Matrix-bound Tenascin-C directs neuronal differentiation through stiffness-tuned MeHA hydrogels mimicking the spinal cord microenvironment.

Spinal cord injury (SCI) leads to a complex remodeling of the extracellular matrix (ECM), where Tenascin-C (TNC) is strongly upregulated during the early phases of the injury cascade. While TNC is known to influence neural cell behavior, its functional role and mode of presentation in guiding neuronal differentiation remains unclear. In this study, we developed a stiffness-controlled methacrylated hyaluronic acid (MeHA) hydrogel platform that mimics the mechanical properties of the spinal cord and enables defined matrix immobilization of TNC. In vivo analyses showed elevated TNC expression from day 1, with the strongest perilesional signal during the subacute period (1 week-1 month). Using this temporal insight, we investigated the role of matrix-bound versus soluble TNC in directing neuronal differentiation of induced spinal cord progenitor cells in vitro. Immobilized TNC presented with naïve spinal cord stiffness matched MeHA substrates significantly enhanced neuronal and motor neuron differentiation, as evidenced by increased βIII-tubulin and ISL1 expression, compared to soluble TNC or unmodified controls. These effects were strongly dependent on both ligand concentration and matrix stiffness, highlighting a narrow bioactive window for TNC-mediated signaling (effective window: 100-200 nM; reduced responses at ≥300 nM). Furthermore, bulk 3D MeHA hydrogels functionalized with TNC supported cell viability and sustained neuronal differentiation, demonstrating translational relevance for future scaffold-based neural repair. These findings identify TNC as a matrix-bound bioactive cue that interacts with the mechanical environment to regulate neuronal lineage commitment, providing a framework for designing next-generation biomaterials for neural repair.