用高度取向石墨烯/聚乳酸(Gr/PLLA)复合超细纤维构建神经导管

doi:10.7503/cjcu20150958

摘要/Abstract

摘要：

制备了一种高度取向的石墨烯(Gr)/聚乳酸(PLLA)复合超细纤维, 并构建了神经导管, 研究了Gr/PLLA促进神经细胞生长与分化的协同诱导作用. 研究结果表明, Gr/PLLA具有较好的纤维形貌与取向度; Gr的引入提高了纤维的热性能及力学性能; Gr加入量(≤1%)的增加及纤维取向度的提高使雪旺细胞(SCs)的黏附数量及伸展比例均呈增加趋势; Gr/PLLA纤维可促进SCs的增殖, 雪旺细胞在96 h时达到最佳生长状态, 表明Gr/PLLA纤维具有较好的细胞相容性. 基于细胞形貌及轴突数量统计发现, Gr/PLLA纤维也能促进大鼠肾上腺嗜铬细胞瘤(PC12细胞)的神经分化. 直径为2 mm的Gr/PLLA纤维导管具有较好的纤维取向度和抗压能力, 能促进细胞沿管轴方向取向生长.

关键词: 静电纺丝, 取向纤维, 石墨烯/聚乳酸复合超细纤维, 协同效应, 神经组织工程

Abstract:

Highly aligned ultrafine fibers of graphene/poly(L-lactic acid)(Gr/PLLA) composite were prepared for studying the synergistic effect of fiber orientation degree and Gr incorporation on nerve cell growth and differentiation, from which nerve conduits by reeling-heating the aligned Gr/PLLA fibers were fabricated for potential neural tissue engineering applications. The results showed that the Gr/PLLA fibers made by stable jet electrospinning(SJES) had higher degree of fiber alignment. Incorporation of Gr improved the thermal and mechanical properties of the PLLA fibers. Increasing the Gr content and fiber orientation degree gave rise to increased adhesion number and elongation percentage of the Schwann cells(SCs). All the fiber scaffolds could promote SCs to proliferate well and cells growth reached the best state at 96 h, indicating appropriate cytocompatibility of the Gr/PLLA fibers. In addition, it was found the aligned Gr/PLLA fibers also promoted the neural differentiation of PC12 cells. The nerve conduits with inner diameter of 2 mm made of Gr/PLLA fibers showed good axial fiber orientation degree and pressure-resistance ability, and could promote cell growth directionally.

Key words: Electrospinning, Aligned fiber, Ultrafine fibers of grapheme/poly(L-lactic acid) composite, Synergistic effect, Neural tissue engineering

中图分类号:

TrendMD:

张会兰, 易兵成, 王先流, 李碧云, 余哲泡, 娄向新, 张彦中. 用高度取向石墨烯/聚乳酸(Gr/PLLA)复合超细纤维构建神经导管. 高等学校化学学报, 2016, 37(5): 972.

ZHANG Huilan, YI Bingcheng, WANG Xianliu, LI Biyun, YU Zhepao, LOU Xiangxin, ZHANG Yanzhong. Highly Aligned Ultrafine Fibers of Graphene/Poly(L-lactic acid)(Gr/PLLA) Composite for the Construction of Nerve Conduit^†. Chem. J. Chinese Universities, 2016, 37(5): 972.

图/表 14

Fig.1 Preparation of the oriented nerve conduit(A) and schematic of cell seeding into nerve conduit(B)

Fig.2 SEM images(A1—E1), pixel intensity plots against the angle of acquisition(A2—E2) and histograms of diameter distribution(A3—E3) of electrospun fibers through CES(A1—A3) and SJES(B1—E3)(A1—A3) Semi-oriented; (B1—B3) PLLA; (C1—C3) 0.1%Gr/PLLA; (D1—D3) 0.5%Gr/PLLA;(E1—E3) 1%Gr/PLLA. Insets in (A1—E1) are FFT output images.

