Control of microorganisms of oral health interest with Arctium lappa L. (burdock) extract non-cytotoxic to cell culture of macrophages (RAW 264.7)

ObjectivesTo evaluate the antimicrobial activity of Arctium lappa L. extract on Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Candida albicans, C. tropicalis and C. glabrata. In addition, the cytotoxicity of this extract was analyzed on macrophages (RAW 264.7).DesignBy broth microdilution method, different concentrations of the extract (250–0.4 mg/mL) were used in order to determine the minimum microbicidal concentration (MMC) in planktonic cultures and the most effective concentration was used on biofilms on discs made of acrylic resin. The cytotoxicity A. lappa L. extract MMC was evaluated on RAW 264.7 by MTT assay and the quantification of IL-1β and TNF-α by ELISA.ResultsThe most effective concentration was 250 mg/mL and also promoted significant reduction (log₁₀) in the biofilms of S. aureus (0.438 ± 0.269), S. epidermidis (0.377 ± 0.298), S. mutans (0.244 ± 0.161) and C. albicans (0.746 ± 0.209). Cell viability was similar to 100%. The production of IL-1β was similar to the control group (p > 0.05) and there was inhibition of TNF-α (p < 0.01).ConclusionsA. lappa L. extract was microbicidal for all the evaluated strains in planktonic cultures, microbiostatic for biofilms and not cytotoxic to the macrophages.     

Cytotoxicity and potential anti-inflammatory activity of velutin on RAW 264.7 cell line differentiation: Implications in periodontal bone loss

ObjectivesHypoxia-inducible factor-1α (HIF-1α) has been implicated in periodontal tissue inflammation and possibly in osteoclast differentiation, while polyphenols are known to be anti-inflammatory natural compounds that are capable of regulating the NF-κB protein complex pathway. The objective of this study was to investigate cytotoxicity and HIF-1α expression through the NF-κB pathway by polyphenol velutin (Euterpe oleracea Mart.), found in the pulp of acai fruit, during inflammatory RAW 264.7 differentiation.DesignRAW 264.7 mouse monocyte macrophage cells were stimulated with RANKL (30 ng/mL) and Porphyromonas gingivalis lipopolysaccharide (1 μg/mL). Cells were treated with various concentrations of velutin (0.5–2 μM) to check for viability, morphology, osteoclast differentiation, and HIF-1α expression (Western blot).ResultsAlamar blue cell viability assay showed no toxicity to RAW cells with the use of velutin in all concentrations tested (p > 0.05). Velutin did not induce cell apoptosis based on caspase 3/7 assay (p > 0.05). Fluorescence images stained by DAPI showed no alteration in the morphology of RAW cell nuclei (p > 0.05) treated with velutin. TRAP assays demonstrated a dose-dependent reduction in osteoclast formation by velutin when compared with control (p < 0.05). Velutin showed a reduction in HIF-1α expression related to IκB phosphorylation when compared with control (p < 0.001).ConclusionsAt the tested concentrations, velutin was not cytotoxic to RAW 264.7 and differentiated cells. Velutin reduced osteoclast differentiation and downregulated HIF-1α through the NF-κB pathway.     

Salivary pellicles equalise surfaces’ charges and modulate the virulence of Candida albicans biofilm

IntroductionNumerous environmental factors influence the pathogenesis of Candida biofilms and an understanding of these is necessary for appropriate clinical management.AimsTo investigate the role of material type, pellicle and stage of biofilm development on the viability, bioactivity, virulence and structure of C. albicans biofilms.MethodsThe surface roughness (SR) and surface free energy (SFE) of acrylic and titanium discs was measured. Pellicles of saliva, or saliva supplemented with plasma, were formed on acrylic and titanium discs. Candida albicans biofilms were then generated for 1.5 h, 24 h, 48 h and 72 h. The cell viability in biofilms was analysed by culture, whilst DNA concentration and the expression of Candida virulence genes (ALS1, ALS3 and HWP1) were evaluated using qPCR. Biofilm metabolic activity was determined using XTT reduction assay, and biofilm structure analysed by Scanning Electron Microscopy (SEM).ResultsWhilst the SR of acrylic and titanium did not significantly differ, the saliva with plasma pellicle increased significantly the total SFE of both surface. The number of viable microorganisms and DNA concentration increased with biofilm development, not differing within materials and pellicles. Biofilms developed on saliva with plasma pellicle surfaces had significantly higher activity after 24 h and this was accompanied with higher expression of virulence genes at all periods.ConclusionInduction of C. albicans virulence occurs with the presence of plasma proteins in pellicles, throughout biofilm growth. To mitigate such effects, reduction of increased plasmatic exudate, related to chronic inflammatory response, could aid the management of candidal biofilm-related infections.     

Effect of different pre-irradiation times on curcumin-mediated photodynamic therapy against planktonic cultures and biofilms of Candida spp

ObjectivesThe aim of this study was to evaluate the effects of pre-irradiation time (PIT) on curcumin (Cur)-mediated photodynamic therapy (PDT) against planktonic and biofilm cultures of reference strains of Candida albicans, Candida glabrata and Candida dubliniensis.Materials and methodsSuspensions and biofilms of Candida species were maintained in contact with different concentrations of Cur for time intervals of 1, 5, 10 and 20 min before irradiation and LED (light emitting diode) activation. Additional samples were treated only with Cur, without illumination, or only with light, without Cur. Control samples received neither light nor Cur. After PDT, suspensions were plated on Sabouraud Dextrose Agar, while biofilm results were obtained using the XTT-salt reduction method. Confocal Laser Scanning Microscopy (CLSM) observations were performed to supply a better understanding of Cur penetration through the biofilms after 5 and 20 min of contact with the cultures.ResultsDifferent PITs showed no statistical differences in Cur-mediated PDT of Candida spp. cell suspensions. There was complete inactivation of the three Candida species with the association of 20.0 μM Cur after 5, 10 and 20 min of PIT. Biofilm cultures showed significant reduction in cell viability after PDT. In general, the three Candida species evaluated in this study suffered higher reductions in cell viability with the association of 40.0 μM Cur and 20 min of PIT. Additionally, CLSM observations showed different intensities of fluorescence emissions after 5 and 20 min of incubation.ConclusionPhotoinactivation of planktonic cultures was not PIT-dependent. PIT-dependence of the biofilm cultures differed among the species evaluated. Also, CLSM observations confirmed the need of higher time intervals for the Cur to penetrate biofilm structures.     

Impact of titanium ions on osteoblast-, osteoclast- and gingival epithelial-like cells

PurposeTo investigate the effects of titanium (Ti) ions on the cell viability, the cell differentiation and the gene expressions related to bone resorption including Receptor Activator of NF-κB Ligand (RANKL) and Osteoprotegerin (OPG) in the tissues around dental implants, the osteoblast-, osteoclast-, and gingival epithelial-like cells were exposed to Ti ions.MethodsAn MTS assay was carried out to evaluate the viabilities of osteoblast-like MC3T3-E1, osteoclast-like RAW264.7 and epithelial cell-like GE-1 cells. The gene expressions in these cells were analyzed by the use of RT-PCR and real-time quantitative RT-PCR.ResultsTi ions in the concentration range 1–9 ppm had little effect on the viabilities of MC3T3-E1, RAW264.7 and GE-1, whereas 20 ppm Ti ions significantly decreased the viabilities of all cells. Analyses of RT-PCR and real-time quantitative RT-PCR data revealed that Ti ions at 9 ppm remarkably inhibited the expressions of Runx2, Osterix and type I collagen in MC3T3-E1. In RAW264.7, Ti ions showed no effects on the levels of mRNAs for TRAP and cathepsin K enhanced by RANKL. Ti ions at the range of 1–9 ppm showed no effects on the levels of mRNAs for RANKL and OPG in GE-1, while Ti ions at 9 ppm enhanced the expression of these genes in MC3T3-E1.ConclusionsThese results, taken together, suggested that Ti ions show the biological effects, both on the viabilities of osteoblast and osteoclast and on the differentiation of either the osteoblastic or osteoclastic cells, which may influence the prognosis of dental implants.     

