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Copyright by Mary Gazell Mapili Call 2007 The Dissertation Committee for Mary Gazell Mapili Call Certifies that this is the approved version of the following dissertation: Microfabrication of Spatially-Patterned, Polymer Scaffolds for Applications in Stem Cell and Tissue Engineering Committee:
Krishnendu Roy, Supervisor Shaochen Chen Wolfgang Frey Anshu Mathur Christine Schmidt Microfabrication of Spatially-Patterned, Polymer Scaffolds for Applications in Stem Cell and Tissue Engineering by Mary Gazell Mapili Call, B.S.; M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August 2007 Dedication I would like to dedicate this dissertation and Ph.D. studies to my husband, Seth, my family, and close friends for their unconditional love, support, and constant encouragement while growing up and throughout the years at the University of Texas. v Acknowledgements I would like to thank Dr. Krishnendu Roy, my Ph.D. adviser, for giving me the invaluable opportunity to research in his lab over the past five years. My experience at the University of Texas will always be a memorable one. You taught me how to correctly approach science and served as an excellent mentor for balancing life in general with the challenges of research and courses. Thank you for your guidance and constant encouragement throughout the process of my doctoral studies. My sincere thanks go to Dr. Shaochen Chen for giving me the opportunity to work in collaboration in using their stereolithography systems for creating the scaffolds. I would also like to thank Yi Lu for his help in using these systems and for our numerous research discussions. Thanks to Curt Deister for his help with tissue histology. I would like to also thank Dr. Andrea Gore and Sarah Dickerson from the College of Pharmacy for their help with real time PCR. Thanks to the rest of my committee members, Drs. Wolfgang Frey, Anshu Mathur, and Christine Schmidt, for their advice and invaluable input on the direction of my Ph.D. project. I would also like to thank Julie Rytlewski, Myung Hee Kim, and Sindura Bandi, my undergraduate research assistants, who all helped me with my research from start to finish. Especially to Julie, I would like to thank her for her commitment, sincere interest, and eagerness to learn all aspects of my project. Thanks to Sabia Taqvi, Sonia Kumar, and Hunter Lauten for always being such wonderful friends throughout these past five years. Everyone else in the lab, I appreciate all of our discussions and for making the lab environment an enjoyable place to work. I would also like to thank everyone at the vi ICMB Core Facility for their help with instruments: specifically Dr. Angela Bardo and John Mendenhall for their help with confocal and SEM microscopy; Shawn Tucker and Cecil Harkey for their help in using the Biomek robots and real time PCR instruments. My special thanks also go to my parents, Rey and Dia, and immediate family, whose never-ending prayers and encouragement have always reminded me that loved ones are the most important in life. Most of all, I would like to thank my husband and best friend, Seth, for his unconditional love, support, and patience through even the most difficult times of being a graduate student. vii Microfabrication of Spatially-Patterned, Polymer Scaffolds for Applications in Stem Cell and Tissue Engineering Publication No._____________ Mary Gazell Mapili Call, Ph.D. The University of Texas at Austin, 2007 Supervisor: Krishnendu Roy Tissue engineering is a recently developed field that combines material science, cell biology, and engineering to create or improve functional tissues/organs. The field of tissue engineering has progressed from a fledgling science to an emerging technology, in large part due to parallel advances in the application of biomaterials and understanding stem cell behavior. Current studies have evaluated certain types of natural and synthetic biomaterials for feasibility of replicating the physio-chemical microenvironments of stem cells. Furthermore, technologies derived from micro-machining and solid free-form fabrication industries have utilized these biomaterials to create scaffolds that resemble tissue-like structures. Recent scaffold fabrication methods have attempted to overcome certain challenges in engineering tissues and organs. One of the fundamental limitations in current tissue engineering efforts has been the inability to develop multiple tissue types (i.e. bone, cartilage, muscles, ligaments) within a single scaffold structure in a pre- designed manner. The differentiation of multiple cells within a three-dimensional (3D) viii scaffold using a single stem cell population has yet to be developed due to challenges in integrating various biochemical factors in a spatially-patterned method. This dissertation discusses scaffold micro-fabrication techniques that use layer- by-layer, ultraviolet-based (UV) stereolithography systems. These approaches in micro- fabricating scaffolds provide an optimal, biomimetic environment for the pre-patterned differentiation of mesenchymal stem cells into skeletal-type tissues. We demonstrated both laser-based and digital micromirror