Biomimetic nerve guidance conduit containing engineered exosomes of adipose-derived stem cells promotes peripheral nerve regeneration

Background Efficient and stable delivery of neurotrophic factors (NTFs) is crucial to provide suitable microenvironment for peripheral nerve regeneration. Neurotrophin-3 (NT-3) is an important NTF during peripheral nerve regeneration which is scarce in the first few weeks of nerve defect. Exosomes are nanovesicles and have been served as promising candidate for biocarrier. In this work, NT-3 mRNA was encapsulated in adipose-derived stem cell (ADSC)-derived exosomes (Exo.sup.NT-3). These engineered exosomes were applied as NT-3 mRNA carrier and then were loaded in nerve guidance conduit (Exo.sup.NT-3-NGC) to bridge rat sciatic nerve defect. Method NT-3 mRNA was encapsulated in exosomes by forcedly expression of NT-3 mRNA in the donor ADSCs. Exo.sup.NT-3 were co-cultured with SCs in vitro; after 24 h of culture, the efficiency of NT-3 mRNA delivery was evaluated by qPCR, western blotting and ELISA. Then, Exo.sup.NT-3 were loaded in alginate hydrogel to construct the nerve guidance conduits (Exo.sup.NT-3-NGC). Exo.sup.NT-3-NGC were implanted in vivo to reconstruct 10 mm rat sciatic nerve defect. The expression of NT-3 was measured 2 weeks after the implantation operation. The sciatic nerve functional index (SFI) was examined at 2 and 8 weeks after the operation. Moreover, the therapeutic effect of Exo.sup.NT-3-NGC was also evaluated by morphology assay, immunofluorescence staining of regenerated nerves, function evaluation of gastrocnemius muscles after 8 weeks of implantation. Results The engineered exosomes could deliver NT-3 mRNA to the recipient cells efficiently and translated into functional protein. The constructed NGC could realize stable release of exosomes at least for 2 weeks. After NGC implantation in vivo, Exo.sup.NT-3-NGC group significantly promote nerve regeneration and improve the function recovery of gastrocnemius muscles compared with control exosomes (Exo.sup.empty-NGC) group. Conclusion In this work, NGC was constructed to allow exosome-mediated NT-3 mRNA delivery. After Exo.sup.NT-3-NGC implantation in vivo, the level of NT-3 could restore which enhance the nerve regeneration. Our study provide a potential approach to improve nerve regeneration. Keywords: Peripheral nerve, Regeneration, Exosomes, Neurotrophin-3, Nerve guidance conduit