Integration of a Fluoride- and Mint-Based Spray in Nighttime Aligner Therapy: Effects on Salivary Concentration and Biofilm

Abstract

Continuous use of clear aligners modifies the oral environment and may favor bacterial colonization. Integration of topical fluoride-based agents could strengthen enamel and reduce biofilm formation. This study evaluated the effects of a galenic fluoride-mint spray (225–250 ppm fluoride and 1–2% peppermint essential oil) on salivary fluoride concentration and bacterial biofilm during orthodontic treatment. Ten patients using 3D-printed nighttime aligners were enrolled. Saliva samples were analyzed with an ion-selective electrode (ISE) at baseline, immediately after inserting the sprayed aligners and after 15, 30, 45 min post application. Biofilm morphology was qualitatively assessed by scanning electron microscope (SEM) in three aligners: unused, worn 14 nights without spray, worn 14 nights with spray. Salivary fluoride increased from 0.7–0.8 mg/L at baseline to 5.96 mg/L when the spray was applied on a new aligner and 8.42 mg/L on a used aligner, then progressively decreased, returning close to baseline at 45 min with the new aligner and remaining higher with the used aligner. SEM images showed mature and heterogeneous biofilm on used aligners without the spray, while aligners with nightly spray application exhibited qualitatively reduced and less organized surface deposits. The fluoride- and mint-based spray rapidly increases salivary fluoride and reduces biofilm formation on nighttime clear aligners, improving preventive oral health during orthodontic treatment.

1. Introduction

In recent years, orthodontic treatment has increasingly relied on removable aligners, which are appreciated for their esthetics, comfort and the ability to maintain better oral hygiene compared with fixed appliances [1]. Among the various materials used for clear aligners, 3D-printed polyamide has gained interest due to its mechanical stability and design flexibility [2]. Nighttime aligners manufactured through selective laser sintering (SLS) of polyamide (PA) are designed for limited daily wear and exhibit reduced stress relaxation, enabling higher and more sustained orthodontic forces over time [3].

In addition, the 3D-printing process allows for differential thicknesses across different regions of the arch, including variable thickness between cervical and incisal areas. This design flexibility enhances fit accuracy and enables selective force modulation, particularly increasing the force applied to specific teeth where greater movement is required [4,5,6]. These mechanical advantages justify the clinical rationale for nighttime use, offering a discreet and patient-friendly alternative to full-time aligner wear.

Despite these advantages, the prolonged use of aligners can reduce the natural self-cleansing of saliva, leading to stagnation of fluids and accumulation of bacterial plaque. This condition creates a microenvironment favorable to bacterial adhesion, enamel demineralization, and white spot lesions [7], which remain one of the most common side effects in orthodontic patients. Previous studies have shown that biofilm forms readily on removable orthodontic appliances and retainers, even when hygiene practices are adequate, highlighting the need for adjunctive preventive strategies [8].

Fluoride remains fundamental in caries prevention because it enhances enamel remineralization, enhances the formation of fluorapatite and inhibits acid-induced demineralization caused by bacterial metabolism [9,10]. Among different compounds, sodium monofluorophosphate, in particular, demonstrates superior enamel penetration compared with other fluorides, making it particularly suitable for topical applications [11].

Recent reviews have highlighted the development of modern antimicrobial agents for dental applications, including plant-derived extracts, nanoparticles and hybrid biomaterials, showing promising antibacterial and antifungal effects in oral environments [12]. These findings further support the rationale for investigating natural bioactive additives in aligner therapy.

