Spatially controlling cardiac fibroblast-to-myofibroblast transition using Young's modulus patterned GelMA hydrogels

The spatial organisation of mechanical cues is increasingly recognised as a key regulator of tissue development and disease, yet in vitro systems capable of replicating such environments remain limited. Fibroblast migration and the fibroblast-to-myofibroblast transition, which affect scar formation, are examples of mechanisms affected by mechanical cues. We report a photolithographic platform that enables precise, spatial patterning of the Young's modulus of gelatin methacryloyl (GelMA) hydrogels, using a ruthenium/sodium persulfate (SPS) photoinitiation system. By altering light intensity, we achieved Young's modulus tunability between 4 kPa and 46 kPa in non-patterned gels. By displaying a binary light intensity pattern over the gel, the Young's modulus could be switched from 10 kPa to 45 kPa, representing healthy and fibrotic Young's moduli, over a distance of ∼20 μm (2.3MPa·mm⁻¹). This system could also generate relatively linear Young's modulus gradients that are more physiologically relevant, ∼10 kPa⋅mm^-1. Non-patterned and binary-patterned gels confirmed that increasing the Young's modulus drives the fibroblast-to-myofibroblast transition, observed by significantly greater cell volumes and α-smooth muscle actin (α-SMA) stress fibre formation, from encapsulated cardiac fibroblasts after 7 days. In gradient-patterned gels, fibroblasts exhibited a progressive, modulus-dependent increase in both cell volume and α-SMA stress fibre formation, alongside expressing a durotactic response, moving from healthy to fibrotic environments, and also aligning themselves between ±45 degrees to the Young's modulus gradient. Beyond cardiac fibrosis, this versatile platform enables rapid generation of biologically relevant mechanical landscapes for diverse mechanobiological applications, bridging the gap between simplified, mechanically uniform models and the heterogeneous microenvironments of native tissues. STATEMENT OF SIGNIFICANCE: This study developed a platform for spatially controlling the Young's modulus of GelMA using a digital light projection system. Using this system, a change in Young's modulus from 10 kPa to 45 kPa was achieved with a resolution of 20 µm. Natural Young's modulus gradients observed in tissues (∼10 kPa·mm⁻¹) can also be achieved. The fibroblast-to-myofibroblast transition was controlled spatially in the patterned gels, evidenced by changes in cell volume and α-SMA fibrillation. Fibroblasts demonstrated alignment and migration to the Young's modulus gradient. Our photopatterning system demonstrates ease of pattern/gradient definition with adaptability to a diverse range of systems, highlighting the impact of mechanical properties on cardiac scar formation, offering avenues for studying these effects.