Inhibitory effect of resin composite containing S-PRG filler on Streptococcus mutansglucose metabolism

Abstract

Objectives

Resin composites containing surface pre-reacted glass-ionomer (S-PRG) fillers have been reported to inhibit Streptococcus mutans growth on their surfaces, and their inhibitory effects were attributed to BO 3 3− and F ions. The aim of this study was to evaluate S. mutans acid production through glucose metabolism on resin composite containing S-PRG fillers and assess inhibitory effects of BO 3 3− and F on S. mutans metabolic activities.

Methods

The pH change through S. mutans acid production on experimental resin composite was periodically measured after the addition of glucose. Inhibitory effects of BO 3 3− or F solutions on S. mutans metabolism were evaluated by XTT assays and measurement of the acid production rate.

Results

The pH of experimental resin containing S-PRG fillers was significantly higher than that of control resin containing silica fillers ( p < 0.05). OD 450 values by XTT assays and S. mutans acid production rates significantly decreased in the presence of BO 3 3− and F compared with the absence of these ions ( p < 0.05).

Conclusions

pH reduction by S. mutans acid production was inhibited on resin composite containing S-PRG fillers. Moreover, S. mutans glucose metabolism and acid production were inhibited in the presence of low concentrations of BO 3 3− or F .

Clinical significance

BO 3 3− or F released from resin composite containing S-PRG fillers exhibits inhibitory effects on S. mutans metabolism at concentrations lower than those which inhibit bacterial growth.

Introduction

Resin composites are more susceptible to bacterial colonization than other dental restorative materials, such as metallic alloys or ceramics [ ]. Bacterial attachment or biofilm formation adjacent to restoration margins lead to secondary caries, shortening the longevity of restorative materials. To provide resin-based restorative materials with the ability to inhibit bacterial attachment or growth on their surfaces, it is beneficial to incorporate antimicrobial agents, such as quaternary ammonium compounds (QACs), silver nanoparticles, or fluoride [ ].

Surface pre-reacted glass-ionomer (S-PRG) fillers, prepared by an acid-base reaction between fluoroboroaluminosilicate glass and a polyacrylic acid, are unique particles that can be incorporated into various dental resinous materials. The pre-reacted glass-ionomer phase on the surface of the glass core of S-PRG filler has the capability to release multiple ions such as fluoride (F ), borate (BO 3 3− ), aluminum (Al 3+ ), sodium (Na + ), silicate (SiO 3 2− ), and strontium (Sr 2+ ) ions [ , ]. Thus, resinous materials containing S-PRG filler effectively prevent demineralization of dentin [ ] or impart acid resistance to enamel [ ]. Due to the release of F or other ions, S-PRG filler incorporated into resinous materials demonstrates these protective effects by acid-buffering abilities.

Several studies have demonstrated that resin composites containing S-PRG fillers effectively reduce bacterial adherence and prevent biofilm formation on their surfaces [ ]. To elucidate the mechanism of their antibacterial activities, we previously assessed inhibitory effects on Streptococcus mutans growth by preparing experimental resin composites containing different ratios of S-PRG fillers [ ]. In addition, the association between the release of six ions from S-PRG filler and antibacterial activity of the filler was evaluated. This in vitro study confirmed that resin composites containing S-PRG filler at 13.9 (vol)% or greater inhibited S. mutans growth on their surfaces, and inhibitory effects of the experimental resins were mainly attributed to BO 3 3− and F . Based on these previous findings [ ], it is considered that release of BO 3 3− and F from resin composites containing S-PRG filler can inhibit planktonic S. mutans growth prior to biofilm formation on resin composite surfaces.

Several components with strong antibacterial activities show inhibitory effects against bacteria even at low concentrations. It has been reported that antibiotics or QACs showed inhibitory effects on bacterial growth at subminimal inhibitory concentrations (sub-MICs) [ ]. Nakajo et al. [ ] investigated inhibitory effects of glass-ionomer cements (GICs) on the acid production of streptococci and identified F , released from GICs, inhibited S. mutans glucose metabolism at concentrations lower than those that had bacteriostatic or bactericidal effects.

Therefore, we hypothesized that resin composite containing S-PRG filler also inhibits S. mutans metabolic activity via the release of BO 3 3− and/or F from S-PRG fillers. To verify this hypothesis, planktonic S. mutans acid production through glucose metabolism on resin composite containing S-PRG filler was evaluated. In addition, inhibitory effects on planktonic S. mutans metabolism and acid production in the presence of low concentrations of BO 3 3− and F were assessed in vitro .

Materials and methods

pH changes through bacterial acid production on cured resin composite containing S-PRG fillers

Sample preparation

An experimental resin composite containing S-PRG fillers was prepared as previously reported [ ]. Briefly, S-PRG fillers (mean diameter: 1 μm) were loaded to monomer compositions containing 70 (wt)% 2,2-bis [4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane (Bis-GMA) and 30 (wt)% triethylene glycol dimethacrylate (TEGDMA). As a control resin composite, silica fillers (mean diameter: 1 μm) were loaded to the same monomer compositions. Based on the maximum amount of S-PRG filler loaded, the total filler contents for both experimental and control resin composites were unified at 55.9 (vol)%. For the photo-initiator system, 0.3 (wt)% camphorquinone and 0.3 (wt)% ethyl 4-dimethylaminobenzoate were used.

