Protection against Dental Erosion and the Remineralization Capacity of Non-Fluoride Toothpaste, Fluoride Toothpaste and Fluoride Varnish

Abstract

Introduction: The oral cavity and the teeth are frequently subjected to numerous physiological pH variations, mainly due to the type of diet. These changes are the main cause of enamel demineralization and consequent breakage under mechanical forces. This causes severe sensitivity and sometimes pain, which endures unless the hard tissue is remineralized. Since enamel does not self-repair, the application of alloplastic materials that have the property of releasing remineralizing ions is strongly recommended. The aim of this in vitro study is to evaluate the effectiveness of two different toothpastes and a fluoride varnish in the prevention of demineralization, and their ability to remineralize enamel after it has undergone several acidifications. Methods: Eight human teeth with no caries or defects were prepared. The acid attack simulations were performed using a commercial cola carbonated drink. Samples were immersed in 5 mL of soft drink for two minutes at room temperature, and then were rinsed with distilled/deionized water. The immersion process lasted about 2 min, and was repeated four times for a total of eight minutes. Then, two different types of toothpaste, one with and one without fluoride, and a fluoride varnish were applied to the surfaces of the samples and rinsed off with demineralized water. A second acidification cycle was then carried out, with the subsequent reapplication of the three different products to evaluate their ability to protect against demineralization. SEM and profilometer analysis were then carried out to evaluate the results. Results: The statistical analysis showed a good remineralization capacity for all three products, especially in the fluoride-varnish-treated samples. However, regarding protection from demineralization, non-fluoride toothpaste was found to be ineffective, while fluoride toothpaste and varnish produced positive results. Conclusions: Dental professionals should advise fluoride toothpastes and varnishes over non-fluoride toothpastes. Fluoride toothpastes and varnishes represent a valid treatment for surface remineralization after a first acid attack and for protection from subsequent demineralization in an acid environment, such as that which can develop in the oral cavity.

1. Introduction

Dental erosion is considered a partial chemical dissolution of the hard tissues of the tooth surface. This phenomenon is caused mainly by repeated exposure to acids of both microbiological and non-microbiological origin. Simultaneous and/or subsequent exposure to mechanical forces might result in tooth surface loss [1]. Furthermore, the dissolution of enamel is closely associated with some precise chemical parameters, such as pH, saliva buffering capacity, and quantity, and external factors, such as acidity, viscosity, calcium, phosphate and fluoride ion concentration in drinks and food [2]. It is widely demonstrated that these parameters have a direct influence on tooth surface strength, and this is the reason why they are normally used to calculate the degree of saturation of various substances, so as to quantify the demineralizing force in hard tissues [3].

Substances with a low pH, high titratable capacity and high buffering capacity have the greatest erosive force; conversely, substances with high concentrations of the Ca²⁺ ion and phosphate cause the least dissolution of the tooth surface, and can enhance its chemical stability and integrity [4].

At the present time, dental erosion is a daily problem, accentuated by the consumption of acidic drinks, which have a high demineralizing potential. Unfortunately, a lot of chemicals, additives and sugar are hidden in most food and drinks of regular intake, such as carbonated, alcoholic, non-alcoholic and sports drinks and fruit juices. These substances have the capability to decrease the pH of the saliva, which enhances the ongoing process of dental erosion [5]. As far as the origin of the acid is concerned, extrinsic and intrinsic erosions can be distinguished. In fact, the extrinsic ones are generally caused by acids in food, and the severity of the erosion is associated with several factors, such as nutrition, saliva and mechanical and frictional stress [6]. An important factor is contributed by the digestive enzymes released by the salivary glands, and, in particular, by their response to hunger and various food nutrients. In fact, these enzymes were found to increase their activity in response to food, revealing the dependence of their production on food [7].

