ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10005-2622
International Journal of Clinical Pediatric Dentistry
Volume 16 | Issue S-1 | Year 2023

Comparative Evaluation of the Remineralizing Potential of Silver Diamine Fluoride, Casein Phosphopeptide-amorphous Calcium Phosphate, and Fluoride Varnish on the Enamel Surface of Primary and Permanent Teeth: An In Vitro Study

Samarpreet Kaur¹ https://orcid.org/0000-0002-9808-9570, Meenu Bhola², Nitika Bajaj³, Gurlal S Brar⁴

^1-4Department of Pedodontics and Preventive Dentistry, Dasmesh Institute of Research and Dental Sciences, Faridkot, Punjab, India

Corresponding Author: Samarpreet Kaur, Department of Pedodontics and Preventive Dentistry, Dasmesh Institute of Research and Dental Sciences, Faridkot, Punjab, India, Phone: +91 9646130125, e-mail: drsamarpreet@gmail.com

ABSTRACT

Introduction: With the paradigm shift in the management of dental caries, the focus is now laid on remineralization therapies that can arrest the progression of the disease and remineralize the subsurface lesions.

Objectives: The purpose of this study was to determine and compare the remineralizing potential of silver diamine fluoride (SDF), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and fluoride varnish (FV) on enamel surfaces in primary and permanent teeth.

Materials and methods: A total of 120 primary anterior teeth and 120 premolars were used to prepare enamel blocks in acrylic resin. The mean baseline surface microhardness (SMH) for each sample was determined using a microvickers hardness testing machine. Thereafter, the samples were randomly and equally distributed into groups and subgroups based on the materials used, that is, SDF, CPP-ACP, FV, and distilled water (control). After subjecting the samples to a pH cycling regime, SMH was determined again and the percentage change in SMH was calculated.

Results: The data were tabulated and subjected to statistical analysis using an independent t-test and one-way analysis of variance (ANOVA). In primary teeth, the least mean percentage reduction in SMH was observed after the application of FV followed by SDF, CPP-ACP, and control. In permanent teeth, both SDF and FV showed the least percentage reduction of enamel SMH followed by CPP-ACP and control.

Conclusion: Silver diamine fluoride (SDF), FV, and CPP-ACP showed remineralizing potential in both primary and permanent teeth.

How to cite this article: Kaur S, Bhola M, Bajaj N, et al. Comparative Evaluation of the Remineralizing Potential of Silver Diamine Fluoride, Casein Phosphopeptide-amorphous Calcium Phosphate, and Fluoride Varnish on the Enamel Surface of Primary and Permanent Teeth: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(S-1):S91–S96.

Source of support: Nil

Conflict of interest: None

Keywords: Casein phosphopeptide-amorphous calcium phosphate, Fluoride varnish, In vitro study, Remineralizing agents, Remineralizing efficacy, Silver diamine fluoride, Surface microhardness

INTRODUCTION

Despite recent advancements in oral healthcare, dental caries remain a significant health problem for individuals of all age-groups.¹ Ernest Newbrun best explained dental caries as a cyclic process, with periods of demineralization interspersed with periods of remineralization.² The cycles of demineralization and remineralization continue with only one progressing at one time, depending on various pathological and protective factors.³ With the paradigm shift for the management of dental caries, emphasis is now laid on remineralization therapies that can halt the progression of caries and repair the subsurface lesions rather than restoring the tooth and controlling the disease when obvious cavitation occurs. Among the plethora of remineralization therapies available, the professional application of fluoride varnish (FV) is regarded as a safe and effective caries preventive approach for use in children with special healthcare needs.⁴ In the present study, Fluor protector (Ivoclar Vivadent), a 0.9% difluorsilane, ethyl acetate, and isoamyl propionate solvents in a polyurethane varnish base was used. It is known to effectively incorporate fluoride ions in high concentrations on the enamel surface by adhering to the tooth surface for longer periods.⁵

