Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal ß-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

Antimicrobial efficacy of DJK-5 peptide in combination with EDTA against biofilms in dentinal tubules: Primary irrigation, recovery and re-irrigation.

AIM: To investigate the dynamic recovery of biofilms within dentinal tubules after primary irrigation with different protocols, and to evaluate the efficacy of various re-irrigation protocols on recovered biofilm, considering factors such as smear layer, nutrient conditions, and primary irrigants. METHODOLOGY: A total of 416 mono or multi-species biofilms samples were prepared from human teeth and incubated for 3 weeks. After inducing a smear layer on half of the samples, all specimens were irrigated with one of the following irrigant sequences: (1) 6% NaOCl +17% EDTA, (2) 6% NaOCl +8.5% EDTA, (3) 6% NaOCl and (8.5% EDTA +10 µg/mL DJK-5 antimicrobial peptide), or (4) sterile water. Thirty-two samples were used to assess immediate effect, whilst the rest were re-incubated to assess biofilms recovery. Nutrient conditions were defined based on whether culture media were changed (nutrient-rich) or not (nutrient-poor) during re-incubation. After 16 weeks, recovered biofilms underwent re-irrigation using four additional protocols, with or without DJK-5 peptide, based on primary irrigants. Confocal laser scanning microscopy was employed to evaluate immediate irrigant effects, biofilms recovery intervals (1, 3, 5, 8, 12, and 16 weeks after primary irrigation), and re-irrigation effects at the 16-week. Statistical analysis included one-way anova and two-way mixed anova tests. RESULTS: The DJK-5 peptide irrigation protocols demonstrated the highest killing rates during primary irrigation and resulted in a longer biofilms recovery time of 16 weeks compared to non-peptide protocols (p < .001). Both primary irrigation type and smear layer presence significantly influenced biofilms recovery (p < .001). In the absence of smear layer, re-irrigation efficacy didn't significantly differ from primary irrigation, regardless of primary irrigation type or nutrient conditions. However, with a smear layer present, re-irrigation led to significantly higher proportion of dead bacteria compared to primary irrigation (p < .05). Inclusion of the DJK-5 peptide into the re-irrigation protocol displayed superior killing rate compared to other protocols (p < .001). CONCLUSIONS: Biofilms exhibited susceptibility to both peptide and non-peptide protocols during re-irrigation, irrespective of nutrient conditions or primary irrigation protocols. The DJK-5 peptide irrigation protocols consistently displayed superior effectiveness compared to non-peptide protocols.

The effect of laser therapy for the treatment of dentin hypersensitivity on surface roughness and bacterial adhesion.

The aim of the study was to measure the degree of dentine surface roughness caused by five distinct lasers used to treat dentine hypersensitivity, as well as to evaluate the subsequent bacterial colonization on these irradiated surfaces. Sixty human maxillary premolar teeth without caries or restoration which were extracted for periodontal reasons were used in this study. Five different types of lasers were applied to the root dentin surface. Tested samples were divided into six groups of 10 samples each; control, diode (810 nm), diode (980 nm), Nd: YAG, Er: YAG, and Er, Cr: YSGG laser groups. The arithmetic mean of the surface roughness values (Ra) and the average roughness over a measurement area (Sa) were measured pre- and post-application using any of the laser types. Swab samples were then collected from the dentin surface. Following a 24-hour incubation period at 37 °C, the colony forming units were counted using a stereoscope. The results demonstrated a statistically significant difference in the surface roughness values pre- and post-application (Ra and Sa, respectively) in the Er, Cr: YSGG laser group (p = 0.037,p = 0.007). No significant difference was observed in the other groups (p > 0.05). There was no statistically significant difference in the number of bacterial colonies observed between the test and control groups. Diode and Nd: YAG lasers showed either a decrease or no change in surface roughness; however, the hard tissue lasers (Er: YAG, Er, Cr: YSGG) showed an increase. The Er: YAG and Nd: YAG laser groups exhibited decreased bacterial adhesion compared to the other groups.

In-vitro effects of novel periodontal scalers with a planar ultrasonic piezoelectric transducer on periodontal biofilm removal, dentine surface roughness, and periodontal ligament fibroblasts adhesion.

OBJECTIVES: To compare ultrasonic scaler prototypes based on a planar piezoelectric transducer with different working frequencies featuring a titanium (Ti-20, Ti-28, and Ti-40) or stainless steel (SS-28) instrument, with a commercially available scaler (com-29) in terms of biofilm removal and reformation, dentine surface roughness and adhesion of periodontal fibroblasts. MATERIALS AND METHODS: A periodontal multi-species biofilm was formed on specimens with dentine slices. Thereafter specimens were instrumented with scalers in a periodontal pocket model or left untreated (control). The remaining biofilms were quantified and allowed to reform on instrumented dentine slices. In addition, fibroblasts were seeded for attachment evaluation after 72 h of incubation. Dentine surface roughness was analyzed before and after instrumentation. RESULTS: All tested instruments reduced the colony-forming unit (cfu) counts by about 3 to 4 log10 and the biofilm quantity (each p < 0.01 vs. control), but with no statistically significant difference between the instrumented groups. After 24-hour biofilm reformation, no differences in cfu counts were observed between any groups, but the biofilm quantity was about 50% in all instrumented groups compared to the control. The attachment of fibroblasts on instrumented dentine was significantly higher than on untreated dentine (p < 0.05), with the exception of Ti-20. The dentine surface roughness was not affected by any instrumentation. CONCLUSIONS: The planar piezoelectric scaler prototypes are able to efficiently remove biofilm without dentine surface alterations, regardless of the operating frequency or instrument material. CLINICAL RELEVANCE: Ultrasonic scalers based on a planar piezoelectric transducer might be an alternative to currently available ultrasonic scalers.

Assessment of multispecies biofilm growth on root canal dentin under different radiation therapy regimens.

OBJECTIVES: To assess the growth of a multispecies biofilm on root canal dentin under different radiotherapy regimens. MATERIALS AND METHODS: Sixty-three human root dentin cylinders were distributed into six groups. In three groups, no biofilm was formed (n = 3): NoRT) non-irradiated dentin; RT55) 55 Gy; and RT70) 70 Gy. In the other three groups (n = 18), a 21-day multispecies biofilm (Enterococcus faecalis, Streptococcus mutans, and Candida albicans) was formed in the canal: NoRT + Bio) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. The biofilm was quantified (CFUs/mL). Biofilm microstructure was assessed under SEM. Microbial penetration into dentinal tubules was assessed under CLSM. For the biofilm biomass and dentin microhardness pre- and after biofilm growth assessments, 45 bovine dentin specimens were distributed into three groups (n = 15): NoRT) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. RESULTS: Irradiated specimens (70 Gy) had higher quantity of microorganisms than non-irradiated (p = .010). There was gradual increase in biofilm biomass from non-irradiated to 55 Gy and 70 Gy (p < .001). Irradiated specimens had greater reduction in microhardness after biofilm growth. Irradiated dentin led to the growth of a more complex and irregular biofilm. There was microbial penetration into the dentinal tubules, regardless of the radiation regimen. CONCLUSION: Radiotherapy increased the number of microorganisms and biofilm biomass and reduced dentin microhardness. Microbial penetration into dentinal tubules was noticeable. CLINICAL RELEVANCE: Cumulative and potentially irreversible side effects of radiotherapy affect biofilm growth on root dentin. These changes could compromise the success of endodontic treatment in oncological patients undergoing head and neck radiotherapy.

Antimicrobial action, cytotoxicity and erosive potential of hypochlorous acid obtained from an electrolytic device compared with sodium hypochlorite.

