Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites

Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites

Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 780-787, Copyright © 2011 Elsevier Ltd

Abstract

Objectives

The aim of this study was to evaluate the acid production, acid tolerance and composition of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites.

Methods

S. mutans biofilms were formed on saliva-coated discs prepared from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing materials (Filtek Z350, GRADIA DIRECT and hydroxyapatite). To assess the level of acid production and acid tolerance, glycolytic pH drop and proton permeability assays were performed using 94 h old S. mutans biofilms. To evaluate the biofilm composition, the biomass (total dry-weight), colony forming unit (CFU), water-insoluble extracellular polysaccharides (EPS), water-soluble EPS and intracellular iodophilic polysaccharides (IPS) of 94 h old S. mutans biofilms were analysed. The amount of fluoride of old culture medium released from the materials during the experimental period was also determined. Each assay was performed in duplicate in at least four different experiments ( n = 8).

Results

All biofilms showed similar initial rates of acid production (0.083–0.089 pH drop/min) and proton permeability (0.025–0.036 pH increase/min), irrespective of fluoride release from the materials. On the other hand, the amount of biomass, water-insoluble EPS and IPS of the biofilms on Unifil Flow, which releases a larger amount of fluoride in the early stages of biofilm formation, were significantly lower than those on the other materials (up to 27%, 38% and 36% reduction in biomass, water-insoluble and IPS, respectively).

Conclusions

Our finding suggests that fluoride releasing resin composites might contribute to the decrease in cariogenic composition of S. mutans biofilms if an appropriate amount of fluoride is released in the early stages of biofilm formation.

Introduction

Tooth-coloured dental restorative materials, such as resin composites and compomers, are used frequently to restore decayed tooth structures owing to their low relative thermal conductivity, aesthetics and improved performance. However, many tooth-coloured restorations have been replaced because of secondary caries, which is responsible for 60% of all restoration replacements in typical dental practice. One of the important causative factors in the development of secondary caries is the accumulation of dental biofilms adjacent to the margins of restorations, which causes dissolution of the adjacent tooth surfaces and finally shortens the serving life of the restorations.

If dental biofilms are allowed to remain on tooth surfaces or margins of restorations with the frequent consumption of a high carbohydrate diet, Streptococcus mutans , as a constituent of the biofilm community, will continue to synthesize extracellular polysaccharides (EPS) and metabolize the sugars to organic acids. The elevated amounts of EPS increase the bulk and structural stability of the biofilms. In addition, the ability of S. mutans to utilise some of the EPS and intracellular iodophilic polysaccharides (IPS) as short-term storage compounds offers an additional ecological benefit, which simultaneously increases the amount of acid production and the extent of acidification within the biofilms. The persistence of this aciduric environment leads to the selection and dominance of highly acid tolerant and acidogenic microorganisms, such as S. mutans . The low pH environment within the matrix results in the development of caries.

Recently, several studies have examined the pattern of bacterial adherence and biofilm formation on resin composites. Some investigations demonstrated that the surface roughness and composition of resin composites influence the level of bacterial adherence in the process of biofilm formation. Furthermore, biofilms on resin composites can degrade the material and roughen their surfaces, which in turn, promote biofilm formation and more surface deterioration of the material. Nevertheless, surprisingly little information is available on the cariogenicity of biofilms formed on resin composites.

To prevent secondary caries, resin composites that release fluoride ions as well as calcium and phosphate ions have been developed. Initial fluoride release into deionized water within 24 h amounted to 0.04–2.7 ppm for different composite brands (1.5 mm thick, 6 mm in diameter), but decreased to 0.02–2 ppm within 30–60 days. A variety of mechanisms are involved in the anti-cariogenic effects of fluoride, including the reduction of demineralization, the enhancement of remineralization, the interference of pellicle and biofilm formation and the inhibition of microbial growth and metabolism. Although resin composites are being used extensively, it is unclear if the cariogenicity of biofilms on the materials are affected by the amount of fluoride released from the materials.

Considering the continued widespread use of resin composites for dental restorations and the occurrence of secondary caries by dental biofilms, it would be meaningful to test the hypothesis that the cariogenicity of biofilms formed on resin composite may depend on the amount of fluoride released from the materials. Therefore, the aim of this study was to evaluate the acid production, acid tolerance and composition of S. mutans biofilms formed on various types of resin composites, particularly on fluoride releasing and non fluoride releasing resin composites.

Materials and methods

Disc preparation for biofilm formation

Table 1 lists the materials used in the present study. Three commercially available resin composites (flowable, nanohybride or microhybride) were selected. Compomer (polyacid-modified composite resins) was also included because of its chemical similarity to composite resins. The flowable resin composite (Unifil Flow) and compomer (F2000) are fluoride releasing materials. The nanohybride and microhybride resin composite (Filtek Z350 and GRADIA DIRECT, respectively) are non fluoride releasing materials. The shade of all materials was A2.

Table 1
Characteristics of restorative materials used in this study.
Type of material Product Fluoride containing * Composition Filler level Manufacturer
Flowable resin composite UniFil Flow Yes Aluminofluorosilicate glass
Urethane dimethacrylate
Dimethacrylate
Silica fine powder
Photoinitiator
67 wt% GC Corporation, Japan
Nanohybrid resin composite Filtek Z350 No Bisphenol A-glycidyl methacrylate
Bisphenol A-polyethylene glycol diether dimethacrylate
Urethane dimethacrylate with small amounts of triethylene glycol dimethacrylate
78.5 wt% 3M ESPE, USA
Microhybrid resin composite GRADIA DIRECT Anterior No Urethane dimethacrylate
Dimethacrylate
Silica powder
Organic filler
Pigment
Camphorquinone
73 wt% GC Corporation, Japan
Compomer F2000 Yes Aluminofluorosilicate glass
Colloidal silica
Dimethacrylate functional oligomer derived from citric acid
Glyceryl dimethacrylate
High molecular weight hydrophilic polymer
Camphorquinone/amine
84 wt% 3M ESPE, USA

* Based on information provided by the manufacturers.

