3D打印USPIO/EGCG功能化双层水凝胶胆道支架的构建及体外功能评价
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1中南大学湘雅医学院附属海口医院 肝胆胰外科,海南 海口 570208;2中南大学湘雅医学院附属海口医院 中心实验室,海南 海口 570208;2湖南妇女儿童医院 外科, 湖南 长沙 410208;3暨南大学生命科学技术学院,广东 广州 510632;5海南省海口市消化病临床研究 与转化重点实验室,海南 海口 570208

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彭柏铭,中南大学湘雅医学院附属海口医院硕士研究生,主要从事肝胆疾病基础和临床方面的研究。

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海南省海口市重点科技创新基金资助项目 2022-032国家自然科学基金资助项目 82260136海南省海口市重点科技创新基金资助项目(2022-032);国家自然科学基金资助项目(82260136)。


Fabrication and in vitro functional evaluation of a 3D-printed USPIO/EGCG-functionalized bilayer hydrogel biliary stent
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1Department of Hepatobiliary and Pancreatic, Affiliated Haikou Hospital of Xiangya Medical College, Central South University, Haikou 570208, China;2Surgery Central Laboratory, Affiliated Haikou Hospital of Xiangya Medical College, Central South University, Haikou 570208, China;2Department of Surgery, Women & Children's Hospital of Hunan, Changsha, 410208, China;3School of Life Science and Technology, Jinan University, Guangzhou 510632, China;5Haikou Key Laboratory of Clinical Research and Translational Medicine of Digestive Diseases, Haikou 570208, China

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    摘要:

    背景与目的 胆道狭窄的治疗仍面临支架力学性能、术后再狭窄及术后监测等问题。为兼顾胆道支架的机械支撑、局部药物递送和影像学可视化,本研究构建了一种基于3D打印的超小超顺磁性氧化铁(USPIO)/表没食子儿茶素没食子酸酯(EGCG)功能化双层水凝胶胆道支架,并评价其理化性质、药物释放特性、磁共振成像可视化性能及体外生物学性能。方法 以聚己内酯甲基丙烯酸酯(PCLMA)构建具有机械支撑作用的内层,以甲基丙烯酰化明胶(GelMA)构建生物活性外层,并采用介孔二氧化硅纳米颗粒(MSN)负载EGCG和USPIO,通过3D打印及光交联技术制备PCLMA/GelMA/MSN@USPIO@EGCG双层水凝胶支架。采用透射电子显微镜、扫描电子显微镜、傅里叶变换红外光谱、核磁共振氢谱、动态光散射及流变学等方法表征材料结构与性能;通过体外降解及EGCG释放实验评价支架的降解和药物递送特性;采用磁共振成像评价USPIO赋予支架的磁共振可视化能力;以人脂肪来源间充质干细胞(hADSCs)评价细胞相容性、细胞增殖、活性氧(ROS)、线粒体膜电位及细胞衰老,并通过溶血和凝血实验评价其血液相容性。结果 所制备MSN及其复合纳米颗粒具有均一的球形形貌和较好的分散性,PCLMA/GelMA/MSN@USPIO@EGCG支架呈规则的多孔网格结构。PCLMA内层具有较高的压缩模量,而GelMA及其复合水凝胶具有良好的黏弹性。复合支架在体外表现出可控的降解特性,且EGCG在MSN负载及GelMA包埋后呈现持续释放特征。USPIO的引入使复合材料具有明显的T2加权磁共振成像负性对比特性,弛豫率与Fe浓度呈良好的线性关系。细胞实验显示,各组支架均具有较好的细胞相容性,并可支持hADSCs黏附和铺展;EGCG负载后细胞增殖活性进一步提高,同时表现出较低的氧化应激水平和较好的线粒体膜电位维持作用。复合支架在不同浓度及孵育时间下均未引起明显的溶血或凝血异常。结论 本研究成功构建了一种具有机械支撑、EGCG缓释及磁共振成像可视化功能的3D打印双层水凝胶胆道支架。该支架具有良好的体外降解性能、生物相容性及细胞保护作用,为胆道狭窄的局部微环境调控及支架影像学监测提供了新的材料学策略,但其体内安全性及抗再狭窄作用仍需进一步验证。

    Abstract:

    Background and Aims Biliary stricture remains challenging to treat because of limitations related to stent mechanical performance, restenosis, and postoperative monitoring. This study aimed to develop a 3D-printed ultrasmall superparamagnetic iron oxide (USPIO)/epigallocatechin gallate (EGCG) functionalized bilayer hydrogel biliary stent integrating mechanical support, local drug delivery, and MRI visibility, and to evaluate its physicochemical and in vitro biological properties.Methods Polycaprolactone methacrylate (PCLMA) was used to construct the mechanically supportive inner layer, while gelatin methacryloyl (GelMA) was used as the bioactive outer layer. Mesoporous silica nanoparticles (MSN) loaded with EGCG and USPIO were incorporated into the hydrogel system. A PCLMA/GelMA/MSN@USPIO@EGCG bilayer hydrogel biliary stent was fabricated by 3D printing and photocrosslinking. The structural and physicochemical properties of the materials were characterized using transmission electron microscopy, scanning electron microscopy, Fourier-transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and rheological analysis. In vitro degradation and EGCG release were evaluated, and MRI was used to assess the imaging capability conferred by USPIO. The cytocompatibility, cell proliferation, reactive oxygen species (ROS), mitochondrial membrane potential, and cellular senescence of human adipose-derived mesenchymal stem cells (hADSC) were evaluated. Hemolysis and coagulation assays were performed to assess hemocompatibility.Results The prepared MSN and composite nanoparticles exhibited relatively uniform spherical morphology and good dispersion, while the PCLMA/GelMA/MSN@USPIO@EGCG stent showed a regular porous grid-like structure. The PCLMA inner layer exhibited high compressive strength, whereas the GelMA-based composite hydrogel showed favorable viscoelastic properties. The composite stent exhibited controllable degradation and sustained EGCG release in vitro. USPIO incorporation conferred a distinct T2-weighted negative MRI contrast effect, with a strong linear relationship between the relaxation rate and iron concentration. All stent groups showed good cytocompatibility and supported hADSCs adhesion and spreading. EGCG incorporation further enhanced cell proliferation, reduced oxidative stress, and helped maintain mitochondrial membrane potential. The composite stent caused no obvious hemolytic or coagulation abnormalities over the tested concentration and incubation ranges.Conclusion A 3D-printed bilayer hydrogel biliary stent with mechanical support, sustained EGCG release, and MRI visibility was successfully developed. The stent exhibited favorable biodegradability, cytocompatibility, and cytoprotective properties in vitro, providing a potential biomaterial strategy for local microenvironment modulation and imaging-assisted monitoring of biliary strictures. Its in vivo safety and anti-restenotic efficacy require further investigation.

