Motility and alignment of human umbilical vein endothelial cells (HUVEC) on 3d scaffolds

MOTILITY AND ALIGNMENT OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) ON 3D SCAFFOLDS ZHENG ZHONG NATIONAL UNIVERSITY OF SINGAPORE 2005 MOTILITY AND ALIGNMENT OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) ON 3D SCAFFOLDS ZHENG ZHONG (B.Sc) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2005 To my PARENTS To my DREAM iii Table of Contents Table of Contents iv List of Tables vi List of Figures vii Acknowledgements x Summary xiii Introduction 1.1 Tissue Engineering and 3D Scaffolds 1.2 Endothelial Cells and Angiogenesis 1.3 Understanding Angiogenesis Physically and Biologically 1.4 Chapter Outline Fabrication of 3D Scaffolds Using P-beam 2.1 CIBA and Instruments 2.2 Proton Beam Stage Scanning 2.3 Materials Fabricated by P-beam Writing 2.3.1 Thick PMMA Substrates 2.3.2 PMMA Resist Writing Cell Culture Processes and Migration Experiments 3.1 Introduction to Cell Culture 3.2 Facilities and Methods Used to Record Cell Movement 3.2.1 Introduction to the Time-lapse Microscope 3.2.2 Program for Cell Speed Measurement 3.3 Experiments for Cell Motility Study iv 1 9 14 18 19 22 25 25 27 27 29 31 3.3.1 3.3.2 3.3.3 Movement on Grooves and Ridges Cell speed Comparison on Different Structure Movement on Smooth Muscle Cell Pattern Discussion and Summary of Cell Motility and alignment on 3D scaffolds 4.1 Cell Migration on Plain Surface 4.1.1 Background of Cell Migration 4.1.2 Materials for Cell Migration Experiment 4.2 Cell Motility and Alignment on Microstructure 4.2.1 Cell Movement on Grooves and Ridges 4.2.2 Cell Movement on Smooth Muscle Cell Pattern 4.3 Discussion behavior Fabrication of multiple 3D scaffolds and RNA studies of cells large area scaffolds 5.1 Introduction to Electroplating 5.2 Ni Electroplating of PMMA Resist Structures 5.3 Process of Hot Embossing and Bonding 5.3.1 Introduction to Hot Embossing 5.3.2 Hot Embossing of PMMA 5.3.3 Bonding Technique 5.4 RNA Studies of the Cells on the Large Area Scaffolds 5.4.1 Experiment Process of Gene Detection 5.4.2 Results and Discussion for the Microarray Detection Conclusion and further development 6.1 Conclusion 6.2 Further Development 6.2.1 Cell Migration on Smooth Muscle Cell like Pattern 6.2.2 Endothelial Cell Gene Expression according to Different terns of Scaffolds Bibliography 31 40 43 49 49 50 52 55 56 59 61 on Pat 64 65 67 70 70 71 74 76 76 78 81 81 83 83 84 85 v List of Tables 2.1 Depth of the structure in PMMA with specific energy 21 3.1 Average speed on 5µm wide grooves and ridges 35 3.2 Average speed on 10µm wide grooves and ridges 36 3.3 Average speed on 15µm wide grooves and ridges 36 3.4 Average speed on 20µm wide grooves and ridges 36 3.5 Average speed on 25µm wide grooves and ridges 37 3.6 Threshold of t test 40 3.7 P value for Vx comparison 41 3.8 P value for Vy comparison 42 3.9 P value for V comparison 42 3.10 Total turning index 47 4.1 Types of PMMA for cell migration experiments 52 4.2 The results of contact angle test 54 4.3 Biological reactions to topography 56 5.1 Bonding parameters refer to different types of PMMA 75 5.2 Angiogenesis Superarray 79 5.3 Extracellular Matrix and Adhesion Molecules Microarray 79 5.4 Control Genes 80 vi List of Figures 1.1 Micrograph of HUVE Cells plated on a flat substrate 1.2 A diagram of the artery 1.3 The process of angiogenesis 2.1 Schematic diagram of the beam line facilities at CIBA 10 2.2 P-beam writing end station set-up 11 2.3 Interview of P-beam exposure station 12 2.4 Scanning and control hardware setup 13 2.5 The Ionscan software graphical user interface 15 2.6 Magnetic plus stage scan mode 16 2.7 Too fast magnetic scanning result 17 2.8 The beam path representation 18 2.9 The structure of PMMA: (C5 O2 H8 )n 19 2.10 Schematic representation of the fabrication process of micro patterns 20 2.11 Mechanism of radiation-induced chain scission in PMMA 20 2.12 Micro patterns fabricated in thick PMMA substrates 22 2.13 Spin-coating for PMMA 950 resist 24 3.1 Zeiss Axiovert 200M Light Microscope 28 3.2 Top view of the culture dish with medium and scaffold 29 3.3 The interface of the IDL program software 30 3.4 Image of cell movement on the plain surface 32 3.5 Bonding Process 33 vii 3.6 Top view of the bonded scaffold 34 3.7 Image of cell movement on the structures 35 3.8 Average Speed: Vx , Vy , V on 5µm grooves and ridges 37 3.9 Average Speed: Vx , Vy , V on 10µm grooves and ridges 38 3.10 Average Speed: Vx , Vy , V on 15µm grooves and ridges 38 3.11 Average Speed: Vx , Vy , V on 20µm grooves and ridges 39 3.12 Average Speed: Vx , Vy , V on 25µm grooves and ridges 39 3.13 Distributions for treated and comparison group values 40 3.14 Formula for the t-test 41 3.15 Smooth muscle view in longitudinal section 43 3.16 Typical smooth muscle with fibers inside 44 3.17 Smooth muscle cell like pattern 45 3.18 The pattern of SMC simulated in software Ionutils 45 3.19 The pattern of SMC fabricated by PBW 46 3.20 The top view of the bonded scaffold with SMC pattern 47 4.1 Illustration of Different Forces Involved in Cell Migration 50 4.2 Cells cannot adhere to the 1.5mm PMMA substrate properly 53 4.3 Software for measuring the contact angle 54 4.4 Cells can adhere to the 3mm PMMA substrate properly 55 4.5 Cells can adhere to the 3mm PMMA substrate properly 57 4.6 The speed on Y direction of different width of grooves or ridges 58 4.7 Angles between cells movement and the direction of grooves 59 4.8 Possible migration path of cells in the SMC pattern 60 4.9 Schematic of the movement path within the blocks 61 5.1 Ni electroplating setup 66 5.2 Process Scheme for achieving a Ni stamp from electroplating PMMA 5.3 resist 68 SEM image of Ni stamp 69 viii 5.4 Schematic view of hot embossing system 71 5.5 Interface of the hot embossing control software: Imprinting 72 5.6 Processes of applied temperature 73 5.7 Processes of applied pressure 73 5.8 Optical image of PMMA substrate hot embossed with the Ni stamp 74 5.9 The outlook of the scaffold for the RNA detection experiments 75 6.1 New dimensions of the smooth muscle like pattern 83 ix Acknowledgements I would like to thank Prof Frank Watt, my supervisor, for his many suggestions and constant support during this work I am also thankful to Shao and Jeroen for their guidance through the early years of chaos and confusion And the close cooperation from A/Prof Ge Ruowen and Sun Feng makes these two years of research go fluently and fruitful I would also like to thank Mark for writing the recommendation letter for me last year to assist and support my application for further study And thanks for Thomas’s module helping me to get familiar with our honey–3.5Mev accelerator I had the pleasure of meeting Min, Jennifer, Liping, Huang Long They are wonderful people and their passion and kindness make me feel at home, especially during the lonely years The discussion on the LATEXwith Reshmi makes the writing of thesis so funny Besides, I want to thank to Kambiz for discussing with me on the plating process and his keen on physics leaves me a deep impression I also cherish the time playing basketball with Chammika, Chorng Haur, Ah Fook, and x 76 5.4 RNA Studies of the Cells on the Large Area Scaffolds Studies on the molecular control of endothelial cell (EC) morphogenesis during angiogenesis or vasculogenesis have revealed many insights into how blood vessels participate in complex biological processes such as development, wound repair and tumorigenesis [8] [51] [52] [53] Identifying new molecular targets that block specific steps in EC morphogenesis may become crucial in efforts to inhibit angiogenesis in human diseases where angiogenesis is a pathogenic component (i.e cancer, diabetic, retinopathy, arthritis, atherosclerosis) [7] [52] In our work, cells are harvested for gene detection to explore whether geometric constraints have any effects on the level of related genes These results will provide additional information for the control of genes to inhibit or promote angiogenesis 5.4.1 Experiment Process of Gene Detection When the cells have been cultured and raised on the bonded scaffolds (the whole pattern is 5mm×5mm, the width and the depth of the ridges are 20µm, 9µm respectively) for several days, they are harvested for gene detection experiments by our collaborators Dr Ge Ruowen and Sun Feng from DBS, NUS The experiment procedure is as follows [54]: A RNA Preparation 77 The mRNA (messenger RNA) need to be isolated from the cells mRNA is synthesized from a DNA template during transcription, that mediates the transfer of genetic information from the cell nucleus to ribosomes in the cytoplasm, where it serves as a template for protein synthesis The most important prerequisite for any gene expression analysis experiment is consistent, high-quality RNA from every experimental sample Therefore, the sample handling and RNA isolation procedures are critical to the success of the experiment The mRNA is extracted from the treated cells followed the process of ArrayGrade mRNA purification Kit [54] B cDNA Synthesis Second, combine the RNA sample with Component G1 and RNase-Free H2 O, and incubate at 70◦ C for 10 min; this combination is named Annealing Mixture After that, prepare the cDNA Synthesis Master Mix, then add 10 µl of cDNA Synthesis Master Mix to each tube containing 10 µl of Annealing Mixture Incubate at 42 ◦ C for 50 minutes followed by 75 ◦ C for minutes then cool to 37 ◦ C Finally, centrifuge briefly to collect the mixture at the bottom of the tube and return to 37 ◦ C C cRNA Synthesis, Labeling, and Amplification Add 20 µl Amplification Master Mix to each tube containing 20 µl of cDNA Synthesis Reaction Mix well and Incubate for at least one hour or up to overnight at 37 ◦ C After that, SuperArray ArrayGrade cRNA Cleanup Kit is used for the cRNA Purification 78 D Oligo GEArray Hybridization The appropriate volume of the purified cRNA preparation is added to the hybridization solution described in the Oligo GEArray User Manual After overnight hybridization, pour the Target Hybridization Mix from the hybridization tube into a clean microcentrifuge tube Then add ml Wash Solution to the hybridization tube Wash the membrane for 15 minutes at 60 ◦ C with 20 to 30 rpm agitation, and discard the wash solution E.Chemiluminescent Detection The Chemiluminescent Detection Kit is used to obtain the chemiluminescent array image F.Image and Data Acquisition and Analysis The chemiluminescent signal can be collected through a cooled CCD camera and can aslo be recorded using X-ray film and a flatbed desktop scanner The data extraction and analysis is conducted with the GEArray Expression Analysis Suite 5.4.2 Results and Discussion for the Microarray Detection Following the experimental procedure mentioned above, several groups of microarray have been tested for related genes Some selected data are shown in the following tables: In the tables, the 2D column stands for the level of gene expression from the 79 Angiogenesis Superarray Gene No Code Name 2D 3D 67 MMP2 Matrix metalloproteinase 27030 26682 103 TIMP1 Tissue inhibitor of metalloproteinase 22454 30714 112 VEGFB Vascular endothelial growth factor B 17449 18912 2D/3D 1/0.99 1/1.37 1/1.08 Table 5.2: Angiogenesis Superarray Extracellular Matrix and Adhesion Molecules Gene No Code Name CNTN1 Contactin 32 CTGF Connective tissue growth Factor 38 FN1 Fibronectin Microarray 2D 3D 2D/3D 5450 967 1/0.18 10542 — 14760 399 1/0.03 Table 5.3: Extracellular Matrix and Adhesion Molecules Microarray cells seeded on the plain surface, while the 3D column data from 3D scaffolds For the Angiogenesis Superarray detection in Table 5.2, the listed genes’ expression does not change much from 2D to 3D environment That means our 3D structures seems to have little influence on those genes related to the angiogenesis process For the ECM and Adhesion Molecules Microarray detection in Table 5.3, the listed genes’ expression changes a lot The CNTN1 decreases around times, the FN1 decreases to around 33 times CTGF has zero expression in the cells on the 3D structures, while on plain surface the gene still remain at a relatively high level These results indicated that the 3D pattern can change the genes expression related to adhesion Table 5.4 is the detection results for control genes The control genes are relatively stable and will not change to much under normal conditions However, from the data, HSPCB expression decreases as much as times Also ACTB and RPS27a decrease slightly These results shows that the status of the cell on 3D scaffolds may change 80 Control Genes Gene No Code Name 2D 3D 2D/3D RPS27a Ribosomal protein S27a 47815 33849 1/0.71 124 HSPCB Heat shock 90kDa protein 1, beta 30138 7967 1/0.26 125 ACTB Actin, Beta 50877 30973 1/0.61 Table 5.4: Control Genes somewhat, with the major changes occurring in the CTGF expression Chapter Conclusion and further development 6.1 Conclusion The migration experiments of the endothelial cells on different dimensions of ridges and within patterns that simulate smooth muscle cells has shown that proton beam writing is a potential and suitable patterning technique to understand cell response to different kinds of geometrical constraints In addition, it is efficient and relatively low cost to produce multiple scaffolds through the hot embossing technique, which make the RNA detection experiments possible Grooves and ridges can align the cells and guide their movement direction, compared to the random walk on a plain surface While previous results [13] have indicated that cells move faster on 20 µm ridges than on 12µm ridges, our results show that the total average speed of the cells are not significantly different between each 81 82 ridge geometry The previous measurements were made with different substrates using different sets of cells The surface treatment each time might not be the same and the condition of the cells may also have varied From our new results, it seems that the 3D pattern does not change the cells’ average speed, but confirms our previous work showing that ridges can act as alignment and guidance structures For the migration on the smooth muscle like pattern, the cells have almost the same possibility to turn a corner and as to move in a straight path When the front filaments change their orientation along the corner, the cells can turn around However, if the width of gap at the corner is not the same, the cells may have a preference in the direction of their movement This hypothesis still need further experiments to confirm Multiple copies of larger area scaffolds are made by hot embossing, and the RNA detection of cells grown on these scaffolds has been conducted by our collaborators Dr Ge Ruowen and Sun Feng from Dept of Biological Sciences, NUS The results show that the genes related to geometric constraints involved in the angiogenesis process have not been affected too much, while those genes related to adhesion have changed significantly The control genes also change a little, which infers that living cells on the 3D