Lipopeptide biosurfactant as a potential root canal irrigation agent: Antimicrobial and anti-biofilm evaluation.

OBJECTIVES: Lipopeptide Biosurfactant (LB) is a bacteria derived compound able to reduce surface tension between water and hydrophobic substances and exhibit antimicrobial and anti-biofilm properties. This study aimed to investigate the antimicrobial and anti-biofilm effect of a Lipopeptide Biosurfactant (LB) on Enterococcus faecalis, and its potential use in root canal treatment, either as a standalone irrigation solution or in conjunction with sodium hypochlorite (NaOCl). METHODS: LB was extracted from Bacillus clausii isolate and the dry extract was diluted in deionized water. The antimicrobial effect of LB against planktonic E. faecalis was evaluated by determining the Minimal Inhibitory Concentration (MIC50). The anti-biofilm effect was evaluated by Minimal Biofilm Inhibitory Concentration (MBIC50) and Minimal Biofilm Eradication Concentration (MBEC50) assays on biofilm grown on dentin specimen surface. To evaluate the effectiveness of LB as a single irrigation solution and as a pre-irrigation prior to NaOCl, live and dead bacterial cells were quantified using Confocal Laser Scanning Microscopy (CLSM), and cell biomass was assessed. RESULTS: LB exhibited an MIC50 and MBIC50 of 100 ppm, with an MBEC50 of 1000 ppm, resulting in 52.94 % biofilm inhibition and 60.95 % biofilm eradication on dentin specimens. The effectiveness was concentration-dependent, at 500 ppm, LB demonstrated comparable antimicrobial efficacy to 2.5 % NaOCl. Pre-irrigation with LB resulted in lower biofilm biomass compared to NaOCl alone. CONCLUSION: Pre-irrigation with LB enhanced the antimicrobial effect when followed by NaOCl irrigation. Consequently, LB shows promise as both a standalone root canal irrigation solution and as an adjunct to NaOCl in root canal treatment. CLINICAL SIGNIFICANCE: The study highlights the potential of Lipopeptide Biosurfactant (LB) as an environmentally friendly irrigation solution for root canal treatment, demonstrating potent antimicrobial and anti-biofilm properties against Enterococcus faecalis. LB exhibits concentration-dependent efficacy comparable to 2.5 % NaOCl and can be used as a standalone irrigation solution or in conjunction with NaOCl.

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.

Presence of host and bacterial-derived collagenolytic proteases in carious dentin: a systematic review of ex vivo studies.

Introduction and aim: The presence of host collagenases in the degradation of the protein matrix at later stages of carious dentin lesions development, as well as the potential involvement of bacterial collagenases, have been suggested but lack conclusive evidence. This study aims to conduct a systematic review to comprehensively assess the profile of host and bacterial-derived collagenolytic proteases in both root and coronal dentin carious lesions. Methods: The search was performed in eight databases and the grey literature. Studies evaluating ex vivo dentin, extracted teeth, or biofilms from natural caries lesions were included. The methodological quality of studies was assessed using the Joanna Briggs Institute tool. Synthesis of the results and the certainty of evidence were performed following the Synthesis without Meta-analysis (SWiM) checklist and GRADE approach for narrative synthesis, respectively. Results: From 935 recovered articles, 18 were included. Although the evidence was very uncertain, it was possible to suggest that 1) MMP-2, MMP-9, MMP-13, and CT-B may be increased in carious dentin when compared to sound dentin; 2) there is no difference in MMP-2 presence, while MMP-13 may be increased in root when compared to coronal carious dentin; 3) there is no difference of MMP-2 and MMP-9 expression/activity before and after cavity sealing; 4) MMP-8 may be increased in the dentin before cavity sealing compared to dentin after cavity sealing; 5) there is no difference of MMP-20 in irradiated vs. non-irradiated carious dentin. MMP-20 probably reduces in carious outer dentin when compared to carious inner dentin (moderate certainty). Genes encoding bacterial collagenolytic proteases and protein-degrading bacteria were detected in coronal and root carious lesions. Conclusion: Trends in the direction of the effect were observed for some collagenolytic proteases in carious dentin, which may represent a potential target for the development of new treatments. (Protocol register-PROSPERO: CRD42020213141).

