Dual‐Component Spidroin‐Based Hydrogels: A Mechanically Tunable Platform for 2D and 3D Cell Culture

ABSTRACT Hydrogels that recapitulate the biochemical and mechanical complexity of the extracellular matrix (ECM) are critical for advanced cell culture and tissue engineering. Here, we develop spider silk protein (spidroin)‐based hydrogels from the recombinant miniature spidroin NT2RepCT mixed with VN‐NT, a vitronectin peptide fused to the spidroin N‐terminal (NT) domain. These hydrogels undergo spontaneous gelation at 37°C under physiological conditions and exhibit tunable stiffness. Structural and functional characterization confirmed stable fibril networks. Inclusion of VN‐NT in the gels improved epithelial cell adhesion, monolayer organization, and integrin‐linked adhesion formation, while human fetal mesenchymal stem cells exhibited robust viability and migration. Traction force microscopy demonstrated protein concentration‐dependent force transmission from cells to the hydrogel matrix. Collectively, these results establish NT2RepCT‐based hydrogels as non‐toxic substrates with adjustable mechanics and straightforward, modular functionalization with bioactive motifs and proteins, providing a promising platform for mechanobiology studies, stem cell culture, and tissue engineering.