Erosion protection of low-concentration fluoride on human tooth enamel: results from surface morphology and nanomechanical and anti-wear properties

: Dental erosion results in excessive tooth wear. The contribution of low-concentration fluoride used daily in the prevention and treatment of erosion has not been fully understood. In this study, the effects of fluoride (225 ppm F − ) on the surface morphology and nano-mechanical and anti-wear properties of human tooth enamel were investigated to explore whether low-concentration NaF solution could help protect tooth enamel from erosion. In total, 40 enamel samples were divided into 5 groups, viz. group O: original surface with no treatment, group F: fluoride treatment (NaF, 225 ppm F − , pH 6.3), group E: erosion treatment (0.001 M citric acid, pH 3.2, 3 min), group EF: erosion treatment and then fluorination and group FE: fluoride treatment and then erosion. The mechanical and anti-wear properties of enamel samples were examined using a nano-indentation/scratch technique. Both surface morphology and scratch morphology of enamel samples were observed with scanning electron microscopy. The results showed that, from the perspectives of surface morphology and anti-wear properties, fluorination with low-concentration fluoride (225 ppm F − ) before erosion has a certain potential for protection against dental erosion. Fluoride treatment after erosion has no obvious impact on the remineralisation of eroded enamel.


Introduction
For decades, tooth wear has been regarded as a triad of attrition, abrasion and erosive tooth wear [1]. Recently, however, the contribution of erosive tooth wear has increased considerably, especially with the worldwide increase in soft-drink consumption [2,3]. Therefore, the studies focus for tooth wear has steadily shifted towards erosive tooth wear since the mid-1990s [4]. Among these studies, preventing dental erosion might be the initial objective and ultimate goal of both researchers and dentists. Various approaches have been proposed to prevent dental erosion; they vary from obeying lifestyle advice to oral hygiene or clinical therapy to soft-drink modifications [4,5].
Fluoride successfully inhibits dental caries. Therefore, the effect of fluoride formulations on dental erosion has been investigated widely in previous studies [6][7][8][9][10]. The fluoride concentration used in these studies varied. There are more evidencees regarding the effect of intensive fluoridation against erosion, however, a low fluoride concentration did not show a clear resistance to acid attacks [11,12]. In fact, high-concentration fluoride might only be usable in clinical operations, rather than as a choice for daily use. Moreover, in numerous in vitro studies, a cycling model was often used, in which fluoride treatments and erosion attacks were alternated repeatedly before the total erosion amount was finally quantified [12,13]. The effectiveness of fluoride in the in vitro cycling model might be a cumulative effect. In fact, due to oral activities, e.g. swallowing, speaking or eating, the amount of functional fluoride may be far below that accumulated during treatment times. Hence, it is important to make clear whether the low-concentration fluoride can protect our teeth from dental erosion.
Dental erosion results in a direct loss of enamel and a softening of the tooth surface, with a substantial decrease in mechanical properties and wear resistance. Eroded enamel is susceptible to wear even under normal conditions and has an increase in attrition and abrasion [14,15]. Usually, erosive tooth wear is not caused by attrition and/or abrasion, but by dental erosion [16]. It is, therefore, necessary to recognise that, in studies on dental erosion, not only is the loss of enamel important, but the reduction of the enamel's mechanical properties and wear resistance is also relevant.
In this study, the effects of short-term (3 min) exposure to low-concentration fluoride (225 ppm F − ) on the morphology, mechanical, and anti-wear properties of human tooth enamel were investigated using scanning electron microscopy (SEM) and nanoindentation and nanoscratch tests. We aimed to explore whether low-concentration fluoride can protect tooth enamel from erosion and clarify if the time of fluoride application has an impact on the fluoride effect. The study results will provide a useful theoretical basis and practical support for using fluoride daily to reduce dental erosion.

Sample preparation
Enamel samples were prepared from human non-carious third permanent molars, which were stored in distilled water at 4°C before use. The roots of 40 teeth were cut off using a diamond saw. The crowns were then embedded in self-setting plastic to obtain enamel samples with exposed occlusal surfaces. The specimens were first ground using 800, 1200 and 1500-grit abrasive paper, in turn, and then polished with 2.5, 1 and 0.5 μm diamond paste, in turn. Only 0.2-0.3 mm of each specimen was ground and polished to obtain a surface similar to the original occlusal surface of the enamel in the oral cavity. The cutting, grinding and polishing of enamel specimens were conducted under

Research Article
Biosurf. Biotribol., 2020, Vol. 6, Iss. 3, pp. 87-91 a water-cooling condition to avoid local overheat which can result in dehydration and may denature the tooth. After the polishing, the specimens were stored in distilled water at 4°C. The teeth were partly dehydrated during the preparation; however, efforts were made to both shorten the drying time and keep the preparation time approximately the same for all specimens. All specimens were cleaned with cotton swabs dipped in alcohol and then rinsed immediately with an abundant spray of distilled water before testing.

