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Fluoride concentration and amount of dentifrice influence enamel demineralization in situ
Journal of Dentistry xxx (xxxx) xxx–xxx
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Fluoride concentration and amount of dentifrice influence enamel demineralization in situ M.F. Paivaa, A.C.B. Delbema, M. Danelona, M.E. Nagataa, F.R.N. Moraesa, G.E.G. Cocletea, ⁎ R.F. Cunhaa, M.A.R. Buzalafb, J.P. Pessana, a b
São Paulo State University (Unesp), School of Dentistry, Araçatuba, Department of Pediatric Dentistry and Public Health, Brazil University of São Paulo, Bauru School of Dentistry, Department of Biological Sciences, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Dentifrices Dental enamel Fluorides Dental plaque
Objectives: This study evaluated the effect of conventional (CD, 1100 ppm F) and low-fluoride (LFD, 550 ppm F) dentifrices, applied in different quantities, on enamel demineralization, and on fluoride (F) concentrations in the dental biofilm formed in situ. Methods: Five combinations of dentifrices and quantities were tested: placebo (P–F-free) applied on all brush bristles; LFD applied by the transversal technique (0.3 g–T1) or on all bristles (0.6 g–T2); and CD applied in a pea-sized amount (0.15 g–T3) or by the transversal technique (0.3 g–T4), in order to produce comparable intensities (F concentration in the dentifrice × amount applied to the brush). Volunteers (n = 13, 20–36 years old) wore palatal devices containing 4 bovine enamel blocks, and performed cariogenic challenges (30% sucrose solution) 6×/day, and brushing 3×/day, following a double-blind, cross-over and randomized protocol. On the 8th day, biofilm was collected 5 and 60 min after brushing. The percentage of surface hardness loss (%SH), integrated loss of subsurface hardness (ΔKHN) and biofilm F concentrations (solid and fluid phases) were determined. Data were analyzed by repeated-measures ANOVA, Student-Newman-Keuls test, and Pearson’s correlation coefficient (p < 0.05). Results: Significantly lower ΔKHN was observed for treatments with higher intensity (T2 and T4) in comparison with the lower intensity (T1 and T3). A strong correlation was observed between ΔKHN and F concentrations in total biofilm (r = −0.71) and biofilm fluid (r = −0.72) 5 min after brushing. Conclusions: The treatment intensity has a significant influence on the development of caries lesions in situ. Clinical significance: The intensity of treatment (amount of dentifrice × concentration) during brushing seems to be a more relevant parameter of clinical efficacy than simply observing the F concentration of the product. The use of a small amount of CD significantly reduced the protective effects against enamel demineralization.
1. Introduction A comprehensive Cochrane review assessing the relative cariesprotective effectiveness of dentifrices with different fluoride (F) concentrations concluded that toothbrushing with dentifrices with concentrations of 1000 ppm F or above is an effective measure in preventing caries in children and adolescents [1]. Given that the early use of fluoridated dentifrices has been shown to be a potential risk factor for the development of dental fluorosis [2], professionals and health authorities have advocated the use of dentifrices with F concentrations above 1000 ppm F, but applied in very small quantities for young children [3,4], under the assumption that this measure would minimize systemic F exposure from this source without compromising the clinical
efficacy of the product. Considering that the clinical effects of F are known to be dose-dependent, this strategy would be valid except for the fact that the dentifrice is diluted in saliva during toothbrushing. It could be anticipated, therefore, that reducing the amount of dentifrice on the brush would reduce the F concentration in the natural dentifrice-saliva slurry formed during toothbrushing, which, in turn, could affect the clinical efficacy of the dentifrice [5]. While previous studies have addressed the impact of the amount of dentifrice used during toothbrushing on salivary F concentrations, enamel remineralization and enamel F uptake [6,7], the effect of the amount of dentifrice as a function of F concentration in the product has only recently been assessed. It was demonstrated that brushing with a low-F dentifrice (LFD, 550 ppm F) applied using the
⁎ Corresponding author at: São Paulo State University (Unesp), School of Dentistry, Araçatuba Department of Pediatric Dentistry and Public Health José Bonifácio 1193, 16015-050 Araçatuba–SP, Brazil. E-mail address: [email protected] (J.P. Pessan).
http://dx.doi.org/10.1016/j.jdent.2017.09.004 Received 25 July 2017; Received in revised form 2 September 2017; Accepted 11 September 2017 0300-5712/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: MF, P., Journal of Dentistry (2017), http://dx.doi.org/10.1016/j.jdent.2017.09.004
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from bovine incisors previously stored in 2% neutral formaldehyde solution (pH 7.0) for 1 month. They were sequentially polished (600, 800, 1200 grit), followed by polishing with felt paper and 1 μ diamond suspension. Four enamel blocks were fixed in 4-mm deep spaces of custom-made acrylic palatal devices, approximately 1 mm below the level of the acrylic surface, in order to allow biofilm accumulation on the enamel specimens. A plastic mesh covered the enamel blocks to prevent biofilm disturbance by mechanical forces [12].
transversal technique (i.e., perpendicular to the long axis of the brush) led to significantly higher salivary F concentrations than a conventional dentifrice (CD, 1100 ppm F) using a pea-sized amount [8], confirming the hypothesis that the dilution of the dentifrice in saliva during toothbrushing has a direct impact on intraoral F concentrations. Based on dose-response considerations, the above-mentioned results could have important clinical implications, raising questions on the appropriateness of the current recommendation of dentifrices to children. Nonetheless, as only one intraoral variable was assessed, and given the short-term nature of that study [8], these results cannot be fully extrapolated to a clinical situation. Considering that dental caries is the net result of the slow mineral loss occurring in dental enamel covered by a cariogenic biofilm [9], the aim of this study was to assess the effects of treatment intensity (i.e., F concentration in the dentifrice versus the amount used during toothbrushing) on enamel mineral loss, as well as on biofilm F concentrations. The null hypothesis was that the treatment intensity would not influence the variables assessed.
2.5. Intraoral procedures Two drops of a 30% sucrose solution were dispensed on each enamel block ex vivo, 6 times/day, at previously established times (8:00, 11:00, 14:00, 17:00, 19:00, 21:00 h). The appliances were left to rest during 5 min before being returned to the oral cavity [12]. The treatment of the blocks with the dentifrices was performed 3 times/day (7:30, 13:00 and 22:00 h), during 7 days. The amount of dentifrice to be used was demonstrated individually to all volunteers, in each experimental phase. They also received photographs via cell phone showing the quantity to be applied on the brush. Volunteers were instructed to brush their natural teeth with the appliance in the oral cavity for 1 min, and to swish the natural suspension of dentifrice/saliva for 30 s. Volunteers then completed the cleaning of their natural teeth without the appliance in their mouths, and rinsed the appliances (20 mL) and their mouths (10 mL) with tap water. They were instructed to remove the appliances only during meals and ingestion of liquids, with no restrictions on their diet. Prior to each experimental phase (lead in/wash out), the volunteers used a F-free dentifrice for 7 days.
