Effects of brief sodium fluoride treatment on the growth of early and mature cariogenic biofilms

doi:10.21203/rs.3.rs-586115/v1

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Effects of brief sodium fluoride treatment on the growth of early and mature cariogenic biofilms

https://doi.org/10.21203/rs.3.rs-586115/v1

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Although fluoride has been widely used in the prevention of dental caries, the effect of fluoride on the activity of biofilm in different stages of cariogenic biofilm formation is less studied. This study aimed to investigate the antibiofilm activity of sodium fluoride during early and mature Streptococcus mutans (S. mutans) biofilms formation. S. mutans biofilms were formed on saliva-coated hydroxyapatite disks. In the early (0 ~ 46 h) and mature (46 ~ 94 h) biofilm stages, the biofilm was treated with different concentrations of fluoride (250, 500, 1000, 2000 ppm; 5 times in total, 1 min/treatment). Acidogenicity, dry weight, colony-forming units, water-soluble/insoluble extracellular polysaccharides (EPS), and intracellular polysaccharides were analyzed and confocal laser scanning microscopy images were obtained of the two stages of biofilms (early and mature biofilms). To determine the antibiofilm activity of sodium fluoride during the formation of early and mature biofilms, and to evaluate the relationship between different concentrations of sodium fluoride and antibiofilm activity. In the early cariogenic biofilm formation stage, all fluoride concentration test groups (250, 500, 1000, 2000 ppm) significantly inhibited the growth of S. mutans biofilm. The antibiofilm and anti-EPS formation activities of the brief fluoride treatment increased in a concentration-dependent pattern. At the mature biofilm stage, only the 2000 ppm fluoride treatment group significantly inhibited biofilm accumulation, activity, and intracellular/extracellular polysaccharide content compared with the control and other fluoride treatment groups. The antimicrobial activity of fluoride is related to the formation stage of cariogenic biofilm. The early formation stage of cariogenic biofilm is more susceptible to the inhibition of fluorine than the mature stage. The fluoride treatment in the early formation stage of cariogenic biofilm may be an effective means to control the development of cariogenic biofilm and prevent caries.

Bacteriology

Applied & Industrial Microbiology

General Microbiology

sodium fluoride

dental caries

dental biofilms

Streptococcus mutans

cariogenic

Dental caries is an oral disease associated with dental biofilm¹. The increase of acidogenic and aciduric bacteria in the microorganism plays an important role in the pathogenesis of dental caries¹. When biofilms exist on the surface of teeth and expose to dietary sugar, acidogenic bacteria reduce pH in the dental biofilm utilizing glycolysis¹. The low pH environment further increased the growth of acidity and acidogenic bacteria including mutans streptococci, other acidogenic streptococci, lactobacilli, and bifidobacteria². The composition of cariogenic biofilm microflora is complex³. Although there may be other acidogenic and aciduric bacteria⁴, S. mutans is considered one of the important etiological agents for the development of dental caries^{5, 6}. S. mutans can produce glucosyltransferases (GTFs) and can synthesize extracellular polysaccharides (EPSs) and intracellular polysaccharides (IPSs) with sucrose as substrate. EPSs promote bacterial adhesion on the tooth surface, colonization, and formation of plaque biofilm, which is an important cariogenic factor of S. mutans⁷. The other major cariogenicity of S. mutans is determined by acid production (using carbohydrates to produce acid) and acid tolerance (living at low pH), resulting in the loss of local hard tissue and the start of the cariogenic process⁷. Reducing the adhesion of S. mutans to tooth surfaces, acid production, and EPSs formation of S. mutans biofilms will reduce the incidence of dental caries.

Fluoride is a widely used dental caries preventive agent. Fluoride products, such as fluoride water, mouthwash, ordinary toothpaste, and prescription toothpaste, play an important role in preventing dental caries⁸. The anti-caries mechanism of fluoride is to reduce the enamel solubility and promote enamel remineralization^{9, 10}. Some studies have shown that fluoride can inhibit the adhesion of S. mutans to the surface of hydroxyapatite¹¹, acid production, acid tolerance, and glucosyltransferase (GTF) production of S. mutans biofilms^{12, 13, 14}. Previous studies have reported the effect of fluoride on cariogenic biofilm virulence. When fluoride concentration ≥ 300 ppm, a single brief fluoride treatment (1 minute) can affect cariogenic characteristics of S. mutans biofilm¹⁵. However, a single brief fluoride treatment (1 minute) did not provide sustained antiviral activity because S. mutans can recover from fluoride shock over time¹⁶. Periodical brief fluoride treatment (1 minute) can reduce the dry weight and acid production of S. mutans biofilms in a concentration-dependent manner¹⁷.

