Comparative analysis of Enterococcus faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37°C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (μm3), live bacteria biovolume (μm3), and substrate coverage (%) were quantified by using the BioImage-L software. Results obtained were analyzed by nonparametric tests (P =.05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P <.05) and hydroxyapatite the highest at 21 days (P <.05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P <.05). The percentages of coverage were similar among all substrates at 21 days (P >.05), but at 14 days, bovine bone presented the highest coverage (P <.05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated. © 2013 American Association of Endodontists.

Photodynamic inactivation of biofilm: Taking a lightly colored approach to stubborn infection

Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents. © 2013 Informa UK Ltd.

Caracterização e avaliação da citocompatibilidade de nanocompositos baseados em celulose bacteriana e fibroína para aplicação em regenração tecidual

Pós-graduação em Ciências Odontológicas - FOAR

Streptococcus Mutans Adhesion to Titanium After Brushing with Fluoride and Fluoride-Free Toothpaste Simulating 10 Years of Use

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expansão ex vivo de células uroteliais em scaffolds 3D

Malformations and possible damages to the urogenital system can be originated in the embryonic period. Moreover, fire guns, knives and accidents, where there is the disruption of the urethra, also cause these lesions. The objective was to analyze the contribution of tissue engineering in the construction of neo-urethra, developed by bioengineering. We performed an urothelial ex vivo expansion of cells in 3D scaffolds (platelet gel matrix and acellular porcine aorta) to assess the contribution of this technique in the construction of a neo-urethra. Mechanical dissociation was made of the inner wall of 10 North Folk rabbit’s bladder, weighing 2.5 to 3.0 kg. After dissociation the cell content was centrifuged and obtained a pellet of urothelial cells. The pellet was ressuspended in culture medium DMEM F12 and cells were maintained in culture for 15 days. Immunohistochemical analysis characterized the urothelial culture. The cells were then implanted in the scaffold - platelet gel. In a second experiment using aortic porcine acellular matrix were implanted urothelial cells alone and urothelial cells on platelet gel, on the inner wall of the scaffold - aorta, with space for setting bordered by a urethral probe. The complex probe - cells - aorta and probe - cells in platelet gel - aorta, were sealed with suture material and culture were maintained in a humidified 37ºC incubator with 5% CO2 in air for 12 days to subsequent histological analysis of urothelium cell adhesion to the scaffolds. By observation under an optical microscope, we could see the growth of cells in the scaffold platelet gel, from a monolayer in to a three-dimensional structure. In the acellular porcine aortic matrix containing the platelet gel, we could observe a few quantity of urothelial cells adhered. However with the acellular porcine aortic matrix in which was implanted only the urothelial cells, we have obtained adhesion to the wall

Preparation and characterization of a bacterial cellulose/silk fibroin sponge scaffold for tissue regeneration

Bacterial cellulose (BC) and silk fibroin (SF) are natural biopolymers successfully applied in tissue engineering and biomedical fields. In this work nanocomposites based on BC and SF were prepared and characterized by scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). In addition, the investigation of cytocompatibility was done by MTT, XTT and Trypan Blue dye technique. Cellular adhesion and proliferation were detected additionally. The evaluation of genotoxicity was realized by micronucleus assay. In vitro tests showed that the material is non-cytotoxic or genotoxic. SEM images revealed a greater number of cells attached at the BC/SF:50% scaffold surface than the pure BC one, suggesting that the presence of fibroin improved cell attachment. This could be related to the SF amino acid sequence that acts as cell receptors facilitating cell adhesion and growth. Consequently, BC/SF:50% scaffolds configured an excellent option in bioengineering depicting its potential for tissue regeneration and cultivation of cells on nanocomposites.

Confocal laser scanning microscopy investigation of bond interfaces involved in fiber glass post cementation

Objective: This confocal microscopy study evaluated the cement/dentin and cement/post interfaces along theroot canalwallswhenfiberglasspostswerebonded to dentin using different types of cements. Material & Methods: Thirty endodontically treated premolars were divided into 3 groups according to the adhesive materials used in the bonding procedure: Prime & Bond 2.1/Self Cure + Enforce, RelyX Unicem and RelyX Luting. Rhodamine B dye was incorporated in the luting materials for the cementation of the fiber glass posts (Exacto, Angelus) to dentin. Three transversal slices (apical, middle and coronal) were examined under confocal laser scanning microscopy. Statistical analysis was performed using the Kappa, Kruskal-Wallis and Dunnet tests, in a significance level of 5%. Results: The Prime & Bond 2.1/Self Cure + Enforce presented a uniform formation of tags in the dentin but gaps in the cement/dentin interface. The RelyX Unicem and RelyX Luting presented an adhesive interface with a fewer amount of gaps, but showed shorter tag formation than the Enforce system. All cements presented the same pattern of bubbles inside the cements. The RelyX Luting presented a greater amount of cracks inside the cement in comparison with the other cements in the coronal third, while no difference was observed between RelyX Unicem and Enforce. The RelyX Luting showed the lowest quantity of cement penetration into the post. Conclusion: In general, the quality of bonding interfaces of fiber posts luted to root canals was affected by both location and type of cement.

Immune receptors and adhesion molecules in human pulmonary leptospirosis

Pulmonary involvement in leptospirosis has been increasingly reported in the last 20 years, being related to the severity and mortality of the disease. The pathogenesis of pulmonary hemorrhage in leptospirosis is not understood. Lung endothelial cells have been proposed as targets in the pathogenesis of lung involvement in leptospirosis through the activation of Toll-like receptor 2 or the complement system, which stimulates the release of cytokines that lead to the activation of adhesion molecules. The aim of this study was to investigate the involvement of immune pathways and of the intercellular and vascular cell adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule, respectively) in the lungs of patients with pulmonary involvement of leptospirosis. We studied the lungs of 18 patients who died of leptospirosis and compared them with 2 groups of controls: normal and noninfectious hemorrhagic lungs. Using immunohistochemistry and image analysis, we quantified the expression of the C3a anaphylatoxin receptor, intercellular adhesion molecule, vascular cell adhesion molecule, and Toll-like receptor 2 in small pulmonary vessels and in the alveolar septa. There was an increased expression of intercellular adhesion molecule (P <.03) and C3a anaphylatoxin receptor (P <.008) in alveolar septa in the leptospirosis group compared with the normal and hemorrhagic controls. In the vessels of the leptospirosis group, there was an increased expression of intercellular adhesion molecule (P=.004), vascular cell adhesion molecule (P=.030), and Toll-like receptor 2 (P=.042) compared with the normal group. Vascular cell adhesion molecule expression in vessels was higher in the leptospirosis group compared with the hemorrhagic group (P=.015). Our results indicate that immune receptors and adhesion molecules participate in the phenomena leading to pulmonary hemorrhage in leptospirosis. (C) 2012 Elsevier Inc. All rights reserved.

Hybrid Scaffolds Built From PET and Collagen as a Model For Vascular Graft Architecture

A hybrid material with excellent mechanical and biological properties is produced by electrospinning a co-solution of PET and collagen. The fibers are mapped using SEM, confocal Raman microscopy and collagenase digestion assays. Fibers of different compositions and morphologies are intermingled within the same membrane, resulting in a heterogeneous scaffold. The collagen distribution and exposure are found to depend on the PET/collagen ratio. The materials are chemically and mechanically characterized and biologically tested with fibroblasts (3T3-L1) and a HUVEC culture in vitro. All of the hybrid scaffolds show better cell attachment and proliferation than PET. These materials are potential candidates to be used as vascular grafts.

Comparative analysis of Enterococcus Faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37 C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (mm3), live bacteria biovolume (mm3), and substrate coverage (%) were quantified by using the BioImage_L software. Results obtained were analyzed by nonparametric tests (P = .05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P < .05) and hydroxyapatite the highest at 21 days (P < .05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P < .05). The percentages of coverage were similar among all substrates at 21 days (P > .05), but at 14 days, bovine bone presented the highest coverage (P < .05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated

Molecular analysis of Sigma-1 receptor modulation of the dopamine transporter

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).

New insight on a Group A Streptococcus protective antigen contributing to bacterial cell division

Bioinformatic analysis of Group A Streptococcus (GAS) genomes aiming at the identification of new vaccine antigens, revealed the presence of a gene coding for a putative surface-associated protein, named GAS40, inducing protective antibodies in an animal model of sepsis. The aim of our study was to unravel the involvement of GAS40 in cell division processes and to identify the putative interactor. Firstly, bioinformatic analysis showed that gas40 shares homology with ezrA, a gene coding for a negative regulator of Z-ring formation during cell division process. Both scanning and transmission electron microscopy indicated morphological differences between wild-type and the GAS40 knock-out mutant strain, with the latter showing an impaired capacity to divide resulting in the formation of very long chains. Moreover, when the localization of the antigen on the bacterial surface was analyzed, we found that in bacteria grown at exponential phase GAS40 specifically localized at septum, indicating a possible role in cell division. Furthermore, by ELISA and co-sedimentation assays, we found that GAS40 is able to interact with FtsZ, a protein involved in Z-ring formation during cell division process. These data together with the co-localization of GAS40/FtsZ at bacterial septum demonstrated by by confocal microscopy, strongly support the hypothesis for a key role of GAS40 in bacterial cell division.

Analyse der intrazellulären Freisetzung von Wirkstoffmodellen via konfokaler Laser-Raster-Mikroskopie

