Haruaki Kitagawa

Evolution of resistance to cationic biocides in Streptococcus mutans and Enterococcus faecalis

OBJECTIVES The aim of this study was to investigate whether Streptococcus mutans and Enterococcus faecalis develop resistance to the cationic biocides chlorhexidine (CHX), cetylpyridinium chloride (CPC), and 12-methacryloyloxydodecylpyridinium bromide (MDPB). METHODS The minimum inhibitory concentrations (MICs) of CHX, CPC, and MDPB were assessed after repeated exposure of S. mutans and E. faecalis to these biocides. Cell-surface hydrophobicity and protein expression profiles of bacterial cells were examined to elucidate possible resistance mechanisms. RESULTS The MIC of CHX against E. faecalis showed constant increases up to 10 passages. No changes in the MICs of CPC and MDPB against E. faecalis were observed. The MICs of CHX, CPC, and MDPB against S. mutans did not increase. The surface hydrophobicity of E. faecalis significantly increased with increasing exposure to CHX and CPC. However, changes in protein expression profiles were only found in CHX-adapted E. faecalis, as evidenced by the emergence of a novel, approximately 19-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CONCLUSIONS While E. faecalis and S. mutans did not exhibit increased resistance to CPC or MDPB, repeated exposure of E. faecalis to CHX led to resistance. It is likely that the acquisition of resistance is related to an altered protein composition. CLINICAL SIGNIFICANCE Alkyl pyridinium compounds, such as CPC and MDPB, could have a lower risk to cause adaptation of E. faecalis, which is advantageous compared with CHX.

Development of an Antibacterial Root Canal Filling System Containing MDPB

An antibacterial monomer 12-methacryloyloxydodecylpyridinum bromide (MDPB)-containing experimental, chemically cured primer was prepared to develop a new resin-based root canal filling system. This study investigated the antibacterial effects of the MDPB-containing primer (experimental primer [EP]) against Enterococcus faecalis and assessed the in vitro bonding and sealing abilities of the filling system, consisting of EP and a Bis-GMA-based sealer resin. Antibacterial effects of EP were evaluated by contact with planktonic or adherent bacteria for 30 or 60 sec, and the viable bacterial number was counted. The antibacterial effects against E. faecalis in dentinal tubules were also assessed, according to a root canal infection model. Bonding and sealing abilities of the experimental filling system were examined by microtensile bond strength tests and leakage tests based on fluid filtration methods. Significantly greater reduction in viable bacteria in planktonic and adherent form was obtained by short-period contact with EP compared with the control primer (without MDPB) or with the proprietary (Epiphany) primer (p < .05). Significantly greater bactericidal effects of the EP inside the dentinal tubule of root, as opposed to the control primer or Epiphany primer, were confirmed according to a root canal infection model (p < .05), and 100% killing of E. faecalis could be obtained by the application of EP after irrigation with a 5% sodium hypochlorite solution. The experimental endodontic filling system demonstrated significantly greater bond strength to root dentin than Epiphany sealer system (Epiphany primer and Epiphany Root Canal Sealant; p < .05), showing formation of resin tags and a hybridized layer. Leakage tests clarified that the experimental system provided excellent sealing. This study confirmed that the MDPB-containing experimental antibacterial primer has the ability to effectively disinfect the root canal. Additionally, the experimental root canal filling system employing this primer and the Bis-GMA-based sealer resin is useful for achieving good sealing, suggesting its possible benefit for successful endodontic treatments.

Mechanism of detoxification of the cationic antibacterial monomer 12-methacryloyloxydodecylpyridiniumbromide (MDPB) by N-acetyl cysteine

OBJECTIVES The protective effects of N-acetyl cysteine (NAC) against cytotoxicity induced by conventional dental resin monomers have been widely documented. However, its effectiveness to detoxify cationic antibacterial monomers has not yet been elucidated. The aim of the present study was to investigate the possible protective effects of NAC against the cytotoxicity of 12-methacryloyloxydodecylpyridiniumbromide (MDPB) and explore the role of adduct formation in NAC-directed detoxification. METHODS The influences of NAC on the cytotoxicity of MDPB were studied in mouse osteoblast-like MC3T3-E1 cells using the MTT assay. Ultra-performance liquid chromatography (UPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis were performed to investigate the possible chemical reaction between NAC and MDPB. RESULTS While only slight reduction in the cytotoxicity of MDPB by NAC was observed immediately after mixing with MDPB, remarkable protection against MDPB-induced cell death was detected when the mixture was tested after 24h of pre-incubation. UPLC and LC-MS analysis revealed that chemical binding of MDPB and NAC occurred under neutral conditions after 24h of pre-incubation. SIGNIFICANCE Our findings suggest that NAC reduces the toxicity of the cationic antibacterial monomer MDPB, and adduct formation is partially responsible for the detoxification ability of NAC against MDPB-induced cell damage.