Although there are many studies on the mechanism of fluoride inhibiting cariogenic biofilm growth of S. mutans, there are few studies on the relationship between anti-cariogenic biofilms activity of fluoride and biofilms growth stage, and the effect of continuous brief fluoride treatment on the virulence characteristics of early and mature cariogenic biofilm. It is necessary to test the hypothesis that brief fluoride treatment can show anti-cariogenic biofilm activity, which may depend on the early stage of biofilm formation. Therefore, to demonstrate the effect of fluoride on the development of S. mutants biofilms depend on the biofilm formation stage, two stages were used in this study: early biofilm formation stage (0 ~ 46h) and mature biofilm stage (46h ~ 94h)^{18, 19}. S. mutans biofilm was treated with different concentrations of fluoride, and the effects of different concentrations of fluoride on the development of S. mutans biofilm in two stages were compared.

This study aimed to evaluate the anti-cariogenic biofilm activity treated with brief fluoride (1 min/treatment, five treatments were performed) during early and mature cariogenic biofilm formation using the S. mutans biofilm model. Also investigated changes in anti-cariogenic biofilm activity associated with treatment concentrations (0, 250, 500, 1000, 2000 ppm).

Effects of brief fluoride treatments during early S. mutans biofilm formation.

The microbiological and biochemical analyses in early S. mutans biofilms formation (0 ~ 46 h). The effects of brief fluoride treatment on biofilm dry weight, bacterial activity, and polysaccharides were related to the fluoride concentration. As shown in Fig. 1, the dry weight (all biofilm components except water) (Fig. 1A; p < 0.05), CFUs (Fig. 1B; p < 0.05), water-insoluble extracellular polysaccharides (ASP) (Fig. 1C; p < 0.05), water-soluble extracellular polysaccharides (WSP) (Fig. 1D; p < 0.05), and intracellular polysaccharides (IPS) (Fig. 1E; p < 0.05) values of 46-h-old S. mutans biofilms decreased with the increase of brief fluoride concentration and showed the lowest value in the 2000 ppm fluoride treatment group.

Relationship between fluoride concentration and acid production of early S. mutans biofilms. Figure 2A showed the pH changes in the culture medium during the formation of early S. mutans biofilms (0 ~ 46 h) after brief fluoride treatment. During the formation of early S. mutans biofilms, brief fluoride treatment affected its acidogenicity (P < 0.05). The acidogenicity of the biofilm was inhibited in all fluoride concentration test groups during fluorine treatment (2 ~ 31 h; P < 0.05; 1 min/treatment, a total of five times) and without fluorine treatment (31 ~ 46 h; P < 0.05). The inhibition of acidogenicity of biofilms was enhanced with the increase of brief fluoride concentration, and the highest inhibition ability was shown in the 2000 ppm fluoride treatment group. The glycolytic pH drop assay was used to determine the effect of brief fluoride treatment on the acid production and acid tolerance of 46-h-old S. mutans biofilm. As shown in Fig. 3A and 3B, the initial acid production rate and total acid production concentration of the 46-h-old S. mutans biofilm were affected by the brief fluoride treatment, which was closely related to the fluoride treatment concentration. Compared with the control group, acid production was inhibited in all fluoride concentration treatment groups. With the increase of fluoride concentration, the anti-acidogenic activity was gradually enhanced. The 2000 ppm fluoride treatment group showed the lowest initial rate of H⁺ production and total produced concentrations of H⁺ (Fig. 3B). Also, the final pH value of the 2000 ppm fluoride treatment group was the highest, reflecting the decrease of acid tolerance of cells (Fig. 3A).

Confocal laser scanning microscope study of early S. mutans biofilms. To further evaluate the effect of brief fluoride treatment on early S. mutans biofilm formation components and structure, the CLSM analysis was performed. As shown in Fig. 4, the live/dead cells bacterial biovolume, bacterial thickness, and total biovolume of 46-h-old S. mutans biofilms decreased with the increase of brief fluoride treatment concentration, showing the lowest values in the 2000 ppm fluoride treatment group (Fig. 4A, 4B, 4C; P < 0.05). The three-dimensional image of bacterial microcolonies of 46-h-old S. mutans biofilms showed that with the increase of brief fluoride concentration, the morphology of the live/dead cells microcolonies changed, the volume and number decreased, and the arrangement was loose (Fig. 4D). Brief fluoride treatment also affected EPS formation of S. mutans biofilms, as shown in Figs. 5A and 5B. Brief fluoride treatment reduced EPS biovolume (P < 0.05) and thickness (P > 0.05) (Fig. 5A, 5B). With the increase of fluoride concentration, the anti-EPS formation increased in a concentration-dependent manner after transient fluoride treatment. EPSs and bacterial microcolony images of 46-h-old S. mutans biofilms showed minimal concentrations of homogeneous structures of EPSs covering and surrounding bacterial microcolonies in the 2000 ppm fluoride treatment group (Fig. 5C).

