Comparative Evaluation of the Enamel Remineralization Potential and Antibacterial Activity of Pediatric Toothpastes Containing Fluoride, Hydroxyapatite, Ozone, Propolis, and Postbiotics: An in vitro Study

Abstract

Objective

This study aimed to evaluate the remineralization potential and antibacterial activity of 5 commercially available pediatric toothpastes with different active ingredients.

Subject and Methods

The evaluated toothpastes were Kids Ozon, Oral-B Pro-Kids, Dentiste Junior Postbiotics, Curasept Biosmalto, and Zubio. To assess remineralization efficacy, 48 specimens from extracted third molars were subjected to baseline microhardness testing. The samples were divided into six groups: five experimental and one negative control (n = 8). After 4 days in a demineralizing solution, microhardness was remeasured, followed by a 14-day pH-cycling protocol at 37°C with twice-daily toothpaste applications. Final microhardness values were recorded and statistically analyzed. For antibacterial assessment, the agar diffusion method was applied using Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, and Candida albicans.

Results

All the tested toothpastes produced statistically significant remineralization of initial enamel carious lesions (p < 0.05). The microhardness ranking after pH-cycling was as follows: Kids Ozon > Dentiste Junior Postbiotics > Curasept Biosmalto > Zubio > Oral-B Pro-Kids > negative control. However, intergroup differences weren’t statistically significant (p > 0.05). All formulations showed antibacterial activity against the tested microorganisms. For S. mutans, Curasept Biosmalto had a significantly larger inhibition zone than Kids Ozon (p < 0.05). Although Curasept Biosmalto showed relatively higher antibacterial activity, differences among the other formulations were not significant (p > 0.05).

Conclusion

Fluoridated and hydroxyapatite-based toothpastes exhibited comparable remineralization potential and antibacterial activity under in vitro conditions. The addition of other agents (ozone, propolis, and postbiotics) showed statistically nonsignificant improvements under the present experimental conditions.

Highlights of the Study

• This study addresses a gap in the literature, as studies comparing the antibacterial activity and enamel remineralization potential of pediatric toothpastes within the same design are limited.

• Fluoridated and hydroxyapatite-based toothpastes have comparable remineralizing and antibacterial properties under in vitro conditions.

• The addition of ozone, propolis, or postbiotics to fluoridated toothpaste won’t result in significant improvement.

Introduction

Dental caries is a common oral disease clinically characterized by the chronic and progressive destruction of the tooth’s hard tissues [1]. The prevention of early carious lesions and the arrest of their progression are of significant importance in preventive dentistry. A physiological balance exists between the dental hard tissues and the bacterial biofilm. Disruption of this balance leads the process to progress toward demineralization. In the early stages of the demineralization process, an adequate availability of calcium, phosphate, and fluoride ions enables the reversal of demineralization and promotes the initiation of remineralization [2]. Fluoride toothpastes have long been the most widely used products for the prevention of caries and the management of initial lesions. Fluoride promotes the remineralization process by transforming the hydroxyapatite (HAP) in enamel into fluorapatite [3]. Frequent exposure to low doses of fluoride is more effective at preventing tooth decay than occasional high doses. For this reason, fluoride is incorporated into toothpastes, allowing patients to use it easily and regularly [4]. In recent years, an increasing encounter with fluoride-hesitant families, advancements in bioactive materials, and their increased commercial availability have raised interest in supportive biocompatible agents. Accordingly, ingredients like HAP, ozone, and propolis are incorporated into pediatric toothpastes to enhance remineralizing efficiency [5, 6].

Ozone has become an important therapeutic modality in dentistry because of its antimicrobial efficacy, anti-inflammatory effects and biocompatibility [7]. The increasing use of propolis in dentistry is attributed to its multifaceted biological activities, including antimicrobial, anti-inflammatory, antioxidant and wound-healing effects [8].

Postbiotics are defined as biological agents composed of inactivated microbial cells or their metabolites that do not contain live microorganisms but exert beneficial effects on host health. Recently, they have emerged as potential bioactive agents capable of modulating the oral microbiome through nonviable microbial metabolites [9].

