Karan M. Shah

Effects of cobalt and chromium ions at clinically equivalent concentrations after metal-on-metal hip replacement on human osteoblasts and osteoclasts: implications for skeletal health

Metal-on-metal hip replacement (MOMHR) using large diameter bearings has become a popular alternative to conventional total hip arthroplasty, but is associated with elevated local tissue and circulating levels of chromium (Cr) and cobalt (Co) ions that may affect bone health. We examined the effects of acute and chronic exposure to these metals on human osteoblast and osteoclast formation and function over a clinically relevant concentration range previously reported in serum and within hip synovial fluid in patients after MOMHR. SaOS-2 cells were cultured with Co(2+), Cr(3+) and Cr(6+) for 3 days after which an MTS assay was used to assess cell viability, for 13 days after which alkaline phosphatase and cell viability were assessed and for 21 days after which nodule formation was assessed. Monocytes were isolated from human peripheral blood and settled onto dentine disks then cultured with M-CSF and RANKL plus either Co(2+), Cr(3+) or Cr(6+) ions for 21 days from day 0 or between days 14 and 21. Cells were fixed and stained for TRAP and osteoclast number and amount of resorption per dentine disk determined. Co(2+) and Cr(3+) did not affect osteoblast survival or function over the clinically equivalent concentration range, whilst Cr(6+) reduced osteoblast survival and function at concentrations within the clinically equivalent serum range after MOMHR (IC(50) =2.2 μM). In contrast, osteoclasts were more sensitive to metal ions exposure. At serum levels a mild stimulatory effect on resorption in forming osteoclasts was found for Co(2+) and Cr(3+), whilst at higher serum and synovial equivalent concentrations, and with Cr(6+), a reduction in cell number and resorption was observed. Co(2+) and Cr(6+) within the clinical range reduced cell number and resorption in mature osteoclasts. Our data suggest that metal ions at equivalent concentrations to those found in MOMHR affect bone cell health and may contribute to the observed bone-related complications of these prostheses.

Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two –omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three –omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.

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.

Cobalt and chromium exposure affects osteoblast function and impairs the mineralization of prosthesis surfaces in vitro.

Cobalt (Co) and chromium (Cr) ions and nanoparticles equivalent to those released through tribo‐corrosion of prosthetic metal‐on‐metal (MOM) bearings and taper junctions are detrimental to osteoblast activity and function in vitro when examined as individual species. Here we examined the effects of Co2+:Cr3+ and Co2+:Cr6+ combinations on osteoblast‐like SaOS‐2 cellular activity, alkaline phosphatase (ALP) activity and mineralization to better reflect clinical exposure conditions in vivo. We also assessed the effect of Co2+:Cr3+ combinations and Co:Cr nanoparticles on SaOS‐2 cell osteogenic responses on grit‐blasted, plasma‐sprayed titanium‐coated, and hydroxyapatite‐coated prosthesis surfaces. Cellular activity and ALP activity were reduced to a greater extent with combination treatments compared to individual ions. Co2+ and Cr3+ interacted additively and synergistically to reduce cellular activity and ALP activity, respectively, while the Co2+ with Cr6+ combination was dominated by the effect of Cr6+ alone. Mineralization by osteoblasts was greater on hydroxyapatite‐coated surfaces compared to grit‐blasted and plasma‐sprayed titanium‐coated surfaces. Treatments with Co2+:Cr3+ ions and Co:Cr nanoparticles reduced the percentage mineralization on all surfaces, with hydroxyapatite‐coated surfaces having the least reduction. In conclusion, our data suggests that previous studies investigating individual metal ions underestimate their potential clinical effects on osteoblast activity. Furthermore, the data suggests that hydroxyapatite‐coated surfaces may modulate osteoblast responses to metal debris.

