Biofilm Challenge Research Articles

Assessment of the Antibiofilm Performance of Silver-Containing Wound Dressings: A Dual-Species Biofilm Model.

Background It is commonly accepted that microorganisms found within hard-to-heal wounds are present in biofilm form. Biofilms are often polymicrobial in nature, which increases their virulence and tolerance to antimicrobial agents. The aim of this study was to compare the antibiofilm activity of silver-containing antimicrobial wound dressings in a dual-species simulated wound biofilm model. Materials and methods Four silver-containing wound dressings were evaluated in vitro: Aquacel® Ag+ Extra™ dressing, KerraContact® Ag dressing, Durafiber* Ag dressing, and UrgoClean Ag dressing. Each dressing was applied to a simulated wound assembly containing biofilm-gauze inoculated with Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). Each biofilm-inoculated gauze was incubated at 35±3ºC for 6, 24, 48 and 72 hours. Enumeration of surviving biofilm bacteria at each time point was performed in triplicate for each test dressing and its equivalent control. Results Aquacel® Ag+ Extra™ dressing was observed to reduce the biofilm population within 24 hours with a >4 log10 kill observed for K. pneumoniae and >6 log10 for MRSA from an initial biofilm challenge of 4.16×109 CFU/mL. This kill rate was sustained for the duration of the challenge period, with Aquacel®Ag+ Extra™ dressing reducing the biofilm population to non-detectable levels (<30 Colony Forming Units (CFU) per test) by 72 hours for K. pneumoniae and by 48 hours for MRSA. KerraContact® Ag dressing demonstrated an initial reduction at 6 hours of ~2 log10 in both K. pneumoniae and MRSA. Durafiber* Ag dressing exhibited a slight, gradual reduction in biofilm population over the course of the test period, reducing each challenge organism by ~2.5 log10 by 72 hours. UrgoClean Ag was shown to have little to no impact on the dual-species biofilm with levels remaining similar or greater than that recovered prior to dressing application. The no-dressing biofilm-colonised gauze control demonstrated that the biofilm bacteria remained viable throughout the test period and species population proportionality was maintained. Conclusion Using a dual-species simulated wound biofilm model comprising the pathogens K. pneumoniae and MRSA, Aquacel® Ag+ Extra™ dressing demonstrated significantly greater antibiofilm activity than the other silver-containing dressings. The enhanced antibiofilm activity of Aquacel® Ag+ Extra™ dressing in this study may be attributed to the additional antibiofilm agents, ethylenediaminetetraacetic acid and benzethonium chloride, contained within the dressing.

The impact of Streptococcus mutans biofilms on the color stability and topographical features of three lithium disilicate ceramics.

Secondary caries around ceramic restorations is the most common reason for the replacement of fixed dental prostheses (FDPs). Therefore, it is important to examine the susceptibility of different ceramic materials to biofilm formation. This study aimed to evaluate biofilm development and associated roughness and color alterations in three lithium disilicate ceramics: Emax CAD (EC), Emax Press (EP), and LiSi Press (LP). Streptococcus mutans biofilms were grown on the three ceramics (n = 10 per group) for 7 days. Surface roughness values and color alteration were assessed before and after the biofilm using a non-contact profilometer and spectrophotometer, respectively. Biofilm growth was evaluated using colony-forming units (CFUs) and scanning electron microscope (SEM) images. The data were analyzed using one-way ANOVA and Tukey tests. There was a significant (p≤0.001) growth of S. mutans colonies on EC (6.75 ± 0.56) and EP (6.72 ± 0.54) specimens compared to LP, which showed no biofilm growth. The change in average surface roughness (∆Ra, nm) was significantly lower (p < 0.001) in the EC specimens (0.029 ± 0.003) compared to the EP (0.055 ± 0.012) and LP (0.041 ± 0.010). When the changes in the Rv and Rt values were investigated, no significant difference was observed among the groups. Following the biofilm challenge, the change in color (∆E00) was significantly lower (p = 0.005) in the LP group (1.68 ± 1.45) compared to the EC group (3.89 ± 1.50) and no significant difference was observed between the EP group (2.74±1.01) and the other two ceramics (p ≥ 0.05). LP ceramics exhibited superior resistance to S. mutans biofilm formation and associated changes in surface roughness and color compared to the Emax CAD and Emax Press ceramics. These findings suggest that the LiSi Press material may be more favorable to mitigate the risk of secondary caries.

