Cryo-shocked neural stem cells as factor pool-functionalized nerve guidance conduits for enhanced peripheral nerve regeneration

Peripheral nerve injuries (PNI) often induce severe motor and sensory impairments, subsequent muscle atrophy, and even permanent disability. Autologous nerve grafting, the clinical gold standard for PNI repair, is limited by donor site morbidity and graft shortage, while conventional nerve guidance conduits (NGCs) only provide physical guidance and fail to reconstruct the bioactive microenvironment essential for nerve regeneration. To address these limitations, we fabricated chitosan NGCs functionalized with cryo-shocked neural stem cells (Cryo-NSC NGCs) as a tissue-specific factor pool via seeding NSCs onto the conduit lumen for short-term expansion followed by liquid nitrogen cryo-shock. Cryo-NSC NGCs sustainably release neurotrophic factors, enhance neural cell adhesion and neuronal differentiation, with the average axon length reaching 9.42-fold that of the blank control, outperforming cryo-shocked MSC-functionalized NGCs as validated by proteomic enrichment of neural regeneration-related proteins. In a rat 10-mm sciatic nerve defect model, Cryo-NSC NGCs effectively promoted axonal regeneration, myelination and vascularization, restored motor and sensory functions with nerve conduction recovery approaching autologous grafts, and exhibited excellent biocompatibility without excessive inflammation or organ damage. Collectively, Cryo-NSC NGCs provide a safe, efficient and translational platform for peripheral nerve repair. STATEMENT OF SIGNIFICANCE: 1. Cryo-shocked neural stem cells (NSCs) retain a complete, native repertoire of bioactive factors to deliver sustained, tissue-specific bioactive support directly to injured peripheral nerves. 2. Neuro-specific bioactive proteins in Cryo-NSCs synergistically enhance neural cell adhesion and drive a 9.42-fold increase in axonal elongation, significantly outperforming cryo-shocked mesenchymal stem cells (Cryo-MSCs). 3. Cryo-NSC-functionalized nerve guidance conduits (NGCs) effectively repair 10-mm sciatic nerve defects, with favorable behavioral, electrophysiological, and histological outcomes, offering a promising translational strategy for peripheral nerve injury treatment.