHODS Twelve volunteers took part in this cross-over and double-blind study performed in 4 phases of each 5 days. Bovine dentin samples were worn in palatal appliances and subjected to extraoral erosion (4 times/day, Coca-Cola, 5 min) or erosion plus abrasion (2 times/day, fluoride-free toothpaste and electrical toothbrush, 15s/sample). Immediately after each erosion, the appliances were reinserted in the mouth and the oral cavity was rinsed for 60s with: 250 ppm F solution (SnF(2)/AmF, pH 4.5, Meridol-Gaba, Switzerland), 0.12% chlorhexidine digluconate (0.06% chlorhexidine, pH 6.0, Periogard-Colgate, Brazil), 0.61% green tea extract solution (OM24, 100% Camellia Sinensis leaf extract, catechin concentration: 30+/-3%, pH 7.0, Omnimedica, Switzerland) or deionized water (pH 6.0, control). Dentin loss was assessed by profilometry (microm). The data were analysed by two-way repeated measures ANOVA and Bonferroni post hoc test. RESULTS There was a significant difference between the conditions (EroxEro+Abr, p<0.001) and among the solutions (p<0.001). All solutions (F: 1.42+/-0.34; 1.73+/-0.50, chlorhexidine: 1.15+/-0.26; 1.59+/-0.32, green tea: 1.06+/-0.30; 1.54+/-0.55) significantly reduced the dentin wear when compared to control (2.00+/-0.55; 2.41+/-0.83) for both conditions. There were not significant differences among green tea extract, chlorhexidine and F solutions. CONCLUSIONS Thus, the possible MMP-inhibitors tested in this study seem to be a promising preventive measure to reduce dentin erosion-abrasion, but their mechanism of action needs to be investigated in further studies.

Gels Containing MMP Inhibitors Prevent Dental Erosion in situ

Matrix metalloproteinase (MMP) inhibition has been shown to reduce dentin caries progression, but its role in dental erosion has not yet been assessed. This study tested the hypothesis that gels containing MMP inhibitors (epigallocatechin gallate-EGCG and chlorhexidine) can prevent dental erosion. Volunteers (n = 10) wore palatal devices containing bovine dentin blocks (n = 10/group) treated for 1 min with EGCG at 10 (EGCG10) or 400 µM (EGCG400), chlorhexidine at 0.012%, F at 1.23% (NaF), and no vehicle (placebo). Erosion was performed with Coca-Cola® (5 min) 4X/day during 5 days. The wear, assessed by profilometry (mean ± SD, µm), was significantly reduced by the gels containing MMP inhibitors (0.05 ± 0.02a, 0.04 ± 0.02a, and 0.05 ± 0.02a for EGCG10, EGCG400, and chlorhexidine, respectively) when compared with NaF (0.79 ± 0.35b) and placebo gels (1.77 ± 0.35b) (Friedman and Dunn’s tests, p < 0.01). The use of gels delivering MMP inhibitors was shown to prevent erosion and opens a new perspective for protection against dental erosion.

Impact of Protease Inhibitors on Dentin Matrix Degradation by Collagenase

This proof-of-concept study assessed whether the reduction of the degradation of the demineralized organic matrix (DOM) by pre-treatment with protease inhibitors (PI) is effective against dentin matrix loss. Bovine dentin slices were demineralized with 0.87 M citric acid, pH 2.3, for 36 hrs. In sequence, specimens were treated or not (UT, untreated) for 1 min with gels containing epigallocatechin 3-gallate (EGCG, 400 µM), chlorhexidine (CHX, 0.012%), FeSO4 (1 mM), NaF (1.23%), or no active compound (P, placebo). Specimens were then stored in artificial saliva (5 days, 37°C) with the addition of collagenase (Clostridium histolyticum, 100 U/mL). We analyzed collagen degradation by assaying hydroxyproline (HYP) in the incubation solutions (n = 5) and evaluated the dentin matrix loss by profilometry (n = 12). Data were analyzed by ANOVA and Tukey’s test (p < 0.05). Treatment with gels containing EGCG, CHX, or FeSO4 led to significantly lower HYP concentrations in solution and dentin matrix loss when compared with the other treatments. These results strongly suggest that the preventive effects of the PI tested against dentin erosion are due to their ability to reduce the degradation of the DOM.

Protective effect of green tea on dentin erosion and abrasion

Objective: This in situ study evaluated the protective effect of green tea on dentin erosion (ERO) and erosion-abrasion (ABR). Material and methods: Ten volunteers wore intraoral palatal appliances with bovine dentin specimens subjected to ERO or ERO + toothbrushing abrasion performed immediately (ERO+I-ABR) or 30 min after erosion (ERO+30-min-ABR). During 2 experimental 5-day crossover phases, the volunteers rinsed with green tea or water (control, 1 min) between each erosive (5 min, cola drink) and abrasive challenge (30 s, toothbrushing), 4x/day. Dentin wear was measured by profilometry. Results: The green tea reduced the dentin wear significantly for all conditions compared to control. ERO+I-ABR led to significantly higher wear than ERO, but it was not significantly different from ERO+30-min-ABR. ERO+30-min-ABR provoked significant higher wear than ERO, only for the placebo treatment. Conclusions: From the results of the present study, it may be concluded that green tea reduces the dentin wear under erosive/abrasive conditions.

The Role of Matrix Metalloproteinases in Dental Erosion

This review discusses the role of matrix metalloproteinases (MMPs) in the development of dentin erosion and the protective effects of MMP inhibitors, based on recent evidence from in vitro and in situ studies. MMPs are present in both dentin and saliva and play an important role in dentin erosion progression. Enzymatic removal of the organic matrix by MMPs increases the demineralization process, since the demineralized organic matrix has been shown to hamper ionic diffusion after an acidic challenge. Recent evidence from in vitro and in situ studies has shown a protective role of MMP inhibitors against dentin erosion and erosion plus abrasion. The inhibitors tested were green tea and its active epigallocatechin-gallate (EGCG), ferrous sulfate, and chlorhexidine. They have been tested in dentifrices, solutions, and gels. The latter led to a more pronounced protective effect against dentin erosion and erosion plus abrasion. The protection was long-lasting and could be observed after up to 10 days of severe erosive and erosive-plus-abrasive challenges in situ. Thus, the use of MMP inhibitors has emerged as an important preventive tool against dentin erosion. Clinical studies should be conducted to confirm the results obtained and to give support to the establishment of clinical protocols of use.

Effect of Iron on Matrix Metalloproteinase Inhibition and on the Prevention of Dentine Erosion

It is known that some metal salts can inhibit matrix metalloproteinase (MMP) activity, but the effect of iron has not been tested yet. On the other hand, it has recently been suggested that MMP inhibition might influence dentine erosion. Based on this, the aims of this study were: (1) to test in vitro the effect of FeSO4 on MMP-2 and -9 activity, and (2) to evaluate in situ the effect of FeSO4 gel on dentine erosion. MMP-2 and -9 activities were analysed zymographically in buffers containing FeSO4 in concentrations ranging between 0.05 and 1.5 mmol/l or not. Volunteers (n = 10) wore devices containing bovine dentine blocks (n = 60) previously treated with the following gel treatments: FeSO4 (1 mmol/l FeSO4), F (NaF 1.23%; positive control) and placebo (negative control). The gels were applied once and removed after 1 min. Erosion was performed extraorally with Coca-Cola 4 times per day for 5 min over 5 days. Dentine wear was evaluated by profilometry. The data were analysed by Kruskal-Wallis and Dunn’s tests (p < 0.05). FeSO4 inhibited both MMP-2 (IC50 = 0.75 mmol/l) and MMP-9 (IC50 = 0.50 mmol/l) activities. In the in situexperiment, the mean wear (± SD) found for the F gel (0.79 ± 0.08 µm) was significantly reduced in more than 50% when compared to the placebo gel (1.77 ± 0.33 µm), but the FeSO4 gel completely inhibited the wear (0.05 ± 0.02 µm). Since FeSO4 was able to inhibit MMP in vitro, it is possible that the prevention of dentine wear by the FeSO4 gel in situ might be due to MMP inhibition, which should be investigated in further studies.

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.

Fluoride modulates preosteoblasts viability and matrix metalloproteinases-2 and -9 activities.

This study evaluated the influence of fluoride on cell viability and activity of matrix metalloproteinases (MMP) -2 and -9 secreted by preosteoblasts. Preosteoblasts (MC3T3-E1 murine cell line) were cultured in MEM medium supplement with 10% Fetal Bovine Serum (FBS) and nucleosides/ribonucleosides without ascorbic acid. Adherent cells were treated with different concentrations of F (as sodium fluoride-NaF) in medium (5 x 10(-6) M, 10(-5) M, 10(-4) M and 10(-3) M) for 24, 48, 72 and 96 h at 37ºC, 5% CO(2). Control cells were cultivated in MEM only. After each period, preosteoblast viability was assessed by MTT assay. MMP-2 and -9 activities were performed by gel zymography. Also, alkaline phosphatase (ALP) activity was quantified by colorimetry in all experimental groups. It was shown that cultured cells with the highest dose of F (10(-3) M) for 96 h decreased preosteoblast viability while lower doses of F did not alter it, when compared to untreated cells. No differences were observed in ALP activity among groups. Moreover, compared to control, the treatment of cells with F at low dose slightly increased MMP-2 and -9 activities after 24 h. It was concluded that F modulates preosteoblast viability in a dose-dependent manner and also may regulate extracellular matrix remodeling.

Preventive effect of toothpastes with MMP inhibitors on human dentine erosion and abrasion in vitro

ABSTRACT The use of gels and mouthrinses with MMP inhibitors (chlorhexidine, and green tea extract) was shown to prevent erosive wear. The aim of this study was to analyze the protective effect of toothpastes containing MMP inhibitors on dentine loss induced by erosion in vitro. Material and Methods Five groups each containing 12 specimens of human root dentine were prepared. The specimens were subjected to 1 min erosion by immersion in a cola drink, 4 times a day, for 5 d. Each day, after the first and last erosive challenges, the specimens were brushed for 15 s with a slurry of dentifrice and water (1:3) containing placebo, 1,100 ppm fluoride, 0.61% green tea extract, 0.12% chlorhexidine or 0.004% chlorhexidine (commercial toothpaste). Between the acid challenges, the specimens were stored in artificial saliva with remineralizing potential until the next treatment. Dentine loss was determined using profilometry. Data were analyzed using one-way ANOVA after log transform (p<0.05). Results The mean wear values (μm) were as follows: placebo 1.83±0.53; 0.61% green tea extract 1.00±0.21; fluoride 1.27±0.43; 0.12% chlorhexidine 1.19±0.30; and 0.004% chlorhexidine 1.22±0.46. There was a significant difference in wear between placebo and all the treatment toothpastes, which did not differ from each other. Conclusion The results suggest that toothpastes containing MMP inhibitors are as effective as those based on NaF in preventing dentine erosion and abrasion.