HAP is a natural component of teeth and bones, sharing both structural and chemical similarities with human enamel. These biomimetic properties make it a valuable ingredient in preventive dentistry, as HAP particles can adhere to damaged enamel and fill microscopic defects, helping to restore surface integrity [10].

Nevertheless, the clinical performance of complete toothpaste formulations depends on multiple factors beyond the biological properties of their individual ingredients, including formulation composition, active agent concentration, bioavailability and interactions among components. Various methods such as microhardness testing, microradiography, micro-computed tomography and Raman spectroscopy are used to assess the remineralization efficacy of toothpastes. Among these techniques, microhardness testing is frequently preferred due to its ease of application, repeatability, and its documented reliability in the literature [11]. For the evaluation of antibacterial activity, agar diffusion and dilution methods are commonly employed. The agar diffusion method is the most widely used approach, owing to its rapid and straightforward application as well as its ability to visually demonstrate inhibitory effects [12].

Although various studies have compared different pediatric toothpastes, relatively few investigations have evaluated enamel remineralization and antimicrobial activity within the same experimental design. Therefore, the aim of this study was to evaluate and compare the antibacterial activity and remineralization potential of 5 commercially available pediatric toothpastes containing different active ingredients using agar diffusion testing and Vickers microhardness analysis. The null hypothesis was that pediatric toothpastes formulated with different active ingredients would not differ significantly in their antibacterial effects or their capacity to promote enamel remineralization.

Materials and Methods

Microhardness Analysis

Sample Preparation

The sample size of the study was calculated using the G*Power 3.1.9 (GPower, Universität Düsseldorf, Germany) software. Considering the primary hypothesis as “the change in remineralization across 6 different solutions and 3 within-time comparisons,” the effect size was projected to be medium-to-large (f = 0.30) based on a similar study in the literature [13]. With a type I error rate of α = 0.05 (95% confidence level) and a target statistical power of 1-β = 0.90, the minimum sample size required for statistical analyses (ANOVA: repeated measures, within-between interaction) was calculated as 48, and it was planned to include 8 teeth (48/6) as samples for each group (“Table 1”).

Table 1.

Toothpastes and their contents used in the study

Toothpaste/Manufacturer	Contents
Oral-B Pro-kids (Procter&Gamble, Germany)	Sorbitol, water, hydrated silica, cocamidopropyl betaine, cellulose gum, flavor, trisodium phosphate, sodium phosphate, sodium fluoride (1,000 ppm), carbomer, sodium chloride, glycerin, sodium saccharin, limonene, benzyl alcohol, sodium benzoate, cinnamal, Polysorbate 80, colorant (C.I. 42090)
Kids Ozon (Pharmaozon, Turkey)	Water, glycerin, hydroxyethylcellulose, flavor, vitamin B5, sodium benzoate, colorant, Stevia leaf extract, propolis extract, ozonated olive oil, sodium fluoride (1,000 ppm)
Dentiste Junior Postbiotics (Siam Cosmeceutical Co, Thailand)	Sorbitol, water, hydrated silica, xylitol, sodium cocoyl glutamate, xanthan gum, Lactobacillus ferment filtrate (probiotic component), flavor, sodium fluoride (1,000 ppm), grapefruit seed oil, cinnamon oil, ascorbic acid, anise oil, sage leaf extract, acacia, myrrh resin, chamomile extract, aloe vera leaf juice, fennel extract, ratanhia root extract, Licorice root extract, Echinacea root extract, benzyl alcohol, limonene
Zubio (Dar Cosmetic, Turkey)	Water, glycerin, aloe vera leaf juice, xylitol, hydrated silica, xanthan gum, flavor (strawberry), HAP, lauryl glucoside, calcium carbonate
Curasept Biosmalto (Curasept S.p.A., Italy)	Purified water, glycerin, hydrated silica, MgSr carbonate HAP chitosan conjugate (bioactive complex), fluorohydroxyapatite, cellulose gum, xylitol, cocamidopropyl betaine, sodium monofluorophosphate (1000 ppm), xanthan gum, potassium acesulfame, ethylhexylglycerin, sodium saccharin, sodium benzoate, citric acid, colorant (C.I. 19140, C.I. 17200)

Following baseline microhardness measurements, all teeth were numbered. Since there was no statistically significant difference in baseline microhardness values among the groups and the specimens showed a homogeneous distribution, the samples were assigned to the groups in numerical order.

