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Understanding the tissue effects of tribo‐corrosion: Uptake, distribution, and speciation of cobalt and chromium in human bone cells

Cobalt and chromium species are released in the local tissues as a result of tribo‐corrosion, and affect bone cell survival and function. However we have little understanding of the mechanisms of cellular entry, intracellular distribution, and speciation of the metals that result in impaired bone health. Here we used synchrotron based X‐ray fluorescence (XRF), X‐ray absorption spectroscopy (XAS), and fluorescent‐probing approaches of candidate receptors P2X7R and divalent metal transporter‐1 (DMT‐1), to better understand the entry, intra‐cellular distribution and speciation of cobalt (Co) and chromium (Cr) in human osteoblasts and primary human osteoclasts. We found that both Co and Cr were most highly localized at nuclear and perinuclear sites in osteoblasts, suggesting uptake through cell membrane transporters, and supported by a finding that P2X7 receptor blockade reduced cellular entry of Co. In contrast, metal species were present at discrete sites corresponding to the basolateral membrane in osteoclasts, suggesting cell entry by endocytosis and trafficking through a functional secretory domain. An intracellular reduction of Cr6+ to Cr3+ was the only redox change observed in cells treated with Co2+, Cr3+, and Cr6+. Our data suggest that the cellular uptake and processing of Co and Cr differs between osteoblasts and osteoclasts. © 2014 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:114–121, 2015.

Tribo-corrosion is the degradation of material surfaces under the combined action of mechanical loading and electrochemical corrosion that occurs at the bearing surfaces and modular taper junctions of total hip arthroplasty (THA) components, resulting in the elevation of cobalt (Co) and chromium (Cr) concentrations in the patient synovial fluid and peripheral circulation. In asymptomatic patients with well-functioning metal-onmetal (MOM) bearings representative of the majority of patients, Lass et al recently reported joint fluid aspirate median Co and Cr concentrations of 113.4 mg/L (range: 3.9-176 mg/L) and 54 mg/L (range: 1.5-334 mg/L), 1 respectively, in a population with serum metal concentrations of <1.0 mg/L at minimum follow up of 18 years. In symptomatic patients with failing MOM bearings various investigators have reported joint fluid aspirate median Co and Cr concentrations of up to 1,496 mg/L (range: 11-24,262 mg/L) and 5,072 mg/L (range: ,731 mg/L) respectively, and serum concentrations of 17.8 mg/L (range: 4.6-110 mg/L) and 33.9 mg/L (range: 5.3-93 mg/L), respectively. [2][3][4] We, and others, have previously shown that these concentrations of Co and Cr affect bone cell survival and function in-vitro, [5][6][7] and associate with systemically measurable effects on bone mass and bone turnover in patients. 8 In order to understand the adverse effects of metal debris exposure on bone health and how these effects may be mitigated, it is necessary to understand how metal enters the relevant cell populations, and their intracellular distribution and speciation characteristics. In this study we have used microfocus X-ray spectroscopy, an analytical technique that uses highenergy X-ray beams derived by synchrotron radiation, to determine the chemical form and oxidation state of an element in microscopic samples. [9][10][11] Specifically, we have used Microfocus X-ray fluorescence (m-XRF) to measure the elemental distribution and micro-X-ray absorption near-edge structure (m-XANES) to characterize the chemical form of the metals in human bone cells.
We also investigated the role of two candidate metal transporters, the P2X7 receptor (P2X7R) and the divalent metal transport-1 (DMT-1), in the cellular entry of Co and Cr in human bone cells. The P2X7R is expressed in both human osteoblasts and osteoclasts and plays an important role in bone homeostasis. 12,13 The DMT-1 is a ubiquitously expressed transporter of ferrous iron and a broad range of other divalent cations, including Co 2þ , in mammalian cells. 14 However, its contribution to metal transport in bone cells is unknown. The overall goal of these studies was to better understand the cellular entry, trafficking, and processing of Co and Cr in bone cells, and identify possible tractable targets to mitigate their adverse effects after THA.

