Effect of Diameters of Silk Fibroin Nanofibers on the Growth and Migration of Olfactory Ensheathing Cells
|Keywords||Olfactory ensheathing cells Silk fibroin Nanofiber Cell growth Cell migration Tissue engineering|
Objective: To investigate the growth and migration of OECs on diameters of silk fibroin nanofibers (SFS), which would provide basic theory for repairing spinal cord injury(SCI).Methods: (1) SFS were prepared by electrospinning technique, then they were observed by scanning electron microscope (SEM). (2) The OECs were isolated and purified by the modified differential adherent velocity method(.3)The purified OECs were seeded on the poly-L-lysine (control group) and (400nm,1200nm)SFS (experiment group)coated coverslips in Petri dish.(4) At desired time points, the morphological features, growth, distribution and adhesion of the cells were detected using phase contrast inverted microscopy. The phenotype of OECs was confirmed by the presence of cell-specific markers such as nerve growth factor receptor p75 (NGFR p75) and glial fibrillary acidic protein (GFAP). The proliferation of OECs were evaluated by Thiazoyl Blue Tetrazolium Bromide(MTT) assay. The viability of OECs was examined by live/dead and flow cytometric assay. The migration tracks, turning behavior, migration distances, migration speeds and forward migration indices of OECs were observed and calculated by Leica AF6000.Results: (1) The SEM showed that the average diameter of the fibers was about (395±3)nm、(1210±7)nm and the nanofibers constituted a three-dimensional structure with porous network and smooth surface. The morphology of OECs on the SFS group was similar to that on the poly-L-lysine (PLL) group. (2) Fluorescence microscopy showed the OECs in the experimental group stained positive for both NGFR p75 and GFAP similar to the control group, indicating that the cells still maintained the OECs characteristic phenotype in the experimental group 4 days after culture. (3) In addition, on the days 4, MTT assay showed that there was significant difference between the 1200nmSFS and the control group (P<0.05); on the days7, there was significant difference between the experiment group and the control group on the proliferation rate (P<0.05); what’s more, the proliferation rate was significant different between the 400nmSFS and 1200nmSFS (P<0.05). (4) However, On the days 4 and 7, live/dead staining assay showed that the cells form in the experiment group was similar to the control group, the survival rate was higher, the death rate was no significant difference between the two groups. (5)The flow cytometric assay showed that there was no significant difference between the two groups in the apoptosis activity on the day 4(P>0.05). (6) Dynamic observation of living cells demonstrated that there was no obvious difference between the experimental group and the control group on the migrating speed, efficiency and distance (P>0.05).Conclusion: Diameters of SFS could support and guide the migration of OECs, the transplantation of OECs combined with SFS could be a novel method for repairing spinal cord injury.