- Email: [email protected]
Fluoride gel supplemented with sodium hexametaphosphate reduces enamel erosive wear in situ
Journal of Dentistry 43 (2015) 1255–1260
Contents lists available at ScienceDirect
Journal of Dentistry journal homepage: www.intl.elsevierhealth.com/journals/jden
Fluoride gel supplemented with sodium hexametaphosphate reduces enamel erosive wear in situ J.M. Conceiçãoa , A.C.B. Delbema , M. Danelona , D.M. da Camaraa , A. Wiegandb , J.P. Pessana,* a b
Araçatuba Dental School, Univ. Estadual Paulista (UNESP), Araçatuba, SP, Brazil University of Göttingen, Göttingen, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2015 Received in revised form 5 August 2015 Accepted 14 August 2015
Objective: This study evaluated the effect of fluoride gels, supplemented or not with sodium hexametaphosphate (HMP), on enamel erosive wear in situ. Methods: Twelve healthy volunteers wore palatal appliances containing four bovine enamel discs. Subjects were randomly allocated into four experimental phases (double-blind, crossover protocol) according to the gels: Placebo (no fluoride or HMP), 1% NaF, 2% NaF, and 1% NaF + 9% HMP. Enamel discs were selected after polishing and surface hardness analysis, and treated only once with the respective gels prior to each experimental phase. Erosion (ERO) was performed by extra-oral immersion of the appliance in 0.05 M citric acid, pH 3.2 (four times/day, five minutes each, 5 days). Additional abrasion (ERO + ABR) was produced on only two discs by toothbrushing with fluoridated dentifrice after ERO (four times/day, 30 s, 5 days). The specimens were submitted to profilometry and hardness analysis. The results were analyzed by two-way ANOVA and the Student–Newman–Keuls test (p < 0.05). Results: The 1% NaF + 9% HMP gel promoted significantly lower enamel wear for ERO compared to the other groups, being statistically lower than 1% NaF and Placebo for ERO + ABR. Similarly, the lowest values of integrated lesion area were found for 1% NaF + 9% HMP and 2% NaF, respectively, for ERO and ERO + ABR. Conclusion: The addition of HMP to the 1% NaF gel promoted greater protective effect against ERO and ERO + ABR compared to the 1% NaF gel, achieving similar protective levels to those seen for the 2% NaF gel. Clinical significance: Gel containing 1% NaF + 9% HMP showed a high anti-erosive potential, being a safer alternative when compared to a conventional 2% NaF gel. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: Dental erosion Dental abrasion Topical fluorides Dental enamel Sodium hexametaphosphate
1. Introduction Topical fluoride application has an inhibitory effect on dental erosion [1,2], which depends on the concentration of fluoride and the frequency of application. The addition of inorganic polyphosphate salts to fluoridated products has been shown to be effective in increasing their effectiveness against caries [3,4]. One of the most promising candidate is sodium hexametaphosphate (HMP), which is able to inhibit the formation of dental calculus [5], has antimicrobial action [6], and also prevents the formation of extrinsic stains [7]. HMP consists of a cyclic polyphosphate, presenting strong attraction to calcium in the hydroxyapatite in relation to other phosphates [8], being mixable in water and
* Corresponding author at: Araçatuba Dental School, Univ. Estadual Paulista (UNESP), Department of Pediatric Dentistry and Public Health, Rua José Bonifácio 1193, 16015-050 Araçatuba, SP, Brazil. E-mail address: [email protected] (J.P. Pessan). http://dx.doi.org/10.1016/j.jdent.2015.08.006 0300-5712/ ã 2015 Elsevier Ltd. All rights reserved.
insoluble in organic solvents [9,10]. It is a variant of the longer pyrophosphate chain and presents greater possibility of binding to the enamel surface, protecting it from acid dissolution [11]. In vitro and in situ studies demonstrated that the association of fluoride and HMP in conventional dentifrices (1100 mg F/g) promoted greater protection against enamel demineralization compared to their counterparts without HMP [12–14]. A synergistic effect was also demonstrated when HMP was added to a dentifrice with reduced fluoride concentration (250 mg/g), achieving mineral loss values similar to those observed for the positive control (1100 mg F/g) [15]. Moreover, the addition of HMP to a dentifrice containing stannous fluoride promoted lower erosive wear in enamel and dentin compared to the positive control [16]. Concerning the addition of HMP to fluoridated gels, only one study has evaluated the effects of this association against dental caries in vitro, demonstrating that the 1% NaF gel supplemented with 9% HMP presented greater ability to inhibit enamel demineralization compared to a 1% NaF gel without HMP, as well
1256
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
as similar results to the 2% NaF gel [17]. However, the effect of gels containing fluoride and HMP on erosion has not yet been investigated. Considering that gels are widely used in dental practice and for normal oral hygiene in some countries, it would be interesting to analyze their effectiveness against erosion as an adjunct to normal oral hygiene with a fluoridated dentifrice. Therefore, this in situ/ex vivo study analyzed the effects of fluoridated gels supplemented or not with HMP on enamel erosion, followed or not by abrasion. The null hypothesis was that the use of a fluoridated gel supplemented with HMP is not different from a gel with the same fluoride concentration without HMP in preventing erosive wear and mineral loss of enamel. 2. Material and methods 2.1. Volunteers’ selection and ethical aspects This study was approved by the Institutional Review Board of Araçatuba Dental School, UNESP (CAAE n. 5421712.2.0000.5420, report n. 154.616). Twelve healthy adult volunteers were selected [18], aged 20 to 33 years old (4 male, 8 female) living in areas with fluoridated water supply and presenting good general and oral health. Sample size was determined based on a previous study with similar methodology [19] considering the mean difference among the groups of 0.30 and standard deviation of 0.20 from enamel wear data, and an a-error of 5% and a b-error of 20%. The inclusion criteria comprised normal salivary flow (1–3 mL/min, stimulated saliva, determined as described by Pessan et al. [20]) and no use of drugs that might interfere with salivary composition and flow [21]. The exclusion criteria were smokers, individuals presenting active caries lesions, receiving fluoride applications up to two weeks before the study, using antidepressants, narcotics, diuretics and antihistamine drugs in the last two months, submitted to radiation therapy, practicing water activities, working in environments polluted by compounds with low pH, and presenting systemic diseases (xerostomia, type I diabetes, autoimmune diseases, malnutrition, gastroesophageal problems, regurgitation disorders and vomiting). Exclusion criteria during the study comprised volunteer dropout, health changes with consequent alteration in salivary flow or need of antibiotics, utilization of mouthrinses or dentifrices different than those supplied by the investigators, or failure to follow the experimental design. If any volunteer was excluded, a new volunteer would be selected to complete all study phases. Before the experimental phase, the volunteers received written and verbal instructions on the study protocol, and were supervised to follow the study conditions. All volunteers used the same standard 1100 mg F/g dentifrice (NaF1100 mg F/g-Sorriso Fresh Plus Gel, Colgate) throughout the study. 2.2. Experimental protocol This in situ/ex vivo study was conducted in 4 experimental phases of 5 days each, with a lead in and wash out periods of 7 days. Volunteers wore palatal appliances containing 4 enamel discs, which were treated with Placebo, 1% NaF, 2% NaF and 1% NaF + 9% HMP gels, following a double-blind, crossover protocol. Enamel discs were subjected to ex vivo erosive challenges (immersion in 0.05 M citric acid), followed or not by abrasion by toothbrushing with a silica-based NaF toothpaste (1100 ppm F). Subjects were instructed to keep the palatal appliances in the mouth during all times throughout the experimental phases, except for eating and drinking and for performing oral hygiene procedures. In each phase, all volunteers received a kit containing one tube of 1100 mg F/g dentifrice, one soft bristle toothbrush (Sorriso Kolynos Original Macia—Colgate), 5 disposable cups with visible indication of 50 mL, one orthodontic appliance case, gauze, one
dropper bottle containing the dentifrice suspension (ratio 1 g of dentifrice: 3 mL of deionized water) and one liter of citric acid pH 3.2. 2.3. Preparation of enamel discs Permanent mandibular central incisors were used, obtained from bovines aged 2 to 3 years and maintained in plastic flasks with 2% formalin solution pH 7.0 for 30 days [4]. Enamel discs (n = 192, approximate 4.5 mm diameter) were obtained from the flattest portion on the buccal surface of crowns, using a bur (DS 07882457) connected to a bench drill (MOD. FGC-16, reamer 5/8). The discs were then marked with a round diamond bur to identify the cervico-occlusal direction. The dentin was adjusted (thickness 2 mm) for achievement of parallel surfaces between enamel and dentin. Then, enamel surfaces were turned upwards, and polished with sandpaper grits 400 (20 s), 600 (30 s), 800 (30 s) and 1200 (30 s), under water cooling. The surface was then polished with polishing cloth (Polishing Cloth BUEHLER 40-7618) and diamond suspension (METADI Diamond Suspension 1 micron Blue Color Polish Spray, Water Base 40-653). Finally, the discs were rinsed with deionized water for 20 s and stored in a humid environment with gauze soaked in deionized water, for the initial enamel surface microhardness analysis [22–24]. 2.4. Determination of initial enamel hardness (SHi) and sample selection The surface microhardness of enamel discs was evaluated using the microhardness meter Shimadzu Micro Hardness Tester HMV2.000 (Shimadzu Corporation—Kyoto—Japan) using Knoop indenters, static load of 25 grams and time of 10 s, connected to the image analysis software CAMS-WIN (NewAge Industries, USA). For the initial surface microhardness (initial SH), five indents were made on the central region of the enamel disc, equidistant at 100 mm. Discs presenting mean hardness values within the confidence interval 353.7 (2.7) to 354.2 (1.4) were selected. The discs were identified according to their initial microhardness value. 2.5. Formulation and assessment of fluoride and pH in the experimental gels The gels were prepared at the laboratory of Pediatric Dentistry of Araçatuba Dental School, São Paulo State University (Brazil), containing the following ingredients: carboxymethyl cellulose, saccharin, glycerin, mint oil and deionized water. The sodium fluoride concentrations were 1% and 2% (NaF, Merck, Germany). Ssodium hexametaphosphate (HMP, Sigma, USA) was added to the 1% NaF gel at a concentration of 9%, based on a previous study assessing the effects of gels on enamel demineralization in vitro [17]. Also, a gel without NaF or HMP was prepared and used as negative control (Placebo). For fluoride analysis, approximately 100 mg of each gel was weighed and sequentially diluted in distilled/deionized water. Following, the flask contents were transferred to volumetric flasks and the volume was completed up to 100 mL with distilled/ deionized water. Three dilutions were made for each product. Thereafter, two samples of 1 mL were obtained and buffered with TISAB II [25]. The solutions were then analyzed using a fluoride ion-specific electrode (9609 BN—Orion) connected to an ion analyzer (Orion 720 A+), previously calibrated with the five standards (2.0; 4.0; 8.0; 16.0 and 32 mg F/mL). The total fluoride (TF) and ionic fluoride (IF) were analyzed. Data obtained in mV were converted into mg F/mL. The pH of the gels was assessed in
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
1257
triplicate using a pH meter (Orion 4 Star, Thermo Scientific) and a pH electrode, as previously described [26].