2. Materials and methods 2.1. Ethical aspects and inclusion criteria This study was approved by the IRB of School of Dentistry, Araçatuba–UNESP (CAAE 44712715.5.0000.5420). Participants signed an informed consent form and received written and verbal instructions on the research protocol prior to the beginning of the study. Thirteen individuals (20–36 years old), living in the city of Araçatuba-SP, Brazil (0.6–0.8 mg F/L in the public water supply) [10], were enrolled. The inclusion criteria involved participants in good general and oral health. Individuals who used drugs that could interfere with the formation of the dental biofilm or salivary flow, smokers, those who received fluoride applications 2 weeks before the experiment, were using orthodontic appliances or had systemic diseases could not participate in the study [11].
2.6. Sample collection On the morning of the 8th day, the volunteers attended the laboratory of Pediatric Dentistry, School of Dentistry, Araçatuba, while fasting, having been instructed not to brush their teeth and not to perform the cariogenic challenge on the specimens. Volunteers then performed the brushing procedures as previously described, but with the dentifrices weighed by the researchers (Shimadzu Balance AUY220, Kyoto–Japan). Biofilm from 2 blocks was randomly collected at 5 and 60 min after brushing, being immediately transferred to a microcentrifuge tube filled with mineral oil. The tube was weighed (before and after collection) and centrifuged (21,000g, 5 min, 4 °C) to separate the solid and fluid phases of the biofilm. Following, a small fraction of the fluid phase was collected, using a micropipette.
2.2. Experimental design Sample size was based on a previous study assessing the effects of dentifrices containing 500 and 1100 ppm F on enamel demineralization and biofilm F concentrations [12]. A sample of 14 volunteers was calculated considering α-error of 5%, β-error of 20% [13], and dropout rate of 20%. The protocol used was in situ, double-blind and crossover. The volunteers were randomly divided into 5 groups, totaling 4 combinations of dentifrice and quantity, namely: 550 ppm F applied by the transversal technique (0.3 g) or on all bristles of the brush (0.6 g), and 1100 ppm F applied in a pea-size amount (0.15 g) or by the transversal technique (0.3 g). A placebo dentifrice (F-free) applied on all bristles of the brush (0.6 g) was also included. The dentifrice tubes were coded and the quantities used during brushing were determined by an examiner not involved in sample collection and analysis.
2.7. Fluoride analysis Biofilm fluid samples were transferred to the surface of an inverted ion-specific electrode (Orion 9409), immersed in mineral oil, and placed on drops of TISAB III previously placed on the electrode membrane, at a 10:1 ratio (sample:TISAB III), as described by Vogel et al. [16]. F was determined in the total biofilm after HCl extraction and buffering with TISAB III, using a F ion-specific electrode (Orion 9409), and a reference electrode (Orion 900200), both coupled to an ion analyzer (Orion 720A+) [17].
2.3. Formulation and determination of F in the experimental dentifrices The experimental dentifrices were produced in the laboratory of Pediatric Dentistry from School of Dentistry, Araçatuba, using the same basic formulation with the following components: titanium dioxide, carboxymethylcellulose, methyl p-hydroxybenzoate, sodium saccharine, oil peppermint, glycerin, silica abrasive, sodium lauryl sulfate, water and sodium fluoride (NaF, Merck®, Germany 550 e 1100 μg F/g). A F-free formulation without F (placebo) was also used. The determination of the ionic and total F concentrations [14] and pH [15] of the dentifrices was performed prior to the beginning of the study.
2.8. Hardness analysis Surface hardness was determined prior to the experiments (SHi) using the Micromet 5114 hardness tester (Buehler, Lake Bluff, USA and Mitutoyo Corporation, Kanagawa, Japan), under 25 g for 10 s (five indentations, spaced 100 μm from each other, made at the center of the block) [18]. Blocks included in the study had SHi between 330–370 KHN, and were randomly distributed among the study groups. After the experimental phases, final surface hardness (SHf) was determined as described for SHi, 100 μm from the initial indentations. The percentage of SH loss (%SH) was calculated using the formula: %SH = 100 x (SHf –
2.4. Enamel blocks and appliance preparation Enamel blocks (n = 260) measuring 4 × 4 x 2 mm were obtained 2
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SHi)/SHi. Blocks were then longitudinally sectioned at the center, and one of the halves was embedded in acrylic resin with the cut surface exposed and polished. One sequence of 14 indentations was created at the center of the blocks at 5, 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220, and 330 μm from the enamel surface, with a Knoop diamond indenter under a 5 g load for 10 s [19]. The integrated area of the hardness (ΔKHN × μm) of the lesion to the sound enamel was calculated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated area of the enamel hardness, obtaining the integrated loss of subsurface hardness (ΔKHN) [20].
Table 2 Mean percentage of surface hardness loss (%SH) and integrated loss of subsurface hardness (ΔKHN) according to the different treatment intensities (fluoride concentration in the dentifrice x amount applied on the toothbrush). Dentifrices Amount applied on the toothbrush %SH ΔKHN
2.9. Statistical analysis
Placebo 550 ppm F 1100 ppm F
18.5 (3.3) 545.1 (4.0) 1075.5 (14.6)
13.5 (8.4) 543.5 (16.8) 1095.6 (22.7)
0.6 g
0.15 g
0.3 g
−51.3a (26.1) 7378.6a (1123.0)
−25.0bc (13.2) 3113.4b (411.7)
−23.4bc (8.7) 2604.0c (453.4)
−30.2b (13.0) 3224.7b (845.5)
−20.2c (8.7) 2431.6c (379.4)
A recent in vivo study assessing salivary F concentrations in children brushing with CD and LFD applied in different quantities showed that the treatment intensity (i.e., amount of dentifrice × F concentration) is a relevant parameter when comparing dentifrices with different F concentrations [8]. The present study demonstrated that treatment intensity had a direct impact on the mineral loss of enamel covered by dental biofilm formed in situ under highly cariogenic conditions, as well as on the resulting biofilm F concentrations, thus leading to the rejection of the study’s null hypothesis. Brushing with the placebo dentifrice promoted significantly higher %SH and ΔKHN when compared with the fluoridated dentifrices, thus validating the in situ protocol employed (i.e., in vivo exposure to the dentifrice slurries and ex vivo exposure to sucrose). Interestingly, while brushing with the LFD using larger quantities (transversal technique or full bristles) led to similar %SH values to that observed for the CD applied using the transversal technique, the use of the CD in a pea-size amount promoted the highest %SH among the fluoridated dentifrices, being significantly higher than the CD applied with the transversal technique. Given that the use of a pea-size amount of a CD was also shown to significantly reduce salivary F when compared with the transversal technique [8], these data taken together reinforce the concept that the use of very low quantities of a CD might reduce the cariesprotective effect of the product under clinical conditions. It is noteworthy that the interplay between fluoride dose and concentration had already been demonstrated in an in situ study with fluoridated milk, regarding surface hardness recovery and enamel fluoride uptake [22]. The authors concluded that both variables appear to impact the cariostatic properties of fluoride in milk, what reinforces the present data related to fluoride dentifrices. A more evident effect of the treatment intensity on enamel mineral loss was observed for ΔKHN data. The highest treatment intensity (i.e., 0.6 g of 550 ppm F or 0.3 g of 1100 ppm F) led to significantly lower ΔKHN when compared with the lowest treatment intensity (i.e., 0.3 g of 550 ppm F or 0.15 g of 1100 ppm F), with no significant differences between CD and LFD within each intensity. These findings are important from a clinical standpoint considering the current recommendations of dentifrices to children [3,4]. Despite reducing the amount of dentifrice is indeed an effective measure in minimizing F intake during toothbrushing, especially by younger children [21], the present study clearly demonstrates that (1) this recommendation significantly reduces the protective effect of the dentifrice, and (2) the protective effect of a LFD can be similar or higher than that of a CD depending on the amount of dentifrice used during toothbrushing. Based on the above and considering the scarcity of well-controlled
Table 1 Mean (SD) fluoride concentrations and pH in the experimental dentifrices (n = 3).