In parallel, there is a growing interest in natural active ingredients with antimicrobial properties to complement or enhance traditional fluoride-based products. Among these, peppermint essential oil has been widely incorporated into oral formulations. It exhibits antibacterial and refreshing properties [13] and has shown antimicrobial activity against several oral microorganisms such as Streptococcus mutans, Lactobacillus acidophilus and Candida albicans [14,15,16]. Its mechanism of action involves disruption of bacterial membranes, interference with enzymatic pathways, and inhibition of quorum sensing, thereby limiting bacterial adhesion and early biofilm development [17,18,19,20]. Additional evidence supports the antimicrobial potential of plant-derived essential oils in dentistry: poly-herbal extracts have been shown to reduce S. mutans adhesion to enamel [14], blended essential oil formulations demonstrate antibacterial action against periodontal pathogens such as Porphyromonas gingivalis [15], and essential oils from the Mentha genus, including Mentha spicata and Mentha piperita, exhibit strong inhibitory effects on S. mutans biofilm formation [16]. The mechanism of action of peppermint essential oil involves disruption of bacterial membranes, interference with metabolic and enzymatic pathways, and inhibition of quorum sensing, thereby limiting bacterial adhesion and early biofilm development [17,18,19,20].

From a clinical standpoint, salivary fluoride concentration is a key marker of oral fluoride bioavailability. Higher fluoride levels can maintain a mineralizing environment at the tooth surface, which is critical during orthodontic treatment [21].

The combination of fluoride and mint essential oil is particularly innovative for aligner users because it integrates two synergistic mechanisms: (1) chemical reinforcement of enamel via fluoride, and (2) biological modulation of biofilm formation through essential oils. This dual action may be especially relevant in aligner therapy, where prolonged appliance wear alters salivary flow and clearance.

Although removable aligners facilitate hygiene compared with fixed appliances, previous in vitro investigations have shown that clear aligner and retainer materials can support adhesion and biofilm formation by microorganisms such as Streptococcus mutans, Porphyromonas, Prevotella, Fusobacterium, Actinomyces, Lactobacillus spp. and Candida albicans, especially when worn for extended periods [22,23]. This highlights the need for adjunctive preventive measures capable of both maintaining optimal fluoride availability and limiting bacterial adhesion.

In this context, the use of a spray containing sodium monofluorophosphate and mint essential oils may offer dual benefits: chemical protection through fluoride and biological modulation through the essential oil. However, no studies have evaluated its effect on salivary fluoride concentration or on biofilm accumulation in patients wearing polyamide aligners.

Therefore, the present study aimed to investigate the preventive potential of a fluoride–mint spray during aligner therapy,

(1)

measuring the salivary fluoride concentration over time with a fluoride ion-selective electrode (ISE);

(2)

assessing the biofilm formation in aligners with a Scanning Electron Microscopy (SEM).

It was initially hypothesized that the application of a fluoride–mint spray would not significantly alter salivary fluoride concentration nor reduce biofilm accumulation compared with untreated conditions.

Accordingly, the null hypothesis stated that the spray would not modify salivary fluoride levels and would not reduce biofilm deposition.

Based on the mechanism of action of the spray, it was further hypothesized that fluoride concentration would transiently increase after application and that aligners treated with the spray would exhibit reduced biofilm accumulation compared with untreated ones.

Considering that polyamide aligners undergo material changes after intra-oral exposure, a secondary hypothesis was formulated, suggesting that the magnitude of these effects could differ between new aligners and those aged in the oral environment.

2. Materials and Methods

2.1. Study Design and Participants Selection

Ten patients (aged 18–30 years; 6 females and 4 males) undergoing orthodontic treatment with 3D-printed nighttime aligners—namely, NOXI aligners (Sweden & Martina, Due Carrare, Padua, Italy)—at the University of Ferrara were enrolled in the study according to the following inclusion and exclusion criteria (

Table 1. Inclusion and exclusion criteria.

Inclusion Criteria	Exclusion Criteria
Age > 18 years	Systemic diseases or medications affecting salivary flow
Ongoing orthodontic treatment with nighttime 3D-printed polyamide aligners	Untreated active caries, gingivitis or periodontitis in acute phase
Good general hygiene	Use of fluoride-containing products during observation
Normal salivary flow	Known allergy to fluorides or peppermint essential oil
No use of fluoride products in the previous 24 h	Pregnancy or breastfeeding
Ability and willingness to follow the protocol	Poor compliance with study requirements
Signed informed consent

).

