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Effect of Decellularisation Methods on Methacryloyl‐Substituted Placental ECM Hydrogels I

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Effect of Decellularisation Methods on Methacryloyl‐Substituted Placental ECM Hydrogels I EFFECT OF DECELLULARISATION METHODS ON METHACRYLOYL‐SUBSTITUTED PLACENTAL ECM HYDROGELS David Pershouse Bachelor Applied Science/ Bachelor of Mathematics Submitted in fulfilment of the requirements for the degree of Master of Applied Science (Research) School of Mechanical, Medical and Process Engineering Science and Engineering Faculty Queensland University of Technology 2020 Keywords 3D cell culture Placenta Cell instructivity Chorion Crosslinking Decellularisation Extra‐cellular matrix Functionalisation, functionalization Hydrogel Methacrylation Photopolymerisation, photopolymerization Effect of Decellularisation Methods on Methacryloyl‐Substituted Placental ECM Hydrogels i Abstract Hydrogels based on solubilized extracellular matrices (ECMs) represent a promising source for the creation of cell‐instructive scaffolds for tissue engineering. The diverse range of biochemical cues retained by placental ECM drive cell proliferation, differentiation, and function for all tissue types in utero while providing mechanical support for tissue growth. However, the formation of ECM‐derived hydrogels typically relies on the thermally‐induced self‐assembly of collagenous polypeptides, severely limiting the control over the resulting biochemical and mechanical hydrogel properties. Decellularisation methods of the biomaterial may alter these properties of the final hydrogel. Here the properties of photocrosslinkable hydrogels derived from different decellularisation methods, based on human placental ECM digests, has been explored to overcome aforementioned limitations and allow for precise control over physicochemical properties, while still retaining cell‐ instructive bioactivity. Briefly, human full‐term placenta was obtained following caesarean delivery from consenting donors with ethics approval. Chorionic villi tissue was decellularised using N‐lauroyl sarcosine and Triton X‐100 detergent‐based methods, respectively, and enzymatically digested. Each resulting ECM digest was then functionalized with methacrylic anhydride to result in methacryloyl‐substituted placental ECM (PlacMA). Precursor solutions of PlacMA were liquid at room temperature and formed mechanically stable and transparent hydrogels by visible light‐induced photocrosslinking via cytocompatible free radical chain polymerisation. The mechanical properties of the resulting hydrogels could be tailored through the variation of PlacMA concentration and crosslinking parameters. Methacryloyl‐ functionalisation of native ECM digests from N‐lauroyl sarcosine and Triton X‐100 detergent based methods yield different properties in the final hydrogel. In particular, Triton X‐100 decellularised material provided less batch‐to‐batch variation in hydrogel physicochemical properties and cell viability. Both methods still allowed for the formation of mechanically stable hydrogels with improved control over physicochemical properties. This method can be applied to a large variety of native tissue digests, allowing researchers to mimic native cellular microenvironments in vitro and develop cell‐instructive scaffolds for tissue engineering ii Effect of Decellularisation Methods on Methacryloyl‐Substituted Placental ECM Hydrogels Table of Contents Keywords ...................................................................................................................................... i Abstract ........................................................................................................................................ ii List of Figures ............................................................................................................................... v List of Tables ............................................................................................................................... vi List of Abbreviations .................................................................................................................. vii Preface ........................................................................................................................................ ix Statement of Original Authorship ............................................................................................... ix Acknowledgements ...................................................................................................................... x CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ................................................ 1 1.1 Hydrogels .......................................................................................................................... 1 1.2 Native Tissue as a Source for Biomaterials ....................................................................... 9 1.3 Placenta .......................................................................................................................... 11 1.4 Hypothesis and Objectives ............................................................................................. 16 CHAPTER 2: MATERIALS AND METHODS ..................................................................... 17 2.1 Decellularisation ............................................................................................................. 17 2.2 Determination of DNA and sGAG Content ..................................................................... 19 2.3 dECM Solubilisation ........................................................................................................ 19 2.4 Functionalisation of sECM with Methacryloyl Groups ................................................... 20 2.5 1Hydrogen Nuclear Magnetic Resonance ....................................................................... 20 2.6 Determination of the Degree of sECM Functionalisation ............................................... 20 2.7 Preparation of PlacMA hydrogels ................................................................................... 21 2.8 Effective Swelling ............................................................................................................ 21 2.9 Mechanical Testing ......................................................................................................... 22 2.10 Cell Expansion .............................................................................................................. 23 2.11 Cell Encapsulation and Culture .................................................................................... 24 2.12 Cell Viability ................................................................................................................. 24 2.13 Cell Morphology ........................................................................................................... 25 Effect of Decellularisation Methods on Methacryloyl‐Substituted Placental ECM Hydrogels iii 2.14 Statistical Analysis ........................................................................................................ 25 CHAPTER 3: RESULTS ................................................................................................. 27 3.1 Decellularisation Efficacy ................................................................................................ 27 3.2 Solubilisation of dECM and Functionalisation with Methacrylic Anhydride .................. 29 3.3 Mechanical Testing ......................................................................................................... 32 3.4 Effective Swelling ............................................................................................................ 35 3.5 Cell Culture ..................................................................................................................... 36 3.6 Cell Morphology ............................................................................................................. 37 CHAPTER 4: DISCUSSION ............................................................................................ 41 4.1 Decellularisation Assessment ......................................................................................... 41 4.2 Functionalisation Assessment ........................................................................................ 43 4.3 Mechanical Assessment ................................................................................................. 45 4.4 Cell Viability and Morphological Assessment ................................................................. 47 4.5 further work ................................................................................................................... 48 CHAPTER 5: CONCLUSION .......................................................................................... 51 BIBLIOGRAPHY .................................................................................................................. 53 APPENDICES ..................................................................................................................... 67 Appendix A Decellularisation Data ............................................................................................ 6 7 Appendix B Mechanical Data ..................................................................................................... 68 Appendix C Cellular Viability Data ............................................................................................. 70 Appendix D ImageJ Code ........................................................................................................... 71 ANNEX 1 ..........................................................................................................................
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