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Amino Acid-Based Polymeric Scaffold Fabrication And AMINO ACID-BASED POLYMERIC SCAFFOLD FABRICATION AND MODIFICATION FOR BONE REGENERATION APPLICATIONS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Shan Li May, 2018 i AMINO ACID-BASED POLYMERIC SCAFFOLD FABRICATION AND MODIFICATION FOR BONE REGENERATION APPLICATIONS Shan Li Dissertation Approved: Accepted: Advisor Department Chair Dr. Matthew L Becker Dr. Coleen Pugh Committee Member Dean of the College Dr. Yu Zhu Dr. Eric J. Amis Committee Member Dean of the Graduate School Dr. Darrell H. Reneker Dr. Chand Midha Committee Member Date Dr. Toshikazu Miyoshi Committee Member Dr. Rebecca Kuntz Willits ii ABSTRACT Bone tissue engineering has evolved into an inter-disciplinary field of chemistry, engineering, and biology to regenerate defective tissues or organs since its emergence. Polymeric scaffolds represent one of the key components in bone tissue engineering and are widely used due to the low cost, unlimited supply, biointegrity, biodegradability, bioresorbability, tunable mechanical properties, non-toxicity and processability. It is designed to act as a 3D template to provide mechanical support and guide cells to form new tissue by mimicking ECM. Amino acid based poly(ester urea)s (PEUs) are high modulus, biodegradable, and non-toxic thermoplastic polymers, which have been synthesized and characterized by the Becker lab for their applications in tissue engineering. However, their inherent radiolucent and bioinert properties limit clinical application. Efforts have been made to modify PEUs with radiopacity for in vivo detection with X-rays and osteoinductivity via growth factor delivery for bone inducement. The advances in new technologies bring 3D printing to the scaffold fabrication. Unlike the traditional fabrication methods, 3D printing adds materials layer by layer under the guide of a computer, which saves the cost of mold and ensures the high reproducibility of the scaffold. Via CAD design, the architecture of scaffolds could be controlled to meet the criteria for cell penetration and tissue growth. Especially when coupled with medical imaging such as computerized tomography (CT) and magnetic resonance imaging (MRI), the patient specific scaffolds could be 3D printed, which is useful in the clinical aspect. iii Unfortunately, one challenge with 3D printing is the delivery of growth factors since their structure and activity will be affected under the 3D printing processing conditions such as photochemical crosslinking or high temperature. Here the post- printing PEU scaffold surface modification with OGP [10-14] and BMP-2 [73-92] via copper-catalyzed azide alkyne cycloaddition (CuAAC) is reported to resolve this problem. The in vitro hMSCs osteogenic differentiation study demonstrated the enhancement effect of both peptides in terms of ALP activity, gene expression, protein expression, and calcium deposition. The in vivo study with a 8 mm rat cranial critical size defect model confirms the results from in vitro that more new bone was formed in the peptide functionalized samples using µ-CT 3D scanning, H&E staining and Goldner’s trichrome staining. Additionally, under X-ray, the iodinated PEUs rendered clear images showing 3D architecture while the non-iodine functionalized groups were undetectable. iv ACKNOWLEDGEMENTS This dissertation would not have been possible without the help from other people. First and foremost I would like to express the deepest appreciation to my advisor, Dr. Matthew L. Becker, for all his constant support, valuable help, and patient guidance during my graduate study. He has been a very good example as researcher and advisor and it has been my great honor to work with him. I am also very grateful to my dissertation committee: Dr. Yu Zhu, Dr. Darrell H. Reneker, Dr. Abraham Joy and Dr. Rebecca Kuntz Willits for their comments and help in the final stage of work. I appreciate the help from Dr. Rebecca Kuntz Willits in the animal study and the student in College of Polymer Science and Engineering, especially Dr. Fei Lin, Dr. Yanyi Xu, Dr. Jukuan Zheng, Dr. Kun Yang, Jiayi Yu, Karissa Hagen, Derek Luong and all my group members for their collaboration, discussion, and encouragement in my research. Finally, special thanks go to my family. For my parents and grandparents who raised me with love and encouragement. For my little brother who gave me understanding. For my husband, Tian Liang, for his love and support. v TABLE OF CONTENTS Page TABLE OF CONTENTS .............................................................................................. vi LIST OF FIGURES ....................................................................................................... x LIST OF SCHEMES ................................................................................................... xvi LIST OF TABLES ..................................................................................................... xvii CHAPTER I. INTRODUCTION ...................................................................................................... 1 1.1 Bone defect repair ................................................................................................ 1 1.2 Scaffold ................................................................................................................ 3 1.2.1 Synthetic flexibility ....................................................................................... 5 1.2.2 Chemical functionality .................................................................................. 6 1.2.3 Mechanical properties .................................................................................... 9 1.2.4 Degradation property ................................................................................... 10 1.3 Scaffold fabrication by 3D printing.................................................................... 11 1.4 Scaffolds modification ....................................................................................... 17 1.4.1 Radiopacity .................................................................................................. 17 1.4.2 Osteoinductivity........................................................................................... 21 1.4.2.1 Bone morphogenic proteins (BMPs) .................................................. 21 1.4.2.2 Osteogenic growth peptide (OGP) ..................................................... 25 II. MATERIALS AND INSTRUMENTS ................................................................... 29 2.1 Materials ............................................................................................................. 29 2.2 Instruments ......................................................................................................... 31 III. RADIOPAQUE, IODINE FUNCTIONALIZED PHENYLALANINE-BASED POLY(ESTERUREA)S ........................................................................................ 36 3.1 Abstract .............................................................................................................. 36 3.2 Introduction ........................................................................................................ 37 vi 3.3 Experimental Section ......................................................................................... 40 3.3.1 Materials ...................................................................................................... 40 3.3.2 Characterization of chemical structure and thermal properties ................... 41 3.3.3 Synthesis of di-p-toluene sulfonic acid salt of bis-L-phenylalanine-1,6- hexanediol-diester (1-PHE-6 monomer) and di-p-toluene sulfonic acid salt of bis- 4-I-L-phenylalanine-1,6-hexanediol-diester (1-iPHE-6 monomer) 41 3.3.4 Synthesis of bis-L-phenylalanine-1,6-hexanediol-diester PEU (poly(1-PHE- 6)), bis-4-I-L-phenylalanine-1,6-hexanediol-diester PEU (poly(1-iPHE-6)), co- polymers of 1-iPHE-6 monomer and 1-PHE-6 monomer (1:4 molar ratio, poly(1- iPHE-6)0.24-co-poly(1-PHE-6)0.76) and co-poly(ester urea) of 1-iPHE-6 monomer and 1-PHE-6 monomer (3:4 molar ratio, poly(1- iPHE-6)0.44-co- poly(1-PHE-6)0.56). ........................................................ 43 3.3.5 PEU films and 3D porous scaffold preparation and characterization .......... 46 3.3.6 In vitro cell viability and spreading characterization................................... 47 3.4 Results and discussion ........................................................................................ 49 3.4.1 Synthesis of L-phenylalanine-based and 4-I-L-phenylalanine-based poly(ester urea)s ........................................................................................... 49 3.4.2 Thermal properties of PEUs ........................................................................ 53 3.4.3 Mechanical properties of bulk PEU films ................................................... 55 3.4.4 Radiopacity of PEUs.................................................................................... 56 3.4.5 PEU 3D porous scaffolds analysis ............................................................... 59 3.4.6 Cell viability and spreading assay ............................................................... 62 3.5 Conclusion .......................................................................................................... 64 3.6 Acknowledgement .............................................................................................. 65 IV. ENHANCED
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