All participants provided written informed consent, and the study was approved by Ethics Committee of the University of Ferrara (5/2024).

This study was designed as an exploratory pilot investigation. A formal sample size calculation was not performed because no previous data were available on fluoride kinetics in 3D-printed polyamide aligners. A sample of ten participants was considered appropriate based on feasibility and in accordance with similar pilot studies evaluating fluoride release and biofilm accumulation in orthodontic appliances.

Each subject participated in two experimental sessions performed on separate days, involving two aligner conditions:

• NEW aligner: a freshly delivered aligner that had never been worn;
• USED aligner: an aligner previously worn by the same subject for 14 consecutive nights.

Including both NEW and USED aligners was necessary because the PA material employed in the nighttime aligners considered experiences relevant properties changes after prolonged exposure to the oral environment. Immersion in saliva can alter the material’s surface characteristics, hydration level, and porosity, potentially influencing its ability to absorb and retain the applied liquid. By analyzing both new and intra-orally aged aligners, we were able to more accurately evaluate how clinical use modifies their absorption behavior and to ensure that the findings reflect real clinical conditions rather than ideal laboratory conditions.

During both sessions, the fluoride-based spray was applied only once, immediately after collecting the baseline saliva sample and before inserting both aligners.

To avoid order effects and minimize bias, an intra-individual crossover design (NEW vs. USED aligners) was adopted, allowing each participant to serve as their own control. This approach increased statistical efficiency and reduced inter-subject variability. The sample size was therefore considered adequate to detect clinically relevant trends and to provide preliminary data for future, fully powered studies.

The sequence in which participants used NEW and USED aligners was randomly assigned through computer-generated simple randomization. Half of the participants began with the NEW aligner condition followed by the USED condition, whereas the remaining participants completed the protocol in the opposite order. A 24 h washout period separated the two conditions to prevent carry-over effects in salivary fluoride measurements.

2.2. Spray Formulation and Application Protocol

The spray was a galenic formulation containing sodium monofluorophosphate (225–250 ppm F⁻) and mint essential oil (1–2%) in a hydroalcoholic base. As characteristic of compounded preparations, no chromatographic (HPLC or GC-MS) characterization of the essential oil is provided by the pharmacist. For clarity, peppermint essential oil is known to typically contain menthol, menthone, menthyl acetate and other monoterpenes, as reported in the phytochemical literature.

Commercial fluoride mouth-rinses commonly contain about 0.05% NaF (~230 ppm F) for daily use in individuals older than 6 years, while higher strength solutions (0.2% NaF ~920 ppm F) are reserved for weekly supervised use in high caries-risk populations [24]. Given this context, the choice of a 225–250 ppm fluoride concentration in our nighttime spray places it within the range of home-use rinses, ensuring safety and compatibility with nightly aligner wear without approaching the high concentrations used in professional treatments [24]. The concentration of peppermint essential oil used in the spray (1–2%) is supported by previous evidence showing that peppermint oil at similar concentrations (1–2.5%) enhances fluoride ion release in topical fluoride formulations, demonstrating a dose-dependent interaction between the essential oil and the fluoride matrix [25]. On this basis, the present formulation was considered clinically relevant and safe for in vivo use during aligner therapy.

The formulation was dispensed using a standard multidose mechanical pump delivering approximately 0.12 mL per actuation. Six sprays were applied uniformly to the internal surface of the upper and lower aligners (three per arch), corresponding to approximately 212 µg of fluoride per application. After spraying, aligners were left for 5 min to allow evaporation of the volatile components before insertion.

2.3. Saliva Sampling and Fluoride Measurement (ISE Analysis)

Unstimulated whole saliva was collected from each participant under both experimental conditions (NEW and USED aligners). Sampling was performed at five time points relative to spray application:

• Baseline: before applying the spray to the aligners;
• T0: immediately after the insertion of sprayed aligners;
• T15, T30 and T45: 15, 30, and 45 min after spray application.