Effects of brief fluoride treatments during mature S. mutans biofilm formation.

The microbiological and biochemical analyses in mature S. mutans biofilms formation. As shown in Fig. 1, mature S. mutans biofilm formation (46 ~ 94 h) was also affected by the 2000 ppm fluoride treatment group (1 min/treatment, a total of five times). There was no significant difference in the dry weight of 94-h-old biofilm in the 250 ppm, 500 ppm, and 1000 ppm fluoride treatment groups compared with the control group. In the 2000 ppm fluorine treatment group, the dry weight of 94-h-old biofilm was significantly lower than that in the control group and other fluoride treatment groups. Compared with the control group, biofilm dry weight was 12% less in the 2000 ppm fluoride treatment group (Fig. 1A; p > 0.05). Also, the values of extracellular polysaccharides (ASP) (Fig. 1C; p > 0.05), water-soluble extracellular polysaccharides (WSP) (Fig. 1D; p < 0.05), and intracellular polysaccharides (IPS) (Fig. 1E; p < 0.05) in 94-h-old biofilm showed the lowest values in the 2000 ppm fluorine treatment group. The CFUs count in 94-h-old S. mutans biofilm decreased with the increase of brief fluoride concentration (Fig. 1B; p > 0.05). Compared with the control group, the CFUs count of biofilm was reduced by about 80% in the 2000 ppm fluoride treatment group (Fig. 1B).

Relationship between fluoride concentration and acid production of mature S. mutans biofilms. Figure 2B showed the pH changes in the culture medium during the formation of mature S. mutans biofilms after brief fluoride treatment. In the mature S. mutans biofilm stage, the acidogenicity of the biofilm was inhibited after brief fluoride treatment (46 ~ 94 h; P < 0.05). The acidogenicity of the biofilm was inhibited in all fluoride concentration test groups during fluoride treatment (46 ~ 55 h; 70 ~ 79 h; 1 min/treatment, a total of five times; P < 0.05) and without fluoride treatment (55 ~ 70 h; 79 ~ 94 h; P < 0.05). The inhibition of acidogenicity of biofilms was enhanced with the increase of brief fluoride concentration, and the highest inhibition ability was shown in the 2000 ppm fluoride treatment group. As shown in Fig. 3C and 3D, the initial acid production rate and total acid production concentration of the 94-h-old S. mutans biofilms were affected by the brief fluoride treatment, which was closely related to the fluoride treatment concentration. Compared with the control group, acid production was inhibited in all fluoride concentration treatment groups. With the increase of fluoride concentration, the anti-acidogenic activity was gradually enhanced. The 2000 ppm fluoride treatment group showed the lowest initial rate of H⁺ production and total produced concentrations of H⁺ (Fig. 3D; P < 0.05). In addition, the final pH value of the 2000 ppm fluoride treatment group was the highest, reflecting the decrease of acid tolerance of cells (Fig. 3C).

Confocal laser scanning microscope study of mature S. mutans biofilms. As shown in Fig. 4, the live/dead cells bacterial biovolume, bacterial thickness, and total biovolume of 94-h-old S.mutans biofilms decreased with the increase of brief fluoride treatment concentration, showing the lowest values in the 2000 ppm fluoride treatment group (Fig. 4A, 4B, 4C; P < 0.05). Three-dimensional images of 94-h-old S. mutans biofilms showed that the live/dead cells of the 94-h-old biofilms showed larger microcolonies than the 46-h-old biofilms, with uniform morphology and compact arrangement. With the increase of brief fluoride concentration, the number of live/dead cells microcolonies decreased and the arrangement of microcolonies was loose, but the morphology of microcolonies did not change significantly, and there were still some microcolonies in each treatment group similar to the control group (Fig. 4E). Brief fluoride treatment also affected EPS formation of S. mutans biofilms, as shown in Figs. 5A and 5B. Brief fluoride treatment reduced EPS biovolume and thickness (Fig. 5A, 5B; P < 0.05). With the increase of fluoride concentration, the anti-EPS formation increased in a concentration-dependent manner after brief fluoride treatment. EPSs and bacterial microcolony images of 94-h-old S. mutans biofilms showed minimal concentrations of homogeneous structures of EPSs covering and surrounding bacterial microcolonies in the 2000 ppm fluoride treatment group (Fig. 5D).

