[Biocompatibility of silk fibroin nanofibers scaffold with olfactory ensheathing cells].
OBJECTIVE
To investigate the biocompatibility 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 x 10(4) cells/cm2) were seeded on the poly-l-lysine (control group) and the silk fibroin nanofibers (experimental group) coated coverslips 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 viability of OECs was examined by live/dead assay. The proliferation 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 solid 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 > 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 viability was observed in both groups 3 days after culture. There was no significant difference on cell viability between two groups. The proliferation 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 < 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.


CONCLUSIONS
Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI.
Qian
Yuqiang
Y
Department of Orthopedics, Second Affiliated Hospital, Soochow University, Suzhou Jiangsu 215004, PR China.
Shen
Yixin
Y
Lu
Zhengfeng
Z
Fan
Zhihai
Z
Liu
Tun
T
Zhang
Junke
J
Zhang
Feng
F
D000818:Animals; D001672:Biocompatible Materials; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005260:Female; D005349:Fibroins; D008297:Male; D008422:Materials Testing; D057139:Nanofibers; D009830:Olfactory Bulb / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds
D001672:Biocompatible Materials; D005349:Fibroins