University of Wurzburg 18 articles published in JoVE Bioengineering Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts Ana Luísa Graça1, Niklas Kroner-Weigl2, Viviana Reyes Alcaraz1, Sigrid Müller-Deubert1, Maximilian Rudert3, Denitsa Docheva1 1Department of Musculoskeletal Tissue Regeneration, Orthopaedic Hospital König-Ludwig-Haus, University of Würzburg, 2Laboratory for Experimental Trauma Surgery, Department of Trauma Surgery, University Hospital Regensburg, 3Department of Orthopaedics, Orthopaedic Hospital König-Ludwig-Haus, University of Würzburg Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering. Medicine The Transition to an Anterior-Based Muscle Sparing Approach Improves Early Postoperative Function but is Associated with a Learning Curve Alexander J. Nedopil1,2 1Department of Orthopaedic Surgery, König-Ludwig-Haus, University of Würzburg, 2Adventist Health Lodi Memorial The anterior-based muscle-sparing approach for total hip arthroplasty (THA) is associated with a learning curve. However, the improved clinical outcome in the early post-operative phase makes the consideration to transition worthwhile. Bioengineering Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates Mahnaz Sabeti Azad*1, Angelo Cardoso Batista*1, Jean-Loup Faulon1, Chase L. Beisel2,3, Jerome Bonnet4, Manish Kushwaha1 1INRAe, AgroParisTech, Micalis Institute, Université Paris-Saclay, 2Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 3Medical Faculty, University of Würzburg, 4Centre de Biochimie Structurale, INSERM U1054, CNRS UMR 5048, University of Montpellier Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (ΔrecBCD and ΔrecB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates. Engineering Evaluating Usability Aspects of a Mixed Reality Solution for Immersive Analytics in Industry 4.0 Scenarios Burkhard Hoppenstedt1, Thomas Probst2, Manfred Reichert1, Winfried Schlee3, Klaus Kammerer1, Myra Spiliopoulou4, Johannes Schobel1, Michael Winter1, Anna Felnhofer5, Oswald D. Kothgassner6, Rüdiger Pryss7 1Institute of Databases and Information Systems, Ulm University, 2Department for Psychotherapy and Biopsychosocial Health, Danube University Krems, 3Department of Psychiatry and Psychotherapy, University of Regensburg, 4Faculty of Computer Science, Otto von Guericke University Magdeburg, 5Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, 6Department of Child and Adolescent Psychiatry, Medical University of Vienna, 7Institute of Clinical Epidemiology and Biometry, University of Würzburg This protocol delineates the technical setting of a developed mixed reality application that is used for immersive analytics. Based on this, measures are presented, which were used in a study to gain insights into usability aspects of the developed technical solution. Immunology and Infection Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Leo M. Endres1, Sarah F. Hathcock2, Alexandra Schubert-Unkmeir1, Brandon J. Kim1,2 1Institute for Hygiene and Microbiology, University of Würzburg, 2Department of Biological Sciences, University of Alabama The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses. Medicine Cardiac Magnetic Resonance Imaging at 7 Tesla Daniel Stäb1,2, Aiman Al Najjar1, Kieran O'Brien1,3, Wendy Strugnell4, Jonathan Richer3, Jan Rieger5, Thoralf Niendorf5, Markus Barth1 1The Centre for Advanced Imaging, The University of Queensland, Brisbane, Australia, 2Department of Diagnostic and Interventional Radiology, University Clinic Würzburg, Würzburg, Germany, 3Siemens Healthcare Pty Ltd, Brisbane, Australia, 4Richard Slaughter Centre of Excellence in CVMRI, The Prince Charles Hospital, Brisbane, Australia, 5MRI.Tools GmbH, Berlin, Germany The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept. Bioengineering Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry Claudia Siverino1, Barbara Tabisz2, Tessa Lühmann3, Lorenz Meinel3, Thomas Müller4, Heike Walles1,2, Joachim Nickel1,2 1Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik (IGB), Translationszentrum Würzburg 'Regenerative Therapien für Krebs- und Muskuloskelettale Erkrankung', Institutsteil Würzburg, 2Lehrstuhl für Tissue Engineering und Regenerative Medizin, Universitätsklinikum Würzburg, 3Lehrstuhl für Pharmazeutische Technologie und Biopharmazie, Universität Würzburg, 4Lehrstuhl für molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs Institut für Biowissenschaften, Universität Würzburg Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities. Medicine Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment Kerstin Hadamek1,2, Angelika Keller1,2, Antje Gohla1,2 1Institute of Pharmacology and Toxicology, University of Würzburg, 2Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized α4β1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points. Bioengineering Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications Felix M. Wunner1, Onur Bas1, Navid T. Saidy1, Paul D. Dalton2, Elena M. De-Juan Pardo1, Dietmar W. Hutmacher1,3,4 1ARC ITTC in Additive Biomanufacturing, Institute for Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), 2Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, 3Institute for Advanced Study, Technical University of Munich (TUM), 4George W Woodruff School of Mechanical Engineering, Georgia Institute of Technology This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures. Medicine Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video Zuzanna Rowinska1,2, Simone Gorressen3, Marc W. Merx4, Thomas A. Koeppel5, Alma Zernecke6, Elisa A. Liehn2 1Department of Vascular Surgery and Interdisciplinary Vein Center, St. Josef-Hospital, Ruhr- University Bochum, 2Institute of Molecular Cardiovascular Research, University Hospital, RWTH Aachen University Department of Medicine, 3Institute of Pharmacology and Clinical Pharmacology, Düsseldorf University Hospital, 4Department of Cardiology, Vascular Medicine and Intensive Care Medicine, Hospital Robert Koch Gehrden, 5Division of Vascular Surgery, Asklepios Klinik St. Georg, 6Institute of Experimental Biomedicine, Würzburg University We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease. Medicine In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging Angelika Hoffmann1,2, Xavier Helluy2,3, Manuel Fischer2, Ann-Kristin Mueller4,5, Sabine Heiland2, Mirko Pham1,6, Martin Bendszus1, Johannes Pfeil4,5,7 1Department of Neuroradiology, Heidelberg University Hospital, 2Division of Experimental Radiology, Department of Neuroradiology, Heidelberg University Hospital, 3NeuroImaging Centre Research, Department of Neuroscience, Ruhr-University Bochum, 4Centre for Infectious Diseases, Parasitology Unit, Heidelberg University Hospital, 5German Centre for Infection Research (DZIF), 6Department of Neuroradiology, University of Würzburg, 7Center for Childhood and Adolescent Medicine, General Pediatrics, Heidelberg University Hospital We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging. Genetics Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair Jonas Gross*1, Nicolas Wirth*1, Ingrid Tessmer1 1Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described. Neuroscience Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells Sandra Meyer1,2, Philipp Wörsdörfer1, Katharina Günther1, Marc Thier2,3, Frank Edenhofer1,2 1Institute of Anatomy and Cell Biology, University of Würzburg, 2Institute of Reconstructive Neurobiology, University of Bonn, 3German Cancer Research Center, Heidelberg Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages. Environment A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements Vicenta Salvador-Recatalà1, W. Freddy Tjallingii2 1Department of Plant Physiology and Biophysics, University of Würzburg, 2EPG Systems, Wageningen, The Netherlands Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants. Bioengineering High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy Titiwat Sungkaworn1, Finn Rieken1, Martin J. Lohse1, Davide Calebiro1 1Institute of Pharmacology and Toxicology and Bio-Imaging Center/Rudolf Virchow Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Germany This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins. Neuroscience Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees Martin Fritz Brill1, Maren Reuter1, Wolfgang Rössler1, Martin Fritz Strube-Bloss1 1Department of Behavioral Physiology and Sociobiology (Zoology II) Biozentrum, University of Würzburg Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal. Medicine Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier Ellaine Salvador1, Winfried Neuhaus1,2, Carola Foerster1 1Klinik und Poliklinik für Anästhesiologie, Zentrum für operative Medizin der Universität Würzburg, 2Department of Medicinal Chemistry, Faculty of Life Sciences, University of Vienna In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro TBI model. Bioengineering Tissue Engineering of a Human 3D in vitro Tumor Test System Corinna Moll*1, Jenny Reboredo*1, Thomas Schwarz1, Antje Appelt1, Sebastian Schürlein1, Heike Walles1, Sarah Nietzer1 1Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.
