Extracellular matrix cues as drivers of liver zonation: A framework for in vitro biomaterial design

The liver's complex microenvironment and spatially zonated functions present major challenges for in vitro modeling, particularly in drug development and disease research. While oxygen and nutrient gradients have been used to create zonation, the potential of the surrounding biomaterial, i.e. the extracellular matrix (ECM), remains relatively underexplored. Recently, native ECM components and/or binding motifs, such as decellularized ECM (dECM) or arginine-glycine-aspartate (RGD) peptides, are increasingly integrated to improve in vitro hepatocyte functionality. However, the biological underpinning of ECM-cell interactions and resulting hepatocyte behavior are often poorly understood, which hampers to exploit the full potential of biomaterial-based strategies for relevant liver tissue modeling. Within this context, the spatial ECM characteristics within the Space of Disse are of critical importance. In this review, we therefore first outline how ECM-receptor interactions influence hepatocyte function and then review how biomaterial composition and mechanics steer zone-specific cell functionality. We propose six practical design principles to guide biomaterial engineering for future applications, including mechanical tuning, affinity-guided selection, zone-specific ligand selection, enhanced ligand diversity, binding site accessibility, and native molecular environment preservation. These strategies will not necessarily increase model complexity, but may support intentional biology-driven biomaterial design beyond conventional scaffold macro-engineering, and enhance the physiological relevance of 3D liver models without sacrificing scalability, simplicity, or reproducibility. STATEMENT OF SIGNIFICANCE: There is an urgent demand for in vitro liver models that accurately reflect human biology for drug testing, disease modeling, and regenerative medicine. Existing systems rarely capture the liver's complexity, while animal models face ethical and translational constraints. A crucial but largely neglected feature is liver zonation, the region-specific variation in hepatocyte function. While oxygen and nutrient gradients are known to influence zonation, the role of extracellular matrix (ECM) cues remains underexplored. This review integrates evidence from hepatology, ECM-receptor biology, and biomaterials to propose an evidence-based, zonation-aware design framework. By translating biological principles into actionable material guidelines, it offers researchers the tools to develop next-generation liver models that better predict human outcomes and accelerate progress across the whole field.