OBJECTIVES: This study aimed to compare the antimicrobial action, cytotoxicity, cleaning ability, and erosion of dentine of hypochlorous acid (HClO) obtained from an electrolytic device at two different concentrations (Dentaqua) and three concentrations of sodium hypochlorite (NaOCl). METHODS: Microbiological test-The root canals of sixty single-rooted extracted human teeth were inoculated with Enterococcus faecalis and divided into 6 groups (n = 10), according to decontamination protocol: DW (control); 1% NaOCl; 2.5% NaOCl; 5.25% NaOCl; 250 ppm HClO and 500 ppm HClO. The colony-forming units were counted to evaluate the decontamination potential of each group, calculating the reduction in bacterial percentage. Cytotoxicity test-Cytotoxicity was evaluated after inoculation of the same tested protocols in fibroblastic cells for 3 min, calculating the cell viability percentages. Specifical statistical analysis was performed (α = 5%). Cleaning ability and erosion-Fifty-six single-rooted bovine lower incisors were divided into seven groups of 8 roots each, being the test groups 1% NaOCl; 2.5% NaOCl; 5,25% NaOCl; 250 ppm HClO and 500 ppm HClO, and a negative and positive control. Negative control was not contaminated, and the other groups were inoculated with Enterococcus faecalis. SEM images were ranked as from the cleanest to the least clean. Erosion was also assessed, being ranked from the least to the most eroded dentine. RESULTS: The highest bacterial reduction was observed in experimental groups, with no statistical differences between them (p > 0.05). The highest number of viable cells was observed in control group, followed by 250 ppm HClO and 500 ppm HClO groups, with statistical differences between them (p < 0.05). 1% NaOCl; 2.5% NaOCl; 5.25% NaOCl and 500 ppm HClO displayed the cleanest areas. All sodium hypochlorite groups displayed erosion with higher ranks with greater concentration, while hypochlorous acid did not display any erosion regardless the concentration. CONCLUSIONS: It is possible to conclude that HClO obtained from an electrolytic device presented high antimicrobial activity and low cytotoxicity in both tested concentrations. 500 ppm HClO did not display erosion and showed great cleaning ability. CLINICAL RELEVANCE: The use of 500 ppm hypochlorous acid may reduce unfavorable behavior of sodium hypochlorite whilst maintaining its antimicrobial action.

Sodium hypochlorite against Enterococcus faecalis biofilm in dentinal tubules: effect of concentration, temperature, and exposure time.

This study aimed to evaluate the effectiveness of two sodium hypochlorite concentrations at different exposure times and temperatures against Enterococcus faecalis biofilms of varying ages in human dentinal tubules. Dentin blocks were infected with E. faecalis for either 3 days or 3 weeks of incubation. Subsequently, the samples were exposed to sterile water, 2%, and 5.25% sodium hypochlorite for 3 and 10 min at 20 °C and 60 °C . Viability staining and confocal laser scanning microscopy were used to assess the proportion of killed bacteria in the dentinal tubules after exposure. There are no significant differences in the efficacy of E. faecalis killing between 2% sodium hypochlorite at 60 °C for various exposure times and 5.25% sodium hypochlorite at different temperatures or exposure times (P > 0.05). When both solutions were compared at the same temperatures with a 10-min exposure time, no significant differences in the effectiveness of E. faecalis killing between 2% and 5.25% sodium hypochlorite were observed (P > 0.05). To optimize the effectiveness of sodium hypochlorite in killing E. faecalis while minimizing potential damage to root dentin and soft tissue, clinicians should prioritize increasing the temperature or exposure time of sodium hypochlorite, rather than raising its concentration.

The effect of the enzymes trypsin and DNase I on the antimicrobial efficiency of root canal irrigation solutions.

The purpose of this study was to evaluate the antibacterial efficacy of using 2.5% NaOCl, 2% chlorhexidine (CHX), Irritrol, and chitosan-coated silver nanoparticles (AgCNPs) alone or in combination with deoxyribonuclease I (DNase I) and trypsin pre-enzyme applications in dentin samples contaminated with Enterococcus faecalis (E. faecalis) by CLSM. 144 dentin blocks with confirmed E. faecalis biofilm formation were divided randomly according to the irrigation protocol (n = 12): NaOCl, CHX, Irritrol, AgCNPs, trypsin before NaOCl, CHX, Irritrol, AgCNPs, and DNase I before NaOCl, CHX, Irritrol, AgCNPs. Dentin blocks were stained with the Live/Dead BacLight Bacterial Viability Kit and viewed with CLSM after irrigation applications. The percentage of dead and viable bacteria was calculated using ImageJ software on CLSM images. At a significance level of p < 0.05, the obtained data were analyzed using one-way Anova and post-hoc Tukey tests. In comparison with NaOCl, CHX had a higher percentage of dead bacteria, both when no pre-enzyme was applied and when DNase I was applied as a pre-enzyme (p < 0.05). There was no difference in the percentage of dead bacteria between the irrigation solutions when trypsin was applied as a pre-enzyme (p > 0.05). AgCNPs showed a higher percentage of dead bacteria when trypsin was applied as a pre-enzyme compared to other irrigation solutions (p < 0.05), while the pre-enzyme application did not affect the percentage of dead bacteria in NaOCl, CHX, and Irritrol (p > 0.05). No irrigation protocol tested was able to eliminate the E. faecalis biofilm. While the application of trypsin as a pre-enzyme improved the antimicrobial effect of AgCNPs, it did not make any difference over other irrigation solutions.

