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It is widely recognized that innate macrophage immune reactions to implant debris are central to the inflammatory responses that drive biologic implant failure over the long term. Less common, adaptive lymphocyte immune reactions to implant debris, such as delayed type hypersensitivity (DTH), can also affect implant performance. It is unknown which key patient factors, if any, mediate these adaptive immune responses that potentiate particle/macrophage mediated osteolysis. The objective of this investigation was to determine to what degree known adaptive immune responses to metal implant debris can affect particle-induced osteolysis (PIO); and if this pathomechanism is dependent on: 1) innate immune danger signaling, i.e., NLRP3 inflammasome activity, 2) sex, and/or 3) age. We used an established murine calvaria model of PIO using male and female wild-type C57BL/6 vs. Caspase-1 deficient mice as well as young (12–16 weeks old) vs. aged (18–24 months old) female and male C57BL/6 mice. After induction of metal-DTH, and Cobalt-alloy particle (ASTM F-75, 0.4um median diameter) calvaria challenge, bone resorption was assessed using quantitative micro-computed tomography (micro-CT) analysis and immune responses were assessed by measuring paw inflammation, lymphocyte transformation test (LTT) reactivity and adaptive immune cytokines IFN-gamma and IL-17 (ELISA). Younger aged C57BL/6 female mice exhibited the highest rate and severity of metal sensitivity lymphocyte responses that also translated into higher PIO compared to any other experimental group. The absence of inflammasome/caspase-1 activity significantly suppressed DTH metal-reactivity and osteolysis in both male and female Caspase-1 deficient mice. These murine model results indicate that young female mice are more predisposed to metal-DTH augmented inflammatory responses to wear debris, which is highly influenced by active NLRP3 inflammasome/caspase-1 danger signaling. If these results are clinically meaningful for orthopedic patients, then younger female individuals should be appropriately assessed and followed for DTH derived peri-implant complications.

BACKGROUND Recent studies indicate that females demonstrate an increased risk of experiencing adverse local tissue reactions, aseptic loosening, and revision after primary metal-on-metal hip resurfacing arthroplasty compared with males; the underlying biological mechanisms responsible for sex discrepancies in implant failure remain unclear. In addition to anatomical and biomechanical sex differences, there may be inherent immunological disparities that predispose females to more aggressive adaptive immune reactivity to implant debris, i.e., metal sensitivity. METHODS In this retrospective study, we analyzed sex-associated rates and levels of metal sensitization in 1,038 male and 1,575 female subjects with idiopathic joint pain following total joint arthroplasty (TJA) who were referred for in vitro metal-sensitivity testing. RESULTS Females demonstrated a significantly higher rate and severity of metal sensitization compared with males. The median lymphocyte stimulation index (SI) among males was 2.8 (mean, 5.4; 95% confidence interval [CI], 4.9 to 6.0) compared with 3.5 (mean, 8.2; 95% CI, 7.4 to 9.0) among females (p < 0.05). Forty-nine percent of females had an SI of ≥4 (reactive) compared with 38% of males, and the implant-related level of pain was also significantly (p < 0.0001) higher among females (mean, 6.8; 95% CI, 6.6 to 6.9) compared with males (mean, 6.1; 95% CI, 6.0 to 6.3). CONCLUSIONS In a select group of patients who had joint pain following TJA and no evidence of infection and who were referred for metal-sensitivity testing, females exhibited a higher level of pain and demonstrated a higher rate and severity (as measured by lymphocyte SI) of metal sensitization compared with males. LEVEL OF EVIDENCE Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Purpose There appears to be little consensus on how to evaluate and manage patients reporting a possible allergic reaction to joint replacement devices containing metal. This article presents an analysis of the current orthopedic literature in this regard as it relates to diagnosis, testing, and management. Based on that evidence, a management algorithm for metal hypersensitivity in the patient receiving an alloplastic temporomandibular joint replacement is proposed. Materials and Methods Orthopedic surgery has recognized metal sensitivity as a problem in joint replacement; using a PubMed search for this topic, the pertinent orthopedic literature was reviewed. Results Metal hypersensitivity response to implant materials is often a diagnosis of exclusion. The 2 most commonly used tests are the in vivo skin patch test and in vitro lymphocyte transformation test. Initially, conservative management is indicated and other more common causes of symptomatic total joint replacement should be fully explored. Device removal should be considered a last resort. Conclusions Before a primary total joint replacement, testing could be helpful when a patient reports a history of intolerance to jewelry or of an allergic reaction to a prior metal implant. However, to date, routine testing is not supported by the literature.

Metal sensitivity testing is generally the diagnosis method of last resort for aseptic painful implants with elevated inflammatory responses. However, the relationship between implant-related pain and implant-debris-related metal sensitization remains incompletely understood. Although a sensitivity to nickel alone has been used as a general measure of metal allergy, it may lack the specificity to correlate sensitivity to specific implant metals and thus to select a biologically appropriate implant material. In this retrospective study, we report the incidence of pain and nickel sensitivity in patients with total joint arthroplasties (TJAs) referred for metal sensitivity testing (n=2018). We also correlated the degree of nickel hypersensitivity to implant pain levels (none, mild, moderate, and high, using a scale of 0-10) and the incidence of sensitivity to alternative implant metals in highly nickel-reactive subjects. Most patients (79%) reported pain levels that were moderate to high regardless of implant age, whereas patients with severely painful TJAs had a statistically greater incidence of nickel sensitivity over the short-term post-operative period (≤4 years). Patients with moderate pain scores (4-7) and high pain scores (≥8) also exhibited significantly higher sensitivity to nickel compared to patients with no pain and no implant (controls) (p0.05). Highly nickel-sensitive subjects (SI8) also showed incidences of sensitization to alternative materials such as cobalt, chromium, or molybdenum (57%) or aluminum or vanadium alloy (52%). These data suggest that painful TJAs caused by metal sensitivity more likely occur relatively early in the post-operative period (≤4 years). The incidences of sensitivity to alternative implant metals in only a subset of nickel-reactive patients highlights the importance of testing for sensitization to all potential revision implant materials.

Recent investigations indicate that innate immune “danger-signaling” pathways mediate metal implant debris induced-inflammatory responses, e.g. NALP3 inflammasome. How the physical characteristics of particles, (size, shape and chemical composition) affect this inflammatory reactivity remains controversial. We examined the role of Cobalt-Chromium-Molybdenum (CoCrMo) alloy particle shape and size on human macrophage phagocytosis, lysosomal destabilization, and inflammasome activation. Round/smooth vs. irregularly shaped/rough CoCrMo-alloy particles of ~1µm and 6 to 7µm diameter were investigated for differential lysosomal damage and inflammasome activation in human monocytes/macrophages. While spherical/smooth 1µm CoCrMo-alloy particles did not measurably affect macrophage IL-1β production, irregular 1µm CoCrMo-alloy particles induced significant IL-1β increases over controls. Both round/smooth particles and irregular CoCrMo-alloy particles that were 6 to 7µ min size induced >10-fold increases in IL-1β production compared to similarly shaped smaller particles (p 3-fold increase in IL-1β vs. similarly sized round/smooth particles (at an equal dose, particles/cell). CoCrMo-alloy particle-size-induced IL-1β production was dependent on the lysosomal protease Cathepsin B, further supporting lysosomal destabilization as causative in inflammation. Phagocytosable larger/irregular shaped particles (6µm) demonstrated the greatest lysosomal destabilization (observed immunofluorescently) and inflammatory reactivity when compared on an equal dose basis (particles/cell) to smaller/spherical 1µm particles in vitro.

Some tissues from metal-on-metal (MoM) hip arthroplasty revisions have shown evidence of adaptive-immune reactivity (i.e., excessive peri-implant lymphocyte infiltration/activation). We hypothesized that, prior to symptoms, some people with MoM hip arthroplasty will develop quantifiable metal-induced lymphocyte reactivity responses related to peripheral metal ion levels. We tested three cohorts (Group 1: n = 21 prospective longitudinal MoM hip arthroplasty; Group 2: n = 17 retrospective MoM hip arthroplasty; and Group 3: n = 20 controls without implants). We compared implant position, metal-ion release, and immuno-reactivity. MoM cohorts had elevated (p 2), compared with 76% of Group 2, and 15% of Group 3 controls (patch testing was a poor diagnostic indicator with only 1/21 Group 1 positive). Higher cup-abduction angles (50° vs. 40°) in Group 1 were associated with higher serum Cr (p < 0.07). However, sub-optimal cup-anteversion angles (9° vs. 20°) had higher serum Co (p < 0.08). Serum Cr and Co were significantly elevated in reactive versus non-reactive Group-1 participants (p < 0.04). CD4+CD69+ T-helper lymphocytes (but not CD8+) and IL-1β, IL-12, and IL-6 cytokines were all significantly elevated in metal-reactive versus non-reactive Group 1 participants. Our results showed that lymphocyte reactivity to metals can develop within the first 1-4 years after MoM arthroplasty in asymptomatic patients and lags increases in metal ion levels. This increased metal reactivity was more prevalent in those individuals with extreme cup angles and higher amounts of circulating metal.