Disc-shaped specimens (12 mm in diameter and 1.2 mm in thickness) were prepared using a polytetrafluoroethylene (Teflon) moulds with glass microscope slides to cover each face. The specimens were light-cured for 20 s with a light curing unit (Optilux, Kerr, Danbury, USA). After curing, all specimens were polished sequentially from # 600 to # 2000 sand paper and buffered clothes with 0.3 μm alumina powder. The specimens were sonicated for 5 min to remove the remaining powder and then stayed in a desiccator at room temperature. Hydroxyapatite discs (12 mm in diameter and 1.2 mm in thickness; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) were also included as a control.

Biofilm formation and experimental scheme

The microorganism used in the present study was S. mutans UA159. Fig. 1 shows S. mutans biofilm preparation and experimental scheme for the present study. S. mutans UA159 biofilms were formed on saliva-coated resin composites, compomer or hydroxyapatite discs placed in a vertical position in 24-well plates, as detailed elsewhere. Briefly, the saliva-coated discs were generated by incubation with filter-sterilized (0.22 μm low protein binding filter) and clarified human whole saliva for 1 h at 37 °C. For biofilm formation, the saliva-coated discs were transferred to a 24 well plate containing 1% sucrose (v/v) ultrafiltered (10 kDa molecular-weight cut-off) tryptone yeast-extract (UTE) broth with S. mutans UA159 (2–5 × 10 6 CFU/ml). The biofilms were grown undisturbed for 22 h to allow initial biofilm growth. From this point (22 h old), the culture medium was changed twice daily (9 AM and 6 PM) until it was 94 h old. The culture medium was changed a total of 6 times.

Biofilm preparation and experimental scheme. This study examined the acid production, acid tolerance and composition of 94 h old S. mutans biofilms formed on fluoride releasing or non fluoride releasing resin composites.
Fig. 1
Biofilm preparation and experimental scheme. This study examined the acid production, acid tolerance and composition of 94 h old S. mutans biofilms formed on fluoride releasing or non fluoride releasing resin composites.

To evaluate the acid production and acid tolerance of S. mutans biofilms formed on the test materials, glycolytic pH drop and proton permeability assays were performed using the 94 h old biofilms. To evaluate the biofilm composition, the 94 h old biofilms were analysed using microbiological and biochemical methods. The amount of fluoride in the old culture medium was also determined during the experimental period. Each assay was performed in duplicate in at least four different experiments ( n = 8).

Evaluation of acid production and acid tolerance of biofilms

Glycolytic pH drop assay

The glycolytic pH drop of S. mutans biofilms on the test materials was measured, as described elsewhere. Briefly, the 94 h old biofilms, which had been incubated in 20 mM potassium phosphate buffer (pH 7.2) for 1 h to deplete the endogenous catabolites, were transferred to a salt solution (50 mM KCl plus 1 mM MgCl 2 , pH 7.0). The pH was adjusted to 7.0 with a 0.01 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 a period of 120 min (Futura Micro Combination pH electrode, 5 mm diameter; Beckman Coulter Inc., CA, USA). The initial rate of acid production was calculated using the pH values in the linear portion (0–30 min incubation period) because the rate of the decrease in pH reflects the acid production capacity of the biofilms.

Proton permeability assay

The proton permeability of S. mutans biofilms on the test materials was assessed using the procedures described previously. Basically, the 94 h old biofilms, which had been incubated in 20 mM potassium phosphate buffer (pH 7.2) for 1 h to deplete the endogenous catabolites, were incubated initially at a constant pH of approximately 4.6. HCl was then added to decrease the pH values in approximately 0.4 units. The subsequent increase in pH associated with proton movements across the cell membrane into the cytoplasm was monitored using a glass electrode. Butanol (10%, v/v) was added to the suspensions at 80 min to disrupt the cell membrane. The initial rate of proton entry was estimated because changes in initial rates of proton uptake give the best measure of disruptive effects. The initial rate of proton entry was calculated using the pH changes of 0–10 min incubation period.

Evaluation of biofilm composition

For microbiological and biochemical composition analyses, the 94 h old biofilms on the test materials were removed and sonicated at 7 W for 3 periods of 30 s each (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA). The colony forming unit (CFU), biomass (dry-weight), and polysaccharide composition in the homogenized suspension was examined. The water-soluble EPS, water-insoluble EPS and IPS were extracted and quantified by colorimetric assays, as detailed elsewhere. The decrease in biomass, water-insoluble EPS or IPS of the biofilms on a material ( A ) was calculated using the following formula: reduction (%) = (the amount of the biomass, water-insoluble EPS or IPS of biofilms on A /the amount of the biomass, water-insoluble EPS or IPS of biofilms on the other materials) × 100.

Determination of fluoride amount of old culture medium

During biofilm development, as soon as the biofilms on the test materials were transferred to the new culture medium twice daily (9 AM and 6 PM), the amount of fluoride released from the materials into the old medium was measured using a fluoride ion selective electrode (Thermo Electron Corporation, OH, USA). A total of 2.8 ml of each medium was transferred into a 15 ml tube and the concentration of fluoride was measured after vortex. The fluorometer was calibrated using three standard solutions (0.1, 1 and 10 ppm). To calculate the fluoride releasing rate, the concentration of fluoride from the test materials was divided by the incubation time.

Statistical analysis

The data are presented as mean ± standard deviation. The intergroup differences were estimated by one-way analysis of variance (ANOVA), followed by a post hoc multiple comparison (Tukey test) to compare the multiple means. Values were considered statistically significant when P value was <0.05. The statistical analyses were performed using SPSS 12 software (SPSS Inc., Chicago, IL, USA).

Results

Initial rates of acid production and proton permeability of biofilms

Fig. 2 shows the glycolytic pH drop of S. mutans biofilms on the test materials. There was no significant difference in the initial rates of acid production (amount of pH drop/min) between the biofilms on fluoride releasing and non fluoride releasing materials ( p > 0.05). The initial rates of acid production ranged from 0.083 to 0.089 pH drop/min. Fig. 3 shows the result of the proton permeability assay. In general, the initial rates of proton entry (amount of pH increase/min) of the biofilms on fluoride releasing and non fluoride releasing materials were also similar ( p > 0.05), ranging from 0.025 to 0.036 pH increase/min.

Pattern of glycolytic pH drop and initial rate of pH drop of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. N/S: non-significant difference between the test materials at 30 min.
Fig. 2
Pattern of glycolytic pH drop and initial rate of pH drop of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. N/S: non-significant difference between the test materials at 30 min.
Pattern of proton permeability and initial rate of proton entry of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. N/S: non-significant difference between the test materials at 10 min.
Fig. 3
Pattern of proton permeability and initial rate of proton entry of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. N/S: non-significant difference between the test materials at 10 min.

Biofilm composition

Fig. 4 A shows the result of the biomass evaluation of S. mutans biofilms on the test materials. Of the materials tested, the lowest amount of biomass was recovered from the biofilms on UniFil Flow. The biomass on UniFil Flow was 6.19 mg/disc, which was 78% of Filtek Z350, 77% of GRADIA DIRECT and 73% of hydroxyapatite ( p < 0.05). Interestingly, although F2000 is also one of fluoride releasing materials, the amount of biomass on the material was not significantly different from those on non fluoride releasing materials ( p > 0.05).

Biomass (A) and CFU (B) of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. * P < 0.05: significantly different from each other. N/S: non-significant difference between the test materials.
Fig. 4
Biomass (A) and CFU (B) of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. * P < 0.05: significantly different from each other. N/S: non-significant difference between the test materials.

Fig. 4 B presents the number of S. mutans CFU in the biofilms on the test materials. Although the amount of biomass on UniFil Flow was lower than those of non fluoride releasing materials, the number of S. mutans CFU on UniFil Flow was not significantly different from those on non fluoride releasing materials ( p > 0.05). In general, the number of S. mutans CFU on fluoride releasing and non fluoride releasing materials was not significantly different ( p > 0.05).

Fig. 5 shows the amount of polysaccharide (water-insoluble EPS, water soluble EPS and IPS) of S. mutans biofilms on the test material. As shown in Fig. 5 A, the amount of water-insoluble EPS on UniFil Flow was significantly lower than those on non fluoride releasing materials ( p > 0.05). The water-insoluble EPS of UniFil Flow was 1.09 mg/disc, which was 69% of Filtek Z350, 70% of GRADIA DIRECT and 62% of hydroxyapatite ( p < 0.05). However, the amount of water-insoluble EPS on F2000 was not significantly different from those on non fluoride releasing resin composites ( p > 0.05). The amount of IPS on UniFil Flow (0.31 mg/disc) was significantly lower than those of Filtek Z350 and hydroxyapatite ( Fig. 5 B, p < 0.05). However, there was no significant difference in the amount of water-soluble EPS between fluoride releasing and non fluoride releasing materials ( Fig. 5 C, p > 0.05).

Water-insoluble extracellular polysaccharides (EPS, A), intracellular iodophilic polysaccharides (IPS, B) and water-soluble EPS (C) of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represents the mean ± standard deviation. * P < 0.05: significantly different from each other. N/S: non-significant difference between the test materials.
Fig. 5
Water-insoluble extracellular polysaccharides (EPS, A), intracellular iodophilic polysaccharides (IPS, B) and water-soluble EPS (C) of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represents the mean ± standard deviation. * P < 0.05: significantly different from each other. N/S: non-significant difference between the test materials.

Fluoride release from the materials

Fig. 6 shows the amount of fluoride and fluoride releasing rates from the test materials during the incubation period. In general, the amount of fluoride from UniFil Flow and F2000 in old culture medium was higher than those from non fluoride releasing materials ( Fig. 6 , p < 0.05) but these materials showed a different pattern of fluoride release. UniFil Flow released a higher amount of fluoride than F2000 during the first experimental day (22 and 31 h) but F2000 released a higher amount of fluoride than UniFil Flow after late second day to the end of the experiment (55–94 h). In general, UniFil Flow showed the highest rate of fluoride release in the early stages of biofilm formation. On the other hand, F2000 showed the highest rate of fluoride release in the late stages of biofilm formation.

Amount of fluoride and fluoride releasing rate from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite) during S. mutans biofilms formation. The data represent mean ± standard deviation. * (S) P < 0.05: significant difference between Unifil Flow and F2000. In the table of the fluoride releasing rate, the values in the same row followed by the same letters are not significantly different from each other.
Fig. 6
Amount of fluoride and fluoride releasing rate from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite) during S. mutans biofilms formation. The data represent mean ± standard deviation. * (S) P < 0.05: significant difference between Unifil Flow and F2000. In the table of the fluoride releasing rate, the values in the same row followed by the same letters are not significantly different from each other.

Discussion

Caries is a biofilm-related dental disease associated with the increased consumption of dietary sugar. The bacterial composition of dental biofilms remains relatively stable when exposed to minor environmental changes. On the other hand, if environmental perturbations occur, such as an increase in sucrose availability, the development of cariogenic biofilms would be induced by triggering EPS matrix formation, and the growth and dominance of cariogenic streptococci. Although additional acidogenic and aciduric bacteria can be involved, S . mutans is one bacterial species that has been implicated most frequently in the establishment of cariogenic biofilms and the initiation of caries because this bacterium is highly acidogenic and acid tolerant, and utilizes dietary sucrose to synthesize large amounts of EPS. In this context, S . mutans biofilm grown under sucrose-rich conditions (1%) was chosen to examine the virulence and composition of cariogenic biofilms on resin composites. Although the S. mutans biofilm model does not mimic the complex microbial community found in dental biofilms precisely, the biofilm is advantageous for examining the cariogenic process of biofilms.

