Effect of two different fluoride varnishes on enamel de-mineralization and re-mineralization: an in vitro study

INTRODUCTION

Interdental stripping, mechanical grinding of the proximal tooth surface, is a common practice to reduce mesiodistal diameter for orthodontic management of minor crowding and tooth shape or size disharmony [1]. Stripping methods affect proximal integrity by leaving roughness that cannot be eliminated by subsequent polishing procedures [2, 3]. Enamel roughness facili­tate plaque accumulation even after intensive oral hygiene [4]. Therefore, the stripped enamel surface is more susceptible to de-mineralization and subsequent caries, and any protective agent is worth investigating [5].
On the other hand, the enamel surface is exposed over time to acidic challenges and consequent changes in mineral content [6]. Exposure to acidic challenges de-mineralizing the enamel surface and oral fluid becomes super-saturated with calcium and phosphate ions. Conversely, when acidic attacks subside, calcium and phosphate ions gradients invert to ultimately re-mineralize the enamel surface [6]. Although de-minera­lization and re-mineralization processes occur parallelly at the same time, their ratio determine the end state of enamel [6, 7]. Early stages of the de-mineralization process are called ‘initial enamel lesions’ that have relatively intact surface overlying de-mineralized sub-surface [7]. Fortunately, initial enamel lesions can be prevented, treated, or even stopped with appropriate non-invasive me­thods [7, 8].
Topical fluoride applications are effective non-invasive management methods for initial enamel lesions [9, 10]. Fluoride varnishes contain 5% sodium fluoride salt solubilized in an alcohol-based solution of natural or synthetic resins that prolong the adhesion of varnish to tooth surfaces [11]. Fluoride varnishes are widely used in the prevention and treatment of initial enamel lesions [10, 11]. The effect of fluoride vanishes is attributed mainly to the formation of calcium fluoride deposits on the enamel surface [12]. These calcium fluoride deposits become incorporated into de-mineralized tissues, and transform hydroxyapatite crystals into more stable acid-resistant fluorapatite or fluor hydroxyapatite crystals [13, 14]. In addition, these calcium fluoride-like deposits may act as physical barriers for underlying enamel against further acidic challenges or slow-releasing fluo­ride reservoirs that discharge cariostatic quantities of fluoride ions over at least 24 hours [13, 15].
The first commercial 5% sodium fluoride varnish product introduced into the market was Duraphat varnish in 1964 by Woelm Company from Germany [13]. Recently, many ingredients with promising re-minera­lizing efficacy have been added to sodium fluoride-based varnishes in an attempt to enhance their therapeutic effects and improve mechanical properties of the enamel [16, 17]. Among these products is Clinpro White varnish of 3M ESPE containing 5% sodium fluoride and innovative tricalcium phosphate modified by fumaric acid. The manufacturer of Clinpro varnish states that the presence of a fumaric acid protective barrier allows for calcium ions to co-exist with fluoride and phosphate ions without premature reaction throughout its shelf-life. After the application of varnish on the tooth surface, the protective barrier slowly breaks down to deliver calcium ions along with phosphate and fluoride ions to the enamel surface [18].
Although Duraphat varnish is the gold standard and its anticaries effect is based on evidence, the effect of addition of other ingredients to sodium fluoride varnish is not settled yet in literature [11]. In addition, very limited investigations have evaluated the efficacy of fluoride varnish in improving the integrity of stripped enamel. The aim of the present study was to evaluate the efficacy of Duraphat and Clinpro varnishes in protecting stripped enamel against acidic challenge and re-mine­ralizing initial enamel lesions.