Aseptic loosening is the devastating long term complication of total hip arthroplasty and orthopedic implant debris has been shown to trigger an intense inflammatory reaction leading to resorption of the bone matrix. Inflammatory cytokines, such as tumor necrosis factor-α (TNFα), have been implicated in this process and osteocytes may play a role in its production. We previously demonstrated that cobalt-chromium-molybdenum (CoCrMo) particles upregulate TNFα production by MLO-Y4 osteocytes in vitro, but the underlying mechanism has not been elucidated. Based on previous studies by others, we hypothesized that the calcineurin-nuclear factor of activated T cells (NFAT) pathway mediates CoCrMo particle-induced TNFα production in MLO-Y4 osteocytes. MLO-Y4 osteocytes exposed to CoCrMo particle treatment resulted in a rapid and significant increase in calcineurin activity. We also demonstrate that CoCrMo particle-induced upregulation of TNFα is reduced to control levels with calcineurin-NFAT inhibitors and this was also confirmed at mRNA level. Moreover, we demonstrate the localization of NFATs in MLO-Y4 osteocytes and that NFAT1 and 2 translocate to the nucleus upon CoCrMo particle treatment. Our results suggest that calcineurin-NFAT signaling is involved in TNFα production by MLO-Y4 osteocytes after CoCrMo particle treatment.

Wear debris-induced osteolysis is purportedly the limiting problem affecting the long term results of joint arthroplasty. Pathogenic effects of wear debris in peri-implant cells such as macrophages, osteoblasts and osteoclasts have been well studied. In contrast, the effects of wear debris on osteocytes, which make up over 90% of all bone cells, remain unknown. We hypothesized that metal implant debris can induce the pro-inflammatory response in osteocytes. This study demonstrated the effects of cobalt-chromium-molybdenum alloy (Co-Cr-Mo) particles on a well-characterized MLO-Y4 osteocyte cell line. Co-Cr-Mo alloy particle treatment significantly (p0.05) up-regulated tumor necrosis factor alpha (TNFalpha) gene expression after 3 and 6 h and TNFalpha protein production after 24 h, but down-regulated interleukin-6 (IL-6) gene expression after 6 h. Co-Cr-Mo alloy particle treatment also induced osteocyte apoptosis after 24 h. This apoptotic effect was partially (40%) dependent on TNFalpha. Therefore, our results suggest that osteocytes play a role in particle-induced inflammation and bone resorption following total joint arthroplasty by inducing pro-inflammatory cytokines and inducing osteocyte apoptosis.

Immune reactivity to soluble and particulate implant debris remains the primary cause of aseptic inflammation and implant loosening. However, the intracellular mechanisms that trigger immune cells to sense and respond to exogenous nonbiological agents such as metal particles or metal ions released from orthopedic implants remain unknown. Recent studies in immunology have outlined the importance of the intracellular inflammasome complex of proteins in sensing danger/stress signals triggered by nonbiological agents in the cytosol of macrophages. We hypothesized that metal implant debris can activate the inflammasome pathway in macrophages that causes caspase-1-induced cleavage of intracellular pro-IL-1β into its mature form, resulting in IL-1β secretion and induction of a broader proinflammatory response. We tested this hypothesis by examining whether soluble cobalt, chromium, molybdenum, and nickel ions and Co-Cr-Mo alloy particles induce inflammasome- mediated macrophage reactivity. Our results demonstrate that these agents stimulate IL-1β secretion in human macrophages that is inflammasome mediated (i.e., NADPH-, caspase-1-, Nalp3-, and ASC-dependent). Thus, metal ion- and particle-induced activation of the inflammasome in human macrophages provides evidence of a novel pathway of implant debris-induced inflammation, where contact with implant debris is sensed and transduced by macrophages into a proinflammatory response. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 847–854, 2009

It remains unclear how metal released from implants affects cells of the immune system and, in particular, cells of the adaptive immune system, that is, T-helper lymphocytes. In this study, we investigated the effects of aluminum, chromium, cobalt, copper, iron, molybdenum, nickel, niobium, vanadium, and zirconium ions at concentrations from 0.05 to 5.0 mM on human CD4+ T lymphocytes. The DNA damage, apoptosis, necrosis, and proliferation responses of a human T-helper lymphocyte (Jurkat) cell line were evaluated to test our hypothesis that some metals will preferentially induce genotoxicity (DNA damage). Our results demonstrated that metal ions did not preferentially induce Jurkat T-lymphocyte DNA damage prior to other forms of toxicity, that is, apoptosis and/or direct necrosis. Nickel and vanadium induced the most DNA damage and were the most apoptotic metals tested, inducing >50% caspase-9 positive T cells at 0.05 mM and 0.1 mM concentrations, respectively. Cobalt and niobium were the most toxic metals, inducing 50% apoptosis (i.e.

