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负载抗生素铜掺杂羟基磷灰石微球的制备及抗菌成骨评价

蒋波波 李世宏 郑伟

蒋波波, 李世宏, 郑伟. 负载抗生素铜掺杂羟基磷灰石微球的制备及抗菌成骨评价[J]. 四川大学学报(医学版), 2021, 52(5): 799-806. doi: 10.12182/20210960209
引用本文: 蒋波波, 李世宏, 郑伟. 负载抗生素铜掺杂羟基磷灰石微球的制备及抗菌成骨评价[J]. 四川大学学报(医学版), 2021, 52(5): 799-806. doi: 10.12182/20210960209
JIANG Bo-bo, LI Shi-hong, ZHENG Wei. Preparation of Antibiotic-Loaded Copper-Doped Hydroxyapatite Microspheres and Evaluation of Their Antibacterial and Osteogenic Effect[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2021, 52(5): 799-806. doi: 10.12182/20210960209
Citation: JIANG Bo-bo, LI Shi-hong, ZHENG Wei. Preparation of Antibiotic-Loaded Copper-Doped Hydroxyapatite Microspheres and Evaluation of Their Antibacterial and Osteogenic Effect[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2021, 52(5): 799-806. doi: 10.12182/20210960209

负载抗生素铜掺杂羟基磷灰石微球的制备及抗菌成骨评价

doi: 10.12182/20210960209
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Preparation of Antibiotic-Loaded Copper-Doped Hydroxyapatite Microspheres and Evaluation of Their Antibacterial and Osteogenic Effect

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  • 摘要:   目的   探讨负载万古霉素(vancomycin, Van)的铜(Cu)掺杂羟基磷灰石(hydroxyapatite, HA)微球制备方法,并于体外评价其抗菌及成骨作用。  方法   采用水热合成法制备摩尔掺杂比例1%、5%、10%、20%的Cu掺杂HA微球(Cu-HA),通过扫描电镜观察其微观形貌变化,X射线衍射仪(X-ray diffractometer, XRD)研究其相组成并分析样品的结晶度。选择摩尔掺杂比例为10%的Cu-HA,利用X射线能谱仪(energy dispersive X-ray spectroscopy, EDS)分析样品的元素成分信息后,以聚多巴胺(polydopamine, PDA)为介质涂覆制备Cu-HA-PDA,采用XRD和傅里叶红外光谱仪检测样品涂覆效果。在Cu-HA-PDA装载Van,制备成Cu-HA-PDA-Van。将均为10 mg/mL的HA、Cu-HA、HA-PDA、Cu-HA-PDA-Van分别加入α培养基中制备各组浸提液,检测浸提液主要成分,检查Van质量浓度。探究材料浸提液对成骨前体细胞的毒性作用及骨髓间充质干细胞的增殖、分化作用,并检测成骨相关基因骨钙素(osteocalcin, OCN)、Runt相关转录因子2(runt-related transcription factor 2, RUNX-2)、碱性磷酸酶(alkaline phosphatase, ALP)表达。备好灭菌的HA、Cu-HA、HA-PDA、Cu-HA-PDA、Cu-HA-DPA-Van微球材料,采用菌落计数法评价材料对金黄色葡萄球菌的抗菌效果。  结果  成功合成各种Cu-HA,随着Cu比例的增加,其形貌从条带状逐渐变成均匀球状,其中摩尔掺杂比例为10%的Cu-HA显示良好的微球形状及表面花瓣状多孔微纳形貌。EDS及XRD分析表明材料主体结构依然为羟基磷灰石晶体且Cu已成功掺杂HA。傅里叶红外光谱仪显示PDA成功涂覆在材料表面。浸提液主要成分检测再次佐证Cu元素成功进入并替代部分HA中的Ca元素。10 mg/mLCu-HA-PDA-Van浸提液中含Van 0.27 mg/mL。体外细胞实验和成骨相关基因检测表明,Cu-HA-Van具有良好的生物活性和促骨分化作用。Cu-HA-PDA-Van微球最小抑菌浓度(minimum inhibitory concentration, MIC)为16 μg/mL,Cu-HA-Van微球相对于Cu-HA、HA-PDA和纯HA有显著和持久的抗菌效果。  结论   利用Cu元素调控HA微观形貌、成功涂覆PDA并装载Van制备的Cu-HA-PDA-Van微球具有良好的抗菌性能和生物活性。
  • 图  1  Cu掺杂对HA形貌及晶体结构的影响

