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Caries is still prevalent despite the current understanding and use of fluoride. Faster-dissolving crystals of sodium fluoride (NaF) in a fluoride treatment could improve the efficacy of current preventative approaches.
In this study, crystals of NaF with a high surface area-to-volume ratio were prepared by adding an aqueous solution of NaF to an ethanol-water antisolvent mixture. Dissolution of these high surface area to volume crystals was measured using a conductivity probe.
Key variables in the preparation of hopper crystals included the amount of water in the antisolvent, the ratio of NaF solution to antisolvent, and the rate of stirring. The dissolution rate of these crystals was significantly greater than NaF crystals produced without a hopper morphology.
Including NaF hopper crystals in fluoride treatments could increase the bioavailability of fluoride from a fluoride treatment and potentially improve the remineralization of enamel.
Improving the release rate of fluoride from varnish could improve the efficacy of the treatment if the fluoride in the matrix is more completely released to the oral environment within the recommended time that patients are told not to brush or eat. Another challenge of preventative remineralization products is the difficulty of delivering incompatible bioactive agents from the same moiety. It has been shown that fluoride treatments tend to be more effective in the presence of a calcium ion source. However, in products where fluoride is already in its ionic form, the addition of a calcium ion would precipitate calcium fluoride in the package, rendering the formulation unstable. If fluoride and calcium ion sources could be kept as discrete salts in a continuous phase of a preventative treatment, the ability to rapidly dissolve both salts in situ when applied in the oral cavity could provide a pathway to improved caries prevention. In this study, the authors have prepared sodium fluoride in new hopper crystal morphologies. These morphologies have a higher surface area to volume than conventional sodium fluoride crystals leading to a greater rate of dissolution, potentially giving a pathway to improving fluoride treatments.
The bioavailability of fluoride in the oral environment is critical for a fluoride treatment to be effective. Caries is multifactorial, with the demineralization and remineralization process, diet, and oral hygiene being contributing factors. Despite dental health care providers having extensive knowledge of the etiology, caries is the most commonly occurring noncommunicable disease globally, according to the World Health Organization.
The mechanism of fluoride in conjunction with calcium ions works in 2 ways to prevent caries. First, fluoride makes enamel resistant to demineralization while also promoting remineralization. When fluoride is introduced to the oral cavity from topical sources (eg, fluoridated water), it is incorporated into the salivary pellicle. Calcium ions in plaque fluid attract fluoride ions, ultimately facilitating fluoride to adsorb to the enamel surface. Fluoride is incorporated into the demineralized surface, forming a fluorapatite crystal. Fluorapatite has a lower solubility in solution than hydroxyapatite, making the remineralized portion of enamel more resistant to acid challenges.
Fluoride varnishes are usually prepared as 5% wt/wt sodium fluoride (NaF). Varnishes are painted directly on the teeth by the dental care practitioner. However, unlike dental sealants, they are not meant to stick with any permanency. Rather, a small amount of product with a high fluoride concentration is in contact with the tooth for an ideal period. Compared with gels or foams, the advantages of varnishes include easier application and desensitization of exposed root surfaces.
Fluoride is considered a reference standard in preventing caries by the dental community and nearly every major public health organization. However, there are challenges to its effective delivery. Children and adolescents are considered the most at-risk population for developing caries.
These groups tend to comply poorly with at-home oral hygiene and clinical fluoride treatments. In addition, data from 2019 have shown caries prevalence plateauing or increasing in certain population groups, even with fluoride-containing dentifrices.
This indicates the need for more effective fluoride delivery.
One of the challenges around patient compliance with varnish formulations is that the full concentration of NaF is not effectively dissolved and released from the continuous phase from noncompliant patients. Although the varnish is purposefully designed for the slow release of the NaF compared with a formulation in which the fluoride is already in an ionic form, the potential to improve the efficacy of the treatment is possible if the fluoride could be dissolved in the matrix and released faster to the oral environment. Another challenge of preventative remineralization products is the difficulty of delivering incompatible bioactive agents from the same moiety. Fluoride treatments are more effective in the presence of a calcium ion source.
