Assessing the Impact of Treatment Duration and Demineralization on Enamel Fluoride Uptake

Abstract

Objectives

This study aimed to determine the effect of time span between exposing the enamel to calcium lactate and sodium fluoride and of a demineralized surface on the uptake of alkali-soluble fluorides.

Materials and Methods

A total of 20 non-carious human wisdom teeth, were used. First 10 teeth, were cut into four slabs each and allocated to one of four groups: two treatment groups A) Calcium lactate followed by sodium fluoride with minimal delay (just drying between the two treatments); B) Calcium lactate followed by sodium fluoride with a delay of one hour; one of two control groups C) Sodium fluoride, as the positive control; D) Negative control group without treatment. The other 10 teeth were used for the second part of the research, each tooth was cut into 4 slabs and divided into one of two groups - the first group underwent two weeks of demineralization liquid treatment, the second group, as a control, underwent two weeks in deionized water. After two weeks, all slabs were treated with calcium lactate and followed with sodium fluoride 226ppm F-.Enamel fluoride uptake was determined by the fluoride extraction method by Caslavska and analyzed using a fluoride ion-specific electrode.

Results

Enamel fluoride uptake showed no statistical difference regarding various durations of treatment between exposing enamel slabs to calcium lactate and sodium fluoride, as well as demineralization of the surface of the enamel.

Conclusions

Time span between exposing enamel to calcium lactate and sodium fluoride and the demineralization process did not significantly affect the enamel uptake of alkali-soluble fluorides.

Introduction

Dental caries, a complex disease caused by the interaction of dietary sugars, oral bacteria, and the host's oral environment, continues to pose a major public health challenge worldwide. Despite progress in dental care, a significant proportion of the population still suffers from caries, resulting in discomfort, tooth loss, and a lower quality of life. Implementing effective preventive strategies is crucial to reduce the incidence of this condition and improve overall oral health (1-3).

Preventive strategies are essential to protect against dental caries and maintain oral health. The use of fluoride is a widely recognized approach to strengthen enamel, making it more resistant to demineralization while also aiding in the remineralization process (4-8). Fluorides help prevent dental caries by suppressing the activity of bacterial enzymes that produce acids and encouraging the development of fluorapatite crystals, which are more resistant to acid dissolution than hydroxyapatite. Recent research has examined the potential for enhancing fluoride’s cariostatic effects by combining it with other agents. (9).

One such agent is calcium, which has been shown to work synergistically with fluoride to enhance enamel remineralization as well, thereby offering better protection against caries (10, 11). Researchers have explored how calcium application prior to fluoride exposure can enhance fluoride uptake by dental enamel, thereby increasing the overall efficacy of fluoride treatments in preventing dental caries. This approach leverages calcium's chemical affinity for fluoride, potentially leading to better protection against tooth decay (12). However, the optimal time span between applying calcium and fluoride treatments to maximize fluoride uptake remains unclear.

Studies have shown that using a calcium pre-rinse before applying a low-concentration fluoride rinse (0.05% NaF, 226 ppm F) can significantly increase fluoride levels in saliva and dental biofilm, reduce fluoride toxicity, minimize mineral loss, and promote the formation of calcium fluoride-like deposits in dental plaque (13).

This study aimed to investigate the effect of demineralized surface and the time span between exposing enamel slabs to calcium lactate and sodium fluoride on the uptake of alkali-soluble fluorides.

Null Hypothesis (H0): The time span between calcium and fluoride exposure and the demineralization process does not significantly alter the enamel's ability to uptake fluoride when compared to the control.

Materials and methods

All experimental procedures in this blind and randomized, in vitro study were carried out in accordance with the Declaration of Helsinki, which outlines ethical principles for medical research involving human subjects. The study received approval from the Ethics Committee of the School of Dental Medicine, University of Zagreb, under protocol number (05-PA-30-17-4/2023).

The study included 20 human non-carious wisdom teeth extracted for orthodontic reasons, from Department of Oral Surgery of the School of Dental Medicine, from patients aged 21 to 30. All patients signed written informed consent of donation of biological materials to scientific purposes, with prior approval of the Ethics Committee. After the extraction, teeth were mechanically cleaned from the debris, disinfected with 5% NaOCl, and stored in 0,25% chloramine solution, in sealed cup, refrigerated on 4 – 7 °C until the experimental procedures. Each tooth was brushed with manual toothbrush and universal polishing fluoride-free paste, and thoroughly washed with tap water and deionized water afterwards. Each tooth crown was inspected for sound and smooth enamel surface and then cut and divided into four slabs marked with a number and letter (a, b, c, d) and randomly allocated into one of the treatment groups. Each slab was covered in laboratory dental wax (Iskra ltd chemical industry, Sv. Ivan Zelina, Croatia), leaving only the upper enamel surface open. Orthodontic wire was placed at the back of each slab for easier handling and covered with transparent neutral varnish to prevent any interaction with experimental solutions. Experimental solutions used in this study included calcium lactate 150mM (Farmalabor, GLJZ - Croatia), sodium fluoride 226 ppm F (Farmalabor, GLJZ - Croatia), and deionized water (E&H Renfert, Singen, Germany). The solution used for demineralization purposes was as described by Buskes et al. 1985)

