Effect of fluoride gels with nano-sized sodium trimetaphosphate on the in vitro remineralization of caries lesions

Abstract Objective To evaluate the effects of fluoride (F) gels supplemented with micrometric or nano-sized sodium trimetaphosphate (TMPmicro and TMPnano, respectively) on the in vitro remineralization of caries-like lesions. Methodology Bovine enamel subsurface lesions (n=168) were selected according to their surface hardness (SH) and randomly divided into seven groups (n=24/group): Placebo (without F/TMP), 4,500 ppm F (4500F), 4500F + 2.5% TMPnano (2.5% Nano), 4500F + 5% TMPnano (5% Nano), 4500F + 5% TMPmicro (5% Micro), 9,000 ppm F (9000F), and 12,300 ppm F (Acid gel). The gels were applied in a thin layer for one minute. Half of the blocks were subjected to pH cycling for six days, whereas the remaining specimens were used for loosely- (calcium fluoride; CaF2) and firmly-bound (fluorapatite; FA) fluoride analysis. The percentage of surface hardness recovery (%SHR), area of subsurface lesion (ΔKHN), CaF2, FA, calcium (Ca), and phosphorus (P) on/in enamel were determined. Data (log10-transformed) were subjected to ANOVA and the Student-Newman-Keuls’ test (p<0.05). Results We observed a dose-response relation between F concentrations in the gels without TMP for %SHR and ΔKHN. The 2.5% Nano and 5% Micro reached similar %SHR when compared with 9000F and Acid gels. For ΔKHN, Placebo and 5% Nano gels had the highest values, and 5% Micro, 2.5% Nano, 9000F, and Acid gels, the lowest. All groups had similar retained CaF2 values, except for Placebo and Acid gel. We verified observed an increase in Ca concentrations in nano-sized TMP groups. Regarding P, TMP groups showed similar formation and retention to 9000F and Acid. Conclusion Adding 2.5% nano-sized or 5% micrometric TMP to low-fluoride gels lead to enhanced in vitro remineralization of artificial caries lesions.


Introduction
Caries prevention programs focus on children and usually considered as a priority for dental public health since they are less expensive than treatment. 1 Topically applied fluoride (F) products, especially at high concentrations, have served as a low-cost and easily implemented measure to prevent and treat caries lesions, 2 most commonly in solutions, gels, and varnishes. 3 Because of the risk of overingestion and thus acute toxicity, recommendations usually suggest the use of F gels for children older than six years old. 4 Considering the extensive benefits and wide use of these products, the search for strategies that increase their clinical effectiveness without increasing possible side-effects in young children is highly desirable.
We find considerable in vitro and in situ evidence on the efficacy of fluoridated products supplemented with phosphate salts on enamel de-and remineralization processes, including sodium trimetaphosphate (TMP), 5,6 sodium hexametaphosphate (HMP), 7,8 and calcium glycerophosphate (CaGP). 9,10 A randomized clinical trial has recently confirmed laboratory evidence, showing that the supplementation of a low-F (500 ppm F) toothpaste with micrometric TMP or CaGP resulted in lower or similar caries progression, respectively, than a conventional dentifrice formulation (1100F). 11 In addition, the use of nano-sized TMP or HMP further enhanced the effects of F on enamel de-and remineralization when added to dentifrices. 12,13 Such additional effects could be due to the high ratio of surface area to volume, as well as a higher percentage of atoms on surfaces than on larger particles, which makes nano-sized particles more reactive. 13 Despite the promising results obtained for dentifrice formulations described above, no evidence is available for F vehicles for professional application supplemented with nano-sized phosphate salts.
Considering the relevance of this issue for clinical practice, this study aimed to evaluate the effects of F gels supplemented with two sizes of sodium trimetaphosphate (TMP) particles (conventional: TMPmicro or TMP nanoparticles: TMPnano) on the in vitro remineralization of caries-like enamel lesions.
Our null hypotheses suggested that (1) fluoride gels supplemented with TMP would promote similar remineralization to gels containing the same F concentrations without this phosphate and that (2) varnishes containing TMPmicro or TMPnano would promote similar remineralization.

Methodology Experimental Design
Bovine enamel blocks (4×4 mm, n=168) were obtained from the flattest portion of the vestibular face of crowns. The blocks were selected by surface hardness (SH) and caries-like lesions were induced.
Synthesis and characterization of nano-sized (TMP) particles Nano-sized TMP were synthesized and characterized at the Federal University of São Carlos, as previously described. 13 The particles were prepared by ball milling 70 g of pure (micrometric) sodium trimetaphosphate (Na 3 O 9 P 3 , Aldrich, purity ≥ 95% CAS 7785-84-4) using 500 g of zirconia spheres (diameter 2 mm) in 1 L of isopropanol. After 48 h, the resulting powder was separated from the alcoholic medium and ground in a mortar. Powder crystallinity was characterized by X-ray diffraction (XRD) using a Rigaku Dmax 2500 PC diffractometer in the 2θ range from 10° to 80° at a scanning rate of 28/min. The coherent crystalline domains (crystallite size) were estimated using the Scherrer equation: L = ƛK / B cos θ B , in which L is the linear dimension of a monocrystalline nano-particle, ƛ is the wavelength of the incident X-ray, B is the diffraction line width of the diffraction peak, θ B is the Bragg angle obtained from the XRD pattern, and the shape factor K is a numerical constant whose value is 0.9. Scanning electron microscopy (SEM) images were The blocks were subjected to post-demineralization surface hardness measurement (SH 1 ) by producing five indentations spaced 100-μm apart from the five initial ones (SH).

