Dental caries is one of the most prevalent chronic diseases affecting the oral cavity. It is a multifactorial disease characterized by the progressive dissolution of dental hard tissues due to bacterial acid production and changes in the oral environment. Enamel is composed primarily of hydroxyapatite crystals, which undergo continuous cycles of demineralization and remineralization. When the balance shifts towards demineralization, subsurface mineral loss occurs, resulting in the formation of initial enamel lesions, commonly recognized clinically as white spot lesions. Early enamel lesions are potentially reversible through non-invasive remineralization strategies. Fluoride has traditionally been considered the cornerstone of preventive dentistry because of its ability to reduce enamel solubility and promote remineralization [1]. However, alternative calcium-phosphate-based remineralizing agents have gained considerable attention. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) CPP-ACP is a bioactive remineralizing agent derived from milk protein casein. Casein phosphopeptides stabilize amorphous calcium phosphate and maintain calcium and phosphate ions in a bioavailable state.These complexes attach to the enamel surface and can accumulate within dental plaque and on the tooth surface. They act as reservoirs of calcium and phosphate ions, thereby promoting remineralization during periods of reduced oral pH [2,3] Sodium fluoride Sodium fluoride is widely used in preventive dentistry because of its ability to enhance enamel resistance against acid dissolution.Fluoride ions facilitate the formation of fluoridated apatite and promote remineralization by enhancing mineral deposition at partially demineralized enamel surfaces. It also reduces mineral loss during subsequent acid challenges [1,4] Nano-hydroxyapatite Nano-hydroxyapatite consists of calcium phosphate particles with a chemical composition similar to natural enamel mineral. Its small particle size and large surface area facilitate interaction with enamel surfaces. These particles may deposit within surface irregularities, act as mineral reservoirs, and promote the formation of a mineral-rich protective layer [5,6] A comparative in vitro study by Geeta et al. reported the highest post-treatment surface microhardness in the nano-hydroxyapatite group, followed by CPP-ACP and fluoride[7] A systematic review and meta-analysis published in 2025 also reported promising surface-hardness findings for nano-hydroxyapatite, although the authors highlighted substantial methodological limitations and heterogeneity among the included studies [8,9] Rationale for the study Although all three agents have demonstrated remineralizing potential, differences in their mechanisms of action, composition, application protocols, and experimental conditions may influence their effectiveness. Therefore, a standardized comparative investigation is warranted to evaluate their relative ability to restore the surface microhardness of artificially demineralized enamel. Aim and objectives Aim To evaluate and compare the remineralization potential of CPP-ACP, sodium fluoride, and nano-hydroxyapatite on artificially induced enamel lesions. Objectives • To assess baseline enamel surface microhardness. • To evaluate the reduction in surface microhardness following artificial demineralization. • To determine changes in surface microhardness following application of CPP-ACP. • To evaluate the remineralization potential of sodium fluoride. • To assess the remineralization potential of nano-hydroxyapatite. • To compare the effectiveness of all three remineralizing agents statistically

