Effects And Mechanisms Of Cobalt Nanoparticles On Human T Cells In Vitro

Background: Modern metal-on-metal (MOM) total hip replacement system hasimproved the resistance to wear and fatigue and demonstrated excellent effects on earlyand mid-term clinical outcomes. The implanted materials were degraded caused by theinevitable mechanism and electrochemical corrosion, then formed the products of chemicalactivities. Numerous studies showed that the blood and urine metal ion levels increased inpatients after MOM hip arthroplasty. It is worth mentioning that the ultrafine metalparticles (6-834nm) were found in periprosthetic tissue, mainly including cobalt-basednanoparticles. Perivascular accumulations of T cells and/or B cells as well as plasma cells,within periprosthetic tissues adjacent to MOM implants, have recently been reported in theliteratures. Moreover, various authors have recently reported the clinical cases ofdestructive soft-tissue masses relating to the hip joint in patients following MOM hipresurfacing arthroplasty, such as those described as pseudotumours. The commonhistological features of pseudotumours are extensive necrosis and lymphocytic infiltration.Furthermore, recent study has indicated that over time,patients with MOM hip arthroplastydemonstrate serum cobalt, chromium-specific immunoglobulin E, increased levels of bothchromosome translocations and aneuploidy in peripheral blood lymphocytes, anddecreased levels of T cells, leukocyte and bone marrow cells. In vitro studies, metalnanoparticles showed the biological effects different from the macro-particles orcorresponding ions. Therefore, it is significant to investigate the effects and mechanisms ofcobalt nanoparticles on immune system. Part One The cytotoxicity and genotoxicity of cobalt nanoparticleson human T cells in vitroObjective: To investigate the cytotoxicity and genotoxicity of cobalt nanoparticles(CoNPs) on human T cells, comparing with cobalt ions (Co²⁺), and to evaluate the etiologyof complex reactions in patients following MOM hip arthroplasty.Methods: Human peripheral blood T cells were selected as test system. We used theMTT assay and LDH assay to evaluate the cell viability, and the comet assay and themicronucleus test (MNT) to study the genotoxic effects of CoNPs, comparing with cobaltions. Moreover, the Co²⁺levels released from CoNPs were analyzed by inductivelycoupled plasma-mass spectrometry (ICP-MS).Results: CoNPs and Co²⁺inhibited T cell viability in a concentration andtime-dependent manner. CoNPs at5μM had no obvious influence on cell viability(P>0.05), but a statistically significant reduction of cell viability was induced by CoNPsabove10μM (P<0.05). In contrast, Co²⁺inhibited cell viability at concentrations of50μM and higher (P<0.05). The CC50values were approximately10and50μM for CoNPsand Co²⁺respectively, suggesting that NPs are more toxic than Co²⁺. Similar to the MTTresults, LDH levels increased in a concentration-and time-dependent manner in T cellsexposed to CoNPs and Co²⁺. The release of Co²⁺from Co-NPs in culture medium wasanalyzed by ICP-MS after4,24, and48h of incubation. A time-dependent increase in therelease of Co²⁺from CoNPs were found in both CoNPs groups (P<0.001). The release of50μΜ CoNPs was5.12±1.44,14.22±3.17,32.78±2.70after4,24, and48h of incubation,and100μΜ was6.27±2.49,18.78±4.29,38.81±2.11respectively. Our data demonstratedthat the amount of ions released from NPs in the culture medium were low and did notreach any effective concentrations,therefore, under no condition could ionic cobalt aloneexplain the toxicity of Co-NPs. In comet assay, a statistically significant, concentrationdependent increase in primary DNA damage was induced directly by nanoparticles at3and6μM. The tail DNA percent were8.73±4.60and9.64±4.77for CoNPs at3and6μMrespectively, and the olive tail moment were2.16±1.19and9.66±4.73for CoNPs at3and 6μM respectively. In contrast, a4-h treatment with Co²⁺(10–30μM) did not induce DNAdamage. In micronucleus test, a statistically significant increase in BNMN(‰) wasinduced directly by nanoparticles at6μM (P<0.05). BNMN(‰) was13.69±1.85,comparing with control (5.46±1.28). In contrast, a4-h treatment with Co²⁺(10-30μM) didnot show any statistically significant increase in BNMN(‰). Our data evidenced genotoxiceffects in T cells exposed to CoNPs, not to Co²⁺.Discussion: This study clearly showed the cytotoxicity and genotoxicity of CoNPs onT cells and the effects of NPs were stronger than that of Co²⁺. The levels of Co²⁺releasedfrom NPs in the culture medium were not high enough to induce cell damage, whichsuggested that the increased NPs toxicity in T cells may be due to particles themselves. Thetoxicity, determined in the present in vitro study, raises the possibility to associate withpseudotumours, chromosome translocations and aneuploidy and reduction of peripheralblood T cells. Moreover, we could not exclude the synergistic effects with ions. Part Tow Biological mechanisms of cobalt nanoparticles onhuman T cells in vitroObjective: To investigate size distribution, dispersion and cellar uptake of CoNPs,and the effects of CoNPs on oxidant-antioxidant system.Methods: CoNPs were dissolved in PBS and serum-containing medium in order toinvestigate size distribution and dispersion. T cells were exposed to CoNP for24h at aconcentration of5μM. After this incubation period, exposed cells were centrifuged,washed, fixed, postfixed and stained, subsequently dehydrated, embedded and stained withlead citrate. The location and distribution of particles within the cells were then analyzedby TEM. Moreover, T cells were exposed to CoNP and Co²⁺respectively, then investigatethe intracellular reactive oxygen species (ROS) level through fluorescentspectrophotometric analysis, the superoxide dismutase (SOD) activity through auto-oxidation of catechol, the vitality of catalase (CAT) through H2O2reduction, the glutathione (GSH) levels through glutathione reductase assay.Results: The depolymerization and dispersion of CoNPs, mean50nm, showed betterunder the conditions of serum-containing medium than that of PBS. Uptake of suspendedCoNP into cells was observed by TEM after24h exposure. The particles were located inthe cytosol as aggregates as well as single particles as compared to the untreated cells. Themicrographs organised in the column below show incorporated nanoparticles within thecytoplasm; no particles were found in the nuclei of cells. In oxidant system, a statisticallysignificant increase in RUF was induced directly by nanoparticles at6μM. In antioxidantsystem, statistically significant decreases in GSH, SOD, CAT, GPx were induced by6μMnanoparticles, and values were8.74±2.41μM/mg protein,8.92±0.57U/mg protein,6.33±0.46U/mg protein,6.15±0.64U/mg protein respectively. In contrast, a4h-treatmentwith30μM of Co²⁺did not show statistically significant changes in oxidant-antioxidantsystem.Discussion: Stable CoNPs serum suspension system could be in favor oftransportation through blood and lymph, subsequently exert biological activity. Our studyfounded that nanoparticles entered T cells quickly and easily, suggesting that CoNPs couldenter human cells successfully, irrespective of the phagocytosis of cells. CoNPs couldinduce oxidative stress in T cells, which is the result of an imbalance in oxidant/antioxidantsystem, and could lead to tissue degeneration. The short-term exposure of Co²⁺ did notinduce oxidative stress effects at the subtoxic concentrations (<CC50).