摘要
背景:脊髓组织工程的基本思路是将体外分离培养的种子细胞种植到具有三维结构的生物材料支架上,并加入生物活性因子保持一定浓度,构成具有生物活性的细胞-支架复合物。然而如何构建合理的细胞生长微环境以及维持生物活性因子的最佳浓度一直面临诸多问题。该实验创新性地提出了双交联缓释体系,持续稳定地释放神经营养因子,以便更好地促进种子细胞的生长与分化。目的:利用冷冻干燥法,将包载音猬因子的壳聚糖微球填充于纤维蛋白胶,构建具有三维结构的复合生物工程支架,探讨该支架对外胚层间充质干细胞向神经元样细胞分化的影响。方法:①采用离子凝胶法制备包载音猬因子的壳聚糖微球,而后将其与纤维蛋白胶混合,冷冻干燥成型,即得到包埋载音猬因子壳聚糖微球的纤维蛋白支架,采用扫描电子显微镜观察支架微观结构和ELISA检测音猬因子缓释效果,并制备包载音猬因子的纤维蛋白支架和包载音猬因子的壳聚糖微球作为对照;②采用悬浮抗贴壁法获取外胚层间充质干细胞来源的神经球样细胞分别种植于上述支架和经多聚赖氨酸修饰的圆玻片上,加入神经诱导培养基培养。14d后免疫荧光标记神经细胞相关蛋白β3-Tubulin、MAP-2和MBP,采用Westernblot方法测定神经细胞相关蛋白表达水平。结果与结论:①扫描电子显微镜观察纤维蛋白胶经冷冻干燥后呈海绵型网状结构,载音猬因子的壳聚糖微球均匀分散在其中;②ELISA检测结果显示复合纤维蛋白支架缓释速度较为平缓且持续时间较长,形成更稳定、持续的缓释体系;③免疫荧光染色显示复合纤维蛋白支架组高表达神经细胞相关蛋白;同时,Western blot检测结果显示复合纤维蛋白支架组表达的神经细胞相关蛋白水平明显高于其他组(P<0.05);④结果表明,壳聚糖复合纤维蛋白支架缓释效果较好,该复合支架对
BACKGROUND:The basic idea of spinal tissue engineering is to plant seed cells isolated and cultured in vitro onto a biomaterial scaffold with three-dimensional structure,where bioactive factors are added to maintain a certain concentration,eventually forming a biologically active cell-scaffold complex.However,how to construct a suitable cell growth microenvironment and maintain the optimal concentration of bioactive factors has been faced with many problems.This experiment innovatively proposed a double-crosslinking release system,which continuously released neurotrophic factors and promoted the growth and differentiation of seed cells.OBJECTIVE:To fabricate a fibrin glue scaffold incorporated with sonic hedgehog(SHH)-loaded chitosan microspheres to construct a biologically active cell-scaffold complex with three-dimensional structure,and to investigate its effect on the differentiation of nasal mucosa-derived ectomesenchymal stem cells(EMSCs)into neuron-like cells.METHODS:(1)Firstly,the SHH-containing chitosan microspheres were prepared via ionic gel method,and then mixed with fibrin glue;thus,the fibrin glue scaffolds embedded with SHH-loaded chitosan microspheres were prepared after freeze-drying.The structure of composite scaffold was observed with scanning electron microscope and the ELISA kit was employed to study the SHH releasing behavior.Meanwhile,the SHH-loaded chitosan scaffold and the SHH/fibrin scaffold were prepared as control.(2)The EMSCs-derived neurospheres cultured by suspension culture methods were transplanted on the above scaffolds and poly-L-lysine-coated glass,respectively.After co-cultured for 14 days,the differentiation of EMSCs was investigated with immunofluorescence staining(β3-Tubulin,MAP-2 and MBP)and western blot.RESULTS AND CONCLUSION:(1)Under the scanning electron microscope,the fibrin glue/chitosan/SHH scaffold after lyophilization had reticular structure,which looked like sponge,and SHH-loaded chitosan microspheres were dispersed uniformly.(2)ELISA results showed that the relea
作者
崔学文
杨开元
杨文静
陆浩
史文涛
陈平波
毕士奇
沈元昊
张志坚
Cui Xuewen;Yang Kaiyuan;Yang Wenjing;Lu Hao;Shi Wentao;Chen Pingbo;Bi Shiqi;Shen Yuanhao;Zhang Zhijian(Department of Orthopedics,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,Jiangsu Province,China;School of Medicine,Jiangsu University,Zhenjiang 212013,Jiangsu Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第29期4694-4700,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(51403086),项目负责人:杨文静
江苏省自然科学基金(BK20140544),项目负责人:杨文静
江苏大学高级人才启动基金(13JDG089),项目负责人:杨文静~~
关键词
外胚层间充质干细胞
壳聚糖
音猥因子
纤维蛋白胶
复合纤维蛋白支架
缓释
神经球样细胞
神经元
国家自然科学基金
ectomesenchymal stem cells
chitosan
sonic hedgehog
fibrin glue
composite fibrin scaffold
sustained release
neurosphere-like cells
neurons
National Natural Science Foundation of China
