Biomaterials生物材料Word格式.docx

1、Metal ions were released from thesurface of nanoparticles, particularly from larger （80 nm） particles generated by thermal plasma technology. 金属离子被释放纳米颗粒的表面,特别是从大（80海里）粒子产生的热等离子体技术。Exposure of fibroblasts to these nanoparticles triggered rapid （2 h） generation of reactive oxygen species （ROS） that c

2、ould be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. 成纤维细胞接触到这些纳米粒子快速触发（2 h）代的活性氧物种（ROS）,可以消除NADPH氧化酶的抑制作用,表明这是介导吞噬作用的粒子。The exposure also caused a more prolonged, MitoQ sensitive production of ROS （24 h）, suggesting involvement of mito

3、chondria曝光也引起了更长期,MitoQ敏感的生产ROS（24小时）,这表明线粒体的参与Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. 因此,我们记录的水平升高非整倍性染色体聚集,线粒体的分裂和破坏细胞骨架尤其是微管网络。Exposure to the nanoparticles

4、resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. 暴露于纳米颗粒导致畸形细胞核,中断成熟的核纤层蛋白B1和核质增加桥梁、MitoQ可以预防的。In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the inciden

5、ce of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. 此外,增加微核的数据观察,这些只是部分预防 MitoQ,微核发生率和离子释放的纳米颗粒呈正相关 纳米粒子的大小,虽然细胞遗传学变化,核形状和修改 ROS没有。These results su

6、ggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential. 这些结果表明,细胞表现出多样化的线粒体ROS-dependent 和独立应对CoCr粒子,纳米颗粒大小和金属离子的数量发布是有影响力的。1. IntroductionOrthopaedic patients with meta

7、l-on-metal （MoM） hip replacements are exposed to CoCr nanoparticles as a result of wear of the implant 1e4. 骨科金属对金属介面人工髋关节（MoM）髋关节置换患者暴露于CoCr纳米颗粒由于穿的吗e4植入1。Approximately 6.7 _ 1012e2.5 _ 1014 particles （generally 50 nm） are generated by articulating CoCr surfaces in each patient every year 5,6. 大约6.

8、7 1012 e2.5 1014粒子 （一般 50 nm）由阐明CoCr表面生成每个病人每年（5、6）。Calls have been made for the establishment and validation of material characterisation protocols and biological testing methodologies to understand the potential toxicity of these nanoparticles. 的电话了建立和验证的材料描述协议和生物测试方法了解的潜力这些纳米颗粒的毒性。There is also a

9、n increase of circulating metal ions in the blood of these patients 5,7e17, who may be exposed to metal for up to 60 years after surgery. This internal surgical exposure to nanoparticles is different from the external exposure （such as environmental pollution/inhalation） as it bypasses many of the b

10、odys natural defences, for example by macrophage uptake or contact with fluids in the airways before entering the body. 还有一个增长循环这些病人的血液中金属离子（5、7 e17）,可能是谁接触到金属长达60年手术后。这个内部手术暴露于纳米粒子不同于外部风险（如环境污染/吸入）,因为它绕过许多身体的自然防御系统,例如巨噬细胞吸收或接触液体的航空公司在进入身体。While it is not known whether the release of Co and Cr ions

11、is essentially from the whole implant, or by corrosion of wear debris and/or both; the mechanisms of Co2 18e21, and Cr ions（trivalent and hexavalent） 22e27 toxicity in vitro is well understood with oxidative stress mediated cyto-and genotoxicity playing a major role. We have previously observed incr

12、eases in aneuploidy in peripheral blood lymphocytes of patients with CoCr-on-CoCr wear debris 28. We also demonstrated that CoCr nanoparticles （30 nm） which were generated by a pin-on-plate tribometer were significantly more toxic than commercially available micron sized （2.9 mm） CoCr particles in v

13、itro 29, demonstrating that CoCr particle size may be a key factor governing toxicity. Importantly, the CoCr wear debris generated in situ from MoM implants is often a heterogeneous mix of nanoscale particles. 虽然不知道Co和铬离子的释放基本上整个植入或腐蚀磨损碎片和/或两者;机制Co2万国宫e2118,和铬离子（三价和六价）（22 e27）体外毒性是很好理解的与氧化应激介导cyto-a

14、nd基因毒性一个主要的角色。我们曾观察到非整倍性的增加CoCr-on-CoCr患者的外周血淋巴细胞磨屑28。我们还表明,CoCr纳米颗粒 （30 nm）是由pin-on-plate生成摩擦计毒性比商用微米大小的（2.9毫米）CoCr粒子体外29,证明CoCr粒子管理毒性大小可能是一个关键因素。重要的是, CoCr磨屑经常从妈妈植入原位生成异构的混合纳米粒子。Size dependent but concentration independent toxicity ofnanoparticles has been demonstrated previously for other metalox

15、ides such as TiO2, CuO, ZnO 30,31. There is very little knownabout the mechanisms of toxicity of differentially sized CoCr particles within the nano scale range. The toxicity of metal oxides may also be dependent on the extent of oxidative stress 32e34. In this study, we have investigated whether th

16、ere is a relationship between the size of the CoCr nanoparticle and the amount of genotoxicity. We have used a variety of assays to explore this. We have also explored the role of oxidative stress in the genotoxic response. 独立的毒性大小依赖但浓度为其他金属纳米粒子已经证明之前氧化物如二氧化钛、措,氧化锌（30、31）。很少有机制的差异大小的CoCr粒子的毒性在纳米尺度范围

17、内。金属氧化物的毒性可能也依赖于氧化应激的程度32 e34。在这研究中,我们调查是否有关系CoCr纳米粒子的大小和数量的基因毒性。我们使用各种各样的分析探讨。我们有也探讨了作用的氧化应激基因毒性反应。2. Materials and methods2.1. Preparation and sterilisation of nanoparticles2.1.1. Preparation of thermal plasma （ThP） particlesA water-cooled tungsten tip and copper crucible were used as cathode and

