| Objective:To establish the methods to get high activated, purified and adequate Schwann cells (schwann cells, SCs),and provide sufficient seed cells for peripheral nerve repairment. To find a material which support SCs adhesion and proliferation and to provide a three diamensions bioscaffold,which has ideal biocompatibility and can guide the growth of SCs.Methods:1. Six Sprague‐Dawley rats(5 days old)were divided into sciatic nerve group (control group) and DRG (dorsal root ganglia, DRG) group (experime‐ ntal group,then the sciatic nerves and DRGs were harvested respectively. They were digested by co‐enzyme and dispersed by medium containing serum. Freshly isolated SCs from rats were cultured, purified and subcultured. 1st generation of SCs were choosed. Growth curve of SCs were drawed by the counting method and the proliferation of SCs was detected by MTT assay in 8 days in culture. Immunocyto‐ chemistry of Anti‐S100 was used to detected the purity of SCs. ELISA (enzyme‐linked immunosorbent assay, ELISA) was used to detected BDNF(brain‐derived neurotrophic factor, BDNF) concentration. 2. Silk fibrion nanofibers, which diameter is 1000nm, were prepared by using electrospinning techniques. 3. Freshly isolated SCs from 5 days SD rats's DRG were purified by the modifided differential adherent velocity method and cultured. 1st generation of SCs were seeded on the poly‐l‐ lysine(PLL group) and tussah silk fibrioin nanofibers(TSF group).The coverslips were coated in Petri dish. At different time points, the morphological features, growth and adhesion of cells were observed using phase contrast inverted microscopy. Immunocytochemistry of Anti‐S100 was used to identitied the purity of SCs in two group. The proliferation of SCs was determined by MTT assay. The cytoxicity of TSF was evaluated. ELISA was used to detected BDNF concentration.Results: 1. Each rat could get 36～43 DRGs. Single cells were counted after digestion. A total of(7.5±0.6)×10~6 SCs could be obtained in experimental group higher than (3.5±0.4)×10~6 SCs in control group .There was significant difference between two groups (P< 0.05). SCs reach logarithm proliferation phase in 3 days. The cells's quantity and A value of two groups all show upward trend along of time proceed. The number and A value of experimental group is higher than control group. There were significant difference between two groups(P<0.05). Immunocytochemist‐ ry of Anti‐S‐100 detects SCs. The purity of experimental group (92.08%±3.45% ) is higher than control group (77.50%±3.57%) . The difference is significant (P<0.05). Elisa results showed that the density of BDNF at 3d and 5d in experimental group was higher than control group. The difference is significant (P<0.05). 2. The morphology of SCs on the TSF group was better than PLL group. The cells distributed along the fibers, and the cell protrusions were in the same as that of the fibers. Immunofluorescence showed that the purity was 91.25%±2.57% in the PLL group and 91.51%±1.34% in the TSF group 3 days after subculture(P>0.05). SCs still maintained the characteristic phenotype in TSF nanofibers. The axons's length and diversity of SCs in TSF group was higher than PLL group. The A value at1,3,5,7,9 days was detected by MTT assay and didn't have significant differences by contrast in two growps(P>0.05). The relative growth rates were above 90% at different time points. The cytotoxicity of the material was grade 1 and nonevenomous according to GB/T 16886 standard. Elisa results showed that the density of BDNF at 3 days and 5 days in SF group was similar to PLL group. No significant difference (P>0.05).Conclusion: The sufficient amount, higher purity and viability of SCs which drawing from DRG can meet the needs of studies on peripheral nerve repairment. Three diamensions scaffold which was made of TSF nanofibers have good compatibility and nonvenomous. It's a promising tissue engineered scaffold for the repair of peripheral nerve injury. |
PDF Full Text Request