Comparison of the Caries Remineralizing Effects of Dentifrices Based on Natural Hydroxyapatite, Synthetic Hydroxyapatite, and Fluoride: A pH Cycling Study

TL;DR

This study compared how well different toothpaste ingredients like natural and synthetic hydroxyapatite and fluoride can repair early tooth decay in a lab setting.

Contribution

The study provides a direct comparison of remineralization efficacy between natural and synthetic hydroxyapatite and fluoride in an in vitro model.

Findings

All tested dentifrices significantly remineralized initial caries compared to artificial saliva.

Natural and synthetic hydroxyapatite formulations showed comparable remineralization to fluoride toothpaste.

No significant differences were found among the natural hydroxyapatite formulations or between natural and synthetic hydroxyapatite.

Abstract

Objective: In vitro study compared the efficacy in remineralizing initial caries of dentifrice containing natural hydroxyapatite (natHAP), synthetic HAP (synHAP), and fluoride. Methods: Initial carious lesions were created on 105 bovine enamel blocks by 4-day demineralization in a microbial caries model inoculated and fed 3× daily with 10% sucrose (6 min/episode). The caries-bearing blocks were stratified across seven treatment groups (N = 15/group); 20% nat-nHAP tooth powder, 20% nat-nHAP toothpaste, 30% nat-nHAP toothpaste, 20% nat-microHAP toothpaste, 15% syn-nHAP, fluoride (1100 ppm) toothpaste (NaF), and artificial saliva (AS) were used, and the groups were subjected to 28-day remineralization using a standardized pH cycling model with a daily regimen consisting of three 2 min toothpaste slurry treatments and one 2 h acidic challenge, and AS storage for the rest of the day. Surface microhardness of each block was measured after demineralization and after remineralization. Statistical analyses were performed using a paired t-test and Tukey’s multiple comparison test. Results: All groups induced significant (p < 0.001) remineralization. The %Rem was significantly lower for AS (9.61 ± 6.17%) than 30% nat-nHAP (29.21 ± 16.47%) and fluoride (27.05 ± 10.9%) toothpastes. There were no significant differences in %Rem among the natHAP formulations or between natHap and synHAP formulations. Conclusions: Within the limitations of the present in vitro study, dentifrices based on natHAP or synHAP have comparable efficacy to standard fluoride toothpaste in remineralizing initial caries.

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1. Introduction

Caries disease is a substantial global health burden. Around 2.3 billion people are estimated to have caries of the permanent teeth and approximately 530 million children have decayed primary teeth [1]. The implications of caries include toothache, dental infection, premature tooth loss, malnutrition, malocclusion, and speech impediments. They contribute to diminishing the quality of life, adversely influencing general and emotional health of children and adults, and even may lead to social isolation [2]. Therefore, dental caries is associated with high social and economic costs [3].

Caries develops as the loss of minerals from the dental tissues occurs in an acidic environment [4]. The acids result from the bacterial metabolism of fermentable carbohydrates in the oral biofilm [2,4]. However, the development of dental caries is a dynamic, preventable, and reversible condition that alternates between phases of demineralization and remineralization [5]. This alternation stems from an interplay between protective factors that facilitate mineral gain and pathological factors that encourage mineral loss [6].

Under physiological conditions, saliva takes on a protective role in preventing demineralization by buffering acids and providing a source of calcium and phosphate ions that are capable of diffusing into demineralized tissues and promoting remineralization [7,8]. Nevertheless, saliva alone cannot inhibit caries development. In individuals who have a preponderance of cariogenic factors such as salivary dysfunction, carbohydrate-rich diet, and plaque accumulation, the scale tips towards demineralization and caries progress [5,6]. Numerous techniques have been developed over the years with the goal to boost the protective effects of factors that prevent caries with a particular focus on enhancing the natural ability of saliva to remineralize caries lesions, and the most applied evidence-based techniques are those based on fluoride [5,9,10,11].

However, products using fluoride alone have several limitations. First, the effectiveness of fluoride products relies on the availability and bioavailability of calcium and phosphate ions in saliva [5]. Additionally, studies have shown that the caries remineralization process initiated by fluoride is not homogenous as it is limited to the lesion’s surface and does not extend into the body of the carious lesion [12]. Finally, the efficacy of fluoride is proportional to its dose, which means that greater efficacy can be achieved at higher dosages [13]. Yet, high dosages of fluoride are more likely to cause undesirable side effects, namely fluorosis in children and toxicity in adults [14,15]. Thus, for safety reasons, it is recommended to limit the amount of toothpaste applied to the toothbrush in children, but also to use higher fluoride concentrations. However, the in vitro study has shown that the amount of toothpaste applied may affect its ability to remove dental plaque, and a better cleaning effect is achieved with a larger volume of toothpaste [16]. This constitutes another limitation as the recommended non-toxic dose of fluoride in children may not reach optimal levels for caries prevention [10].

Based on the above discussion, new remineralization materials that can be as effective as fluoride while compensating for its limitations are being developed and studied [5]. Hydroxyapatite (HAP) particles are a promising active ingredient in oral care products with a great resemblance in structure, solubility, and thermal stability to crystallites found in the dental hard tissues [17,18,19]. Formulations based on synthesized HAP particles have been studied and their efficacy in caries inhibition [20,21,22,23,24] and caries remineralization [21,25,26,27] in comparison to fluoride formulations was demonstrated. Additionally, HAP was shown to have a multitude of other dental indications, and are now used in mouthwashes, toothpastes, gels, and lotions [19,24,28,29]. All these formulations are appropriate for use in different age groups due to the excellent biocompatibility of HAP and its ability to be used in effective doses without undesirable toxic effects [30,31].

Hydroxyapatite has been established as a bioactive and biomimetic material in oral care products as a result of its chemical similarity to natural enamel and dentin as well as its ability to release calcium and phosphate ions under acidic conditions, thereby enhancing remineralization and reducing demineralization [31,32,33,34]. One of the main mechanisms through which HAP promotes remineralization is by increasing the bioavailability of calcium and phosphate ions present in saliva and the dental biofilm [32]. Following acid production by bacteria in the biofilm, hydroxyapatite (HAP) particles dissociate into calcium and phosphate ions [29,31,32]. The resulting supersaturation of the saliva and biofilm with the mentioned ions will tip the scale in favor of remineralization [31]. In addition to this mechanism, HAP particles have the unique ability of penetrating in the micropores between the enamel crystallites of the demineralized lesion and directly stimulating the deposition and growth of crystals inside these porosities [9,25,31,33]. Fluoride acts by superficially causing the hypermineralization of the superficial surface of the carious lesion and thus hinders deeper diffusion of calcium and phosphate ions into the lesion center, while hydroxyapatite induces uniform, homogeneous, and profound remineralization of the entire caries lesion [35].

Currently, two forms of hydroxyapatite (HAP) are used in oral care products: micro-clustered particles (5–10 µm) and nanocrystalline particles (20–80 nm in diameter). Nanoparticles are similar in size to hydroxyapatite which builds the dental hard tissues and, therefore, nHAP is able to penetrate deeper into the initial enamel defects and dentinal tubules. Furthermore, HAP in nanoscale dimensions has a much greater surface area per unit volume compared with the larger particles and it results in its higher chemical reactivity [19]. A recent in vitro study revealed that nanohydroxyapatite (nHAP) markedly increased the effectiveness of a toothpaste in remineralizing early carious lesions compared to microcrystalline hydroxyapatite (mHAP) and fluoride [36]. However, the randomized clinical trials (RCTs) displayed a comparable clinical efficacy of both types of HAP in caries prevention and remineralization [22,23,24].

Furthermore, based on the method of extraction, two forms of HAP can be distinguished: synthetic (synHAP) and natural (natHAP) hydroxyapatite. A commercial synthetic hydroxyapatite (synHAP) is characterized by an optimized calcium-to-phosphate (Ca/P) ratio of 1.67, which has been shown to be most effective in promoting bone regeneration [37]. It can be produced from chemically available calcium and phosphorus precursors using a variety of relatively simple and cost-effective methods. Nevertheless, the production process is long-lasting and leads to the loss of various biologically valuable trace elements such as sodium, silicon, potassium, strontium, magnesium, and iron. Moreover, synHAP is much less biodegradable than the natural one [38]. On the other hand, the naturally extracted HAP (natHAP) is considered as a low-cost alternative, achievable in a faster production process and, most of all, widespread, readily available, and environmentally friendly [39]. Elementary sources of natHAP are biowaste of plant or animal origin such as mammalian bones or fish bones and scales, poultry egg shells, seashells, algae, wood, calendula flower, papaya leaves, and orange and potato peels. Converting these by-products into natHAP can reduce debris and contribute to the development of more sustainable dental denouements [40]. The natHAP has a structure and CA/P ratio that depends on the biological material from which it is extracted, and ranges from 1.19 to 2.63 [38]. It is characterized by lower crystallinity, mechanical strength, and hardness than the synHAP, which is due to its porosity, organic residues, and other mineral components. Nevertheless, the superiority of the natHAP lies in its “innate” content of important trace ions (zinc, sodium, magnesium, strontium, potassium, barium, carbonates), which makes it similar to the chemical composition of human hard tissues (bones, enamel, dentin). These trace elements may affect the properties of natHAP and modulate its bioactivity [41].

In recent years, numerous RCTs (including long-term studies) have confirmed the clinical effectiveness of oral hygiene products containing synHAP in caries prevention and caries remineralization [22,23,24]. Currently, natHAP has been attracting great interest among scientists. To the best of our knowledge, there is still an insufficient number of studies that examine the efficacy of products based on natHAP in dental applications, namely, in the remineralization of caries lesions [40,42]. Therefore, the objective of this in vitro study was to investigate the efficacy of natHAP in tooth powder and toothpaste (Natural Tooth Health LLC, Davidson, NC, USA) and in remineralizing initial caries and to compare it with those of toothpastes based on synHAP or fluoride. Our null hypotheses were as follows.

H0.  The group means for the experimental products (natHAP) and the reference (synHAP or fluoride) products are equal with respect to percentage remineralization (%Rem) following 28 days of treatment (no significant difference).

H1.  The group means for the experimental product and the reference product are not equal with respect to %Rem following 28 days of treatment (significant difference).

2. Materials and Methods

2.1. Sample Preparation

Sound bovine maxillary incisors were collected, cleaned to remove soft tissue debris, and brushed with a pumice slurry using a Colgate manual toothbrush (Colgate Palmolive, Piscataway, NJ, USA). The teeth were subsequently examined by transillumination to assess structural integrity. Specimens exhibiting cracks, hypo mineralization, white spot lesions, or other developmental defects were excluded, and only intact teeth were selected and stored in 0.1% thymol solution until use. A total of 105 enamel blocks (approximately 3 mm in length × 3 mm in width × 1.5 mm in thickness) were prepared from the labial crown surfaces using a water-cooled diamond wire saw (Walter Ebner, 2400 Le Locle, Switzerland). The enamel surface and the base of each block were polished to obtain flat, plane-parallel surfaces suitable for surface microhardness (SMH) measurements, using adhesive-backed lapping films with decreasing grit sizes (30 µm to 1 µm) in a MultiPrep™ Precision Polishing machine (Allied High Tech, Cerritos, CA, USA). Subsequently, all surfaces of each block were coated with two layers of acid-resistant nail varnish, except for the buccal enamel surface, which was exposed for acid challenge to induce initial caries formation.

2.2. Creation of Initial Caries Lesion

Initial caries lesions were induced on each tooth block over a 4-day demineralization period using a microbial caries model previously described in our publication [43]. The microbial model comprised Todd Hewitt broth inoculated with a mixed culture of Streptococcus mutans and Lactobacillus casei at a broth-to-inoculum ratio of 10:1. This system generates a natural cariogenic biofilm that is subjected to a daily fasting–feasting cycle, closely simulating conditions within the oral cavity. Specifically, the biofilm was exposed to a sucrose solution for 6 min, three times daily (representing three meals per day), followed by incubation in growth medium for the remainder of the day over a 4-day period [43,44].

2.3. Measurement of Baseline Surface Microhardness (SMHd) of the Lesions

Baseline surface microhardness (SMHd) of the initial carious lesions was assessed using a Vickers diamond indenter on a microhardness tester (Tukon 2100; Wilson-Instron, Norwood, MA, USA). Each enamel specimen was mounted onto a 1-inch square acrylic block using sticky wax and then positioned on the microhardness testing device. Five baseline indentations were made on the buccal surface of the flattened and polished sound enamel block using a Vickers diamond indenter under a load of 50 g applied for 15 s, with indentations spaced 100 µm apart. The Vickers Hardness Number (VHN) was calculated by measuring the diagonal lengths of the indentations using the Wilson 2100–Wolpert Image Analysis Software (version 3.5.032; BUEHLER, Lake Bluff, IL, USA), and the mean value was recorded for each block. Prior to initiation of the daily remineralization treatment, all bovine enamel blocks demonstrated a minimum of 40% demineralization.

2.4. Specimen Allocation

The list and composition of the tested oral products are presented in Table 1. The 105 blocks exhibiting initial caries lesions were categorized into 7 treatment groups (N = 15) described below according to their SMHd values. The blocks were assigned to ensure that there would be no statistically significant difference between the mean SMHd values among the groups.

2.5. Power Analysis and Sample Size Calculation

A power analysis was performed using the G*Power statistical software 3.1.9.7 for Window (Heinrich-Heine-Universität, Düsseldorf, North Rhine-Westphalia, Germany) to establish the required sample size. Effect sizes derived from previously published studies [21,27,36] were used, with a confidence level of 95% (α = 0.05) and a statistical power of 80% (1 − β = 0.80) set as the analytical parameters. The results of the analysis demonstrated that a sample size of 15 specimens per group was adequate to detect a statistically significant difference among groups.

