Leveraging protein-engineered biomaterials and bioorthogonal chemistries to elucidate the role of non-elastic matrix properties in regulating cell fate

PROJECT SUMMARY Cells are exquisitely sensitive to the mechanical properties of their environment, altering fundamental processes like adhesion, migration, cell division, and cell fate specification in response to how the polymers comprising the extracellular matrix (ECM) and surrounding cells react to applied forces. Thus, environmental mechanical cues

serve as crucial drivers of development, homeostasis, tissue regeneration, and disease progression. To understand the molecular mechanisms underpinning cellular mechanosensing, engineered systems are required that can decouple the influence of multiple confounding parameters, such as matrix stiffness (elasticity), viscous

force dissipation, plastic deformation, microstructure, and adhesive cues. While there is an extensive body of literature exploring how cells sense and respond to stiffness and, increasingly, viscous force dissipation, present materials systems used in these studies are only capable of independently controlling one or two mechanical

parameters at a time and make use of chemistries that can react with biologically relevant molecules, leading to altered material properties over time and potential off-target effects on cells. My lab leverages interdisciplinary expertise in bioorthogonal chemistries, protein engineered biomaterials, and stem cell biology to develop new

platforms to study fundamental mechanisms of cellular mechanosensing under physiologically relevant conditions. This includes developing new chemistries and hydrogel materials to enable simultaneous, independent, and dynamic tuning of matrix stiffness, viscous force dissipation, and presentation of cell adhesive

cues within 3D organotypic ensembles of cells. Recent efforts have focused on the development of highly- selective, stimuli-responsive chemistries to alter the stiffness and force dissipation rate of hydrogel materials on demand to model changes that occur in various diseases and during aging. We have also developed new protein

engineered materials with genetically encoded viscoelasticity and are applying these materials to develop chemically-defined and highly tunable 3D organotypic culture platforms. In this proposal, we extend our work by developing new bioorthogonal chemistries that will enable tuning of viscoelastic force dissipation without off-

target chemical reactivity, permitting casual relationships to be identified in complex systems over long culture durations without deterioration of material properties. We will also introduce new mechanically-labile crosslinking chemistries to provide additional modes of plastic deformation induced by cells. Finally, we will address a

limitation of cellular force generation measurement techniques in native-like viscoelastic materials by developing new force sensors through protein engineering and chemoenzymatic modifications. The platforms developed in this proposal will be broadly useful to elucidate the molecular mechanisms by which cells sense and respond to

changes in their mechanical microenvironment and to study how these changes drive desired phenotypes during development and tissue regeneration and undesired phenotypes during aging and disease progression.