The effect of magnesium hydroxidecontaining dentifrice using an extrinsic and intrinsic erosion cycling model

(1)

Contents lists available atScienceDirect

Archives of Oral Biology

journal homepage:www.elsevier.com/locate/archoralbio

Research paper

The e

_ﬀ

ect of magnesium hydroxide-containing dentifrice using an extrinsic

and intrinsic erosion cycling model

Vanara Florêncio Passos, Lidiany Karla Azevedo Rodrigues, Sérgio Lima Santiago

⁎

Universidade Federal do Ceará–UFC, Faculty of Pharmacy, Dentistry and Nursing, Monsenhor Furtado St., Fortaleza, CE, Brazil

A R T I C L E I N F O

Keywords:

Magnesium hydroxide Toothpastes Hydrochloric acid Sodiumﬂuoride Citric acid

A B S T R A C T

Objective:To evaluate,in vitro, the effect of Mg(OH)2dentifrice, and the influence of the number of experimental days, on the extrinsic (citric acid–CA) and intrinsic (hydrochloric acid–HCl) enamel erosion models. Design:Human enamel slabs were selected according to surface hardness and randomly assigned to 3 groups (n = 9) as follows: non-fluoridated (negative control), NaF (1450 ppm F- positive control) and Mg(OH)2(2%) dentifrices. The slabs were daily submitted to a 2-h period of pellicle formation and, over a period of 5 days, submitted to cycles (3×/day) of erosive challenge (CA 0.05 M, pH = 3.75 or HCl 0.01 M, pH = 2 for 30 s), treatment (1 min−1:3 w/w of dentifrice/distilled water) and remineralization (artificial saliva/120 min). Enamel changes were determined by surface hardness loss (SHL) for each day and mechanical profilometry analysis. Data were analyzed by two-way ANOVA followed by Tukey’s test to % SHL and one-way ANOVA to profilometry (p < 0.05).

Results:The number of experimental days influenced the erosion process for the two types of erosion models (p < 0.001). Mg(OH)2-containing dentifrices were effective in reducing enamel extrinsic acid erosion as de-termined by % SHL (p < 0.001) when compared to the control group, being better than positive control (p < 0.001); however, the dentifrices were not effective for the intrinsic model (p = 0.295). With regards to surface wear, no statistically significant differences were found among the groups for CA (p = 0.225) and HCl (p = 0.526).

Conclusion:Theﬁndings suggest that Mg(OH)2dentifrices might protect enamel against slight erosion, but protection was not eﬀective for stronger acid erosion.

1. Introduction

The frequent ingestion of citrus fruits, acidic juices, carbonated drinks or sports drinks, as well as gastrointestinal disorders that cause the presence of gastric acid in the oral cavity may lead to loss of dental hard tissues (Lussi, Schlueter, Rakhmatullina, & Ganss, 2011; Johansson, Omar, Carlsson, & Johansson, 2012). According toDuﬀey et al. (2012)adolescents consume an average of 360 ml of acid drinks per day, which indicates a high level of ingestion of acid-containing products. Furthermore, a systematic literature review demonstrated a median prevalence of 24% for tooth erosion in adult patients with gastro-esophageal reﬂux disease (Pace, Pallotta, Tonini, Vakil, & Bianchi Porro, 2008), proving that gastric acid is also an important etiological factor for dental erosion.

Avoiding a lifelong contact of erosive acidic contents with dental surfaces is an impossible task. Therefore, the development of early di-agnostic methods and adequate preventive measures should be re-searched (Lussi et al., 2011). Many preventive measures are based on

the release of fluoride compounds by oral solutions or dentifrices through daily use and easy access to over the counter products (Ganss, Lussi, Grunau, Klimek, & Schlueter, 2011; Passos, de Vasconcellos, Pequeno, Rodrigues, & Santiago, 2015; Scaramucci, Borges, Lippert, Frank, & Hara, 2013). The protective effect of ionicfluoride from NaF (sodiumfluoride) dentifrices on enamel erosion has been showed in some researches, once it forms CaF2-like products, which can protect this tissue against acid challenges (Ganss et al., 2011;Passos, Santiago, Tenuta, & Cury, 2010;Passos et al., 2015; Soares et al., 2017).