Comparative analysis of microbiome in coronal and root caries.

BACKGROUND: The global rise in the elderly population has increased the prevalence of root caries. Streptococcus mutans, Lactobacilli and Actinomyces are considered the primary pathogens of dental caries in culture-based studies. This study aimed to investigate bacterial profiles in coronal and root caries lesions and determine the association of specific bacterial genera at each site. METHODS: Dentine samples from carious lesions were collected from 22 extracted teeth using an excavator. Microbial DNA was extracted from the samples using a protocol developed for this study. 16S rRNA gene amplicon sequencing was employed for microbial analysis. PCR amplification targeted the V3-V4 region of the bacterial 16S rRNA, and the amplicon sequencing used an Illumina MiSeq system (2 × 300 bp paired-end reads). Statistical analysis was performed by the Phyloseq and DESeq2 packages in R. RESULTS: In coronal caries, Olsenella, Lactobacillus and Prevotella were the most prevalent genera, comprising approximately 70% of the microbiome community. In the root caries, however, although Olsenella, Prevotella and Lactobacillus remained the dominant genera, they accounted for only half of the microbiome community. This study identified significant differences in alpha diversity indices between the coronal and root caries. LEfSE analysis revealed several unique genera in each caries lesion. CONCLUSION: The microbiome of root caries lesions was richer and more complex than the coronal caries microbiota. The results suggest that lesion-related variations in the oral microflora may be detected in carious dentine.

Tracing of Microbes in Prepared Cavity Following Different Minimally Invasive Caries Removal Protocols.

AIMS: The conventional caries removal technique has been replaced with minimally invasive (MI) techniques to preserve healthy natural teeth and to provide durable dental restorations. Each of these MI caries removal protocols is reported to be favorable in dealing with different caries conditions. The current study aimed to trace the residual bacteria that may remain in a prepared cavity following a visual-tactile (VT), caries detection dye (CDD), and chemo-mechanical caries removal (CMCR) protocol. MATERIALS AND METHODS: A total of 29 extracted human molar teeth with visible caries lesions were randomly divided into three groups. The cavity preparation and caries removal of each group was accomplished following one of the MI caries removal protocols. Swab samples (one from each specimen) were taken and inoculated onto a blood agar plate and incubated for 48 hours. The growth of the bacterial colony was observed under a microscope and the specific genome of the bacteria was identified by polymerase chain reaction (PCR) test. RESULTS: The maximum number of traceable bacteria was observed following the chemo-mechanical caries removal group followed by the caries detection dye group and the least in the visual-tactile group. The PCR test revealed the presence of Streptococcus mutans in all the observed colonies; however, Streptococcus sobrinus was absent completely. The Chi-square test reveals a statistically insignificant (p = 0.646) difference among the tested groups. CONCLUSION: All of the MI caries removal protocols used in this study showed a trace of microbes in certain teeth. The cavity prepared following a visual tactile protocol showed the least amount of traceable bacteria in the prepared cavity. CLINICAL SIGNIFICANCE: Cavity that is prepared following individual MI protocol has a risk of leaving microbes in it.

Effect of high-fluoride dentifrice on root dentine and bacterial composition in a multispecies biofilm model.

The present study evaluated the effect of high-fluoride dentifrice on dentine demineralization and bacterial composition in a multispecies biofilm model in vitro. A seven-organism bacterial consortium was grown on bovine dentine discs in a high-throughput active attachment model. The biofilms were submitted twice per day to the following dentifrices treatments: 5,000 ppm F, 1,100 ppm F, with placebo as a negative control. After 5 days of biofilm growth, dentine samples were assessed by transversal microradiography, the biofilm was collected for bacterial counts and the pH of the media was determined. Lower integrated mineral loss values were observed when 5,000 ppm F-treatment was used compared to the other treatments. Overall microbiological counts decreased with increasing F-concentration as well the pH of the media throughout the experiment. The 5,000 ppm F-treatment caused a shift in microbial composition and reduced dentine demineralization in the in-vitro experimental model.

A modified dentine infection model with fluorescent lipopolysaccharide and lipopolysaccharides sampling technique to compare XP-Endo finisher and passive ultrasonic irrigation.

