Ti-Zr-Cu合金的抗菌性能和体外生物相容性
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Antibacterial Property and in vitro Biocompatibility of a Ti-Zr-Cu Alloy
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通讯作者: 战德松,教授,zhandesong@126.com,研究方向为新型种植体材料的生物学基础应用
收稿日期: 2021-01-05 修回日期: 2021-05-24 网络出版日期: 2021-11-30
基金资助: |
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Corresponding authors: ZHAN Desong, Tel:
Received: 2021-01-05 Revised: 2021-05-24 Online: 2021-11-30
作者简介 About authors
于佳莹,女,1995年生,硕士生
采用平板共培养法测定Ti-Zr-Cu合金表面的菌落数,通过CCK8细胞增殖检测、鬼笔环肽细胞染色观察细胞核和细胞骨架形貌、细胞凋亡和黏附能力测定,研究了Ti-Zr-Cu合金的抗菌性能和体外生物相容性。结果表明,空白样品和Ti-Zr合金表面上的菌落密集,而Ti-Zr-Cu合金表面上的菌落数极少。Ti-Zr-Cu合金对金黄色葡萄球菌和大肠杆菌的抗菌率分别达到98.28%和97.67%,表现出优异的抗菌性能。MC3T3-E1在Ti-Zr-Cu合金表面培养1、4、7 d的相对增值率分别为168.8%、109.8%和106.5%,均高于100%,表明这种合金没有细胞毒性。细胞在Ti-Zr-Cu合金表面上的附着和铺展良好,有利于贴壁细胞在其表面黏附和进一步增值,表明Ti-Zr-Cu合金具有良好的生物相容性。Ti-Zr-Cu合金对细胞凋亡没有不良影响。从Ti-Zr-Cu合金表面细胞的SEM照片可见细胞的粘附正常,也表明其具有良好的生物相容性。
关键词:
The antibacterial property and in vitro biocompatibility of a novel Ti-Zr-Cu alloy were investigated. Meanwhile, the number of bacterial colonies on the surface of Ti-Zr-Cu alloy was determined by plate co-culture method, and the morphology of nuclear and cytoskeleton, cell apoptosis and adhesion ability on Ti-Zr-Cu alloy were assessed by means of CCK8 cell proliferation detection and phyllopeptide cell staining observation. The results show that the density of bacterial colonies on the blank sample and Ti-Zr alloy surface was very high, while the number of bacterial colonies on the Ti-Zr-Cu alloy surface was very low. The antibacterial rate of Ti-Zr-Cu alloy against Staphylococcus aureus and Escherichia coli reached 98.28% and 97.67%, respectively, showing excellent antibacterial properties. The relative productivity of MC3T3-E1 cultured on Ti-Zr-Cu alloy surface for 1, 4 and 7 days were 168.8%, 109.8% and 106.5%, respectively, which were all higher than 100%, indicating that this alloy had no cytotoxicity. The adhesion and spread of cells on the surface of Ti-Zr-Cu alloy are good, which is conducive to the adhesion and further growth of adherent cells on the surface of Ti-Zr-Cu alloy, indicating that Ti-Zr-Cu alloy has good biocompatibility. Ti-Zr-Cu alloy had no adverse effect on cell apoptosis. The SEM images of the cells on the surface of Ti-Zr-Cu alloy showed that the adhesion of the cells was normal, which also indicated that the cells had good biocompatibility.
Keywords:
本文引用格式
于佳莹, 杨希祥, 战德松, 杨柯, 任玲, 王敬人, 徐嘉蔚.
YU Jiaying, YANG Xixiang, ZHAN Desong, YANG Ke, REN Ling, WANG Jingren, XU Jiawei.