protected specimen area (reference surface) and the peak wear. Five scans were performed on the entire enamel surface [32].
2.6. Preparation of palatal appliances
2.9.2. Determination of final enamel hardness For the final surface microhardness test (SHf), five indents were made on the central region of the eroded enamel, equidistant at 100 mm, with a static load of 25 g for 10 s. For the microhardness test in longitudinal section, the enamel discs were sectioned through the half, and the portion not submitted to ERO or ERO + ABR, respectively, was used for comparison. A microhardness meter Micromet 5114 hardness tester (Buehler, Lake Bluff, USA) and the software Buehler OmniMet (Buehler, Lake Bluff, USA) were used, with a load of 5 g for 10 s with 1000 magnification. Nine indents were made at 5, 10, 15, 20, 25, 30, 40, 50, 70 mm from the external enamel surface, in eroded and intact areas on the center of each disc half. The integrated hardness area (KHN mm) of the lesion up to the intact enamel was calculated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated area of intact enamel hardness, thus achieving the integrated hardness loss (DKHN).
Alginate impressions were taken from the maxillary arch of all volunteers for fabrication of acrylic palatal appliances. In each experiment, four bovine enamel discs were included the appliances divided in two rows (A and B). Half of the disc surfaces were isolated with nail polish (Revlon), so that only half of the disc surfaces would be submitted to treatment and the other half, used for comparison after treatment. The test surfaces of the specimens were leveled with the surface of the palatal appliance. 2.7. Treatment Volunteers inserted the appliance in the mouth two hours before study onset, to allow formation of acquired pellicle [27]. Thereafter, the appliances were removed for application of gels on the four discs of each appliance outside the mouth. Gels were applied with disposable syringes, allowing standardization of the quantity to be applied (approximately 1 mL on each disc surface, for one minute). Then, the gel was removed with gauze and the appliance was placed in the oral cavity of the volunteer. Simultaneously, the same gels were applied on all teeth of volunteers using disposable trays. Therefore, in each experimental phase, the volunteers were randomly distributed according to the four possible treatments: (1) Placebo (gel without NaF or HMP); (2) 1% NaF gel; (3) 2% NaF gel; and (4) 1% NaF + 9% HMP gel. After completion of each experimental phase, the discs were prepared for the analyses. 2.8. Erosive and abrasive challenges The erosive challenge (ERO) was performed during five days for each group, with a seven-day interval prior to the beginning of the study (lead in) between each phase (washout) [28,29]. The challenge was performed with 0.05 M citric acid (Synth, P.A—A. C.S, P.M 192,13), pH 3.2, adjusted with saturated NaOH (Dinâmica, P.A—A.C.S, P.M 40,00), and the appliances were immersed in the acid four times a day for five minutes [30,31]. In each appliance, the enamel discs in row “A” were submitted to erosion associated with abrasion (ERO + ABR), while discs in row “B” were only submitted to ERO. Abrasion was performed by the volunteers after each erosive challenge using a soft bristle toothbrush during 30 s (1 stroke/second), simultaneously in the two discs in row “A”. For this procedure, subjects were instructed to hold the toothbrush with the fingertips, in order to minimize excessive load during toothbrushing. The discs in row “B”, in turn, were treated with a dentifrice suspension during 30 s, immediately after each erosive challenge. 2.9. Analyses 2.9.1. Determination of surface wear—profilometry At completion of each experimental phase, the nail polish was carefully removed from the disc surfaces using an acetone-soaked cotton pellet, prior to analysis of surface wear using a profilometer Surftest SJ 401—Mitutoyo (Mitutoyo American Corporation). The roughness meter was connected to a microcomputer, which expressed and stored the testing information using a specific software (Surftest). The active tip of the roughness meter scanned the specimen surfaces from the control (untreated) portion to the affected area. Wear was determined as the distance (micrometers) between the mean line on the graph corresponding to the
2.9.3. Statistical analysis Statistical analysis was performed on the software SigmaPlot version 12.0, at a significance level of 5%. Data analysis considered the types of experimental gels and type of challenge as variation factors (ERO and ERO + ABR). The variables included the final surface and cross sectional hardness, as well as enamel surface wear (mm). After transformation (Log10), the wear data passed normality (Shapiro–Wilk) and homogeneity tests (Bartlett), fulfilling the ideal conditions for the use of parametric tests. The final surface hardness outcomes did not pass the normality and homogeneity tests even after logarithmic transformation, while for the variable DKHN the distribution was normal, yet did not present homogeneity after logarithmic transformation. Thus, considering the different types of gels and the erosive challenge, all data were submitted to two-way ANOVA followed by the Student–Newman– Keuls test. The hardness data according to depth were separately analyzed for each type of challenge, also using the two-way ANOVA after transformation (Log10) followed by the Student–Newman– Keuls test. 3. Results Total fluoride and ionic fluoride (SD, mg F/g) amounted to: Placebo: 47.0 (3.1) and 8.8 (3.6); 1% NaF: 4895.9 (57.5) and 4773.4 (20.7); 2% NaF: 9737.2 (24.2) and 9314.7 (27.8) and 1% NaF + 9% HMP: 5118.0 (5.1) and 4807.6 (31.5). Mean (SD) pH values were 6.71 (0.13), 6.41 (0.02), 6.47 (0.06) and 6.06 (0.04), respectively for the Placebo, 1% NaF, 2% NaF and 1% NaF + 9% HMP. The initial mean value (standard deviation) of surface hardness (SHi) considering all groups was 354.0 0.4 kgf/mm2, ranging from 353.7 2.7 to 354.2 1.4 (p > 0.05). The 1% NaF + 9% HMP gel promoted the lowest enamel wear after ERO compared to other gels, with significant differences among all groups (Table 1). For ERO + ABR, the lowest wear was observed for the group treated with 2% NaF gel, followed by groups 1% NaF + 9% HMP, 1% NaF and Placebo, with significant differences among all groups. Surface hardness values were significantly greater for the 2% NaF group for both challenges, followed by groups 1% NaF + 9% HMP, 1% NaF and Placebo, with significant differences among all groups (Table 1). Concerning the integrated lesion area, the 2% NaF and 1% NaF + 9% HMP gels promoted significantly higher values compared to the other groups. For ERO, the 1% NaF + 9% HMP gel yielded significantly higher DKHN values than the 2% NaF gel, and
1258
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
Table 1 Enamel erosive wear, surface hardness (SH) and integrated subsurface hardness loss (DKHN) after treatment with the experimental gels and erosive challenges (ERO), followed or not by toothbrushing (ERO + ABR). Gels
Wear (mm) EROA
Placebo 1% NaF 2% NaF 1% NaF + 9% HMP
a
3.32 (0.27) 2.29b (0.12) 1.71c (0.06) 1.28d (0.04)
DKHN (Kgf/mm2 mm)
SH (Kgf/mm2) ERO + ABRB a
4.99 (0.14) 3.56b (0.12) 2.17c (0.06) 2.36d (0.12)
ERO
ERO + ABR
Aa
91.6 (19.9) 219.7Ab (13.3) 263.7Ac (5.5) 240.7Ad (24.8)
Aa
77.3 (13.2) 103.9Bb (11.7) 226.9Bc (2.5) 194.1Bd (35.9)
EROA
ERO + ABRB a
4282.0 (558.8) 2059.7b (113.6) 1024.7c (96.6) 904.8d (113.9)
4096.9a (354.4) 2036.8b (112.7) 872.4c (107) 1052.2d (142.4)
Mean (SD), n = 12. Upper and lower case letters indicate significant differences between challenges and among treatments, respectively, (Student–Newman–Keuls; p < 0.05).
the opposite pattern occurred for ERO + ABR. All groups presented significantly lower final hardness values for ERO + ABR compared to ERO, except for the Placebo group. The mean hardness profiles according to depth for each treatment and challenge are presented in Fig.1. Hardness values are very similar and mostly without significant differences between groups 1% NaF + 9% HMP and 2% NaF for each distance analyzed. These, in turn, presented significant differences in relation to the other gels at the three most external depths analyzed, reaching more similar values in deeper regions of enamel.
