Microscopic local stiffening in a supramolecular hydrogel network expedites stem cell mechanosensing in 3D and bone regeneration

Dynamic hydrogels cross-linked by weak and reversible physical interactions enhance the 3-dimensional (3D) spreading and mechanosensing abilities of encapsulated cells in a matrix. However, the highly dynamic nature of these physical cross-links also results in low mechanical stiffness in the hydrogel network and high tether compliance of the cell adhesion motifs attached to the network. The resulting low force feedback of the soft hydrogel network impedes the efficient activation of mechanotransduction signalling in the encapsulated cells. Herein, we demonstrate that the chemical incorporation of acryloyl nanoparticle-based cross-linkers creates regionally stiff network structures in the dynamic supramolecular hydrogels without compromising the dynamic properties of the cell-adaptable inter-nanoparticle hydrogel network. The obtained dynamic hydrogels with a heterogeneous hydrogel network topology expedite the development of adhesion structures, 3D spreading, and mechanosensing of the encapsulated stem cells, as evidenced by the upregulated expression of key biomarkers such as vinculin, FAK, and YAP. This enhanced spreading and mechanotransduction promotes the osteogenic differentiation of the encapsulated stem cells. In contrast, doping with physically entrapped nanoparticles or molecular cross-linkers (PEGDA) cannot locally reinforce the dynamic hydrogel network and therefore fails to facilitate cell mechanosensing or differentiation in the 3D hydrogels. We further show that the dynamic hydrogels with a locally stiffened network promote the in situ regeneration of bone defects in an animal model. Our findings provide valuable insights into the design of the supramolecular dynamic hydrogels with biomimetic hierarchical biomechanical structures as the optimized carrier material for stem cell-based therapies.