The acid production and acid tolerance of S. mutans biofilms were examined using a well-established glycolytic pH-drop and proton permeability assay. As shown in Figs. 2 and 3 , the initial rates of acid production and proton permeability of S. mutans biofilms were not affected by the support materials. Interestingly, the initial rates of the biofilms on the fluoride releasing materials (UniFil Flow and F2000) were similar to those on the non fluoride releasing materials. It is well known that fluoride has biological effects on the critical virulence of cariogenic streptococci, particularly on S. mutans . Furthermore, the physiological ability of S. mutans biofilms was reported to be affected by fluoride. On the other hand, in the present study, although UniFil Flow and F2000 released significantly higher amounts of fluoride than non fluoride releasing materials ( Fig. 6 ), these materials did not inhibit the initial rates of acid production and proton permeability of S. mutans biofilms ( Figs. 2 and 3 ), suggesting that the amount of fluoride released by the materials is too low to inhibit the physiological ability of S. mutans biofilms. These finding are supported by a previous study showing that fluoride concentrations ≤10 ppm cannot reduce the initial rates of acid production and proton permeability of S. mutans biofilms.

However, our result indicated that the biomass of S. mutans biofilms can be affected by the amount of fluoride released from the test materials, particularly in the early stages of biofilm formation. As shown in Fig. 4 A, the biomass on UniFil Flow, which released the highest amount of fluoride in the early stages of biofilm formation ( Fig. 6 ), was significantly lower than that on non fluoride releasing materials. On the other hand, the biomass on F2000, which released the highest amount of fluoride in the late stages of biofilm formation ( Fig. 6 ), was not significantly different from those on the non fluoride releasing materials. These finding suggest that the biomass of dental biofilms on dental restorative materials can be affected if the restorative materials release an appropriate amount of fluoride in the early stages of biofilm formation.

In the present study, although the amount of biomass of S. mutans biofilm was affected by UniFil Flow, there was no significant difference in the CFU numbers of S. mutans in the biofilms between the fluoride releasing and non fluoride releasing materials ( Fig. 4 ), which suggests that the amount of fluoride from the test materials did not affect the growth and viability of S. mutans in the biofilms. Furthermore, this finding indicates that the biomass reduction of the biofilms on UniFil Flow is related to the decrease in the other main constituents (e.g. EPS) of the biofilms, not a decrease in the number of bacterial cells.

It is well known that EPS, synthesized by microbial glucosyltransferases (GTFs), contribute to the formation and structural integrity of dental biofilm matrix , and the concentration of EPS in dental biofilms is associated with dental caries. Therefore, the decrease in EPS can affect cariogenic biofilm formation and accumulation, and the subsequent initiation of dental caries. In the present study, the amount of water-insoluble EPS of S. mutans biofilms on UniFil Flow were reduced by up to 27%, compared to the non fluoride-releasing materials ( Fig. 5 A). On the other hand, the amount of water-soluble EPS of S. mutans biofilms on UniFil was not affected ( Fig. 5 C). Although the precise mechanisms by which the water-insoluble EPS were reduced and water-soluble EPS were not affected were not determined in this study, a recent study reported that GTF for the synthesis of water-insoluble EPS is more sensitive to fluoride than that for water-soluble EPS. The study also showed that fluoride at low concentrations (3.8 ppm) partially inhibits the secretion of GTFs by S. mutans , which support the present result that there is less EPS formation on UniFil Flow than that on the non fluoride releasing materials. In addition, the amount of IPS on the fluoride releasing materials used in this study (UniFil Flow and F2000) was lower than that of the non fluoride releasing material ( Fig. 5 B). This suggests that fluoride releasing materials might reduce the level of acid production by dental biofilms during the periods of carbohydrate limitation in the oral cavity because IPS are glycogen-like storage polymers and serves as a metabolic substrate for acid production of S. mutans when exogenous fermentable substrate have been depleted in the oral cavity.

It has been well reported that despite the variations in the amount of fluoride from restorative materials, the largest amount of fluoride release occurs during the first day with a decrease thereafter. On the other hand, in the present study, F2000 showed a higher fluoride releasing rate over time ( Fig. 6 ). In general, the release of fluoride from restorative materials is a very complex process, and can be affected by intrinsic variables, i.e. fillers and formulations, and experimental factors, i.e. storage media, frequency of storage medium change and, composition of pH of saliva and dental plaque. Furthermore, the relationship of fluoride ion exchange between dental biofilms, and oral fluid remains to be determined. Therefore, a more sophisticated and long-term study will be needed to identify the fluoride release pattern of the fluoride releasing materials coated with biofilms.

Although fluoride is known to inhibit the metabolism of cariogenic and other bacteria in the mouth, it is unclear if the fluoride released from restorative materials, particularly resin composites, affects the physiological ability and composition of dental biofilms. To our knowledge, this is the first report describing the fluoride released from resin composites can affects the formation of polysaccharide composition (EPS and IPS) in cariogenic biofilms. However, it is still unclear if the decreases in EPS and IPS correlate with the clinical anti-dental biofilm effects and subsequent development of secondary caries. Furthermore, the possible effect of the chemical components released from the test materials on the composition of biofilms was not examined in the present study. These components might have a significant influence on dental biofilms because various components are released from different resin composites filling after polymerization.

