Comparative Evaluation of the Effect on Enamel Solubility and Remineralizing Potential Following Surface Treatment with Fluoride-alone Varnish and Two Fluoride Varnishes Containing Calcium and Phosphate Additives: An In Vitro Study

Abstract

Background

Fluoride Varnishes have incorporated calcium and phosphate in various forms, such as casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and xylitol-coated calcium and phosphate (CXP).

Aim

The aim of this in vitro study was to assess and compare the effect on enamel solubility and remineralizing potential following the application of Embrace^TM Varnish, MI Varnish^TM, and Profluorid Varnish.

Design

For assessing both enamel solubility and remineralization potential, 40 enamel specimens were randomly divided into four groups (n = 10/group) each, according to surface treatment: group I—no treatment (control); group II—Embrace^TM Varnish; group III—MI Varnish^TM; group IV—Profluorid Varnish. The specimens were processed according to the respective experimental protocols. The percentage decrease of surface microhardness (SMH) and surface hardness recovery percentage were calculated. For statistical analysis, a paired sample t-test and an analysis of variance (ANOVA) with Tukey's Post Hoc test were applied.

Results

Embrace^TM Varnish demonstrated the lowest mean percentage decrease of SMH, followed by MI Varnish^TM, Profluorid Varnish, and the control group. MI Varnish^TM demonstrated the highest mean SHR%, followed by Embrace^TM Varnish, Profluorid Varnish and the control group.

Conclusion

Embrace^TM Varnish and MI Varnish^TM demonstrated superior acid resistance and higher remineralizing potential compared to Profluorid Varnish.

How to cite this article

Karanth SK, Naidu J. Comparative Evaluation of the Effect on Enamel Solubility and Remineralizing Potential Following Surface Treatment with Fluoride-alone Varnish and Two Fluoride Varnishes Containing Calcium and Phosphate Additives: An In Vitro Study. Int J Clin Pediatr Dent 2025;18(5):553–559.

Introduction

Numerous strategies have been utilized to preserve the structural integrity of enamel. The application of fluoride in various forms is a well-established approach that strengthens the enamel structure by forming stronger fluorapatite crystals, thereby increasing resistance to demineralization.¹

Topically applied fluoride Varnishes are professionally applied adherent materials that consist of a high concentration of fluoride as a salt or saline preparation in a fast-drying, alcohol or resin-based solution.² The caries-preventive effect of topical fluoride therapy depends on an adequate supply of calcium and phosphate ions.³ To increase the amount of calcium and phosphate in saliva and to further improve remineralization efficacy, fluoride Varnishes with added calcium and phosphate have been introduced recently.⁴ Several fluoride Varnish formulations incorporate calcium and phosphate in various forms, including functionalized tricalcium phosphate (fTCP), amorphous calcium phosphate (ACP), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and xylitol-coated calcium and phosphate (CXP). These additions enhance mineral adherence to the tooth surface, reduce demineralization, promote remineralization, stabilize fluoride-calcium-phosphate ion phases, and help prevent the formation of unstable phases during storage.^4,5 Although the anticaries effectiveness of fluoride Varnishes supplemented with calcium and phosphate,^1,5–7 as well as those with xylitol-coated calcium phosphate,³ has been previously studied in comparison to traditional topical fluoride Varnishes, a thorough review of both print and electronic sources found limited research evaluating the impact of MI Varnish and Embrace Varnish on enamel solubility.⁸ One study has investigated the remineralization ability of these two Varnishes.³

In view of the paucity of evidence, the present study was conducted to assess and compare the remineralizing potential and effect on enamel solubility of conventional topical fluoride Varnish (Profluorid^® Varnish) and fluoride Varnish with casein phosphopeptide stabilized amorphous calcium phosphate (MI Varnish^TM) and xylitol coated calcium phosphate-based fluoride Varnish (Embrace^TM Varnish).

Materials and Methods

Experimental Design

The study was carried out in compliance with the Declaration of Helsinki, and approval was obtained from the Institutional Ethics Committee. The material description, composition and Manufacturer details are given in Table 1.

Table 1:

Material description, composition, and manufacturer details of the materials used in the study

Material	Material description	Chemical composition	Manufacturer	Batch no.
Embrace Varnish9	Sodium fluoride with CXP™ [CXP™ = Xylitol-coated calcium and phosphate] Varnish	Sodium fluoride: 5% with CXP™ [CXP™ = Xylitol-coated calcium and phosphate] Hydrogenated rosin <35%. Ethanol <20% Amorphous fumed silica <3%.	PulpdentTM Corporation	LOT: 191002
MI Varnish 10	Sodium fluoride, with casein phosphopeptide stabilized amorphous calcium phosphate Varnish	Sodium fluoride: 5% (2.26% or 22600 ppm fluoride ion), Casein phosphopeptide stabilized amorphous calcium phosphate, Polyvinyl acetate, hydrogenated rosin: 10–20%, Ethanol: 25–50%, Silicon dioxide, flavor.	GC Corporation	LOT: 1912051
Profluorid Varnish11	Sodium fluoride Varnish	Sodium fluoride: 5% (22600 ppm fluoride), Xylitol, Ethanol: 10–25%, Flavors: Melon/mint/cherry/caramel	VOCO	LOT: 2043090

Methodology

Enamel blocks obtained from forty noncarious therapeutically extracted premolars were used. Teeth were cleaned with an ultrasonic scaler to remove blood and adherent tissues with an ultrasonic scaler. The teeth were examined under a stereomicroscope at 10× magnification to ensure the absence of caries, white spot lesions, dental restorations, fluorosis, enamel hypoplasia, and enamel cracks. The selected teeth were stored following the International Standardization Organization (ISO) specification TS 11405:2015 (E)¹² guidelines. They were immersed in a 1.0% chloramine T trihydrate solution for up to one week, and subsequently stored in deionized distilled water at approximately 4°C in a refrigerator. The storage medium was replaced at least once every 2 months.¹² All stored teeth were used for sample preparation and testing within 6 months of storage.

Evaluation of the Effect on Enamel Solubility

Preparation of Tooth Specimen

The crowns of the collected tooth specimens were sectioned from the roots at a point 2 mm below the cementoenamel junction (CEJ). The crowns were then longitudinally sectioned in the mesiodistal direction to produce buccolingual sections. The resulting tooth sections were horizontally embedded in cold-cure acrylic resin, with the sectioned buccal or lingual surface facing upward, using a custom-made standardized cylindrical metal mould (2.0 ± 0.1 cm in diameter and 1.0 ± 0.1 cm in height).

All specimens were ground flat and manually polished using a sequential series of wet silicon carbide abrasive papers (#800, #1000, and #1200). The specimens were finely polished with a water-based diamond paste to eliminate any scratches. The specimens were sonicated in deionized water to clean the test surfaces. After grinding and polishing, the cleaned specimens were visualized under a stereomicroscope at 10× magnification to eliminate any specimens with obvious cracks/defects on the enamel surface. The prepared specimens were kept at 100% relative humidity and 4°C in a refrigerator and tested within a maximum period of 7 days.

Enamel Surface Microhardness Testing

Enamel surface microhardness (SMH represented by the Vickers hardness number–VHN) assessment was carried out using a Vickers Microhardness Tester [Mitutoyo Vickers hardness tester (Mitutoyo Corporation, Tokyo, Japan. 810 - 125 HM 102)] with a 1360 diamond indenter and 50× objective lens, by applying 25 gm load for 20 seconds. The length of the indentation was determined optically by measuring the two diagonals of the square indent, microscopically with 50× magnification.

The VHN value displayed by the Vickers microhardness tester was calculated using the following formula:¹³

VHN =185.4 × P/d²

Where P is the testing load in grams and d is the length of the diagonal line across the indentation in microns.

Baseline Enamel Microhardness Testing

The initial baseline SMH (VHN1) of the sound enamel specimens was assessed using a Vickers microhardness indenter, which created indentations in the enamel by applying a load of 25 gm for 20 seconds. Five indentations, spaced 1 mm apart, were made on the left upper, left lower, center, right upper, and right lower areas of the enamel block. The average of these five indentations was calculated to represent the specimen's surface hardness number. Only specimens with SMH values ranging between 300 ≤ VHN1 ≤ 400, corresponding to the average microhardness value of sound human enamel, were accepted into the study.

