Regulation of hepatic stellates cell activation by biomimetic fibrotic hydrogels with uniform and heterogeneous matrix stiffness

Abstract Current research on liver fibrosis predominantly relies on culture matrices with uniform stiffness, largely overlooking the pathophysiological relevance of stiffness heterogeneity-the spatially uneven stiffness distribution characteristic of fibrotic liver tissue. To address this, we engineered biomimetic fibrotic hydrogels using decellularized liver matrix (DLM) from fibrotic tissues and methacrylated hyaluronic acid (MeHA). These hydrogels replicated the distinct stiffness profiles of normal, earlystage fibrotic, and late-stage fibrotic liver. Using a custom honeycomb-shaped mold, we integrated them into a unified platform featuring both uniform and heterogeneous mechanical regions. By co-culturing hepatic stellate cells (HSCs) on this platform, we investigated how matrix stiffness regulates HSCs proliferation, migration, and activation. Results demonstrated that on uniformly stiff substrates, particularly those mimicking late-stage fibrosis, ECM components and high stiffness promoted HSCs activation, upregulated the Piezo1-YAP signaling axis. In contrast, although the lowstiffness region of the mechanically heterogeneous platform exhibited increased HSC proliferation and activation, the proliferation and activation levels in the medium-and high-stiffness regions were significantly reduced. The expression patterns of the Piezo1-YAP signaling pathway further corroborated this phenomenon. These findings suggest that mechanical heterogeneity may exert a negative regulatory effect on HSC activation. This study provides new insights into the mechanobiological mechanisms underlying hepatic fibrosis and reveals potential therapeutic targets.