Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (22): 3464-3471.doi: 10.12307/2024.486
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Wang Zhaofei1, He Guoyun1, Tian Fangcan2, Li Guangfeng1, Cao Zhonghua1, Liu Xiangfei1
Received:
2023-08-02
Accepted:
2023-09-28
Online:
2024-08-08
Published:
2024-01-20
Contact:
Liu Xiangfei, Associate chief physician, Department of Orthopedic Surgery, Shanghai ZhongYe Hospital, Genertec Universal Medical Group, Shanghai 200941, China
About author:
Wang Zhaofei, Doctoral candidate, Attending physician, Department of Orthopedic Surgery, Shanghai ZhongYe Hospital, Genertec Universal Medical Group, Shanghai 200941, China
Supported by:
CLC Number:
Wang Zhaofei, He Guoyun, Tian Fangcan, Li Guangfeng, Cao Zhonghua, Liu Xiangfei. Antibacterial properties of silver nanoparticle-coated stainless steel prepared via active screen plasma surface modification in vitro[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(22): 3464-3471.
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2.1 各组Ag-ASPSM@SS的表征结果 扫描电镜下观察3组样品的表面形貌,见图2A。在1 h-Ag-ASPSM@SS和2 h-Ag-ASPSM@SS样品中,纳米银颗粒几乎是球形的,颗粒分散良好;在4 h-Ag-ASPSM@SS样品中,纳米银颗粒聚集形成了一个链状的岛状和层状结构,大小不一,呈弥散分布。从X射线能谱图来看,随着攻击时间的延长Ag-ASPSM@SS表面的银含量增加,1 h-Ag-ASPSM@SS、2 h-Ag-ASPSM@SS、4 h-Ag-ASPSM@SS分别约含有8.90%,32.98%与49.28%的银,组间比较差异有显著性意义(P < 0.01),见图2B。在1 h-Ag-ASPSM@SS样品中大多数纳米银颗粒的直径约为60 nm,然而随着Ag-ASPSM轰击时间增加到2,4 h,纳米银颗粒的直径逐渐增大到约80 nm和400 nm,组间比较差异有显著性意义(P < 0.001),见图2C。 样品内部的腐蚀区域是不锈钢基体,样品边缘的白色光亮部分是渗碳层,腐蚀金相检测显示,1 h-Ag-ASPSM@SS和2 h-Ag-ASPSM@SS样品的平均渗碳厚度分别为5 μm和6 μm,4 h-Ag-ASPSM@SS样品的渗碳层的平均厚度为6 μm,与2 h轰击的样品接近,见图2D。 X射线衍射分析显示,除不锈钢外,3组Ag-ASPSM@SS样品基本上都没有杂乱的峰,这些峰主要在2θ范围内,即38.11°(111),44.28°(200),64.43°(220),77.47°(311)和81.54°(222)。结果还显示随着轰击时间的增加,银涂层的厚度增加,不锈钢衍射峰强度开始下降。然而随着沉积时间的增加,银涂层的(111)取向趋于减弱,取向系数从66.5%下降到40.0%,其他取向(200)(220)(311)和(222)的取向系数逐渐提高,有上升趋势,见图2E。"
2.2 各组Ag-ASPSM@SS体外抗菌能力与杀菌性能 抗菌能力:对于金黄色葡萄球菌ATCC25923菌株与铜绿假单胞菌ATCC27853菌株,材料A5(空白对照不锈钢)没有显示出明显的抑菌环;对于金黄色葡萄球菌ATCC25923菌株,材料A1、A2、A3和A4的抑菌环直径分别为(6.70±0.02),(7.73±0.12),(7.54±0.01),(17.56±0.76) mm,材料组间比较差异有显著性意义(P <0.05,P < 0.000 1);对于铜绿假单胞菌ATCC27853菌株,材料A1、A2、A3和A4的抑菌环直径分别为(6.25±0.02),(7.76±0.03),(7.26±0.02),(17.24±1.70) mm,材料组间比较差异有显著性意义(P < 0.05,P < 0.000 1),见图3A-C。 综合来看,材料A2(2 h-Ag-ASPSM@SS)对金黄色葡萄球菌ATCC25923和铜绿假单胞菌ATCC27853呈现出更好的抑制效果,比万古霉素和多黏菌素药敏纸片的效果略差。 杀菌性能:将各组样品浸入金黄色葡萄球菌悬液中,观察细菌悬浮液底部的絮状物质沉积程度,可见A2组底部有明显的絮状物沉积,这代表了死亡细菌的沉积;A4组底部有明亮和清晰的物质,表明A2组材料在24 h内具有更好的有效杀菌效果;此外,革兰染色结果与上述现象一致,见图3D,E。"
2.3 2 h-Ag-ASPSM@SS与ACNs的体外长效抗菌能力比较 图4展示了各组样品与细菌培养后特定时间点的涂布平板计数结果。在所有的时间节点(1,7,21,42,84 d),2 h-Ag-ASPSM@SS样品表面的金黄色葡萄球菌数量明显低于无涂层不锈钢组,表明2 h-Ag-ASPSM@SS样品对抑制金黄色葡萄球菌的定植有长期效果,其在84 d后仍能够显著减少金黄色葡萄球菌和铜绿假单胞菌的黏附。意外的是,培养84 d后,ACNs样品表面黏附的细菌菌落数量明显多于无涂层不锈钢。涂布平板计数定量分析结果显示,2 h-Ag-ASPSM@SS在1,7,21,42和84 d对金黄色葡萄球菌的抑菌率分别为100%,98.29%,88.81%,80.87%和71.39%;ACNs在1,7,21,42和84 d对金黄色葡萄球菌的抑菌率的抑菌率为100%,99.18%,50.47%,24.16%和-21.13%。与2 h-Ag-ASPSM@SS相比,ACNs呈现出强大的初始杀菌效果,然而随着时间的推移,ACNs的抑菌能力明显下降,甚至促进了细菌的黏附。"
2.4 2 h-Ag-ASPSM@SS与ACNs的体外细胞相容性 如图6A所示,与无涂层不锈钢组比较,2 h-Ag-ASPSM@SS组培养24,72 h的细胞活力无明显变化(P > 0.05),ACNs组培养24,72 h的细胞活力降低(P < 0.001)。Calcein-AM/PI双重染色显示,与无涂层不锈钢组比较,2 h-Ag-ASPSM@SS组培养24,72 h的活细胞、死细胞数量无明显变化,ACNs组培养24,72 h的活细胞数量减少、死细胞数量增加,见图6B。如图6C所示,与无涂层不锈钢组比较,2 h-Ag-ASPSM@SS组培养24,72 h的细胞上清液内乳酸脱氢酶活性无明显变 (P > 0.05),ACNs组培养24,72 h的细胞上清液内乳酸脱氢酶活性升高(P < 0.001)。"
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