Several studies have reported that fluoride can affect acidogenicity, acid tolerance, and EPS formation of cariogenic biofilms, such as S. mutans biofilms^{20, 21, 22}. Although widespread studies of the effects of fluoride on the biological activity of oral microorganisms, the effects of fluoride on the activity of biofilm at different stages of cariogenic biofilm formation are rarely studied. The S. mutans biofilm model was used in this study. The effects of different fluoride concentrations on the growth, virulence (EPSs and acidogenicity), and activity of early and mature S. mutans biofilms were compared. Although the S. mutans biofilms model used in the present study does not mimic the complex microbial community found in dental biofilms precisely, the mono-species biofilm is advantageous in examining the mechanisms of actions of S. mutans in biofilms, also the model could provide significant benefit in establishing the reproducibility of data and reducing variance^{10, 23, 24}.

Biofilms are typically characterized by dense, highly hydrated clusters of bacterial cells. Bacterial adhesion and early biofilm formation are the first steps of cariogenic biofilm formation^{25, 26}. In the early formation stage of biofilm (0 ~ 46h), the bio-volume and average thickness of bacterial microcolonies and extracellular polysaccharides of biofilm continued to increase¹⁹. Since biomass density of dental biofilms increases from the saliva-biofilm interface inwards²⁷. At the mature biofilm stage (≥ 46 h), the CFU count and acid production capacity of the biofilm remained stable, as did the bacterial microflora of the biofilm and the bio-volume and average thickness of the extracellular polysaccharides. the standard deviation of bio-volume (bacteria or EPS) and mean biofilm thickness (bacteria or EPS) did not change over time¹⁹.

According to some reports, the effect of antibacterial agents applied in the mature biofilm stage is lower than that in the early biofilm stage, and the antibacterial activity of mature biofilm is limited to the surface layer of the biofilm substrate^{28, 29}. Mature biofilms have thicker and more complex biofilm structures, which may affect the diffusion and penetration of antibacterial agents^{27, 30}. Therefore, the antimicrobial membrane activity of S. mutans treated with brief fluoride during biofilm formation was studied in this study. As shown in Fig. 1, in the 46-h-old S. mutans biofilms, brief fluoride treatment reduced biofilm dry weight accumulation and bacterial activity at all fluoride test concentrations. The dry weight accumulation of biofilm decreased in a concentration-dependent manner, and there were significant differences between each treatment group and the control group (Fig. 1A; P < 0.05). When the fluoride concentration ≥ 500 ppm, the activity of biofilm bacteria was significantly affected compared with the control group (Fig. 1B; P < 0.05). In the 94-h-old S. mutans biofilms, only the 2000 ppm fluoride treatment group significantly inhibited biofilm dry weight accumulation and bacterial activity compared to the control and other fluoride treatment groups (Fig. 1A, 1B; P > 0.05).

In the studies of confocal laser scanning scope, the 46-h-old and 94-h-old biofilms live/dead cells bacterial biovolume, total biovolume, and bacterial thickness showed a concentration-dependent decrease in brief fluoride treatment. The lowest values were found in the 2000 ppm fluoride treatment group. There were significant differences between each treatment group and the control group at 46-h-old biofilms, while there were significant differences between the treatment group and the control group at 94-h-old biofilms when fluoride concentration ≥ 500 ppm (Fig. 4A, 4B, 4C; P < 0.05). The morphology of 46-h-old biofilms live/dead cell microcolonies was uniform and tightly arranged in the control group. With the increase of brief fluoride concentration, the morphology of live/dead cell microcolonies changed, and the size and number of microcolonies decreased, and the arrangement of microcolonies was loose. These results indicate that high fluoride treatment at the early stage of biofilm can affect the structural integrity of S. mutans biofilm cells (Fig. 4D). The live/dead cells of the 94-h-old biofilms showed larger microcolonies than those of the 46-h-old biofilms with uniform morphology and compact arrangement. With the increase of brief fluoride concentration, the number of live/dead cell microcolonies decreased and the arrangement of microcolonies was loose, but the morphology of microcolonies did not change significantly, and there were still some microcolonies in each treatment group similar to the control group (Fig. 4E). These results indicate that brief fluoride treatment is closely related to antibacterial biofilm activity. The antimicrobial biofilm activity of brief fluoride treatment depends on the biofilm formation stage and fluoride treatment concentration. In the early formation stage of biofilm, it is more susceptible to the influence of fluoride than in the mature stage. The antibacterial biofilm activity of the high-concentration fluoride treatment group is higher than that of the low-concentration fluoride treatment group. Also, according to the results of live/dead cell microcolonies, the brief fluoride concentration used in this study had no bactericidal or killing effect on the growth of S. mutans biofilms. Studies have reported that fluoride concentration of 3040–5700 ppm or fluoride concentration of > 5000 ug/ml has a bactericidal effect on S. mutans^{31, 32}. In this study, the highest fluoride concentration was 2000 ppm, which was lower than the fluoride concentration with bactericidal activity.