• Group 1: Oral-B Pro-kids toothpaste containing 1000-ppm sodium fluoride.

• Group 2: Kids Ozon toothpaste containing propolis, ozone, and 1000-ppm sodium fluoride.

• Group 3: Dentiste Junior Postbiotics toothpaste containing 1000-ppm sodium fluoride, Lactobacillus ferment filtrate.

• Group 4: Zubio toothpaste containing HAP.

• Group 5: Curasept Biosmalto toothpaste containing 1000-ppm sodium monofluorophosphate, HAP.

• Group 6: Distilled water (no toothpaste treatment control group).

In total, 48 permanent third molars extracted for orthodontic reasons were collected. Before inclusion in the study, the teeth were visually examined, and those with caries, cracks, or defects were excluded. Until the time of the experiment, the specimens were stored in deionized water at +4°C and used within 2 months. The crowns of the teeth were separated from the roots at the cementoenamel junction using a diamond disc under water cooling. Each tooth was sectioned buccolingually with a low-speed cutting device (Mod Dental Esetron Mekatronik, Turkey). The obtained specimens were embedded in acrylic resin using silicone molds (10 mm height×10 mm width), leaving the buccal surfaces exposed for demineralization and remineralization procedures. To prevent thermal damage during polymerization of the acrylic, the specimens were kept in water throughout the process. The measurement surfaces were then flattened and polished using silicon carbide abrasive papers of 400; 800; 1,200; and 2,000 grit on a grinding and polishing machine (Saphir 530, Germany). After polishing, windows measuring 3 × 3 mm² were created on the enamel surfaces by applying labels, and the remaining surfaces were coated with nail varnish. The measurements were performed by a single examiner. To ensure intra-observer reliability and minimize measurement error, the final microhardness value at each stage was determined by averaging three sequential measurements taken from different points within the same area. Furthermore, the use of a microhardness tester equipped with automated calculation features based on diagonal lengths minimized subjective examiner bias, thereby enhancing intra-observer consistency.

pH Cycling and Treatment with Toothpastes

The baseline, post-demineralization, and post-pH cycling microhardness measurements of the prepared samples were performed using a Vickers microhardness tester (HWMMT-X3B, TTS Unlimited, Japan). After the samples were placed under the microscope, a clear image was obtained at ×40 magnification by moving the microscope stage within an area measuring 3 × 3 mm². A load of 200 grams was applied for 15 s, and the diagonal lengths of the indentation created by the diamond tip on the enamel surface were measured. The microhardness values were automatically calculated by the device. Measurements were repeated three times at different points within the same area measuring 3 × 3 mm², and the mean values were recorded. Following the initial microhardness measurement, the samples were immersed in the demineralization solution and incubated at 37°C for 4 days. At the end of the 4-day period, the microhardness measurement was repeated. The samples were subjected to a pH cycling regimen for 14 days to simulate the oral environment. The undiluted toothpastes were applied twice daily, each time using 1 gram measured on a precision balance and gently spread over the tooth surface with a standard applicator for 2 min. The samples were then kept in the demineralization solution for 6 h and subsequently in the remineralization solution for 16 h at 37°C to complete the daily cycle. After completion of the 14-day pH cycling period, the final microhardness measurement was performed following the same procedures, and the data were recorded.

Demineralization and remineralization solutions prepared in accordance with ISO 20795 and ISO/TS 11405 standards were used during the initial caries lesion formation and pH cycling stages. According to the manufacturer’s specifications, the demineralization solution contained 2.2-mM calcium chloride (CaCl₂), 2.2-mM sodium dihydrogen phosphate (NaH₂PO₄), and 0.05-M acetic acid, with the pH adjusted to 4.4. The remineralization solution contained 1.5-mM calcium chloride, 0.9-mM sodium dihydrogen phosphate, and 0.15-M potassium chloride (KCl), with the pH adjusted to 7.0. A new bottle of each solution was prepared daily, using separate containers for the demineralization and remineralization processes. The containers were thoroughly washed and dried during each solution replacement.

