Original article
Effect of F:Ca:P ratio on fluoride deposition by fluoride-calcium-phosphate complex
2023 年 23 巻 1 号 p. 5-8
詳細
2023 年 23 巻 1 号 p. 5-8
Purpose: The aim of this study was to determine fluoride deposition by a fluoride-calcium-phosphate (FCP) of varying F:Ca:P ratios with the goal of maximizing the fluoride deposition at a fixed fluoride concentration of 12 mmol/L. Methods: The FCP solutions, prepared using NaF, CaCl2, H3PO4 and de-ionized water, had a calcium concentration of (7, 20, or 50) mmol/L, a phosphate concentration of (0.5, 2, or 10) mmol/L, and pH in the range of 2.73 to 3.43. A 12 mmol NaF solution was used as a control. A previously reported in vitro model was used for accessing fluoride deposition. The results were analyzed using multiple comparison with calcium and phosphate concentrations as the independent variables. Results: All FCP solutions were stable as expected. Both the calcium and phosphate concentrations effects and their interactions were significant (P < 0.01). Fluoride deposition, ranging from (3.08 to 4.57) µg/cm2, were at least 3.5 times greater than that produced by the NaF control. Higher calcium and phosphate concentrations did not necessarily increase fluoride deposition. Conclusion: FCP has the potential for use in fluoride rinses and dentifrices that will produce greater fluoride deposition without increasing the F dose.
There is a general consensus that the predominant anticaries effects of fluoride are not systemic, e.g., pre-eruptively changing the enamel structure, but mainly local, via interfering with the caries process. Hence, fluoride must be present in the right places, biofilm fluid, saliva, etc., and at the right time, sugar exposure, to interact with de- and remineralization events.
Fluoride is widely used in various compositions in the form of rinses, toothpastes, and the like. Fluoride-containing mouth rinses that are formulated for daily self-applications typically contain between 250 to 1,000 ppm. Fluorine, which most commonly is present as sodium fluoride or stannous fluoride. Office-administered topical fluoride gels, such as acidulated phosphate fluoride (APF) typically contain about 12,000 ppm (1.2%) fluorine. The concentration of fluorine in oral fluids has a profound effect on the de-/remineralization process [1,2,3]. The cariostatic effects of the various fluorine regimens are believed to derive from their ability to deposit fluorine in plaque and saliva and onto the surfaces of teeth and other tissues in the mouth. Although the deposited fluorine is labile and leached out with time, daily applications of fluorine can maintain an elevated level of fluorine in the mouth.
Calcium fluoride (CaF2) is recognized as a significant labile oral fluoride reservoir. Several previous developed fluoride rinses have successfully raised oral fluorine retention by forming CaF2. These very large increases in salivary fluoride and, more importantly, in plaque fluid fluoride suggest that a calcium pre-rinse or another way of supplying the calcium, may increase the effectiveness of fluoride-containing therapeutic agents [4,5]. However, these approaches required the use of two solutions applied either simultaneously or sequentially.
Previous studies have shown that a fluoride-calcium-phosphate (FCP) complex is stable in aqueous solutions for an indefinite length of time. A fluoride rinse containing an FCP with F:Ca:P molar ratio of 6:10:1 produced higher fluorine deposition than NaF in an in vitro model. FCP complex can form in a wide range of fluoride concentration that encompasses the entire range of the fluoride concentration found in currently used regiments. Complex formation allows high concentrations of fluorine and calcium to coexist in the solution. FCP, which can be easily destabilized to precipitate CaF2, may have applications in topical fluoride treatments [6]. The aim of this study was to determine fluoride deposition by the FCP complex of varying F:Ca:P ratios with the goal of maximizing the fluoride deposition at a fixed fluoride concentration of 12 mmol/L.
Reagent grade chemicals (NaF, CaCl2, and H3PO4,) and de-ionized water were used to prepare the FCP solutions. These solutions were prepared with a fixed fluoride concentration of 12 mmol/L, calcium concentration from 7 to 50 mmol/L, and phosphate concentration from 0.5 to 10 mmol/L. A 12 mmol/L fluorine solution without calcium and phosphate was prepared and used as the control (Table 1).
