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  • A Novel Thiolated Hyaluronic acid Hydrogel for

    Spinal Cord Injury Repair

    Ruifu Li

    A thesis submitted to the

    Faculty of Graduate and Postdoctoral Studies

    In partial fulfillment of the requirements

    For the MASc degree in Chemical Engineering

    Department of Chemical and Biological Engineering

    Faculty of Engineering

    University of Ottawa,

    © Ruifu Li, Ottawa, Canada, 2014

  • ii

    Acknowledgments

    I would like to extend my sincerest thanks and appreciation to my supervisor Dr.

    Xudong Cao, for his patience, background knowledge, guidance and support over

    the course of this research. He was a constant inspiration, and his assistance and

    suggestions were invaluable towards the completion of this work.

    I would also like to thank Dr. Erik Suuronen and Donna Padavan for granting access

    to the rheometer, and the help in rheological experiment. To the other members of

    my group, especially Yan Du, Chunyu Lu and Jian Tang, thank you for the assistance

    when I needed it.

    Finally, I would like to express my deepest gratitude to my parents, for their support,

    patience and encouragement during my pursuit of the Master’s degree.

  • iii

    Table of contents Acknowledgments .......................................................................................................... ii Table of contents .......................................................................................................... iii List of Figures ................................................................................................................. v List of Schemes and Tables ........................................................................................... vi Abstract ....................................................................................................................... vii Résumé ........................................................................................................................ viii Nomenclature ............................................................................................................... ix 1. Introduction ........................................................................................................... 1

    1.1 A short history of biomaterials ........................................................................ 2

    1.2 Biodegradable polymeric materials ................................................................. 3

    2. Literature survey ........................................................................................................ 6 2.1 Spinal cord injury .............................................................................................. 6

    2.1.1 Overview of SCI ................................................................................... 6 2.1.2 Inhibitory factors ................................................................................. 7 2.1.3 Nerve growth factor ............................................................................ 9 2.1.4 Development of neurotrophic factors therapeutic methods ........... 10

    2.2 Hydrogels ....................................................................................................... 12

    2.2.1 Properties of hydrogels ..................................................................... 12 2.2.2 Preparation of hydrogel .................................................................... 15

    2.3 Naturally derived materials ............................................................................ 17

    2.3.1 Collagen ............................................................................................. 17 2.3.2 Hyaluronic acid (HA) .......................................................................... 17 2.3.3 Alginate ............................................................................................. 19 2.3.4 Chitosan............................................................................................. 20

    2.4 Cell adhesion .................................................................................................. 22

    2.4.1 Cell adhesive proteins and peptides ................................................. 22 2.4.2 Most commonly used cell adhesive peptides ................................... 23

    3 Hypothesis and Objectives ........................................................................................ 25 3.1 Project goals ................................................................................................... 25

    3.2 Hypothesis ...................................................................................................... 25

    3.3 Objectives ....................................................................................................... 25

    4. Experimental Methods ............................................................................................. 27 4.1 Materials ........................................................................................................ 27

    4.2 Synthesis of thiolated-hyaluronic acid (HA-SH) ............................................. 27

  • iv

    4.3 Determination of thiol groups ....................................................................... 30

    4.3.1 Fourier transform infrared (FT-IR) spectroscopy of HA-SH .............. 30 4.3.2 Ellman’s test ...................................................................................... 31 4.3.3 Stability of thiol groups in the lyophilized product ........................... 32

    4.4 Differential scanning calorimetry (DSC) analysis ........................................... 32

    4.5 Cross-linking of hydrogel ................................................................................ 32

    4.6 Rheological analysis ....................................................................................... 33

    4.7 Water content of hydrogel............................................................................. 33

    4.8 In vitro enzymatic degradation assay ............................................................ 34

    4.9 Statistics ......................................................................................................... 35

    5. Results and Discussion ............................................................................................. 36 5.1 Synthesis of Thiolated HA .............................................................................. 36

    5.2 FT-IR characterization .................................................................................... 38

    5.3 Experiment design .......................................................................................... 39

    5.3.1 Fractional factorial design ................................................................. 39 5.3.2 Solubility of lyophilized HA-SH samples ............................................ 42

    5.3 Ellman’s test ................................................................................................... 43

    5.4 Oxidation of thiol groups ............................................................................... 45

    4.5 DSC analysis .................................................................................................... 46

    5.6 BM(PEG)2 cross-linked HA-SH hydrogels ....................................................... 48

    5.6.1 Attempted crosslinking ..................................................................... 49 5.6.2 Transparency of hydrogels ................................................................ 50

    5.7 Rheology ......................................................................................................... 51

    5.8 Water content ................................................................................................ 54

    5.9 Enzymatic degradation ................................................................................... 56

    6. Conclusions and Future work ................................................................................... 59 Reference ..................................................................................................................... 61

  • v

    List of Figures Figure 1.1 Different types of polymer degradation. .............................................. 5 Figure 2.1 The chemical structure of hyaluronic acid consisting of GlcUA and

    GlcNAc. ......................................................................................................... 19 Figure 2.2 The chemical structure of sodium alginate......................................... 20 Figure 2.3 The chemical structure of chitosan ..................................................... 21 Figure 3.1 An ideal 3D micro-channel cell patterning scaffold to stimulate and

    direct neural cell growth in injured spinal cord. .......................................... 26 Figure 4.1 EDC/NHS coupling reaction to create amine-reactive intermediates

    that can improve the coupling yield. ........................................................... 28 Figure 4.3 DTT added to cleave the disulfide ....................................................... 29 Figure 5.1 Lyophilized HA-SH. The sample is white, appeared fibrous structure.36 Figure 5.2 FTIR spectrums of hyaluronic acid and HA-SH. ................................... 38 Figure 5.3 Standard curve of absorbance at 412nm for solution of varying

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