Prevention of secondary caries using fluoride-loaded chitosan nanoparticle-modified glass-ionomer cement.

To study the effect of incorporating chitosan and fluoride-loaded chitosan nanoparticles into a glass-ionomer cement (GIC) to prevent secondary caries. A standard cervical cavity (mesio-distal width 6mm, cervico-occlusal width 2mm, and depth 2mm) was prepared on 30 molars for the following restoration groups: group 1, conventional GIC restoration; group 2, chitosan (10%) modified GIC restoration; group 3, fluoride loaded chitosan nanoparticles (10%) modified GIC restoration. The restored teeth were subjected to 1,500 thermal cycles before undergoing a multi-species cariogenic biofilm challenge. The restored teeth were examined by micro-computed tomography (micro-CT), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX). Data were analyzed by the one-way ANOVA, Tukey HDS, Kruskal Wallis, and Dunn's test. Micro-CT determined outer lesion depths for groups 1-3 were: 614 ± 20μm, 589 ± 17μm, and 560 ± 19μm respectively. Both modifications with chitosan and fluoride-loaded chitosan nanoparticles significantly affected outer lesion depth (p < 0.05). The modification with fluoride-loaded chitosan nanoparticles statistically significantly decreased the outer lesion depth compared to all other groups (p < 0.05). SEM/EDX showed an increase of calcium, phosphorus, and fluoride at the root dentine adjacent to the restoration in groups 2 and 3 (modified GIC). This increase was statistically significantly higher in the group modified with fluorine-loaded nano chitosan particles compared to the other groups (p < 0.05). Incorporation of 10% chitosan and 10% fluoride-loaded chitosan nanoparticles into GIC restorative material can prevent secondary root caries development. 10% fluoride-loaded chitosan nanoparticles were more effective. Glass ionomer cement modified with fluoride-loaded chitosan nanoparticles may be a promising restorative material in pediatric and preventive dentistry due to their controlled release properties.

Biofilm contamination in confined space stations: reduction, coexistence or an opportunity?

The prolonged human permanence in confined environments in space, such as in the case of the International Space Station, has inadvertently fostered conditions leading to uncontrolled microbial proliferation on surfaces, known as biofilm. Biofilm presence represents a challenge in critical spacecraft systems, that can lead to contamination issues and systems loss of function due to biofouling phenomena. This scenario is further complicated by microgravity that has a controversial role on biofilm growth and formation. Biocontamination can be a limiting factor in human long-term mission in outer Earth orbit and an economic and health issue on ISS. This study addresses the pressing need for effective antimicrobial strategies against such resilient biofilms in confined environments where the usage of biocidal chemical compounds is strictly controlled due to toxicity dangers. Traditional methods can be complemented by advanced antimicrobial coatings techniques. A promising approach is based on the oxygen plasma as coating platform. The technology can be potentially extended to a wide range of antibiofilm agents (e.g., peptides, bacteriophages, nanoparticles, quorum sensing disrupting agents, etc.) and substrates (e.g., metal, plastic, ceramic) showing an exceptional flexibility. An alternative vision of the biofilm challenge can be inspired by the dual nature of biofilms, addressed as “good” or “bad” depending on the specific application. Indeed, biofilm have a great potential in closed systems as small space habitat (e.g., ISS) that can be inspired by their role in “large space habitat” as planet Earth itself. The replication of such a complex biological equilibrium is an open challenge.

