Arterial Tissue Engineering: Influence of Shear Stress
Total Page：16
File Type：pdf, Size：1020Kb
“Arterial tissue engineering: Influence of shear stress on endothelial progenitor cells” “Arterielles Tissue Engineering: Einfluss von Scherstress auf Endotheliale Progenitor Zellen” Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Dipl.-Biol. Sebastian Łukasz Olszewski aus Wołomin (Polen). Berichter: Universitätsprofessor Dr.med. Stefan Jockenhövel Universitätsprofessor Dr.rer.nat. Lothar Elling Tag der mündlichen Prüfung: 09.04 2013 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. 2 For my beloved family. 3 4 List of Abbreviations ................................................................................................ 8 1. Introduction ........................................................................................................... 9 1.1. Endothelial progenitor cells (EPCs) .......................................................................12 1.1.1 Origins of EPCs ................................................................................................................... 14 1.1.2 Phenotypes of EPCs ............................................................................................................ 15 1.1.2.1 Early outgrowth EPCs .................................................................................................. 16 1.1.2.2 Late outgrowth EPCs ................................................................................................... 16 1.1.3 Clinical application of EPCs ................................................................................................. 16 1.1.3.1 EPCs as clinical markers .............................................................................................. 17 1.1.3.2 Homing of EPCs in angiogenesis and infarction models ............................................. 20 1.1.3.3 EPCs as cell source for grafts or as lining for grafts in tissue engineering .................. 20 1.1.4 Shear stress and EPCs ....................................................................................................... 22 1.2 Shear stress devices ................................................................................................28 1.2.1 Shear stress - short definition .............................................................................................. 28 1.2.2 Generation of shear stress with a Taylor-Couette system .................................................. 29 1.2.3 Other shear stress devices .................................................................................................. 35 1.2.3.1 Parallel plate flow chamber-based device .................................................................... 35 1.2.3.2 Rotating cone and plate-based device ......................................................................... 36 1.2.3.3 Tissue-engineered vessel-based device ...................................................................... 38 1.3 Aim of this study ......................................................................................................39 2. Materials and Methods ....................................................................................... 41 2.1 Materials (Please see appendix) ..............................................................................41 2.2 Methods ............................................................................................................. 42 2.2.1 Isolation of EPCs ................................................................................................................. 42 2.2.2 Cultivation of EPCs .............................................................................................................. 44 2.2.3 Flow cytometry analysis of EPC .......................................................................................... 45 2.2.4 Staining of cells with DiI - labelled acetylated low density lipoprotein (DiI ac-LDL) ............ 47 2.2.5 Staining with FITC labelled UEA - lectin .............................................................................. 47 2.2.6 Isolation of HUVECs (human umbilical vein endothelial cells) ............................................ 48 2.2.7 Cultivation of HUVECs ......................................................................................................... 49 2.2.8. Isolation of myofibroblasts from umbilical cord arteries ...................................................... 50 2.2.9. Cultivation of myofibroblasts ............................................................................................... 50 2.2.10. Real-time PCR analysis .................................................................................................... 51 2.2.10.1. RNA isolation ............................................................................................................. 55 2.2.10.2. Synthesis of cDNA .................................................................................................... 57 2.2.10.3. Primer design ............................................................................................................ 57 2.2.10.4. Real-time PCR setup protocol ................................................................................... 60 2.2.11. Immunohistology staining ................................................................................................. 63 5 2.2.12. Nitrite oxide measurement ................................................................................................ 64 2.2.13 Assembly of the Taylor-Couette system ............................................................................ 68 2.2.13.1 Exposure of cells to laminar shear stress and turbulent flow in the Taylor-Couette system ...................................................................................................................................... 72 2.2.13.2 Short versus long time exposure in the Taylor-Couette system ................................ 72 2.2.13.3 Nitrogen oxide production under laminar shear stress and turbulent flow ................. 72 2.2.13.4 Homing of EPCs to different surfaces and at different non-pulsatile flow conditions . 73 2.2.14 Assembly of the parallel plate flow chamber with pulsatile flow pattern ............................ 73 2.2.14.1 Exposure to different levels of pulsatile shear stress ................................................. 75 2.2.14.2 Short versus long time exposure to pulsatile shear stress ......................................... 76 2.2.14.3 Nitrogen oxide production under pulsatile shear stress ............................................. 76 2.2.14.4 Homing of EPCs to different surfaces under pulsatile conditions .............................. 76 2.2.15 BioStent with EPCs and/or HUVECs ................................................................................. 77 2.2.16 Development of a novel pulsatile Taylor-Couette system ................................................. 79 2.2.16.1 System requirements - pulsatile Taylor-Couette shear stress device ........................ 79 2.2.16.2 Geometry of the systems ........................................................................................... 80 2.2.16.3 Design and assembly of module 1 ............................................................................. 80 2.2.16.3 Design and assembly of module 2 ............................................................................. 86 3. Results ................................................................................................................. 88 3.1 Validation of the EPC phenotype .............................................................................88 3.1.1 Validation of the EPC phenotype by DiI-ac-LDL and FITC - UEA 1 stain ........................... 88 3.1.2 Flow cytometry analysis of EPC .......................................................................................... 89 3.1.3 Optimal cultivation time for generation of human peripheral blood late outgrowth EPC (n=3) ...................................................................................................................................................... 89 3.2 Laminar shear stress and turbulence experiments in the Taylor-Couette system .........................................................................................................................................91 3.2.1 Short versus long time exposure to laminar shear stress of 0.85 Pa .................................. 91 3.2.2 Exposure of EPCs to steady laminar shear stress of 0.85 Pa, low and high turbulence in the Taylor-Couette system ............................................................................................................ 94 3.2.3 Nitrogen oxide production at 0.85 Pa, low and high turbulence ........................................ 104 3.2.4 Homing of early EPCs under laminar flow conditions........................................................ 104 3.3 Pulsatile shear stress experiments with EPCs ..................................................... 104 3.3.1 Short versus long time exposure to pulsatile shear stress ................................................ 105 3.3.2 Exposure to pulsatile flow and shear stress levels 0.45, 0.7 and 1 Pa ............................. 107 3.3.3 Nitrogen oxide production under pulsatile shear stress....................................................