中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (8): 1508-1512.doi: 10.3969/j.issn.1673-8225.2010.08.043

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

以海藻酸盐及异种骨复合技术制备组织工程化骨及体内成骨

孙  磊1,2，孟国林2，陈  磊1，陶剑锋1，江  健1，张柏青3，窦榆生2，徐建强2，刘丹平2，胡蕴玉2，张仲文3   

  1. 1 北京积水潭医院，北京市  100035；2 解放军第四军医大学全军创伤骨科研究所，陕西省西安市  710032；3 武警总医院，北京市  100039
  • 出版日期:2010-02-19 发布日期:2010-02-19
  • 作者简介:孙 磊☆，男，1966年生，黑龙江省牡丹江市人，汉族，2001年解放军第四军医大学毕业，博士，副研究员，主要从事骨组织工程、骨移植相关研究。
  • 基金资助:

    中国博士后研究基金资助项目(199910) ；北京市科技新星培养计划资助项目(H013610310113)。

Preparation of tissue engineered bone and in vivo osteogenesis using alginate and xenograft bone composite technology

Sun Lei 1,2, Meng Guo-lin2, Chen Lei1, Tao Jian-feng1, Jiang Jian1, Zhang Bo-qing3, Dou Yu-sheng2,  Xu Jian-qiang2, Liu Dan-ping2, Hu Yun-yu2, Zhang Zhong-wen3   

  1. 1 Beijing Jishuitan Hospital, Beijing   100035, China; 2 Orthopedic Trauma Research Institute, Fourth Military Medical University of Chinese PLA, Xi’an   710032, Shaanxi Province, China; 3 Chinese People’s Armed Police General Hospital, Beijing   100039, China
  • Online:2010-02-19 Published:2010-02-19
  • About author:Sun Lei☆, Doctor, Associate researcher, Beijing Jishuitan Hospital, Beijing 100035, China; Orthopedic Trauma Research Institute, Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China
  • Supported by:

    Chinese Post-doctoral Research Fund, No. 199910*; Beijing Technology Star Cultivation Program, No.H013610310113*

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摘要：

背景：海藻酸有相对温和的凝胶条件与良好的生物相容性，已广泛应用于生物组织工程。
目的：采用海藻酸钠凝胶复合异种骨的方法，构建骨组织工程载体，观察载体中细胞的生物性能及体内成骨能力。
方法：取2 只2 周龄新西兰兔的骨髓，以1×10-8 mol/L重组人骨形态发生蛋白2诱导骨髓间充质干细胞。取诱导后第2 代骨髓间充质干细胞接种于1%海藻酸钠凝胶中，培养4 d 苏木精-伊红染色观察凝胶中细胞形态。将第2 代骨髓间充质干细胞分为单纯DMEM凝胶组和含1%海藻酸钠的DMEM凝胶组，分别培养7 d 后行骨形态发生蛋白2免疫组织化学染色观察。取24 只裸鼠，随机分为2 组，于两侧股部肌袋中分别植入骨髓间充质干细胞/海藻酸钠凝胶/牛松质骨复合体作为实验组，骨髓间充质干细胞/牛松质骨复合体作为对照组。术后2，4 周后组织学观察复合体成骨情况，图像分析系统分析各组成骨或软骨的面积百分比。
结果与结论：海藻酸钠凝胶中骨髓间充质干细胞形态饱满，细胞悬浮于凝胶中，可见细胞分裂和核分裂相。单纯DMEM凝胶组和含1% 海藻酸钠的DMEM凝胶组免疫组织化学观察，细胞分裂增殖正常，伸出多种形态的突起，胞核大，核仁清晰。单纯DMEM凝胶组和含1%海藻酸钠的DMEM凝胶组的骨形态发生蛋白2 表达阳性率差异无显著性意义(P > 0.05)。扫描电镜观察海藻酸钠凝胶均匀地复合于牛松质骨微孔中，不同平面均有细胞生长。动物实验显示术后2，4 周实验组和对照组的成骨或软骨的面积百分比差异有显著性意义（P < 0.05）。提示以海藻酸钠凝胶/牛松质骨构建骨组织工程载体，合乎组织工程载体的超结构原理，能最大限度地承载细胞，生物性能好，对骨髓间充质干细胞增殖和成骨表型及相关的生物性能无不良影响，在体内成骨效率较高。

关键词: 组织工程骨, 海藻酸钠凝胶, 异种骨, 载体, 支架材料

Abstract:

BACKGROUND: Alginic acid has a relatively mild gel condition and good biocompatibility, and it has been widely used in bio-tissue engineering.
OBJECTIVE: To construct bone tissue engineering scaffolds using alginate gel composite bone xenograft approach, and to observe the cell biological properties and in vivo osteogenic potential in scaffolds.
METHODS: The bone marrow was harvested from two 2-week-old New Zealand rabbits, 1×10-8 mol/L recombinant human bone morphogenetic protein-2 was used to induce bone marrow mesenchymal stem cells. The induced bone marrow mesenchymal stem cells at the second generation were incubated into 1% sodium alginate gel, after cultured for 4 days, the cell morphology in gel was observed by hematoxylin-eosin staining. Bone marrow mesenchymal stem cells at the second generation were divided into simple DMEM gel group and DMEM containing 1% sodium alginate gel group, followed by a culture of 7 days. Then bone morphogenic protein-2 immunohistochemical staining was performed. A total of 24 nude mice were randomly divided into two groups, both sides of the thigh muscle pockets were implanted with bone marrow-derived mesenchymal stem cells/alginate gel/bovine cancellous bone complex as an experimental group, with bone marrow-derived mesenchymal stem cells/bovine cancellous bone as a control group. At 2 and 4 weeks post-operation, the osteogenesis in the composite was observed by histological examination, the percentage area of new bone or cartilage was determined using image analysis system.
RESULTS AND CONCLUSION: The bone marrow-derived mesenchymal stem cells in the sodium alginate gel exhibited a well-stacked morphology, they suspended in a gel, showing cell division and mitosis phase. In the simple DMEM gel group and DMEM gel containing 1% sodium alginate group, the immunohistochemical results showed that, cell division and proliferation were normal, with prominence at a variety of forms, large nucleus, and clear nucleolus. The bone morphogenetic protein-2 expression had no significant difference between the simple DMEM gel group and DMEM gel containing 1% sodium alginate group (P > 0.05). Scanning electron microscopy revealed that, the alginate gel evenly composited in bovine cancellous bone micropores, cell grew at different planes. Animal experiments showed that there were significant differences regarding the percentage of new bone or cartilage area between the experimental group and control group at 2 and 4 weeks postoperation (P < 0.05). It is indicated that constructing bone tissue engineering scaffolds by using alginate gel/bovine cancellous bone, complies with the ultra-structural principle of tissue engineering scaffolds, can maximize the cell loads, achieve good bio-performance, without adverse affects on the proliferation, osteogenic phenotype and related biological properties of bone marrow-derived mesenchymal stem cells, the in vivo osteogenic efficiency was high. 

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