AIM: The lipopolysaccharides-dentine-infection (LPS-dentine-infection) models and sampling techniques frequently used to evaluate LPS disinfection have limitations. In this study, a LPS-dentine-infection model was devised using fluorescent conjugate LPS. Secondly, a sampling technique using cryogenic grinding for intraradicular LPS analysis was evaluated. Thirdly, the effectiveness of the XP-endo Finisher (XP-EF) was compared with passive ultrasonic irrigation (PUI) in removing LPS from root canal system. METHODOLOGY: Sixty-nine mandibular premolars were submitted to dentine pretreatment and inoculated with fluorescent LPS conjugate (Alexa Fluor® 594). Twenty-three teeth were analysed under confocal laser scanning microscopy (CLSM) to validate this modified LPS-dentine-infection model. Forty-six teeth were randomly divided into two experimental groups: XP-EF (n = 23) and PUI (n = 23). All teeth were instrumented with XP-endo shaper (XPS; FKG Dentaire) and 2.5% NaOCl. The root canals were sampled with paper points before (s1) and after (s2) instrumentation and after supplemental treatment (s3) with XP-EF and PUI. After s3, all roots were cryogenically ground for intraradicular LPS analysis (s4). Limulus amebocyte lysate assay was used for LPS quantification. The Friedman test was used for differences in LPS among four time-points (s1, s2, s3, and s4). Dunn's test was used for pairwise testing of time-points. The significance level was set at 5% (p < .05). RESULTS: Fluorescent LPS conjugate was detected in 100% of the samples under CLSM with a penetration depth of approximately 400 µm into dentine. Chemo-mechanical preparation using XPS files significantly reduced LPS levels (p < .05). Both the XPS and PUI improved the LPS disinfection (p < .05), with no difference between them (p > .05). LPS was recovered from all samples after cryogenic grinding. The residual amount of LPS detected using the cryogenically sampling technique at s4 was approximately three times greater than with the paper-point sampling technique at s3. CONCLUSION: This study established a modified LPS-dentine-infection model using fluorescent conjugate LPS, and validated a LPS sampling technique for using cryopulverization intraradicular LPS analysis. Moreover, both the XP-EF and PUI further improved LPS disinfection from the root canals, and the innovative XP-EF was as effective as PUI.

The dentine-sealer interface: Modulation of antimicrobial effects by irrigation.

AIM: Assess whether sodium hypochlorite (NaOCl) or chlorhexidine (CHX) and two irrigation protocols may alter the antibacterial properties of dentine and three endodontic sealers using a novel ex vivo tooth model. METHODOLOGY: Prior to antibacterial testing, the tooth model was validated by means of scanning electron microscopy (SEM) to evaluate the separation between dentine and sealer surfaces. Root blocks prepared from extracted human roots were pre-treated with 17% EDTA + 0.9% saline and subsequently treated with 1% NaOCl (G1), 2% CHX (G2) or no irrigant (G3). Two irrigation protocols were further investigated, "1% NaOCl + 17% EDTA" (P1) and "1% NaOCl + 17% EDTA + 2% CHX" (P2). Following irrigation, the root blocks were either filled with AH Plus, BioRoot RCS and Pulp Canal Sealer (PCS), or left empty. All groups were incubated for 1, 7 and 28 days. Direct contact tests for planktonic E. faecalis and 48 h E. faecalis biofilms were performed at the level of dentine and sealer surfaces. Statistical analysis was performed on the bacterial survival between irrigants (G1, G2 and G3) and between irrigation protocols (P1 and P2); p < .05. RESULTS: The model was considered reproducible as SEM examination of dentine samples indicated consistent separation between dentine and sealer surfaces. Irrigation with CHX (G2) and irrigation protocol P2 enhanced the antibacterial properties of dentine without sealer application as well as dentine in contact with all three sealers tested, especially against planktonic E. faecalis. G2 and P2 also improved the antibacterial effect of AH Plus surfaces for all three incubation times. No irrigation groups (G1, G2) or irrigation protocols (P1, P2) altered the antibacterial properties of BioRoot RCS surfaces against planktonic bacteria or biofilms. Only BioRoot RCS surfaces eliminated the planktonic E. faecalis in all irrigation groups (G1, G2, G3) and protocols (P1, P2) investigated whilst PCS surfaces eliminate E. faecalis in biofilms in all groups up to 7 days. CONCLUSIONS: The tooth model was reproducible. CHX improved the antibacterial activity upon both sealer and dentine surfaces. Amongst sealers, BioRoot RCS was less affected by NaOCl and CHX, and exhibited high antibacterial properties regardless the irrigation applied.

