New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action

Abstract A new sugarcane-derived cystatin (CaneCPI-5) showed anti-erosive properties when included in solutions and strong binding force to enamel, but the performance of this protein when added to gel formulations and its effect on surface free energy (SFE) requires further studies. Objective 1) to evaluate the protective effect of gels containing different concentrations of CaneCPI-5 against initial enamel erosion (Experiment 1); and 2) to analyze the SFE (γS) after treating the enamel surface with CaneCPI-5 solution (Experiment 2). Methodology In Experiment 1, 75 bovine enamel specimens were divided into five groups according to the gel treatments: placebo (negative control); 0.27%mucin+0.5%casein (positive control); 0.1 mg/mL CaneCPI-5; 1.0 mg/mL CaneCPI-5; or 2.0 mg/mL CaneCPI-5. Specimens were treated with the gels for 1 min, the AP was formed (human saliva) for 2 h and the specimens were incubated in 0.65% citric acid (pH=3.4) for 1 min. The percentage of surface hardness change (%SHC) was estimated. In Experiment 2, measurements were performed by an automatic goniometer using three probing liquids: diiodomethane, water and ethylene glycol. Specimens (n=10/group) remained untreated (control) or were treated with solution containing 0.1 mg/mL CaneCPI-5, air-dried for 45 min, and 0.5 µL of each liquid was dispensed on the surface to measure contact angles. Results Gels containing 0.1 and 1.0 mg/mL CaneCPI-5 significantly reduced %SHC compared to the other treatments (p<0.05). Treated enamel showed significantly lower γS than control, without changes in the apolar component (γSLW), but the polar component (γSAB=Lewis acid-base) became more negative (p<0.01). Moreover, CaneCPI-5 treatment showed higher γS - (electron-donor) values compared to control (p<0.01). Conclusions Gels containing 0.1 mg/mL or 1.0 mg/mL CaneCPI-5 protected enamel against initial dental erosion. CaneCPI-5 increased the number of electron donor sites on the enamel surface, which may affect AP formation and could be a potential mechanism of action to protect from erosion.


Introduction
Dental erosion is the chemical loss of mineralized tooth substance due to exposure to non-bacterial acids. 1 Saliva is the main patient-related factor that interferes with dental erosion, since it is saturated regarding apatite, buffers the acids and is the main source of proteins that form the acquired pellicle (AP).
This proteinaceous layer acts as a mechanical barrier to the acids, thus reducing erosion. 2 Not all proteins found in the AP protect the tooth surface from acid dissolution. Studies have suggested that the proteins present in the basal layer have a greater participation in this regard. 3 Thus, the concept of "acquired pellicle engineering", which involves changing the AP by adding molecules, has a strong potential to increase its protective effect on the tooth surface. 4,5 A series of in vivo studies used proteomic approaches to identify acid-resistant proteins in the AP that would be candidates for inclusion in dental products to reduce erosive demineralization. [6][7][8] Among them, cystatin-B is a good alternative, 6 but the cost of the human recombinant protein is prohibitive. Therefore, our group recently cloned sugarcanederived cystatin  that has a strong binding force to hydroxyapatite and can protect from initial erosion in vitro 4 and in vivo 5 when added to rinse solutions. Moreover, incorporating molecules in the AP may change the enamel reactivity and the surface free energy (SFE), which might guide protein binding to the AP, thus changing its composition.
Regarding the application vehicle, the use of gels in studies involving the inhibition of matrix metalloproteinases in dentin a offered better protection against dentin erosion when compared with their inclusion in solutions. 9,10 Possibly due to the prolonged contact time of the gel with the tooth surface, due to its viscosity. We hypothesize that the same could happen with CaneCPI-5 gels. If the protection conferred by CaneCPI-5-containing gels is better than that conferred by solutions, the frequency of application of the first can be lower, which is an advantage from the clinical point of view. Therefore, our study evaluates the protective effect of gels containing different concentrations of CaneCPI-5 against enamel initial erosion in vitro.
Since little is known about the mechanisms by which CaneCPI-5 interacts with the enamel surface, we also analyzed the ability of CaneCPI-5 to alter the SFE of enamel by measuring the contact angle using the sessile drop method. The null hypotheses tested were: 1) gels containing CaneCPI-5 do not protect from initial dental erosion and 2) CaneCPI- 5  Preparation of the enamel specimens A total of 95 bovine enamel specimens were prepared (4 mm×4 mm×4 mm), being 75 specimens for "Experiment 1" and 20 specimens for "Experiment 2". They were obtained from the buccal-cervical region of bovine incisors and stored in 2% thymol solution (pH 7.0) for 30 days. Besides, the specimens were visually analyzed to assess possible stains and cracks. In these cases, the teeth were excluded. Then, the enamel surface was sequentially polished using water-cooled silicon carbide paper disks (320, 600, and 1200 grit, Extec, Enfield, CT, USA). A felt polishing cloth (Extec Corp. Polishing cloth; Buehler, Lake Bluff, IL, USA), moistened with a 1-μm diamond solution (Extec Corp. Buehler, Lake Bluff, IL, USA), was used on the surface of interest to finalize the polishing. After polishing, the specimens were immersed in an ultrasonic bath (T7 Thornton, Unique Ind. E Com. Ltda., São Paulo, SP, BR) with deionized water for seven min at 25°C. Lastly, they were stored (with wet gauze) at 4°C prior to the experiment.
All gels were prepared as described by Kato,et al. 9 (2010) and had the same composition, except for the presence of casein + mucin or CaneCPI-5.
The amount of gel applied was controlled by a dispenser (pipette, 20 µl per specimen), then the gel was added on the microbrush and applied on the enamel surface of each specimen for 1 minute, and the excess was removed with a cotton swab. 9 The specimens were then incubated in saliva for 2 h at 37°C under agitation to form the AP. 14 Then, the specimens were washed in deionized water (10 s) and air-dried was also estimated to determine the hydrophobicity/ hydrophilicity of the enamel surface: DG iwi > 0 indicated a hydrophilic surface and DG iwi < 0 indicated a hydrophobic surface. 16,19 Statistical Analysis All the data were analyzed using the GraphPad InStat (version 3.10 for Windows) and GraphPad Prism (GraphPad Software Inc., La Jolla, CA) software. Data were checked for normality (Kolmogorov-Smirnov test) and homogeneity (Bartlett test) to select the appropriate statistical test. In the first experiment, the data were analyzed using Kruskall-Wallis and Dunn's tests. In the second experiment, the data were analyzed using ANOVA and Student-Newman-Keuls's test and by Pearson's correlation coefficient.
The significance levels of both experiments were considered as p<0.05.

