纺织学报, 2026, 47(03): 70-76 doi: 10.13475/j.fzxb.20250901701

生物医用材料

导电各向异性复合心脏补片的熔体静电纺丝/直写构建及体外评价

李好义1,2, 田鑫哲1,2, 张毅1,2, 牟文英3, 张超,1,2, 赵千龙1,2, 杨卫民1,2

1 北京化工大学 生物医用材料北京实验室, 北京 100029

2 北京化工大学 机电工程学院, 北京 100029

3 首都医科大学附属北京安贞医院, 北京 100029

Fabrication and in vitro evaluation of conductive anisotropic composite cardiac patch via melt electrospinning/electrowriting

LI Haoyi1,2, TIAN Xinzhe1,2, ZHANG Yi1,2, MOU Wenying3, ZHANG Chao,1,2, ZHAO Qianlong1,2, YANG Weimin1,2

1 Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China

2 College of Mechanical and Electrical Engineering, Beijing University of Chemical Technology, Beijing 100029, China

3 Beijing Anzhen Hospital of Capital Medical University, Beijing 100029, China

通讯作者: 张超(1986—),男,讲师,博士。研究方向为高分子材料加工及复合材料绿色制造。E-mail:2014500015@buct.edu.cn

收稿日期: 2025-09-5   修回日期: 2026-01-29  

Received: 2025-09-5   Revised: 2026-01-29  

作者简介 About authors

李好义(1987—),男,副教授,博士。主要研究方向为微纳米纤维的先进制备及其应用。

摘要

针对现有心脏补片难以同时模拟天然心肌复杂的力学各向异性与电生理传导功能,从而限制心肌修复效果这一难题,采用熔体微分静电纺丝与熔体静电纺直写相结合的双重成形工艺,开发了一种集结构仿生与功能集成于一体的复合补片。首先,采用微分静电纺构建具有高孔隙率的微米级聚己内酯(PCL)纤维膜作为基底,以防止细胞渗漏;随后,利用静电直写技术在基底上沉积具有特定取向的菱形PCL骨架,通过调控网格角度实现力学各向异性定制;最后,经碳纳米管(CNTs)超声分散涂层处理赋予支架导电属性,并对其进行理化性能与生物学评价。结果表明,超细纤维基底膜(平均孔径为9.36 μm)可有效防止细胞渗漏;菱形PCL支架通过调控结构参数实现力学各向异性(70°支架纵横向弹性模量比达3.55,接近天然心肌);结合超声分散CNTs后处理赋予支架导电性(纵向电导率为1.15×10-3 S/cm);体外实验结果显示,该补片细胞存活率大于90%,且能有效诱导大鼠H9c2心肌细胞沿纤维方向定向黏附与生长。

关键词: 心脏补片; 熔体静电纺直写; 熔体微分静电纺丝; 导电支架; 聚己内酯; 碳纳米管; 医用纺织材料

Abstract

Objective Current cardiac patches often fail to simultaneously replicate the complex mechanical anisotropy and electrical conductivity of native myocardium, limiting their efficacy in tissue repair. This study aims to develop a hierarchical composite patch integrating structural biomimicry with functional electrical properties. By combining structural design with material modification, the research provides a multi-dimensional platform that supports cell retention, mimics heart tissue mechanics, and facilitates electrical signal propagation for myocardial regeneration.

Method A dual-process manufacturing strategy combining melt differential electrospinning and melt electrowriting (MEW) was employed using polycaprolactone (PCL) and carbon nanotubes (CNTs). First, a PCL microfiber base membrane was fabricated via differential electrospinning to serve as a cell barrier. Subsequently, a rhombus-patterned PCL backbone was deposited onto the membrane using MEW, with grid angles adjusted to customize mechanical anisotropy. Finally, multi-walled CNTs were coated onto the scaffold via ultrasonic dispersion to confer conductivity. The patches underwent physicochemical characterization and in vitro evaluation with H9c2 cardiomyocytes.

