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Surface-enhanced Raman spectroscopy (SERS) with pyramidal gold nanostructures increases the signal of Raman active analytes, since hotspots form at the edges and tip of a nanopyramid under illumination. 2D hexagonal arrays of pyramidal nanostructures with a quadratic base are fabricated through cost-effective nanosphere lithography and transferred onto elastomeric polydimethylsiloxane. By making use of the {111} crystal plane of a silicon (100) wafer, an inverted pyramidal array is etched, which serves as the complementary negative for the gold nanostructures. Either a continuous gold thin-film with protruding pyramids or separate isolated nanopyramids are produced. Three basic fabrication strategies are presented. The SERS enhancement is verified by Raman mapping of 4-mercaptobenzoic acid (4-MBA) molecules. Fabrication on a flexible substrate paves the way for future applications on curved surfaces orin situtunable resonances.
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BACKGROUND: Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology. METHODS: We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains. RESULTS: We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible. CONCLUSION: The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.
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Barreira Hematoencefálica/virologia , Líquido Cefalorraquidiano/virologia , Plexo Corióideo/virologia , Surtos de Doenças , Enterovirus Humano B/isolamento & purificação , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/ultraestrutura , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células Cultivadas , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/metabolismo , Plexo Corióideo/ultraestrutura , Enterovirus Humano B/metabolismo , Humanos , Filogenia , Especificidade da EspécieRESUMO
AIMS: To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. METHODS: Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. RESULTS: 3View-SEM image stacks as large as 59 × 59 × 17 µm3 (xyz) at a resolution of 16 × 16 × 50 nm3 and high resolution (5 × 5 × 10 nm3 ) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. CONCLUSIONS: Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.
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Células Epiteliais/ultraestrutura , Junções Intercelulares/ultraestrutura , Microscopia/métodos , Mucosa/ultraestrutura , Bexiga Urinária/ultraestrutura , Urotélio/ultraestrutura , Células Epiteliais/citologia , Feminino , Humanos , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mucosa/citologia , Bexiga Urinária/citologia , Urotélio/citologiaRESUMO
Drug eluting stents (DES) have shown efficacy in reducing restenosis after angioplasty followed by application of a coronary stent. However, polymer matrices typically used for immobilizing drugs on the stent surface may cause irritation and have limited drug loading capacity. In contrast, drug loading into micro- or nanopores created within the stent material could avoid these problems. We present a technology based on electrochemically induced pitting corrosion to form pores in medical grade steel, followed by loading with rapamycin. This process is applied to pore formation and drug loading in coronary stents consisting of L605 medical steel. Sustained release of the drug over 28 days at rates comparable to established DES was demonstrated. This technology is capable of creating pores with well-defined pore size and filling of these pores by a drug employing a crystallization process thus completely avoiding polymer matrices to immobilize drugs. Electrochemically induced pitting corrosion provides a generic means to introduce micro-pores suitable as drug reservoirs into medical grade steel without the need for any further matrix material. Further research will expand these findings to other materials and types of implants that could benefit from the additional function of drug release and/or improved implant/tissue integration.
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Ligas de Cromo/química , Stents Farmacológicos , Técnicas Eletroquímicas , Desenho de Prótese , Antibacterianos/química , Cinética , Teste de Materiais , Sirolimo/químicaRESUMO
Bioactive cations, including calcium, copper and magnesium, have shown the potential to become the alternative to protein growth factor-based therapeutics for bone healing. Ion substitutions are less costly, more stable, and more effective at low concentrations. Although they have been shown to be effective in providing bone grafts with more biological functions, the precise control of ion release kinetics is still a challenge. Moreover, the synergistic effect of three or more metal ions on bone regeneration has rarely been studied. In this study, vaterite-calcite CaCO3 particles were loaded with copper (Cu2+) and magnesium (Mg2+). The polyelectrolyte multilayer (PEM) was deposited on CaCuMg-CO3 particles via layer-by-layer technique to further improve the stability and biocompatibility of the particles and to enable controlled release of multiple metal ions. The PEM coated microcapsules were successfully combined with collagen at the outmost layer, providing a further stimulating microenvironment for bone regeneration. The in vitro release studies showed remarkably stable release of Cu2+ in 2 months without initial burst release. Mg2+ was released in relatively low concentration in the first 7 days. Cell culture studies showed that CaCuMg-PEM-Col microcapsules stimulated cell proliferation, extracellular maturation and mineralization more effectively than blank control and other microcapsules without collagen adsorption (Ca-PEM, CaCu-PEM, CaMg-PEM, CaCuMg-PEM). In addition, the CaCuMg-PEM-Col microcapsules showed positive effects on osteogenesis and angiogenesis in gene expression studies. The results indicate that such a functional and controllable delivery system of multiple bioactive ions might be a safer, simpler and more efficient alternative of protein growth factor-based therapeutics for bone regeneration. It also provides an effective method for functionalizing bone grafts for bone tissue engineering.
