Abstract
Objectives
The aim of this study was to evaluate the difference between anti-cariogenic biofilm activities of glass ionomers (G-Is) during the initial and second fluoride release phases and to define relationships between the anti-biofilm activities and fluoride release.
Methods
Fluoride release of three commercially available G-Is in a buffer was evaluated for 770 h, and then 70-h-old Streptococcus mutans UA159 biofilms were formed on the G-Is that had been immersed in the buffer for 0, 100, 200, or 700 h. The dry weight, bacterial cell number, water-insoluble extracellular polysaccharides (EPSs), and accumulated fluoride concentration of the 70-h-old biofilms and fluoride release and acid production rates during biofilm formation were determined. Relationships between the experimental variables and fluoride release rate were also evaluated using linear regression analysis.
Results
In this study, fluoride release of the tested G-Is did not exhibit a biphasic pattern during biofilm formation. The release was sustained or did not rapidly decrease even over long immersion periods and was strongly correlated with an increase in accumulated fluoride concentration of the biofilms ( R = 0.99, R 2 = 0.98) and reductions in dry weight, water-insoluble EPSs, and acid production rate of the biofilms ( R = −0.99 to −0.96, R 2 = 0.92–0.98).
Conclusions
This study suggests that G-Is can effectively affect acid production, EPS formation, and accumulation of cariogenic biofilms even during the second fluoride release phase, and that the anti-cariogenic biofilm activity is strongly correlated with fluoride release, which may be enhanced by acid production of cariogenic biofilms.
Clinical significance
G-Is can affect cariogenic biofilm formation even during the second fluoride release phase.
1
Introduction
Similar to dental caries, secondary caries results from the interaction of specific bacteria with constituents of the diet within cariogenic biofilms, which develop from environmental perturbations such as pH decrease and increase in sucrose availability in dental biofilms . Streptococcus mutans , a caries-related bacterium, has been regarded as a primary etiologic agent of secondary caries and dental caries . This bacterium is highly acidogenic and aciduric and efficiently utilizes sucrose to synthesize large amounts of extracellular polysaccharides (EPSs) using glucosyltransferases (GTFs) . Combining these virulence properties, S. mutans becomes a prominent member of cariogenic biofilms and subsequently causes development of secondary caries.
Fluoride-releasing materials have been developed to prevent secondary caries. These materials include glass ionomers (G-Is), which have been widely used in dentistry because of their biocompatibility and cariostatic properties . Many previous studies have demonstrated that G-Is can reduce demineralization of adjacent hard tooth tissues via fluoride release . However, although secondary caries results from cariogenic biofilms, little research has been conducted on the anti-cariogenic biofilm activity of G-Is, especially as it relates to fluoride release.
It has been well reported that the fluoride release from fluoride-releasing materials occurs in a biphasic pattern, with an initial rapid and a second slow release phase . G-Is release the largest amount of fluoride during the first day and demonstrate a continuous decrease thereafter . This suggests that, if anti-cariogenic biofilm activity of G-Is is determined by fluoride release, G-Is may not sustain their activity over time. We recently reported that G-Is effectively reduce acidogenicity, bacterial bio-volume, and EPS formation of S. mutans biofilms during the initial rapid release phase, and the fluoride release rate is closely correlated with the anti-cariogenic biofilm activity of G-Is . However, it has been unclear whether i) G-Is can maintain anti-cariogenic biofilm activity during the second slow release phase and ii) if the anti-cariogenic biofilm activity is related to the fluoride release rate of the materials during the second slow release phase.
In this study, we evaluated the difference between anti-cariogenic biofilm activities of G-Is, especially with regard to composition and acid production, during initial and second release phases and defined the relationships between the anti-cariogenic biofilm activity and the fluoride release rate of the materials using an S. mutans biofilm model.