Each resin composite paste was placed in a mold (diameter: 9 mm, thickness: 2 mm), 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 850 mW/cm 2 for 40 s. The cured discs were sterilized with ethylene oxide gas. Three specimens were prepared for each resin composite.

Measurement of pH changes through S. mutans acid production on cured discs

pH changes through acid production from S. mutans glucose metabolism on the cured discs were periodically measured as previously reported [ ]. S. mutans NCTC 10449 was pre-cultured in TYG medium containing 1.7% tryptone (Becton Dickinson, Sparks, MD, USA), 0.3% yeast extract (Becton Dickinson), 0.5% NaCl, and 0.5% glucose at pH 7.0 and 37 °C under anaerobic conditions (80% N 2 , 10% H 2 , and 10% CO 2 ). The pre-cultures were then transferred to fresh TYG medium (inoculum size, 5%) and incubated under the same conditions. When the cells reached exponential growth phase (optical density at 660 nm of about 0.5), they were harvested by centrifugation (21,000 × g for 15 min at 4 °C), washed with 2 mM potassium phosphate buffer (PPB, pH 7.0), and suspended in PPB. Bacterial cells were incubated at 37 °C for 1 h to exhaust intracellularly accumulated polysaccharide and washed with 2 mM PPB (pH 7.0). The cell suspension was distributed into 1.5-mL tubes and centrifuged (16,000 × g for 7 min at 4 °C). The bacterial pellet [1 × 10 11 colony-forming units (CFU)/g] was stored at 4 °C until use.

Fig. 1 shows a schematic drawing of the experimental apparatus with a specimen of resin composite at the bottom. A groove (4.5 mm long × 1.0 mm wide × 1.0 mm deep) was prepared on the surface of a cured disc. An ISFET pH electrode (H + ion-sensitive area, 2.0 mm long × 1.0 mm wide × 0.2 mm thick; model PH-60T1; Nihon Kohden, Tokyo, Japan) was placed in the prepared groove. A plate was made of polymethyl methacrylate (PMMA) with a well (diameter: 4.0 mm, depth: 1.0 mm) for pH monitoring and placed on the cured disc such that the sensitive area of the pH electrode coincided with the position of the well.

Fig. 1
Schematic drawing of the experimental apparatus used to measure pH changes through S. mutans acid production on cured resin composite.
A groove (4.5 mm long × 1.0 mm wide × 1.0 mm deep) was prepared on the surface of a cured disc and a pH electrode was placed in the prepared groove. Then, a plate made of PMMA with a well (diameter: 4.0 mm, depth: 1.0 mm) for pH monitoring was placed on the cured resin composite. Finally, the S. mutans pellet was placed into the well to contact the pH electrode.

The adjusted pellet of S. mutans (16 mg, 1.6 × 10 9 CFU) was placed into the well by means of a spatula and a syringe to contact the pH electrode. Then, 100 μL of PPB was added to the pellet and kept at 37 °C for 60 min. Next, 500 μL of 0.5% glucose (Nacalai Tesque, Kyoto, Japan) was added to the pellet, and the pH was monitored at 37 °C every 5 min for 120 min with a pH meter (ISFET mV/pH METER, BAS, Tokyo, Japan) attached to a chart recorder (LR 4220, Yokogawa Electric Corporation, Tokyo, Japan) in an incubator (Merck KGaA, Darmstadt, Germany). The tests were repeated three times.

Evaluation of S. mutans metabolic activities in the presence of BO 3 3− or F

Inhibitory effects on S. mutans metabolic activities were assessed by XTT assay in the presence of BO 3 3− or F at concentrations lower than those reported to inhibit S. mutans growth [ ]. BO 3 3− solutions containing various concentrations of BO 3 3− were prepared by dissolving H 3 BO 3 (Wako, Osaka, Japan) in distilled water and adjusted to 135.5, 271, and 542 ppm (pH = 6.13) in PPB. F solutions containing various concentrations of F were prepared by dissolving NaF (Sigma-Aldrich, Tokyo, Japan) in distilled water and adjusted to 6.8, 13.5, and 27 ppm (pH = 6.13) in PPB. S. mutans NCTC 10449 was pre-cultured in brain-heart infusion (BHI) broth (Becton Dickinson) supplemented with 0.5% yeast extract. Then, 50 μL of each prepared ion solution was added to each well of a 96-well microplate containing 50 μL of S. mutans suspension at 2.0 × 10 6 or 2.0 × 10 5 CFU/mL. The plate was incubated under anaerobic conditions at 37 °C. After 16 h, 50 μL of XTT reagent solution (AppliChem GmbH, Darmstadt, Germany) was added to each well, and the plate was further incubated at 37 °C for 2 h. The optical density (OD) at 450 nm was measured using a microplate reader (Model 680 microplate reader, Bio-Rad Laboratories, Hercules, CA, USA).

