Differentiation and extracellular matrix interactions of chondrocytes in response to signaling molecules and biomaterials for cartilage repair = Differenzierung und Interaktionen von Chondrozyten mit der extrazellulären Matrix als Reaktion auf Signalmoleküle und Biomaterialien für die Wiederinstandsetzung von Knorpel [[Elektronische Ressource]] / Natalia Sánchez-Fernández

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Sujets

• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2012
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 12 Mo

Extrait

Differentiation and extracellular matrix interactions of
chondrocytes in response to signaling molecules and
biomaterials for cartilage repair


Differenzierung und Interaktionen von Chondrozyten mit
der extrazellulären Matrix als Reaktion auf
Signalmoleküle und Biomaterialen für die
Wiederinstandsetzung von Knorpel






Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaften
Am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz



Natalia Sánchez-Fernández
geb. am 21.02.1977 in Sevilla
Mainz, Juli 2011


























Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündliche Prüfung: i

Table of Contents

Table of Contents ........................................................................................................................ i
Acknowledgements ................................................................................................................... vi
Abbreviations .......................................................................................................................... viii
Summary ................................................................................................................................. xiv
1. Introduction ......................................................................................................................... 1
1.1. Cartilage ..................................................................................................................... 1
1.1.1 Cartilage Embryology ................................................................................................ 2
1.1.2. Cartilage Biochemistry and Morphology .................................................................. 3
1.2.Chondrocyte ...................................................................................................................... 5
1.2.1 Chondrogenesis .......................................................................................................... 5
1.2.1.1. Mesenchymal Stem Cell Condensation and Determination of
Chondroprogenitors ........................................................................................................ 5
1.2.1.2. Chondrocyte Differentiation ............................................................................. 6
1.2.1.3. Chondrocyte Proliferation and Hypertrophy ..................................................... 7
1.2.2. Morphology and Functions ....................................................................................... 9
1.3. Cartilage Diseases .......................................................................................................... 10
1.4. Cartilage Injuries .... 13
1.5. Treatment of Cartilage Injuries ...................................................................................... 16
1.5.1. Total Knee Arthoplasty ........................................................................................... 16
1.5.2. Repair Strategies ..................................................................................................... 17
1.5.3. Tissue Engineering .................................................................................................. 20
1.5.3.1. Scaffolds .......................................................................................................... 21
1.5.3.2. Cells ................................................................................................................. 23
1.5.3.3. Signaling Molecules ........................................................................................ 24
1.5.3.4. Future Perspectives ......................................................................................... 27
1.6. Aim of the Study ............................................................................................................ 29
2. Materials and Methods ......................................................................................................... 30
2.1. Materials ........................................................................................................................ 30
2.1.1. Stains ....................................................................................................................... 30
2.1.2. Buffers and Solutions .............................................................................................. 30
2.1.3. Media ...................................................................................................................... 31
2.1.4. Instruments .............................................................................................................. 32 ii

2.1.5. Consumables ........................................................................................................... 34
2.1.6. Kits .......................................................................................................................... 35
2.1.7. Antibodies ............................................................................................................... 35
2.1.8. Primers .................................................................................................................... 35
2.1.8.1. RT-PCR Primers ............................................................................................. 35
2.1.8.2. Quantitative real-time RT-PCR ....................................................................... 36
2.1.9. Vectors .... 37
2.1.10 Competent Cells ..................................................................................................... 37
2.1.11. Restriction Enzymes 37
2.1.12. Biomaterials .......................................................................................................... 37
2.1.12.1. Gelatin Sponges ............................................................................................. 37
2.1.12.2. Collagen type I Hydrogels ............................................................................. 38
2.1.13. Softwares ............................................................................................................... 38
2.2. Methods ......................................................................................................................... 39
2.2.1. Primary Human Articular Chondrocyte Isolation and Expansion .......................... 39
2.2.2. Analysis of Morphology ......................................................................................... 39
2.2.2.1. Histology ......................................................................................................... 39
2.2.2.2 Calcein-AM Live Staining ............................................................................... 40
2.2.3. Cytotoxicity Assays ................................................................................................ 41
2.2.3.1. MTS Conversion Assay .................................................................................. 41
2.2.3.2. Crystal violet Staining ..................................................................................... 41
2.2.4. Analysis of Gene Expression 41
2.2.4.1. RNA Isolation ................................................................................................. 41
2.2.4.1.1. Isolation with TriZol ................................................................................ 42
2.2.4.1.2. Isolation with RNeasy Micro Kit ............................................................. 42
2.2.4.2. Reverse Transcription 43
2.2.4.3. RT-PCR ........................................................................................................... 43
2.2.4.4 Agarose Gel Electrophoresis ............................................................................ 44
2.2.4.5 Quantitative RT-PCR ....................................................................................... 44
2.2.5. Analysis of Protein Expression ............................................................................... 45
2.2.5.1. TGF- 1 ELISA ............................................................................................... 45
2.2.6. Statistical Analysis .................................................................................................. 45
3. Human Articular Chondrocyte Characterization .................................................................. 46
3.1. Experimental Design and Execution ............................................................................. 46
3.1.1. Cell Culture ............................................................................................................. 46
3.1.2. Morphological Analysis .......................................................................................... 46
iii

3.1.3. Gene Expression Analysis ...................................................................................... 46
3.2. Results ........................................................................................................................... 47
3.2.1. HACs De-differentitiation Process ......................................................................... 47
3.2.2