Table 1 Fluoride-calcium-phosphate (FCP) complex solutions and fluoride deposition
| Solution | NaF | CaCl2 | H3PO4 | pH | Shapiro-Wilk | F-deposition µg/cm2 | Welch | |
|---|---|---|---|---|---|---|---|---|
| mmol/L | mmol/L | mmol/L | P -value | Mean | SD | Grouping* | ||
| 1 | 12 | 7 | 0.5 | 3.43 | 0.626 | 3.06 | 0.07 | A |
| 2 | 12 | 7 | 2 | 3.26 | 0.108 | 3.93 | 0.25 | |
| 3 | 12 | 7 | 10 | 3.18 | 0.642 | 3.45 | 0.04 | |
| 4 | 12 | 20 | 0.5 | 2.93 | 0.141 | 3.08 | 0.08 | A |
| 5 | 12 | 20 | 2 | 2.79 | 0.776 | 4.30 | 0.04 | B |
| 6 | 12 | 20 | 10 | 2.73 | 0.295 | 4.10 | 0.05 | B |
| 7 | 12 | 50 | 0.5 | 2.42 | 0.466 | 3.08 | 0.02 | A |
| 8 | 12 | 50 | 2 | 2.30 | 0.723 | 4.57 | 0.22 | C |
| 9 | 12 | 50 | 10 | 2.23 | 0.741 | 4.52 | 0.01 | C |
| Control | 12 | 0 | 0 | 5.38 | 0.85 | 0.04 | ||
* α = 0.05
Fig. 1 The schematic illustration of fluoride uptake
Fig. 2 Powder X-ray diffraction analysis of solutions 1 and 9
In vitro procedure for evaluating fluoride depositionA previously reported in vitro model was used for accessing fluoride deposition [7]. A 6.0 mm-diameter (0.565 cm2 in surface area) cellulosed-based filter disk (0.2 µm pore size, GSWP, Millipore, Bedford, MA, USA), which is relatively inert with respect to fluorine, was used as a substrate for fluorine uptake. Three filter disks were immersed for 1 min in 30 mL of each of the 9 FCP solutions (Table 1). The discs were then washed by immersion for 20 s in 50 mL of a rapidly stirred (300 ppm) calcium fluoride-saturated solution (0.25 mmol/L CaCl2, 0.5 mmol/L NaF) to remove residual fluoride rinse solution, and then blotted dry with tissue papers. Each disc was then placed in a plastic test tube containing 1 mL of 0.5 mol/L HClO4 for 30 min to dissolve deposited fluoride. TISAB (Orion Research, Cambridge, MA, USA) containing 0.5 mol/L NaOH was added 1 mL, and the solution was thoroughly mixed. A fluorine ion-selective electrode and a reference electrode (Orion Research) that had been calibrated in fluoride standards were placed in this solution, and the fluoride concentration was measured (Fig. 1) [8]. The fluoride deposition values, normalized to unit substrate surface area, were calculated from the measured fluoride concentration in the etchant solutions.
In order to identify the products formed by the FCP solutions, the precipitates formed in the solutions with the highest (solution 9) and lowest (solution 1) concentrations of both calcium and phosphate, after raising pH to pH5 with NaOH were collected characterized by powder X-ray diffraction (XRD, Rigaku DMAX 2200, Rigaku/USA, Wilmington, MA, USA). Approximately 200 mg of the sample was placed in an aluminum sample holder for the XRD analysis.
Statistical analysisNumber of specimens were three in each experimental group. Distribution and variance of fluoride deposition values were analyzed by Shapiro-Wilk test and Levene’s test. Then, multiple comparisons were carried out by Welch t -test with Bonferroni correction among experimental groups. All statistical tests were performed using statistical software (SPSS Ver. 22.0, IBM, Armonk, NY, USA), and P -value was set at 0.05.
All FCP solutions were stable and no precipitation was observed. As shown in Table 1, the amounts of fluoride deposition by the FCP solutions ranged from 3.06 ± 0.07 to 4.57 ± 0.22 (µg/cm2), which are about 3.6 to 5.4 times greater than the fluoride deposition (0.85 ± 0.04 µg/cm2) produced by the control 12 mmol/L NaF solution that did not contain either calcium or phosphorus.
