Ultrastructural and Proteomic Signatures of Mechanoadaptive Fibroblast Remodeling across Microphysiological and Mesoscale Shear Platforms

Abstract

Mechanobiological cues in the tissue microenvironment increasingly drive pathological fibroblast activation in inflammatory bowel disease (IBD), yet engineered platforms modeling this transition remain limited. Here, a microfluidic gut-on-a-chip microphysiological system and a mesofluidic rotary shaker are used to demonstrate that sustained fluid shear stress alone is necessary and sufficient to drive an irreversible, profibrotic phenotypic switch in primary normal human intestinal fibroblasts. Across both platforms, normal fibroblasts from small and large intestine reproducibly self-organize into three-dimensional (3D) multicellular aggregates within 72 h, independent of shear delivery format, indicating that the transition is governed by mechanical dose rather than device geometry. The resulting aggregates acquire robust -smooth muscle actin (-SMA) expression with aligned stress fibers, contrasting with the -SMA-negative parental population. Scanning electron microscopy (SEM) resolves densely packed cellular microarchitecture embedded in a microfibrillar extracellular network, while ultrastructural serial block-face 3D EM reveals expansive intercellular spaces, stochastic fibrillar extrusions, and electron-dense cytoplasmic material at cell boundaries. Proteomic profiling confirms enrichment of core matrisome components, including collagen subtypes and matrix metalloproteinases. Together, these results establish fluid shear stress as a platform-independent, sufficient mechanical trigger for fibroblast-to-mechanoadaptive transition, positioning microphysiological shear platforms as tractable tools for modeling and targeting early fibrogenesis in IBD.