Local and systemic concentrations of cobalt (Co) and chromium (Cr) ions may be elevated in patients with accelerated tribo-corrosion at prosthesis bearing surfaces and modular taper junctions. Previous studies by us and others have shown that exposure to these metals negatively affect the viability and function of osteoblasts and osteoclasts in vitro, with implications for bone health. More recently, we have observed an increase in total bone mineral density and reduced bone turnover (TRAP5b and osteocalcin) in patients with well-functioning metal-on-metal hip resurfacing (MOMHR). Here, we provide data to support the hypothesis that osteoclast differentiation and function is altered in this patient population, and that this effect is transferrable through their serum. Patients with well-functioning MOMHR (n=18) at median follow-up of 8 years were individually matched for gender, age and time-since-surgery to a low-exposure group consisting of patients with metal-on-polyethylene total hip arthroplasty (THA). The median circulating concentrations of Co and Cr for the MOMHR group were 2.53µg/L and 2.5µg/L respectively, compared to 0.02µg/L and 0.03µg/L for the THA group. Monocyte fraction of peripheral blood was isolated from these patients, seeded onto dentine wafers and differentiated into osteoclasts using media supplemented with RANKL and M-CSF (osteoclastogenic media, OM). Cultures were monitored for the onset of resorption, following which they were treated with OM, autologous serum or serum from the other individual within the matched MOMHR - THA pair, all supplemented with RANKL and M-CSF. At the end of the culture, cells were TRAP stained and quantified for total osteoclast number, number of resorbing osteoclasts and percentage resorption using the CellD Software Package (Olympus, Southend-on-Sea, U.K.). For cells differentiated in osteoclastogenic media, the resorbing ability of osteoclasts derived from MOMHR patients was reduced by 30% (P=0.046) compared to THA. Correlation analyses showed that chronic exposure to Co and Cr trends towards negative association with resorption ability of these osteoclasts (r = −0.3, P=0.06). Furthermore, the resorbing ability of osteoclasts generated from MOMHR patients and differentiated in autologous serum was reduced 33% (p < 0 .0001), whilst matched THA serum caused a smaller reduction of 14% (p < 0 .01). When cells derived from THA patients were differentiated in autologous serum, the resorbing ability of osteoclasts was similarly reduced by 35% (p < 0 .0001), whilst the matched MOMHR serum also caused a reduction of 21% (p < 0 .0001). Reduced osteoclastogenic response of precursor cells from patients with higher circulating Co and Cr suggests an inherent change in their potential to differentiate into functional osteoclasts. The data also suggests that functional response of mature osteoclasts generated from patient precursor cells are dependent on the prior systemic metal concentrations and the presence of higher circulating CoCr in patients with MOMHR. These effects are modest, but may explain the subtle increase in systemic bone mineral density and decreased bone turnover observed in patients after 8 years exposure compared to age, sex, and exposure-time matched patients who received a conventional THA.
Commonly used alterations of prosthetic surfaces include grit-blasting (GB), plasma-sprayed titanium (Ti) or hydroxyapatite (HA) coating. Systemic concentrations of cobalt (Co) and chromium (Cr) are elevated in patients with metal-on-metal hip replacement, but can occur for all modular hip replacements. Here, we use whole genome microarrays to assess differential gene expression in primary human osteoblasts grown in vitro and on these prosthesis surfaces following exposure to clinically relevant concentrations of Co and Cr. Mesenchymal cells obtained from bone-fragments of 3 patients undergoing joint replacement surgery were differentiated into osteoblasts. Subsequently, cells were cultured in vitro on tissue-culture plates (TCP), or on GB, Ti and HA surfaces (JRI Orthopaedics Ltd, Sheffield, UK). Following 24hr exposure to a combination of clinically equivalent concentrations of Co2+:Cr3+, RNA was extracted and hybridized to SurePrint-G3 Gene Expression Microarray. Probe signals were normalised using ‘Limma’ package on R-Bioconductor and differential gene expression assessed with empirical Bayes approach (Log2FC>1.00, P<0.001 for differentially expressed genes). For cells grown on TCP, 11 genes were upregulated with 500μg/L Co2+:Cr3+. Of these, 4 were associated to HIF-1 signalling based on KEGG pathway analysis (P=5.4e-5). Exposure to 1000μg/L Co2+:Cr3+ altered expression at 164 loci for HA surfaces, and a separate 50 loci for Ti surfaces compared to GB surfaces. Genes for osteoblast differentiation (BMP2 and RGS2) were downregulated on HA surfaces compared to GB, whilst genes for cell-adhesion (ESAM), vesicular trafficking (RAB37) and protection against oxidative damage (NRF2) were upregulated. Ti surfaces caused an upregulation in ERBB3 and CNTF, which are associated with inhibition of osteoblast differentiation and mineralisation, when compared to GB surfaces. This study confirms the role of HIF-1 signalling in response to prosthesis generated metal ions, and is the first to provide a comprehensive genome-wide insight into transcriptional response of osteoblasts at prosthesis surface to clinically equivalent metal exposure.
