Highly Aligned Ultrafine Fibers of Graphene/Poly(L-lactic acid)(Gr/PLLA) Composite for the Construction of Nerve Conduit†

doi:10.7503/cjcu20150958

Abstract

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

CLC Number:

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2016, 37(5): 972.

Figures/Tables 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.

References 40

[1]	Gu Y., Zhu J. B., Xue C. B., Li Z. M. Y., Ding F., Yang Y. M., Gu X. S., Biomaterials, 2014, 35(7), 2253—2263
[2]	Chen M. B., Zhang F., Lineaweaver W. C., Annals of PlasticSurgery, 2006, 57(4), 462—471
[3]	Meek M. F., Coert J. H., Journal of ReconstructiveMicrosurgery, 2002, 18(2), 97—109
[4]	Koh H. S., Yong T., Teo W. E., Chan C. K., Puhaindran M. E., Tan T. C., Lim A., Lim B. H., Ramakrishna S., Journal of NeuralEngineering, 2010, 7(4), 046003
[5]	Schmidt C. E., Leach J. B., Annual Review of Biomedical Engineering, 2003, 5, 293—347
[6]	Prabhakaran M. P., Venugopal J., Chan C. K., Ramakrishna S., Nanotechnology, 2008, 19(45), 455102
[7]	Murugan R., Ramakrishna S., TissueEngineering, 2007, 13(8), 1845—1866
[8]	Bao M., Zhou Y.H., Yuan H. H., Lou X. X., Zhang Y. Z.,Acta Polymerica Sinica, 2014, (5), 604—612
	(包敏, 周雅慧, 袁卉华, 娄向新, 张彦中. 高分子学报, 2014, (5), 604—612)
[9]	Zhong S., Teo W. E., Zhu X., Beuerman R. W., Ramakrishna S., Yung L. Y. L., Journal of Biomedical Materials Research, Part A, 2006, 79A(3), 456—463
[10]	He W., Yong T., Ma Z. W., Inai R., Teo W. E., Ramakrishna S., TissueEngineering, 2006, 12(9), 2457—2466
[11]	Koh H. S., Yong T., Chan C. K., Ramakrishna S., Biomaterials, 2008, 29(26), 3574—3582
[12]	Chew S. Y., Mi R., Hoke A., Leong K. W., Biomaterials, 2008, 29(6), 653—661
[13]	Yin Z., Chen X., Chen J. L., Shen W. L., Nguyen T. M. H., Gao L., Ouyang H. W., Biomaterials, 2010, 31(8), 2163—2175
[14]	Wang B., Cai Q., Zhang S., Yang X. P., Deng X. L., Journal of the Mechanical Behavior of Biomedical Materials, 2011, 4(4), 600—609
[15]	Schneider T., Kohl B., Sauter T., Kratz K., Lendlein A., Ertel W., Schulze-Tanzil G., Clinical Hemorheology and Microcirculation, 2012, 52(2-4), 325—336
[16]	Whited B. M., Rylander M. N., Biotechnology and Bioengineering, 2014, 111(1), 184—195
[17]	Quigley A. F., Razal J. M., Thompson B. C., Moulton S. E., Kita M., Kennedy E. L., Clark G. M., Wallace G. G., Kapsa R. M. I., AdvancedMaterials, 2009, 21(43), 4393—4397
[18]	Ghasemi-Mobarakeh L., Prabhakaran M. P., Morshed M., Nasr-Esfahani M. H., Ramakrishna S., Tissue EngineeringPart A, 2009, 15(11), 3605—3619
[19]	Shao S. J., Zhou S. B., Li L., Li J. R., Luo C., Wang J. X., Li X. H., Weng J., Biomaterials, 2011, 32(11), 2821—2833
