中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (27): 4283-4287.doi: 10.12307/2022.857

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

丝素蛋白/Ⅱ型胶原/羟基磷灰石三相复合体支架的生物相容性

孙  凯1，李瑞欣2，高丽兰3，范  猛1，张西正4，李  晖5   

  1. 1天津市第一中心医院，天津市  300192；2军事医学科学院卫生装备研究所，天津市  300161；3天津理工大学机械工程学院，天津市  300382；4解放军军事科学院系统工程研究院，卫勤保障技术研究所，天津市  300161；5天津医科大学总医院，天津市  300070
  • 收稿日期:2021-08-13 接受日期:2021-11-03 出版日期:2022-09-28 发布日期:2022-03-12
  • 通讯作者: 李瑞欣，副研究员，军事医学科学院卫生装备研究所，天津市 300161
  • 作者简介:孙凯，男，1989年生，山东省济宁市人，汉族，天津医科大学在读博士，主治医师，主要从事骨关节及生物材料研究。
  • 基金资助:
    天津市自然科学基金(20JCYBJC00990)，课题名称：新型拓扑结构外周神经导管大动物实验研究，项目负责人：范猛

Biocompatibility of silk fibroin/type II collagen/hydroxyapatite three-phase composite scaffold

Sun Kai1, Li Ruixin2, Gao Lilan3, Fan Meng1, Zhang Xizheng4, Li Hui5   

  1. 1Tianjin First Central Hospital, Tianjin 300192, China; 2Institute of Medical Equipment, PLA Academy of Military Medical Sciences, Tianjin 300161, China; 3School of Mechanical Engineering, Tianjin University of Technology, Tianjin 300382, China; 4Institute of System Engineering, PLA Academy of Military Sciences, Institute of Health Support Technology, Tianjin 300161, China; 5General Hospital, Tianjin Medical University, Tianjin 300070, China
  • Received:2021-08-13 Accepted:2021-11-03 Online:2022-09-28 Published:2022-03-12
  • Contact: Li Ruixin, Associate researcher, Institute of Medical Equipment, PLA Academy of Military Medical Sciences, Tianjin 300161, China
  • About author:Sun Kai, Doctoral candidate, Attending physician, Tianjin First Central Hospital, Tianjin 300192, China
  • Supported by:
    Tianjin Natural Science Foundation, No. 20JCYBJC00990 (to FM)

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摘要：

文题释义：
三相复合体：既往打印采用超声波粉碎羟基磷灰石，均匀分布于打印基质中，适合软骨层修复，但对于中间层及软骨下骨层没有明确条件设置。此次研究采用不均匀分布打印，提供了一种三相复合软骨支架，包括从上至下依次层叠排布的软骨支架层、钙化支架层及软骨下骨支架层。
双相支架：依据天然骨软骨组织的生物结构，有研究员设计和制备了包含软骨层和成骨层在内的一体化双相支架，用于同时修复软骨和软骨下骨。但是双相支架在设计上并没有复合中间层这一理念，即存在于天然软骨与软骨下骨支架的软骨钙化层，致使骨软骨界面存在结合欠佳，在体内研究中可出现结合部位分离的现象，导致修复效果不理想。

背景：为了同时修复软骨、软骨钙化层和骨组织，需要设计一种多相支架，加入模仿天然骨软骨组织软骨钙化层的中间层。三相支架的3层结构可以同时重建软骨、钙化软骨和软骨下骨，实现骨软骨修复的一体化。
目的：构建丝素蛋白/Ⅱ型胶原/羟基磷灰石三相复合体，研究其理化性质和生物相容性。
方法：通过不均匀沉降技术，采用低温3D打印构建丝素蛋白/Ⅱ型胶原/羟基磷灰石三相复合体，检测复合体的孔隙率、吸水膨胀率及力学性能。将小鼠软骨细胞ADTC-5接种于复合支架表面，扫描电镜下观察软骨细胞在支架上的分布、黏附和伸展形态，MTT法检测细胞增殖，苏木精-伊红染色观察细胞-支架复合体形态。
结果与结论：①复合支架具有延展性和黏弹性，孔隙率为(97.7±1.5)%，吸水膨胀率为(1 410.95±66.55)%，弹性模量为(35.3±2.5) kPa；扫描电镜下可见复合体孔隙网状结构，各孔隙连通性好，大孔径直径(210.5±20.5) μm，小孔径直径(75.5±16.5) μm，孔隙率为(97.7±1.5)%，平均壁厚(92.5±18.5) μm；②共培养3 d后，扫描电镜下可见软骨细胞黏附在支架内部孔隙中，并分泌出大量细胞外基质，细胞形态明显；③MTT实验显示，随着培养时间的延长，支架上的软骨细胞不断增殖；④苏木精-伊红染色显示，共培养3 d后，软骨细胞沿着支架材料内部空隙贴壁生长，形态呈梭形，分布较分散，数量较少；共培养14 d后，细胞数量明显增加，呈集落样生长，可明显看到细胞间连接；⑤结果表明，利用不均匀沉降技术制备的丝素蛋白/Ⅱ型胶原/羟基磷灰石复合体具有良好的理化性能及生物相容性。

https://orcid.org/0000-0002-9432-8386 (孙凯) 

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料;口腔生物材料;纳米材料;缓释材料;材料相容性；组织工程

关键词: 三相复合体, 不均匀, 沉降, 打印, 构建, 理化性质, 生物相容性

Abstract: BACKGROUND: To repair cartilage, cartilage calcification layer, and bone tissue at the same time, it is necessary to design a heterogeneous scaffold and add an intermediate layer that mimics the cartilage calcification layer of natural bone cartilage tissue. The three-layer structure of three-phase scaffold can reconstruct cartilage, calcified cartilage, and subchondral bone at the same time, so as to realize the integration of bone and cartilage repair.
OBJECTIVE: To construct silk fibroin/type II collagen/hydroxyapatite three-phase composite, and to study its physicochemical properties and biocompatibility. 
METHODS: Silk fibroin/type II collagen/hydroxyapatite three-phase composite was constructed by uneven sedimentation and low-temperature 3D printing. The porosity, water absorption expansion rate, and mechanical properties of the composite were detected. Mouse chondrocyte ADTC-5 was seeded on the surface of the scaffold. The distribution, adhesion, and extension of chondrocytes on the scaffold were observed under a scanning electron microscope. MTT assay was used to detect cell proliferation. Hematoxylin-eosin staining was used to observe the cell-scaffold composite morphology. 
RESULTS AND CONCLUSION: (1) The composite scaffold was malleable and viscoelastic, with a porosity of (97.7±1.5)%, a water absorption expansion rate of (1 410.95±66.55)%, and an elastic modulus of (35.3±2.5) kPa. Under the scanning electron microscope, the pore network structure of the composite could be seen, with good connectivity of each pore, large pore diameter of (210.5±20.5) μm, small pore diameter of (75.5±16.5) μm, porosity (97.7±1.5)%, and average wall thickness (92.5±18.5) μm. (2) After 3 days of culture, chondrocytes adhered to the internal pores of the scaffold and secreted a large amount of extracellular matrix under the scanning electron microscope, and the cell morphology was distinct. (3) MTT assay exhibited that with the extension of the culture time, the chondrocytes on the scaffold continued to proliferate. (4) Hematoxylin-eosin staining demonstrated that after 3 days of co-culture, chondrocytes grew adherently along the internal voids of the scaffold material, with a fusiform shape, dispersed distribution and a small number. After 14 days of co-culture, the number of cells increased significantly. Cells grew like a colony, and the connections between cells could be clearly seen. (5) These findings verify that silk fibroin/type II collagen/hydroxyapatite composite prepared by uneven sedimentation technology has good physical and chemical properties and biocompatibility. 

Key words: three-phase composite, uneven, sedimentation, printing, construction, physical and chemical properties, biocompatibility

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