Effect of pH on Galla chinensis extract's stability and anti-caries properties in vitro

ObjectivesConsidering that Galla chinensis extract (GCE) solution has a low pH, which might dissolve dental enamel, we investigated the effects of elevation of pH on GCE stability, and on its anti-caries properties.DesignsStability of GCE solutions, either in H₂O (pH less than 4.0) or when buffered at pH 5.5, 7.0 and 10.0, was assessed from UV–VIS spectra. Inhibition of enamel demineralization was determined in a pH-cycling set up, comprising treatments with either GCE solutions or negative control buffers and acid and neutral buffer immersions. Demineralization was assessed by calcium in the acetate buffers. To determine antimicrobial properties, polymicrobial biofilms were formed after saliva inoculation on glass surfaces which were treated after 48 h. Treatment output parameters were lactic acid formation and viability, the latter by colony forming unit (CFU) counts.ResultsAt pH 7.0 and higher GCE solutions changed colour and absorption spectra in UV–VIS, indicative of chemical changes. Regarding enamel demineralization, significant inhibitions (P < 0.05) were found for all GCE treatments when compared with corresponding controls. In polymicrobial biofilms, GCE reduced the acid production, compared with the negative controls (P < 0.05). However, this difference was only significant at the lower pH values.ConclusionsGCE solutions were unstable under neutral and alkaline conditions. pH did not significantly influence the inhibiting effect of GCE on enamel demineralization. However, GCE was not effective on polymicrobial biofilms at alkaline pH (8.5). To avoid enamel damage due to acidic treatment, GCE solutions should be used at about pH 5.5.     

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.     

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.     

Effect of iRoot SP and mineral trioxide aggregate (MTA) on the viability and polarization of macrophages

ObjectiveThis study was performed to investigate the effect of iRoot SP and mineral trioxide aggregate (MTA) on the viability and polarization of macrophages.MethodsThe effect of iRoot SP and MTA on the viability of RAW 264.7 macrophages was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 1 and 2 days of culture. The gene expression levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), interleukin 12p40 (IL-12p40) were measured by quantitative real time polymerase chain reaction (qRT-PCR) after stimulation of the RAW 264.7 macrophages with iRoot SP and MTA. The expression levels of CD11c and CD206 in RAW 264.7 macrophages were examined by immunofluorescence and flow cytometry after stimulation with iRoot SP and MTA. The data were analyzed by one-way analysis of variance and the Tukey test.ResultsBoth iRoot SP and MTA were non-toxic to the RAW 264.7 macrophages. The use of iRoot SP and MTA increased the expression of IL-1β, TNF-α, IL-10, IL-12p40 on the first day of culture and could promote macrophage M1 and M2 polarization.ConclusionsMTA and iRoot SP have good biocompatibility with macrophages, and they induced both M1 and M2 polarization of the RAW 264.7 macrophages.     

Effect of culture media and nutrients on biofilm growth kinetics of laboratory and clinical strains of Enterococcus faecalis

ObjectiveEnterococcus faecalis is a bacterial pathogen that is often associated with endodontic infections. Biofilm formation is a key virulence attribute in the pathogenicity of E. faecalis. In the present study, we comprehensively examined the effect of various culture media and nutrients on the development of E. faecalis biofilms.DesignA reference strain and a clinical isolate of E. faecalis were used in all experiments for comparison. Commonly used liquid culture media with different nutrient compositions were used to support the development of E. faecalis biofilms in a time-dependent assay. E. faecalis biofilms were quantified by colony forming unit (CFU) and crystal violet (CV) assays. Biofilm architecture and cellular viability were evaluated by scanning electron microscopy and confocal laser scanning microscopy.ResultsGrowth kinetics evaluated by CFU and CV assays and by microscopy showed that E. faecalis biofilms reached maturity at 72 h. “Pg broth” (Tryptic Soy Broth with yeast extract, hemen and vitamin K) promoted E. faecalis biofilm formation more than Brain Heart Infusion broth or Tryptic Soy Broth. Addition of 2% glucose enhanced biofilm formation. Thus, it seems that nutrients such as hemen, vitamin K and glucose are important for E. faecalis for the formation of biofilms.ConclusionThe present study demonstrated that nutrient-rich media containing glucose enhances the formation of E. faecalis biofilms, which exhibit maturation at 72 h.     