At each time point, approximately 4 mL of unstimulated saliva was collected into sterile polypropylene tubes while participants remained seated, minimizing oral movement and avoiding swallowing during collection. The samples were delivered to the Chemistry Laboratory of the University of Ferrara for analysis. Prior to measurement, each sample was treated with ultrapure distilled water (resistivity ≥ 18 MΩ·cm, obtained with a Milli-Q system, Waters Corp., Milford, MA, USA) and mixed 1:1 with TISAB IV (Total Ionic Strength Adjustment Buffer; Sigma-Aldrich, St. Louis, MO, USA) to stabilize fluoride ions and control ionic strength and pH.

Fluoride concentration (mg/L) was determined using a fluoride ion-selective electrode (ISE), model F-9655C (Hach, Loveland, CO, USA), which employs a solid-state lanthanum fluoride (LaF₃) crystal membrane selective for fluoride ions (F⁻). Before each measurement session, the electrode was carefully calibrated using certified fluoride standard solutions (potassium fluoride, KF, Titrisol^®, MERCK, Darmstadt, Germany). During analysis, the electrical potential generated by the electrode was recorded and converted into fluoride concentration (mg/L) through the calibration curve. Each measurement was performed in triplicate, and the mean value was used for statistical analysis.

2.4. SEM Analysis of Biofilm on NOXI Aligners

Three aligner specimens were analyzed:

• Aligner A (new control);
• Aligner B (used 14 nights without spray);
• Aligner C (used 14 nights with nightly spray application).

These three samples were selected to provide representative morphological information for each experimental condition.

Aligner specimens were rinsed in phosphate-buffered saline (PBS) and fixed in a 2.5% glutaraldehyde solution for 4 h, followed by post-fixation in phosphate buffer at room temperature for 2 h to ensure optimal preservation of the biofilm structure. After fixation, samples were dehydrated in ethanol and air-dried under a laminar flow hood. Once completely dry, the specimens were mounted on aluminum stubs using conductive carbon tabs and sputter-coated with a thin (≈5 nm) platinum layer using a sputter coater.

SEM analysis was qualitative and aimed at describing the presence and morphology of biofilm on the inner surface of the aligners. One representative internal-surface area was imaged for each specimen. Micrographs were acquired at standardized magnifications of ×50, ×500, ×5000 and ×10,000 to document surface topography and bacterial deposition at increasing levels of detail.

Biofilm accumulation was assessed using predefined descriptive criteria:

A.

Increased deposition was defined as continuous organic layers, confluent bacterial aggregates, or dense clusters covering the underlying polymer texture.

B.

Reduced deposition was defined as sparse bacterial micro-colonies, isolated clusters, or largely clean regions with visible polymer texture.

This approach allowed comparison of biofilm morphology among the three conditions despite the qualitative nature of the analysis. All images were examined independently by two calibrated evaluators, and discrepancies were resolved by consensus.

2.5. Statistical Analysis

The statistical analysis was designed with two primary objectives: to evaluate changes in salivary fluoride concentration over time within each experimental condition (NEW and USED aligners) and to compare fluoride concentrations between the two conditions at each sampling time. A non-parametric test was employed since the assumption of normal distribution was not met.

For intragroup comparisons (changes over time within each aligner type), the Friedman test for repeated measures was applied as the non-parametric alternative to repeated-measures ANOVA. When significant differences were found (p < 0.05), pairwise post hoc comparisons were conducted using the Wilcoxon signed-rank test, with Bonferroni correction for multiple testing.

For intergroup comparisons (NEW vs. USED aligners at each time point), the Fisher– Pitman permutation test for paired data was used. This test is robust for small sample sizes and does not assume normal data distribution. To control type I error, both Bonferroni and Benjamini–Hochberg false discovery rate (FDR) corrections were applied.

All analyses were performed using R (version 4.4.3) and statistical significance was set at p < 0.05. This analytical framework was chosen to directly test the study hypothesis by evaluating whether the fluoride–mint spray produced measurable changes over time (intragroup effects) and whether aligner wear condition (NEW vs. USED) influenced fluoride retention (intergroup effects).