Dentin disinfection and adhesive bond strength using modified photoactivated carbon nanoparticles.

AIMS: The present study aimed to investigate the bond integrity and disinfection efficacy of Methylene blue(MB) alone, MB-PDT (Photodynamic therapy), MB@ carbon nanoparticles (CP)-PDT, and Cr, Cr: YSGG (ECL) against lactobacilli in Caries-affected dentin (CAD) MATERIAL AND METHODS: Methods consisted of Shear bond strength (SBS), Scanning electron microscopy (SEM), energy dispersive X-ray (EDX), methods of disinfection, and failure analysis. CAD samples were prepared and biofilm formed on the specimens randomly allocated into five groups based on disinfection. Group 1: CHX; Group 2: MB; Group 3: MB-PDT: group 4: MB@CP-PDT and group 5: ECL. After disinfection Colony forming units were measured and specimens were restored and positioned under a universal testing machine (UTM). Failure analysis was performed using a stereomicroscope. The difference in survival rate was assessed using the Kruskal-Walis test. Mean and standard deviation for bond values after different methods of disinfection was evaluated using analysis of variance (ANOVA) and Post Hoc Tukey. The significance level was p<0.05 RESULTS: Morphological analysis revealed that CPs under SEM are flat discs with edged irregular shapes. EDX analyses show a spike indicating carbon particles by more than 95%. MB@CP-PDT displayed the highest reduction in lactobacillus levels in comparison to the other disinfection methods. The highest SBS was exhibited by the CAD sample disinfected with ECL. The lowest SBS values in CAD specimens after cavity cleansing with MB alone. The predominant failure type in CAD disinfected with MB alone, ECL CHX, MB-PDT, and MB@CP-PDT was adhesive. CONCLUSION: The use of MB@CP-PDT showed high antibacterial potency against lactobacillus but demonstrated bond values similar to CHX. Use of Er, Cr: YSGG showed considerable effectiveness against lactobacillus along with the highest bond values.

Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 ß), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1ß correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1ß and IL6 and the chemokine CXCL8.

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.

Impact of Dentin Conditioning and Sealer Modification With Chitosan-Hydroxyapatite Nanocomplexes on the Antibacterial and Mechanical Characteristics of Root Dentin.

INTRODUCTION: This study aimed to characterize the effectiveness of dentin conditioning with bio-mineralizable chitosan-hydroxyapatite precursor (CS-HA) nanocomplexes alone or associated with tricalcium silicate sealer (TCS/CS-HA) on the mechanical property and antibiofilm efficacy in root dentin. METHODS: Flow tests were performed following ISO6876:2012 specifications. Solubility was measured. Micromorphology was assessed using scanning electron microscopy (SEM). Nanohardness/elastic modulus were also determined. Fracture resistance was determined on lower premolars that were prepared, and randomly distributed among 7 groups (n = 8/group), including the control, CS-HA dentin-conditioning, and root canal-filled groups. Similar canal preparation/distribution procedure was followed to test the antibacterial effect on Enterococcus faecalis-infected roots. Descriptive statistics was used to report SEM findings. Flowability results were analyzed using paired t test. Multiple comparisons from solubility, fracture, and antibacterial assays were assessed by one-way analysis of variance-Tukey's tests. RESULTS: TCS/CS-HA showed optimal flow and no effect on solubility after immersion for 4 weeks (P > .05). TCS/CS-HA significantly increased nanohardness and elastic modulus (210 ± 11.3 MPa, 7.9 ± 0.9 GPa) compared with TCS (44.5 ± 7.8 MPa, 2.1 ± 0.3 GPa, P < .05). SEM revealed needle-shaped mineralized structures resulting in fewer voids and a well-adapted sealer-dentin interface in the TCS/CS-HA groups. NaOCl-EDTA irrigation resulted in reduced fracture resistance (444.34 N), whereas CS-HA dentin conditioning alone (928.28 N, P < .05) and CS-HA dentin-conditioning plus CS-HA/TCS resulted in higher fracture resistance (1134.06 N, P < .05). CS-HA dentin conditioning also reduced bacteria by 2.04 log (4.50 ± 0.43) from the initial bacterial load (6.54 ± 0.07, P < .05). There was further bacterial reduction when CS-HA-conditioned root canals were filled with TCS or TCS/CS-HA (0.00 to 0.98 ± 0.57, P > .05). CONCLUSION: Dentin modification with CS-HA increased the fracture resistance of root dentin, and decreased the residual bacterial burden. TCS/CS-HA potentiated the nanomechanical and physical properties of TCS.