In conclusion, despite the similar initial rates of acid production and proton-permeability of S. mutans biofilms formed on the fluoride releasing and non fluoride releasing materials, the biomass, water-insoluble EPS and IPS of the biofilms on the fluoride releasing materials were lower than those on non fluoride releasing materials, which depends on the amount of fluoride or fluoride releasing rate in the early stages of the biofilm formation. This finding suggests that fluoride releasing resin composites may play an important role in the decrease in the cariogenic composition of dental biofilms and subsequent development of secondary caries if an appropriate amount of fluoride is released in the early stages of the biofilm formation.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ( 2009-0071090 ).

c These authors contributed equally to this study.

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Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites Santosh Pandit , Ga-Ram Kim , Min-Ho Lee and Jae-Gyu Jeon Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 780-787, Copyright © 2011 Elsevier Ltd Abstract Objectives The aim of this study was to evaluate the acid production, acid tolerance and composition of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites. Methods S. mutans biofilms were formed on saliva-coated discs prepared from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing materials (Filtek Z350, GRADIA DIRECT and hydroxyapatite). To assess the level of acid production and acid tolerance, glycolytic pH drop and proton permeability assays were performed using 94 h old S. mutans biofilms. To evaluate the biofilm composition, the biomass (total dry-weight), colony forming unit (CFU), water-insoluble extracellular polysaccharides (EPS), water-soluble EPS and intracellular iodophilic polysaccharides (IPS) of 94 h old S. mutans biofilms were analysed. The amount of fluoride of old culture medium released from the materials during the experimental period was also determined. Each assay was performed in duplicate in at least four different experiments ( n = 8). Results All biofilms showed similar initial rates of acid production (0.083–0.089 pH drop/min) and proton permeability (0.025–0.036 pH increase/min), irrespective of fluoride release from the materials. On the other hand, the amount of biomass, water-insoluble EPS and IPS of the biofilms on Unifil Flow, which releases a larger amount of fluoride in the early stages of biofilm formation, were significantly lower than those on the other materials (up to 27%, 38% and 36% reduction in biomass, water-insoluble and IPS, respectively). Conclusions Our finding suggests that fluoride releasing resin composites might contribute to the decrease in cariogenic composition of S. mutans biofilms if an appropriate amount of fluoride is released in the early stages of biofilm formation. 1 Introduction Tooth-coloured dental restorative materials, such as resin composites and compomers, are used frequently to restore decayed tooth structures owing to their low relative thermal conductivity, aesthetics and improved performance. However, many tooth-coloured restorations have been replaced because of secondary caries, which is responsible for 60% of all restoration replacements in typical dental practice. One of the important causative factors in the development of secondary caries is the accumulation of dental biofilms adjacent to the margins of restorations, which causes dissolution of the adjacent tooth surfaces and finally shortens the serving life of the restorations. If dental biofilms are allowed to remain on tooth surfaces or margins of restorations with the frequent consumption of a high carbohydrate diet, Streptococcus mutans , as a constituent of the biofilm community, will continue to synthesize extracellular polysaccharides (EPS) and metabolize the sugars to organic acids. The elevated amounts of EPS increase the bulk and structural stability of the biofilms. In addition, the ability of S. mutans to utilise some of the EPS and intracellular iodophilic polysaccharides (IPS) as short-term storage compounds offers an additional ecological benefit, which simultaneously increases the amount of acid production and the extent of acidification within the biofilms. The persistence of this aciduric environment leads to the selection and dominance of highly acid tolerant and acidogenic microorganisms, such as S. mutans . The low pH environment within the matrix results in the development of caries. Recently, several studies have examined the pattern of bacterial adherence and biofilm formation on resin composites. Some investigations demonstrated that the surface roughness and composition of resin composites influence the level of bacterial adherence in the process of biofilm formation. Furthermore, biofilms on resin composites can degrade the material and roughen their surfaces, which in turn, promote biofilm formation and more surface deterioration of the material. Nevertheless, surprisingly little information is available on the cariogenicity of biofilms formed on resin composites. To prevent secondary caries, resin composites that release fluoride ions as well as calcium and phosphate ions have been developed. Initial fluoride release into deionized water within 24 h amounted to 0.04–2.7 ppm for different composite brands (1.5 mm thick, 6 mm in diameter), but decreased to 0.02–2 ppm within 30–60 days. A variety of mechanisms are involved in the anti-cariogenic effects of fluoride, including the reduction of demineralization, the enhancement of remineralization, the interference of pellicle and biofilm formation and the inhibition of microbial growth and metabolism. Although resin composites are being used extensively, it is unclear if the cariogenicity of biofilms on the materials are affected by the amount of fluoride released from the materials. Considering the continued widespread use of resin composites for dental restorations and the occurrence of secondary caries by dental biofilms, it would be meaningful to test the hypothesis that the cariogenicity of biofilms formed on resin composite may depend on the amount of fluoride released from the materials. Therefore, the aim of this study was to evaluate the acid production, acid tolerance and composition of S. mutans biofilms formed on various types of resin composites, particularly on fluoride releasing and non fluoride releasing resin composites. 