Despite reports associating tissue necrosis with implant failure, the degree to which processes, such as metal toxicity, negatively impact implant performance is unknown. We evaluated representative human peri-implant cells (i.e., osteoblasts, fibroblasts, and lymphocytes) when challenged by Al+3, Co+2, Cr+3, Fe+3, Mo+5, Ni+2, and V+3 chloride solutions (and Na+2 as a control) over a wide range of concentrations (0.01-10.0 mM). Cell responses were measured using proliferation assays, viability assays, and microscopic cell morphology assessments. Differential effects were found to be less a function of the cell type than of the composition and concentration of metal challenge. No preferential immunosuppression was demonstrated. Below 0.01 mM, no metal was toxic. The most toxic metals (i.e., Co, Ni, and V) reduced proliferation (IC50), and viability (LC50) and cell morphology of osteoblasts, fibroblasts, and lymphocytes by50% at challenge concentrations1 mM. All other metals tested required5 mM to exact the same responses. Below 1 mM, these toxic metals also induced alterations in all cell morphology consisting of loss of filopodia or lamellipodia or changes in cell shape. Metals that were toxic at clinically relevant concentrations (less than previously reported values in peri-implant tissues/fluids) include Co (0.6 mM), Ni (0.8 mM), V (0.5 mM) for lymphocytes and Co (0.8 mM), V (0.3 mM), Al (1-5 mM), Fe (1-5 mM) for fibroblasts, and Co (0.8 mM), Ni (0.7 mM), V (0.1 mM) for osteoblasts. Only Co and V were toxic in vitro at concentrations below that detected in vivo in synovial fluid (V at 0.1 mM and Co at 0.8 mM for fibroblasts, and V at 0.4 mM and Co at 0.8 mM on osteoblasts). Thus, soluble Co and V released from Co- and Ti-based alloys, respectively, could be implicated as the most likely to mediate cell toxicity in the periprosthetic milieu.

Cell-mediated hypersensitivity associated with metal components may be related to levels of implant debris. We tested this hypothesis by comparing lymphocyte reactivity to soluble Co, Cr, Ni, and Ti of patients with metal-on-polyethylene and metal-on-metal arthroplasties with healthy controls, and patients with osteoarthritis. The metal-on-metal group (n=9) demonstrated significantly elevated serum Co and Cr concentrations (13- and 58-fold, P.05, respectively) and significantly elevated lymphocyte reactivity to Co (SI5, P.004) and Ni (SI2.5, P.01) when compared to controls (n=12) and subjects with metal-on-poly implants (n=7). These elevated in vivo metal levels demonstrated positive linear correlation with lymphocyte reactivity supporting our hypothesis that lymphocyte metal-induced reactivity increases with increased metal exposure. These results represent the first direct link between in vivo metal exposure and lymphocyte reactivity. Whether this lymphocyte reactivity to metal debris is etiologically linked to poor implant performance remains uncertain.

Background Metal-on-metal prostheses undergo wear and corrosion, releasing soluble ions and wear particles into the surrounding environment. Reports described early failures of the metal-on-metal prostheses, with histologic features similar to a Type IV immune response. Mechanisms by which metal wear products and metal ion causing this reaction are not completely understood, and the effects of metal ions on osteocytes, which represent more than 95% of all the bone cells, have not been also studied. We hypothesized that soluble metal ions released from the cobalt-chromium-molybdenum (Co-Cr-Mo) prosthesis may have cytotoxic effect on osteocytes. Methods MLO-Y4 osteocytes were treated with various metal ion solutions for 24 and 48 h. The effect of ion treatment on cytotoxicity was assessed by WST-1 reagents and cell death ELISA. Morphological changes were analyzed by a phase-contrast microscope or fluorescent microscope using Hoechst 33342 and propidium iodine staining. Results Cr and Mo ions did not cause cell death under 0.50 mM, highest concentration studied, whereas Co and Ni ions had significant cytotoxic effect on MLO-Y4 cells at concentrations grater than 0.10 mM and at 0.50 mM, respectively, in a dose-dependent manner. According to the ELISA data, osteocytes treated with Co ions were more susceptible to necrotic than apoptotic cell death, while Ni ions caused osteocyte apoptosis. The morphological assays show that cells treated with Co and Ni ions at high concentration were fewer in number and rounded. In addition, fluorescent images showed a marked reduction in live cells and an increase in dead osteocytes treated with Co and Ni ions at high concentration. Conclusions Metal ions released from metal-on-metal bearing surfaces have potentially cytotoxic effects on MLO-Y4 osteocytes, in vitro.