Reynolds et al. have strongly emphasized the key role of calcium and phosphate ions in the remineralization process by augmenting the diffusion of minerals into the tooth surface.⁶ With ongoing research on nonfluoride-based remineralizing agents, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) based Recaldent technology provides the strongest level of clinical evidence in tooth remineralization.⁷^,⁸ CPP is a milk protein-derived nanocomplex that stabilizes ACP and forms multiphosphorylated peptides. This helps to maintain a continuous supply of free calcium and phosphate ions in solution form, for penetration into the tooth matrix resulting in the remineralization of hypomineralized enamel areas.⁷^-⁹ CPP-ACP is also known to deliver and localize ACP at the tooth surface, thereby enhancing the subsurface remineralization in situ.¹⁰ Apart from maintaining a state of supersaturation to enhance the remineralization phase, CPP substantially reduces the adhesion of Streptococcus mutans on salivary pellicle making the plaque noncariogenic.¹¹ Hence, GC Tooth Mousse (with Recaldent), GC Corporation, Tokyo, Japan was used in the present study.

With the focus on preventive strategies and minimally invasive dentistry for caries management in young children, silver diamine fluoride (SDF) has emerged as a novel method on the horizon. One of the hassles encountered by a dental practitioner when dealing with very young children or children with special needs is the risk of triggering anxiety and fear with the use of rotary handpieces or sharp spoon excavators. For such patients, SDF can be a successful alternative.¹²

Aptly named a “silver-fluoride bullet,” SDF acts by the synergic effect of silver and fluoride ions.¹³^,¹⁴ Rosenblatt et al. advocated that the silver ions inhibit the colonization of cariogenic bacteria either by blocking the electron transport system in bacteria or by deactivating the thiol group of enzymes or by mutation of bacterial DNA. Its antienzymatic property can also be attributed to the silver ions. SDF penetrates the enamel and accumulates more fluoride ions in the subsurface layer than any other fluoride solution.¹³ It also increases the microhardness of the enamel surface by the formation of fluorapatite.¹⁴ Owing to its benefits, 38% SDF (containing 44800 ppm of fluoride) has been proposed for the prevention of new carious lesions and management of existing carious lesions in both primary and permanent dentition.¹⁵ Hence, in the present study, FAgamin (38% SDF solution), Tedequim, Argentina was used.

In pediatric patients, primary teeth are vital for the growth and development of jaws and face as well as essential for normal eruption and growth of the permanent teeth.¹² Primary teeth are more susceptible to dental caries than permanent teeth by virtue of the structural differences between the two.¹⁶ Hicks et al. have stated that in primary teeth, the enamel is less mineralized and has more organic content as compared to permanent teeth, rendering the former more susceptible to caries. Once established in the enamel of primary teeth, caries progresses to the underlying dentin at a faster rate as compared to permanent teeth. Also, the diffusion coefficient is greater in primary enamel and hence it is more susceptible to demineralization than the permanent enamel.¹⁷

Hence, the present study was carried out with the objective of analyzing the remineralization potential of SDF, CPP-ACP, and FV on the enamel surface of both primary and permanent teeth.

MATERIALS AND METHODS

Around 120 over-retained primary anterior teeth and 120 premolars which were extracted for orthodontic purposes, were collected for this study. Sound and caries-free teeth, lacking any signs of decalcification, cracks, or developmental defects were included in the study.

Preparation of Enamel Blocks for Microhardness Testing

Using a tooth-sectioning diamond disk, a transverse section was made to separate the crown portion of the tooth from its root portion. The intact enamel area on the buccal/labial side of the crown portion was sectioned longitudinally to prepare enamel blocks which were then embedded in the acrylic resin. After polishing the top surface using 400, 800, and 1,000 grit emery paper, a 4 × 4 mm window was created on the buccal/labial surface of the enamel by using adhesive tape and acid-resistant nail varnish.