2 Materials and methods 2.1 Disc preparation for biofilm formation Table 1 lists the materials used in the present study. Three commercially available resin composites (flowable, nanohybride or microhybride) were selected. Compomer (polyacid-modified composite resins) was also included because of its chemical similarity to composite resins. The flowable resin composite (Unifil Flow) and compomer (F2000) are fluoride releasing materials. The nanohybride and microhybride resin composite (Filtek Z350 and GRADIA DIRECT, respectively) are non fluoride releasing materials. The shade of all materials was A2. Table 1 Characteristics of restorative materials used in this study. Type of material Product Fluoride containing * Composition Filler level Manufacturer Flowable resin composite UniFil Flow Yes Aluminofluorosilicate glass Urethane dimethacrylate Dimethacrylate Silica fine powder Photoinitiator 67 wt% GC Corporation, Japan Nanohybrid resin composite Filtek Z350 No Bisphenol A-glycidyl methacrylate Bisphenol A-polyethylene glycol diether dimethacrylate Urethane dimethacrylate with small amounts of triethylene glycol dimethacrylate 78.5 wt% 3M ESPE, USA Microhybrid resin composite GRADIA DIRECT Anterior No Urethane dimethacrylate Dimethacrylate Silica powder Organic filler Pigment Camphorquinone 73 wt% GC Corporation, Japan Compomer F2000 Yes Aluminofluorosilicate glass Colloidal silica Dimethacrylate functional oligomer derived from citric acid Glyceryl dimethacrylate High molecular weight hydrophilic polymer Camphorquinone/amine 84 wt% 3M ESPE, USA * Based on information provided by the manufacturers. Disc-shaped specimens (12 mm in diameter and 1.2 mm in thickness) were prepared using a polytetrafluoroethylene (Teflon) moulds with glass microscope slides to cover each face. The specimens were light-cured for 20 s with a light curing unit (Optilux, Kerr, Danbury, USA). After curing, all specimens were polished sequentially from # 600 to # 2000 sand paper and buffered clothes with 0.3 μm alumina powder. The specimens were sonicated for 5 min to remove the remaining powder and then stayed in a desiccator at room temperature. Hydroxyapatite discs (12 mm in diameter and 1.2 mm in thickness; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) were also included as a control. 2.2 Biofilm formation and experimental scheme The microorganism used in the present study was S. mutans UA159. Fig. 1 shows S. mutans biofilm preparation and experimental scheme for the present study. S. mutans UA159 biofilms were formed on saliva-coated resin composites, compomer or hydroxyapatite discs placed in a vertical position in 24-well plates, as detailed elsewhere. Briefly, the saliva-coated discs were generated by incubation with filter-sterilized (0.22 μm low protein binding filter) and clarified human whole saliva for 1 h at 37 °C. For biofilm formation, the saliva-coated discs were transferred to a 24 well plate containing 1% sucrose (v/v) ultrafiltered (10 kDa molecular-weight cut-off) tryptone yeast-extract (UTE) broth with S. mutans UA159 (2–5 × 10 6 CFU/ml). The biofilms were grown undisturbed for 22 h to allow initial biofilm growth. From this point (22 h old), the culture medium was changed twice daily (9 AM and 6 PM) until it was 94 h old. The culture medium was changed a total of 6 times. Fig. 1 Biofilm preparation and experimental scheme. This study examined the acid production, acid tolerance and composition of 94 h old S. mutans biofilms formed on fluoride releasing or non fluoride releasing resin composites. To evaluate the acid production and acid tolerance of S. mutans biofilms formed on the test materials, glycolytic pH drop and proton permeability assays were performed using the 94 h old biofilms. To evaluate the biofilm composition, the 94 h old biofilms were analysed using microbiological and biochemical methods. The amount of fluoride in the old culture medium was also determined during the experimental period. Each assay was performed in duplicate in at least four different experiments ( n = 8). 2.3 Evaluation of acid production and acid tolerance of biofilms 2.3.1 Glycolytic pH drop assay The glycolytic pH drop of S. mutans biofilms on the test materials was measured, as described elsewhere. Briefly, the 94 h old biofilms, which had been incubated in 20 mM potassium phosphate buffer (pH 7.2) for 1 h to deplete the endogenous catabolites, were transferred to a salt solution (50 mM KCl plus 1 mM MgCl 2 , pH 7.0). The pH was adjusted to 7.0 with a 0.01 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 a period of 120 min (Futura Micro Combination pH electrode, 5 mm diameter; Beckman Coulter Inc., CA, USA). The initial rate of acid production was calculated using the pH values in the linear portion (0–30 min incubation period) because the rate of the decrease in pH reflects the acid production capacity of the biofilms. 2.3.2 Proton permeability assay The proton permeability of S. mutans biofilms on the test materials was assessed using the procedures described previously. Basically, the 94 h old biofilms, which had been incubated in 20 mM potassium phosphate buffer (pH 7.2) for 1 h to deplete the endogenous catabolites, were incubated initially at a constant pH of approximately 4.6. HCl was then added to decrease the pH values in approximately 0.4 units. The subsequent increase in pH associated with proton movements across the cell membrane into the cytoplasm was monitored using a glass electrode. Butanol (10%, v/v) was added to the suspensions at 80 min to disrupt the cell membrane. The initial rate of proton entry was estimated because changes in initial rates of proton uptake give the best measure of disruptive effects. The initial rate of proton entry was calculated using the pH changes of 0–10 min incubation period. 2.4 Evaluation of biofilm composition For microbiological and biochemical composition analyses, the 94 h old biofilms on the test materials were removed and sonicated at 7 W for 3 periods of 30 s each (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA). The colony forming unit (CFU), biomass (dry-weight), and polysaccharide composition in the homogenized suspension was examined. The water-soluble EPS, water-insoluble EPS and IPS were extracted and quantified by colorimetric assays, as detailed elsewhere. The decrease in biomass, water-insoluble EPS or IPS of the biofilms on a material ( A ) was calculated using the following formula: reduction (%) = (the amount of the biomass, water-insoluble EPS or IPS of biofilms on A /the amount of the biomass, water-insoluble EPS or IPS of biofilms on the other materials) × 100. 2.5 Determination of fluoride amount of old culture medium During biofilm development, as soon as the biofilms on the test materials were transferred to the new culture medium twice daily (9 AM and 6 PM), the amount of fluoride released from the materials into the old medium was measured using a fluoride ion selective electrode (Thermo Electron Corporation, OH, USA). A total of 2.8 ml of each medium was transferred into a 15 ml tube and the concentration of fluoride was measured after vortex. The fluorometer was calibrated using three standard solutions (0.1, 1 and 10 ppm). To calculate the fluoride releasing rate, the concentration of fluoride from the test materials was divided by the incubation time. 2.6 Statistical analysis The data are presented as mean ± standard deviation. The intergroup differences were estimated by one-way analysis of variance (ANOVA), followed by a post hoc multiple comparison (Tukey test) to compare the multiple means. Values were considered statistically significant when P value was <0.05. The statistical analyses were performed using SPSS 12 software (SPSS Inc., Chicago, IL, USA). 