Fabrication of proton beam micromachined scaffolds for cell behaviour studies

FABRICATION OF PROTON BEAM MICROMACHINED SCAFFOLDS FOR CELL BEHAVIOUR STUDIES ZHANG FANG NATIONAL UNIVERSITY OF SINGAPORE 2004 FABRICATION OF PROTON BEAM MICROMACHINED SCAFFOLDS FOR CELL BEHAVIOUR STUDIES ZHANG FANG (B.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2004 for mum, dad and my nephew/niece who is probably going to born on Feb 8th, 2005 Acknowledgements Life is always amazing Two years ago, I could not imagine that I was going to join this group, meet these lovely and lively people, and a little bit research which will benefit human being in the future (remember Will Smith’s left arm & hand in the movie ?) But now I know I have done these The first person I would like to thank is my supervisor Frank When I saw him the first time, in our coffee room, I changed my mind that Beckham was the most handsome British guy in the world I like the way that he turned his fingers to mimic cells movement Since that moment I have been attracted to this project His overly enthusiasm and integral view on research and his mission for providing “only highquality work”, has made a deep impression on me I owe him lots of gratitude of having shown me this way of research Besides of being an excellent supervisor, Frank is always as close as a good friend to students His encouragements and optimism always give me more confidence and fun at work He could not even realize how much I have learned from him I feel a deep sense of gratitude for my promoter Jeroen who kept an eye on the progress of my work, taught me many invaluable skills in micromachining and always was available when I needed his help and advices He is one of the people who science with pure and honest passion I appreciate the critical atmosphere during many experiments and discussions we had He can’t imagine how happy and proud I was when he said one of my samples was PERFECT And also, my special gratitude goes to Shao Thanks for sharing many late night runs with me I have enjoyed greatly the innovative and challenging atmosphere created by him, as well as the fried iii rice in Dover Food Centre I give a sincere gratitude to Kambiz for teaching me bonding, developing and many practical skills It was my pleasure to complain about thesis with him Don’t forget Choo, the accelerator wizard, without his patience and kind help, this master’s thesis is not possible Needless to say, that I am grateful to Min for her care and encouragements in the past two years Without her accompanying, my academic life could have been very lonely Her wonderful kids, Yiran and Yilin colored my life I think she will not understand, in a number of years, why I wanna be a lady like her, because of her passion for work, for family and for everybody A journey is easier when you travel together Interdependence is certainly more valuable than independence It is a pleasant aspect that I have now the opportunity to express my gratitude for many, many people I would like to thank Thomas for buying fruits, juice and cookies for us, and lending me his knowledge on nuclear physics and statistics Special thanks are due to Mark (Mr Bean?), whose charm and humor always cheer me up I am grateful for the DVD man, Andrew for offering me the micromachining software and the figure of hardware I thank him and Ee Jin for providing happy memory in my life in S’pore Don’t forget to send me photographs about little Andrew/Ee jin Many thanks go to Chammika (Charley?) whose excellent basketball skill and naughty punch on the court make me stronger to run experiment and appreciate my life I would like to thank Sum and Debbie for your kind advices about my thesis I am grateful for Frederic, Mukhtar and Huang Long for always treating me like a gentleman I also acknowledge Reshmi for encouraging me to turn my dreams into reality Special thanks go to Soma for taking pictures for my stamp And I thank Mangai for her persistence at work which impressed me a lot I have furthermore to thank the young generation I thank Earnest for his continuous iv working on this project Believe me he is a smart and potential guy I also thank Liping for giving me the feeling of being at home in this foreign country Who can me a favour to push this skinny girl to have dinner everyday? Our collaborators, Sun Feng and A/P Ge Ruowen from Biological Sciences Dept supported me greatly in my research work Their help in culturing cells and taking movies was vital for this project I greatly thank the energetic people from Colloidal Lab, Chorng Haur and Chiong for teaching me IDL to track the cute cells They are the best rivals to play basketball with And last but not least, my deepest gratitude flies to my mum and dad Thank them for always requiring me to be healthy and happy instead of working hard I’m always proud to be your little girl ☺ In a word, doing a Master in this group is definitely one of the best decisions of my life Do you know how hard to say good-bye to all of you? But trust me, it’s not an end, it’s a new start So, let’s celebrate! Yours, Bunny/Jenny/Jennifer/Zhennifer/Jane/J….Zhang Fang/ 芳 v Contents Acknowledgements…………………………………………………………… ……iii Contents……………………………………………………………………………….vi Summary…………………………………………………………………………… ix List of Figures……………………………………………………………………… xi List of Tables…………………………………………………………………………xv Introduction 1.1 Background of tissue engineering…………………………………… …….1 1.1.1 Angiogenesis and related diseases….…………………… …………1 1.2 Research scope and aim……………….………… …………….….… … 1.3 Thesis outline …………………….… ……………………….………….5 Fabrication of 3D scaffolds using p-beam writing 2.1 Centre for Ion Beam Applications and instrumentations…….…………… 2.2 Proton Beam Interactions with materials……………….……………… 10 2.3 Proton beam stage scanning…………………………………………….…13 2.4 Polymers fabrication by proton beam writing……………………… ……17 2.4.1 Thick PMMA………….……… ……….…………………… 17 2.4.2 PMMA resist………….…………………….…………………… 21 2.4.3 SU-8 resist………………………………….…………………… 25 Fabrication of multiple copies of 3D scaffolds using hot embossing 3.1 30 Ni electroplating…………….…………………………………………… 30 vi 3.1.1 Electroplating of SU-8 resist structures.……….……………… …32 3.1.2 Electroplating of PMMA resist structures…………………… … 37 3.1.3 Large area stamp fabrication………………………………………40 3.2 Hot embossing……………………………………….………………….…42 3.2.1 Introduction……………………….…………………………….…42 3.2.2 Hot embossing of polymers……………………………………… 43 3.3 Bonding techniques……………………………………….…………….…47 Cell culture processes and migration experiments 50 4.1 Cell culture………………………………….………………………….….50 4.2 Using time-lapse video camera to record cell movement……………… 51 4.2.1 Introduction of time-lapse microscope…………….….………… 51 4.2.2 Cell speed analysis program: IDL………………….………… …53 4.2.3 4.3 Cell migration experiments………………………… ………… 54 Cell speeds comparison on different surfaces………….…………….……60 Discussion and summary of geometric influences on cell behaviour 67 5.1 Background of cell behaviour on micro grooved and ridged surfaces … 67 5.2 Cells adhesion and migration…………………………….……………….70 5.2.1 Introduction…………………………………………………….…70 5.2.2 Cells on plain surface…………………………………………… 72 5.2.3 Cells on grooves and ridges…………….……………………… 74 5.3 Discussion………………………………………………….…………… 77 Conclusion and further development 6.1 80 Conclusion………………………………………………….…………… 80 vii 6.2 Further development……………………………………………….…… 80 6.2.1 Cells migration on PMMA substrates with different dimensional grooves and ridges………………………………….…….……….80 6.2.2 Endothelial cell gene expression associated with micro grooves and ridges………………………………….………………………… 81 Bibliography 83 viii Summary Tissue engineering is a rapidly developing and highly interdisciplinary field that applies the principles of cell biology, engineering and material science In natural tissues, the cells are arranged in a three dimensional (3D) matrix which provides the appropriate functional, nutritional and spatial conditions [1] In scaffold-guided tissue engineering, 3D scaffolds provide the critical function of acting as extracellular matrices onto which cells can attach, grow, and form new tissue The main focus of this thesis is to understand cell behavior in micro-grooved and ridged substrates and to study the effects of geometrical constraints on cell motility and cell function In this study, we found that BAE (Bovine Aortic Endothelial) cells naturally align with and are guided along 3D ridges and grooves machined into polymethylmethacrylate (PMMA) substrates Average cell speeds on micro grooves and ridges ranged from 0.015µm/s (for 12µm wide and 20µm deep ridges) to 0.025µm/s (for 20µm wide and 20µm deep ridges) This compares with the cell motility rates on a flat PMMA surface where the average cell speed is around 0.012µm/s In this work we used scaffolds which were directly written with a focused proton beam, typically MeV protons with a beam spot size of 1ì1àm2 To analyze the genes involved in the endothelial cell responses associated with geometrical constraints, large area scaffolds are needed In order to extend the potential of proton beam writing, studies are underway to implement the hot embossing process which will allow faster replication of 3D structures A Ni stamp with a 5ì5mm2 pattern of 20, 20, 10 àm for widths of grooves, ridges and their depth respectively will be presented ix CHAPTER DISCUSSION AND SUMMARY OF GEOMETRIC INFLUENCE ON CELL BEHAVIOUR Calculation based on cells growing on 20 µm grooves and ridges has shown that the Speed X was significantly greater than Speed Y See Figure 5.6 For cell migration on 12 µm ridges and 18 µm grooves, the same phenomenon is observed, see Figure 5.7 These data and their correlation suggest that ECs basically migrated along the direction of the grooves/ridges The comparison in Figure 5.8 also indicates that cell migrated predominantly in the direction of the grooves/ridges This marked behaviour is associated with migrating on the grooved and ridged substrates Speed X Migration Speed (um/sec) 0.03 0.025 0.02 Speed X 0.015 Speed X Speed Y 0.01 Speed Y Speed Y 0.005 plain surface 20µm grooves/ ridges 12µm ridges, 18µm grooves Figure 5.8 The average speeds in the direction of horizontal (Speed X) and vertical (Speed Y) of cells on a plain surface, 20 µm grooves/ridges and 12 µm ridges, 18 µm grooves The grooves and ridges are parallel to the horizontal direction Based on the T-test of cells speeds in Chapter IV, we learned that the endothelial cells on 20 µm grooves and ridges substrate move significantly faster than those on plain PMMA surface and the other scaffold which has 12 µm ridges and 18 µm grooves, see Figure 5.9 75 CHAPTER DISCUSSION AND SUMMARY OF GEOMETRIC INFLUENCE ON CELL BEHAVIOUR average migration speed (um/sec) 0.035 0.03 0.025 0.02 0.015 0.01 0.005 Plain surface 20µm grooves/ 12µm ridges, 18µm ridges grooves Figure 5.9 The average cell speed on plain surface, 20 µm grooves/ridges and 12 µm ridges, 18 µm grooves 60 angles (deg) 50 40 30 20 10 Plain surface 20µm grooves/ 12µm ridges, 18µm ridges grooves Figure 5.10 The average angles between cell movement and the direction of grooves on plain surface, 20 µm grooves/ridges and 12 µm ridges, 18 µm grooves Also the average angle between cell migration and the groove/ridge direction proves that the cells on 20 µm grooves/ridges are better aligned than those on 12 µm ridges, 18 µm grooves, see Figure 5.10 According to the findings, we can hypothesize that the better the cells are aligned with micro grooves/ridges, the faster they move on the substrates The evident response of endothelial cells associated with micro grooves and ridges shows that cells are inhibited in crossing grooves/ridges, the degree of inhibition or 76 CHAPTER DISCUSSION AND SUMMARY OF GEOMETRIC INFLUENCE ON CELL BEHAVIOUR the probability of crossing is probably dependent on the height of the step and the sharpness of the edge Cells move by extending the membrane that surrounds them, and some of these protuberances touch the surface and adhere to it, thus giving a means whereby the cell can exert force and move itself A cell’s response to the topography is depending on to what extent the feature alters the probability of a cell making a successful protrusion and contact in a given direction The ability to make protrusions is a function of the cytoskeleton—the framework of microfilaments, microtubules, and intermediate filaments that are present inside the membrane These microfilaments not form a rigid skeleton but supply a degree of inflexibility that works during motion of the cell [64] 5.3 Discussion Adhesive interactions between cells and the extracellular environment play a crucial role in affecting cell morphology, gene expression, and the rates of cell proliferation [65] For example, we have shown that cell migration rates vary depending on the various dimensions of micro grooves and ridges fabricated by p-beam micromachining This reaction of the cells to the grooves and ridges is probably reflected in their cytoskeleton The internal structure of the cell works to transmit force to be able to move Consisting of an internal frame of microfilaments, microtubules and intermediate filaments, the cytoskeleton plays this role [54] 77 CHAPTER DISCUSSION AND SUMMARY OF GEOMETRIC INFLUENCE ON CELL BEHAVIOUR Figure 5.11 Schematic of cytoskeleton (a) shows a cell on a flat surface; (b) shows the reorganisation of the micro-filaments that occurs on a grooved surface—the dashed lines show the line of discontinuity between the ridges and grooves The marked cellular behaviour associated with micro grooves/ridges, such as alignment and elongation, can be well explained in terms of the reformation of the cytoskeleton Figure 5.11 (a) displays a schematic of a cell on a flat surface The microfilaments as shown are mainly going from the region close to the nucleus towards the periphery of the cell, with only a few microfilaments being lateral On the other hand, when the cell is on a grooved/ridged substrate, as shown schematically in Figure 5.11 (b), the microfilament bundles form predominately along the direction of the groove Wojciak-Stothard et al proved this by staining using antibodies for Factin, the main constituent of the microfilaments, and using antibodies to reveal the constituent parts of the assembly that links the end of a microfilament to the membrane Once a cell comes to the end of groove/ridge, in the part of the cell that emerges onto the flat surface, this linear organisation of the microfilaments to the direction of the groove is lost, as the microfilaments reform to a configuration more like that shown in Figure 5.11 (a) [54, 66] From the experiments we can see that cells move faster and are aligned better on 20µm grooves/ridges than cells on 12um ridges and 18um grooves This difference suggests that speed of cellular movement relates to the dimensions of the micro 78 CHAPTER DISCUSSION AND SUMMARY OF GEOMETRIC INFLUENCE ON CELL BEHAVIOUR features on the surface Endothelial cells change their shapes when they are aligned on the grooves and ridges; this is also why they move faster in that direction We hypothesize that cells have more suitable contact areas and constraints when they migrated on 20 µm grooves/ridges than the 12 µm ridges and 18 µm grooves 79 Chapter Conclusion and further development 6.1 Conclusion The endothelial cell behaviour studies on the micro grooved and ridged substrates has proved that proton beam writing is a potential and suitable patterning technique to understand cell response associated with geometrical constraints In addition, it is efficient and relatively cheap to produce multiple scaffolds by the hot embossing technique We also find that when growing on the micro grooves and ridges, the endothelial cells elongate and orient along the channel direction On the scaffold which has 20 µm grooves and ridges, endothelial cells migrate faster than cells on plain PMMA surface, while on 12 µm ridges and 18 µm grooves, the cell speed is almost the same compared with cell speed on plain surface Cells on 20 µm groove/ridge substrate have better alignment than those on 12 µm ridge, 18 µm groove substrate More studies are needed to confirm our hypothesis that the better cells are aligned with micro grooves/ridges, the faster they are able to move 6.2 Further development 6.2.1 Cells migration on PMMA dimensional grooves and ridges substrates with different The assumption that the better the endothelial cells are aligned with the micro grooves and ridges, the faster they move, can be tested if a substrate shown in Figure 6.1 which has grooves and ridges with different dimensions, like µm, 15 µm, 25 µm and 35 µm is fabricated to culture endothelial cells The results will be especially useful to prove the correlation between cell speed and orientation Since endothelial cells are 80 CHAPTER CONCLUSION AND FURTHER DEVELOPMENT the explorers for blood vessel formation, the understanding of this study will also contribute to the inhibition or stimulation of angiogenesis which will be essential for organ culture or curing many diseases, like heart disease, ulcer, diabetes and tumor etc grooves / ridges 25 µm µm 15 µm 35 µm Figure 6.1 Schematic view of PMMA substrate with different dimensional grooves/ridges 6.2.2 Endothelial cell gene expression associated with micro grooves and ridges Studies on the responsive genes of human endothelial cells will be performed and their roles in EC behaviour associated with geometric constraints will be analyzed from the gene function information available from the literature and the human genome project Gene array, differential display and cDNA (complementary DNA) library screening will be used to identify both known and novel differentially expressed genes This data will further elucidate the genetic events regulating capillary tube formation in a 3D scaffold environment [67] This gene expression study is dependent on producing a large number of endothelial cells which will provide enough genes for analysis Therefore a large area Ni stamp with a micro grooved and ridged substrate area up to 1×1 cm2 will be more preferred than the previous one with 5×5 mm2 pattern By means of the multiple scaffolds with suitable dimensional grooves and ridges, novel genes can potentially be identified and 81 CHAPTER CONCLUSION AND FURTHER DEVELOPMENT their functional role in endothelial cells as well as angiogenesis will promote further studies in the continuation of this project 82 Bibliography [1] J.L Sanchez, G Guy, J.A van Kan, T Osipowicz and F Watt, Proton micromachining of substrate scaffolds for cellular and tissue engineering, Nucl Instr and Meth B 158 (1999) 185-189 [2] N Barrera, Tissue Engineering, www: http://serendip.brynmawr.edu/biology/b103/f00/web3/barrera3.html [3] J Marsh, Angiogenesis: Trailblazer of the Body or Harmful caner-causing agent? 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SCAFFOLDS FOR CELL BEHAVIOUR STUDIES ZHANG FANG (B.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSICS NATIONAL... conclusion of this project: proton beam micromachined scaffolds for cell behaviour Some suggestions for further development are discussed Chapter Fabrication of 3D scaffolds using p -beam writing... ……………………….………….5 Fabrication of 3D scaffolds using p -beam writing 2.1 Centre for Ion Beam Applications and instrumentations…….…………… 2.2 Proton Beam Interactions with materials……………….……………… 10 2.3 Proton beam