Matrix stiffening toolbox: dynamic hydrogels for three-dimensional cell culture with real-time cell response

Extracellular matrix (ECM) mechanical properties regulate tissue homeostasis and disease progression, with persistent ECM stiffening serving as a hallmark of fibrosis; yet, the early transition from healthy to diseased tissue remains poorly understood. Dynamic three-dimensional (3D) tissue models that capture early-stage stiffening are needed to investigate cellular responses during disease initiation. This work presents an innovative platform for studying cell responses in 3D environments undergoing active matrix stiffening. A bioinspired synthetic ECM incorporates collagen-mimetic peptides and employs sequential, non-terminal strain-promoted azide–alkyne cycloaddition (SPAAC) reactions to enable controlled increases in matrix stiffness over physiologically relevant timescales. Alternating polymer incubations produce a 2.5-fold increase in storage modulus over 72 hours, modeling the mechanical transition from healthy to early-stage fibrotic lung tissue. Live-cell reporter fibroblasts enable real-time monitoring of alpha-smooth muscle actin (αSMA) expression, revealing significant upregulation during matrix stiffening that remains transient and difficult to detect via traditional endpoint assays. Active stiffening also modulates fibroblast motility, transiently increasing migration speed while persistently enhancing directional persistence. Complementary computational reaction-diffusion modeling provides mechanistic insight into modulus gradient formation and reaction kinetics. This versatile toolbox enables investigation of early mechanobiological responses to matrix stiffening and may aid identification of markers of fibrotic disease onset. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, DP2-HL152424, P30 GM110758-02 U.S. National Science Foundation, https://ror.org/021nxhr62, DMR-2011824