[20]	Pinto A. M., Moreira S., Goncalves I. C., Gama F. M., Mendes A. M., Magalhaes F. D., Colloids and SurfacesB-Biointerfaces, 2013, 104, 229—238
[21]	Zhou K., Thouas G. A., Bernard C. C., Nisbet D. R., Finkelstein D. I., Li D., Forsythe J. S., ACS Applied Materials & Interfaces, 2012, 4(9), 4524—4531
[22]	Jin G. Z., Kim M., Shin U. S., Kim H. W., NeuroscienceLetters, 2011, 501(1), 10—14
[23]	Yuan H. H., Zhao S. F., Tu H. B., Li B. Y., Li Q., Feng B., Peng H. J., Zhang Y. Z., Journal of MaterialsChemistry, 2012, 22(37), 19634—19638
[24]	Yuan H.H., Tu H. B., Li B. Y., Li Q., Zhang Y. Z.,Acta Polymerica Sinica, 2014, (1),131—140
	(袁卉华, 屠红斌, 李碧云, 李芹, 张彦中. 高分子学报, 2014, (1), 131—140)
[25]	Dang T.T., Pham V. H.,, Hur S. H., Kim E. J., Kong B. S., Chung J. S ., Journal of Colloid and InterfaceScience, 2012, 376, 91—96
[26]	Wei T., Luo G. L., Fan Z. J., Zheng C., Yan J., Yao C. Z., Li W. F., Zhang C., Carbon, 2009, 47(9), 2296—2299
[27]	Ayres C. E., Jha B. S., Meredith H., Bowman J. R., Bowlin G. L., Henderson S. C., Simpson D. G., Journal of Biomaterials Science-Polymer Edition, 2008, 19(5), 603—621
[28]	Ayres C., Bowlin G. L., Henderson S. C., Taylor L., Shultz J., Alexander J., Telemeco T. A., Simpson D. G., Biomaterials, 2006, 27(32), 5524—5534
[29]	Zhang C., Yuan H. H., Liu H. H., Chen X., Lu P., Zhu T., Yang L., Yin Z., Heng B. C., Zhang Y. Z., Ouyang H. W., Biomaterials, 2015, 53, 716—730
[30]	Zhang J. G., Qiu K. X., Sun B. B., Fang J., Zhang K. H., Ei-Hamshary H., Al-Deyab S. S., Mo X. M., Journal of Materials Chemistry B, 2014, 2(45), 7945—7954
[31]	Lamastra F. R., Nanni F., Camilli L., Matassa R., Carbone M., Gusmano G., Chemical EngineeringJournal, 2010, 162(1), 430—435
[32]	Xia M. G., Su Z. D., Zhang S. L., AipAdvances, 2012, 2(3), 032122
[33]	Zhao X., Zhang Q. H., Chen D. J., Lu P., Macromolecules,2010, 43(5), 2357—2363
[34]	Zhao Y. F., Xiao M., Wang S. J., Ge X. C., Meng Y. Z., Composites Science and Technology, 2007, 67(11/12), 2528—2534
[35]	Uhl F.M., Yao Q., Nakajima H., Manias E., Wilkie C. A., Polymer Degradation and Stability, 2005, 89(1), 70—84
[36]	Fukushima H., Drzal L. T., Rook B. P., Rich M. J., Journal of Thermal Analysis and Calorimetry, 2006, 85(1), 235—238
[37]	Murariu M., Dechief A. L., Bonnaud L., Paint Y., Gallos A., Fontaine G., Bourbigot S., Dubois P., Polymer Degradation and Stability, 2010, 95(5), 889—900
[38]	Guenard V., Kleitman N., Morrissey T. K., Bunge R. P., Aebischer P., Journal ofNeuroscience, 1992, 12(9), 3310—3320
[39]	Bunge M. B., Pearse D., Journal of Rehabilitation Research and Development, 2003, 40(4), 55—62
[40]	Zhang K. H., Wang C. Y., Fan C. Y., Mo X. M., Journal of Biomedical Materials Research, Part A, 2014, 102(8), 2680—2691