In this way, the statistical outcomes allowed verification or rejection of the secondary hypothesis that the spray would not alter salivary fluoride concentration or lead to differences between NEW and USED aligners.

3. Results

3.1. Salivary Fluoride Concentration (ISE)

Fluoride concentrations for NEW and USED aligners at all time points are reported in the combined

Table 2. Salivary fluoride concentrations (mg/L) for NEW and USED aligners. Values are expressed as Mean ± SD. Different superscript letters within each column denote significant intragroup differences (p < 0.05). Asterisks indicate significant intergroup differences at the same time point.

Time Point	NEW Aligner (Mean ± SD)	USED Aligner (Mean ± SD)
Baseline	0.76 ± 0.14 a	0.83 ± 0.27 a
T0	5.96 ± 4.41 b	8.42 ± 4.05 b
T15	1.37 ± 0.71 c *	2.29 ± 0.59 c *
T30	0.91 ± 0.19 c *	1.40 ± 0.30 c *
T45	0.81 ± 0.17 ac *	1.18 ± 0.17 ac *

Data expressed as mean ± standard deviation of salivary fluoride concentration measured by ion-selective electrode (ISE). T0 = immediately after spray application; T15, T30, and T45 = 15, 30, and 45 min after application. Superscript letters (a, b, c) denote significant intragroup differences across time points within each aligner type (p < 0.05). Values sharing at least one letter (e.g., ac) are not significantly different from either group indicated by those letters. Asterisks (*) denote significant intergroup differences (NEW vs. USED) at the same time point (p < 0.05).

. This table summarizes the descriptive data (Mean ± SD) and the intragroup statistical differences using superscript letters. Detailed Wilcoxon post hoc comparisons for each condition are provided in Appendix A 

Table A1. Post hoc pairwise comparisons between time points for NEW aligners (Wilcoxon signed-rank test with Bonferroni correction).

Comparison	Statistic	p-Value	p-Bonferroni
Baseline vs. T0	0	0.002 **	0.02 *
Baseline vs. T15	1	0.013 *	0.129
Baseline vs. T30	18	0.359	1.0
Baseline vs. T45	22	0.625	1.0
T0 vs. T15	55	0.006 **	0.059
T0 vs. T30	55	0.002 **	0.02 *
T0 vs. T45	55	0.002 **	0.02 *
T15 vs. T30	55	0.002 **	0.02 *
T15 vs. T45	55	0.002 **	0.02 *
T30 vs. T45	39	0.058	0.58

* p < 0.05; ** p < 0.01.

(NEW) and

Table A2. Post hoc pairwise comparisons between time points for USED aligners (Wilcoxon signed-rank test with Bonferroni correction).

Comparison	Statistic	p-Value	p-Bonferroni
Baseline vs. T0	0	0.002 **	0.02 *
Baseline vs. T15	0	0.002 **	0.02 *
Baseline vs. T30	0	0.002 **	0.02 *
Baseline vs. T45	0	0.002 **	0.02 *
T0 vs. T15	55	0.002 **	0.02 *
T0 vs. T30	55	0.002 **	0.02 *
T0 vs. T45	55	0.002 **	0.02 *
T15 vs. T30	55	0.002 **	0.02 *
T15 vs. T45	55	0.002 **	0.02 *
T30 vs. T45	44	0.013	0.129

* p < 0.05; ** p < 0.01.

(USED). All analyses refer to n = 10 subjects for each condition (each subject evaluated with NEW and USED aligners). Fluoride concentrations are expressed in mg/L.

3.1.1. Intragroup Analysis

NEW Aligner

Descriptive analysis (Table 2) showed that salivary fluoride concentration in the NEW aligner group markedly increased immediately after spray application (T0: 5.96 ± 4.41 mg/L) compared with baseline (0.76 ± 0.14 mg/L). This was followed by a rapid decrease at T15 (1.37 ± 0.71 mg/L) and a stabilization at T30 and T45 (0.91 ± 0.19 mg/L and 0.81 ± 0.17 mg/L, respectively), reaching values close to baseline. The pattern indicates a sharp but transient increase in fluoride concentration after topical exposure, with dilution and clearance occurring within 30–45 min.

