pH-responsive ion release kinetics of a chitosan-based hydrogel incorporating nanohydroxyapatite and sodium fluoride for enamel remineralization

Abstract

Background

Ion-releasing dental varnishes vary in composition and pH responsiveness, influencing remineralization efficacy. This study aimed to compare pH-responsive multi-ion release profiles of (1) sodium fluoride (NaF) varnish, (2) casein phosphopeptide–amorphous calcium phosphate (CPP-ACP)/NaF varnish, and (3) a newly synthesized NaF/nanohydroxyapatite (nHA)‑loaded chitosan (CS) hydrogel.

Methods

Seventy-two sound primary canine specimens were prepared with standardized enamel windows and allocated to eight groups (n = 9) by material and buffer pH (5.5 or 7.0). Test materials included NaF varnish, CPP-ACP/NaF varnish, and experimentally formulated NaF/nHA-loaded CS hydrogel. Samples were individually immersed in buffer solutions at pH 5.5 and pH 7.0. Solutions were renewed at 4, 24, 72, and 168 h, and analyzed by ion chromatography for fluoride, phosphate, and calcium concentrations. pH stability was monitored. Data were evaluated by 3-way repeated measures ANOVA and one-way ANOVA with Tukey’s post hoc (p < 0.05).

Results

At pH 5.5, CPP-ACP/NaF varnish yielded the highest cumulative fluoride release with peak fluoride output in the first four hours (22.40 ± 0.23 ppm). NaF/nHA-loaded CS hydrogel exhibited the highest calcium release in the first four hours (11.40 ± 0.20 ppm) and sustained both calcium (30.20 ± 0.20 ppm) and phosphate (36.20 ± 0.20 ppm) through 168 h. Phosphate release peaked at 24 h for both the NaF/nHA-loaded CS hydrogel and the CPP-ACP/NaF varnish under both pH conditions. At pH 7.0, all ion releases were significantly reduced (p < 0.001), though relative material rankings remained the same. Buffer pH remained stable throughout (p > 0.05).

Conclusion

While CPP-ACP/NaF varnish maximized early fluoride output, the NaF/nHA-loaded CS hydrogel demonstrated the most sustained multi-ion release under acidic conditions. These distinct pH-dependent kinetics suggest rapid fluoride delivery for early lesion resistance and sustained multi-ion release for extended remineralization.

Introduction

Dental caries is a biofilm-mediated condition in which dental tissues undergo continuous phases of mineral loss and repair [1]. Preventive strategies currently integrate dietary control of fermentable carbohydrates, early lesion detection, and the use of fluoride as the principal preventive agent against dental caries [1–5]. Compared with gels or foams, varnishes adhere to tooth surfaces for longer periods. This extended contact sustains higher oral fluoride levels and slows the rapid post-application decline seen in other delivery forms [6–8]. Additionally, it facilitates the formation of calcium fluoride (CaF₂) globules that act as fluoride reservoirs during cariogenic pH drops [1, 5, 6, 9].

Currently, different types of varnishes are utilized based on their active ingredients. First-generation varnishes, characterized by conventional fluoride-only formulations (e.g., 5% sodium fluoride(NaF)), reduce apatite dissolution effectively. However, they provide minimal bioavailable calcium or phosphate, which restricts complete mineral reestablishment [1, 10]. To address this limitation, adjunctive therapies now include non-fluoride agents such as tricalcium phosphate, bioactive glass (calcium sodium phosphosilicate), casein phosphopeptide–amorphous calcium phosphate (CPP-ACP), and nanohydroxyapatite (nHA) to replace lost minerals actively [1, 10]. Second-generation varnishes incorporate calcium and phosphate compounds, such as amorphous calcium phosphate or nHA, to enhance retention, increase fluoride bioavailability, and accelerate hydroxyapatite deposition, with added antibacterial benefits [9, 11–13]. nHA closely matches the composition of the natural enamel. This similarity allows tight surface bonding, effective defect filling, and remineralization at both surface and subsurface levels [5, 10]. Among second-generation materials, hybrid systems such as MI Varnish™ combine NaF varnish with CPP-ACP to exploit synergistic effects to sustain ion reservoirs and enhance remineralization kinetics. As a result, they offer greater protection against demineralization than fluoride-only varnishes [14].

As a promising additional approach, polymers such as hydrogels have been used in localized drug delivery for decades, especially in situ forming systems that transition from sol to gel under physiological stimuli [5]. Among natural polymers suitable for such systems, chitosan (CS) has emerged as a material of considerable interest in strategies for dental caries prevention. It is derived from chitin through deacetylation or alkaline hydrolysis. It possesses intrinsic antibacterial capacity, combined with excellent biocompatibility, biodegradability, and non-toxicity [5, 8, 15, 16].

