Cellular phenotype is heavily influenced by the extracellular matrix (ECM), a complex and tissue-specific three-dimensional structure with distinct biophysical and biochemical properties. As naturally derived cell culture platforms are difficult to controllably modulate, engineered synthetic ECMs have facilitated our understanding of how specific matrix properties direct cell behavior. However, synthetic approaches typically lack fibrous topography, a hallmark of stromal and interstitial ECMs in vivo. To construct tunable biomimetic models with physiologic microstructure, we developed a versatile approach to generate modular fibrous architectures in 3D. Photo-cross-linkable polymers were electrospun, photopatterned into desired lengths, and coencapsulated alongside cells within natural biopolymer, semisynthetic, and synthetic hydrogels. Cells encapsulated within fiber-reinforced hydrogel composites (FHCs) demonstrated accelerated spreading rates compared to in gels lacking such fibrous topography. Furthermore, increases in fiber density at constant bulk hydrogel elastic modulus produced morphologically distinct cell populations and modulated cellular mechanosensing in 3D, as evidenced by increased nuclear localization of the mechanosensitive transcription factor, Yes-associated protein (YAP). This work documents the impact of physical guidance cues in 3D and establishes a novel approach to generating more physiologic tissue-and disease-specific biomimetic models.

Statistics. More information about statistics is available in the Supplemental Methods. Study approval. All animal procedures were carried out in accordance with the guidelines provided in the Guide for the Use and Care of Laboratory Animals (National Academies Press, 2011) and were approved by the IACUC of the University of Michigan (PRO0007930).

Fibrosis, characterized by aberrant tissue scarring from activated myofibroblasts, is often untreatable. Although the extracellular matrix becomes increasingly stiff and fibrous during disease progression, how these physical cues affect myofibroblast differentiation in 3D is poorly understood. Here, we describe a multicomponent hydrogel that recapitulates the 3D fibrous structure of interstitial tissue regions where idiopathic pulmonary fibrosis (IPF) initiates. In contrast to findings on 2D hydrogels, myofibroblast differentiation in 3D was inversely correlated with hydrogel stiffness but positively correlated with matrix fibers. Using a multistep bioinformatics analysis of IPF patient transcriptomes and in vitro pharmacologic screening, we identify matrix metalloproteinase activity to be essential for 3D but not 2D myofibroblast differentiation. Given our observation that compliant degradable 3D matrices amply support fibrogenesis, these studies demonstrate a departure from the established relationship between stiffness and myofibroblast differentiation in 2D, and provide a new 3D model for studying fibrosis and identifying antifibrotic therapeutics.

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