It is known that tooth enamel is made up mostly of inorganic material, which represents about 96%, while the remaining 4% is organic material and water [8]. The inorganic material consists of hydroxyapatite crystals, which comprise a mineral with the chemical composition Ca₁₀(PO₄)₆(OH)₂ with a hexagonal prismatic structure. Enamel, which is predominantly inorganic, is not composed of living cells with specific repair capabilities with regard to diseases such as caries, abrasions and/or fractures [9]. When this happens, erosive tooth wear might occur, especially in the cervical region, just above the gingival margin, on the incisal edge of anterior teeth or on the cusps of posterior teeth. It is in particular the mechanical forces imposed on anterior teeth that can ultimately lead to the eventual breakage of a tooth in the esthetic region. A similar situation could cause an impairment, not only from an esthetic point of view, but also in terms of the masticatory function, and speech and labial support might be highly compromised. In the case of a growing child, changes in the occlusal vertical dimension may lead to an incorrect development of bones, muscles and temporomandibular joints [10,11].

To sum up, when an acidic environment is formed inside the oral cavity and the surfaces of the enamel are consequently exposed, a process of demineralization begins, which may vary from person to person. To slow down this process, the use of specific products, such as toothpastes and fluoride varnish, allows repair by remineralization [12]. Moreover, the remineralization process can be also induced by products such as glass ionomer cements, which have the ability to release fluorine ions and composite materials that can release CaPO₄ anions. These materials have the capability to induce apatite formation through the presence of bioactive glass and they are able to absorb fluoride ions from toothpastes and fluoride varnishes and subsequently release them in the oral cavity [13,14]. Through them, the enamel can be protected and enhanced in resistance to acid attacks; therefore, these products offer protection against the erosion of the enamel when they contain certain fluoride concentrations [15]. The hydroxyapatite is dissolved in acidic environment. However, if fluoride ions are available in the oral environment, this element can form with HA-fluorohydroxyapatite. The fluoride is absorbed by the crystals on the surface, thus attracting calcium ions. Apatite without carbonates is less soluble, so in the case of demineralization it replaces the original mineral [16]. The new layer is characterized by a lower solubility due to the replacement of carbonate with fluoride, making the surface of the enamel less subject to acid erosion. Fluoridated toothpaste, gel enriched with fluoride and fluoride varnishes still represent the best options available on the market: they are easy to obtain and even easier to use or apply. Gel and toothpaste are widely recommended by pediatric dentists, and they can even be used at home on a daily basis. Apart from these materials, several others have been developed in the last few years: nowadays, conventional flowable and high-strength universal injectable composites represent a concrete solution for the pediatric population for both deciduous and permanent dentition. The importance of these restorative materials is their capability to release fluoride and other compounds that improve hard tissue remineralization [17,18]. In fact, current research aims to identify various elements that make dental tissues harder and more effective in contrasting demineralization [19].

The Aim of the Study

The purpose of this study is to investigate the effectiveness of two different types of toothpaste (one with fluoride and one without) and a fluoride varnish in preventing erosion caused by an acidic environment present in the oral cavity and to protect hard tissues from acids.

The first null hypothesis is that, as far as erosion prevention is concerned, there is no difference between the two toothpastes and the fluoride varnish.

The second null hypothesis is that, as far as protection from a second acid attack is concerned, there is no difference between different toothpastes and the fluoride varnish.

2. Materials and Methods

2.1. Sample Preparation

Eight healthy human teeth with no caries and/or defects were extracted for periodontal reasons. Ethical approval was waived by the ethic committee. Following the extraction, the soft tissue residues were removed from the teeth, which were then analyzed to find any fractures. Fracture analysis was conducted with an optical microscope (Leica Microsystems DM300, Germany) using 4X, 10X, 40X and 100X magnification. Then, the teeth were disinfected in sodium hypochlorite titrated at 5% in active chlorine for 1 h, and were stored in a 0.9% NaCl solution containing 0.1% thymol throughout the treatment so that they remained hydrated. The samples were cut with a high-speed diamond bur (KG^®) and water irrigation at the level of the enamel–dentin junction, and then sectioned, so that the flat surface was opposite to the treated one. To make the surfaces uniform, the samples were polished with silicon abrasive tips [5]. According to the adopted procedure, samples were divided into 8 groups, which are illustrated below in

Table 1. Groups divided by surface treatment.