Distribution and variance of the fluoride deposition data were analyzed using Shapiro-Wilk test and Levene’s test. The fluoride deposition data indicated normal distribution in each calcium concentrations and phosphate concentrations from the results of Shapiro-Wilk test (Table 1). However, variance among the fluoride deposition data indicated unequal. Multiple comparisons of the cell mean values showed that calcium concentration did not have an effect at the lowest phosphate concentration, 0.5 mmol/L, but increased the fluoride deposition at both the mid and the highest levels of phosphate concentration of 2 mmol/L and 10 mmol/L, respectively. The phosphate concentration had a significant effect at any calcium concentration studied, but the highest fluoride deposition generally occurred at the mid phosphate concentration of 2 mmol/L. As a result, the highest fluoride deposition was produced by the FCP solution with the highest level of calcium concentration and mid or highest level of phosphate concentration.
XRD results showed that precipitate formed from solution 1 was CaF2 (Fig. 2). In contrast, precipitate formed from solution 9 contained both CaF2 and apatite.
Results from the present study confirms previous reports [9] that FCP solutions were able to deposit several times more fluoride on inert substrates than did a NaF solution of the same fluoride concentration. The present study further shows that although the calcium concentration and phosphorus concentration in the FCP solutions did have statistically significant effects on fluoride deposition, the effects are relatively small. In the present study, the calcium concentration and phosphorus concentration in the FCP solutions were varied by a factor of 7 and 20, respectively, but the observed highest and lowest fluoride deposition differed only by about 50% (Table 1). Similarly, the pH of the FCP solution did not seem to have a clear effect on fluoride deposition. The FCP solutions used were quite acidic with pH ranging from 2.23 to 3.43. These low pH values were in part a result of the compounds used to prepare the FCP solutions. In spite of the low pH, immersion of an inert substrate such as the filter disc was able to trigger significant amounts of precipitation from the solution. The present study showed that when filter discs were inserted into any FCP solution, significant F depositions were always observed. This suggests that the ability of FCP solutions to deposit fluoride on inert substrates does not require the solution to be highly metastable.
A mass balance calculation showed that mineral deposition on the filter discs would lead to negligibly small reductions in the fluorine concentration, calcium concentration, and phosphorus concentration of the FCP solution. Thus, the fluoride deposition was not limited by depletion of these ions in the FCP solution. Further studies are warranted to understand what other factors may constrain the fluoride deposition so that further enhancements in fluoride deposition by FCP solutions may be achieved.
The present study showed that the phase composition of the fluoride deposits was affected by the calcium concentration and phosphorus concentration in the FCP solution. The apatitic mineral deposition (Fig. 2) produced by FCP solution 9 is likely fluoridated hydroxyapatite (FHAp) and not hydroxyapatite because the former is much more stable in the presence of fluorine and at pH 5 [10]. These findings are clinically important. Because the fluorine in FHAp is non-labile, FCP solutions designed for remineralization effects should avoid FHAp formation. On the other hand, for dentinal tubule obturation applications, both FHAp and CaF2 are desirable products. FHAp, being less soluble than tooth mineral itself under acidogenic oral conditions, can be expected to produce long lasting desensitizing effects. Although CaF2 is soluble in normal and acidogenic oral conditions, its dissolution would elevate local fluoride concentration, leading to further precipitation of fluoridated apatite.
It was concluded that FCP complex has the potential for use in fluoride rinses and dentifrices that will produce a greater fluoride deposition without increasing the fluorine dose. However, all FCP solutions are acidic. Therefore, further studies are needed to develop FCP solutions with higher pH while maintaining their efficacies.
GI: conceptualization, investigation, methodology, visualization, and writing original draft. YS: conceptualization, methodology, supervision, formal analysis, writing, review, and editing. All authors read and approved the final version of the manuscript.
None
All data generated during this study are available from the corresponding author on reasonable request.
This research was supported by Dr. Lawrence Chow and Dr. Shozo Takagi with National Institute of Health grant DE16416, conducted by the National Institute of Standards and Technology in cooperation with the American Dental Association Foundation.
