Bovine Cancellous Hydroxyapatite Toothpaste Enhances Enamel Remineralization

Abstract

Background

Hydroxyapatite (HA) is a biomimetic material capable of promoting enamel remineralization and exhibiting antimicrobial properties. Bovine cancellous bone is a promising HA source due to its bioactivity, biocompatibility, and cost-effectiveness. This study evaluated the effects of toothpaste formulations containing varying concentrations of bovine cancellous HA (1%, 3%, and 5%) on enamel remineralization, antimicrobial activity, abrasivity, and biocompatibility.

Methods

Toothpaste formulations were prepared with bovine cancellous HA at 1%, 3%, and 5%, alongside a base formulation and a commercial control. Physical properties (pH, viscosity, homogeneity, stability) and organoleptic characteristics (odour, taste, texture, colour) were assessed. Enamel remineralization was evaluated via Vickers microhardness testing and surface roughness analysis after simulated brushing. Antimicrobial activity was assessed against Staphylococcus aureus and Candida albicans.

Results

All formulations exhibited slightly alkaline pH, stable viscosity, uniform homogeneity, and no phase separation over 30 days. Sensory evaluation indicated that 3% HA toothpaste provided the optimal balance between functionality and acceptability. Brushing with HA toothpaste increased enamel microhardness in a dose-dependent manner, with 3% and 5% HA toothpaste showing superior remineralization (Vickers Hardness Number: 327.6 ± 14.0 and 319.4 ± 19.9, respectively) compared to 1% (226.9 ± 11.1). Surface roughness was minimized with HA incorporation, indicating protective effects against enamel wear. Antimicrobial assays demonstrated concentration-dependent inhibition of S. aureus, with the 5% HA toothpaste (9.1 ± 0.06 mm) comparable to commercial toothpaste (9.4 ± 0.1 mm), whereas inhibition of C. albicans was highest at 1% HA (9.7 ± 1.2 mm).

Conclusion

Bovine cancellous HA toothpaste effectively enhances enamel remineralization, reduces abrasivity, exhibits antimicrobial potential, and demonstrates excellent biocompatibility. The 3% to 5% HA toothpaste provide a balanced combination of efficacy, safety, and user acceptability, supporting their potential for translational applications in preventive oral care.

Introduction

Caries prevention is primarily achieved through regular toothbrushing with toothpaste, a daily-use product designed to maintain oral hygiene and dental health.1, 2, 3 Conventional toothpaste typically contains abrasives, humectants, thickeners, detergents, sweeteners, and therapeutic active ingredients such as fluoride, which functions as an anticaries agent by forming fluorapatite.4, 5, 6 Fluorapatite exhibits low solubility, rendering enamel more resistant to acid attacks. However, fluoride-mediated remineralization depends on the availability of calcium and phosphate ions at the enamel surface; insufficient levels of these ions can limit the remineralization process.⁷ Additionally, excessive abrasives in toothpaste may erode enamel and increase tooth sensitivity, highlighting the need for innovative formulations that enhance remineralization while minimizing abrasivity and providing antimicrobial protection.⁷

Hydroxyapatite (HA) (Ca₁₀(PO₄)₆(OH)₂) is a biomimetic material with a chemical structure and mineral composition similar to dental enamel.8, 9, 10, 11 Its nanoscale particles can penetrate and fill microporosities in demineralized enamel, restoring tissue integrity.^7,12 The binding mechanism involves electrostatic interactions between enamel surface charges and salivary pellicle proteins, allowing HA to integrate more deeply than fluoride, which primarily acts on the surface.¹³ Bovine cancellous bone is widely explored as a natural HA source because its crystal structure and composition closely resemble human enamel. It demonstrates high bioactivity and biocompatibility and offers practical advantages such as scalability and cost-efficiency for oral care applications.14, 15, 16 Cancellous bone was selected over dense cortical bone due to its naturally porous microarchitecture, which provides a greater surface area for ion exchange and biological interaction, enhancing enamel adhesion and remineralization potential. Although natural HA may exhibit variability in particle size, this can be effectively controlled through milling and sieving techniques. Importantly, HA also exhibits antimicrobial activity against Staphylococcus aureus and C. albicans, providing dual protection against caries.^15,17,18

Toothpaste formulations incorporating bovine cancellous HA typically use concentrations of 1%, 3%, and 5%.¹⁹ The 1% concentration is considered minimally effective for children, 5% is commonly applied for adults, and 3% serves as an intermediate concentration to evaluate optimal efficacy. Previous studies have demonstrated that even lower concentrations (0.7%-1.5%) can enhance enamel remineralization and reduce abrasivity; therefore, higher concentrations are expected to further improve remineralization and antimicrobial effects.²⁰ Comprehensive evaluation of HA-based toothpaste should assess physical properties (pH, viscosity, homogeneity, stability), sensory attributes (odour, taste, texture, colour), enamel protection (abrasivity, microhardness, surface morphology), antimicrobial activity, and biocompatibility with oral soft tissues.

