Biofilm Matrixome: Extracellular Components in Structured Microbial Communities.

Biofilms consist of microbial communities embedded in a 3D extracellular matrix. The matrix is composed of a complex array of extracellular polymeric substances (EPS) that contribute to the unique attributes of biofilm lifestyle and virulence. This ensemble of chemically and functionally diverse biomolecules is termed the 'matrixome'. The composition and mechanisms of EPS matrix formation, and its role in biofilm biology, function, and microenvironment are being revealed. This perspective article highlights recent advances about the multifaceted role of the 'matrixome' in the development, physical-chemical properties, and virulence of biofilms. We emphasize that targeting biofilm-specific conditions such as the matrixome could lead to precise and effective antibiofilm approaches. We also discuss the limited knowledge in the context of polymicrobial biofilms, and the need for more in-depth analyses of the EPS matrix in mixed communities that are associated with many human infectious diseases.

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.

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.

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.

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.

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.

Comparative assessment of the incidence of vertical root fractures between conventional versus surgical endodontic retreatment.

OBJECTIVES: Vertical root fractures (VRFs) are a common cause of tooth loss. Little evidence exists though, relating the incidence of VRFs to the type of endodontic retreatment. This retrospective study aimed at evaluating the impact of conventional versus surgical endodontics on root canal-filled teeth with VRFs. MATERIALS AND METHODS: Over a period of 13 years, 200 endodontically retreated teeth from 192 patients with VRFs were extracted and further examined. VRFs were assessed in relation to age, gender, tooth group, clinical signs, extension on the root surface, patency, as well as type of endodontic retreatment and restoration. Statistical analysis was conducted using a Cox PH Model, Chi-squared, Wilcoxon rank-sum, and Log rank tests at a significance level of 5 %. RESULTS: The majority of teeth with VRFs (62.31 %) had undergone the combination of conventional root canal retreatment and apical surgery. Women (64.06 %) presented VRFs more frequently than men (35.94 %) at the mean age of 51.1 and 55.1 years, respectively. Maxillary first (17.5 %) and second (16.5 %) premolars, restored by a resin-based material without a post (56.28 %) were more susceptible to VRFs. Apically initiated (84.1 %) VRFs could be diagnosed more easily on radiographs. CONCLUSIONS: The type of endodontic treatment strongly correlated with VRFs. The prevalence of VRFs in teeth having undergone both conventional and surgical endodontic retreatment could be attributed, among others, to additive dentin damage related to the aforementioned endodontic procedures. CLINICAL RELEVANCE: The possible involvement of endodontic retreatment in the multifactorial etiology of VRFs needs to be taken into consideration in clinical practice.

Antimicrobial photodynamic therapy using visible light plus water-filtered infrared-A (wIRA).

The aim of this study was to investigate the effectiveness of antimicrobial photodynamic therapy (APDT) using visible light together with water-filtered infrared-A (VIS+wIRA) to eradicate single species of planktonic bacteria and micro-organisms during initial oral bacterial colonization in situ. A broadband VIS+wIRA radiator with a water-filtered spectrum in the range 580-1400 nm was used for irradiation. Toluidine blue (TB) was utilized as a photosensitizer at concentrations of 5, 10, 25 and 50 µg ml(-1). The unweighted (absolute) irradiance was 200 mW cm(-2) and it was applied for 1 min. Planktonic cultures of Streptococcus mutans and Enterococcus faecalis were treated with APDT. Salivary bacteria harvested by centrifugation of native human saliva were also tested. In addition, initial bacterial colonization of bovine enamel slabs carried in the mouths of six healthy volunteers was treated in the same way. Up to 2 log(10) of S. mutans and E. faecalis were killed by APDT. Salivary bacteria were eliminated to a higher extent of 3.7-5 log(10). All TB concentrations tested proved to be highly effective. The killing rate of bacteria in the initial oral bacterial colonization was significant (P=0.004) at all tested TB concentrations, despite the interindividual variations found among study participants. This study has shown that APDT in combination with TB and VIS+wIRA is a promising method for killing bacteria during initial oral colonization. Taking the healing effects of wIRA on human tissue into consideration, this technique could be helpful in the treatment of peri-implantitis and periodontitis.

Microscope-based imaging platform for large-scale analysis of oral biofilms.

A microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grown in situ on BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria, Streptococcus spp., and Fusobacterium nucleatum were stained using specific fluorescence in situ hybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.