中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (2): 228-233.doi: 10.3969/j.issn.2095-4344.0011

• 纳米生物材料 nanobiomaterials • 上一篇    下一篇

可注射性纳米羟基磷灰石/壳聚糖复合支架与骨髓间充质干细胞、骨形态发生蛋白2修复骨缺损的体外实验

刘广涛，高  峰，徐  军，郑伟卓，林效宗，周长林，郭亚山，田  军
  

  1. 哈尔滨医科大学附属第二医院骨科九病房，黑龙江省哈尔滨市  150086
  • 收稿日期:2017-08-01 出版日期:2018-01-18 发布日期:2018-01-18
  • 通讯作者: 田军，主任医师，哈尔滨医科大学附属第二医院骨九科病房，黑龙江省哈尔滨市 150086
  • 作者简介:刘广涛，男，1987年生，汉族，硕士，主要从事骨组织工程研究。
  • 基金资助:
    黑龙江省教育厅科学技术研究项目(12541333)

Injectable nano-hydroxyapatite/chitosan composite scaffolds combined with bone marrow mesenchymal stem cells and bone morphogenetic protein 2 for bone defect repair in vitro  

Liu Guang-tao, Gao Feng, Xu Jun, Zheng Wei-zhuo, Lin Xiao-zong, Zhou Chang-lin, Guo Ya-shan, Tian Jun
  

  1. Ninth Ward, Department of Orthopedic Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China
  • Received:2017-08-01 Online:2018-01-18 Published:2018-01-18
  • Contact: Tian Jun, Chief physician, Ninth Ward, Department of Orthopedic Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China
  • About author:Liu Guang-tao, Master, Ninth Ward, Department of Orthopedic Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China
  • Supported by:
    the Science and Technology Research Project of Heilongjiang Provincial Education Department, No. 12541333

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摘要：

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文题释义：
可注射性纳米羟基磷灰石/壳聚糖复合支架：纳米羟基磷灰石不但是骨骼中主要的无机成分，并且其具有纳米级直径与骨磷灰石大小相当，是很好的骨修复材料。壳聚糖是具有正电荷的天然碱性多糖，因而具有较强的吸附作用，在常温(25-38 ℃)下具有凝胶的特点，即注射性。将纳米羟基磷灰石和壳聚糖复合在一起，使二者的优势叠加，既具有纳米特性又有可注射性，达到了很强的互补作用，既安全又方便。
理想的可注射性骨组织工程材料：应具有以下特点：①良好的注射性及可塑性；②良好的骨传导及诱导活性；③可降解性，为新骨的形成提供空间；④达到要求的机械强度；⑤合理的结构及孔隙率；⑥良好的生物安全性。
 
背景：骨形态发生蛋白2对间充质干细胞的分化及成骨能力有很强的诱导和促进作用。
目的：评估骨形态发生蛋白2对骨髓间充质干细胞在可注射性纳米羟基磷灰石/壳聚糖复合支架上增殖及成骨分化的影响。
方法：①实验1：取生长良好的第3代骨髓间充质干细胞，分2组培养，实验组与可注射性纳米羟基磷灰石/ 壳聚糖复合支架共培养，对照组单独培养骨髓间充质干细胞，培养1，3，5，7，14 d后，CCK-8法检测细胞增殖；培养7 d后，扫描电镜观察细胞在支架上的黏附情况；②实验2：取生长良好的第3代骨髓间充质干细胞，接种于可注射性纳米羟基磷灰石/壳聚糖复合支架表面，分2组培养，实验组加入含骨形态发生蛋白2的细胞培养基，对照组不加入，培养3，6，9，12，15 d，检测细胞碱性磷酸酶活性；培养1，3，5，7，14 d，CCK-8法检测细胞增殖；培养1，2周，茜素红染色观察成骨分化情况。
结果与结论：①实验1：随着培养时间的延长，两组细胞增殖A值逐渐增加，两组间A值比较差异无显著性意义；培养7 d后，骨髓间充质干细胞紧紧黏附于支架表面；②实验2：随着培养时间的延长，两组碱性磷酸酶活性逐渐升高，实验组培养不同时间点的碱性磷酸酶活性高于对照组(P < 0.05)；随着培养时间的延长，两组细胞增殖A值逐渐升高，实验组培养不同时间点的A值高于对照组(P < 0.05)；实验组培养1，2周的钙结节数多于对照组；③结果表明，骨形态发生蛋白可促进骨髓间充质干细胞在可注射性纳米羟基磷灰石/壳聚糖复合支架上的增殖与成骨分化。

关键词: 生物材料, 可注射性, 纳米羟基磷灰石/壳聚糖, 骨形态发生蛋白2, 骨髓间充质干细胞, 组织工程

Abstract:

BACKGROUND: Bone morphogenetic protein 2 (BMP-2) has a strong ability to induce and promote the osteogenic differentiation of mesenchymal stem cells.
OBJECTIVE: To evaluate the BMP-2 effect on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on an injectable nano-hydroxyapatite/chitosan (nHA/CS) composite scaffold.
METHODS: (1) Experiment 1: Passage 3 BMSCs were divided into two groups and cultured with the nHA/CS scaffold or cultured alone. Cell counting kit-8 was used to detect cell proliferation at 1, 3, 5, 7, 14 days of culture. (2) Experiment 2: Passage 3 BMSCs were seeded onto the nHA/CS scaffold and cultured in culture medium containing BMP-2 or not. Alkaline phosphatase activity in cells was detected at 3, 6, 9, 12, 15 days of culture. Cell counting kit-8 was used to detect cell proliferation at 1, 3, 5, 7, 14 days of culture. Alizarin red staining was used to observe the osteogenic differentiation of cells at 1 and 2 weeks of culture.
RESULTS AND CONCLUSION: (1) Experiment 1: With the prolongation of culture time, the absorbance values in the two groups were gradually increased, but there was no significant difference between the two groups. At 7 days of culture, the BMSCs adhered tightly to the scaffold surface. (2) Experiment 2: With the prolongation of culture time, the alkaline phosphatase activities in the two groups were gradually increased, and moreover, the alkaline phosphatase activity in the experimental group was higher than that in the control group at different culture time (P < 0.05). The absorbance values in the two groups were also gradually increased, and the value in the experimental group was higher than that in the control group at different culture time (P < 0.05). At 1 and 2 weeks of culture, the number of calcified nodules was higher in the experimental group than the control group. To conclude, BMP-2 has a promotion role in the proliferation and differentiation of BMSCs cultured on the injectable nHA/CS scaffold.

Key words: Bone Morphogenetic Proteins, Hydroxyapatites, Chitosan, Cell Proliferation, Tissue Engineering

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