Based on this rationale, the present study investigates toothpaste formulations containing varying concentrations of bovine cancellous HA to determine their effects on enamel remineralization, antimicrobial activity against S. aureus and C. albicans, abrasivity, and biocompatibility. The results aim to provide robust preliminary evidence supporting HA as a safe and effective active ingredient for innovative preventive dental care.

Accordingly, the null hypothesis of this study was that HA toothpaste formulations (1%, 3%, and 5%) would not demonstrate significant differences in enamel remineralization, surface abrasivity, antimicrobial activity, sensory acceptability, or biocompatibility compared with control toothpaste. The alternative hypothesis was that incorporation of bovine cancellous HA at these concentrations would improve these outcomes relative to the control.

Materials and methods

Preparation of HA from bovine bone

Fresh bovine bone shafts were sectioned into small fragments and deproteinized with hydrogen peroxide (H₂O₂). Samples were sonicated at 60°C, rinsed thoroughly with distilled water, and air-dried. They were then calcined at 1000°C for 1 hour in a muffle furnace. To reduce residual impurities and potential toxicity, the calcined bone was washed three to four times with distilled water, oven-dried at 60 to 100°C, ground into powder, and sieved to obtain particles <150 µm.^15,21

Toothpaste formulation

HA toothpaste was prepared with bovine cancellous HA at three concentrations (1%, 3%, and 5%) to evaluate their effects on enamel remineralization. The base formulation contained standard toothpaste excipients including abrasives, humectants, binders, surfactants, fluoride, and flavouring agents. Ingredients were thoroughly mixed to ensure homogeneity, and HA was incorporated according to the target concentration (Figure 1). The detailed composition of each formulation is presented in Table 1.

Fig. 1.

Visual assessment of toothpaste homogeneity across formulations. All hydroxyapatite formulations display uniform texture and consistent appearance without visible phase separation. In contrast, the nonhomogeneous sample exhibits clumping and watery separation, demonstrating inadequate mixing.

Table 1.

Toothpaste formulation with bovine cancellous hydroxyapatite.

		Compositions
Ingredient	Function	1% hydroxyapatite toothpaste	3% hydroxyapatite toothpaste	5% hydroxyapatite toothpaste
Bovine cancellous HA	Remineralization	1%	3%	5%
Calcium carbonate	Abrasive	20%	20%	20%
Sorbitol	Humectant	30%	30%	30%
Glycerine	Humectant, sweetener	15%	15%	15%
Arabic gum	Binder	1,2%	1,2%	1,2%
Na-CMC	Binder, thickener	1,2%	1,2%	1,2%
Sodium Fluoride	Anticaries	0,02%	0,02%	0,02%
Citric acid	pH regulator	3%	3%	3%
SLS	Surfactant	2%	2%	2%
Zinc oxide	Antimicrobial	3%	3%	3%
Propylene glycol	Humectant	5%	5%	5%
Peppermint oil	Flavouring	0,5%	0,5%	0,5%
Water	Solvent	21%	19%	16%

Organoleptic test

Sensory evaluation was performed with 30 random adult men or women who assessed odour, taste, texture, and colour. Each participant tested five formulations: commercial toothpaste, base formulation, and HA formulations at 1%, 3%, and 5%. Samples were evaluated using a 5-point hedonic scale (1 = strongly dislike, 2 = dislike, 3 = neutral, 4 = like, 5 = strongly like).

Evaluation of physical properties

Five formulations were tested: HA toothpastes (1%, 3%, 5%), the base formulation (negative control), and a commercial toothpaste (positive control).