The Friedman test for repeated measures confirmed a statistically significant variation across time points (χ² = 31.071, df = 4, p = 2.96 × 10⁻⁶).

Detailed Wilcoxon post hoc comparisons for NEW aligners are reported in Appendix A Table A1, that revealed significant differences between Baseline and T0 (p Bonferroni = 0.02) and between T0 and both T30 and T45 (p Bonferroni = 0.02). The comparison between T0 and T15 showed a near-significant trend (p Bonferroni = 0.059). Significant differences were found between T0 and both T30 and T45, while the comparison between T0 and T15 showed a trend toward significance.

No significant differences were observed among T15, T30, and T45, indicating that salivary fluoride levels returned close to baseline values within 30–45 min. These results support the hypothesis of a rapid but transient fluoride bioavailability following spray application.

USED Aligner

Similarly, in the USED aligner group (Table 2), fluoride concentration increased sharply at T0 (8.42 ± 4.05 mg/L) compared with baseline (0.83 ± 0.27 mg/L), followed by a progressive reduction over time (T15: 2.29 ± 0.59 mg/L; T30: 1.40 ± 0.30 mg/L; T45: 1.18 ± 0.17 mg/L). Concentrations at later time points remained slightly above baseline, suggesting greater fluoride retention in USED aligners.

The Friedman test again indicated a highly significant effect (χ² = 39.176, df = 4, p = 6.407 × 10⁻⁸).

Full Wilcoxon post hoc results for USED aligners are provided in Appendix A Table A2, that confirmed significant differences between Baseline and all subsequent time points, as well as between T0 and each later time (all p Bonferroni < 0.05). Only the comparison between T30 and T45 was not significant. These findings confirm an acute but transient fluoride increase, with slower clearance in USED aligners.

3.1.2. Intergroup Analysis

Intergroup comparisons were performed using the Fisher–Pitman permutation test. As shown in

Table 3. Fisher–Pitman permutation test comparing salivary fluoride concentrations between NEW and USED aligners at each time point (Bonferroni and Benjamini–Hochberg FDR).

Time Point	Z Statistic	p-Value	p-Bonferroni	p-FDR
Baseline	−0.595	0.582	1.000	0.582
T0	−1.30	0.222	1.000	0.278
T15	−2.51	0.0035	0.0175 *	0.00583 **
T30	−2.82	0.0017	0.0085 **	0.005 **
T45	−2.84	0.002	0.010 **	0.005 **

* p < 0.05; ** p < 0.01. Significance was maintained for T15, T30, and T45 after both corrections.

, no significant differences were detected between NEW and USED aligners at baseline or T0. However, fluoride concentrations were significantly higher in USED aligners at T15 (p = 0.0035), T30 (p = 0.0017), and T45 (p = 0.002), indicating slower fluoride clearance in previously worn appliances. These differences remained significant after Bonferroni and Benjamini–Hochberg FDR corrections. This indicates that USED aligners retained fluoride longer, maintaining higher salivary concentrations over time.

These findings were visually confirmed by the permutation distributions shown in Figure 1, where the observed intergroup differences fell within the extreme tails of the null distribution at T15, T30 and T45.

Taken together, these results demonstrate that the fluoride- and peppermint-based spray produced a rapid, significant increase in salivary fluoride concentration immediately after application, followed by a progressive decline to near-baseline levels within 30–45 min. Both aligner types exhibited this transient kinetic profile, though USED aligners retained higher fluoride levels over time, likely due to increased surface roughness and porosity acquired through use.

3.2. SEM Observations

SEM analysis was performed on three aligner specimens representing the different clinical conditions:

• NEW aligner (unused);
• USED aligner worn for 14 nights without spray;
• USED aligner worn for 14 nights with spray.

