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.
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