(540f) Tuning Hydrogel Viscoelasticity and Cell–Cell Signaling to Direct Neural Maturation

Neural progenitor cells (NPCs) hold immense potential as therapeutic candidates for nervous system regeneration as well as in vitro models of neurodevelopment and disease. However, it is not well understood how NPCs simultaneously incorporate biochemical and biophysical cell–matrix interactions to regulate differentiation and fate acquisition. In particular, the native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force, yet most conventional biomaterials are formed via static covalent crosslinks and are therefore elastomeric and non-stress-relaxing. By incorporating dynamic covalent hydrazone bonds in a chemically defined, hyaluronan (HA)–elastin-like protein (ELP) hydrogel called HELP, we develop a three-dimensional (3D) biomaterials platform with tunable cell-adhesive ligands and tunable stress relaxation that can be remodeled by cell-imposed forces. To tune biochemical matrix signaling, modularly designed ELPs were engineered to present one of the following sequences: 1) an N-cadherin mimic sequence (HAVDI) to artificially induce cell–cell signaling, 2) a fibronectin-derived, integrin-binding sequence (RGD) to mimic cell–matrix signaling, or 3) a scrambled, non-integrin-binding sequence (RDG) to which cells cannot bind. We then tuned the mechanical properties of our hydrogels to resemble the stiffness and stress relaxation rates of native brain tissue more closely. The hydrogel stiffness is controlled by altering the total number of hydrazone crosslinks to reach a similar shear modulus for all conditions (800-1000 kPa). The stress relaxation rate is controlled by altering the kinetics of crosslink dynamics, by replacing benzaldehyde-functionalized HA (slow kinetics) with aldehyde-functionalized HA (fast kinetics). Additionally, we develop HELP gels with permanent static bonds (norbornene/tetrazine) as an elastic, non-stress-relaxing control.

We demonstrate that these dynamically crosslinked HELP hydrogels facilitate gel remodeling and support NPC viability. Furthermore, NPCs encapsulated within these gels underwent relaxation rate dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections and expressed higher levels of genes associated with more mature neuronal expression profiles. Conversely, NPCs within static, non-stress-relaxing matrices exhibited limited neurite outgrowth and adopted transcriptomic profiles indicative of intermediate progenitors. By altering the adhesive ligand presented (integrin-binding RGD vs. non-integrin-binding RDG), we identified RGD-initiated integrin signaling to be necessary for mechanosensitive neurite outgrowth. By inhibiting actin polymerization within fast-relaxing gels, we observed decreased neuritic projections and a concomitant decrease in the expression of neural maturation genes. Through independent manipulation of both external biophysical signaling (i.e. matrix stress relaxation rate) and intracellular cytoskeletal regulation (i.e. inhibition of actin polymerization), we identified the ability to extend neurites as a crucial morphological cue guiding NPC fate acquisition. Taken together, these results suggest that cell-mediated strain within remodelable, fast stress-relaxing gels drives neural maturation and that tuning biomechanical signaling cues within engineered hydrogels has significant potential to advance models of the neural microenvironment in vitro and improve neural regeneration in vivo.