To confirm S. mutans growth under the conditions described above, the bacterial suspension (2.0 × 10 6 or 2.0 × 10 5 CFU/mL) was added to each ion solution and anaerobically incubated. After 18 h, the OD at 550 nm was measured. PPB without ion solutions was used as a control. Five samples were prepared for each solution, and the experiments were repeated five times.

Evaluation of S. mutans acid production in the presence of BO 3 3− or F

S. mutans acid production rates in the presence of BO 3 3− or F were evaluated as previously reported [ ]. Ion solutions containing BO 3 3− or F as prepared above were adjusted to 813 or 40.5 ppm in PPB, respectively. S. mutans NCTC 10449 was pre-cultured in BHI broth supplemented with 0.5% yeast extract. Then, 2 mL of S. mutans suspension at 1.5 × 10 8 CFU/mL was added to 1 mL of each ion solution and incubated at 37 °C. After 4 min, 100 μL of 0.5% glucose solution was added to the suspension, and the amount of acid production from S. mutans glucose metabolism was periodically recorded by the titration volume of 50 mM KOH with a pH-stat (AUTO pH-stat, model AUT-211S, TOA Electronics, Tokyo, Japan). Then, acid production rates were calculated by the inclination of the acid production curve at 10 or 30 min after the addition of glucose. PPB without ion solutions was used as a control. Three samples were prepared for each solution, and the experiments were repeated three times.

Statistical analysis

Statistical analyses were performed using SPSS Statistics 21 (IBM, Chicago, IL, USA). The homogeneity of variances was initially confirmed. pH changes on cured discs were statistically analyzed by Student’s t -test with a significance level of p < 0.05. The results for XTT assays and measurements of the OD to confirm bacterial growth and acid production rates were statistically analyzed by analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test with a significance level of p < 0.05.

Materials and methods

pH changes through bacterial acid production on cured resin composite containing S-PRG fillers

Sample preparation

An experimental resin composite containing S-PRG fillers was prepared as previously reported [ ]. Briefly, S-PRG fillers (mean diameter: 1 μm) were loaded to monomer compositions containing 70 (wt)% 2,2-bis [4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane (Bis-GMA) and 30 (wt)% triethylene glycol dimethacrylate (TEGDMA). As a control resin composite, silica fillers (mean diameter: 1 μm) were loaded to the same monomer compositions. Based on the maximum amount of S-PRG filler loaded, the total filler contents for both experimental and control resin composites were unified at 55.9 (vol)%. For the photo-initiator system, 0.3 (wt)% camphorquinone and 0.3 (wt)% ethyl 4-dimethylaminobenzoate were used.

Each resin composite paste was placed in a mold (diameter: 9 mm, thickness: 2 mm), 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 850 mW/cm 2 for 40 s. The cured discs were sterilized with ethylene oxide gas. Three specimens were prepared for each resin composite.

Measurement of pH changes through S. mutans acid production on cured discs

pH changes through acid production from S. mutans glucose metabolism on the cured discs were periodically measured as previously reported [ ]. S. mutans NCTC 10449 was pre-cultured in TYG medium containing 1.7% tryptone (Becton Dickinson, Sparks, MD, USA), 0.3% yeast extract (Becton Dickinson), 0.5% NaCl, and 0.5% glucose at pH 7.0 and 37 °C under anaerobic conditions (80% N 2 , 10% H 2 , and 10% CO 2 ). The pre-cultures were then transferred to fresh TYG medium (inoculum size, 5%) and incubated under the same conditions. When the cells reached exponential growth phase (optical density at 660 nm of about 0.5), they were harvested by centrifugation (21,000 × g for 15 min at 4 °C), washed with 2 mM potassium phosphate buffer (PPB, pH 7.0), and suspended in PPB. Bacterial cells were incubated at 37 °C for 1 h to exhaust intracellularly accumulated polysaccharide and washed with 2 mM PPB (pH 7.0). The cell suspension was distributed into 1.5-mL tubes and centrifuged (16,000 × g for 7 min at 4 °C). The bacterial pellet [1 × 10 11 colony-forming units (CFU)/g] was stored at 4 °C until use.

Fig. 1 shows a schematic drawing of the experimental apparatus with a specimen of resin composite at the bottom. A groove (4.5 mm long × 1.0 mm wide × 1.0 mm deep) was prepared on the surface of a cured disc. An ISFET pH electrode (H + ion-sensitive area, 2.0 mm long × 1.0 mm wide × 0.2 mm thick; model PH-60T1; Nihon Kohden, Tokyo, Japan) was placed in the prepared groove. A plate was made of polymethyl methacrylate (PMMA) with a well (diameter: 4.0 mm, depth: 1.0 mm) for pH monitoring and placed on the cured disc such that the sensitive area of the pH electrode coincided with the position of the well.

Jun 17, 2018 | Posted by in General Dentistry | Comments Off on Inhibitory effect of resin composite containing S-PRG filler on Streptococcus mutansglucose metabolism
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