Application of cuminaldehyde and ciprofloxacin for the effective control of biofilm assembly of Pseudomonas aeruginosa: A combinatorial study

Pseudomonas aeruginosa is widely associated with biofilm-mediated antibiotic resistant chronic and acute infections which constitute a persistent healthcare challenges. Addressing this threat requires exploration of novel therapeutic strategies involving the combination of natural compounds and conventional antibiotics. Hence, our study has focused on two compounds; cuminaldehyde and ciprofloxacin, which were strategically combined to target the biofilm challenge of P. aeruginosa. The minimum inhibitory concentration (MIC) of cuminaldehyde and ciprofloxacin was found to be 400 μg/mL and 0.4 μg/mL, respectively. Moreover, the fractional inhibitory concentration index (FICI = 0.62) indicated an additive interaction prevailed between cuminaldehyde and ciprofloxacin. Subsequently, sub-MIC doses of cuminaldehyde (25 μg/mL) and ciprofloxacin (0.05 μg/mL) were selected for an array of antibiofilm assays which confirmed their biofilm inhibitory potential without exhibiting any antimicrobial activity. Furthermore, selected doses of the mentioned compounds could manage biofilm on catheter surface by inhibiting and disintegrating existing biofilm. Additionally, the test combination of the mentioned compounds reduced virulence factors secretion, accumulated reactive oxygen species and increased cell-membrane permeability. Thus, the combination of cuminaldehyde and ciprofloxacin demonstrates potential in combating biofilm-associated Pseudomonal threats.

Development, characterization, and evaluation of a simple polymicrobial colony biofilm model for testing of antimicrobial wound dressings.

Simple biofilm models are the first step to testing of any antimicrobial and wound dressing; therefore, the aim of this study was to develop and validate a simple polymicrobial colony biofilm wound model comprised of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans on RPMI-1640 agar. The model was then used to evaluate the topical disinfectant chlorohexidine and four commercially available wound dressings using the polymicrobial model. The model used was as a starting point to mimic debridement in clinical care of wounds and the effectiveness of wound dressings evaluated afterwards. Planktonic assessment using AATCC100-2004 demonstrated that all antimicrobial wound dressings reduced the planktonic microbial burden below the limit of detection; however, when challenged with polymicrobial colony biofilms, silver wound dressings showed limited effectiveness (1-2 log CFU reductions). In contrast, a single iodine releasing wound dressing showed potent antibiofilm activity reducing all species CFUs below the limit of detection (>6-10 log) depending on the species. A disrupted biofilm model challenge was performed to represent the debridement of a wound and wound silver-based wound dressings were found to be marginally more effective than in whole colony biofilm challenges while the iodine containing wound dressing reduced microbial recovery below the limit of detection. In this model, silver dressings were ineffective versus the whole colony biofilms but showed some recovery of activity versus the disrupted colony biofilm. The iodine wound dressing reduced the viability of all species below the level of detection. This suggests that mode of action of wound dressing should be considered for the type of biofilm challenge as should the clinical use, e.g. debridement.

Comparison of biofilm models for producing artificial active white spot lesions.

This study compared three protocols for developing artificial white spot lesions (WSL) using biofilm models. In total, 45 human enamel specimens were sterilized and allocated into three groups based on the biofilm model: Streptococcus sobrinus and Lactobacillus casei (Ss+Lc), Streptococcus sobrinus (Ss), or Streptococcus mutans (Sm). Specimens were incubated in filter-sterilized human saliva to form the acquired pellicle and then subjected to the biofilm challenge consisting of three days of incubation with bacteria (for demineralization) and one day of remineralization, which was performed once for Ss+Lc (four days total), four times for Ss (16 days total), and three times for Sm (12 days total). After WSL creation, the lesion fluorescence, depth, and chemical composition were assessed using Quantitative Light-induced Fluorescence (QLF), Polarized Light Microscopy (PLM), and Raman Spectroscopy, respectively. Statistical analysis consisted of two-way ANOVA followed by Tukey's post hoc test (α=0.05). WSL created using the Ss+Lc protocol presented statistically significant higher fluorescence loss (ΔF) and integrated fluorescence (ΔQ) in comparison to the other two protocols (p<0.001). In addition, Ss+Lc resulted in significantly deeper WSL (137.5 µm), followed by Ss (84.1 µm) and Sm (54.9 µm) (p<0.001). While high mineral content was observed in sound enamel surrounding the WSL, lesions created with the Ss+Lc protocol showed the highest demineralization level and changes in the mineral content among the three protocols. The biofilm model using S. sobrinus and L. casei for four days was the most appropriate and simplified protocol for developing artificial active WSL with lower fluorescence, higher demineralization, and greater depth.

