Background
Peripheral neuropathy is a chronic neurological disorder characterized by inflammation, nerve damage, and impaired function. It arises owing to various factors, including traumatic nerve injury, neuropathic pain due to cancer, and diabetic neuropathy. Although conventional two-dimensional culture-based mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) demonstrate therapeutic potential for neuropathy, their regenerative efficacy and consistency are limited. Therefore, the present study proposes a nanobiology-based therapeutic strategy for neuroregenerative medicine.

Results
In this study, MSCs were preconditioned using transforming growth factor beta-3 and cultured in a three-dimensional dynamic culture system to produce highly functional mechanochemically primed regenerative EVs (MCR-EVs). In an ex vivo organotypic spinal cord slice injury model with demyelination, MCR-EVs were internalized by slice-resident cells and significantly attenuated cell death compared to conventional 2D-EVs (Con-EVs). MCR-EVs also promoted the recovery of axonal integrity and pro-survival signaling in injured spinal cord slices, as indicated by increased neurofilament-M immunoreactivity, restored protein kinase B phosphorylation, and growth-associated protein 43 expression. MCR-EVs exhibited enhanced therapeutic effects compared with Con-EVs in a mouse model of peripheral neuropathy induced by chronic constriction injury. The MCR-EV-treated group exhibited downregulated expression of proinflammatory genes, including tumor necrosis factor-α, interleukin-1β, interleukin-6, and cyclooxygenase-2, accompanied by inhibition of microglial activation and cell death. Additionally, the axonal structure was restored, as demonstrated by increased expression of neurofilament heavy chain, neuron-specific class III β-tubulin, and neuronal nitric oxide synthase. The MCR-EV-treated group also exhibited increased expression of Schwann cell-related markers (S100β, myelin basic protein, and myelin-associated glycoprotein), maintenance of neuromuscular structure, and upregulated platelet endothelial cell adhesion molecule 1 expression levels. Moreover, in MCR-EVs, small RNA sequencing confirmed the presence of several miRNAs that are potentially associated with nerve regeneration, inflammation, and pain modulation.

Conclusions
These results suggest that MCR-EVs contribute to the recovery of myelinated axons and regeneration of peripheral tissues, thus protecting endothelial cell components. MCR-EVs improved therapeutic outcomes compared to current Con-EV treatments and may promote peripheral neuropathic recovery by modulating anti-inflammatory and nerve regeneration pathways.