中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (22): 3521-3527.doi: 10.12307/2022.280

• 组织工程骨材料Tissue-engineered bone • 上一篇    下一篇

制备具有促成骨作用改性葡聚糖复合水凝胶的体外实验

唐振州，顾  勇，陈  亮   

  1. 苏州大学附属第一医院骨科，江苏省苏州市   215008
  • 收稿日期:2020-12-07 修回日期:2021-01-23 接受日期:2021-06-27 出版日期:2022-08-08 发布日期:2022-01-11
  • 通讯作者: 陈亮，主任医师，教授，博士生导师，苏州大学附属第一医院骨科，江苏省苏州市 215008
  • 作者简介:唐振州，男，1994年生，江苏省苏州市人，汉族，苏州大学附属第一医院在读硕士，主要从事骨修复材料研究。
  • 基金资助:
    国家自然科学基金项目(81972078，81772312)，项目负责人：陈亮；国家青年科学基金(81601891)，项目负责人：顾勇；苏州市临床重点病种诊疗技术专项(LCZX201701)，项目负责人：陈亮

Preparation of modified dextran composite hydrogel with osteogenetic effect and in vitro experiment

Tang Zhenzhou, Gu Yong, Chen Liang   

  1. Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215008, Jiangsu Province, China
  • Received:2020-12-07 Revised:2021-01-23 Accepted:2021-06-27 Online:2022-08-08 Published:2022-01-11
  • Contact: Chen Liang, Chief physician, Professor, Doctoral supervisor, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215008, Jiangsu Province, China
  • About author:Tang Zhenzhou, Master candidate, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215008, Jiangsu Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81972078, 81772312 (to CL); the National Youth Science Fund, No. 81601891 (to GY); the Special Diagnosis and Treatment Technology for Key Clinical Diseases in Suzhou, No. LCZX201701 (to CL)

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摘要：

文题释义：
葡聚糖：是一种生物性的多糖，其主链上还有很多游离的羟基，易于被修饰和交联。葡聚糖水凝胶具有良好的溶胀能力、可降解性和生物相容性等特点，因此被广泛应用于生物医学工程。
介孔生物活性玻璃：是一种骨科常用的无机材料，主要构成元素为硅、钙、磷，通常具有纳米尺度的孔径，其在生理条件下可释放硅钙离子，促进骨组织再生。

背景：葡聚糖水凝胶因具有良好的生物相容性、可注射性等优势成为热门的组织工程材料，被广泛运用于硬组织、软组织的修复中，然而在骨缺损的修复中，单纯的葡聚糖水凝胶在成骨方面仍存在促成骨能力不足的问题。
目的：构建葡聚糖和改性介孔生物活性玻璃的复合水凝胶材料，对其进行表征，同时对其细胞黏附和体外促成骨能力进行研究。
方法：通过高碘酸钠对葡聚糖进行氧化改性使其富含醛基，对介孔生物活性玻璃进行氨基化改性，将氨基化的介孔生物活性玻璃粉末加入氧化葡聚糖溶液中混合均匀，再加入明胶溶液制得氧化葡聚糖-氨基化介孔生物活性玻璃纳米颗粒(dextrangel-aminated mesoporous bioactive glass nanoparticles，GelDex-AMBGN)复合水凝胶。将骨髓间充质干细胞分别接种于氧化葡聚糖水凝胶与GelDex-AMBGN复合水凝胶中，以单纯培养的细胞为对照，通过Live/Dead荧光染色、扫描电镜与CCK-8实验检测细胞生物相容性，通过碱性磷酸酶染色、茜素红染色与骨钙素免疫荧光染色检测成骨性能。
结果与结论：①共培养3 d后的Live/Dead荧光染色显示，两组水凝胶中的骨髓间充质干细胞生长良好，保持了较高的细胞活性，细胞存活率均高于90%；②共培养3 d后的扫描电镜显示，骨髓间充质干细胞在两种水凝胶表面铺展良好；③CCK-8实验显示，培养1，3，5，7 d时，3组细胞增殖活性比较差异无显著性意义(P > 0.05)；④复合水凝胶组共培养7 d的碱性磷酸酶染色强于氧化葡聚糖水凝胶组、对照组，共培养21 d的钙含量高于氧化葡聚糖水凝胶组、对照组(P < 0.05)，共培养21 d的骨钙素免疫荧光染色和钙含量检测强于氧化葡聚糖水凝胶组、对照组；⑤结果表明，GelDex-AMBGN复合水凝胶具有良好的生物相容性与促成骨能力。
缩略语：氧化葡聚糖-氨基化介孔生物活性玻璃纳米颗粒：dextrangel-aminated mesoporous bioactive glass nanoparticles，GelDex-AMBGN

https://orcid.org/0000-0002-8046-6358 (唐振州) 

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: 水凝胶, 葡聚糖, 氧化葡聚糖, 介孔生物活性玻璃, 生物相容性, 成骨, 骨缺损

Abstract: BACKGROUND: Dextran hydrogel has become a popular tissue engineering material because of its good biocompatibility and injectability, and is extensively used in the repair of hard and soft tissues. However, in the repair of bone defects, pure dextran hydrogel still has insufficient bone formation ability in osteogenesis.  
OBJECTIVE: To construct a hydrogel material with dextran and modified mesoporous bioactive glass, characterize the material, and investigate its cell adhesion and bone formation ability in vitro.
METHODS: The dextran was oxidized and modified by sodium periodate to make it rich in aldehyde groups. After aminating and modifying mesoporous bioactive glass, the aminated mesoporous bioactive glass powder was added to the oxidized dextran solution and mixed evenly. Gelatin solution was added to prepare dextrangel-aminated mesoporous bioactive glass nanoparticles (GelDex-AMBGN) composite hydrogel. The bone marrow mesenchymal stem cells were separately inoculated in the oxidized dextran hydrogel and GelDex-AMBGN composite hydrogel. Simply cultured cells were consisted as controls. The cell biocompatibility was detected by Live/Dead fluorescent staining, scanning electron microscope, and CCK-8 assay. The Osteogenic property was detected by alkaline phosphatase staining, alizarin red staining, and immunofluorescence staining for osteocalcin.  
RESULTS AND CONCLUSION: (1) Live/Dead fluorescence staining results after co-culture for 3 days displayed that bone marrow mesenchymal stem cells in the two hydrogels grew well, maintained high cell viability, and the cell survival rate was higher than 90%. (2) After co-culture for 3 days, scanning electron microscopy results exhibited that bone marrow mesenchymal stem cells spread well on the surface of the two hydrogels. (3) CCK-8 assay results demonstrated that there was no significant difference in the cell proliferation viability of the three groups at 1, 3, 5, and 7 days after culture (P > 0.05). (4) The alkaline phosphatase staining showed stronger results in the composite hydrogel group compared with the oxidized dextran hydrogel and control groups after co-culture for 7 days. The calcium content after co-culture for 21 days was higher in the composite hydrogel group than that of the oxidized dextran hydrogel and control groups (P < 0.05). The immunofluorescence staining for osteocalcin and calcium content detection after co-culture for 21 days revealed stronger results in the composite hydrogel group compared with the oxidized dextran hydrogel group and the control group. (5) The results indicate that GelDex-AMBGN composite hydrogel has good biocompatibility and the osteogenesis ability.

Key words: hydrogel, dextran, oxidized dextran, mesoporous bioactive glass, biocompatibility, osteogenesis, bone defect

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