2.6. Remineralization Treatment Procedure

Using dental heavy-duty putty, the 15 samples in each group were embedded within oblong grooves carved into a cylindrical acrylic rod attached to the lid of a 250 mL treatment tube. The sample surfaces were aligned flush with the surface of the acrylic rod to allow a streamlined flow of fluids across the specimens, while only the exposed enamel surface was available for treatment. The acrylic rod served as sample holder to suspend the samples in the middle of the treatment solutions. The 7 groups were subjected to remineralization using a pH cycling (demineralization/remineralization) model, simulating the activities within the oral environment as close as possible. Artificial saliva [MgCL_2.6_H_2_O (0.03 g/L), K_2_HPO_4_ (0.804 g/L), KH_2_PO_4_ (0.326 g/L), KCl (0.625 g/L), Calcium lactate (0.385 g/L), Carboxy-Methylcellulose (0.4 g/L), Methyl-4-hydroxybenzoate (2 g/L)], pH adjusted to 7.2 using KOH [26] served as the control group as well as the storage medium between treatments for other groups, while an acidified buffer composed of 2.2 mmol/L KH_2_PO_4_, 2.2 mmol/L CaCl_2_, and 50 mmol/L Acetic Acid, with pH raised to 4.5 with KOH [26] was used as the demineralizing solution and serves as the acidic challenging medium (AC). A slurry of each toothpaste sample was prepared by mixing one part toothpaste (g) and three parts sterile deionized distilled water (mL) using a laboratory stand mixer until homogenous (for the tooth powder the ratio was 1 g powder to 1 mL water). Fresh medium for each group was prepared each day and was vortexed vigorously prior to each treatment episode. The cyclic treatment regimen for each day, as shown in Table 2, consisted of 2 h acidic challenge (AC), three 2 min toothpaste/powder slurry treatment (TST) periods, and then storage in artificial saliva (AS) for the rest of the time without agitation. For treatment, 200 mL of the treatment medium (AC, AS, or TST) was placed into each 250 mL treatment tube and each acrylic rod bearing the samples was immersed into the treatment tube containing their respective product. AS and TSTs were magnetically stirred at 350 rpm, while the AC was static. All treatments were performed inside an incubator at 37 °C. The pH of each medium was measured once daily before treatment. After treatment with one medium, the specimens were rinsed with running deionized water and dried with a paper towel before immersion into the next agent. The daily regimen was repeated for 28 days as established in our previous publication [45]. On termination of the experiment, the blocks were harvested and processed for remineralization assessment using a Surface Microhardness Tester (Tukon 2100; Wilson-Instron, Norwood, MA, USA).

2.7. Post-Remineralization Surface Microhardness (SMHr) Measurement

Post-remineralization surface microhardness (SMHr) of the treated surface was reassessed as previously described by placing five indentations 100 µm to the right of the baseline indentations, as illustrated below, and calculating the mean value for each block. At this stage, both the post-demineralization (SMHd) and post-remineralization (SMHr) surface microhardness values of the lesions were available for statistical analysis.

2.8. Efficacy Measurement

The primary efficacy was determined by the percentage change in SMH (%ΔSMH), which is a measure of the percentage remineralization (%Rem) achieved with each product following the 28 days of treatment. This was calculated as follows:

$$[eqn]$$

The comparisons of interest were pairwise comparisons between experimental and reference products. For each of these pairwise treatment comparisons, the null hypothesis tested was as follows. H0: The group means for the experimental products (natHAP) and the reference (synHAP or fluoride) products are equal with respect to percentage remineralization (%Rem) following 28 days of treatment (no significant difference). The corresponding alternative hypothesis was as follows. H1: The group means for the experimental products and the reference products are not equal with respect to %Rem following 28 days of treatment (significant difference). These treatment comparisons were performed through two statistical analyses using SPSS v28, with a p-value of ≤0.05 considered statistically significant. The first analysis aimed to compare the Vickers Hardness Number (VHN) between demineralized and remineralized teeth for each product to determine whether the difference was significantly greater than zero. A one-sided paired samples t-test was used to evaluate this research question. Prior to conducting the t-test, assumptions were assessed, including the normality of the difference scores, to ensure the appropriateness of the test. The second analysis evaluated the percentage remineralization across seven different products. Given that more than two groups were being compared, a one-way ANOVA was conducted. A statistically significant omnibus F-test (p ≤ 0.05) indicated overall group differences, prompting post hoc tests to identify which specific group means differed significantly from one another.