Ionic fluoride
0.3 g
4. Discussion
Table 1 shows mean total and ionic F concentrations in the experimental dentifrices, which presented a variation lower than 3% when compared with the expected values. Mean pH of the dentifrices ranged from 7.4 (1100 ppm F) to 7.7 (placebo). Significantly lower %SH and ΔKHN were observed for all fluoridated dentifrices when compared with the placebo (p < 0.001) (Table 2). For %SH, the only significant difference among the fluoridated groups was observed for CD applied using a pea-size amount or the transversal technique (p = 0.040). A more marked effect of the treatment intensities was observed for ΔKHN, given that values obtained for the lowest intensity (i.e., 0.30 g of 550 ppm F and 0.15 g of 1100 ppm F) were significantly higher than those for the highest intensity (i.e., 0.60 g of 550 ppm F and 0.30 g of 1100 ppm F), without significant differences between CD and LFD within each intensity. A dose-response relationship was observed between treatment intensity and total biofilm F concentrations for both times of sample collection, despite differences among the fluoridated dentifrices were not significant 5 min after brushing, except for the CD applied using the transversal technique (Table 3). At 60 min, F concentrations remained significantly higher than the placebo for all fluoridated dentifrices, except for the LFD applied using the transversal technique. For biofilm fluid F concentrations, all fluoridated dentifrices led to significantly higher F concentrations than the placebo at both times of sample collection, without significant differences among the dentifrices regardless the amount applied on the toothbrush. A strong correlation was observed between ΔKHN and F concentrations in the total biofilm (r = −0.71) and biofilm fluid (r = −0.72) 5 min after brushing, while lower coefficients were observed 60 min after brushing. A moderate correlation was seen between F concentrations in the total biofilm 5 and 60 min after brushing (r = 0.67), whereas a strong correlation was seen for F concentrations in the fluid phase of the biofilm (r = 0.78). ΔKHN and%SH were
Total fluoride
1100 ppm F
moderately correlated (r = 0.60). Other comparisons are displayed in Table 4.
3. Results
Fluoride concentration (ppm)
550 ppm F
Superscript letters indicate significant differences among the means. Data analyzed by one-way, repeated measures ANOVA after cubic and log10 transformation, respectively for%SH and ΔKHN, followed by Student-Newman-Keuls test (p < 0.05, n = 13).
Statistical analysis was performed on the software SigmaPlot version 12.0, at a significance level of 5%. Data of%SH (cubic transformed), ΔKHN, F concentrations in the total biofilm and biofilm fluid (Log10 transformed) passed normality (Shapiro-Wilk) and homogeneity tests (Bartlett). Data were then submitted to one-way (hardness data) or two-way (biofilm data), repeated-measures ANOVA, followed by the Student-Newman-Keuls test. The relationship among the variables was assessed by Pearson’s correlation coefficient.
Dentifrices
Mean (SD) Mean (SD)
Placebo (Ffree) 0.6 g
pH
7.69 (0.01) 7.46 (0.13) 7.40 (0.14)
3
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Table 3 Fluoride concentration in the total biofilm and biofilm fluid (μg F/g) formed in situ according to the different treatment intensities (fluoride concentration in the dentifrice x amount applied on the toothbrush) and time after brushing. Placebo (F-free)
550 ppm F
Amount used during brushing
0.6 g
0.3 g
Time after brushing (min)
5
Total biofilm Biofilm fluid
Mean (SD) Mean (SD)
60 a
1.9 (1.1) 0.12a (0.06)
1100 ppm F 0.6 g
5 A
2.1 (1.3) 0.06A (0.03)
60 b
5 ABC
5.2 (2.6) 4.37b (5.30)
4.0 (3.6) 0.27B (0.21)
0.15 g 60
b
8.6 (6.0) 5.32b (4.28)
5 BD
6.6 (6.9) 0.67B (0.65)
0.3 g 60
b
10.4 (6.3) 5.38b (5.56)
5 CD
8.9 (8.9) 0.40B (0.45)
60 c
22.9 (27.3) 5.38b (5.91)
9.0D (6.3) 0.54B (0.61)
Lowercase and uppercase superscript letters indicate significant differences among the means, respectively for samples collected at 5 and 60 min after brushing (Two-way, repeated measures ANOVA after log10 transformation, followed by Student-Newman-Keuls test (p < 0.05, n = 13). Table 4 Correlation coefficients among the variables assessed in the study. VARIABLES
%SH ΔKHN F in the total biofilm 5 min after brushing F in the total biofilm 60 min after brushing F in the biofilm fluid 5 min after brushing F in the biofilm fluid 60 min after brushing
r p r p r p r p r p r p
ΔKHN
F in the total biofilm 5 min after brushing
F in the total biofilm 60 min after brushing
F in the biofilm fluid 5 min after brushing
F in the biofilm fluid 60 min after brushing
0.60 p < 0.001 *
−0.56 p < 0.001 −0.71 p < 0.001 *
−0.48 p < 0.001 −0.44 p = 0.003 0.67 p = 0.0013 *
−0.35 p = 0.004 −0.72 p < 0.001 0.53 p = 0.005 0.17 p = 0.2 *
−0.31 p = 0.013 −0.55 p = 0.003 0.48 p = 0.005 0.09 p = 0.5 0.78 p < 0.001 *
−0.71 p < 0.001 −0.44 p = 0.003 −0.72 p < 0.001 −0.55 p = 0.003
0.67 p = 0.0013 0.53 p = 0.005 0.48 p = 0.005
0.17 p = 0.2 0.09 p = 0.5
0.78 p < 0.001
%SH = Percentage of surface hardness loss; ΔKHN = Integrated area of subsurface loss. Pearson’s correlation coefficients (r) determined on the Log10 transformed outcomes (n = 65 for each analysis, p < 0.05).