Biologic reactivity to orthopedic implant debris mediates long-term clinical performance of total joint arthroplasty implants. However, the reasons that some facets of implant debris (e.g., particle size, shape, base material, etc.) are more pro-inflammatory remain controversial. This precludes accurate prediction and optimal design of modern total joint replacements. We hypothesized that debris particle size can influence adsorbed protein film composition and affect subsequent bioreactivity. We measured size-dependent proteinfilm adsorption, and adsorbed protein-film-dependent cytokine release using equal surface areas of different sized cobalt-chromium alloy (CoCr-alloy) particles and in vitro challenge of human macrophages (THP-1 and human primary). Smaller (5 μm diameter) versus larger (70 μm diameter) particles preferentially adsorbed more serum protein in general (p0.03), where higher molecular weight serum proteins consistent with IgG were identified. Additionally, 5-μm CoCr-alloy particles pre-coated with different protein biofilms (IgG vs. albumin) resulted in a difference in cytokine expression in which albumin-coated particles induced more TNF-α release and IgG-coated particles induced more IL-1β release from human monocytes/macrophages. In these preliminary in vitro studies, we have demonstrated the capability of equal surface areas of different particle sizes to influence adsorbed protein composition and that adsorbed protein differences on identical particles can translate into complex differences in bioreactivity. Together, these findings suggest that adsorbed protein differences on different-sized particles of the same material may be a contributing mechanism by which certain particles induce different reactivities.

The historical success of orthopedic implants has been recently tempered by unexpected pathologies and early failures of some types of Cobalt-Chromium-Molybdenum alloy containing artificial hip implants. Hypoxia-associated responses to Cobalt-alloy metal debris were suspected as mediating this untoward reactivity at least in part. Hypoxia Inducible Factor-1α is a major transcription factor involved in hypoxia, and is a potent coping mechanism for cells to rapidly respond to changing metabolic demands. We measured signature hypoxia associated responses (i.e. HIF-1α, VEGF and TNF-α) to Cobalt-alloy implant debris both in vitro (using a human THP-1 macrophage cell line and primary human monocytes/macrophages) and in vivo. HIF-1α in peri-implant tissues of failed metal-on-metal implants were compared to similar tissues from people with metal-on-polymer hip arthroplasties, immunohistochemically. Increasing concentrations of cobalt ions significantly up-regulated HIF-1α with a maximal response at 0.3 mM. Cobalt-alloy particles (1 um-diameter, 10 particles/cell) induced significantly elevated HIF-1α, VEGF, TNF-α and ROS expression in human primary macrophages whereas Titanium-alloy particles did not. Elevated expression of HIF-1α was found in peri-implant tissues and synovial fluid of people with failing Metal-on-Metal hips (n = 5) compared to failed Metal-on-Polymer articulating hip arthroplasties (n = 10). This evidence suggests that Cobalt-alloy, more than other metal implant debris (e.g. Titanium alloy), can elicit hypoxia-like responses that if unchecked can lead to unusual peri-implant pathologies, such as lymphocyte infiltration, necrosis and excessive fibrous tissue growths.