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.

Assessment of dental caries lesion activity status using quantitative parameters obtained from the quantitative light-induced fluorescence method and difference of microbial distribution in primary molars.

BACKGROUND: This study aimed to assess differences in quantitative measures obtained from the quantitative light-induced fluorescence method and microbial composition of carious dentin and saliva according to the activity status of caries lesions in primary molars. METHODS: A total of 34 teeth from 34 children were evaluated in this study. The activity status of carious lesions was classified using the International Caries Classification and Management System criteria (active or inactive). Images of the carious lesions were captured using a quantitative light-induced fluorescence device for quantitative analyses. Carious dentin and saliva were collected to detect and quantify selected bacterial species (S. mutans, S. sobrinus, Lactobacillus species, F. nucleatum, P. nigrescence, P. intermedia) and C. albicans by quantitative polymerase chain reaction. Mann-Whitney U tests were performed to evaluate differences in quantitative measures from quantitative light-induced fluorescence, the microbial composition of carious dentin, and saliva according to the activity status of carious lesions. RESULTS: Red fluorescence values (∆R, ∆Rmax) from the quantitative light-induced fluorescence method were significantly higher in active lesions (∆R, p = 0.009; ∆Rmax, p = 0.014). The quantitative mean levels of Lactobacillus species (p = 0.010) in carious dentin and S. sobrinus (p = 0.017) in saliva were significantly higher in the active-lesion group. CONCLUSIONS: Quantitative measures related to red fluorescence from the quantitative light-induced fluorescence method, levels of Lactobacillus species from carious dentin, and levels of S. sobrinus from saliva were associated with caries lesion activity.

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.

Laser-fluorescence assessment of sodium hypochlorite quenching reversal agents on human dentine.

BACKGROUND: Laser-fluorescence diagnostic technology for real-time clinical assessment of residual bacteria could help assist in determining the endpoints for root canal debridement. Sodium hypochlorite (NaOCl) can however quench fluorescence and lead to false low reading. This study aims to evaluate various antioxidant for their ability to recover quenched fluorescence in dentine treated with NaOCl. METHODS: Human dentine fluorescence was measured using 655 nm laser at baseline and again after a 2 min application of 4% NaOCl. The putative recovery agents were then applied, and the fluorescence measured after 5, 10, 20, 30 and 60 min. Recovery from quenching was also assessed using laser confocal scanning microscopy (CLSM) with a bound tetracycline fluorophore using 488 nm excitation. RESULTS: A 5 min application of vitamin E oil or buffered 2% lignocaine solution (1:80,000 adrenaline) was effective in regaining quenched fluorescence within the following 5 mins. Distilled water, sodium thiosulfate, unbuffered 2% lignocaine with 1:80000 adrenaline and phosphate buffered saline were less effective, and of equal performance. Ascorbic acid and butylated hydroxyanisole were not effective and had deleterious effects on the levels of dentine fluorescence. CLSM provided confirmation of recovery from quenched fluorescence using vitamin E oil. CONCLUSION: Based on these findings, reversal agents should be employed when assessing the fluorescence of dentine that has been exposed to NaOCl or other quenching agents.

Modified photoactivated methylene blue-incorporated quartz particles for dentin disinfection: A scanning electron microscope and spectroscopic analysis.