However, in products in which fluoride is already in its ionic form, a calcium ion would precipitate calcium fluoride in the package, rendering the formulation unstable. If fluoride and calcium ion sources could be kept as discrete salts in a continuous phase of preventative treatment, the ability to rapidly dissolve both salts in situ when applied in the oral cavity could provide a pathway to improved caries prevention.
Particle morphology is 1 of the fundamental properties that controls the rate of crystal dissolution. The solid phase of a substance formed by inorganic ions can assemble into different structures while maintaining the same chemical composition.
The shape of a crystal is often determined by thermodynamic equilibrium; that is, the underlying crystalline structure and surface energy. However, kinetics also involves crystal growth, resulting in different morphologies. One such morphology dictated by kinetics is the hopper crystal. Desarnaud et al
3 major factors for hopper crystal formation were identified: the concentration gradient, temperature gradient, and use of a capping agent. Most experiments have validated hopper crystal growth by modifying the concentration gradient and using capping agents.
The formation of hopper crystals can be achieved through control of the solute concentration gradient with various methods, including crystal growth in the vapor phase, the solution phase, gel systems, and electrocrystallization. The concentration gradient in solution is the most important factor in crystal formation, as many crystals can only grow from an aqueous solution.
A solution is said to be saturated when the solvent has dissolved the maximum amount of solute. Supersaturation occurs when the solute is added to a solution beyond the ability of the solvent to solvate the particles. Under certain conditions, regular cubic crystals precipitate from a supersaturated solution. However, conditions can be modified to induce hopper-shaped crystals, including using an antisolvent or rapid evaporation of the solvent in confinement.
Ions in solution form a primary nucleation site at which precipitation of a crystal occurs. Crystal growth occurs rapidly in a supersaturated solution because of the excess ions available. As ions are added to the growing structure, solute concentration decreases in certain areas of the bulk solution. Specifically, solute concentration near the center of the crystal is less than the concentration near the outer edges. Growth then becomes limited by how quickly ions can be incorporated. For growth to continue rapidly, ions are added to the corners and edges of the crystal more quickly than the face centers. The nucleation and growth of a crystal on the corner or edge of an existing crystal gives rise to the hopper morphology.
dissolved different morphologies of NaCl crystals in artificial saliva and correlated it to dissolution speed, affecting the perception of saltiness. They found that salt crystals with a larger surface to volume ratio dissolve faster. For example, it was found that the dissolution rate of Kosher salt was 3.8 times faster than that of extra coarse salt because of the morphologic differences of the crystal structures.
It is well established that the structure of a solid-state material affects its physical properties.
Although hopper crystals of NaF have never been investigated, the same logic can be applied given that NaCl and NaF are both water-soluble inorganic salts assembled in a cubic close-packed structure. The use of hopper crystals in dentistry would be beneficial if NaF crystals within a topical product could dissolve faster, providing greater bioavailability. Given the limitations in topical fluoride delivery, making dental products available with a faster-dissolving active ingredient would be advantageous. The prevalence of caries could be further reduced if this technology was used in dental fluoride treatments. This study tested the hypothesis that NaF crystals can be grown with a hopper morphology. It was further examined if the hopper crystal morphology affected the dissolution rate of NaF.
Hopper crystal synthesis
The synthesis of hopper-shaped crystals was tested using an antisolvent crystallization method. The conditions tested varied the vol% of ethanol (EtOH) used as the antisolvent (water was mixed with EtOH as the antisolvent), the ratio of antisolvent to NaF solution added, and the stir rate. The experiments reported in the Table highlight the importance of each of these variables.
TableThe conditions for selected experiments for hopper crystal growth are reported.
The composition of the antisolvent (% ethanol), the ratio of antisolvent to sodium fluoride solution used, and the stirring speed is reported for each given trial. The percentage of hopper crystals produced under each condition is reported.
Antisolvent (% Ethanol)
Ratio of Antisolvent to Sodium Fluoride Solution
Stirrer Speed (rpm)
% Hopper Crystals (SD)
∗ The composition of the antisolvent (% ethanol), the ratio of antisolvent to sodium fluoride solution used, and the stirring speed is reported for each given trial. The percentage of hopper crystals produced under each condition is reported.