Influence of acidic pH on antimicrobial activity of different calcium silicate based-endodontic sealers.

OBJECTIVE: To investigate the antibacterial activity of calcium silicate-based sealers (CSBSs) against Enterococcus faecalis biofilm in a neutral or acidic condition. MATERIALS AND METHODS: Dentin cylinders (4 mm length) were prepared and infected with 3-week-old E. faecalis. The samples were filled with BioRoot RCS (BR), EndoSequence BC (ES), and NeoMTA Plus (NMTA) and incubated in either neutral or acidic conditions for 7 days (n=10/group). Sterile or infected samples alone were used as the positive and negative control. The root canal sealers were removed after 7 days, and the remaining bacteria on dentinal walls were determined by colony-forming units (CFUs/ml), and three samples from each group were visualized under a confocal laser scanning microscope (CLSM). The pH was also measured (n=3/group) after 4 h and 7 days of incubation at 37°C in both conditions. RESULTS: In the neutral condition, all sealers significantly decreased the log-CFU values (p<0.05), while in the acidic condition, the log-CFU reduction was less for ES and NMTA, but a higher reduction was observed in BR (p<0.05). The antibacterial activity of CSBSs was similar in neutral conditions (p>0.05), and BR showed a greater antibacterial effect than ES and NMTA in the acidic condition (p<0.05). The pH of BR, ES, and NMTA ranged from 8.2 to 8.8 in the neutral condition in the presence of dentin after 7 days. However, acidic conditions reduced the pH values to 7.8 for BR, 6.0 for ES, and 5.8 for NMTA. CONCLUSIONS: All CSBSs showed similar antibacterial activity in neutral conditions, while acidic pH had a reducing antibacterial effect on CSBSs. CLINICAL RELEVANCE: Inflammatory pH decreased the antibacterial properties of CSBSs depending on the sealer type.

Visualization and characterization of Enterococcus faecalis biofilm structure in bovine dentin using 2D and 3D microscopic techniques.

Bacterial biofilms are related to various dental and periodontal infectious diseases, and the characterization of this biological structure with micro-computed tomography (micro-CT) may offer valuable information for clinical and research applications. In this study, we aimed to develop a model to visualize three-dimensionally the biofilm structure on dentin using micro-CT. Dentin blocks were prepared and incubated in tryptic soy broth with Enterococcus faecalis (ATCC 29212). The control group did not receive any staining procedure, while groups 1 and 2 were stained with 100% and 50% barium sulfate, respectively. Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to detect biofilm formation, barium sulfate penetration, and microbial cell density in the biofilm. Micro-computed tomography (micro-CT) (SkyScan 1172, Bruker Co., Belgium) was used to visualize biofilm formation on the dentin blocks. Biofilm thicknesses were measured from 10 different locations on the specimen surfaces, using CTAn v.1.14.4 software. Obtained data were statistically analyzed using Kruskal-Wallis and Dunn's tests. TEM photomicrographs showed that barium sulfate could penetrate the biofilm structure. CLSM analysis showed that viable and total cell densities were similar between the control and barium sulfate-treated groups (P > 0.05), indicating barium sulfate had no significant influence on cell density. In barium sulfate-treated blocks, biofilm could be discriminated from the dentin, and its thickness could be measured with micro-CT. This study showed that bacterial biofilm on dentin could be characterized by micro-CT after barium sulfate staining without causing any significant side effect on viable and total cell densities.

Effect of Propolis Nanoparticles against Enterococcus faecalis Biofilm in the Root Canal.

