The Study On Differentiation Of Msenchymal Stem Cells Into Neuron-Like Cells On PHBV Nanofibers Films

Objective:To investigate the impact of nanomaterials PHBV on attached、 proliferation and differentiation of BMSCs into neuron-like cells, and to provide a new basis for the treatment of spinal cord injury with combined transplantation contains BMSCs and PHBV.Methods:The BMSCs were got from the SD rats marrow after their death immediately through cervical amputation.aligned and random-oriented PHBV nanofibrous scaffolds (A-NF and R-NF) were fabricated through electrospinning technique. Scanning electron microscope was used to observe the surface morphology changes of the cells. Cell proliferation was tested by CCK-8 assay at 1.4 and 7 days after cell seeding. After the cells adhere to the membrane, we added 200uL retinoic acid (RA) as the proliferation and differentiation-inducing factor into each well. Then immunofluorescent analysis was employed to detect the expression of sternness markers in cells cultured in experiment and control groups after 2、4、6 days. The cells were fixed with 0.5% formaldehyde containing 0.2% Triton X-100 in PBS buffer at pH 7.4 for 5min at room temperature, rinsed once with PBS, fixed again in 4% formaldehyde in PBS for 20min, and rinsed three times with PBS. Then closed in 1% bovine serum albumin(BSA) at 37℃ for 1h, the samples were incubated with rabbit anti-rat nestin, glial fibrillary acidic protein (GFAP) and Microtubule-associated protein-2 (MAP-2) primary antibodies at 4℃ for at least 12 h to detect differentiated neural cells. After washing with PBS, the samples were incubated with the appropriate secondary antibody (goat anti-rabbit Alexa-Fluor 488) for 1h at 37℃ while protected from light. Then, the samples were washed twice with PBS, the nuclei were detected by Hoechst 33342 for 30min while protected from light, and images were obtained using a Revolution XD confocal laser scanning microscope.Results:The CCK-8 test indicated that the BMSCs attached and proliferated more favorably on R-NF than A-NF. Day 1,the cells on the A-NF and R-NF slightly more than on the film, The difference between the slide and the A-NF was no significant(P >0.05). Day 4 and 7,the proliferation of the cells on A-NF and R-NF was statistically significant (P<0.05). The PHBV groups and control group were statistically significant difference (P<0.05).The aligned of nanofibers showed a distinct effect on cell morphology with guiding cell skeleton extension. Neural stem cells were detected with immunostaining for Nestin after 2 days of adding in the RA medium, neuron cells and neuron glial cells were detected with immunostaining for MAP-2 and GFAP after 4 days in experiment group While the same appear at 4 and 6 days in control group.Conclusion:The BMSCs attached and proliferated more favorably on R-NF than A-NF and the A-NF could guide the cytoskeleton extends significantly. PHBV nanofiber membrane can promote the differentiation of BMSCs into neuron-like cells. Therefore, we believe the aligned nanofiber scaffold may have an advantage in the nerve tissue engineering.