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Development of an Osteoblast Cell Culture System for Functional TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Experimentelle Genetik Development of an osteoblast cell culture system for functional characterization and comparative analyses of mouse models with bone phenotypes and systemic investigation of an Osteogenesis imperfecta disease model Frank Thiele Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. A. Gierl Prüfer der Dissertation: 1. Univ.-Prof. Dr. M. Hrabé de Angelis 2. apl. Prof. Dr. J. Adamski Die Dissertation wurde am 24.07.2009 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 26.10.2009 angenommen. Für Käte und Erich, Gudrun, Peter, Monique und Alexander I. Table of contents I. TABLE OF CONTENTS............................................................................................ I II. FIGURES AND TABLES ....................................................................................... IV III. ABBREVIATION LIST........................................................................................... VI IV. PUBLICATIONS................................................................................................... IX V. CURRICULUM VITAE........................................................................................... XI VI. AFFIRMATION.................................................................................................... XII VII. ACKNOWLEDGMENT ...................................................................................... XIII 1. INTRODUCTION .................................................................................................... 1 1.1. Bone function ................................................................................................................................. 1 1.2. Classification of bones .................................................................................................................. 1 1.3. Composition and structure of bone ............................................................................................. 3 1.4. Bone cells........................................................................................................................................ 5 1.5. ECM and mineralization of bone................................................................................................... 8 1.6. Development and formation of bone..........................................................................................10 1.7. Bone modeling and homeostasis ............................................................................................... 13 1.8. Bone diseases .............................................................................................................................. 15 1.9. Osteogenesis imperfecta – the “brittle bone disease”............................................................. 17 1.10. Mouse models for human diseases.......................................................................................... 19 1.11. Mouse models for skeletal disorders.......................................................................................20 1.12. Aga2 - a mouse model for Osteogenesis imperfecta ............................................................. 21 1.13. The German Mouse Clinic ......................................................................................................... 21 1.14. In vitro cell culture...................................................................................................................... 22 1.15. Goal.............................................................................................................................................. 24 I. In vitro analysis of osteoblasts................................................................................................... 24 II. Heart and lung investigation in the Aga2 OI mouse model .................................................... 24 - I - 2. MATERIAL AND METHODS ............................................................................... 25 2.1. In vitro analysis of osteoblasts................................................................................................... 25 2.1.1. General remarks.................................................................................................................... 25 2.1.1.1. Workflow......................................................................................................................... 25 2.1.1.2. Scheme of analysis, mating of mice and genotyping................................................ 25 2.1.1.3. Measurement days and assay analysis....................................................................... 27 2.1.1.4. Cell preparation and cultivation................................................................................... 28 2.1.1.5. Important considerations.............................................................................................. 30 2.1.1.6. Statistical analysis......................................................................................................... 31 2.1.2. Material and methods ........................................................................................................... 32 2.1.2.1. Cell preparation and cultivation................................................................................... 32 2.1.2.2. Proliferation / Metabolic activity / Protein content / ALP activity (A1) ..................... 34 2.1.2.3. Collagen secretion / Collagen deposition (A2)........................................................... 40 2.1.2.4. Matrix mineralization (A3)............................................................................................. 45 2.1.2.5. Nodule quantification (A4) ............................................................................................ 48 2.1.2.6. Gene expression (A5).................................................................................................... 50 2.2. Heart and lung investigation in the Aga2 OI mouse model ..................................................... 55 2.2.1. Animal keeping and handling .............................................................................................. 55 2.2.2. Genotyping ............................................................................................................................ 55 2.2.3. Cardiovascular phenotyping ............................................................................................... 55 2.2.4. pO2 measurement.................................................................................................................. 57 2.2.5. Histology and SEM................................................................................................................ 57 2.2.6. In vitro cell culture and TEM ................................................................................................ 59 2.2.7. RNA Isolation......................................................................................................................... 61 2.2.8. Expression profiling ............................................................................................................. 62 2.2.9. qRT-PCR................................................................................................................................. 64 2.2.10. Statistical analysis .............................................................................................................. 65 3. RESULTS............................................................................................................. 66 3.1. In vitro analysis of osteoblasts................................................................................................... 66 3.1.1. Establishment of the cell culture system ........................................................................... 66 3.1.1.1. Growth and differentiation of the osteoblasts............................................................ 66 3.1.1.2. Proliferation / Metabolic activity / Protein content / ALP activity (A1) ..................... 68 3.1.1.3. Collagen secretion / Collagen deposition (A2)........................................................... 72 3.1.1.4. Matrix mineralization (A3)............................................................................................. 73 3.1.1.5. Nodule quantification (A4) ............................................................................................ 73 3.1.1.6. Gene expression (A5).................................................................................................... 74 3.1.2. Validation of the cell culture system................................................................................... 75 3.1.2.1. Selection of suitable mouse mutants with bone phenotype..................................... 75 3.1.2.2. Analysis of Aga2 and ABE2 within the cell culture system ...................................... 78 - II - 3.2. Heart and lung investigation in the Aga2 OI mouse model ..................................................... 82 3.2.1. Phenotypic classification of heterozygous Aga2 .............................................................. 82 3.2.2. Cardiovascular phenotyping ..............................................................................................
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