For each condition, two representative images were acquired at different magnifications, in order to highlight both the general surface morphology and the detailed organization of bacterial deposits.

3.2.1. ALIGNER A—NEW Aligner

The NEW aligner exhibited a smooth and uniform internal surface without visible bacterial colonization or surface irregularities. At higher magnification, the polymer microstructure appeared intact, confirming the pristine condition of the unused material and the absence of detectable biofilm deposition (Figure 2).

3.2.2. ALIGNER B—USED Aligner Without Spray

The USED aligner worn without spray showed clear signs of surface wear and topographic irregularities. Multiple bacterial clusters and confluent organic deposits were visible, forming discontinuous but extensive accumulations across the examined field. In several areas, the underlying polymer texture was partially obscured, indicating increased biofilm deposition compared with the NEW control. At higher magnification, aggregates of bacterial cells embedded within amorphous material were observed, consistent with more advanced surface contamination (Figure 3).

3.2.3. ALIGNER C—USED Aligner with Spray

The USED aligner treated nightly with the fluoride–mint spray displayed a qualitatively different pattern. Although surface wear consistent with intraoral use was present, biofilm accumulation appeared reduced compared with the untreated USED aligner. Deposits were mainly sparse and discontinuous, consisting of isolated micro-colonies or small aggregates, while large portions of the polymer surface remained visible. At higher magnification, the micrographs showed scattered bacterial clusters with limited surrounding extracellular material (Figure 4).

These SEM observations indicate a qualitatively lower level of biofilm deposition on aligners treated nightly with the fluoride–mint spray compared with untreated USED aligners. Although the evaluation was qualitative, the consistent morphological differences suggest that the spray may help limit early microbial accumulation on the internal surface of aligners.

Overall, the results showed that salivary fluoride concentrations increased sharply after a single spray application, with higher fluoride retention observed in USED aligners than in NEW aligners. SEM analysis further revealed minimal surface contamination on NEW aligners, pronounced biofilm deposition on USED aligners worn without spray, and visibly reduced deposition on USED aligners treated nightly with the spray.

Taken together, these findings indicate that the fluoride–mint spray produced measurable changes in salivary fluoride concentration and reduced biofilm accumulation on aligners, thereby rejecting the null hypothesis and supporting the alternative hypothesis proposed in the Introduction.

Since the application of the fluoride–mint spray resulted in significantly higher salivary fluoride levels and lower biofilm accumulation compared with untreated conditions, the null hypothesis was rejected.

4. Discussion

Despite the limitations of this study, the results support the hypothesis that a spray containing fluoride and mint essential oil can effectively increase salivary fluoride concentration even in the presence of an intraoral device such as an orthodontic aligner.

The determination of salivary fluoride concentration represents a key indicator for evaluating the local availability of the active ingredient following application of the experimental spray. This parameter provides insight into the potential efficacy of the product in ensuring anticaries protection, particularly in patients wearing orthodontic aligners, who are more susceptible to plaque retention, reduced salivary clearance, and pH alterations [26]. Nighttime aligner wear, in particular, creates a microenvironment conducive to enamel demineralization; therefore, a prolonged salivary fluoride availability may offer a significant clinical advantage in maintaining enamel integrity and preventing early demineralization [27].

Although biofilm accumulation on aligners does not directly equate to dental-enamel biofilm, the large surface area and extended intraoral wear of aligner surfaces may facilitate microbial retention and offer a source of microorganisms contributing to tooth-surface colonization, especially under conditions of reduced salivary clearance or overnight wear [28]. For this reason, understanding aligner biofilm is clinically relevant as it reflects broader changes in the oral microbial environment during treatment.

The rapid rise in fluoride levels followed by a gradual decline is consistent with previous findings on topical fluoride absorption and release in saliva [22,29,30,31]. These data confirm the spray’s ability to deliver fluoride efficiently in a short time frame. However, the clinical relevance of this short-term increase should be interpreted cautiously, as conventional topical products such as toothpaste, gels, and varnishes can provide higher or more sustained fluoride availability [32,33,34,35]. The spray should therefore be considered a supportive adjunct, rather than a replacement, within the overall preventive strategy.