Groups	Procedure
G1	Rubber polisher
G2	Rubber polisher + soft drink
G3	Rubber polisher + soft drink + non-fluoride toothpaste
G4	Rubber polisher + soft drink + fluoride toothpaste
G5	Rubber polisher + soft drink + fluoride varnish
G6	Rubber polisher + soft drink + non-fluoride toothpaste + soft drink
G7	Rubber polisher + soft drink + fluoride toothpaste + soft drink
G8	Rubber polisher + soft drink + fluoride varnish + soft drink

.

Table 2. The composition of the products used for the process.

Product	Composition
Non-fluoride toothpaste	Aloe vera, chamomile, echinacea, horse chestnut, mint, carboxymethylchitosan, hydrated silica and xylitol
Fluoride toothpaste	Calcium carbonate, water, sorbitol, aroma*, poloxamer 407, sodium monofluorophosphate, cocamidopropyl betaine, zinc oxide, benzyl alcohol, cellulose gum, zinc citrate, sodium bicarbonate, tetrasodium pyrophosphate, xanthan gum, sodium saccharin, sucralose, limonene, CI 77891
Fluoride varnish	Active ingredient: sodium fluoride: 1 mL = 22.6 mg of fluoride excipients: 96% ethanol, white wax (E901); shellac (E904), rosin, mastic, saccharin (E954), raspberry essence

shows the product composition.

2.2. Demineralization and Remineralization

In order to simulate the demineralization process, a carbonated soft drink (CocaCola^®, Milano, Italy) was chosen. The pH at 20 °C, buffering capacity, concentration of calcium and phosphate of the drink were measured. Measurements were performed in triplicate (5).

Samples G2, G3, G4, G5, G6, G7 and G8 were immersed in 5 mL of soft drink for two minutes at room temperature, and then they were rinsed with distilled/deionized water.

The immersion process lasted about 2 min and was repeated four times for a total of eight minutes. The three different products were applied, without brushing, to cover the entire surface of the enamel of some samples, and then they were rinsed with distilled water.

Sample G1 did not have any chemical changes; the only treatment was polishing with silicon abrasive tips (3M^®). No products were applied to sample G2; the only procedure was the initial acidification. The different products were applied in the same way: they were placed in contact with the enamel surface for three minutes at zero hour, and again after 8, 24 and 36 h. (5)

Samples “3” and “6” were treated with a non-fluoride toothpaste; samples “4” and “7” were treated with a 1450 ppm of fluoride toothpaste. Finally, samples “5” and “8” were treated with single 0.5 mL doses of professional fluoride varnish specific for dental hypersensitivity and containing 22,600 ppm of fluoride. Samples “6”, “7” and “8” were subjected to another acidification cycle for the evaluation of the effectiveness protection of the products following a second acid attack.

2.3. Scanning Electron Microscope (SEM)

The morphological characteristics of the samples were studied using an SEM (Leica Microsystems srl, Milan, Italy). In order to conduct a proper observation, conductive samples were required, so a metal layer was firstly placed on top of them. In detail, the samples of this study were coated with a gold layer of approximately 20 nm. The microscope used for the experimental activity was a Cambridge Stereoscan 440 (Leica Microsystems srl, Milan, Italy) equipped with a Philips PV9800 EDS microanalysis (Leica Microsystems srl, Milan, Italy), available at the Industrial Engineering Department of the University of Padua. The images were taken using the secondary electron detector [17].

2.4. Profilometer

The surface topography of the teeth was analyzed using a Sensofar Plu Neox optical profilometer (Barcelona, Spain). Scans with lateral dimensions of 1.3 × 0.6 mm² were acquired using a 20× confocal objective. After the acquisition, the topographies were subjected to the removal of the shape by subtraction of a plan. Three scans were performed in different areas for each tooth, and three surface profiles were extracted for each topography, from which the roughness was calculated according to ISO 4288 [20]. Two filters, λ and λc, equal to 2.5 μm and 0.025 mm, were applied for the roughness calculation [18].