用种植体替代缺失牙已经成为一种常规的治疗选择,其适应症不断扩大。纯钛(Ti)和钛合金是应用最为广泛的种植体材料。单颗牙或前牙的缺失,往往使牙间隙和牙根间隙不足或颌骨改建后其宽度很窄[1]。对此除了进行常规的附加骨增量[2],还可选用窄直径种植体。但是,与常规直径的种植体相比,相同强度水平的窄直径种植体的承载能力会下降,应力水平的提高使疲劳折裂的风险大大增加[3]。因此,不推荐用窄直径种植体修复单独的尖牙、前磨牙和磨牙。为了提高种植体的机械强度,新近发展出的高强度钛锆(Ti-Zr)合金的窄直径种植体已经临床应用。例如,Straumann公司推出的Roxolid钛锆合金种植体,其机械性能较高,体外细胞实验、动物实验和临床实验结果表明其耐蚀性能、骨亲和力和生物相容性等都不低于纯钛[3-5],其硬度、弹性模量,拉伸和抗曲强度甚至优于纯钛[6]。
1 实验方法
1.1 样品的制备
实验用材料为Ti-Zr合金和Ti-Zr-Cu合金,其化学成分列于表1。将实验材料(包括空白)切割成直径为10 mm、厚度为2 mm的圆片,依次用150#、400#、800#、1200#碳化硅砂纸打磨。实验前,将空白样品(blank)、Ti-Zr合金和Ti-Zr-Cu合金样品依次用去离子水、75%无水乙醇和100%无水乙醇超声充分清洗,并进行高温高压灭菌。
表1 Ti-Zr合金和Ti-Zr-Cu合金的化学成分
Table 1
Alloy | Ti | Zr | Cu | Fe | C | O | N | H |
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Ti-Zr | Bal. | 15.20 | 0.01 | 0.04 | 0.05 | 0.07 | 0.004 | 0.002 |
Ti-Zr-Cu | Bal. | 14.50 | 3.00 | 0.04 | 0.04 | 0.08 | 0.007 | 0.002 |
1.2 抗菌性能的表征
采用平板共培养计数法,使用感染性细菌菌株——革兰氏阳性金黄色葡萄球菌(ATCC 6538)和革兰氏阴性大肠杆菌(ATCC 8739)评价实验材料的抗菌性能。
用蒸馏水配置LB培养基(牛肉膏5 g/L,氯化钠5 g/L,蛋白胨10 g/L)和琼脂培养皿(牛肉膏5 g/L,氯化钠5 g/L,蛋白胨10 g/L,琼脂23 g/L),调整其pH值为7.2~7.4,并进行高温高压处理。先将两种细菌菌株的冷冻粉溶解在LB培养基中,在温度为37℃的摇床中培养24 h得到细菌悬液。将细菌悬浮液涂布在营养琼脂平皿上,置于温度为37℃、湿度高于90%的恒温培养箱中培养24 h。然后用PBS将细菌菌落配制成浓度为108 cfu/mL的细菌悬液,分别调整大肠杆菌(OD0.150±0.02)和金黄色葡萄球菌(OD0.325±0.02)的光密度,将其浓度稀释为105 cfu/mL。
将50 μL稀释后的菌液分别滴加在空白、Ti-Zr合金和Ti-Zr-Cu合金样品的表面,使菌液均匀铺开完全覆盖样品的表面。每组设置4个平行样品,将其放在恒温培养箱中培养24 h。到达时间后, 将样品连同其上的菌液一同移至离心管中,加入3 mL的无菌PBS溶液,涡旋震荡1 min得到洗脱液。取100 μL洗脱液均匀涂布在琼脂培养基的平板上,在恒温培养箱中培养24 h后取出平板拍照。计数每个样品平板上的菌落数,并计算每组样品表面上的平均菌落数。实验材料的杀菌率为
式中N为对照样品表面上的菌落数;N1为实验样品表面上的菌落数。如果R≥90%,则表明实验样品具有强烈的杀菌性能。
1.3 生物相容性的表征
1.3.1 CCK8细胞增殖
使用空白、Ti-Zr合金和Ti-Zr-Cu合金样品表征细胞增殖,每种材料设置四组平行样品。将小鼠胚胎成骨细胞前体细胞 MC3T3-E1配制成浓度为2×104个/mL的细胞悬液。将空白样、Ti-Zr-Cu合金和Ti-Zr合金各四组平行样品置于48孔板中。用PBS充分清洗样品弃净后,将500 μL/孔细胞悬液均匀滴加在其表面。在37℃、5% CO2条件下,在恒温培养箱(Thermo 公司)中分别培养1 d、4 d和7 d。到达时间点后弃净培养基并用PBS充分冲洗样品表面。在每孔滴加含10%CCK-8的无血清αMEM培养基300 μL,再将其在37℃培养箱中避光孵育4 h,然后每孔取100 μL溶液用酶标仪(Infinite M200型),测定其在450 nm波长处的光密度(OD)。
细胞的相对增殖率(Relative growth rate,RGR)为
表2 细胞毒性的分级标准和评定结果
Table 2
Level | RGR/% | Evaluation results |
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0 | ≥100 | Qualified |
1 | 75~99 | Qualified |
2 | 50~74 | Overview |
3 | 25~49 | Failed |
4 | 0~24 | Failed |
1.3.2 鬼笔环肽细胞骨架染色
在相同条件下将细胞接种在Ti-Zr合金和Ti-Zr-Cu合金样品表面,然后放置在37℃、5%CO2培养箱中培养24 h。到达时间点后弃培养基,将样品取出并移入新的24孔板中,用PBS充分清洗后使用4%多聚甲醛固定10 min。再用PBS清洗并用0.2%TritonX-100 透膜处理 6 min,然后加入罗丹明-鬼笔环肽避光染色40 min。到时间点后用PBS再次充分清洗,随后在避光条件下用DAPI染色5 min。用PBS清洗后,将已固定好细胞的样品置于载玻片上,在Olympus荧光显微镜下观察细胞核及细胞骨架形貌并拍照。
1.3.3 细胞凋亡的检测
使用流式细胞仪采用FITC/PI双染法测定细胞凋亡。按照1×104个/cm2的密度将细胞接种在空白、Ti-Zr合金和Ti-Zr-Cu合金样品表面,然后放在细胞培养箱中培养72 h。到达时间点后,用不含EDTA的胰蛋白酶消化并收集细胞。用冷PBS缓冲溶液轻吹重悬细胞后,在低于4℃以1000 r/min的转速离心分离5 min后弃液,重复两次后在每管中加入100 μL的Binding Buffer轻吹重悬。然后在每管中依次加入AnnexinV-FITC和PI各5 μL对细胞染色,避光反应15 min后在每管加入400 μL的Binding Buffer,轻吹重悬后在1 h内检测流式细胞凋亡。
1.3.4 细胞黏附能力的表征
使用ZEISS场发射扫描电子显微镜(HITACHI-3400N)观察合金样品上的细胞粘附能力。实验接种方法和样品设置,与上述CCK8实验相同。在相同条件下将细胞接种在样品表面,然后放在细胞培养箱中培养168 h,到达时间点后弃去孔板内的培养基并用PBS充分清洗。加入4%多聚甲醛使其没过孔板内样品,然后将其置于4℃的冰箱中固定4 h。到时间后弃去多聚甲醛,再用PBS充分清洗。然后依次用浓度为30%、50%、70%、80%、90%和95%的乙醇梯度脱水,每次10 min。最后用100%无水乙醇脱水5 min,重复一次。将其在阴凉处过夜风干,喷金后用扫描电镜观察细胞粘附情况。所有实验都重复3次,取其结果的平均值。
采用Graphpad prism 8和IBM SPSS Statistics 23分析实验结果,用单因素方差进行统计学分析,p<0.05表示具有统计学意义。
2 结果和讨论
2.1 Ti-Zr-Cu合金的抗菌性能
金黄色葡萄球菌(S. aureus)和大肠杆菌(E. coli)分别属于革兰氏阳性菌和革兰氏阴性菌,是有代表性的两个常见菌种,也是引起骨科感染的常见细菌,因此常选用的菌种评价材料的抗菌性能。采用平板计数法检测Ti-Zr-Cu合金对S. aureus和E. coli的抗菌率,以表征其抗菌性能。图1给出了S. aureus和E. coli分别与Ti-Zr合金和Ti-Zr-Cu合金共培养24 h后的存活细菌菌落照片,可见空白(blank)对照组和Ti-Zr合金对照组的菌落密集,而Ti-Zr-Cu实验组的菌落数极少。表3列出了各实验组的菌落数和根据
图1
图1
金黄色葡萄球菌(S. aureus)和大肠杆菌(E. coli)分别与Ti-Zr合金和Ti-Zr-Cu合金共培养24 h后存活细菌菌落的照片
Fig.1
Photos of colonies of S. aureus and E. coli after co-culture with Ti-Zr alloy and Ti-Zr-Cu alloy for 24 h (a) blank group (S. aureus); (b) Ti-Zr (S. aureus); (c) Ti-Zr-Cu (S. aureus); (d) blank group (E. coli); (e) Ti-Zr (E. coli); (f) Ti-Zr-Cu (E. coli)
表3 不同材料对金黄色葡萄球菌和大肠杆菌的杀菌率(n=4)
Table 3
Group | S. aureus | E. coli | ||
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Colony mean | Sterilizing rate | Colony mean | Sterilizing rate | |
Ti-Zr-Cu | 5±4.99 | (98.28±1.58)% | 21±4.69 | (97.67±0.49)% |
Ti-Zr | 294±16.44 | — | 900±12.96 | — |
blank | 325±18.52 | — | 1087±43.60 | — |
Ti-Zr-Cu合金的抗菌性能,源于铜的杀菌作用。铜具有优异的杀菌性能,属于长效广谱的杀菌剂,已经作为一种固体无机抗菌材料注册并列为医用材料[20,21]。因此,铜的加入显著抑制了细菌在Ti-Zr-Cu合金表面的粘附和增殖。研究表明[22],当细菌与含铜合金共培养时微量铜离子从合金中溶出,在浓度梯度驱动下从合金表面扩散到细菌液中。铜离子通过电荷作用与带负电荷的细菌表面紧密结合,使细菌内外膜电位失衡[23, 24],从而破坏了细菌的细胞膜,导致细菌死亡。也有研究认为,铜破坏细菌细胞膜的原因,是铜离子通过芬顿反应Cu++H2O2→Cu2++OH-+·OH[25]产生活性氧物种(ROS),ROS抑制了细菌中的16SrRNA复制并使其死亡[26]。
2.2 Ti-Zr-Cu合金的生物相容性
2.2.1 Ti-Zr-Cu合金对贴壁细胞增殖的影响
图2给出了CCK8细胞增殖的检测结果。可以看出,培养24 h(1 d)后Ti-Zr合金和Ti-Zr-Cu合金的OD值都为0.2-0.4,培养96 h后增加到1.5,培养168 h后增加到2.8。由此可见,随着培养时间的增加二种样品的OD值逐渐增大。这表明,MC3T3-E1细胞在Ti-Zr和Ti-Zr-Cu合金表面都能良好地生长。第1天Ti-Zr合金组与Ti-Zr-Cu合金组的细胞增值差异非常显著(p<0.01),第4天和第7天的差异都不具有统计学意义(p>0.05)。MC3T3-E1在Ti-Zr-Cu合金表面1、4、7 d的相对增殖率分别为168.8%、109.8%和106.5%,均大于100%,表明这种合金没有细胞毒性。
图2
图2
CCK8细胞增值检测给出的MC3T3-E1细胞的 OD值(在450 nm处的吸光度)
Fig.2
OD values of MC3T3-E1 cells measured by CCK-8 cell proliferation assay (absorbance at 450 nm)
铜是人体中必需的微量元素之一,是许多物理化学反应和代谢所必需的元素,包括蛋白质合成、酶和红细胞的形成[27]。铜还具有多种生物功能,如刺激血管生成和成骨,以及抑制血栓形成,防止支架再狭窄、减小骨质疏松[28]、抗菌和抗炎[29]等。但是,作为一种重金属元素,过量的铜在体外有细胞毒性,在体内可能会导致肝脏和神经方面的疾病[30, 31]。因此,优化含铜金属的铜含量极为重要。世界卫生组织建议成人每日铜摄入量上限为0.03 mg/kg[32]。铜的质量分数达到0.0141%的铜基合金溶出液,会产生较为明显的细胞毒性[33]。文献[34]的结果表明,Ti-Zr-Cu合金的铜释放量约为0.047 mg/L/day,远小于上述标准。口腔内种植体的体积很小,铜的释放量远低于人体所能承受的每日最大摄入量。因此,Ti-Zr-Cu合金在人体中使用是安全的。
2.2.2 Ti-Zr-Cu合金表面的细胞骨架形态
图3
图3
MC3T3-E1细胞在Ti-Zr合金(a)和Ti-Zr-Cu合金(b)表面培养24 h后的罗丹明鬼笔环肽骨架染色
Fig.3
Rhodamine phalloidin backbone staining of MC3T3-E1 cells on surfaces of Ti-Zr (a) and Ti-Zr-Cu (b) alloys after co-culture for 24 h
2.2.3 Ti-Zr-Cu合金表面的细胞凋亡
图4
图4
MC3T3-E1 细胞在Ti-Zr合金和Ti-Zr-Cu合金表面培养72 h后的流式散点图和柱状图统计分析
Fig.4
Flow scatters and bar graph statistical analyses of MC3T3-E1 cells on Ti-Zr and Ti-Zr-Cu alloys after co-culture for 72 h (a) Ti-Zr alloy; (b) Ti-Zr-Cu alloy; (c) normal cell viability; (d) apotoic cell viability
2.2.4 Ti-Zr-Cu合金表面的细胞黏附能力
使用扫描电子显微镜(SEM)表征Ti-Zr合金和Ti-Zr-Cu合金样品的细胞粘附能力,图5给出了培养72 h后样品表面细胞的SEM照片。可以看出,细胞覆盖了二种合金样品的大部分表面,细胞的形态完整,伪足清晰纤长,有大量的丝足和片状脂壁。细胞间通过丝状足孔连接在一起,在样品表面的铺展表现出特定的方向性,且沿表面划痕扩散。这表明Ti-Zr-Cu合金表面有利于MC3T3-E1细胞的黏附和伸展。细胞在Ti-Zr-Cu合金表面的正常粘附,表明其具有良好的生物相容性。
图5
图5
MC3T3-E1细胞在Ti-Zr合金和Ti-Zr-Cu合金表面培养72 h后的扫描电镜照片
Fig.5
Images of scanning electron microscopy of MC3T3-E1 cells on surfaces of Ti-Zr (a) and Ti-Zr-Cu (b) alloys after co-culture for 72 h (200× and 1000×)
3 结论
Ti-Zr-Cu合金对金黄色葡萄球菌和大肠杆菌都具有优异的抗菌功能,抗菌率分别高达98.3%和97.7%。Ti-Zr-Cu合金的抗菌功能,源于在合金中添加了适量的铜。Ti-Zr-Cu合金的生物相容性良好,对成骨细胞无细胞毒性,满足临床应用的要求。
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Effects of copper content on the antibacterial performance and corrosion
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铜含量对钴铬钼铜合金抗菌性和耐腐蚀性的影响
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In vitro evaluation of cytotoxicity and genotoxicity of a commercial titanium alloy for dental implantology
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Elastic moduli of cast Ti-Au, Ti-Ag, and Ti-Cu alloys
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Bactericidal activity and mechanism of action of copper-sputtered flexible surfaces against multidrug-resistant pathogens
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Metabolism and functions of copper in brain
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Killing of enteric bacteria in drinking water by a copper device for use in the home: laboratory evidence
