Expanding the Versatility of Dynamic Covalent Hydrogels with Static Covalent Spot-Welding

Hydrogels cross-linked through dynamic covalent chemistry (DCC) can mimic the viscoelastic properties of native biological tissues; however, these materials often suffer from rapid erosion, greatly limiting their application in biological studies. To address this challenge, we developed a DCC hydrogel with enhanced stability by sparsely distributing static covalent bonds, termed “spot-welds,” throughout the network. These spot-welds served as anchor points to prevent polymer erosion and significantly improved gel stability without compromising viscoelasticity. Specifically, our single-network system (termed HELP) consisted of two recombinant biopolymers, hyaluronic acid (HA) and an engineered elastin-like protein (ELP), each modified to cross-link through both dynamic hydrazone bonds and static strain-promoted azide–alkyne cycloaddition (SPAAC) bonds. Gels with and without sparsely distributed spot-welds had similar stiffness (G′ ∼ 800 Pa), stress relaxation rates (τ1/2 ∼ 6000 s), and shear-thinning behavior, resulting in gels that were viscoelastic and extrudable through a 3D printing syringe. Importantly, the spot-welds significantly improved gel stability, with DCC-only gels suffering complete erosion by day 4, while spot-welded gels remained stable for at least 14 days. This combination of enhanced gel stability with viscoelastic mechanics enabled the 3D culture and maturation of human stem cell-derived cardiomyocytes. While elastic control gels resulted in loss of cardiomyocyte phenotype, the spot-welded viscoelastic gels supported cardiomyocyte spreading, spontaneous beating, and expression of α-actinin and troponin T. In summary, sparsely distributing static cross-links on each biopolymer within a dynamic covalent network results in an injectable and printable single-network hydrogel with viscoelastic mechanics and significantly enhanced stability, supporting 3D cardiomyocyte culture and maturation.