Effects of heat-inactivated Bifidobacterium BB12 on cariogenicity of Streptococcus mutans in vitro

ObjectiveSince some probiotic bacteria are cariogenic themselves, their suitability for caries management is questionable. Inactivated bacteria or their supernatants have been found to exert probiotic effects, whilst having several advantages compared with living bacteria. We hypothesized that viable and heat-inactivated Bifidobacterium animalis BB12 reduces the cariogenicity of Streptococcus mutans (SM) in vitro.DesignWe assessed mono- and mixed species biofilms of SM and viable or heat-inactivated BB12. Biofilms were grown in a continuous-culture-system under cariogenic conditions on smooth proximal enamel or cavitated dentine. For each of eight experimental subsets (4 biofilms × 2 hard-tissue conditions), a total of 32 specimens was used. After 10 days, bacterial numbers of 12 biofilms per group were analysed, and all specimens submitted to transversal microradiography.ResultsMineral loss was higher in cavitated dentine than smooth enamel for all biofilms (p < 0.001, t-test). BB12-monospecies biofilms induced significantly less mineral loss than SM in both enamel (p < 0.05) and dentine (p < 0.001). Viable BB12 did not significantly reduce cariogenicity of SM (p > 0.05), whilst heat-inactivated BB12 decreased cariogenicity of SM in dentinal cavities (p < 0.01). Bacterial numbers were higher on dentine than enamel (p < 0.05), but not significantly influenced by biofilm species (p > 0.05).ConclusionsHeat-inactivated BB12 reduced the cariogenicity of SM in dentinal cavities in vitro. Inactivated probiotics might be suitable for caries control.     

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.     

De novo synthetic short antimicrobial peptides against cariogenic bacteria

ObjectiveAntimicrobial peptides (AMPs) have shown the ability to inhibit planktonic bacteria and biofilms. The objectives of this study were to de novo design and synthesize a series of cationic, amphipathic α-helical AMPs that would be shorter, less cytotoxic, and more potent than existing AMPs against cariogenic bacteria.DesignThree short AMPs (GH8, GLLWHLLH-NH₂; GH12, GLLWHLLHHLLH-NH₂; and GH16, GLLWHLLHHLLHLLHH-NH₂) were designed, synthesized and characterized structurally. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against eight major cariogenic bacteria were tested to select the most promising peptide. Scanning electron microscopy (SEM) was used to observe the bacterial membrane after treatment with selected peptides. The bactericidal kinetics, effects on biofilm and cytotoxity were further investigated.ResultsOf the three AMPs, GH12 had the most balanced structural parameters and a high content of α-helical structure. GH12 had a MIC of 4.0-8.0 μg/mL and MBC of 8.0-32.0 μg/mL. The corresponding values for the other two AMPs were 2- to 64- fold higher. In time-kill assays, GH12 killed all bacterial strains within 60 min at 4- fold MBC. SEM observed lysis and pore formation of the cytomembrane after treatment with GH12. 8.0 μg/mL GH12 inhibited Streptococcus mutans biofilm formation. Confocal laser scanning microscopy showed that GH12 effectively reduced the biomass of 1-day-old S. mutans biofilm. Cytotoxicity assays indicated that GH12 showed little toxic effect on the viability of human gingival fibroblasts.ConclusionThese results indicate that GH12 shows antimicrobial activity against cariogenic bacteria and biofilms in vitro.     

