The oral microbiota is a reservoir for antimicrobial resistance: resistome and phenotypic resistance characteristics of oral biofilm in health, caries, and periodontitis.

BACKGROUND: Antimicrobial resistance (AMR) is an ever-growing threat to modern medicine and, according to the latest reports, it causes nearly twice as many deaths globally as AIDS or malaria. Elucidating reservoirs and dissemination routes of antimicrobial resistance genes (ARGs) are essential in fighting AMR. Human commensals represent an important reservoir, which is underexplored for the oral microbiota. Here, we set out to investigate the resistome and phenotypic resistance of oral biofilm microbiota from 179 orally healthy (H), caries active (C), and periodontally diseased (P) individuals (TRN: DRKS00013119, Registration date: 22.10.2022). The samples were analysed using shotgun metagenomic sequencing combined, for the first time, with culture technique. A selection of 997 isolates was tested for resistance to relevant antibiotics. RESULTS: The shotgun metagenomics sequencing resulted in 2,069,295,923 reads classified into 4856 species-level OTUs. PERMANOVA analysis of beta-diversity revealed significant differences between the groups regarding their microbiota composition and their ARG profile. The samples were clustered into three ecotypes based on their microbial composition. The bacterial composition of H and C samples greatly overlapped and was based on ecotypes 1 and 2 whereas ecotype 3 was only detected in periodontitis. We found 64 ARGs conveying resistance to 36 antibiotics, particularly to tetracycline, macrolide-lincosamide-streptogramin, and beta-lactam antibiotics, and a correspondingly high prevalence of phenotypic resistance. Based on the microbiota composition, these ARGs cluster in different resistotypes, and a higher prevalence is found in healthy and caries active than in periodontally diseased individuals. There was a significant association between the resistotypes and the ecotypes. Although numerous associations were found between specific antibiotic resistance and bacterial taxa, only a few taxa showed matching associations with both genotypic and phenotypic analyses. CONCLUSIONS: Our findings show the importance of the oral microbiota from different niches within the oral cavity as a reservoir for antibiotic resistance. Additionally, the present study showed the need for using more than one method to reveal antibiotic resistance within the total oral biofilm, as a clear mismatch between the shotgun metagenomics method and the phenotypic resistance characterization was shown.

Cytotoxicity and estrogenicity in simulated dental wastewater after grinding of resin-based materials.

OBJECTIVE: This study evaluated the cytotoxic and estrogenic effects of dust and eluates released into simulated wastewater after grinding of dental resin-based materials. METHODS: Four materials were used: ceram.x® universal, Filtek™ Supreme XTE, Lava™ Ultimate and Core-X™ flow. From each composite material, samples (5 × 2 mm, n = 50) were prepared according to the manufacturers' instructions. Lava™ Ultimate was used as blocks. All samples were ground to dust with a diamond bur (106 µm) and suspended in distilled water at 60 mg/mL. After storage for 72 h, the suspensions were separated into a soluble (eluate) and a particulate (dust) fraction. Eluates and dusts were evaluated for inhibition of Vibrio fischeri bioluminescence and cytotoxicity on human A549 lung cells (WST-1-Assay). The estrogenic activity was assessed by YES-Assay using Saccharomyces cerevisiae. Additionally, dental monomers (BisGMA, BisEMA, UDMA, TEGDMA, HEMA) and Bisphenol A were investigated. RESULTS: All eluates showed inhibition of V. fischeri bioluminescence at concentrations above 1.1 mg/mL (p < 0.05). The activity of the eluates of ceram.x® universal and Filtek™ Supreme XTE was significantly higher than Lava™ Ultimate and Core-X™ flow (p < 0.05). In the WST-1-Assay, all materials induced cytotoxic effects at concentrations of 0.1 mg/mL (p < 0.05), while no significant differences were detected among them. The tested materials revealed no estrogenic activity. All dental monomers and Bisphenol A showed concentration dependent cytotoxic effects (p < 0.05), whereas only Bisphenol A induced an estrogenic effect (p < 0.01). SIGNIFICANCE: Dust and eluates of resin-based dental materials released into wastewater exert bactericidal and cytotoxic effects in vitro. However, they reveal no estrogenic effect.

