DESIGNING IONIC-COMPLEMENTARY HYDROGELS FOR BONE TISSUE REPAIR A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Chemical Engineering and Analytical Science 2014 MSc. LUIS ALBERTO CASTILLO DIAZ School of Chemical Engineering and Analytical Science CONTENTS List of figures……………………………………………………………….................4 List of abbreviations…………………………………………………………….……..5 Declaration…………………………………………………………………………….8 Copyright………………………………………………………………………………9 Acknowledgements…………………………………………………………………..10 Thesis Structure……………………………………………………………….…..….11 Abstract………………………………………………………………………………12 Objectives of the thesis………………………………………………………………13 1 Chapter 1 - Introduction. Versatile peptide hydrogels for bone and dental tissue regeneration………………….…………………….………………………..…….….14 2 Chapter 2- Human osteoblasts within soft peptide hydrogels promote mineralisation in vitro…………………………………………………………………….….70 3 Chapter 3 - Functional peptide hydrogels for bone formation applications………..71 4 Chapter 4 - Osteogenic differentiation of human mesenchymal stem cells promotes mineralisation within an octa-peptide hydrogel……………………...…....99 5 Chapter 5- Materials and methods……………………….………………………129 5.1 Materials…………………………………………………………………….…..129 2 5.2 Methods…………………………………………………………………………130 5.3 References………………………………………………………………………139 6 Chapter 6 – Conclusions, outlook and recommendations for future work………..140 6.1 Conclusions……………..……………………………………………………....140 6.2 Outlook………………………………………………………………………….141 6.3 Recommendations for future work……………………………………………...141 Appendix……………………………………………………………………………143 3 LIST OF FIGURES Figure 1. Representative of a peptide hydrogel containing cell culture media. Gel has been plated into a 12 well insert of 15 mm of inner diameter and 3 µm of pore diameter……………………………………………………………………………..132 Figure 2. Representative of the flow of media in between the cell culture insert and the well plate to aid the gelation of the FEFEFKFK gel. The media flows from the bottom to the top of the gel………………………………………………………....132 Figure 3. Oscillatory rheometer……………………………………………………135 4 LIST OF ABBREVIATIONS 2-D two-dimensional 3-D three-dimensional AB alveolar bone ALP or alkphos alkaline phosphatase AP-1 activator protein-1 ATR-FTIR attenuated total reflectance-Fourier transform infrared spectroscopy BGs bioactive glasses bFGF basic fibroblast growth factor BMP’s bone morphogenetic proteins BMUs basic multicellular units BSA bovine serum albumin BSP bone sialoprotein CD circular dichroism CDs clusters of differentiation COL-I1 type I collagen or collagen I COX-2 cyclooxygenase-2 CPC cetylpyridinium chloride DCs dendritic cells ddH2O double distilled water DMEM Dulbecco’s modified Eagle’s medium DPBS Dulbecco’s phosphate-buffered saline DPSCs dental pulp stem cells ECM extracellular matrix EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor EthD-1 ethidium homodimer FBS fetal bovine serum FN fibronectin FSH follicle-stimulating hormone GAGs glycosaminoglycans GCs glucocorticoids GMSCs gingival mesenchymal stem cells GPa gigapascal HA hyaluronic acid HA hydroxyapatite HCA hydroxycarbonate apatite hMSCs human mesenchymal stem cells HOBs or hOBs human osteoblasts HPLC high-performance liquid chromatography HSCs hematopoietic stem cells ICC immunocytochemistry IEC ion-exchange chromatography IGF-1 insulin growth factor-1 IFN-γ interferon gamma IFR infrared spectroscopy 5 IL interleukin MCGS mesenchymal cell growth supplement MCP-1 monocyte chemoattractant protein-1 M-CSF macrophage colony-stimulator factor MIP-1α macrophage inflammatory protein-1 alpha MMPs metalloproteinases of matrix MPa megapascal MSCs mesenchymal stem cells NF-κB nuclear factor-κB NKs natural killer cells NMR nuclear magnetic resonance NOD nucleotide-binding oligomerisation domain OBs osteoblasts OCN osteocalcin OCs osteoclasts ODP osteopontin-derived peptide OPF oligo poly (ethylene glycol) fumarate OPG osteoprotegerin OPN osteopontin ONN osteonectin pAb primary antibody PBS phosphatase-buffered saline PCL poly (ε-caprolactone) PDFG platelet-derived growth factor PDL periodontal ligament PDLSCs periodontal ligament stem cells PEG polyethylene glycol PEG-2 prostaglandin-2 PEGDMA polyethylene glycol dimethacrylate PEO poly (ethylene oxide) PFA paraformaldehyde PGA polyglicolic acid PLA polylactic acid PLLA poly-L-lactide acid PLGA poly (lactic-coglycolide) PMNs polymorphonuclear cells PNIPAAm poly (N-iso-propylacrylamide) pNPP p-Nitrophenyl phosphate PPF poly (propylene fumarate) PTH parathormone PVA poly (vinyl alcohol) RANK receptor activator of NF-κB RANKL receptor activator of NF-κB ligand ROS reactive oxygen species 6 RT