In Vitro Enamel Remineralization by Low-Fluoride Toothpaste with Calcium Citrate and Sodium Trimetaphosphate

(450 μgF/g, NaF) combined with calcium citrate (Cacit) and sodium trimetaphosphate (TMP) on enamel remineralization. Bovine enamel blocks had the enamel surface polished sequentially to determine the surface hardness. After production of artificial carious lesions, the blocks selected by their surface hardness were submitted to remineralization pH cycling and daily treatment with dentifrice suspensions (diluted in deionized water or artificial saliva): placebo, 275, 450, 550 and 1,100 μgF/g and commercial dentifrice (positive control, 1,100 μgF/g). Finally, the surface and cross-section hardness was determined for calculating the change of surface hardness (%SH) and mineral content (%DZ). Fluoride in enamel was also determined. The data from %SH, %DZ and fluoride were subjected to two-way analysis of variance followed by Student-Newman-Keuls’s test (p<0.05). The mineral gain (%SH and %DZ) was higher for toothpastes diluted in saliva (p<0.05), except for the 450 mgF/g dentifrice with Cacit/TMP (p>0.05). The 450 Cacit/ TMP toothpaste and the positive control showed similar results (p>0.05) when diluted in water. A dose-response was observed between fluoride concentration in toothpastes and fluoride present in enamel, regardless of dilution. It was concluded that it is possible to enhance the remineralization capacity of low F concentration toothpaste by of organic (Cacit) and inorganic (TMP) compounds with affinity to hydroxyapatite. In Vitro Enamel Remineralization by Low-Fluoride Toothpaste with Calcium Citrate and Sodium Trimetaphosphate

The objective of this study was to evaluate in vitro the effect of a low fluoride toothpaste (450 µgF/g, NaF) combined with calcium citrate (Cacit) and sodium trimetaphosphate (TMP) on enamel remineralization.Bovine enamel blocks had the enamel surface polished sequentially to determine the surface hardness.After production of artificial carious lesions, the blocks selected by their surface hardness were submitted to remineralization pH cycling and daily treatment with dentifrice suspensions (diluted in deionized water or artificial saliva): placebo, 275, 450, 550 and 1,100 µgF/g and commercial dentifrice (positive control, 1,100 µgF/g).Finally, the surface and cross-section hardness was determined for calculating the change of surface hardness (%SH) and mineral content (%DZ).Fluoride in enamel was also determined.The data from %SH, %DZ and fluoride were subjected to two-way analysis of variance followed by Student-Newman-Keuls's test (p<0.05).The mineral gain (%SH and %DZ) was higher for toothpastes diluted in saliva (p<0.05),except for the 450 mgF/g dentifrice with Cacit/TMP (p>0.05).The 450 Cacit/ TMP toothpaste and the positive control showed similar results (p>0.05) when diluted in water.A dose-response was observed between fluoride concentration in toothpastes and fluoride present in enamel, regardless of dilution.It was concluded that it is possible to enhance the remineralization capacity of low F concentration toothpaste by of organic (Cacit) and inorganic (TMP) compounds with affinity to hydroxyapatite.

