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Baker Uta 2502D 12310.Pdf (2.933Mb) THE PIVOTAL ROLE OF FIBROCYTES ON FOREIGN BODY REACTIONS by DAVID WILLIAM BAKER Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON August 2013 Copyright © by David W Baker 2013 All Rights Reserved ii Acknowledgements I would like to take this opportunity to thank a number of individuals who have helped to guide and shape my development as a researcher here at UT Arlington. I would first like to thank my dissertation committee, Dr. Kytai Truong Nquyen, Dr. Chi-Chun Tsai, Dr. Ramesh Saxena, Dr. Lei Shi, and Dr. Liping Tang, for their guidance, insight, and suggestions during the development of this dissertation. I would like to further express my gratitude to my advisor Dr. Liping Tang for his encouragement, his demand for excellence, and his wisdom in guiding prior works and the outline of this dissertation. In addition, I would like to thank several members of the regenerative medicine lab group whose assistance has been instrumental for the development of this dissertation and my prior works. I would like to acknowledge Dr. Hong Weng and Dr. Jinhui Shen for their assistance with animal studies. In addition, I would also like to acknowledge the work of Dr. Jun Zhou for his fabrication of the imaging probes and other microparticles used in this work. My sincerest thanks to Dr. Yi-Ting Tsai for her technical assistance with, and time spent on, in vivo imaging, as well as Dr. Ashwin Nair for his patience and insight in discussing problems and solutions. Lastly from the laboratory I would like to thank Dr. Paul Thevenot for his help and guidance in getting me started during the early part of my research. Finally, I would like to thank my family for their support and understanding. To my wife, Jordan Baker, I would like to express my deepest appreciation for her patience and companionship. I would also like to thank my parents, David and Janice Baker, who have always believed in me. Without the support and love of my family this work would not have been possible. July 12, 2013 iii Abstract THE PIVOTAL ROLE OF FIBROCYTES ON FOREIGN BODY REACTIONS David W Baker, PhD The University of Texas at Arlington, 2013 Supervising Professor: Liping Tang Fibrocytes are circulating connective tissue precursor cells that were first described in 1994 by Bucala et al. as mediators of the innate immune response [1]. Recently the notion of a fibrocyte has not only gained widespread acceptance, but these cells have been identified to participate in a number of disease and pathologic states including aberrant wound repair, fibrosis of organs, cardiovascular disease, and even normal aging. Despite these advancements, little was known about the response of these dynamic cells to biomaterial implants and foreign body reactions. The main focus of my thesis work has therefore focused on investigating fibrocyte-mediate responses, and identifying cellular interactions and the role of fibrocytes during the foreign body response. Our results support that fibrocytes are vital and primary contributors to collagen deposition and expansion of encapsulating fibrotic matrix and scar formation. This thesis summarizes our efforts on the development of several strategies to alleviate fibrocyte driven fibrotic responses. In the first approach macrophage interactions were investigated through the development of a dual, near infrared, imaging probe modality to non-invasively investigate the role of macrophage polarization on implant-associated fibrotic tissue reactions in real time. Two polyethylene glycol based probes were fabricated to detect iv M1 and M2 macrophages around biomaterial implants and infection. Using antagonistic compounds to block specific cytokine activators, we find that fibrocyte accumulation is linked to macrophages and responsive to transforming growth factor beta (TGF-β) signaling related to alternative macrophage activation. In addition, we found that stabilizing mast cells drastically reduce fibrocyte and fibrocyte-derived collagen production around PLGA implants. Deeper investigation using mast cell depleted and reconstituted mice irrevocably demonstrates that the degree of mast cell activation can dictate subsequent fibrocyte responses. In addition to cellular mediate mechanisms, micropillar implants are used to passively modify fibrocyte activation. Very interestingly fibrocytes were found to be more responsive to topographical changes, specifically spacing and height, of PDMS micropillar implants than fibroblasts or macrophages. The cellular responses were found to correlate with the histological outcomes of fibrosis including granulation tissue formation and collagen production. Lastly, it has recently been shown that fibrocytes possess a multi-potency for several lineages including adipocytes. The potential for differentiation however had not been investigated in vivo. Through the use of mini- osmotic pumps, the potential for directing fibrocyte differentiation in vivo was assessed by delivering specific differentiation agents. The results presented in this thesis provide strong support for novel strategies to resolve or treat complications of the foreign body response and excessive fibrosis to existing medical implants. v Table of Contents Acknowledgements .............................................................................................................iii Abstract .............................................................................................................................. iv List of Illustrations ............................................................................................................... x List of Tables ......................................................................................................................xii Chapter 1 Introduction......................................................................................................... 1 1.1 Background ............................................................................................................... 1 1.2 Acute Inflammatory Responses and Fibrosis ........................................................... 3 1.2.1 Macrophage Polarization and Tissue Responses ............................................. 4 1.2.2 Fibroblasts and Myofibroblasts .......................................................................... 6 1.2.3 Fibrotic Tissue Formation .................................................................................. 7 1.3 Fibrocytes ................................................................................................................. 8 1.3.1 Origin and Properties of Fibrocytes ................................................................... 8 1.3.2 Identification and Isolation of Fibrocytes ........................................................... 9 1.3.3 Fibrocytes in Disease Models and Wound Healing ......................................... 10 1.4 Strategies and Aims of This Dissertation ............................................................... 11 1.4.1 Mast Cells Responses and the Alteration of Fibrocyte Recruitment ............... 12 1.4.2 Macrophage Polarization and the Influence on Fibrocytes ............................. 12 1.4.3 Topographical Cues and Passive Alteration of Fibrocyte Responses ................................................................................................................ 13 1.4.4 Fibrocyte to Adipocyte Differentiation and the Fibrotic Response .................. 13 Chapter 2 Fibrocytes and Implant-Mediated Fibrotic Tissue Responses ......................... 14 2.1 Introduction ............................................................................................................. 14 2.2 Fibrocytes at the Interface and their Role in Biomaterial-mediated Responses .................................................................................................................... 14 vi 2.2.1 Purpose ........................................................................................................... 14 2.2.2 Materials and Methods .................................................................................... 15 2.2.3 Results ............................................................................................................. 18 2.2.4 Conclusion ....................................................................................................... 25 2.3 Mast Cell Stimulation Dictates Fibrocyte and Fibrotic Responses ......................... 26 2.3.1 Purpose ........................................................................................................... 26 2.3.2 Materials and Methods .................................................................................... 27 2.3.3 Results ............................................................................................................. 29 2.3.4 Discussion ....................................................................................................... 32 2.4 Mast Cell Deficiency and Reconstitution on Implant-Associated Fibrotic Reactions ......................................................................................................... 32 2.4.1 Purpose ........................................................................................................... 32 2.4.2 Materials and Methods .................................................................................... 33 2.4.3
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