Up to 40% of the dry weight of dental biofilms is composed of polysaccharides, which are mostly synthesized by microbial GTFs³³. Therefore, the reduction of biofilm biomass is directly related to the reduction of polysaccharides in the whole biofilm matrix. The complex structural integrity of biofilm is mainly determined by the density of extracellular polysaccharides in the biological matrix, and extracellular polysaccharides contribute to the formation, volume, structural integrity, and stability of the biofilm matrix^{7, 34}. In this study, the effects of brief fluoride treatment on the synthesis of water-soluble/insoluble extracellular and intracellular polysaccharides during the formation of S. mutans biofilms were investigated. In the 46-h-old S. mutans biofilm, the synthesis of water-soluble/insoluble extracellular and intracellular polysaccharides was significantly inhibited in all fluoride treatment groups compared with the control group. The anti-EPS formation activity increased in a concentration-dependent manner (Fig. 1C, 1D, 1E; P < 0.05). In the 94-h-old S. mutans biofilm, only the 2000 ppm fluoride treatment group significantly reduced the synthesis of water-insoluble extracellular and intracellular polysaccharides compared with the control group (Fig. 1C, P > 0.05; Fig. 1E, P < 0.05). In terms of water-soluble extracellular polysaccharides, 1000 ppm and 2000 ppm fluoride treatment groups significantly inhibited the synthesis of water-soluble extracellular polysaccharides compared with the control group (Fig. 1D; P < 0.05). Since the biofilms are mainly composed of bacterial cells and polysaccharides⁷, the decrease in dry weight in all fluoride concentrations tested in the 46-h-old S. mutans biofilms and the 2000 ppm fluoride treatment group in the 94-h-old S. mutans biofilms may be due to the reduction in the number of bacterial cells and EPS formation. This result was also supported by the bio-volume and thickness of EPS of biofilm studied by the confocal laser scanning microscope. Brief fluoride treatment has an obvious damage effect on EPS formation of biofilm, and the decrease of bio-volume and thickness of EPS may affect the growth height of bacterial cells (Fig. 5). These results indicate that brief fluoride treatment can inhibit the synthesis of biofilm polysaccharides and is dependent on the biofilm formation stage and fluoride treatment concentration. Compared with the early stage of biofilm, the cariogenic biofilm maturation stage requires a higher concentration of fluoride (≥ 1000 ppm) to significantly inhibit the biofilm polysaccharide synthesis. The inhibition ability of the high concentration fluoride treatment group was higher than that of the low concentration fluoride treatment group. The mechanism of brief fluoride treatment inhibiting biofilm polysaccharide synthesis may be to inhibit GTFs activity or reduce GTFs production in S. mutans biofilm cells. However, further studies are needed to determine the mode of action of transient fluoride treatment on GTF activity and production in S. mutans biofilm cells.

Acid production (using carbohydrates to produce acid) and acid tolerance (living at low pH) are one of the main toxicity characteristics of S. mutans⁷, which are closely associated with enamel demineralization and the formation of dental caries^{35, 36, 37}. It is well known that fluoride has inhibitory effects on the acidogenicity of cariogenic biofilms¹⁴. In this study, the acid production and acid tolerance of S. mutans biofilms formation by brief fluoride treatment were studied. The results showed that fluoride inhibited the acid production and acid tolerance of S. mutans biofilm cells (Fig. 2, 3). In the pH changes of the old culture medium, it was found that after brief fluoride treatment, all fluoride concentration test groups inhibited the acid-producing capacity of the biofilm in the early formation and mature stages of the biofilm during fluoride treatment and without fluoride treatment. The concentration of acid in the old medium decreased in a concentration-dependent manner, and the highest inhibition ability was shown in the 2000 ppm fluoride treatment group (Fig. 2A, 2B). The lower the acid concentration may be related to the decrease of biofilm cell viability or physiological capacity. In the glycolytic pH drop experiment, the initial pH value reflects the acid-producing capacity of the cell, and the final pH value reflects the acid-tolerant capacity of the cell³⁷. As shown in Fig. 4, brief fluoride treatment inhibited acid production in the early and mature stages of cariogenic biofilms. The acidification activity of the biofilms was inhibited in all fluoride treatment groups, and the 2000 ppm fluoride treatment group showed the lowest initial rate and total acid production (Fig. 3B, 3D). Also, brief fluoride treatment reduced the acid resistance of cariogenic biofilm cells, and the 2000 ppm fluoride treatment group showed the highest final H⁺ concentration, which showed the highest effect on the acid resistance of biofilm cells (Fig. 3A, 3C). The change of H⁺ concentration may be due to the inhibition of glycolytic acid production by fluoride. These results indicate that the acid production and acid tolerance of the biofilm can be reduced by brief fluoride treatment during the early biofilm formation and mature biofilm. The anti-acidification activity was related to the concentration of fluoride, and the highest anti-acidification activity was shown in the 2000 ppm fluoride treatment group.

