Dental caries is among the most common oral diseases of childhood, which is considered as a serious public health dilemma.^[1,2] Dental caries often occurs due to an imbalance between the demineralization and remineralization cycles in the enamel following the formation of microbial biofilm on the tooth surface and acid production over time.^[3] Proper oral hygiene and a healthy diet are the main requirements for the prevention of microbial biofilm formation, and caries progression. However, in high-risk individuals, application of remineralizing agents may be recommended as an adjunct. Fluoride is among the most commonly used remineralizing agents. The optimal clinical efficacy of fluoride for caries cessation, especially in the enamel, has been well documented.^[3]

Among the fluoride products, application of 2.26% sodium fluoride (NaF) varnish (containing 22600 ppm fluoride) has been recommended in children under 6 years of age at high risk of caries.^[4] A recent clinical study and a meta-analysis of clinical studies indicated the optimal efficacy of fluoride varnishes for caries prevention.^[5-7] Comparison of the efficacy of NaF varnishes with other fluoride-containing products has shown the comparable or superior efficacy of NaF varnishes.^[8-12] However, some other studies reported that application of NaF varnishes alone did not increase the enamel microhardness.^[13] Accordingly, researchers tried to compare the efficacy of different NaF varnishes. Some short-term studies assessed the increase in fluoride uptake by the enamel, or increase in microhardness of incipient enamel lesions over a 7–10-day period.^[14-18] A long-term study evaluated fluoride release from the enamel surface over 6 months.^[19] Considering the shortcomings of previous studies on the long-term efficacy of different NaF varnishes, this study aimed to assess the primary enamel microhardness following the application of three 5% NaF varnishes with and without tricalcium phosphate (commonly used in Iran), over a long period of time.

This experimental study was approved by the ethics committee of Tehran University of Medical Sciences (IR.TUMS.DENTISTRY.REC.1403.009). The sample size was calculated to be 15 teeth in each group according to a study by Virupaxi et al.,^[19] using one-way ANOVA feature of Minitab 18 software, and assuming alpha = 0.05, beta = 0.20, the largest mean difference between the groups to be 55, and similar standard deviations of the groups equal to 46.80.

A total of 52 primary molars that had been extracted due to dental abscess and had a sound buccal surface with no hypocalcification, fracture, or crack were collected. The teeth were immersed in Chloramine T solution for the purpose of disinfection. After disinfection, they were stored in distilled water to preserve their moisture, and prevent structural changes. Next, the roots were cut 1 mm below the cementoenamel junction, and the crowns were mounted in acrylic resin, such that the sound buccal surface remained exposed for testing. To achieve a smooth surface for the microhardness test, the buccal surface of the teeth was wet-polished with rough-(800-grit), medium-(1000-grit), and fine-grit (1200-grit) abrasive papers.

To measure the enamel microhardness, the standard Vickers test was performed in a Vickers hardness tester (160V-Test II; Baresiss, Germany). In this test, a 300 g load was vertically applied to three points on the buccal surface with a minimum of 100 μm distance from each other for 15 s. The mean of the three values was calculated and recorded as the baseline microhardness of the teeth (Vickers hardness number). To induce artificial caries, the teeth were immersed in a demineralizing solution with a pH of 5 for 10 h followed by immersion in a solution with a pH of 4.5 for 15 h. The demineralizing solution contained 2.2 mM CaCl₂, 2.2 mM NaH₂PO₄, and 0.05 M acetic acid; the pH was adjusted to 5 using 1 M KOH. The composition of solution and its duration of use were determined in a pilot study, which showed that the abovementioned condition led to development of white spot lesions on primary enamel and decreased the microhardness by approximately 30%–40%. The microhardness was measured again after demineralization.