Response Surface Methodology Application for Bacteriophage-Antibiotic Antibiofilm Activity Optimization.

Phage-antibiotic combination-based protocols are presently under heightened investigation. This paradigm extends to engagements with bacterial biofilms, necessitating novel computational approaches to comprehensively characterize and optimize the outcomes achievable via these combinations. This study aimed to explore the Response Surface Methodology (RSM) in optimizing the antibiofilm activity of bacteriophage-antibiotic combinations. We employ a combination of antibiotics (gentamicin, meropenem, amikacin, ceftazidime, fosfomycin, imipenem, and colistin) alongside the bacteriophage vB_AbaP_AGC01 to combat Acinetobacter baumannii biofilm. Based on the conducted biofilm challenge assays analyzed using the RSM, the optimal points of antibiofilm activity efficacy were effectively selected by applying this methodology, enabling the quantifiable mathematical representations. Subsequent optimization showed the synergistic potential of the anti-biofilm that arises when antibiotics are judiciously combined with the AGC01 bacteriophage, reducing biofilm biomass by up to 80% depending on the antibiotic used. The data suggest that the phage-imipenem combination demonstrates the highest efficacy, with an 88.74% reduction. Notably, the lower concentrations characterized by a high maximum reduction in biofilm biomass were observed in the phage-amikacin combination at cA = 0.00195 and cP = 0.38 as the option that required minimum resources. It is worth noting that only gentamicin antagonism between the phage and the antibiotic was detected.

Evaluation of Acid Resistance of Demineralized Dentin after Silver Diamine Fluoride and Potassium Iodide Treatment

This study investigated the effects of silver diamine fluoride (SDF) and potassium iodide (KI) treatments on the acid resistance of dentin exposed to secondary caries. Sixteen bovine dentin specimens with artificially induced caries were assigned to the following four groups: untreated negative control, untreated positive control, SDF-treated (SDF), and SDF and KI-treated (SDFKI). Multispecies cariogenic biofilms containing &lt;i&gt;Streptococcus mutans&lt;/i&gt;, &lt;i&gt;Lactobacillus casei&lt;/i&gt;, and &lt;i&gt;Candida albicans&lt;/i&gt; were cultured on the specimens for 28 days, except for the negative control group. Specimens from the negative control group were stored in phosphate-buffered saline for that period. After a cariogenic biofilm challenge, the degree of demineralization was evaluated using micro-computed tomography (micro-CT). As a result of data analysis using micro-CT, the demineralization depths of the negative control, positive control, SDF, and SDFKI groups were 149.0 ± 7 μm, 392.0 ± 11 μm, 206.0 ± 20 μm, and 230.0 ± 31 μm, respectively. The degree of demineralization was significantly reduced in the SDF and SDFKI groups compared with that in the untreated positive control group. There were no significant differences between the SDF and SDFKI groups. This study confirmed that SDF and SDFKI treatments increase the acid resistance of dentin to secondary caries. KI did not significantly affect the cariesarresting effect of the SDF.

Optimal Surface Pre-Reacted Glass Filler Ratio in a Dental Varnish Effective for Inhibition of Biofilm-Induced Root Dentin Demineralization.