In general, brief fluoride treatment significantly inhibited early S. mutans biofilm formation (0 ~ 46 h). At the early stage of biofilm formation, biofilm dry weight, bacterial activity, water-soluble/insoluble polysaccharides, intracellular polysaccharides, and acid-producing capacity were significantly inhibited in all fluoride treatment groups compared with the control group (Fig. 1, 2, 3; P < 0.05). The inhibition degree of brief fluoride treatment on early biofilm growth of cariogenic bacteria was proportional to fluoride concentration. At the mature biofilm stage, only the 2000 ppm fluoride treatment group significantly inhibited the growth of biofilm compared with the control group. These results indicate that brief fluoride treatment can exhibit anti-S. mutans biofilm activity, which depends on the stage of biofilm formation. In the early stage of biofilm formation, low fluoride concentration treatment can significantly inhibit the growth of biofilm, while in the mature stage, higher fluoride concentration treatment can significantly inhibit the growth of biofilm. At the same time, the experimental results showed that the inhibition ability of fluoride on the growth of early and mature biofilm of S. mutans was related to the concentration of fluoride treatment. Compared with other fluoride treatment groups and the control group, the inhibition ability of the 2000 ppm treatment group was the strongest. This study provides a theoretical basis for the timing of fluoride in preventing caries. However, further research is needed to reveal the exact mechanism by which brief fluoride therapy affects early cariogenic biofilm formation.

The antimicrobial activity of fluoride is related to the stage of biofilm formation. Brief fluoride treatment significantly inhibited the growth of early S. mutans biofilms. The growth of S. mutans biofilm is more easily inhibited by fluoride in the early formation stage than in the mature stage. The fluoride treatment in the early stage of cariogenic biofilm formation may be an effective means to control cariogenic biofilm development and prevent dental caries.

Streptococcus mutans biofilms formation, fluoride, and experimental scheme. Figure 6 shows S. mutans biofilm preparation and experimental scheme for the present study. Streptococcus mutans UA159 (ATCC 700610; serotype c) biofilms were formed on saliva-coated hydroxyapatite (sHA) discs (2.93 cm²; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) placed in a vertical position in 24-well plates. Briefly, an adult male was selected for oral saliva collection. HA discs were incubated in filter-sterilized (0.22-µm low protein-binding filter) saliva (3 ml/disc) for 1 h at 37°C. For biofilms formation, the sHA discs were transferred to a 24-well plate containing brain heart infusion (BHI; D-ifco, Detroit, MI, USA) broth with 1% (w/v) sucrose and S. mutans UA159 (5–7×10⁶ colony-forming unit (CFU)/ml) (3 ml/disc). The biofilms were grown at 37°C with 5% CO² for biofilms growth. After 22 h of biofilm growth, the culture medium was changed twice daily (9 a.m. and 6 p.m. Oral sugar levels rise after 9 a.m. for breakfast and 6 p.m. for dinner) until it was 46 h (0 ~ 46 h early biofilm formation) or 94 h (46 h ~ 94 h mature biofilm formation) of age. In the present study, the biofilms of ≥ 46 hours were defined as mature biofilms¹⁸. This study is approved by the ethics committee/institutional review board of the Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience, Jeonbuk National University. All experimental protocols were approved by the Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience, Jeonbuk National University. The author confirms that all methods were carried out in accordance with relevant guidelines and regulations. The author confirms that informed consent had been obtained from all subjects.

The fluoride source in this study was NaF. NaF was purchased from Sigma-Aldrich (St Louis, MO, USA). The solutions were made with NaF and purified water. Fluoride products at concentrations between 1 to 2000 ppm are recommended⁸. In this study, four concentrations of fluoride were used for the experiment, respectively: 250 ppm, 500 ppm, 1000 ppm, 2000 ppm.

