Effect of different citrus sweets on the development of enamel erosion in vitro

Abstract The increased consumption of citrus sweets can contribute to the development of erosive tooth wear (ETW). Objective This in vitro study evaluated the erosive potential of citrus sweets on bovine enamel samples regarding the quantification of wear. Methodology Ninety bovine crowns were prepared and samples were randomly distributed into 6 groups (n=15): 0.1% citric acid solution (pH 2.5); Coca-Cola ® Soft Drink (pH 2.6); Fini ® Diet (lactic and citric acid, pH 3.3); Fini ® Jelly Kisses (lactic and citric acid, pH 3.5); Fini ® Fruit Salad Bubblegum (maleic acid, pH 2.6); Fini ® Regaliz Acid Tubes (maleic and citric acid, pH 3.1). Sweets were dissolved in the proportion of 40 g/250 mL of deionized water. Enamel samples were submitted to erosive challenges for 7 days (4 daily acid immersion cycles for 90 s each). Enamel wear was measured using contact profilometry (μm), and data (median values [interquartile range]) were submitted to Kruskal-Wallis/Dunn’s test (p<0.0001). Results All citrus sweets tested present a high erosive potential, Fini Diet ® (2.4 [1.2]) and Fini Regaliz Tubs ® (2.2 [0.5]) show the highest erosive potential, similar to 0.1% citric acid (2.3 [0.7]); Fini Regaliz Tubs ® is more erosive than Coca-Cola ® (1.4 [0.9]). Conclusion The evaluated citrus sweets have great erosive potential and play a key role in the development of ETW.


Introduction
Tooth erosion is a well-known condition of tooth wear caused by exposure to acid (erosive tooth wear -ETW) that has a multifactorial etiology, involving chemical, biological, and behavioral factors. 1 It starts as tooth softening and further progresses into wear, due to the cumulative effects of erosive challenges associated with mechanical forces such as abrasion and attrition. 2 Sensitivity to pain, poor functioning and aesthetics, and inflammation of the dental pulp are examples of consequences of ETW. 2 Interest in this topic has grown exponentially among researchers and clinicians 3 due to its increasing prevalence, especially among children and young people, and its severity, which tends to increase with increasing age. 4-6 Tschammler, et al. 6 (2016) showed that the prevalence of tooth erosion increased from 25% to 50% in 10 years among 3-6-year-old German children. 6 This finding corroborates systematic reviews that have shown a prevalence of such condition of about 50% in deciduous dentition, and of 30% in permanent dentition. 5,7,8 The most alarming finding is that erosion in deciduous dentition increases about four times the chance its development in permanent dentition; 9 and, even in permanent dentition, the likelihood for erosion progression is higher among individuals with previous experience than among those who have never had this condition. 10 Acid exposure may occur due to intrinsic sources (gastric hydrochloric acid) and/or extrinsic (food and beverages containing citric, phosphoric, and/or maleic acids). 1,11 Dietary habits involving frequent intake of acidic food and beverage between meals may increase the risk of developing ETW. 5,[12][13][14] Although soft drinks are considered the villains for tooth erosion development, an important systematic review showed that the odds ratio for the development of the condition is higher for individuals who often consume citrus sweets (2.24 x)   To determine enamel wear after erosive challenges, the nail varnish was removed using an acetone solution (1:1 -acetone: water) to avoid any interference on the measurement. Then, 5 readings were performed at the same areas of the baseline readings. For calculating dental wear, baseline profiles were superposed to final profiles using a specific software (Marh Surf XCR 20) and average height differences were calculated (μm), considering 0.1 μm as the limit of detection. 19 The average enamel wear was calculated using the five readings of each sample and then used to compare the erosive potential of different citric sweets in relation to the controls (soft drink and citric acid).

Statistical analysis
The average erosive enamel wear (μm) of each sample was tabulated in Excel. Graph Pad Prism (USA) was used for the statistical analysis. The data passed the normality test (Komogorov-Smirnov test), but they were not homogeneous (Bartlett test). Data were compared using Kruskal-Wallis test followed by post-hoc Dunn's test. Significance level was set at 5%.

Results
All citric sweets evaluated induced enamel wear at different degrees. Among them, Fini ® Diet and Fini ® Regaliz Tubs showed the highest erosive potentialsimilar to 0.1% citric acid. Erosion was significantly greater with Fini ® Regaliz Tubs than with Coca-Cola ® .
Fini ® Kisses and Fini ® Fruit Salad caused the lowest erosive wear, which, although comparable to Coca-Cola ® , was significantly lower than Fini ® Diet and Fini ® Regaliz Tubs (Table 1).

Discussion
This study null hypothesis was that there would be no significant difference between the tested sweets and soft drink on the development of erosive enamel wear. However, our results refuted it, as one of the sweets was more erosive than Coca-Cola ® (phosphoric acid), but comparable to the control (0.1% citric acid). The evaluated sweets contained maleic, citric, and/or lactic acids, but their label did not describe acids concentrations. Our results suggest that sweets containing citric acid combined with other acid (lactic or maleic) tend to be more erosive than those containing only maleic acid, regardless of the pH values. Besides pH values, other factors may determine foods/beverages erosive potential, such as acid type, acid concentration, titratable acidity, buffering capacity, molecular weight (influencing acid diffusion), and dissociated acid concentration. 25 Citric acid is highly aggressive not only because it releases H + at different steps of its cycle (pK a -values: 3.1, 4.74, and 6.42), but also because it entails an additional effect caused by citrate ion, a calcium chelator, whose effect is more significant at pH > 4. [26][27][28] It is also considered a complex chemical compound, hampering the prediction of its erosive potential at different concentrations. 25 In our study, erosive effect was not associated with its chelating action, but rather with the high H + release, which was probably even more aggressive in the presence of other acid.
Our results are in line with those found in the systematic review, which indicates that acidic sweets have a higher impact on tooth erosion than soft drinks. 5  sweets are scarce. The two in vitro studies mentioned throughtout this article, evaluated the impact of sweets on enamel softening (early stage of tooth erosion), but not on enamel wear. Carvalho, et al. 16 (2017) showed that a sweet (containing maleic, citric, and tartaric acid) presented the lowest pH value among different foods, and yet caused an enamel softening 6 times greater than an orange juice. Shen,et al. 17 (2017) tested the erosive potential of 30 sugar-free sweets and found that, from 19 acidic sweets, 17 induced enamel softening, especially those containing citric acid, corroborating our results. In our study, we found that the most erosive sweet was a diet one; however, even the sweets that contained sugar (considered cariogenic) caused erosive enamel wear.

Conclusion
Citrus sweets cause erosive enamel wear at different degrees. Our results raise a red flag about the consumption of this type of sweets -especially because its main consumers are children and teenagers, who present a high prevalence of this condition. 5,6,8,30