CS-based fluoride and nHA carriers have demonstrated combined acid resistance and biomimetic mineralization [1, 5]. Experimental models link its anti-demineralization effect to reduced phosphorus loss and effective acid-barrier formation [15], with performance comparable to that of advanced nHA systems [10]. Loading CS hydrogels with both NaF and nHA yielded the greatest remineralization effect among tested formulations [1], and CS nanoparticle varnishes achieve enhanced mineral recovery through multi-ion release compared with fluoride-only products [13]. HA-CS composite gels likewise improved surface hardness and mineral content in early enamel lesions [17]. Beyond remineralization, CS serves as an effective controlled-release matrix. Release profiles often feature an initial burst followed by sustained diffusion [7, 18–20]. Fluoride release dynamics are influenced by formulation composition, carrier matrix, and immersion time. While certain varnishes released substantial ion concentrations during the first hours, others sustained or increased release over days to weeks [4, 21–23]. Acidic environments frequently accelerate fluoride liberation but also trigger mechanical degradation of certain matrices [11, 24]. Combining fluoride with calcium and phosphate aims for synergistic remineralization. Multiple investigations have demonstrated that CPP-ACP varnishes yielded higher total ion release than fluoride-only systems, while bioactive glass or functionalized tricalcium phosphate further elevated outputs [6, 9, 12]. Despite oral cavity pH fluctuations, only a limited number of studies have investigated their effects on varnish ion release [11, 24–26]. Existing evidence shows peak fluoride release from both first- and second-generation varnishes within hours of application, with magnitude strongly modified by ambient acidity [11].

Taken together, these observations reveal the paucity of focused investigations into pH-driven ion release dynamics. Specifically, there are no studies directly comparing the concurrent release of fluoride, calcium, and phosphate across conventional NaF varnishes, multi-ion-releasing varnishes, and CS-based hydrogels. Addressing this gap, the present study quantifies the concurrent release of fluoride, calcium, and phosphate from a first-generation varnish (FluoroDose^® NaF varnish), a second-generation varnish (MI varnish™), and an experimental NaF/nHA-loaded CS hydrogel at pH 5.5 and pH 7. Furthermore, this comparison evaluates the potential synergistic benefits of CS-mediated delivery. The null hypotheses tested were that there are no significant differences in the release of fluoride, calcium, and phosphate among the tested materials, and that pH variations do not significantly affect these release dynamics.

Materials and methods

Ethical approval and sample size calculation

A total of 80 sound primary canine teeth, extracted for orthodontic purposes, were collected in accordance with the Ethics Review Committee protocol of Shiraz University of Medical Sciences (Approval No. IR.SUMS.DENTAL.REC.1403.041). Written informed consent was obtained from the parents or guardians of participants aged 8–10 years, permitting the use of these teeth for research purposes.

The required sample size was determined from the study by Piesiak-Panczyszyn et al. [11] using G*Power (Heinrich Heine University Düsseldorf, Düsseldorf, Germany) with a 95% confidence level (α = 0.05), 80% statistical power (1 - ß = 0.8), and adjustment for multiple comparisons. This calculation yielded a minimum of nine specimens per group, and thus a total of 72 specimens were prepared for the eight experimental groups. Primary teeth were specifically selected as the testing substrate to accurately reflect the anatomical and physical interface encountered during pediatric varnish applications for Early Childhood Caries (ECC) management. The experimental design evaluated the concurrent release of fluoride, calcium, and phosphate ions (the response variables). The study analyzed three independent factors: (1) varnish type/material, (2) storage medium pH, and (3) immersion time.

Materials

Intermediate molecular-weight CS, tripolyphosphate (TPP), poloxamer 407 (Pluronic^® F 127), glacial acetic acid, and glycerophosphate disodium salt hydrate, all analytical-grade reagents from Merck (Darmstadt, Germany), were used as the primary components for formulation preparation. nHA (Merck, Darmstadt, Germany) and NaF powder (Solarbio^®, Beijing, China) served as formulation enhancers. For comparative and control analyses, two commercial fluoride varnishes were included: 5% NaF varnish (FluoroDose^®, Centrix Inc., Shelton, CT, USA) and a 2% CPP-ACP/ 5% NaF varnish (MI varnish™, GC Corporation, Tokyo, Japan). Buffer solutions at pH 5.5 and pH 7.0 (both, Merck, Darmstadt, Germany) were prepared to support pH-dependent assessments, simulating acidic and neutral oral environments.

Synthesis of NaF/nHA-loaded CS hydrogel

The CS-based hydrogel formulation was prepared according to the protocol reported in a previously reported protocol [5]. Briefly, 1% w/v intermediate-molecular-weight CS (75–85% deacetylated) was dispersed in 0.1 N glacial acetic acid under continuous magnetic stirring at room temperature. Poloxamer 407 (22% w/v) was then incorporated into the CS solution to form the base hydrogel matrix. For bioactive loading, 10% w/w nHA and 5% w/w NaF were gradually dissolved into the dispersion while stirring at 125 rpm. The mixtures were cooled in an ice bath for approximately 30 min. To induce ionic interaction, 0.5% w/v TPP and 14.5% w/v glycerophosphate disodium salt hydrate were added dropwise, followed by magnetic stirring for one hour. Gelation was verified by transferring samples from the ice bath to a water bath at 37 °C, where the sol–gel transition occurred within 30 s, confirming the hydrogel readiness for experimental application.

