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Molecular and Cellular Mechanisms of the Angiogenic Effect of Poly(Methacrylic Acid-Co-Methyl Methacrylate) Beads

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Molecular and Cellular Mechanisms of the Angiogenic Effect of Poly(Methacrylic Acid-Co-Methyl Methacrylate) Beads Molecular and Cellular Mechanisms of the Angiogenic Effect of Poly(methacrylic acid-co-methyl methacrylate) Beads by Lindsay Elizabeth Fitzpatrick A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto © Copyright by Lindsay Elizabeth Fitzpatrick 2012 Molecular and Cellular Mechanisms of the Angiogenic Effect of Poly(methacrylic acid-co-methyl methacrylate) Beads Lindsay Elizabeth Fitzpatrick Doctorate of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto 2012 Abstract Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF- α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, ii suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles. iii Acknowledgments I thank my supervisor, Prof. Michael Sefton, for the guidance, support and encouragement he has provided me over the past six years. His patience and steady encouragement was very much appreciated and it has been a privilege to have his mentorship. I thank my committee members, Yu-Ling Cheng and Dan Dumont, for their time, advice and encouragement. I also thank Brad Saville, Milica Radisic and Peter Zandstra for their time and advice as part of my supervisory and PhD transfer exam committees. I would also like to thank Heather Sheardown, from McMaster University, for the research opportunities she gave me as an undergraduate student and her mentorship over the past eleven years. I thank Sasha, Josephine and Laura for their help and shared ideas while working together on the MAA project. I would also like to thank everyone in the Sefton group for their help over the years, Rohini, Mark, Brendan, Omar, Dean, Ema, and Derek. In particular, I would like to thank Chuen Lo for his invaluable skill and help with the wound healing model and venipuncture, and Carl Walkey and Derek Voice for repeatedly donating blood for my primary macrophage experiments. I acknowledge funding from the Canadian Institutes of Health Research (CIHR), National Science and Engineering Research Council (NSERC), Ontario graduate scholarship (OGS), and the University of Toronto (Doctoral Completion Award). Finally, I would like to thank my family and friends for their support. My heart-felt thanks go out to my parents, Norma & Wayne, for being such wonderful people whom I will always look up to, and for always being there to support and encourage me. Thank you to Erin & Mike, Jamie, Jaclene & Max, and John, Maya & Kyle for brightening my days and the constant support; you are the best family one could want. Thank you Chris & Dave for all your support, and for being such great examples of living life to the fullest. Finally and most of all, I want to thank my husband Scott; going through graduate school with you has made every part of it better. Thank you for understanding the highs and lows of research, pushing me to succeed and keeping me balanced. Congratulations on completing you PhD. iv Table of Contents Abstract .......................................................................................................................... ii Acknowledgments ........................................................................................................ iv Table of Contents .......................................................................................................... v List of Tables ................................................................................................................ xi List of Figures ............................................................................................................. xiii List of Abbreviations ................................................................................................. xxii Chapter 1: Introduction ................................................................................................. 1 1 Poly(methacrylic acid-co-methyl methacrylate) beads ................................................. 1 2 Scope of thesis .................................................................................................................. 2 3 Specific Aims ..................................................................................................................... 2 4 Thesis overview ................................................................................................................ 3 5 References ......................................................................................................................... 5 Chapter 2: Angiogenesis and scaffold vascularization ............................................ 6 1 Introduction ....................................................................................................................... 6 1.1 The native vascular network ........................................................................................ 6 1.2 Angiogenesis: the formation of new blood vessels ...................................................... 6 2 Vascularization of biomaterial scaffolds ...................................................................... 10 2.1 Growth factor delivery ................................................................................................ 12 2.1.1 Single growth factor delivery ............................................................................... 12 2.1.2 Multiple growth factor delivery ............................................................................. 14 2.2 Cell delivery and transplantation ................................................................................ 15 2.2.1 Scaffolds seeded with cells ................................................................................. 15 2.2.2 Modular tissue engineering ................................................................................. 18 2.2.3 Genetically Modified Growth Factor-Producing Cells .......................................... 20 2.2.4 Scaffold-free cell delivery approaches ................................................................ 21 2.3 In situ vascularization with endogenous cells ............................................................ 23 2.4 Scaffold prevascularization ........................................................................................ 25 2.5 Decellularized scaffolds ............................................................................................. 27 v 2.6 Angiogenic biomaterials ............................................................................................. 29 2.7 Microfabrication methods ........................................................................................... 31 3 Limitations and the road ahead ..................................................................................... 34 4 Summary .......................................................................................................................... 35 5 References ....................................................................................................................... 36 Chapter 3: Wound healing, diabetes and the foreign body reaction ...................... 45 1 Cutaneous wound healing ............................................................................................. 45 2 Wound healing and diabetes ......................................................................................... 50 3 The
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