A critical analysis of research methods and experimental models to study irrigants and irrigation systems.

Irrigation plays an essential role in root canal treatment. The purpose of this narrative review was to critically appraise the experimental methods and models used to study irrigants and irrigation systems and to provide directions for future research. Studies on the antimicrobial effect of irrigants should use mature multispecies biofilms grown on dentine or inside root canals and should combine at least two complementary evaluation methods. Dissolution of pulp tissue remnants should be examined in the presence of dentine and, preferably, inside human root canals. Micro-computed tomography is currently the method of choice for the assessment of accumulated dentine debris and their removal. A combination of experiments in transparent root canals and numerical modeling is needed to address irrigant penetration. Finally, models to evaluate irrigant extrusion through the apical foramen should simulate the periapical tissues and provide quantitative data on the amount of extruded irrigant. Mimicking the in vivo conditions as close as possible and standardization of the specimens and experimental protocols are universal requirements irrespective of the surrogate endpoint studied. Obsolete and unrealistic models must be abandoned in favour of more appropriate and valid ones that have more direct application and translation to clinical Endodontics.

Ultrasonic Irrigant Activation during Root Canal Treatment: A Systematic Review.

INTRODUCTION: The aim of this study was to systematically review the evidence on the cleaning and disinfection of root canals and the healing of apical periodontitis when ultrasonic irrigant activation is applied during primary root canal treatment of mature permanent teeth compared with syringe irrigation. METHODS: An electronic search was conducted of the Cochrane Library, Embase, LILACS, PubMed, SciELO, and Scopus databases using both free-text key words and controlled vocabulary. Additional studies were sought through hand searching of endodontic journals and textbooks. The retrieved studies were screened by 2 reviewers according to predefined criteria. The included studies were critically appraised, and the extracted data were arranged in tables. RESULTS: The electronic and hand search retrieved 1966 titles. Three clinical studies and 45 in vitro studies were included in this review. Ultrasonic activation did not improve the healing rate of apical periodontitis compared with syringe irrigation after primary root canal treatment of teeth with a single root canal. Conflicting results were reported by the in vitro microbiological studies. Ultrasonic activation was more effective than syringe irrigation in the removal of pulp tissue remnants and hard tissue debris based on both clinical and in vitro studies. Ultrasonic activation groups were possibly favored in 13 studies, whereas syringe irrigation groups may have been favored in 3 studies. CONCLUSIONS: The level of the available evidence was low, so no strong clinical recommendations could be formulated. Future studies should focus on the antimicrobial effect and healing of apical periodontitis in teeth with multiple root canals.

Plasminogen coating increases initial adhesion of oral bacteria in vitro.

Plasminogen is a major plasma protein and the zymogen of the broad spectrum protease plasmin. Plasmin activity leads to tissue degradation, direct and through activation of metalloproteinases. Infected tooth root canals, as a consequence of the inflammatory response and eventual necrosis, contain tissue fluid and blood components. These will coat the root canal walls and act as conditioning films that allow bacterial biofilms to grow and be a potential source of hematogenously spreading bacteria. We investigated the effect of in vitro surface conditioning with human plasminogen on the initial adhesion of bacteria. Four bacterial species, L. salivarius, E. faecalis, A. naeslundii, and S. gordonii, isolated from dental root canals, and three other oral streptococci, S. oralis, S. anginosus, and S. sanguinis, were grown in albumin- or plasminogen-coated flow chambers and studied by confocal laser scanning microscopy using the cell viability staining LIVE/DEAD and 16S rRNA fluorescence in situ hybridization (FISH). A. naeslundii, L. salivarius and in particular S. gordonii showed a higher initial adhesion to the plasminogen-coated surfaces. E. faecalis did not show any preference for plasminogen. Four-species biofilms cultured for 96 h showed that streptococci increased their proportion with time. Further experiments aimed at studying different streptococcal strains. All these adhered more to plasminogen-coated surfaces than to albumin-coated control surfaces. The specificity of the binding to plasminogen was verified by blocking lysine-binding sites with epsilon-aminocaproic acid. Plasminogen is thus an important plasma component for the initial adhesion of oral bacteria, in particular streptococci. This binding may contribute to their spread locally as well as to distant organs or tissues.

Decreased bacterial adherence and biofilm growth on surfaces coated with a solution of benzalkonium chloride.