Enamel fluoride uptake subject to the duration of the treatment

The first treatment group “a” was pretreated with calcium lactate 150mM followed by sodium fluoride 226 ppm F (pH – 7.6) with minimal delay (just a short drying between the two treatments). The second treatment group “b” was pretreated with calcium lactate 150mM followed by sodium fluoride 226 ppm F with a delay of one hour (leaving time for calcium to eventually adhere or penetrate the enamel) between those two treatments. The third treatment group “c” was treated with sodium fluoride only, as the positive control. The last group “d” was the negative control group – the group without treatment. Treatments with calcium lactate and/or sodium fluoride were done by slow shaking in 5 mL of selected solution and lasted for 5 minutes each.

After the treatments, the slabs were placed in polystyrene plastic cups with lids and alkali-soluble fluoride extraction was conducted by immersing the enamel slabs in a 1 M KOH solution (0.25 ml per sample) for 24h under agitation (160 rpm) at room temperature, using the method outlined by Caslavska (14). After the 24h period, all extracts were buffered with 0.5 ml of TISAB III solution containing 1.0 M HCl, and the samples were carefully removed from the tubes

Fluoride uptake in the solutions were measured using a fluoride ion-selective electrode (ISE) (Thermo Scientific™ Orion™), coupled with an ion analyzer (Orion Dual Star; Thermo Scientific) that had been previously calibrated using fluoride standards prepared to match the samples. Fluoride uptake was expressed in micrograms of fluoride per square centimeter (µg F/cm²) of enamel surface. The surface area of each slab was measured prior to treatment. The study design of the research is schematically presented in Table 1.

Table 1. Schematic representation of the study design of exploring the comparative efficacy of calcium pre-treatment in modulating enamel reactivity with the time span from exposure to calcium lactate to fluoride.

Group a           (n = 10)	Group b           (n = 10)			Group c           (n = 10)			Group d           (n = 10)
Pretreated with calcium lactate followed by sodium fluoride with minimal delay (just drying between the two treatments)	Pretreated with calcium lactate followed by sodium fluoride with a delay of one hour (leaving time for calcium to eventually adhere or penetrate the enamel)		Treated with sodium fluoride only, as the positive control					Negative control group – the group without treatment.
Fluoride extraction was conducted by immersing the enamel slabs in a 1 M KOH solution for 24h under agitation at room temperature. After the 24h period, all extracts were buffered with TISAB III solution containing 1.0 M HCl,
The extracts were analyzed using fluoride ion-specific electrode

Enamel fluoride uptake subject to demineralization of the surface

The second part of the study was conducted using another 40 prepared enamel slabs divided into two groups - the first group of 20 slabs underwent the demineralization process for two weeks in demineralization liquid (procedure described by Buskes et al.) (15). The second group of 20 slabs were left in deionized water for two weeks and served as a control. After two weeks, all slabs were treated with calcium lactate and followed with sodium fluoride 226ppm F-. After the treatments, alkali-soluble fluoride extraction was conducted in the same way as described in the first part of this study.

All collected data were entered, imputed and arranged in a Microsoft Office Excel file. The normality of data distribution was checked, and alkali-soluble fluoride data was transformed using log₁₀ Wilcoxon Matched Pairs Test and Friedman ANOVA was employed to analyze the impact of both the substrate and treatments. The study design of this part of the research is schematically presented in Table 2.

Table 2. Schematic representation of the study design of exploring the comparative efficacy of calcium pre-treatment in modulating enamel reactivity during the enamel demineralization process.

Group I           (n=20)	Group II           (n=20)
Treated for two weeks in demineralization liquid. After two weeks, all slabs were treated with calcium lactate and followed with sodium fluoride 226ppm F-	The second group was two weeks in deionized water, as a control. After two weeks, all slabs were treated with calcium lactate and followed with sodium fluoride 226ppm F-
Fluoride extraction was conducted by immersing the enamel slabs in a 1 M KOH solution for 24h under agitation at room temperature. After the 24h period, all extracts were buffered with TISAB III solution containing 1.0 M HCl,
The extracts were analyzed using fluoride ion-specific electrode

Results

Treatment duration: In all comparisons of observed enamel slab groups (0.635±0.443vs0.661±0.838vs 0.382±0.18vs 0.7052±0.241μg F/cm2, for groups Ca lactate with minimum delay - NaF, Ca lactate with delayed NaF, NaF only, no treatment) there were no statistically significant differences observed between any pair of treatment groups as indicated by the p-values being greater than 0.05. The only statistically significant difference was observed between enamel slabs treated with sodium fluoride only(group c) and those in the negative control group(group d), with a p-value of 0.028, which was expected effect of sodium fluoride.

The mean value and standard deviation (SD) of alkali-soluble fluoride after evaluating the impact of the time span between exposing enamel slabs to calcium lactate and sodium fluoride are presented in Figure 1.

