On the Mechanobiology of Collagen Growth and Remodelling

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On the Mechanobiology of Collagen Growth and Remodelling ON THE MECHANOBIOLOGY OF COLLAGEN GROWTH AND REMODELLING A Dissertation Presented By Seyed Mohammad Siadat to The Department of Bioengineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Bioengineering Northeastern University Boston, Massachusetts August 2020 ii ABSTRACT How organized collagenous structure can arise and grow from a cluster of cells remains one of the most important basic science questions associated with connective tissue research. Despite more than a century of research, there are currently no widely accepted mechanistic models of formation, growth, and remodeling of collagen fibrils. It has been hypothesized in our research group that collagen monomers and enzymes are in a dynamic equilibrium with existing fibrils. Tensile forces on fibrils can shift this equilibrium and change the balance between molecular association and dissociation. Here, we sought to answer this question: Does fibril strain promote the molecular assembly of collagen? To investigate this question, individual collagen fibrils were stretched to 0%, 4%, and 6% strain between two microneedles and exposed to a subthreshold concentration of fluorescently labeled collagen molecules to quantify molecular association onto the stretched fibrils. It was shown that labeled monomers rapidly incorporate onto all tested fibrils and reach a plateau. The time to reach plateau was significantly faster for the stretched fibrils (15.6, 7.0, and 6.0 minutes for fibrils under 0%, 4%, and 6% strain, respectively). Analysis of the fibril intensity and photobleaching data indicated that the association rate was significantly higher for fibrils under 6% strain compared to fibrils under 0% and 4% strain, increasing the association rate by 100%. It was concluded that mechanical stresses and strains could increase fibril growth by decreasing the activation energy required for reaction between monomers and fibrils and also by setting fibrils in a lower state of energy, increasing the association rate of monomers and fibrils. iii ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Jeffrey Ruberti who taught me how to pursue science properly. I learned to develop a hypothesis and try my best to prove it wrong. Thank you for giving me the opportunity to work in your research group even though I was a mechanical engineer and didn’t know much about biology when I started. I would like to thank my parents who have always supported and encouraged me to keep learning. I would like to thank my friends and coworkers - JJ, Ramin, Monica, Ebraheim, Alex, Isabel and everyone else - which made my life as a graduate student fun and exciting. I learned so much from you and had lots of adventures. I would like to especially thank Dr. Paten and Dr. Susilo. I always benefited and developed ideas from our lunchtime discussions. I would like to thank the Bioengineering department and all the amazing people who work there, especially Susan Wilcox which who was always helpful and her love for soccer was also pleasing. I would like to thank Dr. Michael Jaeggli and Dr. Timothy Lannin who I had the opportunity to work with as a teaching assistance during which I discovered a passion for teaching. Last, but not least, I would like to thank my committee, Dr. Charles Dimarzio and Dr. Chiara Bellini who I benefitted from their expertise and knowledge. Thank you all. iv TABLE OF CONTENTS 1 INTRODUCTION ......................................................................................................... 1 1.1 COLLAGEN ............................................................................................................... 3 1.2 SELF-ASSEMBLY ...................................................................................................... 7 1.3 FIBRILLOGENESIS, GROWTH, AND REMODELLING .................................................. 10 1.3.1 The Site and Mechanism of Initial Fibril Formation ....................................... 10 1.3.2 Fibril Growth Mechanism ................................................................................ 43 1.3.3 Role of Mechanics in Fibrillogenesis, Growth, and Remodeling of Collagenous Tissue ........................................................................................................................ 71 2 DYNAMIC TRACKING OF FLUORESCENTLY LABELED TYPE I COLLAGEN MOLECULES; DIRECT QUANTIFICATION OF MOLECULAR ASSOCIATION WITH NATIVE FIBRILS .............................................................................................. 75 2.1 INTRODUCTION ....................................................................................................... 75 2.1.1 Radioactive and Nonradioactive Isotopically-Labeled Amino Acids ............. 76 2.1.2 Non-collagen Based Probes ............................................................................. 77 2.1.3 Endogenous Labeling with GFP ...................................................................... 79 2.1.4 Exogenous Labeling ........................................................................................ 81 2.2 OBJECTIVES AND APPROACHES .............................................................................. 84 v 2.3 EXPERIMENTAL METHODS ..................................................................................... 88 2.3.1 Collagen Labeling ............................................................................................ 88 2.3.2 Purifying the Labeled Collagen ....................................................................... 89 2.3.3 Determining Collagen Concentration and Degree of Labeling ....................... 90 2.3.4 Spectrophotometry ........................................................................................... 91 2.3.5 Scleral Fibril Extraction ................................................................................... 91 2.3.6 Proteoglycan Quantification ............................................................................ 93 2.3.7 Imaging ............................................................................................................ 96 2.3.8 Fibril Diameter Measurement using TEM and DIC ........................................ 97 2.3.9 Molecular Association of Labeled Monomers with Native Fibrils Experiment .................................................................................................................................. 98 2.3.10 Image Analysis for Fluorescence ................................................................... 99 2.3.11 Arrhenius Plot and Activation Energy Measurement .................................. 100 2.3.12 Orientation of Collagen Molecules .............................................................. 101 2.3.13 Statistical Information .................................................................................. 102 2.4 RESULTS .............................................................................................................. 102 2.4.1 The Degree of Labeling (DOL) ..................................................................... 102 2.4.2 De Novo Fibrillogenesis Experiments with Labeled Collagen...................... 104 vi 2.4.3 Single Molecule, Multi-label Fluorescence Orientation Microscopy (SMO Microscopy) ............................................................................................................ 109 2.4.4 Labeled Collagen/Native Fibril Association Experiments ............................ 114 2.5 DISCUSSION ........................................................................................................ 118 2.6 SUMMARY ............................................................................................................ 121 3 A CORRELATIVE METHOD TO MEASURE COLLAGEN FIBRIL DIAMETER IN DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY ........................... 123 3.1 INTRODUCTION ..................................................................................................... 123 3.2 CURRENT METHODS FOR MEASURING COLLAGEN FIBRIL DIAMETER .................. 124 3.2.1 SAXS ............................................................................................................. 124 3.2.2 TEM ............................................................................................................... 125 3.2.3 SEM ............................................................................................................... 127 3.2.4 AFM ............................................................................................................... 128 3.2.5 SHG ............................................................................................................... 130 3.3 DIC MICROSCOPY ................................................................................................ 131 3.4 EXPERIMENTAL METHODS ................................................................................... 135 3.4.1 Fibril Preparation for TEM-DIC imaging ...................................................... 135 3.4.2 DIC Imaging .................................................................................................. 136 vii 3.4.3 TEM Processing ............................................................................................. 136 3.4.4 Microfabrication of Trenches in PDMS Sheets ............................................. 136 3.4.5 Fibril Preparation for SEM-DIC imaging ...................................................... 137 3.4.6 SEM Processing ............................................................................................
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