INTRODUCTION: Secondary biofilm formation by oral bacteria after breakdown/fracture of temporary or permanent restorations imposes a challenge to the outcome of root canal treatment. This study focuses on benzalkonium chloride (BAK) coating on dentin or polystyrene surfaces and its influence on the early adhesion and biofilm formation by oral and root canal bacteria. METHODS: Microbial adhesion and biofilm growth on surfaces coated with BAK were analyzed qualitatively with a dentin disk model and quantitatively with a mini-flow cell biofilm model. Cell viability and total biovolume were analyzed by the LIVE/DEAD technique. The repelling effect of surfaces coated with BAK was compared with NaOCl. Uncoated surfaces were used as controls. RESULTS: Scanning electron microscope images in the dentin disk model revealed that very sparse biofilms were formed on NaOCl- and BAK-coated dentin surfaces. In contrast, biofilms formed on uncoated dentin were clearly visible as numerous irregularly distributed aggregates of rods and cocci. In the mini-flow cell system, confocal laser scanning microscope analysis confirmed that biofilms formed on NaOCl- and BAK-coated surfaces showed significantly less adhesion (2 hours) and biovolume accumulation (24 and 96 hours) compared with the uncoated controls (P < .01). Furthermore, cell viability assessments showed that on uncoated controls the viability measurements were high (>89%) as well as on BAK-coated surfaces (88% viable cells). However, cell viability was significantly reduced on NaOCl-coated surfaces (59% viable cells). CONCLUSIONS: This study illustrates that surface coating with a surfactant solution containing BAK does not cause cell membrane damage but might interfere with cell mechanisms of adhesion. Investigations into the clinical utility of BAK as an antibiofilm medication are warranted.

Development of a multispecies biofilm community by four root canal bacteria.

INTRODUCTION: The development of multispecies biofilm models are needed to explain the interactions that take place in root canal biofilms during apical periodontitis. The aim of this study was to investigate the ability of 4 root canal bacteria to establish a multispecies biofilm community and to characterize the main structural, compositional, and physiological features of this community. METHODS: Four clinical isolates isolated from infected root canals, Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus gordonii, and Enterococcus faecalis, were grown together in a miniflow cell system. Simultaneous detection of the 4 species in the biofilm communities was achieved by fluorescence in situ hybridization in combination with confocal microscopy at different time points. The LIVE/DEAD BacLight technique (Molecular Probes, Carlsbad, CA) was used to assess cell viability and to calculate 3-dimensional architectural parameters such as biovolume (µm(3)). Redox fluorescence dye 5-cyano-2,3-ditolyl tetrazolium chloride was used to assess the metabolic activity of biofilm bacteria. RESULTS: The 4 species tested were able to form stable and reproducible biofilm communities. The biofilms formed in rich medium generally showed continuous growth over time, however, in the absence of glucose biofilms showed significantly smaller biovolumes. A high proportion of viable cells (>90%) were generally observed, and biofilm growth was correlated with high metabolic activity of cells. The community structure of biofilms formed in rich medium did not change considerably over the 120-hour period, during which E. faecalis, L. salivarius, and S. gordonii were most abundant. CONCLUSIONS: The ability of 4 root canal bacteria to form multispecies biofilm communities shown in this study give insights into assessing the community lifestyle of these microorganisms in vivo. This multispecies model could be useful for further research simulating stresses representative of in vivo conditions.

Effects of saliva or serum coating on adherence of Streptococcus oralis strains to titanium.

The use of dental implants to treat tooth loss has increased rapidly over recent years. 'Smooth' implants showing high long-term success rates have successively been replaced by implants with rougher surfaces, designed to stimulate rapid osseointegration and promote tissue healing. If exposed in the oral cavity, rougher surfaces may promote bacterial adhesion leading to formation of microbial biofilms which can induce peri-implant inflammation. Streptococcus oralis is an early colonizer of oral surfaces and has been recovered from titanium surfaces in vivo. The purpose of this study was to examine the adherence of clinical strains of S. oralis to titanium with smooth or moderately rough surface topography and to determine the effect of a saliva- or serum-derived coating on this process. Adherence was studied using a flow-cell system with confocal laser scanning microscopy, while putative adhesins were analysed using proteomics of bacterial cell wall proteins. This showed that adherence to moderately rough surfaces was greater than to smooth surfaces. Serum did not promote binding of any of the studied S. oralis strains to titanium, whereas a saliva coating increased adherence in two of three strains tested. The higher level of adherence to the moderately rough surfaces was maintained even in the presence of a saliva coating. The S. oralis strains that bound to saliva expressed an LPXTG-linked protein which was not present in the non-adherent strain. Thus strains of S. oralis differ in their capacity to bind to saliva-coated titanium and we propose that this is due to differential expression of a novel adhesin.