    图1 不同纳米颗粒的TEM图与粒径分布图 A:依次为MSN、MSN@EGCG和MSN@USPIO@EGCG的TEM图;B:依次为MSN、MSN@EGCG和MSN@USPIO@EGCG的粒径分布图Fig.1 TEM images and particle size distributions of different nanoparticles A: TEM images of MSN, MSN@EGCG, and MSN@USPIO@EGCG; B: Particle size distributions of MSN, MSN@EGCG, and MSN@USPIO@EGCG
    图2 胆道支架的外观及SEM图Fig.2 Macroscopic appearance and SEM images of the biliary stents
    图3 FTIR分析 A:PCL和PCLMA的FTIR谱图;B:Gel和GelMA的FTIR谱图Fig.3 FTIR analysis A: FTIR spectra of PCL and PCLMA; B: FTIR spectra of Gel and GelMA
    图4 核磁共振氢谱图 A:PCL和PCLMA;B:Gel和GelMAFig.4 Proton nuclear magnetic resonance spectra A: PCL and PCLMA; B: Gel and GelMA
    图5 水凝胶支架压缩性能图 A-B:水凝胶的压缩应力-应变曲线图,用于评估其机械强度;C:压缩模量通过单轴压缩测试确定Fig.5 Compressive properties of the hydrogel scaffolds A-B: Compressive stress-strain curves of the hydrogels for evaluation of their mechanical strength; C: Compressive modulus determined by uniaxial compression testing
    图6 水凝胶支架材料的流变学性能 A:储能模量与时间的关系;B:储能模量与频率的关系Fig.6 Rheological properties of the hydrogel scaffolds A: Time-dependent storage modulus; B: frequency-dependent storage modulus
    图7 水凝胶支架体外降解性能图 A:水凝胶支架在降解第28天SEM图;B:水凝胶支架降解性能测试图Fig.7 In vitro degradation of the hydrogel scaffolds A: SEM images of the hydrogel scaffolds after 28 d of degradation; B: Degradation profiles of the hydrogel scaffolds
    图8 EGCG体外释放图 A:EGCG释放标准曲线;B:体外EGCG在MSN@USPIO@EGCG和GelMA/MSN@USPIO@EGCG的释放曲线Fig.8 In vitro release of EGCG A: Standard curve for EGCG quantification; B: in vitro release profiles of EGCG from MSN@USPIO@EGCG and GelMA/MSN@USPIO@EGCG
    图9 MSN@USPIO@EGCG的磁共振成像特性 A:MSN@USPIO@EGCG T2加权图像;B:样品的Fe浓度与1 000/T2(s-1)的直线关系Fig.9 MRI characteristics of MSN@USPIO@EGCG A: T2-weighted MRI images of MSN@USPIO@EGCG at different concentrations; B: linear relationship between Fe concentration and 1 000/T2 (s-1)
    图10 细胞相容性试验 A:活死细胞荧光染色图像;B:CCK-8细胞增殖活性统计图;C-D:hADSCs在24 h和48 h与不同材料共培养的细胞骨架染色图Fig.10 Cytocompatibility analysis A: Live/dead fluorescence staining images; B: quantitative analysis of cell proliferation assessed by the CCK-8 assay; C-D: Cytoskeletal staining of hADSCs cocultured with different materials for 24 and 48 h, respectively
    图11 溶血分析Fig.11 Hemolysis analysis
    图12 凝血分析 A:PT;B:APTTFig.12 Coagulation analysis A: PT; B: APTT
    图13 不同材料对hADSCs MMP水平的影响 A:采用JC-1染色法,直观地显示了不同材料对hADSCs MMP的影响(绿色荧光代表MMP降低,红色荧光代表MMP正常);B:各组MMP定量分析Fig.13 Effects of different materials on MMP in hADSCs A: Representative JC-1 staining images showing MMP in hADSCs (green fluorescence indicates JC-1 monomers associated with lower MMP, whereas red fluorescence indicates JC-1 aggregates associated with higher MMP); B: Quantitative analysis of MMP
    图14 细胞衰老检测图 A:细胞衰老形态图;B:各组SA-β-gal阳性细胞数定量分析Fig.14 Assessment of cellular senescence A: Representative images of cellular senescence morphology; B: quantitative analysis of the number of SA-β-gal positive cells
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彭柏铭,张潇予,冯龙宝,高元慧,张剑权,向杨.3D打印USPIO/EGCG功能化双层水凝胶胆道支架的构建及体外功能评价[J].中国普通外科杂志,2026,35(8):1591-1606.
DOI:10.7659/j. issn.1005-6947.250627

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  • 收稿日期:2025-11-10
  • 最后修改日期:2026-03-31
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  • 在线发布日期: 2026-09-29
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