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

The effects of metal ion exposure on osteocytes, the most abundant cell type in bone and responsible for coordinating bone remodeling, remain unclear. However, several studies have previously shown that exposure to cobalt (Co2+) and chromium (Cr3+), at concentrations equivalent to those found clinically, affect osteoblast and osteoclast survival and function. In this study, we tested the hypothesis that metal ions would similarly impair the normal physiology of osteocytes. The survival, dendritic morphology, and response to fluid shear stress of the mature osteocyte‐like cell‐line MLO‐Y4 following exposure to clinically relevant concentrations and combinations of Co and Cr ions were measured in 2D‐culture. Exposure of MLO‐Y4 cells to metal ions reduced cell number, increased dendrites per cell and increased dendrite length. We found that combinations of metal ions had a greater effect than the individual ions alone, and that Co2+ had a predominate effect on changes to cell numbers and dendrites. Combined metal ion exposure blunted the responses of the MLO‐Y4 cells to fluid shear stress, including reducing the intracellular calcium responses and modulation of genes for the osteocyte markers Cx43 and Gp38, and the signaling molecules RANKL and Dkk‐1. Finally, we demonstrated that in the late osteoblasts/early osteocytes cell line MLO‐A5 that Co2+ exposure had no effect on mineralization, but Cr3+ treatment inhibited mineralization in a dose‐dependent manner, without affecting cell viability. Taken together, these data indicate that metal exposure can directly affect osteocyte physiology, with potential implications for bone health including osseointegration of cementless components, and periprosthetic bone remodeling.

The Genetic Epidemiology of Developmental Dysplasia of the Hip: A Genome-Wide Association Study Harnessing National Clinical Audit Data

Background Developmental dysplasia of the hip (DDH) is a common, heritable condition characterised by abnormal formation of the hip joint, but has a poorly understood genetic architecture due to small sample sizes. We apply a novel case-ascertainment approach using national clinical audit (NCA) data to conduct the largest DDH genome-wide association study (GWAS) to date, and replicate our findings in independent cohorts. Methods We used the English National Joint Registry (NJR) dataset to collect DNA and conducted a GWAS in 770 DDH cases and 3364 controls. We tested the variant most strongly associated with DDH in independent replication cohorts comprising 1129 patients and 4652 controls. Results The heritable component of DDH attributable to common variants was 55% and distributed similarly across autosomal and the X-chromosomes. Variation within the GDF5 gene promoter was strongly and reproducibly associated with DDH (rs143384, OR 1.44 [95% CI 1.34-1.56], p=3.55x10−22). Two further replicating loci showed suggestive association with DDH near NFIB (rs4740554, OR 1.30 [95% CI 1.16-1.45], p=4.44x10−6) and LOXL4 (rs4919218, 1.19 [1.10-1.28] p=4.38x10−6). Through gene-based enrichment we identify GDF5, UQCC1, MMP24, RETSAT and PDRG1 association with DDH (p<1.2x10−7). Using the UK Biobank and arcOGEN cohorts to generate polygenic risk scores we find that risk alleles for hip osteoarthritis explain <0.5% of the variance in DDH susceptibility. Conclusion Using the NJR as a proof-of-principle, we describe the genetic architecture of DDH and identify several candidate intervention loci and demonstrate a scalable recruitment strategy for genetic studies that is transferrable to other complex diseases. Key Messages We report the first genome-wide scan for DDH in a European population, and the first to use national clinical audit data for case-ascertainment in complex disease. The heritable component of DDH attributable to common variants is 55% and is distributed similarly across autosomal and the X-chromosomes. Variation within the GDF5 gene promoter is strongly and reproducibly associated with DDH, with fine-mapping indicating rs143384 as the likely casual variant. Enrichment analyses implicate GDF5, UQCC1, MMP24, RETSAT and PDRG1 as candidate targets for intervention in DDH. DDH shares little common genetic aetiology with idiopathic osteoarthritis of the hip, despite sharing variation within the GDF5 promoter as a common risk factor.

The effects of chronic cobalt and chromium exposure after metal-on-metal hip resurfacing: An epigenome-wide association pilot study.

Metal‐on‐metal (MOM) hip resurfacing has recently been a popular prosthesis choice for the treatment of symptomatic arthritis, but results in the release of cobalt and chromium ions into the circulation that can be associated with adverse clinical effects. The mechanism underlying these effects remains unclear. While chromosomal aneuploidy and translocations are associated with this exposure, the presence of subtle structural epigenetic modifications in patients with MOM joint replacements remains unexplored. Consequently, we analyzed whole blood DNA methylation in 34 OA patients with MOM hip resurfacing (MOM HR) compared to 34 OA patients with non‐MOM total hip replacements (non‐MOM THR), using the genome‐wide Illumina HumanMethylation 450k BeadChip. No probes showed differential methylation significant at 5% false‐discovery rate (FDR). We also tested association of probe methylation levels with blood chromium and cobalt levels directly; there were no significant associations at 5% FDR. Finally, we used the “epigenetic clock” to compare estimated to actual age at sample for all individuals. We found no significant difference between MOM HR and non‐MOM THR, and no correlation of age acceleration with blood metal levels. Our results suggest the absence of large methylation differences systemically following metal exposure, however, larger sample sizes will be required to identify potential small effects. Any DNA methylation changes that may occur in the local periprosthetic tissues remain to be elucidated.

