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Our result confirmed the PVA hydrogel biocompatibility with high hepatocytes survival. Fibronectin (0.01 mg/mL) promoted optimal migratory behavior for single hepatocytes. However, for confluent hepatocytes, a stiff substrate promoted hepatocellular migration compared with the soft and moderate groups via enhancing the formation of actin- and tubulin-rich structures. and protein expression analysis showed that the stiff substrate altered the phenotype of hepatocytes and induced apoptosis. Hepatocytes in stiff 3D hydrogel showed a higher proportion of cell d