However, allergic patients toﬂuoride products (de Groot, Tupker, Hissink, & Woutersen, 2017;Van Baelen, Kerre, & Goossens 2016) re-quire other strategies for prevention of dental erosion. Recently, Abdallah et al. (2016)showed that magnesium ions react with enamel during dissolution and precipitation process improving the physical properties of enamel–hardness increase. To the best of the current authors’knowledge, there is no published data on the eﬀects of denti-frices containing magnesium ions on enamel surfaces exposed to si-mulated exogenous or endogenous acids.

https://doi.org/10.1016/j.archoralbio.2017.11.006

Received 13 June 2017; Received in revised form 23 October 2017; Accepted 13 November 2017

⁎_{Correspondence to: Sergio Lima Santiago, Faculty of Pharmacy, Dentistry and Nursing, Monsenhor Furtado St., s/n, Rodolfo Teo}_ﬁ_{lo, 60.430-355, Fortaleza, CE, Brazil.}

E-mail addresses:vanarapassos@hotmail.com(V.F. Passos),lidianykarla@yahoo.com(L.K.A. Rodrigues),sergiosantiago@yahoo.com(S.L. Santiago).

(2)

Different neutralizing products have been assessed to reduce erosion (Lindquist, Lingström, Fändriks, & Birkhed, 2011; Messias, Serra, & Turssi, 2008;Messias, Turssi, Hara, & Serra, 2010;Turssi et al., 2012). The protective effect of antacid products on dental erosion has been shown in some studies (Messias et al., 2010; Turssi et al., 2012). Ac-cording toLindquist et al. (2011)these products increase the intra-oral pH after an erosive process, presenting a buffering effect (Meurman, Kuittinen, Kangas, & Tuisku, 1988). Furthermore, this effect might be caused by these products reacting with the acid and forming a salt (Turssi et al., 2012).Messias et al. (2010)indicated that oral rinsing containing sodium bicarbonate helped to minimize the destructive ef-fect caused by erosive challenges.

The aim of the present study was to assess the effect of magnesium hydroxide,fluoride- and non-fluoride-based dentifrices on reducing the progression of enamel surface erosion originated by extrinsic acid (ex-periment one) or intrinsic acid (ex(ex-periment two). In addition, the in-fluence of the number of experimental days on enamel surface softening was evaluated. The null hypothesis tested was that there is no differ-ence among the tested dentifrices against both kinds of acid challenges.

2. Material and methods

2.1. Preparation of enamel samples

The study protocol was reviewed and approved by the local Research and Ethics Committee (protocol #75/12). Enamel slabs were obtained from caries-free human third molars that had been stored in 0.01% (w/v) thymol solution at 4 °C (Passos et al., 2015). Enamel slabs (4 × 4 × 2 mm) were cut from the middle third of the coronal surface. Each slab was sequentially ground using a water-cooled mechanical grinder (Ecomet/Automet 250 Grinder-Polisher; Buehler Ltd, Lake Bluﬀ, IL, USA) with 400-, 600-, and 1200-grit Al2O3papers and po-lished using cloths with a 1μm diamond suspension– Alpha Micro-polish; Buehler Ltd, Lake Bluﬀ, IL, USA).

A total offifty-four enamel slabs were randomly divided into ex-perimental groups based upon their baseline surface hardness values (SHbas), using a computer generated list (Microsoft Excel 2007). The SHbas values were determined by placing five indentations, 100μm apart from each other, at the center of the specimens using a Knoop indenter with a load of 50 g and a dwell time offive seconds (FM100, Future Tech, Tokyo, Japan). Enamel specimens presenting a mean hardness of 328.1 ± 13.1 kg/mm2_{were selected and allocated to three} groups for experiment one and experiment two (n = 9), generating balanced groups.

