Engineering cell adhesive surfaces that direct integrin α5β1 binding using a recombinant fragment of fibronectin

Integrin receptors mediate cell adhesion to extracellular matrices and trigger signals that direct cell function. While many integrins bind to the arginine–glycine–aspartic acid (RGD) motif present in numerous extracellular proteins, integrin α5β1 requires both the PHSRN synergy site in the 9th and the RGD site in the 10th type III repeat of fibronectin (FN). Binding of α5β1 to FN is critical to many cellular processes, including osteoblast and myoblast differentiation. This work focused on engineering integrin-specific bioadhesive surfaces by immobilizing a recombinant FN fragment (FNIII7–10) encompassing the α5β1 binding domains of FN. Model hybrid surfaces were engineered by immobilizing FNIII7–10 onto passively adsorbed, non-adhesive albumin. Homo- and hetero-bifunctional crosslinkers of varying spacer-arm length targeting either the cysteine or lysine groups on FNIII7–10 were investigated in ELISA and cell adhesion assays to optimize immobilization densities and activity. FN-mimetic surfaces presenting controlled densities of FNIII7–10 were generated by varying the concentration of FNIII7–10 in the coupling solution at a constant crosslinker concentration. Cells adhered to these functionalized surfaces via integrin α5β1 and blocking with integrin-specific antibodies completely eliminated adhesion. In addition, adherent cells spread and assembled focal adhesions containing α5β1, vinculin, and talin. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of biomimetic materials.