Considering that in practical applications, the secretion, swallowing, and spitting of saliva after fluoride products enter the oral cavity may reduce the initial fluoride concentration³⁸, the 1:3 dilution method is used to simulate the concentration of fluoride products in the oral cavity^{39, 40}. The four fluoride concentrations selected in this study can simulate the oral concentrations of four commonly used fluoride products after 1:3 dilution, respectively: 250 ppm (fluoride content in mouth rinse after a 1:3 dilution), 500 ppm (fluoride content in regular toothpaste after a 1:3 dilution), 1000 ppm (fluoride content in prescription toothpaste after a 1:3 dilution), 2000 ppm (fluoride content in the topical dental gel after a 1:3 dilution)⁴¹.

To determine the anti-cariogenic biofilm activity of brief fluoride treatments during early S. mutans biofilm formation, the saliva-coated HA disks were treated with 0 ppm (control group), 250 ppm, 500 ppm, 1000 ppm, 2000 ppm for 1 min, a total of 5 times treated during the early S. mutans biofilm formation (at 2, 7, 22, 26, 31 hours; The corresponding time is 9 a.m., 1, 6 p.m.). Then, the treated saliva-coated HA disks were transferred into the original 24-well plates containing a 1% sucrose culture medium. The incubated time of the S. mutans biofilms was 46 hours.

To determine the anti-cariogenic biofilm activity of brief fluoride treatments during mature S. mutans biofilm formation, S. mutans biofilms were not treated at the early biofilm growth stage (0 ~ 46h). After mature biofilm formation, the saliva-coated HA disks were treated with 0, 250, 500, 1000, 2000 ppm for 1 min, a total of 5 times treated during the mature S. mutans biofilm formation (at 50, 55, 70, 74, 79 hours; The corresponding time is 9 a.m., 1, 6 p.m.). Then, the treated saliva-coated HA disks were transferred into the original 24-well plates containing a 1% sucrose culture medium. The incubated time of the S. mutans biofilms was 94 hours.

The effects of different concentrations of fluoride on the dry weight, colony-forming unit, water-soluble/insoluble extracellular polysaccharide, intracellular polysaccharide, and acidogenicity of early (46-h-old biofilms) and mature biofilms (94-h-old biofilms) were compared. The images of biofilms at 46-h-old and 94-h-old were obtained by confocal laser scanning microscopy.

Microbiological and biochemical biofilm analyses. The dry weight and colony-forming units (CFUs) in the homogenized suspension were analyzed. Briefly, the 46/94-h-old biofilms on the sHA disc were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath for 10 min to disperse the biofilms. The dispersed solution was re-sonicated at 7W for 30 s after adding 3 ml of 0.89% NaCl (VCX 130PB; Sonics and Materials, Inc., Newtown, CT, USA). For the determination of CFUs count, an aliquot (0.1 ml) of the homogenized solution (5 ml) was serially diluted, plated onto brain heart infusion (BHI; Difco, Detroit, MI, USA) agar plates, and then incubated under aerobic conditions at 37°C to determine the CFUs count^{15, 42}.

For the determination of the dry weight and amount of water-insoluble extracellular polysaccharides (water-insoluble EPSs) (ASP), water-soluble extracellular polysaccharides (water-soluble EPSs) (WSP), intracellular polysaccharides (IPS), the remaining solution (4.9 ml) was centrifuged (3000 ×g) for 20 min at 4°C. The biofilm pellet was resuspended and washed twice in the same volume of water. Mix the water washed the biofilms pellet with 95% alcohol and put it in a refrigerator at -20°C for at least 18 hours to precipitate the water-soluble EPS. Then calculate the content of water-soluble EPS in the biofilms. The washed biofilms pellet was evenly divided into two portions, lyophilized, and weighed to determine the dry weight. One part used 1 N sodium hydroxide to extract water-insoluble EPS from the dried precipitate. The other part was used to calculate the content of intracellular polysaccharides, as detailed elsewhere²³.