Metal Ion Preparation
Co(II) hexahydrate (CoCl 2 .6H 2 0) and Cr(III) chloride hexahydrate (CrCl 3 .6H 2 0) (Fluka, Gillingham, UK) served as salts for Co 2þ and Cr 3þ respectively. Cr(VI) oxide (CrO 3 ) (BDH Laboratory Supplies, Poole, UK) was used as source for Cr 6þ . The salts were dissolved in double distilled water (ddH 2 0) to a concentration of 10 7 mg/L, sterile filtered, aliquoted and stored at À20˚C. Prior to cell culture treatment, all stock solutions were diluted to 100Â of the final working concentrations in sterile distilled water. These were further diluted 1:100 in appropriate feeding media to reach the final working concentrations. Control treatment contained equivalent volume of sterile distilled water to maintain conditions. The concentrations used in this study are based on previously reported clinical metal concentrations in patient synovial fluid and peripheral circulation. [1][2][3][4]15,16 Osteoblast Cell Culture Human osteosarcoma derived osteoblast cells (SaOS-2) were seeded, cultured, and maintained in a 24 well-plate to observe intracellular distribution and speciation of metal ions, as previously described. 5 After the first 24 h, the cells were washed with phosphate buffered saline (PBS) and treated with 5,000 mg/L Co 2þ , 5,000 mg/L Cr 3þ , or 250 mg/L Cr 6þ for 3 days in Dulbecco's MEM GlutaMAX TM containing 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.5% foetal bovine serum (Gibco 1 , Invitrogen, Paisley, UK). At the end of the treatment, cells were fixed in 10% electron-microcroscopygrade formalin (TAAB Laboratories, Aldermaston, UK).

Osteoclast Cell Culture
Primary human osteoclasts were generated as described previously using CD14 þ enriched monocyte population from human peripheral blood of healthy volunteers. 5 To observe the intracellular state of metal ions, cells were treated with 500 mg/L Co 2þ , 500 mg/L Cr 3þ , or 50 mg/L Cr 6þ from day 3 till the onset of resorption (typically day 14) and 500 mg/L Co 2þ , 5,000 mg/L Cr 3þ , or 500 mg/L Cr 6þ for cells that had differentiated into multinuclear resorbing osteoclasts (from day 14) till day 21. Osteoclastogenic media with metal ion treatments was replaced every 2-3 days and cells fixed with 10% electron microscopy-grade formalin at the end of the experiment.

m-XRF Mapping and m-XANES Spectroscopy
The I18 microfocus spectroscopy beamline at the synchrotron facility Diamond Light Source (Harwell Science and Innovations Campus, Oxfordshire, UK) was used to perform the m-XRF and m-XANES scans. 17 A two-dimensional m-XRF elemental distribution map was generated for 2 or 3 cells per treatment by raster scanning samples with 4 mm Â 2 mm stepsize, 1000 ms collection per step with an incident X-ray energy of 8.5 keV. The XRF maps were analyzed using PyMCA 4.4.1. 18 m-XANES spectroscopy was used to determine the oxidation state, electronic configuration and site symmetry for metal ion treated samples at sites with high signal within the m-XRF maps. The X-ray absorption spectra were collected to 200 eV beyond the absorption edge and compared to known metal standards of different oxidation states. Data were analyzed using Athena and PySpline, 19,20 and plotted using GraphPad Prism version 5.04 for Windows (GraphPad Inc, La Jolla, CA).