N-Acetylcysteine modulates the effects of composites on human gingival keratinocytes.

OBJECTIVE: The aim of this study was to evaluate, if antioxidants, like N-Acetylcysteine, can modulate effects of composite eluates on human gingival keratinocytes. METHODS: Composite samples of ceram.x® universal, Filtek™ Supreme XTE, and Admira® Fusion were stored 72h in cell culture medium to prepare eluates, according to ISO 10993-12:2012. Human gingival keratinocytes were exposed to these eluates with or without 3mM N-Acetylcysteine. Following cell observation by iCELLigence®, exposure periods were determined at 1d and 4d. Cell morphological analysis combined with live/dead staining was performed. Tissue-specific biomarkers of terminal differentiation, Involucrin and Filaggrin, were analyzed by indirect immunofluorescence (IIF) and Western blot (WB). qPCR profiling was performed on genes encoding for: inflammation, apoptosis, turn-over of extracellular matrix, adhesion, proliferation and differentiation. For statistical analysis one-way Anova was used (p<0.05). RESULTS: Cells exposed to N-Acetylcysteine exhibited morphological changes but no cell death. After adding 3mM N-Acetylcysteine to HGK cultures, increased fluorescence intensity and protein amounts of Involucrin and Filaggrin indicated enhanced differentiation (p<0.05). Gene expression was modulated by: (i) composition of the composite eluates, (ii) NAC and (iii) exposure time. Filtek™ Supreme XTE showed a significant increased gene expression in inflammatory genes (p<0.05), which was amplified by the addition of NAC at 1d. Concerning exposure time, modulated gene expression showed eluate dependency, substantiated by Filtek™ Supreme XTE modulation at day 1 and Admira® Fusion at day 4. SIGNIFICANCE: N-Acetylcysteine-emerging effects on gingival keratinocytes were threefold: (i) increase of differentiation, (ii) modulation of composite-related effects and (iii) in parts counteraction of eluate-induced effects.

Influence of the long-term use of oral hygiene products containing stannous ions on the salivary microbiome - a randomized controlled trial.

Oral hygiene products containing tin are suitable to prevent erosive tooth wear, yet effects on the oral microbiota are not known yet. Therefore, this study determined the salivary microbiome of 16 participants using products with stannous ions for three years (TG) compared with a control group (CG) to assess their influence on the microbiota. Participants were included in a randomized controlled clinical trial (RCT) with biannual visits. Illumina Miseq sequencing revealed as most abundant genera: Streptococcus (TG 14.3%; CG 13.0%), Veillonella (TG 11.3%; CG 10.9%), Prevotella (TG 7.0%; CG 9.8%), Haemophilus (TG 6.6%; CG 7.2%), Porphyromonas (TG 5.9%, CG 5.1%), Leptotrichia (TG 5.8%; CG 4.9%), Actinomyces (TG 4.0%; CG 4.6%) and Neisseria (TG 5.4%; CG 4.2%). Beta-Diversity was not significantly different between groups at both time points, although significant differences between groups were found for certain taxa after three years. The genus Prevotella was found in higher abundance in CG whereas Neisseria and Granulicatella, health-associated taxa, were found more abundantly in TG. Salivary microbiota after three years reflected a composition associated with oral health, hence continual use as a preventive measure for dental erosion can be considered safe and benefitting oral health for patients with a high risk of erosion.

Comparative differentiation analysis of distinct oral tissue-derived cells in response to osteogenic stimulation.