instructive to confirm the above-mentioned hypothesis, as well as to determine the most suitable timing of biofilm collection after brushing when using this biomarker as an indicator of clinical performance. It was also noteworthy that enamel mineral loss (ΔKHN) was strongly correlated with F concentrations in the biofilm (solid and fluid phases) collected 5 min after brushing, but lower correlation coefficients were observed for samples collected 60 min after brushing. Given that ΔKHN and biofilm F concentrations are variables related to the long- and short-term effects of F, respectively, the higher correlation coefficients observed for biofilm collected at 5 min indicates that the effect of fluoridated dentifrices on the biofilm immediately after brushing is an utmost aspect when assessing the efficacy of dentifrices on enamel demineralization, as it represents the maximum boosting effect of the treatment in interfering with the de- and re-mineralization processes of enamel. These findings also highlight the importance of brushing frequency, as a higher frequency of exposure to the dentifrice will raise biofilm F levels more often during the day, ultimately having a greater impact on the caries dynamics Data from a systematic review assessing the effects of fluoridated dentifrices on dental caries support this conclusion [27]. In addition to brushing frequency, the rinsing procedure after brushing is another important factor that affects intraoral F levels, and consequently the process of enamel demineralization. Increasing the amount of water and rinsing frequency were shown to significantly reduce intraoral F concentrations [28], reason by which these variables were carefully standardized in the present study. Two other aspects related to the correlations deserve comment. First, the coefficients obtained between %SH and total biofilm were shown to be moderate (total biofilm) or weak (biofilm fluid), regardless of the time of biofilm sampling. This seems to be related to the nature of the enamel caries lesion (i.e., subsurface lesion), what seems to justify the lower correlation coefficients between these variables in comparison to those obtained for ΔKHN and biofilm F concentrations. Second,
studies assessing the real benefits of dentifrices containing 500–550 ppm F on caries control, especially in the deciduous dentition [1], the present data reinforce the need to discuss and re-evaluate the current recommendation of dentifrices to children. The present in situ model was designed to produce thick biofilms, as typically found in stagnation sites, what has some implications regarding biofilm F concentrations in the solid and fluid phases. While F uptake by total biofilm was shown to be related to both the F concentration in the dentifrice and amount, the two treatment intensities did not produce comparable results, given that higher values were observed for the CD regardless of the amount applied on the toothbrush. On the other hand, the corresponding values in the biofilm fluid reached a plateau at 5 min after brushing, for all combinations of dentifrices and quantities. Despite the study protocol does not allow extrapolations regarding the interactions between the treatments and the biofilm, it is possible that the salivary F concentrations immediately after the initial brushing strokes would be largely influenced by the F concentration in the product (considering the reduced dilution of the product in the mouth at the beginning of toothbrushing), what could explain the higher values observed for CD. This natural dentifrice-saliva slurry would then bathe all oral surfaces, allowing F to retain in the dental biofilm mainly through calcium bridging [23,24], especially at the biofilm-saliva interface, given the reduced F penetration into deeper layers of thick biofilms [25,26]. Unlike for total biofilm, however, biofilm fluid represents a compartment without any mechanism of “retention” [24], so that F concentrations in this compartment would be related to a complex interplay among the residual salivary F concentration, the release of F from the biofilm biomass retained in more labile forms, as well as salivary clearance. In this sense, it is likely that the plateau observed for biofilm fluid F would result from a saturation state obtained immediately after brushing. Time-course studies assessing biofilm F concentrations in the solid and fluid phases would be 4
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the correlation between%SH and ΔKHN was shown to be moderate, what raises concerns regarding the suitability of surface hardness as a surrogate method for the assessment of the mineral changes in caries models. In fact, hardness does not necessarily measure the mineral content of the enamel, but instead its “structural integrity”, which may or not indicate changes in the mineral content [29]. Therefore, from the present results, it seems that surface hardness should only be used as an adjunct analysis to other methods such as cross-sectional hardness, transverse micro radiography or micro-computed tomography, so that mineral changes at both the surface and subsurface are analyzed together. In addition, it is important to note that caries lesion formed in an in situ study (bovine enamel) is structurally different from the lesion formed in in vivo (human enamel). Thus, despite of bovine enamel can be considered a good substitute for human enamel under in vitro conditions [30], the present results cannot be fully extrapolated to in vivo conditions. To conclude, the amount of dentifrice used during toothbrushing and the F concentration in the product were shown to have a direct impact on the development of enamel subsurface lesions and on F concentrations in the biofilm and biofilm fluid, emphasizing the concept that treatment intensity is a more relevant parameter of efficacy than merely observing the F concentration of the dentifrice.
[8] K.B. Hall, A.C. Delbem, M.E. Nagata, T.Y. Hosida, F.R. de Moraes, M. Danelon, R.F. Cunha, J.P. Pessan, Influence of the amount of dentifrice and fluoride concentrations on salivary fluoride levels in children, Pediatr. Dent. 38 (2016) 379–384. [9] M.A. 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. [10] S.A. Moimaz, O. Saliba, F.Y. Chiba, D.H. Sumida, C.A. Garbin, N.A. Saliba, Fluoride concentration in public water supply: 72 months of analysis, Braz. Dent. J. 23 (2012) 451–456. [11] A.C.B. Delbem, M. Danelon, K.T. Sassaki, A.E.M. Vieira, E.M. Takeshita, F.L. Brighenti, E. Rodrigues, 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. [12] J.G. Amaral, K.T. Sassaki, C.C.R. Martinhon, A.C.B. Delbem, Effect of low-fluoride dentifrices supplemented with calcium glycerophosphate on enamel demineralization in situ, Am. J. Dent. 26 (2013) 75–80. [13] DSS Research Tools and Resources for Marketing Researches, (2017) http://www. dssresearch.com/toolkit/default.asp . (Accessed 8 February 2015). [14] A.C. Delbem, K.T. Sassaki, A.E. Vieira, E. Rodrigues, M. Bergamaschi, S.R. Stock, M.L. Cannon, X. Xiao, F. De Carlo, Comparison of methods for evaluating mineral loss: hardness versus synchrotron microcomputed tomography, Caries Res. 43 (2009) 359–365. [15] 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. [16] G.L. Vogel, Y. Mao, C.M. Carey, L.C. Chow, Increased overnight fluoride concentrations in saliva plaque, and plaque fluid after a novel two-solution rinse, J. Dent. Res. 76 (1997) 761–767. [17] J.A. Cury, M.A. Rebello, A.A. Del Bel Cury, In situ relationship between sucrose exposure and the composition of dental plaque, Caries Res. 31 (1997) 356–360. [18] E.M. Takeshita, L.P. Castro, K.T. Sassaki, A.C.B. Delbem, In vitro evaluation of dentifrice with low fluoride content supplemented with trimetaphosphate, Caries Res. 43 (2009) 50–56. [19] M. Danelon, J.P. Pessan, N.S. Neto, E.R. Camargo, A.C.B. Delbem, Effect of toothpaste with nano-sized trimetaphosphate on dental caries: in situ study, J. Dent. 43 (2015) 806–813. [20] M.H. Spiguel, M.F. Tovo, P.F. Kramer, K.S. Franco, K.M. Alves, A.C. Delbem, Evaluation of laser fluorescence in the monitoring of the initial stage of the de-/ remineralization process: an in vitro and in situ study, Caries Res. 43 (2009) 302–307. [21] C.A. Kobayashi, M.R. Belini, F. de, M. Italiani, A.R. Pauleto, J.J. Araújo, V. Tessarolli, L.T. Grizzo, J.P. Pessan, M.A. Machado, M.A. Buzalaf, Factors influencing fluoride ingestion from dentifrice by children, Community Dent. Oral Epidemiol. 39 (2011) 426–432. [22] F. Lippert, E.A. Martinez-Mier, D.T. Zero, An in situ caries study on the interplay between fluoride dose and concentration in milk, J. Dent. 42 (2014) 890–893. [23] R.K. Rose, R.P. Shellis, A.R. Lee, The role of cation bridging in microbial fluoride binding, Caries Res. 30 (1996) 458–464. [24] G.L. Vogel, Oral fluoride reservoirs and the prevention of dental caries, Monogr. Oral. Sci. 22 (2011) 146–157. [25] P.S. Watson, H.A. Pontefract, D.A. Devine, R.C. Shore, B.R. Nattress, J. Kirkham, C. Robinson, Penetration of fluoride into natural plaque biofilms, J. Dent. Res. 84 (2005) 451–455. [26] J.P. Pessan, K.M. Pinto Alves, F. de, M. Italiani, I. Ramires, J.R. Lauris, G.M. Whitford, K.J. Toumba, C. Robinson, M.A.R. Buzalaf, Distribution of fluoride and calcium in plaque biofilms after the use of conventional and low-fluoride dentifrices, Int. J. Paediatr. Dent. 24 (2014) 293–302. [27] V.C.C. Marinho, J.P.T. Higgins, A. Sheiham, S. Logan, Fluoride toothpastes for preventing dental caries in children and adolescents, Cochrane Database Syst. Rev. (1) (2003) CD002278. [28] K. Sjögren, D. Birkhed, J. Ruben, J. Arends, Effect of post-brushing water rinsing on caries-like lesions at approximal and buccal sites, Caries Res. 29 (1995) 337–342. [29] W. Buchalla, T. Imfeld, T. Attin, M.V. Swain, P.R. Schmidlin, Relationship between nanohardness and mineral content of artificial carious enamel lesions, Caries Res. 42 (2008) 157–163. [30] F. Lippert, K. Juthani, Fluoride dose-response of human and bovine enamel artificial caries lesions under pH-cycling conditions, Clin. Oral Invest. 19 (2015) 1947–1954.
Acknowledgements The authors thank the volunteers for taking part in the study. This study was supported by CAPES (scholarship to the first author), FAPESP (grant #2014/16443-1) and CNPq, (grant #458997/2014-5), which played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors also attest that there are no conflicts of interest that might introduce bias or affect the manuscript’s judgment. References [1] T. Walsh, H.V. Worthington, A.M. Glenny, P. Appelbe, V.C. Marinho, X. Shi, Fluoride toothpastes of different concentrations for preventing dental caries in children and adolescents, Cochrane Database Syst. Rev. (1) (2010) CD007868. [2] M.C. Wong, A.M. Glenny, B.W. Tsang, E.C. Lo, H.V. Worthington, V.C. Marinho, Topical fluoride as a cause of dental fluorosis in children, Cochrane Database Syst. Rev. (1) (2011) CD007693. [3] European Academy of Paediatric Dentistry, Guidelines on the use of fluoride in children: an EAPD policy document, Eur. Arch. Paediatr. Dent. 10 (3) (2009) 129–135. [4] American Academy of Pediatric Dentistry, Policy on use of fluoride, Pediatr. Dent. 38 (6) (2016) 45–46. [5] R.M. Duckworth, S.N. Morgan, R.J. Gilbert, Oral fluoride measurements for estimation of the anti-caries efficacy of fluoride treatments, J. Dent. Res. 71 (1992) 836–840. [6] P. DenBesten, H.S. Ko, Fluoride levels in whole saliva of preschool children after brushing with 0 25 g (pea-sized) as compared 1.0 g (full-brush) of a fluoride dentifrice, Pediatr. Dent. 18 (1996) 277–280. [7] D.T. Zero, J.E. Creeth, M.L. Bosma, A. Butler, R.G. Guibert, R. Karwal, R.J.M. Lynch, E.A. Martinez-Mier, C. González-Cabezas, S.A. Kelly, The effect of brushing time and dentifrice quantity on fluoride delivery in vivo and enamel surface microhardness in situ, Caries Res. 44 (2010) 90–100.
5
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Full length article
Fluoride concentration and amount of dentifrice influence enamel demineralization in situ M.F. Paivaa, A.C.B. Delbema, M. Danelona, M.E. Nagataa, F.R.N. Moraesa, G.E.G. Cocletea, ⁎ R.F. Cunhaa, M.A.R. Buzalafb, J.P. Pessana, a b
São Paulo State University (Unesp), School of Dentistry, Araçatuba, Department of Pediatric Dentistry and Public Health, Brazil University of São Paulo, Bauru School of Dentistry, Department of Biological Sciences, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Dentifrices Dental enamel Fluorides Dental plaque
Objectives: This study evaluated the effect of conventional (CD, 1100 ppm F) and low-fluoride (LFD, 550 ppm F) dentifrices, applied in different quantities, on enamel demineralization, and on fluoride (F) concentrations in the dental biofilm formed in situ. Methods: Five combinations of dentifrices and quantities were tested: placebo (P–F-free) applied on all brush bristles; LFD applied by the transversal technique (0.3 g–T1) or on all bristles (0.6 g–T2); and CD applied in a pea-sized amount (0.15 g–T3) or by the transversal technique (0.3 g–T4), in order to produce comparable intensities (F concentration in the dentifrice × amount applied to the brush). Volunteers (n = 13, 20–36 years old) wore palatal devices containing 4 bovine enamel blocks, and performed cariogenic challenges (30% sucrose solution) 6×/day, and brushing 3×/day, following a double-blind, cross-over and randomized protocol. On the 8th day, biofilm was collected 5 and 60 min after brushing. The percentage of surface hardness loss (%SH), integrated loss of subsurface hardness (ΔKHN) and biofilm F concentrations (solid and fluid phases) were determined. Data were analyzed by repeated-measures ANOVA, Student-Newman-Keuls test, and Pearson’s correlation coefficient (p < 0.05). Results: Significantly lower ΔKHN was observed for treatments with higher intensity (T2 and T4) in comparison with the lower intensity (T1 and T3). A strong correlation was observed between ΔKHN and F concentrations in total biofilm (r = −0.71) and biofilm fluid (r = −0.72) 5 min after brushing. Conclusions: The treatment intensity has a significant influence on the development of caries lesions in situ. Clinical significance: The intensity of treatment (amount of dentifrice × concentration) during brushing seems to be a more relevant parameter of clinical efficacy than simply observing the F concentration of the product. The use of a small amount of CD significantly reduced the protective effects against enamel demineralization.
1. Introduction A comprehensive Cochrane review assessing the relative cariesprotective effectiveness of dentifrices with different fluoride (F) concentrations concluded that toothbrushing with dentifrices with concentrations of 1000 ppm F or above is an effective measure in preventing caries in children and adolescents [1]. Given that the early use of fluoridated dentifrices has been shown to be a potential risk factor for the development of dental fluorosis [2], professionals and health authorities have advocated the use of dentifrices with F concentrations above 1000 ppm F, but applied in very small quantities for young children [3,4], under the assumption that this measure would minimize systemic F exposure from this source without compromising the clinical
efficacy of the product. Considering that the clinical effects of F are known to be dose-dependent, this strategy would be valid except for the fact that the dentifrice is diluted in saliva during toothbrushing. It could be anticipated, therefore, that reducing the amount of dentifrice on the brush would reduce the F concentration in the natural dentifrice-saliva slurry formed during toothbrushing, which, in turn, could affect the clinical efficacy of the dentifrice [5]. While previous studies have addressed the impact of the amount of dentifrice used during toothbrushing on salivary F concentrations, enamel remineralization and enamel F uptake [6,7], the effect of the amount of dentifrice as a function of F concentration in the product has only recently been assessed. It was demonstrated that brushing with a low-F dentifrice (LFD, 550 ppm F) applied using the
⁎ Corresponding author at: São Paulo State University (Unesp), School of Dentistry, Araçatuba Department of Pediatric Dentistry and Public Health José Bonifácio 1193, 16015-050 Araçatuba–SP, Brazil. E-mail address: [email protected] (J.P. Pessan).
