Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2011 Characterization of Novel Akermanite:Poly-E- Caprolactone Scaffolds for Bone Tissue Engineering Aapplications Combined with Human Adipose-Derived Stem Cells Andre S. Zanetti Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Engineering Science and Materials Commons Recommended Citation Zanetti, Andre S., "Characterization of Novel Akermanite:Poly-E-Caprolactone Scaffolds for Bone Tissue Engineering Aapplications Combined with Human Adipose-Derived Stem Cells" (2011). LSU Doctoral Dissertations. 2797. https://digitalcommons.lsu.edu/gradschool_dissertations/2797 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. CHARACTERIZATION OF NOVEL AKERMANITE:POLY-E-CAPROLACTONE SCAFFOLDS FOR BONE TISSUE ENGINEERING APPLICATIONS COMBINED WITH HUMAN ADIPOSE-DERIVED STEM CELLS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Interdepartmental Program in Engineering Sciences by Andre S. Zanetti D.V.M., Sao Paulo State University, 2006 M.S. in Veterinary Medical Sciences, Louisiana State University, 2009 December, 2011 Acknowledgments It is a pleasure to thank the many people who made this dissertation possible. It is difficult to overstate my gratitude to my Ph.D. supervisor, Dr. Daniel J. Hayes. With his passion, his inspiration, and his great efforts to provide available resources, he helped to make the completion of this section of my academic life pleasant and straightforward. I would like to thank the many people who provided ideal conditions for the development of the research presented in this dissertation: Ted Garthier for his assistance with the lyophilized and the plate reader as well as for letting me use some of his RT-PCR reagents during my pilot study; Julia Y. Chang and Gregory McCandless for the synthesis of akermanite and heat treatment of all ceramic scaffolds used in this dissertation; Marc J Boudreaux for teaching me how to run and analyze qPCR data; Matthew Brown for teaching me how to use the confocal and the fluorescent microscopy at the Biological Sciences Building; Ying Xiao for teaching me how to use the scanning electron microscopy (SEM); Dr. Kenneth Matthews for teaching me how to use the micro-computer tomography machine (μ-CT) in the Physics Department; Dr. Ram Devireddy for letting me use his cell culture facility during my pilot study and for teaching me how to use the cooling machine when we were investigating cryopreservation of scaffolds loaded with human adipose-derived stem cells; and my laboratory colleagues (Lekeith Terrell and Mark Hoppens) for helping me with the fabrication of scaffolds using the unidirectional freezing technique and for assisting me with the analysis of the scaffolds collected from mice, respectively; and for Dr. David Burk for assisting with the histological processing and staining of the scaffolds. I am indebted to my many graduate student colleagues for providing a stimulating and fun environment in which to learn and grow. I am especially grateful to Jeffrey Cardinale, Britton ii Grasperge, Paula Mischler, Sunita Seemanapalli, Rebecca Christofferson, Lekeith Terrell, Niranjan Butchi and Lie Xie. I am grateful for the College of Engineering Sciences and the Department of Biological Engineering for giving me a home to finish my Ph.D. research project and the administrative assistants (Claudia E. Hawkins, Angela Singleton, Donna T. Elisar and Rhonda D. Shepard) for helping with the deadlines and for assisting me in many different ways. I would like to thank Dr. Bruce Bunnell and Marjorie McCantes for the human bone marrow- derived stem cells used in the in vivo experiment. I wish to thank Dr. Barry Roberts and Cynthia Kloster for assisting Dr. Daniel J. Hayes and I with the animals and orientation at the Pennington Biomedical Research Center. I would like to thank Dr. Jeffrey M. Gimble for the adult stem cells and for the countless advices; for sharing his passion to biomedical research; and for believing in my potential during the most difficult times. To him I dedicate most of this work. I would like also to thank Drs. Cristina Sabliov, Michael J. Keenan and Ram Devireddy for accepting to be part of my Ph.D. research committee. I wish to thank my entire extended family for providing a loving environment for me. My siblings: Marcello and Fernanda; and my deceased grandparents: Elydia and Americo that, unfortunately, did not physically survive to treasure this moment of happiness with us. Lastly, and most importantly, I wish to thank my parents, Giuseppe Americo Zanetti and Marilene C. Serrano Zanetti. They born me, raised me, supported me, taught me, and loved me. To them I dedicate this entire dissertation. This study was supported by the Longwell Family Foundation and the Pennington Biomedical Research Foundation. iii Table of Contents Acknowledgments........................................................................................................................... ii List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix Abstract ........................................................................................................................................ xiii Chapter 1. Human Adipose-Derived Stem Cells and 3D Scaffold Constructs: A Review of the Biomaterials and Models Currently Used to Regenerate Bone…..………………………………..1 1.1. Introduction ................................................................................................................ 1 1.2. hASC Osteogenesis: A Retrospective Analysis ......................................................... 3 1.2.1. Isolation, Characterization and Problems with Nomenclature ..................... 3 1.2.2. Culture Requirements ................................................................................... 6 1.2.3. hASC Osteogenic Differentiation ................................................................. 7 1.3. hASC and Scaffolds for Bone Tissue Engineering .................................................... 9 1.3.1. The Tissue Engineering Decade ................................................................... 9 1.3.2. Current Biomaterials Used in hASC Bone Tissue Engineering Strategies . 11 1.3.3. Current Bone Tissue Engineering Models: A Retrospective Analysis on hASC-Scaffolds .................................................................................................... 19 1.4. Conclusions .............................................................................................................. 35 1.5. References ................................................................................................................ 35 Chapter 2. Characterization of Novel Akermanite:Poly-E-Caprolactone Scaffolds for Human Adipose-Derived Stem Cells Bone Tissue Engineering ............................................................... 41 2.1. Introduction .............................................................................................................. 41 2.2. Materials and Methods ............................................................................................. 44 2.2.1. Synthesis of Akermanite ............................................................................. 44 2.2.2. Characterization of Akermanite by Powder X-Ray Diffraction ................. 45 2.2.3. Synthesis of Ceramic Scaffolds .................................................................. 46 2.2.4. Fabrication of PCL and Akermanite:PCL Scaffolds .................................. 46 2.2.5. Scaffolds Ultrastructural Characteristics .................................................... 47 2.2.6. Mechanical Test .......................................................................................... 47 2.2.7. Degradation Rate in Stromal Medium ........................................................ 47 2.2.8. Isolation of hASC and Culture .................................................................... 48 2.2.9. MTT Assay and hASC-Cytotoxicity to Medium Extracts .......................... 48 2.2.10. hASC Loading on Scaffolds and Culture.................................................. 49 2.2.11. Quantification of DNA on Scaffolds ........................................................ 49 2.2.12. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) ................ 49 2.2.13. Statistical Analysis .................................................................................... 50 2.3. Results ...................................................................................................................... 50 2.3.1. Synthetic Akermanite Purity Confirmation ................................................ 50 iv 2.3.2. Ultrasctructure Characterization of the Scaffolds ....................................... 51 2.3.3. Compression