The concentration of peppermint essential oil used in the spray (1–2%) is supported by previous evidence showing that adding peppermint oil at similar concentrations (1–2.5%) enhances fluoride ion release in topical fluoride formulations, indicating a dose-dependent interaction between the essential oil and fluoride [25].

The greater fluoride retention in USED aligners may also be explained by intraoral aging, which increases surface roughness and porosity and may temporarily enhance fluid retention.

The difference observed between NEW and USED aligners may reflect interactions between the polyamide material and the spray formulation. However, the current literature lacks clear evidence regarding the potential fluoride adsorption or release from 3D-printed orthodontic materials, indicating the need for further studies to better understand these material-fluoride interactions.

The qualitative SEM analysis confirmed the presence of complex bacterial colonies on untreated aligners, in line with findings reported by Lombardo et al. [28]. In contrast, treated aligners exhibited a visibly lower bacterial density and a less organized biofilm structure, suggesting that the spray may interfere with the maturation and organization of oral biofilm. This observation is consistent with previous studies showing that essential-oil-based formulations can reduce early microbial adhesion and biofilm accumulation on removable prostheses. For example, denture cleansers or dentifrices containing tea tree oil or plant-derived essential oils significantly reduced microbial load and Candida biofilm on acrylic denture bases or removable partial dentures [36].

Similarly, research on fluoride-releasing removable materials has shown that they can act as fluoride reservoirs. For example, removable partial dentures containing S-PRG fillers demonstrated an initial fluoride burst followed by long-term rechargeability, resulting in increased salivary fluoride levels [37]. Although biofilm outcomes were not measured in that study, the findings support the concept that removable devices can contribute to sustained fluoride availability.

These findings have broader implications for preventive orthodontics. Maintaining optimal fluoride bioavailability while reducing biofilm accumulation is fundamental for preventing caries and demineralization in patients wearing removable aligners. The dual action of this spray, chemical protection through fluoride and biological modulation via mint essential oil, could provide an effective and patient-friendly strategy to improve oral health outcomes during orthodontic treatment.

Moreover, the high level of acceptability and absence of adverse reactions reported in this study align with previous investigations demonstrating the tolerability and pleasant sensory profile of mint essential oil in dental applications [38,39,40], reinforcing its potential for regular home use.

Nevertheless, certain limitations must be acknowledged. The relatively small sample size, the lack of quantitative microbiological evaluation, and the short duration of fluoride monitoring limit the generalizability of the results. The monitoring interval was limited to 45 min to minimize participant burden during the crossover design. Future studies should extend fluoride sampling beyond 60–120 min to better characterize the long-term retention profile. Although all USED aligners were worn for exactly 14 nights to ensure standardization, individual variability in wear patterns cannot be completely eliminated and represents a minor limitation of the study.

Future research should focus on quantitative analyses of the spray’s antimicrobial performance, long-term fluoride retention, and nanoscale surface characterization and the impact of aligner material composition on fluoride release and biofilm behavior. Expanding the investigation to different fluoride-essential oil combinations may also provide insights into optimized formulations for orthodontic use.

Overall, the findings of this study indicate a clear effect of the fluoride–mint spray on both salivary fluoride and biofilm accumulation. Therefore, the null hypothesis was rejected, confirming that the spray produced measurable and clinically relevant changes.

5. Conclusions

The integration of a fluoride- and mint-based spray into orthodontic therapy with 3D-printed Nighttime Aligners proved to be an effective strategy for increasing salivary fluoride concentration, qualitatively interfering with biofilm formation. Used aligners showed greater fluoride retention and slower clearance, suggesting that material properties may play a role in fluoride release preventive adjunct for maintaining oral health during aligner therapy, particularly in nighttime wear protocols. However, further studies on larger samples, with extended follow-up periods and quantitative microbiological analyses, are needed to confirm these findings and to better evaluate the long-term cariostatic activity and impact on the oral microbiota.