Effect of vegetable oils applied over acquired enamel pellicle on initial erosion

Abstract Objective The prevalence of dental erosion has been recently increasing, requiring new preventive and therapeutic approaches. Vegetable oils have been studied in preventive dentistry because they come from a natural, edible, low-cost, and worldwide accessible source. This study aimed to evaluate the protective effect of different vegetable oils, applied in two concentrations, on initial enamel erosion. Material and Methods Initially, the acquired pellicle was formed in situ for 2 hours. Subsequently, the enamel blocks were treated in vitro according to the study group (n=12/per group): GP5 and GP100 – 5% and pure palm oil, respectively; GC5 and GC100 – 5% and pure coconut oil; GSa5 and GSa100 – 5% and pure safflower oil; GSu5 and GSu100 – 5% and pure sunflower oil; GO5 and GO100 – 5% and pure olive oil; CON− – Deionized Water (negative control) and CON+ – Commercial Mouthwash (Elmex® Erosion Protection Dental Rinse, GABA/positive control). Then, the enamel blocks were immersed in artificial saliva for 2 minutes and subjected to short-term acid exposure in 0.5% citric acid, pH 2.4, for 30 seconds, to promote enamel surface softening. The response variable was the percentage of surface hardness loss [((SHi - SHf) / SHf )×100]. Data were analyzed by one-way ANOVA and Tukey’s test (p<0.05). Results Enamel blocks of GP100 presented similar hardness loss to GSu100 (p>0.05) and less than the other groups (p<0.05). There was no difference between GP5, GC5, GC100, GSa5, GSu100, GSa100, GSu5, GO5, GO100, CON− and CON+. Conclusion Palm oil seems to be a promising alternative for preventing enamel erosion. However, further studies are necessary to evaluate a long-term erosive cycling.


Introduction
The prevalence of dental erosion has been increasing in recent years 17 . Dental erosion is defined as a chemical process that involves gradual loss of dental hard tissue by intrinsic or extrinsic acids of non-bacterial origin 22 . Advanced stages of this condition may impair esthetics and function, affecting the patient's quality of life 16 . Therefore, establishing effective preventive and therapeutic approaches focused on the etiopathogenesis of the lesion is required. Preventive measures should start as early as possible and involve causal measures, such as dietary advice, to reduce the erosive challenges. In addition, the development of strategies to enhance biological protective factors may help preventing dental erosion. Saliva has been considered the most important biological factor on the pathogenesis of dental erosion 3,14 . The protective mechanism of saliva includes the formation of the acquired enamel pellicle (AEP) 9,25 , a non-bacterial organic film formed over the enamel surface by the adsorption of proteins, peptides, lipids, and other macromolecules available in saliva 6,9 . The AEP plays an important role on the prevention of dental erosion, working as a mediator that diminishes the direct contact of acids with the enamel surface 9 . The protective potential of the AEP depends on its physical properties, including thickness and maturation time 9 . Studies have shown that pellicles formed during two hours or less offer maximum protection against erosive demineralization, without any increase in enamel erosion prevention with longer periods of maturation 10,26 . One possible strategy to increase AEP protection may be the modification of its composition, to improve the protective effect during an erosive challenge by the maintenance of the AEP on the enamel. Lipids consist of about 25% of the dry weight of acquired pellicle 10 , and it is known that lipophilic components are able to modulate the composition and ultrastructure of the AEP 12 . Therefore, it is believed that lipid-rich AEPs are more resistant to acid challenges, protecting against enamel erosion 12 .
The preventive potential of vegetable oils has been widely studied, since they are a natural, edible, low-cost, and worldwide accessible source 2,8,12

Sample size
A pilot study was conducted with six enamel blocks of 100% palm oil and negative control (deionized Effect of vegetable oils applied over acquired enamel pellicle on initial erosion water) per group. Thus, a standard deviation of 8.5% was obtained. Twelve samples per group were set, considering 12 groups with a minimally detectable difference of 15% in hardness loss and 8.5% of standard deviation, with alpha and beta errors of 5% and 20%, respectively.

Enamel blocks preparation
Enamel blocks (4×4×3 mm 3   Bartlett's and Shapiro-Wilk tests, respectively. Once the assumptions were satisfied, two-way ANOVA (for the factors "volunteers" on two levels and "treatments" on 12 levels) and Tukey's post hoc test were applied.
The significance level was set at 5%.

Results
We found no statistically significant difference for the factor "volunteers" (p=0.911), and no interaction between "volunteers" and "treatments" (p=0.634), but we found significant difference between "treatments" (p=0.002). The percentage of hardness loss of the evaluated groups is shown in Table 1. Only GP100 (pure palm oil) was statistically different from the control group, showing the lowest surface hardness loss (p<0.05). All the other studied oils presented surface hardness loss similar to the control groups (p>0.05). We found no significant difference between GP100 and GSu100 (pure sunflower oil) (p>0.05). Our results showed that pure palm oil was capable to protect enamel against initial erosion demineralization, but the same was not found for the 5% palm oil emulsion. No protective effect was observed to 5% emulsion and pure form of coconut,  To our knowledge, palm oil has never been investigated for the prevention of erosion. Palm oil is the second largest produced and consumed vegetable oil in the world, due to its high productivity, low production cost, and rich nutritional content 21 .
It is rich in tocotrienols that have presented health In previous studies, the outer layer of the AEP was modified by the increase of lipids micelles 4,7 .
However, the outer layers of the AEP are supposed to be easily removed after an erosive challenge, in contrast to the basal layer that might not be affected 6 .
In this study, despite the ultrastructure of the AEP not being analyzed, it is hypothesized that palm oil might have modified the basal layer of the acquired pellicle, increasing its protective potential. The tocotrienols contained in the palm oil might have allowed its penetration and distribution into the basal layers of the acquired pellicle, increasing its protective role 1 . We also highlight that we found no differences between the protective effect of pure palm oil and pure sunflower oil. This result can be explained by the tocotrienols content of the sunflower oil, but in a lesser extent when compared to palm oil 1 , which enables a borderline behavior between palm oil and the other tested oils (coconut, safflower, and olive oil).

Conclusion
Considering our study design, palm oil seems to be a promising alternative for the prevention of initial enamel erosion.