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THE DEVELOPMENT of a MESENCHYMAL STEM CELL BASED BIPHASIC OSTEOCHONDRAL TISSUE ENGINEERED CONSTRUCT Scott Am Xson Clemson University, [email protected]

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THE DEVELOPMENT of a MESENCHYMAL STEM CELL BASED BIPHASIC OSTEOCHONDRAL TISSUE ENGINEERED CONSTRUCT Scott Am Xson Clemson University, Smaxson@Clemson.Edu Clemson University TigerPrints All Dissertations Dissertations 1-2010 THE DEVELOPMENT OF A MESENCHYMAL STEM CELL BASED BIPHASIC OSTEOCHONDRAL TISSUE ENGINEERED CONSTRUCT Scott aM xson Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Maxson, Scott, "THE DEVELOPMENT OF A MESENCHYMAL STEM CELL BASED BIPHASIC OSTEOCHONDRAL TISSUE ENGINEERED CONSTRUCT" (2010). All Dissertations. 684. https://tigerprints.clemson.edu/all_dissertations/684 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. THE DEVELOPMENT OF A MESENCHYMAL STEM CELL BASED BIPHASIC OSTEOCHONDRAL TISSUE ENGINEERED CONSTRUCT A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Bioengineering by Scott Anthony Maxson May 2010 Accepted by: Dr. Karen J. L. Burg, Committee Chair Dr. Martine LaBerge Dr. Ted Bateman Dr. James Kellam ABSTRACT The ability of human articular cartilage to respond to injury is poor. Once cartilage damage has occurred, an irreversible degenerative process can occur and will often lead to osteoarthritis (OA). An estimated 26.9 million of U.S. adults are affected by OA. Osteochondral grafting is currently used to treat OA and osteochondral defects; however, complications can develop at the donor site and defect area. Osteochondral tissue engineering provides a potential treatment option and alternative to osteochondral grafting. The long term goal of this work is to develop a tissue engineered mesenchymal stem cell (MSC) based osteochondral construct to repair cartilage damage. The first set of studies evaluated the interactions between skeletal tissue cells and the effectiveness of conditioned media (CM) to modulate mesenchymal stem cell differentiation. The results showed that CM can enhance the differentiation of MSCs towards several different cell types depending on the type of CM. The next set of studies involved the design and characterization of a novel biphasic osteochondral tissue engineered construct. The osteochondral constructs were designed to address some of the current limitations that osteochondral tissue engineering currently faces; i.e., providing an alternate method of anchoring the construct and providing the ability to generate large viable constructs. Based on the results of several studies, a composite of polylactide/polycaprolactone and hydroxyapatite was used for the bone phase, a barbed pin was used to create a stable implant, and agarose was chosen for use in the cartilage phase. In addition, capillary channel fibers were incorporated in the bone phase to provide nutrient and cell delivery. The bundles of capillary channel fibers ii embedded in large tissue engineering scaffolds were shown to facilitate a uniform cell distribution through large volume scaffolds. In the next study, a modified bioreactor was designed and incorporated a pneumatically controlled syringe to generate hydrostatic pressure (HP) on both phases of the biphasic construct. The bioreactor was used to demonstrate synergistic effects of CM and HP on the chondrogenic and osteogenic differentiation of MSCs. Ultimately, in a final study, the combination of CM, bioreactor cultivation, and the unique construct design generated a biphasic construct with a cartilage-like upper phase attached to a bone-like bottom phase. iii DEDICATION This work is dedicated to those closest to me: First of all to my amazing wife, Nicole, who has brought so much happiness and adventure to my life. Without her love, support, and patience the completion of this work would not have been possible. To my parents for their continual love and sacrifice throughout my life that has provided me with so much. To my brother for his friendship and for his hard work that always served as my example to follow. iv ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Karen J. L. Burg, for her guidance and encouragement. Additionally, I would like to acknowledge my committee members, Dr. Martine LaBerge, Dr. Ted Bateman, and Dr. James Kellam for their involvement in the work. I would also like to thank the following people for their help: All the members of the Tissue Engineering Laboratory and the Institute for Biological Interfaces of Engineering for answering my questions and helping me with various problems along the way; Linda Jenkins for her help with histology; Kim Ivey for her help with gel permeation chromatography; Dr. Ken Webb for his help with primer design; and all of the other students in the Bioengineering department for their help on numerous occasions. I appreciate the department administrative staff, including Maria Martin, Maranda Arnold, Sherri Morrison, and Leigh Humphries for always happily assisting me whenever I asked. This work was funded by the AO Foundation, the Clemson University Hunter Endowment Fund, and the Department of Defense, Era of Hope Scholars Award. v TABLE OF CONTENTS TITLE PAGE ................................................................................................................... i ABSTRACT .................................................................................................................... ii DEDICATION ............................................................................................................... iv ACKNOWLEDGEMENTS ............................................................................................. v LIST OF TABLES ......................................................................................................... ix LIST OF FIGURES ......................................................................................................... x PREFACE .................................................................................................................... xiv CHAPTER 1. LITERATURE REVIEW ................................................................................... 1 Native Osteochondral Tissue .......................................................................... 1 Significance of Problem ................................................................................ 16 Current Treatment Options ........................................................................... 20 Osteochondral Tissue Engineering ............................................................... 26 Bioreactors in Tissue Engineering ............................................................... 28 2. INTERACTIONS BETWEEN SKELETAL TISSUE CELLS AND THE EFFECTIVENESS OF CONDITIONED MEDIA TO MODULATE MESENCHYMAL STEM CELL DIFFERENTIATION .................................. 43 Introduction .................................................................................................... 43 Conditioned Media Cause Increases in Select Osteogenic and Adipogenic Differentiation Markers in Mesenchymal Stem Cell Cultures ....................................................................................................... 45 Methods and Materials ..................................................................... 45 Results................................................................................................ 51 Conditioned Media Enhance the Osteogenic and Chondrogenic Differentiation of Mesenchymal Stem Cells ............................................ 56 Methods and Materials ..................................................................... 56 Results................................................................................................ 65 Discussion .............................................................................................. 71 Conclusion ..................................................................................................... 83 vi 3. DESIGN AND CHARACTERIZATION OF A NOVEL BIPHASIC OSTEOCHONDRAL TISSUE ENGINEERING CONSTRUCT ...................... 91 Introduction .................................................................................................... 91 Design of Novel Osteochondral Constructs................................................. 95 Construction of Novel Osteochondral Constructs ................................... 99 Characterization of the Newly Designed Osteochondral Constructs and Evaluation of the Stability of Anchoring Systems .......................... 103 Methods and Materials ................................................................... 103 Results.............................................................................................. 106 Discussion ............................................................................................ 110 Conclusion ........................................................................................... 113 4. EVALUATION AND OPTIMIZATION OF THE NEWLY DESIGNED OSTEOCHONDRAL CONSTRUCTS ........................................................... 117 Introduction .................................................................................................. 117 The Effect of Hydroxyapatite on MSC Attachment and Osteogenic Differentiation in a Bone Phase Polymer Scaffold ................................ 117 Methods and Materials ................................................................... 117 Results.............................................................................................
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