Agar Diffusion Test

To evaluate the antibacterial efficacy of the toothpastes, four lyophilized microorganism strains obtained from the American Type Culture Collection (ATCC) and stored in an inactive state at 4 ± 10°C were used: Lactobacillus acidophilus ATCC 4356, Streptococcus mutans ATCC 25175, Enterococcus faecalis ATCC 29212, and Candida albicans ATCC 10231. Although the agar diffusion method does not fully reflect clinical conditions and is influenced by diffusion capacity, it was selected due to its reproducibility and widespread use in preliminary antibacterial screening studies.

For the cultivation and verification of the lyophilized bacterial strains used in this study, each strain was inoculated onto its appropriate culture medium. Pure colonies obtained were subsequently transferred into tryptic soy broth and incubated at 36°C to prepare bacterial suspensions. Tryptic soy agar plates, used in the antibacterial tests, were poured to a uniform thickness of approximately 5 mm. The concentration of the bacterial suspensions was adjusted to the 0.5 McFarland standard using a VITEK^® DensiChek device and subsequently diluted at a ratio of 1:10. Each microbial suspension was evenly spread onto individual tryptic soy agar plates using a sterile spreader. On each plate, three wells (6 mm in diameter) were created using a sterile Pasteur pipette: two wells were filled with the test toothpastes, and the third, serving as the negative control, was filled with Vaseline. While liquid-phase distilled water is prone to leakage and rapid diffusion across the agar surface, the semi-solid and hydrophobic nature of Vaseline ensures its stability within the well. Consequently, it effectively mimics the physical and mechanical presence of the undiluted toothpaste formulations. The toothpastes were applied directly into the 6-mm wells in their undiluted form, with 100 mg introduced per well. All toothpastes and the control group were evaluated using independent replicate plates. For each microorganism, the experiments were performed in triplicate (independent plates per group), and the inhibition zone diameters were calculated as mean values with standard deviations. Plates containing L. acidophilus were incubated under 5% CO₂ conditions, whereas the other strains were incubated aerobically at 36°C for 48 h. Following incubation, the diameters of the inhibition zones formed around each toothpaste were measured at two different points using a caliper, and the mean values were calculated for statistical analysis. The 6-mm well diameter was included in the calculation of the overall inhibition zone diameter.

Statistical Analysis

All data were recorded and analyzed using SPSS (Statistical Package for the Social Sciences) for Windows, version 22. Prior to data analysis, the assumptions required for selecting either parametric or nonparametric tests were examined. For repeated measures comparisons, the Friedman test was used, while the Kruskal-Wallis H test was applied for comparisons involving more than two independent groups. Pairwise comparisons were performed using the Bonferroni correction. A significance level of 0.05 was used as the threshold for determining statistical significance.

Results

Results of Microhardness Analysis

Intergroup statistical analysis revealed no significant differences in surface microhardness values among the groups at the baseline, after demineralization and after pH cycling stages (p > 0.05). Following remineralization, the highest mean microhardness value was observed in the Kids Ozon group, while the lowest mean value was recorded in the distilled water group; however, this difference among the groups was not statistically significant. In contrast, intragroup comparisons demonstrated statistically significant differences in surface microhardness values between the baseline, after demineralization and after pH cycling stages within each toothpaste group. In all groups, the microhardness values measured at the initial and after remineralization stages were higher than those recorded after demineralization (“Table 2”).

Table 2.

Intergroup and intragroup comparison of surface microhardness values at baseline, after demineralization, and after pH cycling stages

Groups	Baseline	After demineralization	After pH cycling	p values
Oral-B Pro-kids	333.31±43.64	264.43±40.13	292.76±34.86	0.002*
	326.65	262.60	290.84
Kids Ozon	329.81±32.19	250.71±67.17	304.83±39.75	0.001*
	331.20	273.12	316.85
Dentiste Junior Postbiotics	344.81±30.42	256.35±44.60	303.97±38.43	0.003*
	341.85	263.69	309.67
Zubio	333.45±29.02	249.21±32.82	295.15±35.04	0.001*
	331.35	261.30	295.02
Curasept Biosmalto	329.82±44.92	256.26±32.97	295.75±23.19	0.001*
	319.93	260.44	300.17
Distilled water	340.06±28.37	268.74±28.65	274.83±17.1	0.001*
	342.47	257.77	272.75
p values	0.97	0.96	0.31	​

Data are presented as mean ± standard deviation in the upper row, and as median in the lower row for each group. p < 0.05.

KW, Kruskal-Wallis H test; X2, Friedman test.

Results of Antibacterial Activity

As a result of the antibacterial activity evaluation, all the toothpastes used exhibited antimicrobial effects. No inhibition zone was observed in the negative control group. The mean and standard deviation values of the inhibition zone diameters, based on statistical analyses, are presented in “Table 3.” In the antibacterial activity evaluation, Curasept Biosmalto demonstrated a statistically significantly higher antibacterial effect against the S. mutans strain compared to only the Kids Ozon group (p < 0.05). Although it showed higher antibacterial activity compared to the other toothpastes, the differences were not statistically significant (p > 0.05). When antibacterial activity was evaluated against L. acidophilus, E. faecalis, and C. albicans strains, no statistically significant differences were found among the toothpastes (p > 0.05).

Table 3.

The mean and standard deviation values of the inhibition zone diameters

Groups	Streptococcus mutans	Lactobacillus acidophilus	Candida albicans	Enterococcus faecalis
Oral-B Pro-kids	25.06±0.39	19.22±0.62	10.98±0.67	15.98±0.39
Kids Ozon	6.73±0.18*	15.78±0.81	6.48±0.32	6.45±0.14
Dentiste Junior	28.05±0.40	21.00±1.63	7.31±0.06	10.44±0.17
Postbiotics
Zubio	18.52±0.23	14.57±0.95	14.40±1.48	16.19±0.44
Curasept Biosmalto	41.05±0.41*	31.84±0.40	30.85±0.55	6.65±0.18
Vaseline	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
p values	0.03*	0.06	0.06	0.06

Kruskal-Wallis H test. p < 0.05.

Discussion

The present study showed that all evaluated pediatric toothpastes enhanced enamel remineralization and exhibited inhibitory effects against cariogenic microorganisms under in vitro conditions. The null hypothesis was accepted. While no statistically significant differences were detected among the groups, the results suggest that similar effects can occur through different biological and chemical mechanisms, depending on the active ingredients.

Fluoride is well recognized as a key agent in caries prevention because it promotes remineralization and reduces demineralization. When incorporated into enamel, fluoride helps form fluorapatite, which is more resistant to acid than natural HAP. In this study, fluoride-containing toothpastes increased enamel surface microhardness after pH cycling, and these results are consistent with previous studies showing that regular exposure to low levels of fluoride supports remineralization in early carious lesions [3, 4].

In the present study, the comparable remineralization outcomes exhibited by the ozone- and propolis-containing toothpastes can be primarily attributed to the well-established remineralization efficacy of fluoride, which serves as the base active ingredient in these formulations. Although the Kids Ozon toothpaste exhibited the highest numerical remineralization values, no statistically significant difference was found compared with the other groups. This suggests that while the addition of ozone and propolis may offer a potential synergistic effect, it does not provide a statistically significant additional advantage beyond fluoride alone. Similarly, Kade et al. [14] reported that fluoride-containing, herbal toothpastes and ozonated oil all produced remineralization, with no significant differences among the groups. The comparable outcomes observed between Oral-B Pro-Kids and Kids Ozon suggest that the addition of ozone and propolis does not provide an additional advantage beyond fluoride alone. Consistently, Nie et al. [15] demonstrated that fluoride is more effective than ozone in promoting remineralization. Erçin et al. [16] reported that combining ozone with high-fluoride toothpastes may enhance remineralization, suggesting that the effectiveness of ozone may depend on the accompanying active ingredients and the fluoride concentration. Kids Ozon showed remineralization outcomes comparable to those of the HAP containing toothpastes Curasept Biosmalto and Zubio. Ribeiro et al. [17] reported that the combination of fluoride, n-HAP, propolis, and xylitol produced the highest remineralization effect, underscoring the importance of the synergistic use of active ingredients. The fluoride-free Zubio toothpaste provided remineralization outcomes comparable to the other toothpastes. This finding aligns with the results of Amaechi et al. [18] and Uysal et al. [19], who reported that HAP-containing toothpastes can exhibit remineralization effectiveness similar to fluoride. Amaechi et al. [20] also noted that HAP may produce a more pronounced effect on more porous surfaces, such as root surfaces. Hegazy et al. [21] demonstrated that HAP-containing products provide greater remineralization in early carious lesions. In the present study, Zubio also promoted remineralization; however, no significant differences were observed between the groups.