BACKGROUND: Mixed cell populations from oral tissues may be superior to pure stem cells for regenerative approaches. Therefore, the aim of the present study was to explore the osteogenic potential of mixed cells derived from oral connective tissues compared to alveolar osteoblasts. MATERIALS AND METHODS: Primary cells were isolated from the alveolar bone, periodontal ligament and gingiva. Following characterization by colony formation, growth capacity and flow cytometry, all cells were subjected to osteogenic differentiation induction and screened for a large panel of osteogenic markers using western blots, qPCR arrays, and matrix mineralization and alkaline phosphatase quantification. RESULTS: Non-induced mixed cells from gingiva showed higher colony formation efficiency but decreased proliferation compared to non-induced periodontal mixed cells, while both entities revealed similar surface markers tested in this setup. Following osteogenic induction, all cell populations individually expressed receptors with distinctively activated downstream effectors. Gene expression of induced periodontal mixed cells was similar to alveolar osteoblasts, but was differently modulated in gingival mixed cells. The latter failed to achieve osteogenic differentiation in terms of matrix mineralization and alkaline phosphatase activity, which was well observed in periodontal mixed cells and osteoblasts. CONCLUSION: Mixed cells from periodontal ligament but not from gingiva feature an inherent osteogenic capacity in vitro. From these results, it can be concluded that periodontal cells do not require further stem cell enrichment in order to qualify for bone regeneration. CLINICAL RELEVANCE: Our data contribute to the development of novel cell-based therapies using mixed cells from the periodontal ligament in regenerative periodontics.

Evaluation of the bioactivity of fluoride-enriched mineral trioxide aggregate on osteoblasts.

AIM: To investigate whether a combination of mineral trioxide aggregate (MTA) and fluoride compounds affects bone cells. METHODOLOGY: Mineral trioxide aggregate (MTA) discs (ProRoot® , Dentsply Sirona, Ballaigues, Switzerland) with and without the addition of 0.1%, 0.25% and 0.5% sodium fluoride were characterized for their surface roughness by laser scanning microscopy and for the adhesion of human alveolar osteoblasts by scanning electron microscopy. Using eluates from fluoride-enriched MTA discs, the cell proliferation was measured by monitoring the DNA incorporation of 5-bromo-2'-deoxyuridine. Further, gene expression was evaluated by qPCR arrays, extracellular matrix mineralization was quantified by absorption measurement of Alizarin red stains, and effects were calculated with repeated measures analysis and post hoc P-value adjustment. RESULTS: Irrespective of fluoride addition, cell adhesion was similar on MTA discs, of which the surface roughness was comparable. Control osteoblasts had a curvilinear proliferation pattern peaking at d5, which was levelled out by incubation with MTA. The addition of fluoride partly restored the MTA-related reduction in the cellular proliferation rate in a dose-dependent manner. At the mRNA level, both fluoride and MTA modulated a number of genes involved in osteogenesis, bone mineral metabolism and extracellular matrix formation. Although MTA significantly impaired extracellular matrix mineralization, the addition of fluoride supported the formation of mineralized nodules in a dose-dependent manner. CONCLUSION: The addition of fluoride modulated the biocompatibility of MTA in terms of supporting bone cell proliferation and hard tissue formation. Hence, fluoride enrichment is a trend-setting advancement for MTA-based endodontic therapies.

Modulation of gingival cell response towards dental composites.

OBJECTIVE: This aim of this study was to investigate the cell behavioural response on clinically applied dental composites in exposition-relevant target cells, i.e. human gingival fibroblasts (HGF) and epithelial keratinocytes (HGK). METHODS: HGF and HGK were exposed to eluates of Ceram X™, Filtek™ Silorane, Filtek™ Supreme XTE, Fusio™ Liquid Dentin and Vertise™ Flow. Eluates were created by storing material samples in respective cell culture medium, for 24h and 72h (n=17), according to ISO 10993-12:2012. Cell response was evaluated at eluate exposure periods of 24h and 72h by (i) impedance analysis-based real-time monitoring of adhesion and proliferation, (ii) semi-quantitative indirect immunofluorescence (sq-IIF) detection of tissue-specific biomarkers, and (iii) ELISA-detection of pro-inflammatory interleukin (IL)-6. RESULTS: Generally, cell behavioural response towards the eluates was gradual in HGK and HGF, the latter exhibiting a less pronounced modulation per se. In HGK, ERK 1/2 was mainly activated after 24h by Fusio™ Liquid Dentin and Vertise™ Flow, while an increase in biomarker expression occurred time-delayed. A 72h exposure of HGK to eluates of Filtek™ Supreme XTE, Fusio™ Liquid Dentin and Vertise™ Flow significantly decreased secreted IL-6 amounts. In HGK, the impedance analysis revealed less proliferation and/or adhesion in case of Fusio™ Liquid Dentin and Vertise™ Flow with matched other composites. SIGNIFICANCE: In detail, protein expression and secretion is modulated particularly in terms of signal transduction, differentiation and inflammation. On cell biological level, all tested materials modulated the analysed features of cell behaviour with emphasis on the self-adhering composites.