To determine the antibacterial effect of propolis nanoparticles (PNs) as an endodontic irrigant against Enterococcus faecalis biofilm inside the endodontic root canal system. Two-hundred-ten extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into seven groups, with 30 dentinal blocks in each group including: group I-saline; group II-propolis 100 µg/mL; group III-propolis 300 µg/mL; group IV-propolis nanoparticle 100 µg/mL; group V-propolis nanoparticle 300µg/mL; group VI-6% sodium hypochlorite; group VII-2% chlorhexidine. Dentin shavings were collected at 200 and 400 µm depths, and total numbers of CFUs were determined at the end of one, five, and ten minutes. The non-parametric Kruskal-Wallis and Mann-Whitney tests were used to compare the differences in reduction in CFUs between all groups, and probability values of p < 0.05 were set as the reference for statistically significant results. The antibacterial effect of PNs as an endodontic irrigant was also assessed against E. faecalis isolates from patients with failed root canal treatment. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to PNs. A Raman spectroscope, equipped with a Leica microscope and lenses with curve-fitting Raman software, was used for analysis. The molecular interactions between bioactive compounds of propolis (Pinocembrin, Kaempferol, and Quercetin) and the proteins Sortase A and ß-galactosidase were also understood by computational molecular docking studies. PN300 was significantly more effective in reducing CFUs compared to all other groups (p < 0.05) except 6% NaOCl and 2% CHX (p > 0.05) at all time intervals and both depths. At five minutes, 6% NaOCl and 2% CHX were the most effective in reducing CFUs (p < 0.05). However, no significant difference was found between PN300, 6% NaOCl, and 2% CHX at 10 min (p > 0.05). SEM images also showed the maximum reduction in E. faecalis with PN300, 6% NaOCl, and 2% CHX at five and ten minutes. CLSM images showed the number of dead cells in dentin were highest with PN300 compared to PN100 and saline. There was a reduction in the 484 cm-1 band and an increase in the 870 cm-1 band in the PN300 group. The detailed observations of the docking poses of bioactive compounds and their interactions with key residues of the binding site in all the three docking protocols revealed that the interactions were consistent with reasonable docking and IFD docking scores. PN300 was equally as effective as 6% NaOCl and 2% CHX in reducing the E. faecalis biofilms.

Shear Bond Strength and Film Thickness of a Naturally Antimicrobial Modified Dental Luting Cement.

Although several natural plants and mixtures have been known and used over the centuries for their antibacterial activity, few have been thoroughly explored in the field of dentistry. Thus, the aim of this study was to enhance the antimicrobial activity of a conventional glass ionomer cement (GIC) with natural plant extracts. The effect of this alteration on the bond strength and film thickness of glass ionomer cement was evaluated and related to an 0.5% chlorohexidine modified GIC. Olive leaves (Olea europaea), Fig tree (Ficus carica), and the leaves and roots of Miswak (Salvadora persica) were used to prepare an alcoholic extract mixture. The prepared extract mixture after the evaporation of the solvent was used to modify a freeze-dried glass ionomer cement at three different extracts: water mass ratios 1:2, 1:1, and 2:1. An 0.5% chlorhexidine diacetate powder was added to a conventional GIC for the preparation of a positive control group (CHX-GIC) for comparison. The bond strength to dentine was assessed using a material-testing machine at a cross head speed of 0.5 mm/min. Failure mode was analyzed using a stereomicroscope at 12× magnification. The cement film thickness was evaluated in accordance with ISO standard 9917-1. The minimum number of samples in each group was n = 10. Statistical analysis was performed using a Kruskal-Wallis test followed by Dunn's post hoc test for pairwise comparison. There was a statistically insignificant difference between the median shear bond strength (p = 0.046) of the control group (M = 3.4 MPa), and each of the CHX-GIC (M = 1.7 MPa), and the three plant modified groups of 1:2, 1:1, 2:1 (M = 5.1, 3.2, and 4.3 MPa, respectively). The CHX-GIC group showed statistically significant lower median values compared to the three plant-modified groups. Mixed and cohesive failure modes were predominant among all the tested groups. All the tested groups (p < 0.001) met the ISO standard of having less than 25 µm film thickness, with the 2:1 group (M = 24 µm) being statistically the highest among all the other groups. The plant extracts did not alter either the shear bond strength or the film thickness of the GIC and thus might represent a promising additive to GICs.

Effect of Silver Diamine Fluoride on Reducing Candida albicans Adhesion on Dentine.