3. Results

Paired samples t-tests were performed for each product to determine whether the mean VHN differed significantly before and after lesion remineralization. The mean VHN was statistically significantly higher (p < 0.001) in all groups following remineralization (Figure 1, Table 3).

A one-way variance analysis was conducted to compare the %Remineralization (%Rem) achieved with each of the seven test products (Table 4). The analysis results showed a statistically significant difference in %Rem among the seven groups F(6,77) = 4.87, p < 0.001, at the alpha level of 0.05. Pairwise comparisons of groups using the Tukey HSD multiple comparisons test were conducted to identify the specific group means that differed significantly from one another. There was no statistically significant difference in %Rem among the products with active ingredients (interventions). However, the result indicated that 30% nat-nHAP toothpaste (29.21 ± 16.47) and fluoride (1100 ppm) toothpaste (27.05 ± 10.90) had statistically significantly higher %Rem compared to artificial saliva (9.61 ± 6.17, at the alpha level of 0.05 (Figure 2, Table 5).

4. Discussion

Despite significant progress in cariology, dental caries remains a major concern for individuals globally [3]. The initial caries lesion appears as a white spot, resulting from a decrease in enamel translucency due to mineral loss in its subsurface layer [46]. This increases the porosity of the enamel and decreases its hardness, while the outer layer of the enamel remains intact [47]. Hence, the early caries lesion is referred to as subsurface demineralization, where the loss of calcium and phosphate ions within the lesion can reach up to 50% compared to healthy enamel. At this stage, the enamel lesion is still reversible owing to the unimpaired enamel surface zone acting as a diffusion channel, through which calcium and phosphorus ions may penetrate the lesion center when saliva is sufficiently saturated with these minerals [48]. Detecting a caries lesion at this stage and taking appropriate measures can prevent its progression to cavitation. This knowledge has led to the introduction of one of the non-invasive methods of caries treatment—remineralization. Remineralization refers to the process by which minerals, primarily calcium and phosphate, are transported from the surrounding environment (i.e., saliva, biofilm, and oral mucosa) into partially demineralized tooth structures, resulting in mineral deposition through the appositional growth of hydroxyapatite crystals [7,49]. In addition to fluoride, the literature mentions numerous non-fluoride systems for remineralization of caries lesions, such as casein phosphopeptide–amorphous calcium phosphate, calcium ammonium phosphate, bioactive glass containing calcium sodium phosphosilicate, self-assembling oligopeptide SAP-P11-4, and biomimetic hydroxyapatite [50].

Today, remineralization is an integral part of caries management. Therefore, the goal of this study was to compare the remineralization potential of selected commercially available dentifrices (powder and toothpastes) on microbiologically produced initial caries lesions in bovine teeth. Bovine teeth represent a valid experimental model for evaluating remineralization treatments, as their enamel structure closely approximates that of human teeth [51]. Accordingly, bovine enamel was selected as a surrogate for human enamel because of similarities in mineral composition, microstructure, and hardness [52,53,54]. In addition, bovine teeth are larger in size, in a relatively good state with a more uniform composition, and most of all, they are more easily accessible in greater quantities than human teeth [55]. Additionally, bovine teeth are more ethically and logistically accessible, making them a preferred alternative in laboratory studies [55]. However, it is well established that there are differences in remineralization rates between human and bovine enamel, with studies showing that bovine enamel often remineralizes more readily or intensely than human enamel, especially in surface and subsurface areas [56,57]. This phenomenon has been attributed to the greater porosity and distinct structural characteristics of bovine enamel, including a more pronounced interprismatic matrix, which are thought to facilitate faster and deeper penetration of remineralizing agents and consequently promote more robust mineral deposition [57,58]. However, it is pertinent to mention that under pH cycling bovine enamel’s structure can weaken more significantly, even with good remineralization [57].

One may wonder about the rationale for creating initial caries lesions using a microbial caries model, but remineralization was subsequently evaluated using a non-microbial pH cycling model. We want to be as close as possible to clinical situations, so we used a microbial caries model to create our initial caries lesion to generate data that more closely reflect the clinical remineralization potential of the study products. It is acknowledged that natural lesions have complex, varied structures, due to varying bacterial influence while artificial ones often show more uniform demineralization and lack the intricate microstructural patterns of natural lesions, making them useful models but not perfect replicas of clinical situations [59]. Furthermore, the study has shown that the remineralization of natural caries lesions and artificially created caries-like lesions appears to occur through different mechanisms due to variations in lesion microstructure, organic content, and depth. Artificial lesions generally exhibit greater and more predictable remineralization compared with natural lesions, which possess more complex structural characteristics that influence mineral deposition [59]. Consequently, artificial lesions may be used as a simplified and standardized model for assessing the in vitro remineralization potential of remineralizing materials; however, they may be less accurate predictors of clinical outcomes under real-world conditions [59].

