Tuning hydrogels by mixing dynamic cross-linkers: enabling cell-instructive hydrogels and advanced bioinks

Rational design of hydrogels that balance processability and ECM biomimicry remains a challenge for tissue engineering and biofabrication. Hydrogels which are suitable for biofabrication techniques, yet can be tuned to match mechanical (static and dynamic) properties of native tissues remain elusive. Dynamic covalent hydrogels possessing their shear thinning/self-healing (processability) and time-dependent cross-links (mechanical properties) provide a potential solution, yet can be difficult to rationally control. Here, we explore the straightforward modular mixing of dynamic cross-links with different timescales (hydrazone and oxime) to enable fine control over hydrogel properties. We explore the ability of this modular approach to facilitate fabrication of 3D bioprinted constructs and cell behavior using rheology, self-healing tests, extrusion printing, and culture of primary human dermal fibroblasts. Maintaining a constant polymer content and cross-linker concentration, the stiffness and stress relaxation can be tuned across two orders of magnitude. All formulations demonstrate a similar flow profile after network rupture, allowing the separation of initial mechanical properties from flow behavior during printing. Furthermore, the self-healing nature of hydrogels with high hydrazone content enables recyclability of printed structures. Lastly, a distinct threshold for cell spreading and morphology is observed within this hydrogel series, even in multi-material constructs. Simple cross-linker mixing enables fine control and is of general interest for bioink development, targeting viscoelastic properties of specific cellular niches, and as an accessible and flexible platform for designing dynamic networks.