Jennifer Young (Dual gradient hydrogel systems for mechanobiology applications): The spatial presentation of mechanical information is a key parameter for cell behavior. We have previously developed a method for creating tunable stiffness gradient polyacrylamide hydrogels with values spanning the in vivo physiological and pathological mechanical landscape. Importantly, we created gradients that do not induce durotaxis in human adipose-derived stem cells (hASCs), thereby allowing for the presentation of a continuous range of stiffnesses in a single sample without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied, providing high resolution data on stiffness-dependent expression and localization. Expanding upon this work, we are utilizing these gradient hydrogel systems to study cancer cell-ECM interactions. Interactions with the surrounding microenvironment have been shown to positively influence cancer cell survival and invasion by conferring adhesion-based resistance in response to chemotherapeutic drugs, and subsequently driving metastasis into surrounding tissues. In order to study a wide range of ECM environments, we produce dual-gradient systems by fabricating a gradient of ligands on top of our previously described stiffness gradient hydrogels. Ligand gradients are produced by either a gradient photomask to which proteins can be coupled to the substrate via a UV-sensitive crosslinker or by depositing a gradient of gold nanoparticles onto the hydrogel to which thiolated peptides can readily attach. Using these dual gradient hydrogels, we can better understand the interplay of substrate stiffness, ligand type, and ligand spacing in regulating adhesion-conferred chemoprotection in cancer cells.
Andrew W. Holle (Under pressure: the role of multidimensional confinement in mechanobiology): As bioengineers systematically move from simple 2D substrates to more complex 3D microenvironments, the role of cellular and nuclear volume adaptation in response to these substrates is becoming more appreciated. Long, narrow PDMS microchannels, which recapitulate porous extracellular matrix (ECM) networks found in vivo, confine cells to a single axis of migration and require them to utilize a complex synergy of traction force, mechanosensitive feedback, and subsequent cytoskeletal rearrangement. This process exhibits characteristics of the poorly understood mesenchymal-to-amoeboid transition, in which cells alter their migratory phenotype in order to traverse narrow constrictions and more successfully metastasize. During channel permeation, the volume of the nucleus changes, suggesting that nuclear reorganization and volume adaptation is a key step for successful permeation. Volume adaptation is also an important phenomena in stem cell mechanobiology. 3D GelMA hydrogel scaffolds with linear stiffness gradients were used to confine stem cells in three dimensions, with cells in the soft end more able to deform the matrix and increase their cell volume, while those on the stiff end were more confined. Cells on the soft end, which were able to adapt their volume more efficiently, exhibited markers for osteogenesis, while those on the stiff end became more adipogenic. This trend, which is opposite to what is observed on 2D hydrogels, suggests that volume adaptation, not stiffness, is sufficient for mechanosensitive differentiation in 3D. Ultimately, as volume adaptation is ubiquitous in 3D microenvironments in vivo, new tools will lead the way in analyzing and understanding mechanobiology.
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