Abstract

Three-dimensional (3D) intestinal models require physiologically relevant microarchitectures and mechanically supportive matrices to accurately replicate epithelial behavior; however, most existing in vitro systems lack villus–crypt topography and do not provide the material cues needed to guide epithelial organization. In this work, we developed biomimetic silk fibroin (SF) membranes that reproduce the native villus–crypt structure using two complementary cross-linking and processing strategies: chemical cross-linking with BDDE to form soft hydrogels and ethanol-induced β-sheet formation to generate stiff, dimensionally stable membranes. These routes were selected because they produce distinct material classes with nonoverlapping ranges of crystallinity, hydrophilicity, and stiffness, enabling access to mechanical regimes unattainable through a single cross-linking method. Villus–crypt architectures were replicated with high fidelity using customized molds. By culturing Caco-2 cells on these patterned SF membranes, we systematically examined how matrix stiffness and β-sheet content influence epithelial adhesion, proliferation, and differentiation. The physically cross-linked membranes (∼20 MPa) supported robust spreading, confluent monolayer formation, and relatively high ALP activity, whereas the softer hydrogels (∼15 kPa) limited adhesion and proliferation. Collectively, this study establishes a tunable SF-based platform that provides both physiological topography and mechanical support, offering a promising foundation for advanced 3D in vitro intestinal epithelial models.