A Dissertation Entitled Mechanically-Conditioned Biphasic

A Dissertation entitled Mechanically-Conditioned Biphasic Composite Scaffolds to Augment Healing of Tendon-Bone Interface by Gayathri Subramanian Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Engineering ________________________________________ Dr. Eda Yildirim-Ayan, Committee Chair ________________________________________ Dr. Beata Lecka-Czernik, Committee Member ________________________________________ Dr. Yakov Lapitsky, Committee Member ________________________________________ Dr. Ronald Fournier, Committee Member ________________________________________ Dr. Arunan Nadarajah, Committee Member ________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2017 Copyright 2017, Gayathri Subramanian This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Mechanically-Conditioned Biphasic Composite Scaffolds to Augment Healing of Tendon-Bone Interface by Gayathri Subramanian Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Engineering The University of Toledo August 2017 Rotator cuff injuries are very common among people over the age of 60, with more than 600,000 surgeries performed annually in the United States for rotator cuff repairs. However, in 20-80% of the cases, the repair fails due to re-rupture of the tendon at the tendon-bone insertion site. The complexity of the tendon tissue in terms of their structure, composition, and function at the tendon-to-bone interface demands for a combinatorial tissue-engineering approach in which cell maturation and function can be directed using bioactive proteins encapsulated within a biomaterial with appropriate material stiffness. Further, since tendons experience routine mechanical strains in their native environment, providing suitable mechanical cues to the engineered scaffold was considered important for the success of rotator cuff repair strategies. The objective of this dissertation was to synthesize and characterize a mechanically- conditioned biphasic composite collagen scaffold to enhance rotator cuff regeneration with (1) controlled delivery of adipose-derived stem cells (ASCs) and platelet-derived growth factor (PDGF) to augment and accelerate tendon healing, and (2) spatial material stiffness to promote gradient mineralization and matrix directionality at the tendon-bone interface. iii To this end, a mechanical loading bioreactor consisting of unique silicone loading chambers was designed that was capable of applying homogenous uniaxial tensile strains over 60% of the length of cell-encapsulated 3D collagen scaffolds. Uniaxial tensile mechanical loading at 2% strain with 0.1 Hz frequency was identified to be the appropriate loading modality to induce pure ASC tenogenic differentiation, along with enhanced matrix directionality and ECM gene expression within ASC-encapsulated 3D collagen scaffolds. Next, the poor protein retention and matrix stiffness properties of collagen were improved by synthesizing a composite collagen scaffold (PNCOL) interspersed with functionalized polycaprolactone (PCL) nanofibers. The PDGF-conjugated PNCOL scaffolds demonstrated controlled release of bioactive proteins (0.5% per day) under uniaxial tensile mechanical loading. Finally, the PDGF/ASC-encapsulated COLPNCOL biphasic composite scaffold with gradient matrix stiffness was engineered and subjected to uniaxial tensile mechanical loading to mimic the tissue at the tendon-bone insertion point. Significantly, the COLPNCOL biphasic scaffold demonstrated two major morphological and biochemical characteristics which is representative of the tendon- fibrocartilage region of the native tendon-bone interface tissue: (1) A gradient increase in mineral deposits and a gradient decrease in the matrix alignment. (2) Elevated tenogenic expression with COL region and higher chondrogenic expression within PNCOL region. This mechanically-conditioned PDGF/ASC-encapsulated COLPNCOL biphasic scaffold is expected to provide sustained cells and growth factor delivery to induce tenogenic differentiation and ECM secretion, and promote gradient matrix mineralization and collagen alignment of the de novo tissue, thereby aiding and accelerating the natural tendon-bone interface healing process for functional regeneration of the rotator cuff. iv To my friends and family, by blood and soul, To them, who inspired me far beyond my goals, To the magic of science & the mysteries it holds, I dedicate this study, to each one and all... Acknowledgements I would like to express my sincere and warm gratitude to Dr. Eda Yildirim-Ayan for providing me with the opportunity and resources to pursue my passion in research. Her technical and editorial expertise along with her constant guidance and encouragement were instrumental in the successful completion of this study. I am truly fortunate to have had her as my advisor. Her mentorship has contributed immensely to my growth, both as an individual and a professional. I also would like to thank my committee members: Dr. Beata-Lecka Czernik, Dr. Yakov Lapitsky, Dr. Ronald Fournier, and Dr. Arunan Nadarajah for their valuable inputs and feedback that improved the content of this dissertation. A big thank you to all my previous and current Engineered Biosystems Lab members. In particular, Mostafa Elsaadany, for being a friend and a partner in crime during the last 5 years – right from setting up the lab, mentoring students, troubleshooting experiments, managing ‘crisis’ situations, attending conferences, going through the grind of I-Corps, to graduating together! Special thanks to Maggie Ditto – for the warm welcome I received when I first joined the lab; Callan Bialorucki – for those effective brainstorming sessions every time I hit a roadblock, and Andrew Trumbull – for being a good sounding board during the progress of my work. Finally, my sincere thanks to Tammy Phares, for her unconditional help and support during the course of my research, and for making my 5-year stint as the Teaching Assistant of the Bioprocessing Lab enjoyable and memorable. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................. xvi List of Figures ................................................................................................................. xvii List of Abbreviations ...................................................................................................... xxii 1 Background and Overview ..................................................................................... 1 1.1 Rotator cuff tendon injuries ........................................................................ 1 1.2 Surgical repair of rotator cuff tendon .......................................................... 2 1.3 Tendon anatomy and physiology ................................................................ 3 1.4 Tendon healing process............................................................................... 5 1.5 Commercial solutions for rotator cuff repair .............................................. 7 1.6 Cells or growth factor delivery for tendon repair ....................................... 8 1.7 Tissue engineering approach for tendon repair ........................................... 9 1.8 State-of-the-art in tissue-engineered scaffolds for tendon repair .............. 11 1.8.1 Hydrogel-based scaffolds to augment tendon healing .................. 11 1.8.2 Synthetic and composite scaffolds for partial tendon repair ......... 13 vi 1.8.3 Mechanically-conditioned scaffolds for tendon reconstruction .... 16 1.8.4 Stratified scaffolds for tendon-bone interface repair..................... 17 2 Objective and Thesis Outline ................................................................................ 19 2.1 Rationale ................................................................................................... 19 2.2 Objective ................................................................................................... 20 2.3 Thesis outline ............................................................................................ 25 3 A Mechanical Loading Bioreactor to Apply Uniaxial Tensile Strains to Cell- encapsulated 3D Collagen Scaffolds ................................................................................ 28 3.1 Introduction ............................................................................................... 28 3.2 Materials and Methods .............................................................................. 34 3.2.1 Design and fabrication of the uniaxial tensile strain bioreactor .... 34 3.2.1.1 Components of the uniaxial tensile strain bioreactor ............ 34 3.2.1.2 Loading chamber of the uniaxial tensile strain bioreactor .... 37 3.2.1.3 Material selection .................................................................

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