Sample preparation

The inclusion criteria for tooth selection were intact, sound primary canines extracted from pediatric patients as part of orthodontic treatment plans. Following extraction, teeth were cleaned and debrided with a prophylaxis brush, then disinfected by immersion in 0.1% chloramine T solution for one month to ensure complete disinfection. Absolute sterility was required to prevent any microbial interference during the highly sensitive ion chromatography analysis. Samples were subsequently stored in deionized water at room temperature, with the storage medium replaced weekly until use. The exclusion criteria comprised any teeth presenting developmental enamel defects, caries, or fracture lines when examined under ×20 magnification using a stereomicroscope (Motic K, Wetzlar, Germany). Based on these criteria, eight teeth were eliminated, yielding a final sample size of 72 sound specimens for experimental analysis. The roots were sectioned approximately 2 mm apical to the cemento-enamel junction, and the crowns were embedded in cold-curing acrylic resin, ensuring the labial surface was oriented parallel to the mold base to facilitate enamel block preparation. The exposed enamel surfaces were sequentially polished using 600-, 800-, 1200-, 2400-, and 4000-grit waterproof silicon carbide papers (Buehler, Lake Bluff, IL, USA), followed by final polishing with 1-µm aluminum oxide paste. The specimens then underwent ultrasonic cleaning for 5 min, rinsing in distilled water for 20 s, and air drying. Two layers of nail varnish were applied to each specimen, leaving a standardized 4 × 4 mm exposed enamel window on the flattest surface [1].

Group allocation

The study utilized a 4 × 2 factorial designs comprising four material conditions (experimental NaF/nHA-loaded CS hydrogel, NaF varnish, CPP-ACP/NaF varnish, and a no-material control) and two storage medium pH levels (pH 5.5 and pH 7.0). Seventy-two specimens were randomly allocated into eight experimental groups (n = 9) according to the material applied and storage medium pH, as follows:

• Group 1: NaF/nHA-loaded CS hydrogel, pH 5.5

• Group 2: NaF varnish, pH 5.5

• Group 3: CPP-ACP/NaF varnish, pH 5.5

• Group 4: Control (no material), pH 5.5

• Group 5: NaF/nHA‑loaded CS hydrogel, pH 7.0

• Group 6: NaF varnish, pH 7.0

• Group 7: CPP-ACP/NaF varnish, pH 7.0

• Group 8: Control (no material), pH 7.0

For varnish application during the distinct phases of the experimental duration, a sterile microbrush was used for each specimen, immediately discarded after use to mimic clinical disposal practices. Two coats of the respective experimental or control varnishes (Groups 1, 2, 3, 5, 6, and 7) were applied to specimens in the designated groups [4]. Following a four-minute application period at 37^∘C, the specimens were immersed individually in 5.5 mL of buffer solution at either pH 5.5 or pH 7.0. This procedure yielded a specimen volume-to-solution ratio of approximately 3 mm³/mL, which is consistent with previous studies [24, 26, 27]. For the control groups (Groups 4 and 8), no material was applied to the enamel windows. However, to ensure reproducibility and standardize the methodology across all groups, the control specimens were subjected to the exact same handling protocol and were left undisturbed at 37^∘C for the same four-minute duration prior to immersion.

Ion release assay

Following the four-minute application or handling period, each specimen was placed in a separate polyethylene vial to avoid cross-contamination during the ion-release assay. The samples were maintained in a calibrated incubator at 37^∘C for the entire experimental period to simulate intraoral conditions.

At 4, 24, 72, and 168 h, each specimen was transferred to a fresh 5.5 mL aliquot of buffer solution [9, 11]. The previously used solutions were filtered through sterile 0.45 μm mixed cellulose ester (MCE) syringe filters to remove residual varnish particles. Filtered samples were stored under refrigeration until analysis.

Fluoride, calcium, and inorganic phosphate concentrations were quantified using ion chromatography (930 Compact IC Flex, Metrohm AG, Herisau, Switzerland). Calibration curves were generated using combined seven-anion (#56933; Dionex, Sunnyvale, CA, USA) and six-cation (#046070; Dionex, Sunnyvale, CA, USA) standards. These standards were serially diluted with distilled deionized water to obtain a range of known concentrations. Results were expressed as cumulative release (ppm) for each ion at all time points.

pH measurement

In this study, pH served a dual purpose: the initial buffer pH (5.5 or 7.0) was established as an independent experimental factor, while the pH measured at subsequent intervals acted as a secondary response variable. The pH of the buffer solution was monitored to assess any potential changes associated with ion release, while accounting for the stabilizing effect of the medium’s buffering capacity. Measurements were performed with a digital pH meter (BADÍ model 177, China) at the same time points as the ion release measurements (4, 24, 72, and 168 h). The pH electrode was calibrated before each measurement session against certified external standards (pH 4.01, pH 7.00, and pH 10.01). Calibration was repeated for each measurement occasion to ensure accuracy. Each sample was measured three consecutive times, with the mean value recorded as the representative pH for that time point [4].

Statistical analysis

Statistical analyses were performed using SPSS software (version 26.0; SPSS Inc., Chicago, IL, USA). Descriptive statistics were calculated for all measured variables at each sampling interval.

