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 [Elektronische Ressource] / Frank Thiele

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. pO measurement.................................................................................................................. 57 2
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 .........................