Comparative evaluation of different re-mineralizing agents for enamel protection after pH-cycling

Introduction

Health promotion programs should focus on comprehensive strategies, providing tools for self-management, fair availability of information, and promoting healthy behaviors [1]. Frequent brushing, flossing, a balanced diet, and dental examinations can prevent oral health issues and reduce the risk of disease developing. Recent dental caries therapies are shifting towards preventative strategies [2].
Whenever a pH of bio-film falls under 5.5 in value, the enamel gets de-mineralized, which causes calcium and phosphate loss and a white lesion occurring beneath the surface. If an elevated or neutral pH can be reached, the cycle may be reversed [3]. Saliva’s buffering effect that involves calcium and phosphate ions return to de-mineralized surface, is the natural means of reducing de-mineralization activity [4]. Contemporary re-minera­lization strategies aim to restore lost ions directly to de-mineralized enamel surfaces. Despite advancements in bio-materials, an ideal re-mineralizing agent for long-lasting effect and effective re-mineralization remains unavailable [5].
Recent developments in nano-technology have led to a reduction in particle size as well as changes in form, producing more bio-active calcium phosphate compounds with greater capacity to penetrate de-mineraliz­ed porous area, operating as re-mineralizing agents [6]. As re-mineralizing agents, contemporary bio-materials, such as nano-hydroxyapatite (N-HAp) and nano-bioactive glass (N-BAG), were created. N-BAG, containing calcium, sodium, phosphorous, and silica, is a synthesized mineral combination that can successfully enhance re-mineralization. When tiny N-BAG particles are subjected to moisture, millions of mineral ions are released, forming a significant coating of hydroxyapatite on enamel and dentin surfaces [7]. Furthermore, N-HAp is regarded as one of the greatest bio-compatible and bio-active substances, and it received a lot of attention in the medical and dental fields in recent years. The use of N-HAp directly for bio-mimetic re-minerali­zation of de-mineralized enamel is of particular interest in dentistry research due to its morphological and chemical resemblance to enamel minerals [8].
Moreover, deacetylation of chitin produces chitosan, a non-toxic and bio-compatible polymer. It has antibacterial properties and deposits on the bacterial cell wall, encouraging re-mineralization of initial dental caries because of its positive charge [9]. N-HAp may help promote re-mineralization, if it is used with another effective non-fluoride substance, such as chitosan, which does not interfere with N-HAp. Complete re-minerali­zation might then be anticipated [10].
Indeed, nano-silver nano-particles (NS) can enter their cell walls by coming into touch with bacteria, disrupting the reproduction of DNA, and preventing cellular respiration by reducing the oxidation of lipids. Nano-particles increase bactericidal action, generate free radicals, induce oxidative stress, and liberate silver ions when they are within the bacterial cell [11]. Because of this, adding NS to fluoride varnishes is a practical way to incorporate antibacterial and preventative qualities without discoloring the teeth. Nano-silver fluoride (NSF), a novel formulation, has been shown as a potent anti-caries agent [12]. Furthermore, dental hard tissues (i.e., dentin or enamel) are subjected to multiple processes of de-mineralization and re-mineralization in pH-cycling models. One significant benefit of pH-cycling models is that these combined studies are made to replicate the dynamics of mineral uptake and loss, which influence caries development [13].

Objectives

This research aimed to compare the effect of three different re-mineralizing agents, such as N-BAG, chitosan-loaded N-HAp (CS/N-HAp), and NSF on the prevention ability of sound enamel from de-mineralization after pH-cycling. The null hypothesis was that the three re-minera­lizing agents have the same prevention potential against enamel de-mineralization after pH-cycling.

Material and methods

Three re-mineralizing agents, including 10% N-BAG, CS/N-HAp, and NSF, were utilized in the current research to investigate their capability for enamel’s protection against de-mineralization, and for inducing re-mineralization. Additionally, two solutions were used, such as de-minera­lization solution (Coca-Cola; pH = 2.5) and artificial saliva (pH = 7) as a storage medium for the premolars.

Sample size calculation

According to a previous study conducted by Said et al. [12], a minimum total sample size of 40 samples was sufficient to detect the effect size of 0.32, with a power of 1–β = 0.90, and significance probability level of p ≤ 0.05, to compare energy dispersive X-ray (EDX) results between studied groups. According to sample size calculations, there was a 90% chance of correctly rejecting the null hypothesis of no significant effect, if each sub-group consist of 10 samples. The sample size was calculated according to G*Power software version 3.1.9.7.

Ethical approval

Ethical Committee of the Faculty of Dental Medicine, Al-Azhar University, Cairo, Egypt, provided clearance for this study, with reference approval number of 639/3626.

Teeth selection

A total of 40 human premolars, which were freshly extracted for orthodontic purposes, were gathered. Inclusion criteria were: 1) intact teeth; 2) non-caries teeth; 3) permanent maxillary premolars with complete root formation; and 4) no developmental and formative anomalies. Exclusion criteria were: 1) carious teeth; 2) previous restorations and fractures/cracks; 3) prior endodontic treatments; and 4) presence of non-carious lesions, such as attrition, fluorosis, etc. The teeth were rinsed under tap water to remove any blood or debris, scaled to eliminate calculus and any remaining soft tissues, and then polished. To make sure the teeth utilized in the current research were free of any developmental or deformative flaws, they were verified with a 10× magnifying lens.

Teeth sample grouping

The premolars (n = 40) were equally divided into four main groups (n = 10) according to a re-mineralizing agent used. Group 1: N-BAG, group 2: CS/N-HAp, group 3: NSF, and group 4 (control group): no re-mineralizing agent used. The premolars were numbered (1-40) on their lingual surfaces using round bur with a high-speed contra-angle tool beneath copious amounts of water coolant to recognize their group number (group 1: 1-10, group 2: 11-20, group 3: 21-30, and group 4: 31-40). The entire crown surfaces were coated by double layers of nail varnish, except for a window (3 × 3 mm) on buccal surface. After that, all windows were assessed using energy-dispersive X-ray analysis (baseline data).

Preparation of re-mineralizing agents

All re-mineralizing agents were prepared by Nano Gate (Cairo, Egypt). The 10% N-BAG solution was prepared by dissolving 100 grams of N-BAG powder in 1 liter of distilled water. By adding a 2.5 grams of N-HAp powder into 10 ml of 2.5 mg/ml chitosan solution, a modified version of chitosan bio-glass material (manufacturing process outlined by Zhang et al. [14] was used to create chitosan-loaded nano-hydroxyapatite. Similarly, the NSF solution was prepared by dissolving 100 grams of NSF powder in 1 liter of distilled water [15].

pH-cycling regimen

For seven days, the specimens were exposed to a pH-cycling model to mimic normal pH variations found in oral cavity. Two occurrences of 3-hour de-mine­ralization in 200 ml of Coca-Cola and 18-hour incubation in artificial saliva were included in each cycle [16]. After removing from Coca-Cola, the premolars were rinsed with distilled water and patted dry with gauze. For every group and cycle, a fresh 320 ml can of Coca-Cola was used to ensure sufficient carbonation of the liquid. Also, Coca-Cola was stored at 20°C because the temperature could impact the results [17]. Three times daily, re-mine­ralizing protocol was performed both prior to and after the initial and secondary de-mineralization cycles. The specimens were carefully washed with de-ionized water for 60 seconds after subsequent stages [18]. Since the samples from the control group (group 4) were not treated with any re-mineralizing agent, the same pH-cycling processes were performed. Different containers were utilized for every group, and each day during every session, all solutions were replaced [16].

Application of re-mineralizing agents

Re-mineralizing agents were applied on the exposed enamel windows as follows: group 1 (N-BAG): few drops of N-BAG solution were applied with a micro-brush on the enamel surface for 4 minutes [19]. Group 2 (CS/N-HAp): utilizing a metal spatula, the white powder was combined with sterile saline solution (a single scoop of powder into a single drop of saline) on a glass slab to create a homogenous, thick paste. Then, the paste was applied with a sponge on the enamel surface, and kept for 3 mi­nutes. After that, the specimens were rinsed with de-ionized water for 20 seconds [14]. Group 3 (NSF): few drops of NSF solution were placed on the enamel surface using a micro-brush for 2 minutes. After that, the teeth were rinsed with deionized water for about 1 minute [20]. Group 4 (control group): the samples were not exposed to any re-mineralizing agent.