Fig.3 TEM images of Gr/PLLA fibers containing varied amounts of Gr prepared by SJES(A) PLLA; (B) 0.1%Gr/PLLA; (C) 0.5%Gr/PLLA; (D) 1%Gr/PLLA.

Fig.4 Raman spectra of Gr/PLLA fibers containing varied amounts of Gr prepared by SJESa. Gr; b. PLLA; c. 0.1%Gr/PLLA; d. 0.5%Gr/PLLA; e. 1%Gr/PLLA.

Fig.5 Typical tensile stress-strain curves(A), tensile strength and Young’s modulus(B) of the well-aligned Gr/PLLA fibersa. PLLA; b. 0.1%Gr/PLLA; c. 0.5%Gr/PLLA; d. 1%Gr/PLLA.

Fig.6 DSC thermogram(A), TGA curves(B), and DTG curves(C) of well-aligned Gr/PLLA fibers a. PLLA; b. 0.1%Gr/PLLA; c. 0.5%Gr/PLLA; d. 1%Gr/PLLA.

Table 1 Thermal data of well-aligned Gr/PLLA fibers

Sample	DSC		TGA
Sample	T_g/℃	Temperature of 20% mass loss/℃	Temperature of 50% mass loss/℃	Temperature of maximum rate of degradation/℃
PLLA	61.6	259	288	286.6
0.1%Gr/PLLA	62.1	278	307	312.6
0.5%Gr/PLLA	62.5	296	319	319.1
1%Gr/PLLA	63.6	304	325	329.6

Fig.7 Fluorescent images of SCs cultured on TCP(A1—A5), semi-oriented fibers(B1—B5), PLLA fibers(C1—C5), 0.1%Gr/PLLA fibers(D1—D5), 0.5%Gr/PLLA fibers(E1—E5) and 1%Gr/PLLA fibers(F1—F5) for 2 h(A1—F1), 5 h(A2—F2), 8 h(A3—F3), 24 h(A4—F4) and 96 h(A5—F5)F-actin(red) and nuclei(blue) of cells were stained by Rhodamine-phalloidin and DAPI, respectively.

Fig.8 Adherent cell density(A) and elongation percentage(B) on TCP, semi-oriented, PLLA, 0.1%Gr/PLLA, 0.5%Gr/PLLA and 1%Gr/PLLA substrates after seeding SCs for 2, 5 and 8 h

Fig.9 Proliferation of SCs on TCP, semi-oriented, PLLA, 0.1%Gr/PLLA, 0.5%Gr/PLLA and 1%Gr/PLLA substrates after culturing the SCs for 24, 96 and 168 h* p<0.05.

Fig.10 Fluorescent images of GFAP stained SCs cultured on TCP(A), semi-oriented fibers(B), PLLA fibers(C), 0.1%Gr/PLLA fibers(D), 0.5%Gr/PLLA fibers(E) and 1%Gr/PLLA fibers(F) for 96 hGreen: GFAP; blue: nuclei of cells.

Fig.11 Fluorescent images of PC12 cells cultured on TCP(A), semi-oriented fibers(B), PLLA fibers(C), 0.1%Gr/PLLA fibers(D), 0.5%Gr/PLLA fibers(E) and 1%Gr/PLLA fibers(F) for 96 hF-actin(red) and nuclei of cells(blue) were stained by Rhodamine-phalloidin and DAPI, respectively. The arrows were pointed to differentiated neurites.

Fig.12 Percentage of neurite-bearing PC12 cells after being induced on different substrates for 96 ha. TCP; b. Semi-oriented; c. PLLA; d. 0.1%Gr/PLLA; e. 0.5%Gr/PLLA; f. 1%Gr/PLLA. ** p<0.01.

Fig.13 Digital photograph of different types of nerve conduits fabricated by reeling-heating method(A) and morphologies of Schwann cells after being cultured with Semi-oriented(B), PLLA(C) and 1%Gr/PLLA conduits(D) for 96 hInsets of (B—D): SEM images of the nerve conduits. F-actin(red) and nuclei of cells(blue) were stained by Rhodamine-phalloidin and DAPI, respectively.

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