Marco S. Caicedo

Upregulation of prostaglandin E2 and interleukins in the central nervous system and peripheral tissue during and after surgery in humans.

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 1&bgr; and tumor necrosis factor &agr; were undetectable. Hip surgical site fluid evidenced an increase in prostaglandin E2, interleukin 6, interleukin 8, and interleukin 1&bgr;; tumor necrosis factor &agr; 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.

Soluble and particulate Co-Cr-Mo alloy implant metals activate the inflammasome danger signaling pathway in human macrophages: a novel mechanism for implant debris reactivity.

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.

Soluble ions more than particulate cobalt-alloy implant debris induce monocyte costimulatory molecule expression and release of proinflammatory cytokines critical to metal-induced lymphocyte reactivity.

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.

Co-Cr-Mo alloy particles induce tumor necrosis factor alpha production in MLO-Y4 osteocytes: A role for osteocytes in particle-induced inflammation

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 (p<0.05) 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.

Increasing both CoCrMo‐alloy particle size and surface irregularity induces increased macrophage inflammasome activation in vitro potentially through lysosomal destabilization mechanisms

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 induced >10‐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 a >3‐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.

Cobalt-alloy implant debris induce HIF-1α hypoxia associated responses: a mechanism for metal-specific orthopedic implant failure.

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.

In vitro reactivity to implant metals demonstrates a person-dependent association with both T-cell and B-cell activation.

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.

Asymptomatic prospective and retrospective cohorts with metal‐on‐metal hip arthroplasty indicate acquired lymphocyte reactivity varies with metal ion levels on a group basis

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 < 0.01) amounts of serum Co and Cr compared to controls as early as 3 months post‐op (Group 1:1.2 ppb Co, 1.5 ppb Cr; Group 2: 3.4 ppb Co, 5.4 ppb Cr; Group 3: 0.01 ppb Co, 0.1 ppb Cr). However, only after 1–4 years post‐op did 56% of Group 1 develop metal‐reactivity (vs. 5% pre‐op, metal‐LTT, SI > 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.

Cytotoxic effects of cobalt and nickel ions on osteocytes in vitro

BackgroundMetal-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.MethodsMLO-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.ResultsCr 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.ConclusionsMetal ions released from metal-on-metal bearing surfaces have potentially cytotoxic effects on MLO-Y4 osteocytes, in vitro.

Calcineurin/nuclear factor of activated T cells (NFAT) signaling in cobalt–chromium–molybdenum (CoCrMo) particles‐induced tumor necrosis factor‐α (TNFα) secretion in MLO‐Y4 osteocytes

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.