Genome-wide association study of developmental dysplasia of the hip identifies an association with GDF5

Developmental dysplasia of the hip (DDH) is the most common skeletal developmental disease. However, its genetic architecture is poorly understood. We conduct the largest DDH genome-wide association study to date and replicate our findings in independent cohorts. We find the heritable component of DDH attributable to common genetic variants to be 55% and distributed equally across the autosomal and X-chromosomes. We identify replicating evidence for association between GDF5 promoter variation and DDH (rs143384, effect allele A, odds ratio 1.44, 95% confidence interval 1.34–1.56, P = 3.55 × 10−22). Gene-based analysis implicates GDF5 (P = 9.24 × 10−12), UQCC1 (P = 1.86 × 10−10), MMP24 (P = 3.18 × 10−9), RETSAT (P = 3.70 × 10−8) and PDRG1 (P = 1.06 × 10−7) in DDH susceptibility. We find shared genetic architecture between DDH and hip osteoarthritis, but no predictive power of osteoarthritis polygenic risk score on DDH status, underscoring the complex nature of the two traits. We report a scalable, time-efficient recruitment strategy and establish for the first time to our knowledge a robust DDH genetic association locus at GDF5.Konstantinos Hatzikotoulas et al. report the largest genome-wide association study to date for developmental dysplasia of the hip using national clinical audit data from the UK. They find a significant association with the GDF5 locus and evidence for shared genetic architecture with hip osteoarthritis.

The 2018 Otto Aufranc Award: How Does Genome-wide Variation Affect Osteolysis Risk After THA?

Background Periprosthetic osteolysis resulting in aseptic loosening is a leading cause of THA revision. Individuals vary in their susceptibility to osteolysis and heritable factors may contribute to this variation. However, the overall contribution that such variation makes to osteolysis risk is unknown. Questions/purposes We conducted two genome-wide association studies to (1) identify genetic risk loci associated with susceptibility to osteolysis; and (2) identify genetic risk loci associated with time to prosthesis revision for osteolysis. Methods The Norway cohort comprised 2624 patients after THA recruited from the Norwegian Arthroplasty Registry, of whom 779 had undergone revision surgery for osteolysis. The UK cohort included 890 patients previously recruited from hospitals in the north of England, 317 who either had radiographic evidence of and/or had undergone revision surgery for osteolysis. All participants had received a fully cemented or hybrid THA using a small-diameter metal or ceramic-on-conventional polyethylene bearing. Osteolysis susceptibility case-control analyses and quantitative trait analyses for time to prosthesis revision (a proxy measure of the speed of osteolysis onset) in those patients with osteolysis were undertaken in each cohort separately after genome-wide genotyping. Finally, a meta-analysis of the two independent cohort association analysis results was undertaken. Results Genome-wide association analysis identified four independent suggestive genetic signals for osteolysis case-control status in the Norwegian cohort and 11 in the UK cohort (p ⩽ 5 x 10-6). After meta-analysis, five independent genetic signals showed a suggestive association with osteolysis case-control status at p ⩽ 5 x 10-6 with the strongest comprising 18 correlated variants on chromosome 7 (lead signal rs850092, p = 1.13 x 10-6). Genome-wide quantitative trait analysis in cases only showed a total of five and nine independent genetic signals for time to revision at p ⩽ 5 x 10-6, respectively. After meta-analysis, 11 independent genetic signals showed suggestive evidence of an association with time to revision at p ⩽ 5 x 10-6 with the largest association block comprising 174 correlated variants in chromosome 15 (lead signal rs10507055, p = 1.40 x 10-7). Conclusions We explored the heritable biology of osteolysis at the whole genome level and identify several genetic loci that associate with susceptibility to osteolysis or with premature revision surgery. However, further studies are required to determine a causal association between the identified signals and osteolysis and their functional role in the disease. Clinical Relevance The identification of novel genetic risk loci for osteolysis enables new investigative avenues for clinical biomarker discovery and therapeutic intervention in this disease.

A study on the use of metasurface synthesis using electric and magnetic susceptibility

This paper presents various case studies for meta surface design for plane-wave to plane-wave scattering using electric and magnetic susceptibilities. The case studies look at the practical implications and potential simplifications to ideal meta surface proprieties and how these impact on the scattering patterns of such structures. Specifically we address normal and oblique incidence scattering as well as scattering multiple plane-waves from a surface.