There are various techniques which are available for evaluating the remineralizing effect of various agents on tooth enamel such as mineral content measurement techniques, scanning electron microscopy, surface microhardness assessment, transverse microradiography, and micro-computed tomography [27,60,61,62]. In the present study, remineralization was assessed by surface microhardness measurement (SMH) due to its several advantages. It represents a simple, rapid, and non-destructive technique that enables repeated measurements, thereby permitting the longitudinal monitoring of mineral changes due to therapeutic interventions [61]. However, it is pertinent to mention that SMH primarily reflects surface changes and does not provide information on subsurface or lesion body remineralization.

In the present study, natural hydroxyapatite (natHAP), synthetic hydroxyapatite (synHAP), and fluoride were used as remineralizing agents, and an in vitro pH cycling model was employed to assess the effects of these dentifrices on initial enamel caries lesions. A pH cycling protocol was applied to simulate the dynamic processes of demineralization and remineralization that occur in the oral cavity [45]. The pH cycling caries model serves as an intermediary between laboratory and clinical investigations, as it reflects clinical conditions in which demineralization and remineralization occur continuously, with only brief interruptions during the application of the investigational products. Over time, various pH cycling models have been developed and reported in the literature, and they have been widely accepted and used in industry and dental research as a suitable alternative to animal testing [63]. Although this model does not incorporate a cariogenic microbial biofilm, which is a critical component of the caries process, the alternating feast and famine conditions characteristic of biofilm activity in the oral cavity are simulated through cyclical exposure of the samples to demineralization and remineralization solutions. While the data were generated under laboratory conditions, the pH cycling model provided a more accurate simulation of the caries process and more closely approximated the oral environment than separate demineralization and remineralization studies.

Although previous in vitro studies have used different lengths of days for remineralization varying from 7 to 28 days, the choice of a 28-day cycle in the present study was based on our established and published protocol, which we have long used in several studies [45].

Following demineralization, all groups exhibited a significant overall reduction in microhardness, reflecting substantial mineral loss. After application of the remineralizing agents, all test groups, along with the control group (artificial saliva) demonstrated a statistically significant increase in microhardness, indicating successful remineralization, with the 30% nat-nHAP toothpaste (29%) and the standard fluoride toothpaste (27%) achieving statistically significantly higher remineralization than the control group (9%). Thus, the H0 hypothesis was rejected and H1 was accepted. Other groups showed higher but not statistically significant remineralization than the control, and as such the H0 hypothesis was accepted and H1 was rejected.

The remineralization of the carious lesions observed with the artificial saliva after 28 days of pH cycling suggests that the artificial saliva used in this study possesses remineralizing potential, which agrees with the study of Huang et al. [64]. Furthermore, the artificial saliva was able to, to some extent, mimic natural saliva in its remineralization abilities and, consequently, was capable of inducing the remineralization of the initial caries lesion [65,66]. However, the artificial saliva used in the present study, with a pH of 7.2, exhibited properties more comparable to the natural saliva of individuals at low caries risk, who typically maintain a higher resting salivary pH that enhances the remineralization potential compared with individuals at high caries risk [67,68]. Thus, it is considered that the relatively high baseline pH of the artificial saliva may have influenced the magnitude of differences in the remineralization observed between artificial saliva and treatment groups, which is a potential confounding factor.

Although the application of HAP in caries prevention is on the rise, there are many high-quality studies documenting the cariostatic and remineralizing potential of this mineral [35,69,70]. However, all these publications focus on synthetic HAP, and there is still a shortage of well-designed studies on the remineralizing effects of HAP derived from natural sources [40,42]. Therefore, it is believed that the present study can help to fill this gap. Nevertheless, it is pertinent to mention that other researchers have reported the remineralizing properties of hydroxyapatite derived from natural sources which have been investigated in previous studies. Mathirat et al. [71] employed surface microhardness testing and SEM–EDX analysis to assess the remineralization potential of natural nanohydroxyapatite (nat-nHAP) extracted from fish bone (Epinephelus chlorostigma) in the artificially induced enamel demineralization of human premolars with GC tooth mousse (casein phosphopeptide-based product) serving as a control. The SMH test revealed that fish bone-derived nHAP promoted enamel remineralization more effectively than GC tooth mousse. Moreover, in the fish bone nHAP-treated enamel, the CA/P ratio ranged from 1.63 to 1.99, depending on the method of nHAP extraction, and was comparable to the GC tooth mousse-treated enamel (Ca/P ratio 1.80).

The results of the present study are in agreement with those reported by El Din Eliwa et al. [72], who evaluated the remineralization potential of nano-seashell paste (10%), nano-pearl paste (10%), and synthetic nanohydroxyapatite (syn-nHAP) toothpaste (10%), using fluoride toothpaste (850–1150 ppm F) as a positive control, in human premolars through enamel surface microhardness assessment over a 28-day period. Topical application of these toothpastes resulted in a statistically significant increase in the enamel surface microhardness of artificially induced early caries lesions. These findings indicate that the remineralization efficacy of nano-seashell, nano-pearl, and synthetic nHAP toothpastes is comparable to one another and similar to that of fluoride-based toothpaste.

Furthermore, the remineralizing effect of natural hydroxyapatite (nat-HAP) observed in the present study is supported by findings from another investigation. Mohamad et al. [73] assessed the remineralization potential of eggshell powder (10%) and Novamin (a calcium sodium phosphosilicate-based toothpaste containing 1450 ppm sodium fluoride) on initial caries-like lesions in young permanent teeth and compared their effects with those of a fluoride toothpaste containing 5000 ppm sodium fluoride using a Vickers microhardness tester. Both eggshell powder and Novamin demonstrated effectiveness in remineralizing initial caries-like lesions. However, in contrast to the results of the present study, the authors reported only a minimal increase in enamel surface microhardness in samples treated with fluoride toothpaste compared with untreated control specimens. The discrepancy between their findings and those of the current study may be attributed to differences in the experimental design and study duration, treatment regimens, and the origin of the enamel samples.

Nevertheless, the findings of the present study regarding the surface microhardness (SMH) achieved with fluoride toothpaste are consistent with the results reported by Srinivasan et al. [74] and El-Zayat [75], who demonstrated that casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) combined with 900 ppm fluoride significantly enhanced the remineralization of softened enamel, with the increased remineralization potential attributed to the presence of fluoride. In addition, Satou et al. [76] reported on the development of a dental caries prevention strategy involving eggshell-derived nanosized bioapatite (BioHap) used in combination with a high-concentration fluoride tooth surface application (acidulated phosphate fluoride, APF; 9048 ppm F). In that study, enamel acid resistance and Vickers surface microhardness following application of the proposed method were compared with those obtained using a conventional topical application of APF gel on bovine enamel. The combined use of BioHap and APF resulted in the formation of a thick coating layer on the enamel surface, which provided greater resistance to demineralization compared with the use of APF alone.

In the present study, the effectiveness of fluoride in the remineralization of early caries lesions further supports the substantial body of evidence identifying fluoride as a reliable agent for caries prevention and the remineralization of initial carious lesions. However, it is important to recognize that fluoride does not serve as a direct source of calcium, unlike hydroxyapatite; instead, it facilitates remineralization by utilizing the calcium and phosphate ions available in the saliva and dental plaque. It has been demonstrated that the formation of one unit of fluorapatite crystal requires two fluoride ions in conjunction with ten calcium ions and six phosphate ions [61]. Consequently, the remineralizing action of fluoride is limited by the bioavailability of calcium and phosphate ions within the oral environment [77,78].

The literature frequently compares the properties of nHAP derived from varied natural sources. Several in vitro studies showed that nanohydroxyapatite (nHAP) extracted from eggshells exhibited higher crystallinity, enhanced buffering capacity, and smaller particle size compared with nHAP derived from fish bones and scales, rendering it a more favorable material for dental remineralization [79,80]. However, both types of nHAP are considered promising, effective, and low-cost remineralizing materials for enamel regeneration [40,42].

Although the present study demonstrated some remineralization potential with 20% natHAP, the 30% natHAP formulation produced a significantly greater remineralization of demineralized enamel compared with the control. Our results were confirmed by several previous studies. Wahyuni et al. [81] investigated the effect of hydroxyapatite-based remineralization toothpaste derived from snakehead fish bones (Channa striata) at different concentrations (10%, 30%, 50%) on dental enamel hardness in extracted human teeth with a Vickers hardness tester. The authors showed that all concentrations of HAP toothpaste significantly enhance the SMH of the enamel samples compared to the control (a HAP-free toothpaste). Furthermore, they observed the statistically significantly higher microhardness value in enamel samples treated with 50% HAP toothpaste compared to those treated with 10% and 30% HAP toothpaste. Devitasari et al. [82] determined the effect of hydroxyapatite toothpaste from the bone of tilapia fish (Oreochromis niloticus) at three different concentrations (5%, 10%, 15%) on enamel surface microhardness in the extracted human premolars. The highest value of microhardness was recorded in the enamel samples treated with 15% natHAP followed by 10% and 5% concentrations. There was a statistically significant difference in the results between groups with varying natHAP concentrations. The increased concentration of natHAP in toothpaste results in a greater bioavailability of calcium and phosphorus in saliva and thus enhances the remineralization of decalcified enamel. This indicates that natHAP behaves in a concentration-dependent manner (dose–response relationship) similar to fluoride [83,84] and higher concentrations of natHAP may be more effective in the remineralization of carious lesions.

In this current study, almost all tested products containing natHAP (with the exception of 30% nat-nHAP toothpaste) had similar remineralization potential, which did not differ from that of the syn-nHAP toothpaste, and this agrees with the previous study [85]. Concurrently, our study did prove no differences in remineralization capacity between products with nat-nHAP and nat-mHAP, although there are some reports in the literature that nHAP particles are characterized by greater bioactivity due to their much smaller particle size and higher surface energy and solubility compared to mHAP [19,36,64].

Generally, the findings of the present study, demonstrating comparable remineralization between the 30% nat-nHAP toothpaste and the 1100 ppm NaF fluoride toothpaste, are consistent with reports from two recent clinical non-inferiority studies evaluating HAP-containing toothpastes [21,86]. One of these investigations was a randomized controlled clinical trial that assessed enamel caries progression in highly caries-susceptible orthodontic patients and demonstrated the non-inferiority of a toothpaste containing 10% microcrystalline hydroxyapatite (mHAP) compared with a toothpaste containing 1400 ppm fluoride, delivered as amine fluoride and stannous fluoride [87]. The second study was an in situ investigation that showed non-inferiority between two children’s toothpaste formulations containing 10% mHAP and 500 ppm fluoride, respectively, in promoting caries remineralization and inhibiting tooth demineralization [21].

The outcomes of these previous studies, together with the findings of the present investigation, may be explained by the well-established mechanisms of action of hydroxyapatite as an anti-caries and remineralizing agent [34]. Hydroxyapatite is known to exhibit excellent biocompatibility and a strong affinity for tooth tissues, resulting in its adsorption onto tooth surfaces [78]. Upon adhesion, particulate HAP facilitates the biomimetic remineralization of early carious lesions by effectively filling the microporosities within demineralized dental tissue [88]. In this capacity, HAP acts as a crystal nucleation site, promoting mineral deposition and crystal growth through the continuous attraction of calcium and phosphate ions from the surrounding remineralizing environment [88]. Furthermore, a recent study demonstrated that the HAP oral care toothpaste evaluated in the present study releases calcium and phosphorus upon application to tooth surfaces [29], further highlighting the potential of this formulation to influence remineralization and demineralization processes at the tooth surface.

It is important to acknowledge that a primary limitation of the present study is that, although the pH cycling models were designed to simulate clinical conditions, they did not incorporate the biological processes occurring in the oral cavity, particularly those associated with dental plaque, which play a critical role in influencing the remineralization potential of oral care products. In addition, the demineralization process employed in the pH cycling model is more prolonged during each cycle than the acid challenges typically encountered in the oral environment. Therefore, future investigations should emphasize well-designed clinical trials to further substantiate the effectiveness of hydroxyapatite-based oral care toothpastes in caries remineralization and prevention.

5. Conclusions

The present study demonstrated the lack of statistically significant differences between fluoride-free oral care products containing either natural or synthetic hydroxyapatite and standard fluoride (1100 ppm) toothpaste in remineralizing microbiologically produced initial caries lesions. Although there was no statistically significant difference in percentage remineralization achieved with the product, numerically the toothpaste containing 30% natural nanohydroxyapatite induced the highest remineralization (29.21 ± 16.47%), followed by the fluoride toothpaste (27.05 ± 10.9%), then the toothpaste containing 15% synthetic nanoHAP (22.2795 ± 10.48708) and 20% natural nanohydroxyapatite tooth powder (22.0750 ± 10.46350), while the artificial saliva produced the lowest (9.61 ± 6.17%) percentage remineralization after the toothpaste containing 20% natural nanohydroxyapatite (18.6441 ± 8.05422). All dentifrices tested were significantly effective in remineralizing initial caries lesions and more than the artificial saliva alone. The remineralization of the microbiologically produced initial caries lesions in the current study demonstrates the promising potential of all the tested dentifrices to remineralize natural caries lesions in vivo.