Subsequently, two parts of each specimen were covered with a dark-colored acid-resistant varnish (Jordana Cosmetics Corp., Los Angeles, CA, USA) to serve as the reference area for proﬁlometry analysis. The exposed area of 2 × 4 mm in the center of each specimen was subjected to the treatments. In experiment 1, the acid challenge was performed using 0.05 M citric acid (citric acid dehydrated, pH 3.75; Dinâmica®_,

Diadema, SP, Brazil), while in experiment 2, 0.01 M hydrochloric acid (pH 2.0; Merck, Darmstadt, Germany) was used. The experimental groups were: non-ﬂuoridated (negative control, pH = 6.86; basic in-gredients: sorbitol, sodium lauryl sulfate, sodium hydroxide, hydrated silica), NaF (1450 ppm F; positive control; pH = 7.36; basic in-gredients: sorbitol, sodium lauryl sulfate, copolymer, sodium hydro-xide, sodiumﬂuoride, triclosan, hydrated silica) and Mg(OH)2(0 ppm F; 2%; pH = 9.96; basic ingredients: calcium carbonate, magnesium hydroxide, sodium lauryl sulfate, magnesium sulfate) dentifrices.

2.2. Pellicle formation

Fresh saliva samples were collected each day from groups of 15–20 volunteers without active carious lesions, erosions, or salivary dys-function. The subjects did not eat or smoke for the 8-h period before sampling. Saliva was stimulated with paraﬃn wax forﬁve min. Saliva

from the first minute of chewing was swallowed, and the rest was collected and deposited into 50-ml centrifuge tubules. The saliva sam-ples were centrifuged for 10 min at 2000 rpm in a pre-cooled centrifuge (4 °C) (5415R, Eppendorf, Brazil) (de-Melo et al., 2011). The clearfluid above the sediments was pooled and used for pellicle formation (Nekrashevych & Stösser, 2003). Each group of enamel slabs was in-dependently immersed in the clarified saliva and incubated for a period of two hours each day before the erosive challenges, under agitation at 100 rpm (5 ml per slab) and 37 °C to simulate the oral cavity tem-perature.

2.3. Experimental procedures

The study consisted of two separate experiments. Both experiments were cyclic procedures, repeated over afive–day period, and included pellicle formation, erosion, treatments with the dentifrices and re-mineralization using artificial saliva (1.5 mM Ca; 0.9 mM PO4; 150 mM KCl and 0.1 M Tris buffer, pH 7.0–5 ml per specimen) (Queiroz, Hara, Paes Leme, & Cury, 2008).

For each experimental day, all procedures were performed under agitation at 100 rpm and at 37 °C. All specimens were immersed in clarified saliva for 2 h prior to experimentation to allow for the for-mation of pellicle. Subsequently, each slab was submitted to a citric acid or hydrochloric acid solution (5 ml per specimen) for 30 s. The specimens were then treated with fresh dentifrice slurry (5 ml per specimen) for one minute that had been prepared from non-fluoridated, magnesium hydroxide or sodiumfluoride dentifrices (1 part toothpaste to 3 parts distilled water solution, by weight). The slurries were freshly prepared at the beginning of each experimental day. Next, each slab was rinsed with distilled water and immersed in artificial saliva for two hours. This cycle was repeated three times a day forfive days. At the end of each experimental day, the slabs were evaluated for surface hardness (Table 1).

2.4. Measurement of enamel surface loss

Measurements of enamel surface loss were performed using the stylus profilometer, Hommel Tester T1000 (Hommelwerke GmbH, Germany) after the last experimental day. The difference between the heights (H) of the surfaces of the reference and the treated areas was evaluated. Before the analysis, the nail varnish was carefully removed, exposing the untreated reference areas. On each sample, at intervals of 100μm,five profile traces (1.5 mm in length) were recorded, the levels of enamel wear, in micrometer (μm), were determined in relation to the reference surfaces. For each sample, the mean values obtained from the five traces were calculated.

Table 1

Daily cycling.