Antibacterial activity of resin composites containing surface pre-reacted glass-ionomer (S-PRG) filler.

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 18h to examine the growth of bacteria in contact with the surface of the experimental resins. The concentrations of Al(3+), BO3(3-), F(-), Na(+), SiO3(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 BO3(3-), F(-), Al(3+), and SiO3(2-). Among these four ions, BO3(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. BO3(3-), F(-), Al(3+) and SiO3(2-) released from S-PRG filler have the ability to inhibit S. mutans growth, and the inhibitory effects are mainly attributed to release of BO3(3-) and F(-).

Development of a Cavity Disinfectant Containing Antibacterial Monomer MDPB

An experimental cavity disinfectant (ACC) that is intended to be used for various direct and indirect restorations was prepared by adding an antibacterial monomer 12-methacryloyloxydodecylpyridinum bromide (MDPB) at 5% into 80% ethanol. The antibacterial effectiveness of ACC and its influences on the bonding abilities of resin cements were investigated. To examine the antibacterial activity of unpolymerized MDPB, the minimum inhibitory and bactericidal concentrations (MIC and MBC) were determined for Streptococcus mutans, Lactobacillus casei, Actinomyces naeslundii, Parvimonas micra, Enterococcus faecalis, Fusobacterium nucleatum, and Porphyromonas gingivalis. Antibacterial activities of ACC and the commercial cavity disinfectant containing 2% chlorhexidine and ethanol (CPS) were evaluated by agar disk diffusion tests through 7 bacterial species and by MIC and MBC measurement for S. mutans. The effects of ACC and CPS to kill bacteria in dentinal tubules were compared with an S. mutans–infected dentin model. Shear bond strength tests were used to examine the influences of ACC on the dentin-bonding abilities of a self-adhesive resin cement and a dual-cure resin cement used with a primer. Unpolymerized MDPB showed strong antibacterial activity against 7 oral bacteria. ACC produced inhibition zones against all bacterial species similar to CPS. For ACC and CPS, the MIC value for S. mutans was identical, and the MBC was similar with only a 1-step dilution difference (1:2). Treatment of infected dentin with ACC resulted in significantly greater bactericidal effects than CPS (P < 0.05, analysis of variance and Tukey’s honest significant difference test). ACC showed no negative influences on the bonding abilities to dentin for both resin cements, while CPS reduced the bond strength of the self-adhesive resin cement (P < 0.05). This study clarified that the experimental cavity disinfectant containing 5% MDPB is more effective in vitro than the commercially available chlorhexidine solution to eradicate bacteria in dentin, without causing any adverse influences on the bonding abilities of resinous luting cements.

Development of sustained antimicrobial-release systems using poly(2-hydroxyethyl methacrylate)/trimethylolpropane trimethacrylate hydrogels

Reconstructive materials with sustained antimicrobial effects could be useful for preventing infectious diseases in an environment containing indigenous bacteria or fungi such as the oral cavity. With the objective of applying a non-biodegradable hydrogel to resin-based materials as a reservoir for water-soluble antimicrobials, novel hydrogels consisting of 2-hydroxyethyl methacrylate (HEMA) and trimethylolpropane trimethacrylate (TMPT) were fabricated. Cetylpyridinium chloride (CPC) was loaded into five hydrogels comprising different ratios of HEMA/TMPT, and their ability to release as well as to be recharged with CPC was examined in vitro. A polyHEMA/TMPT hydrogel comprising 50% HEMA/50% TMPT could be effectively loaded and recharged with CPC by immersion into a CPC solution, demonstrating the longest release of CPC, above the concentration required to inhibit bacteria and fungi. The binding of CPC to the hydrogels was mainly through hydrophobic interaction. Loading of CPC into a hydrogel by mixing CPC powder with the HEMA/TMPT monomer before polymerization resulted in marked extension of the initial CPC-release period. The CPC-pre-mixed hydrogel was confirmed to exhibit antibacterial activity by agar diffusion tests. It is possible to achieve a sustained release system for antimicrobials by pre-mix loading and recharging CPC into a 50% HEMA/50% TMPT hydrogel.

