中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (34): 5461-5467.doi: 10.12307/2022.456

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

载银纳米羟基磷灰石/聚己内酯复合纳米纤维支架的制备及成骨抗菌性能

曹  飞，惠  敏，董西玲，王  乐，王祖旭，张  敏，张晓明，刘童斌   

  1. 滨州医学院附属医院口腔修复科，山东省滨州市  256600
  • 收稿日期:2021-05-22 接受日期:2021-07-03 出版日期:2022-12-08 发布日期:2022-04-15
  • 通讯作者: 张晓明，硕士，主任医师，硕士生导师，滨州医学院附属医院口腔修复科，山东省滨州市 256603 刘童斌，硕士，滨州医学院附属医院口腔修复科，山东省滨州市 256603
  • 作者简介:曹飞，男，1995年生，山东省威海市人，汉族，滨州医学院在读硕士，主要从事口腔修复学研究。
  • 基金资助:
    山东省医药卫生科技发展计划项目(2016WS0121)，项目参与者：刘童斌；滨州医学院科技计划项目(BY2017KJ05)，项目负责人：刘童斌

Preparation of silver-loaded nanohydroxyapatite/polycaprolactone composite nanofiber scaffold and its osteogenic and antibacterial properties

Cao Fei, Hui Min, Dong Xiling, Wang Le, Wang Zuxu, Zhang Min, Zhang Xiaoming, Liu Tongbin   

  1. Department of Prosthodontics, Affiliated Hospital of Binzhou Medical University, Binzhou 256600, Shandong Province, China
  • Received:2021-05-22 Accepted:2021-07-03 Online:2022-12-08 Published:2022-04-15
  • Contact: Zhang Xiaoming, Master, Chief physician, Master’s supervisor, Department of Prosthodontics, Affiliated Hospital of Binzhou Medical University, Binzhou 256600, Shandong Province, China Liu Tongbin, Master, Department of Prosthodontics, Affiliated Hospital of Binzhou Medical University, Binzhou 256600, Shandong Province, China
  • About author:Cao Fei, Master candidate, Department of Prosthodontics, Affiliated Hospital of Binzhou Medical University, Binzhou 256600, Shandong Province, China
  • Supported by:
    the Medical and Health Science and Technology Development Plan Project of Shandong Province, No. 2016WS0121 (to LTB); the Science and Technology Program of Binzhou Medical University, No. BY2017KJ05 (to LTB)

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摘要：

文题释义：
骨感染：骨感染的常见致病菌主要是金黄色葡萄球菌和大肠杆菌，菌斑生物膜的形成是骨感染发生的主要诱因，生物膜可保护其内的致病菌免受抗生素和药物等的破坏而持续入侵正常骨组织，因此要想消除骨感染必须彻底根除菌斑生物膜。
银纳米颗粒的抗菌机制：目前的研究认为银主要通过两种机制发挥抗菌作用，一是银具有较高还原电位，可以在其周围产生原子氧，原子氧的强氧化性具有杀灭细菌的能力；二是银与携带负电荷的细菌细胞膜接触时可穿透进入细胞膜，并与胞质内蛋白质上的巯基发生反应，进而影响细菌DNA的合成，导致细菌裂解死亡。


背景：骨组织具有自我更新重建并修复组织缺损的功能，然而当发生大面积严重的骨缺损时往往需要骨移植物或骨替代物来重建缺损，这就加大了植入物周围骨感染的风险。
目的：制备出载有不同银浓度的纳米羟基磷灰石/聚己内酯复合纳米纤维支架，确定既促进细胞增殖又具有抗菌特性的银浓度。
方法：利用静电纺丝技术制备出载有不同银浓度(0，0.001，0.01，0.1 mol/L)的纳米羟基磷灰石/聚己内酯复合纳米纤维支架，通过扫描电镜、能量色散光谱仪及透射电镜表征4组支架的微观形貌与元素组成。将MC3T3-E1成骨细胞与4组支架共培养，检测支架对细胞增殖与成骨分化的影响。将4组支架分别与金黄色葡萄球菌(ATCC25923)和大肠杆菌(ATCC25922)共培养，通过形成的抑菌圈和抑菌率观察支架的抗菌性能。
结果与结论：①扫描电镜下可见，4组静电纺丝纳米纤维较为连续，直径较为均匀，电纺丝呈现网状结构，其中分散着大小各异的孔隙结构，随着支架中载银含量的增加，纳米纤维的直径逐渐增大。透射电镜下可见，载银支架纳米纤维上均匀分散着黑色的银纳米颗粒，颗粒的大小直径为(46±24) nm。②CCK-8实验显示，与未载银支架相比，载银0.001，0.01 mol/L的纳米纤维支架可促进成骨细胞的增殖，载银0.1 mol/L的纳米纤维支架抑制了成骨细胞的增殖。细胞核与细胞骨架染色显示，载银0，0.001，0.01 mol/L的纳米纤维支架有利于成骨细胞形态的伸展，载银0.1 mol/L的纳米纤维支架不利于成骨细胞形态的伸展。③碱性磷酸酶染色显示，载银0，0.001，0.01 mol/L的纳米纤维支架有利于成骨细胞的分化，载银0.1 mol/L的纳米纤维支架抑制了成骨细胞的分化。④抑菌圈实验显示，未载银支架无抗菌性能，随着支架中载银含量的增加，载银支架的抗菌性能增强。抑菌率实验证明载银量为0.01 mol/L的纳米纤维支架具有较强的抗菌活性。⑤结果表明，载银量0.01 mol/L的纳米羟基磷灰石/聚己内酯复合纳米纤维支架既具有良好的生物学活性又具备优秀的抗菌性能。