An oral health optimized diet can reduce gingival and periodontal inflammation in humans - a randomized controlled pilot study.

BACKGROUND: The aim of this pilot study was to investigate the effects of four weeks of an oral health optimized diet on periodontal clinical parameters in a randomized controlled trial. METHODS: The experimental group (n = 10) had to change to a diet low in carbohydrates, rich in Omega-3 fatty acids, and rich in vitamins C and D, antioxidants and fiber for four weeks. Participants of the control group (n = 5) did not change their dietary behavior. Plaque index, gingival bleeding, probing depths, and bleeding upon probing were assessed by a dentist with a pressure-sensitive periodontal probe. Measurements were performed after one and two weeks without a dietary change (baseline), followed by a two week transitional period, and finally performed weekly for four weeks. RESULTS: Despite constant plaque values in both groups, all inflammatory parameters decreased in the experimental group to approximately half that of the baseline values (GI: 1.10 ± 0.51 to 0.54 ± 0.30; BOP: 53.57 to 24.17 %; PISA: 638 mm(2) to 284 mm(2)). This reduction was significantly different compared to that of the control group. CONCLUSION: A diet low in carbohydrates, rich in Omega-3 fatty acids, rich in vitamins C and D, and rich in fibers can significantly reduce gingival and periodontal inflammation. TRIAL REGISTRATION: German Clinical Trials Register; https://www.germanctr.de (DRKS00006301). Registered on 2015-02-21.

Listerine® Products: An Update on the Efficacy and Safety.

UNLABELLED: In the 19th century, the mouthwash Listerine® was formulated from four essential oils. Later, the oils were replaced by their marker substances. To keep them in solution, 24-27% ethanol was added as a vehicle. This is an update of our previous review on the efficacy and safety of Listerine®. METHOD: PubMed was searched for clinical studies on the therapeutic benefits and safety of Listerine® from the end of 2011 to the end of October 2015. RESULTS: Sixteen studies were found and extracted. Three of the four 6-month studies were of sound confirmatory design. Two of these investigated Listerine® and one Listerine Zero®. The evidence of effectiveness for Listerine®, based on the bulk of three confirmatory studies and numerous exploratory studies carried out so far, is strong, but only moderate for Listerine® Zero and poor for Listerine® Cool Blue. In the three safety studies identified, we found methodological flaws that biased the results. CONCLUSIONS: Evidence is accumulating that Listerine® is effective in improving oral health, but the absence of systematic toxicological studies means that an accurate safety assessment cannot be made.

Prevalence of Candida albicans and Candida dubliniensis in caries-free and caries-active children in relation to the oral microbiota-a clinical study.

OBJECTIVES: The correlation between caries and the oral prevalence of Candida spp. in children is contradictory in literature. Thereby, authors focused on Candida albicans as the most isolated Candida species from the oral cavity. Therefore, the aim of the present study was to compare caries-free and caries-bearing children regarding their oral carriage of Candida spp. MATERIAL AND METHODS: Twenty-six caries-free (CF group) and 26 caries-active children (CA group) were included into this study. Three different types of specimens were assessed, saliva and plaque, and in the case of caries, infected dentine samples were microbiologically analyzed for aerobic and anaerobic microorganisms and their counts. Special attention was given to the differentiation between C. albicans and Candida dubliniensis. Additionally, different biochemical tests, VITEK 2 (VITEK®2, bioMérieux, Marcy-l'Etoile, France) and 16S and 18S ribosomal DNA (rDNA) sequencing, were applied for identification. RESULTS: The detection of C. albicans did not differ between the CF and CA groups. C. dubliniensis was never detected in any specimen of the CF group, but occurred in one quarter of the CA group (27 % in plaque, 23 % in saliva), thus leading to a statistically significant difference between the two groups (p < 0.05). In six of these cases, C. dubliniensis was detected concomitantly in saliva and plaque and once only in plaque. CA group harbored statistically more Streptococcus mutans than the control group revealing a correlation between S. mutans and C. dubliniensis regarding the caries group. CONCLUSIONS: This is the first study reporting a frequent detection of C. dubliniensis in caries-active children, which could have been underestimated so far due to difficulties in differentiation between this yeast species and C. albicans. CLINICAL RELEVANCE: Microbiological diagnostic-especially of oral Candida species-is an important determinant for identifying etiological factors of dental caries in children.

The effects of various mixing solutions on the biocompatibility of mineral trioxide aggregate.

AIM: To evaluate the effects of various mixing solutions on the biocompatibility of mineral trioxide aggregate (MTA). METHODOLOGY: Human alveolar osteoblasts (hOAs) were incubated with eluates of 24 h-set cement discs of MTA mixed with sterile H2 O, 3% sodium hypochlorite (NaOCl), 4% articaine (Ultracain(®) D-S), 0.9% NaCl, Ringer's solution or citrated blood, respectively. The cell proliferation in the presence of eluates was assessed by real-time cell analysis, and the expression of genes associated with proliferation (histone H3, HistH3), inflammation (interleukin-6, IL-6, matrix metalloproteinases 1 and 3, MMP1, MMP3) or apoptosis (caspase 3, Casp3) was analysed by qPCR after 24 and 72 h. The ultrastructure of cells grown on cement discs was visualized by scanning electron microscopy (SEM), whilst actin cytoskeleton was monitored by fluorescence staining in the presence of eluates after 7 and 14 days. A repeated-measure analysis was performed, and P-values were adjusted by Tukey. RESULTS: Whilst articaine-MTA sustained hOA proliferation patterns similar to H2 O-MTA, NaOCl-MTA reduced hOA proliferation and significantly increased the expression of MMP1 and MMP3. The addition of H2 O and articaine modulated the gene expression of Casp3 or Hist3H3. The use of NaCl, Ringer and blood induced mRNA levels comparable to matched controls. With the exception of NaOCl-MTA, SEM and FM revealed regular hOA morphology for all mixing solutions. CONCLUSIONS: NaOCl was highly cytotoxic for hOAs whilst all other mixing solutions can be considered as convenient biocompatible mixing solutions as alternatives to H2 O for clinical use.

Decision criteria for replacement of fillings: a retrospective study.

The exchange of restorations goes along with the loss of healthy tooth structure. Therefore, it is important to investigate helpful decision criteria for the replacement of fillings. Five hundred forty-four filling replacements were evaluated retrospectively. Thereby, different clinical parameters were correlated with the clinical finding of caries directly after removal of the existing filling. The parameters checked for correlations were amalgam and composite, age, and size of the filling, morphology, condition of the filling, type of caries, oral hygiene, anamnesis of the respective tooth, and the decisive factor to replace the restoration. Statistical evaluation was performed by chi-squared-test (P < 0,05) and by regression analysis (Power: 80%). A percentage of 69.8% of all cavities showed softened dentin if exploring with the probe after the removal of the restoration, 7.6% were stainable with caries detector, and 22.6% of the cavities were caries free. Significant indicators for a carious lesion were high age of restoration, imperfections at the margin of the filling, a positive pain sensation in correlation with composite fillings, and multi-surface amalgam fillings. On suspicion of caries, the following decision criteria should encourage the dentist to remove a filling: High age of the filling, imperfections at the margin of the filling, especially fillings with marginal cracks, visible secondary caries, a positive pain sensation in composite filled teeth, and multi-surface amalgam fillings. Filling removals only performed due to the patient's desire for removal should be critically regarded, as most of these fillings are caries free.

