Hydrogel-mediated local PTK2 inhibition promotes ordered axonal regeneration associated with cytoskeletal remodeling after spinal cord injury.

Disorganized axonal regrowth after spinal cord injury (SCI) may compromise the spatial organization and directional alignment of regenerating axons. Our previous COL6-modified hydrogel model revealed physical guidance-independent, self-organized spinal cord regeneration, whose neuron-intrinsic mechanisms remain unclear. Using proteomic screening of a COL6-modified hydrogel model that promotes ordered spinal cord regeneration, we identified protein tyrosine kinase 2 (PTK2) as a candidate intracellular regulator associated with axonal organization. In vitro, PTK2 downregulation by siRNA or pharmacological inhibition with PF-573228 reduced neurite tortuosity and branching and increased radial directional coherence. Exploratory long-read transcriptomic analysis, together with Western blotting, revealed coordinated changes in integrin-, RhoA-, cofilin-, and actin-related signaling after PTK2 inhibition, consistent with cytoskeleton-associated remodeling. To achieve localized PTK2 inhibition in vivo, PF-573228 was incorporated into an injectable thiol-modified hyaluronan/gelatin hydrogel, termed HaG-PF, which exhibited porous microarchitecture and hyaluronidase responsiveness. In a mouse model of complete spinal cord transection, HaG-PF was associated with reduced p-PTK2 at the 4-week endpoint, increased NF200+ axonal presence, improved two- and three-dimensional axonal organization, increased CGRP+ fibers within the lesion site, and the presence of 5-HT+ fibers in the caudal stump. Improved axonal organization was accompanied by enhanced locomotor and electrophysiological recovery. Collectively, these findings identify local PTK2 inhibition as a molecular strategy for promoting more organized axonal regeneration after SCI.