Safety and In Situ Antierosive Effect of CaneCPI-5 on Dental Enamel.

The sugarcane cystatin (CaneCPI-5) was recently cloned and showed strong binding force to dental enamel and protection against initial erosion. However, evaluations on its safety and efficacy in a situation closer to the clinical condition are necessary. In the present study we analyzed 1) the cytotoxicity of CaneCPI-5 on human gingival fibroblasts (HGFs); 2) the ability of CaneCPI-5 to reduce enamel erosion and erosion+abrasion in situ. In part 1, HGFs were treated with CaneCPI-5 (0.025, 0.05, 0.1, 0.5 or 1.0 mg/mL) or no treatment (control). The cytotoxicity was assessed after 60 s and 24 h by mitochondrial activity (MTT), confocal microscopy, and hematoxylin/eosin staining. In part 2, 15 volunteers participated in a double-blind crossover protocol consisting of 3 phases, according to the following treatments: 1) 0.1 mg/mL CaneCPI-5; 2) SnCl2/NaF/AmF (Elmex; positive control); 3) water (negative control). The volunteers wore an appliance containing 4 bovine enamel specimens for 5 d. Each day, the specimens were individually treated with 50 µL of the tested solutions per 60 s and then subjected to erosive challenges (0.1% citric acid, pH 2.5, for 90 s, 4 times per day). After the first and last erosive challenge each day, 2 samples were abraded (toothbrushing, 15 s). Enamel wear was measured by contact profilometry. One or two-way analysis of variance (ANOVA)/Tukey's or Sidak's tests (P < 0.05) were applied. Regardless of the concentration and the experimental time, CaneCPI-5 did not decrease the cell viability compared to the negative control (P < 0.05). Erosion+abrasion led to significantly greater wear compared to erosion only. For both conditions, the lowest wear was found for SnCl2 and CaneCPI-5, which did not differ significantly from each other, but showed significant protection when compared to the negative control. In conclusion, CaneCPI-5 is safe on HGFs and reduces enamel erosive wear to the same extent as a commercial solution used to control erosive tooth wear (ETW).

A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion.

Cystatin B was recently identified as an acid-resistant protein in acquired enamel pellicle; it could therefore be included in oral products to protect against caries and erosion. However, human recombinant cystatin is very expensive, and alternatives to its use are necessary. Phytocystatins are reversible inhibitors of cysteine peptidases that are found naturally in plants. In plants, they have several biological and physiological functions, such as the regulation of endogenous processes, defense against pathogens, and response to abiotic stress. Previous studies performed by our research group have reported high inhibitory activity and potential agricultural and medical applications of several sugarcane cystatins, including CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4. In the present study, we report the characterization of a novel sugarcane cystatin, named CaneCPI-5. This cystatin was efficiently expressed in Escherichia coli, and inhibitory assays demonstrated that it was a potent inhibitor of human cathepsins B, K, and L ( Ki = 6.87, 0.49, and 0.34 nM, respectively). The ability of CaneCPI-5 to bind to dental enamel was evaluated using atomic force microscopy. Its capacity to protect against initial enamel erosion was also tested in vitro via changes in surface hardness. CaneCPI-5 showed a very large force of interaction with enamel (e.g., compared with mucin and casein) and significantly reduced initial enamel erosion. These results suggest that the inclusion of CaneCPIs in dental products might confer protection against enamel erosion.

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.

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 FeSO(4) on MMP-2 and -9 activity, and (2) to evaluate in situ the effect of FeSO(4) gel on dentine erosion. MMP-2 and -9 activities were analysed zymographically in buffers containing FeSO(4) 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: FeSO(4) (1 mmol/l FeSO(4)), 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). FeSO(4) inhibited both MMP-2 (IC(50) = 0.75 mmol/l) and MMP-9 (IC(50) = 0.50 mmol/l) activities. In the in situ experiment, the mean wear (+/- SD) found for the F gel (0.79 +/- 0.08 microm) was significantly reduced in more than 50% when compared to the placebo gel (1.77 +/- 0.33 microm), but the FeSO(4) gel completely inhibited the wear (0.05 +/- 0.02 mum). Since FeSO(4) was able to inhibit MMP in vitro, it is possible that the prevention of dentine wear by the FeSO(4) gel in situ might be due to MMP inhibition, which should be investigated in further studies.

Effect of an iron mouthrinse on enamel and dentine erosion subjected or not to abrasion: an in situ/ex vivo study.

OBJECTIVES: This in situ/ex vivo study evaluated whether a rinse with an iron solution could reduce wear and the percentage of microhardness change of human enamel and dentine submitted to erosion followed by brushing after 1 or 30min. DESIGN: During 2 experimental 5-day crossover phases (wash-out period of 10 days), 10 volunteers wore intraoral palatal devices, with 12 specimens (6 of enamel and 6 of dentine) arranged in 3 horizontal rows (4 specimens each). In one phase, the volunteers immersed the device for 5min in 150mL of cola drink, 4 times a day. Immediately after immersion, no treatment was performed in one row. The other row was brushed after 1min using a fluoride dentifrice and the device was replaced into mouth. After 30min, the remaining row was brushed. In the other phase, the procedures were repeated, but after immersion the volunteers rinsed for 1min with 10mL of a 10mM ferrous sulphate solution. Changes in surface microhardness (%SMH) and wear (profilometry) of enamel and dentine were measured. Data were tested using ANOVA and Tukey's tests (p<0.05). RESULTS: The enamel presented more wear than dentine, under all experimental conditions. The iron solution caused a significant reduction on the %SMH in enamel, and a significant reduction on the wear in dentine, regardless the other conditions. CONCLUSIONS: Rinsing with an iron solution after an erosive attack, followed or not by an abrasive episode, may be a viable alternative to reduce the loss of dental structure.