2
Materials and methods
2.1
G-I disc preparation
Three commercially available G-Is were selected for this study: Ketac Fil Plus Aplicap (Keta), Riva self-cure HV (Riva) and GC Fuji Filling LC (GC). The characteristics of the G-Is and disc-shaped preparation (12 mm in diameter and 1.2 mm in thickness) were previously described . Briefly, each G-I was placed in a polytetrafluoroethylene (Teflon) mold with a metal holder and glass slides to cover each face. After curing, all specimens were polished sequentially with #800–#1200 sand papers. Hydroxyapatite discs (HA 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
Experimental design and fluoride release rate in the absence of biofilm formation
Fig. 1 shows the experimental design for this study and the fluoride release rate of G-I discs without biofilms. To determine the fluoride release rate from G-Is without biofilms, HA or G-I discs were immersed in individual 24-well plates that contained 50 mM potassium phosphate (PP) buffer at pH 7.0 (2.8 ml/well). After 1 h incubation at 37 °C, each disc was removed from the buffer and placed in new PP buffer. This was repeated every hour for 12 h. From 12 h to 240 h, the buffer change was repeated every 12 h. From 432 h to 768 h, the buffer change was repeated every 24 h. The fluoride concentration was measured after adding 280 μl of total ionic strength adjustment buffer (TISAB III) to 2.8 ml of old buffer. To calculate the fluoride release rate, the fluoride concentration in the old buffer was divided by the incubation time. Based on the fluoride release rate, G-I discs immersed in PP buffer for 0, 100, 200, or 700 h were selected for 70-h-old S. mutans biofilm formation. In addition, the fluoride release rate in the buffer of biofilm formation periods (0–70, 100–170, 200–270, and 700–770 h) was also calculated.
2.3
S. mutans biofilm formation
S. mutans UA159 (serotype c) biofilms were formed on sterilized HA or G-I discs that had been immersed in PP buffer for 0, 100, 200, or 700 h and placed in a vertical position in 24-well plates, as detailed elsewhere . Briefly, the test discs were transferred to a 24-well plate containing 1% sucrose (v/v) ultrafiltered (10 kDa molecular-weight cut-off) tryptone-yeast extract broth with S. mutans UA159 (2–5 × 10 6 colony forming units (CFUs)/ml). The biofilms were grown undisturbed for 22 h. From this time point (22 h), the culture medium was changed twice daily (9 AM, 6 PM) until it was 70 h old. The culture medium was changed a total of four times (22, 31, 46, and 55 h).
2.4
Acid production and fluoride release rates during biofilm formation
To determine the acid production and fluoride release rates during S. mutans biofilm formation on HA or G-I discs, the pH value and fluoride concentration of old culture medium were measured during biofilm formation (22, 31, 46, 55, and 70 h) using a pH electrode and the method described above, respectively. To calculate the acid production and fluoride release rates, the change in concentration of H + or fluoride at 0–22, 22–31, 31–46, 46–55, and 55–70 h was divided by the respective incubation time, and then the mean was calculated.
2.5
Composition and accumulated fluoride concentration of 70-h-old biofilms
For biofilm composition analysis, the 70-h-old biofilms on HA or G-I discs were removed and homogenized by sonication at 7 W for 30 s (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA). The homogenized suspension was analyzed for number of colony forming units (CFUs), dry weight, amount of water-insoluble EPSs, and accumulated fluoride concentration, as described in Jeon et al. . Briefly, an aliquot (0.1 ml) of the dispersed solution (5 ml) was serially diluted and plated onto Brain Heart Infusion (BHI) agar plates to determine CFU count. The remaining dispersed solution (4.9 ml) was centrifuged, and the supernatant was collected. The centrifuged biofilm pellet was used to determine dry weight and amount of water-insoluble EPSs . The fluoride concentration of the supernatant was measured by adding 490 μl of TISAB III to 4.9 ml of the supernatant. To calculate accumulated fluoride concentration of the biofilms (ppm F − /mg), the fluoride concentration of the supernatant (4.9 ml) was changed to the concentration in 2.8 ml and then divided by the respective dry weight.
2.6
Statistical analysis
To determine the relationships between the experimental variables during the initial rapid and second slow release phases (fluoride release rates in buffer and culture medium, acid production rate during biofilm formation, and composition and accumulated fluoride concentration of the 70-h-old biofilms), a linear regression analysis was performed using the data of 0–70 h (for initial rapid release phase) and 700–770 h biofilm formation periods (for second slow release phase). The correlation coefficient ( R ), which explains the strength of the relationship between two variables, and determination coefficient ( R 2 ), which describes the percentage of the variation in one variable that can be explained by the linear relationship with another variable, of each fit line were calculated .
All experiments were performed in duplicate, and at least five different experiments were conducted. The data are presented as mean ± standard deviation. Intergroup differences were estimated using one-way analysis of variance. Values were considered statistically significant when the p value was <0.05.
