Nano-calcium phosphate and dimethylaminohexadecyl methacrylate adhesive for dentin remineralization in a biofilm-challenged environment

ObjectiveDentin remineralization at the bonded interface would protect it from external risk factors, therefore, would enhance the longevity of restoration and combat secondary caries. Dental biofilm, as one of the critical biological factors in caries formation, should not be neglected in the assessment of caries preventive agents. In this work, the remineralization effectiveness of demineralized human dentin in a multi-species dental biofilm environment via an adhesive containing nanoparticles of amorphous calcium phosphate (NACP) and dimethylaminohexadecyl methacrylate (DMAHDM) was investigated.MethodsDentin demineralization was promoted by subjecting samples to a three-species acidic biofilm containing Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii for 24 h. Samples were divided into a control group, a DMAHDM adhesive group, an NACP group, and an NACP + DMAHDM adhesive group. A bonded model containing a control-bonded group, a DMAHDM-bonded group, an NACP-bonded group, and an NACP + DMAHDM-bonded group was also included in this study. All samples were subjected to a remineralization protocol consisting of 4-h exposure per 24-h period in brain heart infusion broth plus 1% sucrose (BHIS) followed by immersion in artificial saliva for the remaining period. The pH of BHIS after 4-h immersion was measured every other day. After 14 days, the biofilm was assessed for colony-forming unit (CFU) count, lactic acid production, live/dead staining, and calcium and phosphate content. The mineral changes in the demineralized dentin samples were analyzed by transverse microradiography.ResultsThe in vitro experiment results showed that the NACP + DMAHDM adhesive effectively achieved acid neutralization, decreased biofilm colony-forming unit (CFU) count, decreased biofilm lactic acid production, and increased biofilm calcium and phosphate content. The NACP + DMAHDM adhesive group had higher remineralization value than the NACP or DMAHDM alone adhesive group.SignificanceThe NACP + DMAHDM adhesive was effective in remineralizing dentin lesion in a biofilm model. It is promising to use NACP + DMAHDM adhesive to protect bonded interface, inhibit secondary caries, and prolong the longevity of restoration.     

Curcumin reduces Streptococcus mutans biofilm formation by inhibiting sortase A activity

BackgroundSortase A is an enzyme responsible for the covalent attachment of Pac proteins to the cell wall in Streptococcus mutans. It has been shown to play a role in modulating the surface properties and the biofilm formation and influence the cariogenicity of S. mutans. Curcumin, an active ingredient of turmeric, was reported to be an inhibitor for Staphylococcus aureus sortase A. The aim of this study was to investigate the inhibitory ability of curcumin against S. mutans sortase A and the effect of curcumin for biofilm formation.MethodsThe antimicrobial activity of the curcumin to the S. mutans and inhibitory ability of the curcumin against the purified sortase A in vitro were detected. Western-blot and real-time PCR were used to analysis the sortase A mediated Pac protein changes when the S. mutans was cultured with curcumin. The curcumin on the S. mutans biofilm formation was determined by biofilm formation analysis.ResultsCurcumin can inhibit purified S. mutans sortase A with a half-maximal inhibitory concentration (IC₅₀) of (10.2 ± 0.7) μmol/l, which is lower than minimum inhibitory concentration (MIC) of 175 μmol/l. Curcumin (15 μmol/l) was found to release the Pac protein to the supernatant and reduce S. mutans biofilm formation.ConclusionsThese results indicated that curcumin is an S. mutans sortase A inhibitor and has promising anti-caries characteristics through an anti-adhesion-mediated mechanism.     

