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Merrett, Christopher G. (2024) User-defined biomimetic scaffolds through "click" modification of self-assembling peptide hydrogels. PhD thesis, University of Nottingham.

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Abstract

There is increasing demand for in vitro methods to recapitulate the complexity of the extracellular matrix to further our understanding of developmental and disease biology. It is essential to transition away from 2D cell culture, which poorly represents the biochemistry and biomechanics of native tissue, to 3D culture. It is equally critical to reduce the dependency on animal-derived materials such as Matrigel®, which are expensive, ill-defined and inconsistent in quality. Despite these well-established shortcomings, few synthetic models have been developed to offer accessible and bespoke customisation of matrix composition and stiffness.

The self-assembling peptide hydrogel based on the FEFEFKFK octapeptide gelator has emerged as a promising 3D scaffold that is biocompatible, provides structural homogeneity and is of sustainable, non-biological origin. Through its adoption of physical crosslinks and β-sheet supramolecular assembly, the peptide hydrogel emulates the innate hierarchical architecture of extracellular matrix proteins. Importantly, the hydrogel facilitates the exogenous delivery of soluble matrix material alongside independent modification of the matrix stiffness to model tissue-specific microenvironments. However, to expand on the technology, the peptide has been functionalised with a non-proteinogenic, L-azidohomoalanine to afford the matrix with a “click” chemistry handle. By utilising the copper-free strain-promoted azide-alkyne cycloaddition, covalent immobilisation of desired matrix material can be bio-orthogonally introduced during live cell culture experiments.

The design, synthesis and installation of bio-orthogonal linkers onto bioactive molecules such as cell adhesion motifs (RGD, IKVAV and YIGSR) and glycosaminoglycans (hyaluronic acid) has laid the foundation for a “toolbox” of on-demand “clickable” bioconjugates to customise the hydrogel into user-defined tissue-relevant models. In addition, recombinant peptide synthesis has been explored for the sustainable and economical production of the self-assembling peptide to establish a library of chemical handles and motifs.

The potential sophistication of the model has been demonstrated through the 3D culture of mouse embryonic stem cells and fibroblasts, which displayed phenotypically correct morphologies upon in situ chemical modification of the cell-laden hydrogel. The model also supports the installation of concentration gradients and multi-functionalisation to create highly complex mimics of tissue microenvironments. This initial work has shown promise in creating biologically representative models with user-defined levels of precision and reproducibility rarely observed and highly demanded within in vitro research.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Thomas, Neil Merry, Cathy
Keywords:	biomimetic scaffolds; peptide hydrogels; 3D culture;
Subjects:	Q Science > QD Chemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Chemistry
Item ID:	78708
Depositing User:	Merrett, Christopher
Date Deposited:	13 Dec 2024 04:40
Last Modified:	13 Dec 2024 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/78708

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