Wirkung von Silizium und Strontium dotierten Hydroxylapatitnanopartikel auf Mauszellen in vitro

Beim Knochen-Tissue Engineering werden dreidimensionale Matrices (Scaffolds) zusammen mit osteogenen Zellen und bioaktiven Substanzen entwickelt, die zur Regeneration von Knochendefekten eingesetzt werden sollen. Die im Knochen-Tissue Engineering verwendeten Biomaterialien bestehen meist aus Keramik, Polymeren oder Metallen und sie sollten osteoinduktive und osteokonduktive Eigenschaften besitzen. Um die Osseointegration sowie die knochenaufbauende Wirkung der Biomaterialien zu verbessern, können Hydroxylapatitnanopartikel integriert werden oder als Überzugsmaterial eingesetzt werden. Bei Nanopartikel handelt es sich um Partikel, die kleiner als 100 nm sind. Sie können aufgrund ihrer hohen Bioaktivität und -affinität oder aufgrund der Bildung nanotopographischer Muster die osteoinduktiven und osteokonduktiven Eigenschaften des Biomaterials verbessern. Außerdem kann durch eine Dotierung der Nanopartikel mit bioaktiven Ionen das Knochenregenerationspotenzial der Biomaterialien weiter gesteigert werden. Die vorliegende Diplomarbeit untersucht die Effekte von Silizium und Strontium dotierten Hydroxylapatitnanopartikel auf primäre Mausosteoblasten. Zunächst wurde die Wirkung der Nanopartikel auf die Zellviabilität überprüft. Anschließend wurde geprüft, ob sie einen induzierenden Effekt auf die Differenzierung sowie die Aktivität der OB haben. Zuletzt wurden Veränderungen in der Genexpression der Zellen untersucht. Dabei wurden die beiden Nanopartikelsorten in vier unterschiedlichen Konzentrationen - nämlich 250 g/ml, 100 g/ml, 50 g/ml und 20 g/ml - getestet. Selbst in der höchsten Konzentration beeinträchtigten die Nanopartikel die Zellviabilität nicht. Im Gegenteil wurde die Zellproliferation durch den Zusatz der Nanopartikel erhöht. Die Zelldifferenzierung wurde durch beide Nanopartikelsorten gefördert. Das Ausmaß der Mineralablagerung und die Expression von Genen, die an der Zelldifferenzierung beteiligt sind wurde durch die siHA-NP stärker erhöht als durch srHA-NP. Folglich besitzen beide Nanopartikelpräparationen das Potenzial zu Knochenersatzmaterialien weiterverarbeitet zu werden. In Bezug auf die Wirkung auf OB zeigten sich die siHA-NP von Vorteil. Da allerdings der Erfolg eines Knochenersatzmaterials nicht nur von einer fördernden Wirkung auf OB, sondern auch von einer hemmenden Wirkung auf knochenabbauende OC abhängt, wären weitere Untersuchungen der Nanopartikel für eine endgültige Bewertung erforderlich.Bone-Tissue Engineering is a scientific field that deals with the production of scaffolds in combination with osteogenic cells and bioactive substances for the purpose of regenerating bone defects. Biomaterials used for Bone-Tissue Engineering are made of ceramics, polymers or metals and should possess osteoinductive and osteoconductive properties. Hydroxyapatite nanoparticles can be integrated or used as a bioactive coating in order to enhance osseointegration and bone formation. Nanoparticles are smaller than 100 nm. The particles' high bioactivity and bioaffinity or their ability to form nanotopographic patterns facilitate the improvement of the osteoinductive and osteoconductive properties of a biomaterial. Moreover, ion doping can be used as a strategy to improve the bone regeneration potential of biomaterials. This diploma thesis examines the effects of silicon or strontium doped hydroxyapatite nanoparticles on primary mouse osteoblasts. First the particles` effect on cell viability was analyzed. Subsequently, their influence on the differentiation and activity of osteoblasts was tested. Finally, changes in the gene expression of the cells were analyzed. The nanoparticles were tested in four concentrations (250 g/ml, 100 g/ml, 50 g/ml und 20 g/ml). Even in the most potent concentration the nanoparticles did not reduce the cell viability. On the contrary, the cell proliferation was promoted. Additionally, both kinds of nanoparticles enhanced the cell differentiation. Under the influence of silicon-doped hydroxyapatite nanoparticles the mineral deposition as well as the expression of proteins which are important for the cell differentiation increased at a higher extend. As a consequence, both of the nanoparticles have the potential to be used as bone replacement material. The silicon doped nanoparticles proved to be more efficient regarding the positive effects on OB. Nevertheless, a successful bone replacement requires not only a stimulation of bone forming OB, but also an inhibition of bone resorbing OC. Therefore, more studies on nanoparticles are essential for a final assessment

Microbiological and Molecular Diagnosis of Mucormycosis: From Old to New

Members of the order Mucorales may cause severe invasive fungal infections (mucormycosis) in immune-compromised and otherwise ill patients. Diagnosis of Mucorales infections and discrimination from other filamentous fungi are crucial for correct management. Here, we present an overview of current state-of-the-art mucormycosis diagnoses, with a focus on recent developments in the molecular field. Classical diagnostic methods comprise histology/microscopy as well as culture and are still the gold standard. Newer molecular methods are evolving quickly and display great potential in early diagnosis, although standardization is still missing. Among them, quantitative PCR assays with or without melt curve analysis are most widely used to detect fungal DNA in clinical samples. Depending on the respective assay, sequencing of the resulting PCR product can be necessary for genus or even species identification. Further, DNA-based methods include microarrays and PCR-ESI-MS. However, general laboratory standards are still in development, meaning that molecular methods are currently limited to add-on analytics to culture and microscopy

Effects of biological, aerobic substrate pre-treatment of standard organic waste on the anaerobic digestion process

Nina LacknerZusammenfassung in englischer und deutscher SpracheUniversität Innsbruck, Masterarbeit, 2015(VLID)76053

pH and Phosphate Induced Shifts in Carbon Flow and Microbial Community during Thermophilic Anaerobic Digestion

pH is a central environmental factor influencing CH4 production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0–8.5, phosphate buffered) and the application of a phosphate buffer per se induce shifts in the microbial community composition and the carbon flow during nine weeks of thermophilic batch digestion. Beside monitoring the methane production as well as volatile fatty acid concentrations, amplicon sequencing of the 16S rRNA gene was conducted. The presence of 100 mM phosphate resulted in reduced CH4 production during the initial phase of the incubation, which was characterized by a shift in the dominant methanogenic genera from a mixed Methanosarcina and Methanoculleus to a pure Methanoculleus system. In buffered samples, acetate strongly accumulated in the beginning of the batch digestion and subsequently served as a substrate for methanogens. Methanogenesis was permanently inhibited at pH values ≤5.5, with the maximum CH4 production occurring at pH 7.5. Adaptations of the microbial community to the pH variations included shifts in the archaeal and bacterial composition, as less competitive organisms with a broad pH range were able to occupy metabolic niches at unfavorable pH conditions

Hydrogenotrophic Methanogenesis and Autotrophic Growth of Methanosarcina thermophila

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability of Methanosarcina thermophila to use carbon dioxide (CO2) for catabolic and anabolic metabolism was not proven until now. Here, we show that M. thermophila used CO2 to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO2 consecutively, resulting in a biphasic methane production. Growth exclusively from CO2 occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from 13CO2 into 13CH4 molecules was measured to validate the obtained data. New insights into the physiology of M. thermophila can serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.(VLID)3381161Version of recor

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.Video Article(VLID)4466098Version of recor