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).

Quantitative proteomic analysis in symptomatic and asymptomatic apical periodontitis.

AIM: To quantitatively and qualitatively compare the host proteomic profile in samples of symptomatic and asymptomatic apical periodontitis (AP) using nano-liquid chromatography-electron spray tandem mass spectrometry. METHODOLOGY: Samples were obtained from 18 patients with radiographically evident AP, divided into symptomatic and asymptomatic groups (nine per group) according to clinical characteristics. After sample collection, protein extraction, purification and quantification of the samples were performed, which were analysed by reverse-phase liquid chromatography coupled to mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in expression of proteins between the groups were calculated using the Monte Carlo algorithm, considering P < 0.05 for down-regulated proteins and 1 - P > 0.95 for up-regulated proteins. Proteins were identified with the embedded ion accounting algorithm in the software and a search of the Homo sapiens UniProt database. RESULTS: A total of 853 individual human proteins were identified. In the quantitative analysis, common proteins to both groups accounted for 143 proteins. Differences in expression between groups resulted in 51 up-regulated proteins (1 - P > 0.95) in the symptomatic group, including alpha-1-antitrypsin, protein S100-A8, myeloperoxidase, peroxiredoxin and lactotransferrin. This group also had 43 down-regulated proteins (P < 0.05), comprising immunoglobulin, neutrophil defensin, pyruvate kinase and alpha-enolase. The qualitative analysis considered only the exclusive proteins of each group. For the symptomatic group, 318 complete proteins and 29 fragments were identified, such as dedicator of cytokinesis protein, intersectin, prostaglandin, phospholipase DDHD2 and superoxide dismutase. For the asymptomatic group, 326 complete proteins and 37 fragments were identified, including azurocidin, C-reactive protein, collagen alpha, cathepsin, heat shock and laminin. CONCLUSIONS: Quantitative differences in the expression of common proteins in cases of symptomatic and asymptomatic AP were found, which were mostly related to host immune response in both groups. Exclusive proteins in the symptomatic group were mainly related to the host response to the presence of viruses in endodontic infections, oxidative stress and proteolytic enzymes. The results provide a basis for a better understanding of cellular and molecular pathways involved in AP, establishing specific proteomic profiles for symptomatic and asymptomatic conditions.

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.