Testing procedures

Environmental scanning electron microscopy (ESEM) with a built-up EDX unit (ZEISS, EVO 15, UK) (Figure 1) was employed for the assessment of surface morphology. ESEM was utilized to evaluate the surface topography of the tested re-mineralizing agents. Images were captured at magnifications of 8000× for each group. Additionally, the assessment of mineral content (i.e., carbon, calcium, and phosphorous) was performed by EDX, and these assessments were accomplished at baseline before pH-cycling regimen and after pH-cycl­ing regimen.

Statical analysis

Utilizing normality tests, i.e., Kolmogorov-Smirnov and Shapiro-Wilk tests, the distribution of numerical data was examined for normality. For inter-group comparison among four groups, the results of EDX analysis (interactions on mean carbon, calcium, and phosphorous weight percentage) were statistically analyzed using one-way analysis of variance (ANOVA), followed by a post-hoc test. For intra-group comparison between the two measuring points (before and after pH-cycling), a paired t-test was used to compare between the two-time intervals. A significant threshold of p < 0.05 was established. IBM SPSS Statistics for Windows, version 23.0 (Armonk, NY: IBM Corp., USA) was employed for statistical analysis.

Results

Results of environmental scanning electron microscope

At baseline (before pH-cycling), the sound enamel displayed a smooth surface with some scratches and pits. After pH-cycling and application of re-mineraliz­ing agents (in groups 1-3), in comparison with their baseline data, some signs of erosion started to appear on the enamel, losing some of its smoothness. The ESEM results indicated that group 1 (N-BAG) showed the best preservation of enamel structure (Figure 2). In the group 2 (CS/N-HAp), slightly more signs of erosion started to appear on the enamel compared with group 1 (N-BAG), losing some of its smoothness and a slight fish scale appearance (Figure 3). While in the group 3 (NSF), the signs of erosion were more evident than in groups 1 and 2, with many areas appearing degenerated and some losing their structure, showing a very unsmooth surface (Figure 4).
After pH-cycling in group 4 (control group), the ESEM examination revealed signs of erosion, and showed the highest aggression compared with all groups with re-mineralizing agents. Most areas showed dissolution, indicating heavy de-mineralization; the samples lost their structure, showing an extremely rough surface with almost no areas of visible prisms (Figure 5). The ESEM results indicated that group 1 (N-BAG) demonstrated the best preservation of enamel structure, while group 4 (control group) displayed the most erosion.

Results of energy dispersive X-ray spectroscopy (EDAX)

The findings displayed that at baseline (before pH-cycling), there were no significant differences between all groups regarding the minerals (carbon, phosphorous, and calcium) (Table 1 and Figure 6). After pH-cycling, there was a significant increase in calcium and phosphorus levels, and significant decrease in carbon level. The results revealed significant differences in carbon and calcium/phosphorus levels between groups, with group 1 (N-BAG) showing a significantly higher range of calcium and phosphorus gain and carbon loss, when compared with other groups. While comparing group 2 (CS/N-HAp) with groups 3 (NSF) and 4 (control group), there were significant differences in carbon and calcium/phosphorus levels between groups, with group 2 (CS/N-HAp) displaying a significantly higher range of calcium and phosphorus gain and carbon loss. Comparison between groups 3 (NSF) and 4 (control group) demonstrated a significant difference in carbon and calcium levels, with group 3 (NSF) indicating a higher range of calcium/ phosphorus gain and carbon loss (Table 2 and Figure 7).
The comparison between before (baseline) and after pH-cycling outcomes revealed that there were significant differences between all groups. Group 1 (N-BAG) showed the greatest calcium and phosphorus levels, and the greatest carbon reduction, followed by group 2 (CS/N-HAp) and group 3 (NSF). While group 4 (control group) displayed some calcium reduction, minimal carbon decrease, and the lowest phosphorus gain (Table 3 and Figure 8).

