Saudi Cultural Missions Theses & Dissertations
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Item Restricted Stiffness-induced EMT and Cancer Stemness in Glioblastoma Using Polyacrylamide Hydrogels(University of Glasgow, 2024) Alotaibi, Mohammed; Salmeron-Sanchez, ManuelGliomas are the most common type of brain and are considered one of the most fatal cancer forms due to their detrimental and aggressive behaviour. Amongst these types of brain cancer is glioblastoma (GBM), classified by the World Health Organization (WHO) as grade IV, known to have characteristics like high malignancy, rapid growth and aggressiveness. As these tumours progress, the extracellular matrix (ECM) stiffness increases, influencing their growth, survivability and treatment outcomes. The induction of Epithelial to Mesenchymal transition (EMT) was associated with the production of Cancer stem-like cells (CSCs), a small subpopulation with self renewal capabilities that generates phenotypic heterogeneity comparable to the original tumour. CSCs are responsible for sustaining tumour growth and metastasis formation to other body tissues. The main focus of this study was assessing the role of brain tissue mechanical stiffness in promoting EMT and cancer of glioblastoma cells. The surface of PAAm hydrogels was modified to overcome the non-adhesiveness via covalently linked to collagen type I to facilitate the attachment of glioblastoma cells. The stiffness of Polyacrylamide Hydrogels (PAAm hydrogels) was measured using Rheology and Nanoindentation. The three stiffnesses fabricated and used were soft 305.9±16.9 Pa, which is similar to normal brain tissue, medium 10.5±0.4 kPa, comparable to glioblastoma stiffness and rigid 34.9±5.1kPa which is stiffer than glioblastoma tumours. The nanoindentation measurements were for soft 321.72±59.83 Pa, medium 8.01±0.37kPa and rigid 39.19±2.58kPa, illustrating that the stiffnesses are unfirmed across the surface and reproducible. EMT markers like N-CAD, VIMENTIN and TGF-β showed increased protein levels in the medium and rigid hydrogels compared to soft hydrogels. This response was further by increased protein expression of the EMT transcription factor SNAI1(SNAIL), which showed a significant increase in levels of SNAI1(SNAIL) (p≤0.05) on the medium and rigid hydrogels. II CSC markers showed increased protein levels highlighted by the significant increase in the protein levels of NESTIN (P≤0.001), CD133 (P≤0.0001), POU5F1(OCT-4) (P≤0.05), and EGFR (P≤0.05), respectively on the rigid hydrogels compared to soft hydrogels. Medium hydrogels showed significant increases in the protein levels of CD133 (P≤0.0001) and POU5F1(OCT-4) (P≤0.05), respectively. The findings of this research suggest that mechanical stiffness promoted EMT and cancer stemness in glioblastoma cells, underlining the influence of microenvironment stiffness in promoting invasion capabilities in glioblastoma cells.7 0Item Restricted The Utilisation of GelMA-based Surface-patterned Scaffolds for Craniofacial Muscle Regeneration Applications(University College London (UCL), 2024-03-28) Aljaber, Mohammad; Knowles, JonathanIn the circumstances of volumetric muscle loss of craniofacial muscles, which can happen due to accidents, for instance, the functionality of muscle tissue could be completely lost. Although muscle flap surgery is the current standard treatment utilised, a novel approach is the use of “tissue engineering” in which scaffolds, cells and additional biomolecules are exploited to aid the muscle regeneration process. In this project, gelatin methacryloyl (GelMA) was synthesised and optimised for muscle regeneration applications. Various parameters involved in the reaction, such as reaction time and methacrylic anhydride (MA) concentration, GelMA concentration, photo-initiator concentration and ultraviolet (UV) exposure time were optimised. The mechanical and biological properties were evaluated and reported for a number of formulations/processing conditions. The data suggested that using 10-15% (w/v) GelMA mixed with 0.1% (w/v) LAP and crosslinked for 2 minutes had the optimum balance between mechanical and biological properties for skeletal muscle regeneration applications. The investigation also thoroughly examined the impact of both the source and the bloom number of gelatin on the properties of the laboratory-synthesised GelMA. Specifically, porcine, bovine, and fish-derived gelatin sources were evaluated, along with porcine gelatin of 175 versus 300 bloom number. Extensive assessment of the mechanical and biological characteristics was carried out using various techniques. The results concluded that bovine-derived GelMA demonstrated superior mechanical properties compared to other groups, while no significant differences were observed in terms of biological properties. All groups showed relatively high metabolic activity and low LDH release which suggests high cell viability and a low cytotoxic response. Additionally, the ability of GelMA hydrogels (derived from porcine, bovine or fish) to promote myoblast differentiation of C2C12 muscle cells and myotube formation was evaluated. Results showed that all groups succeeded in promoting differentiation of myoblasts into myocytes and myotubes, and presented similar myogenesis ability with no significant differences after 21 days of differentiation. A key challenge in skeletal muscle regeneration is enhancing the alignment of the myocytes and myotubes. Therefore, three-dimensional (3D) printed moulds with specific sizes of grooves and ridges (300 µm, 600 µm or 900 µm) were printed using biocompatible commercial resin before GelMA hydrogels and C2C12 cells were cast in the moulds and crosslinked via UV. The influence of these patterns was characterised using DAPI and phalloidin F-actin staining to study the impact of the surface patterns on the alignment of myotubes. The Z-stack images obtained by confocal microscope illustrated that using surface patterns of 300 µm helped in improving the alignment of myotubes in comparison with the other groups. To this end, the investigation of GelMA hydrogels from different species concluded that bovine-GelMA (B-GelMA) presented the greatest mechanical properties in which all groups presented excellent biological properties in terms of promoting cell growth and attachment, as well as cellular differentiation into myocytes and myotubes. The surface pattern of 300 µm grooves and ridges demonstrated the highest cell and myotube alignment compared to larger-sized grooves and ridges. In addition, this myotube alignment, as documented by the RT-qPCR, led to enhanced gene expression of MyoG and MyoD-1.9 0