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A full understanding of the relationship between surface properties, protein adsorption, and immune responses is lacking but is of great interest for the design of biomaterials with desired biological profiles. In this study, polyelectrolyte multilayer (PEM) coatings with gradient changes in surface wettability were developed to shed light on how this impacts protein adsorption and immune response in the context of material biocompatibility. The analysis of immune responses by peripheral blood mononuclear cells to PEM coatings revealed an increased expression of proinflammatory cytokines tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1ß, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 and the surface marker CD86 in response to the most hydrophobic coating, whereas the most hydrophilic coating resulted in a comparatively mild immune response. These findings were subsequently confirmed in a cohort of 24 donors. Cytokines were produced predominantly by monocytes with a peak after 24 h. Experiments conducted in the absence of serum indicated a contributing role of the adsorbed protein layer in the observed immune response. Mass spectrometry analysis revealed distinct protein adsorption patterns, with more inflammation-related proteins (e.g., apolipoprotein A-II) present on the most hydrophobic PEM surface, while the most abundant protein on the hydrophilic PEM (apolipoprotein A-I) was related to anti-inflammatory roles. The pathway analysis revealed alterations in the mitogen-activated protein kinase (MAPK)-signaling pathway between the most hydrophilic and the most hydrophobic coating. The results show that the acute proinflammatory response to the more hydrophobic PEM surface is associated with the adsorption of inflammation-related proteins. Thus, this study provides insights into the interplay between material wettability, protein adsorption, and inflammatory response and may act as a basis for the rational design of biomaterials.
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Anti-Inflamatórios/química , Materiais Revestidos Biocompatíveis/química , Citocinas/imunologia , Inflamação/imunologia , Polieletrólitos/química , Adsorção , Anti-Inflamatórios/farmacologia , Células Cultivadas , Materiais Revestidos Biocompatíveis/farmacologia , Citocinas/análise , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Humanos , Interações Hidrofóbicas e Hidrofílicas , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Tamanho da Partícula , Polieletrólitos/farmacologia , Propriedades de Superfície , MolhabilidadeRESUMO
It has been widely shown that biomaterial surface topography can modulate host immune response, but a fundamental understanding of how different topographies contribute to pro-inflammatory or anti-inflammatory responses is still lacking. To investigate the impact of surface topography on immune response, we undertook a systematic approach by analyzing immune response to eight grades of medical grade polyurethane of increasing surface roughness in three in vitro models of the human immune system. Polyurethane specimens were produced with defined roughness values by injection molding according to the VDI 3400 industrial standard. Specimens ranged from 0.1 µm to 18 µm in average roughness (Ra), which was confirmed by confocal scanning microscopy. Immunological responses were assessed with THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs), and whole blood following culture on polyurethane specimens. As shown by the release of pro-inflammatory and anti-inflammatory cytokines in all three models, a mild immune response to polyurethane was observed, however, this was not associated with the degree of surface roughness. Likewise, the cell morphology (cell spreading, circularity, and elongation) in THP-1-derived macrophages and the expression of CD molecules in the PBMC model on T cells (HLA-DR and CD16), NK cells (HLA-DR), and monocytes (HLA-DR, CD16, CD86, and CD163) showed no influence of surface roughness. In summary, this study shows that modifying surface roughness in the micrometer range on polyurethane has no impact on the pro-inflammatory immune response. Therefore, we propose that such modifications do not affect the immunocompatibility of polyurethane, thereby supporting the notion of polyurethane as a biocompatible material.
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Materiais Biocompatíveis/química , Imunidade , Poliuretanos/química , Anti-Inflamatórios/imunologia , Citocinas/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/ultraestrutura , Macrófagos/imunologia , Macrófagos/ultraestrutura , Masculino , Microscopia Confocal , Microscopia Eletrônica de Varredura , Monócitos/imunologia , Monócitos/ultraestrutura , Propriedades de Superfície , Linfócitos T/imunologia , Linfócitos T/ultraestrutura , Células THP-1RESUMO
Electrophysiological activities of neuronal networks can be recorded on microelectrode arrays (MEAs). This technique requires tight coupling between MEA-surfaces and cells. Therefore, this study investigated the interface between DRG neurons and MEA-surface materials after adsorption of neurite promoting proteins: laminin-111, fibronectin, L1Ig6 and poly-l-lysine. Moreover, substrate-induced effects on neuronal networks with time were analyzed. The thickness of adsorbed protein layers was found between approximately 1 nm for poly-l-lysine and approximately 80 nm for laminin-111 on platinum, gold and silicon nitride. The neuron-to-substrate interface was characterized by Scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and SEM after in situ focused-ion-beam milling demonstrating that the ventral cell membrane adhered inhomogeneously to laminin-111 or L1Ig6 surfaces. Tight areas of 20-30 nm and distant areas <1 microm alternated and even tightest areas did not correlate with the physical thickness of the protein layers. This study illustrates the difficulties to predict cell-to-material interfaces that contribute substantially to the success of in vitro or in vivo systems. Moreover, focused ion beam (FIB)/SEM is explored as a new technique to analyze such interfaces.