Cytotoxicity,genotoxicity and antibacterial activity of poly(vinyl alcohol)-coated silver nanoparticles and farnesol as irrigating solutions

ObjectiveTo evaluate the cytotoxicity, genotoxicity and antibacterial activity of poly(vinyl alcohol)-coated silver nanoparticles (AgNPs-PVA) and farnesol (FAR).DesignThe cytotoxicity (% of cell viability) was evaluated by MTT assay and the genotoxicity (% of DNA in the tail) was evaluated by Comet assay. Root canal disinfection with different irrigating protocols was evaluated ex vivo in human teeth contaminated with Enterococcus faecalis for 21 days. Three microbiological samples were collected: initial (after contamination); post-irrigation (after irrigation); and final (after 7 days). After each sample, the number of log 10 CFU mL⁻¹ was determined. Statistical analyses was performed using two-way ANOVA and Bonferroni post-hoc tests for MTT assay, Kruskal-Wallis and Dunn post-hoc tests for Cometa and antibacterial assays (α = 0.05).ResultsThe MTT assay showed that AgNPs and FAR were less cytotoxic that sodium hypochlorite (NaOCl) and showed a lower% of DNA in the tail, in comparison with H₂O₂ (positive control − C+). In the post-irrigation microbiological sample, all the irrigating protocols were more effective than C+ (without irrigation). NaOCl/saline, NaOCl/saline/AgNPs-PVA and NaOCl/saline/FAR led to complete bacterial elimination (p > 0.05). In comparison with the initial sample, both the post-irrigation and the final samples showed microbial reduction (p < 0.05).ConclusionsAgNPs-PVA and FAR showed low cytotoxicity and genotoxicity, and exhibit potential for use as a final endodontic irrigation protocols.     

Evaluation of the Candida albicans removal and mechanical properties of denture acrylics cleaned by a low-cost powered toothbrush

PurposeTo investigate the effects of using a low-cost powered toothbrush for cleaning on dental prostheses made of heat polymerized poly(methyl methacrylate), PMMA.MethodsHeat cured PMMA specimens beam with the dimensions of 45.0 mm × 6.5 mm × 4.5 mm were fabricated. The specimens were kept in water storage at 37 °C constant temperature for 0, 1, 7, 15, 30 and 60 days and randomly assigned for testing or control. Test specimens underwent brushing by using a powered toothbrush at an applied force of 2.00 N for 22 min with water as medium. Surface roughness measurement (R_a), flexural strength and efficacy of brushing to remove coated Candida albicans biofilm were investigated.ResultsThe results of the mean surface roughness value and the flexural strength were analysed by using two-way ANOVA and Tukey post hoc test at 5% significance level. In general, the specimens showed no significant changes in flexural strength after brushing. However, the flexural strength and the surface roughness value were significantly lower in specimens group after 7 days in water storage compared to the control. SEM micrographs of post-brushed specimens revealed satisfactory removal of C. albicans biofilm.ConclusionA low-cost powered toothbrush together with a liquid medium successfully removed C. albicans biofilm on dental acrylic PMMA-based prostheses, without compromising the mechanical properties.     

The effect of subinhibitory concentrations of gentian violet on the germ tube formation by Candida albicans and its adherence to oral epithelial cells

ObjectiveThis study investigated the effect of subinhibitory concentrations of gentian violet on the germ tube formation by Candida albicans and its adherence ability to oral epithelial cells.MethodsThirty strains of C. albicans isolated from denture wearers, normal healthy individuals and HIV positive patients were used in the study. The antifungal property (Minimum Fungicidal Concentration) of gentian violet was determined at various time intervals using a microdilution technique. The effect of subinhibitory concentrations of gentian violet on the adherence ability (0.000244%) and on germ tube formation ((0.000244%, 0.000122%, 0.000061% and 0.000031%) was determined. In both experiments, water was used as a control. The test results were compared using the Kruskal-Wallis test.ResultsAt 60 min a high concentration (0.0078%) of gentian violet was required to completely kill C. albicans. Subinhibitory concentrations of gentian violet significantly reduced the adherence ability of C. albicans by 57% (p < 0.01) and equally inhibited germ tube formation (p < 0.01) compared with the controls. The inhibition was concentration dependent, with up to 98% reduction at a concentration of 0.000244%. Germ tube reduction was significantly higher in the isolates from the HIV positive patients than in the isolates from denture wearers.ConclusionAt high concentrations, gentian violet killed C. albicans, whereas at subinhibitory concentrations it reduced its virulence by preventing the adherence ability and germ tube formation. This suggests that the beneficial effects of gentian violet would last beyond the fungicidal concentrations in the treatment of candidiasis.     

