Abstract
Objective
A surface pre-reacted glass-ionomer (S-PRG) filler is a technology of interest for providing bio-functions to restorative materials. Resin composites containing S-PRG filler have been reported to show less plaque accumulation and reduced bacterial attachment. In this study, experimental resin composites containing S-PRG filler at various concentrations were fabricated, and the inhibitory effects on bacterial growth on their surface and the association of ions released from S-PRG filler with antibacterial activity were evaluated.
Methods
Five kinds of experimental resin composites containing S-PRG filler at 0, 13.9, 27.3, 41.8, or 55.9 (vol.%) were fabricated. Streptococcus mutans was cultured on the cured discs for 18 h to examine the growth of bacteria in contact with the surface of the experimental resins. The concentrations of Al 3+ , BO 3 3− , F − , Na + , SiO 3 2− , or Sr 2+ released from each experimental resin into water were measured. The standardized solutions of each ion were prepared at the concentrations determined to be released from the experimental resin, and their inhibitory effects of single ion species on S. mutans growth were evaluated by using each standardized solution.
Results
Resin composites containing S-PRG filler at 13.9 (vol.%) or greater inhibited S. mutans growth on their surface. When S. mutans was incubated in the presence of six kinds of ions at the concentrations released from the resin composite containing S-PRG filler at 55.9 (vol.%), a significant reduction in bacterial number was observed for BO 3 3− , F − , Al 3+ , and SiO 3 2− . Among these four ions, BO 3 3− and F − demonstrated the strongest inhibitory effect on S. mutans growth.
Significance
Our findings suggest that resin composites containing S-PRG filler inhibit the growth of S. mutans on their surface. BO 3 3− , F − , Al 3+ and SiO 3 2− released from S-PRG filler have the ability to inhibit S. mutans growth, and the inhibitory effects are mainly attributed to release of BO 3 3− and F − .
1
Introduction
As the concept of minimal intervention has become popular, applications of resin composites have been widely expanded. However, resin composites do not show any biologically beneficial effects. It has been reported that increased plaque accumulation occurs on resin composites compared with other restorative materials and tooth surfaces , and much attention has been focused on imparting the ability to inhibit bacteria to resin composites.
A surface pre-reacted glass-ionomer (S-PRG) filler, prepared via an acid–base reaction between fluoroboroaluminosilicate glass and a polyacrylic acid, is a technology of interest for providing bio-functions to restorative materials. The pre-reacted glass-ionomer phase on the surface of the glass core allows S-PRG filler to release and recharge fluoride ions . Moreover, S-PRG filler has a unique characteristic of releasing other ions such as aluminum (Al 3+ ), borate (BO 3 3− ), sodium (Na + ), silicate (SiO 3 2− ), and strontium (Sr 2+ ) ions since fluoroboroaluminosilicate glass is used as the glass core. Incorporation of S-PRG filler into dental materials has been reported to effectively prevent demineralization of dentin , and to impart acid resistance to enamel . Release of multiple ions from S-PRG filler also promotes mineralization to induce apatite formation .
Among several ions released from S-PRG filler, F − is known to show some antibacterial activity against Streptococcus mutans . BO 3 3− , one of the ionization states of boric acid, is used as a preservative, and inhibits growth of Staphylococcus aureus and Escherichia coli at high concentrations . A few studies reported less plaque accumulation and reduced bacterial attachment on resin composites containing S-PRG filler . Hahnel et al. examined the release of fluoride ion and the biofilm formation on experimental resin composites containing different fractions of S-PRG fillers at 0%, 10%, 30%, 50%, 70% (w/v). This study suggested that the fluoride ion released from S-PRG filler was influenced on the early phases of biofilm formation on the resin composites containing S-PRG filler. Yoneda et al. demonstrated that eluate from S-PRG filler suppressed adherence of S. mutans to polystyrene . However, it has not yet been clarified if resin composites containing S-PRG filler inhibit bacterial growth. Moreover, the relationship between ions released from S-PRG filler and the antibacterial activity of S-PRG filler-containing resin composites also remains unclear.
In this study, experimental resin composites containing S-PRG filler at various concentrations were fabricated, and the hypothesis that the experimental composites inhibit the growth of S. mutans was examined. We also determined the relevance of six ions released from S-PRG filler to inhibitory effects against bacteria by using standardized solutions of each ion.