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Metallic copper as an antimicrobial surface
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Contact killing of bacteria on copper is suppressed if bacterial-metal contact is prevented and is induced on iron by copper ions
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Toward a molecular understanding of the antibacterial mechanism of copper-bearing titanium alloys against Staphylococcus aureus
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Influence of Cu content on the cell biocompatibility of Ti-Cu sintered alloys
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In vitro study on cytocompatibility and osteogenesis ability of Ti-Cu alloy
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Antibacterial activity of copper-bearing 316L stainless steel for the prevention of implant-related infection
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A novel nano-copper-bearing stainless steel with reduced Cu(2+) release only inducing transient foreign body reaction via affecting the activity of NF-kappaB and Caspase 3
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Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis
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Copper supplementation of adult men: effects on blood copper enzyme activities and indicators of cardiovascular disease risk
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Cytotoxicity of dental metal material composition
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牙科金属材料的组成对细胞毒性影响的研究
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Preliminary study of microstructure, mechanical properties and corrosion resistance of antibacterial Ti-15Zr-xCu alloy for dental application
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Antibacterial properties and biocompatibility of SLM-fabricated medical titanium alloys
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3D打印医用钛合金的抗菌性能和体外生物相容性
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Research Advances on Apoptosisa
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细胞凋亡研究进展
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Anti-apoptosis and cell survival: a review
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Mitochondria-mediated apoptosis in mammals
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