Peripheral nerve injury requires optimal conditions in both macro-environment and microenvironment for promotion of axonal regeneration. However, most repair strategies of traumatic peripheral nerve injury often lead to dissatisfying results in clinical outcome. Though various strategies have been carried out to improve the macro-environment, the underlying molecular mechanism of axon regeneration in the microenvironment provided by nerve conduit remains unclear. In this study, we evaluate the effects of from adipose-derived mesenchymal stem cells (adMSCs) originating exosomes with respect to sciatic nerve regeneration and neurite growth. Molecular and immunohistochemical techniques were used to investigate the presence of characteristic exosome markers. A co-culture system was established to determine the effect of exosomes on neurite elongation in vitro. The in vivo walking behaviour of rats was evaluated by footprint analysis, and the nerve regeneration was assessed by immunocytochemistry. adMSCs secrete nano-vesicles known as exosomes, which increase neurite outgrowth in vitro and enhance regeneration after sciatic nerve injury in vivo. Furthermore, we showed the presence of neural growth factors transcripts in adMSC exosomes for the first time. Our results demonstrate that exosomes, constitutively produced by adMSCs, are involved in peripheral nerve regeneration and have the potential to be utilised as a therapeutic tool for effective tissue-engineered nerves.

No abstract

Adipose-derived stem cells (AScs) can easily be obtained and expanded in vitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted AScs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated AScs on nerve regeneration is currently only partially understood. In the present study, undifferentiated AScs and cutaneous primary afferent dorsal root ganglion (dRG) neurons were co-cultured in order to investigate their interaction. AScs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co-culture system with dRG cells was established to determine the effect of undifferentiated pluripotent AScs on neurite elongation. Neurite outgrowth, length and number was examined in the co-culture and compared with single-culture cells and cells stimulated with nerve growth factor (NGF). In ASc cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers cd44, cd73 and cd90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASc differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of dRG neurons was enhanced following co-culture with AScs, resulting in increased neurite length after 24 h of cultivation. Furthermore, neurite outgrowth of dRG neurons was directed towards the undifferentiated ASc and direct cell-to-cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASc optimizing tissue-engineered constructs may be promising for peripheral nerve repair.