ABSTRACT DESIGN, FABRICATION, AND ANALYSIS OF POLYMER SCAFFOLDS FOR USE IN BONE TISSUE ENGINEERING by Joshua Minton Bone tissue engineering is an emerging field that seeks to improve the treatment of bone defects by restoring the functions of bone using the body’s natural healing processes. Polymer scaffolds seeded with osteoblast and growth factors is one technique that has shown the potential to speed the healing process and decrease the rehabilitation time from bone defects. The goal of this study is to create viable polymer/ceramic scaffolds through melt processing of polycaprolactone and hydroxyapatite and using polyethylene oxide as porogen. The results of this study show that melt processing of these materials is an effective method for creating stable scaffolds. The properties of these scaffolds can be altered by changing several factors including polymer ratio, ceramic and salt content, and the pressure applied during the fabrication process. Biological analysis shows that the scaffolds seeded with MC3T3-E1 cells are capable of facilitating cell attachment and proliferation in vitro over time. DESIGN, FABRICATION, AND ANALYSIS OF POLYMER SCAFFOLDS FOR USE IN BONE TISSUE ENGINEERING A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Department of Chemical, Paper, and Biomedical Engineering by Joshua Minton Miami University Oxford, Ohio 2013 Advisor _________________________________ Dr. Azizeh Yousefi Moshirabad Advisor _________________________________ Dr. Paul James Reader _________________________________ Dr. Paul Urayama Reader _________________________________ Dr. Jason Berberich Table of Contents Title Page i Table of Contents ii List of Tables iv List of Figures v Acknowledgements vi Chapter 1: Introduction 1 1.1 Bone Structure and Function 2 1.2 Traditional Repair Methods 3 1.3 Tissue Engineering 4 1.3.1 Solvent Casting/Particulate Leaching 8 1.3.2 Gas Foaming/Particulate Leaching 8 1.3.3 Melt Processing 8 1.4 Materials 10 1.4.1 Polymers 10 1.4.2 Ceramics 12 1.5 Scaffold Fabrication 13 1.6 Porosity and Interconnectivity 14 1.7 Mechanical Properties 15 1.8 Cell Attachment and Growth 15 1.7.1 Scanning Electron Microscopy 17 1.7.2 Proliferation 18 1.9 Project Statement 19 1.8.1 Hypothesis 19 1.8.2 Objectives 20 1.10 Thesis Outline 20 1.11 References 22 Chapter 2: Design and Fabrication of Polymer/Ceramic Scaffolds for Bone Tissue 27 Engineering 2.1 Abstract 27 2.2 Introduction 27 2.3 Scaffold Materials 29 2.4 Materials 31 2.5 Scaffold Characterization 32 2.6 Statistical Analysis 32 2.7 Results and Discussion 32 2.7.1. Effect of Polymer Ratio 32 2.7.2. Effect of HA concentration 36 2.7.3. Effect of Pressure 38 2.8 Conclusions 39 ii 2.9 References 41 Chapter 3: Polymer/Ceramic Scaffolds for Bone Tissue Engineering: Fabrication, 43 Analysis, and Cell Growth 3.1 Abstract 43 3.2 Introduction 44 3.3 Materials and Methods 46 3.3.1. Materials 46 3.3.2. Fabrication of PCL and PCL/HA scaffolds 47 3.3.3. Characterization of porous scaffolds 47 3.3.4. Cell Culture 48 3.3.5. Proliferation Assay 48 3.3.6. Preparation for SEM imaging 48 3.3.7. Statistical Analysis 49 3.4 Results and Discussion 49 3.4.1. Characterization of porous scaffolds 49 3.4.2. Proliferation 54 3.4.3. SEM 54 3.5 Conclusions 55 3.6 References 56 Chapter 4: Significance and Future Work 59 4.1 Significance 59 4.2 Future Work 60 iii List of Tables Table 1.1: Scaffold fabrication techniques in tissue engineering applications 5 Table 1.2: Methods of preparation of porous scaffolds and their characteristics 7 Table 1.3: Properties of commonly-used biomaterials 11 Table 1.4: Properties of PCL and PEO 12 Table 1.5: Optimum pore size for bone regeneration 14 Table 1.6: Optimum parameters for grinding 1 gram of polymer 15 Table 1.7: Comparison of different SEM sample preparation methods 17 Table 2.1: Properties of PCL and PEO 30 Table 2.2: Mechanical properties of human tissues 31 Table 2.3: T-test results of the various salt concentrations and particle sizes tested 35 Table 2.4: T-test results of the mechanical properties of the scaffolds with different HA 38 Table 2.4: concentrations Table 3.1: Mechanical properties of human tissue 45 Table 3.2: Properties of PCL and PEO 46 Table 3.3: Results of physical characterization of scaffolds 52 Table 3.4: Porosity information determined by microCT analysis 53 Table 3.5: Pore size distribution determined by microCT analysis 53 iv List of Figures Figure 1.1: Stages of bone repair 3 Figure 1.2: Fabrication of porous scaffolds by gas foaming/particulate leaching 8 Figure 1.3: SEM micrographs of NaCl particles (a) before mixing and (b) after mixing 9 Figure 1.4: SEM images of the surface of PCL/PEO 50/50 (% vol) sample 9 Figure 1.5: The effect of wt.