Role of (p)ppGpp in biofilm formation by Enterococcus faecalis.

Enterococcus faecalis strain OG1RF and its (p)ppGpp-deficient ΔrelA, ΔrelQ, and ΔrelA ΔrelQ mutants were grown in biofilms and evaluated for growth profiles, biofilm morphology, cell viability, and proteolytic activity. E. faecalis lacking (p)ppGpp had a diminished capacity to sustain biofilm formation over an extended period of time and expressed abundant proteolytic activity.

Antimicrobial effect of chitosan nanoparticles on streptococcus mutans biofilms.

Nanoparticle complexes were prepared from chitosans of various molecular weights (MW) and degrees of deacetylation (DD). The antimicrobial effect was assessed by the Live/Dead BacLight technique in conjunction with confocal scanning laser microscopy (CSLM) and image analysis. Nanocomplexes prepared from chitosans with high MW showed a low antimicrobial effect (20 to 25% of cells damaged), whereas those prepared from low-MW chitosans showed high antimicrobial effect (>95% of cells damaged).

Effects of clinical isolates of Pseudomonas aeruginosa on Staphylococcus epidermidis biofilm formation.

Pseudomonas aeruginosa is often found in chronic infections, including cystic fibrosis lung infections and those related to chronic wounds and venous ulcers. At the latter sites, P. aeruginosa can be isolated together with Staphylococcus epidermidis, and we have therefore explored the effect of clinical isolates and laboratory strains of P. aeruginosa strains on colonization by S. epidermidis in dual-species biofilms. Biofilm formation was assayed using 16S rRNA FISH and confocal laser scanning microscopy. Among the six P. aeruginosa strains tested, one particular strain, denoted 14:2, exerted a significant inhibitory effect, and even after 6 h, S. epidermidis levels in dual-species biofilms were reduced by >85% compared with those without P. aeruginosa. Interestingly, strain 14:2 was found to be negative for classical virulence determinants including pyocyanin, elastase and alkaline protease. Therefore, we suggest that less virulent phenotypes of P. aeruginosa, which may develop over time in chronic infections, could counteract colonization by S. epidermidis, ensuring persistence and dominance by P. aeruginosa in the host micro-habitat. Further studies are required to explain the inhibitory effect on S. epidermidis, although extracellular polysaccharides produced by P. aeruginosa might play a role in this phenomenon.

Differential effects of Pseudomonas aeruginosa on biofilm formation by different strains of Staphylococcus epidermidis.

Pseudomonas aeruginosa and Staphylococcus epidermidis are common opportunistic pathogens associated with medical device-related biofilm infections. 16S rRNA-FISH and confocal laser scanning microscopy were used to study these two bacteria in dual-species biofilms. Two of the four S. epidermidis strains used were shown to form biofilms more avidly on polymer surfaces than the other two strains. In dual-species biofilms, the presence of P. aeruginosa reduced biofilm formation by S. epidermidis, although different clinical isolates differed in their susceptibility to this effect. The most resistant isolate coexisted with P. aeruginosa for up to 18 h and was also resistant to the effects of the culture supernatant from P. aeruginosa biofilms, which caused dispersal from established biofilms of other S. epidermidis strains. Thus, different strains of S. epidermidis differed in their capacity to withstand the action of P. aeruginosa, with some being better equipped than others to coexist in biofilms with P. aeruginosa. Our data suggest that where S. epidermidis and P. aeruginosa are present on abiotic surfaces such as medical devices, S. epidermidis biofilm formation can be inhibited by P. aeruginosa through two mechanisms: disruption by extracellular products, possibly polysaccharides, and, in the later stages, by cell lysis.

The effects of antimicrobials on endodontic biofilm bacteria.

