A bilayer nerve conduit integrating aligned guidance with an RGI peptide/retinoic acid-loaded hydrogel for peripheral nerve regeneration.

The repair of long-gap peripheral nerve defects remains challenging, as regeneration depends on temporally coordinated bioactive regulation and spatially organized guidance. To address this, we developed a spatiotemporally coordinated nerve conduit integrating an injectable thermosensitive poly(amino acid) hydrogel with an aligned-random bilayer poly(ε-caprolactone) (PCL) wall. A brain-derived neurotrophic factor (BDNF)-mimetic RGI peptide was covalently tethered to mPEG-PMet-PLysMA (P), and retinoic acid (RA) was physically incorporated to form P-RGI@RA. Aligned luminal fibers promoted Schwann cell orientation and spreading, whereas random outer fibers increased transverse tensile strength and elastic modulus by 4.4- and 6.0-fold, respectively, relative to fully aligned membranes. In vitro, the RGI-conjugated formulation increased the mean longest neurite length in PC12 cells, whereas the RA-containing formulation enhanced Schwann cell migration, with the respective responses reaching 1.9- and 1.7-fold the control values. In a 10 mm rat sciatic nerve defect model, P-RGI@RA-filled conduits produced the most favorable repair profile among conduit-treated groups. At 12 weeks, the compound muscle action potential (CMAP) amplitude ratio and injured-to-contralateral gastrocnemius wet weight ratio were 2.3- and 1.5-fold those of hollow conduits, reaching 88% and 92% of the corresponding autograft values, respectively. These outcomes were accompanied by improved axon- and Schwann cell-associated regeneration, myelin ultrastructure, and target muscle preservation. Proteomics associated P-RGI@RA treatment with cytoskeletal regulation, extracellular matrix remodeling, axon guidance, and inflammation-related pathways, while immunoblotting showed increased phosphorylation of phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK). This conduit-hydrogel system provides a potential strategy for peripheral nerve regeneration.