The Effects Of Transplanting Olfactory Ensheathing Cells On Stereo Silk Nanofibers Scaffolds In Vitro And In Vivo After Spinal Cord Injury In Rats

Objectives:The therapy of spinal cord injury（SCI）by transplanting olfactory ensheathing cells（OECs）to the lesioned spinal cord has been hot research topic in recent years.The curative effect of traditional intrathecal transplantation（IT）is limited by many factors,such as the migratory ability of OECs and structural and chemical inhibitory scar within the lesioned spinal cord.Here,we demonstrated a new method of transplanting functional cells which overcomes the limitations of existing cell therapies for SCI,with a new tissue-engineered scaffold:stereo silk nanofibers scaffolds（SSNS）.We studied the effects of SSFN to OECs in vitro and in vivo and provided a new approach of transplanting OECs as a strategy for the treatment of SCI.Methods:SSNS was sliced into small pieces（0.5 mm thickness 2D-SSNS）to conduct studies in vitro.We observed cell morphology and numbers under fluorescence microscope.CCK-8,Annexin V-FITC apoptosis assay and immunocytochemistry are performed to visualize effects of SSNS on OECs proliferation,survival and differentiation.Continuous observation under fluorescence microscope was conducted after OECs were transplanted into SSNS（SSNS-OEC）.By western blot,RhoA/ROCK1 signaling pathways are analyzed in SSNS-OECs.Spinal cord of OEC-transplanted rats were made into sections to observe the number of transplanted OECs and the area of lesioned scar.BBB scales were used to evaluate motor ability after OECs transplanted.Results:Experiments in vitro reveals 2D-SSNS made OECs more irregular and tend to spread out along fiber bundles.The results of standard CCK-8 assay revealed that on the 3^rd day the absorbance value was not significantly different but on the 5^th and 7^th day,the cells cultured on SSNS had a significantly higher absorbance value compared with the control group（P<0.01）.The results of flow cytometry showed SSNS group had lower apoptosis and death rate（3.57%）compared with the the control group（5.75%）.After transplanted into SSNS,OECs showed higher level of RhoA and ROCK1while downstream proteins of ROCK1,cyclin D1 and cyclin A2,were higher while that of p27 was lower than control group.In vivo,in SSNS group,more GFP-positive OECs were revealed along both the lesioned side than IT group and the GFAP-devoid area of SSNS group was shorter than that of PBS and IT groups（P<0.05）.The locomotory assessment revealed that SSNS group had higher scores than PBS and IT group after OECs transplanted（P<0.05）.Conclusions:All in vitro or in vivo date sets reveal that SSNS supports OECs survival with a stronger proliferative ability and a lower apoptosis rate,which is highly probably associated with RhoA/ROCK1 signaling pathway.Most importantly,compared with the intrathecal injection,more OECs can be transplanted into the lesioned site via SSNS in vivo,which improved the rate of OECs’localization in SCI and provides a new approach of transplanting OECs as a strategy for the treatment of SCI.