Qian Yuqiang, Shen Yixin, Lu Zhengfeng, Fan Zhihai, Liu Tun, Zhang Junke, Zhang Feng
Department of Orthopedics, Second Affiliated Hospital, Soochow University, Suzhou Jiangsu 215004, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Nov;23(11):1365-70.
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).
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.
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.
Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI.
研究丝素蛋白纳米纤维支架与嗅鞘细胞（OECs）的生物相容性，为脊髓损伤（SCI）修复提供理想的组织工程支架。
采用静电纺丝技术制备丝素蛋白纳米纤维，并用扫描电子显微镜（SEM）观察。培养通过改良的差速贴壁法从SD大鼠新鲜分离并纯化的OECs。将第1代细胞（1×10⁴个细胞/cm²）接种于培养皿中涂有多聚-L-赖氨酸的盖玻片（对照组）和丝素蛋白纳米纤维的盖玻片（实验组）上。在预定时间点，使用相差倒置显微镜观察细胞的形态特征、生长和黏附情况。通过神经生长因子受体p75（NGFR p75）免疫荧光染色鉴定OECs。通过活/死检测法检测OECs的活力。通过MTT检测法检测OECs的增殖情况。评估纳米纤维的细胞毒性。
SEM显微照片显示，纳米纤维表面光滑，纤维间有实心孔隙，相互连接形成多孔网络，构成纤维状三维结构，纤维平均直径约为（260±84）nm。实验组OECs的形态与对照组细胞形态相似，细胞沿纤维分布，细胞突起方向与纤维方向一致。荧光显微镜显示，培养5天后，实验组OECs纯度为74.21%±2.48%，对照组为79.05%±2.52%。两组细胞纯度无显著差异（P>0.05）。与对照组相比，实验组OECs的NGFR p75染色呈阳性，表明实验组细胞仍保持OECs特征性表型。活/死染色显示，培养3天后两组细胞活力均较高。两组细胞活力无显著差异。通过MTT检测法检测1、3、5、7和10天的增殖活性。培养3天和5天后，对照组和实验组的吸光度值有显著差异（P<0.05）。相对生长率分别为95.11%、90.35%、92.63%、94.12%和94.81%。根据GB/T 16886标准，该材料的细胞毒性为1级，无毒。
丝素蛋白纳米纤维支架与OECs具有良好的相容性，是一种有前景的用于SCI修复的组织工程支架。