Evaluation of antibiofilm and mechanical properties of new nanocomposites based on acrylic resins and silver vanadate nanoparticles

ObjectiveThe purpose of this study was evaluate, for the first time, the impact of incorporation of nanostructured silver vanadate (β-AgVO₃) in antibiofilm and mechanical properties of dental acrylic resins (poly(methyl methacrylate), PMMA).DesignThe β-AgVO₃ was synthesized and characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy, and microanalysis (SEM/EDS). Resins specimens were prepared with 0–10% wt.% β-AgVO₃ and characterized by SEM, XRD and optical microscopy_. The antibiofim activity of the samples against Candida albicans and Streptococcus mutans was investigated by XTT reduction test, colony-forming units (CFUs), and confocal laser scanning microscopy (CLSM). The flexural strength, hardness, and surface roughness of the samples containing β-AgVO₃ were compared with the pure PMMA matrix.ResultsThe incorporation of 10% β-AgVO₃ significantly reduced the metabolic activity of C. albicans and S. mutans (p < 0.05). There was a reduction in microbial load (CFU/mL) of microorganisms for the different concentrations used (p < 0.05), which was confirmed by confocal microscopy. The addition of β-AgVO₃ did not change the mechanical properties of hardness and surface roughness of the resins (p > 0.05). However, flexural strength decreased with the addition of amounts greater than 1% (p < 0.05).Conclusionsβ-AgVO₃ additions in dental acrylic resin may have an impact on inhibition of biofilm of main microorganisms associated with dental prostheses. However, the viability of clinical use should be evaluated in function of changed promoted in some mechanical properties.     

An in vitro study on the anti-adherence effect of Brucea javanica and Piper betle extracts towards oral Candida

ObjectiveThe adherence of Candida to mucosal surfaces is the initial step for successful invasive process of the oral cavity. The study aimed to investigate the effect of two plant extracts on the non-specific and specific bindings of oral candida.MethodsIn the former, adsorption to hexadecane was used to measure the hydrophobic interaction of the candida cells. In the later, glass beads coated with saliva represented the experimental pellicles in specific adhesion of oral candida to hard tissue surface.ResultsCandida krusei, Candida dubliniensis and Candida tropicalis showed the highest adsorption to hexadecane at 30.23%, 26.19% and 19.70%, respectively, while the others within the range of 7–10%. All candidal species were significantly affected by the extracts (P < 0.05) with Brucea javanica exhibited more than 60% reduction of CSH than Piper betle. Candida parapsilosis showed the highest affinity in specific-bindings to pellicle with 18.72 ± 0.71 × 10⁵ CFU/ml. Exposing to P. betle-treated pellicle has drastically reduced the adherence of C. tropicalis, Candida albicans and C. krusei by 86.01%, 61.41% and 56.34%, respectively. B. javanica exhibited similar effect on C. tropicalis (89.86%), Candida lusitaniae (88.95%), C. albicans (79.74%), Candida glabrata (76.85%) and C. krusei (67.61%).ConclusionThe extracts demonstrated anti-adherence activities by modifying the CSH and the characteristics of the experimental pellicle.     

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.     

Cytotoxicity and proliferative effects of Iodoform-containing root canal-filling material on RAW 264.7 macrophage and RKO epithelial cell lines

ObjectiveThe present study investigated the effect of the Iodoform-containing root canal filling material on the viability of cultured macrophages and epithelial cells, and on cytokine secretion.DesignThe effect of Endoflas F.S. on the proliferation of a RAW 264.7 macrophage cell line and on a RKO epithelial cell line, and on the production of tumour necrosis factor alpha (TNFα) from macrophages was examined. Cell vitality was evaluated using a colourimetric XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) assay. The presence of cytokines was determined by two-site enzyme-linked immunosorbent assay (ELISA).ResultsDirect exposure of Endoflas F.S. and its media, up to a dilution of 1/8, decreased the viability of macrophages and epithelial cells by ～70% compared to control media (P < 0.05). Media dilution from 1/16 to 1/1024 demonstrated a proliferative effect, increasing cell viability by about 60% compared to media without Iodoform-containing root canal filling material.ConclusionsDirect and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation.