A MULTI-PHASE APPROACH TO TISSUE ENGINEERING OF THE TEMPOROMANDIBULAR JOINT by Catherine Hagandora Bachelor of Science, Presbyterian College, 2009 Submitted to the Graduate Faculty of Swanson School of Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2014 UNIVERSITY OF PITTSBURGH SWANSON SCHOOL OF ENGINEERING This dissertation was presented by Catherine Kunkle Hagandora It was defended on July 11, 2014 and approved by Stephen Badylak, D.V.M, Ph.D., M.D., Professor, Department of Surgery Charles Sfeir, D.D.S., Ph.D., Associate Professor, Department of Oral Biology Yadong Wang, Ph.D., Professor, Department of Bioengineering Dissertation Director: Alejandro J. Almarza, Ph.D., Assistant Professor, Department of Oral Biology, Department of Bioengineering ii Copyright © by Catherine Kunkle Hagandora 2014 iii A MULTI-PHASE APPROACH TO TISSUE ENGINEERING OF THE TEMPOROMANDIBULAR JOINT Catherine Kunkle Hagandora, PhD University of Pittsburgh, 2014 It is estimated that 10 million Americans are affected by temporomandibular joint (TMJ) disorders, a class of disorders encompassing symptoms such as jaw pain, functional disparities, and degenerative changes. In advanced cases, there is irreversible degradation of the articulating tissues of the joint. Due to the frequency and severity of these conditions, and the inadequacies of current replacement options, it is necessary to formulate tissue engineering strategies in order to restore TMJ anatomy and function. The objectives of this thesis were to further characterize the native TMJ fibrocartilage and identify effective methods and materials for tissue regeneration. As a first step, characterization of the native goat TMJ disc and condylar cartilage was performed. Bioactive magnesium ions and poly (glycerol sebacate) (PGS) were then investigated for their in-vitro potential to increase fibrocartilage production of goat costal fibrochondrocytes. Then, the response of human progenitor cells seeded in ECM scaffolds to mechanical stimulation was explored to better understand the in-vivo effectiveness of these scaffolds. Biomechanical, biochemical, and histological assessments were made to characterize both native and regenerated tissues. Gene expression analysis was also performed to determine the cellular response to mechanical stimulation within ECM scaffolds. The collagen content of the TMJ disc was found to be significantly greater than the condylar cartilage, while the opposite held for the glycosaminoglycan (GAG) and DNA content. The mandibular condylar cartilage, despite having significantly higher GAG content, is significantly less stiff than the TMJ disc iv under compression. At high concentrations, magnesium ions allowed for fibrocartilage regeneration in-vitro, with constructs cultured in MgSO4 exhibiting a significantly higher collagen type II/I ratio than the control. PGS was investigated as a mechano-transductive scaffold material for TMJ tissue engineering. It was shown that PGS is a substrate conducive to fibrochondrocyte infiltration and ECM regeneration, with scaffolds exhibiting near-native compressive properties after a culture period of 4 weeks. The mechanical properties of PGS also allowed for the transmission of compressive forces from the scaffold to the cells, impacting the patterning of collagen type I deposition. Similarly, within the bioactive environment of ECM scaffolds, compressive mechanical loading resulted in increased fibrochondrogenic gene expression of human bone marrow stromal cells. The results demonstrate that there is a set of culture conditions, including bioactive ions and/or compressive loading, and scaffolds that will stimulate cells to produce fibrocartilage, providing the foundation for tissue-engineered solutions to TMJ disorders. v TABLE OF CONTENTS TABLE OF CONTENTS ........................................................................................................... VI LIST OF TABLES ...................................................................................................................... XI LIST OF FIGURES .................................................................................................................. XII PREFACE .................................................................................................................................. XV 1.0 INTRODUCTION ........................................................................................................ 1 2.0 TMJ DISC REMOVAL: COMPARISON BETWEEN PRECLINICAL STUDIES AND CLINICAL FINDINGS ................................................................... 6 2.1 INTRODUCTION ............................................................................................... 6 2.2 MID-SIZE ANIMAL MODELS ....................................................................... 10 2.3 LARGE ANIMAL MODELS ........................................................................... 11 2.4 CLINICAL FINDINGS ..................................................................................... 16 2.4.1 Recent Investigations (past 10 years) ........................................................... 16 2.4.2 Imaging to assess surgical outcomes ............................................................ 21 2.5 INFERENCES FROM ANIMAL MODELS TO HUMAN FINDINGS ...... 23 3.0 A COMPARISON OF THE MECHANICAL PROPERTIES OF THE GOAT TEMPOROMANDIBULAR JOINT DISC TO THE MANDIBULAR CONDYLAR CARTILAGE IN UNCONFINED COMPRESSION ..................... 25 3.1 INTRODUCTION ............................................................................................. 25 3.2 METHODS ......................................................................................................... 28 3.2.1 Mechanical testing ......................................................................................... 28 3.2.2 Compression Analysis ................................................................................... 31 vi 3.2.3 Biochemistry................................................................................................... 34 3.2.4 Histology ......................................................................................................... 34 3.2.5 Statistical Analysis ......................................................................................... 35 3.3 RESULTS ........................................................................................................... 36 3.3.1 Simple Analysis .............................................................................................. 36 3.3.2 Transversely Isotropic Biphasic Model ....................................................... 40 3.3.3 Biochemical and Histological Analysis ........................................................ 43 3.4 DISCUSSION ..................................................................................................... 46 4.0 THE EFFECT OF MAGNESIUM ION CONCENTRATION ON THE FIBROCARTILAGE REGENERATION POTENTIAL OF GOAT COSTAL CHONDROCYTES ................................................................................................... 50 4.1 INTRODUCTION ............................................................................................. 50 4.2 MATERIALS AND METHODS ...................................................................... 53 4.2.1 Cell Culture .................................................................................................... 53 4.2.2 Histology ......................................................................................................... 54 4.2.3 Biochemistry................................................................................................... 55 4.2.4 Mechanical Testing ........................................................................................ 56 4.2.5 Statistical Analysis ......................................................................................... 57 4.3 RESULTS ........................................................................................................... 57 4.3.1 Gross Morphology ......................................................................................... 57 4.3.2 Histology ......................................................................................................... 59 4.3.3 Biochemistry................................................................................................... 60 4.3.4 Mechanical Properties ................................................................................... 62 4.4 DISCUSSION ..................................................................................................... 67 vii 5.0 POLY (GLYCEROL SEBACATE): A NOVEL SCAFFOLD MATERIAL FOR TEMPOROMANDIBULAR JOINT DISC ENGINEERING ............................... 71 5.1 INTRODUCTION ............................................................................................. 71 5.2 METHODS ......................................................................................................... 73 5.2.1 Cell Isolation and Culture ............................................................................. 73 5.2.2 Cell Seeding .................................................................................................... 74 5.2.3 Histology ......................................................................................................... 75 5.2.4 Biochemistry................................................................................................... 75 5.2.5