In the agar diffusion test, all toothpastes demonstrated antibacterial activity, with the largest inhibition zones observed in Curasept Biosmalto, which contains fluoride and HAP. The antibacterial effect of Kids Ozon was lower in comparison. Çakir et al. [22] reported that ozone-containing toothpaste showed lower antibacterial activity than fluoride-based formulations, while Düzyol et al. [23] reported that ozone therapy applied to deep dentin carious lesions significantly reduced the counts of S. mutans and Lactobacillus species, demonstrating the antimicrobial effect of ozone against cariogenic bacteria. These findings align with the relatively limited antibacterial effect of Kids Ozon in the present study. Antibacterial efficacy of propolis-containing products has been shown to vary depending on formulation and concentration [24]. The fluoride-free Zubio toothpaste produced inhibition zones against all tested microorganisms. Previous research indicates that HAP may exert antibacterial effects indirectly by reducing biofilm adhesion and altering mineral-surface interactions [25, 26].

Based on the literature review, studies on postbiotics have predominantly focused on their effects on periodontal diseases and plaque control. To the authors’ knowledge, this is among the first studies to evaluate a postbiotic-containing pediatric toothpaste in terms of both antibacterial activity and enamel remineralization.

This study has some limitations. The present research is an in vitro study and cannot fully replicate oral conditions due to the absence of many factors found in the human mouth, such as saliva and bacteria, which have significant effects on remineralization and demineralization processes. Additionally, the remineralization activity in the present study was evaluated over a period of only 14 days; therefore, only the short-term effects of the tested materials could be assessed. Further studies are required to evaluate the long-term effects of these materials and the durability of their remineralization potential. Another limitation is that primary enamel differs from permanent enamel in mineral content, thickness, and acid solubility. As the structural characteristics of primary teeth can lead to sample loss or measurement instability during experimental procedures, third molars were preferred for sample preparation. Therefore, clinical extrapolation of these findings to primary dentition should be interpreted with caution.

Conclusion

The present study supports the established evidence that fluoridated toothpastes exert remineralizing and antibacterial effects on artificially induced caries lesions in vitro. The addition of bioactive components such as ozone, propolis, and postbiotics may have a potential synergistic effect with fluoride; however, their added benefit appeared marginal and was not statistically significant. Additionally, HAP-based toothpastes showed comparable remineralizing and antibacterial effects to fluoridated toothpastes under the conditions of this in vitro study. While fluoride remains the gold standard for remineralization, alternative and bioactive formulations may serve as supportive agents in early caries prevention and oral health maintenance in children. Nevertheless, further in vivo studies are required to confirm these findings and evaluate their true efficacy under clinical conditions.

Statement of Ethics

The ethical approval required for this study was obtained from the Non-Interventional Clinical Research Ethics Committee of Aydin Adnan Menderes University, Faculty of Dentistry (File No: 2024/07, Decision No: 01, Date: 21.08.2024).

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was supported by Aydin Adnan Menderes University Scientific Research Projects Coordination Unit (Project No: DHF-24012).

Author Contributions

Işıl Sönmez and Rabia Kadriye Özçiftci conceived the ideas, analyzed the data, and led the writing. Rabia Kadriye Özçiftci collected the data.

Funding Statement

This study was supported by Aydin Adnan Menderes University Scientific Research Projects Coordination Unit (Project No: DHF-24012).

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.