Antimicrobial Photoinactivation Using Visible Light Plus Water-Filtered Infrared-A (VIS + wIRA) Alters In Situ Oral Biofilms.

Recently, growing attention has been paid to antimicrobial photodynamic therapy (aPDT) in dentistry. Changing the microbial composition of initial and mature oral biofilm by aPDT using visible light plus water-filtered infrared-A wavelengths (VIS + wIRA) has not yet been investigated. Moreover, most aPDT studies have been conducted on planktonic bacterial cultures. Therefore, in the present clinical study we cultivated initial and mature oral biofilms in six healthy volunteers for 2 hours or 3 days, respectively. The biofilms were treated with aPDT using VIS+wIRA (200 mW cm(-2)), toluidine blue (TB) and chlorine e6 (Ce6) for 5 minutes. Chlorhexidine treated biofilm samples served as positive controls, while untreated biofilms served as negative controls. After aPDT treatment the colony forming units (CFU) of the biofilm samples were quantified, and the surviving bacteria were isolated in pure cultures and identified using MALDI-TOF, biochemical tests and 16S rDNA-sequencing. aPDT killed more than 99.9% of the initial viable bacterial count and 95% of the mature oral biofilm in situ, independent of the photosensitizer. The number of surviving bacterial species was highly reduced to 6 (TB) and 4 (Ce6) in the treated initial oral biofilm compared to the 20 different species of the untreated biofilm. The proportions of surviving bacterial species were also changed after TB- and Ce6-mediated aPDT of the mature oral biofilm, resulting in a shift in the microbial composition of the treated biofilm compared to that of the control biofilm. In conclusion, aPDT using VIS + wIRA showed a remarkable potential to eradicate both initial and mature oral biofilms, and also to markedly alter the remaining biofilm. This encourages the clinical use of aPDT with VIS + wIRA for the treatment of periimplantitis and periodontitis.

A Preliminary Investigation on the Antimicrobial Activity of Listerine®, Its Components, and of Mixtures Thereof.

Listerine® is one of the most popular mouthwashes worldwide and claims to combat harmful bacteria. In the past century, its recipe was changed from an essential oil mouthwash to a five-component mixture (thymol, menthol, eucalyptol, and methyl salicylate dissolved in 27% ethanol). The aim of this study was to get preliminary information about the antimicrobial activities of individual Listerine® components and their mixtures. We tested the bacterial strains Streptococcus mutans, Enterococcus faecalis, and Eikenella corrodens and the yeast Candida albicans. The established minimum inhibitory concentration (MIC) assay and the minimum bactericidal/fungicidal concentration (MBC/MFC) assay were applied. None of the combinations of two phenols at the concentrations contained within Listerine® were associated with either an additive or synergistic effect. Thymol had lower MIC and MBC/MFC values than the other Listerine® components and Listerine® against E. corrodens and C. albicans. The mixtures consisting of eucalyptol, methyl salicylate, and thymol were the most effective against S. mutans and E. faecalis and more effective than Listerine®. Our results demonstrate that the phenols and their concentrations as contained within Listerine® could be further optimized in terms of selecting those which increase their general effectiveness, at concentrations that do not induce harm.

Ultrasonic activation and chemical modification of photosensitizers enhances the effects of photodynamic therapy against Enterococcus faecalis root-canal isolates.

