Dose-effect relationship and molecular mechanism by which BMSC-derived exosomes promote peripheral nerve regeneration after crush injury

BACKGROUND: The development of new treatment strategies to improve peripheral nerve repair after injury, especially those that accelerate axonal nerve regeneration, is very important. The aim of this study is to elucidate the molecular mechanisms of how bone marrow stromal cell (BMSC)-derived exosomes (EXOs) participate in peripheral nerve regeneration and whether the regenerative effect of EXOs is correlated with dose. METHOD: BMSCs were transfected with or without an siRNA targeting Ago2 (SiAgo2). EXOs extracted from the BMSCs were administered to dorsal root ganglion (DRG) neurons in vitro. After 48 h of culture, the neurite length was measured. Moreover, EXOs at four different doses were injected into the gastrocnemius muscles of rats with sciatic nerve crush injury. The sciatic nerve functional index (SFI) and latency of thermal pain (LTP) of the hind leg sciatic nerve were measured before the operation and at 7, 14, 21, and 28 days after the operation. Then, the number and diameter of the regenerated fibers in the injured distal sciatic nerve were quantified. Seven genes associated with nerve regeneration were investigated by qRT-PCR in DRG neurons extracted from rats 7 days after the sciatic nerve crush. RESULTS: We showed that after 48 h of culture, the mean number of neurites and the length of cultured DRG neurons in the SiAgo2-BMSC-EXO and SiAgo2-BMSC groups were smaller than that in the untreated and siRNA control groups. The average number and diameter of regenerated axons, LTP, and SFI in the group with 0.9 × 10(10) particles/ml EXOs were better than those in other groups, while the group that received a minimum EXO dose (0.4 × 10(10) particles/ml) was not significantly different from the PBS group. The expression of PMP22, VEGFA, NGFr, and S100b in DRGs from the EXO-treated group was significantly higher than that in the PBS control group. No significant difference was observed in the expression of HGF and Akt1 among the groups. CONCLUSIONS: These results showed that BMSC-derived EXOs can promote the regeneration of peripheral nerves and that the mechanism may involve miRNA-mediated regulation of regeneration-related genes, such as VEGFA. Finally, a dose-effect relationship between EXO treatment and nerve regeneration was shown.