Sample Distribution

Samples were distributed into groups and subgroups as follows:

Group I: Primary teeth (n = 120).
Subgroup IA: Included 30 primary teeth on which no agent was applied (control).
Subgroup IB: Included 30 primary teeth treated with SDF.
Subgroup IC: Included 30 primary teeth treated with CPP-ACP.
Subgroup ID: Included 30 primary teeth treated with FV.
Group II: Permanent teeth (n = 120).
Subgroup IIA: Included 30 premolars on which no agent was applied (control).
Subgroup IIB: Included 30 premolars treated with SDF.
Subgroup IIC: Included 30 premolars treated with CPP-ACP.
Subgroup IID: Included 30 premolars treated with FV.

Experimental Design

Surface microhardness (SMH) of the prepared enamel blocks was tested with the microvickers hardness testing machine (HM series, Mitutoyo, Europe) by applying a load of 25 gm force or 245 N to the enamel surface for five seconds. Vickers hardness number (VHN) was assessed at random, equally spaced at five different points, and a mean value was calculated and recorded as a baseline for each sample (denoted by SMH).

Silver diamine fluoride (SDF), CPP-ACP, FV, and distilled water (control) were applied only on the exposed window (4 × 4 mm). The agents were applied according to the manufacturer’s instructions. Using a microbrush, two-three drops of SDF solution were applied to the enamel blocks in subgroups IB and IIB. For the subgroups IC and IIC, a generous layer of CPP-ACP was applied using a microbrush. The subgroups ID and IID were treated with a thin layer of FV using a microbrush. Thereafter, all the prepared samples were washed with distilled water for 30 seconds and lightly dried with absorbent paper.

After the treatment, every sample was subjected to a pH cycling regime for 7 days.¹⁸ The demineralizing solution was prepared using 2.0 mmol/L calcium, 2.0 mmol/L phosphate in 0.075 mol/L acetate buffer, and 0.02 µmol/mL fluoride at pH 4.7. The remineralizing solution was prepared using 1.5 mmol/L calcium, 0.9 mmol/L phosphate, 150 mmol/L KCl in 0.1 mol/L tris buffer, and 0.03 µmol/mL fluoride at pH 7.0

The enamel blocks were kept in the demineralizing solution for 3 hours, 35.5 mL per block, and in the remineralizing solution for 21 hours, 17.75 mL per block for 5 days. To prevent the depletion or saturation of the solution, both demineralizing and remineralizing solutions were changed daily. After 5 days of repeated cycling, the enamel blocks were kept in the remineralizing solution for 2 days.

After the completion of pH cycling, the SMH of each sample was measured again using the same microhardness tester. Five indentations equally spaced from each other were made and a mean value was calculated and recorded (denoted by SMH₁). The percentage change in SMH (%SMHC) was also calculated in (Fig. 1) for all the groups and subgroups (namely, IA, IB, IC, ID, IIA, IIB, IIC, and IID) as follows:

Fig. 1: Percentage of SMH loss (%SML) for subgroups IA, IB, IC, ID, IIA, IIB, IIC, and IID

%SMHC = [(SMH₁–SMH)/SMH] × 100

herein,

%SMHC: Percentage change in SMH.
SMH₁: Enamel SMH after treatment and pH cycling.
SMH: Enamel SMH at baseline.

Statistical Analysis

The data were entered in Microsoft Excel 2007 and analyzed using the Statistical Package for the Social Sciences statistical software 19.0 version. The descriptive statistics included mean and standard deviation. The level of significance was fixed at 5%. The intergroup comparison for the difference in mean scores between independent groups was done using the unpaired/independent t-test and one-way analysis of variance (ANOVA).

RESULTS

In the present study, the mean baseline SMH was within the normal range of enamel hardness (Tables 1 and 2). As seen in Table 1, the percentage loss of SMH for primary teeth, was highest in the subgroup IA, followed by subgroups IC, IB, and ID. The difference was statistically significant when analyzed using one-way ANOVA at p ≤ 0.001. In permanent teeth (Table 2), the highest percentage of SMH loss was noted for subgroup IIA, followed by subgroups IIC, IIB, and IID.