Results Characterization revealed that the melt-electrospun substrate membrane were 9.36 μm, effectively preventing cell leakage while maintaining permeability. The MEW process successfully modulated mechanical properties, the stent with a 70° grid angle exhibited a non-linear J-shaped stress-strain behavior and a longitudinal-to-transverse elastic modulus ratio of 3.55, falling within the physiological range of native myocardium (1.9-3.9). Decreasing the grid angle enhanced longitudinal strength, with the 50° stent achieving peak longitudinal modulus. Following ultrasonic CNT treatment, the stent achieved a longitudinal conductivity of 1.15×10-3 S/cm. Stability tests in physiological conditions showed a slight initial conductivity decay, stabilizing at 1.09×10-3 S/cm after 21 d. Biologically, the composite patch demonstrated excellent biocompatibility, with cell viability exceeding 90% after 3 d culture. Crucially, fluorescence staining indicated that the anisotropic rhombic topology and conductive cues synergistically induced H9c2 cardiomyocytes to adhere, spread, and align along the fiber direction, significantly improving morphological maturation compared to isotropic controls.

Conclusion This study successfully constructed a hierarchical PCL/CNTs cardiac patch that overcomes the limitations of conventional isotropic stent. By innovating the anti-leakage substrate + anisotropic skeleton + conductive coating strategy, the patch achieves precise matching of myocardial mechanics and restores electrical connectivity. The 70° diamond-shaped structure provides effective contact guidance cues, promoting cardiomyocyte alignment, while the CNT integration facilitates electrical functionality. These results suggest that the composite patch offers a promising biomimetic strategy for preventing ventricular remodeling and promoting functional recovery in myocardial infarction treatment. Future work will focus on in vivo implantation to assess tissue integration, vascularization, and long-term biodegradation kinetics.

Keywords: cardiac patch; melt electrowriting; melt differential electrospinning; conductive stent; polycaprolactone; carbon nanotube; medical textile material

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本文引用格式

李好义, 田鑫哲, 张毅, 牟文英, 张超, 赵千龙, 杨卫民. 导电各向异性复合心脏补片的熔体静电纺丝/直写构建及体外评价[J]. 纺织学报, 2026, 47(03): 70-76 doi:10.13475/j.fzxb.20250901701

LI Haoyi, TIAN Xinzhe, ZHANG Yi, MOU Wenying, ZHANG Chao, ZHAO Qianlong, YANG Weimin. Fabrication and in vitro evaluation of conductive anisotropic composite cardiac patch via melt electrospinning/electrowriting[J]. Journal of Textile Research, 2026, 47(03): 70-76 doi:10.13475/j.fzxb.20250901701

尽管现代医学在心血管疾病防控领域已取得长足进展,但该类疾病长期以来在全球范围内有着高死亡率[1]。心脏的核心功能依赖心肌组织,其主要由心肌细胞(CMs)及支持性非肌细胞构成[2-3]。面对终末期心力衰竭,心脏移植虽是目前最有效的手段,但受限于供体极度匮乏,难以普及,因此,探索促进受损心肌再生及阻断心衰进程的新型治疗策略迫在眉睫[2]

心脏组织工程为心肌修复提供了新希望。虽然干细胞疗法通过旁分泌机制有助于改善心梗后心功能[4],但直接注射往往面临细胞在炎症及缺血微环境中存活率低的问题。相比之下,心脏补片技术作为一种前沿方案,通过植入仿生支架不仅能为梗死区提供类似细胞外基质(ECM)的力学支撑,防止心室重构,还能作为载体递送细胞或生物活性因子[5-6]

为此,本研究融合熔体微分静电纺丝、熔体静电纺直写及导电改性技术,以“防渗漏基底构建-力学各向异性设计-导电功能赋予”为技术主线,构建层级化聚己内酯/碳纳米管(PCL/CNTs)复合心脏补片。首先通过熔体微分静电纺丝技术制备微孔基底膜,阻隔细胞渗漏;进而利用熔体静电纺直写技术成形菱形拓扑结构,精准调控力学各向异性;最后通过超声分散碳纳米管实现导电功能。三者协同,旨在实现心肌组织结构与功能的仿生整合,为心肌细胞定向排列与电信号传导提供多维调控。该技术路线有望突破传统心脏补片在结构仿生与功能集成方面的瓶颈,为心肌梗死后的心肌再生提供新策略。