Effect of arginine on the growth and biofilm formation of oral bacteria

BackgroundAlkali production via arginine deiminase system (ADS) of oral bacteria plays a significant role in oral ecology, pH homeostasis and inhibition of dental caries. ADS activity in dental plaque varies greatly between individuals, which may profoundly affect their susceptibility to caries.ObjectiveTo investigate the effect of arginine on the growth and biofilm formation of oral bacteria.Methods and resultsPolymicrobial dental biofilms derived from saliva were formed in a high-throughput active attachment biofilm model and l-arginine (Arg) was shown to reduce the colony forming units (CFU) counts of such biofilms grown for various periods or biofilms derived from saliva of subjects with different caries status. Arg hardly disturbed bacterial growth of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus gordonii in BHI medium, but only inhibited biofilm formation of S. mutans. Scanning electron microscope (SEM) showed S. mutans biofilms harboured fewer cells grown with Arg than that without Arg, even in the initial 2 h and 8 h phase. Confocal laser scanning microscope (CLSM) images of poly-microbial dental and S. mutans biofilms revealed the biofilms grown with Arg had lower exopolysaccharide (EPS)/bacteria ratios than those without Arg (P = 0.004, 0.002, respectively). Arg could significantly reduce the production of water-insoluble EPS in S. mutans biofilms (P < 0.001); however, quantitative real-time PCR (qRT-PCR) did not show significantly influence in gene expression of gtfB, gtfC or gtfD (P = 0.32, 0.06, 0.44 respectively).ConclusionsArg could reduce the biomass of poly-microbial dental biofilms and S. mutans biofilms, which may be due to the impact of Arg on water-insoluble EPS. Considering the contribution to pH homeostasis in dental biofilms, Arg may serve as an important agent keeping oral biofilms healthy thus prevent dental caries.     

Experimental composites containing quaternary ammonium methacrylates reduce demineralization at enamel-restoration margins after cariogenic challenge

ObjectiveThis study evaluated the influence of experimental composites containing quaternary ammonium monomers (QAM) at different concentrations and alkyl chains on demineralization at enamel-composite margins after cariogenic challenge.MethodsStandardized 4 × 4 mm cavities were cut into 35 bovine enamel blocks, which were randomly divided into seven groups (n = 5) and restored with the following experimental composites and commercial materials: (G12.5) – 5% dimethylaminododecyl methacrylate (DMADDM) with a 12-carbon alkyl chain (G12.10) – 10% DMADDM, (G16.5) – 5% dimethylaminohexadecyl methacrylate (DMAHDM) with a 16-carbon alkyl chain (G16.10) – 10% DMAHDM, (CG) – control group (without QAM), (GZ250) – commercial composite (Filtek Z250^®), and (GIC) – glass ionomer cement (Maxxion R^®). After restorative procedures, initial microhardness was measured and experimental composites were subjected to Streptococcus mutans biofilm formation for 48 h. After cariogenic challenge, the samples were washed and microhardness was reassessed. A 3D non-contact profilometer was used to determine surface roughness and enamel demineralization was assessed by micro-CT. Microhardness results were analyzed by the Kruskal–Wallis and Mann-Whitney tests and micro-CT results were analyzed by Tukey’s HSD test (95% confidence interval).ResultsNone of the materials could prevent mineral loss at the enamel-restoration margins. The addition of 10% DMAHDM yielded the lowest, albeit statistically significant, mineral loss (p < 0.05). 3D non-contact profilometry showed enamel surface roughness modification after biofilm exposure. The CG had the highest roughness values. Micro-CT analysis revealed mineral loss, except for GIC.SignificanceThe addition of 10% QAM with a 16-carbon chain in experimental composites reduced mineral loss at the enamel-restoration margins after cariogenic challenge.     

Novel magnetic nanoparticle-containing adhesive with greater dentin bond strength and antibacterial and remineralizing capabilities

Objectives

A nanoparticle-doped adhesive that can be controlled with magnetic forces was recently developed to deliver drugs to the pulp and improve adhesive penetration into dentin. However, it did not have bactericidal and remineralization abilities. The objectives of this study were to: (1) develop a magnetic nanoparticle-containing adhesive with dimethylaminohexadecyl methacrylate (DMAHDM), amorphous calcium phosphate nanoparticles (NACP) and magnetic nanoparticles (MNP); and (2) investigate the effects on dentin bond strength, calcium (Ca) and phosphate (P) ion release and anti-biofilm properties.

Novel root canal sealer with dimethylaminohexadecyl methacrylate,nano-silver and nano-calcium phosphate to kill bacteria inside root dentin and increase dentin hardness

ObjectivesRoot canal re-infection and weakening of roots are two main challenges in endodontics. The objectives of the study were: (1) to develop a novel root canal sealer containing dimethylaminohexadecyl methacrylate (DMAHDM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP), and (2) to investigate the effects on the physical, anti-biofilm, remineralizing ions, and hardness of human dentin for the first time.MethodsMethacrylate-resin dual-cured root canal sealer contained 5% DMAHDM, 0.15% NAg, and NACP at 10%, 20% and 30% mass fractions. The flow, film thickness, and Ca and P ions release were investigated. The effects of NACP on radicular dentin hardness after treatment with sodium hypochlorite (NaOCL) and ethylenediaminetetraacetic acid (EDTA) were assessed. Antibacterial properties were measured against Enterococcus faecalis (E. faecalis)-impregnated dentin blocks; colony-forming units (CFU) and live/dead assays were measured.ResultsIncorporating DMAHDM, NAg and NACP did not adversely influence the flow and film thickness properties. Sealer with 30% NACP neutralized the acid and increased the solution pH (p < 0.05). Sealer containing 30% NACP regenerated dentin minerals lost due to NaOCL and EDTA treatment, and increased the dentin hardness to match that of sound dentin (p > 0.1). Incorporating 5% DMAHDM and 0.15% NAg reduced biofilm CFU of E. faecalis-impregnated dentin blocks by nearly 3 logs when compared control group (p < 0.05).SignificanceThe novel therapeutic root canal sealer with triple bioactive agents of DMAHDM, NAg and NACP neutralized acid, raised the pH, regenerated dentin minerals, increased root dentin hardness, and reduced dentin-block-impregnated biofilm CFU by 3 logs. This new sealer with highly desirable antibacterial and remineralization properties are promising to increase the success rate of endodontic therapy and strengthen the tooth root structures.     

Self-cured resin modified by quaternary ammonium methacrylates and chlorhexidine: Cytotoxicity,antimicrobial, physical,and mechanical properties

ObjectiveTo evaluate the addition of dimethylaminohexadecyl methacrylate (DMAHDM) and chlorhexidine diacetate on cytotoxicity, antimicrobial activity, physical, and mechanical properties of a self-cured resin.Methods132 disk-shaped and 48 rectangular specimens were divided into four experimental groups as described: Control Group (CG – no addition), dCHX (1%), DMAHDM (5%), and DMAHDM + dCHX (5% + 1%). The biofilm viability, flexural strength (FS - ISO 20795-1:2013), surface roughness (SR), and color stability (ΔE) were analyzed after being stored for 4 weeks in distilled water and immersed for 72 h in coffee. Cytotoxicity was measured after 24 h, 3, and 7 days of elution using an MTT test on L929 cells (ISO 10993-5:2009). SR and ΔE were measured by a contact profilometer and a spectrophotometer using the CIELab parameter. Data were submitted to ANOVA and Bonferroni’s/Tukey’s tests (p ≤ 0.05).ResultsSignificant antimicrobial activity against Streptococcus mutans and Candida albicans was detected in all groups when compared to the CG (p < 0.05). Only the dCHX group, in 24 h of elution, demonstrated no cytotoxicity effects. There was a statistical difference for FS on the tested groups (p < 0.05). No differences were detected in the initial roughness’ measurements among the groups (p > 0.05). However, after storage and immersion in coffee, the groups containing DMAHDM presented with rougher surfaces and significantly lower color stability compared to the control (p < 0.05).SignificanceThe addition of dCHX and DMAHDM in self-cured resin presented antimicrobial properties; however, cytotoxicity, physical, and mechanical properties were compromised.     

Concentration dependence of quaternary ammonium monomer on the design of high-performance bioactive composite for root caries restorations

ObjectivesDental plaque build-up on the cervical area adjacent to gingival margins is a trigger factor for secondary caries around restored root caries lesions. Dimethylaminohexadecyl methacrylate (DMAHDM) and amorphous calcium phosphate nanoparticles (NACP) impart anti-caries effect by reducing the bacterial growth and releasing high concentrations of calcium and phosphate ions, respectively. The present study explored the optimization and formulation of dental composite with increased concentration of DMAHDM combined with NACP and its effect on mechanical behavior and antibacterial response.MethodsDMAHDM was incorporated into dental composite formulation at 3% and 5% with 20% NACP fillers. Mechanical properties were assessed by flexural strength and elastic modulus. The cationic charge density of the samples was determined using fluorescein staining assay. A human saliva-derived microcosm biofilm model was used to assess antibacterial response via colony-forming units, metabolic activities, lactic acid production, and live/dead assay. Surface roughness was measured after 48 h-biofilm formation.ResultsThe viability of human saliva microcosm biofilms was DMAHDM concentration-dependent, where all the microbiological assays were substantially reduced in the presence of 5%DMAHDM. The increased DMAHDM concentration mirrors an increased surface charge density of composites by 8–12 folds and reduced the growth of cariogenic species by 2–5 log (p ≤ 0.05). Metabolic activity and lactic acid were reduced by 70–90% and 48–99%, respectively. Increasing DMAHDM concentration up to 5% and its association with NACP fillers did not adversely affect the mechanical properties.SignificanceA highly potent antibiofilm bioactive composite for root caries restorations having DMAHDM-NACP could be flexibly tailored during formulation without detrimental outcome for mechanical function. The enhanced antibacterial performance of the novel bioactive composite has great potential to suppress the dental plaque build-up that triggers secondary caries around the restored root caries lesions.     

Dual-functional adhesive containing amorphous calcium phosphate nanoparticles and dimethylaminohexadecyl methacrylate promoted enamel remineralization in a biofilm-challenged environment

ObjectiveThe cariogenic biofilm on enamel, restoration, and bonding interface is closely related to dental caries and composite restoration failure. Enamel remineralization at adhesive interface is conducive to protecting bonding interface and inhibiting secondary caries. This study intended to assess the remineralization efficiency of adhesive with dimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP) on initial caries lesion of biofilm-coated enamel.MethodsArtificial initial carious lesion was created via 72-hour immersion in demineralization solution and cariogenic biofilm was formed after 24-hour culture of Streptococcus mutans (S. mutans). Specimens were then divided into 4 groups: enamel control, enamel treated with NACP, DMAHDM and NACP+DMAHDM respectively. Samples next underwent 7-day cycling, 4 h in BHIS (brain heart infusion broth containing 1 % sucrose) and 20 h in AS (artificial saliva) per day. The pH of BHIS was tested daily. So did the concentration of calcium and phosphate in BHIS and AS. Live/dead staining, colony-forming unit (CFU) count, and lactic acid production of biofilms were measured 7 days later. The enamel remineralization efficiency was evaluated by microhardness testing and transverse microradiography (TMR) quantitatively.ResultsEnamel of NACP+DMAHDM group demonstrated excellent remineralization effectiveness. And the NACP+DMAHDM adhesive released a great number of Ca²⁺ and PO₄^3- ions, increased pH to 5.81 via acid neutralization, decreased production of lactic acid, and reduced CFU count of S. mutans (P < 0.05).SignificanceThe NACP+DMAHDM adhesive would be applicable to preventing secondary caries, strengthening enamel-adhesive interface, and extending the lifespan of composite restoration.     

Resin composite blocks for dental CAD/CAM applications reduce biofilm formation in vitro

