ELECTROSPUN PLLA NANOFIBER COATING OF SCAFFOLDS FOR APPLICATIONS IN BONE TISSUE ENGINEERING A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment Of the requirements for the Degree Doctor of Philosophy Phillip E. McClellan December, 2015 ELECTROSPUN PLLA NANOFIBER COATING OF SCAFFOLDS FOR APPLICATIONS IN BONE TISSUE ENGINEERING Phillip E. McClellan Dissertation Approved: Accepted: ______________________________ ______________________________ Advisor Department Chair Dr. William J. Landis Dr. Coleen Pugh ______________________________ ______________________________ Committee Member Dean of the College Dr. Darrell H. Reneker Dr. Eric J. Amis ______________________________ ______________________________ Committee Member Interim Dean of the Graduate School Dr. Nita Sahai Dr. Chand Midha ______________________________ ______________________________ Committee Member Date Dr. Nic Leipzig ______________________________ Committee Member Dr. Edward Evans ii ABSTRACT In the field of tissue engineering, electrospun nanofibers gained notoriety for their capability to mimic the extracellular matrix of native tissues and organs. However, few reports have been published that detail methods of producing electrospun nanofibrous materials with macroscopic three-dimensional complexity. There is a potential method of incorporating the benefits of electrospun nanofibers into prefabricated tissue engineering scaffolds in the form of a thin coating. Nanofibers of poly-L-lactic acid (PLLA) were applied successfully to tissue engineering scaffolds composed of polycaprolactone/poly-L-lactic acid (PCL/PLLA, 75/25) or sterile, human allograft bone by a modified electrospinning method. The electrospun PLLA nanofibers conform to the shape of the scaffolds, resulting in a thin layer of nanofibers over all the surfaces of the material. These scaffolds were then wrapped with human periosteal tissue and implanted in athymic (nude) mice. The mice, then, acted as bioreactors for growing and developing over various time periods the engineered electrospun PLLA nanofiber-coated constructs. Specimens containing PCL/PLLA as the underlying scaffold material were implanted for 10 weeks in vivo and specimens containing iii allograft bone as scaffolds were implanted for 20 and 40 weeks. Harvested specimens were analyzed using histochemical and immunohistochemical methods to examine proliferation of cells, growth of new tissue, presence of mineral within the tissue, and presence of osterix, a bone-specific transcription factor necessary for osteoblast differentiation. Mineralized tissue was present in the electrospun PLLA nanofiber-coated PCL/PLLA constructs wrapped with periosteum after 10 weeks of implantation in vivo. Hematoxylin and eosin stains showed presumably new layers of tissue present between the layers of electrospun nanofibers and the underlying allograft bone in the allograft bone scaffolds coated with PLLA and then wrapped with human periosteal tissue. Osterix was identified by immunohistochemical staining and thereby verified presence of osteoblasts and preosteoblasts within the periosteal tissue and the electrospun PLLA nanofiber layers after 10, 20, and 40 weeks of implantation in vivo. The summary of these novel results suggests that electrospinning nanofibers such as PLLA on polymeric scaffolds or allograft bone can enhance tissue ingrowth from a periosteal wrap over such scaffolds or allografts for wider applications in bone tissue engineering. iv DEDICATION To my mother, father, and sister who have encouraged and supported me. v ACKNOWLEDGMENTS First, I want to thank Dr. William J. Landis, my advisor, for his time and dedication in helping me to achieve my goals in graduate research. His dedication to students is astounding. I also thank Robin Jacquet for her assistance in developing the immunohistochemistry protocol for osterix as well as her help with the implantation of the constructs into nude mice. She devotes a considerable portion of her time and effort to ensuring the students in Dr. Landis’ laboratory have the tools and knowledge they need in order to succeed. Beth Lowder was instrumental in providing knowledge of histological and cell culture techniques essential throughout the course of this research. I thank Dr. Bojie Wang for providing access to and teaching me proper use of the scanning electron microscope in his laboratory. I appreciate the help I have received from all of my lab group members as well. Dr. Hitomi Nakao assisted with the nude mouse implantation surgeries. Qing Yu and Dr. Nakao helped with the harvesting of the constructs. I am especially grateful for the aid Dr. Darrell Reneker provided through the course of the electrospinning portions of this project. I also thank the other members of my thesis committee, Dr. Nita Sahai, Dr. Nic Leipzig, and Dr. Edward Evans, for providing insights which directed the vi course of my research. Finally, I thank Dr. Susan Chubinskaya (Rush University, Chicago, IL), the Gift of Hope Organ and Tissue Donor Network (Itasca, IL), and donor families for tissue access. vii TABLE OF CONTENTS Page LIST OF TABLES .................................................................................................xi LIST OF FIGURES .............................................................................................. xii CHAPTER I. BACKGROUND LITERATURE REVIEW.......................................................... 1 1.1 Bone fracture repair and regeneration ........................................................ 1 1.2 Segmental bone defects ............................................................................. 3 1.3 Emergence of tissue engineering ............................................................... 6 1.4 Periosteal tissue as a source of osteoprogenitor cells ................................ 8 1.5 Brief history of electrospinning .................................................................. 10 1.6 Basic principles of the electrospinning process ........................................ 12 1.7 Electrospun nanofibers used in biological systems .................................. 13 1.8 Biodegradable nanofibers for tissue engineering ...................................... 15 1.9 Emulsion and coaxial electrospinning ....................................................... 18 1.10 Fabricating three-dimensional nanofiber scaffolds ................................. 19 1.10.1 Mechanically expanded nanofiber mats ............................................ 20 1.10.2 Nanofiber collector alteration ............................................................ 20 1.10.3 Polymer solution manipulation .......................................................... 22 viii II. APPLICATION OF PLLA NANOFIBERS TO SCAFFOLDS OF PCL/PLLA ... 23 2.1 Materials and methods ............................................................................. 25 2.1.1 Preparation of PLLA nanofiber-coated PCL/PLLA scaffolds ................ 25 2.1.2 Scaffold drying and immersion in liquid ............................................... 26 2.1.3 Nanofiber morphology ......................................................................... 27 2.2 Results ...................................................................................................... 27 2.3 Figures ...................................................................................................... 30 2.4 Discussion ................................................................................................ 37 III. TESTING OF PLLA NANOFIBER-COATED PCL/PLLA SCAFFOLDS IN VIVO ............................................................................................................ 45 3.1 Materials and methods ............................................................................. 46 3.1.1 Material and chemical sources ............................................................ 46 3.1.2 Human periosteal tissue collection ...................................................... 47 3.1.3 Scaffold sterilization and construct preparation ................................... 47 3.1.4 Contruct harvest, fixation, processing, and embedding ....................... 48 3.1.5 Construct sectioning and staining ........................................................ 49 3.2 Results ...................................................................................................... 49 3.3 Figures ...................................................................................................... 51 3.4 Discussion ................................................................................................ 59 IV. EXAMINATION OF PLLA NANOFIBER-COATED HUMAN ALLOGRAFT BONE SCAFFOLDS IN VIVO ...................................................................... 67 4.1 Materials and methods ............................................................................. 69 4.1.1 Human allograft bone scaffold preparation .......................................... 69 4.1.2 Preparation of periosteum-wrapped allograft constructs ...................... 70 ix 4.1.3 Construct implantation, harvest, and analysis...................................... 70 4.1.4 Slide preparation and histochemical staining ....................................... 71 4.2 Results ...................................................................................................... 72 4.3 Figures ...................................................................................................... 74 4.4 Discussion ...............................................................................................