S-PRG-based toothpastes compared to NaF toothpaste and NaF varnish on dentin permeability in vitro

Abstract Objectives To analyze the effect of 5 toothpastes containing different percentages of S-PRG fillers compared to NaF toothpaste and NaF varnish on the dentin hydraulic conductance (Lp). Methodology Dentin disks (1.0±0.2 mm thickness) were cut from third molars, and their Lp values were evaluated using Flodec. The specimens were allocated into 7 groups (n=8). The minimum (smear layer) and the maximum (after acid etching) Lp values were recorded. Lp was also assessed after treatment with either a 0wt.%, 1wt.%, 5wt.%, 20wt.%, or 30wt.% S-PRG toothpaste, a NaF toothpaste, or a NaF varnish. Toothpastes were applied by brushing for 15 s, allowing it to settle for 1 min, and rinsing with deionized water. The NaF varnish was applied for 4 min and was removed with a probe. Specimens were exposed to citric acid (6%, pH 2.1, 1 min) and their final Lp was recorded. The pH of all products was recorded (n=3) and specimens from each group were analyzed by Laser Scanning Confocal Microscopy (LSCM). Data were subjected to 2-way repeated measures ANOVA and post-hoc Bonferroni (a=0.05). Results The highest Lp reduction was noticed for the 5wt.% S-PRG toothpaste, NaF toothpaste, and NaF varnish. However, the toothpastes containing 5wt.%, 20wt.%, and 30wt.% of S-PRG were similar to all toothpastes but differed from the NaF varnish. After erosion, all groups retrieved their maximum Lp values, except for the NaF varnish. The LSCM evidenced deposits on the surface of specimens treated with 5%, 20%, and 30% S-PRG-based toothpastes and NaF toothpaste. Even more deposits were observed for the NaF varnish. After the erosive challenge, the deposits were diminished in all groups. Conclusion Toothpastes containing 5wt.%, 20wt.%, and 30wt.% of S-PRG fillers behaved similarly to a conventional NaF toothpaste, even after an erosive challenge. The NaF varnish promoted better reduction of the Lp, but its effect was also diminished after erosion.


Introduction
The Surface Pre-Reacted Glass (S-PRG) fillers are manufactured using milled pre-reactive fluoro-boroaluminosilicate glass, which is treated with an alcoholic polysiloxane solution to create a porous external layer. This glass is then sprayed with a polyacrylic acid aqueous solution, creating an intermediate phase between the external treated surface and the inner glass core. 1 Due to its manufacturing technology, these trilaminar particles are able to interact with the external environment by releasing six different ions (Sr 2+ , F -, SiO 3
The dentin is a mineralized tissue morphologically constituted by tubules, which vary in number and diameter according to the proximity of the pulp. 13 These tubules are usually filled with fluids and extensions of odontoblastic and nerve cells found in the dental pulp. When dentin is exposed to the oral environment, chemical, thermal, tactile, and osmotic stimuli excite these nerves and generate a painful response known as dentin hypersensitivity. [14][15][16][17][18] So far, there is no consensus on the real mechanism for dentinal sensitivity. The most accepted theory is the hydrodynamic theory formulated by Brännström and Åström 14 (1964). This theory assumes that these chemical, thermal, tactile, and osmotic stimuli can cause an abrupt movement of the fluid within the dentinal tubules and stimulate the intra-pulpal nerve cells, leading to dentin hypersensitivity. Therefore, most desensitizing materials work by suppressing the effect, reducing the diameter of the dentinal tubules and, consequently, the movement of dentinal fluids. 17,19-23 Among these materials, fluoridated salts are often used since fluoride allows the formation of calcium fluoride (CaF 2 ) deposits. 16,17,22,24,25 Most of these products, however, are limited to the effects of time, especially due to erosive challenges within the oral cavity. Since the prevalence of dental erosion is increasing, approaches that can detain the consequences of tooth wear are desired. 17,22,25 Therefore, strategies that could provide long-lasting prevention and/or therapeutic efficacy to help control the impact of dentin hypersensitivity on the quality of life of individuals are needed.
Up to now, studies using the S-PRG fillers have focused mainly on evaluating their remineralizing effect on enamel samples in an artificial caries environment. The results mainly address the role of ions such as Sr 2+ , which can reinforce the mineral structure of the enamel by forming the strontiumapatite; 6,9 F -, proving to be effective at reducing demineralization and promoting remineralization; [7][8][9][10][11] and Al 3+ , which can promote tubule occlusion and reduce dentin permeability in vitro. 26 Considering these properties, it seems reasonable to suggest that this technology could potentially contribute to the relief of dentin hypersensitivity, given the multiple ions composing this material. Since dentin permeability is a validated way to assess this potential, it can serve as a method to infer this clinical ability. 24 Thus, this in vitro study aims to investigate the potential of experimental toothpastes containing S-PRG fillers in five different concentrations on dentin permeability and to compare their efficacy with a conventional NaF-based toothpaste and a NaF varnish.
The tested null hypothesis is that there is no difference in dentin permeability after treatment with S-PRGbased toothpastes, NaF toothpaste, or a NaF varnish.