Figure 1.

The effect of treatment duration between exposing enamel slabs to calcium lactate and to sodium fluoride, on enamel uptake of alkali soluble fluorides.

Demineralization process: The comparison between the control group and demineralized samples showed that the demineralization process did not significantly alter the enamel's ability to uptake fluoride when compared to the control (15.1696±4.1059 vs 12.9853 ± 6.0061μg F/cm2) since there was no significant difference found. The mean value and standard deviation (SD) of alkali-soluble fluoride after investigating the comparative efficacy of calcium pretreatment, followed by fluoride, all following the demineralization process of enamel are presented in Figure 2.

Figure 2.

Impact of demineralization process on enamel fluoride uptake.

Discussion

Fluoride plays a vital role in preventing dental caries, which remains the most prevalent preventable chronic disease globally. Its key function lies in halting the process of demineralization and enhancing remineralization of tooth enamel. Recognizing the growing burden of dental caries, the World Health Organization (WHO) promotes fluoride use as a cornerstone of its Global Oral Health Strategy for the 21st century. This approach includes population-wide fluoridation measures, such as water fluoridation, which WHO endorses as one of the most cost-effective methods to reduce tooth decay. Additionally, the regular use of fluoride-containing toothpastes complements these efforts, particularly in areas with limited access to dental care (16, 17).

The present study investigated the efficacy of calcium pretreatment followed by fluoride application on enamel fluoride uptake, focusing on the impact of the demineralization process of the surface and the treatment period.

The results showed in all comparisons of observed enamel slab groups (0.635±0.443vs0.661±0.838vs 0.382±0.18vs 0.7052±0.241μg F/cm2, for groups Ca lactate with minimum delay - NaF, Ca lactate with delayed NaF, NaF only, no treatment) there were no statistically significant differences observed between any pair of treatment groups. This is consistent with previous research (18), which also reported no significant differences in fluoride uptake with varying time intervals between calcium and fluoride treatments. Both studies suggest that the timing of calcium application relative to fluoride does not critically influence fluoride uptake. These findings offer valuable insights for clinicians in preventive dentistry, thus emphasizing that the timing of fluoride application is less important than the use of calcium pretreatment and the correct application sequence. Such confirmed results are particularly beneficial for patients at higher risk of caries and non-cooperative patients who cannot apply fluoride over a specific period, providing a more adaptable approach to caries prevention.

However, our study found a statistically significant difference between enamel slabs treated with sodium fluoride only (group C) and those in the negative control group (group D), with a p-value of 0.028. This expected result aligns with the study by Mellberg and Chomicki (19), which demonstrated a significant increase in fluoride uptake in enamel treated with sodium fluoride compared to untreated controls. The effectiveness of sodium fluoride in promoting enamel remineralization is well-documented also in the literature (4), (20).

On the demineralization part of the study, findings showed no significant difference in fluoride uptake between the control group and the demineralized samples. This suggests that the demineralization process did not significantly alter the enamel's ability to incorporate fluoride. This result is consistent with the study by González-Cabezas et al. (2012) (21), which also found that demineralized enamel can effectively uptake fluoride, supporting the potential of fluoride treatments in re-mineralizing early carious lesions. We expected an increased uptake due to a significant lack of minerals in demineralized enamel, but this was not the case since the uptake is regulated by the presence of calcium ions, the integrity of the enamel surface, and the concentration of fluoride in the surrounding environment. Calcium ions are essential for forming stable calcium-fluoride complexes on the enamel surface (22).

These findings hold important clinical relevance. They indicate that sodium fluoride on its own is effective at increasing fluoride uptake in enamel, and the timing of calcium lactate application does not play a major role in this process. This insight can be used to refine fluoride treatment protocols in dental practice, maximizing remineralization outcomes for patients, which is key for long-term prevention and sustained oral health. Modern fluoride protocols in dentistry aim to prevent dental caries by using various ion-releasing dental materials and fluoride products that cater to different patient needs (23, 24). Common fluoride protocols include fluoride toothpastes, fluoride mouth rinses, fluoride gels and varnishes, and fluoridated water (25, 26). Silver diamine fluoride (SDF) is a new protocol, which is used to arrest active dental caries. It is commonly used in patients who cannot undergo traditional restorative procedures, especially in children and elderly populations (27).

This study has several limitations. Since it was conducted in vitro, the results may not entirely reflect the conditions of the oral cavity. Factors such as saliva flow, oral microbiota, and dietary habits can affect fluoride absorption and enamel remineralization in vivo (28). Additionally, the study assessed the treatments over a short duration, and further long-term research is needed to evaluate the sustainability and enduring effects of these treatments on enamel remineralization.

Conclusions: The time span between exposing enamel slabs to calcium lactate and sodium fluoride did not significantly affect enamel uptake of alkali-soluble fluorides. Based on these findings, it is suggested that the sodium fluoride products may be applied immediately after calcium lactate. Findings from demineralization processes indicate that the demineralization process did not significantly alter the enamel's ability to uptake fluoride when compared to the control.