On the inflammatory response in metal-on-metal implants

Five patients undergoing revision surgery due to a hip resurfacing implant (metal-on-metal, ASR Hip System, DePuy) and six patients suffering from aseptic loosening of a total hip replacement (metal-on-polyethylene or ceramic-on-polyethylene) were included in the study. Diagnosis of loosening was based on patient’s complaints, clinical examination and by conventional x-ray and/or CT-scan (patients’ clinical data are summarised in Table 1). The study was approved by the local ethic committee, and informed consent was obtained from the patients.

During surgery, soft tissue samples were taken from the cup, the capsule, the femur and for comparison from muscle in a standardized fashion. The tissue samples were divided, a part was fixed in formalin and conserved for histological analysis, and another was placed into RNAlater (Ambion, Lifetechnologies, Heidelberg, Germany) for quantitative PCR analysis. Immediately before surgery, and 7 to 10 days thereafter, peripheral blood was collected into heparinized tubes and cells were subjected to cytofluorometry (see below).

For routine histological evaluation, the samples were embedded in paraffin, decalcified, and sections of 3 to 4 μM were prepared for eosin-haematoxylin staining.

The tissue samples were disrupted with RiboLyser devices (ThermoHYBAID, Heidelberg, Germany) containing 400 μl lysis buffer from the MagnaPure mRNA Isolation Kit containing 1%DTT (v/w) (ROCHE Diagnostics, Mannheim, Germany). mRNA was isolated with the MagnaPure-LC device using the mRNA- standard protocol for cells. An aliquot of mRNA was reversely transcribed using AMV-RT and oligo- (dT) as primer (First Strand cDNA synthesis kit, ROCHE Diagnostics, Mannheim, Germany) according to the manufactures protocol in a thermocycler. Primer sets optimized for the LightCycler® (RAS, Mannheim Germany) were purchased from SEARCH-LC GmbH (http://​www.​Search-LC.​com). The PCR was performed with the LightCycler® FastStart DNA Sybr GreenI kit (RAS) according to the protocol provided. To control for specificity, a melting curve analysis was performed. The copy number was calculated from a standard curve, obtained by plotting known input concentrations of four different plasmids at log dilutions to the PCR-cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. To correct for differences in the content of mRNA, the calculated transcript numbers were normalized according to the expression of the housekeeping gene peptidylprolyl isomerase B (PPIB). Values were thus given as transcripts per 1000 transcripts of PPIB.

The following antibodies were used: CD4 PerCP, CD8 PerCP, CD11b PE, CD28 FITC (all Becton Dickinson, Heidelberg, Germany), and the respective isotype: mouse IgG1-PerCP, mouse IgG1-PE (all Becton Dickinson, Heidelberg, Germany) and IgG1-FITC (Beckman Coulter, Marseille, France). Whole heparinized blood (100 μl) was incubated with the respective antibodies (20 min, room temperature), erythrocytes were then lysed by Facs Lysing solution and cells were washed and fixed with 1% paraformaldehyde. Cells were analysed by FacsCalibur using CellquestPro 3.0 as software (Becton and Dickinson, Heidelberg, Germany).

Differences between groups were calculated using Friedman test and Mann–Whitney test, respectively (as indicated in the figure legends or tables).

Five patients with metallosis and six patients with aseptic loosening were included in the study (patients’ data are summarised in Table 1). The implant used in patients developing metallosis is shown in Figure 1A. In comparison to other forms of endoprostheses, the femur is better preserved, but because of the metal-on-metal contact, metal wear particles accumulate at the site, macroscopically seen as greyish colouring (Figure 1B). In this example, also pseudotumour formation occurred (Figure 1B), as did osteolysis (Figure 1C). Biopsies taken from the site showed metal wear particles, and also a cellular infiltrate, consisting mainly of mononuclear cells (Figure 1D,E).

Tissue samples from the cup and from the femoral bone were taken from areas that were exposed after removal of the implant. Moreover, samples from the capsule and when present from the pseudotumour, were gathered and for comparison, from distant muscle as an unaffected site. Gene expression of CD14 as marker for monocytes/macrophages, of CD3 as marker for T cells and of cathepsin K, characteristic for osteoclasts, was determined by quantitative PCR. At the primary osteolytic site, the cup, expression of cathepsin K, CD14 or CD3 was considerably higher compared to expression in muscle (Figure 2). The absolute numbers varied widely among the patients, but the expression pattern was similar.

In the same tissues, gene expression of proinflammatory cytokines was analysed. CXCL8 (interleukin (IL)-8), IL-1ß, CXCL2 (macrophage inflammatory protein, MIP2α) and MRP14 (S100A9) were highly expressed in tissue of the cup, again with a wide variation among the patients (Table 2). Gene expression of TNFα (tumour necrosis factor alpha), RANK (receptor activator of NfκB) or RANK ligand (RANKL) was rather low in all tissues. The monocyte chemotactic protein, MCP-1 (CCL2) was found in all tissues with no apparent preference, as was CXCL10 (IP-10).

To address the question whether aspects of the cytokine expression pattern were typical for metallosis, we compared the data with those obtained from patients requiring replacement of a hip implant because of aseptic loosening.

Because for both patient groups tissue from the capsule and from the femoral bone was available, gene expression in these tissue were used, although these were not the primary osteolytic sites, particularly not in patients with metallosis. As summarised in Table 3, for metallosis patients essentially the same expression pattern was seen: in tissue from either capsule or femoral bone, expression of CXCL8, IL-1ß, CXCL2 and MRP14 was higher compared to muscle tissue, as was expression of CD14. In patients with aseptic loosening, in contrast, no major differences in cytokine expression were observed between muscle and the other tissues; only CD14 was expressed to a higher extent in femoral bone. In patients with metallosis, CXCL8 and IL-1ß expression was higher by trend than in patients with aseptic loosening; CXCL2 and CXCL10 were significantly higher expressed in patients with metallosis (Table 3).

Gene expression of CD3, CD14 and of the cytokines was also determined in peripheral blood cells of the patients. No major differences were observed between blood cells from patients with metallosis or with aseptic loosening (data not shown). Only expression of CXCL10 was higher in blood cells of metallosis patient compared to patients with aseptic loosening (9.4 ± 4.1 copies versus 1.7 ± 1.75).

Peripheral blood T cells were analysed for expression of activation-associated receptors. As sensitive markers, down-modulation of CD28 and up-regulation of CD11b on CD4+ and CD8+ T cells was measured (example in Figure 3A). Because the percentage of CD4 + CD28- and CD8 + CD28- cells varies greatly among donors (mean ± SD of n = 18: 6.51 ± 5.59% CD4 + CD28- and 37.4 ± 20.3% CD8 + CD28-)[26], and because the activation of T cells is transient, we determined the T cell population in the patients immediately before surgery and 7 to 10 days thereafter. There were no major differences regarding CD8 + CD28- cells, whereas CD4 + CD28- cells were seen in patients with metallosis (and not in patients with aseptic loosening) (Figure 3B). CD11b upregulation occurred in both, CD4+ and CD8+, and the percentage declined after surgery, as did the mean values of CD11b (Figure 3C).