A unique type of dental varnish (DV) containing surface pre-reacted glass (S-PRG) fillers of different concentrations was evaluated to determine the unpresented optimal ratio for inhibiting root dentin bio-demineralization. S-PRG DVs (10% to 40%)-10%-S, 20%-S, 30%-S, and 40%-S-were applied to bovine root dentin blocks and compared with controls-0%-f (no S-PRG) and 5%-NaF (5%-NaF). The Streptococcus mutans biofilm challenge was executed inside and outside an oral biofilm reactor for 7 days. The specimens were examined using a confocal laser scanning microscope and swept-source optical coherence tomography. Furthermore, they were observed using a scanning electron microscope and analyzed using energy-dispersive X-ray spectroscopy. The roughness (SzJIS) due to leaching of DV materials and demineralization depth were significantly less in the S-PRG groups than the control groups (p &lt; 0.05). Complete or partially plugged dentinal tubules (DTs) were observed in 20%-S, 30%-S, and 40%-S, while wide-open DTs were observed more in controls. Cylindrical tags were present in groups containing more than 20% S-PRG. F, Na, Al, and Sr were detected in a higher percentile ratio in the 20%-S, 30%-S, and 40%-S groups compared to 0%-f and 10%-S. Nonetheless, it is suggested that incorporating 20% to 30% S-PRG fillers in DVs would be effective enough as an anti-demineralization coating, together with supplementing minerals; further evaluation is required to validate these findings.

Synbiotic-fluoride synergism on enamel remineralization, cytotoxicity and genotoxicity

Objective(s)The objectives of the present study were to examine the – a) enamel remineralization potential of synbiotic-fluoride (SF) therapy using a multi-species bacterial pH-cycling model; and b) cytotoxic and genotoxic effects of SF therapy extracts. Materials and methodsThe SF therapy group comprised of 2% arginine (Arg), 0.2% NaF, and a probiotic Lactobacillus rhamnosus GG (LRG). The intervention groups studied were: 1) No treatment; 2) 2% Arg; 3) 0.2% NaF; 4) LRG; 5) 2% Arg+0.2% NaF; 6) 2% Arg+LRG; 7) 0.2% NaF+LRG; and 8) 2% Arg+0.2% NaF+LRG (SF therapy). The enamel remineralization potential of SF therapy was investigated under cariogenic biofilm challenge; while the cytotoxicity and genotoxicity of SF therapy extracts were examined on HGF-1 and Chinese hamster fibroblast V79, respectively. To determine the remineralization effect, the specimens were subjected to mineral density (MD) assessment using micro-CT, Ca/P molar ratio with SEM-EDX, and enamel fluoride uptake (EFU) estimates. The HGF-1 proliferation assessment was quantified using MTT/CCK-8 assays with qualitative analysis by nuclei staining Hoechst-based fluorescence imaging. The genotoxicity was determined by micronuclei formation test. ResultsMineral gain and %remineralization derived from MD assessment for the SF therapy were significantly higher than the other groups (p<0.05). The %ΔCa/P for the SF and 2% Arg+0.2% NaF were significantly higher than the other groups (p<0.05). The SF and 2% Arg+0.2% NaF groups had the highest EFU compared to the other groups (p<0.05). No significant difference in the %viable HGF-1 cells were observed between the treatment interventions and no treatment group (p>0.05). Compared to the EMS-positive control, the micronuclei formation for all the intervention groups was significantly lower (p<0.05), with no significant difference among the treatment groups (p>0.05). ConclusionThe SF therapy enhanced enamel remineralization with no biocompatibility concerns. Clinical significanceWith the enhanced enamel remineralization potential discerned in the present study, the SF therapy can be used as a promising caries-preventive agent targeted for high caries-risk individuals.