For these experiments, a stock solution of 0.9 M NaF (MP Biomedicals) was prepared and used for all of the following experiments. The concentration was close to the solubility limit of NaF to maximize yield. Ten mL of the antisolvent selected for a given formulation was measured and transferred to a 50-mL beaker. If the conditions called for stirring, a stir flea was added to the beaker, and stirring commenced at the targeted speed. Next, the NaF solution was added dropwise with a pipette into the antisolvent. The beaker containing the NaF-antisolvent solution was then covered with parafilm and stirred for 24 hours.
After 24 hours, the beakers were uncovered, and the top layer of the remaining antisolvent was pipetted out and discarded. The solution was left uncovered to dry at room temperature for 24 hours. A spatula was then used to transfer the crystallized sample to a round-bottomed flask, and it was vacuum dried to evaporate any remaining EtOH-water mixture. The sample flask was placed in a silicone oil bath set at 110 °C for 48 hours. The synthesis of each formulation was prepared in triplicate. There was no variance in the morphologic outcome of the results determined by scanning electron microscope (SEM).
Scanning electron microscopy
Samples were imaged using SEM (Hitachi TM3000). Each specimen was sputter coated with gold before imaging.
Conductivity measurements of samples with different morphologies were recorded and compared. The Thermo Scientific Orion Star A222 conductivity meter (Fisher Scientific), Orion 013005MD conductivity cell (Fisher Scientific), and Starcom software were used to collect conductivity values. The conductivity meter was calibrated with 1,413 μS and 12.9 mS standards (Thermo Fischer Scientific) each time before measurements were taken. A mass of 56 mg of the dried NaF crystals was placed in a beaker. A stir flea was then added with the conductivity probe ready to go. Forty mL of nanopure water was added to the beaker submerging the probe. The solution was stirred at a rate of 30 rpm. The software collected data every 3 seconds. Therefore, the first data point represents the conductivity of the solution 3 seconds after the experiment commenced.
Hopper crystal formulation tables
The Table shows the conditions for potential hopper crystal growth of NaF. The antisolvent (% EtOH) column represents what antisolvent was used. One hundred percent EtOH refers to an antisolvent with no water intentionally added. Ninety percent EtOH refers to an antisolvent that intentionally had 5% vol/vol water purposefully added to the EtOH. The ratio of antisolvent to NaF solution column refers to the ratio of the antisolvent volume relative to the volume of NaF solution that was ultimately dripped into it. The stirrer speed refers to the speed at which the solution was stirring as the NaF was dripped into the antisolvent. The stirring speed was maintained for the length of the experiment. The corresponding percentage of hopper crystals synthesized is also reported for each formulation (if applicable). This was done by observation of SEM images. If no hopper crystals were present, it was reported as 0% in the Table.
The results in the Table show a series of experiments that showed the important variables for hopper crystal growth for NaF. Looking at formulations 1 through 6, the importance of the percentage of EtOH in the antisolvent is apparent. The ratio of antisolvent to NaF solution and stir speed was held constant for this data set. Although only formulation 1 is reported here for brevity, there were numerous variations in which 100% EtOH was used as the antisolvent. In no cases did hopper crystals form when no water was added to the EtOH before the precipitation of the NaF solution. Looking at formulations 2 through 4, the amount of hopper crystals formed increased as the amount of water added to the EtOH antisolvent before adding the NaF solution increased from 5 to 10 to 15 vol/vol%. However, as the amount of water added to the antisolvent increased past 15% vol/vol, as seen in formulations 5 and 6, the hopper crystals formed significantly dropped off at 20% vol/vol, and no hopper crystals were formed when 25% vol/vol of water was added.
The importance of the ratio of antisolvent used relative to the volume of NaF solution added is seen in formulations 3, 7, 8, and 9. In these experiments, the antisolvent and stir speed were held constant. In formulation 7, in which the ratio of antisolvent to NaF solution was 1.25:1, no hopper crystals were formed. This was true for every formulation tested in our experiments. However, once the ratio of antisolvent to NaF solution was 5:1 or greater (formulations 3, 8, 9), a significant amount of hopper crystals were grown in experiments in which the percentage of antisolvent was from 85% through 95% EtOH, and the stirring speed was more than 100 rpm.
