Nerve Injury-on-a-Chip Uncovers Extracellular Matrix and Mitochondrial Roles in Axonal Regeneration.

Peripheral nerve regeneration depends on interactions among extracellular matrix cues, neuronal bioenergetics, and therapeutic signal presentation, yet progress is limited by the lack of physiologically relevant quantitative in vitro models to evaluate biomaterials and mitochondria-targeted therapies. Here, we present a nerve injury-on-a-chip (NI-Chip) that integrates dorsal root ganglion explants, aligned nanofibers, compartmentalized microfluidics, and precisely controlled axotomy to enable real-time, quantitative analysis of axonal regeneration dynamics. Extracellular matrix composition significantly influences axonal regeneration velocity, with laminin-coated nanofibers supporting faster growth than fibronectin-coated ones. Pharmacological promotion of mitochondrial transport using the small-molecule compound M1 enhances axonal regeneration in a dose-dependent manner, revealing that M1 increases mitochondrial motility within regenerating axons, with a selective enhancement of retrograde transport. Furthermore, M1 treatment remains effective in older sensory neurons, highlighting mitochondrial dynamics as an age-independent target for regeneration. Leveraging the compartmentalized design of the NI-Chip, we demonstrate that localized axonal delivery of M1 enhances regeneration, and sustained delivery via M1-loaded nanofibers further promotes axonal regrowth. This work establishes the NI-Chip as a versatile platform for dissecting the interplay between biomaterials, mitochondrial dynamics, and axonal regeneration, and provides a foundation for accelerating the development of clinically translatable therapies for peripheral nerve repair. STATEMENT OF SIGNIFICANCE: Peripheral nerve repair remains challenging due to limited functional recovery and a lack of reliable in vitro models to evaluate biomaterials and therapies. This study introduces a nerve injury-on-a-chip (NI-Chip) that combines aligned nanofibers, controlled injury, and real-time imaging to quantitatively measure axonal regeneration. Using this platform, we show that extracellular matrix coatings and mitochondrial fusion modulation distinctly regulate regeneration dynamics, and that sustained drug delivery from nanofibers further enhances repair. Unlike conventional assays that measure static neurite growth, this system enables dynamic and mechanistic evaluation of regeneration. This work provides a versatile tool for designing and optimizing biomaterials and therapeutic strategies for peripheral nerve repair.