MATERIAL AND METHODS

This study was conducted after obtaining ethical approval from the Dental Research Ethics Committee of the Faculty of Dentistry, Mansoura University, approval code No. M0309023PP. For anticipating a large effect size of 0.40 with an 80% power and a 0.05 significance level, a total sample size of sixty-six proximal surfaces was deemed adequate for comparing the micro-hardness of three groups of samples using ANOVA.
Sixty-six sound and freshly extracted for orthodontic reasons permanent premolars were collected. Proximal surfaces of premolars were visibly free from any carious lesions or macroscopic defects. Premolars with any restorations, enamel malformations, cracks, fluorosis, or hypo-mineralization were excluded from the study. The premolars were cleaned under tap water and stored in a thymol solution until used. The premolars were randomly assigned into 2 equally divided groups (n = 33) based on proximal enamel surface preparation as follows: group 1 (stripped enamel) and group 3 (initial enamel lesion).
In group 1, thirty-three premolars were aligned in an arch form with interproximal contact in a plaster block. After that, premolars were mesially and distally stripped at their contact areas using safe-side diamond-coated metal hand strips under water cooling. To standardize procedures, twenty-five abrasions in one direction were performed by one operator on each proximal surface in a random fashion, and a new strip was used for each surface [2]. Then, the premolars were removed from the plaster block, their roots were cut 3 mm apically to cemento-enamel junction, and coronal portions were sectioned buccolingually with water-cooled diamond disc to obtain 66 stripped mesial and distal enamel halves. Subsequently, 66 stripped proximal surfaces were covered with acid-resistant nail varnish, except for 3 × 4 mm enamel windows on the middle third.
In group 2, roots of thirty-three premolars were cut 3 mm apically to cemento-enamel junction, and coronal portions were sectioned buccolingually with water-cooled diamond discs to obtain 66 mesial and distal halves. Proximal surfaces of 66 halves were covered with different color acid-resistant nail varnish, except for 3 × 4 mm enamel windows on the middle third. The specimens were subjected for 10 days to a de-mine­ralizing solution composed of 2.2 mM calcium chloride, 0.05 M acetic, 2.2 mM sodium dihydrogen phosphate, and 1 M potassium hydroxide, with adjusted pH of 4.7 [19]. The solution was changed every day to prevent pH change from accumulation of dissolution products. By this procedure, 66 specimens with initial enamel lesions on exposed windows were obtained. The specimens were rinsed with distilled water to remove any traces from de-mineralizing solution. Each main group was then divided into 3 equal sub-groups (n = 22) according to varnish treatment of exposed enamel windows: sub-group A (no varnish treatment, control), sub-group B (Duraphat varnish, Colgate-Palmolive Co., USA), and sub-group C (Clinpro White varnish, 3M ESPE Company, USA). Before varnish application, Vickers micro-hardness test (Micro-hardness tester, FM-700, Future-Tech. Co., Tokyo, Japan) for 20 specimens from each sub-group was performed at exposed windows to obtain sub-group baseline micro-hardness. Three indentations under a 100 gram static load for 10 seconds were performed, and mean value was taken for statistics.
Each varnish was applied to the cleaned and dried enamel window of its respective sub-groups, according to the manufacturer’s instructions. Specimens of all sub-groups were stored in de-ionized water for 24 hours and then immersed in the same de-mineralizing solution for 10 days. The specimens were removed from the de-mine­ralizing solution and washed with de-ionized water. Vickers micro-hardness test was performed again for the same 20 specimens from each sub-group at exposed windows as a baseline measurement. The remaining two specimens from each sub-group were prepared and examined under SEM (JEOL, JSM-6510LV, Tokyo, Japan) at magnifications above x1000 to observe ultra morphological changes on enamel surface with or without varnish treatment after exposure to de-mineralizing solution (signs of de-mineralization or re-mineralization).
Data were collected and analyzed statistically using SPSS package software program version 25 at a 5% level of significance to investigate any substantial difference. Student t test was used to compare between means of the same group at baseline and after treatment, while ANOVA test was employed to compare between means of sub-groups and Tukey post-hoc test was used when significance was found.

RESULTS

The mean and standard deviations of Vickers micro-hardness for the studied sub-groups at baseline and without treatment or after treatment with either Duraphat or Clinpro varnish are illustrated in Table 1. The highest mean of Vickers micro-hardness was the baseline of stripped enamel sub-groups (339.1 ± 16.7), and the lowest mean was found in the initial enamel lesion control sub-group (158.6 ± 13.3). A high statistically significant difference was found between control sub-groups and their respective sub-groups treated with Duraphat or Clinpro varnishes (p < 0.05). However, no statistically significant difference was found in the comparison between Duraphat- and Clinpro-treated sub-groups, or between treated sub-groups and their respective baseline values (p > 0.05).
The representative SEM photo-micrographs for different sub-groups after exposure to de-mineralizing solution are illustrated in Figure 1. SEM images for control sub-groups showed a typical honeycomb-like de-mineralized surface, with severely affected enamel prisms in the stripped enamel sub-group (Figure 1A), and plenty of deep pitting with variable sizes of porosities in the initial enamel lesion sub-group (Figure 1B). This revealed early signs of enamel de-mineralization on stripped enamel and more advanced state of de-mineralization on initial enamel lesion after exposure to de-mineralizing solution without Duraphat or Clinpro treatments.
The SEM images after varnish treatment showed deposition of a well-defined uniform protective layer of crystals for Duraphat sub-groups (Figures 1C-D) compared with a well-defined irregular protective layer of crystals for Clinpro sub-groups (Figures 1E-F), with inter-prismatic and prismatic enamel structures as well as surface microporosities completely hidden by this protective layer. This revealed efficacy of both Duraphat and Clinpro varnishes to protect and repair stripped enamel and re-mineralize initial enamel lesion by filling up defects and micropores on the enamel surface with well-adherent nano-crystals deposit.

