Diurnal and nocturnal salivary fluoride bioavailability following brushing with high- or regular-fluoride dentifrices.

INTRODUCTION

The use of fluoride (F) remains the most effective strategy for preventing and controlling dental caries at both individual and population levels [1]. Its mechanism of action is physicochemical, promoting enamel and dentin remineralization while reducing demineralization [2]. Among various fluoride delivery methods, dentifrices are considered the most rational approach, as they combine mechanical biofilm disruption with the topical availability of fluoride to support de- and remineralization processes [3, 4]. The widespread use of fluoride dentifrices has been identified as a major contributor to the global decline in dental caries in recent decades [5].

For fluoride to be effective, it must be free and soluble in the aqueous oral environment (saliva or biofilm fluid), and its anticaries activity therefore depends on its bioavailability in these fluids [1, 6]. Following topical fluoride application, such as toothbrushing with fluoridated dentifrice, salivary fluoride concentrations increase proportionally to the fluoride content of the product used [7]. Although salivary flow dilutes fluoride levels post-brushing, fluoride can be retained in enamel, residual biofilm, and oral soft tissues (such as the tongue and mucosa) [3, 8, 9]. Fluoride stored in these reservoirs can later be released into the biofilm fluid, particularly during pH drops caused by sugar intake, thereby prolonging its protective effect.

The salivary bioavailability of fluoride after toothbrushing is limited to a relatively short period. While fluoride concentrations can exceed pre-brushing levels by more than 100-fold within the first 3 min after brushing, they typically return to pre-brushing within 120 min [10, 11]. This dynamic is influenced by factors such as the form and solubility of fluoride in the formulation, post-brushing rinsing practices, and salivary flow rate [10, 12]. During sleep, when salivary flow is markedly reduced, fluoride availability in the oral cavity may be significantly altered [13].

Dentifrices containing 1450 ppm F represent the standard formulation recommended for daily use by the general population, providing effective caries prevention through regular topical fluoride exposure [14]. In contrast, high-fluoride dentifrices (5000 ppm F) are indicated for individuals at elevated risk of caries, such as those with reduced salivary flow, orthodontic appliances, root exposure, or a history of active carious lesions [15]. Indeed, high-fluoride dentifrices (5000 ppm F) have been shown to increase fluoride levels in saliva, biofilm fluid, and solids; reduce mineral loss and lesion depth in demineralized dentin; reverse non-cavitated lesions; and potentially promote greater calcium fluoride deposition compared to regular dentifrices containing 1000–1500 ppm F) [9, 16, 17].

Despite the proven clinical benefits of high-fluoride dentifrices, particularly in high-risk groups, limited data exist regarding their pharmacokinetics under different physiological conditions, such as during nocturnal periods with reduced salivary flow. Comparing these two formulations under both diurnal and nocturnal conditions offers valuable insights into their respective capacities to maintain therapeutic fluoride levels in the oral cavity over time, which is crucial for optimizing individualized caries prevention strategies. Therefore, this study aimed to evaluate the salivary fluoride bioavailability during diurnal and nocturnal periods following a single brushing with a high-fluoride dentifrice, and to compare it with the fluoride bioavailability achieved using a regular 1450 ppm F dentifrice.

MATERIAL AND METHODS

Study design and participants

A randomized short-term clinical trial with a crossover design was conducted with 15 healthy adult participants (6 males and 9 females; mean age: 24.4 ± 2.5 years), all residing in a municipality with fluoridated public water supplies (0.6–0.8 ppm F), which were submitted at four experimental conditions derived from the combination of two dentifrice formulations (1450 and 5000 ppm F) and two brushing periods (diurnal and nocturnal). Participants were allocated to treatment sequences according to a Williams Latin square design. Specifically, each participant completed the four experimental phases in a unique order, and all possible treatment sequences were arranged so that each treatment appeared once in each period position, and each treatment was preceded equally often by each of the other treatments.

Eligible participants were adults in good general and oral health (i.e., free from chronic systemic diseases) with a normal stimulated salivary flow rate >2 mL in 2 min. Exclusion criteria were smoking, active caries lesions, periodontal disease, orthodontic appliances, cardiovascular disease, pregnancy, lactation, and the use of medications known to reduce salivary flow (e.g., anticonvulsants, tricyclic antidepressants, and hormonal contraceptives) and the use of antibiotics for the last month before study. All participants underwent a dental examination and completed a questionnaire addressing general health status and medication use.