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Neurônios/fisiologia , Adsorção , Animais , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Embrião de Galinha , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Gânglios Espinais/ultraestrutura , Microeletrodos , Rede Nervosa/citologia , Rede Nervosa/ultraestrutura , Neurônios/citologia , Neurônios/ultraestrutura , Análise Serial de Proteínas , Propriedades de SuperfícieRESUMO
Carbon nanotube (CNT) membranes were produced from multi-walled CNTs by a filtration technique and used for the removal of the betablocker metoprolol by adsorptive and reactive processes. The reactivity of CNT membranes was enhanced by nanoparticulate zero-valent iron (NZVI) which was deposited on the CNT membranes by pulsed voltammetry applying defined number of pulses (Fe-CNT (100) and Fe-CNT (400) membranes). Surface analysis with SEM showed iron nanoparticle sizes between 19 and 425 nm. Pore size distribution for the different membranes was determined by capillary flow porometry (Galwick fluid). Pore size distribution for all membranes was similar (40 nm), which resulted in a water permeability typical for microfiltration membranes. Metoprolol was removed by the CNT membrane only by sorption, whereas the Fe-CNT membrane revealed also metoprolol degradation due to Fenton type reactions. Further application of electrochemical potentials on both the CNT and the Fe-CNT membranes improved the removal efficiencies to 74% for CNT membranes at 1 V and to 97% for Fe-CNT (400) membranes at 1 V. Seven transformation products have been identified for metoprolol by high-resolution mass spectrometry when electrochemical degradation was performed with CNT and Fe-CNT membranes. Additionally, two of the identified transformation products (TPs) were also observed for Fe-CNT membranes without the application of electrochemical potential. However, only 10% of the degraded metoprolol could be explained by the formation of TPs.
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Ferro/química , Nanotubos de Carbono/química , Adsorção , Filtração , NanopartículasRESUMO
Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.
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Hélio , Íons , Microscopia/métodos , Animais , Arabidopsis/ultraestrutura , Bactérias/ultraestrutura , Células HeLa/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura/métodos , Nematoides/ultraestruturaRESUMO
A concept for a partially implantable hearing device, for which the power supply and signal transmission are provided by an optical transmission path, is evaluated. The actuator is designed to fit into the round-window niche and to couple directly to the round-window membrane. Implantable hearing aids can be a suitable solution in the case of severe hearing loss, where conventional hearing aids often fail. However, the surgical effort for an implantation is comparatively high. Therefore, the objective of our work was to provide a hearing system which combines reliable coupling to the auditory system with an easy implantation technique. The actuator was designed as a piezoelectric thin-film cantilever. The optical transmission path was realised using an infrared light-emitting diode combined with an active receiver circuit. For a voltage of 1V, the unloaded actuator presents displacement amplitudes of 1µm up to a stimulus frequency of 25kHz and forces up to 0.2mN. Proportionally larger forces can be achieved by stacking single actuators. The overall transmission loss from the electrical input of the light-emitting diode driver to the mechanical output of the unloaded actuator was less than 25dB at 1kHz and maximum output.
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Meato Acústico Externo , Prótese Ossicular , Desenho de Prótese , Raios InfravermelhosRESUMO
Automats for patch clamping suspended cells in whole-cell configuration must (1) bring isolated cells in contact with patch contacts, (2) form gigaseals, and (3) establish stable intracellular access that allows for high quality recording of ionic currents. Single openings in planar substrates seem to be intriguing simple solutions for these problems, but due to the low rate of formation of whole-cell configurations we discarded this approach. Single openings are not suited for both attracting cells to the opening by suction and forming gigaseals with subsequent membrane rupture. To settle the three tasks with a mechanical microstructure we developed the socalled CYTOCENTERING technique to apply to suspended cells the same operation sequence as in conventional patch clamping. With this method we immobilized selected cells from a flowing suspension on the tip of a patch pipette by suction with a success rate of 97% and formed gigaseals with a success rate of 68%. Subsequent whole-cell recordings and intracellular staining with Lucifer yellow proved the stable access to the cytoplasm. Currently, a chip with an embedded suction opening in glass surrounding the microstructured contact pipette is under development. The processing of this CYTOPATCH chip is compatible to large-volume production. The CYTOPATCH automat will allow for fully automated, parallel, and asynchronous whole-cell recordings.