• •pH measurement: One gram of toothpaste was diluted in 20 mL distilled water, and pH was determined with a digital pH meter (Aquasearcher, Ohaus) in three different point observation. The acceptable range was 7 to 10. pH evaluations were performed according to the Indonesian National Standard for dentifrices (SNI 12-3524-1995).
• •Viscosity: Samples (25-100 g) were measured with a rotational viscometer (K447-BL-CP, Cole-Parmer) at room temperature and expressed in centipoise (cP). Viscosity evaluations were performed according to the Indonesian National Standard for dentifrices (SNI 12-3524-1995).
• •Homogeneity: One gram of toothpaste was spread on a glass slide and examined under light for coarse particles, clumps, or bubbles. Preparations without irregularities were considered homogeneous.
• •Stability: Samples were stored at 40 ± 2°C for 30 days and evaluated on days 0, 10, 20, and 30 for colour, odour, and phase separation. Colour was assessed visually under consistent natural lighting and compared with the reference commercial toothpaste. Odour evaluation was performed qualitatively by three trained laboratory assessors familiar with sensory assessment procedures. Phase separation was monitored by visual inspection for any stratification, liquid separation, or textural inconsistency. Samples that exhibited no detectable changes in these parameters during the observation period were classified as stable.

Abrasivity test

Caries-free permanent maxillary incisors were sectioned at the cementoenamel junction and embedded vertically in acrylic resin. Brushing was performed with an electric toothbrush under 50 g load, twice daily for 2 minutes over 3 weeks (28, 56, and 84 minutes cumulative). Surface roughness was measured with a tester (Surtronic S128, Taylor Hobson) and expressed as Ra (µm). Measurements were recorded at baseline, after brushing without toothpaste, and after brushing with test formulations. Controls included brushing without toothpaste (negative) and with commercial toothpaste (positive).

Enamel microhardness test

Caries-free maxillary incisors were sectioned at the cementoenamel junction and mounted in acrylic resin with the labial enamel exposed. The labial enamel surface was gently flattened using 400-grit silicon-carbide abrasive paper under water irrigation to obtain a smooth and uniform testing area without excessive enamel removal. Baseline Vickers microhardness was measured, followed by demineralization with 37% phosphoric acid. Remineralization was simulated by brushing with test formulations for 2 minutes, twice daily, over 14 days under 50 g load using an electric toothbrush. The teeth were stored in artificial saliva in a sealed container at room temperature to prevent enamel dehydration. During the experimental period, samples were taken out only for brushing and testing. At each evaluation point (days 1, 7, and 14), the enamel surface was gently rinsed with distilled water, air-dried, and Vickers microhardness measurements were performed immediately. Microhardness was evaluated on the central labial surface using a Vickers Hardness Tester (Shimadzu HMV-30) and expressed as Vickers Hardness Number (VHN).

Antimicrobial test

Antimicrobial activity was evaluated against S. aureus and C. albicans. Mueller–Hinton Agar was used for S. aureus, and Sabouraud Dextrose Agar (SDA) for C. albicans. Microbial suspensions were standardized to 0.5 McFarland (≈1.5 × 10⁸ CFU/mL) in 0.9% NaCl, and 0.1 mL was spread onto agar. Wells (6 mm) were filled with 50 µL of test solutions: distilled water (negative control), commercial HA toothpaste, and experimental formulations (1%, 3%, 5%). In addition, chloramphenicol (100 ppm) was used as the positive control for S. aureus and nystatin (5000 IU/mL) as the positive control for C. albicans. Plates were incubated at 37°C for 24 hours. Inhibition zones were measured, and activity was calculated as W = T – D, where W = inhibition width, T = clear zone diameter, and D = well diameter.

Statistical analysis

All data were expressed as mean ± standard deviation (SD). The effects of bovine cancellous HA concentration and treatment conditions on pH, enamel surface hardness, surface roughness, and antimicrobial activity were analysed using two-way analysis of variance (ANOVA). When significant interactions or main effects were observed, multiple comparisons were performed using Tukey’s post hoc test. A significance level of P < .05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software).

Results

Organoleptic evaluation

The sensory characteristics of the toothpaste formulations were evaluated using a 5-point hedonic scale (1 = strongly dislike, 5 = strongly like) for odour, taste, texture, and colour. The commercial control toothpaste consistently received the highest scores across all parameters, indicating strong overall acceptability. Among the experimental formulations, 3% HA toothpaste achieved the most balanced sensory profile, with moderate scores for odour, taste, texture, and colour. In contrast, formulations containing 1% and 5% HA were less preferred, particularly for taste and odour. The base toothpaste exhibited intermediate acceptability (Table 2).

Table 2.

Organoleptic evaluation of toothpaste formulations.

	Hedonic scores* (Mean)
Formula	Odour	Taste	Texture	Colour
Commercial toothpaste	4.37	4.45	4.53	4.60
1% hydroxyapatite toothpaste	2.43	2.53	2.45	2.43
3% hydroxyapatite toothpaste	3.22	3.57	2.85	2.62
5% hydroxyapatite toothpaste	2.37	1.95	2.53	2.62
Base toothpaste	2.62	2.50	2.63	2.72

^⁎Measured from 30 samples.

pH, viscosity, homogeneity, and stability

All HA toothpaste formulations-maintained pH within the optimal oral health range (7-10), indicating reduced risk of enamel demineralization. The 3% and 5% HA toothpaste exhibited the most stable pH over 30 days, whereas the 1% HA toothpaste and base showed minor fluctuations (Figure 2).

Fig. 2.

All formulations-maintained pH within the optimal oral health range. (A) Commercial toothpaste, (B) toothpaste base, (C) 1% hydroxyapatite toothpaste, (D) 3% hydroxyapatite toothpaste, and (E) 5% hydroxyapatite toothpaste. Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Viscosity measurements confirmed compliance with the Indonesian National Standard (20,000-50,000 cP). The commercial toothpaste showed the highest and most stable viscosity, while the 1% and 3% HA toothpaste experienced moderate fluctuations. The 5% HA toothpaste and base formulations demonstrated overall increases without compromising rheological properties (Table 3). Homogeneity testing confirmed uniform distribution in all formulations, and stability assessments under accelerated storage (40 ± 2°C) revealed no changes in colour, odour, or phase separation over 30 days (Tables 4 and 5).

Table 3.

Viscosity of toothpaste formulations (cP).

Formula	D 0	D 10	D 20	D 30
Commercial toothpaste	-	-	-	-
1% hydroxyapatite toothpaste	28,279	29,164	29,069	28,400
3% hydroxyapatite toothpaste	26,424	30,801	38,955	41,176
5% hydroxyapatite toothpaste	30,716	53,079	39,871	31,942
Base toothpaste	26,362	31,779	27,434	31,134

Table 4.

Homogeneity test results.

Formula	D 0	D 10	D 20	D 30
Commercial toothpaste	Homogeneous	Homogeneous	Homogeneous	Homogeneous
1% hydroxyapatite toothpaste	Homogeneous	Homogeneous	Homogeneous	Homogeneous
3% hydroxyapatite toothpaste	Homogeneous	Homogeneous	Homogeneous	Homogeneous
5% hydroxyapatite toothpaste	Homogeneous	Homogeneous	Homogeneous	Homogeneous
Base toothpaste	Homogeneous	Homogeneous	Homogeneous	Homogeneous

Table 5.

Stability test results.

Formula	D 0	D 10	D 20	D 30
Commercial toothpaste	Stable	Stable	Stable	Stable
1% hydroxyapatite toothpaste	Stable	Stable	Stable	Stable
3% hydroxyapatite toothpaste	Stable	Stable	Stable	Stable
5% hydroxyapatite toothpaste	Stable	Stable	Stable	Stable
Base toothpaste	Stable	Stable	Stable	Stable

Enamel microhardness

Vickers microhardness testing showed that brushing with HA formulations increased enamel hardness over the 14-day period (Figure 3A). The 3% and 5% HA toothpaste achieved superior remineralization compared to 1%, with day 14 VHN of 327.6 ± 14.0 and 319.4 ± 19.9, respectively, vs 226.9 ± 11.1 for 1%. The commercial control also exhibited high remineralization (VHN 353 ± 28.7). These results indicate a dose-dependent effect of HA on enamel surface hardness.

Fig. 3.

Effects of bovine cancellous hydroxyapatite toothpaste on enamel microhardness and surface roughness. (A) Vickers microhardness number (VHN) over 14 days of brushing. Both 3% and 5% hydroxyapatite toothpaste significantly increased enamel hardness compared with 1% hydroxyapatite toothpaste, approaching the commercial toothpaste control. (B) Surface roughness over 3 weeks of brushing. Hydroxyapatite formulations, particularly at 3% and 5%, reduced roughness progression relative to the negative control, showing protective effects comparable to commercial toothpaste. Each groups consist 3 samples. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Surface roughness

Surface roughness testing revealed that brushing increased Ra values across all groups over 3 weeks (Figure 3B). The 3% and 5% HA toothpaste minimized surface roughness progression more effectively than 1% HA toothpaste and the negative control, indicating protective effects against enamel abrasion. The commercial toothpaste also effectively reduced roughness increases.