BACKGROUND: The aim of this study was to evaluate the effect of photodynamic therapy (PDT) on Enterococcus faecalis biofilms in artificially infected root canals using modified photosensitizers and passive ultrasonic activation. METHODS: Two hundred and seventy extracted human teeth with one root canal were instrumented utilizing ProTaper files, autoclaved, infected with E. faecalis T9 for 72 h and divided into different groups: irrigation with 3% sodium hypochlorite (NaOCl), 20% ethylenediaminetetraacetic acid (EDTA), or 20% citric acid, PDT without irrigation, PDT accompanied by irrigation with NaOCl, EDTA, or citric acid, PDT using an EDTA-based photosensitizer or a citric-acid-based photosensitizer and PDT with ultrasonic activation of the photosensitizer. A 15 mg/ml toluidine blue served as the photosensitizer, activated by a 100 mW LED light source. Sterile paper points were used for sampling the root canals and dentin chips were collected to assess the remaining contamination after treatment. Samples were cultured on blood agar plates and colony forming units were quantified. RESULTS: PDT alone achieved a reduction in E. faecalis counts by 92.7%, NaOCl irrigation alone and combined with PDT by 99.9%. The antibacterial effects increased by the combination of irrigation using EDTA or citric acid and PDT compared to irrigation alone. More than 99% of E. faecalis were killed using PDT with the modified photosensitizers and ultrasonic activation. CONCLUSIONS: NaOCl based disinfection achieved the highest antimicrobial effect. Using PDT with an EDTA-based or citric-acid-based phozosensitizer or activating the photosensitizer with ultrasound resulted in a significantly higher reduction in E. faecalis counts compared to conventional PDT.

Pre-clinical endodontic training with artificial instead of extracted human teeth: does the type of exercise have an influence on clinical endodontic outcomes?

AIM: To evaluate whether artificial resin teeth could replace extracted human teeth in pre-clinical endodontic training and if this teaching approach influences the outcome of root canal treatment on patients. METHODOLOGY: In a pre-clinical training course, students of group 1 (n = 44) performed simulated endodontic exercises on four plastic blocks and three extracted human teeth. Students of group 2 (n = 45) performed their exercises on plastic blocks and artificial resin teeth (Real-T Endo, Acadental, Lenexa, KS, USA). Both groups performed their first root canal treatments on patients in the following term. Radiographs taken during root canal treatment were used for the evaluation of treatment outcome. Distances between the master cone or the root filling and the radiographic apex as well as iatrogenic errors were assessed, and comparisons were made using Fischer's exact test. RESULTS: In the pre-clinical course root canal treatments performed by students of group 2 were more often classified as acceptable and a higher number of iatrogenic errors were observed in group 1. When root canal treatments were performed on patients for the first time, no significant difference was observed between the groups in terms of radiographic technical quality of root fillings. CONCLUSIONS: The application of artificial teeth instead of extracted human teeth had no effect on the technical quality of root fillings in terms of position in relation to the root apex or the creation of aberrations.

Supplementary sampling of obturation materials enhances microbial analysis of endodontic treatment failures: a proof of principle study.

OBJECTIVES: Root canal treatment failures often correlate with persistent biomaterial-associated endodontic infections. The aim of the present study was to assess the impact of endodontic obturation material sampling from root canals with posttreatment apical periodontitis on improving standard study protocols. MATERIALS AND METHODS: Samples from previously filled root canals and their corresponding endodontic filling materials were obtained from five root-filled teeth with posttreatment periradicular lesions. After cultivation, the isolated microorganisms were quantified and biochemically identified. Moreover, clone libraries were constructed after the amplification of bacterial 16S ribosomal DNA (rDNA) from the same samples. DNA from selected clones was sequenced to identify microbial species. Transmission electron microscopy (TEM) aided visualization of the detected bacteria. RESULTS: Overall, 22 taxa of the phyla Firmicutes, Actinobacteria, and Bacteroidetes were detected in both obturation and root canal samples by culture-dependent and culture-independent methods. Root canal fillings sheltered 17 species (3.30-7.50 × 10(3) CFU/ml). Of these, nine were detected solely in the retrieved obturation materials. The reinfected root canals harbored 13 taxa (3.48-7.36 × 10(3) CFU/ml). Obligate and facultative anaerobic bacteria prevailed. The number of different species ranged from 1 to 5 within a single sample. Fungi were not detected. CONCLUSIONS: Bacteria can colonize both root canals and endodontic fillings in vivo. CLINICAL RELEVANCE: Integrating the sampling of obturation materials with standard root canal sample collection offers a clearer insight into the actual microbial flora of reinfected root canals and improves the study protocols of secondary/persistent endodontic infections.

