BONE MARROW DERIVED MESENCHYMAL STEM CELL (BM MSC) APPLICATION IN ARTICULAR CARTILAGE REPAIR HOSSEIN NEJADNIK (M.D., Isfahan University of Medical Sciences, Isfahan, Iran) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ORTHOPEDIC SURGERY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENTS It is my pleasure to thank all the kind people who made this thesis possible with their great help and support I would like to thank my supervisor, Associate Professor James Hui, Department of Orthopedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore (NUS), for his critical supervision and active support during my PhD study I am also deeply indebted to my co-supervisor Professor Shih-Chang Wang, Head of Discipline of Medical Imaging at Sydney Medical School, University of Sydney, for his constant support and encouragement My gratitude is also towards Professor Wong Hee Kit, the Head of Orthopedic Surgery Department, for giving me this chance to pursue my PhD degree and use the facilities in the department, and Professor Lee Eng Hin and Professor James Goh Cho Hong, Head of National University of Singapore Tissue Engineering Program (NUSTEP), for giving me the opportunity to use the NUSTEP facilities I also would like to thank Professor Vincent Chong, Head of Radiology Department, and Associate Professor Sudhakar Venkatesh for their help and support I am most grateful to Professor Roger Kamm, lead investigator of BioSystems and Micromechanics (BioSyM) at Singapore-MIT Alliance for Research & Technology (SMART) and Cecil and Ida Green Distinguished Professor of Biological and Mechanical Engineering at MIT University, for his mentorship and support and Dr Amirreza Aref, and Dr Choong Kim for their support in performing the bioengineering parts of my projects ii   I also would like to extend my sincere gratitude to Professor Kishore Bhakoo, Head of Translational Molecular Imaging Group at Singapore Bioimaging Consortium (SBIC), who provided me a great support in the MR imaging I owe my deepest gratitude to my colleagues and students in the NUSTEP lab: Dr Kenon Chua, Dr Xiafei Ren, Dr Zheng Yang, Dr Sintje Böhrensen, Afizah Binte Mohd Hassan, and Dr Sari Panjang, who have taught me a lot They have been wonderful friends I am thankful to all the colleagues, students and staff members of Department of Orthopedics Surgery, specially Ms Perumal Premalatha, and Ms Sarojeni Shanmugam, Orthopaedic Surgery Department management assistant officers, Yong Loo Lin School of Medicine for their timely help I would like to thank A*STAR and NUS for granting me graduate student scholarship This work was supported by grants from Singapore Bioimaging Consercium (to A/P James Hui) I wish to thank my great friends who made my PhD life a great memory for me and I owe my loving thanks to my wife, Pooneh, and my parents and I dedicate this thesis to them iii   TABLE OF CONTENTS ACKNOWLEDGEMENTS ii   Summary x   LIST OF TABLES AND FIGURES xiii   LIST OF ABBREVIATIONS xvii   LIST OF PUBLICATIONS xx   Chapter Background   1.1 Basic Science of Articular Cartilage   1.2 Cartilage injuries   1.2.1 Focal cartilage defects   1.2.2 Osteochondritis dissecans (OCD)   1.3 Importance of cartilage repair   1.4 Different methods of cartilage repair   1.4.1 Palliative technique: Arthroscopic lavage and debridement   1.4.2 Intrinsic repair enhancement: Microfracture   1.4.3 Whole tissue transplantation: Osteochondral Autograft Transplantation (OAT)   1.4.5 Cell based cartilage repair 11   1.4.5.1 Autologous chondrocyte implantation (ACI) 11   1.4.5.2 Other Cell-based therapy methods 12   1.4.5.3 Stem Cells in Articular Cartilage repair 13   iv   1.5 Types of stem cells 14   1.5.1 Embryonic stem cells (ESCs) 15   1.5.2 Induced pluripotent stem (iPS) cells 15   1.5.3 Adult stem cells 16   1.5.4 Heterologous stem cells 17   1.6 MSCs in cartilage repair 17   1.7 Monitoring of Cell Therapy 18   1.8 Histological Methods 19   1.9 Imaging Modalities 19   1.10 Contrast agents 22   1.10.1 T1 Contrast Agents (Table 1-7) 23   1.10.2 T2 Contrast Agents 23   1.10.3 Iron Oxide Particles 24   1.11 Cellular MRI 24   1.11.1 Cellular Imaging with Iron Oxide Particles 25   1.11.2 Mechanisms of Cellular Uptake 28   1.12 Cell migration 30   1.12.1 Stem cell migration mechanism 30   1.12.2 MSC migration and homing 31   1.12.3 Cell migration evaluation systems 32   Chapter Overall hypothesis and objectives 36   Chapter In vivo monitoring of the intra articular injected SPIO-labeled stem cells for cartilage repair 39   3.1 Abstract 40   v   3.2 Introduction 43   3.3 Methods 45   3.3.1 Expansion of MSCs 45   3.3.2 Flow cytometry assessment 45   3.3.3 Labeling of the MSCs with SPIO 46   3.3.4 Prussian blue staining 46   3.3.5 Transmission electron microscopy (TEM) 47   3.3.6 Atomic Absorption Spectroscopy (AAS) 47   3.3.7 Viability and proliferation evaluation 48   3.3.8 Differentiation of MSCs 48   3.3.9 Histological evaluation 50   3.3.10 Animal model 51   3.3.11 Surgical procedure 51   3.3.12 Preliminary MR imaging experiments 52   3.3.13 MR imaging of live animals 53   3.3.14 Postmortem analysis 55   3.3.15 Statistical analysis 57   3.4 Results 58   3.4.1 Characterization of MSCs 58   3.4.2 Prussian blue staining of SPIO-labeled MSCs 60   3.4.3 Transmission Electron Microscopy (TEM) 60   3.4.4 Iron content quantification in labeled-MSCs 61   3.4.5 Viability and proliferation of labeled MSCs 62   3.4.6 Differentiation potential of labeled MSC 64   3.4.6.1 Adipogenic differentiation 64   vi   3.4.6.2 Osteogenic differentiation 65   3.4.6.3 Chondrogenic differentiation 66   3.4.7 MR imaging of animals 68   3.4.7.1 Preliminary experiments 68   3.4.7.2 MRI of the mini-pigs’ knee 70   3.4.8 Postmortem analysis 73   3.5 Discussion 76   Chapter Simulating Injured Articular Cartilage Environment for Mesenchymal Stem Cell Migration Evaluation in A Three Dimensional Microenvironment 78   4.1 Abstract 79   4.2 Introduction 82   4.3 Methods 84   4.3.1 Design of microfluidic device 87   4.3.2 Computational modeling of concentration gradient 88   4.3.3 Fabrication of microfluidic device 89   4.3.4 MSC characterization and culture in microfluidic devices 90   4.3.5 Microfluidic device migration validation 91   4.3.6 Injured and uninjured sample preparation 91   4.3.7 MSCs migration toward injured cartilage conditioned media 92   4.3.8 Tissue placement and device assembly 93   4.3.9 MSCs migration toward injured tissue 94   4.3.10 Quantification of the MSCs migration 94   vii   4.3.11 Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) 95   4.3.11 Statistical analysis 98   4.4 Results 99   4.4.1 Computational modeling of concentration gradient 99   4.4.2 MSC characterization 100   4.4.3 Microfluidic device migration validation 101   4.4.4 MSCs migration stimulated by conditioned media 103   4.4.5 MSCs migration toward injured tissue 105   4.4.6 Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) 108   4.5 Discussion 110   Chapter Autologous Bone Marrow Derived Mesenchymal Stem Cell versus Autologous Chondrocyte Implantation: An Observational Cohort Study 115   5.1 Abstract 116   5.2 Introduction 118   5.3 Methods 120   5.3.1 Participants 120   5.3.2 Cell Sources 120   5.3.2.1 Chondrocyte (ACI) preparation 120   5.3.2.2 MSCs preparation 121   5.3.3 Operation techniques 123   5.3.4 Rehabilitation 123   viii   5.3.5 Post operation evaluation 124   5.3.6 Statistical analysis 125   5.4 Results 126   5.4.1 ICRS package SF-36 components clinical outcomes 127   5.4.2 IKDC subjective knee evaluation outcomes 129   5.4.3 Tegner activity level outcomes 132   5.4.4 Lysholm knee scale outcomes 132   5.4.5 Second-look arthroscopy and histological outcomes 133   5.5 Discussion 135   Chapter Conclusions and Future Direction 139   Chapter References 142   ix   Summary Articular cartilage repair is one the most challenging issues in orthopedic surgery due to the avascular and aneural nature of the cartilage, which affects its self-repair capability Cell therapy shows a great potential for cartilage defect repair Brittberg et al in 1994 used autologous chondrocytes as a cell source to repair the injured knee cartilage, which remains a promising method even to date However, this method has some limitations such as inadequate cell number, age dependent quality of the chondrocytes and donor site morbidity