Methodology
Experimental design of NaF was also fabricated using the abovementioned composition but with no S-PRG fillers (positive control).
A pH electrode (2A09E, Analyser, São Paulo, Brazil) calibrated with standard pH levels of 7.0 and 4.0 was used to measure the pH levels of the toothpastes as slurries (1:3 with deionized water) (n=3). 27 The pH of the NaF varnish was measured using a universal pH indicator strip (Merck, Darmstadt, Germany).

Sample size estimation
Sample size was estimated using the G*Power 3.1 software (Aichach, Germany). Based on the results of the pilot study, the effect size was estimated to be 0.752, with an a=0.05 and power (1-b)=0.8. The correlation among repeated measures was set at 0.5. Based on this information, the total sample size estimated was 28 (n=4 per group).
However, based on a previously published study, 24 a sample size of n=8 per group was determined to achieve power (1-b)>0.9.

Sample preparation
This study was approved by the local Ethics Committee. In total, 56 sound third molars were used, which were stored in 0.1% thymol solution (pH 7.0) during the experiment.
Crowns were sectioned using a diamond wafer blade (XL-12205, Extec, Enfield, USA) in an automatic Isomet machine (Buehler, Lake Bluff, USA) at 300 rpm and under cooling with deionized water. The teeth were cut transversely right below the amelodentin junction and above the pulp horns, resulting in 1.0±0.2 mmthick discs. One single disk was obtained from each tooth. Thickness was controlled using a digital caliper (Digimatic Caliper Absolute, Mitutoyo, Kawasaki, Japan). When prepared, the specimens were stored in deionized water at 10 °C during the experiment.

Treatment protocols
After cutting, the specimens were immersed in 37% phosphoric acid (pH 1.0) for 15 s, and rinsed with deionized water to remove the smear layer formed during cutting, allowing the measurement of the specimen's maximum permeability values (LpMax). 24 Dentin permeability was evaluated as hydraulic conductance (Lp) using a measurement device (Flodec, DeMarco Engineering, Geneva, Switzerland). 28 The LpMax values were used to randomize and allocate the specimens into seven groups (n=8) allowing the baseline permeability to be similar among all groups.
Treatment was conducted using one of the seven materials after the specimens were gently dried with an absorbent paper. The toothpaste was applied with a disposable microbrush, by actively brushing for 15s, allowing it to settle for 1 min, and then rinsing with deionized water. The varnish was applied on the specimens by allowing it to settle for 4 min and then gently removed with an explorer probe.

Dentin permeability measurement
The Flodec device contains a split chamber with two o-rings to allow water passage only to a standardized circular area of each specimen (16.9 mm 2 ). The split chamber is attached to a capillary tube containing an air bubble (internal diameter: 0.83 mm; external 4 mm; detectable volume 2.71 nL) and a water column with a reservoir. The water reservoir was filled with deionized water at room temperature and placed 140 cm above the Flodec to standardize the pressure at 2 psi. Only

Dentin condition analyses
Four consecutive readings of Lp for each sample were conducted during the study, being one for each dentin condition, 24 as described: Minimum permeability (LpMin): with the smear layer, which was created by standardized polishing of the occlusal surface of each dentin disc with #600 silicon carbide (SiC) sandpapers (Buehler, Lake Bluff, paper and treated with one of the tested materials, and then rinsed with deionized water. Permeability after erosive challenge (LpEro): after treatment, the specimens were subjected to an erosive challenge (6% citric acid, pH 2.1, 1 min), then rinsed with deionized water.
As conducted in a previously published study, 30 the LpMax value was considered as 100% permeability; the LpMin, LpTreat, and LpEro were estimated based the LpMax value (e.g. if LpMax was 20 µLmin -1 , it was considered to be 100%, so the value of 1 was given. If, after treatment with one of the designated products, the LpTreat value was 5 µLmin -1 , the LpTreat would be estimated at 25% of LpMax, so the value of 0.25 would be attributed to LpTreat). This method allowed for the estimation of the specimen's Lp in comparison to itself before the application of the products, allowing each specimen to be used as its own control group.
Laser scanning confocal microscopy Specimens of each group were also evaluated by a laser scanning confocal microscope (LSCM) (Leica TCS SPE, Leica Microsystems, Mannheim, Germany).
For this analysis, 15 new dentin disks were prepared and a central groove was made using a spherical carbide bur (KG Sorensen, São Paulo, Brazil) to divide the surface into two halves. 30 The specimens were prepared exactly as performed in the "dentin conditions analyses" section, but one half represented the dentin after treatment with one of the designated product (pTreat), while the other was immersed in citric acid to represent the dentin after the 1-min erosive challenge (pEro) (n=2/group). One specimen represented the dentin with (pMin) and without smear layer (pMax) and was used as a control for all groups.

Results
The pH of all toothpastes ranged from 7.21 to 7.63, and the pH of the NaF varnish was 4.0. Table   1 shows the means and standard deviation of each tested toothpaste.  The specimen in Figure 2a is covered by smear layer, and this layer also seems to occlude the aperture of the dentinal tubules (Figure 2c). After a 15-second acid etching, the smear layer was removed from the dentin surface (Figure 2b) and the aperture of the tubules is seen unclogged (Figure 2d).

Statistical analysis evidenced that both Material
When the specimens were treated with the toothpaste free of S-PRG fillers, few solid deposits can be seen on the dentin surface ( Figure 3a) and inside the dentinal tubules (Figure 3c). Yet, after the erosive challenge, these deposits were mostly removed from the surface (Figure 3b) and from the dentinal tubules ( Figure 3d). The same occurred for the specimens treated with toothpastes containing 1 wt.% ( Figure 4) and 5 wt.% of S-PRG fillers, with a few more deposits in the latter ( Figure 5).
For the specimens treated with toothpastes containing 20 wt.% ( Figure 6) and 30 wt.% ( Figure   7) of S-PRG fillers, the solid deposits on the dentin surface was higher than the previous groups. After the erosive challenge, however, these deposits were almost completely removed from the dentin surface (Figures 6b and 7b) and from the aperture of the dentinal tubules (Figures 6d and 7d).
Moreover, the amount of deposits on the surface was slightly greater for the specimens treated with the 1,450 ppm NaF toothpaste (Figure 8a). Yet, the amount of deposits decreased after an erosive  (2a) and at the aperture of the dentinal tubules (2c). After etching with 37% phosphoric acid, the smear layer was removed from the surface (2b) and from the dentinal tubules (2d) Figure 3-After the application of a toothpaste without S-PRG fillers, the dentin surface (3a) and the aperture of dentinal tubules (3c) presented few solid deposits, which were almost completely removed from the surface (3b) and from the tubules (3d) after the erosive challenge J Appl Oral Sci. 2022;30:e20220082 7/14 Figure 4-The toothpaste containing 1 wt.% of S-PRG fillers could not promote the formation of solid deposits on the surface (4a) and at the aperture (4c) of dentinal tubules. After the acid challenge, the surface (4b) and the aperture of the tubules (4d) were mostly free of deposits Figure 5-The toothpaste with 5 wt.% of S-PRG was able to promote the formation of some solid deposits on the dentin surface (5a) and at the aperture of dentinal tubules (5c). After the erosive challenge, however, these deposits were almost completely removed from the surface (5b) and from the dentinal tubules (5d) 2022;30:e20220082 8/14 Figure 6-The 20 wt.% S-PRG-based toothpaste promoted some solid deposits on the dentin surface (6a) and at the dentinal tubules (6c). Yet, the number of particles on the surface (6b) and at the aperture of the tubules (6d) decreased significantly after the erosive challenge  After being treated with the 22,600 ppm NaF varnish (Figure 9), the specimens presented a much higher amount of solid deposits on the dentin surface ( Figure 9a) and at the aperture of the dentinal tubules ( Figure 9c). The amount of deposits decreased after the erosive challenge both on the surface (Figure 9b) and at the apertures (Figure 9d), but it was still higher than the previous groups.