Steps Sequence Treatment

1 Pellicle formation Human saliva (2 h)

2 Erosive challenge Citric acid (30 s)–Experiment 1 Hydrochloric acid (30 s)–

Experiment 2 3 Treatment with dentifrice/destilled

water

Non-ﬂuoride (1 min) Magnesium Hydroxide (1 min) Sodium Fluoride (1 min) 4 Remineralization Artiﬁcial saliva (2 h)

Repeat step 2 Repeat step 3 Repeat step 4 Repeat step 2 Repeat step 3 Repeat step 4

(3)

2.5. Percentage of surface hardness loss assessment

Immediately after each experimental day, the slabs were placed in the hardness machine andﬁve new indentations were made using a Knoop diamond under a 50-g load for 5 s (SHafter), in the method described for the baseline determinations and with the indentations spaced 100μm apart from the previous measurements (SHbas). The percentage of SH loss (% SHL) was then calculated for each day, ac-cording to the equation:

% SHL = [(SHbas.−SHafter) × 100/SHbas.]

2.6. Statistical analysis

Mean values of wear and % SHL were calculated. A Kolmogorov-Smirnov test was applied to all groups to test for the normal distribution of errors. Because the values were normally distributed across all groups, two-way ANOVA was performed to analyze the percentage of SH loss to evaluate the influence of treatment and number of experi-mental days. One-way ANOVA was used to evaluate the profilometry among the groups. Tukey’spost hoctest was applied, when necessary, in cases where ANOVA revealed significant differences. Statistical ana-lyses were performed with the Statistical Package for Social Sciences (SPSS 17.0) for Windows. The level of significance was set at 5%.

3. Results

In experiment one, to % SHL, two-way ANOVA revealed a sig-nificant difference among the dentifrices tested (p < 0.001; F = 26.0), as well as the duration of demineralization represented by the number of experimental days (p < 0.001; F = 68.0). However, the interaction between the factors was not significant (p = 0.982; F = 0.24). The results showed that magnesium hydroxide (30.5 ± 7.24) significantly reduced the % SHL when compared to the non-fluoridated (38.3 ± 7.95; p = 0.0001) and fluoridated dentifrices (34.3 ± 4.4; p = 0.001). Thefluoridated dentifrice reduced the percentage of sur-face hardness loss when compared to the non-fluoridated dentifrice (p = 0.001) [Fig. 1].

In experiment two, to % SHL, two-way ANOVA did not show a significant difference among the tested dentifrices (p = 0.295; F = 1.23; non-fluoridated – 64.9 ± 12; NaF – 56.9 ± 8.8; Mg (OH)2–62.6 ± 10.4). However, the % SHL according to the number of experimental days differed significantly (p < 0.001; F = 48.43). The interaction between factors (tested dentifrices and number of experi-mental days) was not significant (p = 0.326; F = 1.16). In both ex-periments, the increase in the number of experimental days yielded a statistically significant surface softening from day 1 to day 3, which

reached a plateau at days 4 and 5 (Figs. 1 and 2). Moreover, the tested dentifrices showed no significant protective effects for reduce surface loss when compared to the non-fluoride dentifrice after citric acid or hydrochloric acid erosion (Fig. 3).

4. Discussion

This in vitro cycling de-remineralization model investigated two products with respect to their capacity to protect enamel from intrinsic or extrinsic acids. This present study conﬁrmed the expected surface softening and enamel tissue loss due to the action of citric acid or hy-drochloric acid, even after pellicle formation for two hours before each experimental procedure. The use of anin vitromultiple-exposure acid model allows for a better understanding of the erosive challenges faced by the dentition. Additionally, a controlled investigation, such as this current study, reduces the experimental time and cost (Shellis, Ganss, Ren, Zero, & Lussi, 2011).

