New-type nano-scaffolds and neural stem cell apoptosis

doi:10.3969/j.issn.2095-4344.2013.03.007

Abstract

Abstract:

BACKGROUND: As a good material for spinal cord tissue engineering scaffold, collagen is conducive to adhesion and growth of nerve cells and nerve fibers. But it needs to be improved in the preparation process because of its poor mechanical properties, not only to improve its basic performance, but also to have a positive impact on the biological behavior of the seed cells.
OBJECTIVE: To observe the properties of new nano-scaffolds for tissue engineering and to detect the changes of neural stem cell apoptosis and related gene expression affected by the nano-scaffold.
METHODS: Aligned and randomly oriented nanofibrous scaffolds were made of collagen by using electrospinning technology. Superficial morphous, porosity, mechanical property, swelling coefficient, degradation disposition were tested. Spinal cord derived neural progenitor cells were cultured and identified, and then the cells were cultured on aligned and randomly oriented collagen nanofibrous scaffolds. Cells cultured under normal conditions served as control group. The changes of neural stem cell apoptosis and related gene expression were tested.
RESULTS AND CONCLUSION: Superficial morphous of electrospun aligned and randomly oriented collagen nanofibrous scaffolds were in accordance with contrivable requisition, their porosities were supernal, mechanical properties were fine, swelling coefficients were satisfactory. Compared with the control group, apoptosis rate of cells cultured on aligned and randomly oriented collagen nanofibrous scaffolds decreased significantly (P < 0.05), expression level of apoptosis-related gene bcl-2 significantly increased, bax and Caspase-3 significantly decreased. There was no significant difference between the cells on aligned and randomly oriented collagen nanofibrous scaffolds. These findings indicate that properties and histocompatibility of new nano-scaffolds for tissue engineering are satisfactory, which can inhibit and regulate cell apoptosis from the level of gene expression.

Key words: biomaterials, nano-biological materials, collagen, nano-scaffolds, fibrous membrane, tissue engineered scaffolds, proliferation, apoptosis, differentiation, gene, neural stem cells, oriented arrangement, National Natural Science Foundation of China, biomaterial photographs-containing paper

CLC Number:

R318

Zhou Ji-hui, Yao Meng, Wang Yan-song, Liu Yu-gang, Sui Fu-ge, Zhao Cong-ran, Tian Fei-peng, He Xiao-feng. New-type nano-scaffolds and neural stem cell apoptosis[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(3): 419-426.

Figures/Tables 3

2.2 纤维定向及非定向胶原纳米纤维膜的孔隙率和溶胀系数定向及非定向纳米纤维膜的孔隙率均大于70%，分别为(79.31±2.87)%及(84.53±1.65)%，溶胀系数分别为386.11%和334.30%。 2.3 纤维定向及非定向胶原纳米纤维支架的机械力学性能定向纳米纤维膜的弹性模量、拉伸强度及断裂伸长率分别为(1.93±0.82) MPa、(31.02±3.22) MPa、(5.90±0.85)%；非定向纳米纤维膜的弹性模量、拉伸强度及断裂伸长率分别为(1.73±0.33) MPa、(14.64±3.23) MPa、(5.23±3.04)%。 2.4 神经干细胞的培养和鉴定结果接种后的神经干细胞在显微镜下多为圆形，原代培养1 d后，多数细胞悬浮并单个存在，细胞边缘及细胞核折光性好，可见少量细胞聚集形成不规则团块，细胞碎片及活性不佳的细胞沉于瓶底，少量细胞贴壁。原代培养2 d后，仍以单个细胞为主，细胞聚集形成较大的不规则的细胞团块，并非神经球，需将其吹散。原代培养二三天后，细胞分裂增殖形成神经球，大小不等，最初由几个细胞组成，随生长逐渐增大，细胞数逐渐增多，神经球悬浮于培养液中，多呈椭圆形，边界清楚，在高倍显微镜下观察，可见神经球中部细胞形态良好，胞核及细胞边缘轮廓清晰，折光性好，若细胞数目过多导致中心部细胞营养供应不良则神经球中部细胞形态欠佳，胞核及细胞边缘轮廓模糊且折光性差。原代培养第7天神经球已增大至包含一百乃至数百个细胞，部分已增大至肉眼可见，神经球中心细胞出现营养不良表现，神经球仍以悬浮为主，部分过大的神经球贴壁，在瓶壁可见细胞碎片，梭形帖壁的成纤维细胞，此时应进行机械传并更换培养瓶，传代后细胞的增殖速度与原代细胞基本相同。双标免疫荧光组化鉴定结果显示，培养获得的脊髓源性神经干细胞均表达神经干细胞的特有标志物巢蛋白，在荧光显微镜下，标记细胞为圆形，显明亮的绿色荧光。同时表达5-溴脱氧尿嘧啶核苷，在荧光显微镜下，标记细胞为圆形，显明亮的橙红荧光。荧光合成图像可见两种荧光同时表达。 2.5 MTT实验检测定向及非定向纳米纤维膜与神经干细胞的生物相容性实验结果共培养第1天，与定向及非定向纳米纤维膜共培养的神经干细胞A值低于对照组，自第3天起，与定向及非定向纳米纤维膜共培养的神经干细胞A值均明显高于对照组，并以定向纳米纤维膜组A值明显高于非定向纳米纤维膜组。经统计学分析，自第3天起，3组之间A值差异有显著性意义(P < 0.05)；定向纳米纤维膜组明显大于非定向纳米纤维膜组及对照组(P < 0.05)，非定向纳米纤维膜组明显大于对照组(P < 0.05)，见表1。"

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