The sample size was calculated based on previous studies [18] using a similar experimental design. The parameters considered included four experimental groups, an estimated standard deviation of the salivary fluoride concentration of 0.55 µg F/mL, a minimum detectable salivary fluoride concentration difference of 0.45 µg F/mL between groups, an alpha error of 5%, and a beta error of 20%. The calculated sample size was 13 participants; anticipating a potential 20% dropout rate, the final sample size was set at 15.

Each participant underwent four experimental phases in which they randomly performed a single brushing with one of the two dentifrices (1450 or 5000 ppm F) during either the diurnal (8:00 a.m. to 4:00 p.m.) or nocturnal (10:00 p.m. to 6:00 a.m.) periods (Figure 1). They were advised to avoid fluoride-rich foods (e.g., tea and fish) during the saliva collection period but were not restricted from drinking water. Due to the crossover design of the study, no dietary restrictions were imposed. A 3-day lead-in and washout period with fluoride-free dentifrice was applied before and between each experimental phase. Randomization of participants to sequences was performed prior to the start of the study by an investigator not involved in data collection.

RESULTS

Table 2 presents the median and the interquartile range of salivary fluoride concentrations (µg F/mL) following the use of dentifrices containing 1450 and 5000 ppm F during the diurnal and nocturnal periods. During the diurnal period, statistically significantly higher fluoride concentrations were observed at 0 min, 5 min, and 2 h after brushing with the 5000 ppm F dentifrice than with the 1450 ppm F dentifrice (p < 0.05). In the nocturnal period, salivary fluoride concentrations were statistically significantly higher for the 5000 ppm F dentifrice at all time points except pre-brushing (p < 0.05). Pre-brushing salivary fluoride concentrations did not differ across experimental conditions (p > 0.05), indicating comparable starting levels before brushing and supporting the effectiveness of the fluoride-free washout period in minimizing residual fluoride carry-over between phases. However, for the 1450 ppm F dentifrice, significantly higher fluoride levels were detected during the nocturnal period than during the diurnal period at 0 min, 5 min, 2 h, and 4 h (p < 0.05). For the 5000 ppm F dentifrice, higher salivary fluoride concentrations were also found during the nocturnal period than during the diurnal period at 2, 4, and 8 h post-brushing (p < 0.05).

Figure 2 displays the kinetic curves of salivary fluoride concentration (µg F/mL) over time following brushing with both dentifrices during diurnal and nocturnal periods. For all groups, the peak fluoride concentration occurred immediately after brushing, followed by a progressive decline. The highest salivary fluoride concentrations were recorded after using the 5000 ppm F dentifrice during the nocturnal period, with elevated levels persisting for up to 8 h (p < 0.05). This figure also illustrates the time required for salivary fluoride levels to return to pre-brushing. For the 1450 ppm F dentifrice, fluoride concentrations returned to pre-brushing after 5 min (diurnal) and 2 h (nocturnal). In contrast, for the 5000 ppm F dentifrice, pre-brushing levels were reached at 2 h (diurnal) and 8 h (nocturnal) post-brushing (p < 0.05).

Figure 3 shows the AUC for salivary fluoride concentration over time. Statistically significant differences were found between the diurnal and nocturnal periods, regardless of the dentifrice used (p < 0.05). Additionally, the 5000 ppm F dentifrice showed significantly higher AUC values than the 1450 ppm F dentifrice in both periods (p < 0.05), indicating greater overall fluoride bioavailability, especially during the nocturnal period.

DISCUSSION

In this study, both dentifrices increased salivary fluoride concentrations following brushing; however, significantly higher concentrations were observed after using the high-fluoride (5000 ppm F) dentifrice. These findings align with previous reports demonstrating greater fluoride bioavailability with high-fluoride formulations [11, 19, 20]. Notably, this is the first study to compare the salivary fluoride kinetics of regular and high-fluoride dentifrices across both diurnal and nocturnal periods, providing new insights into circadian effects on fluoride retention in the oral cavity.

The differences in salivary fluoride concentrations observed between the regular and high-fluoride dentifrices support the dose-response relationship of fluoride and confirm that saliva is a sensitive indicator capable of detecting variations between treatments and across time points following dentifrice use [18]. These results can be attributed in part to the formulations of the products. Both NaF/silica-based dentifrices demonstrated total soluble fluoride concentrations consistent with their labeled content, ensuring effective delivery of bioavailable fluoride [21], as fluoride must remain chemically soluble within the formulation to exert its preventive effects [1]. Nevertheless, it should be acknowledged that dentifrices differ not only in fluoride concentration but also in other excipients and active compounds such as abrasives, humectants, and detergents, which may influence fluoride availability, salivary clearance, and even interaction with oral surfaces. Although both formulations tested here were silica-based, such compositional variations may have contributed to the observed differences in peak salivary fluoride concentrations and in the rate and duration of fluoride retention over time, particularly under nocturnal conditions, and warrant further investigation.

The analysis of the AUC for salivary fluoride confirmed that the 5000 ppm F dentifrice maintained significantly higher fluoride levels over time compared to the regular formulation, in both diurnal and nocturnal periods. Additionally, peak salivary fluoride concentrations were higher for participants using the high-fluoride dentifrice. These findings underscore the superior potential of high-fluoride products to protect dental substrates, especially root surfaces, which are more susceptible to caries and require higher fluoride concentrations for effective remineralization [22]. This is clinically relevant for high-risk populations, such as individuals with active caries, reduced salivary flow, or those undergoing orthodontic treatment, where high-fluoride dentifrices have demonstrated efficacy in reducing demineralization around brackets [17, 23, 24].

The nocturnal period warrants particular consideration due to the physiological reduction in salivary flow during sleep, which limits both mechanical clearance and buffering capacity in the oral cavity. While both dentifrices were found to elevate salivary fluoride levels at night, only the high-fluoride dentifrice sustained elevated concentrations up to 8 h post-brushing. In contrast, fluoride concentrations returned to pre-brushing within 2 h following the use of the regular formulation. This extended fluoride bioavailability during nocturnal hyposalivation may provide crucial protection when the risk of demineralization is heightened due to reduced salivary defense mechanisms [13].

The clinical implication of these findings is the reinforcement of nightly toothbrushing with fluoridated dentifrices, particularly those with high fluoride concentration. During sleep, oral clearance and the natural protective mechanisms against acid challenges are impaired [13]. Under these conditions, residual fermentable carbohydrates in dental biofilm or late-evening acidic exposures may lead to prolonged low pH episodes, favoring demineralization. The prolonged fluoride availability provided by the 5000 ppm F formulation before bedtime may therefore enhance protection against nocturnal demineralization. In addition, extended intraoral fluoride retention favors the formation of fluoride reservoirs [7, 9], which can gradually release ions into saliva and biofilm, sustaining cariostatic activity throughout the night.

It is important to address some limitations of the present study. Although the experimental design involved awakening participants during the night for saliva collection, allowing accurate and consistent sampling, this simulated sleep condition may have temporarily increased salivary flow compared to continuous sleep, possibly underestimating the extent of fluoride retention under real nocturnal hypofunction. Additionally, interindividual differences in brushing technique, dentifrice foam removal, and post-brushing rinsing, despite instructions and standardization and the crossover design, may have contributed to variability in salivary fluoride levels. These should be considered when generalizing the findings.

Another methodological limitation concerns the use of TISAB II in fluoride analysis. Although there is a recommendation to use TISAB III in samples with low concentrations of F, there is no consensus in the literature on which TISAB to use to buffer saliva samples to determine F concentration, since some used TISAB II [6, 18] and others used TISAB III [9, 20]. Although TISAB II has been routinely employed in our laboratory and is well-documented in the literature, it may present reduced sensitivity for detecting very low fluoride concentrations, particularly at later collection times when salivary fluoride levels approach pre-brushing. This methodological choice, even though internally validated and consistent with previous studies, could have influenced the accuracy of results under critical low-fluoride conditions.

In conclusion, the high-fluoride dentifrice significantly enhanced salivary fluoride bioavailability compared to the regular formulation, during both daytime and nighttime use. These results support its clinical indication for managing dental caries, particularly in individuals at elevated risk. The prolonged fluoride retention observed during the night further reinforces the recommendation of its use before bedtime as part of a targeted caries-prevention strategy, particularly when salivary protection is naturally reduced.

AUTHOR CONTRIBUTIONS

Conceptualization: Vale G. Investigation: Carvalho G. Methodology: Carvalho G; Santana G; Macena N. Supervision: Vale G. Writing—original draft: Carvalho G; Vale G. Writing—review and editing: Vale G.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.