Effect of fibrin glue scaffolds embedded with sonic hedgehog-loaded chitosan microspheres on the differentiation of nasal mucosa-derived ectomesenchymal stem cells

doi:10.3969/j.issn.2095-4344.1807

Abstract

Abstract:

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 release of SHH from the fibrin glue/chitosan/SHH scaffold was relatively gentle and could last for a longer time to form a more stable and sustained release system. (3) Immunofluorescence staining indicated that EMSCs could highly expressed neuron-related proteins after co-cultured with the fibrin glue/chitosan/SHH scaffold. Western blot results indicated that the expression of neuron-related proteins in the fibrin glue/chitosan/SHH scaffold was better than that in the control group (P < 0.05). These results indicate that the fibrin glue/chitosan/SHH scaffold exhibits good sustained drug release effect, and certainly promote the differentiation of EMSCs towards neuron-like cells.

Key words: ectomesenchymal stem cells, chitosan, sonic hedgehog, fibrin glue, composite fibrin scaffold, sustained release, neurosphere-like cells, neurons, National Natural Science Foundation of China

CLC Number:

Cui Xuewen, Yang Kaiyuan, Yang Wenjing, Lu Hao1, Shi Wentao, Chen Pingbo, Bi Shiqi, Shen Yuanhao, Zhang Zhijian. Effect of fibrin glue scaffolds embedded with sonic hedgehog-loaded chitosan microspheres on the differentiation of nasal mucosa-derived ectomesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(29): 4694-4700.

Figures/Tables 3

2.3 外胚层间充质干细胞的形态学和表面标志鉴定结果第1代大鼠鼻黏膜细胞形成小集群分布，早期扁平多边形上皮样细胞为优势细胞，呈鹅卵石状，巢样分布。随传代次数增加上皮样细胞逐渐消失，梭形的外胚层间充质干细胞增殖较快，当细胞铺满瓶底时，可见大量长条形纺锤状细胞平行排列。免疫荧光染色可观察到传代培养的外胚层间充质干细胞表达vimentin、nestin，见图3，且这2种表面标志物的表达率都大于98%，这表明分离培养的外胚层间充质干细胞纯度较高。 2.4 外胚层间充质干细胞来源神经球的形态学和表面标志鉴定结果当更换为神经球诱导培养基后，外胚层间充质干细胞迅速聚合成球状。培养7 d后，逐渐形成外表透亮，悬浮在培养基中的神经球样细胞。免疫荧光染色可观察到外胚层间充质干细胞来源的神经球表达nestin、CD133，见图3。 2.5 免疫荧光染色检测支架中神经再生相关蛋白的表达将外胚层间充质干细胞来源神经球分别接种于经多聚赖氨酸修饰的圆玻片(BLANK)、壳聚糖微球(CS-SHH)、纤维蛋白支架(FG-SHH)、复合纤维蛋白支架(FG-CS-SHH)上，用Neurobasal培养基培养后细胞进入分化状态。神经球在纤维蛋白支架和多聚赖氨酸修饰的玻璃片上均贴壁良好，3 d后从神经球边缘伸出许多细小突起。培养过程中动态观察，可见复合纤维蛋白支架组神经球内细胞逐渐向周围迁移并长出丝状突起，而壳聚糖微球组以及纤维蛋白支架组神经球内细胞迁移不明显，空白对照组几乎没有细胞迁移。14 d后行β3-Tubulin、MAP-2、MBP免疫荧光染色结果显示：复合纤维蛋白支架组 β3-Tubulin、MAP-2染色可见细胞自神经球向四周辐射状生长出细长突起，神经纤维连织成网。纤维蛋白支架组与壳聚糖微球组神经球内迁移出的细胞所形成的突起较少，未能形成完整的神经网络。空白对照组中几乎没有突起形成。复合纤维蛋白支架组MBP染色可见神经球逐渐分化形成髓鞘样结构，而纤维蛋白支架组与壳聚糖微球组髓鞘样结构形成较少，空白对照组几乎没有髓鞘样结构形成，见图4。"

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