干细胞工程化双面异性静电纺丝膜促进硬脊膜修复的体外实验  

In vitro experiment of stem cell engineered two-sided anisotropic electrospun membranes for promoting dural repair

作　　者：徐敬之 王文博 孙慧雯 顾勇[1] Xu Jingzhi;Wang Wenbo;Sun Huiwen;Gu Yong(Department of Orthopedics,First Affiliated Hospital of Soochow University,Suzhou 215000,Jiangsu Province,China)

机构地区：[1]苏州大学附属第一医院骨科,江苏省苏州市215000

出　　处：《中国组织工程研究》2024年第10期1540-1546,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(82072438);项目负责人:顾勇;江苏省杰出青年基金(BK20211504);项目负责人:顾勇。

摘　　要：背景:目前临床上采用自体组织或明胶海绵等材料对硬脊膜进行修补,但都存在不同程度的缺陷,因此亟需一种可以促进硬脊膜修复的生物材料。目的:利用定向静电纺丝及胶原自组装技术构建双面异性静电纺丝膜,并将其负载骨髓间充质干细胞构建人工硬脊膜,探讨该人工硬脊膜的各项理化性能以及生物特性。方法:采用静电纺丝技术及胶原自组装技术制备有序聚乳酸静电纺丝纤维的双面(一面为胶原蛋白,另一面为聚乳酸)异性静电纺丝膜(胶原组)、无序的聚乳酸静电纺丝膜(无序纤维组)、有序定向的聚乳酸静电纺丝膜(有序纤维组),通过扫描电镜、力学拉伸、水接触角测试、降解实验等来表征静电纺丝膜的理化性能。将胶原组(在胶原蛋白面接种骨髓间充质干细胞,即得到干细胞工程化静电纺丝膜)、无序纤维组、有序纤维组静电纺丝膜分别与骨髓间充质干细胞共培养,采用CCK-8法和活/死染色评估静电纺丝膜的生物相容性,整合素β1免疫荧光染色评估静电纺丝膜的黏附特性。将干细胞工程化静电纺丝膜及胶原组静电纺丝膜分别与骨髓巨噬细胞共培养,通过诱导型一氧化氮合酶(M1型)、CD206(M2型)免疫荧光染色及qRT-PCR检测炎症相关基因的表达来评估免疫调控性能。结果与结论:①定向静电纺丝纤维膜可模拟天然硬脊膜的纵向排列结构,胶原蛋白加入后,纤维膜的亲水性提高约2倍,力学性能提升了1.2倍;②与骨髓间充质干细胞共培养时,CCK-8和活/死染色显示,胶原组静电纺丝膜的细胞生物活性明显高于无序纤维组、有序纤维组;免疫荧光染色显示,胶原组整合素β1表达约为无序纤维组、有序纤维组的2.6倍,且细胞铺展形态良好;③与骨髓巨噬细胞共培养时,免疫荧光染色显示,干细胞工程化静电纺丝膜组M1型巨噬细胞荧光强度低于胶原组(P<0.01),M2型巨噬细胞荧光强度高于�BACKGROUND:Currently,the dura mater is clinically repaired using autologous tissue or materials such as gelatin sponge,but all of them have their inherent defects.Therefore,there is an urgent need for a biomaterial that can promote dural repair.OBJECTIVE:The two-sided anisotropic electrospun membrane was constructed by using directional electrospinning technology and collagen self-assembly technology,and was used as a carrier for bone marrow mesenchymal stem cells to investigate various physicochemical properties and biological characteristics of the artificial dura mater.METHODS:Ordered polylactic acid electrospun fibers with double-sided(collagen protein on one side and polylactic acid on the other side)anisotropic electrospun membranes(collagen group),disordered polylactic acid electrospun membranes(disordered fiber group),and ordered oriented polylactic acid electrospun membranes(ordered fiber group)were prepared by electrospinning technique as well as collagen self-assembly technique.Scanning electron microscopy,mechanical stretching,water contact angle testing,and degradation experiments were used to characterize the physicochemical properties of the electrospun membranes.Electrospun membranes in the collagen group(bone marrow mesenchymal stem cells were inoculated on the collagen surface to obtain the stem cell-engineered electrospun membranes),disordered fiber group and ordered fiber group were cocultured with bone marrow mesenchymal stem cells.The biocompatibility of electrospun membranes was evaluated using CCK-8 assay and live/dead staining.Integrinβ1 immunofluorescence staining was used to evaluate the adhesion characteristics of electrospun membranes.The stem cell-engineered electrospun membrane and the electrospun membrane in the collagen group were cocultured with bone marrow macrophages respectively.Immunomodulatory properties were assessed by detecting the expression of inflammation-related genes using inducible nitric oxide synthase(M1 type),CD206(M2 type)immunofluorescence staining,and qRT-PCR

关 键 词：硬脊膜修补 人工硬脊膜 静电纺丝 胶原蛋白 骨髓间充质干细胞 聚乳酸 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学] R686.3