The hypothesis that there is no difference in the preventive effect for the tested dentifrices on enamel erosion caused by acidic challenges was rejected, due to an observed difference in the percentage change of surface hardness. Magnesium hydroxide and sodiumfluoride reduced the surface softening when compared with a non-fluoridated dentifrice. The results of this erosion model for extrinsic acid reinforces the relevance of magnesium hydroxide and sodium fluoride-containing dentifrices in the reduction of enamel erosion caused by dietary acids. However, magnesium hydroxide was more effective than sodium fluoride in protecting the human enamel against citric acid erosion. This result may be explained through the acid buffering that occurs immediately after the contact of Mg(OH)2and citric acid, which pro-duces a salt (Mg2C6H4O7) and H2O, a neutralization reaction. Furthermore, the abrasive (calcium carbonate) present in the dentifrice can react with magnesium hydroxide and form byproducts of reaction (MgCO3 and Ca(OH)2), making residual acid neutralization possible. The use of a magnesium hydroxide dentifrice may also help saliva to neutralize and remove erosive products from the oral cavity (Messias et al., 2010). In addition, the presence of magnesium ions during de-mineralization and rede-mineralization reactions, change crystallographic enamel structure, since Mg ions are incorporated in the surface layer of enamel, reducing the apatite crystallite size (Abdallah et al., 2016). As consequence, the tooth hardness increases, thus suggesting that mag-nesium dentifrice could be used to leave the tooth less susceptible to erosive challenges.

Furthermore, the relevance of sodium ﬂuoride-containing denti-frices in the reduction of enamel erosion caused by dietary acids was described by otherin vitro(Ganss et al., 2011; Rochel et al., 2011) and in situ(Passos et al., 2010;Hara, Barlow, Eckert, & Zero, 2014) studies. White et al. (2012) and Scaramucci et al. (2013) veriﬁed that low

(4)

concentrations (10 ug/g F−_{to 275 ppmF) of sodium}_{fluoride solutions} are capable of reducingin vitrohydroxyapatite dissolution with citric acid. Therefore, the 1450 ppm F present in the NaF dentifrice, even after dilution in saliva, can be effective. The preventive effect of a so-diumfluoride dentifrice in erosive lesions is mainly based on the de-position of CaF2-like products on the enamel surface, which can protect against acid attack (Saxegaard & Rölla, 2008). However, according to Hjortsjö et al. (2015)NaF products, in nonacidulated conditions, sug-gest a slow formation of CaF2. Furthermore, the protective action is still reduced after repeated and excessive acid contact. Another point to consider is the pH of environment, which presents an important role in the efficacy of the preventive agent, since acidic conditions increase the formation of CaF2-like deposits. In the present study, the sodium fluoride dentifrice might have been less effective than magnesium hy-droxide in the protection of enamel erosion because the NaF dentifrice presented a neutral pH.

Meanwhile, sodiumfluoride and magnesium hydroxide dentifrices are not effective in reducing enamel loss. In fact, surface profilometry analysis is more indicated for evaluating advanced erosion (Schlueter, Hara, Shellis, & Ganss, 2011), which was not performed in experiment 1. Based on the present results, enamel surface loss was only of ap-proximately 0.2μm afterfive experimental days of extrinsic acid ex-posure, whereas intrinsic acid exposure produced a surface loss se-venfold more severe, proving a moderate action of acidic beverages. However, profilometric analysis is widely accepted as a technique to evaluate erosive challenge (Jordão et al., 2016; Passos, Melo, Vasconcellos, Rodrigues, & Santiago, 2013; Passos et al., 2015). Al-though profilometric analysis does not properly assess the prejudicial effect of erosion in the case of experiment 1, this analysis serves as a complementary assessment to prove the loss of tooth structure at the end of the experiment.

In both analyses of experiment two, the tested dentifrices failed to reject the null hypothesis. A possible explanation for the lack of beneﬁt for the sodiumﬂuoride and magnesium hydroxide dentifrices used in thisin vitroreport may be due to the severe action of hydrochloric acid compared to citric acid, which may have decreased the preventive ef-fect of these products. In addition, this strong acid (pKa =−6.3–HCl)

dissolves and removes the mineral surface more quickly than weaker acids such as citric acid (pKa1 = 3.15; pKa2 = 4.77; pKa3 = 6.40). However, the observed wear was not too severe when compared with a recent study, once the formation of a protective salivaryﬁlm was not reproduced, and the erosion protocol used consisted of longer and more frequent period where enamel surface was put in contact with HCl (2 min–4 times per day–during 5 days) (Oliveira et al., 2017).