Discussion

Mineral loss is a key phase in enamel de-mineralization, and bio-active materials can reduce de-mineralization caused by bacterial acids by gaining minerals [21]. Two types of de-mineralization/re-mineralization models are used in laboratory studies, including chemical and micro-biological models. In the present study, a chemical model was employed due to its simplicity of use, affordability, effectiveness, and experimental reliability [22].
Furthermore, it was demonstrated that using an acidic solution for seven days might cause a histological, clinical, and radiological distortion, which resembled an initial enamel lesion [23]. In the current research, after applying re-mineralizing agents to healthy enamel surfaces, the specimens were switched for seven days amongst de-mineralization and re-mineralization cycl­ing to replicate the oral environment’s continuous pH changes [20].
The 7-day pH-cycling model was utilized to simu­late fluctuating mineral loss and gain in the oral cavity, allowing for close modeling of pH variations linked to caries formation. The samples were then evaluated with ESEM and EDX units for surface morphology and mineral content (carbon, calcium, and phosphorous), respectively [10]. Coca-Cola was used as a de-mineralizing solution due to its high frequency usage as a soft drink beverage and low pH values, while also causing the highest effect of de-minerali­zation on the enamel. This was used twice daily for 7 minutes, mimicking the widespread use of acidic soft drinks [24].
The outcomes of the present research showed that, following pH-cycling in groups 1, 2, and 3 (application of re-mineralizing agents), there was a significantly higher mineral content of calcium and phosphate than at baseline and in group 4 (control group). This confirms that the bio-mimetic agents may improve the mine­ral content of early enamel lesions and re-mineralize them, resulting in more mineral gain [25]. Also, nano-sized particles, through their scaffolding function, can seal microscopic porosities in a de-mineralized region, thereby creating a new apatite layer by drawing calcium and phosphate ions from saliva [26]. In group 4 (control group), where no re-mineralizing agent was applied, there was a decrease in calcium and phosphate content when compared with baseline results; development of porous surfaces during SEM scanning was also observed. This may be because the acid causes minerals loss from the surface, which is consistent with the results of previous investigations [15, 18, 25].
The findings of SEM indicated that Coca-Cola application gradually affected the enamel, with resistance varying depending on a re-mineralizing agent used. Group 1 (N-BAG) showed the most resistance, followed by group 2 (CS/N-HAp) and group 3 (NSF), while group 4 (control group) presented minimal to no resistance, as no re-mineralizing agent was applied. The authors observed that Coca-Cola’s low pH value of 2.67 with phosphoric acid contains, lead to a high erosive effect in group 4 (control group) compared with other groups where re-mineralizing agents were applied [27]. Under acidic conditions, the balance of calcium and phosphate ions in saliva shifts from supersaturation to undersaturation, facilitating enamel de-mineralization. Re-mineralizing agents, such as N-BAG, restore this balance by supplying ions, which enhance enamel hardness and reduce structural porosity [28].
Following pH-cycling, EDAX tests in group 4 (control group) demonstrated a significant increase in carbonate, but a substantial decrease in calcium and phosphate ions. It is assumed that carbonate substitute calcium and phosphorus to create enamel that dissolves more readily. The crystal becomes unstable, and the rate of apatite dissolution increases when carbon ions substitute phosphate ions and, at elevated concentrations, hydroxyl ions [29]. This is consistent with Mahfouz et al. [30], who found that carbonate ions disrupt the hydroxyapatite structure of enamel.
Moreover, the outcomes of the current study show that the calcium and phosphate contents increased, while the carbon content decreased in groups 1-3 (re-mineralizing agents used). In order to fill the empty spaces of the enamel calcium crystals, a significant amount of Ca2+ and PO43– from the re-mineralization agents were drawn to the enamel surface by the calcium and phosphate ions in these types of re-mineralizing agents, which were able to enter the enamel pores and act as a template during the procedure of precipitation [31].
Group 1 (N-BAG) had a significantly largest rise in calcium and phosphate, followed by group 2 (CS/N-HAp) and group 3 (NSF), and there were significant diffe­rences between all groups. Also, the ESEM results indicated that group 1 (N-BAG) showed the best preservation of enamel structure. The null hypothesis was rejected, as the three re-mineralizing agents did not have the same prevention potential against enamel de-mineralization after pH-cycling.
