BIOMATERIALS FOR TISSUE ENGINEERING FOR RHEUMATOID ARTHRITIS BASED ON CONTROLLING DENDRITIC CELL PHENOTYPE A Dissertation Presented to The Academic Faculty By Jaehyung Park In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy in Bioengineering Georgia Institute of Technology August 2009 BIOMATERIALS FOR TISSUE ENGINEERING FOR RHEUMATOID ARTHRITIS BASED ON CONTROLLING DENDRITIC CELL PHENOTYPE Approved by: Dr. Julia Babensee, advisor Dr. Andrés García College of Engineering College of Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Barbara Boyan Dr. Robert Guldberg College of Engineering College of Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Todd McDevitt College of Engineering Georgia Institute of Technology Date Approved: June 25, 2009 ii ACKNOWLEDGEMENTS I especially wish to express my gratitude to my advisor, Dr. Julia Babensee, who from the outset, encouraged me in my research work and provided me with many details and suggestions for the research progress. During my years at Georgia Tech, as ever, I benefited greatly from her vast knowledge, intelligence, and originality of mind. Her sincere guidance and encouragement inspired me to strive during my difficult period. This unforgettable support and trust I have received from her will continue to be indispensable for my future career in whichever place or position I might be. Deepest gratitude is also due to the members of the thesis committee, Dr. Barbara Boyan, Dr. Andrés García, Dr. Robert Guldberg, and Dr. Todd McDevitt, without whose contributions and commitment this thesis study would not have been successful. There are other kinds of help that make this thesis research possible: I also would like to record my gratitude to Dr. Brani Vidakovic and Dr. Melissa Kemp for their invaluable advice. I am very grateful to the staff at Georgia Tech including Johnafel Crowe, Steve Woodard, and Sha’aqua Asberry for their helpful technical support. I also would like to extend my sincere appreciation to a number of individuals who have given me help and encouragement over the period in which this research work was done. In the successful completion of my doctoral thesis study, the in vitro study using human blood would never have been completed without the help of the staff at the Phlebotomy Laboratory (Georgia Tech Student Health Center): Jack Horner, Erica Waller, LaShonna Stokes, Lisa Carr, and Patricia Dureja. My highest appreciation also goes to all blood donors for their constant support for my research work. The completion of the in vivo rabbit study involved the labor and support of Dr. Laura O’Farrell, Angela Lin, and all staff members at the Physiological Research Laboratory (PRL). iii I would like to recognize the creative discussion and helpful input of my remarkable cohorts: Dr. Mutsumi Yoshida, Dr. Stacey Rose, Dr. Sucharita Shankar, Dr. Richard Payne, Dr. Lori Norton, Todd Rogers, Peng Meng Kou, Nathan Hotaling, Inn Inn Chen, Christina Duden, Michael Gerber, Rishi Patel, and Carrie Oliver. I am especially grateful to my undergraduate research assistants, Michael Gerber, Rishi Patel, and Carrie Oliver, for their faith and patient encouragement in this project. I also have greatly appreciated the help and pleasant moments from my friends in school and the Institute of Biosciences and Bioengineering (IBB), especially the Wing 1D. I would like to take this opportunity to thank my family, my parents, and my sisters. My parents instilled in me the belief that intellectual pursuit is the highest calling, and that ideas do have the power to change people’s lives. As always, they have been there, providing all sorts of tangible and intangible support to achieve my academic and personal goals, even from afar. It is a particular pleasure to acknowledge my debt to my lovely daughter, Jasmine (Yunjae), for her patience and understanding. My wife, Yunhee, has patiently supported me in innumerable ways; whatever I might say here cannot do full justice to the extent and the value of her contribution. This work has been supported by funding from Arthritis Foundation (Arthritis Investigator Grant), National Institutes of Health (NIBIB/NHLBI, 1RO1EB004633-01A1), National Science Foundation (CAREER Award), and Georgia Tech/Emory Center for the Engineering of Living Tissues (GTEC - Critical Animal Model). iv TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………………....iii LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix SUMMARY………………………………………………………………………...…...xiv CHAPTER 1: INTRODUCTION.......................................................................................1 CHAPTER 2: RESEARCH SIGNIFICANCE....................................................................7 CHAPTER 3: LITERATURE REVIEW............................................................................9 Immunology of RA and role of dendritic cells......................................................9 Rheumatoid Arthritis (RA) and tissue engineering..............................................12 Dendritic cells......................................................................................................15 Innate and adaptive immune response.................................................................17 Adjuvant..............................................................................................................17 Biomaterials in tissue engineering and dendritic cells.........................................19 Biomaterials in combination products.................................................................22 Summary..............................................................................................................27 CHAPTER 4: DIFFERENTIAL FUNCTIONAL EFFECTS OF BIOMATERIALS ON DENDRITIC CELL MATURATION. PART 1. EFFECTS OF BIOMATERIALS IN 2-DIMENSIONAL FILM FORM.........................28 Introduction..........................................................................................................28 Methods...............................................................................................................29 Results..................................................................................................................41 Discussion............................................................................................................57 CHAPTER 5: DIFFERENTIAL FUNCTIONAL EFFECTS OF BIOMATERIALS ON v DENDRITIC CELL MATURATION. PART 2. EFFECTS OF BIOMATERIALS IN 3-DIMENSIONAL SCAFFOLD FORM..............68 Introduction..........................................................................................................68 Methods...............................................................................................................70 Results..................................................................................................................78 Discussion............................................................................................................86 CHAPTER 6: PHENOTYPE AND POLARIZATION OF AUTOLOGOUS T CELLS BY BIOMATERIAL-TREATED DENDRITIC CELLS.................................94 Introduction..........................................................................................................94 Methods...............................................................................................................98 Results................................................................................................................110 Discussion..........................................................................................................122 CHAPTER 7: DIFFERENTIAL INTEGRATION OF BIOMATERIALS IMPLANTED INTO THE KNEE JOINT OF RHEUMATOID ARTHRITIS INDUCED RABBIT..................................................................................................134 Introduction........................................................................................................134 Methods.............................................................................................................137 Results................................................................................................................145 Discussion..........................................................................................................165 CHAPTER 8: CONCLUSIONS AND FUTURE WORK..............................................173 APPENDICES................................................................................................................182 A.1 Water content of biomaterial films...........................................................182 A.2 Differential functional effects of clinical grade biomaterials on DC maturation................................................................................................184 A.3 Differential functional effects of biomaterials on autologous T cell marker expressions when DCs are treated with biomaterials in the absence of model antigen, OVA.................................................................................187 A.4 Differential functional effects of biomaterials on cytokine secretion from vi DCs in almost the same levels even after DCs were isolated from biomaterials..............................................................................................189 REFERENCES...............................................................................................................191 vii LIST OF TABLES Table 1: Low resolution XPS survey scans of biomaterial film surfaces used for DC treatment...............................................................................................................42 Table 2: High resolution XPS scans of biomaterial film surfaces used for DC treatment. ...............................................................................................................................43 Table 3: Samples and controls in 4 different groups used in the Chapter 6...................107 Table 4. Rabbit groups and treatments...........................................................................141 Table 5. X-ray attenuation values obtained from the micro-CT analysis on rabbit groups. .............................................................................................................................157 viii LIST OF FIGURES Figure 1-1: Schematic representation of dendritic cell phenotype changes and T cell- mediated adaptive immunity.............................................................................6 Figure 4-1: Schematic representation of the in vitro experimental procedure of DC culture and treatment with biomaterials......................................................................36 Figure 4-2: Dendritic cell treated with PLGA or chitosan films possess cell morphologies similar to mDC induced with LPS treatment..................................................44 Figure 4-3: Flow cytometry histograms for expression of co-stimulatory molecules, CD40, CD80, CD86, the maturation marker, CD83, and MHC class II molecules, HLA-DQ and HLA-DR in differential levels depending on DCs treated with different biomaterials..................................................................47 Figure 4-4: Dendritic cells treated with PLGA or chitosan film induced CD 86 expression in levels significantly higher than iDCs or other biomaterial treatments.......48 Figure 4-5: Allostimulatory capacities in Mixed Lymphocyte Reaction (MLR) in differential levels depending on DCs treated with different biomaterial films. .........................................................................................................................49 Figure 4-6: Differential levels of tumor necrosis factor (TNF) – α (a) and Interleukin -6 (IL-6) (b) in differential levels upon DC treatment with biomaterial films....51 Figure 4-7: Activation of NF-κB (subunit of p50) upon DC treatment with biomaterials as a function of time (5 and 24 hours).................................................................52 Figure 4-8: Geometric mean fluorescence intensity (gMFI) of flow cytometry analysis of Annexin V and propidium iodide (PI) expression in differential levels upon DCs treated with different biomaterial films..................................................54 ix Figure 4-9: Geometric mean fluorescence intensity (gMFI) of flow cytometry analysis of FITC-dextran uptake by DCs in differential levels upon DCs treated with different biomaterial films..............................................................................55 Figure 4-10: Geometric mean fluorescence intensity (gMFI) of flow cytometry analysis of CD32, CD206, and CD44 expression in differential levels upon DCs treated with different biomaterial films..........................................................56 Figure 4-11: Schematic representation of effects of biomaterials in 2-dimensional film forms on human monocyte-derived DCs........................................................57 Figure 5-1: PLGA and agarose scaffolds exhibited different morphologies of cross- section.............................................................................................................79 Figure 5-2: Schematic representations (view of top surface and cross-section of scaffolds) of cell distributions into 3-D porous scaffolds and cell morphologies of control DCs or upon DC treatment with scaffolds..........................................80 Figure 5-3: Geometric mean fluorescence intensity (gMFI) of flow cytometry analysis on co-stimulatory, MHC class II, and other functional (DC migration) molecules of CD44 on DCs treated with biomaterial scaffolds (PLGA or agarose).......82 Figure 5-4: Allostimulatory capacities in Mixed Lymphocyte Reaction (MLR) in differential levels upon DCs treated with biomaterial scaffolds (PLGA or agarose)...........................................................................................................83 Figure 5-5: Differential levels of pro-inflammatory cytokine (a), chemokine (b), & anti- inflammatory cytokine (c) release upon DC treatment with biomaterial scaffolds (PLGA or agarose)..........................................................................85 Figure 5-6: Schematic representation of effects of biomaterials in 3-dimensional scaffold forms on human monocyte-derived DCs........................................................86 Figure 6-1: Schematic representation of the study procedure (Fig. 6-1a) & time line (Fig. 6-1b). During 14 days, the study has been performed based on three main procedures as shown by color-coded blocks.................................................103 x