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