room temperature sAb secondary antibody SAXS small angle X-ray scattering SEC size exclusion chromatography SPPS solid phase peptide synthesis TCP tissue culture plastic TEOS tetraethylorthosilicate TEM transmission electron microscopy TFA trifluoroacetic acid TGF-β transforming factor-beta Ths helper T lymphocytes TIMPS tissue inhibitors of metalloproteinase TLRs toll-like receptors TNF-α tumor necrosis factor-alpha TRAP tartrate-resistant acid phosphatase Tregs regulator T cells VCAM-1 vascular cell adhesion molecule-1 VEGF vascular endothelial growth factor 7 DECLARATION The University of Manchester PhD by published work candidate declaration Candidate name: Luis Alberto Castillo Díaz Faculty: Chemical Engineering and Analytical Science Thesis title: Designing ionic-complementary hydrogels for bone tissue repair Declaration to be completed by the candidate: I declare that no portion of this work referred to this thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. Signed: Date: 8 COPYRIGHT The author of this thesis (including any appendices and/or schedules to this thesis) owns and copyright in it (“the Copyright”)1 and he has given The University of Manchester the right to use such Copyright for any administrative, promotional, educational and /or teaching purposes. Copies of this thesis, either in full or in extracts, may be made only in accordance with the regulations of the John Rylands University Library of Manchester. Details of these regulations may be obtained from the librarian. This page must form part of any copies made. The ownership of any patents, designs, trademarks and any all other intellectual property rights except for the Copyright (the “Intellectual Property Rights”), and any reproductions of copyright works, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such intellectual Property Rights and Reproductions cannot and must not be made available for use without the prior written permission of the owner (s) of the relevant intellectual Property Rights and/or Reproductions. Further information on the conditions under which disclosure, publication and exploitation of this thesis, the Copyright and any Intellectual Property Rights and/or Reproductions described in it may take place is available from the Head School of Chemistry (or the Vice-President) and the Dean of the Faculty of Engineering and Physical Sciences, for Faculty of Engineering and Physical Sciences candidates. 1This excludes material already printed in academic journals, for which the copyright belongs to said journal and publisher. Pages for which the author does not own the copyright are numbered differently from the rest of the thesis. 9 ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor Prof. Aline Fiona Miller, for giving me the opportunity to work on this fascinating project, and for her generous and unconditional support. Another thanks goes to my co-supervisor, Prof. Julie Elizabeth Gough and to Dr Alberto Saiani for their support and the opportunity to work in an extraordinary interdisciplinary network. I am also proud and grateful to have been part of the Polymers and Peptides, and the Tissue Engineering research groups, where I had the opportunity to work in a great atmosphere meeting extraordinary people. I would like to thank my colleagues, Dr Ayeesha Mujeeb, Dr Claire Tang, Dr Jean Baptiste-Guilbaud, Dr Jonathan Gibbons, Dr Louise Carney, Dr Mohamed Elsawy, Dr Mi Zhou, Dr Kate Alexandra Meade, Dr Richard Balint, Dr Stephen Boothroyd, Dr Natasha Bhuiyan, Simon Wan, Shirley Bentley, Dr Nabilah Russlan, Dr Laura Szkolar, Dr Israa Sabree, Dr Deepak Kumar, Chistopher Hickling, and Dr Dave Roberts for their comradeship and kindly support. A special thanks to CONACyT-Mexico, for the generous fellowship support. Last but not least, another thanks goes to my wife Dr Juana Elizabeth Reyes Martinez, my parents and sisters, María Priscila del Castillo Frías, Dr Benjamín Raziel Jaramillo Ávila and Dr Elsa Aurora Calleja Quevedo, because of without their support, the culmination of this work would not have been possible. 10 THESIS STRUCTURE This thesis describes the design of peptide hydrogels for bone tissue engineering applications. The body of this thesis includes an article that has been already published, and three manuscripts, which will be submitted for publication. The format of both the published paper and the three manuscripts has been modified to fit in the template of this thesis, however the main text was not modified. The references either for the article or manuscripts are at the end of each chapter, as in the submitted version. In this thesis, a review manuscript