We have previously observed an increase in total bone mineral density and reduced bone turnover (TRAP5b and osteocalcin) in patients with well-functioning metal-on-metal hip resurfacing (MOMHR). Here, we provide data to support the hypothesis that osteoclast differentiation and function is altered in this patient population, and that this effect is transferrable through their serum. Patients with well-functioning MOMHR (cases, n=18) at a median follow-up of 8 years were individually matched for gender, age and time-since-surgery to a low-exposure group consisting of patients with THA (controls, n=18). The monocyte fraction of patient peripheral blood was isolated and differentiated into osteoclasts on dentine wafers using RANKL and M-CSF supplemented media (osteoclastogenic media, OM). Cultures were monitored for the onset of resorption, at which point the cells were treated with OM, autologous serum or serum from matched MOMHR/THA donors, all supplemented with RANKL and M-CSF. At the end of the culture, cells were TRAP-stained and quantified using CellD Software Package, Olympus. When cells were differentiated in standard osteoclastogenic media, the resorbing ability of osteoclasts derived from MOMHR patients was reduced 22%(p<0.0079) compared to THA. The resorbing ability of osteoclasts generated from MOMHR patients and differentiated in autologous serum was reduced 33%(p<0.0001), whilst matched THA serum caused a smaller reduction of 14%(p<0.01). When cells derived from THA patients were differentiated in autologous serum, the resorbing ability of osteoclasts was similarly reduced by 35%(p<0.0001), whilst the matched MOMHR serum also caused a reduction of 21%(p<0.0001). This data suggests that prior exposure to higher circulating Co and Cr in patients with MOMHR reduces osteoclastogenesis, and that the detrimental effect on the functionality of mature osteoclasts is transferable through the serum. This has implications for systemic bone health of patients with MOMHR or modular taper junctions.
Systemic concentrations of metal ions (cobalt and chromium) are persistently elevated in patients with metal-on-metal hip resurfacing (MOMHR) compared to conventional total hip arthroplasty (THA). Several studies by us and others have described the detrimental effects of metal exposure on survival and function of various cell types The cohort consisted of 34 patients with a well-functioning MOMHR at a median follow-up of 9.75 years. These were individually matched for gender, age and time-since-surgery to a non-exposure group consisting of patients with THA. Genomic DNA was isolated from blood samples and cell composition estimated using the ‘estimateCellCounts’ function in ‘minfi R-package’. Methylation was assessed using the Illumina 450k BeadChip array analysing 426,225 probes. Logit model was fitted at each probe with case/control status as independent variable and covariates of gender, age, time-since-surgery, smoking, non-arthroplasty metal exposure, and cell composition. DNA methylation age was assessed using an online calculator ( Cell distributions did not differ between the cases and controls (Wilcoxon test In summary, large methylation changes following MOMHR seem to be absent, compared to THA. Future research with larger samples will be needed to clarify the presence and extent of small methylation changes.
Bone related adverse events including failure of implant osseo-integration, periprosthetic fracture, femoral neck narrowing, and unexplained pain occur more frequently following metal-on-metal hip resurfacing (MoMHR) versus total hip arthroplasty (THA). The exact mechanism for the adverse effects is still unclear and may be due to the direct effect on bone cells of metal ions released from the prostheses. The aim of the present study was to determine the effect of clinically relevant combinations of metal ions on osteoblast cell survival and function. To assess cell proliferation and alkaline phosphatase (ALP) activity of osteoblasts, human osteoblast cells (SaOS-2), were cultured in 96-well plates for 24-hours and then treated with metal ions. Cell proliferation was measured at day 3 and day 7 using MTS assay, whilst ALP activity was assessed at day 3 by measuring pNPP substrate hydrolysis by the cell lysate. Mineralisation ability of the cells was assessed in 24-well plates cultured until day 21 and staining the calcium deposits using Alizarin red. All cultures were treated with the IC50 concentration of Co(II) (135μM) and an equivalent Cr(III) concentration (1Co(II):1Cr(III)). After 3 days, Co(II) at an IC50 concentration decreased osteoblast proliferation as expected, but no further decrease in proliferation was observed with the 1Co(II):1Cr(III) combination treatment. However, after 7 days, a further significant decrease (P<0.05) in proliferation was observed with the combination treatment compared to Co(II) IC50. A similar significant decrease (P<0.01) was observed for ALP activity at day 3 with 1Co(II):1Cr(III) compared to Co(II) alone. For mineralization, a significant reduction (P<0.0001) was observed for Co(II) IC50 concentration, however no further reduction was seen with the 1Co(II):1Cr(III) combination treatment. The observed decrease in cell proliferation and ALP activity with combination treatments suggest an additive detrimental effect compared to single ions alone. The mineralisation ability did not show any additive effect due to cell toxicity of chronic exposure to IC50 concentrations calculated from 3 day proliferation cultures. The results suggest that presence of both cobalt and chromium ions in the periprosthetic environment have more severe detrimental effect on osteoblasts than single ions alone and extend our understanding of the periprosthetic bone health.
One possible mechanism by which metal-on-metal hip resurfacing (MOMHR) may be associated with prosthesis loosening, periprosthetic fracture, and femoral neck narrowing is through an increase in bone resorption by osteoclast cells. Whilst it is known that metal ions such as cobalt (Co) and chromium (Cr) ions (that are elevated locally and systemically after MOMHR), may affect osteoblast and macrophage activity in-vitro, little is known about the effect of these ions on osteoclasts. We examined whether these ions have an adverse effect on human peripheral blood derived osteoclasts at levels that are clinically relevant after MOMHR. Peripheral blood mononuclear cells from healthy donors were seeded onto dentine wafers, and treated to transform them into osteoclasts using standard techniques in the presence of various clinically relevant concentrations of Co2+, Cr3+, and Cr6+. After 3 weeks of culture osteoclast number and resorption pit formation was quantified using histological techniques. All 3 metal ions had a dose-dependent effect on both osteoclast formation and resorption activity. At ion levels found in serum after MOMHR, an increase in osteoclast formation and bone resorption was found, but at higher levels found in synovial fluid, osteoclast cell proliferation and resorption activity was decreased, likely due to a direct toxic effect of the ions on the cells (Figure 1). Cr6+ was more toxic than the other ions at higher concentrations. Our data suggest that metal ion release following MOMHR may increase osteoclast activity systemically that might have a deleterious effect on general and local bone health, and may contribute to the observed bone related complications of MOMHR.
