Molecular Stiffness Cues of Interpenetrating Hydrogel Networks for Cell Adhesion

The response of cultured cells to the mechanical properties of hydrogel substrates depends ultimately on the response of single crosslinks to external forces exerted at cell attachment points. We performed single-molecule force spectroscopy experiments using AFM to probe the mechanics of single cross-links which mediate the cell attachment and spreading. We compared hydrogels of varying elastic modulus which exhibited significant differences in fibroblast spreading and derived an effective spring constant for the displacement of single cross-links at the hydrogels’ surface. A factor of ten in the elastic modulus <i>E</i> of the hydrogel corresponded to a factor of five in the effective spring constant <i>k</i> of single crosslinks, indicating a transition in scaling with the mesh size ξ from the macroscopic <i>E</i> ∝ ξ⁻³ to the molecular <i>k</i> ∝ ξ⁻² [1].<br/>We also prepared an interpenetrating network (IPN) design based on a stiff PEGDA host network interlaced within a soft 4-arm PEG-Maleimide/thiol (guest) network. The IPN presents different mechanical cues at the molecular scale, depending on which network is linked to the probe, but the same mechanical properties at the macroscopic length scale as the individual host network. Cells attached to the interpenetrating (guest) network of the IPN or to the single network (SN) PEGDA hydrogel modified with RGD adhesive ligands showed comparable attachment and spreading areas, but cells attached to the guest network of the IPN, with lower molecular stiffness, showed a larger number and size of focal adhesion complexes. The observations indicate that cell adhesion to the IPN hydrogel through the network with lower molecular stiffness proceeds effectively as if a higher ligand density is offered [2].<br/>[1] A. Colak, B. Li, J. Blass, K. Koynov, A. del Campo, R. Bennewitz, The mechanics of single cross-links which mediate cell attachment at a hydrogel surface, Nanoscale, 11 (2019) 11596-11604.<br/>[2] B. Li, A. Çolak, J. Blass, M. Han, J. Zhang, Y. Zheng, Q. Jiang, R. Bennewitz, A.del Campo, Molecular stiffness cues of an interpenetrating network hydrogel for cell adhesion, Materials Today Bio, 15 (2022) 100323.