Recent investigations indicate that innate immune "danger-signaling" pathways mediate metal implant debris induced-inflammatory responses, for example, NALP3 inflammasome. How the physical characteristics of particles (size, shape, and chemical composition) affect this inflammatory reactivity remains controversial. We examined the role of Cobalt-Chromium-Molybdenum (CoCrMo) alloy particle shape and size on human macrophage phagocytosis, lysosomal destabilization, and inflammasome activation. Round/smooth versus irregularly shaped/rough CoCrMo-alloy particles of ∼1 and 6-7 µm diameter were investigated for differential lysosomal damage and inflammasome activation in human monocytes/macrophages. While spherical/smooth 1 µm CoCrMo-alloy particles did not measurably affect macrophage IL-1β production, irregular 1 µm CoCrMo-alloy particles induced significant IL-1β increases over controls. Both round/smooth particles and irregular CoCrMo-alloy particles that were 6-7 µm in size induced10-fold increases in IL-1β production compared to similarly shaped smaller particles (p 0.05). Larger irregular particles induced a greater degree of intracellular lysosomal damage and a3-fold increase in IL-1β versus similarly sized round/smooth particles (at an equal dose, particles/cell). CoCrMo-alloy particle-size-induced IL-1β production was dependent on the lysosomal protease Cathepsin B, further supporting lysosomal destabilization as causative in inflammation. Phagocytosable larger/irregular shaped particles (6 µm) demonstrated the greatest lysosomal destabilization (observed immunofluorescently) and inflammatory reactivity when compared on an equal dose basis (particles/cell) to smaller/spherical 1 µm particles in vitro.

Aseptic loosening is the devastating long term complication of total hip arthroplasty and orthopedic implant debris has been shown to trigger an intense inflammatory reaction leading to resorption of the bone matrix. Inflammatory cytokines, such as tumor necrosis factor-α (TNFα), have been implicated in this process and osteocytes may play a role in its production. We previously demonstrated that cobalt-chromium-molybdenum (CoCrMo) particles upregulate TNFα production by MLO-Y4 osteocytes in vitro, but the underlying mechanism has not been elucidated. Based on previous studies by others, we hypothesized that the calcineurin-nuclear factor of activated T cells (NFAT) pathway mediates CoCrMo particle-induced TNFα production in MLO-Y4 osteocytes. MLO-Y4 osteocytes exposed to CoCrMo particle treatment resulted in a rapid and significant increase in calcineurin activity. We also demonstrate that CoCrMo particle-induced upregulation of TNFα is reduced to control levels with calcineurin-NFAT inhibitors and this was also confirmed at mRNA level. Moreover, we demonstrate the localization of NFATs in MLO-Y4 osteocytes and that NFAT1 and 2 translocate to the nucleus upon CoCrMo particle treatment. Our results suggest that calcineurin-NFAT signaling is involved in TNFα production by MLO-Y4 osteocytes after CoCrMo particle treatment.

Aseptic osteolysis has been associated with excessive immune reactivity to particulate implant debris; however, innate and adaptive immune mechanisms that underlie implant debris reactivity remain incompletely understood. Although particulate debris has been implicated as the major type of implant debris mediating macrophage-induced osteolysis, the degree to which metal ions affect a proinflammatory response (if at all) remains unknown. We hypothesized that both soluble and particulate metal implant debris will induce proinflammatory responses in human monocytes resulting in cytokine production and elevated expression of T cell costimulatory molecules, facilitating adaptive immune responses. We tested this hypothesis by characterizing the response of a human monocyte cell line (THP-1), isolated primary human monocytes and PBMCs challenged with Co-Cr-Mo alloy particles and soluble cobalt, chromium, molybdenum, and nickel ions. Our results indicate that soluble cobalt, nickel, and molybdenum can induce monocyte up-regulation of T cell costimulatory molecules (CD80, CD86, ICAM-1) in human monocytes/macrophages. Furthermore, cobalt, molybdenum ions, and Co-Cr-Mo alloy particles similarly induce elevated secretion of IL-1beta, TNFalpha, and IL-6. Antibody blockade of CD80 and CD86, crucial secondary molecules for adaptive responses, abrogated lymphocyte reactivity to metal challenge in metal reactive subjects. Also the addition of IL-1 receptor antagonist (IL-1ra), (which indirectly blocks pro-IL-1beta and thus IL-1beta release), significantly reduced lymphocyte reactivity in metal-reactive subjects. Thus, both soluble and particulate metal implant debris induce monocyte/macrophage proinflammatory responses that are metal and individual specific. This suggests metal-induced up-regulation of costimulatory molecules and proinflammatory cytokine production is necessary to induce lymphocyte activation/proliferation to metal implant debris.

Hypersensitivity to metallic implants remains relatively unpredictable and poorly understood. We initially hypothesized that metal-induced lymphocyte proliferation responses to soluble metal challenge (ions) are mediated exclusively by early T-cell activation (not B-cells), typical of a delayed-type-hypersensitivity response. We tested this by comparing proliferation (6 days) of primary lymphocytes with early T-cell and B-cell activation (48 h) in three groups of subjects likely to demonstrate elevated metal reactivity: group 1 (n = 12) history of metal sensitivity with no implant; group 2a (n = 6) well performing metal-on-metal THRs, and group 2b (n = 20) subjects with poorly performing metal-on-polymer total joint arthroplasties (TJA). Group 1 showed 100% (12/12) metal reactivity (stimulation index > 2) to Ni. Groups 2a and 2b were 83% (5/6) and 75% (15/22) metal reactive (to Co, Cr, or Ni), respectively. Of the n = 32 metal-reactive subjects to Co, Cr, or Ni (SI > 2), n = 22/32 demonstrated >2-fold elevations in % of T-cell or B-cell activation (CD25+, CD69+) to metal challenge when compared with untreated control. 18/22 metal-activated subjects demonstrated an exclusively T-cell or B-cell activation response to metal challenge, where 6/18 demonstrated exclusively B-cell activation and 12/18 demonstrated a T-cell only response, as measured by surface activation markers CD25+ and CD69+. However, there was no direct correlation (R(2) < 0.1) between lymphocyte proliferation and % T-cell or B-cell activation (CD25+:CD69+). Proliferation assays (LTT) showed greater ability to detect metal reactivity than did subject-dependent results of flow-cytometry analysis of T-cell or B-cell activation. The high incidence of lymphocyte reactivity and activation indicate that more complex than initially hypothesized immune responses may contribute to the etiology of debris-induced osteolysis in metal-sensitive individuals.

Immune reactivity to soluble and particulate implant debris remains the primary cause of aseptic inflammation and implant loosening. However, the intracellular mechanisms that trigger immune cells to sense and respond to exogenous nonbiological agents such as metal particles or metal ions released from orthopedic implants remain unknown. Recent studies in immunology have outlined the importance of the intracellular inflammasome complex of proteins in sensing danger/stress signals triggered by nonbiological agents in the cytosol of macrophages. We hypothesized that metal implant debris can activate the inflammasome pathway in macrophages that causes caspase-1-induced cleavage of intracellular pro-IL-1beta into its mature form, resulting in IL-1beta secretion and induction of a broader proinflammatory response. We tested this hypothesis by examining whether soluble cobalt, chromium, molybdenum, and nickel ions and Co-Cr-Mo alloy particles induce inflammasome- mediated macrophage reactivity. Our results demonstrate that these agents stimulate IL-1beta secretion in human macrophages that is inflammasome mediated (i.e., NADPH-, caspase-1-, Nalp3-, and ASC-dependent). Thus, metal ion- and particle-induced activation of the inflammasome in human macrophages provides evidence of a novel pathway of implant debris-induced inflammation, where contact with implant debris is sensed and transduced by macrophages into a proinflammatory response.

Background The central and peripheral inflammatory response to surgery may influence patient outcomes. This study examines the time course and clinical relevance of changes in prostaglandin E2 and cytokines in cerebrospinal fluid, local tissue (surgical site), and circulating blood during and after total hip replacement. Methods Thirty osteoarthritis patients undergoing primary total hip arthroplasty with spinal anesthesia were randomly allocated to three groups (n = 10/group): placebo for 4 days before surgery and on the morning of surgery; placebo for 4 days before surgery and oral rofecoxib 50 mg on the morning of surgery; oral rofecoxib 50 mg for 4 days before surgery and the morning of surgery. Cerebrospinal fluid and plasma were collected before surgery and up to 30 h after incision for measurement of prostaglandin E2 and interleukins. When hip replacement was complete, a drain was placed in the hip wound and exudates were collected at 3 to 30 h after incision. Results Cerebrospinal fluid showed an initial increase in interleukin 6 and a later rise in prostaglandin E2 concentration after surgery; interleukin 1beta and tumor necrosis factor alpha were undetectable. Hip surgical site fluid evidenced an increase in prostaglandin E2, interleukin 6, interleukin 8, and interleukin 1beta; tumor necrosis factor alpha decreased at 24 and 30 h. Preoperative administration of the cyclooxygenase 2 inhibitor rofecoxib reduced cerebrospinal fluid and surgical site prostaglandin E2 and cerebrospinal fluid interleukin 6. Cerebrospinal fluid prostaglandin E2 was positively correlated with postoperative pain and cerebrospinal fluid interleukin 6 with sleep disturbance. Poorer functional recovery was positively correlated with increased surgical site prostaglandin E2. Conclusions These results suggest that upregulation of prostaglandin E2 and interleukin 6 at central sites is an important component of surgery induced inflammatory response in patients and may influence clinical outcome.

Implant debris remains the major factor limiting the longevity of total joint replacements. Whether soluble implant debris of Zr and Nb containing implant alloys constitute a greater risk than other implant metals remains unknown. We evaluated the relative effects of soluble forms of Zr+4 and Nb+5 (0.001–10.0 mM) relative to Cr+3, Mo+5, Al+3, Co+2, Ni+2, Fe+3, Cu+2, Mn+2, Mg+2, Na+2, and V+3 chloride solutions on human peri-implant cells (i.e., osteoblast-like MG-63 cells, fibroblasts, and lymphocytes). Metals were ranked using a 50% decrease in proliferation and viability to determine toxic concentrations. Lymphocytes, fibroblasts, and osteoblasts were, generally, similarly affected by metals where the most toxic metals, Co, Ni, Nb, and V required 1.0 mM challenge to produce toxicity. Overall, Co and V were the most toxic metals tested, thus Zr and Nb containing implant alloys would not likely be more toxic than traditional implant alloys. Below concentrations of 0.1 mM, neither Zr nor Nb reduced osteoblast, lymphocyte, or fibroblast proliferation. Zr was generally an order of magnitude less toxic than Nb to lymphocytes, fibroblasts, and osteoblasts. Our results indicated that soluble Zr and Nb resulting from implant degradation likely act in a metal- and concentration-specific manner capable of producing adverse local and remote tissue responses to the same degree as metals from traditional implant alloys, e.g., Ti-6Al-4V (ASTM F 138) and Co-Cr-Mo alloys (ASTM F 75).

Background All prostheses with metallic components release metal debris that can potentially activate the immune system. However, implant-related metal hyper-reactivity has not been well characterized. In this study, we hypothesized that adaptive immunity reaction(s), particularly T-helper type 1 (Th1) responses, will be dominant in any metal-reactivity responses of patients with total joint replacements (TJAs). We tested this hypothesis by evaluating lymphocyte reactivity to metal "ions" in subjects with and without total hip replacements, using proliferation assays and cytokine analysis. Methods Lymphocytes from young healthy individuals without an implant or a history of metal allergy (Group 1: n = 8) were used to assess lymphocyte responses to metal challenge agents. In addition, individuals (Group 2: n = 15) with well functioning total hip arthroplasties (average Harris Hip Score = 91, average time in-situ 158 months) were studied. Age matched controls with no implants were also used for comparison (Group 3, n = 8, 4 male, 4 female average age 70, range 49–80). Group 1 subjects' lymphocyte proliferation response to Aluminum+3, Cobalt+2, Chromium+3, Copper+2, Iron+3, Molybdenum+5, Manganeese+2, Nickel+2, Vanadium+3 and Sodium+2 chloride solutions at a variety of concentrations (0.0, 0.05, 0.1, 0.5, 1.0 and 10.0 mM) was studied to establish toxicity thresholds. Mononuclear cells from Group 2 and 3 subjects were challenged with 0.1 mM CrCl3, 0.1 mM NiCl2, 0.1 mM CoCl2 and approx. 0.001 mM titanium and the reactions measured with proliferation assays and cytokine analysis to determine T-cell subtype prominence. Results Primary lymphocytes from patients with well functioning total hip replacements demonstrated a higher incidence and greater magnitude of reactivity to chromium than young healthy controls (p < 0.03). Of the 15 metal ion-challenged subjects with well functioning total hip arthroplasties, 7 demonstrated a proliferative response to Chromium, Nickel, Cobalt and/or Titanium (as defined by a statistically significant >2 fold stimulation index response, p < 0.05) and were designated as metal-reactive. Metals such as Cobalt, Copper, Manganese, and Vanadium were toxic at concentrations as low as 0.5 mM while other metals, such as Aluminum, Chromium, Iron, Molybdenum, and Nickel, became toxic at much higher concentrations (>10 mM). The differential secretion of signature T-cell subsets' cytokines (Th1 and Th2 lymphocytes releasing IFN-gamma and IL-4, respectively) between those total hip arthroplasty subjects which demonstrated metal-reactivity and those that did not, indicated a Th1 type (IFN-gamma) pro-inflammatory response. Conclusion Elevated proliferation and production of IFN-gamma to metals in hip arthroplasty subjects' lymphocytes indicates that a Th1 (vs. Th2) type response is likely associated with any metal induced reactivity. The involvement of an elevated and specific lymphocyte response suggests an adaptive (macrophage recruiting) immunity response to metallic implant debris rather than an innate (nonspecific) immune response.