In der vorliegenden Arbeit wurde gezeigt, wie man das Potential nanopartikulärer Systeme, die vorwiegend via Miniemulsion hergestellt wurden, im Hinblick auf „Drug Delivery“ ausnutzen könnte, indem ein Wirkstoffmodell auf unterschiedliche Art und Weise intrazellulär freigesetzt wurde. Dies wurde hauptsächlich mittels konfokaler Laser-Raster-Mikrokopie (CLSM) in Kombination mit dem Bildbearbeitungsprogramm Volocity® analysiert.rnPBCA-Nanokapseln eigneten sich besonders, um hydrophile Substanzen wie etwa Oligonukleotide zu verkapseln und sie so auf ihrem Transportweg in die Zellen vor einem etwaigen Abbau zu schützen. Es konnte eine Freisetzung der Oligonukleotide in den Zellen aufgrund der elektrostatischen Anziehung des mitochondrialen Membranpotentials nachgewiesen werden. Dabei war die Kombination aus Oligonukleotid und angebundenem Cyanin-Farbstoff (Cy5) an der 5‘-Position der Oligonukleotid-Sequenz ausschlaggebend. Durch quantitative Analysen mittels Volocity® konnte die vollständige Kolokalisation der freigesetzten Oligonukleotide an Mitochondrien bewiesen werden, was anhand der Kolokalisationskoeffizienten „Manders‘ Coefficients“ M1 und M2 diskutiert wurde. Es konnte ebenfalls aufgrund von FRET-Studien doppelt markierter Oligos gezeigt werden, dass die Oligonukleotide weder beim Transport noch bei der Freisetzung abgebaut wurden. Außerdem wurde aufgeklärt, dass nur der Inhalt der Nanokapseln, d. h. die Oligonukleotide, an Mitochondrien akkumulierte, das Kapselmaterial selbst jedoch in anderen intrazellulären Bereichen aufzufinden war. Eine Kombination aus Cyanin-Farbstoffen wie Cy5 mit einer Nukleotidsequenz oder einem Wirkstoff könnte also die Basis für einen gezielten Wirkstofftransport zu Mitochondrien liefern bzw. die Grundlage schaffen, eine Freisetzung aus Kapseln ins Zytoplasma zu gewährleisten.rnDer vielseitige Einsatz der Miniemulsion gestattete es, nicht nur Kapseln sondern auch Nanopartikel herzustellen, in welchen hydrophobe Substanzen im Partikelkern eingeschlossen werden konnten. Diese auf hydrophobe Wechselwirkungen beruhende „Verkapselung“ eines Wirkstoffmodells, in diesem Fall PMI, wurde bei PDLLA- bzw. PS-Nanopartikeln ausgenutzt, welche durch ein HPMA-basiertes Block-Copolymer stabilisiert wurden. Dabei konnte gezeigt werden, dass das hydrophobe Wirkstoffmodell PMI innerhalb kürzester Zeit in die Zellen freigesetzt wurde und sich in sogenannte „Lipid Droplets“ einlagerte, ohne dass die Nanopartikel selbst aufgenommen werden mussten. Daneben war ein intrazelluläres Ablösen des stabilisierenden Block-Copolymers zu verzeichnen, welches rn8 h nach Partikelaufnahme erfolgte und ebenfalls durch Analysen mittels Volocity® untermauert wurde. Dies hatte jedoch keinen Einfluss auf die eigentliche Partikelaufnahme oder die Freisetzung des Wirkstoffmodells. Ein großer Vorteil in der Verwendung des HPMA-basierten Block-Copolymers liegt darin begründet, dass auf zeitaufwendige Waschschritte wie etwa Dialyse nach der Partikelherstellung verzichtet werden konnte, da P(HPMA) ein biokompatibles Polymer ist. Auf der anderen Seite hat man aufgrund der Syntheseroute dieses Block-Copolymers vielfältige Möglichkeiten, Funktionalitäten wie etwa Fluoreszenzmarker einzubringen. Eine kovalente Anbindung eines Wirkstoffs ist ebenfalls denkbar, welcher intrazellulär z. B. aufgrund von enzymatischen Abbauprozessen langsam freigesetzt werden könnte. Somit bietet sich die Möglichkeit mit Nanopartikeln, die durch HPMA-basierte Block-Copolymere stabilisiert wurden, gleichzeitig zwei unterschiedliche Wirkstoffe in die Zellen zu bringen, wobei der eine schnell und der zweite über einen längeren Zeitraum hinweg (kontrolliert) freigesetzt werden könnte.rnNeben Nanokapseln sowie –partikeln, die durch inverse bzw. direkte Miniemulsion dargestellt wurden, sind auch Nanohydrogelpartikel untersucht worden, die sich aufgrund von Selbstorganisation eines amphiphilen Bock-Copolymers bildeten. Diese Nanohydrogelpartikel dienten der Komplexierung von siRNA und wurden hinsichtlich ihrer Anreicherung in Lysosomen untersucht. Aufgrund der Knockdown-Studien von Lutz Nuhn konnte ein Unterschied in der Knockdown-Effizienz festgestellt werden, je nach dem, ob 100 nm oder 40 nm große Nanohydrogelpartikel verwendet wurden. Es sollte festgestellt werden, ob eine größenbedingte, unterschiedlich schnelle Anreicherung dieser beiden Partikel in Lysosomen erfolgte, was die unterschiedliche Knockdown-Effizienz erklären könnte. CLSM-Studien und quantitative Kolokalisationsstudien gaben einen ersten Hinweis auf diese Größenabhängigkeit. rnBei allen verwendeten nanopartikulären Systemen konnte eine Freisetzung ihres Inhalts gezeigt werden. Somit bieten sie ein großes Potential als Wirkstoffträger für biomedizinische Anwendungen.rn

General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds

As opposed to culture on standard tissue-treated plastic, cell culture on three-dimensional scaffolds impedes additional challenges with respect to substrate preparation, cell seeding, culture maintenance, and analysis. We herewith present a general route for the culture of primary cells, differentiated cells, or stem cells on plasma-coated, electrospun scaffolds. We describe a method to prepare and fix the scaffolds in culture wells and discuss a convenient method for cell seeding and subsequent analysis by scanning electron microscopy or immunohistology.

The metalloprotease meprinbeta processes E-cadherin and weakens intercellular adhesion

BACKGROUND: Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used MDCK and Caco-2 cells stably transfected with meprin alpha and or meprin beta to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. CONCLUSIONS/SIGNIFICANCE: By identifying E-cadherin as a substrate for meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression.