Template assisted surface micro microstructuring of flowable dental composites and its effect on the microbial adhesion properties

Despite their various advantages, such as good esthetic properties, absence of mercury and adhesive bonding to teeth, modern dental composites still have some drawbacks, e.g., a relatively high rate of secondary caries on teeth filled with composite materials. Recent research suggests that microstructured biomaterials surfaces may reduce microbial adhesion to materials due to unfavorable physical material–microbe interactions. The objectives of this study were, therefore, to test the hypotheses that (i) different surface microstructures can be created on composites by a novel straightforward approach potentially suitable for clinical application and (ii) that these surface structures have a statistically significant effect on microbial adhesion properties.Peer ReviewedPostprint (author's final draft

Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota

Background and Objective: There are few in vitro models available in the scientific literature for study of the structure, formation and development of the subgingival biofilm. The purpose of this study was to develop and validate an in vitro biofilm model, using representative selected bacteria from the subgingival microbiota. Material and Methods: Six standard reference strains were used to develop biofilms over sterile ceramic calcium hydroxyapatite discs coated with saliva within the wells of presterilized polystyrene tissue culture plates. The selected species represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The structure of the biofilm obtained was studied using a vital fluorescence technique in conjunction with confocal laser scanning microscopy. The biofilm bacterial kinetics were studied by terminal restriction fragment length polymorphism analysis. Results: After 12 h, initial and early colonizers were the first microorganisms detected adhering to the calcium hydroxyapatite discs. The intermediate colonizer F. nucleatum was not detected in the model until 24 h of incubation. Late colonizers A. actinomycetemcomitans and P. gingivalis could be measured inside the biofilm after 48 h. The biofilm reached its steady state between 72 and 96 h after inoculation, with bacterial vitality increasing from the hydroxyapatite surface to the central part of the biofilm. Conclusion: An in vitro biofilm model was developed and validated, demonstrating a pattern of bacterial colonization and maturation similar to the in vivo development of the subgingival biofilm.Depto. de Especialidades Clínicas OdontológicasFac. de OdontologíaTRUEpu

Template assisted surface micro microstructuring of flowable dental composites and its effect on the microbial adhesion properties

Despite their various advantages, such as good esthetic properties, absence of mercury and adhesive bonding to teeth, modern dental composites still have some drawbacks, e.g., a relatively high rate of secondary caries on teeth filled with composite materials. Recent research suggests that microstructured biomaterials surfaces may reduce microbial adhesion to materials due to unfavorable physical material–microbe interactions. The objectives of this study were, therefore, to test the hypotheses that (i) different surface microstructures can be created on composites by a novel straightforward approach potentially suitable for clinical application and (ii) that these surface structures have a statistically significant effect on microbial adhesion properties.Peer Reviewe

3D tracking of extracellular vesicles by holographic fluorescence imaging

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.ISSN:2375-254

Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model

Background and Objectives: Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. Material and Methods: PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 lM). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. Results: When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10. P. gingivalis showed a vitality reduction in the biofilm of 3 log10, while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. Conclusion: These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.Depto. de Especialidades Clínicas OdontológicasFac. de OdontologíaTRUEpu