Design of static and dynamic hydrogel platforms for mechanobiology

Physical cues that cells receive from the extracellular matrix (ECM) are primary drivers of cell and tissue behavior, determining stemness, cell differentiation and proliferation and are often decisive in disease development and progresses. In this context, 2D substrates of controllable mechanical properties are indispensable tools for mechanobiology studies, as they allow to recapitulate biophysical and adhesive cues of natural ECM. In this thesis work I designed and developed tools for cell culture having defined mechanical properties and adhesiveness to study mechanotrasduction events in the context of aging and from a molecular biology perspective. I systematically studied the mechanosignalling properties of these systems, monitoring the activation state of the mechanotrasducers YAP and TAZ for diverse cell types. I optimized a norbornene-terminated Polyethyleneglycol (PEG-NB) based hydrogel, in which stiffnesses were tunable in a range of physiological relevant rigidities. The relevance of these gels is that the physical stimuli of the substrate could be dissected, in particular rigidity from adhesiveness, allowing to separately study their influence on cell behavior. Using the level of localization of YAP/TAZ mechanotrasducers as a beacon to investigate cell responses and human osteosarcoma U2OS cell line as a paradigm, we found that rigidity is an overarching parameter that regulate cell mechanotrasduction. However, at intermediate physiologically relevant stiffness (<1-few kPa), adhesiveness can impair stiffness. Moreover, a threshold of 150 μm2 nuclear projected area is a necessary checkpoint to be surpassed for YAP/TAZ activation. Using polyacrylamide based gels, I developed substrates with different fixed stiffnesses, finely tunable in a broad range of physiological relevant rigidities (static gels), that could also be decreased in time during cell culture (dynamic substrates), without the need of detach cells from the culture substrate. These tools allowed to mimic the dynamic processes occurring in natural ECM, in particular in the context of tissue aging. Using WI38 fibroblasts, it was found that YAP/TAZ activity is impaired in ECM typical of aged tissues. Studying this phenomenon both in vivo and in vitro, we found that YAP/TAZ mechano-activation induced tissue senescence, demonstrating that these aging traits are due to cGAS-STING activation and consequent inflammation processes. Indeed, by YAP/TAZ rescue these processes are inhibited. Finally, to fully recapitulate the dynamicity of the ECM I optimized substrates that undergo to a periodical deformation during cell culture, by means of a stretching device. Studies of cell responses to periodical mechanical cues are on-going. Preliminary results show that YAP/TAZ activity is instrumental in preserving nuclear envelope integrity from damage arising from repeated, acute ii mechanical strains, as such providing a protection mechanism from the onset of an ageing-associated inflammatory phenotype in fibroblasts.