Histocompatibility of new nano-scaffolds for spinal tissue engineering

doi:10.3969/j.issn.2095-4344.2013.21.008

Abstract

Abstract:

BACKGROUND: The properties of tissue engineering materials have been improved obviously with the rise and development of nanotechnology, the prospect of which is broad in spinal cord tissue engineering.
OBJECTIVE: To observe the histocompatibility of new nano-scaffolds for spinal tissue engineering.
METHODS: Electrospun aligned and randomly oriented nano?brous scaffolds were made of collagen. Spinal cord derived neural progenitor cells from Sprague-Dawley rats were cultured and identified, and then the cells were cultured on the aligned and randomly oriented collagen nano?brous scaffolds. Neural stem cells under normal culture served as controls. Scanning electron microscope was used to observe the cells’ adhesion and generation on the scaffold surface, and cell histocompatibility was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Histocompatibility and degradation of nano?brous scaffolds in vivo were determined through histological observation. The survival and movement of neural stem cells in the body were determined by immunohistochemistry experiments.
RESULTS AND CONCLUSION: Superficial morphous of electrospun aligned and randomly oriented collagen nano?brous scaffolds were in accordence with contrivable requisition. The cell adhesion and proliferation on the surface of the collagen nano?brous scaffolds was perfect. Results from 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay showed that the cellular compatibility of nano-scaffolds was good. Nano?brous scaffolds degraded well and their histocompatibility in vivo was good. The 5-bromo-2'-deoxyuridine-labeled cells in vivo survived and migrated well. Histocompatibility of new nano-scaffolds for tissue engineering is satisfactory.

Key words: biomaterials, nanobiomaterials, spinal cord, tissue engineering, nano?brous scaffold, nanofibrous membrane, histocompatibility, neural stem cells, National Natural Science Foundation of China

CLC Number:

R318

Zhou Ji-hui1, Yao Meng, Wang Yan-song, Sui Fu-ge, Liu Yu-gang, Zhao Cong-ran,Tian Fei-peng. Histocompatibility of new nano-scaffolds for spinal tissue engineering[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.21.008.

Figures/Tables 5

2.1 定向及非定向排列纳米纤维膜的表征及性能测试结果扫描电镜见定向纳米纤维膜纤维排列方向大体一致，经专用图像计算系统(哈尔滨林业大学提供)测试，纤维直径为(694±157) nm；非定向纳米纤维膜纤维排列方向纵横交错，纤维直径为(785±177) nm。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%。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)%。 2.2 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100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。双标免疫荧光组织化学鉴定结果显示，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BrdU，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。 2.3 定向及非定向排列纳米支架的体内外生物相容性实验结果 MTT实验结果：第1天，与定向及非定向纳米纤维膜共培养的神经干细胞吸光度值低于对照组，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吸光度值均明显高于对照组，定向膜组吸光度值明显高于非定向膜组。经统计学分析，自第3天起，3组之间吸光度值有明显差异：定向膜组明显大于非定向膜组及对照组(P < 0.05)，非定向膜组明显大于对照组(P < 0.05)，见表1。"

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