The normality of data distribution was evaluated and confirmed using the Shapiro-Wilk test (p > 0.05). To evaluate the main effects and interactions of the three independent factors (material type, immersion pH, and immersion time) on the continuous response variables a three-way repeated measures analysis of variance (ANOVA). Upon identifying significant interactions in the global model, a one-way ANOVA followed by Tukey’s post-hoc test was conducted to analyze the simple main effects. This approach allowed for pairwise comparisons of (i) the cumulative release of fluoride, phosphate, and calcium within each material group at pH 5.5 and pH 7.0 across the 4, 24, 72, and 168 h intervals, and (ii) the pH fluctuations across time points within each material group. Statistical significance was set at p < 0.05.

Results

Ion release at pH 5.5

Cumulative fluoride, phosphate, and calcium releases at 4, 24, 72, and 168 h are presented in Table 1, with corresponding cumulative and non-cumulative patterns illustrated in Figs. 1, 2 and 3.

Table 1.

Cumulative fluoride, phosphate, and calcium ion release in ppm (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 5.5

Ion	Group	4 h	24 h	72 h	168 h	P value
Fluoride	NaF/nHA-loaded CS hydrogel	15.14 ± 0.08	24.32 ± 0.18	29.21 ± 0.08	32.39 ± 0.24	< 0.001
	NaF varnish	10.33 ± 0.25	17.41 ± 0.32	22.80 ± 0.44	25.27 ± 0.31	< 0.001
	CPP-ACP/NaF varnish	22.40 ± 0.23	30.48 ± 0.34	37.52 ± 0.43	42.31 ± 0.04	< 0.001
	Control	0.069 ± 0.000	0.014 ± 0.001	0.005 ± 0.000	0.001 ± 0.000	N/A
Phosphate	NaF/nHA-loaded CS hydrogel	7.80 ± 0.20	23.10 ± 0.30	30.20 ± 0.20	36.20 ± 0.20	< 0.001
	NaF varnish	0.015 ± 0.003	0.02 ± 0.002	0.023 ± 0.000	0.024 ± 0.001	N/A
	CPP-ACP/NaF varnish	3.94 ± 0.12	11.58 ± 0.19	15.56 ± 0.39	19.13 ± 0.15	< 0.001
	Control	0.012 ± 0.000	0.018 ± 0.000	0.021 ± 0.000	0.0231 ± 0.001	N/A
Calcium	NaF/nHA-loaded CS hydrogel	11.40 ± 0.20	19.50 ± 0.30	25.81 ± 0.21	30.20 ± 0.20	< 0.001
	NaF varnish	0.013 ± 0.003	0.017 ± 0.002	0.020 ± 0.000	0.021 ± 0.001	N/A
	CPP-ACP/NaF varnish	5.75 ± 0.17	9.80 ± 0.20	12.66 ± 0.09	15.13 ± 0.15	< 0.001
	Control	0.010 ± 0.000	0.015 ± 0.000	0.02 ± 0.001	0.023 ± 0.001	N/A

NaF Sodium fluoride, nHA nanohydroxyapatite, CS Chitosan, CPP-ACP Casein phosphopeptide-amorphous calcium phosphate

The P values presented in the horizontal rows represent the statistical comparison of ion release across the different time points (4, 24, 72, and 168 h) within each experimental group. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test

Fig. 1.

Cumulative (A) and non‑cumulative (B) fluoride ion release (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 5.5

Fig. 2.

Cumulative (A) and non‑cumulative (B) phosphate ion release (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 5.5

Fig. 3.

Cumulative (A) and non‑cumulative (B) calcium ion release (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 5.5

Fluoride release

CPP-ACP/NaF varnish exhibited the highest fluoride release at every time point, followed by NaF/nHA‑loaded CS hydrogel and NaF varnish (all p < 0.001 vs. control). The largest increase occurred within the first four hours, with cumulative concentrations for CPP-ACP/NaF varnish rising from 22.4 ± 0.23 ppm to 42.31 ± 0.04 ppm at 168 h. Both graphical profiles (Fig. 1) and statistical outcomes confirmed sustained fluoride release over the 7-day period in all active groups.

Phosphate release

NaF/nHA‑loaded CS hydrogel released significantly more phosphate than CPP-ACP/NaF varnish across all intervals (p < 0.001), whereas NaF varnish and control remained near background levels (p = 0.999). Release peaked at 24 h in both NaF/nHA‑loaded CS hydrogel (23.10 ± 0.30) and CPP-ACP/NaF varnish (11.58 ± 0.19) and then plateaued (Fig. 2).

Calcium release

NaF/nHA‑loaded CS hydrogel was also the dominant calcium source, followed by CPP-ACP/NaF varnish (p < 0.001). The greatest rise occurred during the initial four hours, with cumulative concentrations for the NaF/nHA-loaded CS hydrogel increasing from 11.40 ± 0.20 ppm to 30.20 ± 0.20 ppm by 168 h. Both the NaF varnish and the control groups showed minimal release throughout the experiment. Graphical data (Fig. 3) revealed a sharp early release (0–4 h) followed by slower increments.

Ion release at pH 7.0

Cumulative values are shown in Table 2, with release profiles depicted in Figs. 4, 5 and 6.

