| 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 2ã€4ã€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. |