The stirring of the antisolvent during the experiment also seemed to be a critical variable. Formulations 10, 11, and 12 held antisolvent composition and the ratio of antisolvent to NaF solution constant at 85% EtOH and 5:1, respectively. In 1 experiment, there was no stirring (formulation 10). In the other 2 experiments, the experiment was stirred at 100 or 1,000 rpm; as seen in formulation 10, the absence of stirring led to no hopper crystal growth. This was found true for other antisolvent compositions and other ratios of antisolvent to NaF solution ratios tested. For brevity, these experiments are not reported in the manuscript, but the observation of the need for stirring was apparent from the conditions tested.
The images were obtained using an SEM and are representative samples of synthesized crystals. Images are grouped according to the condition being tested. The numbers labeling each image correspond to formulation numbers in the previous tables. Figure 1 shows the morphology of the commercially available NaF crystals purchased for this study compared with 2 examples of NaF hopper crystal morphologies produced. Hopper crystals are either precipitated with a hollow cube or skeletal morphologies. Figure 1 contrasts the morphologies obtained in this study with the structure of the commercial-grade material.
The SEM images in Figure 2 depict the effect of the percentage of EtOH in water by volume used to formulate the antisolvent. Formulations 1 through 6 were selected for this figure to represent the effect of EtOH concentration of the antisolvent on hopper crystal morphology. The ratio of antisolvent to NaF solution used was constant at 10:1. The stir rate was constant at 1,000 rpm.
Figure 3 depicts the effect of the ratio of antisolvent to 0.9 M NaF solution on morphology. Formulation 7 represents the 1.25:1 ratio of 95% EtOH to 0.9 M NaF stirred at 1,000 rpm. No crystals with hopper morphologies were produced. Formulation 8 represents the 5:1 ratio of 90% EtOH to 0.9 M NaF stirred at 1,000 rpm. Formulation 3 represents the 10:1 ratio of 90% EtOH to 0.9 M NaF stirred at 1,000 rpm. Formulation 9 represents the 15:1 ratio of 90% EtOH to 0.9 M NaF stirred at 1,000 rpm.
The SEM images in Figure 4 depict the effect of stir rate on NaF hopper crystal formation. Formulations 10 through 12 were selected to represent the effect of the stir rate of the antisolvent on hopper crystal morphology. No hopper crystal morphology resulted from formulation 10 in the absence of stirring. The percentage of hopper crystals formed significantly increased in formulations 11 (100 rpm) and 12 (1,000 rpm). A trend emerged in our experiments that no hopper crystals were produced without stirring.
After being identified with a unique hopper morphology via SEM, NaF crystal samples were prepared to perform solubility experiments. Measuring conductivity as a function of time allows for the discernment of how fast the crystals dissolve in water. Data were collected for approximately 500 seconds, and a data point was taken every 3 seconds during each trial. Two trials of conductivity measurements were performed on each selected crystal formulation.
Figure 5 represents the conductivity values of NaF crystals dissolving in water as a function of time. The dissolution of salt crystals produced from formulations 2 through 5 was measured. Dissolution of crystals prepared with either 80%, 85%, 90%, or 95% EtOH as the antisolvent was compared, whereas the ratio of antisolvent to NaF solution and stir speed was held constant. Both formulations that produced hopper crystals of NaF led to a greater dissolution rate than NaF without the cubic morphology (typically found in commercially available NaF). In the first 3 seconds of the experiment, the greatest contrast in crystal dissolution rate is observed. In our study, 56 mg of NaF was dissolved in 40 mL of ultrapure water. This solution has a conductivity of 2,700 mS/cm when the NaF is fully dissolved. The first measurement provided by the conductivity probe after 3 seconds was 2,068 mS/cm for formulation 4. This suggests that approximately 76% of the salt dissolved in this time. Contrast this with the NaF produced without a hopper morphology in formulation 5, which had only 2% of the crystals with a hopper morphology. This dissolution of this salt had a conductivity of 1,060 mS/cm after 3 seconds. This conductivity suggests that approximately 39% of the NaF dissolved within 3 seconds. Similarly, in the crystals produced from formulation 3, in which 90% EtOH was used as the antisolvent and approximately 85% of the crystals had a hopper morphology, the conductivity reading at 3 seconds was 2,010 mS/cm, representing 74% of the crystals dissolved in this time. In formulation 2, in which the antisolvent was 95% EtOH and the yield of hopper crystals was approximately 5%, the conductivity of the solution after 3 seconds was 1,140 mS/cm representing 42% of the salt dissolving in that time frame.
