The Role of Cell-Substrate Interactions in ECM Remodeling, Migration, and the Formation of Multicellular Structures Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By James William Reinhardt B.E. Graduate Program in Biomedical Engineering The Ohio State University 2014 Dissertation Committee: Dr. Keith Gooch, Advisor Dr. Richard Hart Dr. Samir Ghadiali Dr. Peter Anderson ©Copyright by James William Reinhardt 2014 Abstract Active, mechanical interactions between cells and their extracellular matrix (ECM) are essential for ECM remodeling and cell migration, two behaviors that support diverse biological processes including embryonic development, wound healing, fibrosis, and cancer progression. Cell-populated reconstituted type I collagen hydrogels are often used as a model system in which to study ECM remodeling and cell migration in vitro. Unfortunately, this system has limitations since it is not possible to independently control individual microstructural properties. This limitation has inspired theoretical models as an alternative way to study ECM remodeling and migration. However, so far no single approach has been able to capture both fibril-level detail and dynamic cell traction force. With the goal of creating a model that can capture both fibril-level detail and dynamic cell traction force, we developed an agent-based model of cell-mediated collagen compaction and migration. With this model we observed behaviors that were not programmed, but emerged from simple rules for cell-fibril interactions. Among these, our model qualitatively reproduced remodeling commonly seen in cell-populated collagen gels: macroscopic, pericellular, and intercellular compaction. Similar to experimental observations, matrical tracks formed between pairs of cells before directional migration ii of nearby cells toward on another. Cells also exhibited durotaxis in the absence of force- strengthening of cell-matrix bonds. This suggests that durotaxis may not involve a complicated mechanism, but may simply be an emergent behavior, the cumulative result of analogous, simple, cell-matrix interactions. We then further developed this model to make collagen fibrils more physically-realistic by modeling them as elastic rods and using parameter values obtained from the experimental literature. Subjecting our fibrils to loading conditions that created tension and bending demonstrated that our simulated fibrils approximated their analytically-predicted deformations and shapes. Modeling of cross-links was also improved to more closely approximate their expected behavior. Together, these changes resulted in a more intentionally constructed network that was stress-free in the absence of external perturbation. Model development culminated in model validation against two sets of data from the experimental literature. Similar to experimental data our computational model showed that collagen displacement decreased linearly with increasing distance from a single cell and that the compaction of collagen between pairs of cells was inversely related to cell-cell distance. In other work we used in vitro experiments to show that PANC-1 cells did not exhibit directed migration toward a central cluster. Using agent-based modeling, we then were able to show that clustering may occur simply due to random migration, relatively high cell-cell adhesion, and low cell-matrix adhesion. Separately, I have contributed to the refinement of an experimental system used to study cell-matrix interactions that provides an alternative to reconstituted type I collagen. iii Dedication To my wife Anne, for her endless love and support. iv Acknowledgments First, I would like to thank my advisor, Dr. Keith Gooch for his mentorship, thoughtful attention to professional development, and admirable commitment toward student success. I would also like to thank my dissertation and candidacy committee members, Dr. Richard Hart, Dr. Samir Ghadiali, Dr. Peter Anderson, Dr. Nicanor Moldovan, Dr. Vincent Pompili, and Dr. John Lannutti. I would like to further thank Dr. Rich Hart and Dr. Samir Ghadiali for their countless letters of recommendation as well as Dr. Vincent Pompili, an interventional cardiologist, who served as my clinical mentor during my HHMI Fellowship. I would also like to thank Dr. Ginny Bumgardner and Dr. Joanna Groden for the valuable opportunity afforded me in the HHMI OSU MED into GRAD Fellowship Program. In addition, I would like to extend my appreciation to all the members of the Moldovan Lab for their hospitality, the training I received, and exposure to different areas of research during the time I volunteered with them. I also want to acknowledge my fellow lab members for their friendship and for their critical feedback on my work. I especially would like to thank Melanie Senitko, for all her help during my time in the BME Department. This research was supported with funds from NSF (CMMI- 0928739, CBET-1067481, CBET/BMMB-1314291), the HHMI OSU MED into GRAD Fellowship Program and the Department of Biomedical Engineering. v Vita June 2002.......................................................Bedford High School May 2006.......................................................B.E. Biomedical Engineering, Vanderbilt University 2010 to present ..............................................Graduate Research Associate, Department of Biomedical Engineering, The Ohio State University Publications 1. Reinhardt, J. W., & Gooch, K. J. (2014). Agent-Based Modeling Traction Force Mediated Compaction of Cell-Populated Collagen Gels Using Physically Realistic Fibril Mechanics. Journal of Biomechanical Engineering, 136(2), 021024. 2. Reinhardt, J. W., Krakauer, D. A., & Gooch, K. J. (2013). Complex Matrix Remodeling and Durotaxis Can Emerge from Simple Rules for Cell-Matrix Interaction in Agent-Based Models. Journal of Biomechanical Engineering, 135(7), 71003, 1–10. vi Fields of Study Major Field: Biomedical Engineering vii Table of Contents Abstract............................................................................................................................... ii Dedication.......................................................................................................................... iv Acknowledgments ...............................................................................................................v Vita .................................................................................................................................... vi Publications........................................................................................................................ vi Fields of Study.................................................................................................................. vii Table of Contents............................................................................................................. viii List of Tables .......................................................................................................................x List of Figures.................................................................................................................... xi Chapter 1: Introduction........................................................................................................1 Chapter 2: Complex Matrix Remodeling and Durotaxis Can Emerge from Simple Rules for Cell-Matrix Interaction in Agent-Based Models .........................................................20 Chapter 3: Agent-Based Modeling Traction Force Mediated Compaction of Cell- Populated Collagen Gels Using Physically Realistic Fibril Mechanics ............................53 viii Chapter 4: Validating an Agent-based Model of Cell Traction Force Induced Collagen Network Remodeling.........................................................................................................92 Chapter 5: Pancreatic Epithelial Cells Form Islet-Like Clusters In The Absence Of Directed Migration...........................................................................................................117 Chapter 6: Conclusions and Future Directions................................................................146 Appendix A: Parameters Explored During Model Development....................................151 References........................................................................................................................152 ix List of Tables Table 2.1 A list of parameter values..................................................................................39 Table 3.1 A summary of values for the elastic modulus of a collagen fibril reported the literature.............................................................................................................................77 Table 3.2 A summary of values for the diameter of a collagen fibril reported in the literature.............................................................................................................................78 Table 5.1 Mean chemotactic index values.......................................................................134 Table A.1 A list of parameter values...............................................................................151 x List of Figures Figure 2.1 Graphical representations of model details. A. Large view of a cell composed of 30 “membranes”, 1 “nucleus” (center), 30 membrane-nucleus links, and inter- membrane links (not visible).