Specimen Grouping

Forty enamel blocks that met the baseline SMH qualification criteria were randomly assigned into four groups of equal size (N = 4), based on the type of fluoride Varnish. There was no statistically significant difference between the baseline SMH values of various treatment groups, which implied all the pretreated enamel samples were similar.

Group I: No Varnish application (control) (n = 10):

Specimens were not treated with Varnish.

Group II: Embrace^TM Varnish (n = 10):

Varnish was dispensed on the mixing pad or mixing well. Using the applicator brush provided by the Manufacturer, a thin coat of Embrace^TM Varnish was applied on the enamel surface with one horizontal swipe of the brush.

Group III: MI Varnish^TM (n = 10):

A thin, uniform layer of MI Varnish was applied on the enamel surface using the disposable brush provided by the manufacturer.

Group IV: Profluorid Varnish (n = 10):

Using the applicator brush provided by the manufacturer, a thin coat of Varnish was applied on the enamel surface.

Specimen Storage:

Specimens were stored in freshly prepared artificial saliva. After 24 hours, the Varnish was removed using a surgical blade, followed by wiping with acetone-soaked cotton swabs. All specimens were then rinsed with deionized water for 1 minute. At this stage, the investigator was blinded to the test groups to prevent any bias during testing.

Enamel Solubility Testing

Solubility of the enamel specimens was assessed by subjecting the specimens to a demineralization-remineralization cycle (pH cycling) simulating a high caries challenge.

pH Cycling

The prepared enamel specimens were subjected to a pH cycling regime (Featherstone laboratory pH cycling model)¹⁴ for 7 days. Each day consisted of a period of demineralization and a period of remineralization. For demineralization, the enamel specimens were placed in a demineralizing solution for 3 hours at 37°C. All the specimens were then immersed in a remineralization solution for 21 hours to remineralize, and this process was continued for an additional 6 days. Between the cycles, the specimens were rinsed with deionized water and dried using blotting paper. Over any weekend, the enamel specimens were stored in the remineralizing solution at 37°C in an incubator. Fresh demineralizing solution was changed two times weekly, and fresh remineralizing solution was changed three times weekly.

Final Surface Microhardness Testing

After pH cycling, the final enamel SMH was determined using a Vickers microhardness tester, following the same procedure outlined for baseline SMH measurement.

Additionally, the percentage reduction in SMH/percentage change/percentage of hardness loss (VHN%) was calculated using the formula: % VHN =100 × (VHN2–VHN1)/VHN1

Where VHN1 is the SMH at baseline, VHN2 is the SMH post-pH cycling.

All the measurements during testing were performed by the same examiner using the same calibrated machine.

Evaluation of Remineralizing Potential

Preparation of Tooth Specimen

The specimen preparation procedure was similar to that of the preparation of specimens for assessment of enamel solubility.

Baseline Enamel Microhardness Testing

Initial baseline SMH (SMH₀ represented by VHN_Sound) was carried out as mentioned for baseline SMH testing for assessment of enamel solubility.

Artificial Caries Formation

One-third of the enamel surface on each test specimen was coated with nail Varnish to act as a control area. The remaining two-thirds were left uncoated and subjected to artificial caries induction by immersion in a demineralization solution for 96 hours. Subsequently, the specimens were rinsed with deionized water. Following demineralization, the outer one-third of the previously demineralized enamel area was coated with nail Varnish to serve as the demineralization control, while the inner one-third remained exposed for treatment.

Baseline Enamel Surface Microhardness of Artificial Lesion

Baseline enamel SMH of the demineralized enamel specimen/artificial lesion (SMH₁ represented by VHN_lesion) was determined using Vickers microhardness indenter under 25 gm load for 20 seconds, at five different points in the demarcated demineralized one-third area. The average of these five indentations was taken to represent SMH₁. Only specimens with values ranging between 25 ≤ VHN_lesion ≤80, postdemineralization, were selected and randomly allotted to four different groups for remineralization based on the type of Varnish.

