Hydrogels with multiple RGD presentations increase cell adhesion and spreading

A key challenge in designing hydrogels for cell culture is replicating the cell-matrix interactions found in tissues. Cells use integrins to bind their local matrix and form adhesions in which integrins dynamically move on the cell membrane while applying significant forces to the local matrix. Identifying the important biomaterial features for these interactions is challenging because it is difficult to independently adjust variables such as matrix stiffness, stress relaxation, the mobility of adhesion ligands, and the ability of these ligands to support cellular forces. In this work, we designed a hydrogel platform consisting of interpenetrating polymer networks of covalently crosslinked poly(ethylene glycol) (PEG) and self-assembled peptide amphiphiles (PA). We can tune the viscoelasticity of the hydrogel by modulating the composition of both networks. Ligand mobility can be adjusted independently of the matrix mechanical properties by attaching the arginine-glycine-aspartic acid (RGD) cell adhesion ligand to either the covalent PEG network, the dynamic PA network, or both networks at once. We find that endothelial cell adhesion formation and spreading is maximized in soft gels in which adhesion ligands are present on both the covalent and non-covalent networks. The dynamic nature of adhesion domains, coupled with their ability to exert substantial forces on the matrix, suggests that having different presentations of RGD ligands which are either mobile or capable of withstanding significant forces is needed to mimic different aspects of complex cell-matrix adhesions. These results will contribute to the design of hydrogels that better recapitulate physiological cell-matrix interactions. STATEMENT OF SIGNIFICANCE: Creating artificial environments that accurately mimic how cells interact with their surrounding matrix in natural tissues remains a fundamental challenge in biomaterials science. This study introduces a dual-network hydrogel platform that independently controls mechanical properties and adhesion ligand mobility by combining stable and dynamic polymer networks. A significant body of work has shown that matrix viscoelasticity and adhesion ligand mobility are important for cell adhesion and spreading. Our work builds on this by showing that endothelial cells function optimally when they can simultaneously engage with both mobile adhesion sites and force-resistant anchoring points, independent of matrix viscoelasticity. These insights will guide the design of more physiologically relevant hydrogels for tissue engineering applications and disease modeling.