BACKGROUND: Candida albicans is the most frequent pathogenic fungus in oral cavities. It adheres to dental tissues as part of dental plaques and contributes to caries formation. OBJECTIVES: To evaluate the effect of silver diamine fluoride (SDF) on reducing C. albicans adhesion on dentine surfaces. METHODS: Flat dentine surfaces were prepared from bovine dental disks, and samples were divided into three groups. The first and second groups were pretreated for 3 min with 299 mM or 2.99 M SDF, respectively, and the third group (control) did not undergo any SDF pretreatment. All samples were washed, inoculated with C. albicans suspension onto their dentine surface, incubated at 30 °C for 6 h, and washed again to remove any nonadherent cells. The abundance of adherent cells was investigated using colorimetric and real-time polymerase chain reaction approaches. Subsequently, the morphological changes in C. albicans by pretreatment with SDF were observed under a scanning electron microscope (SEM). RESULTS: SDF inhibited candidal growth at concentrations as low as 2.99 µM. Dentine disks pretreated with 299 mM or 2.99 M SDF displayed significantly fewer adhered cells as compared with the control group. Upon pretreatment with SDF, SEM images showed severe morphological changes in the cellular walls, in a dose-dependent manner, suggesting a fungicidal effect of SDF against the yeast. CONCLUSION: SDF should be considered for clinical applications aimed at inhibiting dental plaque caused by C. albicans, particularly in children and elderly individuals.

Antibacterial effect of diode laser on different cariogenic bacteria: An In-vitro study.

AIMS: The authors have used an in vitro model to appraise the antimicrobial efficacy of diode lasers with two different power outputs on Streptococcus mutans (SM), Lactobacillus casei (LC), and Actinomyces naeslundii (AN). METHODS AND MATERIALS: The coronal dentin of thirty human mandibular third molars was prepared with four cylindrical cavities left in contact with SM, LC, and AN for 72 h to facilitate bacterial penetration. Diode laser (810 nm for 30 s in two cycles) with 1.5 W (group I), 1 W (group II), and 2% chlorhexidine gluconate solution for 60 s (group III) was applied on three cavities and the fourth cavity was not subjected to any treatment (control). Similar amounts of dentin debris were collected from the cavity into sterile tubes. The bacterial count was determined by serial dilution and plate count method. Percentage of killing was calculated for comparative analysis. RESULTS: The percentage of SM killed after exposure was 73.68 ± 23.37, 51.75 ± 25.45, and 26.78 ± 21.8 in three groups, respectively, (P = 0.002; Kruskal-Wallis) with no significant difference between group I and group II (P = 0.089; Mann-Whitney). The percentage of AN killed after exposure was 37.77 ± 49.52, 22 ± 19.48, and 56.86 ± 23.93 in three groups, respectively, (P = 0.013; Kruskal-Wallis) with significant difference between group II and group III (P = 0.002; Mann-Whitney). The percentage of LC killed after exposure was 51.32 ± 39.07, 36.65 ± 38.48, and 75.41 ± 22.6 in three groups, respectively (P = 0.091; Kruskal-Wallis). CONCLUSIONS: Diode lasers exerted antibacterial effect of varying levels against all the three cariogenic bacteria. Although they are recommended as a supplementary antibacterial surface pretreatment technique for efficient removal of cariogenic bacteria, further clinical studies are required to confirm the in vitro findings.

Oil droplet formation on pellicle covered tooth surfaces studied with environmental scanning electron microscopy.

Lipophilic components are known to modulate the process of bioadhesion on the tooth surface. However, the presence of lipid droplets at the acquired pellicle under oral conditions has not been demonstrated, yet. The purpose of the present study was to establish a method for direct visualisation of lipids on the surface of hydrated, pellicle covered tooth samples by environmental scanning electron microscopy (ESEM), and to use this technique for studying the effects of rinsing with edible oils on the acquired pellicle under in vivo conditions. In situ pellicle formation was performed by 3 min exposure of enamel and dentin specimens in the oral cavity of volunteers. Subsequently, the volunteers rinsed in vivo with safflower oil or linseed oil for 30 s, and the specimens were further carried intraorally for periods from 0 min up to several hours. After intraoral exposure the specimens were treated by osmium tetroxide vapour, and were subsequently analysed by ESEM. This technique was capable to directly visualise the presence of lipid droplets at the pellicle's surface under hydrated conditions. ESEM analyses revealed that surface bound nano- and micro-sized lipid droplets were present at the acquired pellicle's surface even several hours after rinsing with edible oils indicating that these droplets had tightly adhered to the pellicle surface. Pellicle modification by edible oil rinsing as demonstrated in the present study might have the potential to be beneficial as an adjunct in dental prophylaxis.