脱细胞猪皮基质构建组织工程半月板支架  

Decellularized porcine skin matrix for tissue-engineered meniscus scaffold

作　　者：王皓 陈明学[2,3] 李俊康[2] 罗旭江 彭礼庆 李获 黄波 田广招 刘舒云 眭翔 黄靖香[2] 郭全义[2] 鲁晓波[1] Wang Hao;Chen Mingxue;Li Junkang;Luo Xujiang;Peng Liqing;Li Huo;Huang Bo;Tian Guangzhao;Liu Shuyun;Sui Xiang;Huang Jingxiang;Guo Quanyi;Lu Xiaobo(Department of Bone and Joint Surgery,Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China;Institute of Orthopedics,Chinese PLA General Hospital,Beijing Key Lab of Regenerative Medicine in Orthopedics,Key Laboratory of Musculoskeletal Trauma&War Injuries,PLA,Beijing 100853,China;Department of Orthopedics,Beijing Jishuitan Hospital,Beijing 100035,China)

机构地区：[1]西南医科大学附属医院骨与关节外科,四川省泸州市646000 [2]中国人民解放军总医院骨科研究所,骨科再生医学北京市重点实验室,全军骨科战创伤重点实验室,北京市100853 [3]北京积水潭医院矫形骨科,北京市100035

出　　处：《中国组织工程研究》2021年第22期3473-3478,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家重点研发计划(2017YFC1103400),项目参与者:刘舒云;国家自然科学基金(81972070),项目负责人:郭全义。

摘　　要：背景:半月板损伤的再生修复目前仍然是一个科学性难题,构建具有良好生物力学性能与细胞相容性的组织工程支架至关重要。目的:制备脱细胞猪皮基质支架,评价其生物力学性能和细胞相容性。方法:采用胰蛋白酶、Triton X-100、十二烷基磺酸钠组合对猪皮进行脱细胞处理,获得脱细胞猪皮基质,采用DAPI染色观察脱细胞效果,扫描电镜观察其微观结构。通过组织学染色、胶原和糖胺多糖定量及生物力学检测对比脱细胞猪皮基质与天然半月板的差异。将第3代骨髓间充质干细胞接种于脱细胞猪皮基质上,培养3 d后分别进行扫描电镜、死/活细胞染色和鬼笔环肽染色观察。结果与结论:①DAPI染色显示,脱细胞猪皮基质无细胞核结构;扫描电镜显示,脱细胞猪皮基质表面较为粗糙,表面纤维呈拓扑学结构;②组织学染色显示,天然半月板组织含有丰富的细胞外基质,半月板细胞在基质中均匀分布,胶原纤维致密且排列规则,胶原成分主要以Ⅰ型胶原纤维为主,含有糖胺多糖成分;脱细胞猪皮基质无细胞核,呈疏松的纤维条索状,有较多的孔隙结构,胶原成分主要以Ⅰ型胶原纤维为主,含有糖胺多糖成分;③脱细胞猪皮基质的胶原含量、拉伸弹性模量与天然半月板比较差异无显著性意义(P>0.05),糖胺多糖含量、压缩弹性模量低于天然半月板(P<0.05),并且脱细胞猪皮基质的生物力学性能足够承受膝关节的负荷、保持支架的完整;④扫描电镜显示,骨髓间充质干细胞黏附分布在猪皮基质的孔隙结构中,细胞呈椭圆形,聚集生长;⑤死/活细胞染色显示,脱细胞猪皮基质上的骨髓间充质干细胞具有较高的活性(94.0±3.8)%;⑥鬼笔环肽/DAPI染色显示,骨髓间充质干细胞在脱细胞猪皮基质上呈现出很好的铺展状;⑦结果表明,脱细胞猪皮基质与天然半月板具有类似的胶原成分,呈现出良好的生物力学性能和BACKGROUND:Regeneration and repair of the injured meniscus remain scientific challenges.It is of significance to construct a tissue-engineered scaffold with good biomechanical performance and cytocompatibility.OBJECTIVE:To prepare decellularized porcine skin matrix scaffold and evaluate its biomechanical properties and cytocompatibility.METHODS:Porcine skin was decellularized with the combination of trypsin,Triton X-100 and sodium dodecyl sulfate to obtain decellularized porcine skin matrix.DAPI staining was used to observe whether the cells were well removed and scanning electron microscope to observe its microstructure.The decellularized porcine skin matrix was compared with natural meniscus in term of histological staining,contents of collagen and glycosaminoglycans,and biomechanical properties.The third-generation bone marrow mesenchymal stem cells were seeded on the decellularized porcine skin matrix.After 3 days of culture,the scanning electron microscopy,dead/live cell staining and phalloidin staining were used to observe cytocompatibility of the decellularized porcine skin matrix.RESULTS AND CONCLUSION:(1)DAPI staining showed that no cell nucleus was found in the decellularized porcine skin matrix.Scanning electron microscopy showed that the surface of the decellularized porcine skin matrix was rough,and the surface fibers showed a topological structure.(2)Histological staining showed that the natural meniscus tissue was rich in extracellular matrix,and the meniscus cells were evenly distributed in the matrix.The collagen fibers were dense and arranged regularly,and the collagen composition was mainly type I collagen fibers and contained glycosaminoglycan components.The decellularized porcine skin matrix had no cell nucleus and was in the shape of loose fiber strips with more pore structure,and its collagen component was mainly type I collagen fiber and contained glycosaminoglycan components.(3)There was no significant difference in collagen content and tensile modulus between decellularized porcine skin

关 键 词：半月板 组织工程 脱细胞支架 猪皮基质 胶原 细胞外基质 

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