Study design A controlled in vitro experimental study. Sample size A total of 60 extracted human permanent premolars were included and divided into four groups. The proposed sample size is 15 specimens per group. Final sample-size justification should be based on a pilot study, expected effect size, statistical power of 80%, and significance level of 5%. Inclusion criteria • Extracted human permanent premolars. • Intact buccal enamel surface. • No visible caries or developmental defects. • No cracks, fractures, or restorations. • No evidence of enamel hypoplasia. Exclusion criteria • Teeth with carious lesions. • Teeth with fluorosis or hypoplastic defects. • Teeth with cracks or fractures. • Teeth with previous bleaching or chemical treatment. • Teeth with extensive surface irregularities. Specimen preparation The extracted teeth were cleaned to remove adherent soft tissues and deposits. They were stored in an appropriate validated storage medium until specimen preparation. The crowns were separated from the roots, and enamel specimens were prepared from the buccal surfaces using a water-cooled diamond disc. Each specimen were embedded in acrylic resin, leaving the enamel surface exposed. The enamel surfaces were polished using progressively finer abrasive papers to obtain a standardized smooth surface. Experimental grouping Group Remineralizing agent Sample size I CPP-ACP 15 II Sodium fluoride 15 III Nano-hydroxyapatite 15 IV Artificial saliva (control) 15 Total 60 Baseline surface microhardness assessment Baseline surface microhardness recorded using a Vickers microhardness testing machine. Three indentations were made on each specimen under standardized loading conditions. The mean of the three readings were considered the baseline Vickers hardness number (VHN). Preparation of artificial enamel lesions Artificial enamel lesions were produced using a standardized demineralizing solution. The solution will contain: • Calcium chloride. • Sodium phosphate. • Acetic acid. • Deionized water. The pH adjusted to approximately 4.4 using potassium hydroxide. The specimens were exposed to the demineralizing solution under controlled laboratory conditions to produce initial enamel lesions. The reduction in microhardness was subsequently be recorded. This approach is based on the established enamel-lesion preparation protocol described by Geeta et al[7] Application of remineralizing agents The specimens received the respective remineralizing agents according to the manufacturer's instructions. • Group I: CPP-ACP paste. • Group II: Sodium fluoride preparation. • Group III: Nano-hydroxyapatite preparation. • Group IV: Artificial saliva without an active remineralizing agent. All specimens were maintained under standardized storage conditions in artificial saliva between applications. The exposure period, concentration, application frequency, and formulation of each agent must be standardized and documented before the experiment begins. Post-remineralization assessment Following the experimental remineralization period, surface microhardness was measured again using the Vickers microhardness tester. The following three measurements were recorded: • T0: Baseline surface microhardness. • T1: Surface microhardness after demineralization. • T2: Surface microhardness after remineralization. The percentage of surface microhardness recovery was calculated using: SMH recovery (%) = {T2-T1}/{T0-T1} X 100 Where: • T0 = Baseline microhardness. • T1 = Post-demineralization microhardness. • T2 = Post-remineralization microhardness. A higher percentage indicates greater recovery of the initial surface hardness. Statistical analysis Data was entered into a statistical software package such as SPSS. The following tests are proposed: 1. Descriptive statistics: mean and standard deviation. 2. Shapiro-Wilk test: assessment of normality. 3. Repeated-measures ANOVA: comparison of changes over time. 4. One-way ANOVA: comparison between groups. 5. Tukey post-hoc test: pairwise comparisons. 6. Statistical significance: p-value less than 0.05.

Table 1. Comparison of enamel surface microhardness Vickers hardness values reported Group n Baseline (Mean ± SD) After demineralization (Mean ± SD) After remineralization (Mean ± SD) Sodium fluoride 15 236.54 ± 75.60 196.81 ± 63.54 201.86 ± 38.77 CPP-ACP 15 219.70 ± 81.07 233.84 ± 77.01 240.71 ± 58.26 Nano-hydroxyapatite 15 230.39 ± 74.75 232.57 ± 83.59 329.82 ± 69.75 Table 2. Absolute changes in published microhardness values Group Post-demineralization Post-remineralization Absolute change Sodium fluoride 196.81 201.86 +5.05 CPP-ACP 233.84 240.71 +6.87 Nano-hydroxyapatite 232.57 329.82 +97.25 Nano-hydroxyapatite demonstrated the largest numerical increase in surface microhardness, with a change of 97.25 VHN. CPP-ACP demonstrated an increase of 6.87 VHN, whereas sodium fluoride demonstrated an increase of 5.05 VHN. The findings suggest that nano-hydroxyapatite may provide greater surface-hardness recovery under the specific experimental conditions used in that study. However, these numerical differences should not be interpreted as proof of statistically significant superiority between all three agents.