Introduction
From low to high concentrations of fluoride have been tested, but the beneficial effect of fluoride has its limits.One cannot indefinitely increase the concentrations of fluoride dentifrice at risk of adverse effects such as dental fluorosis, even without losing their cariostatic effect (1).A review confirms the benefits of using fluoride toothpaste for caries prevention in children and adolescents when compared with placebo, but significant only for fluoride concentrations of 1,000 ppm and above (2).There is strong evidence that higher levels of fluoride (1,000 ppm or more) in toothpaste are associated with an increased risk of fluorosis if given to children under 5 to 6 years of age (3).Considering that most toothpastes available to the children present more than 1,000 ppm F (4), it would be interesting to use dentifrice with lower fluoride concentration (5), while maintaining the effectiveness of a commercial standard dentifrice (1,100 ppm F).The acidifying (6,7) or addition of organic or inorganic salts (8-10) can improve the effectiveness of fluoride toothpastes.
Combination of active agents with ability to link on enamel can enhance the effect of fluoride.The inorganic phosphates, such as sodium trimetaphosphate (TMP), have great affinity with hydroxyapatite (HA) and great action against enamel demineralization (8)(9)(10)(11)(12).Similar to TMP, the calcium citrate (Cacit, organic salt) can also be adsorbed to HA and influence the demineralization process (10).In in vitro studies, the association of TMP and Cacit to toothpastes improved the effect against caries with only 450 µgF/g (10).However, the effect of Cacit/TMP on remineralization has not been tested yet.
It should be noted that in these studies the dentifrice was diluted in water (6)(7)(8)(9)(10).However, in in situ and in vivo studies with both materials, calcium and phosphate, are available from the saliva, reducing the difference between toothpastes (13).As Cacit, TMP and fluoride have affinity to hydroxyapatite, they can interfere on the saliva mainly in the remineralization process.Thus, the aim of this experiment was to evaluate in vitro the ability of a low fluoride toothpaste (450 µgF/g) containing Cacit/TMP to promote enamel remineralization using saliva and water as dilution means.

Study Design
Enamel blocks (4x4 mm) were obtained from bovine incisors stored in a solution of 2% neutral formalin for 30 days at room temperature (10).The blocks had their enamel surface polished sequentially to determine the initial surface hardness (SH) (325.6 to 348.0 kgf/mm 2 ).

E. Hirata et al.
Enamel blocks with artificial carious lesions presenting surface hardness (SH 1 ) between 22.8 to 130.8 kgf/mm 2 were selected.They were assigned to twelve groups (n=10), according to the concentration of fluoride in dentifrice (placebo, 275, 450, 550 and 1,100 µgF/g) and the dilution media (deionized water and artificial saliva).A commercially available dentifrice was used as positive control (1,100 μg F/g, Crest ™ , Procter & Gamble, Cincinnati, OH, USA).The blocks were subjected to six pH cycling regimens twice a day to assess the ability of dentifrices to promote remineralization and treatment with dentifrice diluted in water or artificial saliva.After pH cycling, the surface (SH 2 ) and cross-sectional hardness were determined as well as the fluoride concentration in enamel.The fluoride concentration range of dentifrices and the dilution were considered as factors while hardness and fluoride in enamel as variables.
Determination of fluoride in toothpaste was made in triplicate, according to Delbem et al. (14).After dilution in deionized water, samples of the suspension (0.25 mL) were hydrolyzed by adding 0.25 mL of HCl 1 M/L and heating at 45 o C for 1 h for total fluoride measurement (TF).For the determination of ionic fluoride (IF), the suspension (0.25 mL) was centrifuged (906 g, 20 min) and added 0.25 mL of HCl 1 M/L.An amount of 0.5 mL TISAB II ("total ionic strength adjustment buffer", Orion Research Inc., Beverly, MA, USA) combined with NaOH (20 g/L) was added to the TF and IF solutions.The reading was performed with the Orion 9609 (Orion Research Inc.,) fluoride ion specific electrode and 720 A+ Orion (Orion Research Inc.) ion analyzer, calibrated with standards containing 0.125 to 4.0 mgF/mL.