Treatment with gels and pH cycling (Re>Des)
In the first day of pH cycling, the exposed enamel area was completely covered by a thin layer of gel, applied using a cotton swab for 1 min. After treatment, the gel was removed and blocks were washed with deionized water and gently dried with absorbent paper.
The specimens were individually subjected to a pH cycling regimen at 37 °C for six consecutive days.  , 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220, and 330 μm from the enamel surface were created in the central region, using the aforementioned microhardness tester with a Knoop diamond indenter under a 5-g load for 10 s (Buehler, Lake Bluff, USA). 18 The integrated hardness area (KHN×μm) for the lesion into sound enamel was estimated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated hardness area of healthy enamel to obtain the integrated recovery of subsurface hardness (ΔKHN). 18 Determination of CaF 2 -like concentrations (formed and retained) The concentration of (CaF 2 ) on enamel was determined immediately after the application of the experimental gels (to determine the formed CaF 2 ) and after pH cycling (to evaluate the retained CaF 2 ). 19 The blocks  Fluoride, calcium, and phosphorus content in enamel (formed and retained) The other halves of the blocks were sectioned again (2×2 mm) and enamel biopsies were performed. 20,21 The blocks were fixed to a mandrel and attached to the top of a modified microscope with a micrometer (Pantec, São Paulo, Brazil) to measure their depth.
Self-adhesive polishing disks (13 mm in diameter) with 400-grit silicon carbide (Buehler) were fixed to the bottom of polystyrene crystal tubes (J-10, Injeplast, São Paulo, Brazil). A 50 μm-deep layer was removed from each enamel block 9,18 and 0.8 mL HCl 0.5 mol/L was added to the resulting enamel powder. The tubes were agitated for 60 min and 0.8 mL NaOH 0.5 mol/L was then added to them, following a modified protocol based on Danelon, et al. 13 (2015). For F analysis, samples were buffered with TISAB II and analyzed with an ion-specific electrode (Orion 9609) connected to an ion analyzer (Orion 720+). A 1:1 ratio (TISAB:sample) was used. Ca analysis was performed using the Arsenazo III colorimetric method. 22  Statistical analysis All data passed normality (Shapiro-Wilk) and homogeneity (Barlett) tests after log 10 -transformation, obeying a normal and homogeneous distribution. Data on SH 1 , %SHR, and ΔKHN were subjected to one-way ANOVA followed by the Student-Newman-Keuls post hoc test, considering our study groups (seven levels) as variation factors. For enamel CaF 2 , F, Ca, and P contents, data were subjected to two-way ANOVA, considering our study groups (seven levels) before (formed) and after (retained) pH cycling (two levels) as All gels, except for the acidic one, had a neutral pH.
We observed a dose-response relation between fluoride concentrations in the experimental gels without TMP and %SHR (Placebo < 4500F < 9000F = acid gel). The lowest %SHR values were observed for placebo, 4500F, and 5% Nano, which significantly differed from the other groups. Conversely, the 5% Micro, 2.5% Nano, 9000F, and Acid gel groups had the highest %SHR values but no significant differences between them. We found a pattern in subsurface lesion areas (ΔKHN) that resembled that we observed for %SHR, with the placebo and 5% Nano gels (p=0.953) showing significantly higher ΔKHN values than the remaining groups, without significant differences among the 5% Micro, 2.5% Nano, 9000F, and acid gel groups. Adding 5% TMPnano to 4500F led to results that resembled those for the placebo gel (p=0.953; Table 1).
Acid gel promoted the highest CaF 2 -like levels out of all groups after topical application (p<0.001).
After pH cycling, all groups had similar CaF 2 -like concentrations, except for placebo and acid gel (p<0.001). The 9,000 and TMP groups showed similar concentrations of formed F in enamel. The amount of F retained in enamel significantly increased after remineralization in all groups (p<0.05) ( Figure 2).
As for enamel Ca concentrations, we observed a marked increase after pH cycling for groups treated with nano-sized TMP, in contrast to the other groups (p<0.05). Regarding formed and retained P in enamel, groups treated with TMP showed similar values to 9000F and acid gel. P levels increased after pH cycling, despite being significant only for the 4500F, 5% Micro, 2.5% Nano, and 5% Nano treatments (p<0.05). Figure   2 shows other comparisons.