Results
In the first experiment, only the treatments with CaneCPI-5 at 0.1 and 1.0 mg/mL significantly reduced the SHC compared to control (p<0.05). The treatment performed with the higher concentration of CaneCPI-5 did not significantly differ from control or from mucin + casein (p>0.05) (Figure 1).
In the second experiment, the SFE (g S ) was significantly lower with CaneCPI-5 (p<0.001) compared to control (  Table 1-Means (SD) of the contact angles of probing liquids, surface free energy (g S ) and interaction free energy (DG iwi ) after treating enamel surface with 0.1 mg/mL CaneCPI-5 or not (n=10).

Discussion
Our study involves the concept of "acquired pellicle engineering" that involves changing the AP by adding molecules or ions that can increase its protection against dental erosion. 20 The change was done by using CaneCPI-5, a sugarcane-derived cystatin that has a strong binding force to hydroxyapatite.  citric acid pH 2.5 for 10 s (increase in keratin, IgG, lactotransferrin, serum albumin, alpha amylase, basic salivary proline-rich protein, carbonic anhydrase). 5 We recognize the limitations of the present in vitro study. Although the protocols suit preliminary studies, they do not accurately simulate the clinical condition due to the absence of oral cavity-specific factors, such as the formation of AP. In Experiment 1, limitation of treatment time (with the gels and CaneCPI-5) for 1 min could be extended for longer periods (e.g., 4 min). Regarding Experiment 2, the presence of saliva, which is the main biological factor involved in the occurrence of dental erosion whose factor is the most determinant for the oral cavity, was not considered.
Moreover, CaneCPI-5 was included in gels in the first experiment, while it was included in solution in the second one, due to the analytical technique used.
These limitations must be addressed in future studies.
We rejected both hypotheses based on the results, since: 1) gels containing CaneCPI-5 at 0.1 and 1.0 mg/ mL protected enamel from initial dental erosion; and 2) CaneCPI-5 altered the enamel SFE. Moreover, change in SFE of enamel after applying CaneCPI-5 may help to partially explain alterations in the AP proteome, with consequent change in its protective ability, induced by this phytocystatin.