Spatial Photopatterning of Substrate Stiffness in Dual-Cure Silicones for Cardiac Mechano-Regulation

The mechanical properties of the extracellular matrix play a key role in regulating cellular functions, yet many in vitro models lack the mechanical complexity of native tissues. Traditional hydrogel-based substrates offer tunable stiffness but are often limited by instability, porosity, and coupled changes in both mechanical and structural properties, making it difficult to isolate the effects of stiffness alone. Here, we introduce a spatially patterned dual-cure polydimethylsiloxane (DC-PDMS) system, a nonporous, mechanically tunable polymer that allows for precise spatial control of stiffness over a range of patho-physiological values. This platform enables the design and creation of in vitro models for studying the influence of spatial mechanical cues on cellular behavior. To demonstrate its utility, we examined primary cardiac fibroblast responses across different substrate stiffness conditions. Fibroblasts on soft regions exhibited rounded morphologies with disorganized actin networks, while those on stiffer regions became more elongated with highly aligned stress fibers, indicating stiffness-dependent cytoskeletal remodeling. Stiff substrates also led to nuclear compression and increased nucleus curvature, correlating with increased nuclear localization of YAP, a key mechanotransduction regulator. By allowing cells to interact with mechanically distinct regions within a single substrate, this system provides a powerful approach for investigating mechanotransduction processes relevant to fibrosis and other mechanically regulated diseases. The ability to create stiffness patterns with subcellular resolution makes DC-PDMS a valuable tool for studying cell-material interactions, enabling new insights into mechanobiology-driven cellular responses and therapeutic targets.