Objective To establ ish the methods to get high activity, high purity, and adequate Schwann cells (SCs), and to provide sufficient seed cells for the peripheral nerve repair. Methods Six 5-day-old, male or female, Sprague Dawley rats were selected and the sciatic nerve (control group) and dorsal root gangl ion (DRG) (ex perimental group) were harvested.Then the sciatic nerves and DRG were digested by co-enzyme and dispersed by medium containing serum to isolate SCs. Freshlyisolated SCs from rats were cultured, purified and subcultured. The 1st generation of SCs were chosen to draw the growth curve of SCs by the counting method and to detect the prol iferation of SCs by MTT assay at 8 days of culture, the purity of SCs by immunocytochemistry of anti-S-100 and the brain-derived neurotrophic factor (BDNF) concentration by ELISA. Results A total of 36-43 DRGs could be obtained in each rat. The number of obtained single SC in experimental group [(7.5 ± 0.6）× 106] was significantly higher than that in control group [(3.5 ± 0.4）× 106 ] (t=13.175, P=0.000). SCs reached logarithm prol iferation phase at 3 days. With time, the cell number and the prol iferation absorbance (A) value of 2 groups all showed upward trend. The number and A value of experimental group were significantly higher than those of control group (P lt; 0.05). The SCs purity of experimental group (92.08% ± 3.45%) was significantly higher than that of control group (77.50% ± 3.57%) (t=6.689, P=0.001).The concentrations of BDNF at 3 days and 5 days in experimental group were significantly higher than those of control group (P lt; 0.05). Conclusion The sufficient amount, high purity, and viabil ity of SCs from DRGs can meet the needs of studies on peripheral nerve repairment.
Objective To investigate the effects of chondroitinase ABC (ChABC) on axonal myelination and glial scar after spinal cord injury (SCI) in rats. Methods Seventy-two adult male Sprague Dawley rats were randomly assigned into ChABC treatment group (group A), saline treatment group (group B), and sham operation group (group C), 24 rats in each group. In groups A and B, the SCI model was established with modified Allen’s method and then the rats of groups A and B were administrated by subarachnoid injection of 6 μL ChABC (1 U/mL) and saline respectively at 1 hour after injury and every day for 1 week; the rats of group C served as control, which canal was opened without damage to spinal cord. At 1, 7, 14, and 28 days after operation, the locomotor functions were evaluated according to the Basso-Beattie-Bresnahan (BBB) score scale; and the spinal cord samples were harvested for HE staining, Nissl staining, and immunohistochemistry analysis to detect the change of myelin basic protein (MBP), growth associated protein 43 (GAP-43), and glial fibrillary acidic protein (GFAP) of the injured spinal cord. Results At different time points, the BBB score of group C was significantly higher than those of groups A and B (P lt; 0.05), and the BBB score of group A was significantly better than that of group B at 14 and 28 days after operation (P lt; 0.05). HE staining and Nissl staining showed that the morphous and the neuron number of the remainant injured spinal cord in group A were better than those in group B. The integral absorbance (IA) values of MBP and GAP-43 and the positive area of GFAP after SCI in groups A and B were significantly higher than those in group C at different time points (P lt; 0.05), and the IA values of MBP and GAP-43 were significantly higher in group A than those in group B at 7, 14, and 28 days after operation (P lt; 0.05), but the positive area of GFAP was significantly smaller in group A than that in group B (P lt; 0.05). Conclusion The ChABC can effectively improve the microenvironment of the injured spinal cord of rats, enhance the expressions of MBP and GAP-43, and inhibit the expression of GFAP, which promotes the axonal regeneration and myelination, attenuate glial scar formation, and promote the recovery of nerve function.
Objective To investigate the biocompatibil ity of silk fibroin nanofibers scaffold with olfactory ensheathing cells (OECs) and to provide an ideal tissue engineered scaffold for the repair of spinal cord injury (SCI). Methods Silk fibroin nanofibers were prepared using electrospinning techniques and were observed by scanning electron microscope (SEM). Freshly isolated OECs from SD rats purified by the modified differential adherent velocity method were cultured. The cells at passage 1 (1 × 104 cells/cm2) were seeded on the poly-l-lysine (control group) and the silk fibroin nanofibers (experimental group) coated coversl ips in Petri dish. At desired time points, the morphological features, growth,and adhesion of the cells were observed using phase contrast inverted microscopy. The OECs were identified by the nerve growth factor receptor p75 (NGFR p75) immunofluorescence staining. The viabil ity of OECs was examined by l ive/dead assay. The prol iferation of OECs was examined by MTT assay. The cytotoxicity of the nanofibers was evaluated. Results The SEM micrographs showed that the nanofibers had a smooth surface with sol id voids among the fibers, interconnecting a porous network, constituted a fibriform three dimensional structure and the average diameter of the fibers was about (260 ± 84) nm. The morphology of OECs on the experimental group was similar to the cell morphology on the control group, the cells distributed along the fibers, and the directions of the cell protrusions were in the same as that of the fibers. Fluorescence microscopy showed that the purity of OECs was 74.21% ± 2.48% in the experimental group and 79.05% ± 2.52% in the control group 5 days after culture. There was no significant difference on cell purity between two groups (P gt; 0.05). The OECs in the experimental group stained positive for NGFR p75 compared to the control group, indicating that the cells in the experimental group still maintained the OECs characteristic phenotype. Live/dead staining showed that high viabil ity was observed in both groups 3 days after culture. There was no significant difference on cell viabil ity between two groups. The prol iferation activity at 1, 3, 5, 7, and 10 days was examined by MTT assay. The absorbency values of the control group and the experimental group had significant differences 3 and 5 days after culture (P lt; 0.05). The relative growth rates were 95.11%, 90.35%, 92.63%, 94.12%, and 94.81%. The cytotoxicity of the material was grade 1 and nonvenomous according to GB/T 16886 standard. Conclusion Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI.