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Bioengineered extracellular matrix (ECM) mimetic materials have tunable properties and can be engineered to elicit desirable cellular responses for wound repair and tissue regeneration. By incorporating relevant cell-instructive domains, bioengineered ECM mimics can be designed to provide well-defined ECM-specific cues to influence cell motility and differentiation. More importantly, bioengineered ECM surfaces are ideal platforms for studying cell–material interactions without the need to genetically alter the cells. Here, we showed that bioengineered ECM mimics can be employed to clarify the role of integrins in keratinocyte migration. Particularly, the roles of α5β1 and α3β1 in keratinocytes were examined, given their known importance in keratinocyte motility. Two recombinant proteins were constructed; each protein contains a functional domain taken from fibronectin (FN-mimic) and laminin-332 (LN-mimic …