A pulsatile bioreactor system for durability testing and compliance estimation of vascular grafts [Elektronische Ressource] / Stefanos-Evangelos Diamantouros

Veligekas

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 30 Mo

Extrait

A Pulsatile Bioreactor System for
Durability Testing and Compliance Estimation
of Vascular Gr afts

Von der Medizinischen Fakultät der Rheinisch-Wescsthefänli
Technischen Hochschule Aachen zur Erlangung deiscs haekan dGeramdes
eines Doktors der Theoretischen Medizin geneihssemritgatetio nD

vorgelegt von
Stefanos-Evangelos Diamantouros
aus
Athen (Griechenland)


Berichter: Herr Universitätsprofessor
Dr.med. Stefan Jockenhövel

Herr Privatdozent
Dr.med. Jörg S. Sachweh

Tag der mündlichen Prüfung: 20. Juli 2011

Diese Dissertation ist auf den Internetseiten dheurlb ibHloiocthesck
online verfügbar. Table of Conten ts






ii
Table of Contents

Table of Contents iii
Abbreviations List viii
1. Introduction 1
1.1 General 1
1.2 Tissue Engineering 3
1.3 Cardiovascular Disease and Treatment Methods 8
1.4 Cardiovascular Tissue Engineering 10
1.5 Bioreactor Systems for Vascular Tissue Engine12e ring
1.6 Compliance of Vascular Grafts 16
1.7 Durability Testing of Vascular Grafts 19
1.8 Aim of the Study 21
2. Development 22
2.1 Bioreactor System Development 22
2.1.1 Overview 2 2
2.1.2 Pressure 23
2.1.2.1 Pressure Calculations 23
2.1.2.2 Circulator Pump 26
2.1.2.3 Pulsatile Pressure Chamber and
Frames 28
2.1.2.4 Pressure Sensors 34Table of Conten ts

2.1.3 Vascular Graft Chamber 35
2.1.3.1 Vascular Graft Chamber #1 35
2.1.3.2 Vascular Graft Chamber #2 37
2.1.3.3 Vascular Graft Chamber #3 37
2.1.3.4 Compliance Measurement Frame 39
2.1.5 Medium Reservoir 4 5
2.1.6 Flow Sensor 4 6
2.1.7 Tubing, Connectors and Consumables 47
2.1.8 Bioreactor System Assembly 48
2.1.9 Medium Change 54
2.2 Monitoring and Control 56
2.2.1 Overview 5 6
2.2.2 Pressure Monitoring 60
2.2.3 Blood Gas Monitoring 6 1
2.2.4 Flow Sensor Monitoring 6 2
2.2.5 Diameter Measurement 64
2.2.6 Actuator Control 6 4
2.2.7 Automatically Controlled Valve 68
2.3 Mechanical Testing 71
2.3.1 Burst Strength Chamber and Measurement 71
2.3.2 Tensile Strength Testing 73
2.3.3 Suture Retention Strength Testing 73
2.3.4 Compliance Measurement 74
3. Materials and Methods 77
3.1 Cell Culture 77
3.1.1 Cell Isolation 77
3.1.2 Medium Change 7 8
3.1.3 Cell Passaging 79

iv
Table of Conten ts

3.1.4 Cell Harvesting 80
3.2 Tissue Engineering of Vascular Grafts 81
3.2.1 Overview 8 1
3.2.2 Preparation of Fibrinogen 82
3.2.3 Preparation of Fibrin Gel 83
3.2.4 Moulding and Extraction 84
3.2.5 Preparation of Porcine Carotid Arteries 86
3.3 Analytical Methods 87
3.3.1 Fixation and Paraffin Embedding of Tissue 87
3.3.2 Haematoxylin – Eosin Staining 89
3.3.3 Trichrome Staining 90
3.3.4 Immunohistochemistry 90
3.4 Evaluation of the Bioreactor System 92
3.4.1 Cultivation of Blood Vessels in the Bioreac tor 92
3.4.2 Measurement of the Diameter and Estimation of
Compliance 95
3.4.3 Testing of Vascular Grafts 96
4. Results 100
4.1 Assembly of the Bioreactor System and Cultfiv ation o
Vascular Grafts 100
4.2 Monitoring of Blood Gases 1 04
4.3 Pressure Pulse Profiles Creation 108
4.4 Compliance Measurement and Mechanical Testin g 111
4.4.1 Static and Dynamic Measurement of the
Compliance 111
4.4.2 Mechanical Testing of Equine Carotid Arter ies 113

v
Table of Conten ts

5. Discussion 117
5.1 Pressure Pulse Profile Creation 117
5.2 Monitoring of Blood Gases 1 19
5.4 Compliance Measurement and Mechanical Testin g 128
5.4.1 Static and Dynamic Measurement of the
Compliance 128
5.4.2 Mechanical Testing of Equine Carotid Arter ies 132
5.5 Conclusions 134
6. Summary 135
7. Appendix 137
7.1 Protocols 137
7.1.1 Bioreactor Medium 1 37
7.1.2 Phosphate Buffered Saline (PBS) 137
7.1.3 Tris Buffered Saline (TBS) 138
7.1.4 Transport Buffer 1 38
7.1.5 Carnoy’s Solution 139
7.1.6 Haematoxylin – Eosin Staining 139
7.1.7 Gomori Trichrome Staining 1 40
7.1.8 Immunehistochemical Staining 141
7.2 Bioreactor Part Drawings 143
7.2.1 Burst Strength Chamber 1 43
7.2.2 Vascular Graft Chamber Frame 146
7.2.3 Vascular Graft Chamber and Connector 149
7.2.4 Medium Reservoir 15 3
7.2.5 Pulse Chamber 1 55
7.2.6 Pulse Chamber Frame 157

vi
Table of Conten ts

7.2.7 Blood Gas Sensors Chamber and Frame 163
7.3 Error Analysis 172
References 174
List of Figures 189
List of Tables 193
Publications List 194
Acknowledgements 196
Data Storage Declaration - Erklärung zur Datenauwfabherung 199
Curriculum Vitae 200


vii
Abbreviations List


CABG coronary artery bypass pCO partial pressure of carbon 2
graft surgery dioxide
CC circumferential PEEK polyether ether ketone
compliance (thermoplastic, used for
bioreactor parts) DAQ data acquisition system
pH the negative logarithm of DAPI 4’,6-diamidino-2-
the molar concentration phenylindole (fluorescent
of hydrogen ions (H+) stain)
PMMA poly (methyl DES drug eluting stent
methacrylate)
DNA deoxyribonucleic acid
(transparent
ECM extracellular matrix thermoplastic, used for
bioreactor parts) FCS foetal calf serum
pO partial pressure of 2FDA United States Food and
oxygen Drug Administration
POM polyoxymethylene FITC fluorescein
(thermoplastic, used for isothiocyanate (reporter
bioreactor parts) molecule in
immunohistochemistry) PVDF polyvinylidene fluoride
(thermoplastic, used for HE Haematoxylin – Eosin
TE-constructs mesh) (histological staining)
RNA ribonucleic acid ISO International
Organization for SMA smooth muscle actin
Standardization
T-125 type of cell culture flask
PBS phosphate-buffered with a cell culture area of
saline (isotonic buffer) 125 cm2 Abbreviations List

T-75 type of cell culture flask Medical Engineering of
with a cell culture area of the RWTH Aachen
75 cm2 University
TBS tris-buffered-saline TRITC tetramethyl-rhodamine
(isotonic buffer) isothiocyanate (reporter
molecule in TE tissue engineering, tissue
immunohistochemistry) engineered
VGC vascular graft chamber TEB Department of Tissue
Engineering and VISA Virtual Instrument
Biomaterials of the Software Architecture (NI
Institute for Applied LabView term)

ix