Project Summary/Abstract Peripheral nerve injuries (PNI) affect millions of people in the US, and PNI with large gaps require surgical repair. Although biological and synthetic grafts are widely used to repair PNI with large gaps, they both can suffer from suboptimal clinical outcomes. Autografts are the gold standard treatment but are limited by availability and defect repair size, while synthetic grafts have poor biodegradability, strength, bioactivity, and functionality. Thus, the long-term objective of this proposal is to engineer grafts with enhanced large-gap nerve regeneration capabilities. Physical and chemical stimulation can enhance nerve regeneration responses, thus, incorporating these modalities into engineered grafts may address some current treatment limitations. Electrical stimulation (ES) can enhance nerve conduction, neurotrophin release, and functional recovery of nerve crush injuries, but these benefits have not been established for large-gap PNI. Chemical stimulation using 4-aminopyridine (4-AP; a potassium channel blocker) appears similar to ES in its effects on neurons and can enhance crush PNI repair, yet may act synergistically with ES. Implementing these physical and chemical cues for effective large-gap PNI repair will require surgical insertion of an electrically conductive scaffold with appropriate mechanical strength, degradation, conductivity, and pore properties. This proposal aims to deliver 4-AP and ES via novel, biodegradable, ionically conducting (IC) chitosan scaffolds and hybrid engineered nerve allografts to repair large- gap nerve defects. Bioengineered IC scaffolds with 4-AP can increase neurotrophin release in vitro and enhance myelination of large-gap PNI in vivo in early-stage repair. Preliminary studies revealed that combined application of 4-AP and ES reduced fiber capsule thickness around subcutaneously implanted scaffolds and increased in vitro neurotrophin expression compared to 4-AP or ES alone. This suggests combining 4-AP and ES improves functionality, biocompatibility, and positive immune responses. Therefore, it was hypothesized that IC scaffolds combined with chemical and electrical cues will modulate cell-material interactions to enhance axon regeneration rate and functional recovery comparable to autografts. This will be tested in three Specific Aims: 1) Develop and characterize IC scaffolds with variations in 4-AP release rate, conductivity, and biodegradation; 2) Assess human and rat Schwann cell responses to IC scaffolds with 4-AP and/or ES in vitro to model in vivo responses and future interventions; and 3) Test safety and efficacy of engineered scaffolds and allografts with 4-AP +/- ES in a critical-sized sciatic nerve defect. Engineered repair of large-gap PNI using bioactive electrical and chemical cues will broadly impact the field. These studies will bridge the knowledge gap between the complex ES- mediated cell-material interaction microenvironment and poorly studied underlying re...