Silk Protein as a Biomaterial for Tissue Engineering Application: Theoretical and Experimental Study A Thesis Submitted to the Faculty of Drexel University by Milind Ramesh Gandhi in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2006 ii ACKNOWLEDGEMENTS I will like to take few moments thanking everyone for helping me throughout. First of all I thank my mother, Dr. Renuka Gandhi and my father, Dr. Ramesh Gandhi for believing in me and supporting my decision to come to Drexel for Ph.D. I would like to express my gratitude to my mentor, Prof. Frank K. Ko. Under his supervision I was able to grow as an engineer. Being trained as a medical doctor I had limited knowledge of engineering and biomaterials. Dr. Ko guided me throughout my studies. I would like to acknowledge the help of my committee members, Dr. Donald L. McEachron, Dr. Peter I. Lelkes, Dr. Fred D. Allen and Dr. Andrzej Fertala. Special thanks to the committee members from Biomedical Sciences, Engineering & Health Systems Department, Dr. McEachron, Dr. Lelkes and Dr. Allen for pushing me to carry out the cell-matrix interaction study on my scaffolds. I learnt a lot from that experience. I am also grateful for the financial support provided by the funding agencies, Pennsylvania Nanotechnoly Institute, Taiwan Textile Research Institute and Nexia Biotechnologies. I would also like to thank the present and past members of the Fibrous Materials Laboratory, Heejae Yang, Donia El-Khamy , Dr. Hoa Lam, Dr. Jonathan Ayutsede, Nick Titchenal, Dr. Jason Lyons, Sharaili Rao, David Heldt, Dr. Afaf El-Aufy, and Jennifer Atchinson. Thank you all for all your suggestions and for being with me not only during my good times but also bad ones. I am grateful to the visiting scholars, Dr. Sachiko Sukigara and Dr. Xiong Jie for their help. iii I wish to thank my friends from Biomed, Dr. Michele Cox, Mai Bui, Nancy Robinson and Dr. Amir Rezvan. I met you guys for the first time when I came to Drexel. You have always supported me since then. I thank you all for being such loyal friends. Special thanks to Michele’s mom, Mrs. Cynthia Cox for being a mom! I am grateful to Dr. Sun from Mechanical Engineering for allowing me to use his lab for cell culture work. Special thanks to Lauren Shor for helping me out with osteoblasts culture. I would like to extend my thanks to the ever helping staff of Materials and Biomed departments, especially Judy Trachtman and Lisa Williams. I am also thankful to all my other friends, Mary Sullivan, Amanda Hunt, Elaine Steinke, Maria Pia Rossi, Jonathan Thomas and Jackie. I am grateful to all my family members in United States as well as back in India for all the support and encouragement. Special thanks to my brother, Sachin Gandhi and his wife Roma Bhansali. Lastly, I will like to thank my ex-wife, Payal Nanavati for being a good friend. I thank her family members for their emotional support and understanding. iv TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………ix LIST OF FIGURES………………………………………………………………………x ABSTRACT……………………………………………………………………………xvi 1. INTRODUCTION…………………………………………………………………….1 2. BACKGROUND AND LITERATURE REVIEW…………………………………...5 2.1. Types of Silk Protein……………………………………………………………..6 2.1.1. Silkworm Silk……………………………………………………………..6 2.1.2. Spider Silk…………………………………………………………………7 2.2. Structure and Properties of Silk…………………………………………………..8 2.2.1. Structure of Silk …………………………………………………………..8 2.2.2. Mechanical Properties……………………………………………………..9 2.2.3. Biological Properties……………………………………………………..10 2.3. Production of Silk Fibers………………………………………………………..12 2.3.1. Natural Silk Spinning Process…………………………………………...12 2.3.2. Artificial Fiber formation using Regenerated Silk………………………13 2.3.3. Electrospinning…………………………………………………………..16 2.4. Genetic Engineering…………………………………………………………….18 2.4.1. Recombinant Silkworm and Spider Silk…………………………………18 2.4.2. Hybrid Silk.………………………………………………………………21 2.4.3. Transgenic Spider Silk …………………………………………………..22 2.5. Biomedical Applications of Silk………………………………………………...23 v 2.5.1. Tissue Engineering……………………………………………………….23 2.5.2. Drug Delivery……………………………………………………………24 2.6. Bone Tissue Engineering………………………………………………………..25 2.6.1. Structure and Functions of Bone…………………………………………25 2.6.2. Bone Tissue Engineering Methods………………………………………27 3. RESEARCH DESIGN AND OBJECTIVES………………………………………...30 3.1. Structural Changes in Natural and Regenerated Silk…………………………...31 3.2. Nanofibrous Scaffolds from Silkworm Silk ……………………………………31 3.3. Modification of Mechanical Properties of Silkworm Silk Nanofibers …………32 3.3.1. Post Spinning Modifications……………………………………………..32 3.3.2. Co-electrospinning with Carbon Nanotubes……………………………..33 3.3.3. Co-electrospinning with Collagen and Polylactic-co-glycolic Acid ……34 3.4. Nanofibrous Scaffolds from Spider Silk and Carbon Nanotubes………………34 3.5. Cell-Scaffolds Interaction………………………………………………………34 4. STRUCTURAL CHANGES IN NATURAL AND REGENERATED SILK ……...35 4.1. Introduction……………………………………………………………………...35 4.2. Materials and Methods………………………………………………………….38 4.2.1. Molecular Dynamics Simulation of Natural Silk Spinning Process……..38 4.2.2. Molecular Dynamics Simulation of Regenerated Silk …………………..40 4.3. Results…………………………………………………………………………...41 4.3.1. Natural Silk Spinning Process…………………………………………...41 4.3.2. Regenerated Silk Process………………………………………………...46 4.4. Summary…………………………………………………...................................55 vi 5. NANOFIBROUS SCAFFOLDS FROM SILKWORM SILK………………………57 5.1. Introduction ……………………………………………………………..............57 5.2. Materials and Methods………………………………………………….............59 5.2.1. Regenerated Silk and Spinning Dope Preparation ………………………59 5.2.2. Electrospinning Process Optimization and Characterization ……………59 5.2.3. Cell-scaffolds Interaction………………………………………………...62 5.3. Results………………………………………………….......................................65 5.3.1. Fiber Diameter Distribution ……………………………..........................65 5.3.2. FT-IR Spectroscopy…………………………….......................................70 5.3.3. Raman Spectroscopy……………………………......................................71 5.3.4. Wide Angle X-ray Diffraction…………………………….......................73 5.3.5. Mechanical Properties……………………………....................................74 5.3.6. Cell Proliferation and Differentiation……………………………............77 5.4. Summary……………………………...................................................................82 6. MODIFICATION OF MECHANICAL PROPERTIES OF SILKWORM SILK NANOFIBERS ……………………………………………………………………...84 6.1. Introduction ……………………………..............................................................84 6.2. Materials and Methods………………………………………………………….86 6.2.1. Post spinning treatment …………………………………………………86 6.2.2. Co-electrospinning Silkworm Silk and Carbon Nanotubes ……………..88 6.2.3. Co-electrospinning Silkworm Silk, Polylactic-co-glycolic Acid and Collagen …………………………………………………………………90 6.2.4. Cell-scaffolds Interaction………………………………………………..90 vii 6.3. Results…………………………….......................................................................90 6.3.1. Effect of Methanol on the Structure of Silk Nanofibers………………....90 6.3.2. Effect of Carbon Nanotubes on the Structure of Silk Nanofibers……….94 6.3.3. Effect of Methanol on Mechanical Properties…………………………...97 6.3.4. Effect of Carbon Nanotubes on Mechanical Properties and Electrical Conductivity…………………………………………………………….101 6.3.5. Effect of Collagen and Polylactic-co-glycolic Acid on Mechanical Properties……………………………………………………………….105 6.3.6. Cell Proliferation and Differentiation…………………………………..107 6.4. Summary……………………………………………………………………….116 7. NANOFIBROUS SCAFFOLDS FROM SPIDER SILK AND CARBON NANOTUBES ……………………………………………………………………..117 7.1. Introduction…………………………….............................................................117 7.2. Materials and Methods………………………………………………………...119 7.2.1. Process Optimization and Characterization…………………………….119 7.2.2. Co-electrospinning MaSp1 and Carbon Nanotubes…………………….120 7.2.3. Cell-scaffolds Interaction……………………………………………….120 7.3. Results………………………………………………………………………….121 7.3.1. Fiber Diameter Distribution ……………………………………………121 7.3.2. FT-IR Spectroscopy…………………………………………………….122 7.3.3. Raman Spectroscopy……………………………………………………125 7.3.4. Mechanical and Electrical Properties…………………………………...126 7.3.5. Cell Proliferation and Differentiation…………………………………..129 viii 7.4. Summary……………………………………………………………………….132 8. DISCUSSION AND CONCLUSION……………………………………………...133 9. FUTURE WORK AND SUGGESTIONS………………………………………….138 9.1. Silk Sponges …………………………………………………………………..138 9.2. Silk Microspheres……………………………………………………………...142 LIST OF REFERENCES……………………………………………………………….145 APPENDIX……………………………………………………………………………..156 VITA……………………………………………………………………………………159 ix LIST OF TABLES Table 1. Summary of recombinant spider silk production efforts………………………20 Table 2. Hybrid silk proteins…………………………………………………………….22 Table 3. Flowchart showing the method employed in this study for simulating natural silk spinning process………………………………………………………….………………40 Table 4. Flowchart showing the method employed in this study for simulating regenerated silk spinning process………………………………………………………..41 Table 5. Comparison between the percentage of β sheets calculated by FT-IR spectroscopy and molecular dynamics simulation……………………………………….54 Table 6. Flowchart showing the research design for making silkworm nanofibers……..65 Table 7. Factorial design of experiment…………………………………………………67 Table 8. The morphology and diameter of fibers at charge density of 3kV/cm at concentrations from 5 to 19.5% w/w with