Moreover, one possible explanation to magnesium hydroxide have had a bigger effect on citric acid erosion than on HCl erosion is that the latter solution has very little buffer capacity, so the pH in the near-surface enamel will rise very quickly when the specimen is removed from the acid solution. While, when the specimen is removed from a citric acid solution, the pH of the acid in the near-surface enamel will rise much more slowly because of the buffering ability of citric acid. Despite the methodological differences, studies using hydrochloric acid to simulate erosion by intrinsic acid and using fluoride or antacid dentifrices agree with the presentfindings (Ganss, Schlueter, Friedrich, & Klimek, 2007;Messias et al., 2008). Moreover, in the case of NaF dentifrice, the time of application (30 s) using a neutral sodiumfluoride may be not sufficient to form a resistant CaF2-layer able to protect si-mulated intrinsic acid. Additionally, the use of diluted dentifrices (1:3) may have also reduced the formation of CaF2, damaging the protective effect of sodiumfluoride against strong acids.

Dental biofilm may serve as a reservoir of magnesium hydroxide. However, in the presentin vitroreport, this complementary action was not observed, probably reducing the protective effect exerted by Mg Fig. 2.Percentage of surface hardness loss after each erosive day using hydrochloric acid (experiment 2) according to two-way Anova and Tukey tests. Different letters imply statistical differences between each day.

(5)

(OH)2. Thus, further studies mimicking in vivo conditions and re-producing intra-oral influences, such as a constant action of saliva, may be needed to verify the real effect of this agent.Turssi et al. (2012) indicated that a magnesium hydroxide suspension can provide a sig-nificant reduction of surface enamel loss. Nevertheless, this effective result may be based on the high concentration of magnesium hydroxide in the suspension used by those authors (80 mg ml−1_{), while the} con-centration used in the present study was approximately 6 mg/ml. The cycling erosive model used in the former study was less aggressive (5 cycles) than the one used in the current study (15 cycles).

A limitation of thisin vitromodel tested is that it does not evaluate the abrasive effect of the dentifrice, as occurs during toothbrushing. This was done intentionally to check the real action of dentifrices in this erosive process. Therefore, the results reflect the neutralizing and re-mineralizing effect of dentifrice slurry. In both of the present experi-ments, the surface hardness and tissue loss results (Figs. 1–3) demon-strated that all dentifrices were unable to avoid the simulated extrinsic or intrinsic erosive challenges on human enamel. Moreover, an increase in the number of experimental days generated a longer exposure time to acids, leading to greater softening of the surface enamel, confirmed the progressive destruction of the enamel structures. According toAmaechi, Higham, and Edgar (1999) andWest, Hughes, and Addy (2000)the frequent contact of the teeth to acidic products leads to the loss of a protective layer on the enamel surface, exposing a more vulnerable surface to future acid exposure. In addition, Creanor, Creanor, and Alharthy (2011)observed that an intermittent erosive protocol created considerably greater lesion depths when evaluating continuous or in-termittent orange juice tooth exposure, which is more realistic. Inter-mittent erosive challenges do not allow mineral ions dissolved from the tooth to influence the under-saturated condition generated by acid beverages. Therefore, new acid solutions constantly remove more cal-cium and phosphate from the tooth surface. Also, the commercial Mg (OH)2dentifrice used contains calcium carbonate as abrasive. Thus, a possible neutralizing effect of this component in reducing erosion may not be discarded and should be further tested.

5. Conclusion

In conclusion, within the limitations of this presentin vitrostudy, the use ofﬂuoride or hydroxide magnesium-containing dentifrices can present a protective role against extrinsic acids. However, erosive le-sions caused by hydrochloric acid were not prevented by these ther-apeutic products. Furthermore, frequent and chronic contact of acids with the dentition progressively increases erosion, even in the presence of protective products.

Conﬂict of interest

None.

Acknowledgments

This research was supported by National Council for Scientific and Technological Development (Process # 620107/2008-1). Thefirst au-thor thanks the National Council for Scientific and Technological Development for concession of scholarships during this study (Proc. # 140643/2011-7).

References

Abdallah, M. N., Eimar, H., Bassett, D. C., Schnabel, M., Ciobanu, O., Nelea, V., et al. (2016). Diagenesis-inspired reaction of magnesium ions with surface enamel mineral modiﬁes properties of human teeth.Acta Biomaterialia, 37, 174–183.

