Thermophoretic fabrication of gradient hydrogels for mechanobiology applications

Cell behavior during development, disease, and homeostasis are regulated by spatially dynamic cues found within their microenvironment, often in the form of stiffness gradients. In the context of mechanobiology, microengineered stiffness gradient hydrogels offer a powerful tool to probe how cells sense and respond to biophysical cues, particularly towards the identification of new mechanistic understanding and therapeutic strategies. However, the need to spatially manipulate the properties of soft hydrogels at the micron scale remains a major fabrication challenge. Therefore, this thesis aims to enhance the accessibility and adoption of gradient hydrogel technology in mechanobiology research, particularly by leveraging temperature-driven thermophoresis phenomenon and microfluidics technology to create a robust fabrication platform. Through extensive optimization and characterization studies, this work demonstrates four key features of the engineered platform that overcome several limitations of existing methods, including 1) highly precise and reproducible patterning, 2) compatibility with a wide range of hydrogel chemistries, 3) broad biologically relevant stiffness gradient regimes, and 4) the ability to create complex gradient patterns. Alongside this is the development of a new class of fluorescently labelled gradient hydrogels which display a stiffness-dependent fluorescence readout. This strategy enables quantitative assessment of the gradient formation process and contactless stiffness mapping via standard microscopy imaging, offering a simpler alternative to the gold standard atomic force microscopy for material characterization. Overall, the technical developments and findings gained from the series of cell-material interaction studies in this thesis have implications for advancing biomaterials technologies in mechanobiology, with potential impacts on cell signaling and how to modulate cell behavior by programming the microenvironment.