TiN涂覆的 Ti-6Al-4V注入Cu~(2+)后的抑菌性与细胞相容性研究
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摘要
在TiN涂覆的Ti-6Al-4V表面分别注入一定剂量的Cu~(2+)或Ag~+,研究并对比所注入表面的抑菌性和细胞相容性。将L929细胞分别接种于4种材料上:空白对照组——Ti-6Al-4V(钛合金组),阴性对照组——TiN涂覆的Ti-6Al-4V(TiN组),阳性对照组——TiN涂覆的Ti-6Al-4V表面注Ag(注Ag组),实验组——TiN涂覆的Ti-6Al-4V表面注Cu(注Cu组)。通过激光共聚焦观察细胞的骨架形态,通过扫描电镜观察细胞铺展黏附,通过CCK-8检测细胞增殖。将金黄色葡萄球菌液滴到各组样品表面,24 h后通过活菌铺板计数法观察材料表面活菌的菌落数目,用激光共聚焦、扫描电镜观察细菌黏附、形态。接触角检测材料亲水性,XPS检测材料表面元素组成,ICP-MS检测材料目标元素离子析出量。激光共聚焦和扫描电镜:注Ag组和注Cu组细胞较为密集,黏附、铺展充分,可见板状伪足与丝状伪足。CCK-8:接种1、3、5天后,注Ag组和注Cu组细胞与钛合金组以及TiN组相比增殖明显,证明这两组材料没有明显毒性。活菌铺板计数:注Cu组和注Ag组菌落少,抑菌率分别为91%±2%和87%±2%。激光共聚焦和扫描电镜:注Cu组和注Ag组的细菌比钛合金组和TiN组的金黄色葡萄球菌黏附少。细胞壁完整性被破坏,部分细菌破碎。研究表明,TiN涂覆Ti-6Al-4V表面注Cu和注Ag均具有较好的细胞相容性和抑菌性。
The aim of this work is to investigate and compare the antibacterial activity and biocompatibility of Ti-6 Al-4 V carrying TiN implanted with Cu~(2+) and Ag~+ respectively. L929 cells were incubated on blank control group-Ti-6 Al-4 V(titanium alloy group), negative control group-TiN coated Ti-6 Al-4 V(TiN group), positive control group-TiN coated Ti-6 Al-4 V surface conjugated with Ag(Ag group), experimental group-TiN coated Ti-6 Al-4 V surface conjugated with Cu(Cu group). The cytoskeleton morphology of the cells was observed by laser confocal scanning. Scanning electron microscope was used to observe the spreading, adhesion and growth of cells on the substrates. Cell proliferation was measured by CCK-8 assay. The straphylococcus aureus solution was dropwise added to the surface of each group of samples. After 24 h incubation, the number of viable bacteria colonies on the surface of the material was determined by the plate count method. The contact angle reflects the hydrophilzicity of the material. XPS reflects the surface elements composition of the material and ICP-MS can reflect the amount of ion precipitation of the target element of the material. CLSM and SEM: The cells in the Ag group and the Cu group were dense, meanwhile spread and adhered on the substrates, showing slab pseudopods and filopodia. CCK-8 result: After 1 day, 3 day, and 5 day incubation, viable cells in Ag group and Cu group were more than those on the titanium alloy group and the TiN group, which proved that the two groups had no apparent toxicity. Plate count method: The Ag group and Cu group had fewer colonies; the bacteriostasis rate was 91%±2% and 87%±2% respectively. CLSM and SEM: The bacteria adhered on Cu group and Ag group was less than those on titanium alloy group and TiN group. The integrity of bacterial wall was damaged and even broken. The Cu group and Ag group showed similar cell compatibility and antibacterial property.