2
Materials and methods
2.1
G-I disc preparation
Three commercially available G-Is were selected for this study: Ketac Fil Plus Aplicap (Keta), Riva self-cure HV (Riva) and GC Fuji Filling LC (GC). The characteristics of the G-Is and disc-shaped preparation (12 mm in diameter and 1.2 mm in thickness) were previously described . Briefly, each G-I was placed in a polytetrafluoroethylene (Teflon) mold with a metal holder and glass slides to cover each face. After curing, all specimens were polished sequentially with #800–#1200 sand papers. Hydroxyapatite discs (HA 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
Experimental design and fluoride release rate in the absence of biofilm formation
Fig. 1 shows the experimental design for this study and the fluoride release rate of G-I discs without biofilms. To determine the fluoride release rate from G-Is without biofilms, HA or G-I discs were immersed in individual 24-well plates that contained 50 mM potassium phosphate (PP) buffer at pH 7.0 (2.8 ml/well). After 1 h incubation at 37 °C, each disc was removed from the buffer and placed in new PP buffer. This was repeated every hour for 12 h. From 12 h to 240 h, the buffer change was repeated every 12 h. From 432 h to 768 h, the buffer change was repeated every 24 h. The fluoride concentration was measured after adding 280 μl of total ionic strength adjustment buffer (TISAB III) to 2.8 ml of old buffer. To calculate the fluoride release rate, the fluoride concentration in the old buffer was divided by the incubation time. Based on the fluoride release rate, G-I discs immersed in PP buffer for 0, 100, 200, or 700 h were selected for 70-h-old S. mutans biofilm formation. In addition, the fluoride release rate in the buffer of biofilm formation periods (0–70, 100–170, 200–270, and 700–770 h) was also calculated.
2.3
S. mutans biofilm formation
S. mutans UA159 (serotype c) biofilms were formed on sterilized HA or G-I discs that had been immersed in PP buffer for 0, 100, 200, or 700 h and placed in a vertical position in 24-well plates, as detailed elsewhere . Briefly, the test discs were transferred to a 24-well plate containing 1% sucrose (v/v) ultrafiltered (10 kDa molecular-weight cut-off) tryptone-yeast extract broth with S. mutans UA159 (2–5 × 10 6 colony forming units (CFUs)/ml). The biofilms were grown undisturbed for 22 h. From this time point (22 h), the culture medium was changed twice daily (9 AM, 6 PM) until it was 70 h old. The culture medium was changed a total of four times (22, 31, 46, and 55 h).
2.4
Acid production and fluoride release rates during biofilm formation
To determine the acid production and fluoride release rates during S. mutans biofilm formation on HA or G-I discs, the pH value and fluoride concentration of old culture medium were measured during biofilm formation (22, 31, 46, 55, and 70 h) using a pH electrode and the method described above, respectively. To calculate the acid production and fluoride release rates, the change in concentration of H + or fluoride at 0–22, 22–31, 31–46, 46–55, and 55–70 h was divided by the respective incubation time, and then the mean was calculated.
2.5
Composition and accumulated fluoride concentration of 70-h-old biofilms
For biofilm composition analysis, the 70-h-old biofilms on HA or G-I discs were removed and homogenized by sonication at 7 W for 30 s (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA). The homogenized suspension was analyzed for number of colony forming units (CFUs), dry weight, amount of water-insoluble EPSs, and accumulated fluoride concentration, as described in Jeon et al. . Briefly, an aliquot (0.1 ml) of the dispersed solution (5 ml) was serially diluted and plated onto Brain Heart Infusion (BHI) agar plates to determine CFU count. The remaining dispersed solution (4.9 ml) was centrifuged, and the supernatant was collected. The centrifuged biofilm pellet was used to determine dry weight and amount of water-insoluble EPSs . The fluoride concentration of the supernatant was measured by adding 490 μl of TISAB III to 4.9 ml of the supernatant. To calculate accumulated fluoride concentration of the biofilms (ppm F − /mg), the fluoride concentration of the supernatant (4.9 ml) was changed to the concentration in 2.8 ml and then divided by the respective dry weight.
2.6
Statistical analysis
To determine the relationships between the experimental variables during the initial rapid and second slow release phases (fluoride release rates in buffer and culture medium, acid production rate during biofilm formation, and composition and accumulated fluoride concentration of the 70-h-old biofilms), a linear regression analysis was performed using the data of 0–70 h (for initial rapid release phase) and 700–770 h biofilm formation periods (for second slow release phase). The correlation coefficient ( R ), which explains the strength of the relationship between two variables, and determination coefficient ( R 2 ), which describes the percentage of the variation in one variable that can be explained by the linear relationship with another variable, of each fit line were calculated .
All experiments were performed in duplicate, and at least five different experiments were conducted. The data are presented as mean ± standard deviation. Intergroup differences were estimated using one-way analysis of variance. Values were considered statistically significant when the p value was <0.05.
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