The teeth were then randomly assigned to four groups. Group 1 served as the control group with no fluoride varnish application. Ariadent varnish (Asia-chemi Teb company, Tehran, Iran) was applied in group 2, Parla varnish (Parla company, Tehran, Iran) was applied in group 3, and Morvabon varnish (Morvabon company, Tehran, Iran) was applied in group 4. In groups 2–4, the specimens were dried with an air spray, and the respective NaF varnish was applied on their buccal surface by a microbrush in two coats. The specimens were subsequently immersed in artificial saliva as the remineralizing solution, composed of 1.5 mM CaCl₂, 0.9 mM NaH₂PO₄, and 0.15 M KCl; the pH was adjusted to 7.0. After 24 h, the residual fluoride was removed by a soft toothbrush in the fluoride varnish groups. The microhardness was then measured again by an examiner blinded to the group allocation of the specimens. Next, the specimens were subjected to remineralization-demineralization cycles (pH cycling) for 45 days, such that the specimens were immersed in the demineralizing solution for 3 h, and the remineralizing solution for 21 h within each 24 h. The specimens were kept in an incubator at 37°C during the entire study period.^[20] After 45 days, the microhardness of the specimens was measured again by a blind examiner.

Normal distribution of data was evaluated by the Shapiro–Wilk test. The trend of change in the mean microhardness over time was analyzed by repeated measures ANOVA, considering the type of varnish as between-subject variable (alpha = 0.05). The Bonferroni correction was used for multiple comparisons in the next step. The trend of change in microhardness (mean difference) was compared among the groups by Tukey’s HSD test using SPSS version 26 at a 0.05 level of significance.

Table 1 presents the measures of central dispersion for the microhardness of the study groups. The mean baseline microhardness was measured to be 349.48 ± 39.75, which was within the normal range reported for sound primary enamel.^[21] The results showed no significant difference in baseline microhardness among the study groups (P = 0.941). This finding indicated that the baseline microhardness of all groups was comparable, with no significant difference. As shown in Table 1, the Parla group showed the highest percentage of increase in microhardness during the time interval between demineralization and 24 h and 45 days after the varnish application, followed by the Ariadent and Morvabon groups. The control group showed the smallest increase in microhardness after demineralization.

Noninvasive treatments and preservation of tooth structure are major priorities in contemporary dentistry. It appears that remineralization of demineralized tooth structure should be prioritized when encountering incipient enamel caries. Accordingly, calcium, phosphorous, fluoride, and Vitamin D are supplied for the tooth structure.^[22]

The fluoride content of drinking water may not be sufficiently high; therefore, application of topical fluoride products has been approved, among which, NaF varnish has been recommended for children under 6 years of age. Evidence shows that 5% NaF varnish contains 22600 ppm fluoride. It adheres to the tooth surface for a couple of hours, and serves as a source of sustained fluoride release during this period. The fluoride release rate from this product is influenced by the varnish formulation and the matrix used, its mechanism of binding to insoluble NaF, its interactions with the tooth structure, and environmental factors such as the salivary flow rate, saliva calcium and phosphate contents, consumption of acidic beverages, and activity of oral bacteria. This topic has been investigated through assessment of the degree of enamel demineralization and remineralization by chemical analysis, evaluation of the hardness of tooth sections, and microradiography after the application of fluoride varnish. The present study assessed the efficacy of three fluoride varnishes available in the Iranian market by evaluating their effects on enamel microhardness. Microhardness measurement is a simple, noninvasive, and fast method for this purpose. It is reproducible, and allows repeated microhardness measurements of the same specimen at different time points. It also possesses optimal accuracy and reliability. Featherstone^[23] (1999) and Lata et al.^[24] stated that enamel microhardness measurement is an efficient method for evaluation of enamel mineral loss or gain. Since formation of hydroxyapatite crystals and recording the microhardness change need time, in the present study, the microhardness of the specimens was evaluated both in the short-term (after 24 h) and also after 45 days, which is longer than the assessment time in previous studies for evaluation of the durability of the effect of varnishes.

The present results showed that Ariadent and Morvabon varnishes improved primary enamel microhardness, but their effects did not last for 45 days. However, Parla varnish increased enamel microhardness in the short-term, and its effect lasted for 45 days.

In a short-term study on primary teeth, Jafari et al.^[14] evaluated fluoride uptake by primary enamel after the application of fluoride varnishes by using the acid-etch enamel biopsy method. They reported the lowest fluoride uptake in the Ariadent group, and the highest in the Sultan and Voco groups. Nonetheless, they added that fluoride uptake was more homogeneous in the Ariadent group, and stated that this varnish is reliable and applicable. In the present study, data dispersion was acceptably low in the Ariadent group.

Some similar short-term studies were conducted on permanent teeth. Molaasadolah et al.^[15] evaluated the enamel microhardness of permanent premolars following the application of Ariadent and Duraflor varnishes in 10 days under pH-cycling. They showed a significantly greater increase in microhardness in both fluoride varnish groups compared to the control group. The other studies on fluoride varnishes conducted within a 1-week period showed that Fluor Protector^[16] and Enamelast^[18] optimally increased the microhardness, and Duraflor and Enamel Pro^[17] effectively enhanced enamel resistance to demineralization.

Virupaxi et al.^[19] (2016) evaluated the long-term effects of three varnishes on fluoride release over 6 months, and reported that application of Clinpro^TM XT on the primary tooth surface resulted in continuous fluoride release over a 6-month period.

NaF varnishes have a low free fluoride content, which is released following saliva penetration into the varnish matrix. Therefore, they need a longer exposure time to the saliva. Aside from the exposure time, difference in the type of matrix and mechanism of binding or dissolution of NaF in the matrix affect the efficacy of the varnish.^[2] Accordingly, the manufacturers of fluoride varnishes need to provide a suitable protocol regarding the application time of their products.

Some previous studies focused on the frequency of effective use of NaF varnish to find the most efficient frequency of use. An in vitro study indicated higher enamel microhardness in the group that received NaF varnish 4 times a year compared with once and twice a year, and the intensive method of 4 times/week.^[25] Salehzadeh et al.,^[26] Farhadian et al.,^[27] and Sivapriya et al.^[28] used NaF varnish with a higher frequency rate in the short-term and reported its improved efficacy for remineralization of demineralized permanent enamel. Some other studies also confirmed the optimal clinical efficacy of varnish application three times within a 2-week period and four times within 4 weeks to control white spot lesions.^[29,30]

Some short-term studies indicated that a fluoride varnish containing tricalcium phosphate had a superior remineralizing capacity than a varnish without tricalcium phosphate, and suggested using an additive such as tricalcium phosphate to improve the protective ability of varnishes on primary enamel.^[31,32] A 30-days in vitro study conducted to assess the long-term effectiveness of fluoride varnish containing tri-calcium phosphate on permanent teeth confirmed the advantages of these products^[33] and was in accordance with the results of the present study on primary teeth.

According to the results obtained in this in vitro study under a controlled environment, the Parla varnish had a longer remineralizing effect on primary enamel. Unlike Ariadnet and Morvabon, the Parla fluoride varnish contains tricalcium phosphate, which can increase and prolong the remineralizing effect of fluoride. It appears that the manufacturers of less effective NaF varnishes need to recommend a 3-time application protocol within a 2-week period to maximize fluoride uptake and enamel microhardness.

In the present study, pH-cycling (demineralization-remineralization cycles) was performed over a longer period of time (45 days) instead of one week to assess the durability of the effect of fluoride varnishes on primary enamel in a setting simulating the oral environment. The artificial saliva and solutions were refreshed daily during this period to prevent the adverse effect of materials released from the tooth structure on the varnish. The fluoride content of the pH-cycling solutions mimics the role of fluoride in the human saliva or the fluoride obtained from foods and drinks. Nonetheless, it should be noted that many influential factors present in the oral environment, such as oral microorganisms, differences in chemical composition of the saliva in different individuals, and oral hygiene practices, cannot be simulated in vitro; therefore, in vitro results cannot be completely generalized to the clinical setting.

Future studies are recommended to assess the efficacy of the available varnishes with different timing protocols. Furthermore, fluoride release and fluoride uptake should be evaluated in addition to microhardness.

The results revealed that the Parla varnish, containing tricalcium phosphate, increased the primary enamel microhardness and had a more durable effect than other tested varnishes. Considering the observed differences in the durability of the effects of different fluoride varnishes, the timing protocol for clinical application of each varnish needs to be further evaluated, and revised, if required.