中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (34): 5436-5441.doi: 10.3969/j.issn.2095-4344.1439

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇    下一篇

人关节软骨细胞外基质来源组织工程支架的制备和评估

彭礼庆1，罗旭江1，张  彬1，沈  师1，黄  波1，高  超2，鲁晓波1，刘舒云2，郭全义1，2
  

  1. 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第一医学中心骨科研究所，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
  • 收稿日期:2019-06-10 出版日期:2019-12-08 发布日期:2019-12-08
  • 通讯作者: 郭全义，博士，主任医师，教授，博士生导师，西南医科大学附属医院骨与关节外科，四川省泸州市 646000；中国人民解放军总医院第一医学中心骨科研究所，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
  • 作者简介:彭礼庆，男，1987年生，四川省达州市人，汉族，西南医科大学在读硕士，主要从事关节软骨修复方向的研究。
  • 基金资助:

    国家重点研发计划(2018YFC1105900)，项目参与人：刘舒云；国家自然科学基金(81772319)，项目负责人：郭全义；北京自然科学基金(7172203)，项目负责人：郭全义

Preparation and evaluation of human articular cartilage-derived extracellular matrix tissue engineering scaffold

Peng Liqing1, Luo Xujiang1, Zhang Bin1, Shen Shi1, Huang Bo1, Gao Chao2, Lu Xiaobo1, Liu Shuyun2, Guo Quanyi1, 2
  

  1. 1Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Institute of Orthopedics, First Medical Center, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries, PLA, Beijing 100853, China
  • Received:2019-06-10 Online:2019-12-08 Published:2019-12-08
  • Contact: Guo Quanyi, MD, Chief physician, Professor, Doctoral supervisor, Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Institute of Orthopedics, First Medical Center, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries, PLA, Beijing 100853, China
  • About author:Peng Liqing, Master candidate, Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:

    National R & D Program of China, No. 2018YFC1105900 (project participant: LSY); the National Natural Science Foundation of China, No. 81772319 (to GQY); the Natural Science Foundation of Beijing, No. 7172203 (to GQY)

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摘要：

文章快速阅读：

 

文题释义：
细胞外基质：通常是指存在组织中、细胞周围的由多种大分子物质构成的具有维持细胞形状、结构，影响细胞增殖、分化及发挥生物学功能的物质。
人关节软骨来源的细胞外基质(HAC-ECM)组织工程支架：HAC-ECM支架采用物理粉碎、不等速离心、冷冻干燥法制备。物理粉碎法不使用有毒性的化学物质而使用机械力将组织粉碎成匀浆，其一可使细胞充分暴露，采用不等速离心法可使不同大小的组织块逐步分离，尽量保留其细胞外基质成分；其二液体匀浆性质易于塑形以构建支架物理形态。冷冻干燥法制备的HAC-ECM支架孔隙率高并为细胞分布提供方向，可使其具有与天然软骨组织同向的软骨纤维和软骨细胞排列方式，在架构上已达到仿生目的，为修复的软骨组织提供了负重结构基础。
 
 
背景：软骨细胞外基质作为一种天然材料，为依赖组织生长的细胞提供了理想的微环境，已成为一种理想的软骨修复支架材料。
目的：将人关节软骨来源的细胞外基质制备成软骨组织工程支架，评价其理化性质及生物学性能。
方法：收集人源性关节软骨组织，予粉碎成匀浆、去细胞处理、冷冻干燥法制备成软骨组织工程支架。观察：①扫描电镜观察支架形态；②支架的孔隙率、吸水率测定，测量支架力学性质；③支架的成分：使用组织化学和免疫组织化学法染色观察；④支架细胞毒性：用人软骨细胞外基质支架浸提液与常规细胞培养液做对比，培养兔软骨细胞不同时间后使用MTT比色法检测；⑤细胞黏附及增殖情况：使用扫描电镜及共聚焦显微镜观察人软骨细胞与支架共培养后细胞黏附增殖情况，支架细胞复合体切片染色观察。
结果与结论：①制备而成的支架呈三维多孔海绵状纵行取向形态，支架孔隙周围有软骨纤维，支架苏木精-伊红染色未见细胞核、番红O及天狼星红染色均证实支架含有胶原和软骨基质成分；②支架的孔隙率为(91.8±2.9)%、吸水率为(93.5±1.4)%；③人软骨细胞外基质支架浸提液经MTT检测，对软骨无细胞毒性；④共培养后，人软骨细胞在支架孔隙周壁上黏附、增殖且分布均匀。结果表明，人软骨细胞外基质多孔取向支架的成分与天然软骨近似，结构可供细胞均匀黏附、增殖良好，组织相容性好，可作为一种组织工程修复软骨缺损的支架材料。

关键词: 组织工程, 细胞外基质, 支架, 软骨, 同种异体

Abstract:

BACKGROUND: Cartilage-derived extracellular matrix as a natural material provides an ideal microenvironment for cells that depend on tissue growth and has become an ideal scaffold material for cartilage repair.
OBJECTIVE: To prepare human articular cartilage-derived extracellular matrix as a cartilage tissue engineering scaffold and to evaluate its physicochemical properties and biological properties.
METHODS: The human articular cartilage tissue was collected, pulverized into homogenate, decellularized, and freeze-dried to prepare a cartilage tissue engineering scaffold. Scaffold morphology was observed under scanning electron microscope. The porosity and water absorption and mechanical properties of the cartilage tissue engineering scaffold were determined. The composition of tissue engineering scaffold was observed by histochemistry and immunohistochemistry. Rabbit chondrocytes were cultured with the leaching liquor of human cartilage-derived extracellular matrix tissue engineering scaffold for different time periods. Routine cell culture fluid was used as control. The cytotoxicity of tissue engineering scaffold was detected by MTT colorimetry. Human chondrocytes and tissue engineering scaffold were co-cultured and cell adhesion and proliferation were observed under the scanning electron microscope and confocal microscope. The cell-scaffold complex sections were stained by histochemistry and immunohistochemistry.
RESULTS AND CONCLUSION: The prepared scaffold had three-dimensional porous sponge-like longitudinally oriented structure. There were cartilage fibers around the scaffold pores. After hematoxylin-eosin staining, no nucleus was observed. Both safranin O and Sirius red staining confirmed that cartilage tissue engineering scaffold contained collagen and cartilage matrix. The porosity and water absorption of the scaffold was (91.8±2.9)% and (93.5±1.4)%, respectively. MTT results showed that the leaching liquor of human cartilage-derived extracellular matrix was non-toxic to chondrocytes. After co-culture, human chondrocytes adhered, proliferated and evenly distributed on the peripheral wall of the scaffold pores. The results showed that human articular cartilage-derived extracellular matrix had similar composition to natural cartilage, provided the structure suitable for cell adhesion and proliferation, and exhibited good histocompatibility. Therefore, human articular cartilage-derived extracellular matrix can be used as a scaffold material for repairing cartilage defects by tissue engineering technique.

Key words: tissue engineering, extracellular matrix, scaffold, cartilage, allogenic

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