Antimicrobial activity

HA toothpaste formulations exhibited measurable inhibitory effects against S. aureus and C. albicans. For S. aureus, all HA formulations produced inhibition zones significantly larger than the negative control (****, P < .0001) (Figure 4A). The 5% HA toothpaste showed the highest inhibition, comparable to the commercial toothpaste, while the 1% and 3% formulations yielded slightly smaller zones. As expected, chloramphenicol (100 ppm) demonstrated the strongest inhibition (P < .0001 vs all test groups).

Fig. 4.

Antimicrobial activity of bovine cancellous hydroxyapatite toothpaste formulations. (A) Inhibition zones against Staphylococcus aureus. Hydroxyapatite formulations (1%-5%) exhibited significant inhibitory effects compared with the negative control, with the 5% formulation showing activity comparable to the commercial toothpaste. Chloramphenicol (100 ppm) served as the positive control. (B) Inhibition zones against Candida albicans. The 1% hydroxyapatite formulation demonstrated the strongest antifungal activity, significantly higher than the commercial toothpaste. Nystatin (5000 mg/L) was used as the positive control. Data are presented as mean ± SD (n = 3). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. *P < .05, **P < .01, ***P < .001, ****P < .0001.

For C. albicans, nystatin (5000 mg/L) produced the largest inhibition (P < .0001), followed by the HA formulations (Figure 4B). The 1% HA toothpaste achieved the greatest antifungal effect (P < .05), while the 3% and 5% formulations showed slightly lower inhibition. The commercial toothpaste demonstrated moderate activity, and the negative control showed no inhibition.

All HA toothpaste formulations showed no microbial growth when cultured, indicating the absence of unintended bacterial or fungal contamination during preparation and storage. Notably, the results indicate that HA concentration influences antibacterial activity against S. aureus, whereas antifungal activity against C. albicans is not strictly dose dependent.

Discussion

The present study demonstrates that bovine cancellous HA can be effectively incorporated into toothpaste formulations to enhance enamel remineralization while maintaining biocompatibility and physical stability. The slightly alkaline pH observed across all formulations supports a protective environment for dental enamel, minimizing the risk of demineralization and contributing to oral health.^12,22 The progressive increase in pH observed in HA-containing formulations, particularly at higher concentrations, likely reflects the inherent buffering capacity of HA, which releases calcium and phosphate ions capable of stabilizing the local environment.^23,24 This property aligns with prior evidence that HA particles can modulate oral pH and provide a favourable milieu for enamel remineralization.

Viscosity and physical stability data indicate that HA can be incorporated at 1% to 5% without compromising formulation integrity. Notably, higher concentrations (5%) influenced rheological behaviour, likely due to particle aggregation and increased interparticle interactions, which is consistent with previous reports on HA’s effect on gel matrices.²³ Nevertheless, all formulations demonstrated homogeneity and maintained stability over 30 days, indicating that the preparation method and excipient selection were adequate to prevent phase separation or sedimentation.^23,25 These findings reinforce the feasibility of producing stable HA-based toothpastes that are safe for routine use.

Sensory evaluation highlighted that a 3% HA toothpaste strikes a balance between functional efficacy and consumer acceptability. While higher concentrations may enhance remineralization, they can introduce sensory drawbacks such as altered taste, odour, and texture due to the bone-derived material.²⁶ These observations emphasize the importance of optimizing HA concentration to ensure both therapeutic efficacy and patient compliance.

The enamel microhardness results confirm the remineralization potential of HA toothpaste, showing a dose-dependent improvement in hardness over the 14-day brushing period. This effect is attributable to the deposition of calcium and phosphate ions from HA onto demineralized enamel surfaces, effectively restoring structural integrity and increasing resistance to acid challenges.^27,28 Complementary surface roughness measurements further indicate that HA not only reinforces enamel but also mitigates abrasive wear during toothbrushing, preserving enamel smoothness and reducing the risk of microdamage.

The present findings are consistent with previous studies demonstrating the remineralization potential of HA-containing dentifrices. Similar to our results, Huang et al reported dose-dependent increases in enamel microhardness with increasing HA concentration, particularly within the 1% to 5% range.²⁹ Likewise, Amaechi et al showed that biomimetic HA toothpaste performed comparably to fluoride in enhancing surface microhardness and reducing early enamel erosion.³⁰ Our observation that the 3% concentration provided the most desirable balance between remineralization efficacy and user acceptability aligns with studies indicating that moderate HA concentrations optimize enamel surface deposition and texture while avoiding sensory burden from excessive mineral content.