https://orcid.org/0000-0002-4104-921X (曹飞) 

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: 静电纺丝, 纳米纤维, 纳米羟基磷灰石, 银纳米颗粒, 骨感染, 成骨性, 抗菌性

Abstract: BACKGROUND: Bone tissue has the function of self-renewal, reconstruction and repair of tissue defects. However, when a large area of severe bone defect occurs, bone grafts or bone substitutes are often needed to reconstruct the defect, which increases the risk of bone infection around the implant. 
OBJECTIVE: To prepare nanohydroxyapatite/polycaprolactone composite nanofiber scaffolds with different silver concentrations to determine the silver concentration that promotes cell proliferation and has antibacterial properties. 
METHODS: Nanohydroxyapatite/polycaprolactone composite nanofiber scaffolds with different silver concentrations (0, 0.001, 0.01, 0.1 mol/L) were prepared by using electrospinning technology. The microscopic morphology and elemental composition of scaffolds of four groups were shown by scanning electron microscopy, energy dispersive spectroscopy and transmission electron microscope. MC3T3-E1 osteoblasts were co-cultured with four groups of scaffolds to study the effect of sample scaffolds on the cell proliferation and osteogenic differentiation. Four groups of scaffolds were co-cultured with Staphylococcus aureus (ATCC25923) and Escherichia coli (ATCC25922) to study the antibacterial properties of scaffolds by the formation of antibacterial circle and antibacterial rate.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that the four groups of electrospinning films were more continuous and uniform in diameter. The electrospinning filaments displayed a mesh-like structure, in which pore structures of different sizes were dispersed. As the silver-loaded content of the scaffold increased, the diameter of the nanofibers gradually increased. Transmission electron microscope showed that the silver-loaded scaffold nanofibers were uniformly dispersed with black silver nanoparticles, and the particle size diameter was (46±24) nm. (2) CCK-8 assay showed that compared with the unloaded silver scaffolds, silver-loaded 0.001, 0.01 mol/L nanofiber scaffolds could promote osteoblast proliferation. Silver-loaded 0.1 mol/L nanofiber scaffolds inhibited osteoblast proliferation. Nucleus and cytoskeleton staining showed that silver-loaded 0, 0.001, and 0.01 mol/L nanofiber scaffolds facilitated the extension of osteoblast morphology. Silver-loaded 0.1 mol/L nanofiber scaffolds were not conducive to the extension of osteoblast morphology. (3) Alkaline phosphatase staining showed that silver-loaded 0, 0.001, and 0.01 mol/L nanofiber scaffolds were beneficial to osteoblast differentiation, and silver-loaded 0.1 mol/L nanofiber scaffolds inhibited osteoblast differentiation. (4) Antibacterial circle experiment showed that the unloaded silver scaffolds had no antibacterial properties. As the silver-loaded content of the scaffold increased, the antibacterial properties of silver-loaded scaffolds increased. Antibacterial rate experiment proved that silver-loaded 0.01 mol/L nanofiber scaffolds had strong antibacterial activity. (5) The results confirm that the silver-loaded 0.01 mol/L nanohydroxyapatite/polycaprolactone composite nanofiber scaffold has both good biological activity and excellent antibacterial properties. 

Key words: electrospinning, nanofiber, nanohydroxyapatite, silver nanoparticle, bone infection, osteogenesis, antibacterial property

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