The aim of this work is to investigate and compare the antibacterial activity and biocompatibility of Ti-6 Al-4 V carrying TiN implanted with Cu~(2+) and Ag~+ respectively. L929 cells were incubated on blank control group-Ti-6 Al-4 V(titanium alloy group), negative control group-TiN coated Ti-6 Al-4 V(TiN group), positive control group-TiN coated Ti-6 Al-4 V surface conjugated with Ag(Ag group), experimental group-TiN coated Ti-6 Al-4 V surface conjugated with Cu(Cu group). The cytoskeleton morphology of the cells was observed by laser confocal scanning. Scanning electron microscope was used to observe the spreading, adhesion and growth of cells on the substrates. Cell proliferation was measured by CCK-8 assay. The straphylococcus aureus solution was dropwise added to the surface of each group of samples. After 24 h incubation, the number of viable bacteria colonies on the surface of the material was determined by the plate count method. The contact angle reflects the hydrophilzicity of the material. XPS reflects the surface elements composition of the material and ICP-MS can reflect the amount of ion precipitation of the target element of the material. CLSM and SEM: The cells in the Ag group and the Cu group were dense, meanwhile spread and adhered on the substrates, showing slab pseudopods and filopodia. CCK-8 result: After 1 day, 3 day, and 5 day incubation, viable cells in Ag group and Cu group were more than those on the titanium alloy group and the TiN group, which proved that the two groups had no apparent toxicity. Plate count method: The Ag group and Cu group had fewer colonies; the bacteriostasis rate was 91%±2% and 87%±2% respectively. CLSM and SEM: The bacteria adhered on Cu group and Ag group was less than those on titanium alloy group and TiN group. The integrity of bacterial wall was damaged and even broken. The Cu group and Ag group showed similar cell compatibility and antibacterial property.
引文
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[4] 黄佳木,徐成俊,张兴元,等.室温直流磁控溅射氮化钛薄膜研究[J].真空科学与技术学报,2005,25(4):297-300.
[5] 王浩.磁控溅射TIN/Ag抑菌纳米复合膜制备及其性能研究[D].重庆:重庆理工大学,2016.
[6] 冷崇燕,周荣,张旭,等.Ag和Ta离子双注入改善Ti6A14V合金耐磨性能[J].金属学报,2009(6):764-768.
[7] Woods EJ,Cochrane CA,Percival SL.Prevalence of silver resistance genes in bacteria isolated from human and horse wounds[J].Veterinary Microbiology,2009,138(3/4):325-329.
[8] Meran Z,Besinis A,De Peralta T,et al.Antifungal properties and biocompatibility of silver nanoparticle coatings on silicone maxillofacial prostheses in vitro[J].Journal of Biomedical Materials Research Part B:Applied Biomaterials,2018,106(3):1038-1051.
[9] 王晓岚.金属离子的抑菌性能及其抑菌机理研究[D].广州:华南理工大学,2015.
[10] Wang Xiaolan,Liu Shaoxiang,Li Mei,et al.The synergistic antibacterial activity and mechanism of multicomponent metal ions-containing aqueous solutions against Staphylococcus aureus[J].Journal of Inorganic Biochemistry,2016,163:214-220.
[11] Heidenau F,Mittelmeier W,Detsch R,et al.A novel antibacterial titania coating:Metal ion toxicity and in vitro surface colonization[J].J Mater Sci Mater Med,2005,16(10):883-888.
[12] 杜娟,姜焕焕,莫嘉骥,等.钛表面形貌和亲水性表面对成骨细胞增殖、分化的影响[J].中国口腔颌面外科杂志,2012,10(3):182-187.
[13] 秦晖.Ag+注入钛的抑菌、成骨作用研究[D].上海:上海交通大学,2015.
[14] Wiedmer D,Petersen FC,Nnstensrud J,et al.Antibacterial effect of hydrogen peroxide-titanium dioxide suspensions in the decontamination of rough titanium surfaces[J].Biofouling,2017,33(6):1-9.
[15] Sudha VBP,Singh KO,Prasad SR,et al.Killing of enteric bacteria in drinking water by a copper device for use in the home:Laboratory evidence[J].Transactions of the Royal Society of Tropical Medicine & Hygiene,2009,103(8):819-822.
[16] Sun Xiuhua,Dong Lei,Zhao Mengli,et al.Modulation period of Ag deposition on co-sputtered TiN-Ag leading to different microstructures:Implication on mechanical properties and living cells growth[J].Surface & Coatings Technology,2016:S0257897216310362.
[17] Grass G,Rensing C,Solioz M.Metallic copper as an antimicrobial surface[J].Appl Environ Microbiol,2011,77(5):1541-1547.
[18] Ansari MI,Malik A.Biosorption of nickel and cadmium by metal resistant bacterial isolates from agricultural soil irrigated with industrial wastewater[J].Bioresour Technol,2007,98(16):3149-3153.