570 Biowissenschaften, Biologie
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The early detection of head and neck cancer is a prolonged challenging task. It requires a precise and accurate identification of tissue alterations as well as a distinct discrimination of cancerous from healthy tissue areas. A novel approach for this purpose uses microspectroscopic techniques with special focus on hyperspectral imaging (HSI) methods. Our proof-of-principle study presents the implementation and application of darkfield elastic light scattering spectroscopy (DF ELSS) as a non-destructive, high-resolution, and fast imaging modality to distinguish lingual healthy from altered tissue regions in a mouse model. The main aspect of our study deals with the comparison of two varying HSI detection principles, which are a point-by-point and line scanning imaging, and whether one might be more appropriate in differentiating several tissue types. Statistical models are formed by deploying a principal component analysis (PCA) with the Bayesian discriminant analysis (DA) on the elastic light scattering (ELS) spectra. Overall accuracy, sensitivity, and precision values of 98% are achieved for both models whereas the overall specificity results in 99%. An additional classification of model-unknown ELS spectra is performed. The predictions are verified with histopathological evaluations of identical HE-stained tissue areas to prove the model’s capability of tissue distinction. In the context of our proof-of-principle study, we assess the Pushbroom PCA-DA model to be more suitable for tissue type differentiations and thus tissue classification. In addition to the HE-examination in head and neck cancer diagnosis, the usage of HSI-based statistical models might be conceivable in a daily clinical routine.
Focal adhesion clusters (FAC) are dynamic and complex structures that help cells to sense physicochemical properties of their environment. Research in biomaterials, cell adhesion or cell migration often involves the visualization of FAC by fluorescence staining and microscopy, which necessitates quantitative analysis of FAC and other cell features in microscopy images using image processing. Fluorescence microscopy images of human umbilical vein endothelial cells (HUVEC) obtained at 63x magnification were quantitatively analysed using ImageJ software. A generalised algorithm for selective segmentation and morphological analysis of FAC, nucleus and cell morphology is implemented. Further, a method for discrimination of FACnear the nucleus and around the periphery is implemented using masks. Our algorithm is able to effectively quantify different morphological characteristics of cell components and shows a high sensitivity and specificity while providing a modular software implementation.
Soft lithography, a tool widely applied in biology and life sciences with numerous applications, uses the soft molding of photolithography-generated master structures by polymers. The central part of a photolithography set-up is a mask-aligner mostly based on a high-pressure mercury lamp as an ultraviolet (UV) light source. This type of light source requires a high level of maintenance and shows a decreasing intensity over its lifetime, influencing the lithography outcome. In this paper, we present a low-cost, bench-top photolithography tool based on ninety-eight 375 nm light-emitting diodes (LEDs). With approx. 10 W, our presented lithography set-up requires only a fraction of the energy of a conventional lamp, the LEDs have a guaranteed lifetime of 1000 h, which becomes noticeable by at least 2.5 to 15 times more exposure cycles compared to a standard light source and with costs less than 850 C it is very affordable. Such a set-up is not only attractive to small academic and industrial fabrication facilities who want to enable work with the technology of photolithography and cannot afford a conventional set-up, but also microfluidic teaching laboratories and microfluidic research and development laboratories, in general, could benefit from this cost-effective alternative. With our self-built photolithography system, we were able to produce structures from 6 μm to 50 μm in height and 10 μm to 200 μm in width. As an optional feature, we present a scaled-down laminar flow hood to enable a dust-free working environment for the photolithography process.
Collagen-based barrier membranes are an essential component in Guided Bone Regeneration (GBR) procedures. They act as cell-occlusive devices that should maintain a micromilieu where bone tissue can grow, which in turn provides a stable bed for prosthetic implantation. However, the standing time of collagen membranes has been a challenging area, as native membranes are often prematurely resorbed. Therefore, consolidation techniques, such as chemical cross-linking, have been used to enhance the structural integrity of the membranes, and by consequence, their standing time. However, these techniques have cytotoxic tendencies and can cause exaggerated inflammation and in turn, premature resorption, and material failures. However, tissues from different extraction sites and animals are variably cross-linked. For the present in vivo study, a new collagen membrane based on bovine dermis was extracted and compared to a commercially available porcine-sourced collagen membrane extracted from the pericardium. The membranes were implanted in Wistar rats for up to 60 days. The analyses included well-established histopathological and histomorphometrical methods, including histochemical and immunohistochemical staining procedures, to detect M1- and M2-macrophages as well as blood vessels. Initially, the results showed that both membranes remained intact up to day 30, while the bovine membrane was fragmented at day 60 with granulation tissue infiltrating the implantation beds. In contrast, the porcine membrane remained stable without signs of material-dependent inflammatory processes. Therefore, the bovine membrane showed a special integration pattern as the fragments were found to be overlapping, providing secondary porosity in combination with a transmembraneous vascularization. Altogether, the bovine membrane showed comparable results to the porcine control group in terms of biocompatibility and standing time. Moreover, blood vessels were found within the bovine membranes, which can potentially serve as an additional functionality of barrier membranes that conventional barrier membranes do not provide.
Human bestrophin-1 protein (hBest1) is a transmembrane channel associated with the calcium-dependent transport of chloride ions in the retinal pigment epithelium as well as with the transport of glutamate and GABA in nerve cells. Interactions between hBest1, sphingomyelins, phosphatidylcholines and cholesterol are crucial for hBest1 association with cell membrane domains and its biological functions. As cholesterol plays a key role in the formation of lipid rafts, motional ordering of lipids and modeling/remodeling of the lateral membrane structure, we examined the effect of different cholesterol concentrations on the surface tension of hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and hBest1/SM Langmuir monolayers in the presence/absence of Ca2+ ions using surface pressure measurements and Brewster angle microscopy studies. Here, we report that cholesterol: (1) has negligible condensing effect on pure hBest1 monolayers detected mainly in the presence of Ca2+ ions, and; (2) induces a condensing effect on composite hBest1/POPC and hBest1/SM monolayers. These results offer evidence for the significance of intermolecular protein–lipid interactions for the conformational dynamics of hBest1 and its biological functions as multimeric ion channel.
Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio‐based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide–alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side‐chain extension. Approximately 84–90% of the amino groups are modified as shown by 1H‐NMR spectroscopy, 2,4,6‐trinitrobenzenesulfonic acid assay as well as Fourier‐transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido‐functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG‐DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG‐DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.