Specimen Grouping

Forty enamel specimens meeting the baseline SMH₁ qualification criteria were randomly assigned into four groups of equal size (N = 4), in the same manner as while testing for enamel solubility, based on the type of fluoride Varnish. A thin layer of fluoride Varnish was applied to one-third of the demineralized enamel surface designated for treatment in all experimental groups (Groups II–IV), following the manufacturer's instructions.

Specimen Storage

The specimens were immersed in freshly prepared artificial saliva for 6 hours and placed in an incubator at 37°C for 6 hours. Fluoride Varnish was carefully removed using a surgical blade, followed by cotton swabs soaked in acetone. All the specimens were washed with deionized water for 1 minute. The investigator was blinded with regard to the test groups at this point to avoid bias during testing.

Remineralization Potential Testing

Remineralization potential of fluoride Varnishes, were assessed by subjecting the specimens to demineralization-remineralization cycle (pH cycling) simulating a high caries challenge.

pH Cycling

The pH cycling protocol was similar to that of pH cycling protocol carried out for enamel solubility assessment.

Final Surface Microhardness Testing

The final enamel SMH₂ was determined using a Vickers microhardness tester. For each specimen, five indentations were made within the defined treatment area using a 25 gm load applied for 20 seconds. The mean value of these five indentations was calculated to represent SMH₂. All measurements were conducted by the same examiner using the same calibrated device.

Percentage of Surface Hardness Change

Percentage of surface hardness change (%SHR) was calculated by using the formula: %SHR = 100 × (SH₂–SH₁)/(SH₀–SH₁)

Where SH₀ is the SMH at the baseline, SH₁ is the SMH of the lesion (after demineralization), and SH₂ is the final SMH post-pH cycling.

Statistical Analysis

The collected data were analyzed with IBM SPSS Statistics for Windows, Version 23.0 (Armonk, NY: IBM Corp). For enamel solubility, to describe about the data, descriptive statistics, the mean and standard deviation (SD) were used. To find the significant difference between the bivariate samples in paired groups, the paired sample t-test was used. For the multivariate analysis, the one-way ANOVA with Tukey's Post hoc test was used. For remineralizing potential, to describe about the data, descriptive statistics, frequency analysis, and percentage analysis was used for categorical variables, and the mean and SD were used for continuous variables. To find the significant difference in the multivariate analysis, the one-way ANOVA with Tukey's Post hoc test was used, and for repeated measures, the repeated measures of ANOVA was used with Bonferroni correction to control the Type I error on multiple comparisons. In all the above statistical tools the probability value 0.05 has been considered as a significant level.

Results

Effect on Enamel Solubility

The enamel SMH values, measured at baseline (VHN1), the post-treatment and pH cycling (VHN2), and the percentage loss of SMH (%VHN) values are shown in Table 2 and Figure 1, respectively. No significant difference was observed in the mean VHN1 between the groups (p = 0.153). Specimens treated with fluoride Varnishes demonstrated significantly higher mean VHN2 when compared to the Control/no treatment group (p-value = 0.0005).

Table 2:

Overall comparison between mean values of baseline surface microhardness (VHN1) and final surface microhardness (VHN2) using paired t-test.

Groups	N	VHN1 Mean± SD	VHN2 Mean± SD	Paired differences					t	df	p-value
				Mean	Std. Deviation	Std. error mean	95% confidence interval of the difference
							Lower	Upper
Group I (Control)	10	311.20 ± 15.23	51.08 ± 5.94	260.120	9.839	3.111	253.082	267.158	83.603	9	0.0005
Group II (Embrace Varnish)	10	312.88 ±11.21	242.10 ±16.93	70.780	21.824	6.901	55.168	86.392	10.256	9	0.0005
Group III (MI Varnish)	10	324.10 ±18.47	246.40 ±7.51	77.700	17.432	5.512	65.230	90.170	14.096	9	0.0005
Group IV (Profluorid Varnish)	10	320.78 ±11.17	224.52 ±9.30	96.260	10.132	3.204	89.012	103.508	30.042	9	0.0005

Fig. 1:

Intergroup comparison of mean percentage loss of surface microhardness (%VHN) of test groups

A highly significant difference was noted between MI Varnish™ and Profluorid Varnish, as well as between Embrace™ Varnish and Profluorid Varnish, with Profluorid Varnish showing a significantly lower VHN2 value compared to both test Varnishes. Additionally, the control group exhibited a markedly lower mean VHN2 value than all the test Varnish groups.

The mean percentage loss or reduction in SMH (%VHN) was significantly lower in specimens treated with fluoride Varnishes compared to the control group that received no treatment (p = 0.0005). Among the fluoride Varnishes, Embrace™ Varnish showed a significantly lower mean% VHN than Profluorid® Varnish (p = 0.001). MI Varnish™ also exhibited a significantly smaller mean % VHN decrease compared to Profluorid® Varnish (p = 0.010). However, the difference in mean % VHN values between Embrace™ Varnish and MI Varnish™ was not statistically significant (p = 0.897).

Remineralization Potential

The enamel SMH values were measured at baseline (SH₀), post-demineralization (baseline enamel SMH of artificial lesion–SH₁) and post treatment and pH cycling (SH₂), and percentage surface hardness recovery (SHR%) values are shown in Table 3 and Figure 2. No significant difference in the baseline enamel SMH of artificial lesion values between the test groups (p-value = 0.475) was observed. Specimens treated with fluoride Varnishes demonstrated significantly higher mean SH₂ when compared to the Control/no treatment group (p-value = 0.0005). MI Varnish^TM group demonstrated the highest mean SHR%, followed by Embrace^TM Varnish group, Profluorid Varnish group, and the control group. The mean percentage surface hardness recovery (SHR%) of specimens treated with fluoride Varnishes was significantly greater than that of the control group, which received no treatment (p = 0.0005). Among the fluoride Varnishes, both MI Varnish™ and Embrace™ Varnish showed significantly higher mean SHR% values compared to Profluorid® Varnish (p = 0.0005). However, the difference in mean SHR% between the MI Varnish™ and Embrace™ Varnish groups was not statistically significant (p = 0.062).

Table 3:

Mean and within-subject effects comparison between mean values of baseline surface microhardness, baseline enamel surface microhardness of artificial lesion, and final surface microhardness values of group I, group II, group III, and group IV test specimens

Group	VHN	Mean	SD	N	Source	Type III sum of squares	df	Mean square	F	p-value
Group I	SH0	322.8	13.0	10	Huynh–Feldt	490,252.066	1.052	465,849.530	1,779.042	0.0005
	SH1	42.7	12.5	10
	SH2	61.5	13.8	10
Group II	SH0	320.9	11.1	10	Huynh–Feldt	404,739.653	1.019	397,104.291	1,837.920	0.0005
	SH1	50.9	13.8	10
	SH2	108.2	12.8	10
Group III	SH0	336.2	14.2	10	Huynh–Feldt	468,269.858	1.212	386,432.638	1,689.973	0.0005
	SH1	43.6	12.8	10
	SH2	112.2	16.3	10
Group IV	SH0	330.3	7.5	10	Sphericity assumed	469,214.402	2	234,607.201	6,017.653	0.0005
	SH1	45.1	11.7	10
	SH2	90.7	11.2	10

Fig. 2:

Intergroup comparison of mean % SHR of test groups

Discussion

Fluoride Varnishes are thought to promote the formation of intraoral fluoride reservoirs through the formation of calcium fluoride.¹ Adding calcium ions to fluoride-containing Varnishes is intended to enhance the retention of both fluoride and calcium within the oral environment. Calcium is crucial, as its concentration in plaque fluid diminishes with frequent sucrose exposure, yet it is essential for supporting the remineralization process. Moreover, the remineralizing capacity of saliva is also limited by its calcium content. Supplementing with calcium and phosphate ions—delivered through Varnish—can help improve the remineralization of initial carious lesions. As a result, many manufacturers have reformulated fluoride Varnishes to include calcium and inorganic phosphate ions to boost their effectiveness.¹⁵

Amorphous calcium phosphate (ACP) is an unstabilized form of a calcium- and phosphate-based remineralization system. Casein phosphopeptides (CPPs) are multiphosphorylated peptides derived from the enzymatic tryptic digestion of casein, which is a milk protein obtained from cow's milk. CPP binds to ACP to create nanoclusters, inhibiting the aggregation of calcium and phosphate ions beyond the critical size required for nucleation and phase transformation.³ Each CPP molecule can bind up to 25 calcium ions, 15 phosphate ions, and five fluoride ions.¹⁶

The stabilization of bioavailable calcium and phosphate ions helps maintain a high concentration gradient, which promotes deeper diffusion of these ions into the body of demineralized enamel subsurface lesions.³ CPP-ACP complexes readily interact with fluoride ions to form CPP-ACFP, a compound that delivers additional fluoride along with calcium and phosphate. This combination aids in preventing demineralization and supports the remineralization of dental caries or erosion.¹⁷ MI Varnish (GC Corporation, Tokyo, Japan) is a 5% sodium fluoride Varnish formulated with casein phosphopeptide-stabilized amorphous calcium phosphate.¹⁰

Xylitol-coated calcium and phosphate have been incorporated into fluoride Varnish formulations to provide sustained, time-released delivery of active ingredients.¹⁸ Xylitol is also thought to interact with calcium in aqueous environments and plays a role in reducing the dissolution of calcium and phosphate ions from enamel.¹⁹ When exposed to saliva, the xylitol coating on the permeable resin matrix dissolves, enabling a continuous reaction between calcium, phosphate, and fluoride ions, which leads to the formation of fluorapatite on the tooth surface.^9,18 This process enhances the remineralization of carious lesions. Embrace Varnish, developed and marketed by Pulpdent™ Corporation, is a fluoride Varnish containing added CXP.

Profluorid Varnish (VOCO), a 5% sodium fluoride Varnish that does not contain calcium phosphate additives but includes xylitol,¹¹ was used as the positive control in the present study.

The percentage loss in SMH provides an indirect measure of mineral loss from enamel after the application of fluoride Varnish.²⁰ Changes in mineral content within the superficial enamel layers are closely linked to variations in microhardness; for instance, remineralization of enamel carious lesions corresponds to an increase in enamel SMH.²¹ This method is effective for assessing the resistance of fluoride-treated enamel and is particularly suitable for enamel testing due to its fine microstructure, heterogeneous composition, and brittle characteristics.²²

Different approaches are used to assess mineral gain or loss, with SMH analysis being the most commonly used. This technique can be employed to evaluate enamel demineralization and remineralization. Microhardness indentation testing is a relatively straightforward, fast, and nondestructive method for monitoring these changes.²² The two most frequently used microhardness tests are the Vickers and Knoop microhardness tests. In tooth hardness studies, the Vickers indenter is generally preferred, as it is less affected by specimen curvature and is more suitable for evaluating more demineralized samples than the Knoop indenter.¹⁶ Additionally, the Vickers indenter penetrates less deeply into enamel, minimizing the risk of enamel cracking.²³

During the evaluation of remineralization potential, a noticeable decrease in microhardness was observed in all enamel specimens following demineralization. Previous studies have reported varying microhardness values after demineralization, which may be attributed to differences in the composition of the demineralizing solutions used. The reduction in SMH values before and after demineralization was statistically significant across all specimens, aligning with findings from earlier research.^19,24 The posttreatment increase in SMH observed in the present study was interpreted as evidence of remineralization and may be considered a clinically relevant outcome.

During the evaluation of enamel solubility and remineralization potential, a single coat of fluoride Varnish was applied in accordance with the manufacturer's instructions to simulate clinical application.²⁵ The Varnish was then removed from the enamel surface using a surgical blade,^26,27 carefully avoiding direct contact with the enamel, followed by cleaning with a cotton swab and acetone.^1–3 This method of removal was designed to replicate intraoral conditions, where fluoride Varnish naturally wears off within a few hours due to continuous contact with buccal musculature and tongue, mastication, salivary flow, and oral hygiene practices.³ Clinical studies have shown that fluoride Varnish typically remains on the tooth surface for up to 24 hours. Therefore, in this study, Varnish removal was performed after 24 hours using a surgical blade and acetone.

The results of the present study indicate that enamel blocks treated with fluoride Varnishes exhibited greater acid resistance and enhanced remineralization potential compared to those in the control group (no treatment). This observation is supported by several studies in the existing literature.^{6,15,17,28,29}

In this study, fluoride Varnishes containing calcium and phosphate additives showed greater acid resistance and remineralization potential compared to the conventional fluoride Varnish.^1,5–7

MI Varnish™ exhibited a higher remineralization potential compared to the other test Varnishes, although the difference between the MI Varnish™ and Embrace™ Varnish groups was not statistically significant. The enhanced remineralization observed with the CPP-ACP-containing fluoride Varnish may be attributed to the synergistic action of fluoride, calcium, and phosphate, which provides greater remineralizing capacity than the fluoride alone Varnish. Embrace™ Varnish showed superior acid resistance relative to the other Varnishes tested, though again, the difference between Embrace™ and MI Varnish™ was not statistically significant. The inclusion of CXP in Embrace™ Varnish is believed to facilitate improved initial fluoride release. However, the assumption that CXP-containing Varnish can lead to supersaturation of calcium and phosphate ions—triggering precipitation and subsequent remineralization—requires further research for confirmation.

All Varnishes used in this study were resin-based. In resin-based fluoride Varnishes, the diffusion of fluoride ions occurs more slowly because sodium fluoride exists in a suspended (insoluble) form rather than in a dissolved (soluble) form. The presence of a viscous agent slows down diffusion, resulting in a more gradual release of fluoride. This leads to higher fluoride availability in the environment over time and a reduction in mineral loss.³⁰

Currently, there is no clinically validated in vitro model to assess the efficacy of fluoride Varnishes; therefore, the findings from both the present and previous in vitro studies should be interpreted with caution. Laboratory models serve as surrogates for clinical caries but do not fully replicate all aspects of the demineralization and remineralization processes.³¹ Additionally, the pH cycling model used in this study cannot entirely reproduce the complex intraoral environment that influences remineralization. It is also important to note that the area subjected to pH cycling in this study was not the original, intact enamel surface.

Microhardness values of treated and polished enamel may not accurately reflect those of the original, intact enamel surface. Polishing removes the hypermineralized outer enamel layer, rendering the enamel more prone to demineralization. Consequently, the reduction in enamel SMH observed after pH cycling may differ from that seen in unaltered, intact enamel.³² In this study, enamel surfaces were polished flat to standardize the specimens and create a uniform area suitable for microhardness testing. However, this preparation method may influence results, as the tested surface becomes more susceptible to deeper demineralization.³³

Surface microhardness testing alone does not assess lesion depth or variations in mineral content and therefore provides only a partial understanding of mineral changes within carious lesions. To evaluate lesion depth, techniques such as transverse microradiography, cross-sectional microhardness, optical coherence tomography, or polarized light microscopy can be employed. For analyzing subsurface mineral content, methods such as transverse microradiography and atomic absorption spectrophotometry are effective.

Structural analysis for detecting small changes in mineral content can be conducted using scanning electron microscopy (SEM), while ultra-structural chemical alterations can be quantified using energy-dispersive X-ray analysis (EDAX). As traditional methods of monitoring mineral gain or loss are not feasible in clinical settings, quantitative techniques such as quantitative light-induced fluorescence (QLF) offer a reliable way to assess the progression or regression of enamel demineralization. Ion chromatography is useful for measuring the release of fluoride, calcium, and phosphate, and laser fluorescence can assist in caries detection and tracking mineral changes in tooth structure.

Hence, incorporating these evaluation methods could offer deeper insights into the effects of fluoride Varnishes. As this study was conducted in vitro on premolar teeth, further in vivo studies involving both primary and permanent teeth are necessary to more comprehensively assess the effectiveness of fluoride Varnishes containing calcium and phosphate additives.

Conclusion

There appears to be a synergistic effect when adding calcium and phosphate additives to fluoride Varnish, which results in increased efficacy. It can be concluded that fluoride Varnishes containing calcium and phosphate additives are more effective in increasing acid resistance of enamel and in remineralizing incipient carious lesions in comparison with fluoride Varnish alone.

Acknowledgments

The authors sincerely thank Mr Yogendra (Foreman), Department of Mechanical Engineering, Ramaiah Institute of Technology, for his selfless support and meticulous help.