INTRODUCTION: In the present study, confocal microscopy, a miniflow cell system, and image analysis were combined to test in situ the effect of antimicrobials and alkali on biofilms of Enterococcus faecalis, Lactobacillus paracasei, Streptococcus anginosus, and Streptococcus gordonii isolated from root canals with persistent infections. METHODS: Biofilms formed for 24 hours were exposed for 5 minutes to alkali (pH = 12), chlorhexidine digluconate (2.5%), EDTA (50 mmol/L), and sodium hypochlorite (1%). The biofilms were then characterized by using fluorescent markers targeting cell membrane integrity (LIVE/DEAD) and metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride and fluorescein diacetate). In addition, the biofilm architecture and the extent to which coating of the substrate surface with collagen influenced the resistance pattern to the chemicals were also analyzed. RESULTS: NaOCl (1%) affected the membrane integrity of all organisms and removed most biofilm cells. Exposure to EDTA (50 mmol/L) affected the membrane integrity in all organisms but failed to remove more than a few cells in biofilms of E. faecalis, L. paracasei, and S. anginosus. Chlorhexidine (2.5%) had a mild effect on the membrane integrity of E. faecalis and removed only 50% of its biofilm cells The effects were substratum-dependent, and most organisms displayed increased resistance to the antimicrobials on collagen-coated surfaces. CONCLUSIONS: The biofilm system developed here was sensitive and differences in cell membrane integrity and removal of biofilm cells after exposure to antimicrobials commonly used in endodontics was discernible.

Image analysis software based on color segmentation for characterization of viability and physiological activity of biofilms.

The novel image analysis software package bioImage_L was tested to calculate biofilm structural parameters in oral biofilms stained with dual-channel fluorescent markers. By identifying color tonalities in situ, the software independently processed the color subpopulations and characterized the viability and metabolic activity of biofilms.

Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation.

The ability of oral bacteria to enter a non-growing state is believed to be an important mechanism for survival in the starved micro-environments of the oral cavity. In this study, we examined the reactivation of nutrient-deprived cells of two oral bacteria in biofilms, Streptococcus anginosus and Lactobacillus salivarius. Non-growing cells were generated by incubation in 10 mM potassium phosphate buffer for 24 h and the results were compared to those of planktonic cultures. When both types of cells were shifted from a rich, peptone-yeast extract-glucose (PYG) medium to buffer for 24 h, dehydrogenase and esterase activity measured by the fluorescent dyes 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and fluorescein diacetate (FDA), respectively, was absent in both species. However, the membranes of the vast majority of nutrient-deprived cells remained intact as assessed by LIVE/DEAD staining. Metabolic reactivation of the nutrient-deprived biofilm cells was not observed for at least 48 h following addition of fresh PYG medium, whereas the non-growing planktonic cultures of the same two strains were in rapid growth in less than 2 h. At 72 h, the S. anginosus biofilm cells had recovered 78 % of the dehydrogenase activity and 61 % of the esterase activity and the biomass mm(-2) had increased by 30-35 %. With L. salivarius at 72 h, the biofilms had recovered 56 % and 75 % of dehydrogenase and esterase activity, respectively. Reactivation of both species in biofilms was enhanced by removal of glucose from PYG, and S. anginosus cells were particularly responsive to yeast extract (YE) medium. The data suggest that the low reactivity of non-growing biofilm cells to the introduction of fresh nutrients may be a survival strategy employed by micro-organisms in the oral cavity.

Redefining the persistent infection in root canals: possible role of biofilm communities.

Current concepts suggest that persisting infections subsequent to endodontic therapy are caused by one or two bacterial species that are "too robust" to be eliminated by conventional treatment measures. As a consequence, numerous studies are exploring the characteristics of these "most" resistant organisms to define an effective treatment strategy to eradicate them from root canals. By taking an ecological perspective, the main objective of this review is to present evidence that the nature of persisting endodontic infections depends not on the robustness of the organisms in the infected site, but on their capability of adapting their physiology to the new environmental conditions set by the treatment. Changes in the environment, such as an increase in pH by calcium hydroxide or the effect of antimicrobials, are capable of triggering genetic cascades that modify the physiological characteristics of bacterial cells. Surface adherence by bacteria to form biofilms is a good example of bacterial adaptation and one that is pertinent to endodontic infections. Increasing information is now available on the existence of polymicrobial biofilm communities on root canal walls, coupled with new data showing that the adaptive mechanisms of bacteria in these biofilms are significantly augmented for increased survival. This ecological view on the persisting infection problem in endodontics suggests that the action of individual species in persisting endodontic infections is secondary when compared to the adaptive changes of a polymicrobial biofilm community undergoing physiological and genetic changes in response to changes in the root canal environment.