The ability of different formulations of artificial saliva to protect dentin from erosive wear

Objective: Evaluate the protective effect of artificial saliva formulations associated or not with mucin on dentin. Materials and Methods: Bovine dentin specimens were randomly allocated to 10 groups (n = 20) according to the artificial saliva tested and the presence or absence of mucin: Amaechi et al. (1998); Klimek et al. (1982); Vieira et al. (2005) and Eisenburger et al. (2001) and deionized water (control). Samples were submitted to an erosive cycle consisting of two immersions of 120 min in the saliva, followed by 1 min in hydrochloric acid solution, and new storage in saliva for 120 min. Surface loss (μm) was measured before and after the cycle. Data were analyzed using 2-way ANOVA and Tukey’s test (p < 0.05). Results: A significant difference was observed for the saliva formulation but not for the presence of mucin. The deionized water provided the highest surface loss and the Eisenburger’s saliva formulation the lowest. The groups testing the Amaechi, Klimek, and Vieira saliva did not present significant differences. Conclusion: Eisenburger’s saliva formulation provides a higher protective effect against dentin erosion. The presence of mucin did not increase the erosion-preventive effect of artificial saliva formulations.


INTRODUCTION
Erosive tooth wear (ETW) is a multifactorial condition resulting from a chemical-mechanical process involving the dissolution of enamel and dentin by non-bacterial acids and modulated by several behavioral and biological aspects [1].Dental surface loss due to erosive conditions is considered a frequent condition, mainly among children and young adults [2][3][4], enhancing the scientific community's interest in this topic and encouraging the development of preventive measures and treatment options.
Saliva is considered the main biological factor responsible for modulating the development of ETW lesions [5].It acts as a reservoir of ions responsible for remineralizing the dental tissues softened by extrinsic or intrinsic acids.It also functions as a diluent of them and promotes their buffering, reducing surface loss [6,7].Most of the evidence in dental erosion is obtained by in vitro studies.These set-ups allow standardization of the study variables under controlled situations, reduce costs compared to clinical studies, and enable a rapid assessment of products or treatments without considering ethical aspects [8].
Ideally, in vitro experiments should mimic the clinical conditions to produce results comparable to the clinical situations [9]; therefore, in 1982, Klimek et al. [10] described an artificial saliva formulation to be used in erosion experiments [10], and later, different formulations were proposed, such as the ones from Amaechi et al. (1998) [11], Vieira et al. (2005) [12] and Eisenburger et al. (2001) [13].To make the artificial saliva consistency more similar to natural human saliva, some formulas add mucin to the mixture, an important component of the salivary pellicle and the main lubricant constituent of saliva [5].This aims to increase the representativity of the in vitro experiments and facilitate the performance of such experiments since there would be no need to collect human saliva from volunteers.However, there are a few suggestions that using artificial saliva formulations and human saliva in in vitro experiments does not reflect the actual intraoral situation [8] and that the presence of mucin in the artificial saliva formulation may increase its remineralizing effect [14].
Therefore, this study aimed to evaluate the dentin erosion-preventive effect of artificial saliva formulations used in previous studies [11][12][13] with or without mucin.The null hypotheses tested were that the different artificial saliva formulation does not influence the dentin erosive surface loss and that the presence of mucin in the formulation does not improve its protective effect.

Specimen preparation and allocation to the groups
Fresh and sound bovine incisors were collected for this study.The crowns were separated from the roots and stored in 0.5% thymol solution at 4 o C until use [15].Two hundred cylindrical dentin specimens (3 mm diameter) were obtained from the roots using a diamond-coated trephine mill.The specimens were embedded in acrylic resin (diameter: 6 mm, height: 3 mm; JET, Classico, Sao Paulo, Brazil).The external surfaces of the specimens were ground flat and polished using silicon carbide paper in sequential grits of 1200, 2400, and 4000 (Extec Corp, CT, USA) in a polishing device (DP-10, Panambra Industrial e Técnica SA, Sao Paulo, SP, Brazil) under water irrigation for 30, 60 and 120 s, respectively.After each paper grit change, specimens were kept in ultrasonic baths in distilled water for 10 min to remove debris and abrasive grains.The prepared samples were examined under a stereomicroscope (Discovery V20, Karl Zeiss, Jena, Germany) to ensure the absence of cracks or other surface defects.After preparation, the specimens were stored at 100% relative humidity at 4 o C to avoid dehydration.[13].Table I shows the composition of the artificial saliva formulations.Each formulation was made with and without mucin, as shown in Figure 1.
After preparing artificial saliva, the specimens were randomly assigned to ten groups (n = 20) according to the storage media (Figure 1).The control group was deionized water (negative control).

Surface loss assessment and erosive cycling
In each specimen, two parallel groves were made to provide a reference for the surface loss   determination (profilometry), as shown in Figure 2.
The baseline profiles of each specimen were obtained using a contact profilometer (MaxSurf XT 20, Mahr-Goettingen, Germany).The diamond stylus moved from the first groove in the acrylic resin to the second one (4.2mm long).Three profile measurements were performed for each specimen at intervals of 0.25 mm.After obtaining the baseline profiles, the erosive cycling was performed under agitation (40 rpm) and at room temperature.The specimens of each group were stored in the respective artificial saliva for 120 min.Then, they were immersed for 1 min in a hydrochloric acid solution (2.5 mmol/l, pH = 2.6).The specimens were washed with deionized water (pH = 5.5) for 10 s.Additional storage in the respective artificial saliva (120 min) and erosive challenge (1 min) were performed [8].After the second erosive challenge, the final profiles of each specimen were obtained using the same parameters previously described.Figure 2 shows a schematic chart illustrating the erosive cycle.
At the end of the cycling, a second profile reading was performed in each specimen using the same parameters described above.The initial and final profiles were overlapped, and the dentin loss was calculated by the difference in height between them using dedicated software (MarSurf XCR 20 4.50-07 SP3, 2011).