3 Results 3.1 Initial rates of acid production and proton permeability of biofilms Fig. 2 shows the glycolytic pH drop of S. mutans biofilms on the test materials. There was no significant difference in the initial rates of acid production (amount of pH drop/min) between the biofilms on fluoride releasing and non fluoride releasing materials ( p > 0.05). The initial rates of acid production ranged from 0.083 to 0.089 pH drop/min. Fig. 3 shows the result of the proton permeability assay. In general, the initial rates of proton entry (amount of pH increase/min) of the biofilms on fluoride releasing and non fluoride releasing materials were also similar ( p > 0.05), ranging from 0.025 to 0.036 pH increase/min. Fig. 2 Pattern of glycolytic pH drop and initial rate of pH drop of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. N/S: non-significant difference between the test materials at 30 min. Fig. 3 Pattern of proton permeability and initial rate of proton entry of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. N/S: non-significant difference between the test materials at 10 min. 3.2 Biofilm composition Fig. 4 A shows the result of the biomass evaluation of S. mutans biofilms on the test materials. Of the materials tested, the lowest amount of biomass was recovered from the biofilms on UniFil Flow. The biomass on UniFil Flow was 6.19 mg/disc, which was 78% of Filtek Z350, 77% of GRADIA DIRECT and 73% of hydroxyapatite ( p < 0.05). Interestingly, although F2000 is also one of fluoride releasing materials, the amount of biomass on the material was not significantly different from those on non fluoride releasing materials ( p > 0.05). Fig. 4 Biomass (A) and CFU (B) of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represent mean ± standard deviation. * P < 0.05: significantly different from each other. N/S: non-significant difference between the test materials. Fig. 4 B presents the number of S. mutans CFU in the biofilms on the test materials. Although the amount of biomass on UniFil Flow was lower than those of non fluoride releasing materials, the number of S. mutans CFU on UniFil Flow was not significantly different from those on non fluoride releasing materials ( p > 0.05). In general, the number of S. mutans CFU on fluoride releasing and non fluoride releasing materials was not significantly different ( p > 0.05). Fig. 5 shows the amount of polysaccharide (water-insoluble EPS, water soluble EPS and IPS) of S. mutans biofilms on the test material. As shown in Fig. 5 A, the amount of water-insoluble EPS on UniFil Flow was significantly lower than those on non fluoride releasing materials ( p > 0.05). The water-insoluble EPS of UniFil Flow was 1.09 mg/disc, which was 69% of Filtek Z350, 70% of GRADIA DIRECT and 62% of hydroxyapatite ( p < 0.05). However, the amount of water-insoluble EPS on F2000 was not significantly different from those on non fluoride releasing resin composites ( p > 0.05). The amount of IPS on UniFil Flow (0.31 mg/disc) was significantly lower than those of Filtek Z350 and hydroxyapatite ( Fig. 5 B, p < 0.05). However, there was no significant difference in the amount of water-soluble EPS between fluoride releasing and non fluoride releasing materials ( Fig. 5 C, p > 0.05). Fig. 5 Water-insoluble extracellular polysaccharides (EPS, A), intracellular iodophilic polysaccharides (IPS, B) and water-soluble EPS (C) of 94 h old S. mutans biofilms formed on fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite). The data represents the mean ± standard deviation. * P < 0.05: significantly different from each other. N/S: non-significant difference between the test materials. 3.3 Fluoride release from the materials Fig. 6 shows the amount of fluoride and fluoride releasing rates from the test materials during the incubation period. In general, the amount of fluoride from UniFil Flow and F2000 in old culture medium was higher than those from non fluoride releasing materials ( Fig. 6 , p < 0.05) but these materials showed a different pattern of fluoride release. UniFil Flow released a higher amount of fluoride than F2000 during the first experimental day (22 and 31 h) but F2000 released a higher amount of fluoride than UniFil Flow after late second day to the end of the experiment (55–94 h). In general, UniFil Flow showed the highest rate of fluoride release in the early stages of biofilm formation. On the other hand, F2000 showed the highest rate of fluoride release in the late stages of biofilm formation. Fig. 6 Amount of fluoride and fluoride releasing rate from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing discs (GRADIA DIRECT, Filtek Z350 and hydroxyapatite) during S. mutans biofilms formation. The data represent mean ± standard deviation. * (S) P < 0.05: significant difference between Unifil Flow and F2000. In the table of the fluoride releasing rate, the values in the same row followed by the same letters are not significantly different from each other. 4 Discussion Caries is a biofilm-related dental disease associated with the increased consumption of dietary sugar. The bacterial composition of dental biofilms remains relatively stable when exposed to minor environmental changes. On the other hand, if environmental perturbations occur, such as an increase in sucrose availability, the development of cariogenic biofilms would be induced by triggering EPS matrix formation, and the growth and dominance of cariogenic streptococci. Although additional acidogenic and aciduric bacteria can be involved, S . mutans is one bacterial species that has been implicated most frequently in the establishment of cariogenic biofilms and the initiation of caries because this bacterium is highly acidogenic and acid tolerant, and utilizes dietary sucrose to synthesize large amounts of EPS. In this context, S . mutans biofilm grown under sucrose-rich conditions (1%) was chosen to examine the virulence and composition of cariogenic biofilms on resin composites. Although the S. mutans biofilm model does not mimic the complex microbial community found in dental biofilms precisely, the biofilm is advantageous for examining the cariogenic process of biofilms. The acid production and acid tolerance of S. mutans biofilms were examined using a well-established glycolytic pH-drop and proton permeability assay. As shown in Figs. 2 and 3 , the initial rates of acid production and proton permeability of S. mutans biofilms were not affected by the support materials. Interestingly, the initial