3. Results

Five measurements were made for each sample, but in different locations. Please check

Table 3. Profilometer measurements.

	Ra1	Ra2	Ra3	Ra4	Ra5	Media	Standard Deviation
1	0.4778	0.4683	0.4508	0.4375	0.513	0.46948	0.02888022
2	1.9845	2.0159	1.9518	2.3981	2.5405	2.17816	0.27144762
3	0.9504	0.8388	0.8195	1.0379	1.0858	0.94648	0.11779426
4	0.9042	1.1652	1.0916	0.9886	0.9196	1.01384	0.11235617
5	0.9337	0.6827	0.5569	0.565	0.5016	0.64798	0.17280751
6	1.118	0.9084	0.8817	1.0521	1.3365	1.05934	0.18347338
7	0.7099	0.7319	0.8403	0.8248	1.002	0.82178	0.11557131
8	1.5126	1.7345	1.4272	0.9311	1.0254	1.32616	0.33846695

for further clarification.

Statistical analysis was carried out using R Statistical Software. Figure 1 shows the boxplot of the eight treatments considered in this study. It is possible to observe that the G2 group differed from the other groups with regard to the roughness, having the highest value. The group with the second highest roughness was G8. Among all groups, G2 and G8 exhibited greater variability than the others. Regarding G1, it can be noted that it had the lowest values, while G5 was the only group that had an outlier point. The other groups had relatively similar behaviors.

In

Table 4. p-value following the Dunnet t-test.

	G1	G2	G3	G4	G5	G6	G7
G2	0.0001
G3	0.4000	0.3632
G4	0.1227	0.9314	1.0000
G5	1.0000	0.0041	1.0000	0.9916
G6	0.0912	1.0000	1.0000	0.9138	0.8695
G7	1.0000	0.0388	1.0000	1.0000	1.0000	1.0000
G8	0.0103	1.0000	1.0000	1.0000	0.1913	1.0000	0.8613

, the results with the p-value of each test are reported. It may be underlined that the values highlighted in red represent the groups that had a statistically significant difference, considering the p-value of α = 0,10.

Therefore, it can be concluded, for example, that the G1 treatment presented a significant difference compared to the G2, G6 and G8 groups. In particular, G1 had on average a lower roughness value than groups “2”, “6” and “8”; however, there was no statistically significant difference between group “1” and groups “3”, “4”, “5” and “7”.

Moreover, G2 was statistically different when groups “5” and “7” were compared, showing on average a higher value than groups “5” and “7”.

Finally, as far as the p-values between the other groups are concerned, it can be noted that these values were greater than 0.10, so the null hypothesis is not rejected, since there was no significant difference in the roughness found in the tooth.

The SEM images show the differences between the surfaces, even at higher magnification levels (Figure 2).

The first null hypothesis is therefore confirmed, as no statistically significant difference between the two toothpastes and the fluoride varnish in terms of erosion prevention was reported.

The second null hypothesis is rejected, as in terms of protection from a second acid attack, the fluoride toothpaste and the fluoride varnish seemed to be more effective than the non-fluoride toothpaste

4. Discussion

In the present study, a scanning electron microscope (SEM) and profilometer were used to test the effectiveness of two different types of toothpaste and a fluoride varnish in preventing erosion caused by an acidic environment present in the oral cavity. The SEM image results were in accordance with the findings of our study, showing differences between the surfaces even at higher magnification levels (Figure 2)

The SEM results were not sufficient to demonstrate the actual change in the enamel surfaces caused by the acid attack; a profilometer was also used for this reason. Data obtained from the roughness analysis showed that the effect of acidification was detectable by the increase in roughness. The values were G1 0.46948 µm < G2 2.17816µm. In accordance with our findings, Mullan et al., 2017, after the acidification of twenty portions of human dental enamel with of orange juice at pH 3.2, found a statistical increase in the roughness of the samples subjected to acidification, showing that soft drinks are capable of increasing enamel surface roughness [21]. Similar results were also reported by Rochel et al., 2011, where the specimens were treated with a regular cola drink at pH 2.3: their surfaces were found to have a noticeable roughness caused by acid compounds presents in the soft drink [22]. To oppose this effect, it is recommended to brush with a fluoridated toothpaste containing between 1000 ppm and 1500 ppm of fluoride. After a sugar-rich or an acid-rich meal, the pH level decreases during the first 40 min, and the risk of the enamel surface degeneration under acid attack is higher; this happens especially if teeth are brushed soon after food or drinks consumption. This phenomenon is described in the Stephen’s Curve diagram, which highlights the trend of acid changes during the first 40 min after eating. For this reason, it is recommended to wait at least 20 min before brushing, and generally 2 to 3 min brushing are enough to allow the spread of fluoride and remineralizing compounds all around the surfaces of the teeth. The combination of the mechanical plaque removal and the remineralizing action of the toothpaste allows the hard tooth tissue to return to its initial surface state: shiny and smooth. In such a manner, the tooth surface is protected from demineralization, erosion and the appearance of carious lesions [23,24].

In our study, the SEM images and profilometer results demonstrate how the three different products (non-fluoride toothpaste, fluoride toothpaste and fluoride varnish) applied to G3, G4 and G5 had remineralization effects as they transformed the enamel surface to a statistically similar condition to G1 (Table 3). In support of the obtained results, the study of Olivan et al., 2020, evaluates the effect of four different products (a fluoride varnish, two different fluoride toothpastes and a non-fluoride toothpaste) on erosion lesions caused by a carbonated drink [25]. Through laser imaging of the speckle, the authors demonstrated that all the products proved able to protect the enamel from erosion. These conclusions are in agreement with the lack of statistical difference obtained by comparing the samples G1, G3, G4 and G5 in our study. These results show the importance of the use of a toothpaste or a varnish to restore the original tooth surface roughness, which, in the long term, can help in preventing more serious lesions, such as caries, teeth erosion and sensitivity.

Analyzing the SEM results, it is easily notable that the G2 surface showed a rougher surface, especially at 3000 magnification, and the enamel surface appeared more uneven, with visible furrows. In contrast with that, images of G3 and G4 at the same magnification showed the typical aprismatic appearance of an intact surface that is remineralizing. The SEM analysis was confirmed by statistical analysis, where both toothpastes, the fluoride and the non-fluoride, had the ability to restore the surfaces of G3 and G4 to a level of roughness statistically similar to the non-acidified G1. Instead, after the second acid attack in the G6 sample, there was a statistical similarity between its roughness and that of the G2 sample, and a statistical difference between its roughness and that of G1 sample, which demonstrated the inability of the non-fluoride toothpaste to protect the enamel from a second acid attack. SEM analysis corroborated the numerical results, as in the G6 images, a rougher surface could be appreciated, as well as the presence of furrows, comparable with the ones found in G2 images. These findings are in accordance with recent literature, which embraces the hypothesis that a dose-dependent effect exists between toothpaste fluoride concentration and the percentage of demineralization due to an acid oral environment [22,26]. Moreover, the G7 sample, treated with fluoride toothpaste and subjected to the second acidification cycle, shows a statistical difference compared to the G2 sample and a statistical similarity with the G1 sample. This result, when compared with the lack of statistical difference between the G2 and G6 samples, defines a better efficacy by the fluoride toothpaste in preventing enamel erosion when teeth are subjected to a second acid attack.

Looking at the literature, other studies reported results comparable to our findings: all the surfaces previously treated with a fluoridated toothpaste proved to be more resistant to enamel stress, such as tooth wear and erosion. In addition to these findings, Rochel et al., 2011, found that the synergy between xylitol and fluoride significantly reduced enamel erosion. In this study, in fact, 10% xylitol- and fluoride-enriched toothpastes proved to have a wider remineralizing action on the enamel surface compared to placebo or common toothpastes filled with xylitol or fluoride [22].

