The wettability of biomaterials determines the protein adsorption and the cellular responses [Elektronische Ressource] / von Rumiana Tzoneva-Velinova

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• Publié par: universitat_potsdam
• Publié le: 01 janvier 2003
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 6 Mo

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Aus dem GKSS Forschungszentrum Geesthacht GmbH, Institute
für Chemie, Teltow
THE WETTABILITY OF BIOMATERIALS DETERMINES THE
PROTEIN ADSORPTION AND THE CELLULAR RESPONSES
Dissertation
Zur Erlangung des akademischen Grades Doktor der Naturwissenschaften
(Dr. rer. nat.)
in der Wissenschaftsdisziplin „Biotechnologie-Biomaterialen“
eingereicht an
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität Potsdam
von
Rumiana Tzoneva-Velinova
Teltow, im Mai 2003To my husband Ivan, my daughter Borislava, and
my parents Liliana and Dimitar Tzonevi
iiPreface
This work was carried out at the GKSS Forschungszentrum Geesthacht GmbH,
Institut für Chemie, Teltow, during the period from November 1999 to April 2003 under
the guidance of Dr. Albrecht and Dr. Groth.
This thesis consists of six parts. Chapter 1 is Introduction and Chapter 2 is Literature
Survey. Chapter 3 is Materials and Methods. Results and Discussion are shown in
Chapter 4. Summary is given in Chapter 5 and Chapter 6 contains Perspectives.
I would like to express my enormous gratitude to the Director of Institute of
Chemistry Prof. Dr. Lendlein for his interest and strong support to my work during the
whole my stay in the Institute.
I would like to thank the Chairman of the Examination Commission Prof. Dr. Micheel for
making possible my defense in Potsdam University.
I would like to thank also Dr. Groth for giving me the chance to work in his
laboratory and for his kind support during my Ph.D. work.
I cannot be thankful enough to Prof. Dr. Nagel for her exceptional kindness and
for the valuable advices during the writing of my Ph.D.
I cannot be thankful enough to Dr. Albrecht and Dr. Hilke for their valuable
advices and lots of encouragements during all the years of my working stay.
Very special thanks to Dr. Faucheux for her enormous encouragements, warm and
friendly support and a lot of very helpful discussions and advices.
I would like to thank very much to Dr. Heuchel for his kind guidance and support
in the field of the physicochemistry.
Many thanks go to Dr. Karola Luetzow and Herr Martin Siegert and the whole
Molecular Modeling Group of Dr. Hoffmann for their friendly helps and nice support
every day.
I am deeply grateful to Prof. Dr. D. Paul (emeritus professor) for having given me the
chance to do my Ph.D. work in Institute of Chemistry and providing me all the necessary
support through the whole my stay in the Institute.
iiiI would like to thank Dr. Jean-Luc Duval from the Universite de Technologie de
Compiegne, France for his kind assistance for ESEM analysis.
I would like to thank to Dr. Kamuzewitz for the valuable discussions for the contact angle
measurements.
I am also grateful to Frau Manuela Keller for the AFM images and Herr Schossig for the
SEM images.
My gratefulness goes to all colleagues and friends from the Institute of Chemistry
for their kind assistance not only for my work but also when I had other problems.
And at least, but not at last I want to thank to my family-my husband and my
daughter and my parents for their patience, encouragements and for the support during all
the years of my work.
ivAbbreviations
AJ adherent junctions
surface free energyτ
surface tensionγ
contact angle at the solid-liquid interfaceθ
β-TG β-thromboglobuline
ABP actin-binding protein
ADP adenosine diphosphate
AFM atomic force microscopy
CA contact angle
cAMP cyclic adenosine monophosphate
Cb solution concentration of the protein (µg/ml)
CE Cuprophan
C limiting value of protein adsorption (adsorption “plateau”)L
CLSM confocal laser scanning microscope
Cs adsorption amount of protein (per surface area)
DTS dense tubular system
EC endothelial cells
ECM extracellular matrix
EDTA ethylenediaminetetraacetic acid
ESEM environmental scanning electron microscopy
FITC fluorescein isothiocyanate
FN Fibronectin
FNG Fibrinogen
GP gap junctions
HMWK High Molecular Weight Kininogen
HUVEC Human Umbilical Vein Endothelial Cells
ICAM-1 Intracellular Adhesion Molecule-1
K binding constant
mAb monoclonal antibody
MMP matrix metalloproteinase
MTS microtubular system
NO nitric oxide
vOCS open canicular system
ODS Dimethyloctadecylchlorosilane
pAb polyclonal antibody
PAI-1 plasminogen activator inhibitor-1
PBS phosphate buffer saline
PC-PE polycarbonate-polyether
PEI polyether imide
PEO polyethylene oxide
PET Polyethyleneterephthalate
PEX MMP-2 termed hemopexin fragment
PGI Prostacyclin2
PSU Polysulfone
PF4 platelet factor 4
PTFE poly(tetrafluoroethylene)
PVDF polyvinylidene fluoride
RGD arginine-glycine-aspartic acid
SDS sodium dodecyl sulphate
TF tissue factor
TJ tight junctions
tumor necrosis factor alfaTNF-α
t-PA tissue plasminogen activator
TxA Thromboxane2
u-PA urokinase type activator
VWF von Willebrand factor
W work of adhesion
viChapter Contents Page
1. Introduction 1
1.1. General introduction 1
1.2. Aim of the work 3
2. Literature survey 4
2.1. Hemocompatibility of polymers 4
2.2. Protein adsorption 6
2.2.1. General aspects 6
2.2.2. Fibrinogen adsorption-role in blood-polymer interactions 6
2.2.2.1. Adsorption isotherms of FNG-amount and affinity 9
112.2.3. Physicochemical properties of the biomaterials influencing
protein adsorption
2.2.3.1. Wettability 11
2.2.3.2. Energetics of wetting 14
2.2.3.3. Surface charge 16
2.2.3.4. Topography and roughness 17
2.3. Platelets 18
2.3.1. General aspects 18
2.3.1.1. Structure 18
2.3.1.2. Function 20
2.3.2. Activation of platelets 21
2.3.2.1. LRG gene family 22
2.3.2.2. Integrins 22
2.3.2.3. Selectins 23
2.3.2.4. Immunoglobulin supergene family 23
2.4. Endothelial cells 24
2.4.1. General aspects-structure and function 24
2.4.2. Role of endothelium 24
2.4.2.1. Anti-thrombogenic function of endothelium 24
2.4.2.2. Prostacyclin (PGI ) 252
262.4.3. Role of EC-substrate interactions
viiChapter Contents Page
2.4.3.1. Integrin-ECM binding 27
2.4.3.2. Remodelling of ECM proteins 29
2.4.3.2.1. Remodelling of synthesized and deposited ECM proteins 29
2.4.3.2.2. ECM breakdown/destruction 33
2.4.4. Role of cell-cell interactions 35
2.4.4.1. Tight junctions (TJ) 35
2.4.4.2. Gap junctions (GJ) 35
2.4.4.3. Syndesmos or complexus adherents 36
2.4.4.4. Adherent junctions (AJ) 36
382.5. Endothelization of polymer membranes
2.5.1. General aspects 38
2.5.2. EC adhesion, spreading and proliferation on polymer membranes 39
2.5.3. Functionality of seeded EC monolayer (newly established 41
endothelium)
3. Materials and methods 42
3.1. Materials 42
3.1.1. Polymer membranes 42
3.1.1.1. Basic polymer membranes 42
3.1.1.2. Modified PEI membranes 44
3.1.1.3. Reference membranes 44
3.1.2. Model surfaces (hydrophilic and hydrophobic glasses) 44
3.1.3. Proteins 44
3.1.4. Fluorescent labeling of the proteins 44
3.1.5. Citrate Human Plasma 45
3.1.6. Cells 45
3.1.6.1. Platelet preparation 45
3.1.6.2. HUVEC 45
3.1.7. HUVEC cell lysates 46
3.2. Methods 46
3.2.1. Characterization of carboxylated PEI membranes 46
viiiChapter Contents Page
3.2.2. Contact angle measurements 46
3.2.2.1. Calculation of surface energy from contact angle 47
3.2.3. Atomic Force Microscopy (AFM) 48
3.2.4. Desorption of plasma proteins by different eluting agents 48
3.2.5. Fluorescent method for protein adsorption (adsorption of FITC- 48
labeled FNG)
3.2.6. Enzyme immunoassay (EIA) 49
3.2.6.1. Adsorption/conformation of FNG adsorbed from plasma to basic 49
membranes
3.2.6.2.FN and FNG adsorbed from single 50
solution to glass and ODS glass
3.2.6.3. Adsorption/conformation of FN and FNG adsorbed from single 50
solution to modified membranes
3.2.7. Substrate and membrane coating 51
3.2.8. Immunofluorescence microscopy 51
3.2.8.1. Platelets 51
3.2.8.2. HUVEC 51
3.2.8.2.1. Vinculin staining 51
3.2.8.2.2. Remodelling of substratum-bound or soluble FN and FNG by 52
HUVEC
3.2.8.2.3. Distribution of integrin receptors on the ventral and dorsal cell 52
surface
3.2.8.2.4. Co-localization experiments 52
3.2.8.2.5. E-Cadherin staining 53
3.2.9. Actin staining 53
3.2.10. Cell attachment on glass and ODS glass 53
3.2.11. Cell attachment and growth on polymer membranes 53
3.2.12. Scanning Electron Microscopy (SEM) 54
3.2.13. Western Blotting 54
3.2.14. Immunoprecipitation 54
ixChapter Contents Page
3.2.15. Zymography 55
3.2.16. In situ Zymography on FITC-labeled Gelatine 55
3.2.17. Prostacyclin assays 55
3.2.18. Environmental Scanning Electron Microscopy (ESEM) 56
3.2.19 Statistical analysis 56
4. Results and Discussion 57
57Part I. The influence of the materials surface properties on
protein adsorption and platelet adhesion/activation
4.1. Materials surface properties 58
4.1.1. Wettability 58
4.1.2. Roughness (AFM measurements) 59
4.1.3. Surface free energy 60
614.2. Protein adsorption
4.2.1. Total protein adsorption 61
4.2.2. FNG adsorption (adsorption isotherms of FNG) 62
4.2.3. Frption/conformation 63
4.3. Platelet adhesion/activation 65
4.3.1. Platelet adhesion 65
4.3.2.