Chondrogenic differentiation of human mesenchymal stem cells and articular cartilage reconstruction [Elektronische Ressource] / vorgelegt von Andrea Heymer

Andrea Heymer

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 13 Mo

Extrait

Chondrogenic Differentiation
of Human Mesenchymal Stem Cells
and Articular Cartilage Reconstruction
Dissertation zur Erlangung des naturwissenschaftlichen
Doktorgrades der Julius-Maximilians-Universität Würzburg
vorgelegt von
Andrea Heymer
aus Königs Wusterhausen
Würzburg, 2008

Eingereicht am: a
Mitglieder der Promotionskommission:
Vorsitzender: a
Gutachter: PD Dr. med. Ulrich Nötha
Gutachter: Prof. Dr. rer. nat. Roland Benza
Tag des Promotionskolloquiums: a
Doktorurkunde ausgehändigt am: a

Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbstständig
angefertigt und keine anderen als die von mir angegebenen Hilfsmittel und Quellen
verwendet habe. Des Weiteren erkläre ich, dass diese Arbeit weder in gleicher noch in
ähnlicher Form in einem Prüfungsverfahren vorgelegen hat und ich noch keinen
Promotionsversuch unternommen habe.
Oberstenfeld, 17. Juli 2008
Andrea Heymer
Table of contents I
Table of contents

1 Summary .......................................................................................................................... 1
1.1 Summary ...................................................................................................................... 1
1.2 Zusammenfassung ....................................................................................................... 2
2 Introduction ...................................................................................................................... 5
2.1 Structure and composition of articular cartilage ............................................................ 5
2.2 Articular cartilage damage and repair ........................................................................... 7
2.3 Mesenchymal stem cells .............................................................................................. 8
2.3.1 Definition of mesenchymal stem cells .................................................................... 8
2.3.2 Chondrogenesis .................................................................................................... 9
2.4 Scaffolds for cartilage tissue engineering ....................................................................10
2.5 Bioreactor cultivation for cartilaginous tissue growth ...................................................12
2.6 Cellular imaging ..........................................................................................................14
2.6.1 Use of superparamagnetic iron oxide particles for MR imaging ............................14
2.6.2 Labeling of cells with very small superparamagnetic iron oxide particles (VSOPs)
for cellular MR imaging .........................................................................................15
2.7 Objective of the study ..................................................................................................16
3 Materials and methods .................................................................................................. 18
3.1 Materials .....................................................................................................................18
3.1.1 Consumables .......................................................................................................18
3.1.2 Chemicals and reagents .......................................................................................18
3.1.3 Cell culture media and additives ...........................................................................20
3.1.4 Components for the fabrication of collagen hydrogels ..........................................21
3.1.5 Antibodies ............................................................................................................21
3.1.6 Enzymes ..............................................................................................................22
3.1.7 Primers .................................................................................................................22
3.1.8 Buffers and other solutions ...................................................................................23
3.1.9 Kits .......................................................................................................................25
3.1.10 Equipment ............................................................................................................25
3.1.11 Software and online sources ................................................................................26
3.2 Methods ......................................................................................................................27
3.2.1 Isolation and culture of human mesenchymal stem cells (hMSCs) .......................27
3.2.2 Phenotypic characterization of hMSCs .................................................................27
3.2.3 Labeling of hMSCs with very small superparamagnetic iron oxide particles
(VSOPs) ...............................................................................................................28
3.2.3.1 Labeling protocol..............................................................................................28 Table of contents II
3.2.3.2 Detection of iron oxide particles within VSOP-labeled hMSCs .........................28
3.2.3.3 Cellular viability and apoptosis .........................................................................29
3.2.3.4 Proliferation assays ..........................................................................................30
3.2.3.5 Differentiation of hMSCs ..................................................................................30
3.2.4 Fabrication and culture of hMSC collagen hydrogels ............................................31
3.2.4.1 Fabrication of hMSC collagen hydrogels ..........................................................31
3.2.4.2 Cell proliferation in hMSC collagen hydrogels ..................................................32
3.2.4.3 Chondrogenic differentiation of hMSCs in collagen hydrogels ..........................32
3.2.5 Bioreactor cultivation of hMSC collagen hydrogels ...............................................32
3.2.6 High-field MR imaging ..........................................................................................34
3.2.7 Histology and immunohistochemistry ...................................................................34
3.2.7.1 Oil red O staining .............................................................................................34
3.2.7.2 Alizarin red S staining ......................................................................................35
3.2.7.3 Hemalaun&Eosin (H&E) staining .....................................................................35
3.2.7.4 Alcian blue staining ..........................................................................................35
3.2.7.5 Immunohistochemical staining for collagen type II ...........................................35
3.2.8 Gene expression analyses ...................................................................................35
3.2.8.1 Total RNA isolation ..........................................................................................35
3.2.8.2 cDNA synthesis ...............................................................................................36
3.2.8.3 Reverse transcriptase polymerase chain reaction (RT-PCR) ...........................36
3.2.8.4 Agarose gel electrophoresis .............................................................................37
4 Results ........................................................................................................................... 38
4.1 Characterization of hMSCs ..........................................................................................38
4.2 Fabrication and culture of hMSC collagen hydrogels ...................................................40
4.2.1 Contraction of hMSC collagen hydrogels ..............................................................40
4.2.2 Cell proliferation in hMSC collagen hydrogels .......................................................42
4.2.3 Chondrogenic differentiation of hMSCs in collagen hydrogels ..............................47
4.3 Mechanical stimulation of hMSC collagen hydrogels ...................................................56
4.3.1 Cellular behavior of hMSCs in collagen hydrogels during bioreactor cultivation ....56
4.3.2 Influence of mechanical stimulation on the gene expression of hMSCs in collagen
hydrogels ..............................................................................................................59
4.4 Labeling of hMSCs with VSOPs for cellular MR imaging .............................................61
4.4.1 Detection of iron oxide particles within VSOP-labeled hMSCs ..............................61
4.4.2 Inf