Our results are also analogous to and confirmed by the study of Gavic et al., 2018, in which 112 teeth without caries were demineralized and then treated with toothpastes containing different amounts of fluoride. The results obtained showed a tendency for micro-hardness (used in this study to measure in vitro remineralization) to increase after treatment with toothpaste containing a higher fluoride amount. Instead, the greatest decrease in micro-hardness was found in the enamel surfaces treated with non-fluoride toothpaste [27]. These results agree with the lack of statistical difference between the surface roughness of the G2 and G6 samples and with the presence of a statistical difference between the G2 and G7 samples found in our study.

The Dunnet test showed a significant statistical difference between the G1 and G8 samples. Although it initially seemed that the fluoride varnish did not provide sufficient protection of the enamel against acid attack, the G5 and G8 samples were statistically similar, which means that after the second acid attack, the roughness remained unchanged. This indicates that the G5 and G8 samples treated with fluoride varnish did not undergo variations in roughness. Therefore, this result highlights the ineffectiveness of the second acid attack. Following the instructions regarding the method of application of the fluoride varnish, an excess was created on the surfaces of the samples (as can be seen from the images taken by the SEM scanning microscope) (Figure 2). These data are interpreted as measurements of the roughness of the fluoride varnish and not of the enamel surfaces of the samples. In support of the results we obtained, Murakami et al., 2009, evaluated the effect of a cola-based drink on the surface of human enamel treated with fluoride varnish. The authors concluded that the fluoride varnish was able to inhibit erosive enamel loss [28].

Similarly, Tumbulaci et al., 2020, found that fluoride toothpaste, fluoride varnish and fluoride-enriched fissure sealant promoted hard tissue remineralization in teeth. These substances are also capable of perpetuating their action, even under an acidic environment. By precipitating calcium and phosphate into demineralized pores from saliva or external sources, remineralization therapies attempt to regain lost mineral content. In this study, fluoride varnish showed a statistically significant difference compared to placebo- and phosphate-treated surfaces. Instead, no statistically significant differences were reported between fluoride application, fluoride varnish and fluoride-enriched fissure sealant [29].

Likewise, Alexandria et al., 2017, carried on an investigation in bovine enamel specimens previously treated with soft drinks and pediatric liquid medicine. After examining 3D and SEM images, it was reported that the application of products containing CPP-ACP and NaF varnish seemed to be a promising treatment for reducing enamel loss from erosion produced by soft drinks. In this study, both varnishes also showed a great capacity to reduce tooth structure loss and surface roughness after erosion by soft drinks. In this specific case, soft drinks were combined with pediatric liquid medicine: in fact, these medications, in order to be more appealing for children, generally contain large amounts of hidden sugar [30].

Beyond everything, it must be taken into account that the density, the amount of fluoride and the method of application of fluoride varnish are different compared to toothpastes. This means that, after application, varnishes remain more consistently on the tooth surface, thus allowing a higher level of protection as well as absorption of fluoride. Therefore, depending on the level of demineralization of the tooth, it could be more convenient to combine the application of fluoride varnish every 3 to 6 months with continuous toothpaste use at home. Eventually, the prescription of gel rich in fluoride might be a good option for those children with severe early childhood caries [31].

In our study, after comparing the surface roughness of the samples treated with only the first acidification cycle and remineralized with the different toothpastes (i.e., samples G3, G4, G5), it was possible to note a statistical difference between sample G5 and G2, but not between G3 and G2 or between G4 and G2, meaning that the G5 sample seemed to be the most similar to the G1 sample. This indicates that, despite the acidification cycle, the fluoride varnish was able to repair the roughness of the enamel surface of the G5 sample to a level statistically similar to G1. Kim et al., 2018, conducted a similar study on the effectiveness of direct and indirect remineralization by fluoride varnish on artificial carious lesions. They evaluated the acid resistance of lesions remineralized by fluoride varnish and artificial saliva [32]. The results of this study corroborate our findings, confirming the remineralization effect of the fluoride varnish.

5. Conclusions

All three applied products showed a good remineralization effect on the enamel surface after the first acid attack. However, regarding protection from a second acid attack, no-fluoride toothpaste proved to be ineffective, while fluoride toothpaste produced positive results. The fluoride varnish seemed to have the ability to both remineralize and protect against acid attacks.