Induction of Subsurface Enamel Demineralization
The enamel blocks were completely isolated with a thin layer of nail polish except for the enamel surface (area=16 mm 2 ).The blocks (n=120) were placed individually in demineralizing solution (1.3 mM/L Ca(NO 3 ) 2 .4H 2 O and 0.78 mM/L NaH 2 PO 4 H 2 O in 0.05 M/L acetate buffer, 0.03 µgF/mL, pH 5.0, 32 mL/block) for 16 h at 37°C to produce subsurface enamel demineralization (15,16).The blocks were assessed for surface hardness after demineralization (SH 1 ), with indentations distanced 100 mm from the initial impressions.

pH Cycling and Treatments with Toothpastes
Enamel blocks were submitted to pH cycling simulating the process of remineralization for six days at 37 o C, based on Vieira et al. (17).The blocks were kept in a remineralizing solution (1.5

Determination of Surface and Cross-Sectional Hardness
Surface and cross-sectional hardness were determined using a microhardness tester (HMV-2000; Shimadzu, Tokyo, Japan) under load of 25 g and for 10 s.For the initial surface hardness (SH) five Knoop indentations were made 100 mm distant from each other in the center of the block, as described by Vieira et al. (17).After induction of subsurface enamel demineralization (SH 1 ) and pH cycling (SH 2 ), five indentations were made spaced 100 mm from each other and from the baseline.The percentage of surface hardness recovery (%SH) was calculated [%SH = ((SH 2 -SH 1 )/(SH -SH 1 )) x 100].
To assess the cross-sectional hardness, the blocks were sectioned in the center and one-half included in acrylic resin and polished sequentially.Three sequences of eight indentations at distances of 10, 30, 50, 70, 90, 110, 220 and 330 mm from the outer surface of enamel were made in the central block, and two 100 mm above and below.The averages were calculated for each distance and the values converted into mineral content (% vol.min.= 4.3*(√KHN)+11.3),according to Featherstone et al. (18).Integrated area (% vol.min.x mm) for sound enamel (Z), post demineralization enamel (Z 1 ) and after pH cycling (Z 2 ) was calculated by the trapezoidal rule (GraphPad Prism, version 3.02; GraphPad Software, Inc. La Jolla, CA, USA) and the percent mineral recovery (%DZ) determined [%DZ = ((Z 2 -Z 1 )/(Z -Z 1 )) x 100].

Determination of Fluoride Concentration in Enamel
Blocks (4x2 mm) were obtained from one of the halves of the longitudinally sectioned blocks.The outer enamel Low-fluoride toothpaste with Cacit/TMP surface (84.5 [13.9] µm) was removed by immersing the enamel blocks in 0.5 M/L HCl for 90 s under agitation (10).The same volume of TISAB II (total ionic strength adjustment buffer) modified with NaOH (20 g/L) was added to the solution.Fluoride measurements were made using a fluoride specific electrode Orion 96-09 and an Orion 720 A+ ion analyzer, previously calibrated with standards containing 0.125 up to 2.0 µgF/mL and 0.25 up to 4.0 µgF/mL.

Statistical Analysis
After proof of homogeneity, %SH, mineral gain (%DZ) and fluoride concentration in enamel (µgF/cm 2 ) data were subjected to two-way ANOVA followed by Student-Newman-Keuls's test.Two-way ANOVA was used to analyze the data of mineral volume as a function of depth in each dilution (water and saliva).A correlation test (Pearson's test) was applied to verify relationship between the variables.Analyses were performed using SigmaPlot version 12.0 and the level of significance was set at 5%.

Results
The mean (SD) of total and ionic fluoride concentration (mg F/g) in the placebo, 275, 450 Cacit/TMP, 550, 1,100 mg F/g and positive control are described in Table 1.The average fluoride concentrations of all the toothpastes were within the expected values, showing a variation of less than 14%.The toothpastes diluted in saliva presented higher values (p<0.001) of %SH and %DZ when compared with water, except for the toothpaste with 450 mgF/g and Cacit/TMP (Table 2).There was correlation between the results for dentifrices diluted in water and saliva for %SH (Pearson's r=0.941,R 2 =0.885, p<0.001) and %DZ (Pearson's r=0.974,R 2 =0.948, p<0.001).With the increase of fluoride concentration in toothpastes there was a dose-response relation to %SH (Pearson's r=0.965,R 2 =0.931, p<0.001) and %DZ (Pearson's r=0.970,R 2 =0.942, p<0.001), regardless of dilution.The toothpaste with 450 mgF/g and Cacit/TMP presented the best results when diluted with water (p<0.05).Profile of cross-sectional hardness showed higher mineral gain at 10-30 µm of depth when diluted in saliva (Fig. 1B).

Discussion
Studies show that phosphate compounds (inorganic or organic) have caries-protective activity related with their ability to interact with enamel (8)(9)(10)(11)19,20).As Cacit, TMP and fluoride have affinity to enamel and may compete for the same sites of linkage, it is important to determine how their association could improve the enamel remineralization.Based on this, one concern in this study was to compare two means of dilution (water and saliva).Diluting the dentifrice in artificial saliva, the experimental model introduces a further condition of the oral cavity, i.e., sources of calcium and phosphate ions.The adsorption of Cacit and TMP on enamel may enhance or not the ion diffusion into the enamel lesion when combined with fluoride.
Although the results for toothpastes diluted in water and saliva present correlation, the data show that the availability of calcium and phosphate in the medium promotes higher remineralization, even in an in vitro study.This is evident in the placebo group when it provided an increase of 86% (%SH) and 174% (%DZ) in enamel remineralization.Added to this, the presence of fluoride in the enamel has not been influenced by the dilution.However, fluoride not only increased the ability of remineralization of toothpastes, but also reduced the difference between the dilution means.In toothpastes with higher fluoride concentrations this difference does not exist (Table 2).
The association of Cacit/TMP with 450 µgF/g improved the effectiveness of the dentifrice.Two data support this statement: (A) enhancing the remineralization ability by 62% (dilution in water) compared to 550 µgF/g dentifrice and (B) similar fluoride concentration in enamel for both groups.This supports the hypothesis of the capacity of Cacit and TMP of binding to the enamel surface and favor the diffusion of ions through the lesion (8)(9)(10)(11)(19)(20)(21)(22).The present study showed that Cacit and TMP are able to bind on the enamel surface and to remain bound for a longer time.After toothpaste applications, the enamel blocks were always washed with deionized water.As there were improvements in the remineralizing capacity, the addition of Cacit/TMP led to greater diffusion of calcium and phosphate ions from pH cycling solutions, since the fluoride concentration did not differ between the toothpastes with 550 and 450 µgF/g.
As described above, the saliva dilution produces a large increase in the capacity of remineralizing toothpastes whereas the suspension remains in contact with the enamel for 1 min only.However, in the saliva dilution, only 450 Cacit/TMP toothpaste showed reduction of remineralization ability.One hypothesis is that the adsorption of Cacit and TMP on enamel could: (A) reduce the reaction of calcium and phosphate from saliva during the treatment; or (B) induce a high precipitation of these ions on the enamel surface, blotting the pores and reducing the diffusion into the lesion.Calcium citrate is an unionized soluble salt, which can interact with surface of hydroxyapatite by two carboxylate groups via hydrogen and calcium sites of HA (21,22).In the presence of a calcium-and phosphate-rich solution (saliva).Cacit can increase the precipitation with respect to HA (22).The cross-sectional profile shows a lower remineralization in the depth of 30-50 µm when compared with 1,100 µgF/g toothpastes (Fig. 1B).The results were reversed when diluted in water (Fig. 1A).This confirms a lower diffusion of calcium and phosphate ions in the depth of the lesion as the fluoride concentration was not different between the dilutions.
Under the experimental conditions, it was concluded that it is possible to increase the remineralization efficacy of a dentifrice with low-F concentration by organic (Cacit) and inorganic (TMP) compounds with affinity to hydroxyapatite.The calcium and phosphate-rich dilution medium was shown to be important, especially when associated with other active substances that may influence the diffusion and precipitation of ions into enamel.

Table 1 .
Mean (SD) concentration of total (TF) and ionic (IF) fluoride in the formulation according to the toothpastes (n=3)