Amaechi, B. T., Higham, S. M., & Edgar, W. M. (1999). Factors inﬂuencing the devel-opment of dental erosion in vitro: Enamel type, temperature and exposure time. Journal of Oral Rehabilitation, 26(8), 624–630.

Creanor, S., Creanor, S., & Alharthy, N. (2011). A comparison of in vitro erosion-like

mineral loss between continuous and intermittent acidic exposure with and without human saliva.Archives of Oral Biology, 56(7), 703–708.

de Groot, A., Tupker, R., Hissink, D., & Woutersen, M. (2017). Allergic contact cheilitis caused by olaﬂur in toothpaste.Contact Dermatitis, 76(1), 61–62.

de-Melo, M. A., Passos, V. F., Alves, J. J., Barros, E. B., Santiago, S. L., & Rodrigues, L. K. (2011). The eﬀect of diode laser irradiation on dentin as a preventive measure against dental erosion: An in vitro study.Lasers in Medical Science, 26(5), 615–621.

Duﬀey, K. J., Huybrechts, I., Mouratidou, T., Libuda, L., Kersting, M., De Vriendt, T., et al. (2012). Beverage consumption among European adolescents in the HELENA study. European Journal of Clinical Nutrition, 66(2), 244–252.

Ganss, C., Schlueter, N., Friedrich, D., & Klimek, J. (2007). Eﬃcacy of waiting periods and topicalﬂuoride treatment on toothbrush abrasion of eroded enamel in situ.Caries Research, 41(2), 146–151.

Ganss, C., Lussi, A., Grunau, O., Klimek, J., & Schlueter, N. (2011). Conventional and anti-erosionﬂuoride toothpastes: Eﬀects on enamel erosion and erosion-abrasion.Caries Research, 45(6), 581–589.

Hara, A. T., Barlow, A. P., Eckert, G. J., & Zero, D. T. (2014). Novel in-situ longitudinal model for the study of dentifrices on dental erosion-abrasion.European Journal of Oral Sciences, 122(2), 161–167.

Hjortsjö, C., Young, A., Kiesow, A., Cismak, A., Berthold, L., & Petzold, M. (2015). A descriptive in vitro electron microscopic study of acidﬂuoride-treated enamel po-tential anti-erosion eﬀect.Caries Research, 49(6), 618–625.

Johansson, A. K., Omar, R., Carlsson, G. E., & Johansson, A. (2012). Dental erosion and its growing importance in clinical practice: From past to present.International Journal of Dentistry, 2012, 632907.

Jordão, M. C., Forti, G. M., Navarro, R. S., Freitas, P. M., Honório, H. M., & Rios, D. (2016). CO2 laser and/orﬂuoride enamel treatment against in situ/ex vivo erosive challenge.Journal of Applied Oral Science, 24(3), 223–228.

Lindquist, B., Lingström, P., Fändriks, L., & Birkhed, D. (2011). Inﬂuence ofﬁve neu-tralizing products on intra-oral pH after rinsing with simulated gastric acid.European Journal of Oral Sciences, 119(4), 301–304.

Lussi, A., Schlueter, N., Rakhmatullina, E., & Ganss, C. (2011). Dental erosion–An over-view with emphasis on chemical and histopathological aspects.Caries Research, 45(Sp. Issue 1), 2–12.

Messias, D. C., Serra, M. C., & Turssi, C. P. (2008). Potential eﬀect of sodium bicarbonate-containing dentifrice in controlling enamel erosion in situ.American Journal of Dentistry, 21(5), 300–302.

Messias, D. C., Turssi, C. P., Hara, A. T., & Serra, M. C. (2010). Sodium bicarbonate solution as an anti-erosive agent against simulated endogenous erosion.European Journal of Oral Sciences, 118(4), 385–388.

Meurman, J. H., Kuittinen, T., Kangas, M., & Tuisku, T. (1988). Buﬀering eﬀect of an-tacids in the mouth–A new treatment of dental erosion?Scandinavian Journal of Dental Research, 96(5), 412–417.

