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Here, we develop spider silk protein (spidroin)\u2010based hydrogels from the recombinant miniature spidroin NT2RepCT mixed with VN\u2010NT, a vitronectin peptide fused to the spidroin N\u2010terminal (NT) domain. These hydrogels undergo spontaneous gelation at 37\u00b0C under physiological conditions and exhibit tunable stiffness. Structural and functional characterization confirmed stable fibril networks. Inclusion of VN\u2010NT in the gels improved epithelial cell adhesion, monolayer organization, and integrin\u2010linked adhesion formation, while human fetal mesenchymal stem cells exhibited robust viability and migration. Traction force microscopy demonstrated protein concentration\u2010dependent force transmission from cells to the hydrogel matrix. Collectively, these results establish NT2RepCT\u2010based hydrogels as non\u2010toxic 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.<\/jats:p>","DOI":"10.1002\/smll.75778","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T15:05:06Z","timestamp":1790262306000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Dual\u2010Component Spidroin\u2010Based Hydrogels: A Mechanically Tunable Platform for 2D and 3D Cell Culture"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1502-4630","authenticated-orcid":false,"given":"Sanna","family":"Korpela","sequence":"first","affiliation":[{"name":"Biosciences Unit Faculty of Medicine and Health Technology Tampere University  Tampere 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Reproductive Medicine Karolinska University Hospital  Stockholm Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1872-1207","authenticated-orcid":false,"given":"Anna","family":"Rising","sequence":"additional","affiliation":[{"name":"Department of Medicine Huddinge Karolinska Institutet Huddinge Sweden"},{"name":"Department of Animal Biosciences Swedish University of Agricultural Sciences  Uppsala Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4351-8697","authenticated-orcid":false,"given":"Teemu O.","family":"Ihalainen","sequence":"additional","affiliation":[{"name":"Biosciences Unit Faculty of Medicine and Health Technology Tampere University  Tampere 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