ObjectivesModern dentistry is increasingly focusing on digital procedures, including CAD/CAM technologies. New materials have to resist in a demanding environment that includes secondary caries occurrence. The current study hypothesized that the microbiological behavior of different RBCs for CAD/CAM applications is better than that of their counterparts for direct restorations due to differences in the surface characteristics.MethodsBoth direct and CAD/CAM RBCs were tested. Specimens were obtained from each group, polished, cleaned, stored in artificial saliva (1 w), then sterilized under UV (24 h). Specimens’ surface was assessed using profilometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction; resin/filler content was assessed using thermogravimetry. After pre-incubation with sterile human saliva (24 h), the microbiological behavior of the materials was assessed using four models: Streptococcus mutans adherence (2 h), S. mutans biofilm formation in an orbital shaking bioreactor (24 h), S. mutans biofilm formation in a continuous-flow bioreactor simulating shear forces (24 h), and mixed-plaque formation in the bioreactor (24 h). The viable biomass adhering to the specimens’ surfaces was measured using a tetrazolium dye-based test. Statistical analysis included verification of normality of distribution and homoscedasticity, then Oneway ANOVA and Tukey's test (α = 5%).ResultsWhen using the bioreactor setup, CAD/CAM RBCs generally yielded lower S. mutans and mixed-plaque biofilm formation compared to direct RBCs. This difference was not evidenced in the first two microbiological models. Differences in manufacturing and curing processes rather than in materials’ surface roughness and composition could explain these results.SignificanceCAD/CAM RBCs are promising materials from a microbiological point of view, featuring reduced biofilm formation on their surfaces when shear conditions similar to in vivo ones are present.     

Influence of a Brazilian wild green propolis on the enamel mineral loss and Streptococcus mutans’ count in dental biofilm

ObjectiveThis study investigated the anti-demineralizing and antibacterial effects of a propolis ethanolic extract (EEP) against Streptococcus mutans dental biofilm.DesignBlocks of sound bovine enamel (n = 24) were fixed on polystyrene plates. S. mutans inoculum (ATCC 25175) and culture media were added (48 h–37 °C) to form biofilm. Blocks with biofilm received daily treatment (30 μL/1 min), for 5 days, as following: G1 (EEP 33.3%); G2 (chlorhexidine digluconate 0.12%); G3 (ethanol 80%); and G4 (Milli-Q water). G5 and G6 were blocks without biofilm that received only EEP and Milli-Q water, respectively. Final surface hardness was evaluated and the percentage of hardness loss (%HL) was calculated. The EEP extract pH and total solids were determined. S. mutans count was expressed by log₁₀ scale of Colony-Forming Units (CFU/mL). One way ANOVA was used to compare results which differed at a 95% significance level.ResultsG2 presented the lowest average %HL value (68.44% ± 12.98) (p = 0.010), while G4 presented the highest (90.49% ± 5.38%HL) (p = 0.007). G1 showed %HL (84.41% ± 2.77) similar to G3 (87.80% ± 6.89) (p = 0.477). Groups G5 and G6 presented %HL = 16.11% ± 7.92 and 20.55% ± 10.65; respectively (p = 0.952). G1 and G4 differed as regards to S. mutans count: 7.26 ± 0.08 and 8.29 ± 0.17 CFU/mL, respectively (p = 0.001). The lowest bacterial count was observed in chlorhexidine group (G2 = 6.79 ± 0.10 CFU/mL) (p = 0.043). There was no difference between S. mutans count of G3 and G4 (p = 0.435). The EEP showed pH 4.8 and total soluble solids content = 25.9 Brix.ConclusionThe EEP seems to be a potent antibacterial substance against S. mutans dental biofilm, but presented no inhibitory action on the de-remineralization of caries process.     

In vitro evaluation of composite containing DMAHDM and calcium phosphate nanoparticles on recurrent caries inhibition at bovine enamel-restoration margins

ObjectiveRecurrent caries is a primary reason for restoration failure caused by biofilm acids. The objectives of this study were to: (1) develop a novel multifunctional composite with antibacterial function and calcium (Ca) and phosphate (P) ion release, and (2) investigate the effects on enamel demineralization and hardness at the margins under biofilms.MethodsDimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP) were incorporated into composite. Four groups were tested: (1) Commercial control (Heliomolar), (2) Experimental control (0% DMAHDM + 0% NACP), (3) antibacterial group (3% DMAHDM + 0% NACP), (D) antibacterial and remineralizing group (3% DMAHDM + 30% NACP). Mechanical properties and Ca and P ion release were measured. Colony-forming units (CFU), lactic acid and polysaccharide of Streptococcus mutans (S. mutans) biofilms were evaluated. Demineralization of bovine enamel with restorations was induced via S. mutans, and enamel hardness was measured. Data were analyzed via one-way and two-way analyses of variance and Tukey’s multiple comparison tests.ResultsAdding DMAHDM and NACP into composite did not compromise the mechanical properties (P > 0.05). Ca and P ion release of 3% DMAHDM + 30% NACP was increased at cariogenic low pH. Biofilm lactic acid and polysaccharides were greatly decreased via DMAHDM, and CFU was reduced by 4 logs (P < 0.05). Under biofilm acids, enamel hardness at the margins was decreased to about 0.5 GPa for control; it was about 1 GPa for antibacterial group, and 1.3 GPa for antibacterial and remineralizing group (P < 0.05).ConclusionsThe novel 3% DMAHDM + 30% NACP composite had strong antibacterial effects. It substantially reduced enamel demineralization adjacent to restorations under biofilm acid attacks, yielding enamel hardness that was 2-fold greater than that of control composites. The novel multifunctional composite is promising to inhibit recurrent caries.     

Comparative effect of a stannous fluoride toothpaste and a sodium fluoride toothpaste on a multispecies biofilm

ObjectivesThis paper aimed to compare the mode of action of a stannous fluoride-containing toothpaste with a conventional sodium fluoride-containing toothpaste on anti-biofilm properties.MethodsA three-species biofilm model that consists of Streptococcus mutans, Streptococcus sanguinis and Porphyromonas gingivalis was established to compare the anti-biofilm properties of a stannous fluoride-containing toothpaste (CPH), a conventional sodium fluoride-containing toothpaste (CCP) and a negative control (PBS). The 48 h biofilms were subjected to two-minute episodes of treatment with test agents twice a day for 5 consecutive days. Crystal violet staining and XTT assays were used to evaluate the biomass and viability of the treated biofilm. Live/dead staining and bacteria/extracellular polysaccharides (EPS) double-staining were used to visualize the biofilm structure and to quantify microbial/extracellular components of the treated biofilms. Species-specific fluorescent in situ hybridization and quantitative polymerase chain reaction (qPCR) were used to analyze microbial composition of the biofilms after treatment.ResultsThe biomass and viability of the biofilms were significantly reduced after CPH toothpaste treatment. The inhibitory effect was further confirmed by the live/dead staining. The EPS amounts of the three-species biofilm were significantly reduced by CCP and CPH treatments, and CPH toothpaste demonstrated significant inhibition on EPS production. More importantly, CPH toothpaste significantly suppressed S. mutans and P. gingvalis, and enriched S. sanguinis in the three-species biofilm. In all experiments CPH had a significantly greater effect than CCP (p < 0.05) and CCP had a greater effect than PBS (p < 0.05).ConclusionsStannous fluoride-containing toothpaste not only showed better inhibitory effect against oral microbial biofilm, but was also able to modulate microbial composition within multi-species biofilm compared with conventional sodium fluoride-containing toothpaste.     

Influence of sucrose and xylitol on an early Streptococcus mutans biofilm in a dental simulator

ObjectivesIn vitro methods to study dental biofilms are useful in finding ways to support a healthy microbial balance in the oral cavity. The effects of sucrose, xylitol, and their combination on three strains of Streptococcus mutans and one strain of Streptococcus sobrinus were studied using a dental simulator.MethodsA simulator was used to mimic the oral cavity environment. It provided a continuous-flow system using artificial saliva (AS), constant temperature, mixing, and hydroxyapatite (HA) surface in which the influence of xylitol was studied. The quantities of planktonic and adhered bacteria were measured by real-time qPCR.ResultsCompared against the untreated AS, adding 1% sucrose increased the bacterial colonization of HA (p < 0.0001) whereas 2% xylitol decreased it (p < 0.05), with the exception of clinical S. mutans isolate 117. The combination of xylitol and sucrose decreased the bacterial quantities within the AS and the colonization on the HA by clinical S. mutans isolate 2366 was reduced (p < 0.05). Increasing the concentration (2%–5%) of xylitol caused a reduction in bacterial counts even in the presence of sucrose.ConclusionsThe continuous-culture biofilm model showed that within a young biofilm, sucrose significantly promotes whereas xylitol reduces bacterial colonization and proliferation. The results indicate that xylitol affects the ability of certain S. mutans strains to adhere to the HA. Clinical studies have also shown that xylitol consumption decreases caries incidence and reduces the amount of plaque. This study contributes to the understanding of the mechanism behind these clinical observations.     