Statistical analysis
The assumption of normal distribution (Kolmogorov-Smirnov test) was checked for the variable tested.Descriptive and inferential statistical analyzes were performed using SigmaPlot 13 (Systat Software Inc, San Jose, CA, EUA).Two-way ANOVA was performed for profilometry analysis, followed by Tukey's test with 5% significance.

RESULTS
The results of 2-way ANOVA showed a significant difference (p < 0.05) for the different artificial saliva formulations (p = 0.0001) and the interaction between the two factors (p = 0.040), but not for the presence of mucin (p = 0.360).Tukey's test revealed that the deionized water without mucin (control group) provided the highest surface loss compared with the other formulations.The lowest surface loss was observed for the Eisenburger's saliva formulation, while the groups testing the formulas from Amaechi, Klimek, and Vieira presented similar surface loss.The mean surface loss (μm), standard deviations, and results of Tukey's test are shown in Figure 3.

DISCUSSION
This study showed that the different artificial saliva formulations significantly influenced the erosive surface loss but not by the presence of mucin.Hence, the first null hypothesis was denied, and the second one was accepted.
Hydrochloric acid is a strong inorganic acid, easily ionizable in an aqueous solution and resistant to buffering by saliva, making it a potent agent to erode dental tissues and cause surface loss.It was used in the present study in short erosive events (1 min) aiming to simulate patients with gastroesophageal disorders, such as GERD or bulimia [16].Also, the storage in artificial saliva for 120 minutes aimed to optimize the remineralization process as proposed previously [17].When acid comes into contact with dentin, it dissolves the mineral phase, but the organic part remains as a spongy and demineralized structure that acts as a physical barrier for all chemical processes, including active ingredients from preventive measures and remineralizing agents [18].It is suggested that in in vivo situations, the exposed collagen matrix can be broken down by collagenases and proteolytic enzymes found in the oral fluids [18,19] or through abrasion such as toothbrushing.However, in this study, salivary collagenases and toothbrushing were not performed, so this collagen barrier resultant from the erosive process might have remained intact and helped to modulate the remineralization by the ions from some of the artificial saliva formulations.Still, it is suggested that the collagen fibrils of organic matrix exposed during erosive or carious events house some prenucleation mineral crystals that aggregate into larger amorphous calcium phosphate (ACP) nanoparticles and induce mineral formation [20] in the presence of remineralizing ions, such as calcium, phosphate, or magnesium.
From the solutions tested, the artificial saliva formulation described by Eisenburger presented the lowest values of surface loss, which might have been due to the presence of-HEPES (C 8 H 18 N 2 O 4 S), an organic chemical buffer known as zwitterionic sulfonic acid and classified as a good buffer [21], thus able to neutralize the H + ions from the dissociation of HCl and promote an anti-erosive effect.The groups testing the formulation from Amaechi, Klimek, and Vieira did not differ from each other, even though the last one also presented a buffer in its composition (C 4 H 11 NO 3 -Tris Buffer), and the possible explanation for that relies on the different degrees of saturation with respect of calcium phosphates in each solution [8].
Regarding the groups in which mucin was included, it has been reported that they contribute to a large extent to the protective effect of the acquired pellicle against enamel erosion [22].However, this study showed that mucin did not significantly reduce dentin erosive surface loss.Even for the comparison between the control group (deionized water) with and without mucin, the absence of significant differences indicated that this protein was not able to protect dentin against erosion.This may be because the surface of eroded enamel presents high mineral content, whereas the mesh of collagen matrix from eroded dentin might have interfered with the deposition of mucin, thus reducing its protective effect against erosion.Although, a previous study found that the anti-erosive protection of the acquired pellicle in dentin is lower than in enamel [23].Moreover, it is essential to consider that the behavior of the synthetic mucin used in in vitro studies may be different from the mucin found in human saliva.Its stability in the formulations and the combination with the other components may be able to cause differences from the natural human saliva.
Thus, the result from the present study suggested that the protection of dentin submitted to erosive challenges is affected by the formulation of the artificial saliva tested, and future studies, including the comparison with human saliva in in situ protocols, are encouraged, aiming to find the best suitable formula for in vitro analysis.It can be concluded that the protective effect of different artificial saliva against erosion must be considered to avoid incorrect inferences in in vitro studies.The presence of mucin did not provide a significant reduction in the erosive surface loss of dentin.

Figure 2 -
Figure 2 -Schematic chart of the steps performed in the study.

Figure 1 -
Figure 1 -Specimen allocation according to the storage media.

Batista
GR et al.The ability of different formulations of artificial saliva to protect dentin from erosive wear Batista GR et al.The ability of different formulations of artificial saliva to protect dentin from erosive wear

Figure 3 -
Figure 3 -The mean surface loss (µm), standard deviations, and results of Tukey's test for the artificial saliva, with or without the presence of mucin.Different letters mean significant difference among groups.

Batista GR et al.
The ability of different formulations of artificial saliva to protect dentin from erosive wear

Table I -
Composition of tested artificial saliva formulations