The present proposed investigation aims to compare three commonly used remineralizing agents based on their ability to restore the surface microhardness of artificially demineralized enamel.The primary outcome will be the change in Vickers microhardness following treatment. Remineralization potential of CPP-ACP CPP-ACP promotes remineralization by stabilizing calcium and phosphate ions in an amorphous state. The phosphopeptides bind to calcium phosphate and prevent its premature precipitation, maintaining a reservoir of bioavailable minerals.In the study conducted by Geeta et al., the CPP-ACP group demonstrated an increase in mean microhardness from 233.84 to 240.71 VHN after remineralization [7]Kumar et al. also investigated the effect of CPP-ACP on artificial caries-like lesions and reported its remineralizing potential [10] These findings support the role of CPP-ACP as a calcium-phosphate delivery system for early enamel lesions. Remineralization potential of sodium fluoride Fluoride promotes remineralization by facilitating mineral deposition and reducing enamel dissolution during acid exposure.It enhances the formation of more acid-resistant fluoridated mineral phases.In the comparative study by Geeta et al., the sodium fluoride group showed a mean microhardness increase from 196.81 to 201.86 VHN following remineralization [7] However, fluoride efficacy is influenced by its concentration, formulation, frequency of application, and the availability of calcium and phosphate ions. Remineralization potential of nano-hydroxyapatite Nano-hydroxyapatite has gained considerable attention because its chemical composition resembles the mineral component of natural enamel.Its particles can deposit on demineralized enamel surfaces and may contribute to the restoration of surface integrity. Geeta et al. reported a post-remineralization mean microhardness of 329.82 ± 69.75 VHN in the nano-hydroxyapatite group, compared with 240.71 ± 58.26 VHN in the CPP-ACP group and 201.86 ± 38.77 VHN in the fluoride group [7] Swarup and Rao also investigated enamel surface remineralization using synthetic nano-hydroxyapatite and reported its potential to improve enamel surface characteristics [11] These findings suggest that nano-hydroxyapatite deserves further investigation as a biomimetic remineralizing agent. Comparison of the three remineralizing agents The three agents have different mechanisms of action: Parameter CPP-ACP Sodium fluoride Nano-hydroxyapatite Primary mechanism Calcium-phosphate stabilization Fluoride-mediated mineral protection Biomimetic mineral deposition Calcium availability Yes Not a primary calcium source Yes Phosphate availability Yes Not a primary phosphate source Yes Enamel interaction Mineral-ion reservoir Fluoridated mineral formation Surface deposition and crystal interaction Research potential High Extensive established evidence Promising emerging evidence A 2025 systematic review and meta-analysis by Santhosh et al. included 19 in vitro studies. The pooled estimate for fluoride versus alternative remineralizing approaches was −1.31 (95% CI: −3.90 to 1.28; p = 0.12), while nano-hydroxyapatite demonstrated a positive pooled estimate of 1.40 (95% CI: 0.65–2.15) in surface-hardness analysis. The review also highlighted substantial heterogeneity and limitations in the available evidence [8] This reinforces the importance of conducting well-standardized comparative experiments rather than relying solely on the results of individual studies. Clinical significance The findings of such investigations may contribute to the selection of appropriate remineralizing products for patients with early enamel demineralization. CPP-ACP may be useful where calcium-phosphate delivery is desirable, fluoride remains an established preventive agent, and nano-hydroxyapatite offers a biomimetic approach to enamel surface repair. However, an in vitro improvement in surface microhardness does not necessarily establish clinical superiority or demonstrate complete restoration of the enamel subsurface. Limitations of the study The proposed investigation may have the following limitations: 1. An in vitro environment cannot completely reproduce the complex oral environment. 2. Artificially induced enamel lesions may differ from naturally occurring carious lesions. 3. The duration of remineralization may influence the observed results. 4. Differences in commercial product formulations and active ingredient concentrations may affect the outcomes. 5. Surface microhardness does not independently measure subsurface mineral recovery. 6. The findings cannot be directly extrapolated to long-term clinical outcomes. Future studies incorporating transverse microradiography, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX) may provide additional information regarding mineral deposition and enamel structural changes.

CPP-ACP, sodium fluoride, and nano-hydroxyapatite represent important approaches to the remineralization of early enamel lesions. Published comparative evidence indicates that nano-hydroxyapatite can produce substantial improvements in enamel surface microhardness. CPP-ACP also demonstrates remineralizing potential through the delivery and stabilization of calcium and phosphate ions. Sodium fluoride continues to play an important role in enamel remineralization and caries prevention. The relative effectiveness of these agents should be determined through a standardized comparative experiment using consistent specimen preparation, treatment concentrations, exposure periods, and microhardness assessment. Further clinical investigations are necessary to establish whether differences observed in vitro translate into meaningful clinical benefits.

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