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Nanoscalar Modifications to Polymeric Tissue Engineering Scaffolds: Effect on Cellular Behavior

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Nanoscalar Modifications to Polymeric Tissue Engineering Scaffolds: Effect on Cellular Behavior NANOSCALAR MODIFICATIONS TO POLYMERIC TISSUE ENGINEERING SCAFFOLDS: EFFECT ON CELLULAR BEHAVIOR DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Heather M. Powell, M.S. ***** The Ohio State University 2004 Dissertation Committee: Professor John J. Lannutti, Adviser Approved by Professor Douglas A. Kniss Professor Derek Hansford Professor David Rigney _______________________________ Adviser Professor William Brantley Graduate Program in Materials Science and Engineering ABSTRACT Polymeric scaffolds provide a surface that can facilitate cell growth and tissue morphogenesis. Of particular interest is the role of nanoscalar features on cell behavior. Nanoscale topographies can be generated on two-dimensional polymeric substrates via reactive ion etching. The magnitude and morphology of the resultant surfaces can be tailored by varying the gas media, etching time and power used. Nanofibrillar surfaces were produced on polyethylene terephthalate films via oxygen-plasma etching. These nanofibrils were dimensionally similar to collagen fibers. Cells cultured on nanofibrillar surfaces were shown to have a disrupted cytoskeleton, lower levels of cell-substrate signaling, reduced strength of adhesion and an inhibition of lipid droplet coalescence. The results suggest that cells can detect nanoscalar surface topographies and alter their function in response to these environmental stimuli. While nanofibrillar surfaces can be considered pseudo-three dimensional, they cannot produce 3-D cell structures. Thus truly three dimensional scaffolds must be fabricated to determine the role of nanoscalar fibers on cell organization and function. Electrospinning was employed to generate 3-D meshes of polycaprolactone, a common biodegradable polymer. These nonwoven meshes were comprised of 500 nm fibers with an average pore size of 5 µm. In addition to forming mats of nonwoven fibers, ii electrospinning technology can also produce tubular scaffolds. These tubular scaffolds were seeded with human vascular smooth muscle cells and cultured for two days. After 2 days in culture, cells assumed a helical orientation around the lumen of the tube, an architecture which closely mimics natural blood vessels. Thus electrospun scaffolds facilitate the growth and organization of cell populations in a manner which imitates the natural tissue. iii Dedicated to Jon, Bill and Sharon for all their support. iv ACKNOWLEDGMENTS I wish to thank my adviser, Dr. John Lannutti and co-adviser, Dr. Douglas Kniss, for their advice on my research. Dr. Lannutti has been indispensable during the course of my study, providing the fundamental engineering and polymer principles. Dr. Douglas Kniss has provided an environment in which I have had the opportunity to increase my knowledge on cell biology immensely. The combination of their expertise has allowed me to research tissue engineering problems from initial scaffold fabrication to final cell seeded product. I also greatly acknowledge the members of the Perinatal Lab Group in Dr. Kniss’ lab for their technical support throughout the years. I specifically would like to thank Dr. Yubing Xie for sharing her knowledge of cell culture, Dr. William Ackerman for discussions on immunocytochemistry technique and lipid bodies, and Taryn Summerfield for help with the initial reactive ion etching. I am very grateful to have had to chance to work with all the members of the Lannutti group and the Perinatal Lab. Financial support from the National Defense Science and Engineering Graduate Fellowship program and the University Fellowship are acknowledged. v VITA August 23, 1978………………………….Born in Warren, Ohio August 1999……………………………...B.S. Geology/Paleobiology Emphasis Bowling Green State University August 2000-present……………………. Graduate Research Associate The Ohio State University September 2000 – September 2001……...University Fellow The Ohio State University September 2001-present…………………National Defense Science and Engineering Graduate Fellow (NDSEG) The Ohio State University January 2003……………………………..M.S. Materials Science and Engineering The Ohio State University PUBLICATIONS Research Publications 1. Kohm, A., H. Powell, I. Wood, J. Dyce, and J. Lannutti “Apparent Skid Damage Controls Implantation Time” Journal of Biomedical Materials Research: Applied Biomaterials, 2004. 2. Wang, H., JK Lee, A. Moursi, D. Anderson, P. Winnard, H. Powell, and J. Lannutti. “Microstructural disassembly of calcium phosphates” Journal of Biomedical Materials Research 2004; 68A(1): 61-70. 3. Powell, H. and J. Lannutti “Nanofibrillar Surfaces Via Reactive Ion Etching” Langmuir 2003, 19: 9071-78. 