Mechanical Behavior of Individual Type I Collagen Fibrils
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MECHANICAL BEHAVIOR OF INDIVIDUAL TYPE I COLLAGEN FIBRILS BY JULIA HONG LIU THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in Aerospace Engineering in the Graduate College of the University of Illinois at Urbana-Champaign, 2016 Urbana, Illinois Adviser: Professor Ioannis Chasiotis ABSTRACT Despite the plethora of studies on the mechanical response of collagen, especially at the molecular scale or at much larger length scales, such as of those of fibers, tendons, and fascicles, there is still limited information about the mechanics of collagen fibrils (50 - 500 nm diameter) that serve as mesoscale building blocks in tissues. In this dissertation research the mechanical behavior of dry individual reconstituted collagen fibrils with different diameters were investigated via monotonic tests at 0.004 s-1, cyclic loading and cyclic loading/recovery experiments, and strain rate tests spanning six decades of strain rates in the range 10-3 – 102 s-1. Experiments were conducted with reconstituted type I calfskin collagen fibrils which were isolated from buffer and after drying were adhesively attached to a microelectromechanical systems (MEMS) device. Experiments were carried out using high-resolution optical microscopy under dry conditions at 20-30% relative humidity (RH) and laboratory temperature. Eight fibrils with diameters of 165±77 nm tested under monotonic loading yielded an average tensile strength of 752±186 MPa, ultimate stretch ratio of 1.3±0.06, and initial stiffness of the stress (σ) vs. stretch ratio (λ) curves, E1 = 5.7±2.3 GPa. These results depended on fibril diameter: fibrils with larger diameters showed increased maximum stretch ratio, λmax, and decreased E1 and decreased stiffness, E2, of the softening regime in the σ - λ curves. Normalizing the applied stress with E1, removed the diameter size effect and provided great consistency in the softening regime of different σ - λ curves. The same process was applied to fibrils tested at nominal strain rates of 10-2 - 102 s-1 showing good agreement between σ/E1 - λ curves obtained at the same strain rate from different fibrils, but also showed a clear increase in E2 with the applied strain rate without a reduction in λ at failure, which implies a gradual linearization of σ-λ curves at higher rates. The mechanical behavior under cyclic loading was studied via experiments in each of the three regimes, with target λmax ~ 1.05 in regime I, λmax ~ 1.25 in regime II, and λmax ~ 1.3 in regime III. In regime I, E1 was unaffected by cycling loading or recovery. The residual strain increased in every cycle, but ~80% of λmax was recovered after resting for 1 hr at zero stress. Regime II was characterized by constant E1, after an initial drop ii between cycles 1 and 2, a slightly increasing value of E2 in every cycle, and increasing residual strain with cycling. Cycling in regime III also resulted in constant E1 and E2 after an initial reduction between cycles 1 and 2 and increased residual strain with cycle order. The experimental results point out to a process of damage accumulation during cycling, as manifested by the very consistent hysteresis loops and the gradually accumulated residual strain, which however, does not affect the mechanical stiffness of regimes I and II. The latter points out to a cross-link network within the collagen fibril that maintains molecular connectivity, as well as material regions that allow for viscous sliding (supported by the increase in E2 and E2/E1 with applied strain rate) in the softening regime of the σ - λ curves without disrupting the cross-link network. The rapid recovery and restoration of the three-regime shape of the σ - λ curves of collagen fibrils also supports the existence of sacrificial bonds which reform upon recovery that is driven by residual stresses in the fiber. iii ACKNOWLEDGMENTS Completing a thesis requires a great deal of support and guidance, and I would like to thank here the people who guided, mentored, and assisted me throughout my research here at the University of Illinois. First and foremost, I would like to thank my primary research advisor Professor Ioannis Chasiotis. After I was accepted into the University of Illinois he offered me the opportunity to work on a project that straddled both biology and nanomechanics. His expertise in nanomechanics has helped me understand my research and develop methods for performing my experiments, and his understanding and support allowed me to create a solid base on which to pursue my dreams. He guided me not only as his student, but also as a person. I cannot express how grateful I am for his mentorship. I would like to acknowledge the funding support by the National Institutes of Health (NIH) under award numbers 1U01EB016422-01A1, 5U01EB016422-02 and 5U01EB016422-03 Revised. I would also like to thank Professor Guy Genin from Washington University in St. Louis and Professor Stavros Thomopoulos from Columbia University in New York City. Their advice was indispensable and their knowledge of biological materials guided me in the directions necessary to move forward with my research. I would also like to thank Professor Thomopoulos’s student, Dr. Annie Schwartz, for reconstituting the collagen used in these experiments and for teaching me how to handle the fibrils. I want to show my gratitude to my colleagues in the Nanomechanics and Materials Research Lab, who taught me everything I know about using the lab and pushed me to perform my best. Mr. Jan Clawson was my first mentor and taught me the basics of experimental nanomechanics. With his assistance, my transition from student to researcher was seamless. Dr. Pavan Kolluru has also been an incredible mentor. I am grateful to have been under his tutelage, as his knowledge and guidance have made me strive for perfection. I would also like to thank Mr. Debashish Das for helping me run experiments and performing data analysis when I was unable. His support and friendship has been instrumental in completing this thesis and in my development as a researcher. iv Mr. Fan Yang and Mr. Korhan Sahin worked with me on this project, and I would like to thank them for being by my side and helping me these last few years. I am also grateful to Mr. Dimitrios Antartis and Mr. Ryan Mott for their friendship and assistance. An incredible portion of my time was spent in Frederick Seitz Materials Research Laboratory Central Facilities at UIUC, and I would like to thank the staff there for training and assisting me. I would like to thank Ms. Honghui Zhou for training me on the Hitachi S4700 and S4800 SEMs, as well as for ensuring that these machines were always operational. Thank you to Mr. Matt Bresin for training me on the FEI DB235 FIB and for being quick to respond to my questions and issues at all hours of the day. I am grateful to the rest of the staff at MRL for assisting me with non-technical questions and issues. The Aerospace Department also deserves a large thanks. Mr. Greg Milner and the others at the Aerospace Department’s machine shop were extremely helpful. I brought them several drawings and they always provided me with perfect materials and parts. I am grateful for their hard work, friendship, and patience. I would also like to thank Ms Staci McDannel and Mrs. Kendra Lindsey for keeping me informed about the state of my degree and addressing any administrative issues for me. Finally, I would like to thank my family and friends. Thank you for always encouraging and supporting me. I would not be here without you. v TABLE OF CONTENTS INTRODUCTION............................................................................................................. 1 1.1. Structure of Collagenous Tissues ........................................................................ 2 Effect of Mineralization on Mechanical Behavior of Collagen Fibrils .............. 6 Effect of Hydration on Mechanical Behavior of Collagen Fibrils ...................... 9 Formation and Mechanical Effects of Chemical Cross-Links in Collagen ...... 11 1.2. Mechanical Properties of Collagen Molecules and Fibers .............................. 14 Mechanical Stiffness and Strength.................................................................... 14 Viscoelastic and Strain Rate Dependent Behavior of Collagen Fibrils ............ 18 1.3. Computational Modeling of Mechanical Behavior of Collagen Fibrils ......... 21 1.4. Objectives of this Dissertation Research........................................................... 24 MATERIALS AND EXPERIMENTAL METHODS ……………………………….26 2.1. Materials .............................................................................................................. 27 Synthesis of Reconstituted Collagen ................................................................ 27 MEMS Devices for Mechanical Testing of Individual Collagen Fibrils .......... 29 2.2. Experimental Methods ....................................................................................... 31 Isolation and Mounting of Individual Collagen Fibrils .................................... 31 Mechanical Experiments with Individual Collagen Fibrils .............................. 34 Mechanical Hysteresis and Recovery of Collagen Fibrils ................................ 35 MECHANICAL BEHAVIOR OF DRY COLLAGEN FIBRILS …………………..38 3.1. Tensile Testing of Individual Collagen Fibrils ................................................. 38 3.2. Strain Rate Dependence of Mechanical Behavior of Collagen Fibrils ........... 45 3.3. Deformation