Fig. 1. Mean hardness (n = 12) according to the depth and erosive challenges (A— erosion; B—erosion + abrasion). Different letters indicate significant difference among hardness values at each depth (Student–Newman–Keuls; p < 0.05). Vertical bars represent the standard deviations of the means. ¥: indicates significant difference between the Placebo gel and the other treatments. C: indicates significant difference between the Placebo gel and the other treatments, as well as between 1% NaF and 1% NaF + 9% HMP gels.
4. Discussion The addition of inorganic polyphosphate salts to fluoridated products for topical use has been shown to be an effective option to increase their effectiveness against both caries and dental erosion in in vitro and in situ trials [4,26,32–34]. In the present study, application of fluoridated gels associated with the use of a conventional fluoridated dentifrice (1100 mg F/g) significantly reduced wear and mineral loss suffered by enamel after the erosive challenges associated or not with abrasion, when compared to the placebo gel. Also, rejecting the null hypothesis, supplementation of a fluoridated gel with HMP led to lower wear compared to a gel with the same fluoride concentration, without addition of HMP, thus demonstrating that a gel with reduced fluoride concentration may be effective against the erosive challenge when associated with a polyphosphate. The use of the 1% NaF + 9% HMP gel promoted significantly lower wear in relation to the other groups for enamel submitted to ERO, including the gel with twice the concentration of fluoride, without HMP. Conversely, the wear suffered by enamel treated with the gel containing HMP was significantly lower only when compared to 1% NaF and placebo groups for ERO + ABR. However, it should be mentioned that the wear values observed for the group treated with HMP gel was very similar to that obtained for the 2% NaF gel (8.6% of difference) for ERO + ABR, and the significant difference might be due to the small variability of outcomes around the means. Thus, it can be concluded that the HMP-containing gel presented a similar protective effect against enamel erosive wear as the standard 2% NaF gel. HMP cannot be considered as a phosphate source to react with tooth enamel, as it does not become spontaneously hydrolyzed [35,36]. This cyclic phosphate forms strong complexes with metal ions [35,37–39] and, in the oral environment, adsorbs to enamel surface and retains charged ions of CaF+ and Ca2+ by replacement of Na+ in the cyclic structure, leading to a reticular formation via Ca2+ binding to one or more HMP molecules [40]. Similarly to sodium trimetaphosphate (TMP), this produces higher fluoride and calcium retention, further releasing with H2PO4, increasing CaF+ and Ca2+ species to the oral environment under acidic conditions; these would subsequently react the ionic activity of neutral ions CaHPO40 and HF0, which are known to have diffusion coefficients much higher than charged species [39]. The above-mentioned hypothesis is supported by the significantly lower subsurface caries-like lesions seen for enamel treated with HMP-containing toothpaste [15] and gel [17], as well as for the present crosssectional data, as discussed below. In order to better understand the effect of HMP associated with F, enamel specimens were also evaluated by hardness analysis. The surface hardness values presented a dose-dependent relationship according to the fluoride concentration in the gels, confirming previous studies demonstrating that the eroded enamel presents a
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
less softened surface when submitted to treatment with fluoride [41,42]. This marked effect of the 2% NaF gel on enamel surface may have accounted for the higher resistance to abrasion in this group after enamel was eroded by citric acid, given that mean enamel wear under ERO + ABR was 26% higher than under ERO only, while the corresponding value for 1% NaF gel was around 55%. As for the HMP-supplemented gel, this difference was even higher (85%), what might suggest that the protective layer formed on enamel surface after gel application was partially lost due to erosive and abrasive challenges. Nonetheless, this gel led to significantly higher hardness value compared to 1% NaF gel under both challenges, what reinforces the synergistic effect of fluoride and HMP and suggests that higher hardness values may be related to greater enamel protection for ERO and ERO + ABR. Despite the trend seen for surface hardness in the present study, this measurement has been considered not to be a good indicator for analysis of erosion, since the final surface analyzed cannot be considered as the same surface that precedes the challenge [43]. Cross-sectional hardness was then determined, allowing quantification of demineralization by calculation of the integrated hardness loss (DKHN), as well as the mean hardness according to enamel depth. The pattern of DKHN for both challenges was the same observed for enamel wear (lowest values for 1% NaF + 9% HMP and 2% NaF, respectively, for ERO and ERO + ABR). Nonetheless, as observed for wear outcomes, the differences between 1% NaF + 9% HMP and 2% NaF gels concerning the DKHN for ERO + ABR were very small (approximately 6%), as shown in Fig. 2-B. Considering that the present in situ protocol was conducted under extreme conditions of ERO and ERO + ABR challenges, thereby maximizing the differences between treatments, it is possible that the small differences observed between 1% NaF + 9% HMP and 2% NaF gels, for both wear and integrated hardness loss, might not be relevant for the clinical practice. This assumption, however, should be confirmed in studies with different research protocols, especially considering the time of each erosive challenge, which could be much lower than that used in the present work. It is well known that when the erosive challenge is associated with enamel abrasion by toothbrushing, the most external enamel layer (previously softened by the erosive agent) is removed by abrasion, exposing an underlying layer with higher degree of mineralization [26,32]. Although DKHN values confirm this, surface hardness values presented the opposite tendency. The reasons for this discrepancy are not evident, yet it may be assumed that they are related to the concomitant use of gels and a fluoridated dentifrice, while previous studies have used a single source of fluoride. Considering that fluoride presents a more marked effect in partially demineralized areas [44], it is possible that fluoride from the dentifrice bound only to the most external enamel layers (due to the short time of enamel exposure to the dentifrice), which would promote re-precipitation of calcium and phosphate ions on enamel. According to this hypothesis, this reprecipitation would promote increased surface hardness for ERO, while the net mineral gain occurring between the erosive challenge (loss) and re-precipitation (gain) might be removed by abrasion for the ERO + ABR condition. The above-mentioned hypothesis seems to be supported by data in Fig. 2-A and 2-B. The hardness values closer to the enamel surface (5 mm) are higher for ERO compared to ERO + ABR, confirming the data obtained for surface hardness. However, when advancing in depth, ERO + ABR values increased in greater proportion compared to ERO, so that the differences observed at 5 mm are minimized when the integrated area is calculated (Fig. 2A). Since this phenomenon had not been previously observed, it would be interesting to conduct studies using different methodologies to confirm this hypothesis.
1259
Finally, it should be mentioned that none of the gels tested was able to completely inhibit the erosion or even promote repair of established lesions, reinforcing the role of fluoride only as coadjutant for reduction of wear and mineral loss suffered by enamel after erosive challenges, associated or not with abrasion. Similarly to carious lesions, this highlights the need of other preventive measures, especially dietary control and modification of hygiene habits. These include reduced intake of acidic foods and beverages, utilization of low-abrasive dentifrices and soft bristle toothbrush [45]. In summary, it was concluded that utilization of a gel with reduced fluoride concentration (1% NaF) supplemented with HMP promoted smaller wear and lower enamel mineral loss compared to a gel with the same fluoride concentration without addition of HMP. This effect was similar to that achieved after utilization of 2% NaF gel, evidencing the synergic effect between fluoride and HMP in the erosion process. The results also demonstrated that, for individuals making regular use of fluoridated dentifrice, additional therapy with fluoridated gels seems a viable option to reduce the wear and enamel mineral loss associated with erosive challenges, associated or not with abrasion. Acknowledgments The authors would like to thank the volunteers for their participation and also the laboratory technician of the Department of Pediatric Dentistry and Public Health, Maria dos Santos Ferreira Fernandes, for the collaboration with the laboratory procedures during this study. References [1] C. Ganss, Definition of erosion and links to tooth wear, Monogr. Oral Sci. 20 (2006) 9–16. [2] M.D. Lagerweij, W. Buchalla, S. Kohnke, K. Becker, A.M. Lennon, T. Attin, Prevention of erosion and abrasion by a high fluoride concentration gel applied at high frequencies, Caries Res. 40 (2006) 148–153. [3] M. Danelon, E.M. Takeshita, K.T. Sassaki, A.C.B. Debem, In situ evaluation of a low fluoride concentration gel with sodium trimetaphosphate in enamel remineralization, Am. J. Dent. 26 (2013) 15–20. [4] M. Danelon, E.M. Takeshita, L.C. Peixoto, K.T. Sassaki, A.C. Delbem, Effect of fluoride gels supplemented with sodium trimetaphosphate in reducing demineralization, Clin. Oral Invest. 18 (2014) 1119–1127. [5] T. Schiff, L. Saletta, R.A. Baker, J.L. Winston, T. He, Desensitizing effect of a stabilized stannous fluoride/Sodium hexametaphosphate dentifrice, Compend. Contin. Educ. Dent. 26 (2005) 35–40. [6] M. Vaara, Agents that increase the permeability of the outer membrane, Microbiol. Rev. 56 (1992) 395–411. [7] H. Liu, V.A. Segreto, R.A. Baker, K.A. Vastola, L.L. Ramsey, R.W. Gerlach, Anticalculus efficacy and safety of a novel whitening dentifrice containing sodium hexametaphosphate: a controlled six month clinical trial, J. Clin. Dent. 13 (2002) 25–28. [8] A. Baig, T. He, J. Buisson, L. Sagel, E. Suszcynsky-Meister, D.J. White, Extrinsic whitening effects of sodium hexametaphosphate—a review including a dentifrice with stabilized stannous fluoride, Compend. Contin. Educ. Dent. 26 (2005) 47–53. [9] S. Budavari, M.J. O’Neil, A. Smith, P.E. Heckelman, An Encyclopedia of Chemicals, Drugs, and Biological, 11, Rahway Merck Co., 19591559. [10] R.J. Lewis, Hazardous Chemicals Desk Reference, 4, Van Nostrand Reinhold, New York, 19931680. [11] A.A. Baig, K.M. Kozak, E.R. Cox, J.R. Zoladz, L. Mahony, D.J. White, Laboratory studies on the chemical whitening effects of a sodium hexametaphosphate dentifrice, J. Clin. Dent. 13 (2002) 19–24. [12] A.M. Pfarrer, C.M. McQueen, M.A. Lawless, M. Rapozo-Hilo, J.D. Featherstone, Anticaries potential of a stabilized stannous fluoride/sodium hexametaphosphate dentifrice, Compend. Contin. Educ. Dent. 26 (2005) 41– 46. [13] J.S. Wefel, C.M. Stanford, D.K. Ament, M.M. Hogan, J.D. Harless, A.M. Pfarrer, L.L. Ramsey, M.S. Leusch, A.R. Biesbrock, In situ evaluation of sodium hexametaphosphate-containing dentifrices, Caries Res. 36 (2002) 122–128. [14] D.M. da Camara, J.P. Pessan, T.M. Francatia, J.A.S. Souza, M. Danelon, A.C.B. Delbem, Synergistic effect of fluoride and sodium hexametaphosphate in toothpaste on enamel demineralization in situ, J. Dent. (2015) , doi:http://dx. doi.org/10.1016/j.jdent.2015.08.007.
1260
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
[15] D.M. da Camara, M. Miyasaki, M. Danelon, K.T. Sassaki, A.C.B. Delbem, Effect of low-fluoride toothpastes combined with hexametaphosphate on in vitro enamel demineralization, J. Dent. 42 (2014) 256–262. [16] S.M. Hooper, R.G. Newcombe, R. Faller, S. Eversole, M. Addy, N.X. West, The protective effects of toothpaste against erosion by orange juice: studies in situ and in vitro, J. Dent. 35 (2007) 476–481. [17] M. Danelon, D.M. Camara, M.L. Miyasaki, J.B. Scarpa, A.C.B. Delbem, K.T. Sassaki, Effect of fluoride gels supplemented with hexametaphosphate in inhibiting demineralization, J. Dent. Res. 91 (Sp Iss B) (2012) 162,761. [18] J.P. Haas, Sample size and power, Am. J. Infect. Control 40 (2012) 766–767. [19] M.J. Moretto, A.C. Delbem, M.M. Manarelli, J.P. Pessan, C.C. Martinhon, Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion: an in situ/ex vivo study, J. Dent. 41 (2013) 1302–1306. [20] J.P. Pessan, K.M.R.P. Alves, I. Ramires, M.F.L. Taga, F.C. Sampaio, G.M. Whitford, M.A.R. Buzalaf, Effects of regular and low-fluoride dentifrices on plaque fluoride, J. Dent. Res. 89 (2010) 1106–1110. [21] M.T. Kato, M.A.R. Buzalaf, Iron supplementation reduces the erosive potential of a cola drink on enamel and dentin in situ, J. Appl. Oral Sci. 20 (2012) 318– 322. [22] A.C. Delbem, D. Pedrini, J.G. França, T.M. Machado, Fluoride release/recharge from restorative materials–effect of fluoride gels and time, Oper. Dent. 30 (2005) 690–695. [23] A.E. Vieira, A.C. Delbem, K.T. Sassaki, E. Rodrigues, J.A. Cury, R.F. Cunha, Fluoride dose response in pH-cycling models using bovine enamel, Caries Res. 39 (2005) 514–520. [24] C.O. Favretto, M. Danelon, F.C.N. Castilho, A.E.M. Vieira, A.C.B. Delbem, In vitro evaluation of the effect of mouth rinse with trimetaphosphate on enamel demineralization, Caries Res. 47 (2013) 532–538. [25] A.C. Delbem, K.T. Sassaki, A.M. Castro, L.M. Pinto, M. Bergamaschi, Assement of the fluoride concentration and pH in different mouthrinses on the brazilian market, J. Appl. Oral Sci. 11 (December) (2003) 319–323. [26] M.J. Moretto, A.C. Magalhães, K.T. Sassaki, A.C. Delbem, C.C. Martinhon, Effect of different fluoride concentrations of experimental dentifrices on enamel erosion and abrasion, Caries Res. 44 (2010) 135–140. [27] A. Cheung, Z. Zid, D. Hunt, J. McIntyre, The potential for dental plaque to protect against erosion using an in vivo-in vitro model—a pilot study, Aust. Dent. J. 50 (2005) 228–234. [28] A.C. Delbem, M. Danelon, K.T. Sassaki, A.E. Vieira, E.M. Takeshita, F.L. Brighenti, et al., Effect of rinsing with water immediately after neutral gel and foam fluoride topical application on enamel remineralization: an in situ study, Arch. Oral Biol. 55 (2010) 913–918. [29] R.P. Shellis, C. Ganss, Y. Ren, D.T. Zero, A. Lussi, Methodology and models in erosion research: discussion and conclusions, Caries Res. 45 (2011) 69–77. [30] J.A. Hughes, K.D. Jandt, N. Baker, D.M. Parker, R.G. Newcombe, M. Eisenburger, M. Addy, Futher modification to so drinks to minimize erosion. A study in situ, Caries Res. 36 (2002) 70–74.
[31] A. Wiegand, T. Attin, Design of erosion/abrasion studies insights and rational concepts, Caries Res. 45 (2011) 53–59. [32] M.M. Manarelli, A.E. Vieira, A.A. Matheus, K.T. Sassaki, A.C. Delbem, Effect of mouth rinses with fluorides and trimetaphosphate on enamel erosion: an in vitro study, Caries Res. 46 (2011) 506–509. [33] E.M. Takeshita, L.P. Castro, K.T. Sassaki, A.C.B. Delbem, In vitro evaluation with low fluoride content supplemented with trimetaphosphate, Caries Res. 43 (2009) 50–56. [34] M.M. Manarelli, A.C. Delbem, T.M. Lima, F.C. Castilho, J.P. Pessan, In vitro remineralizing effect of fluoride varnishes containing sodium trimetaphosphate, Caries Res. 48 (2014) 299–305. [35] I.K. Choi, W.W. Wen, R.W. Smith, Technical note the effect of a long chain phosphate on the adsorption of collectors on kaolinite, Miner. Eng. 6 (1993) 1191–1197. [36] E. Castellini, G. Lusvardi, G. Malavasi, L. Menabue, Thermodynamic aspects of the adsorption of hexametaphosphate on kaolinite, J. Colloid Interf. Sci. 292 (2005) 322–329. [37] G. Kura, S. Ohashi, S. Kura, Complex formation of cyclic phosphate anions with bivalent cations, J. Inorg. Nucl. Chem. 36 (1974) 1605–1609. [38] L. Changgen, L. Yongxin, Selective flotation of scheelite from calcium minerals with sodium oleate as a collector and phosphates as modifiers. II. The mechanism of the interaction between phosphate modifiers and minerals, Int. J. Miner. Process. 10 (1983) 219–235. [39] N.J. Cochrane, S. Saranathan, F. Cai, K.J. Cross, E.C. Reynolds, Enamel subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride, Caries Res. 42 (2008) 88–97. [40] J.R. van Wazer, D.A. Campanella, Structure and properties of the condensed phosphates. IV. Complex ion formation in polyphosphate solutions, J. Am. Chem. Soc. 72 (1950) 655–663. [41] D. Rios, H.M. Honorio, A.C. Magalhães, M.A. Buzalaf, R.E. Palma-Dibb, M.A. Machado, S.M. Silvia, Influence of toothbrushing on enamel softening and abrasive wear of eroded bovine enamel: an in situ study, Braz. Oral Res. 20 (2006) 148–154. [42] D. Rios, A.C. Magalhães, R.O. Polo, A. Wiegand, T. Attin, M.A. Buzalaf, The efficacy of a highly concentrated fluoride dentifrice on bovine enamel subjected to erosion and abrasion, J. Am. Dent. Assoc. 139 (2008) 1652–1656. [43] A.C. Magalhães, D. Rios, A.L. Moino, A. Wiegand, T. Attin, M.A. Buzalaf, Effect of different concentrations of fluoride in dentifrices on dentin erosion subjected or not to abrasion in situ/ex vivo, Caries Res. 42 (2008) 112–116. [44] M.A.R. Buzalaf, J.P. Pessan, H.M. Honório, J.M. ten Cate, Mechanisms of action of fluoride for caries control, Monogr. Oral Sci. 22 (2011) 97–114. [45] B.T. Amaechi, S.M. Higham, Dental erosion: possible approachs to prevent and control, J. Dent. 33 (2005) 243–252.
Contents lists available at ScienceDirect
Journal of Dentistry journal homepage: www.intl.elsevierhealth.com/journals/jden
Fluoride gel supplemented with sodium hexametaphosphate reduces enamel erosive wear in situ J.M. Conceiçãoa , A.C.B. Delbema , M. Danelona , D.M. da Camaraa , A. Wiegandb , J.P. Pessana,* a b
Araçatuba Dental School, Univ. Estadual Paulista (UNESP), Araçatuba, SP, Brazil University of Göttingen, Göttingen, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2015 Received in revised form 5 August 2015 Accepted 14 August 2015
Objective: This study evaluated the effect of fluoride gels, supplemented or not with sodium hexametaphosphate (HMP), on enamel erosive wear in situ. Methods: Twelve healthy volunteers wore palatal appliances containing four bovine enamel discs. Subjects were randomly allocated into four experimental phases (double-blind, crossover protocol) according to the gels: Placebo (no fluoride or HMP), 1% NaF, 2% NaF, and 1% NaF + 9% HMP. Enamel discs were selected after polishing and surface hardness analysis, and treated only once with the respective gels prior to each experimental phase. Erosion (ERO) was performed by extra-oral immersion of the appliance in 0.05 M citric acid, pH 3.2 (four times/day, five minutes each, 5 days). Additional abrasion (ERO + ABR) was produced on only two discs by toothbrushing with fluoridated dentifrice after ERO (four times/day, 30 s, 5 days). The specimens were submitted to profilometry and hardness analysis. The results were analyzed by two-way ANOVA and the Student–Newman–Keuls test (p < 0.05). Results: The 1% NaF + 9% HMP gel promoted significantly lower enamel wear for ERO compared to the other groups, being statistically lower than 1% NaF and Placebo for ERO + ABR. Similarly, the lowest values of integrated lesion area were found for 1% NaF + 9% HMP and 2% NaF, respectively, for ERO and ERO + ABR. Conclusion: The addition of HMP to the 1% NaF gel promoted greater protective effect against ERO and ERO + ABR compared to the 1% NaF gel, achieving similar protective levels to those seen for the 2% NaF gel. Clinical significance: Gel containing 1% NaF + 9% HMP showed a high anti-erosive potential, being a safer alternative when compared to a conventional 2% NaF gel. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: Dental erosion Dental abrasion Topical fluorides Dental enamel Sodium hexametaphosphate
1. Introduction Topical fluoride application has an inhibitory effect on dental erosion [1,2], which depends on the concentration of fluoride and the frequency of application. The addition of inorganic polyphosphate salts to fluoridated products has been shown to be effective in increasing their effectiveness against caries [3,4]. One of the most promising candidate is sodium hexametaphosphate (HMP), which is able to inhibit the formation of dental calculus [5], has antimicrobial action [6], and also prevents the formation of extrinsic stains [7]. HMP consists of a cyclic polyphosphate, presenting strong attraction to calcium in the hydroxyapatite in relation to other phosphates [8], being mixable in water and
* Corresponding author at: Araçatuba Dental School, Univ. Estadual Paulista (UNESP), Department of Pediatric Dentistry and Public Health, Rua José Bonifácio 1193, 16015-050 Araçatuba, SP, Brazil. E-mail address: [email protected] (J.P. Pessan). http://dx.doi.org/10.1016/j.jdent.2015.08.006 0300-5712/ ã 2015 Elsevier Ltd. All rights reserved.
insoluble in organic solvents [9,10]. It is a variant of the longer pyrophosphate chain and presents greater possibility of binding to the enamel surface, protecting it from acid dissolution [11]. In vitro and in situ studies demonstrated that the association of fluoride and HMP in conventional dentifrices (1100 mg F/g) promoted greater protection against enamel demineralization compared to their counterparts without HMP [12–14]. A synergistic effect was also demonstrated when HMP was added to a dentifrice with reduced fluoride concentration (250 mg/g), achieving mineral loss values similar to those observed for the positive control (1100 mg F/g) [15]. Moreover, the addition of HMP to a dentifrice containing stannous fluoride promoted lower erosive wear in enamel and dentin compared to the positive control [16]. Concerning the addition of HMP to fluoridated gels, only one study has evaluated the effects of this association against dental caries in vitro, demonstrating that the 1% NaF gel supplemented with 9% HMP presented greater ability to inhibit enamel demineralization compared to a 1% NaF gel without HMP, as well
1256
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
as similar results to the 2% NaF gel [17]. However, the effect of gels containing fluoride and HMP on erosion has not yet been investigated. Considering that gels are widely used in dental practice and for normal oral hygiene in some countries, it would be interesting to analyze their effectiveness against erosion as an adjunct to normal oral hygiene with a fluoridated dentifrice. Therefore, this in situ/ex vivo study analyzed the effects of fluoridated gels supplemented or not with HMP on enamel erosion, followed or not by abrasion. The null hypothesis was that the use of a fluoridated gel supplemented with HMP is not different from a gel with the same fluoride concentration without HMP in preventing erosive wear and mineral loss of enamel. 2. Material and methods 2.1. Volunteers’ selection and ethical aspects This study was approved by the Institutional Review Board of Araçatuba Dental School, UNESP (CAAE n. 5421712.2.0000.5420, report n. 154.616). Twelve healthy adult volunteers were selected [18], aged 20 to 33 years old (4 male, 8 female) living in areas with fluoridated water supply and presenting good general and oral health. Sample size was determined based on a previous study with similar methodology [19] considering the mean difference among the groups of 0.30 and standard deviation of 0.20 from enamel wear data, and an a-error of 5% and a b-error of 20%. The inclusion criteria comprised normal salivary flow (1–3 mL/min, stimulated saliva, determined as described by Pessan et al. [20]) and no use of drugs that might interfere with salivary composition and flow [21]. The exclusion criteria were smokers, individuals presenting active caries lesions, receiving fluoride applications up to two weeks before the study, using antidepressants, narcotics, diuretics and antihistamine drugs in the last two months, submitted to radiation therapy, practicing water activities, working in environments polluted by compounds with low pH, and presenting systemic diseases (xerostomia, type I diabetes, autoimmune diseases, malnutrition, gastroesophageal problems, regurgitation disorders and vomiting). Exclusion criteria during the study comprised volunteer dropout, health changes with consequent alteration in salivary flow or need of antibiotics, utilization of mouthrinses or dentifrices different than those supplied by the investigators, or failure to follow the experimental design. If any volunteer was excluded, a new volunteer would be selected to complete all study phases. Before the experimental phase, the volunteers received written and verbal instructions on the study protocol, and were supervised to follow the study conditions. All volunteers used the same standard 1100 mg F/g dentifrice (NaF1100 mg F/g-Sorriso Fresh Plus Gel, Colgate) throughout the study. 2.2. Experimental protocol This in situ/ex vivo study was conducted in 4 experimental phases of 5 days each, with a lead in and wash out periods of 7 days. Volunteers wore palatal appliances containing 4 enamel discs, which were treated with Placebo, 1% NaF, 2% NaF and 1% NaF + 9% HMP gels, following a double-blind, crossover protocol. Enamel discs were subjected to ex vivo erosive challenges (immersion in 0.05 M citric acid), followed or not by abrasion by toothbrushing with a silica-based NaF toothpaste (1100 ppm F). Subjects were instructed to keep the palatal appliances in the mouth during all times throughout the experimental phases, except for eating and drinking and for performing oral hygiene procedures. In each phase, all volunteers received a kit containing one tube of 1100 mg F/g dentifrice, one soft bristle toothbrush (Sorriso Kolynos Original Macia—Colgate), 5 disposable cups with visible indication of 50 mL, one orthodontic appliance case, gauze, one
dropper bottle containing the dentifrice suspension (ratio 1 g of dentifrice: 3 mL of deionized water) and one liter of citric acid pH 3.2. 2.3. Preparation of enamel discs Permanent mandibular central incisors were used, obtained from bovines aged 2 to 3 years and maintained in plastic flasks with 2% formalin solution pH 7.0 for 30 days [4]. Enamel discs (n = 192, approximate 4.5 mm diameter) were obtained from the flattest portion on the buccal surface of crowns, using a bur (DS 07882457) connected to a bench drill (MOD. FGC-16, reamer 5/8). The discs were then marked with a round diamond bur to identify the cervico-occlusal direction. The dentin was adjusted (thickness 2 mm) for achievement of parallel surfaces between enamel and dentin. Then, enamel surfaces were turned upwards, and polished with sandpaper grits 400 (20 s), 600 (30 s), 800 (30 s) and 1200 (30 s), under water cooling. The surface was then polished with polishing cloth (Polishing Cloth BUEHLER 40-7618) and diamond suspension (METADI Diamond Suspension 1 micron Blue Color Polish Spray, Water Base 40-653). Finally, the discs were rinsed with deionized water for 20 s and stored in a humid environment with gauze soaked in deionized water, for the initial enamel surface microhardness analysis [22–24]. 2.4. Determination of initial enamel hardness (SHi) and sample selection The surface microhardness of enamel discs was evaluated using the microhardness meter Shimadzu Micro Hardness Tester HMV2.000 (Shimadzu Corporation—Kyoto—Japan) using Knoop indenters, static load of 25 grams and time of 10 s, connected to the image analysis software CAMS-WIN (NewAge Industries, USA). For the initial surface microhardness (initial SH), five indents were made on the central region of the enamel disc, equidistant at 100 mm. Discs presenting mean hardness values within the confidence interval 353.7 (2.7) to 354.2 (1.4) were selected. The discs were identified according to their initial microhardness value. 2.5. Formulation and assessment of fluoride and pH in the experimental gels The gels were prepared at the laboratory of Pediatric Dentistry of Araçatuba Dental School, São Paulo State University (Brazil), containing the following ingredients: carboxymethyl cellulose, saccharin, glycerin, mint oil and deionized water. The sodium fluoride concentrations were 1% and 2% (NaF, Merck, Germany). Ssodium hexametaphosphate (HMP, Sigma, USA) was added to the 1% NaF gel at a concentration of 9%, based on a previous study assessing the effects of gels on enamel demineralization in vitro [17]. Also, a gel without NaF or HMP was prepared and used as negative control (Placebo). For fluoride analysis, approximately 100 mg of each gel was weighed and sequentially diluted in distilled/deionized water. Following, the flask contents were transferred to volumetric flasks and the volume was completed up to 100 mL with distilled/ deionized water. Three dilutions were made for each product. Thereafter, two samples of 1 mL were obtained and buffered with TISAB II [25]. The solutions were then analyzed using a fluoride ion-specific electrode (9609 BN—Orion) connected to an ion analyzer (Orion 720 A+), previously calibrated with the five standards (2.0; 4.0; 8.0; 16.0 and 32 mg F/mL). The total fluoride (TF) and ionic fluoride (IF) were analyzed. Data obtained in mV were converted into mg F/mL. The pH of the gels was assessed in
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
1257
triplicate using a pH meter (Orion 4 Star, Thermo Scientific) and a pH electrode, as previously described [26].
protected specimen area (reference surface) and the peak wear. Five scans were performed on the entire enamel surface [32].
2.6. Preparation of palatal appliances
2.9.2. Determination of final enamel hardness For the final surface microhardness test (SHf), five indents were made on the central region of the eroded enamel, equidistant at 100 mm, with a static load of 25 g for 10 s. For the microhardness test in longitudinal section, the enamel discs were sectioned through the half, and the portion not submitted to ERO or ERO + ABR, respectively, was used for comparison. A microhardness meter Micromet 5114 hardness tester (Buehler, Lake Bluff, USA) and the software Buehler OmniMet (Buehler, Lake Bluff, USA) were used, with a load of 5 g for 10 s with 1000 magnification. Nine indents were made at 5, 10, 15, 20, 25, 30, 40, 50, 70 mm from the external enamel surface, in eroded and intact areas on the center of each disc half. The integrated hardness area (KHN mm) of the lesion up to the intact enamel was calculated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated area of intact enamel hardness, thus achieving the integrated hardness loss (DKHN).
Alginate impressions were taken from the maxillary arch of all volunteers for fabrication of acrylic palatal appliances. In each experiment, four bovine enamel discs were included the appliances divided in two rows (A and B). Half of the disc surfaces were isolated with nail polish (Revlon), so that only half of the disc surfaces would be submitted to treatment and the other half, used for comparison after treatment. The test surfaces of the specimens were leveled with the surface of the palatal appliance. 2.7. Treatment Volunteers inserted the appliance in the mouth two hours before study onset, to allow formation of acquired pellicle [27]. Thereafter, the appliances were removed for application of gels on the four discs of each appliance outside the mouth. Gels were applied with disposable syringes, allowing standardization of the quantity to be applied (approximately 1 mL on each disc surface, for one minute). Then, the gel was removed with gauze and the appliance was placed in the oral cavity of the volunteer. Simultaneously, the same gels were applied on all teeth of volunteers using disposable trays. Therefore, in each experimental phase, the volunteers were randomly distributed according to the four possible treatments: (1) Placebo (gel without NaF or HMP); (2) 1% NaF gel; (3) 2% NaF gel; and (4) 1% NaF + 9% HMP gel. After completion of each experimental phase, the discs were prepared for the analyses. 2.8. Erosive and abrasive challenges The erosive challenge (ERO) was performed during five days for each group, with a seven-day interval prior to the beginning of the study (lead in) between each phase (washout) [28,29]. The challenge was performed with 0.05 M citric acid (Synth, P.A—A. C.S, P.M 192,13), pH 3.2, adjusted with saturated NaOH (Dinâmica, P.A—A.C.S, P.M 40,00), and the appliances were immersed in the acid four times a day for five minutes [30,31]. In each appliance, the enamel discs in row “A” were submitted to erosion associated with abrasion (ERO + ABR), while discs in row “B” were only submitted to ERO. Abrasion was performed by the volunteers after each erosive challenge using a soft bristle toothbrush during 30 s (1 stroke/second), simultaneously in the two discs in row “A”. For this procedure, subjects were instructed to hold the toothbrush with the fingertips, in order to minimize excessive load during toothbrushing. The discs in row “B”, in turn, were treated with a dentifrice suspension during 30 s, immediately after each erosive challenge. 2.9. Analyses 2.9.1. Determination of surface wear—profilometry At completion of each experimental phase, the nail polish was carefully removed from the disc surfaces using an acetone-soaked cotton pellet, prior to analysis of surface wear using a profilometer Surftest SJ 401—Mitutoyo (Mitutoyo American Corporation). The roughness meter was connected to a microcomputer, which expressed and stored the testing information using a specific software (Surftest). The active tip of the roughness meter scanned the specimen surfaces from the control (untreated) portion to the affected area. Wear was determined as the distance (micrometers) between the mean line on the graph corresponding to the
2.9.3. Statistical analysis Statistical analysis was performed on the software SigmaPlot version 12.0, at a significance level of 5%. Data analysis considered the types of experimental gels and type of challenge as variation factors (ERO and ERO + ABR). The variables included the final surface and cross sectional hardness, as well as enamel surface wear (mm). After transformation (Log10), the wear data passed normality (Shapiro–Wilk) and homogeneity tests (Bartlett), fulfilling the ideal conditions for the use of parametric tests. The final surface hardness outcomes did not pass the normality and homogeneity tests even after logarithmic transformation, while for the variable DKHN the distribution was normal, yet did not present homogeneity after logarithmic transformation. Thus, considering the different types of gels and the erosive challenge, all data were submitted to two-way ANOVA followed by the Student–Newman– Keuls test. The hardness data according to depth were separately analyzed for each type of challenge, also using the two-way ANOVA after transformation (Log10) followed by the Student–Newman– Keuls test. 3. Results Total fluoride and ionic fluoride (SD, mg F/g) amounted to: Placebo: 47.0 (3.1) and 8.8 (3.6); 1% NaF: 4895.9 (57.5) and 4773.4 (20.7); 2% NaF: 9737.2 (24.2) and 9314.7 (27.8) and 1% NaF + 9% HMP: 5118.0 (5.1) and 4807.6 (31.5). Mean (SD) pH values were 6.71 (0.13), 6.41 (0.02), 6.47 (0.06) and 6.06 (0.04), respectively for the Placebo, 1% NaF, 2% NaF and 1% NaF + 9% HMP. The initial mean value (standard deviation) of surface hardness (SHi) considering all groups was 354.0 0.4 kgf/mm2, ranging from 353.7 2.7 to 354.2 1.4 (p > 0.05). The 1% NaF + 9% HMP gel promoted the lowest enamel wear after ERO compared to other gels, with significant differences among all groups (Table 1). For ERO + ABR, the lowest wear was observed for the group treated with 2% NaF gel, followed by groups 1% NaF + 9% HMP, 1% NaF and Placebo, with significant differences among all groups. Surface hardness values were significantly greater for the 2% NaF group for both challenges, followed by groups 1% NaF + 9% HMP, 1% NaF and Placebo, with significant differences among all groups (Table 1). Concerning the integrated lesion area, the 2% NaF and 1% NaF + 9% HMP gels promoted significantly higher values compared to the other groups. For ERO, the 1% NaF + 9% HMP gel yielded significantly higher DKHN values than the 2% NaF gel, and
1258
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
Table 1 Enamel erosive wear, surface hardness (SH) and integrated subsurface hardness loss (DKHN) after treatment with the experimental gels and erosive challenges (ERO), followed or not by toothbrushing (ERO + ABR). Gels
Wear (mm) EROA
Placebo 1% NaF 2% NaF 1% NaF + 9% HMP
a
3.32 (0.27) 2.29b (0.12) 1.71c (0.06) 1.28d (0.04)
DKHN (Kgf/mm2 mm)
SH (Kgf/mm2) ERO + ABRB a
4.99 (0.14) 3.56b (0.12) 2.17c (0.06) 2.36d (0.12)
ERO
ERO + ABR
Aa
91.6 (19.9) 219.7Ab (13.3) 263.7Ac (5.5) 240.7Ad (24.8)
Aa
77.3 (13.2) 103.9Bb (11.7) 226.9Bc (2.5) 194.1Bd (35.9)
EROA
ERO + ABRB a
4282.0 (558.8) 2059.7b (113.6) 1024.7c (96.6) 904.8d (113.9)
4096.9a (354.4) 2036.8b (112.7) 872.4c (107) 1052.2d (142.4)
Mean (SD), n = 12. Upper and lower case letters indicate significant differences between challenges and among treatments, respectively, (Student–Newman–Keuls; p < 0.05).
the opposite pattern occurred for ERO + ABR. All groups presented significantly lower final hardness values for ERO + ABR compared to ERO, except for the Placebo group. The mean hardness profiles according to depth for each treatment and challenge are presented in Fig.1. Hardness values are very similar and mostly without significant differences between groups 1% NaF + 9% HMP and 2% NaF for each distance analyzed. These, in turn, presented significant differences in relation to the other gels at the three most external depths analyzed, reaching more similar values in deeper regions of enamel.
Fig. 1. Mean hardness (n = 12) according to the depth and erosive challenges (A— erosion; B—erosion + abrasion). Different letters indicate significant difference among hardness values at each depth (Student–Newman–Keuls; p < 0.05). Vertical bars represent the standard deviations of the means. ¥: indicates significant difference between the Placebo gel and the other treatments. C: indicates significant difference between the Placebo gel and the other treatments, as well as between 1% NaF and 1% NaF + 9% HMP gels.
4. Discussion The addition of inorganic polyphosphate salts to fluoridated products for topical use has been shown to be an effective option to increase their effectiveness against both caries and dental erosion in in vitro and in situ trials [4,26,32–34]. In the present study, application of fluoridated gels associated with the use of a conventional fluoridated dentifrice (1100 mg F/g) significantly reduced wear and mineral loss suffered by enamel after the erosive challenges associated or not with abrasion, when compared to the placebo gel. Also, rejecting the null hypothesis, supplementation of a fluoridated gel with HMP led to lower wear compared to a gel with the same fluoride concentration, without addition of HMP, thus demonstrating that a gel with reduced fluoride concentration may be effective against the erosive challenge when associated with a polyphosphate. The use of the 1% NaF + 9% HMP gel promoted significantly lower wear in relation to the other groups for enamel submitted to ERO, including the gel with twice the concentration of fluoride, without HMP. Conversely, the wear suffered by enamel treated with the gel containing HMP was significantly lower only when compared to 1% NaF and placebo groups for ERO + ABR. However, it should be mentioned that the wear values observed for the group treated with HMP gel was very similar to that obtained for the 2% NaF gel (8.6% of difference) for ERO + ABR, and the significant difference might be due to the small variability of outcomes around the means. Thus, it can be concluded that the HMP-containing gel presented a similar protective effect against enamel erosive wear as the standard 2% NaF gel. HMP cannot be considered as a phosphate source to react with tooth enamel, as it does not become spontaneously hydrolyzed [35,36]. This cyclic phosphate forms strong complexes with metal ions [35,37–39] and, in the oral environment, adsorbs to enamel surface and retains charged ions of CaF+ and Ca2+ by replacement of Na+ in the cyclic structure, leading to a reticular formation via Ca2+ binding to one or more HMP molecules [40]. Similarly to sodium trimetaphosphate (TMP), this produces higher fluoride and calcium retention, further releasing with H2PO4, increasing CaF+ and Ca2+ species to the oral environment under acidic conditions; these would subsequently react the ionic activity of neutral ions CaHPO40 and HF0, which are known to have diffusion coefficients much higher than charged species [39]. The above-mentioned hypothesis is supported by the significantly lower subsurface caries-like lesions seen for enamel treated with HMP-containing toothpaste [15] and gel [17], as well as for the present crosssectional data, as discussed below. In order to better understand the effect of HMP associated with F, enamel specimens were also evaluated by hardness analysis. The surface hardness values presented a dose-dependent relationship according to the fluoride concentration in the gels, confirming previous studies demonstrating that the eroded enamel presents a
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
less softened surface when submitted to treatment with fluoride [41,42]. This marked effect of the 2% NaF gel on enamel surface may have accounted for the higher resistance to abrasion in this group after enamel was eroded by citric acid, given that mean enamel wear under ERO + ABR was 26% higher than under ERO only, while the corresponding value for 1% NaF gel was around 55%. As for the HMP-supplemented gel, this difference was even higher (85%), what might suggest that the protective layer formed on enamel surface after gel application was partially lost due to erosive and abrasive challenges. Nonetheless, this gel led to significantly higher hardness value compared to 1% NaF gel under both challenges, what reinforces the synergistic effect of fluoride and HMP and suggests that higher hardness values may be related to greater enamel protection for ERO and ERO + ABR. Despite the trend seen for surface hardness in the present study, this measurement has been considered not to be a good indicator for analysis of erosion, since the final surface analyzed cannot be considered as the same surface that precedes the challenge [43]. Cross-sectional hardness was then determined, allowing quantification of demineralization by calculation of the integrated hardness loss (DKHN), as well as the mean hardness according to enamel depth. The pattern of DKHN for both challenges was the same observed for enamel wear (lowest values for 1% NaF + 9% HMP and 2% NaF, respectively, for ERO and ERO + ABR). Nonetheless, as observed for wear outcomes, the differences between 1% NaF + 9% HMP and 2% NaF gels concerning the DKHN for ERO + ABR were very small (approximately 6%), as shown in Fig. 2-B. Considering that the present in situ protocol was conducted under extreme conditions of ERO and ERO + ABR challenges, thereby maximizing the differences between treatments, it is possible that the small differences observed between 1% NaF + 9% HMP and 2% NaF gels, for both wear and integrated hardness loss, might not be relevant for the clinical practice. This assumption, however, should be confirmed in studies with different research protocols, especially considering the time of each erosive challenge, which could be much lower than that used in the present work. It is well known that when the erosive challenge is associated with enamel abrasion by toothbrushing, the most external enamel layer (previously softened by the erosive agent) is removed by abrasion, exposing an underlying layer with higher degree of mineralization [26,32]. Although DKHN values confirm this, surface hardness values presented the opposite tendency. The reasons for this discrepancy are not evident, yet it may be assumed that they are related to the concomitant use of gels and a fluoridated dentifrice, while previous studies have used a single source of fluoride. Considering that fluoride presents a more marked effect in partially demineralized areas [44], it is possible that fluoride from the dentifrice bound only to the most external enamel layers (due to the short time of enamel exposure to the dentifrice), which would promote re-precipitation of calcium and phosphate ions on enamel. According to this hypothesis, this reprecipitation would promote increased surface hardness for ERO, while the net mineral gain occurring between the erosive challenge (loss) and re-precipitation (gain) might be removed by abrasion for the ERO + ABR condition. The above-mentioned hypothesis seems to be supported by data in Fig. 2-A and 2-B. The hardness values closer to the enamel surface (5 mm) are higher for ERO compared to ERO + ABR, confirming the data obtained for surface hardness. However, when advancing in depth, ERO + ABR values increased in greater proportion compared to ERO, so that the differences observed at 5 mm are minimized when the integrated area is calculated (Fig. 2A). Since this phenomenon had not been previously observed, it would be interesting to conduct studies using different methodologies to confirm this hypothesis.
1259
Finally, it should be mentioned that none of the gels tested was able to completely inhibit the erosion or even promote repair of established lesions, reinforcing the role of fluoride only as coadjutant for reduction of wear and mineral loss suffered by enamel after erosive challenges, associated or not with abrasion. Similarly to carious lesions, this highlights the need of other preventive measures, especially dietary control and modification of hygiene habits. These include reduced intake of acidic foods and beverages, utilization of low-abrasive dentifrices and soft bristle toothbrush [45]. In summary, it was concluded that utilization of a gel with reduced fluoride concentration (1% NaF) supplemented with HMP promoted smaller wear and lower enamel mineral loss compared to a gel with the same fluoride concentration without addition of HMP. This effect was similar to that achieved after utilization of 2% NaF gel, evidencing the synergic effect between fluoride and HMP in the erosion process. The results also demonstrated that, for individuals making regular use of fluoridated dentifrice, additional therapy with fluoridated gels seems a viable option to reduce the wear and enamel mineral loss associated with erosive challenges, associated or not with abrasion. Acknowledgments The authors would like to thank the volunteers for their participation and also the laboratory technician of the Department of Pediatric Dentistry and Public Health, Maria dos Santos Ferreira Fernandes, for the collaboration with the laboratory procedures during this study. References [1] C. Ganss, Definition of erosion and links to tooth wear, Monogr. Oral Sci. 20 (2006) 9–16. [2] M.D. Lagerweij, W. Buchalla, S. Kohnke, K. Becker, A.M. Lennon, T. Attin, Prevention of erosion and abrasion by a high fluoride concentration gel applied at high frequencies, Caries Res. 40 (2006) 148–153. [3] M. Danelon, E.M. Takeshita, K.T. Sassaki, A.C.B. Debem, In situ evaluation of a low fluoride concentration gel with sodium trimetaphosphate in enamel remineralization, Am. J. Dent. 26 (2013) 15–20. [4] M. Danelon, E.M. Takeshita, L.C. Peixoto, K.T. Sassaki, A.C. Delbem, Effect of fluoride gels supplemented with sodium trimetaphosphate in reducing demineralization, Clin. Oral Invest. 18 (2014) 1119–1127. [5] T. Schiff, L. Saletta, R.A. Baker, J.L. Winston, T. He, Desensitizing effect of a stabilized stannous fluoride/Sodium hexametaphosphate dentifrice, Compend. Contin. Educ. Dent. 26 (2005) 35–40. [6] M. Vaara, Agents that increase the permeability of the outer membrane, Microbiol. Rev. 56 (1992) 395–411. [7] H. Liu, V.A. Segreto, R.A. Baker, K.A. Vastola, L.L. Ramsey, R.W. Gerlach, Anticalculus efficacy and safety of a novel whitening dentifrice containing sodium hexametaphosphate: a controlled six month clinical trial, J. Clin. Dent. 13 (2002) 25–28. [8] A. Baig, T. He, J. Buisson, L. Sagel, E. Suszcynsky-Meister, D.J. White, Extrinsic whitening effects of sodium hexametaphosphate—a review including a dentifrice with stabilized stannous fluoride, Compend. Contin. Educ. Dent. 26 (2005) 47–53. [9] S. Budavari, M.J. O’Neil, A. Smith, P.E. Heckelman, An Encyclopedia of Chemicals, Drugs, and Biological, 11, Rahway Merck Co., 19591559. [10] R.J. Lewis, Hazardous Chemicals Desk Reference, 4, Van Nostrand Reinhold, New York, 19931680. [11] A.A. Baig, K.M. Kozak, E.R. Cox, J.R. Zoladz, L. Mahony, D.J. White, Laboratory studies on the chemical whitening effects of a sodium hexametaphosphate dentifrice, J. Clin. Dent. 13 (2002) 19–24. [12] A.M. Pfarrer, C.M. McQueen, M.A. Lawless, M. Rapozo-Hilo, J.D. Featherstone, Anticaries potential of a stabilized stannous fluoride/sodium hexametaphosphate dentifrice, Compend. Contin. Educ. Dent. 26 (2005) 41– 46. [13] J.S. Wefel, C.M. Stanford, D.K. Ament, M.M. Hogan, J.D. Harless, A.M. Pfarrer, L.L. Ramsey, M.S. Leusch, A.R. Biesbrock, In situ evaluation of sodium hexametaphosphate-containing dentifrices, Caries Res. 36 (2002) 122–128. [14] D.M. da Camara, J.P. Pessan, T.M. Francatia, J.A.S. Souza, M. Danelon, A.C.B. Delbem, Synergistic effect of fluoride and sodium hexametaphosphate in toothpaste on enamel demineralization in situ, J. Dent. (2015) , doi:http://dx. doi.org/10.1016/j.jdent.2015.08.007.
1260
J.M. Conceição et al. / Journal of Dentistry 43 (2015) 1255–1260
[15] D.M. da Camara, M. Miyasaki, M. Danelon, K.T. Sassaki, A.C.B. Delbem, Effect of low-fluoride toothpastes combined with hexametaphosphate on in vitro enamel demineralization, J. Dent. 42 (2014) 256–262. [16] S.M. Hooper, R.G. Newcombe, R. Faller, S. Eversole, M. Addy, N.X. West, The protective effects of toothpaste against erosion by orange juice: studies in situ and in vitro, J. Dent. 35 (2007) 476–481. [17] M. Danelon, D.M. Camara, M.L. Miyasaki, J.B. Scarpa, A.C.B. Delbem, K.T. Sassaki, Effect of fluoride gels supplemented with hexametaphosphate in inhibiting demineralization, J. Dent. Res. 91 (Sp Iss B) (2012) 162,761. [18] J.P. Haas, Sample size and power, Am. J. Infect. Control 40 (2012) 766–767. [19] M.J. Moretto, A.C. Delbem, M.M. Manarelli, J.P. Pessan, C.C. Martinhon, Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion: an in situ/ex vivo study, J. Dent. 41 (2013) 1302–1306. [20] J.P. Pessan, K.M.R.P. Alves, I. Ramires, M.F.L. Taga, F.C. Sampaio, G.M. Whitford, M.A.R. Buzalaf, Effects of regular and low-fluoride dentifrices on plaque fluoride, J. Dent. Res. 89 (2010) 1106–1110. [21] M.T. Kato, M.A.R. Buzalaf, Iron supplementation reduces the erosive potential of a cola drink on enamel and dentin in situ, J. Appl. Oral Sci. 20 (2012) 318– 322. [22] A.C. Delbem, D. Pedrini, J.G. França, T.M. Machado, Fluoride release/recharge from restorative materials–effect of fluoride gels and time, Oper. Dent. 30 (2005) 690–695. [23] A.E. Vieira, A.C. Delbem, K.T. Sassaki, E. Rodrigues, J.A. Cury, R.F. Cunha, Fluoride dose response in pH-cycling models using bovine enamel, Caries Res. 39 (2005) 514–520. [24] C.O. Favretto, M. Danelon, F.C.N. Castilho, A.E.M. Vieira, A.C.B. Delbem, In vitro evaluation of the effect of mouth rinse with trimetaphosphate on enamel demineralization, Caries Res. 47 (2013) 532–538. [25] A.C. Delbem, K.T. Sassaki, A.M. Castro, L.M. Pinto, M. Bergamaschi, Assement of the fluoride concentration and pH in different mouthrinses on the brazilian market, J. Appl. Oral Sci. 11 (December) (2003) 319–323. [26] M.J. Moretto, A.C. Magalhães, K.T. Sassaki, A.C. Delbem, C.C. Martinhon, Effect of different fluoride concentrations of experimental dentifrices on enamel erosion and abrasion, Caries Res. 44 (2010) 135–140. [27] A. Cheung, Z. Zid, D. Hunt, J. McIntyre, The potential for dental plaque to protect against erosion using an in vivo-in vitro model—a pilot study, Aust. Dent. J. 50 (2005) 228–234. [28] A.C. Delbem, M. Danelon, K.T. Sassaki, A.E. Vieira, E.M. Takeshita, F.L. Brighenti, et al., Effect of rinsing with water immediately after neutral gel and foam fluoride topical application on enamel remineralization: an in situ study, Arch. Oral Biol. 55 (2010) 913–918. [29] R.P. Shellis, C. Ganss, Y. Ren, D.T. Zero, A. Lussi, Methodology and models in erosion research: discussion and conclusions, Caries Res. 45 (2011) 69–77. [30] J.A. Hughes, K.D. Jandt, N. Baker, D.M. Parker, R.G. Newcombe, M. Eisenburger, M. Addy, Futher modification to so drinks to minimize erosion. A study in situ, Caries Res. 36 (2002) 70–74.
[31] A. Wiegand, T. Attin, Design of erosion/abrasion studies insights and rational concepts, Caries Res. 45 (2011) 53–59. [32] M.M. Manarelli, A.E. Vieira, A.A. Matheus, K.T. Sassaki, A.C. Delbem, Effect of mouth rinses with fluorides and trimetaphosphate on enamel erosion: an in vitro study, Caries Res. 46 (2011) 506–509. [33] E.M. Takeshita, L.P. Castro, K.T. Sassaki, A.C.B. Delbem, In vitro evaluation with low fluoride content supplemented with trimetaphosphate, Caries Res. 43 (2009) 50–56. [34] M.M. Manarelli, A.C. Delbem, T.M. Lima, F.C. Castilho, J.P. Pessan, In vitro remineralizing effect of fluoride varnishes containing sodium trimetaphosphate, Caries Res. 48 (2014) 299–305. [35] I.K. Choi, W.W. Wen, R.W. Smith, Technical note the effect of a long chain phosphate on the adsorption of collectors on kaolinite, Miner. Eng. 6 (1993) 1191–1197. [36] E. Castellini, G. Lusvardi, G. Malavasi, L. Menabue, Thermodynamic aspects of the adsorption of hexametaphosphate on kaolinite, J. Colloid Interf. Sci. 292 (2005) 322–329. [37] G. Kura, S. Ohashi, S. Kura, Complex formation of cyclic phosphate anions with bivalent cations, J. Inorg. Nucl. Chem. 36 (1974) 1605–1609. [38] L. Changgen, L. Yongxin, Selective flotation of scheelite from calcium minerals with sodium oleate as a collector and phosphates as modifiers. II. The mechanism of the interaction between phosphate modifiers and minerals, Int. J. Miner. Process. 10 (1983) 219–235. [39] N.J. Cochrane, S. Saranathan, F. Cai, K.J. Cross, E.C. Reynolds, Enamel subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride, Caries Res. 42 (2008) 88–97. [40] J.R. van Wazer, D.A. Campanella, Structure and properties of the condensed phosphates. IV. Complex ion formation in polyphosphate solutions, J. Am. Chem. Soc. 72 (1950) 655–663. [41] D. Rios, H.M. Honorio, A.C. Magalhães, M.A. Buzalaf, R.E. Palma-Dibb, M.A. Machado, S.M. Silvia, Influence of toothbrushing on enamel softening and abrasive wear of eroded bovine enamel: an in situ study, Braz. Oral Res. 20 (2006) 148–154. [42] D. Rios, A.C. Magalhães, R.O. Polo, A. Wiegand, T. Attin, M.A. Buzalaf, The efficacy of a highly concentrated fluoride dentifrice on bovine enamel subjected to erosion and abrasion, J. Am. Dent. Assoc. 139 (2008) 1652–1656. [43] A.C. Magalhães, D. Rios, A.L. Moino, A. Wiegand, T. Attin, M.A. Buzalaf, Effect of different concentrations of fluoride in dentifrices on dentin erosion subjected or not to abrasion in situ/ex vivo, Caries Res. 42 (2008) 112–116. [44] M.A.R. Buzalaf, J.P. Pessan, H.M. Honório, J.M. ten Cate, Mechanisms of action of fluoride for caries control, Monogr. Oral Sci. 22 (2011) 97–114. [45] B.T. Amaechi, S.M. Higham, Dental erosion: possible approachs to prevent and control, J. Dent. 33 (2005) 243–252.