Discussion
Proposals to add inorganic phosphate salts to topical F products aim to improve the efficacy of the prevention and treatment of early caries lesions (i.e., white spot lesions) and reduce F levels in formulations to minimize possible acute side-effects. In vitro and in situ studies showed that micrometric TMP Distinct superscript lowercase letters indicate statistical significance in each column (Student-Newman-Keuls' test, n=12, p<0.05). Data were log 10 -transformed for statistical analyses. associated with fluoridated gels significantly enhanced the remineralization of artificial caries lesions 24 and prevented enamel demineralization. 25,26 This study showed that adding TMP to a low-F gel (4500F) resulted in a higher remineralizing capacity then its counterpart without TMP, achieving levels similar to those of conventional neutral (9,000 ppm F) and acid (12,300 ppm F) gels. Nonetheless, we achieved no additional benefit by using nano-sized TMP, when we compared it with micrometric particles, thus leading us to accept our second null hypothesis.
The current method and the product tested were chosen based on promising results observed for dentifrices associated with nano-sized TMP and HMP particles. Danelon, et al. 27 (2017) found that in vitro treatment with a 1100 ppm F toothpaste supplemented with 3% TMPnano reduced mineral loss in ~44%, compared to its micrometric counterpart. In an in vitro study, Dalpasquale,et al. 12 (2017) showed that adding 0.5% HMPnano to a 1100F toothpaste significantly enhanced its effects against enamel demineralization compared to its counterpart without HMPnano. Furthermore, adding 0.05% nano-sized TMP to a low-F toothpaste (250 µg F/g) promoted significantly lower ΔKHN among all groups, including a 1100 ppm F toothpaste. 28 In this study, while 2.5% TMPnano, 5% TMPmicro, 9,000F, and acid gel promoted similar %SHR and ΔKHN, the addition of 5% TMPnano to 4500F resulted in %SHR and ΔKHN values similar to those achieved for the placebo formulation. These findings seem to confirm that TMP:F molar ratios have a strong influence on the resulting effect against enamel demineralization. 27,29-31 The TMPnano concentrations tested in this study (2.5% and 5%) were based on TMP:F ratio from previous studies which reported that adding micrometric particles of TMP at 5% to a 4,500 ppm F gel promoted a significantly higher effect against enamel demineralization and on the remineralization of caries-like lesions than their counterparts without TMP. 5,23,25 Furthermore, the studies with nano-sized TMP/HMP in dentifrices were decisive for our choice of 2.5% TMPnano since we expected that a lower concentration of nano-sized TMP (compared with micrometric particles) could result in greater efficacy, as observed in previous studies. 12,28 Despite most clinical studies adopting professional topical gel application times ranging from 2 to 10 min, 4 this study used a time of 1 min based on Delbem, et al. 18 (2010) and Villena, Tenuta, and Cury 32 (2009).
In the latter study, results showed that applying acidulated phosphate fluoride for either one or four minutes equally increased enamel F concentrations and reduced enamel demineralization. Another recommendation associated with gel application is to refrain from eating and drinking for at least 30 min after application. 4 In this study, however, the blocks were washed with deionized water immediately after application, based on previous findings showing that water rinsing after professionally applied topical fluoride gel or foam will have no adverse effect on the therapeutic benefit of a treatment. 18,33 Moreover, we adopted this step to avoid contaminating cycling solutions with gel treatments and to ensure that no trace of gel remained on the block surface, which could alter study results. (2014), a study with fluoridated varnishes. Also, we observed a marked increase in Ca concentrations in enamel after pH cycling for the nano-sized TMP groups, in contrast with groups without TMP supplementation.
Regarding P results, groups treated with TMPmicro/ nano showed values similar to the 9000F and acid gel groups. Moreover, adding TMP to 4,500F gel had an effect on enamel mineral composition.
Despite the two-to three-fold difference in F content between TMPmicro/nano groups and 9,000F/acid gel, respectively, we observed similar F (formed), Ca, and P values in the enamel treated with these gels, which confirm the results obtained for previous studies with dentifrices. 18,28,35,36 In this study, while TMPnano at 5% increased the amount of F (formed), Ca, and P in enamel, it promoted the lowest %SHR between the fluoridated gels and similar ΔKHN, when compared with the placebo gel. We could suggest that a large percentage of TMPnano would supersaturate the enamel surface and, since the adsorption of polyphosphates to enamel occurs rapidly after exposure and is followed by the adsorption of F, 37 an appropriate molar proportion between TMP and F must be sought to optimize the anticaries action. The suggested molar proportion of TMP/NaF lies from 1.24:1 to 3.72:1. 30 For high-fluoride gels, the 4,500 5% TMPmicro (TMP: NaF/0.7) achieved the greatest effect on enamel demineralization. Conclusion Under the conditions of this in vitro study, we can conclude that adding TMP to low-fluoride gels leads to enhanced remineralization of artificial caries lesions in vitro and that we obtained no additional benefit by using nano-sized particles instead of micrometric ones.