DISCUSSION

Interdental stripping is a common procedure during orthodontic treatments to create extra space to overcome dental crowding or re-shape teeth. However, stripping procedures inevitably affect the integrity of hyper-mineralized aprismatic enamel surface layer, and expose less resistant internal layers to de-mineralization [3]. The current concept for initial enamel lesions is an imbalance between de-mineralization and re-mineralization processes [7]. This study aimed to mimic a single professional application for two different varnishes to protect stripped enamel against acidic challenge as well as their effect on initial enamel lesions using micro-hardness tests and SEM examinations.
Since premolars used in this study came from diffe­rent individuals, they were aligned in an arch-form with correct proximal contact in a plaster block to simulate intra-oral stripping. In addition, the stripping procedure was performed with safe-side diamond-coated metal hand strips under water cooling, as this is one of the most widely used techniques [2].
Artificial enamel lesions represent all histological features of natural caries for in vitro studies [19, 20]. The degree of enamel de-mineralization depends on the pH and calcium phosphate content of de-mineralizing solution [19, 20]. The specimens used in the study were kept in freshly prepared de-mineralizing solution, according to ten Cate and Duijsters formula, for 10 days to induce initial enamel lesions [19]. The use of weak organic acid along with a 50% saturation level of calcium and phosphate was to achieve gradual de-mineralization, thus enamel de-mineralization would occur only on the sub-surface area while maintaining a relatively intact surface as natural initial enamel lesions [6, 20].
Different artificial saliva formulations were used instead of natural saliva in vitro studies [21]. However, all artificial saliva formulations or any formulations containing minerals may induce some degree of re-mineralization, which in turn generate false results for the study [21]. The rationale for employing de-ionized water in this study was to provide some accurate evaluation of the effect of the two varnishes themselves. In addition, specimens were left for only 24 hours to achieve maximum enamel varnish reaction, as it has been previously estimated [13, 22].
Cut and polished surfaces are generally preferred for micro-hardness tests to ensure accuracy, since any tilt or non-flat surface would yield too large variation [23]. This study was planned to simulate the clinical performance of stripped enamel or initial enamel lesions when subjected to de-mineralization and/or re-mineralizing agents. Therefore, the use of a cut polished surface would not provide beneficial or accurate representation of the original enamel surface.
Each specimen was embedded in self-curing acrylic resin, so that its proximal surface lay flat on top and parallel to the horizontal plane. In addition, three indentations were made for each specimen to avoid discrepancies from surface curvature or any operational bias, and an average was taken. Moreover, standardization of baseline micro-hardness for each sub-group made it possible to establish objective comparisons among different sub-groups, and to clarify the effect of both varnishes on stripped enamel and initial enamel lesion.
The results of the present study revealed that the baseline micro-hardness test for stripped and initial lesion control sub-groups was 339.1 ± 16.7 and 269.4 ± 14.1, which became 228.2 ± 14.2 and 158.6 ± 13.3, respectively, after exposure to de-mineralizing solution. This result demonstrated that stripping and initial lesions of enamel decreased its resistance to acidic challenge, and was statistically significant and in line with studies of Hellak et al. [4] and de Carvalho et al. [24].
The mean micro-hardness for sub-groups treated with Duraphat or Clinpro varnishes was significantly high compared with their respective control sub-groups, without any significant difference between Duraphat and Clinpro varnishes. These results demonstrated the efficacy of both varnishes to re-mineralize and protect initial enamel lesions as well as stripped enamel against acidic challenge. Regarding the effect of both varnishes on the initial lesion, the results of the study come in agreement with studies of Mohd Said et al. [25], Torres et al. [26], and Elkassas and Arafa [27].
To date, only one published in vitro study on the effect of fluoride varnish after enamel stripping was conducted by Peng et al. [28]. They reported that Duraphat varnish enhanced surface conditions of stripped enamel in terms of surface micro-hardness and acidic challenge resistance [28]. Morphological analysis for representative SEM specimens confirmed the previous micro-hardness changes at different study stages. The control sub-groups showed evidence of variable degrees of de-mineralization, as typical honeycomb-like surfaces with severely affected enamel prisms and surface porosities were observed. Meanwhile, the sub-groups treated with Duraphat or Clinpro varnishes showed evidence of re-mineralization, as observed by a protective layer of crystals with no evidence of surface porosities. SEM findings for the initial lesion group regarding Duraphat varnish agreed with the report of de Carvalho et al., who observed a protective layer with globular deposits on the enamel surface under atomic force microscopy, which mimics to some extent the protective layer observed in this study under SEM [24].
Daas et al. observed a smooth enamel surface with reduced surface porosities following treatment of the initial enamel lesion with Clinpro varnish [29]. Nonetheless, uncovered porous enamel surface was observed after pH cycling. This contradiction with the present study could be attributed to variation in contact hours between varnish and enamel surfaces. Clinpro remained in contact with specimens of this study for 24 hours compared with only 4 hours in the Daas et al. study [29]. Therefore, 4 hours was not sufficient to affect enamel surface morphology, since varnish enamel interaction is time-dependent and reaches its maximum after 24 hours [13, 22].

CONCLUSIONS

Under the limitations of this in vitro study, it could be concluded that both Clinpro and Duraphat varnishes provide equal protection for stripped enamel against de-mineralization and enhance its surface properties. Both varnishes are effective in re-mineralizing the initial enamel lesions and increasing their resistance against acidic challenge.

DISCLOSURES

1. Institutional review board statement: The study was approved by the Regional Ethics Committee of the Faculty of Dentistry, Mansoura University, with approval number: M0309023PP.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

References