Discussion
The results of this in vitro study indicate that the NaF varnish was able to reduce dentin permeability and differed from all S-PRG-based toothpastes. The NaF toothpaste and the one containing 5 wt.% were able to reduce the Lp after treatment, but their LpTreat values did not differ from the 20 wt.% and 30 wt.%

of S-PRG fillers. Other concentrations of S-PRG fillers
were not able to influence on the dentin permeability in vitro. Therefore, the null hypothesis was rejected.
Notably, our study focused on the S-PRG concentration itself, thus, the same composition was used for all tested toothpaste to avoid biases on the interpretation of the results. Therefore, this study contributes to the scientific literature by evidencing that the S-PRG technology available in different dental materials may contribute to controlling the symptoms of dentin hypersensitivity. Other published studies have been conducted addressing dentin permeability after the application of several fluoridated and nonfluoridated agents; 15,[17][18][19]23,24,28,[30][31][32][33]  In the study performed by et al. 18 (2019), no reduction of dentin permeability was seen after the application of any of the tested desensitizing toothpastes. These toothpastes may induce the deposition of some particles on the tooth surface and reduce the lumen of the tubules, which consequently reduce the flow of fluids inside the tubules to the power of 4, according to the Poiseuille's law. 18 these deposits, however, may not be strongly bonded to the tooth structure and can be removed from the surface by rinsing or by the pressure of the water imposed by the Flodec device. 18 Other studies have also evidenced that tooth brushing promotes the formation of smear layer on the tooth structure, which is also able to interfere on the obliteration of dentinal tubules depending on the abrasiveness of toothpastes and the hardness of toothbrush bristles. 34 Since our study aimed to evidence the effect of the toothpastes on the dentin samples and not the toothbrush effect, toothpastes were applied using a disposable microbrush. Moreover, future investigations addressing the abrasive potential of S-PRG-based toothpastes should be conducted.
One dental alteration that may lead to dentin hypersensitivity is erosive tooth wear, in which layers of sound structure are dissolved. Even after treatment, patients are still commonly subjected to periodical erosive episodes, which result in the dissolution of the clogging particles and consequent opening of the tubules, directly increasing dentin hypersensitivity. 24,30 Additionally, acidic challenges are able to dissolve some fluoride deposits on top of tooth structure, shortening the desensitizing effect of fluoridated products, and some toothpastes are also known to increase the wear of eroded dentin. 35 Therefore, based on the fact that LpEro was statistically similar to LpMax in all groups in which a toothpaste was used, it can be suggested that the tested toothpastes were not able to prevent further unclogging of the dentinal tubules, hence, their desensitizing effect might not last an erosive challenge.
The only material that presented LpEro lower than LpMax was the NaF varnish. This might have occurred because the product was applied for a longer period (4 min), has a lower pH, and has a fluoride concentration more than fifteen times higher than that of the NaF toothpaste. Similarly to a previous study, 24 the NaF varnish was applied onto the specimen for a longer period since it is an in-office product, contrary to As reported by Kaga, et al. 9 (2014), the S-PRG fillers are able to release ions upon pH decrease. 6,9 Among these ions, strontium and fluoride are able to reinforce the tooth structure by creating strontiumapatite and fluorapatite, which are more acidresistant than conventional apatite. 6,9 Nevertheless, the formation of calcium fluoride-like precipitates related to tubules occlusion is dependent on fluoride concentration, pH, and time. 37,38 Thus, given that the pH of these toothpastes are within the neutral range and that the amount of fluoride released might be low, especially in the toothpastes with less than 5 wt.% of S-PRG fillers, this could justify the lack of reduction in dentin Lp. Also, this study applied the toothpaste only once, and subsequent applications of the product could also be responsible to improve its clinical efficacy.