18、anode electrodes, respectively. A piece of CoCr disc of 33 mm diameter and 10 mm thick was placed in thewater-cooled copper crucible. A dynamic flow of Ar gas at 5L/min was applied between the tungsten tip and the CoCr disc maintained with a gap of 2 mm. A plasma was then generated between the tungs

19、ten tip and the CoCr disc using current and voltage listed in Table 1A. The plasma caused the CoCr disc to evaporate and the vaporised gas was quenched onto a water-cooled column, where the nanoparticles were collected for analysis. The composition of the alloy from which the thermal plasma particle

20、s were made, in terms of both weight% and atomic %, is shown in Table 1B. 热等离子体（ThP）粒子的制备 水冷钨的小费和铜坩埚被用作阴极和阳极电极,分别。一块CoCr盘33毫米直径10毫米厚是放置在thewater-cooled铜坩埚。一个动态的基于“增大化现实”技术的气体流动在5 l /钨之间的最小应用技巧和CoCr盘保持空白2毫米。那时等离子体生成钨提示和CoCr盘之间表1中列出使用电流和电压。CoCr盘引起的等离子体蒸发,蒸发气体淬火到水冷列收集纳米粒子进行分析。合金的成分热等离子体粒子,在重量%和原子%,表1 b

21、所示。2.1.2. Preparation of pin-on-plate （PoP） particlesNanoparticles （29.5 _ 6.3 nm） of CoCr alloy were generated using a flat pin-onplate tribometer. The composition of CoCr alloy （ASTM F1537） used to generate particles is listed in Table 1C. Particles were generated with a bi-directional motion of 5

22、.6 _ 10_2 ms_1 and a contact stress of 11 MPa over a 24 h period in water. The particles were recovered by filtration onto 100 nm pore sized 25 mm polycarbonate filter membranes and the mass of particles on the filters determined gravimetrically. The filters were placed in sterile pyrogen free-water

23、 and sonicated in a sonic bath for 1 h in order to resuspend and pool the particles. 粒子制备pin-on-plate（流行）纳米颗粒（29.5 - 6.3海里）CoCr合金使用平pin-onplate生成摩擦计。CoCr合金的成分（ASTM F1537）用于生成粒子是表1中列出。粒子生成的双向运动5.6 102 ms1和接触应力的11 MPa在24小时内的水。的粒子被过滤恢复到100纳米孔隙大小25毫米聚碳酸酯过滤膜和颗粒的质量过滤器重量分析地决定。过滤器被置于无菌热原质自由液和sonicated声波浴1 h

24、粒子以resuspend和池。2.1.3. Sterilisation, sonication and exposuresThe metal nanoparticles were transferred into glass universals, weighed, washed in 100% ethanol and heated at 180 _C for 3 h as a mode of sterilisation. Latex nanoparticles were sterilised by filtering through a 0.2 mm filter. After steril

25、isation, the nanoparticles were resuspended in serum free growth medium by sonication （pulsed mode: 7 pulses each of 1e2 s long） using a titanium probe and Sonics VibraCELL VC130PB sonicator （maximum output power: 130W）. The fibroblasts were exposed to the nanoparticles at doses of 0.0005 mm3/cell t

26、o 500 mm3/cell over a range of time points （2 he5 days）. 冲销,声波降解法和曝光金属纳米粒子被转移到玻璃共性,重,在100%乙醇洗和加热在180 C 3 h作为冲销的模式。乳胶纳米粒子被通过0.2毫米过滤器过滤消毒。消毒后,纳米颗粒在血清resuspended自由生长介质声波降解法脉冲（脉冲模式:7每个1 e2年代长）使用钛调查和超音速VibraCELL VC130PB超声发生器（最大输出功率:130 w）。的成纤维细胞暴露在纳米颗粒在剂量的0.0005 mm3 / 500 / mm3细胞细胞吗一系列的时间点（2 he5天）。2.2. D

27、issolution of metal ions into the growth mediumAll CoCr particles were incubated in growth medium for 24 h, 37 _C. At the end of the incubation period, the particle laden medium was centrifuged at 14,000 rpm for 20 min. The supernatant was analysed for trace metal by inductively coupled plasma mass

28、spectrometry （ICP-MS） Analytica Ltd, Sweden. 所有CoCr粒子在生长介质孵化24 h,37 _C。结束的时候潜伏期,粒子拉登介质centrifuged在14000 rpm20分钟。分析了上层的电感耦合等离子体的微量金属质谱（icp - ms）分析有限公司、瑞典。Table 1AConditions used to generate thermal plasma nanoparticles条件用于生成热等离子体纳米颗粒Sample Current （A ） Voltage （V）20 nm 350 4034.8 nm 410 4080 nm 500 4

29、0 样品 电流（A） 电压（V）Table 1bComposition of alloy from which thermal plasma particles were prepared . Element C Co Cr Fe Mn Mo N Ni SiWt% 0.06 64.3 28.0 0.25 0.50 6.00 0.20 0.20 0.50Atomic% 0.29 62.5 30.8 0.26 0.52 3.58 0.82 0.20 1.022.3. Characterisation of particles by transmission electron microscopy

30、（TEM） and photon correlation spectroscopy （PCS） 描述粒子的透射电子显微镜（TEM）和光子相关光谱（电脑）The sizes of the particles were determined by TEM and PCS. Samples for TEM analysis were prepared by applying a drop of the appropriate dispersion onto a 3 mm diameter, carbon covered copper grid. Specimens were examined on a Jeol 1200EXmkII TEM operated