Table 2.

Cumulative fluoride, phosphate, and calcium ion release in ppm (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 7.0

Ion	Group	4 h	24 h	72 h	168 h	P value
Fluoride	NaF/nHA-loaded CS hydrogel	10.43 ± 0.38	13.53 ± 0.42	15.58 ± 0.36	16.37 ± 0.26	< 0.001
	NaF varnish	6.70 ± 0.30	10.11 ± 0.10	12.87 ± 0.12	14.24 ± 0.25	< 0.001
	CPP-ACP/NaF varnish	14.30 ± 0.26	18.28 ± 0.17	21.54 ± 0.16	23.55 ± 0.38	< 0.001
	Control	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	N/A
Phosphate	NaF/nHA-loaded CS hydrogel	3.01 ± 0.13	8.70 ± 0.27	12.28 ± 0.07	13.64 ± 0.14	< 0.001
	NaF varnish	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	N/A
	CPP-ACP/NaF varnish	2.84 ± 0.17	8.32 ± 0.12	11.69 ± 0.26	13.66 ± 0.20	< 0.001
	Control	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	N/A
Calcium	NaF/nHA-loaded CS hydrogel	4.77 ± 0.11	9.21 ± 0.24	12.65 ± 0.06	16.05 ± 0.20	< 0.001
	NaF varnish	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	N/A
	CPP-ACP/NaF varnish	4.15 ± 0.13	8.91 ± 0.11	11.95 ± 0.07	14.98 ± 0.11	< 0.001
	Control	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	N/A

NaF Sodium fluoride, nHA nanohydroxyapatite, CS Chitosan, CPP−ACP Casein phosphopeptide−amorphous calcium phosphate

The P values presented in the horizontal rows represent the statistical comparison of ion release across the different time points (4, 24, 72, and 168 h) within each experimental group. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test

Fig. 4.

Cumulative (A) and non‑cumulative (B) fluoride ion release (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 7.0

Fig. 5.

Cumulative (A) and non‑cumulative (B) phosphate ion release (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 7.0

Fig. 6.

Cumulative (A) and non‑cumulative (B) calcium ion release (Mean ± SD) of each group at 4, 24, 72, and 168 h in buffering solution at pH 7.0

Fluoride release

CPP-ACP/NaF varnish again led in release rates, followed by NaF/nHA‑loaded CS hydrogel and NaF varnish, with all active groups showing significantly lower values than their pH 5.5 counterparts (p < 0.001). The greatest rise occurred within the first four hours, with cumulative concentrations for CPP-ACP/NaF varnish increasing from 14.30 ± 0.26 ppm to 23.55 ± 0.38 ppm by 168 h. Graphs (Fig. 4) display an early spike at the first four hours with modest increases thereafter.

Phosphate release

Measurable release occurred only in NaF/nHA‑loaded CS hydrogel and CPP-ACP/NaF varnish groups, with no significant difference between them at four hours (p = 0.308), 24 h (p = 0.601), or 168 h (p = 0.999). However, at 72 h, where NaF/nHA‑loaded CS hydrogel exceeded the CPP-ACP/NaF varnish (p < 0.001). Release peaked at 24 h at 8.70 ± 0.27 in the NaF/nHA-loaded CS hydrogel and 8.32 ± 0.12 in the CPP-ACP/NaF varnish before plateauing. The release profile of NaF varnish and control remained at baseline (Fig. 5).

Calcium release

Both NaF/nHA‑loaded CS hydrogel and CPP-ACP/NaF varnish showed detectable calcium release, highest within the first four hours and consistently greater in the NaF/nHA‑loaded CS hydrogel (p ≤ 0.003). Cumulative concentrations for the NaF/nHA-loaded CS hydrogel rose from 4.77 ± 0.11 ppm to 16.05 ± 0.20 ppm by 168 h. NaF varnish and control showed negligible levels (Fig. 6).

Effect of pH

Across all varnish types, fluoride, phosphate, and calcium release rates were significantly higher at pH 5.5 than at pH 7.0 at every time point (all, p < 0.001), consistent with the enhanced dissolution of ion-releasing matrices under acidic conditions.

pH stability

pH measurements over 168 h for both initial conditions are summarized in Table 3 (pH 5.5) and Table 4 (pH 7.0). No statistically significant changes were observed in any group at any time point (all p > 0.05), indicating that the experimental buffers maintained stable pH throughout the testing period.

