Development of Biomimetic Materials for Enhanced Maturation of Engineered Cardiac Tissue
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DEVELOPMENT OF BIOMIMETIC MATERIALS FOR ENHANCED MATURATION OF ENGINEERED CARDIAC TISSUE by Alexander Jay Hodge A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 8, 2016 Copyright 2016 by Alexander Jay Hodge Approved by Elizabeth A. Lipke, Chair, Mary and John H. Sanders Endowed Associate Professor of Chemical Engineering Mark E. Byrne, Daniel F. & Josephine Breeden Associate Professor of Chemical Engineering Jin Wang, Walt and Virginia Woltosz Endowed Associate Professor of Chemical Engineering Rajesh Amin, Assistant Professor of Drug Discovery and Development Abstract The creation of cardiac tissue constructs holds potential in the development of clinical and diagnostic platforms, including the creation of novel treatments for patients with heart disease and cost-effective platforms for drug discovery. Many materials and techniques have been investigated for use in creating engineered cardiac tissue; however, formation of engineered myocardium in vitro, particularly when derived from pluripotent cell sources, often results in the creation of cardiac tissue or cardiomyocytes with functional properties that are markedly different from the native adult myocardium. The use of biomimetic materials, those materials that emulate one or more properties of native tissue, may be necessary for obtaining cardiac tissue demonstrating physiologically- relevant electrophysiological properties. In this document, the ability of biomimetic materials to drive pluripotent stem cell derived cardiac tissue maturation has been investigated. This document first introduces the overall challenges associated with heart disease and drug discovery, which emphasizes the need for biomimetic material-based solutions. A review on current biomaterial-based methods for treating heart disease, as well as background on work utilizing biomimetic materials for cardiac regeneration is presented, as well as current methods for evaluating functional maturity (with an emphasis on quantifying electrophysiology) of cardiomyocytes and cardiac tissue. Next, the design and implementation of an optical mapping platform for assessing calcium wave velocity and calcium transient duration is presented. This combined macroscopic and microscopic ii imaging system was capable of capturing optical mapping data from samples ranging 21 to 0.5 mm in size at rates in excess of 500 frames per second. After, a study utilizing biomimetic nitric oxide donor S-nitrosocysteine (CysNO) to direct maturation of stem cell derived cardiomyocytes (SC-CMs) is discussed. Differentiating embryoid bodies treated with CysNO developed greater numbers of faster contracting cardiomyocytes in comparison to controls; dissociated SC-CMs treated with CysNO demonstrated improvement in calcium handling via faster calcium transient velocities and shorter calcium transient durations. Next, a study utilizing the conductive polymer polypyrrole (PPy) is presented which evaluated cardiomyocyte survival and development when grown on the novel material. HL-1 cardiomyocytes cultured on PPy-polycaprolactone (PPy-PCL) films demonstrated greater numbers of connexin-43 positive cells in conjunction with faster calcium transient velocities and shorter calcium transient durations in comparison to cells grown on PCL alone. Finally, the response of SC-CMs differentiated on the surface of the novel PPy-PCL material is reported; PPy-PCL supported the growth and viability of dissociated SC-CMs at a comparable number to PCL alone. Together, these studies provide insight into how novel biomimetic materials can be implemented in the creation of functionally-mature cardiac tissue. Additionally, this work demonstrates how a custom optical mapping apparatus can be adapted to assess cardiac maturation among a gamut of different platforms. The ability to direct and evaluate maturity of differentiating cardiomyocytes contributes to the development of clinical and research platforms for improving treatment of heart disease. iii Acknowledgements Assembly of this document has been a challenging endeavor that reflects my time, skills, and patience while at Auburn University. Although I would like to claim sole responsibility for all work contained within the manuscript, I cannot alone take credit for this effort. In truth, there have been a number of individuals which have assisted me throughout the course of my graduate studies. These individuals include a number of graduate students that personally invested their time and intellect to assist with many facets of my project: Aaron Seeto, Samuel Chang, Petra Kerscher, and Shantanu Pradhan. During my time at Auburn University, I had the opportunity to mentor several undergraduate students that all contributed in part to the completion of my program. I would like to thank: Benjamin Spearman, James Morris, Wesley Grove, John Porter, and Alec Castinado for their hard work and dedication. I must also thank my PI, Dr. Elizabeth Lipke, for her continued dedication and guidance throughout my project, as well as my other committee members and faculty that have devoted time and knowledge to my work. Furthermore, I could not have completed this document without the support of my friends and family. I would like thank my parents, Pat and Dale Hodge, and my in-laws, Katherine and Jerry Shearin, for all the support they have given me during this process. Lastly, I would like to dedicate this document to my wife Laura for her unyielding support, patience, and motivation for me during this challenging time in my life. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv List of Figures ................................................................................................................... vii List of Tables .................................................................................................................... xii List of Abbreviations ....................................................................................................... xiii 1. THE NEED FOR ENGINEERED MYOCARDIUM ................................................. 1 1.1. Current methods and challenges in the treatment of heart disease .......................2 1.2. The use of engineered myocardium in diagnostic applications ............................5 2. METHODS FOR REPAIRING CARDIAC FUNCTION........................................... 9 2.1. Biomaterials for myocardium regeneration...........................................................9 2.2. Properties of biomimetic materials for cardiac regeneration ..............................12 2.3. Classes of Biomaterials .......................................................................................19 2.4. Cell types for cardiac regeneration......................................................................24 2.7. Biomimetic materials for injectable cardiac therapies ........................................35 2.8. Use of Nanotechnology for Cardiac Regeneration .............................................37 2.9. Ongoing Challenges for Cardiac Tissue Regeneration .......................................41 2.10. The Need for Electrophysiological Characterization of Cardiac Tissue .............42 2.11. Techniques for measuring electrophysiology in cardiomyocytes .......................45 2.12. Considerations for the design of an optical mapping platform ...........................50 2.13. Conclusions .........................................................................................................51 3. DEVELOPMENT OF AN OPTICAL MAPPING PLATFORM FOR ELECTROPHYSIOLOGICAL ANALYSIS OF CULTURED CARDIOMYOCYTES . 69 3.1. Introduction .........................................................................................................69 3.2. Materials and methods ........................................................................................72 3.3. Results .................................................................................................................80 3.4. Discussion ...........................................................................................................88 v 3.5. Conclusions .........................................................................................................93 4. ENHANCED STEM CELL-DERIVED CARDIOMYOCYTE DIFFERENTIATION IN SUSPENSION CULTURE BY DELIVERY OF NITRIC OXIDE USING S- NITROSOCYSTEINE ...................................................................................................... 96 4.1. Introduction .........................................................................................................96 4.2. Materials and Methods ......................................................................................100 4.3. Results ...............................................................................................................108 4.4. Discussion .........................................................................................................125 4.5. Conclusions .......................................................................................................132 5. NOVEL ELECTROACTIVE SUBSTRATE SUPPORTS GROWTH AND PROLIFERATION OF HL-1 CARDIOMYOCYTES ..................................................