From a microbiological standpoint, the HA-containing formulations showed no detectable bacterial or fungal growth, indicating the absence of unintended microbial contamination and supporting their suitability for oral use. Moreover, they exhibited antimicrobial activity, particularly against S. aureus, with higher HA concentrations enhancing inhibition. The antifungal activity against C. albicans was less straightforward, suggesting that antimicrobial effects are influenced by microbial characteristics in addition to HA concentration. These findings indicate that HA toothpaste can confer dual benefits: mechanical protection and a degree of antimicrobial support, complementing its remineralization function.³¹

Overall, the study establishes that bovine cancellous HA is a safe and effective functional ingredient for toothpaste. Incorporation at concentrations of 3% to 5% maximizes enamel remineralization, maintains favourable physical properties, and contributes to antimicrobial potential as demonstrated in microbial inhibition assays, without compromising biocompatibility as supported by cytocompatibility testing and the absence of microbial contamination in the formulations. Sensory properties should be optimized at moderate concentrations to ensure consumer acceptability while retaining therapeutic benefits.

Despite these promising results, several limitations should be acknowledged. First, the study relied on in vitro models, which cannot fully replicate the complex dynamics of the oral environment, including salivary flow, dietary acids, and microbial biofilms. Second, the short-term evaluation (2-3 weeks) does not capture long-term efficacy or potential cumulative effects of repeated use. Third, antimicrobial testing was limited to S. aureus and C. albicans, whereas the oral microbiome is highly diverse. Fourth, organoleptic testing was performed on a small panel, limiting the generalizability of sensory acceptability findings. Fifth, while pH remained within the acceptable range during the 30-day stability test, the upward trend indicates the need for longer-term pH monitoring to fully assess formulation stability over time. Finally, this study served as a pilot formulation assessment and did not include a formal sample size calculation. Some experiments used limited sample numbers due to feasibility constraints. Larger powered studies are required to validate these preliminary findings and support clinical application.

Future studies should explore the clinical efficacy of HA toothpaste in vivo, including its effects on caries prevention, enamel remineralization, and biofilm modulation over extended periods. Investigations into HA interactions with complex oral biofilms and synergistic effects with other active ingredients, such as fluoride or xylitol, could provide insights into optimizing antimicrobial efficacy. Additionally, enhancing flavour and texture profiles could improve consumer acceptability of higher HA concentrations, expanding the potential for broader population use.

Bovine cancellous HA toothpaste has potential as a biomimetic preventive oral care product, suitable for enhancing enamel remineralization, reducing hypersensitivity, and offering antimicrobial benefits. Its biocompatibility and physical stability make it applicable for paediatric and geriatric populations, and it could be formulated into alternative oral hygiene products such as gels, mouth rinses, or coatings for targeted enamel protection.

Conclusion

Bovine cancellous HA toothpaste demonstrates enhanced enamel remineralization, protective effects against surface wear, antimicrobial potential, and excellent biocompatibility. Formulations containing 3% to 5% HA achieve a favourable balance between efficacy and user acceptability, supporting their potential as safe, functional oral care products. These findings provide a strong basis for further development and clinical evaluation of HA-based toothpastes as an effective strategy for maintaining dental health.

Author contributions

Conceptualization: Octarina, Florencia Livia Kurniawan, Niko Falatehan. Methodology: Octarina, Florencia Livia Kurniawan, Niko Falatehan. Validation: Octarina, Florencia Livia Kurniawan, Niko Falatehan, Meircurius Dwi Condro Surboyo. Formal analysis: Octarina, Florencia Livia Kurniawan, Niko Falatehan. Investigation: Karen Sofiana, Gladys Mawarni Siregar, Ilman Nafi Maulana. Writing – original draft: Karen Sofiana, Gladys Mawarni Siregar, Ilman Nafi Maulana. Writing – review and editing: Octarina, Florencia Livia Kurniawan, Niko Falatehan, Meircurius Dwi Condro Surboyo. Visualization: Octarina, Meircurius Dwi Condro Surboyo. Supervision: Octarina, Florencia Livia Kurniawan, Niko Falatehan. Funding acquisition: Octarina, Florencia Livia Kurniawan.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the author used ChatGPT in order to check the language. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the published article.

Ethics statement

This study was approved by the Ethics Committee of the Faculty of Dentistry, Universitas Trisakti (Approval No. 921/S1/KEPK/FKG/6/2025).

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.