structures not grow as well as those on the plain surface 83 6.2 6.2.1 Further Development Cell Migration on Smooth Muscle Cell like Pattern The assumption that the different gap width of the corner may influence the choice of the direction of the cell movement Figure 6.1 displays two new possible pattern of smooth muscle cell like blocks with different width of gaps Figure 6.1: New dimensions of the smooth muscle like pattern In addition, there is a defect in our design: the material we used is rigid PMMA sheet, while in vivo the smooth muscle cells are elastic and placed next to each other with little gap, just as shown in Figure 3.15 Thus the material of the substrate may also need improvement in order to be closer to the real environment 84 6.2.2 Endothelial Cell Gene Expression according to Different Patterns of Scaffolds In this project, we have studied the 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structures are discussed in detail Chapter 3: A general description of the main points of cell culture is given, followed by a series of cell migration experiments, including human umbilical vein endothelial cells (HUVEC) movement on plain PMMA surface, and on. .. new tissue As the result, the motility and alignment of endothelial cells, which form the inner surface of blood vessels, is worth exploring, especially in a three dimensional (3D) environment The aim of this work is to understand the behavior of endothelial cells on polymer substrates with different microfabricated patterns A pattern with different dimensions of grooves and ridges (width: 5µm, 10µm,... research scope and aim is to investigate the motility and alignment of endothelial cells involved in angiogenesis, on 3D scaffolds fabricated using the Proton Beam Writing (PBW) technique 1.1 Tissue Engineering and 3D Scaffolds Tissue engineering may be defined as the use of a combination of cells, engineering materials, and suitable biochemical factors to improve or replace biological functions Probably... conclusion of this project, where some suggestions for further development are discussed Chapter 2 Fabrication of 3D Scaffolds Using P-beam Writing In this chapter, facilities used in this work, and the process of 3D scaffold fabrication is given in detail 2.1 Center for Ion Beam Applications and Instruments The Centre for Ion Beam Applications (CIBA), National University of Singapore, is a state -of- the-art... nutritional, and spatial conditions [3] Although the behavior and function of cells are changed by 1 2 the geometric constraints, very little work has been carried out to explore the motility and alignment of the cells in this 3D environment In addition, this knowledge is particularly important in the newly emerging field of tissue engineering It is becoming increasingly necessary to understand these... the growth and formation of new blood vessels to supply essential oxygen and nutrients In our study, we want to explore the process from the point of view of the physical constraints of the endothelial cells in a simulated 3D environment In a collaboration with scientists in the Department of Biological Sciences, NUS, we have also analyzed potential changes in related gene expressions in cells which... cell adhesion, and migration on both plain and patterned substrates are briefly described to aid the understanding and interpretation of the cell migration experiments Further discussion will be made on the results from the measurements Chapter 5: This chapter shows how to make a metal stamp, and introduces the basic aspects of Ni electroplating including the details of the plating process of PMMA 8... physically constrained by 3D scaffolds In order to collect a large number of cells for analysis, large area 3D scaffolds are required The fabrication of metal stamps, and the process of imprinting these stamps into multiple copies of scaffolds are also discussed 7 1.4 Chapter Outline Chapter 2: This Chapter will introduce the proton beam writing facilities at the Centre for Ion Beam Applications (CIBA)... environment, and combined with the National Instruments NI-DAQ drivers, it allows us to support any of the National Instruments analog output card 13 under Microsoft Windows operation system without requiring any major revisions to the code Another software package, named as Ionutils, is used to support file conversion to and from monochromatic bitmap, ascii and epl, the native file format used by Ionscan .. .MOTILITY AND ALIGNMENT OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) ON 3D SCAFFOLDS ZHENG ZHONG (B.Sc) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSICS NATIONAL... focus on the fabrication of 3D biocompatible scaffolds to study the geometric influence these scaffolds have on motility and alignment of the Human Umbilical Vein Endothelial Cells (HUVEC, Figure... behavior and function of cells are changed by the geometric constraints, very little work has been carried out to explore the motility and alignment of the cells in this 3D environment In addition,