Controlling ligand density and viscoelasticity in synthetic biomimetic polyisocyanide hydrogels for studying cell behaviours: the key to truly biomimetic hydrogels

Abstract

Many cell types within whole living organisms continuously sense external forces from their microenvironment through an extracellular matrix that orchestrates biophysically-cued cellular processes and mechanosensitive cell behaviours. Studying cells in 3D environments rather than in conventional 2D, is increasingly seen as a necessity for obtaining a comprehensive understanding of the dynamic and complex cell–matrix interactions and cellular responses to biophysical stimuli. While many synthetic hydrogels used in 3D cell culture applications do not provide a microenvironment that accurately mimics the structural and mechanical properties of native ECMs, ethylene-glycol grafted isocyanopeptide-based polymers (PIC) have become one of the most promising types of synthetic material for controllably mimicking endogenous ECM microenvironments based on their unique biomimetic strain-stiffening response. However, the precise effects of polymer modification through integrin-binding peptide addition and density on the nanoscale architecture of PIC polymer–peptide conjugates have not yet been comprehensively studied and the stress relaxation behaviours of PIC-based hydrogels have also yet to be comprehensively explored. Here we present new data on the structure–property relationship for PIC-based polymer–peptide conjugates by highlighting the effect of peptide addition and density on the PIC hydrogel physicochemical properties and nanoscale network structure. We show that the addition of a peptide-linker to polymer-bundle forming PIC polymers alters the polymer-bundle network structure, by potentially inhibiting the polymer-bundle formation and reducing the polymer-bundle diameter and density. Furthermore, we demonstrate the synergistic effects of ligand density, strain-stiffening responsiveness and viscoelasticity on fibroblast rapid cell spreading, morphological changes, and cytoskeleton alignment in comparison to a natural fibrin hydrogel. Cells cultured in a 6% (mol mol⁻¹) ligand density PIC hydrogel, with a low critical stress point and fast stress relaxation, facilitates similar spreading kinetics, spreading phenotype and F-actin cytoskeleton alignment to fibroblast cells cultured in a naturally-derived fibrin hydrogel. We find that the viscoelastic high ligand density PIC–peptide conjugate facilitates an accurate replication of fibroblast cellular behaviours in a natural matrix and that this material composition shows considerable promise for directing the optimal design of bioinspired materials for mechanotherapy and 3D cell culture.