电纺纤维的刚度对iPS-MSCs黏附和迁移的影响

doi:10.7503/cjcu20170542

摘要/Abstract

摘要：

采用退火处理方法调控电纺聚左旋乳酸(PLLA)纤维的刚度, 研究了纤维刚度变化对诱导多能干细胞来源的间充质干细胞(iPS-MSCs)的形貌、增殖和迁移行为的影响. 结果表明, 退火处理对纤维的直径影响不大[退火前(1.26±0.25) μm, 退火后(1.24±0.26) μm], 但提高了纤维结晶度和力学性能, 导致纤维刚度增加1.73倍. 将iPS-MSCs在退火处理的纤维支架上培养24 h后的细胞骨架铺展面积及7 d后的细胞增殖分别是未处理前的1.78和1.18倍. 纤维刚度的提高影响iPS-MSCs的迁移并促进迁移相关基因Integrinβ1, RhoA和Rock1的表达上调. 通过退火处理提高电纺PLLA纤维刚度可作为仿生支架构建中调控细胞生物功能的一个重要手段.

关键词: 电纺纤维, 纤维刚度, 诱导多能干细胞来源的间充质干细胞, 细胞黏附, 细胞迁移

Abstract:

The stiffness of as-electrospun poly-L-lactic acid(PLLA) fibers was altered by annealing treatment, and then influence of fiber stiffness on the morphology, proliferation and migration of induced pluripotent stem cell-derived mesenchymal stem cells(iPS-MSCs) were evaluated. The results showed that annealing treatment had negligible influence on the diameter of PLLA fibers[before: (1.26±0.25) μm and after: (1.24±0.26) μm]. However, fiber crystallinity and mechanical properties were enhanced, which resulted in 1.73 times stiffer than that of fibers before annealing treatment. The cell spreading area and proliferation of iPS-MSCs cultured on the treated PLLA scaffold after 1 and 7 d of incubation were 1.78 and 1.18 times higher, respectively, than that of untreated scaffold. The increase of fiber stiffness can affect the migration of iPS-MSCs and promote up-regulated expression of the migration-related genes Integrinβ1, RhoA and Rock1. Collectively, this study demonstrated that increasing fiber stiffness by annealing may be used as an important modality to regulate cell biological functions of the cells in engineering tissues.

Key words: Electrospun fiber, Fiber stiffness, Induced pluripotent stem cell-derived mesenchymal stem cells(iPS-MSCs), Cell adhesion, Cell migration

中图分类号:

O631
R318.08

TrendMD:

余哲泡, 袁卉华, 易兵成, 王先流, 张赵文斌, 张彦中. 电纺纤维的刚度对iPS-MSCs黏附和迁移的影响. 高等学校化学学报, 2018, 39(4): 807.

YU Zhepao, YUAN Huihua, YI Bingcheng, WANG Xianliu, ZHANG Zhaowenbin, ZHANG Yanzhong. Effects of Electrospun Fiber-Stiffness on Adhesion and Migration of iPS-MSCs^†. Chem. J. Chinese Universities, 2018, 39(4): 807.

图/表 12

Scheme 1 Schematic of cell migration experiment

Table 1 Primer sequences of speciﬁc genes used for qRT-PCR

Gene	Forward primer sequence(5'-3')	Reverse primer sequence(5'-3')
Integrinβ1	ATGCCAAATCTTGCGGAGAAT	TTTGCTGCGATTGGTGACATT
RhoA	AGCTTGTGGTAAGACATGCTTG	GTGTCCCATAAAGCCAACTCTAC
Rock1	GACTGGGGACAGTTTTGAGAC	ATCCAAATCATAAACCAGGGCAT
GAPDH	TGACCTCAACTACATGGTCTACA	CTTCCCATTCTCGGCCTTG

Fig.1 SEM images(A, B) and the diameter distribution(C, D) of electrospun PLLA fibers before(A, C) and after(B, D) annealing treatmentInsets of (A) and (B): high magnification SEM images of electrospun PLLA fibers before and after annealing treatment.

Fig.2 DSC(A) and XRD(B) analysis of electrospun PLLA fibers before(a) and after(b) annealing treatment

Table 2 Thermal data of PLLA fibers before and after annealing treatment

Sample	T_g/℃	T_c/℃	T_m/℃	ΔH_c/(J·g^-1)	ΔH_m/(J·g^-1)	X_c(%)
PLLA	56.19	80.23	179.38	1.84	11.96	10.88
Annealing	58.51		177.43		10.50	11.29

Fig.3 Tensile properties and stiffness of the electrospun PLLA fibrous membranes before and after annealing treatment(A) Typical stress-strain curves; (B) tensile strength and fiber stiffness; (C) Young’s modulus; (D) strain at break.* P<0.05; ** P<0.01.

Fig.4 Fluorescent images of iPS-MSCs cultured on as-electrospun(A) and annealed electrospun PLLA fiber scaffolds for 24 h(B) and quantitative results of the spreading area of iPS-MSCs(C)The F-actin(red) and nuclei(blue) of cells were stained by Rhodamine-phalloidin and DAPI, respectively. **P<0.01.

Fig.5 SEM micrographs of iPS-MSCs cultured on as-electrospun(A, C) and annealed electrospun PLLA fiber scaffolds(B, D) for 4 d(A, B) and 7 d(C, D)

Fig.6 Proliferation of iPS-MSCs on as-electrospun and annealed electrospun PLLA fiber scaffolds* P< 0.05, **P< 0.01.

Fig.8 iPS-MSCs migration on as-electrospun(A-C) and annealed electrospun PLLA fiber scaffolds(D-F) for 0 h(A, D), 48 h(B, E) and 72 h(C, F)

Fig.8 Quantitative measurment of iPS-MSCs migration after culturing the cells on as-electrospun and annealed electrospun PLLA fibrous scaffolds for 0, 48 and 72 h

Fig.9 Migration-related genes expression of iPS-MSCs cultured on as-electrospun and annealed electrospun PLLA fiber scaffolds for 1, 3 and 4 d(A) Integrinβ1; (B) RhoA; (C) Rock1.

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