A temporary filling material used for coronal sealing during endodontic treatment may cause tooth fractures in large Class II cavities in vitro.

AIM: To investigate the use of a zinc oxide/zinc sulphate-based cement (Coltosol(®) F, Coltène Whaledent, Cuyahoga Falls, OH, USA) as a temporary filling material during multiple-visit root canal treatments and the occurrence of cracks within the filling material or the tooth. METHODOLOGY: Root canals of one hundred and twenty-two extracted human molars were prepared using ProTaper instruments up to size F2. After root canal preparation, standardized mesial-occlusal-distal cavities were prepared. The buccal-lingual/palatal width of the cavities was 4.5 mm), so that the remaining cavity walls had a mean thickness of 3.5 mm. Teeth were checked for cracks and fracture lines using a stereomicroscope with 10× magnification. A calcium hydroxide slurry was used as an intracanal dressing. The teeth were divided into three groups. In the Coltosol group, the cavity was filled with Coltosol(®) F. In the Coltosol-Clearfil group, a 2-mm layer of Coltosol(®) F was placed into the coronal pulp chamber, the remaining cavity was filled with Clearfil(™) . In the Clearfil group, a foam pellet was placed onto the orifices of the root canals, the remaining cavity was filled with Clearfil(™) . In the control group, the cavities were left without any filling material. The teeth were stored in water at 37 °C for 14 days and examined every 24 h under a stereomicroscope for fracture lines occurring on the tooth surface or in the filling material. RESULTS: In the Coltosol group, fractures within the filling material were observed in 28 (85%) of 33 teeth. 13 (39%) teeth had tooth fractures. Amongst these teeth, 8 (61%) had root fractures, 1 (8%) had a crown fracture and 4 (31%) had a root-crown fracture. CONCLUSION: Coltosol(®) F, when used alone as a restorative material, led to tooth fractures in Class II cavities in teeth undergoing root canal treatment. Tooth fractures may occur 4 days after placement of the filling.

Novel Broad-Spectrum Antimicrobial Photoinactivation of In Situ Oral Biofilms by Visible Light plus Water-Filtered Infrared A.

Antimicrobial photodynamic therapy (APDT) has gained increased attention as an alternative treatment approach in various medical fields. However, the effect of APDT using visible light plus water-filtered infrared A (VIS + wIRA) on oral biofilms remains unexplored. For this purpose, initial and mature oral biofilms were obtained in situ; six healthy subjects wore individual upper jaw acrylic devices with bovine enamel slabs attached to their proximal sites for 2 h or 3 days. The biofilms were incubated with 100 µg ml(-1) toluidine blue O (TB) or chlorin e6 (Ce6) and irradiated with VIS + wIRA with an energy density of 200 mW cm(-2) for 5 min. After cultivation, the CFU of half of the treated biofilm samples were quantified, whereas following live/dead staining, the other half of the samples were monitored by confocal laser scanning microscopy (CLSM). TB- and Ce6-mediated APDT yielded a significant decrease of up to 3.8 and 5.7 log10 CFU for initial and mature oral biofilms, respectively. Quantification of the stained photoinactivated microorganisms confirmed these results. Overall, CLSM revealed the diffusion of the tested photosensitizers into the deepest biofilm layers after exposure to APDT. In particular, Ce6-aided APDT presented elevated permeability and higher effectiveness in eradicating 89.62% of biofilm bacteria compared to TB-aided APDT (82.25%) after 3 days. In conclusion, antimicrobial photoinactivation using VIS + wIRA proved highly potent in eradicating oral biofilms. Since APDT excludes the development of microbial resistance, it could supplement the pharmaceutical treatment of periodontitis or peri-implantitis.