Dynamic nanostructured scaffolds as advanced biomaterials
Creators: name: Boyd-Moss, Mitchell
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 400302
subject: Biomaterials
classificationCode: 401807
subject: Nanomaterials

description: Growing replacement tissues and organs in the laboratory will revolutionise healthcare; however, the maturation of cells into functional tissue constructs requires the controlled presentation of biochemical factors within a mechanically suitable scaffold. In nature, the presentation of such signals is provided through factors and structures existent within the nanoarchitecture of the extracellular matrix (ECM); therefore, in tissue engineering there is significant need to develop dynamic advanced artificial tissue constructs capable of mimicking the complexities of the native ECM. The requirement for bioactive, innervated constructs that contain biologically relevant signals delivered through tuneable mechanisms has yet to be achieved.<br><br>One approach to address this key-challenge is offered through bioprinting, which allows for the controlled spatial distribution of bioinks containing cells, structures and signals within a single printed construct. However, currently bioprinting applications are severely limited by bioink function - with the majority of bioinks either lacking sufficient mechanical properties or biochemical signalling. Therefore, there is a key need to develop bioinks which adequately mimic the native ECM on a nanostructured, chemical level - particularly in establishing effective control over cell fate and tissue innervation.<br><br>Tissue composition and extracellular signalling varies substantially between tissue-types, and therefore, advanced approaches that allow for ease of mechanical and biological tuneability through modular mechanisms would provide a practical avenue for bioink development. Self-assembling peptides (SAPs) are a unique class of biomaterials capable of spontaneously forming simple biomimetic structures which entangle to form highly hydrated, bioactive networks with favourable conditions for cell maturation. These biomaterials are easily tuned through modification of amino acid sequence, enabling tailored control over biochemical signalling between cells and scaffold. This provides the ability to artificially replicate natural signalling in a controlled manner - bringing about desired cell behaviour. Using these peptides, a variety of synergistic ECM-protein analogues have been developed, including Fmoc-FRGDF containing fibronectin's attachment motif RGD, and Fmoc-DIKAV, containing laminin's attachment motif IKVAV. Fmoc-SAPs possess the ability to be further functionalised through macromolecule addition, allowing for the presentation of charged, developmentally or structurally-important macromolecules on the surface of peptide fibrils. These macromolecules can integrate with the peptide networks, facilitating additional signalling and allowing for mechanical tunability. <br><br>Here, we take advantage of these properties to develop an advanced and dynamic bioink for bioprinting applications. Initially, material enhancement is investigated through development of multi-sequence scaffolds. Specifically, Fmoc-FRGDF is combined with a synergistic cell attachment motif PHSRN, either through sequence engineering (Fmoc-FRGSFPHSRN) or through control over assembly properties (Fmoc-FRGDF/Fmoc-PHSRN coassembly). Here, the coassembled (Fmoc-FRGDF/Fmoc-PHSRN) system forms a synergistic network which promotes the attachment, proliferation and migration of muscle cells in vitro. The potential of Fmoc-SAP multi-sequence scaffolds is further investigated through the development of an artificial tumour microenvironment for cancer-cell studies. Here, Fmoc-FRGDF is combined with Fmoc-DIKVAV and used as a spheroid (LLC, NOR-10, LLC + NOR-10) micro-environment. The coassembled Fmoc-FRGDF/Fmoc-DIKVAV microenvironment enhances cancer-cell growth and progression compared to 2D cultures, non-encapsulate spheroids, and spheroids encapsulated in agarose. Agarose was selected as a control owing to the similar physical properties yet lack of biofunctionalisation. Results from this study reinforce the potential of Fmoc-SAPs as advanced microenvironments, and further support the ease of biological functionalisation inherent with this material.<br><br>Further scaffold functionalisation is investigated through macromolecule addition. Here, one of two macromolecules are coassembled into a Fmoc-FRGDF network. The first macromolecule is fucoidan, a seaweed-derived polysaccharide with known anti-inflammatory properties, while the second is versican, a developmentally important proteoglycan which plays a variety of roles in muscle development. Versican was selected owing to its charge similarity to fucoidan, yet vastly different biological function. Fucoidan addition was found to increase fibre bundling and alter hydrogel mechanical properties, while versican addition had no substantial effect on hydrogel mechanics when compared to an Fmoc-FRGDF empty-vector control. Cell morphology was substantially altered by macromolecule addition, with fucoidan samples resulting in smaller, rounder cells with fewer multinucleated syncytia compared to an Fmoc-FRGDF control, while versican hydrogels showed an initial decrease in cell-size and multinucleation after 24h and a comparable cell-size and multinucleation following 72h. Here, it is possible that macromolecule addition perturbs cells attachment, and therefore, macromolecule selection is a key consideration. Interestingly, the regain of cell morphological characteristics in versican-containing hydrogels following 72h indicates the ability of cells to break-down versican, while the maintenance of small, round cells in the fucoidan hydrogels shows an inability for cells to break down fucoidan. <br><br>The ability of Fmoc-SAPs to form components in bioinks is investigated through assembly with gelatin methacryloyl (GelMA) macromolecules. Initially, GelMA nanostructure and mechanical properties are investigated in response to increased degree of methacrylation or increased control. Here, structure-function relationships are drawn, and 18% methacryloyl Gelma (LM-GelMA) is selected for further bioink development owing to favourable thermoresponsive viscoelastic properties and improved strain tolerance. LM-GelMA assembly with coassembled Fmoc-FRGDF/Fmoc-PHSRN is investigated as a potential avenue to develop biologically and mechanically tuneable hydrogels. The incorporation of Fmoc-SAPs allows for control over sequence selection, while control over mechanical properties is offered through GelMA inclusion. LM-GelMA/Fmoc-FRGDF/Fmoc-PHSRN (FPG-Hybrid) bioinks demonstrate enhanced printability and are shown to support primary myoblast differentiation. The potential of Fmoc-SAP/GelMA bioinks to act as a modular bioink toolkit is further investigated through Fmoc-FRGDF/Fmoc-PHSRN substitution with Fmoc-DIKVAV, to develop a neural-suitable bioink (DIKVAV-Hybrid). This DIKVAV-Hybrid bioink demonstrated unique mechanical morphological properties and is shown to support rat cortical neurosphere viability. <br><br>Throughout this project, the networks have been vigorously characterised through various analytical techniques, including micro/nanoimaging (Transmission electron microscopy, Atomic force microscopy, Cryo-scanning electron microscopy), Small-angle X-ray scattering, Small-angle neutron scattering, rheology, and spectroscopy; while the overall effectiveness of these systems have been analysed through in vitro muscle and neural cultures. Work detailed through this thesis aims to vigorously characterise Fmoc-SAP hydrogels and bioinks, providing the foundations for further biological studies and material optimisation.