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Polymer Biomaterial Constructs for Regenerative POLYMER BIOMATERIAL CONSTRUCTS FOR REGENERATIVE MEDICINE AND FUNCTIONAL BIOLOGICAL SYSTEMS by LINGHUI MENG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Gary E. Wnek Department of Macromolecular Science and Engineering CASE WESTERN RESERVE UNIVERSITY May, 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Linghui Meng s candidate for the Ph.D. degree *. (signed) Dr. Gary E. Wnek s (chair of the committee) Dr. David Schiraldi s Dr. Alexander M. Jamieson s Dr. Horst Von Recum s (date) 12/05/2011 s *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Chapter I Crosslinking and Modification of Collagen-based Materials ..................... 1 1.1 Introduction ............................................................................................................................ 1 1.2 Characteristics of Collagen as a Biomaterial ......................................................................... 2 1.2.1 Collagen Native Structures ............................................................................................. 2 1.2.2 Collagen Natural Crosslinks ........................................................................................... 6 1.3 Isolation and Purification of Collagen ................................................................................... 7 1.4 Exogeneous Crosslinking of Collagen ................................................................................. 10 1.4.1 Chemical Crosslinking .................................................................................................. 11 1.4.2 Physical Crosslinking ................................................................................................... 17 1.4.3 Enzymatic Crosslinking ................................................................................................ 18 1.5 Collagen in Tissue Engineering ........................................................................................... 19 1.5.1 Electrospinning Technique ........................................................................................... 19 1.5.2 Electrospinning of Collagen ......................................................................................... 21 1.5.3 Electrospinning of Collagen and Natural Polymer Blends ........................................... 24 1.5.4 Electrospinning of Collagen and Synthetic Polymer Blends ........................................ 26 1.6 Dissertation Overview ......................................................................................................... 29 Chapter II Collagen-PCL Sheath-Core Bicomponent Fibers ..................................... 30 2.1 Introduction .......................................................................................................................... 30 2.2 Coaxial Electrospinning ....................................................................................................... 31 2.3. Fabrication and Characterization of Collagen-PCL Sheath-Core Fibers ............................ 33 2.3.1 Materials ....................................................................................................................... 33 2.3.2 Coaxial Electrospinning of Collagen and PCL ............................................................. 34 2.3.3 Post-crosslinking Treatment ......................................................................................... 35 2.3.4 Characterization of Fiber Morphology ......................................................................... 35 2.3.5 Characterization of Mechanical Properties ................................................................... 36 2.4 Results and Discussion ........................................................................................................ 37 2.4.1 Core-Shell Fiber Formation .......................................................................................... 37 2.4.2 Post-Crosslinking of Collagen-PCL Hybrid Fibers ...................................................... 44 2.4.3 Mechanical Properties of Collagen-PCL Scaffolds ...................................................... 45 2.5 Conclusions .......................................................................................................................... 48 Chapter III In-situ Crosslinked Collagen Nanofibers ................................................. 50 3.1 Introduction .......................................................................................................................... 50 i 3.2 Fabrication and Characterization of In-situ Crosslinked Collagen Nanofibers ................... 53 3.2.1 Materials ....................................................................................................................... 53 3.2.2 Preparation of In-situ Crosslinking Collagen Solution ................................................. 54 3.2.3 Rheological Experiment ............................................................................................... 54 3.2.4 Electrospinning of In-situ Crosslinked Collagen Nanofibers ....................................... 55 3.2.5 Characterization of Fiber Morphology ......................................................................... 55 3.2.6 Fourier Transform Infrared (FTIR) Spectroscopy ........................................................ 56 3.2.7 Characterization of Mechanical Properties ................................................................... 56 3.3 Results and Discussion ........................................................................................................ 57 3.3.1 In-situ Crosslinking Approach ...................................................................................... 57 3.3.2 Formation of In-situ Crosslinked Collagen Nanofibers ................................................ 59 3.3.3 Mechanical Properties of In-situ Crosslinked Collagen Scaffolds ............................... 66 3.3.4 FTIR Spectra of In-situ Crosslinked Collagen Scaffolds .............................................. 68 3.3.5 Plausible Mechanisms of In-situ Crosslinking Reactions ............................................. 70 3.4 Applications of In-situ Crosslinked Collagen Fiber Scaffolds ............................................ 72 3.4.1 Cell Culture Studies ...................................................................................................... 72 3.4.2 Open Wound Healing Test ........................................................................................... 74 3.4.3 Drug Release Studies .................................................................................................... 77 3.5 Conclusions .......................................................................................................................... 81 Chapter IV In-situ Crosslinked Collagen Gels and Sponges ...................................... 84 4.1 In-situ Crosslinked Collagen Gels ....................................................................................... 84 4.1.1 Formation and Characterization of In-situ Crosslinked Collagen Gels ........................ 85 4.1.2 Results and Discussion ................................................................................................. 87 4.1.3 Conclusions ................................................................................................................... 94 4.2 In-situ Crosslinked Collagen Sponges ................................................................................. 95 4.2.1 Formation and Characterization of In-situ Crosslinked Collagen Sponges .................. 96 4.2.2 Results and Discussion ................................................................................................. 97 4.2.3 Conclusions ................................................................................................................. 101 4.3 Biomineralization of In-situ Crosslinked Collagen Gels ................................................... 102 4.3.1 Electrophoresis Experiments ...................................................................................... 103 4.3.2 Results and Discussion ............................................................................................... 104 4.3.3 Conclusions ................................................................................................................. 107 Chapter V Phase Transition of Poly (Acrylic Acid) Nanofibers .............................. 109 5.1 Introduction ........................................................................................................................ 109 ii 5.2 Formation and Characterization of PAA Nanofiber Tubes ................................................ 111 5.2.1 Materials ..................................................................................................................... 111 5.2.2 Preparation of PAA Ethanol Solution ......................................................................... 111 5.2.3 Fabrication of PAA Nanofibers .................................................................................. 111 5.2.4 Thermo-crosslinking of PAA Nanofibers ................................................................... 112 5.2.5 Neutralization of PAA Nanofiber Tubes .................................................................... 112 5.2.6 Characterization of Fiber
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