TY - JOUR A1 - Kasper, J. A1 - Herrmanns, M.I. A1 - Bantz, C. A1 - Utech, S. A1 - Koshkina, Olga A1 - Maskos, Michael A1 - Brochhausen, C. A1 - Pohl, C. A1 - Fuchs, S. A1 - Unger, R. E. A1 - Kirkpatrick, C.J. T1 - Flotillin-involved uptake of silica nanoparticles and responses of an alveolar-capillary barrier in vitro N2 - Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60 µg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560 ± 8 Ω cm². H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake. KW - Silica nanoparticles KW - Alveolar-capillary barrier KW - NP uptake KW - NP-transport KW - Endocytosis KW - Flotillin-1/-2-dependent uptake/trafficking PY - 2013 DO - https://doi.org/10.1016/j.ejpb.2012.10.011 SN - 0939-6411 SN - 1873-3441 VL - 84 IS - 2 SP - 275 EP - 287 PB - Elsevier B.V. CY - Amsterdam AN - OPUS4-28841 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Zehbe, R. A1 - Haibel, A. A1 - Riesemeier, Heinrich A1 - Gross, U. A1 - Kirkpatrick, C.J. A1 - Schubert, H. A1 - Brochhausen, C. T1 - Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells N2 - Current light microscopic methods such as serial sectioning, confocal microscopy or multiphoton microscopy are severely limited in their ability to analyse rather opaque biological structures in three dimensions, while electron optical methods offer either a good three-dimensional topographic visualization (scanning electron microscopy) or high-resolution imaging of very thin samples (transmission electron microscopy). However, sample preparation commonly results in a significant alteration and the destruction of the three-dimensional integrity of the specimen. Depending on the selected photon energy, the interaction between X-rays and biological matter provides semi-transparency of the specimen, allowing penetration of even large specimens. Based on the projection-slice theorem, angular projections can be used for tomographic imaging. This method is well developed in medical and materials science for structure sizes down to several micrometres and is considered as being non-destructive. Achieving a spatial and structural resolution that is sufficient for the imaging of cells inside biological tissues is difficult due to several experimental conditions. A major problem that cannot be resolved with conventional X-ray sources are the low differences in density and absorption contrast of cells and the surrounding tissue. Therefore, X-ray monochromatization coupled with a sufficiently high photon flux and coherent beam properties are key requirements and currently only possible with synchrotron-produced X-rays. In this study, we report on the three-dimensional morphological characterization of articular cartilage using synchrotron-generated X-rays demonstrating the spatial distribution of single cells inside the tissue and their quantification, while comparing our findings to conventional histological techniques. KW - Cartilage KW - Chondrocyte KW - Synchrotron micro-computed tomography KW - Histology KW - Scanning electron microscopy KW - Three-dimensional imaging PY - 2010 DO - https://doi.org/10.1098/rsif.2008.0539 SN - 1742-5689 SN - 1742-5662 VL - 7 SP - 49 EP - 59 PB - The Royal Society CY - London AN - OPUS4-22123 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Zehbe, R. A1 - Riesemeier, Heinrich A1 - Kirkpatrick, C.J. A1 - Brochhausen, C. T1 - Imaging of articular cartilage - data matching using X-ray tomography, SEM, FIB slicing and conventional histology N2 - The study was aimed at demonstrating a true cellular resolution for articular cartilage using synchrotron radiation-based X-ray microcomputed tomography (SR-µCT) with a sample-specific optimization of the phase contrast. The generated tomographic data were later used to prepare a matching histological sample from the full volume specimen. We used highly coherent and monochromatic X-rays from a synchrotron source to image a tissue sample of bovine articular cartilage after deparaffinization. Phase contrast enhancement was achieved by using five different sample to detector distances for the same X-ray energy. After tomography, the sample was re-embedded into resin while retaining a dedicated sample orientation for subsequent sectioning and polishing, which was conducted until a previously defined spatial position was achieved. The protocol for resin embedding was developed to inhibit morphological changes during embedding. Giemsa staining was applied for better structural and morphological discrimination. Data from tomography and lightmicroscopy were exactly matched and finally compared to results from FIB/SEM imaging. Image detail was achieved at a single cell resolution. Image detail was achieved at a single cell resolution, which has been estimated to be 0.833 µm/voxel in the tomographic data. SR-µCT with optimized phase contrast properties represents a method to investigate biological tissues in certain areas of interest, where true cellular resolution or enhanced volumetric imaging is needed. In this study, we demonstrate that this method can compete with conventional histology using light microscopy but even surpasses it due to the possibility of retrieving volumetric data. KW - Synchrotron radiation-based µCT KW - Phase contrast KW - FIB/SEM imaging KW - Histology KW - Articular cartilage KW - Data matching PY - 2012 DO - https://doi.org/10.1016/j.micron.2012.05.001 SN - 0047-7206 SN - 0968-4328 VL - 43 IS - 10 SP - 1060 EP - 1067 PB - Pergamon Press CY - Oxford AN - OPUS4-26435 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -