Hyaluronic acid-alginate hydrazone crosslinked hydrogels support the generation and maturation of V2a interneurons

Injury to the central nervous system (CNS) causes inflammation, cell death, and glial scar formation that inhibits tissue repair. Injectable hydrogels modified with extracellular matrix (ECM)-derived peptides can provide biochemical cues to promote neural tissue repair and serve as a vehicle to deliver therapeutics across the blood-spinal/blood-brain barrier in a minimally invasive manner. We developed an injectable hydrazone crosslinked hyaluronic acid-alginate (HA-Alg) hydrogel for neural tissue repair. We fabricated hydrogels with a range of polymer concentrations and evaluated their physicochemical properties to identify formulations that mimic the stiffness and viscoelastic properties of the CNS tissue environment. Hyaluronic acid was further modified with ECM-derived, cell-adhesive peptides (RGD and IKVAV) to enhance neuronal adhesion and viability. To evaluate the therapeutic potential of our hydrogel platform, we embedded mouse embryonic stem cell aggregates and differentiated them toward mature V2a interneurons. These interneurons are critical for relaying motor signals and represent a promising therapeutic cell population for treating spinal cord injuries. We demonstrated successful enrichment for V2a interneurons in HA-Alg hydrogels containing ECM-derived peptides, with a 2-fold increase in Chx10 expression compared to laminin controls. Interestingly, both our newly described HA-Alg and established crosslinked HA-HA hydrogels containing IKVAV peptides demonstrated significantly increased neurite length in interneuron-enriched cultures compared to hydrogels without peptides, averaging approximately 197.2 ± 53.5 µm for HA-IKVAV + HA and 212.4 ± 75.2 µm for HA-IKVAV + Alg. This study demonstrates that a biomimetic hydrogel platform containing ECM-derived peptides can support the neuronal adhesion and viability required for future applications in functional CNS tissue repair.