DISSERTATION INVESTIGATION OF CATIONIC CONTRAST-ENHANCED COMPUTED TOMOGRAPHY FOR THE EVALUATION OF EQUINE ARTICULAR CARTILAGE Submitted by Bradley B. Nelson Department of Clinical Sciences In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2017 Doctoral Committee: Advisor: Christopher E. Kawcak Co-Advisor: Laurie R. Goodrich C. Wayne McIlwraith Mark W. Grinstaff Myra F. Barrett Copyright by Bradley Bernard Nelson 2017 All Rights Reserved ABSTRACT INVESTIGATION OF CATIONIC CONTRAST-ENHANCED COMPUTED TOMOGRAPHY FOR THE EVALUATION OF EQUINE ARTICULAR CARTILAGE Osteoarthritis and articular cartilage injury are substantial problems in horses causing joint pain, lameness and decreased athleticism resonant of the afflictions that occur in humans. This debilitating joint disease causes progressive articular cartilage degeneration and coupled with a poor capacity to heal necessitates that articular cartilage injury is detected early before irreparable damage ensues. The use of diagnostic imaging is critical to identify and characterize articular cartilage injury, though currently available methods are unable to identify these early degenerative changes. Cationic contrast-enhanced computed tomography (CECT) uses a cationic contrast media (CA4+) to detect the early molecular changes that occur in the extracellular matrix. Glycosaminoglycans (GAGs) within the extracellular matrix are important for the providing the compressive stiffness of articular cartilage and their degradation is an early event in the development of osteoarthritis. Cationic CECT imaging capitalizes on the electrostatic attraction between CA4+ and GAGs; exposing the proportional relationship between the amount of GAGs present within and the amount of CA4+ that diffuses into the tissue. The amount of CA4+ that resides in the tissue is then quantified through CECT imaging and estimates tissue integrity through nondestructive assessment. Despite the emergence of this promising technology to capture quantitative information on articular cartilage quality, cationic CECT has not been ii thoroughly investigated in equine tissues in vitro or in vivo, nor has it been investigated in in vivo in any large animal or human subjects. This compilation of studies was designed to critically examine the capacity of cationic CECT to provide quantitative information on articular cartilage quality across a continuum of disease states in horses while dually serving as a translational model to showcase its potential application in humans. The first experiments successfully characterized the diffusion properties of CA4+ into equine articular cartilage in vitro and in vivo, and also established the femoropatellar joint as the optimum joint for use in the subsequent in vivo experimental models. These initial studies also established preliminary evidence of the safety of CA4+ on articular tissues. The experimental in vivo equine impact model delivered a contusive force to articular cartilage that successfully instituted degeneration of the extracellular matrix components and decreased tissue stiffness. This subtle degradation was discernable from healthy articular cartilage using cationic CECT imaging (microCT). In vivo, the clinical cationic CECT attenuation demonstrated fair correlations with the biochemical, mechanical and histologic staining measures, though there was not sufficient delineation among groups to detect significant differences between disease states. Further investigations are warranted to determine how focal sites of injury can be identified from the resolution limited by current CT technology. The second in vivo experimental study generated reparative and degenerative articular cartilage to provide comparisons with healthy tissue. The cationic CECT method, using microCT and clinical scanners, demonstrated an ability to distinguish between these groups of varying iii articular cartilage quality. Though the results were promising, this study revealed the complexity of detecting subtle articular cartilage injury in a solitary imaging examination owing to the inherent biochemical and biomechanical variation that exists across articular surfaces. However, this study confirmed the successful monitoring of articular cartilage through longitudinal examinations. Overall, this collection of studies established that cationic CECT imaging in vitro and in vivo is capable of distinguishing articular cartilage across a spectrum of disease states exemplifying its utility in the comprehensive evaluation of equine articular cartilage. Further work of how this technique compares to other quantitative diagnostic imaging techniques and optimization strategies for routine use are required. Nonetheless, the results of this work reveal the excellent potential of cationic CECT imaging to be incorporated into research and clinical investigations in horses and highlight the feasible translation of this work into human tissues. iv ACKNOWLEDGEMENTS There are many individuals who have been essential in allowing me to complete this work. First and foremost, I acknowledge my graduate committee especially Drs. Chris Kawcak and Laurie Goodrich for serving as my advisors and permitting me to chase my research and clinical interests in orthopedic surgery and articular cartilage imaging. Their immeasurable efforts in providing me with the opportunities and skills necessary to accomplish this work and for my professional development as a clinician scientist are greatly appreciated. I would also like to thank Dr. C Wayne McIlwraith for his valuable advice regarding the in vivo models and in his overall guidance throughout this graduate work and in supporting my long-term career goals. I thank Dr. Myra Barrett, who was very encouraging and generously welcomed a surgeon into her diagnostic imaging domain. A special thanks to Dr. Mark Grinstaff whose cationic contrast agent provided the cornerstone of this work, and who helped diversify my research training experience by letting me join his laboratory in Boston. A very special thanks to Jen (Suddreth) Daniels and the entire Equine Orthopaedic Research Center staff for their care of the research horses and in providing support for the countless early mornings that were required for the in vivo studies. I would also like to thank the Equine Orthopaedic Research Center laboratory personnel: specifically Nikki Phillips for her guidance and assistance with laboratory assays and troubleshooting histology staining protocols and Dr. John Kisiday for his guidance with the hydroxyproline assay. I also would like to thank Jeff Stewart, Anthony Nguyen and Chris Schmit, the diagnostic imaging technologists who assisted with the clinical CT and MR imaging. v I am incredibly grateful for my colleagues and mentors in Boston directed by Dr. Mark Grinstaff whose assistance and encouragement substantially improved the caliber of this work. Dr. Janne T.A. Mäkelä, Dr. Rachel Stewart, Taylor Lawson, Dr. Benjy Cooper and Dr. Brian Snyder all welcomed me into their laboratory space at Boston University and the Beth Israel Deaconess Medical Center broadening my knowledge of cartilage biomechanics, imaging analysis and chemistry while providing the equipment necessary to accomplish the goals of this work. A particular thanks to Drs. Amit Patwa and Jon Freedman who literally synthesized gallons of CA4+ for use in these experiments. I would also like to thank Dr. Valerie Moorman for performing lameness examinations and for her continued guidance, friendship and encouragement throughout my graduate training. I also acknowledge Dr. Katie Seabaugh for performing lameness examinations and for her general support. I thank Dr. Ali Daniel for his friendship, inspiration and discussions about clinical cases and for reinforcing my interest in clinical practice, and Dr. Holly Stewart for her never-ending optimism and reassurance during these last couple of years. I also extend a special thanks to my parents and brother, Judy, Rod and Tim Nelson for their continued support throughout my veterinary and post-graduate training and for their encouragement as I pursue my career goals. vi TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS .............................................................................................................v CHAPTER 1: THE ROLE OF ADVANCED DIAGNOSTIC IMAGING IN ARTICULAR CARTILAGE EVALUATION ........................................................................................................1 Introduction ..........................................................................................................................1 Pathophysiology of articular cartilage injury ...........................................................2 Diagnostic imaging of articular cartilage .................................................................8 Magnetic resonance imaging .................................................................................10 Compositional assessment .........................................................................13 Contrast-enhanced computed tomography ............................................................16 Contrast media composition ......................................................................17 Contrast media used in computed tomography ..........................................19 Purpose of study .................................................................................................................23