Chemical and Biomolecular Engineering - Theses
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ItemAntibacterial, macroporous chitosan hydrogels for soft tissue engineering applications( 2017)One of the goals of tissue engineering is to be able to develop functional tissue substitutes that can one day replace the function of an organ that is damaged or requires replacement. The development of macroporous scaffolds that can potentially replicate or mimic the functionality and architecture of native extracellular matrix (upon implantation at the site of defect) is one of the major focus in the field of tissue engineering. For a large number of tissue engineering strategies surgical implantation of the scaffold is necessary, yet it also increases the risk of infections. The growing of antimicrobial resistance has necessitated the development of new strategies to tackle drug-resistant bacteria. The focus of this study was to develop porous hydrogel scaffolds for soft tissue engineering and antimicrobial wound healing. This research was first focused on the development of macroporous chitosan hydrogel scaffolds utlilising thermally induced phase separation (TIPS). Two different types of gelation (freeze neutralisation vs freeze crosslinking) were employed for the fabrication of porous chitosan scaffolds. Pore size and porosities are important scaffold parameters for neovascularisation, potentially improving the incorporation of the implant into the host tissue. Hence, various physical parameters such as temperature, chitosan concentration and crosslinking were varied to study their effects on the pore size distribution (60-150 µm) and porosity (65-80%) of the chitosan scaffolds. Mechanical properties of these scaffolds were tunable (compressive modulus: 1-450 kPa) via change of crosslinking and chitosan concentrations. The study was also able to demonstrate scaffold mechanical properties which were relatively similar to the that of the soft tissues (2.5-40 kPa). Mouse 3T3 fibroblasts (relevant cell line for dermis) were shown to adhere, proliferate and penetrate into the scaffolds in vitro. A novel combined mechanical gas foaming and TIPS technique was also developed in order to better control the pore size and morphology of the scaffolds. To establish the range where mechanical foaming results in optimal entrainment of air (hence porous voids) the effects of surfactant (polyvinyl alcohol (PVA)) concentration, foaming speed, foaming time and chitosan concentration were probed and correlated to the foam generating potential. Upon establishing the physical constraints on mechanical foaming for this system, various parameters (PVA concentration, chitosan concentration, mixing speed) that could affect the pore size distribution of these chitosan/PVA scaffolds were studied. This combined technique yielded scaffolds with a more homogeneous spherical morphology. Although pore sizes were generally larger (average pore sizes 120-170 µm) compared to TIPS standalone method, however, pore size distributions were wide and highlighted the need for further improvement of pore size distribution. Scaffolds fabricated with this technique presented soft mechanics (elastic modulus: 2.5-25 kPa), which were similar to those of soft tissues. Experiments with mouse 3T3 fibroblasts revealed that these novel scaffolds supported cell proliferation, attachment without any observable cytotoxicity. Wet chemical synthesis was utilised in order to incorporate selenium (Se) and silver (Ag) nanostructures (separately) into the novel foamed TIPS scaffold, to improve its antimicrobial properties. The morphology of the Ag and Se nanostructures formed in situ on chitosan/PVA scaffolds revealed stark differences which were attributed to the interaction between the chitosan backbone and selenite ions via a hydrogen bonding mechanism. This study also demonstrated the fine control of the loading of both the elements and measured its release in various culture media (complete DMEM, LB media and deionised water). There were significant differences in release of Ag species in all three types of media, indicating the possible role of O2, chloride ions and various organic molecules (e.g., glucose, proteins) that can affect Ag release. Se release was largely unaffected in DMEM and LB media. Extracts from chitosan/PVA scaffolds loaded with various doses of Se (0, 0.06, 0.25, 0.92, 3.67 w/w %) or Ag (0, 0.37, 2.33, 8.97, 17.79 w/w %) showed significant toxicity for Ag based scaffolds. However, no significant cytotoxicity was observed for Se-chitosan/PVA scaffolds. Antibacterial studies carried out on S.aureus, MRSA and E.coli via a Syto9/PI flow cytometric assay revealed significant membrane damage in presence of Se scaffolds and no membrane damage for Ag scaffolds. Colony forming unit (CFU) counts of all three bacterial species tested revealed that CS-Ag caused significant cell death to all bacterial species. However, CS-Se did not lead to significant reduction in bacterial cell numbers, which suggested that while CS-Se extracts caused membrane damage, it did not lead to cell death at the loadings tested. Overall, this thesis focuses on the development of soft scaffolds with tunable pore sizes, morphology and porosities utilising TIPS and gas foaming methodologies. The results also indicate these scaffolds have soft mechanics, support mouse 3T3 fibroblast proliferation, attachment without cytotoxicity. This point towards the possible application of these scaffolds for wound healing. Furthermore, incorporation of Se nanostructures led to enhancement of the scaffold’s bacterial cell permeabilisation properties which could be useful as an adjuvant to enhance the potency of current antimicrobials against drug resistant bacteria.
