AbstractMechanical cues play important roles in a range of biological processes from embryonic development to pathologies such as cancers. This thesis explores the application and development of two fundamental tools often used in these studies – hydrogels and atomic force microscopy (AFM). In particular, we use these tools to help characterise biomechanical changes between healthy and diseased phenotypes in tissue and tissue-like models. To start, we first developed and characterised a novel 3D PEG-Peptide hydrogel. Adhesive and degradable peptide designs were verified for their designed biological function, and we carried out mechanical characterisation by performing both swelling measurements, as well as AFM to determine hydrogel elastic modulus. As expected, good cell adhesion was found when the adhesion peptide was incorporated, and hydrogel degradation could be tuned by altering the percentage of degradable peptide within the gel. As solid content was increased, gels then had a higher Young’s modulus (ranging from 0.6 – 19 kPa) and a lower mass swelling ratio. In performing these AFM measurements a robust protocol was also developed which we have further validated on other hydrogel systems. Moreover, in the context of inflammatory bowel disease (IBD), we used AFM to understand how human intestinal organoids remodel their surroundings in the presence of innate lymphoid immune cells (associated in inflamed tissues of IBD patients). Using a large colloidal probe to indent deep into the hydrogel we found organoids, when stimulated by these immune cells, remodel their surroundings, changing their mechanical properties through both matrix metalloproteinase (MMP) mediated softening and fibronectin driven stiffening.
Along with characterising simple acellular hydrogels, AFM can also be used to understand the complex mechanics of cells and tissues. AFM has been used to understand how tissue mechanics change in diseases such as breast cancer; however, less is known about non-solid tumours such as leukemia. Here, we used AFM to understand how murine bone marrow stiffness changes in acute myeloid leukemia. We performed AFM on both non-chemically fixed cryosections and live tissue. In live tissue preparations, we observed a trend towards an increase in stiffness in leukemic tissue.
In merging this research in both AFM and hydrogel development, we finally explore the application of hydrogels in creating in vitro models of leukemia. Whilst we were unable to culture acute myeloid leukemia cells long term, we did find chronic myeloid leukemia cells to preferentially grow and proliferate in softer hydrogels (600 Pa) compared to stiff (7.5kPa).
Overall, hydrogels and AFM have become two vital tools in understanding cellular mechanobiology. This thesis has successfully explored how they can be applied to answer a variety of biological questions.
|Date of Award||1 Mar 2022|
|Supervisor||Eileen Gentleman (Supervisor)|