Effectiveness of non-biodegradable poly(2-hydroxyethyl methacrylate)-based hydrogel particles as a fibroblast growth factor-2 releasing carrier

OBJECTIVES Dental resin-based restorative materials are used in a variety of dental treatment modalities such as root-end filling, perforation sealing, and adhesion of fractured roots. However, the prognosis after such treatments is not necessarily favorable because they fail to promote healing of the surrounding alveolar tissue. In the present study, non-biodegradable poly-2-hydroxyethyl methacrylate (polyHEMA)-based hydrogel particles were fabricated as a carrier vehicle for drug delivery that is applied to dental resins. METHODS The loading and release characteristics of bovine serum albumin (BSA) and fibroblast growth factor-2 (FGF-2) from the polyHEMA-based hydrogel particles were evaluated over time in culture. The hydrogel particles were immersed into an aqueous FITC-labeled BSA solution and were observed using confocal laser scanning microscopy (CLSM). To determine the activity of the FGF-2 released from the particles, the proliferation of osteoblast-like cells cultured with eluates collected from the particles for up to 14 days was determined. RESULTS CLSM revealed that BSA was adsorbed to the surface of the hydrogel particles. A sustained release of BSA and FGF-2 from the particles was detected for up to 14 days. The eluates from the FGF-2-loaded particles increased the proliferation of the osteoblast-like cells, suggesting that the activity of FGF-2 was maintained for at least 2 weeks within the particles. SIGNIFICANCE These polyHEMA-based non-degradable hydrogel particles may be useful tools that can be applied to dental restorative materials to achieve sustained delivery of drugs that promote tissue regeneration.

Multi-scale analysis of the effect of nano-filler particle diameter on the physical properties of CAD/CAM composite resin blocks

Abstract The objective of this study was to assess the effect of silica nano-filler particle diameters in a computer-aided design/manufacturing (CAD/CAM) composite resin (CR) block on physical properties at the multi-scale in silico. CAD/CAM CR blocks were modeled, consisting of silica nano-filler particles (20, 40, 60, 80, and 100 nm) and matrix (Bis-GMA/TEGDMA), with filler volume contents of 55.161%. Calculation of Young’s moduli and Poisson’s ratios for the block at macro-scale were analyzed by homogenization. Macro-scale CAD/CAM CR blocks (3 × 3 × 3 mm) were modeled and compressive strengths were defined when the fracture loads exceeded 6075 N. MPS values of the nano-scale models were compared by localization analysis. As the filler size decreased, Young’s moduli and compressive strength increased, while Poisson’s ratios and MPS decreased. All parameters were significantly correlated with the diameters of the filler particles (Pearson’s correlation test, r = −0.949, 0.943, −0.951, 0.976, p < 0.05). The in silico multi-scale model established in this study demonstrates that the Young’s moduli, Poisson’s ratios, and compressive strengths of CAD/CAM CR blocks can be enhanced by loading silica nanofiller particles of smaller diameter. CAD/CAM CR blocks by using smaller silica nano-filler particles have a potential to increase fracture resistance.

Development of layered PLGA membranes for periodontal tissue regeneration

OBJECTIVE Various commercial products are available for guided tissue regeneration (GTR) therapy; however, they do not combine biosafety with the ability to control cell function. The purpose of this study was to evaluate the physicochemical and biological characteristics of the novel bilayer biodegradable poly(lactic-co-glycolic acid) (PLGA) membrane, and to assess whether the bilayer PLGA membrane could be used for periodontal tissue regeneration. METHODS Bilayer biodegradable membrane was fabricated thorough a two-step freezing and lyophilization process using PLGA solution. The characteristics of bilayer membranes were evaluated with respect to surface morphology, stability, mechanical strength, and operability for clinical use. Cell proliferation and osteogenic differentiation were investigated on the each surface of bilayer membrane. Then, these membranes were implanted to the rat calvaria bone defect models and evaluated their capability for tissue regeneration. RESULTS Biodegradable membranes composed of the solid and porous layer were successfully prepared and the surface morphologies analyzed. Physicochemical analyses revealed that the membranes possessed enough stability and mechanical properties for clinical use. It was also confirmed that the solid layer inhibited cell proliferation and subsequent connective tissue invasion, while the inner layer promoted proliferation and osteogenic differentiation, thus resulting in bone regeneration in vivo. SIGNIFICANCE The layering technology used to fabricate the bilayer polymer membrane could be applied in the developing of other novel biomaterials. The present study demonstrates that the bilayer biodegradable polymer membranes facilitate tissue regeneration in vivo, and therefore represent a prospective biomaterial for GTR therapy.

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

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 BO33- 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 BO33- 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 BO33- 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). OD450 values by XTT assays and S. mutans acid production rates significantly decreased in the presence of BO33- 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 BO33- or F-. CLINICAL SIGNIFICANCE BO33- 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.