**Table 1:** Comparison of SMH changes after treatment with the agents for group I (primary teeth); one-way ANOVA
Subgroups (n = 30)	Baseline (SMH) (VHN)	Treatment (SMH₁) (VHN)	Percentage loss (%SML)	F value	p-value
IA (control)	311.52 ± 42.324	170.88 ± 7.27	43.992 ± 10.870	8.230	0.001 (significant)
IB (SDF)	318.16 ± 25.533	217.45 ± 4.66	31.325 ± 17.069
IC (CPP-ACP)	303.23 ± 31.490	194.95 ± 5.75	35.692 ± 11.829
ID (FV)	311.53 ± 30.735	223.88 ± 5.61	27.579 ± 13.294

CPP-ACP, casein phosphopeptide-amorphous calcium phosphate; FV, fluoride varnish; SDF, silver diamine fluoride; SMH, surface microhardness at baseline; SMH₁, surface microhardness after treatment and pH cycling regime; %SML, percentage loss of surface microhardness

**Table 2:** Comparison of SMH changes after treatment with the agents for group II (permanent teeth); one-way ANOVA
Subgroups (n = 30)	Baseline (SMH) (VHN)	Treatment (SMH₁) (VHN)	Percentage loss (%SML)	F value	p-value
IIA (control)	318.702 ± 36.604	250.002 ± 51.750	21.6637 ± 13.051	2.069	0.108
IIB (SDF)	314.332 ± 63.457	265.232 ± 69.214	13.8126 ± 22.320
IIC (CPP-ACP)	310.112 ± 27.620	258.002 ± 35.049	16.8953 ± 7.578
IID (FV)	314.922 ± 37.914	272.652 ± 47.098	13.0256 ± 13.002

CPP-ACP, casein phosphopeptide-amorphous calcium phosphate; FV, fluoride varnish; SMH, surface microhardness at baseline; SMH₁, surface microhardness after treatment and pH cycling regime; %SML, percentage loss of surface microhardness; SDF, silver diamine fluoride

The results depicted in Table 3 showed a significantly greater percentage loss of SMH for subgroup IA as compared to subgroup IIA. The percentage loss of SMH was significant in subgroup IB as opposed to subgroup IIB (Table 3). The percentage reduction of SMH was significantly greater in subgroup IC in comparison to subgroup IIC (Table 3). In subgroup ID, the percentage loss of SMH was significantly more when compared to subgroup IID (Table 3). The difference between the subgroups was statistically significant when analyzed using an independent t-test at p ≤ 0.05.

**Table 3:** Intergroup comparison between subgroups IA and IIA, IB and IIB, IC and IIC, and ID and IID
Subgroups (n = 30)	Baseline (SMH) (VHN)	Treatment (SMH₁) (VHN)	Percentage loss (%)	F value	p-value
IA	311.52 ± 42.324	170.88 ± 7.27	43.992 ± 10.870	7.201	0.001 (significant)
IIA	318.702 ± 36.604	250.002 ± 51.750	21.6637 ± 13.051	7.201	0.001 (significant)
IB	318.16 ± 25.533	217.45 ± 4.66	31.325 ± 17.069	3.478	0.001 (significant)
IIB	314.332 ± 63.457	265.232 ± 69.214	13.812 ± 22.320	3.478	0.001 (significant)
IC	303.23 ± 31.490	194.95 ± 5.75	35.692 ± 11.829	3.897	0.001 (significant)
IIC	310.112 ± 27.620	258.002 ± 35.049	16.8953 ± 7.578	3.897	0.001 (significant)
ID	311.53 ± 30.735	223.88 ± 5.61	27.579 ± 13.294	6.897	0.001 (significant)
IID	314.922 ± 37.914	272.652 ± 47.098	13.0256 ± 13.002	6.897	0.001 (significant)

SMH, surface microhardness at baseline; SMH₁, surface microhardness after treatment and pH cycling regime

DISCUSSION

Under experimental conditions, microhardness testing is the most widely used method to determine and quantify the mineral changes in enamel as it provides indirect evidence of mineral loss or gain.¹⁹ It is a rapid and easy method of determining microhardness that requires only a small area of the surface of the specimen to be tested.²⁰^-²² In this study, Vickers microhardness testing was used to determine the SMH.

Accurate measurement of SMH by Vickers microhardness test can be made on flat surfaces only. Therefore, the prepared enamel blocks were polished and smoothened using 400, 800, and 1000-grit emery paper to achieve a flat and homogenous surface.²³^-²⁵ SMH of all the prepared enamel blocks was tested and recorded as a baseline. This was done so as to select the blocks in the same SMH range.²⁴^,²⁵ Hence, the changes seen in the SMH after treatment with agents from its respective baseline values surmised that these changes are attributable to the effect of these agents applied.

To help determine the mineral changes and assess the remineralization potential of FV, CPP-ACP, and SDF, all the samples were subjected to a pH cycling model with the purpose of simulating the dynamic cycles of demineralization and remineralization as seen in the oral environment.²⁴^-²⁶ Addition of fluoride at low concentrations in both the solutions helped preserve the enamel surface for microhardness determination. Also, these fluoride concentrations mimicked the fluoride levels found in saliva after regular tooth brushing or with regular consumption of fluoridated water.¹⁸ Fresh demineralizing and remineralizing solutions were prepared daily to avoid the collection of enamel dissolution products or saturation of both solutions. Enamel blocks were subjected to a pH cycling regime for 5 days daily and then allowed to remain in the remineralizing solution for 2 days. The latter is advocated to preserve the enamel surface for microhardness determination at the end of the experiment.¹⁸^,²³^,²⁵^,²⁷

After completion of the pH cycling, the SMH of each enamel block was measured again (SMH₁) and compared with the SMH at baseline for both groups I and II. Various authors had stated that there is a high correlation between the percentage of mineral loss measured by using microhardness techniques and the progression of net remineralization.²⁸^,²⁹

The results from Table 1 showed a statistically significant difference in the SMH between the control group and the treatment groups at the end of the experimental design. This depicted that with the application of SDF, CPP-ACP, and FV, the net remineralization process in primary teeth was significantly enhanced in comparison to when no agent is applied. In primary teeth, the least reduction in SMH was observed for FV, depicting its maximum remineralizing potential in comparison to SDF and CPP-ACP. Similarly, Babu et al. demonstrated that FV application significantly increased the enamel SMH.²¹ The results of this study are in agreement with the findings of Oliveira et al. and Bandekar et al. who found that FV had superior remineralizing potential compared to CPP-ACP.¹⁹^,³⁰

In the present study, FV exhibited more remineralizing potential in comparison to SDF. This could be because the effect of SDF is more pronounced on dentinal carious lesions.¹⁴ The silver in SDF inhibits the dentin collagenase, cathepsin B and K that help mediate the degradation of collagen during a caries attack. Silver ions have an affinity for proteins due to their large ionic radius, adding to their strong inhibitory effect on matrix metalloproteins and cathepsins.³¹ Since the protein content, carbonates, and phosphates in enamel are much less in comparison to dentin, SDF does not have this protective effect on enamel.¹²^,²⁵ However, in contrast to the findings of the present study, Mohammadi and Far observed that in primary teeth, SDF, and FV showed no statistically significant difference in percentage mineral loss.²⁵

In the present study, CPP-ACP showed a significantly lesser percentage reduction of SMH in primary teeth than in the control. Similar to these findings, Krishnan et al. observed a statistically significant increase in enamel SMH on using GC Tooth Mousse (containing CPP-ACP) than when no remineralizing agent was applied to primary teeth.²⁴

The results from Table 2 indicate that both SDF and FV are equally effective as remineralizing agents in permanent teeth. The results of the present study are in agreement with the clinical findings of Liu et al.³² Another study comparing the anticaries effect of SDF and FV concluded that 38% SDF aids in higher uptake of fluoride in young permanent molars, thereby significantly reducing carious lesions in comparison to FV.³³

As seen in Table 2, CPP-ACP exhibited a greater percentage loss of SMH than SDF and FV in permanent teeth. The control group showed the highest percentage loss of SMH in comparison to all other subgroups depicting that, under experimental conditions, CPP-ACP was efficacious in remineralizing enamel but not as effectively as FV and SDF.