% HA on the strength of HA/polymer composites 13 Figure 2.1: Average Young’s modulus for scaffolds with various PEO/PCL polymer ratios 33 Figure 2.2: Average calculated porosity for scaffolds with various polymer ratios 33 Figure 2.3: Average Young’s modulus for the scaffolds with various salt particles sizes 34 Figure 2.3: and concentrations Figure 2.4: Average calculated porosity for the scaffolds with various salt particles sizes 35 Figure 2.4: and concentrations Figure 2.5: SEM image of a PEO/PCL 60/40 scaffold with 20% salt with particle sizes less 36 Figure 2.5: than 100 µm Figure 2.6: SEM image of PEO/PCL scaffold with 40% salt with particle sizes between 36 Figure 2.6: 150 µm and 250 µm Figure 2.7: Average Young’s modulus for the scaffolds with various HA concentrations 37 Figure 2.8: Average calculated porosity for the scaffolds with various HA concentrations 37 Figure 2.9: SEM image of PEO/PCL 60/40 scaffold 38 Figure 2.10: SEM image of PEO/PCL 60/40 scaffold with 20% HA 38 Figure 2.11: Average Young’s modulus for the scaffolds with various HA concentrations 39 Figure 2.11: and pressure level Figure 2.12: Average calculated porosity for the scaffolds with various HA concentrations 39 Figure 2.12: and pressure level Figure 3.1: SEM image of PCL scaffold 50 Figure 3.2: SEM image of PCL/HA scaffold 50 Figure 3.3: Elemental mapping images of PCL/HA scaffolds 50 Figure 3.4: Overlaid elemental mapping images of PCL/HA scaffolds 50 Figure 3.5: Thermogravimetric analysis of PCL/HA scaffolds fabricated with 20% HA 51 Figure 3.6: Stress-strain curve of PCL/HA scaffolds 52 Figure 3.7: Pore size distribution of PCL and PCL/HA scaffolds 53 Figure 3.8: DNA content of PCL scaffolds at various time points 54 Figure 3.9: SEM images of PCL/HA scaffolds 55 Figure 3.10: SEM images of PCL scaffolds 55 v Acknowledgements There are several people I would like to acknowledge and thank for their help and guidance throughout my time at Miami and my through my Master’s research. I would not be where I am without their support and guidance – my advisors, committee members, faculty and staff at Miami University, and fellow graduate and undergraduate students. It has been my privilege to work with Dr. Yousefi, my advisor, for the last three years in both undergraduate and graduate research. Dr. Yousefi has been so helpful and kind throughout the whole time and I have enjoyed the experience immensely. I have no doubt that the knowledge and skills I have learned under her supervision will continue to serve me well in my career and future endeavors. I would also like to thank my co-advisor, Dr. James, who has been very patient and helpful guiding me through the biological aspects of my research that were new and challenging to me and for sharing his lab and resources so freely. I would like to thank Dr. Urayama and Dr. Berberich for agreeing to be on my committee and for being so accommodating with their schedules. I would also like to thank Dr. Saul for being on my committee for my proposal and for his insight and assistance during my proposal and with my final work. I would like to thank Doug Hart for being a constant source of support and encouragement throughout my time at Miami, for always going above and beyond to help in any way he could, and for being one of the best friends I could have, especially on the bad days. I would like to thank the Instrumentation lab and especially Barry Landrum and Jayson Alexander for their inventive ideas and hard work that designed and built many of the tools I used in my research. Much of my work would not have been possible without their help. I would like to thank Matt Duley and Dr. Edelmann from the Electron Microscopy facility for teaching me how to prepare and image my samples with SEM and for helping me find solutions to the obstacles I faced along the way. I would like to thank Rosa Akbarzadeh, Cara Janney, and Carlie Focke for their immense help in the lab. So much work has been put into this research and I could never have done it alone. Their help and support has made this all possible. vi Chapter 1 1. Introduction Polymer scaffolds have a variety of applications in today’s medical field. For example, tissue regeneration and cardiac function after myocardial infarction can be improved by an injection of primary skeletal myoblasts.