Acid production analysis. The final pH values of the old culture media were also determined during the experimental period using a glass electrode (Beckman Coulter Inc., Brea, CA, USA) to investigate the change in acidogenicity of S. mutans biofilms by the treatments. The effect of brief fluoride treatment on the acidogenic and aciduric activity of early and mature S. mutans biofilms was determined by the glycolytic pH drop assay. Briefly, S. mutans biofilms were not treated with fluoride during the formation stage. the 46/94-h-old S. mutans biofilm was incubated in 20 mM potassium phosphate buffer (pH 7.2) for 1 h to deplete endogenous catabolites. They were then washed with salt solution (50 mM KCl + 1 mM MgCl2, pH 7.0) and treated with fluoride (0, 250, 500, 1,000, 2,000 ppm F-). After the fluoride treatment, the biofilms were dip-washed with salt solution and transferred into a 6-well plate containing salt solution. The pH was adjusted to 7.2 with a 0.2 M KOH solution. Glucose was then added to the mixture to give a final concentration of 1% (w/v). The decrease in pH was assessed using a glass electrode over 120 min (Futura Micro Combination pH electrode, 5 mm diameter; Beckman Coulter Inc., CA, USA). The effect of fluoride on the acid production of the biofilm was determined according to the acid production rate, calculated by the change in pH values over the linear portion (0 ~ 20, 30, 120 min) of the pH drop curves⁴³.

The initial rate (0 ~ 20 min) of H⁺ production (y1) and initial rate (0 ~ 30 min) of H⁺ production (y2) was derived from the equation:

y1 = (H⁺ concentration at 20 min – H⁺ concentration at 0 min)/20.

y2 = (H⁺ concentration at 30 min – H⁺ concentration at 0 min)/30.

The total produced concentration of H⁺ (y3) was derived from the equation:

y3 = H⁺ concentration at 120 min – H⁺ concentration at 0 min.

Confocal Laser Scanning Microscopy Analysis

Live and dead bacterial cells staining. Confocal laser scanning microscopy (CLSM) analysis was performed to confirm the results of microbiological and biochemical studies. To investigate the difference in bacterial cells, the 46/94-h-old biofilms were stained at room temperature in the dark for 30 min using the Film Tracer LIVE/DEAD Biofilm viability kit L10316 (Invitrogen, Molecular Probes Inc., Eugene, OR, USA). The final concentrations of SYTO®9 and propidium iodide (PI) were 6.0 and 30 µM, respectively. This viability kit was based on plasma membrane integrity to determine live and dead cells. In this study, we regarded the cells with intact membranes (green) as live cells, whereas cells with damaged membranes (red) were regarded as dead cells. The excitation/emission wavelengths were 480/500nm for SYTO®9 and 490/635nm for PI for collecting the fluorescence. The stained live and dead bacterial cells were observed with an LSM 510 META microscope (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium-neon lasers. All confocal fluorescence images were taken with an EC Plan-Neofuar 10x/0.30 M27 objective lens. A stack of slices in 6.4 µm step sizes was captured from the top to the bottom of the biofilms. The biovolume and thickness of live and dead cells were quantified from

the entire stack using COMSTAT image-processing software. The biovolume is defined as the volume of the biomass (µm³) divided by the substratum (hydroxyapatite surface) area (µm²). The three-dimensional architecture of the biofilms was visualized using ZEN 2.3 (blue edition) (Carl Zeiss Microscopy GmbH, Jena, Germany). The original confocal data was uploaded to ZEN 2.3 software and the intensity of green and red fluorescence in the full thickness of biofilms layers were captured automatically. The software reconstructed the 2-dimensional intensity of fluorescence in all the layers to a 3-dimensional volume stack⁴⁴.

EPS staining. The EPSs of 46/94-h-old biofilms were also investigated by simultaneous in situ labeling as described elsewhere⁴⁵. Briefly, Alexa Fluor® 647-labeled dextran conjugate (1 µM, 10,000 MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) was added to the culture medium during the formation of S. mutans biofilms (at 0, 22, 31 h of 46-h-old biofilms; at 0, 22, 31, 46, 55, 70, 79 h of 94-h-old biofilms) to label the newly formed EPSs. As described above, the stained EPSs were observed with an LSM 510 META microscope (Carl Zeiss, Jena, Germany) (objective: EC Plan Neofuar 10x/0.30 M27) equipped with argon-ion and helium-neon lasers and visualized using ZEN 2.3. A stack of slices in 7.8 µm step sizes was captured from the top to the bottom of the biofilms. Four independent experiments were performed, and five image stacks per experiment were collected. The EPSs biovolume and thickness were quantified from the confocal stacks using COMSTAT.

Statistical Analysis. All experiments (except CLSM and SEM) were performed in duplicate, and at least six different experiments were conducted. The data are presented as mean ± standard deviation. Inter-group differences were estimated using a one-way analysis of variance, followed by a post hoc multiple comparison (Tukey) test to compare multiple means (SPSS® software, IBM). Values were considered statistically significant when the p-value was < 0.05.

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No competing interests reported.

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Effects of brief sodium fluoride treatment on the growth of early and mature cariogenic biofilms

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