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Angiogenic Potential of Mesenchymal Cells and T Lymphocytes Induced by Mechanical Stimuli That Improve Bone Healing – an in Vitro 2D and 3D Bioreactor Study

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Angiogenic Potential of Mesenchymal Cells and T Lymphocytes Induced by Mechanical Stimuli That Improve Bone Healing – an in Vitro 2D and 3D Bioreactor Study Angiogenic Potential of Mesenchymal Cells and T Lymphocytes Induced by Mechanical Stimuli that Improve Bone Healing – An In Vitro 2D and 3D Bioreactor Study vorgelegt von Diplom-Ingenieurin (FH), Master of Science Friederike H. Bieler, geb. Bleckwehl aus Bremen Von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des wissenschaftlichen Grades Doktorin der Ingenieurwissenschaften Dr.-Ing. genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr.-Ing. Frank-Jürgen Methner Berichter: Prof. Dr. rer. nat. Roland Lauster Berichter: Prof. Dr.-Ing. Georg N. Duda Berichter: Prof. Dr. rer. nat. Leif-Alexander Garbe Tag der wissenschaftlichen Aussprache: 14. Dezember 2010 Berlin 2011 D83 Acknowledgements First of all, I would like to thank Prof. Dr. Georg N. Duda. He was not only a great supervisor supporting and guiding the work my thesis is based on, but he also enabled my research at the Julius Wolff Institute (JWI), Charité – Universitätsmedizin Berlin. I am glad that I got the opportunity to hand in my thesis at the TU Berlin. This would not have been possible without Prof. Dr. Roland Lauster and the other appointed referees. Especially Dr. Mark S. Thompson deserves my gratitude. He initiated my PhD project at the JWI. Although Mark moved on to Oxford University, already at the beginning of my PhD project, he was always present with great advice, support and ideas that helped a lot to proceed. Some of my gratitude is related to the great time at the JWI, which I do not want to miss, and some to the honest support I experienced in the Mechanobiology and the Cell Therapy team. Here I would also like to thank their team leaders, especially Dr. Grit Kasper, who initiated my bioreactor study. I highly appreciate the advice I received, especially regarding newly learned lab-techniques, from Sven Geissler and the experi-mental work performed together with Aline Groothuis, Florian Witt, and Annett Kurtz. I would further like to thank my intern Lauren Ehardt and all technical assistants at the JWI. They were a great help in the laboratory. Additionally, the clinicians and veterinaries who prepared the bone material for mesenchymal cell isolations deserve a big thank you, as well as the MTL at our institute for building the microscope x-y table adaptor. Also, all collaborators, especially from the Institute of Medical Genetics (Charité) and the MPI for Molecular Genetics, should be mentioned. Dr. J. Adjaye and Dr. A. Prigione from the latter enabled and greatly supported my gene expression analysis. In general, I would like to tell the people who reviewed the thesis at hand how much I appreciate their advice and opinion. Their thoughts helped to make this thesis what it is. The encouragement I received from my family and friends helped me a lot in finalizing this work. Without my parents I would not have been able to achieve this. They always supported my goals and enabled my good education. But my deepest gratitude goes to my patient husband Sven Bieler. He had to suffer a lot during the past few years from my moods and from my ambition and still, he always supported me, not only personally but also scientifically. Finally, this study was financially supported by a grant partially from the German Research Foundation (DFG SFB 760, Berlin), partially from the Berlin-Brandenburg Center for Regenerative Therapies (BCRT), and, to a large extent, from the AO Foundation. 2 Content ACKNOWLEDGEMENTS.................................................................................................................................. 2 ABSTRACT........................................................................................................................................................... 5 ZUSAMMENFASSUNG ...................................................................................................................................... 6 1 INTRODUCTION ....................................................................................................................................... 8 1.1 BACKGROUND ...................................................................................................................................... 8 1.2 MOTIVATION ........................................................................................................................................ 9 1.3 GOALS AND OUTLINE OF THESIS ............................................................................................................ 9 1.4 HYPOTHESIS ....................................................................................................................................... 10 1.5 CURRENT KNOWLEDGE : BONE HEALING AND ANGIOGENESIS – CELLS , PROTEINS , AND MECHANICS ... 10 1.5.1 Angiogenesis – involvement of plasminogen activation system and matrix metalloproteases and other proteins .............................................................................................................................................. 10 1.5.2 The course of bone healing and the proteins involved .................................................................. 12 1.5.3 Loading environment occurring during fracture healing.............................................................. 14 1.5.4 Mechanical stimulation of cells..................................................................................................... 16 1.5.4.1 Devices and different kinds of stimuli................................................................................................. 16 1.5.4.2 Mechanotransduction .......................................................................................................................... 18 2 MATERIALS AND METHODS.............................................................................................................. 22 2.1 2D BIOREACTOR STUDY ...................................................................................................................... 22 2.1.1 Materials ....................................................................................................................................... 23 2.1.2 Flexercell characterisation ........................................................................................................... 24 2.1.2.1 Characterisation of applied strains using DIC ..................................................................................... 24 2.1.2.2 Determination of strain transferred to mesenchymal cells................................................................... 25 2.1.2.3 Characterisation of fluid mechanics .................................................................................................... 26 2.1.3 Influence of characterised mechanical environment on mesenchymal cells ................................. 27 2.1.3.1 Cell culture of osteoprogenitor/osteoblast like cells and mechanical stimulation................................ 27 2.1.3.2 Immunohistology................................................................................................................................. 28 2.1.3.3 ELISA ................................................................................................................................................. 29 2.2 3D BIOREACTOR STUDY ...................................................................................................................... 30 2.2.1 Materials ....................................................................................................................................... 32 2.2.2 MSCs’ isolation, culture, and characterisation ............................................................................ 34 2.2.3 T lymphocytes’ isolation, culture, and characterisation ............................................................... 35 2.2.4 Bioreactor experiment – generation of conditioned media and determination of cell viability after mechanical stimulation................................................................................................................................ 36 2.2.5 2D tube formation ......................................................................................................................... 38 2.2.6 ELISAs........................................................................................................................................... 38 2.2.7 Lactate and Glucose level ............................................................................................................. 39 2.2.8 Immunohistology........................................................................................................................... 39 2.2.9 Gene expression array .................................................................................................................. 40 2.2.10 Statistical analysis.................................................................................................................... 42 3 RESULTS................................................................................................................................................... 43 3.1 MECHANICAL STIMULI IN BONE HEALING – 2D IN VITRO APPLICATION ................................................ 43 3.1.1 Flexercell characterisation - Results ............................................................................................ 43 3.1.1.1 Characteristics of applied strains......................................................................................................... 43 3.1.1.2
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