Immunofluorescence
Osteoblasts (SaOS-2), primary human osteoclasts, and CACO-2 (a colorectal adenocarcinoma cell-line used here as a positive control for DMT-1 expression) 21 cells were cultured as previously described on glass coverslips and fixed in 4% PFA in PBS for 20 min. 5 Cells were blocked with 5% normal goat serum (NGS) in PBS for 1 h and subsequently incubated with 10 mg/mL anti-DMT-1 rabbit polyclonal antibody (Abcam, Cambridge, UK) in 1%NGS for 1 h. The cells were washed three times in PBS and incubated with 5 mg/mL Alexa Fluor 1 488 conjugated secondary goat antirabbit IgG (Life Technologies, Paisley, UK) in 1%NGS for 1 h. The cells were washed in PBS and counterstained with Phalloidin and Hoescht nuclear stain, mounted in ProLong Gold 1 Antifade (Life Technologies, Paisley, UK) and imaged using Leica 4000DB.

Assay for Cellular Entry of Co 2þ
SaOS-2 cells were seeded in a 96-well plate at a density of 5 Â 10 3 cells per well in complete media and left overnight to adhere prior to the assay. Multinucleated mature osteoclasts, usually at day 14 of the culture, were generated in 96-well plates from peripheral blood of healthy volunteers, as de- The wells were imaged with the Leica AF6000 time-lapse fluorescent microscope maintained at 37˚C using the L5 filter, with 3 min interval for 60 min. Co 2þ was added to the wells at a concentration range of 5-50,000 mg/L following the first 6 min which served as baseline. Cellular fluorescence of individual cells was measured using ImageJ (NIH: http:// imagej.nih.gov/ij/). The average change in cellular fluorescence relative to baseline was calculated, and plotted as the amount of fluorescence quenching relative to baseline to represent cellular entry of Co. The area under curve (AUC) was calculated using GraphPad Prism and expressed relative to vehicle with no antagonist.

Statistical Analysis
Fluorescence data was analyzed using One-way ANOVA with Dunnett's multiple comparisons post-test or the Kruskal-Wallis test with Dunn's multiple comparison post-test depending on the normality of the data sets. Cellular uptake of Co 2þ for antagonist treated and untreated samples were analyzed using a Student's unpaired t-test with or without Mann-Whitney post-test based on normality of the data. All analyses were conducted 2-tailed with a critical p-value of 0.05 using GraphPad Prism.

RESULTS
Localization and Speciation of Cobalt in Co 2þ Treated Cells Intracellular cobalt was found in all cell samples in XRF elemental maps (Fig. 1). Within osteoblasts cobalt was present throughout the cell body, but the signal was most intense in the area corresponding to the cell nucleus (Fig. 1A, Ob). Within osteoclasts localization of cobalt signals were to discrete areas within both developing (D-Oc) and mature cells (M-Oc), corresponding to the basolateral membrane in phase contrast images (Fig. 1A). K-edge m-XANES spectra from intracellular sites with high cobalt concentrations MECHANISMS FOR COBALT AND CHROMIUM ENTRY INTO BONE CELLS were compared to standards of cobalt at different oxidation states (Co-metal, CoO, Co 2 O 3 , and Co(II) acetate, Fig. 1B). Osteoblasts and mature osteoclasts showed the presence of Co in the þ2 oxidation state corresponding to the Co(II)acetate spectra, indicating that no intracellular redox change occurs following entry of Co 2þ ions into these cell types. Whilst developing osteoclasts demonstrated some cellular entry of Co 2þ , the concentration was insufficient to generate m-XANES spectra, suggesting that uptake of cobalt is less for developing osteoclasts relative to active, mature osteoclasts.

Localization and Speciation of Chromium in Cr 3þ Treated Cells
The XRF maps for all Cr 3þ treated cells showed the presence of intracellular chromium ( Fig. 2A, Ob). Chromium was seen throughout the osteoblast cell body, but was most concentrated at perinuclear sites. Both developing and mature osteoclasts showed chromium localization to the basolateral membrane ( Fig. 2A, D-Oc and M-Oc). Chromium K-edge m-XANES spectra from all cell samples were compared to chromium standards (Cr-metal, Cr(III)OH, Cr(III) PO 4 , Cr 2 O 3 , and CrO 3 ) (Fig. 2B). The spectra from osteoblasts and both osteoclasts samples were similar to Cr(III)OH and Cr(III)PO 4 indicating the presence of Cr in the þ3 oxidation state only.
Localization and Speciation of Chromium in Cr 6þ Treated Cells All Cr 6þ treated osteoblasts and osteoclasts samples showed the presence of intracellular elemental chromium (Fig. 3A). In contrast to the focal distribution of Co 2þ and Cr 3þ within bone cells, chromium was diffusely distributed, suggesting a different mechanism of cellular entry or intra-cellular processing. The characteristic preedge feature for Cr 6þ (seen in CrO 3 ) was absent in m-XANES spectra from all cell samples indicating intracellular reduction of Cr 6þ to Cr 3þ (Fig. 3B).

Calcein-AM Quenching by Co 2þ as a Measure of Cellular Entry
Osteoblasts incubated with 0.25 mM Calcein AM demonstrated a dose-dependent quenching of fluorescence over time in the presence of extracellular Co 2þ over the observed range of patient serum and hip aspirate (5-5,000 mg/L), and also at a concentration of 50,000 mg/L that was used as a positive control ( Fig. 4A and B, data are presented as the inverse of the fluorescence-quenching curve to represent cellular entry of Co 2þ ). Calcein quenching in mature osteoclasts treated with Co 2þ was delayed, non-dose depen-  116 SHAH ET AL. dent and variable over the time course. Only the positive control concentration of 50,000 mg/L resulted in a significant change in fluorescence as determined by the AUC analysis (p < 0.0001). Whilst this is not a concentration of Co 2þ measured in clinical samples, it acted as a positive control to confirm the uptake model was functional in osteoclasts ( Fig. 4C and D).
Role of P2X7R and DMT-1 in Uptake of Co 2þ P2X7R expression in human bone cells is previously established. 12 Here we confirmed expression of the DMT-1 receptor in the studied human bone cells by immunofluorescence prior to treatment with NSC306711 (Fig. 5). Treatment of osteoblasts with Co 2þ for 1 h resulted in an increase in cellular entry of Co 2þ at concentrations of 50 mg/L and above (p < 0.05, Fig. 6A). Treatment with 40 nM P2X7R antagonist A740003 reduced the cellular entry of Co 2þ at concentrations of 500 mg/L and above (p < 0.05, all comparisons). Treatment of osteoblasts with 50 mM DMT-1 antagonist in combination with exposure to Co 2þ in the range 5-5,000 mg/L did not reduce cellular entry of Co 2þ at any concentration (Fig. 6C). Consistent with the time-course data ( Fig. 4C and D), there was no significant change in fluorescence in osteoclasts below 50,000 mg/L Co 2þ (Fig. 6C and D). Addition of the P2X7R or DMT-1 antagonists did not affect cellular entry of Co 2þ into osteoclasts at any concentration (p > 0.05, all concentrations), consistent with a different mechanism of metal entry for osteoclasts.