Effect of bovine lactoferrin on enamel demineralization and acid fermentation by Streptococcus mutans

The purpose of this study was to evaluate the effects of bovine lactoferrin on acid fermentation and enamel demineralization using Streptococcus mutans in a culture system and an artificial mouth model system. The antibacterial activity of bovine lactoferrin (bLF) against S. mutans was analyzed by a radial diffusion assay. In the culture system, the effect of bLF on the synthesis and adherence of water insoluble glucan (WIG) and the adherence of S. mutans to a glass surface was examined by a batch culture. In the artificial mouth model system, cell suspension of S. mutans, heart infusion broth supplemented with sucrose, and PBS or lactoferrin solution were supplied separately and constantly for 21 hours. The following parameters were determined for evaluation: the amount of artificial biofilm, the changes in pH underneath the biofilm; and the changes in enamel microhardness measured by a Vicker's hardness tester. The antibacterial activity of bLF against S. mutans was observed. The amounts of bacterial cells in the total adherent fractions were inhibited by bLF in a dose dependent manner. The amounts of WIG in a firm-adherent fraction were significantly inhibited by 0.1–1.0% bLF. The changes in microhardness on enamel slabs in the bLF group (2.4 ± 0.8) showed significantly less hardness reduction than those in the control group (22.3 ± 2.5) (P < 0.001). The artificial biofilm accumulation was not reduced by bLF. The results of this study suggest that bLF might have inhibitory effects against acid fermentation and demineralization of enamel by S. mutans.     

Novel pit and fissure sealant containing nano-CaF2 and dimethylaminohexadecyl methacrylate with double benefits of fluoride release and antibacterial function

ObjectivePit and fissure sealants with antibacterial and remineralization properties have broad application prospects in caries prevention. The objectives of this study were to: (1) develop a novel pit and fissure sealant containing CaF₂ nanoparticles (nCaF₂) and dimethylaminohexadecyl methacrylate (DMAHDM); and (2) investigate the effects of nCaF₂ and DMAHDM on biofilm response and fluoride (F) ion release for the first time.MethodsHelioseal F was used as a control. Bioactive sealants were formulated with DMAHDM and nCaF₂. Flow properties, enamel shear bond strength, hardness and F ion releases were measured. Streptococcus mutans (S. mutans) biofilms were grown on sealants. Biofilm metabolic activity, lactic acid production, colony-forming units (CFU), and pH of biofilm culture medium were measured.ResultsAdding 5% DMAHDM and 20% nCaF₂ did not reduce the paste flow and enamel bond strength, compared to control (p < 0.05). Hardness of sealants with 20% nCaF₂ and DMAHDM was higher than control (p < 0.05). The F ion release from 20% nCaF₂ was much higher than that of commercial control (p < 0.05). The sealant with DMAHDM reduced the S. mutans biofilm CFU by 4 logs. The pH in biofilm medium of the new bioactive sealant was much higher (pH 6.8) than that of commercial sealant (pH 4.66) (p < 0.05).SignificanceThe new bioactive pit and fissure sealant with nCaF₂ and DMAHDM achieved high fluoride release and strong antibacterial performance. This novel fluoride-releasing and antibacterial sealant is promising to inhibit caries and promote the remineralizaton of enamel and dentin.     

Clotrimazole and econazole inhibit Streptococcus mutans biofilm and virulence in vitro

Objective: The aim of this study was to determine the inhibitory effect of eight antifungal drugs on S. mutans growth, biofilm formation and virulence factors.MethodsThe actions of antifungal drugs on S. mutans were determined by recovery plates and survival kinetic curves. Biofilms were observed by scanning electron microscopy and the viable cells were recovered on BHI plates, meanwhile biofilms were stained by BacLight live/dead kit to investigate the biofilm viability. Bacteria/extracellular polysaccharides staining assays were performed to determine the EPS production of S. mutans biofilms. Acidogenicity and acidurity of S. mutans were determined using pH drop and acid tolerance assays, and the expression of ldh gene was evaluated using qPCR.ResultsWe found that clotrimazole (CTR) and econazole (ECO) showed antibacterial activities on S. mutans UA159 and S. mutans clinical isolates at 12.5 and 25 mg/L, respectively. CTR and ECO could also inhibit S. mutans biofilm formation and reduce the viability of preformed biofilm. CTR and ECO affected the live/dead ratio and the EPS/bacteria ratio of S. mutans biofilms. CTR and ECO also inhibited the pH drop, lactate acid production, and acid tolerance. The abilities of CTR and ECO to inhibit S. mutans ldh expression were also confirmed.ConclusionsWe found that two antifungal azoles, CTR and ECO, had the abilities to inhibit the growth and biofilm formation of S. mutans and more importantly, they could also inhibit the virulence factors of S. mutans.     

Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea

ObjectiveStreptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified.MethodsEffects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography.ResultsAssam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of <10 kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances >10 kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation.ConclusionsThe higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.     

Effects of dual antibacterial agents MDPB and nano-silver in primer on microcosm biofilm,cytotoxicity and dentine bond properties

ObjectivesThe objective of this study was to investigate the effects of dentine primer containing dual antibacterial agents, namely, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and nanoparticles of silver (NAg), on dentine bond strength, dental plaque microcosm biofilm response, and fibroblast cytotoxicity for the first time.MethodsScotchbond Multi-Purpose (SBMP) was used as the parent bonding agent. Four primers were tested: SBMP primer control (referred to as “P”), P + 5% MDPB, P + 0.05% NAg, and P + 5% MDPB + 0.05% NAg. Dentine shear bond strengths were measured using extracted human teeth. Biofilms from the mixed saliva of 10 donors were cultured to investigate metabolic activity, colony-forming units (CFU), and lactic acid production. Human fibroblast cytotoxicity of the four primers was tested in vitro.ResultsIncorporating MDPB and NAg into primer did not reduce dentine bond strength compared to control (p > 0.1). SEM revealed well-bonded adhesive–dentine interfaces with numerous resin tags. MDPB or NAg each greatly reduced biofilm viability and acid production, compared to control. Dual agents MDPB + NAg had a much stronger effect than either agent alone (p < 0.05), increasing inhibition zone size and reducing metabolic activity, CFU and lactic acid by an order of magnitude, compared to control. There was no difference in cytotoxicity between commercial control and antibacterial primers (p > 0.1).ConclusionsThe method of using dual agents MDPB + NAg in the primer yielded potent antibacterial properties. Hence, this method may be promising to combat residual bacteria in tooth cavity and invading bacteria at the margins. The dual agents MDPB + NAg may have wide applicability to other adhesives, composites, sealants and cements to inhibit biofilms and caries.     

Antimicrobial and antibiofilm action of Casbane Diterpene from Croton nepetaefolius against oral bacteria

ObjectiveThe antibacterial activity of Casbane Diterpene (CD) was evaluated in vitro against Streptococcus oralis, S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. The viability of planktonic cells was analysed by susceptibility tests (MIC and MBC) and antibiofilm action was assayed.MethodsThe minimal inhibitory and bactericidal concentrations (MIC and MBC) of oral Streptococcus were evaluated through microdilution tests. To assay antibiofilm activity, biofilms were generated on 96-wells polystyrene plates under the presence of CD and quantified by a crystal violet technique and colonies forming units counting.ResultsThe CD isolated from Croton nepetaefolius showed antimicrobial effect on planktonic forms and biofilms of oral pathogens, with MIC values of 62.5 μg/mL against Streptococcus oralis and values between 125 and 500 μg/mL against S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. CD showed an inhibitory effect on S. mutans biofilm formation at 250 μg/mL, and a decrease on viable cell of 94.28% compared to the normal biofilm growth.ConclusionsThe compound CD can be considered as a promising molecule for the treatment against oral pathogens responsible for dental biofilm.