The aim of this study was to synthesize methylene blue-incorporated quartz particles (MB@QP) and to investigate its anti-bactericidal properties. Methylene blue was incorporated inside QP and characterized for morphology and chemical structure using scanning electron microscope (SEM) and Fourier transformed infrared spectroscopy (FTIR). Specimens were randomly divided into experimental and control groups (n = 9/groups). The dentin specimen infected with Enterococcus faecalis was treated using different treatment modalities: Control groups: treatment with 5.25% of sodium hypochlorite (NaOCl) for 60 s; MB: treatment with 1 ml MB solution and incubated for 60 s; MB-PDT (photodynamic therapy): treatment with 1 ml MB solution followed by irradiation using diode laser for 60 s; MB-QP-PDT: specimens treated with MB@QP and irradiated by the diode laser for 60s, and Er,Cr:YSGG laser alone. MB@PDT therapy showed the highest efficacy in reducing the survival rate of E. faecalis (0.49%) in comparison to control NaOCl (0.78%) and Er,Cr:YSGG laser treatment (2.17%). Encapsulating MB into QP followed by the PDT significantly improved the bactericidal capacity and significantly reduced the bacterial survival rate to 0.11% (p < .05) compared to other groups. The combination of MB incorporated into QP and PDT could be an alternative treatment modality to conventional disinfection method for eliminating bacteria from the tooth dentin. RESEARCH HIGHLIGHTS: Quartz particles are potent in delivering the photosensitizer. Photoactivated MB@QP has a higher efficacy in eliminating bacteria from tooth dentin.

Visualization of initial bacterial colonization on dentin using fluorescence activating headlight for fluorescence enhanced theragnosis.

OBJECTIVE: To evaluate accuracy of caries detection and the application-sensitivity of the new Designs for Vision's REVEAL™ utilizing a fluorescence activating headlight for excitation purpose. MATERIALS AND METHODS: REVEAL dental fluorescence loupes and headlight system were used. Occlusal enamel was removed, and mid-coronal dentine was exposed. Carious artificial lesion was created. Streptococcus mutans, Actinomyces naeslundii, and Streptococcus sanguis were used. The assessment was performed using two diagnostic methods: naked eye and Design for Vision Glasses with inter examiner blinding using two calibrated examiners. After 7 days, Raman measurements were made on dentin disc specimens with 785 nm wavelength. The bacterial counts in colony-forming units (CFU) were used to examine the growth kinetics of biofilms. The collagen fibril structure within the discs was performed using Transmission Electron Microscope. Scanning Electron Microscope was used to image samples at various magnifications. FISH was performed with specimens fixed in 4% paraformaldehyde in phosphate-buffered saline. Reproducibility was measured by Cohen kappa scores, values of which range from 0 for less than chance agreement to 1 for almost perfect agreement (p < 0.05). RESULTS: A significant kappa score of 0.706 showing significant reproducibility for the given diagnostic techniques, as all the teeth included in the study were spotted with the lesions. Most bacteria were detected using the CFU technique. The Raman bands scanned across the dentin surface at 960 cm-1 (P-O peak) are assigned to hydroxyapatite phosphate vibrations. FISH identified nearly all stained bacteria as days and time and dental hard tissue had a significant impact on the number of adherent bacteria. Scanning electron micrographs of polished cross sections of demineralized and non-demineralized specimens with perpendicular each tubule orientation (zone of demineralized dentin inset. CONCLUSION: Fluorescent enhanced theragnosis through Reveal vision glasses can ensure constant monitoring and diagnosis of caries progress . This may allow for a better clinical outcome.

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.

Depth of Penetration and Antimicrobial Activity of 5% and 10% Bamboo Salt, 2% Chlorhexidine Gel and Calcium Hydroxide Against Enterococcus faecalis - An In Vitro Study.

OBJECTIVE: E. faecalis is one of the most commonly found species in persistent and secondary infections associated with endodontic failure. This in vitro study aimed to evaluate the depth of penetration and antimicrobial efficacy of 5% and 10% bamboo salt (BS), 2% chlorhexidine gel (CHX) and calcium hydroxide (CH) against E. faecalis. METHODS: E. faecalis was inoculated in dentine blocks for 21 days following which the antibacterial efficacy of the experimental medicaments were quantatively assessed by harvesting the dentinal debris from depths of 200 µm and 400 µm from the block lumen on the 2nd and 7th day. The depth of penetration of the medicaments was measured using LIVE/DEAD BacLight stain under CLSM. RESULTS: Results showed that the medicaments had varying degrees of antimicrobial efficacy and depth of penetration. Among the medicaments, CHX showed the highest antimicrobial activity on both the time intervals (P<0.05), followed by 10% BS, 5% BS and the least efficacy was observed in CH group. CHX and 10% BS exhibited the highest depth of penetration, which was proximate to the penetration of E. faecalis. CONCLUSION: It can be concluded that 10% bamboo salt can serve as a viable natural antimicrobial in endodontic therapy.