    Figure  1.  The influence of Cu doping on the morphology and crystal structure of HA

    A: ①-⑤ were the scanning electron microscope (SEM) images of HA, 1% Cu-HA, 5% Cu-HA, 10% Cu-HA and 20% Cu-HA, respectively; B: X-ray diffractometer pattern.

    图  2  10Cu-HA的EDS图谱

    Figure  2.  Energy dispersive X-ray spectroscopy (EDS) atlas of 10Cu-HA

    图  3  改性HA的XRD及FTIR图谱

    Figure  3.  FTIR spectrum of modified HA and XRD patterns of each group of materials

    图  4  BMSCs在各组浸提液中培养7 d的增殖活性

    Figure  4.  The proliferation activity of BMSCs in each group after 7 d of culturing in the extract solution

    *P<0.05, n=3.

    图  5  BMSCs在浸提液(10 mg/mL)中培养7 d后的YF555-鬼笔环肽免疫荧光染色图

    Figure  5.  YF555-Phalloidin immunofluorescence staining image after BMSCs being cultured in the extract solution (10 mg/mL) for 7 days

    The scale bar=20 μm.

    图  6  BMSCs在10 mg/mL浸提液中分别培养1、4、7 d后OCNALPRUNX-2基因表达情况

    Figure  6.  The expression of OCN, ALP and RUNX-2 after BMSCs were cultured in 10 mg/mL extract solution for 1, 4, and 7 d

    *P<0.05, vs. HA group; # P<0.05, vs. Cu-HA group. n=3.

    图  7  Cu-HA-PDA-Van微球MIC测定(n=3)

    Figure  7.  The measuring of minimum inhibitory concentration (MIC) of Cu-HA-PDA-Van microspheres (n=3)

    图  8  各组材料菌落生长情况

    Figure  8.  The colony growth of different groups

    A: Day 1; B: Day 4; C: Day 7.

    表  1  各溶液详细配置及理论掺杂比

    Table  1.   The composition of each solution and the theoretical doping ratio

    SampleCa(NO3)2/gNa2HPO4/gCu(NO3)2/gReaction liquid volume/mLTheoretical doping rate/%
    HA 0.709 0.644 0.00 30 0
    1Cu-HA 0.701 0.644 0.007 30 1
    5Cu-HA 0.672 0.644 0.036 30 5
    10Cu-HA 0.637 0.644 0.072 30 10
    20Cu-HA 0.566 0.644 0.145 30 20
    下载: 导出CSV

    表  2  10 mg/mL浸提液中主要无机元素含量(n=2)

    Table  2.   The content of main inorganic elements in 10 mg/mL extract solution (n=2)

    GroupCa/(mg/L)P/(mg/L)Cu/(mg/L)
    HA 85.45 28.08 0
    Cu-HA 58.88 31.82 16.86
    Cu-HA-PDA-Van 59.08 32.53 7.93
    下载: 导出CSV

    表  3  各组在10 mg/mL浸提液中的毒性及ALP检测

    Table  3.   Toxicity in 10 mg/mL extract solution and ALP test of each group

    GroupCytotoxicityALP content
    RGR/%Cytotoxicity grade
    Control 100.00±3.17 0.288±0.045
    HA 99.45±4.33 0 0.294±0.063
    Cu-HA 95.46±4.19 0 0.377±0.041*
    HA-PDA 105.63±4.50 0 0.357±0.044
    Cu-HA-PDA-Van 104.40±7.15 0 0.411±0.053*
     *P<0.05, vs. control group. n=3. RGR: Relative growth rate.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-04-08
  • 修回日期:  2021-08-29
  • 网络出版日期:  2021-12-06
  • 刊出日期:  2021-09-20

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