(326b) Dynamic Recombinant Hydrogels with Degradation-Independent Relaxation Kinetics

Dynamic hydrogels are attractive platforms for 3D cell culture, as they can recapitulate the viscoelastic properties found in the native extracellular matrix (ECM). While hydrogels crosslinked using dynamic covalent chemistries (DCC) can mimic this rheological behavior, these materials often suffer from rapid erosion due to their appreciable DCC off-rates. Thus, many DCC gels erode within 24-48 h, too short of a time to be useful for cell culture. To address this issue, we invented a hydrogel system with enhanced stability based on engineered, recombinant proteins crosslinked via a combination of DCC bonds and static covalent bonds, which we term “spot-welds.” We hypothesized that these spot-welds would serve as anchor points to prevent polymer erosion and significantly improve gel stability. Specifically, the system consists of two recombinant biopolymers, an engineered elastin-like protein (ELP) and hyaluronic acid (HA), chemically modified to enable both DCC and spot-weld crosslinking. Upon mixing the two hydrogel components, a hydrogel network is spontaneously formed in the presence of mammalian cells. For the DCC reaction, lysine residues within the ELP biopolymer are conjugated to display hydrazine moieties, while HA is modified to display aldehyde groups. To enable the spot-weld reaction, the ELP biopolymer is further functionalized through tyrosine-selective conjugation to display azides (ELP-HYD-AZ), while HA is further modified to display bicyclononyne groups (HA-ALD-BCN). All polymers are characterized for chemical identity (NMR), and mechanical properties of the resulting gels are evaluated at 37 °C using oscillatory shear rheology. The use of limited spot-welds did not affect the overall gel stiffness, as gels with and without the static covalent crosslinks had a similar starting stiffness (G’ ~ 1000 Pa) and retained their viscoelastic, stress-relaxing properties. Likewise, these static covalent crosslinks did not inhibit the shear-thinning behavior of the resulting gels, with the gels being easily hand-injectable through a syringe needle after gelation. Importantly, the formulation of gels with spot-welds dramatically improved the gel stability from 2 days to 10+ days and supported the 3D culture of human neural progenitor cells. In summary, the combined use of dynamic and static covalent crosslinking within a biopolymeric hydrogel provides a biomimetic 3D cell culture platform that is injectable and recapitulates the viscoelastic properties of the dynamic native ECM.