Erosion and fluoride treatments
Erosion treatment in vitro was conducted by immersing the enamel samples in 50 ml of 0.001 M citric acid (pH 3.2) for 3 min. The fluoride treatment in vitro was done by soaking the enamel samples in NaF aqueous solution (225 ppm F − , pH 6.3) for 3 min. In addition, both erosion and fluoride were finished at 25°C with gentle stirring [15]. The F − ion concentration (225 ppm) is widely found in mouth rinses for daily use. The specimens were then randomly divided into the following five groups (N = 8). Group O was a control, with no treatment. Group F received fluoride treatment. Group E received erosion treatment. Group EF was first eroded and then fluoridated. Group FE was first fluoridated and then treated with citric acid.

Nanoindentation test
The nanoindentation measurements were carried out with a TI 900 TriboIndenter® (Hysitron Inc., USA) using a Berkovich diamond indenter with a calibrated tip-area function. A single-load function in load-control mode with a maximum load of 500 μN was chosen for the measurement. A 3 × 3 matrix of array indents, separated from each other by 10 μm to avoid interference with adjacent indents, was performed on each sample. For each sample surface, the hardness (H ) and elastic modulus (E) values were determined using the average of nine indentations.

Scratch test and wear measurement
Unidirectional scratch tests were conducted using a nanoscratch tester (CSM Corp., Switzerland) equipped with a 10-μm radius conical diamond tip. Each sample was scratched twice with an interval of >50 μm to avoid any interaction. All samples were scratched for a distance of 300 μm under a constant applied load (10 mN). The scratch-line profile was obtained by mapping the cross-section of the scratch topography using scanning probe microscopy in the TI 900 TriboIndenter® (Hysitron Inc., USA). For each sample, 18 profiles (9 for each scratch) were processed. The volume of the scratch groove was calculated by the OriginPro 7.5 scientific graphing and analysis software. The differences observed in the measured data of each group were evaluated using the Student's paired t-test, with a significance level of 0.05. Morphology examinations were conducted on each enamel surface and its scratch using SEM (QUANTA200, FEI Corp., England). Fig. 1 shows typical surface morphologies of enamel specimens in each group. Obviously, the enamel surfaces in groups O and F had similar flat and compact morphologies, suggesting that fluoride treatment induced no obvious morphological changes in the original enamel surface. However, for enamel surfaces subjected to erosion challenge (groups E, EF and FE), the hydroxyapatite (HPA) particles and voids between them could be seen clearly at high magnification. The enamel treated with fluoride prior to erosion (group FE) had a relatively intact surface with fewer voids; however, the surface fluoridated after erosion (group EF) possessed almost the same morphology as group E. This implies that the fluoride's effect on the eroded enamel morphology is closely related to the timing of its application.    There was no obvious difference in the scratch images of groups O and F, while the scratches of groups E, EF and FE were significantly worse than those of groups O and F. In addition, it seemed that the scratch of group FE was narrower and shallower than those of groups E and EF. This was confirmed in Fig. 4, which illustrates the wear volume and scratch profile of each group. Obviously, the wear losses of scratches in groups O and F were very slight, with no significant difference between them. The wear volumes of groups E, EF and FE were far larger than those of groups O and F. It is worth noting that the wear volume of group FE (44.68 ± 7.59 μm 3 ) was 21.8% less than that of group E (57.12 ± 6.33 μm 3 ), while there was no statistically significant difference ( p = 0.47) in the wear volume between the groups EF (54.48 ± 7.89 μm 3 ) and E (57.12 ± 6.33 μm 3 ).