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ItemEngineering functional amyloid fibrils for biomaterial applications( 2012)The natural ability of biological molecules to self-assemble provides a useful route for the production of nanomaterials with desirable properties. Amyloid fibrils are a class of self-assembling proteins that are defined by their common core structure, which is rich in β-sheet structure. Fibrils are typically ~10 nm in width and have an elongated morphology with a length ~1 µm. These fibrils are historically associated with disease but they are also the functional state of some proteins in nature and can be produced in a controlled way from non-disease proteins. The fibril core structure imparts excellent physical properties such as a flexibility similar to silk and strength similar to steel, which recommend fibrils for applications in materials science. There is growing interest in the production of fibrils with useful properties, although a number of challenges remain before these materials can be applied. This thesis employed the TTR105-115 peptide, also known as TTR1, to drive the assembly of functional peptides into amyloid fibrils. The TTR1 sequence was selected for two reasons. Firstly, this peptide has a high propensity to self-assemble into highly ordered fibrous structures, where the peptides are arranged in a cross-β core structure. The TTR1 peptide also has a proven ability to drive the assembly of functional groups, so that they are displayed away from the fibril core at the C-terminal end of the TTR105-115 based peptide. The influence of these groups on the properties of assembled fibrils was investigated and the role functional groups on the kinetics of fibril assembly were determined. The TTR1-cycloRGDfK peptide was also designed with the aim of producing a fibril that would display specific properties. The peptide incorporated the functional cyclic RGDfK pentapeptide ligand, which has a high affinity and specificity towards the mammalian cell surface αVβ3 integrin receptor. Fibrils assembled from the TTR1-cycloRGDfK peptide were shown to promote the attachment and spreading of adherent mammalian cells when fibrils were presented as a surface coated layer. These findings demonstrated that the selection of functional sequences is paramount to the properties of fibrils based on the TTR1 peptide. The kinetics of functional fibril assembly were characterised using an established set of TTR1-based peptides: TTR1, TTR1-RGD, TTR1-RAD and the novel TTR1-GGK peptide. The functional ligands that are excluded from the fibril core were found to influence both the lag time and elongation rate of fibril formation. The study of TTR1-GGK assembly was further extended to include a wide range of solution conditions including conditions of varying ionic strength, solution pH or solutions containing different salt ions. The addition of salt promoted fibril. The extent of this effect was dependent on the degree of charge shielding, ion selectivity and the Hofmeister effect. The structure of the mature fibril was largely unaltered when fibrils were assembled in the presence of salt ions, indicating that salts may be used to tune fibril formation. Overall, these measurements demonstrated that non-fibril core residues alter the propensity for fibril formation, even with differences of a single amino acid. The impact of non-fibril core groups on assembly should therefore be considered when designing sequences for the production of functional fibrils. Functional amyloid fibrils were also tested for their biocompatibility using cell viability assays and membrane integrity assays. Mature fibrils assembled from the peptides TTR1, TTR1-RGD and TTR1-RAD were the primary focus since these aggregates are the targets for applications in materials science. The overarching conclusions from this work are firstly that the TTR1 peptide is a robust system that can promote the assembly of functional fibrils and secondly that non-fibril core residues greatly influence select properties of assembled fibrils, while other core structural features of fibrils remain intact. Non-core residues determined the extent and specificity of fibril binding to the cell membrane. Layers of TTR1-cycloRGDfK fibrils promoted the attachment and spreading of cells, demonstrating that fibrils can be engineered to have a positive effect on cell processes. A greater understanding of fibril biocompatibility is needed, however, before functional fibrils may be applied to biotechnology applications where cell interactions may be involved.