1 实验部分

1.1 材料与仪器

材料:聚己内酯(PCL,牌号CAPA6500,重均分子量50 ku,熔体流动速率28 g/(10 min),密度1.1 g/cm3),购自美国Perstorp公司;多壁碳纳米管(CNTs,型号NC700,直径9.5 nm、长度1.5 μm),购自上海析研科技服务中心;大鼠H9c2心肌细胞株、杜尔贝科改良伊格尔培养基(DMEM培养基),均由中国科学院分子细胞科学卓越创新中心提供;最低必需培养基(MEM培养基),购自Corning公司;胎牛血清(FBS)、胰酶消化液、细胞计数试剂盒-8(CCK-8试剂盒)、4',6-二脒基-2-苯基吲哚(DAPI)细胞核染料及4%多聚甲醛固定液,购自北京拜尔迪生物科技有限公司;红色鬼笔环肽(Actin-Tracker Red-594),购自上海碧云天生物技术有限公司;磷酸盐缓冲液(PBS),购自深圳逗点生物科技有限公司;十二烷基磺酸钠(SDS,货号S105389),购自上海阿拉丁生化科技股份有限公司;大鼠骨髓间充质干细胞(CP-R131)及其完全培养基(CM-R131),购自武汉普诺赛生命科技有限公司。

仪器:熔体微分静电纺丝设备[7]、熔体静电纺直写系统[8](自主研发);MAIA3型扫描电子显微镜(泰思肯贸易上海有限公司);3H-2000PB型通孔孔径分析仪(贝士德仪器科技北京有限公司);YG001B型单纤维拉伸试验机(常州新纺检测仪器设备有限公司);UT58C型双探针数显万用表(优利德科技(中国)股份有限公司);DZF-6020型真空干燥箱(上海精其仪器有限公司);CX31型金相显微镜(日本奥林巴斯株式会社);MCO-18AC型CO2培养箱(山东博科科学仪器有限公司);BBS-DDC型超净工作台(济南来宝医疗器械有限公司);微量移液器(德国艾本德股份公司);TD5型台式低温超速离心机(上海笃特科学仪器有限公司);48孔培养板(上海荣泰健康科技股份有限公司);ReadMax 1200型酶联免疫检测仪(上海闪谱生物科技有限公司);BX53型荧光显微镜(日本奥林巴斯株式会社)。

1.2 复合补片制备

复合补片的具体制备流程如图1所示。通过熔体微分静电纺丝技术将PCL制成纤维膜,经热压处理使膜表面平整;随后将该纤维膜铺于氧化铟锡导电玻璃基板,采用熔体静电纺直写技术制备特定菱形支架,形成“基底膜-取向支架”双层结构;再将所得补片浸入CNTs(质量分数0.1%)和十二烷基磺酸钠(质量分数0.5% )分散剂的混合水溶液中超声处理1 h,干燥后即得PCL/CNTs复合补片。制备前将PCL原料在40 ℃下干燥4 h。

图1

图1   复合补片制备流程图

Fig.1   Preparation flowchart of composite patch


PCL熔体微分静电纺丝工艺参数为:电压35 kV,温度200 ℃,挤出机转速3 r/min,辊子转速1 000 r/min;所得纤维膜热压温度为60 ℃。PCL熔体静电纺直写工艺参数为:电压2.5 kV,温度160 ℃,气压17.24 kPa,接收距离3 mm,喷头移动速度10 mm/s,空气相对湿度控制在37%~45%之间。通过该工艺制得30 mm×30 mm的菱形心脏补片支架,同时制备30 mm×30 mm的方形心脏补片支架作为对照,打印路径如图2所示。

图2

图2   补片支架形状与打印路径示意图

注:α为网格角度;d为纤维间距;X代表横向;Y代表纵向。

Fig.2   Schematic diagrams of patch stents shape and printing path. (a) Diamond-shaped; (b) Square-shaped


1.3 测试与表征

微观形貌观察:采用扫描电镜对复合支架的微观形貌进行观察。

力学性能测试:采用单纤维拉伸仪测试样品纵向和横向上的应力-应变曲线,设定拉伸速率为5 mm/min。根据应力-应变曲线初始直线段的斜率,分别计算样品在纵向和横向上的弹性模量,二者的比值即为各向异性弹性模量比,简称各向异性模量比。

采用台式双探针数显万用表测试样品的导电性能:将探针平行固定于样品两端,沿纵向多点测量10次取均值;随后旋转样品90°以同法测定横向数据。按照下式计算电导率:

$G=1/\rho =L/RS$

式中:G为电导率,S/m;ρ为电阻率,Ω·m;L为导线长度,m;R为电阻,Ω;S为横截面积,m2

孔径分布测试:采用通孔孔径分析仪(基于泡压法原理),将30 mm×30 mm的方形纤维膜经无水乙醇完全浸润后进行测试。每组测试3次,结果取平均值。

细胞存活率测试:采用CCK-8法进行测试。将补片与大鼠骨髓间充质干细胞共培养,细胞密度为2×104个/mL;培养结束后用PBS清洗3次,加入10% CCK-8溶液,37 ℃下孵育 2 h。分别对培养1 d和3 d的样品,采用酶标仪测试450 nm波长下的吸光度。按照下式计算细胞存活率:

$R=\frac{{D}_{e}-{D}_{c}}{{D}_{c}}\times 100\%$

式中:R为细胞存活率,%;De为实验组的吸光度;Dc为空白组(未添加任何细胞培养基的48孔板本身)的吸光度。

鬼笔环肽细胞骨架染色:将补片与大鼠H9c2心肌细胞共培养在48孔板上,细胞密度为2×104个/mL,每孔1 mL。染色时,用4%多聚甲醛固定1 h,用PBS清洗3次;随后采用鬼笔环肽染液与DAPI染液避光孵育30 min。分别对培养1 d和3 d的样品,在荧光显微镜下观察并拍摄;利用ImageJ 2X 软件对不同荧光通道图像进行合成与结果分析。

2 结果与讨论

2.1 支架结构调控与力学性能分析

2.1.1 网格角度对支架力学性能的影响

图3示出支架在纵横方向上的力学性能。对于90°网格支架,纤维均呈伸直状态,受力即刻响应,导致应力随应变线性激增。而随着网格角度倾斜,应力-应变曲线呈现出典型的“J”型非线性特征,这归因于拉伸初期的几何重排缓冲效应。数据表明,减小网格角度能显著增强纵向性能,同时削弱横向弹性模量(横向强度波动较小)。所有组别的弹性应变范围均覆盖了天然心肌的需求(大于25%)。当网络角度降至50°时,各向异性最为显著:纵向抗拉强度(0.65 MPa)与弹性模量(3.08 MPa)达到峰值,而横向性能则降至最低点(强度为0.23 MPa,弹性模量为0.24 MPa)。

图3

图3   50°~90°网格支架的力学性能

Fig.3   Mechanical properties of stent with grid angle of 50°-90°. (a) Longitudinal stress-strain curve; (b) Transverse stress-strain curve; (c) Longitudinal and transverse elastic modulus


支架的各向异性模量比如图4所示。其中,灰色区域表示天然心肌的允许范围(1.9~3.9)。当网格角度在67°~78°范围内时,支架模量比满足心肌仿生要求。

图4

图4   不同网格角度支架的各向异性模量比

Fig.4   Anisotropic modulus ratios corresponding to stents of different grid angles


2.1.2 纤维间距对支架力学性能的影响

在保持70°取向及15层堆叠结构不变的条件下,对比了4种规格(间距300~600 μm)支架的纵向拉伸性能,结果如图5所示。数据显示,随着网格变得稀疏,材料的抗拉强度与弹性模量均呈现下降趋势。其中,300 μm间距组表现出最高的力学值(强度0.45 MPa,弹性模量1.67 MPa),而600 μm组则降至最低(强度0.3 MPa,弹性模量1.35 MPa)。这种性能衰减归因于实体材料占比的变化:在相同测试条件下,间距越宽,内部实际承担载荷的纤维根数越少,导致支架抵抗拉伸变形的能力随之下降。

图5

图5   不同纤维间距下支架的力学性能

Fig.5   Mechanical properties of stents with different fiber spacings


2.2 导电性及稳定性分析
2.2.1 支架导电性

图6示出PCL/CNTs超声波处理后支架的光学显微照片。可见,CNTs均匀包覆在纤维表面。

图6

图6   PCL/CNTs超声处理后支架的光学显微镜照片

Fig.6   Optical microscope image of PCL/CNTs ultrasonic treated stent


测量了网格角度为70°及90°(各向同性)、纤维间距为300 μm、15层支架的纵向电导率。结果显示:当网格角度为90°时,支架电导率为1.36×10-3 S/cm;而当网格角度为70°时,支架纵向与横向电导率均低于90°支架,分别为1.15×10-3、8.67×10-4 S/cm。可见,该支架的电导率与天然心肌组织(纵向电导率为1.6×10-3 S/cm,横向电导率为5×10-5 S/cm)接近。

2.2.2 时间对支架电导率衰减的影响

图7示出支架在模拟生理环境(水浴环境、37 ℃、湿度50%)下的纵向电导率演变规律。从整体曲线来看,其导电性能呈现先下降后趋于稳定的变化趋势。在浸泡初期(0~3 d),由于部分结合松散的CNTs发生流失,电导率出现一定程度的衰减,于3 d时降至1.11×10-3 S/cm。此后(3~21 d),电导率进入平台期,21 d时测定值为1.09×10-3 S/cm,基本达到平衡状态。综上,该支架在通常的细胞培养周期内电导率损耗极小,具备优异的湿环境稳定性,可确保实验的可靠性。

图7

图7   支架电导率衰减与时间关系

Fig.7   Relationship between stent conductivity decay and time


2.3 基底膜孔径分析

图8示出基底膜的孔径分布。控制纤维膜面密度为40 g/m2左右,基底纤维膜的平均孔径为9.36 μm,因心肌细胞直径约为十几微米[9],理论上基底膜可起到防渗漏的作用。

图8

图8   基底膜孔径分布

Fig.8   Pore size distribution of basement membrane


2.4 体外生物评价
2.4.1 大鼠H9c2心肌细胞的铺展与黏附情况

为评估不同结构支架 (PCL、PCL/CNTs菱形及方形支架)对细胞行为的影响,将大鼠H9c2心肌细胞接种于各组样品表面,并在培养1 d及3 d后进行鬼笔环肽/DAPI双重荧光染色,以观测细胞的黏附铺展状态,测试结果如图9所示。其中红色标记F-actin骨架,蓝色标记细胞核。

图9

图9   荧光显微镜下大鼠H9c2心肌细胞在不同支架的骨架染色情况

Fig.9   Skeleton staining of rat H9c2 cardiomyocytes on different stents by fluorescence microscopy. (a) PCL stents; (b) PCL/CNTs diamond-shaped stent; (c) PCL/CNTs square-shaped stent


图9可知,共培养24 h后,PCL组细胞大都仍维持未铺展的圆形;相比之下,2组掺杂CNTs的复合支架上,部分细胞已呈现出仿生心肌特征的长梭形形态,表明CNTs的引入有助于诱导细胞极性,加速细胞成熟。随着培养时间延长至第3 d,各组细胞增殖显著,骨架伸展更为充分。其中,PCL/CNTs复合组的细胞铺展面积显著优于纯PCL组。尤为关键的是,菱形支架组的H9c2细胞展现出沿着直写纤维定向排列的趋势,证实了菱形拓扑结构能提供有效的“接触导向”线索,诱导心肌细胞实现各向异性生长。

2.4.2 复合补片的生物相容性

采用CCK-8试剂盒测定大鼠骨髓间充质干细胞在纯PCL、PCL/CNTs方形及菱形支架上的活性,以评估各组材料在培养1 d及3 d的细胞毒性,测试结果如图10所示。由图可知,接种1 d后,PCL对照组与方形支架组表现出相似的细胞相容性,存活率均维持在90%左右;相比之下,菱形支架组的数据略低(87%)。随着培养时间延长至3 d,3组样品的细胞活性均呈稳定增长趋势且无显著差异,其中菱形组的回升幅度最大,由87%跃升至94%。鉴于所有实验组在各检测节点的细胞存活率始终高于80%,证实了3类支架均未表现出明显的细胞毒性,均具备优异的细胞相容性。

图10

图10   不同支架在细胞培养1 d和3 d的细胞存活率

Fig.10   Cell survival rates of different stents cultured for 1 d (a) and 3 d (b)


3 结论

本研究以生物可降解聚己内酯(PCL)为基体,导电碳纳米管(CNTs)为功能填料,结合熔体微分静电纺丝与熔体静电纺直写技术,成功构建了一种具有层级结构的导电各向异性心脏补片。研究结果表明,熔体静电纺直写技术可实现菱形PCL支架力学各向异性的精准调控,70°结构的纵横向弹性模量比为3.55,与天然心肌相匹配;CNTs后处理使支架电导率达到10-3 S/cm量级,熔体微分静电纺丝制备的超细纤维基底膜平均孔径为9.36 μm,可有效防止细胞渗漏;体外培养3 d结果显示,细胞存活率大于90%,且补片可促进心肌细胞定向排列。该复合制备技术为仿生心脏补片的开发提供了新思路,未来工作将进一步开展动物体内实验,将补片植入心肌梗死模型动物体内,系统评估其组织整合性、血管化程度及心功能恢复效果;同时,通过长期随访监测材料的降解动力学与电传导稳定性,可优化参数以适配体内环境,为推进补片临床前研究提供参考。

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