device-based stereolithography systems for creating intricate scaffold architectures with multiple bio-factors encapsulated in pre- determined regions. We showed that micro-stereolithography has the powerful capability of building 3D complex scaffolds with specific pore sizes and shapes in a layer-by-layer fashion using photo-crosslinkable monomers. These polymer-based scaffolds were functionalized with specific signaling proteins to create a biomimetic niche in which stem cells can respond, attach, and differentiate. The ultimate goal of this project is to integrate novel concepts of micro-manufacturing along with polymer-controlled release kinetics and stem cell biology to attain pre-designed architectures of tissue structures. ix Table of Contents List of Tables ....................................................................................................... xiii List of Figures ...................................................................................................... xiv C HAPTER ONE 1 Introduction: Specific Aims and Overview .............................................................1 1.1 Introduction...............................................................................................1
1.2 Specific Aims............................................................................................4 1.2.1 Aim 1: To employ a layer-by-layer stereolithography technique to microfabricate scaffolds ...............................................................4 1.2.2 Aim 2: To study the covalent functionalization of specific ECMs on microfabricated scaffolds..............................................................4 1.2.3 Aim 3: To study the growth and osteogenic differentiation of MSCs within polymer scaffolds...............................................................5 1.3 Overview...................................................................................................6
1.4 References.................................................................................................7 C HAPTER T WO 8 Background and Significance ..................................................................................8 2.1 Current Trends in Tissue Engineering ......................................................8
2.2 Stem Cells in Tissue Engineering.............................................................9
2.3 Sources, Characterization, and Differentiation Lineages of MSCs ........10
2.4 Microfabrication of Tissue Engineering Scaffolds .................................12
2.5 Scaffold Biomaterials used for Studying MSCs .....................................15
2.6 Modifications to Scaffold Biomaterials to Mimic Cellular Niche..........17
2.7 Mechanism of In Vivo and In Vitro Bone Formation .............................26
2.8 References...............................................................................................34
x C HAPTER T HREE 47 Laser-layered Microfabrication of Spatially Patterned Functionalized Tissue Engineering Scaffolds...................................................................................47
3.1 Introduction.............................................................................................47
3.2 Materials and Methods............................................................................50 3.2.1 PEGDMA Solutions and Photoinitiator .....................................50
3.2.2 Pre-treatment of Glass Coverslips ..............................................51
3.2.3 Microfabrication of PEG Scaffolds using a Frequency-Tripled Nd:YAG Laser ............................................................................51 3.2.4 RGD and Heparin Conjugation to PEG ......................................52
3.2.5 Cell Culture.................................................................................53
3.2.6 Cell Attachment to RGD-modified Hydrogels ...........................54
3.2.7 FGF-2 Binding to Heparin-modified PEG..................................55 3.3 Results.....................................................................................................56 3.3.1 Microfabrication of Single and Multi-layered Scaffolds ...........56
3.3.2 Pre-designed Spatial Patterning of Scaffold Structures ..............57
3.3.3 Heparin-functionalization Sequesters FGF-2 .............................58
3.3.4 Scaffolds are Conducive of Cell Attachment..............................59 3.4 Discussion...............................................................................................60
3.5 References...............................................................................................75 C HAPTER F OUR 80 A DMD-based System for the Microfabrication of Complex, Spatially Patterned Tissue Engineering Scaffolds .......................................................................80
4.1 Introduction.............................................................................................80
4.2 Materials and Methods............................................................................83 4.2.1 PEGDA Solutions and Photoinitiator ........................................83 4.2.2 DMD

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