Table 3.

pH variation (Mean ± SD) during 168 h of ion release measurements at each time point in buffering solution initially set to pH 5.5

Group	Initial pH	pH at 4 h	pH at 24 h	pH at 72 h	pH at 168 h	P value
NaF/nHA-loaded CS hydrogel	5.5	5.500 ± 0.000	5.500 ± 0.000	5.553 ± 0.046	5.580 ± 0.072	0.123
NaF varnish	5.5	5.500 ± 0.000	5.500 ± 0.000	5.547 ± 0.040	5.567 ± 0.058	0.113
CPP-ACP/NaF varnish	5.5	5.500 ± 0.000	5.500 ± 0.000	5.540 ± 0.036	5.567 ± 0.058	0.111
Control	5.5	5.500 ± 0.000	5.500 ± 0.000	5.500 ± 0.000	5.527 ± 0.046	0.441
P value		N/A	N/A	0.123	0.328

NaF Sodium fluoride, nHA nanohydroxyapatite, CS Chitosan, CPP−ACP Casein phosphopeptide−amorphous calcium phosphate

The P values presented in the horizontal rows indicate the statistical comparison of pH variations for each specific material across different time points. The P values presented in the vertical columns indicate the between-group comparisons of different materials at each specific time point. All statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc test

Table 4.

pH variation (Mean ± SD) during 168 h of ion release measurements at each time point in buffering solution initially set to pH 7.0

Group	Initial pH	pH at 4 h	pH at 24 h	pH at 72 h	pH at 168 h	P value
NaF/nHA-loaded CS hydrogel	7.0	6.857 ± 0.150	6.853 ± 0.156	6.930 ± 0.089	6.857 ± 0.136	0.875
NaF varnish	7.0	6.953 ± 0.045	6.943 ± 0.051	6.900 ± 0.095	6.910 ± 0.123	0.843
CPP-ACP/NaF varnish	7.0	6.880 ± 0.115	6.933 ± 0.076	6.923 ± 0.071	6.943 ± 0.074	0.814
Control	7.0	6.963 ± 0.055	6.957 ± 0.075	6.983 ± 0.029	6.987 ± 0.023	0.852
P value		0.259	0.258	0.240	0.170

NaF Sodium fluoride, nHA nanohydroxyapatite, CS Chitosan, CPP−ACP Casein phosphopeptide−amorphous calcium phosphate

The P values presented in the horizontal rows indicate the statistical comparison of pH variations for each specific material across different time points. The P values presented in the vertical columns indicate the between-group comparisons of different materials at each specific time point. All statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc test

Discussion

This study demonstrated that ion release profiles from NaF/nHA‑loaded CS hydrogel and two commercially available fluoride varnishes were significantly influenced by formulation type, immersion pH, and exposure duration.

Across all conditions, CPP-ACP/NaF varnish (MI varnish™) achieved the greatest cumulative fluoride release, whereas the CS-based hydrogel delivered the highest calcium and phosphate outputs, particularly under acidic conditions. NaF varnish consistently produced the lowest ion release among the active materials, with levels approaching baseline for calcium and phosphate. Acidic pH (5.5) accelerated ion liberation from all formulations, yielding substantially higher cumulative concentrations compared with neutral pH (7.0). These findings help address the existing gap in comparative pH-driven release kinetics between first-generation NaF varnishes, second-generation multi-ion varnishes, and emerging CS-mediated delivery systems, and underscore the potential of material-specific and pH-targeted strategies for caries prevention.

The fluoride release profiles observed in this study reflected distinct formulation-dependent kinetics, with CPP-ACP/NaF varnish exhibiting the highest cumulative fluoride output at both pH conditions, corroborating previous investigations into CPP-ACP systems [9, 12]. The initial burst phase within the first 24 h was more pronounced in CPP-ACP/NaF varnish, which is critical for maximizing ion bioavailability before the inevitable intraoral loss of varnish due to mastication, oral musculature, and hygiene practices, consistent with the high aqueous solubility of CPP-ACP complexes and their capacity to release loosely bound fluoride rapidly [7, 28]. Such early‐phase flux aligns with the clinical necessity of maintaining salivary fluoride above 0.03–0.08 ppm to enhance enamel remineralization and inhibit demineralization [29]. In contrast, NaF varnish demonstrated a lower burst and cumulative fluoride release, matching its classification as a first‐generation rosin‐based system in which fluoride ion liberation is largely governed by surface elution followed by slower diffusion from the matrix [14, 21]. The NaF/nHA-loaded CS hydrogel exhibited intermediate fluoride release, likely attributable to the electrostatic entrapment of fluoride ions within the cationic CS network [7] and the known capacity of nHA crystallites to bind and gradually liberate mineral ions through surface interaction and partial dissolution, as demonstrated in nHA‐based remineralizing systems [1, 3, 10], thus moderating immediate elution while enabling sustained diffusion.

Notably, acidic immersion conditions (pH 5.5) augmented fluoride release across all materials, in agreement with prior observations that proton-mediated dissolution of fluoride reservoirs enhances ion liberation under cariogenic pH [26, 30]. These findings collectively indicate that fluoride release in all groups followed a biphasic pattern, an initial rapid burst followed by a measurable extended release phase, with the highest overall release observed for CPP-ACP/NaF varnish, followed by the NaF/nHA-loaded CS hydrogel, and then NaF varnish, thereby maximizing ion bioavailability in the early period and sustaining gradual delivery thereafter.