Other important ion present in the S-PRG
technology is Al 3+ . This ion promotes tubule occlusion and reduces dentin permeability in vitro. 26 Yet, this potential requires further testing to be confirmed.
Nonetheless, considering that the specimens were rinsed after the application of the toothpaste and that the Flodec device is based on the passage of water through the dentin sample, the S-PRG fillers could have been washed away before the acidic condition (LpEro) was achieved. This is confirmed by figures obtained by LSCM in the groups in which a S-PRGbased toothpaste was applied. To prevent the fillers from being washed from the surface, other bioactive glasses contain polymers on their surface to bind to the dentin surface, increasing their retention and efficacy. 39 In the study of Spinola, et al. 12 (2020), the S-PRG fillers were inserted in different concentrations in a varnish to prevent enamel demineralization. The results have shown that the highly concentrated (30 wt.% and 40 wt.%) S-PRG-based varnishes were able to prevent enamel demineralization similarly to a 5% NaF varnish. The difference between that study and our study may be due to the fact that the varnish contains binding agents that help retain the varnish and the S-PRG fillers on the tooth surface for longer periods, resulting in an improved performance. 12 Moreover, considering that our study evaluated their effect on dentin, its higher complexity might also influence on the performance of these products compared to the enamel.
As observed in the study of Amaechi, et al. 11 (2018), toothpastes containing S-PRG were more effective in preventing tooth demineralization and in improving remineralization in the enamel than a conventional toothpaste containing 1,100 ppm of NaF. Moreover, in their study, the 5 wt.% concentration seemed to be the optimal concentration for toothpastes, given that it was not different from the most concentrated ones (20 wt.% and 30 wt.%). Despite having different objectives and evaluating different outcomes, their results differ from the ones in our study, in which the NaF toothpaste behaved similarly to the 5 wt.%, 20 wt.%, and 30 wt.% S-PRG-based toothpastes.
Furthermore, dentin samples are expected to have different levels of permeability depending on the depth of dentin, on the patient's age, presence of previous tooth erosion or dental caries, as well as other genetic conditions, such as molar incisor hypomineralization, which is also related to the presence of dentin hypersensitivity. 31 Therefore, this study exclusively standardized the dentin depth of sound caries-free molars before the conductance of the study, with no signs of the abovementioned alterations. Nevertheless, our study was conducted on medium-depth dentin, which might exacerbate the clinical scenario regarding the mechanism of dentin hypersensitivity, given that patients often feel pain even under subclinical expositions of dentin to the oral environment. Thus, this in vitro model may not faithfully represent what occurs clinically, but it is relevant to discuss the mechanism of action of these products. This can be corroborated by the fact that the NaF varnish has already shown good results in suppressing the symptoms of dentin hypersensitivity clinically, similarly to other at-home and in-office products. 17 Since this methodology is used to measure and compare permeability against obliteration, it seems relevant to point out this limitation before translating these results to clinical circumstances.
Further studies should include some of the factors disregarded in this study, for instance human saliva, which plays a role in neutralizing acidic conditions and can also have some effect on tubule occlusion due to the formation of the acquired pellicle 18 and also by inducing the precipitation of minerals within the dentinal tubules. 40 Human saliva was not used in our study so as to analyze only the effect of the S-PRG fillers in the presence of deionized water. However, since human saliva works as a source of calcium and phosphate, the presence of these ions could have led to an increased formation of CaF 2 deposits on the dentin surface, and consequent reduction of the specimens' Lp values. 28 As dentin hypersensitivity impairs the quality of life of patients, technologies as S-PRG could enhance the potential and longevity of the products and should be further tested using other experimental models, including clinical studies.

Conclusion
Within the limitation of this study, we can be conclude that a better reduction of the Lp was noticed for the NaF varnish. The toothpastes containing 5wt.%, 20wt.%, and 30wt.% of S-PRG fillers behaved similarly to a conventional NaF toothpaste. Despite also being affected by the erosive challenge, the NaF varnish was the only group that did not reach its LpMax values.