Synergistic interaction of cuminaldehyde and tobramycin: a potential strategy for the efficient management of biofilm caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic pathogen, has been found to cause several chronic and acute infections in human. Moreover, it often shows drug-tolerance and poses a severe threat to public healthcare through biofilm formation. In this scenario, two molecules, namely, cuminaldehyde and tobramycin, were used separately and in combination for the efficient management of biofilm challenge. The minimum inhibitory concentration (MIC) of cuminaldehyde and tobramycin was found to be 150µg/mL and 1µg/mL, respectively, against Pseudomonas aeruginosa. The checkerboard assay revealed that the fractional inhibitory concentration (FIC) index of cuminaldehyde and tobramycin was 0.36 suggesting a synergistic association between them. The sub-MIC dose of cuminaldehyde (60µg/mL) or tobramycin (0.06µg/mL) individually did not show any effect on the microbial growth curve. However, the same combinations could affect microbial growth curve of Pseudomonas aeruginosa efficiently. In connection to biofilm management, it was observed that the synergistic interaction between cuminaldehyde and tobramycin could inhibit biofilm formation more efficiently than their single use (p < 0.01). Further investigation revealed that the combinations of cuminaldehyde and tobramycin could generate reactive oxygen species (ROS) that resulted in the increase of membrane permeability of bacterial cells leading to the efficient inhibition of microbial biofilm formation. Besides, the synergistic interaction between cuminaldehyde (20µg/mL) and tobramycin (0.03µg/mL) also showed significant biofilm dispersal of the test microorganism (p < 0.01). Hence, the results suggested that synergistic action of cuminaldehyde and tobramycin could be applied for the efficient management of microbial biofilm.

Comparison of collagen features of distinct types of caries-affected dentin

ObjectivesTo compare the biodegradability, mechanical behavior, and physicochemical features of the collagen-rich extracellular matrix (ECM) of artificial caries-affected dentin (ACAD), natural caries-affected dentin (NCAD) and sound dentin (SD). MethodsDentin specimens from human molars were prepared and assigned into groups according to the type of dentin: ACAD, NCAD, or SD. ACAD was produced by incubation of demineralized SD with Streptococcus mutans in a chemically defined medium (CDM) with 1% sucrose for 7 days at 37 °C under anaerobic conditions. Specimens were assessed to determine collagen birefringence, biodegradability, mechanical behavior, and chemical composition. Data were individually processed and analyzed by ANOVA and post-hoc tests (α = 0.05). ResultsCDM-based biofilm challenge reduced loss, storage, and complex moduli in ACAD (p < 0.001), while the damping capacity remained unaffected (p = 0.066). Higher red and lower green birefringence were found in ACAD and NCAD when compared with SD (p < 0.001). Differently to ACAD, SD and NCAD presented higher biodegradability to exogenous proteases (p = 0.02). Chemical analysis of the integrated areas of characteristic bands that assess mineral quality (carbonate/phosphate and crystallinity index), mineral to matrix (phosphate/amide I) and post-translational modifications (amide III/CH2, pentosidine/CH2, and pentosidine/amide III) (p<0.05) showed that NCAD was significantly different from SD while ACAD exhibited intermediate values. ConclusionsCDM-based biofilm challenge produced a dentin ECM with decreased mechanical properties and increased collagen maturity. The compositional and structural conformation of the ACAD suggested that CDM-based biofilm challenge showed potential to produce artificial lesions by revealing a transitional condition towards mimicking critical features of NCAD. Clinical significanceThis study highlights the importance of developing a tissue that mimics the features of natural caries-affected dentin ECM for in vitro studies. Our findings suggested the potential of a modified biofilm challenge protocol to produce and simulate a relevant substrate, such as caries-affected dentin.

膜面バイオフィルムを形成する微生物：細菌の特定から制御へ

膜面バイオフィルムを形成する微生物：細菌の特定から制御へ

Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.

Characteristics of biofilm-induced degradation at resin–dentin interfaces using multiple combinations of adhesives and resins