The release of phosphate and calcium ions showed distinct material-dependent patterns, differing from fluoride release. At pH 5.5, the NaF/nHA-loaded CS hydrogel consistently exhibited the highest phosphate and calcium release across all time points, significantly exceeding CPP-ACP/NaF varnish, while NaF varnish and the control remained at baseline levels. The superior performance of the hydrogel compared to CPP-ACP/NaF varnish might be attributable to its multi‐source mineral composition. Phosphorus is supplied by TPP (Na₅P₃O₁₀), glycerophosphate disodium salt (C₃H₉Na₂O₇P), and the phosphate component of crystalline nanohydroxyapatite [Ca₅(PO₄)₃(OH)]. In contrast, calcium is provided exclusively by the nHA phase, which also serves as a structural analog to biological apatite. nHA serves as a biomimetic mineral phase, whose high surface area and partial solubility facilitate the gradual liberation of calcium and phosphate ions, while its crystallographic similarity to biological apatite enables direct lattice integration into subsurface enamel [31, 32].

In CPP-ACP/NaF varnish, calcium is supplied by CPP-ACP, a nanocomplex that stabilizes ACP clusters via multiple phosphoryl residues on the casein backbone, preventing spontaneous crystallization and maintaining a supersaturated state of calcium and phosphate in plaque fluid [31, 33]. While CPP-ACP is inherently more hydrophilic than nHA due to its peptide backbone and ionic phosphoryl groups [34], it is proposed that the higher calcium release observed from the NaF/nHA-loaded CS hydrogel likely reflects multiple factors. First, the hydrogel contains 10% w/w nHA, corresponding to an approximate total calcium content of approximately 3.99% w/w. This estimation is based on the molar mass of nHA (502 g/mol), where its five calcium atoms (200.4 g/mol) account for approximately 39.9% of the Ca by mass. In contrast, CPP-ACP/NaF varnish contains 2% w/w CPP-ACP (35), equating to only about 0.52% w/w calcium (CPP–ACP is about 26% Ca by mass). Thus, the hydrogel provides roughly seven to eight times more total calcium reservoir than CPP-ACP/NaF varnish before pH-dependent solubilization and release kinetics are considered. Second, the CS-poloxamer matrix swells substantially in aqueous conditions, creating hydrated channels that accelerate ion diffusion and expose greater nHA surface area. Third, additional phosphate donors in the hydrogel, such as TPP and glycerophosphate, may chelate calcium and facilitate its transport into the surrounding medium. Finally, unlike the resinous, partially occlusive base of CPP-ACP/NaF varnish [12], the hydrogel presents a fully hydrated ionic environment from the outset, allowing rapid onset of release.

Collectively, these factors explain why the hydrogel outperformed CPP-ACP/NaF varnish in total calcium and phosphate release despite the latter’s more hydrophilic calcium carrier. It must be acknowledged that the conventional NaF varnish lacks calcium and phosphate sources in its chemical formulation; therefore, its minimal release of these ions was entirely expected and should not be interpreted as a functional inferiority in this regard. Calcium and phosphate ions released from the NaF varnish and the control groups at pH 5.5, owing to the absence of calcium-phosphate phases in the varnish formulations, likely originated from partial dissolution of the tooth structure in an acidic environment.

It is proposed that the 24-hour phosphate release maximum observed in both the NaF/nHA-loaded CS hydrogel and CPP-ACP/NaF varnish, in contrast to the rapid calcium and fluoride peaks within the first four hours, reflects shared mechanistic constraints despite differences in composition. While nHA and ACP are indeed the primary sources of both calcium and phosphate, their dissolution and subsequent ion diffusion out of the respective matrices do not guarantee stoichiometric release kinetics. In each system, phosphate originates from relatively low-solubility or gradually hydrolyzing compounds, which release ions more slowly than the free or readily dissociating salts that contribute to calcium and fluoride bursts [35].

Crucially, the hydrogel formulation contains additional phosphate sources independent of calcium: TPP and glycerophosphate disodium salt. The release of phosphate from these water-soluble compounds, particularly from the TPP crosslinker, is governed by the slower processes of hydrogel swelling and matrix relaxation, which can explain the shift in peak phosphate concentration to 24 h.

In the resin-based CPP-ACP varnish, the difference may be attributable to diffusion kinetics. The smaller, divalent Ca²⁺ ions may migrate through the varnish matrix more readily than the larger, trivalent phosphate anions (PO₄³⁻). Furthermore, these phosphate ions are also electrostatically complexed with the casein phosphopeptides, creating a temporal separation in their peak availability. Additionally, transient precipitation of sparingly soluble calcium phosphate phases such as CaHPO₄ under near-neutral pH may temporarily sequester phosphate in both matrices, delaying its maximum availability until these phases dissolve [35, 36]. Diffusion through the hydrated CS network in the hydrogel and through the resin-based varnish medium in CPP-ACP/NaF varnish may both slow phosphate migration relative to small, monovalent fluoride ions. Together, these factors provide a plausible rationale for the synchronized 24-hour phosphate peaks despite different material architectures.

The present findings showed that for all varnish types, fluoride, calcium, and phosphate releases were significantly greater at pH 5.5 than at pH 7.0 at every time point, supporting the concept that acidic conditions enhance ion liberation through increased dissolution of mineral phases. This “smart” release behavior aligns with studies on calcium-, phosphate-, and fluoride-releasing systems, which report accelerated ion release in cariogenic pH ranges where minerals are most needed to counteract demineralization [24, 26]. A plaque pH between 5.5 and 4.0 is considered high‐risk for caries formation, and the increased ion supply under such conditions could serve a protective role [26]. Acid‐enhanced fluoride release has been attributed to both the formation and increased solubility of calcium fluoride reservoirs and to higher fluoride affinity for partially demineralized enamel surfaces [4, 30].

