Biomaterials生物材料.docx

1、Biomaterials生物材料Biomaterials生物材料Influence of particle size and reactive oxygen species on cobalt chromenanoparticle-mediated genotoxicity粒子大小和活性氧对钴铬nanoparticle-mediated基因毒性nanoparticle-mediated毫微粒（纳米粒）调节，促进Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide siz

2、e range of metallic nanoparticles as a result of wear. 患者钴铬(CoCr)金属对金属介面人工髋关节(MOM)植入可能接触到广泛的大小金属纳米粒子由于穿的范围。 In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles (a) from a tribometer (30 nm) and (b) thermal plasma tech

3、nology (20, 35, and 80 nm) in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. 在这项研究中我们有特征的生物综合反应人类成纤维细胞的两种类型的派生CoCr粒子(从摩擦计(a) (30 nm)和(b)热等离子体技术(20,35岁,和80海里)体外,测试的依赖纳米粒子大小或氧自由基的生成,或两者兼而有之。Metal ions were released from thesurface of nanopartic

4、les, 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 could be eliminated by inhibition of NADPH oxidase, sug

5、gesting 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 mitochondria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Consequ

6、ently, 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 resulted in misshapen nuclei, disruption of mature lam

7、in 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 incidence of micronuclei and ion release from the nanoparticl

8、es 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 suggest that cells exhibit diverse mitochondrial ROS-dep

9、endent 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 metal-on-metal (MoM) hip replacements are exposed to CoCr

10、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6.7 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)。Ca

11、lls 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 an increase of circulating metal ions in the blood of t

12、hese 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 bodys natural defences, for example by macrophage uptak

13、e 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 is essentially from the whole implant, or by corrosion

14、 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 increases in aneuploidy in peripheral blood lymphocytes of

15、 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 vitro 29, demonstrating that CoCr particle size may be

16、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-and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。

17、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 metaloxides such as TiO2, CuO, ZnO 30,31. There is very littl

18、e 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 there is a relationship between the size of the CoCr nan

19、oparticle 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

20、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 anode electrodes, respectively. A piece of CoCr disc o

21、f 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 tungsten tip and the CoCr disc using current and voltage li

22、sted 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 particles were made, in terms of both weight% and atomic %, is

23、 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所示。2.1.2. Preparation of pin-on-plate (PoP) particlesN

24、anoparticles (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 5.6 _ 10_2 ms_1 and a contact stress of 11 MPa over a 2

25、4 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 and sonicated in a sonic bath for 1 h in order to res

26、uspend 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粒子以resuspend和池。2.1.3. Sterilisation, sonication and ex

27、posuresThe 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 sterilisation, the nanoparticles were resuspended in serum f

28、ree 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 to 500 mm3/cell over a range of time points (2 he5 days

29、). 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. Dissolution of metal ions into the growth mediumAll CoC

30、r 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 spectrometry (ICP-MS) Analytica Ltd, Sweden. 所有CoCr粒子在

31、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 40 样品 电流(A) 电压(V)Table 1bComposition of alloy from whic

32、h 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 (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