4. Wood, I., H. Powell, A. Kohm, J.J. Lannutti and J. Dyce. “Evaluation of femoral head damage during canine total hip replacement: a comparison of four reduction techniques”. Veterinary Comparative Orthopaedics and Traumatology. 2003, 16: 184- 90. vi 5. Xie, Y., T. Sproule, Y. Li, H. Powell, JJ Lannutti, DA Kniss “Nanoscale modifications of PET polymer surfaces via oxygen-plasma discharge yield minimal changes in attachment and growth of mammalian epithelial and mesenchymal cells in vitro” Journal of Biomedical Materials Research 2002, 61(2):234-245. FIELD OF STUDY Major Field: Materials Science and Engineering Minor Field: Tissue Engineering vii TABLE OF CONTENTS Page Abstract…………………………………………………………………………………....ii Dedication…………………………………………………………………………….......iv Acknowledgements………………………………………………………………......……v Vita………...……………………………………………………………………………..vi List of Tables…………………………………………………………………………....xiii List of Figures…………………………………………………………………………...xiv Chapters: 1. LITERATURE REVIEW 1.1 Introduction................................................................................................................... 1 1.2 The body's natural scaffold……………………………………………………………3 1.3 Replacement Scaffolds: Material Selection.................................................................. 7 1.3.1 Degradable Polymers....................................................................................... 10 1.3.2 Non-degradable Polymers................................................................................ 12 1.3.2 Non-degradable Polymers................................................................................ 13 1.3.2 Non-degradable Polymers................................................................................ 14 1.4 Replacement Scaffolds: Scaffold Processing.............................................................. 15 1.4.2 Solvent Casting and Particulate Leaching ....................................................... 19 1.4.3 Gas Foaming .................................................................................................... 21 1.4.4 Membrane Lamination..................................................................................... 22 1.4.5 Melt Molding ................................................................................................... 22 1.5 Modification of Scaffolds ........................................................................................... 24 1.5 1 Surface Topography......................................................................................... 25 1.5.2 Drug Delivery .................................................................................................. 34 1.6 The Future of Tissue Engineering Scaffolds .............................................................. 38 References......................................................................................................................... 40 viii 2. CELLULAR RESPONSE TO NANOSCALE TOPOGRAPHY; EFFECT OF 20 NM HIGH PROTRUSIONS ....................................................................................................... 50 2.1 Abstract....................................................................................................................... 50 2.2 Introduction................................................................................................................. 51 2.3 Materials and Methods................................................................................................ 53 2.3.1 Polymer discs........................................................................................................... 53 2.3.2 Oxygen plasma treatment ................................................................................ 54 2.3.3 Scanning electron microscopy (SEM) ............................................................. 54 2.2.4 Atomic force microscopy (AFM) .................................................................... 55 2.3.5 Culture and medium......................................................................................... 55 2.3.6 Cell culture experiments .................................................................................. 56 2.3.7 Cell number and hormone secretion ................................................................ 56 2.3.8 Cell morphology .............................................................................................. 57 2.3.9 Immunocytochemistry ..................................................................................... 57 2.3.10 3T3-L1 cell differentiation............................................................................. 58 2.3.11 Cell apoptosis................................................................................................ 58 2.3.12 Statistical analysis.........................................................................................
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