http://dx.doi.org/10.1016/j.jdent.2017.09.004 Received 25 July 2017; Received in revised form 2 September 2017; Accepted 11 September 2017 0300-5712/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: MF, P., Journal of Dentistry (2017), http://dx.doi.org/10.1016/j.jdent.2017.09.004
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from bovine incisors previously stored in 2% neutral formaldehyde solution (pH 7.0) for 1 month. They were sequentially polished (600, 800, 1200 grit), followed by polishing with felt paper and 1 μ diamond suspension. Four enamel blocks were fixed in 4-mm deep spaces of custom-made acrylic palatal devices, approximately 1 mm below the level of the acrylic surface, in order to allow biofilm accumulation on the enamel specimens. A plastic mesh covered the enamel blocks to prevent biofilm disturbance by mechanical forces [12].
transversal technique (i.e., perpendicular to the long axis of the brush) led to significantly higher salivary F concentrations than a conventional dentifrice (CD, 1100 ppm F) using a pea-sized amount [8], confirming the hypothesis that the dilution of the dentifrice in saliva during toothbrushing has a direct impact on intraoral F concentrations. Based on dose-response considerations, the above-mentioned results could have important clinical implications, raising questions on the appropriateness of the current recommendation of dentifrices to children. Nonetheless, as only one intraoral variable was assessed, and given the short-term nature of that study [8], these results cannot be fully extrapolated to a clinical situation. Considering that dental caries is the net result of the slow mineral loss occurring in dental enamel covered by a cariogenic biofilm [9], the aim of this study was to assess the effects of treatment intensity (i.e., F concentration in the dentifrice versus the amount used during toothbrushing) on enamel mineral loss, as well as on biofilm F concentrations. The null hypothesis was that the treatment intensity would not influence the variables assessed.
2.5. Intraoral procedures Two drops of a 30% sucrose solution were dispensed on each enamel block ex vivo, 6 times/day, at previously established times (8:00, 11:00, 14:00, 17:00, 19:00, 21:00 h). The appliances were left to rest during 5 min before being returned to the oral cavity [12]. The treatment of the blocks with the dentifrices was performed 3 times/day (7:30, 13:00 and 22:00 h), during 7 days. The amount of dentifrice to be used was demonstrated individually to all volunteers, in each experimental phase. They also received photographs via cell phone showing the quantity to be applied on the brush. Volunteers were instructed to brush their natural teeth with the appliance in the oral cavity for 1 min, and to swish the natural suspension of dentifrice/saliva for 30 s. Volunteers then completed the cleaning of their natural teeth without the appliance in their mouths, and rinsed the appliances (20 mL) and their mouths (10 mL) with tap water. They were instructed to remove the appliances only during meals and ingestion of liquids, with no restrictions on their diet. Prior to each experimental phase (lead in/wash out), the volunteers used a F-free dentifrice for 7 days.
2. Materials and methods 2.1. Ethical aspects and inclusion criteria This study was approved by the IRB of School of Dentistry, Araçatuba–UNESP (CAAE 44712715.5.0000.5420). Participants signed an informed consent form and received written and verbal instructions on the research protocol prior to the beginning of the study. Thirteen individuals (20–36 years old), living in the city of Araçatuba-SP, Brazil (0.6–0.8 mg F/L in the public water supply) [10], were enrolled. The inclusion criteria involved participants in good general and oral health. Individuals who used drugs that could interfere with the formation of the dental biofilm or salivary flow, smokers, those who received fluoride applications 2 weeks before the experiment, were using orthodontic appliances or had systemic diseases could not participate in the study [11].
2.6. Sample collection On the morning of the 8th day, the volunteers attended the laboratory of Pediatric Dentistry, School of Dentistry, Araçatuba, while fasting, having been instructed not to brush their teeth and not to perform the cariogenic challenge on the specimens. Volunteers then performed the brushing procedures as previously described, but with the dentifrices weighed by the researchers (Shimadzu Balance AUY220, Kyoto–Japan). Biofilm from 2 blocks was randomly collected at 5 and 60 min after brushing, being immediately transferred to a microcentrifuge tube filled with mineral oil. The tube was weighed (before and after collection) and centrifuged (21,000g, 5 min, 4 °C) to separate the solid and fluid phases of the biofilm. Following, a small fraction of the fluid phase was collected, using a micropipette.
2.2. Experimental design Sample size was based on a previous study assessing the effects of dentifrices containing 500 and 1100 ppm F on enamel demineralization and biofilm F concentrations [12]. A sample of 14 volunteers was calculated considering α-error of 5%, β-error of 20% [13], and dropout rate of 20%. The protocol used was in situ, double-blind and crossover. The volunteers were randomly divided into 5 groups, totaling 4 combinations of dentifrice and quantity, namely: 550 ppm F applied by the transversal technique (0.3 g) or on all bristles of the brush (0.6 g), and 1100 ppm F applied in a pea-size amount (0.15 g) or by the transversal technique (0.3 g). A placebo dentifrice (F-free) applied on all bristles of the brush (0.6 g) was also included. The dentifrice tubes were coded and the quantities used during brushing were determined by an examiner not involved in sample collection and analysis.
2.7. Fluoride analysis Biofilm fluid samples were transferred to the surface of an inverted ion-specific electrode (Orion 9409), immersed in mineral oil, and placed on drops of TISAB III previously placed on the electrode membrane, at a 10:1 ratio (sample:TISAB III), as described by Vogel et al. [16]. F was determined in the total biofilm after HCl extraction and buffering with TISAB III, using a F ion-specific electrode (Orion 9409), and a reference electrode (Orion 900200), both coupled to an ion analyzer (Orion 720A+) [17].
2.3. Formulation and determination of F in the experimental dentifrices The experimental dentifrices were produced in the laboratory of Pediatric Dentistry from School of Dentistry, Araçatuba, using the same basic formulation with the following components: titanium dioxide, carboxymethylcellulose, methyl p-hydroxybenzoate, sodium saccharine, oil peppermint, glycerin, silica abrasive, sodium lauryl sulfate, water and sodium fluoride (NaF, Merck®, Germany 550 e 1100 μg F/g). A F-free formulation without F (placebo) was also used. The determination of the ionic and total F concentrations [14] and pH [15] of the dentifrices was performed prior to the beginning of the study.
2.8. Hardness analysis Surface hardness was determined prior to the experiments (SHi) using the Micromet 5114 hardness tester (Buehler, Lake Bluff, USA and Mitutoyo Corporation, Kanagawa, Japan), under 25 g for 10 s (five indentations, spaced 100 μm from each other, made at the center of the block) [18]. Blocks included in the study had SHi between 330–370 KHN, and were randomly distributed among the study groups. After the experimental phases, final surface hardness (SHf) was determined as described for SHi, 100 μm from the initial indentations. The percentage of SH loss (%SH) was calculated using the formula: %SH = 100 x (SHf –
2.4. Enamel blocks and appliance preparation Enamel blocks (n = 260) measuring 4 × 4 x 2 mm were obtained 2
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SHi)/SHi. Blocks were then longitudinally sectioned at the center, and one of the halves was embedded in acrylic resin with the cut surface exposed and polished. One sequence of 14 indentations was created at the center of the blocks at 5, 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220, and 330 μm from the enamel surface, with a Knoop diamond indenter under a 5 g load for 10 s [19]. The integrated area of the hardness (ΔKHN × μm) of the lesion to the sound enamel was calculated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated area of the enamel hardness, obtaining the integrated loss of subsurface hardness (ΔKHN) [20].
Table 2 Mean percentage of surface hardness loss (%SH) and integrated loss of subsurface hardness (ΔKHN) according to the different treatment intensities (fluoride concentration in the dentifrice x amount applied on the toothbrush). Dentifrices Amount applied on the toothbrush %SH ΔKHN
2.9. Statistical analysis
Placebo 550 ppm F 1100 ppm F
18.5 (3.3) 545.1 (4.0) 1075.5 (14.6)
13.5 (8.4) 543.5 (16.8) 1095.6 (22.7)
0.6 g
0.15 g
0.3 g
−51.3a (26.1) 7378.6a (1123.0)
−25.0bc (13.2) 3113.4b (411.7)
−23.4bc (8.7) 2604.0c (453.4)
−30.2b (13.0) 3224.7b (845.5)
−20.2c (8.7) 2431.6c (379.4)
A recent in vivo study assessing salivary F concentrations in children brushing with CD and LFD applied in different quantities showed that the treatment intensity (i.e., amount of dentifrice × F concentration) is a relevant parameter when comparing dentifrices with different F concentrations [8]. The present study demonstrated that treatment intensity had a direct impact on the mineral loss of enamel covered by dental biofilm formed in situ under highly cariogenic conditions, as well as on the resulting biofilm F concentrations, thus leading to the rejection of the study’s null hypothesis. Brushing with the placebo dentifrice promoted significantly higher %SH and ΔKHN when compared with the fluoridated dentifrices, thus validating the in situ protocol employed (i.e., in vivo exposure to the dentifrice slurries and ex vivo exposure to sucrose). Interestingly, while brushing with the LFD using larger quantities (transversal technique or full bristles) led to similar %SH values to that observed for the CD applied using the transversal technique, the use of the CD in a pea-size amount promoted the highest %SH among the fluoridated dentifrices, being significantly higher than the CD applied with the transversal technique. Given that the use of a pea-size amount of a CD was also shown to significantly reduce salivary F when compared with the transversal technique [8], these data taken together reinforce the concept that the use of very low quantities of a CD might reduce the cariesprotective effect of the product under clinical conditions. It is noteworthy that the interplay between fluoride dose and concentration had already been demonstrated in an in situ study with fluoridated milk, regarding surface hardness recovery and enamel fluoride uptake [22]. The authors concluded that both variables appear to impact the cariostatic properties of fluoride in milk, what reinforces the present data related to fluoride dentifrices. A more evident effect of the treatment intensity on enamel mineral loss was observed for ΔKHN data. The highest treatment intensity (i.e., 0.6 g of 550 ppm F or 0.3 g of 1100 ppm F) led to significantly lower ΔKHN when compared with the lowest treatment intensity (i.e., 0.3 g of 550 ppm F or 0.15 g of 1100 ppm F), with no significant differences between CD and LFD within each intensity. These findings are important from a clinical standpoint considering the current recommendations of dentifrices to children [3,4]. Despite reducing the amount of dentifrice is indeed an effective measure in minimizing F intake during toothbrushing, especially by younger children [21], the present study clearly demonstrates that (1) this recommendation significantly reduces the protective effect of the dentifrice, and (2) the protective effect of a LFD can be similar or higher than that of a CD depending on the amount of dentifrice used during toothbrushing. Based on the above and considering the scarcity of well-controlled
Table 1 Mean (SD) fluoride concentrations and pH in the experimental dentifrices (n = 3).
Ionic fluoride
0.3 g
4. Discussion
Table 1 shows mean total and ionic F concentrations in the experimental dentifrices, which presented a variation lower than 3% when compared with the expected values. Mean pH of the dentifrices ranged from 7.4 (1100 ppm F) to 7.7 (placebo). Significantly lower %SH and ΔKHN were observed for all fluoridated dentifrices when compared with the placebo (p < 0.001) (Table 2). For %SH, the only significant difference among the fluoridated groups was observed for CD applied using a pea-size amount or the transversal technique (p = 0.040). A more marked effect of the treatment intensities was observed for ΔKHN, given that values obtained for the lowest intensity (i.e., 0.30 g of 550 ppm F and 0.15 g of 1100 ppm F) were significantly higher than those for the highest intensity (i.e., 0.60 g of 550 ppm F and 0.30 g of 1100 ppm F), without significant differences between CD and LFD within each intensity. A dose-response relationship was observed between treatment intensity and total biofilm F concentrations for both times of sample collection, despite differences among the fluoridated dentifrices were not significant 5 min after brushing, except for the CD applied using the transversal technique (Table 3). At 60 min, F concentrations remained significantly higher than the placebo for all fluoridated dentifrices, except for the LFD applied using the transversal technique. For biofilm fluid F concentrations, all fluoridated dentifrices led to significantly higher F concentrations than the placebo at both times of sample collection, without significant differences among the dentifrices regardless the amount applied on the toothbrush. A strong correlation was observed between ΔKHN and F concentrations in the total biofilm (r = −0.71) and biofilm fluid (r = −0.72) 5 min after brushing, while lower coefficients were observed 60 min after brushing. A moderate correlation was seen between F concentrations in the total biofilm 5 and 60 min after brushing (r = 0.67), whereas a strong correlation was seen for F concentrations in the fluid phase of the biofilm (r = 0.78). ΔKHN and%SH were
Total fluoride
1100 ppm F
moderately correlated (r = 0.60). Other comparisons are displayed in Table 4.
3. Results
Fluoride concentration (ppm)
550 ppm F
Superscript letters indicate significant differences among the means. Data analyzed by one-way, repeated measures ANOVA after cubic and log10 transformation, respectively for%SH and ΔKHN, followed by Student-Newman-Keuls test (p < 0.05, n = 13).
Statistical analysis was performed on the software SigmaPlot version 12.0, at a significance level of 5%. Data of%SH (cubic transformed), ΔKHN, F concentrations in the total biofilm and biofilm fluid (Log10 transformed) passed normality (Shapiro-Wilk) and homogeneity tests (Bartlett). Data were then submitted to one-way (hardness data) or two-way (biofilm data), repeated-measures ANOVA, followed by the Student-Newman-Keuls test. The relationship among the variables was assessed by Pearson’s correlation coefficient.
