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

Abstract

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 Cr 6þ to Cr 3þ was the only redox change observed in cells treated with Co 2-4We, and others, have previously shown that these concentrations of Co and Cr affect bone cell survival and function in-vitro, 5-7 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.9-11 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. 12,13The DMT-1 is a ubiquitously expressed transporter of ferrous iron and a broad range of other divalent cations, including Co 2þ

Advances in element speciation analysis of biomedical samples using synchrotron-based techniques

“…Indeed, Cr(VI) K-Edge spectrum exhibits a typical pre-edge feature in the XANES region. The absence of such feature and the strict resemblance with Cr(III) spectrum were consistent with the intracellular bio-reduction model of Cr(VI) [51].…”

“…The study of inorganic compounds effects on human health is one of oldest branch of applied toxicology. Modern orthopedics offers interesting XAS case studies [49][50][51]. Cobalt, chromium and relative alloys are the typical constituents of hips prosthesis but also the main responsible for hips failure and induced toxicity.…”

Osteocyte physiology and response to fluid shear stress are impaired following exposure to cobalt and chromium: Implications for bone health following joint replacement

“…The lack of any effect of Cr 3+ alone is consistent with the observations of Kanaji and coworkers and the previously held dogma of the relative inability of Cr 3+ to cross cell‐membranes 34. In contrast to this dogma, we have previously shown the intracellular localization of Cr 3+ in osteoblasts,21 as well as an additive effect of Co 2+ and Cr 3+ treatment 22. Thus the increased effect of combined ions at 500 μg/L over the effect of Co 2+ alone observed in this study suggests for the first time potential facilitation of Cr 3+ entry into osteocytes, as well as common downstream signaling.…”

“…Periprosthetic concentrations are several fold higher (median Co, 113 μg/L; median Cr, 54 μg/L),18 while patients with failing prosthesis have reported Co and Cr concentrations as high as 528 μg/L (range: 0–13,000 μg/L) and 1,844 μg/L (range: 0–38,600 μg/L), respectively 19. We recently demonstrated that exposure to Co 2+ and Cr 3+ adversely affects both osteoclast and osteoblast survival and function, including the mineralization of prosthesis surfaces in vitro, at concentrations equivalent to those found clinically 20, 21, 22…”

“…19 We recently demonstrated that exposure to Co 2þ and Cr 3þ adversely affects both osteoclast and osteoblast survival and function, including the mineralization of prosthesis surfaces in vitro, at concentrations equivalent to those found clinically. [20][21][22] Previous studies have suggested that exposure to Co 2þ in vitro has a cytotoxic and pro-inflammatory effect on osteocytes. [23][24][25][26] Here, we develop understanding in this area by examining the effects of exposure to both Co 2þ and Cr 3þ at clinically relevant concentrations on osteocyte physiology-in particular their survival, dendritic morphology, response to fluid shear stress (FSS), and mineralization.…”