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 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.

Ca-Si mesoporous nanoparticles with the optimal Ag-Zn ratio inhibit the Enterococcus faecalis infection of teeth through dentinal tubule infiltration: an in vitro and in vivo study.

Enterococcus faecalis is the main cause of refractory root canal infections in human teeth. The control of root canal infection is one of the conditions necessary for the successful treatment of refractory root canal infections. In the present study, nano-scale silver-zinc-calcium-silica particles loaded with different ratios of silver-zinc were successfully prepared (Ag0.5Zn3-MCSNs and Ag0.5Zn10-MCSNs). The release profiles, antibacterial activity against E. faecalis, infiltration depth into dentinal tubules, biocompatibility and effects on dentin microhardness in vitro were investigated. In addition, the antimicrobial effects of the particles against Enterococcus faecalis reinfection were evaluated in vivo in the teeth of beagle dogs. Ag, Zn, Ca and Si were released from Ag-Zn-MCSNs, and the atomic ratio of silver and zinc released can reach the optimal value of 1 : 12 (Ag0.5Zn10-MCSNs). The particles also showed good biocompatibility and antibacterial activity against Enterococcus faecalis and did not reduce the hardness of dentin. The nanoparticles could be driven into the dentinal tubules of dentin slices by ultrasonic activation. In the root canals of beagle dogs, both Ag0.5Zn3-MCSNs and Ag0.5Zn10-MCSNs demonstrated strong preventive effects against E. faecalis infection. The Ag-Zn-Ca-Si mesoporous nanoparticles may develop into a new effective root canal disinfectant or root canal sealer.

Potential for direct application of blue light for photo-disinfection of dentine.

The direct application of light for photo-disinfection potentially provides a safe and novel modality to inhibit or eliminate cariogenic bacteria residing upon and within dentine. This study aimed to both; characterize the pattern of transmission of 405 nm light through molar dentine at different tooth locations, as well as, determine the irradiation parameters that are antibacterial for Streptococcus mutans under various growth conditions, including lawns, planktonic cultures, and biofilms. To determine the amount of light (405 nm) transmitted at different anatomical tooth locations; irradiance values were recorded after blue light (470-4054 mW/cm2) had traversed through occlusal, oblique, and buccal dentine sections; and three thicknesses - 1, 2 and 3 mm were investigated. To determine tubular density; scanning electron micrographs from 2 mm outer (dentine-enamel junction) and inner (pulp) dentine sections were analysed. For photo-disinfection studies; S. mutans was irradiated using the same 405 nm wavelength light at a range of doses (110-1254 J/cm2) in both biofilm and planktonic cultures. The inhibitory effect of the irradiation on bacterial lawns was compared by measuring zones of inhibition; and for planktonic cultures both spectrophotometric and colony forming unit (CFU) assays were performed. A live/dead staining assay was utilised to determine the effect of irradiation on bacterial viability in mature biofilms. Data indicated that increasing dentine thickness decreased light transmission significantly irrespective of its orientation. Occlusal and oblique samples exhibited higher transmission compared with buccal dentine. Oblique dentine 405 nm light transmission was comparable with that of occlusal dentine independent of section thickness. An increased tubule density directly positively correlated with light transmission. Irradiation at 405 nm inhibited S. mutans growth in both biofilm and planktonic cultures and a dose response relationship was observed. Irradiation at doses of 340 and 831 J/cm2 led to significant reductions in bacterial growth and viability; as determined by CFU counting and live/dead staining. Data suggests that phototherapy approaches utilising a 405 nm wavelength have therapeutic potential to limit cariogenic bacterial infections both at the surface and within dentine.

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.