DISCUSSION
Cobalt and chromium species are released in the local tissues as a result of tribo-corrosion, and affect bone cell survival and function. Here we used a combination of synchrotron radiation and targeted blockade of receptors involved in metal trafficking to explore the uptake, intracellular distribution and speciation of Co and Cr ions in human bone cells.
The intra-cellular localization of Co 2þ to nuclear and perinuclear sites in osteoblasts is consistent with its known interactions with genomic DNA and nuclear proteins associated with DNA repair. 22,23 Our finding that Co 2þ was also distributed throughout the cell body is in keeping with the distribution of other divalent cations, such as Ca 2þ and Zn 2þ that have established plasma membrane channels and transporters, 24 and may suggest use of similar transport machinery. In support of this, our calcein quenching studies suggest that the P2X7R contributes to this transport. In contrast, our data suggest that the proton-coupled divalent metal transporter DMT-1, although expressed in human osteoblasts, does not contribute significantly to Co 2þ transport in human osteoblasts.
Localization of Co 2þ to the basolateral membrane in osteoclasts suggests that cobalt enters the cell by endocytosis and is sequestered in vesicles undergoing exocytosis through a functional secretory domain (FSD). In support of this, previous studies have shown that metal corrosion products are generated and taken up by endocytosis in osteoclasts cultured on metal surfaces and are released into the culture supernatant via the transcytotic pathway. 25 The absence of m-XANES spectra for cobalt in the developing osteoclast is probably due to its presence at relatively low concentrations. The relatively under-developed endocytic and transcytotic machinery in developing osteoclasts compared to mature osteoclasts might explain this difference. 26 A lower concentration of cobalt observed in developing osteoclasts also supports the concept that endocytosis is the dominant mechanism of cobalt uptake into osteoclasts. Although expressed by osteoclasts, as shown previously for P2X7R and for the first time here for DMT-1, neither of these candidate metal transporters are significant mediators of Co 2þ entry into osteoclasts.
The observation that cobalt exists only in its þ2 state within both osteoblasts and mature osteoclasts is consistent with previous ex-vivo findings, 27,28 and suggests that the mechanisms of intracellular toxicity is not driven by valency changes inducing hydrogen peroxide mediated generation of free radicals. 29 Alternate mechanisms of cytotoxicity may include substitution of other bivalent cations, such as calcium and iron, in mitochondrial function and other biological pathways. 29  The finding of chromium throughout the osteoblast cell body, but most concentrated at perinuclear sites following Cr 3þ treatment, contrasts with current dogma that Cr 3þ has low permeability to cell membranes with no specific system for membrane transport. 30 One explanation for the cellular entry of Cr 3þ in osteoblasts is its ability to bind to extracellular proteins, such as albumin, which enter cells via endocytosis. 31 The internalized protein-bound Cr 3þ is then trafficked in lysosomes and returned to the golgi apparatus for recycling. 32 Both developing and mature osteoclasts showed chromium localization to the basolateral membrane ( Fig. 2A, Intracellular Cr 3þ ). A similar mechanism may exist in osteoclasts whereby the recycled proteins undergo exocytosis from the basolateral FSD. Whilst Cr 3þ has a high affinity for DNA in a cell-free system, 33,34 our observation that Cr 3þ was most highly localized to the perinuclear region may suggest that Cr 3þ bound to proteins once internalized is preferentially targeted to the golgi for recycling.
The diffuse localization of chromium in both Cr 6þ treated osteoblasts and osteoclasts suggest a similar mechanism of metal uptake in these cell types. Cr 6þ exists as divalent chromate ion (CrO 4 2À ) at physiological pH that is analogous to phosphates and sulfates, which readily enter cells via their anion transporters. 35,36 Our m-XANES spectra data showing the presence of only Cr 3þ after cell treatment with Cr 6þ is consistent with the intracellular reduction of Cr 6þ and its proposed cytotoxicity through generation of reactive oxygen species and subsequent DNA damage. 37 These data suggest that alternate explanations for the absence of Cr 6þ from clinical explant tissue 38 include rapid reduction to the stable form, consistent with its established chemistry, as well as a lack of Cr 6þ generation during tribocorrosion. We were unable to examine the uptake of chromium through the P2X7 and DMT-1 receptors using the fluorescence approach as suitable fluorescent probes are currently unavailable.
In conclusion, the intracellular distribution of metal ions highlights a cell-and metal-specific mode of cellular entry, whilst speciation confirms redox stability of Co 2þ and Cr 3þ , and reduction of Cr 6þ to Cr 3þ . The difference in cellular distribution between Cr 3þ and Cr 6þ derived chromium may represent a possible approach to identify the parent species in pathological tissue samples. Finally, our data suggests a role of P2X7R in cellular entry of Co 2þ in osteoblasts, but not osteoclasts, identifying it as an investigative candidate for cell-specific targeting to modulate the osteoblast