2
Materials and methods
2.1
Antibacterial activity of eluates from S-PRG filler
S-PRG fillers with average diameter of 3 μm were added to distilled water at 1:1 ratio by weight (1000 g filler in 1000 mL) and stirred at 23 °C for 24 h. The mixing solution was centrifuged to precipitate the S-PRG fillers, and the supernatant was filtered. The eluates from S-PRG filler were diluted with brain–heart infusion (BHI) broth (Becton Dickinson, Sparks, MD, USA) supplemented with 0.5% yeast extract (Becton Dickinson), and solutions containing the eluates from S-PRG filler at 50, 25, and 12.5 (vol.%) were prepared.
S. mutans NCTC10449 was cultured in BHI broth and adjusted to 2 × 10 5 , 2 × 10 4 , or 2 × 10 3 colony-forming units (CFU)/mL. Aliquots of 50 μL of the diluted eluates from S-PRG filler were added to 50 μL of bacterial suspension and incubated anaerobically at 37 °C for 24 h. Bacterial growth was monitored by measuring the absorbance of the aliquot at OD 550 using a spectrophotometer (Model 680 Microplate Reader, BioRad Laboratories, Hercules, CA, USA). Controls consisted of the diluted eluates from S-PRG filler without any inoculum of bacteria. Cultures with absorbance within ±0.010 of the range for the control were determined to have no bacterial growth. The experiments were repeated five times.
2.2
Inhibitory effects of resin composites containing S-PRG filler on S. mutans growth
2.2.1
Sample preparation
Five experimental resin composites were prepared. To the monomer compositions of 70 (wt%) 2,2-bis [4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl]propane (Bis-GMA) and 30 (wt%) triethyleneglycol dimethacrylate (TEGDMA), S-PRG fillers and silica filler (average diameter: 1 μm) were loaded at different ratios. The final contents of S-PRG filler in the resin composites ranged from 13.9% to 55.9% in volume ( Table 1 ). Based on the maximum amount of filler loaded for P4, the total filler contents for all experimental resin composites were unified as 55.9 (vol.%). For photo-initiator system, 0.3 (wt%) camphorquinone and 0.3 (wt%) ethyl 4-dimethylaminobenzoate were used.
| Code | Ratio of S-PRG filler/silica filler (vol.%) | Total filler content (vol.%) | S-PRG filler content | |
|---|---|---|---|---|
| (vol.%) | (wt%) | |||
| P0 | 0/100 | 55.9 | 0 | 0 |
| P1 | 25/75 | 55.9 | 13.9 | 22.3 |
| P2 | 50/50 | 55.9 | 27.9 | 42.7 |
| P3 | 75/25 | 55.9 | 41.8 | 61.4 |
| P4 | 100/0 | 55.9 | 55.9 | 78.7 |
Each resin composite paste was placed in a mold (9 mm diameter, 2 mm thickness), pressed with a celluloid strip and a glass slide, and cured by irradiation with a light activation unit (Optilux 501, Kerr Corporation, Orange, CA, USA) at an intensity of 500 mW/cm 2 for 40 s. The cured discs were sterilized with ethylene oxide gas.
2.2.2
On-disc culture assay
To analyze growth of bacteria in contact with the surface of the experimental resin composites, S. mutans was cultured on the cured disc as previously reported . A suspension of 50 μL of S. mutans , cultured in BHI broth and adjusted to 3 × 10 3 CFU/mL, was placed on the cured disc. After anaerobically incubation at 37 °C for 18 h under the static condition, each disc with S. mutans suspension was immersed in 9.95 mL of 0.01 M phosphate-buffered saline (PBS; pH 7.4). After vigorous shaking, the suspension was serially diluted with PBS and inoculated on BHI agar (Becton Dickinson) plates. The plates were incubated anaerobically at 37 °C for 48 h, and colonies formed were counted. The experiments were repeated three times.
2.3
Release of ions from resin composites containing S-PRG filler
Distilled water (50 μL) was placed on the cured resin composite discs which were incubated under anaerobic conditions at 37 °C. After 18 h, samples containing eluted ions were collected, and the concentration of each ion was determined. For BO 3 3− , Na + , Al 3+ , SiO 3 2− , and Sr 2+ , concentrations of B, Na, Al, Si, and Sr in the eluates were measured using inductively coupled plasma atomic emission spectroscopy (ICPS-8000, Shimadzu Co., Kyoto, Japan). The concentration of F − was determined using a fluoride ion electrode (Model 9609BN, Orion Research Inc., MA, USA). The experiments were repeated four times.
2.4
Inhibitory effects of single ion species on S. mutans growth
Standard solutions containing each of the six ions determined to be released from the experimental resin composites were prepared. For BO 3 3− , Na + , Al 3+ , SiO 3 2− , and Sr 2+ , the solution was prepared by dissolving H 3 BO 3 , NaNO 3 , Al(NO 3 ) 3 ·9H 2 O, Na 2 SiO 3 , Sr(NO 3 ) 2 (Wako, Osaka, Japan) in distilled water. NaF (Sigma–Aldrich, Tokyo, Japan) was dissolved in distilled water to obtain the standard solution of F − ( Table 2 ). To adjust the pH value of Al 3+ and SiO 3 2− solutions beyond 5.0, sodium hydroxide (Nacalai Tesque, Kyoto, Japan) or nitric acid (Wako) were added, respectively.
| BO 3 3− | F − | Na + | Al 3+ | SiO 3 2− | Sr 2+ | |
|---|---|---|---|---|---|---|
| Components | H 3 BO 3 | NaF | NaNO 3 | Al(NO 3 ) 3 ·9H 2 O NaOH |
Na 2 SiO 3 HNO 3 |
Sr(NO 3 ) 2 |
| pH | 6.13 | 7.32 | 5.57 | 5.14 | 6.07 | 5.50 |
| Concentration (ppm) | 1000 | 1000 | 1000 | 100 | 1000 | 1000 |
Each ion solution was diluted with BHI broth to obtain the concentrations previously determined to be released from P4. To 9.95 mL of each single ion solution prepared, 50 μL of S. mutans suspension at 3 × 10 3 CFU/mL was added and incubated under anaerobic conditions at 37 °C. After 18 h, the bacterial suspension was serially diluted with BHI broth and spread onto BHI agar plates. The plates were incubated at 37 °C for 48 h, and the number of colonies was counted. The experiments were repeated three times.
For BO 3 3− , F − , Al 3+ , and SiO 3 2− , the inhibitory effects on S. mutans growth at different concentrations were further determined. Table 3 shows the concentrations of the four ions examined based on the previous release study. Standard ion solutions were diluted with BHI broth and their inhibitory effects on S. mutans growth were examined as described before. The experiments were repeated three times.
| Concentration (ppm) released | ||||
|---|---|---|---|---|
| P1 | P2 | P3 | P4 | |
| BO 3 3− | 26.1 | 52.2 | 88.3 | 131.0 |
| F − | 4.6 | 7.9 | 8.6 | 8.6 |
| Al 3+ | 4.9 | 6.0 | 6.0 | 6.8 |
| SiO 3 2− | 5.7 | 7.8 | 8.4 | 9.7 |
2.5
Statistical analysis
Statistical significance of the data for the bacterial growth and ion release measurements were compared by one way analysis of variance (ANOVA) and Tukey–Kramer post hoc test with a significance level of p < 0.05.
2
Materials and methods
2.1
Antibacterial activity of eluates from S-PRG filler
S-PRG fillers with average diameter of 3 μm were added to distilled water at 1:1 ratio by weight (1000 g filler in 1000 mL) and stirred at 23 °C for 24 h. The mixing solution was centrifuged to precipitate the S-PRG fillers, and the supernatant was filtered. The eluates from S-PRG filler were diluted with brain–heart infusion (BHI) broth (Becton Dickinson, Sparks, MD, USA) supplemented with 0.5% yeast extract (Becton Dickinson), and solutions containing the eluates from S-PRG filler at 50, 25, and 12.5 (vol.%) were prepared.
S. mutans NCTC10449 was cultured in BHI broth and adjusted to 2 × 10 5 , 2 × 10 4 , or 2 × 10 3 colony-forming units (CFU)/mL. Aliquots of 50 μL of the diluted eluates from S-PRG filler were added to 50 μL of bacterial suspension and incubated anaerobically at 37 °C for 24 h. Bacterial growth was monitored by measuring the absorbance of the aliquot at OD 550 using a spectrophotometer (Model 680 Microplate Reader, BioRad Laboratories, Hercules, CA, USA). Controls consisted of the diluted eluates from S-PRG filler without any inoculum of bacteria. Cultures with absorbance within ±0.010 of the range for the control were determined to have no bacterial growth. The experiments were repeated five times.
2.2
Inhibitory effects of resin composites containing S-PRG filler on S. mutans growth
2.2.1
Sample preparation
Five experimental resin composites were prepared. To the monomer compositions of 70 (wt%) 2,2-bis [4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl]propane (Bis-GMA) and 30 (wt%) triethyleneglycol dimethacrylate (TEGDMA), S-PRG fillers and silica filler (average diameter: 1 μm) were loaded at different ratios. The final contents of S-PRG filler in the resin composites ranged from 13.9% to 55.9% in volume ( Table 1 ). Based on the maximum amount of filler loaded for P4, the total filler contents for all experimental resin composites were unified as 55.9 (vol.%). For photo-initiator system, 0.3 (wt%) camphorquinone and 0.3 (wt%) ethyl 4-dimethylaminobenzoate were used.