However, this effect is material dependent; several varnish and restorative products respond variably to pH change, with only certain formulations showing a substantial low-pH release boost [37, 38]. For CPP-ACP/NaF varnish, a pH decrease can shift the balance between free and loosely bound ions within the CPP complex, promoting increased bioavailability [9]. In the NaF/nHA-loaded CS hydrogel, acidic pH not only promotes nHA dissolution but may also partially protonate CS’s amino groups, enhancing its solubility and thereby facilitating ion diffusion [39]. Collectively, these mechanisms explain the consistent pattern in the obtained data of higher ion release at pH 5.5, suggesting that both resin‐ and hydrogel‐based varnishes can function as responsive ion reservoirs in caries‐prone acidic microenvironments.

From a clinical perspective, the differential release kinetics observed in this study suggest that material selection should be tailored to the patient’s caries risk profile and intraoral pH dynamics. For patients at high caries risk who experience frequent and pronounced plaque pH drops, a short-term, high‐fluoride release profile, as shown for CPP-ACP/NaF varnish, may provide a rapid boost in enamel fluoride acquisition and acid resistance, especially during periods of heightened cariogenic challenge. Conversely, the CS-based hydrogel offers the added benefit of a higher calcium and phosphate ion release, which is sustained over several days. Beyond its remineralizing potential, CS possesses intrinsic antimicrobial and biofilm‐suppressive properties, which may further enhance its protective effects [7, 8, 13]. These findings highlight the importance of aligning varnish composition and application strategy with the biological needs and behavioral risk profile of each patient, bridging the gap between laboratory performance and personalized, preventive dental care.

This study evaluated NaF varnish, CPP-ACP/NaF varnish, and NaF/nHA-loaded CS hydrogel under neutral and cariogenic pH over 168 h with complete medium renewal at each interval. Some studies have limited their ion-release assessment to 24 h [22, 37] because most varnish is lost from tooth surfaces within a day. However, in line with other prior work [4, 9, 11], the present study extended the monitoring period to 168 h. This extended duration enabled the capture of both the initial burst and the slower, sustained release phases. Although in vitro conditions lacked chewing or brushing forces, which can reduce varnish retention in clinical settings, the prolonged monitoring still provided insight into potential long-term release trends. This study also included the use of ion chromatography for multi-ion quantification, clinically relevant pH conditions, and complete medium replacement to avoid saturation effects, alongside the omission of artificial saliva to prevent background fluoride, calcium, or phosphate interference.

However, the study had certain limitations. Primarily, it utilized the static model without salivary flow, enzymatic activity, biofilm, mechanical forces, pH cycling. Additionally, the methodology did not involve repeated applications, and tested only one formulation per category. Future work should include pH-cycling or biofilm systems, durability testing under simulated mastication and hygiene forces, and exploration of combined multi-ion and antimicrobial delivery. Clinical trials stratified by caries risk and salivary pH would help translate the hydrogel’s prolonged calcium-phosphate release and CPP-ACP/NaF varnish’s high initial fluoride output into tailored preventive protocols.

Conclusion

This study demonstrated that ion release patterns from NaF varnish, CPP-ACP/NaF varnish, and NaF/nHA-loaded CS hydrogel vary substantially with formulation type, immersion pH, and exposure time. Within the limitations of this in vitro study, the findings demonstrate that the experimental NaF/nHA-loaded CS hydrogel and the commercial varnishes exhibit distinct, pH-dependent ion release profiles. The CPP-ACP/NaF varnish produced the greatest cumulative fluoride release. In contrast, the experimental NaF/nHA-loaded CS hydrogel demonstrated a superior capacity for sustained calcium and phosphate release, which was significantly enhanced under acidic conditions. These findings suggest that short-term, high-fluoride systems and sustained, multi-ion delivery platforms could serve complementary roles in tailored caries-prevention protocols, warranting further investigation under dynamic, clinically relevant conditions.

Authors’ contributions

AR, MM, and AA: conceptualized and designed the study, supervised data collection, interpreted the data, drafted the manuscript, and approved the final manuscript as submitted. HR, NF, TB, NF: conceptualized the study, supervised data collection and interpretation, reviewed the manuscript, and approved the final manuscript as submitted. HR: assisted in data collection, carried out the initial analyses and interpretation of data, reviewed the manuscript, and approved the final manuscript as submitted. All authors agreed to be accountable for all aspects of this work.

Funding

This work was supported by the Vice-Chancellery of Research of Shiraz University of Medical Sciences, Shiraz, Iran (Grant No. 29705).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Ethics Review Committee of the School of Dentistry, Shiraz University of Medical Sciences (IR.SUMS.DENTAL.REC.1403.041). All methods were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki). Written informed consents for the use of the teeth are available from the corresponding author upon reasonable request.

Competing interests

NF is the Editorial Board Member of BMC Oral Health. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.