Recently bone marrow derived mesenchymal stem cells (MSCs) become an alternative cell source for cartilage repair Our group and others had demonstrated the tri-lineage differentiation ability of MSCs to chondrocytes, osteocytes and adipocytes Wakitani et al and our group demonstrated that MSCs could be used as a new source for cartilage repair However, in order to develop this into an effective cell therapy treatment we need to understand the behavior of cells, especially the proliferation, differentiation, migration and engraftment of MSCs after implantation and in vivo The use of MSCs for cartilage repair relies on their homing and engraftment to the injured cartilage tissue Although there is some theory that injured tissue expresses ligands and chemotactic factors that encourage homing of MSCs, these factors and their mechanism are not fully understood In vitro modeling of the in vivo simulation can be challenging Microfluidic platforms allow the study of cell migration in 3D environment, and at the same time, provide live x   The “under agarose system” (173) was performed by having a layer of the agarose gel in a 60mm culture dish and punching out three holes, which are few millimeters apart from each other Control and attractant (e.g., conditioned media) was placed in the outer wells and the cells in the central well The cell can migrate beneath of the gel towards the diffused gradient of the attractants in the agarose Theoretically multiple sets of experiments can be done in this system but because of the cross contamination of the cytokines gradients in the gel, monitoring of the cell migration pattern is difficult over time The advancement in microfabrication provides a new approach to simulate more closely in vivo environment of target cells and monitor their movement with control in vitro conditions The major advantages of the microfluidic systems are: the capability of providing the nutrient in a short distance to cells, a 3-dimentional (3D) scaffold environment for the cells, the ability of using small numbers of multiple cell lines and small amount of the reagents, control of spatial conditions, real-time monitoring and the required optical resolution of the cellular movements Multiple studies had been performed to monitor cell migration towards substrate stiffness (174, 175), biomechanical forces and biochemical gradients (176-180) The migration study of the biochemical gradient in the microfluidic systems were performed on cells cultured in the wall of the device (176, 177) or with 3D scaffold (181, 182) The need for a device that can provide a practical 3D condition and at the same time a stable biochemical gradient and flow control for cell prompted the development of a microfluidic pattern by integrating the hydrogel scaffold in a PDMS device Another group had used a completely different hydrogel device for cell migration assays (183, 184) This approach not only allows monitoring of cell 34   migration towards the biochemical gradients (from the conditioned media) but also provides the ability to co-culture cells and (damaged) tissues at the same time while observing both components This device is low cost but has high resolution and consistent reproducibility as well as operates in 3D condition with low quantity of cells and reagents 35   Chapter Overall hypothesis and objectives 36   Articular cartilage repair is one the most challenging issues in orthopedic surgery due to the avascular and aneural nature of the cartilage, which affect its self-repair capability Cell therapy shows a great potential for cartilage defect repair Autologous chondrocyte is the current clinically approved cell source to repair the injured knee cartilage However, this method has some limitations such as inadequate number, age dependent quality of the cells, donor site morbidity, and some complications such as hypertrophy and calcification of repaired cartilage Recently bone marrow derived mesenchymal stem cells (MSCs) introduced as an alternative cell source for cartilage repair Our group and others showed the differentiation ability of MSCs to chondrocytes, osteocytes and adipocytes Then in this study we hypothesized that MSCs has a role in cartilage repair and can be used as a cell source in developing an injectable method of cartilage repair and BM MSCs migrate to the injured cartilage and help the cartilage repair Also the injured cartilage has the potential to attract MSCs more than uninjured cartilage; and then we hypothesized that using the MSCs can repair the cartilage as good as chondrocytes Then the aims of this thesis / study was: To develop a non invasive repeatable cell tracking method by optimizing MSC iron oxide labeling through systematic evaluation of effects on MSC function and determine the location of injected cells by MRI using an in vivo animal model To evaluate the cell migration, homing/engraftment, and cartilage repair potential of the intra-articular administered labeled stem cells in full thickness cartilage defect in vivo using a large animal model 37   To develop a microfluidic system to evaluate MSC migration against the injured cartilage tissue and to identify candidate chemo-attractants from the damaged tissue To evaluate and compare the clinical outcomes of cartilage repair in patients with full-thickness articular defects treated with ACI or autologous BM MSCs 38   Chapter In vivo monitoring of the intra articular injected SPIO-labeled stem cells for cartilage repair 39   3.1 Abstract Introduction: One the most challenging issues in orthopedic surgery is articular cartilage repair due to poor self-repair capability Cell therapy shows a great potential for cartilage defect repair Using autologous chondrocytes as cell source has limitations such as inadequate cell number, age dependent quality of the cells and donor site morbidity Recently bone marrow derived mesenchymal stem cells (MSCs) attract considerable attention as an alternative cell source for cartilage repair, because of their ability for chondrogenic differentiation Using the iron nanoparticles to label the cells is one of the promising methods to track the cells in vivo by MRI, which is a repeatable and non-invasive approach Purpose: The aim of this study was to optimize MSC iron oxide labeling by systematic evaluation of the methodology on the function of the cells We studied cell migration, homing, and cartilage repair potential of the intraarticular administered labeled MSC in a full thickness cartilage defect (large) animal model Methods: MSCs were characterized by flow cytometry after isolation and expansion The cells were labeled with different concentration (0, 25, 50, 75, 100, 125 µg/ml) of ferucarbotran by simple incubation, and the labeling efficiency and internalization of the particles were confirmed by Prussian blue staining, and transmission electron microscopy (TEM) respectively The effect of the labeling on the viability, proliferation, and differentiation potential of the MSCs were evaluated in female mini-pigs (N=8; 6-months-old, 12-18 kg) Under general anesthesia, the animal’s knee was opened through standard 40   medial para-patellar incision and a 6mm diameter full thickness cartilage defect was made in medial epi-condyle of both knees After one week, 107 labeled cells were mixed with hyaluronan (SYNVISC®) as scaffold and injected in one knee and hyaluronan alone (as control) were injected in the other knee MR imaging of the knee joints were followed over the time (before injection, immediately after as well as and weeks after injection) Animals were euthanized and femoral condyles and surrounding tissues such as surgical scars, synovium membrane, para-patellar fat were excised, fixed, and processed for histological evaluations Statistical analysis (ANOVA test) was performed to compare the multiple groups of the in vitro data, and paired Student’s T-test was used to evaluate the differences of in vivo histological data of both treatment groups Results: MSCs were positive for MSC markers, CD29, CD44, and CD90, and negative for hematopoietic markers, CD14, CD31, CD34, and CD45 Prussian blue staining and TEM confirmed the presence of the particles inside the cells Trypan blue staining showed no significant decrease in viability when labeled with particles in concentrations of ≤ 75 µg/ml Ferucarbotran (P value