ObjectiveWe aimed to evaluate morphological, mechanical and chemical characteristics at resin–dentin interfaces using multiple combinations of adhesives and resins after a short-term biofilm-induced degradation. MethodsCervical cavities were prepared in bovine incisors, treated by Clearfil SE Bond 2 (SE) or FL-Bond II (FL), restored by Clearfil Majesty ES Flow (ES) or Beautifil Flow Plus (BFP) and grouped into SE-ES, SE-BFP, FL-ES and FL-BFP. After biofilm challenge, interfacial gaps and dentin wall lesions were examined by optical coherence tomography (OCT). Gap depth (GD), gap pattern scale (GPS) and dentin wall lesion depth (WLD) were evaluated from confocal laser scanning microscope. Microhardness of dentin lesions was measured with a Vickers microhardness tester. Chemical elements in resins and dentin wall lesions were analyzed by scanning electron microscopy and energy dispersive X-ray spectrometry (SEM/EDS). Morphological structures of interfacial gaps were observed by SEM. ResultsOCT could detect adhesive-dentin-bonded and adhesive-dentin-debonded gaps. SE-containing groups showed significantly lower GPS than FL-containing groups. FL-BFP showed significantly lower WLD than FL-ES. Microhardness of dentin wall lesions was higher than that of outer lesions and they showed significant differences in FL-BFP. SE-BFP showed a lower GPS curve and higher intensities of Ca and P in the upper half of dentin wall lesions than other groups. From SEM, microgaps between filler and matrix, break and loss of matrix, separation of adhesive matrix with hybrid layer occurred at interfacial gaps. SignificanceThe morphological, mechanical and chemical characteristics of resin–dentin interfacial degradation depend on the component and chemistry of restorative materials.

Effect of ionizing radiation and cariogenic biofilm challenge on root-dentin caries.

To evaluate the effect of ionizing radiation and cariogenic biofilm challenge using two continuous flow models, normal and reduced salivary flow, on the development of initial root-dentin caries lesions. Microcosm biofilms were grown under two salivary flow rates (0.06 and 0.03mLmin-1) and exposed to 5% sucrose (3 × daily, 0.25mLmin-1, 6min) dripped over non-irradiated and irradiated root-dentin blocks for up to 7days. The vibration modes of root dentin, matrix/mineral (M/M), and carbonate/mineral (C/M) ratios were evaluated by FTIR. The mineral density was assessed by micro-CT. With normal salivary flow, FTIR revealed an increase in the organic matrix (amide III) and a decrease in the mineral phase (ν4, ν2 PO43-, AII + ν2 CO32-, C/M) in caries lesions. Irradiated dentin exhibited a reduction in the mineral phase (ν1, ν3 PO43-, ν2 CO32-, C/M). Differences in mineral densities were not significant. With reduced salivary flow, FTIR also revealed increased organic matrix (amide III) for irradiated caries lesions and decrease in mineral phase (v4, v2 PO43-, v2 CO32-, and C/M) in caries lesions. ν1, ν3 PO43- precipitated on the surface of irradiated dentin and a lower mineral density was observed. Initial caries lesions differed between non-irradiated and irradiated dentin and between normal and reduced salivary flow rates. Significant mineral loss with exposure of the organic matrix and low mineral density were observed for irradiated dentin with a reduced salivary flow rate. Ionizing radiation associated with a reduced salivary flow rate enhanced the progression of root-dentin caries.

Inhibition of dentine caries using fluoride solution with silver nanoparticles: An in vitro study

ObjectivesTo investigate the remineralising and staining effects of sodium fluoride (NaF) with silver nanoparticles (AgNPs) on artificial dentine caries. MethodsHuman dentine blocks with artificial caries were divided into four groups. Group 1 received 5 % NaF (22,600 ppm fluoride) with 4000 ppm AgNPs; group 2 received 4000 ppm AgNPs; group 3 received 5 % NaF, group 4 received deionised water (negative control). All groups underwent three biochemical cycles. Each cycle included Streptococcus mutans biofilm challenge and remineralisation process. The lesion depth, mineral-organic content, surface morphology and crystal characteristics of dentine blocks were evaluated using micro-computed tomography, Fourier transform infrared spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction. Colour change of dentine blocks was assessed using spectrophotometry. ResultsThe mean lesion depths of groups 1–4 were 151.13 ± 29.13 μm, 172.38 ± 23.44 μm, 190.41 ± 32.81 μm and 221.24 ± 27.91 μm, respectively. The hydrogen phosphate-to-amide I ratios of groups 1–4 were 5.98 ± 0.36, 3.86 ± 0.56, 4.00 ± 0.67 and 2.53 ± 0.40, respectively. There was no significant interaction effect between AgNPs and NaF. SEM showed less exposure of dentine collagen fibres in group 1 when compared to other groups. X-ray diffraction revealed presence of silver chloride and metallic silver in group 1 and 2. There was no significant difference in colour change among the four groups (p = 0.74). ConclusionsNaF solution with AgNPs can remineralise dentine caries without staining. Clinical SignificanceSodium fluoride solutions that include silver nanoparticles have potential uses in the management of caries.