Dentifrices
Mean (SD) Mean (SD)
Placebo (Ffree) 0.6 g
pH
7.69 (0.01) 7.46 (0.13) 7.40 (0.14)
3
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Table 3 Fluoride concentration in the total biofilm and biofilm fluid (μg F/g) formed in situ according to the different treatment intensities (fluoride concentration in the dentifrice x amount applied on the toothbrush) and time after brushing. Placebo (F-free)
550 ppm F
Amount used during brushing
0.6 g
0.3 g
Time after brushing (min)
5
Total biofilm Biofilm fluid
Mean (SD) Mean (SD)
60 a
1.9 (1.1) 0.12a (0.06)
1100 ppm F 0.6 g
5 A
2.1 (1.3) 0.06A (0.03)
60 b
5 ABC
5.2 (2.6) 4.37b (5.30)
4.0 (3.6) 0.27B (0.21)
0.15 g 60
b
8.6 (6.0) 5.32b (4.28)
5 BD
6.6 (6.9) 0.67B (0.65)
0.3 g 60
b
10.4 (6.3) 5.38b (5.56)
5 CD
8.9 (8.9) 0.40B (0.45)
60 c
22.9 (27.3) 5.38b (5.91)
9.0D (6.3) 0.54B (0.61)
Lowercase and uppercase superscript letters indicate significant differences among the means, respectively for samples collected at 5 and 60 min after brushing (Two-way, repeated measures ANOVA after log10 transformation, followed by Student-Newman-Keuls test (p < 0.05, n = 13). Table 4 Correlation coefficients among the variables assessed in the study. VARIABLES
%SH ΔKHN F in the total biofilm 5 min after brushing F in the total biofilm 60 min after brushing F in the biofilm fluid 5 min after brushing F in the biofilm fluid 60 min after brushing
r p r p r p r p r p r p
ΔKHN
F in the total biofilm 5 min after brushing
F in the total biofilm 60 min after brushing
F in the biofilm fluid 5 min after brushing
F in the biofilm fluid 60 min after brushing
0.60 p < 0.001 *
−0.56 p < 0.001 −0.71 p < 0.001 *
−0.48 p < 0.001 −0.44 p = 0.003 0.67 p = 0.0013 *
−0.35 p = 0.004 −0.72 p < 0.001 0.53 p = 0.005 0.17 p = 0.2 *
−0.31 p = 0.013 −0.55 p = 0.003 0.48 p = 0.005 0.09 p = 0.5 0.78 p < 0.001 *
−0.71 p < 0.001 −0.44 p = 0.003 −0.72 p < 0.001 −0.55 p = 0.003
0.67 p = 0.0013 0.53 p = 0.005 0.48 p = 0.005
0.17 p = 0.2 0.09 p = 0.5
0.78 p < 0.001
%SH = Percentage of surface hardness loss; ΔKHN = Integrated area of subsurface loss. Pearson’s correlation coefficients (r) determined on the Log10 transformed outcomes (n = 65 for each analysis, p < 0.05).
instructive to confirm the above-mentioned hypothesis, as well as to determine the most suitable timing of biofilm collection after brushing when using this biomarker as an indicator of clinical performance. It was also noteworthy that enamel mineral loss (ΔKHN) was strongly correlated with F concentrations in the biofilm (solid and fluid phases) collected 5 min after brushing, but lower correlation coefficients were observed for samples collected 60 min after brushing. Given that ΔKHN and biofilm F concentrations are variables related to the long- and short-term effects of F, respectively, the higher correlation coefficients observed for biofilm collected at 5 min indicates that the effect of fluoridated dentifrices on the biofilm immediately after brushing is an utmost aspect when assessing the efficacy of dentifrices on enamel demineralization, as it represents the maximum boosting effect of the treatment in interfering with the de- and re-mineralization processes of enamel. These findings also highlight the importance of brushing frequency, as a higher frequency of exposure to the dentifrice will raise biofilm F levels more often during the day, ultimately having a greater impact on the caries dynamics Data from a systematic review assessing the effects of fluoridated dentifrices on dental caries support this conclusion [27]. In addition to brushing frequency, the rinsing procedure after brushing is another important factor that affects intraoral F levels, and consequently the process of enamel demineralization. Increasing the amount of water and rinsing frequency were shown to significantly reduce intraoral F concentrations [28], reason by which these variables were carefully standardized in the present study. Two other aspects related to the correlations deserve comment. First, the coefficients obtained between %SH and total biofilm were shown to be moderate (total biofilm) or weak (biofilm fluid), regardless of the time of biofilm sampling. This seems to be related to the nature of the enamel caries lesion (i.e., subsurface lesion), what seems to justify the lower correlation coefficients between these variables in comparison to those obtained for ΔKHN and biofilm F concentrations. Second,
studies assessing the real benefits of dentifrices containing 500–550 ppm F on caries control, especially in the deciduous dentition [1], the present data reinforce the need to discuss and re-evaluate the current recommendation of dentifrices to children. The present in situ model was designed to produce thick biofilms, as typically found in stagnation sites, what has some implications regarding biofilm F concentrations in the solid and fluid phases. While F uptake by total biofilm was shown to be related to both the F concentration in the dentifrice and amount, the two treatment intensities did not produce comparable results, given that higher values were observed for the CD regardless of the amount applied on the toothbrush. On the other hand, the corresponding values in the biofilm fluid reached a plateau at 5 min after brushing, for all combinations of dentifrices and quantities. Despite the study protocol does not allow extrapolations regarding the interactions between the treatments and the biofilm, it is possible that the salivary F concentrations immediately after the initial brushing strokes would be largely influenced by the F concentration in the product (considering the reduced dilution of the product in the mouth at the beginning of toothbrushing), what could explain the higher values observed for CD. This natural dentifrice-saliva slurry would then bathe all oral surfaces, allowing F to retain in the dental biofilm mainly through calcium bridging [23,24], especially at the biofilm-saliva interface, given the reduced F penetration into deeper layers of thick biofilms [25,26]. Unlike for total biofilm, however, biofilm fluid represents a compartment without any mechanism of “retention” [24], so that F concentrations in this compartment would be related to a complex interplay among the residual salivary F concentration, the release of F from the biofilm biomass retained in more labile forms, as well as salivary clearance. In this sense, it is likely that the plateau observed for biofilm fluid F would result from a saturation state obtained immediately after brushing. Time-course studies assessing biofilm F concentrations in the solid and fluid phases would be 4
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the correlation between%SH and ΔKHN was shown to be moderate, what raises concerns regarding the suitability of surface hardness as a surrogate method for the assessment of the mineral changes in caries models. In fact, hardness does not necessarily measure the mineral content of the enamel, but instead its “structural integrity”, which may or not indicate changes in the mineral content [29]. Therefore, from the present results, it seems that surface hardness should only be used as an adjunct analysis to other methods such as cross-sectional hardness, transverse micro radiography or micro-computed tomography, so that mineral changes at both the surface and subsurface are analyzed together. In addition, it is important to note that caries lesion formed in an in situ study (bovine enamel) is structurally different from the lesion formed in in vivo (human enamel). Thus, despite of bovine enamel can be considered a good substitute for human enamel under in vitro conditions [30], the present results cannot be fully extrapolated to in vivo conditions. To conclude, the amount of dentifrice used during toothbrushing and the F concentration in the product were shown to have a direct impact on the development of enamel subsurface lesions and on F concentrations in the biofilm and biofilm fluid, emphasizing the concept that treatment intensity is a more relevant parameter of efficacy than merely observing the F concentration of the dentifrice.
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