Figure 6 depicts the conductivity values of NaF salt solutions formulated at different ratios of antisolvent to 0.9 M NaF solution as a function of time. This represents the dissolution of the salt crystals prepared from formulations 7, 8, 3, and 9, in which the ratio increased from 1.25:1 to 5:1 to 10:1 to 15:1, respectively. Formulation 7, in which the ratio was 1.25:1, resulted in no hopper crystals. The dissolution of these crystals after 3 seconds resulted in a conductivity of 1,060 mS/cm, corresponding to 39% of the crystals dissolving. The hopper crystals produced from formulations 3, 8, and 9 resulted in 71%, 74%, and 80% in the same period.
Figure 7 represents the average conductivity values of NaF salt solutions formulated with different stir rates using 85% EtOH as the antisolvent and a ratio of 5:1 for the antisolvent to the NaF solution. NaF crystals formulated at a stir rate of 100 rpm (formulation 11), 1,000 rpm (formulation 12), and 0 rpm (formulation 10) are represented in Figure 7. The hopper crystals produced with stirring had a significantly greater dissolution rate than those without hopper morphology produced without stirring. The crystals dissolving from formulations 11 and 12 had a conductivity of 1,420 and 1,530 mS/cm, respectively, after 3 seconds representing 53% and 57% of the salt dissolving. Formulation 10 without hopper crystals had a conductivity of 1,015 mS/cm after 3 seconds, representing the dissolution of 38% of the salt.
The ultimate goal of this study was to generate a fluoride source that can dissolve faster in the oral environment. This could lead to a significant improvement in fighting caries. The antisolvent approach to precipitation was implemented in these experiments to induce the growth of hopper-shaped crystals. A concentration gradient forms around a growing cubic crystal in a saturated solution, with the solute concentration greater at the corners and edges than at the faces. The growth of a cubic crystal is governed by kinetics. Adding an antisolvent creates a supersaturated solution, amplifying the effects of the concentration gradient. Under the right conditions, growth can then become diffusion limited, leading to the formation of hopper crystals.
To find if NaF hopper crystals could be synthesized, the first variable explored was the ratio of EtOH to water in the antisolvent. Formulations 1 through 6 varied the amount of water added to the antisolvent before adding the NaF solution. The addition of water to the EtOH increases the viscosity of pure EtOH. An increase in viscosity would slow down the diffusion of ions in the mixture and presumptively favor a diffusion-limited growth of crystal formation, which allows the possibility of hopper crystal growth. When 100% EtOH was used as the antisolvent, no conditions were observed for hopper crystal growth in the experiments. In this report, this can be seen in formulation 1. As 5% vol/vol of water was added to the EtOH, conditions for hopper crystal growth were observed, albeit at low yields (formulation 2 produced 5% hopper crystals). As the amount of water added increased to 10% or 15% vol/vol, the hopper crystals produced with the ratio of antisolvent to NaF solution between held constant at 10:1 with stirring at 1,000 rpm, produced a significant yield of hopper crystals as seen in formulations 3 and 4. However, as the water in the antisolvent increased to 20% vol/vol, the hopper crystals produced dropped significantly to just 2%, and at 25% vol/vol of water in the antisolvent, there were no hopper crystals observed in the product.
The next set of experiments (formulations 7, 3, 8, 9) explored the importance of the ratio of the volume of the antisolvent used to the volume of NaF solution used. In this set of experiments, the antisolvent was 90% EtOH, and the experiment was stirred at a rate of 1,000 rpm. If the ratio of antisolvent to NaF solution was too low, hopper crystals were not produced. In formulation 7, when the ratio of antisolvent to NaF solution was 1.25:1, hopper crystals were formed. This was the case in the other experiments not reported here. However, as the ratio of antisolvent to NaF solution was increased in the range of 5:1 through 15:1, hopper crystals were formed in significant yield. The results from these experiments can likely be explained by a phenomenon discussed by Yang et al.