Bacteriologic status of non-cavitated proximal enamel caries lesions. A histologic and histobacteriologic study

ObjectivesThis histologic and histobacteriologic study evaluated non-cavitated interproximal white spot lesions in human teeth for the 1) presence and morphology of bacterial aggregations at the enamel-dentinal junction (EDJ), and 2) reactions in the subjacent pulp tissue. MethodsThe material comprised 16 third molars diagnosed with early interproximal caries lesions obtained consecutively in a single clinical practice. Four third molars with clinically intact proximal surfaces served as controls. Caries activity was categorized as active or arrested. Teeth were prepared for histologic and histobacteriologic analyses. ResultsControl teeth exhibited normal tissue conditions with no bacteria. Macroscopic discoloration of the superficial dentin occurred in 14/16 teeth with early caries lesions. Bacterial aggregations resembling biofilms were observed in 10/16 teeth (six with active and four with arrested lesions). Bacterial cells were observed superficially in some of the underlying dentinal tubules, while in a few cases a deeper penetration was evident. Pathologic changes of varying degrees were observed in the pulps of all 16 teeth, regardless of the caries activity (active or arrested). Pulp reactions varied from disruption of the odontoblast layer, with loss of odontoblasts, to formation of tertiary dentin. ConclusionsBacterial biofilms associated with white-spot caries lesions may traverse the enamel and reach the underlying dentin in both active and arrested lesions. In all teeth with early lesions, the pulps showed changes in response to the very superficial biofilm challenge. Clinical significanceIn non-cavitated, active or arrested, enamel caries lesions, bacteria traverse the enamel and may establish structured biofilms at the enamel-dentinal junction, causing early pulp changes. These new findings may stimulate clinicians to rethink the rationale for treatment methods that are based on the assumption that bacteria are absent in white-spot lesions.

Polymeric nanoparticles protect the resin-dentin bonded interface from cariogenic biofilm degradation

The objective was to assess doxycycline (Dox) and zinc (Zn) doped nanoparticles' (NPs) potential to protect the resin-dentin interface from cariogenic biofilm. Three groups of polymeric NPs were tested: unloaded, loaded with zinc and with doxycycline. NPs were applied after dentin etching. The disks were exposed to a cariogenic biofilm challenge in a Drip-Flow Reactor during 72 h and 7 d. Half of the specimens were not subjected to biofilm formation but stored 72 h and 7 d. LIVE/DEAD® viability assay, nano-dynamic mechanical assessment, Raman spectroscopy and field emission electron microscopy (FESEM) analysis were performed. The measured bacterial death rates, at 7 d were 46% for the control group, 51% for the undoped-NPs, 32% for Dox-NPs, and 87% for Zn-NPs; being total detected bacteria reduced five times in the Dox-NPs group. Zn-NPs treated samples reached, in general, the highest complex modulus values at the resin-dentin interface over time. Regarding the mineral content, Zn-NPs-treated dentin interfaces showed the highest mineralization degree associated to the phosphate peak and the relative mineral concentration. FESEM images after Zn-NPs application permitted to observe remineralization of the etched and non-resin infiltrated collagen layer, and bacteria were scarcely encountered. The combined antibacterial and remineralizing effects, when Zn-NPs were applied, reduced biofilm formation. Dox-NPs exerted an antibacterial role but did not remineralize the bonded interface. Undoped-NPs did not improve the properties of the interfaces. Application of Zn-doped NPs during the bonding procedure is encouraged. STATEMENT OF SIGNIFICANCE: Application of Zn-doped nanoparticles on acid etched dentin reduced biofilm formation and viability at the resin-dentin interface due to both remineralization and antibacterial properties. Doxycycline-doped nanoparticles also diminished oral biofilm viability, but did not remineralize the resin-dentin interface.