The results of the intergroup comparison of samples on which no agent was applied (control) in both primary and permanent teeth demonstrated that without the application of a remineralizing agent, a significant decrease in enamel SMH was observed (Table 3). The percentage loss of SMH was notably higher for the primary teeth in comparison to the permanent teeth. This could be attributable to the lesser mineral content of enamel in primary teeth. The enamel in primary teeth is thinner, that is, about half of the thickness of enamel in permanent teeth. The width of the prisms was also found to be varied in primary and permanent teeth, that is, 4–7 and 6–10 µm, respectively. Thus, enamel in permanent teeth has a higher density of enamel rods than the enamel in primary teeth. Owing to these differences, the response to demineralization and remineralization cycles simulating the oral environment, without the application of a remineralizing agent may vary in primary and permanent teeth.

In the present study, SDF showed a lesser percentage loss of SMH in permanent teeth as compared to primary teeth (Table 3). Similar studies have demonstrated that the application of 38% SDF has a caries preventive effect on young permanent molars in children. This is likely due to the higher uptake of fluoride by young permanent molars that are more porous and not fully mineralized.¹²^,³⁴^,³⁵ On the contrary, Llorda et al. reported that SDF was more efficacious in arresting carious lesions in permanent teeth (100%) than the primary teeth (55.6%).³³

Significant reduction in SMH and mineral loss observed after application of CPP-ACP in primary teeth portrays that although CPP-ACP promotes remineralization in both primary and permanent teeth, its effect is more pronounced on permanent teeth. Studies have shown an increase in enamel microhardness and a reduction of white spot lesions following the application of CPP-ACP.¹⁰^,³⁶^,³⁷ Further studies, however, are required to compare the effect of CPP-ACP on both primary and permanent teeth.

Systematic reviews show that FVs are efficacious in the prevention of dental caries in 33% of primary teeth and 46% of permanent teeth.³⁸ Several other studies had also shown a significant reduction in carious lesions following the use of FV in both primary and permanent teeth.¹⁰^,³⁹^,⁴⁰ This can be attributed to the formation of calcium fluoride that acts as a slow-releasing reservoir of fluoride ions. Under the cariogenic challenge, the dissolution of calcium fluoride occurs and fluoride ions are released that diffuse into the subsurface of the enamel to form fluorapatite crystals which in turn reduces tooth solubility and promotes enamel remineralization.²⁹

However, in the present study, FV exhibited a significantly greater percentage reduction of SMH in primary teeth than in permanent teeth (Table 3). This is likely due to the higher uptake of fluoride enamel in young permanent teeth. It has been established that young enamel is semipermeable and allows the passage of small molecules through the pores in enamel crystals. This permeability of enamel decreases with age.⁴¹

Limitations of the Study

The oral cavity is a complex entity and several factors influence the remineralization and demineralization cycles in the oral environment. Although the pH cycling regime was used in the present study to best simulate the oral environment, it is difficult to attain the oral conditions in vitro. It is also not possible to adequately simulate the topical use and clearance of the remineralizing agents from the oral cavity under in vitro conditions. Hence, conditions similar to the oral environment may not have been reproduced which might have affected the results. Moreover, the sample size in the present study was limited. Hence, studies with larger sample sizes are needed to further compare the remineralizing effect of FV, CPP-ACP, and SDF on both primary and permanent dentition.

CONCLUSION

Within the limitations of the present study, it can be concluded that SDF, CPP-ACP, and FV depicted remineralizing potential in both primary and permanent teeth. However, further long-term clinical trials and research are required to support their enamel remineralizing efficacy.

ORCID

Samarpreet Kaur https://orcid.org/0000-0002-9808-9570

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