Discussion
Compared with high-concentration fluoride treatment, the application of low-concentration fluoridation in protecting teeth from dental erosion seems safer and more practicable because excessive fluorine can pose health hazards, especially for children [17,18]. Investigations of fluoride applications revealed conflicting results, thus the protective effect of using lower-concentration fluoride for several minutes against tooth erosion still needs to understand deeply [10]. This in vitro study investigated the potential of low-concentration fluoride (225 ppm F − ) treatment on enamel for 3 min to prevent citric acid-based erosion of human enamel, considering the aspects of morphology, mechanical properties and frictional behaviour. From the changes of morphology in groups O, F, E, EF and FE, it can be found that the low-concentration fluoride had no significant effect on the surface morphology of a sound enamel surface, and the two applications of low-concentrated fluoride measures, fluoridation before or after dental erosion, show significantly different effects on dental erosion. Fluoridation prior to dental erosion could reduce erosive lesion on the enamel surface to some extent because the arrangement of HAP particles of group FE was much denser than that of group E. However, fluoride treatment after dental erosion seemed no any help to repair erosive lesion from the observation of surface morphology (Fig. 1).
It is necessary for human enamel to have an excellent anti-wear property. Dental erosion can result in not only morphological damage from erosive demineralisation, but decreases in mechanical properties and wear resistance of tooth enamel. Therefore, to a great extent, resistance to dental erosion of enamel can be evaluated from the perspectives of mechanical properties and wear resistance. The nano-indentation/scratch technique has become very popular in studies related to dental erosion, owing to its sensitivity and accuracy [19,20]. In this study, to ensure the reliability of the measurements, the parameters of both the nanoindentation and nano-scratch tests were chosen carefully to keep the tip-penetration depth within the softened layer, the thickness of which was estimated to be over 400 nm [14]. The values of both the hardness and elastic modulus of the enamel surface of every group in this study were indeed within the range reported in other related studies, confirming the rationality of the parameters used in the nanoindentation and nano-scratch tests [14]. The quantitative results of nano-indentation and nano-scratch tests demonstrate that low-concentration fluoridation prior to dental erosion has a positive effect on preventing dental erosion. The E-value of group FE is statistically significantly higher than that of group E. The scratch on the enamel surface of group FE was narrower and shallower than that of group E and the wear volume of group FE was significantly less than that of group E (Fig. 3). However, there existed no significant difference in the wear loss between groups E and EF. It suggests that fluoridation sequence plays an important role in the protective effect of fluoride solutions against erosive wear, and the low-concentration fluoride treatment before erosion shows a certain potential reduce the damage of mechanical and anti-wear properties of sound enamel caused by dental erosion.
It is believed that the anti-erosive effect of fluoride can mainly be attributed to the formation of CaF 2 -like mineral deposits on the original enamel surface, which by its dissolution under erosive conditions temporarily protects the underlying enamel [12,[21][22]. However, CaF 2 -like mineral was not observed in this study, indicating that this measurement has a certain limitation in the investigation of the influence of low-concentration fluoride on the prevention of demineralisation. In this research, it can be found that fluoridation before dental erosion reduces the erosive damage on the surface morphology and anti-wear properties of enamel, using the combination of morphology characterisation with the mechanical properties and wear resistance measurements. It also should be noted that although the enamel in group FE underwent some statistically significant changes, viz. a higher elastic modulus and a lower wear loss compared with the enamel in group E, no same significant rehardening in its surface nano-hardness occurred ( Fig. 2 and Fig. 4). Moreover, both mechanical and anti-wear properties of group FE were far inferior to group O. It means that fluoride applications, using low concentration of NaF, has limited preventive effect on dental erosion, thus it may need frequent application when low-concentration NaF solution (225 ppm F − ) is chosen to prevent dental erosion. Neither erosive morphology nor decreased anti-wear properties of eroded enamel were, obviously, repaired or enhanced after low-concentration fluoride treatment on eroded enamel (Figs. 1 and 3). It is different from fluoridation on caries. Fluoridation on caries is reported to have the potentials use of diffusing fluoride ions into the enamel (sub) surface, adhering fluoride ions to the HPA crystal, or speeding up remineralisation [18]. These differences indicate that the effect of fluoride on enamel is mainly on the protection against erosive attack, not on remineralisation repair.
Groups EF and FE simulated the two different situations when fluoride is ordinarily used, before or after acidic-drink intake. The results of surface morphology and nanomechanical and anti-wear properties in this study confirm that low concentration of NaF solution in the prevention of dental erosion is limited effective and the repair due to fluoridation after erosion is hard to be observed. Maybe protection against dental erosion using a low-concentration fluoride should be conducted frequently before human tooth enamel will suffer an acid attack. It is not be investigated whether the less compromised enamel resulted from the treatment with FE is able to be more resistant than the other groups to a subsequent erosive challenge. Hence, in our future research, the related work will be carried out. Moreover, the effects of fluoride duration, as well as oral environmental factors such as human saliva, on the fluoride protection against dental erosion will be further considered.

Conclusion
In this study, the effect of a short-term application of low-concentration fluoride on tooth enamel to protect against erosion was investigated. Under the present experimental conditions, the following conclusions were drawn: (i) The low-concentration fluoride had no significant effect on the surface morphology, mechanical properties or anti-wear characteristics of a sound enamel surface. (ii) The low-concentration fluoride prior to dental erosion can reduce the erosive lesion of enamel surface morphology to some extent and has a certain potential to prevent human tooth enamel from erosive wear, while fluorination treatment with low-concentration fluoride after dental erosion shows no significant improvement on the surface morphology or wear resistance of eroded enamel. Fluorination treatment can inhibit the development of enamel erosion to a degree, but cannot repair the mechanical properties and wear resistance of the eroded enamel. (iii) The sequence of fluorination with low-concentration fluoride (225 ppm F − fluoride) has an important effect on the protection of enamel from dental erosion. Compared with applying low-concentration fluoride after enamel erosion a low-concentration fluoride treatment in advance is more effective protecting human tooth enamel from erosion.