They stated that in addition to the use of water as a solvent, alcohol can be added to an aqueous crystallization system. The use of alcohol plays 2 major roles in the growth of hopper crystals, including viscosity and solubility. First, the viscosity of the antisolvent solution system increases as alcohol concentration decreases. This is evidenced by the higher yields of hopper crystals in experiments with 85% EtOH and 90% EtOH relative to hopper crystal yields with 95% EtOH or 100% EtOH as the antisolvent, as previously discussed. As the solution becomes more viscous, this inhibits the diffusion of ions to the crystal structure, potentially favoring the formation of a hopper crystal. Second, the solubility of NaF in solution is decreased as alcohol concentration increases; as the solubility of NaF decreases, the growth rate of the crystal increases. An increased growth rate leads to a greater concentration gradient at the interface of the growing crystal, potentially promoting hopper crystal growth. This is evidenced by the higher yield of hopper crystals prepared with ratios more than 5:1 of the antisolvent to NaF solution compared with hopper crystals with ratios less than 5:1.
The final variable explored that could affect the concentration gradient leading to hopper growth of crystals was whether or not the antisolvent was stirred when adding NaF solution. The effect of stirring was explored under many conditions. In this report, formulations 10, 11, and 12 had 85% EtOH as the antisolvent with the antisolvent to NaF solution ratio held constant at 5:1. The addition of NaF solution was added to the antisolvent without stirring (formulation 10) and with stirring at 100 rpm (formulation 11) and 1,000 rpm (formulation 3). There were no hopper crystals formed without stirring in the experimental conditions that were explored. However, hopper crystals were obtained by stirring at 100 and 1,000 rpm.
The dissolution rate of NaF crystals correlates to the conductivity of NaF in solution. That is to say, as salt dissolves in water, the conductivity of the salt solution increases. Figure 5, Figure 6, Figure 7 depict the dissolution of NaF crystals produced under various conditions. The authors hypothesized in this research that NaF crystals with larger surface area to volume ratios can dissolve faster in solution. Syntheses that resulted in hopper-shaped crystals highlighted 3 key variables in forming these morphologies. These variables include the concentration of the antisolvent, the ratio of antisolvent to NaF solution, and the stir rate. When conditions lead to the precipitation of a significant portion of hopper crystal morphologies (formulations 3, 4, 8, 9, 11, 12), a greater dissolution rate was always observed compared with NaF crystals without hopper morphology (formulations 1, 2, 6, 7, 10). Variations in conductivity between the salts produced with different hopper morphologies are likely because of the exact differences in the surface area to volume and particle size obtained under a set of conditions.
There are 2 other significant variables that were not explored here that would have affected hopper crystal growth significantly. First, the concentration of the NaF solution would have to be an important variable. However, practically speaking, anyone interested in producing hopper crystals would want the highest yields possible. Therefore, working at an initial concentration in which the NaF salt solution is more than 90% saturated would yield the highest amount of salt. If the initial concentration of NaF used in the experiment had been 0.5 M instead of 0.9 M, this would likely have affected the yield and surface area to volume ratio of hopper crystals. In the context of other salts, such as NaCl, in which the salt is far more soluble in water, there would likely be a concentration in which the salt solution could be too concentrated to allow a diffusion-controlled growth of hopper crystals. However, with NaF having a maximum solubility of only 0.98 M in water, this was not a variable explored in these experiments. The other variable that would have affected the growth of hopper crystals is the vessel configuration and source of mixing. As this is a diffusion-controlled process, the fluid dynamics of how the antisolvent is mixing and how the salt solution is being added would have to affect the variables in which hopper crystals can be grown. However, the point of our research project was to show the possibility of growing hopper crystals of NaF and not the design of a reactor for which optimum yields of NaF hopper morphologies can be produced.
The possibility of growing hopper crystals of NaF was shown. Hopper crystals of NaF were dissolved in an aqueous environment at a greater rate than precipitated crystals that did not have hopper morphologies. Our efforts are focused on measuring fluoride release from varnish moieties containing NaF with hopper crystal morphologies and the impact on enamel fluoride uptake.