Nekrashevych, Y., & Stösser, L. (2003). Protective inﬂuence of experimentally formed salivary pellicle on enamel erosion.Caries Research, 37(3), 225–231.

Oliveira, G. C., Tereza, G. P. G., Boteon, A. P., Ferrairo, B. M., Gonçalves, P. S. P., Silva, T. C. D., et al. (2017). Susceptibility of bovine dental enamel with initial erosion lesion to new erosive challenges.PLoS One, 12(8), e0182347.

Pace, F., Pallotta, S., Tonini, M., Vakil, N., & Bianchi Porro, G. (2008). Systematic review: Gastro-oesophageal reﬂux disease and dental lesions.Alimentary Pharmacology & Therapeutics, 27(12), 1179–1186.

Passos, V. F., Santiago, S. L., Tenuta, L. M. A., & Cury, J. A. (2010). Protective eﬀect of NaF/triclosan/copolymer and MFP dentifrice on enamel erosion.American Journal of Dentistry, 23(4), 193–195.

Passos, V. F., Melo, M. A., Vasconcellos, A. A., Rodrigues, L. K., & Santiago, S. L. (2013). Comparison of methods for quantifying dental wear caused by erosion and abrasion. Microscopy Research and Technique, 76(2), 178–183.

Passos, V. F., de Vasconcellos, A. A., Pequeno, J. H., Rodrigues, L. K., & Santiago, S. L. (2015). Eﬀect of commercialﬂuoride dentifrices against hydrochloric acid in an erosion-abrasion model.Clinical Oral Investigation, 19(1), 71–76.

Queiroz, C. S., Hara, A. T., Paes Leme, A. F., & Cury, J. A. (2008). pH-cycling models to evaluate the eﬀect of lowﬂuoride dentifrice on enamel de- and remineralization. Brazilian Dental Journal, 19(1), 21–27.

Rochel, I. D., Souza, J. G., Silva, T. C., Pereira, A. F., Rios, D., Buzalaf, M. A., et al. (2011). Eﬀect of experimental xylitol andﬂuoride-containing dentifrices on enamel erosion with or without abrasion in vitro.Journal of Oral Science, 53(2), 163–168.

Saxegaard, E., & Rölla, G. (2008). Fluoride acquisition on and in human enamel during topical application in vitro.Scandinavian Journal of Dental Research, 96(6), 523–535.

Scaramucci, T., Borges, A. B., Lippert, F., Frank, N. E., & Hara, A. T. (2013). Sodium

ﬂuoride eﬀect on erosion-abrasion under hyposalivatory simulating conditions. Archives of Oral Biology, 58(10), 1457–1463.

Schlueter, N., Hara, A., Shellis, R. P., & Ganss, C. (2011). Methods for the measurement and characterization of erosion in enamel and dentine.Caries Research, 45(1), 13–23.

Shellis, R. P., Ganss, C., Ren, Y., Zero, D. T., & Lussi, A. (2011). Methodology and models in erosion research: Discussion and conclusions.Caries Research, 45(1), 69–77.

Soares, G. G., Magalhães, P. A., Fonseca, A. B. M., Tostes, M. A., Silva, E. M. D., & Coutinho, T. C. L. (2017). Preventive effect of CPP-ACPF paste andfluoride tooth-pastes against erosion and erosion plus abrasion in vitro–A 3D profilometric ana-lysis.Oral Health & Preventive Dentistry, 15(3), 269–277.

Turssi, C. P., Vianna, L. M., Hara, A. T., do Amaral, F. L., França, F. M., & Basting, R. T. (2012). Counteractive eﬀect of antacid suspensions on intrinsic dental erosion. European Journal of Oral Sciences, 120(4), 349–352.

Van Baelen, A., Kerre, S., & Goossens, A. (2016). Allergic contact cheilitis and hand dermatitis caused by a toothpaste.Contact Dermatitis, 74(3), 187–189.

West, N. X., Hughes, J. A., & Addy, M. (2000). Erosion of dentine and enamel in vitro by dietary acids: The eﬀect of temperature, acid character, concentration and exposure time.Journal of Oral Rehabilitation, 27(10), 875–880.