Engineering cell adhesive surfaces that direct integrin alpha5beta1 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 alpha(5)beta(1) requires both the PHSRN synergy site in the 9th and the RGD site in the 10th type III repeat of fibronectin (FN). Binding of alpha(5)beta(1) 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 (FNIII(7-10)) encompassing the alpha(5)beta(1) binding domains of FN. Model hybrid surfaces were engineered by immobilizing FNIII(7-10) onto passively adsorbed, non-adhesive albumin. Homo- and hetero-bifunctional crosslinkers of varying spacer-arm length targeting either the cysteine or lysine groups on FNIII(7-10) were investigated in ELISA and cell adhesion assays to optimize immobilization densities and activity. FN-mimetic surfaces presenting controlled densities of FNIII(7-10) were generated by varying the concentration of FNIII(7-10) in the coupling solution at a constant crosslinker concentration. Cells adhered to these functionalized surfaces via integrin alpha(5)beta(1) and blocking with integrin-specific antibodies completely eliminated adhesion. In addition, adherent cells spread and assembled focal adhesions containing alpha(5)beta(1), vinculin, and talin. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of biomimetic materials.
Cutler
Sarah M
SM
Wallace H Coulter School of Biomedical Engineering, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0363, USA.
García
Andrés J
AJ
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001672:Biocompatible Materials; D002417:Cattle; D002448:Cell Adhesion; D003432:Cross-Linking Reagents; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007096:Imidoesters; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D008422:Materials Testing; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013499:Surface Properties; D023822:Tissue Engineering / Q000379:methods*
D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D005353:Fibronectins; D007096:Imidoesters; D039081:Integrin alpha5beta1; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; C007471:dimethyl dithiobispropionimidate