rates of the biofilms on the fluoride releasing materials (UniFil Flow and F2000) were similar to those on the non fluoride releasing materials. It is well known that fluoride has biological effects on the critical virulence of cariogenic streptococci, particularly on S. mutans . Furthermore, the physiological ability of S. mutans biofilms was reported to be affected by fluoride. On the other hand, in the present study, although UniFil Flow and F2000 released significantly higher amounts of fluoride than non fluoride releasing materials ( Fig. 6 ), these materials did not inhibit the initial rates of acid production and proton permeability of S. mutans biofilms ( Figs. 2 and 3 ), suggesting that the amount of fluoride released by the materials is too low to inhibit the physiological ability of S. mutans biofilms. These finding are supported by a previous study showing that fluoride concentrations ≤10 ppm cannot reduce the initial rates of acid production and proton permeability of S. mutans biofilms. However, our result indicated that the biomass of S. mutans biofilms can be affected by the amount of fluoride released from the test materials, particularly in the early stages of biofilm formation. As shown in Fig. 4 A, the biomass on UniFil Flow, which released the highest amount of fluoride in the early stages of biofilm formation ( Fig. 6 ), was significantly lower than that on non fluoride releasing materials. On the other hand, the biomass on F2000, which released the highest amount of fluoride in the late stages of biofilm formation ( Fig. 6 ), was not significantly different from those on the non fluoride releasing materials. These finding suggest that the biomass of dental biofilms on dental restorative materials can be affected if the restorative materials release an appropriate amount of fluoride in the early stages of biofilm formation. In the present study, although the amount of biomass of S. mutans biofilm was affected by UniFil Flow, there was no significant difference in the CFU numbers of S. mutans in the biofilms between the fluoride releasing and non fluoride releasing materials ( Fig. 4 ), which suggests that the amount of fluoride from the test materials did not affect the growth and viability of S. mutans in the biofilms. Furthermore, this finding indicates that the biomass reduction of the biofilms on UniFil Flow is related to the decrease in the other main constituents (e.g. EPS) of the biofilms, not a decrease in the number of bacterial cells. It is well known that EPS, synthesized by microbial glucosyltransferases (GTFs), contribute to the formation and structural integrity of dental biofilm matrix , and the concentration of EPS in dental biofilms is associated with dental caries. Therefore, the decrease in EPS can affect cariogenic biofilm formation and accumulation, and the subsequent initiation of dental caries. In the present study, the amount of water-insoluble EPS of S. mutans biofilms on UniFil Flow were reduced by up to 27%, compared to the non fluoride-releasing materials ( Fig. 5 A). On the other hand, the amount of water-soluble EPS of S. mutans biofilms on UniFil was not affected ( Fig. 5 C). Although the precise mechanisms by which the water-insoluble EPS were reduced and water-soluble EPS were not affected were not determined in this study, a recent study reported that GTF for the synthesis of water-insoluble EPS is more sensitive to fluoride than that for water-soluble EPS. The study also showed that fluoride at low concentrations (3.8 ppm) partially inhibits the secretion of GTFs by S. mutans , which support the present result that there is less EPS formation on UniFil Flow than that on the non fluoride releasing materials. In addition, the amount of IPS on the fluoride releasing materials used in this study (UniFil Flow and F2000) was lower than that of the non fluoride releasing material ( Fig. 5 B). This suggests that fluoride releasing materials might reduce the level of acid production by dental biofilms during the periods of carbohydrate limitation in the oral cavity because IPS are glycogen-like storage polymers and serves as a metabolic substrate for acid production of S. mutans when exogenous fermentable substrate have been depleted in the oral cavity. It has been well reported that despite the variations in the amount of fluoride from restorative materials, the largest amount of fluoride release occurs during the first day with a decrease thereafter. On the other hand, in the present study, F2000 showed a higher fluoride releasing rate over time ( Fig. 6 ). In general, the release of fluoride from restorative materials is a very complex process, and can be affected by intrinsic variables, i.e. fillers and formulations, and experimental factors, i.e. storage media, frequency of storage medium change and, composition of pH of saliva and dental plaque. Furthermore, the relationship of fluoride ion exchange between dental biofilms, and oral fluid remains to be determined. Therefore, a more sophisticated and long-term study will be needed to identify the fluoride release pattern of the fluoride releasing materials coated with biofilms. Although fluoride is known to inhibit the metabolism of cariogenic and other bacteria in the mouth, it is unclear if the fluoride released from restorative materials, particularly resin composites, affects the physiological ability and composition of dental biofilms. To our knowledge, this is the first report describing the fluoride released from resin composites can affects the formation of polysaccharide composition (EPS and IPS) in cariogenic biofilms. However, it is still unclear if the decreases in EPS and IPS correlate with the clinical anti-dental biofilm effects and subsequent development of secondary caries. Furthermore, the possible effect of the chemical components released from the test materials on the composition of biofilms was not examined in the present study. These components might have a significant influence on dental biofilms because various components are released from different resin composites filling after polymerization. In conclusion, despite the similar initial rates of acid production and proton-permeability of S. mutans biofilms formed on the fluoride releasing and non fluoride releasing materials, the biomass, water-insoluble EPS and IPS of the biofilms on the fluoride releasing materials were lower than those on non fluoride releasing materials, which depends on the amount of fluoride or fluoride releasing rate in the early stages of the biofilm formation. This finding suggests that fluoride releasing resin composites may play an important role in the decrease in the cariogenic composition of dental biofilms and subsequent development of secondary caries if an appropriate amount of fluoride is released in the early stages of the biofilm formation. 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