Designing cell-instructive viscoelastic hydrogels to understand and exploit mechanobiology

Tissues exhibit spatially heterogeneous viscoelastic mechanics during development, disease progression, and wound repair, but dissecting how viscoelasticity regulates in vivo phenomena remains challenging. This MIRA tackles this key challenge through the invention of the first hydrogel 3D cell culture platform with photopatterned spatiotemporal control of viscoelasticity and stress relaxation. Theme 1: Enacting spatiotemporal control of hydrogel viscoelasticity to match heterogeneous tissue mechanics. Knowledge Gap: Tissue viscoelasticity varies, with stress relaxation timescales spanning seconds to hours. This heterogeneity influences cell-microenvironment interactions, but current 3D models fail to replicate it. Rationale: Our hydrogel system combines phototunable slow-relaxing dynamic covalent crosslinks and fast- relaxing guest-host interactions, enabling spatiotemporal viscoelasticity control to mimic tissue mechanics. Preliminary data show robust MSC spreading in these hydrogels. Hypothesis: Hydrogels patterned with varying viscoelasticity, guided by nanoindentation of tissues, will differentially regulate stromal cell responses via YAP/TAZ and TRPV4. Outcomes: Rheology and nanoindentation will guide the fabrication of hydrogels mimicking patient tissue mechanics. These models will elucidate how viscoelasticity regulates cell behavior. Theme 2: Determining how immune cells influence stromal cell mechanotransduction in viscoelastic hydrogels. Knowledge Gap: Fibroproliferative diseases involve an inflammatory, stiffened ECM, but the role of mechanics in immune-driven stromal activation is unclear. Rationale: We previously showed M2 macrophages promote IL6-dependent fibroblast activation in 2D culture, but it is unknown how viscoelasticity modulates immune-stromal interactions in 3D hydrogels. Hypothesis: Slower stress relaxation suppresses IL6-driven fibroblast activation in 3D, even with pro-fibrotic immune cells. Outcomes: Using spatial transcriptomics and knockout cell lines, we will uncover how viscoelasticity shapes immune-stromal crosstalk. Theme 3: Creating bioprinted viscoelastic hydrogels capable of both protease-dependent and protease-independent remodeling. Knowledge Gap: Digital light processing (DLP) bioprinting achieves high fidelity but typically requires dense crosslinking that restricts cell functions. Rationale: Building on our hydrogel platform from Theme 1, we will use DLP to print structures with complex architectures. Incorporating MMP- degradable crosslinks will enable enzymatic remodeling alongside viscoelastic relaxation. Hypothesis: Dual remodeling hydrogels (protease-dependent and protease-independent) will support robust cell viability, spreading, and mechanosensing. Outcomes: Advanced hydrogel platforms will model tissue complexity and reveal how cells integrate viscoelastic and enzymatic cues, advancing understanding of cell-ECM interactions. Project Number: 1R35GM162024-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Steven Caliari | Institution: UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, VA | Award Amount: $415,171 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 CDB-N (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11266337