Rationale: Peripheral nerves are unique in their remarkable elasticity. Schwann cells (SCs), important components of the peripheral nervous system (PNS), are constantly subjected to physiological and mechanical stresses from dynamic stretching and compression forces during movement. So far, it is not clear if SCs sense and respond to mechanical signals. It is also unknown whether mechanical stimuli can interfere with the intercellular communications between neurons and SCs, and what role extracellular vesicles (EVs) play in this process. The present study aimed to examine the effect of mechanical stimuli on the EV-mediated intercellular communication between neurons and SCs, explore their effect on axonal regeneration, and investigate the underlying mechanism. Methods: Purified SCs were stimulated using a magnetic force-based mechanical stimulation (MS) system and EVs were purified from mechanically stimulated SCs (MS-SCs-EVs) and non-stimulated SCs (SCs-EVs). The effect of MS-SCs-EVs on axonal elongation was examined in vitro and in vivo. High throughput miRNA sequencing was performed to compare the differential miRNA profiles between MS-SCs-EVs and SCs-EVs. The functional role of differentially expressed miRNAs on neurite extension in MS-SCs-EVs was examined. Also, the putative target genes of differentially expressed miRNAs in MS-SCs-EVs were predicted by bioinformatics tools, and the regulatory effect of those miRNAs on putative target genes was validated both in vitro and in vivo. Results: The MS-SCs-EVs showed an average size of 137.52±1.77 nm, and could be internalized by dorsal root ganglion (DRG) neurons. Compared to SCs-EVs, MS-SCs-EVs showed a stronger ability to enhance neurite outgrowth in vitro and nerve regeneration in vivo. High throughput miRNA sequencing identified a number of differentially expressed miRNAs in MS-SCs-EVs. Further analysis of those EV-miRNAs demonstrated that miR-23b-3p played a predominant role in MS-SCs-EVs since its deprivation abolished their enhanced axonal elongation. Furthermore, we identified neuropilin 1 (Nrp1) in neurons as the target gene of miR-23b-3p in MS-SCs-EVs. This observation was supported by the evidence that miR-23b-3p could decrease Nrp1-3'-UTR-WT luciferase activity in vitro and down-regulate Nrp1 expression in neurons. Conclusion: Our findings suggested that mechanical stimuli are capable of modulating the intercellular communication between neurons and SCs by altering miRNA composition in MS-SCs-EVs. Transfer of miR-23b-3p by MS-SCs-EVs from mechanically stimulated SCs to neurons decreased neuronal Nrp1 expression, which was responsible, at least in part, for the beneficial effect of MS-SCs-EVs on axonal regeneration. Our results highlighted the potential therapeutic value of MS-SCs-EVs and miR-23b-3p-enriched EVs in peripheral nerve injury repair.

Rationale: Local hypoxia is a challenge for fabrication of cellular grafts and treatment of peripheral nerve injury. In our previous studies, we demonstrated that perfluorotributylamine (PFTBA) could provide short term oxygen supply to Schwann cells (SCs) and counteract the detrimental effects of hypoxia on SCs during the early stages of nerve injury. However, the quick release of oxygen in PFTBA compromised its ability to counteract hypoxia over an extended time, limiting its performance in peripheral nerve injury. Methods: In this study, PFTBA-based oxygen carrier systems were prepared through coaxial electrospinning to prolong the time course of oxygen release. Core-shell structures were fabricated, optimized, and the oxygen kinetics of PFTBA-enriched core-shell fibers evaluated. The effect of core-shells on the survival and function of SCs was examined in both 2D and 3D systems as well as in vivo. The system was used to bridge large sciatic nerve defects in rats. Results: PFTBA core-shell fibers provided high levels of oxygen to SCs in vitro, enhancing their survival, and increasing NGF, BDNF, and VEGF expression in 2D and 3D culture systems under hypoxic condition. In vivo analysis showed that the majority of GFP-expressing SCs in the PFTBA conduit remained viable 14 days post-implantation. We found that axons in PFTBA oxygen carrier scaffold improved axonal regeneration, remyelination, and recovery. Conclusion: A synthetic oxygen carrier in core-shell fibers was fabricated by the coaxial electrospinning technique and was capable of enhancing SC survival and nerve regeneration by prolonged oxygen supply. These findings provide a new strategy for fabricating cellular scaffolds to achieve regeneration in peripheral nerve injury treatment and other aerobic tissue injuries.