The superior performance of N-BAG may be due to its ability to rapidly release calcium, phosphorus, and silicate ions in acidic conditions. These ions help neutralize their environment and enhance enamel re-mine­ralization [32], with the alleged increased mineral content, lower particle size, and prolonged accessibility of N-BAG to retain its minerals for a longer period of time [33]. This may also be related to the capacity of N-BAG to react with saliva-induced calcium, phosphate, and silicate ion breakdown at a glass surface and precipitate a polycondensed silica-rich layer that acts as a template for the formation of calcium phosphate, which eventually crystallizes into hydroxyapatite [34].
These outcomes are in line with those of Sherif et al. [15], who concluded that the application of 10% N-HAp and 10% N-BAG resulted in favorable constructive surface alterations, had the capacity to re-mineralize the early enamel caries, and enhanced the de-minera­lized enamel’s micro-hardness. On the other hand, the current results are not consistent with those of Elbakry et al. [31], who found that the NSF toothpaste showed a greater protection and re-mineralization of the enamel surface than N-BAG. This variation in results may be due to the differences in experimental design and concentration of re-mineralizing agent used.
Regarding the CS/N-HAp, the N-HAp functions as an external ion supplier that encourages re-mineralization, while chitosan may serve as an agent carrier. Furthermore, because chitosan is bio-adhesive, it enhances the N-HAp bond to de-mineralized enamel and functions as a reservoir, accumulating calcium and phosphate ions to supply the de-mineralized enamel with an adequate supply of ions. Using chitosan to transport additional re-mineralizing agents, demonstrate the combination’s synergistic effects [34-36]. Similarly, the positive charge of chitosan enables to adhere to negatively charged surfaces, such as the exterior tooth surface, acting as a template for calcium and phosphate ions to rebuild new tissue, e.g., enamel, due to its chelating capacity and high adherence [37]. This is in agreement with Al-Ward et al. [10], who reported that the CS/N-Hap complex has the ability to prevent the enamel surface from acidic challenge.
Concerning the NSF, the enamel protection of silver nano-particles is due to their small size that allows the substance to enter the enamel structure, optimizing its impact and increasing the deposition of calcium and phosphorus ions on the enamel surface [38]. Due to intrinsic ionic stability, silver nano-particles do not impede fluoride action, and their synergistic interaction with fluoride enhances re-mineralization and enamel surface protection [12]. This may be explained by the NSF treatment affecting the enamel surface by supplying fluoride content, creating a calcium fluoride-like layer that acts as a reservoir during de-mineralization phases. This layer gradually releases fluoride, shielding the enamel from dissolving, as fluoride draws calcium and phosphate ions from the re-mine­ralizing fluid, resulting in a heavily mine­ralized layer on the enamel surface [20]. This is in line with El-Desouky et al. [20], who showed that NSF has the ability to protect the enamel surface from de-mineralization when subjected to pH-cycling. However, the current study’s results does not agree with those of Nozari et al. [39], who found that NSF is more effective in protection of the enamel surface against de-mineralization. This variation in results may be due to the differences in percentages of active ingredients of re-mineralize agents used.
The in vitro study may not replicate in vivo results due to limitations when considering oral factors, such as bio-film, flora, dietary habits, and oral hygiene practices. The 7-day study was not sufficient for understanding long-term dynamic de-/re-mineralization processes. Future clinical studies should be performed to mimic plaque fluid ionic concentration and pH in individuals with different risk of caries. They should also investigate the tested re-mineralizing agents’ response in situ, their association with other anti-caries agents, and their safety in clinical settings.

Conclusions

Within the limitations of the present study, we can conclude that the tested re-mineralizing agents demonstrate varying degrees of efficacy in resisting acidic enamel erosion, highlighting their potential to protect enamel health and resist erosive effects from acidic substances. However, the nano-bioactive glass has a superior mineral gain and enamel protection against acidic challenges, followed by CS/N-HAp and NSF re-mine­ralizing agents.

Disclosures

1. Institutional review board statement: Ethical Committee of the Faculty of Dental Medicine, Al-Azhar University, Cairo, Egypt, provided clearance for this study, with reference approval number of 639/3626.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: None.

References