Stimulation of vascular cells by extracellular signals [Elektronische Ressource] : a biophysical analysis / put forward by Sarah A. Biela

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• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 18
• Langue: Deutsch
• Poids de l'ouvrage: 14 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
Put forward by
Master of Science: Sarah A. Biela
Born in: Bonn, Germany
Oral examination: July 8th, 2009Stimulation of vascular cells
by extracellullar signals
- A biophysical analysis
Referees: Prof. Dr. Joachim P. Spatz
Prof. Dr. Rainer H. A. FinkStimulierung von Gef¨aßzellen durch extrazellul¨are Signale
Die Behandlung von Herz-Kreislauf-Erkrankungen erfordert haufig die selektive Stimulie-¨
rungvonEndothel-(ECs)undglattenMuskelzellen(SMCs).DiezweiGefa¨ßzelltypensind
wichtigfurdieEinheilungvonsog.Stents.DiemoderneForschungbefasstsichmitderEnt-¨
wicklung neuer Materialien und Beschichtung fu¨r Stents, um den komplexen Einheilungs-
prozess zu verbessern. Das Ziel meiner Arbeit war es, durch Oberfla¨chen-Chemie, -Topo-
graphie und andere stimulierende Faktoren wie elektrische Felder oder mechanische elas-
tische Dehnung verschiedene Reaktionen der zwei vaskula¨ren Zelltypen (ECs und SMCs)
zu finden und zu untersuchen. Auf verschiedenen Iridium-Oxid Stent-Beschichtungen er-
scheinen ECs empfindlicher als SMCs. Auf micro-nano-Strukturen aus PDMS richten sich
ECsundSMCsnichtsignifikantunterschiedlichaus.Experimentemitbiofunktionalisierten
Nanostrukturenmachendeutlich,dasseseineuniverselleLiganden-Abstand-Abh¨angigkeit
fur beide Zelltypen geben muss. Unter uniaxialer mechanisch elastischer Dehnung oder ei-¨
nemgerichtetenelektrischenFeld,zeigenECsundSMCssignifikantverschiedeneReaktio-
nen. Die allgemeinen Eigenschaften der zwei Zelltypen ko¨nnen mit einem automatischen
Controller-Modell quantitativ beschrieben werden. Die Ergebnisse machen zuversichtlich,
in kommenden Untersuchungen die Implantat-Einheilung zu verbessern und sogar ku¨nst-
liche Formation neuer Blutadern (Angiogenese) zu ermoglichen.¨
Stimulation of vascular cells by extracellullar signals
Treatment of vascular diseases often requires the selective addressing of endothelial (ECs)
andsmoothmusclecells(SMCs). Thetwovascularcelltypesareimportantforthewound
healing after stent implantation. Recent research designs new materials and coatings for
stents to improve the complex healing process. The aim of my work was to find and
investigate different reactions in the two vascular cell types (ECs and SMCs) through
surface chemistry, topography and other stimulating factors like electrical fields or applied
external stretching forces. On various iridium-oxide stent coatings ECs seem to be more
sensitive to chemical differences than SMCs. On PDMS micro-nano-grooves ECs and
SMCs align not significant differently to the structure. Cell assays on nano-structured
and bio-functionalized surfaces reveal a universal ligand distance dependency for both cell
types. Upon application of uniaxial mechanical stretch or an directed electrical field, ECs
and SMCs show significant different responses. General characteristics of the two cell
types can be quantitatively described by an automatic controller model. These findings
are promising for further studies to improve wound healing after implantation and even
more to allow the artificial generation of new blood vessels (angiogenesis).Contents
Introduction and objective 1
I Review on blood vessel physiology 3
1 Cell system and biology 5
1.1 Blood vessel physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1.1 Cell culture of vascular cells . . . . . . . . . . . . . . . . . . . . . . . 6
1.2 Cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2.1 Stent treatment and current stent materials . . . . . . . . . . . . . . 7
2 Recent research 9
2.1 Samples and surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1.1 Topography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1.2 Surface bio-chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2 External signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.1 Mechanical forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.2 Electrical fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
II Experimental design and measurement techniques 15
3 External signal stimulations 17
3.1 Nano-micro grooves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.2 Gold nano structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.2.1 Passivation of the surfaces with polyethylene glycol (PEG2000) . . . 18
3.3 Surface biofunctionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.3.1 Biofunctionalization with cyclic RGD-Thiol . . . . . . . . . . . . . . 19
3.3.2 Biofunctionalization with REDV . . . . . . . . . . . . . . . . . . . . 19
3.3.3 Biofunctionalization with VE-Cadherins . . . . . . . . . . . . . . . . 19
3.4 Mechanical stretch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.5 Electrical field. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
vvi Contents
4 Methods of characterization 23
4.1 Scanning electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.2 Light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2.1 Phase contrast microscopy for cell phenotype analysis . . . . . . . . 24
4.2.2 Fluorescence for internal cell structure analysis . . . . . . . . . . . . 25
4.3 Data aquisition and processing . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.4 Controller model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
III Authentic stent surfaces 31
5 Cell adhesion, spreading and proliferation on stent surfaces 33
5.1 Additional materials and methods . . . . . . . . . . . . . . . . . . . . . . . 33
5.2 Short and long term studies on flat model surfaces . . . . . . . . . . . . . . 35
5.2.1 Initial cell attachment, spreading and cell adhesion . . . . . . . . . . 35
5.2.2 Long term cell growth on flat model surfaces . . . . . . . . . . . . . 36
5.3 In vitro cell adhesion on stents . . . . . . . . . . . . . . . . . . . . . . . . . 37
5.4 Discussion and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
IV Extracellular signals 41
6 Vascular cells on nano-micro topographies 43
6.1 Different sensitivity of human endothelial cells, smooth muscle cells, and
fibroblasts to topography in the nano-micro ranges . . . . . . . . . . . . . . 43
6.2 Discussion and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
7 Behavior of vascular cells on biofunctionalized gold nanostructures 49
7.1 Additional materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
7.2 Universal integrin clustering distance for vascular cells . . . . . . . . . . . . 49
7.3 Discussion and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
8 Cyclic stretched membranes and mechanical forces 55
8.1 Different time and frequency dependence of endothelial and smooth muscle
cells on cyclically stretched substrates . . . . . . . . . . . . . . . . . . . . . 55
8.2 Discussion and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
9 Static and pulsed electrical fields 63
9.1 Vascular cells in direct current and pulsed physiological electrical fields . . . 63
9.1.1 Automatic controller model of cell orientation and migration in di-
rect current electrical fields . . . . . . . . . . . . . . . . . . . . . . . 63
9.1.2 Vacular cells in pulsed electrical fields . . . . . . . . . . . . . . . . . 70
9.2 Discussion and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Summary 75
List of figures 79Contents vii
Bibliography 85
Acknowledgements 103
V Appendix 105
1 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
2 Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
3 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
3.1 Human primary cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
3.2 Live cell staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3.3 Antibody staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3.4 Analysis of actin stress fibers and focal adhesions . . . . . . . . . . . 109
3.5 Automatic controller model . . . . . . . . . . . . . . . . . . . . . . . 109
4 Supplement material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.1 Stent coatings and stents . . . . . . . . . . . . . . . . . . . . . . . . 113
4.2 Nano-micro topography . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.3 Stretching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.4 Electrical fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114