Establishing clinically meaningful ranges of metal hypersensitivity in orthopaedic patients using COVID-19 vaccine-induced adaptive immune responses from fully vaccinated adults.

Background: This study aimed to assess metal sensitization ranges among orthopaedic patients by comparing adaptive immune responses in all-comer pre- and post-operative orthopaedic adults who were COVID-19 unvaccinated or vaccinated vs patients with a painful aseptic implant by lymphocyte transformation test (LTT) to SARS-CoV-2-Spike-Protein (SP) and implant metal(s), respectively. Methods: Data were retrospectively reviewed from three independent groups: unvaccinated COVID-19 adults (n = 23); fully COVID-19 vaccinated adults (n = 35); unvaccinated, painful aseptic implant patients with history of metal allergy (n = 98). Standard in vitro LTT for SP and implant metal(s) (nickel, cobalt) were performed and rated as negative (stimulation index [SI]<2), mild (SI ≥ 2), positive (SI ≥ 4-15), and high sensitization (SI > 15) adaptive immune responses to tested antigen. Results: Overall, 17/23 (74%) of unvaccinated adults showed negative to mild LTT ranges, and 35/35 (100%) of vaccinated showed mild to positive LTT ranges to SP. Vaccinated individuals showed significantly higher median SI (16.1) to SP than unvaccinated (median SI, 1.7; P < 0.0001). Most vaccinated adults (94%) showed a lymphocyte SI > 4 to SP, establishing LTT SI ≥ 4 with >90% sensitivity for diagnosing effective COVID-19 adaptive immune responses. Significantly fewer painful orthopaedic patients (41%) showed comparable elevated levels of lymphocyte metal sensitivity at SI ≥ 4 compared to vaccinated group (P < 0.0001). Conclusions: Vaccinated adults showed significantly higher lymphocyte SI to SP than unvaccinated indicating that SI ranges ≥4 should be set as unequivocally diagnostic of LTT-positive adaptive immune responses to tested antigen. This analysis supports using higher LTT SI ranges (SI ≥ 4) in diagnosing clinical orthopaedic-related Type IV metal-hypersensitivity responses among orthopaedic patients.

Translational Characterization of Macrophage Responses to Stable and Non-Stable CoCrMo Wear and Corrosion Debris Generated In-Situ for Total Hip Replacement.

Metal wear and corrosion debris remain a limiting factor for long-term durability of total hip replacement (THR). Common wear particle production techniques for research differ from the actual tribocorrosion processes at the implant site, potentially causing loss of valuable information. The aim of this study was to investigate reactions to freshly generated and time-stabilized particles and ions released from CoCrMo-alloy using a bio-tribometer, which mimics conditions of the periprosthetic environment. THP-1 macrophages were challenged with freshly produced or time-stabilized wear debris. Wear generation took place in a custom-built bio-tribometer inside a CO2 incubator operating with a reciprocating rotation of an Al2O3 ball against a CoCrMo disc. Two different electrochemical conditions with increasingly forced corrosion rates were tested: +0.45 V (passive domain) and +0.67 V (transition to transpassive domain). Cell viability, proinflammatory cytokines, electrochemical measurements and ICP-MS metal ion content analyses were performed. Cobalt/ chromium concentrations were 6.6/ 1.6 ppm in the passive domain and almost doubled to 11.4/ 3.0 ppm in the passive-transpassive domain. Under those electrochemical conditions, freshly produced and time-stabilized CoCrMo wear decreased cell viability to the same extent. Secretion of proinflammatory cytokines were not significantly different for freshly produced and time-stabilized debris. This study suggests that freshly generated and time-stabilized metal particles/ions cause similar toxicity and inflammatory reactions in macrophages, indicating that standard practices for generating wear debris are valid methods to evaluate wear particle disease. Other cell types, materials, and corrosion potentials need to be studied in the future to solidify the conclusion.

Metal-induced delayed type hypersensitivity responses potentiate particle induced osteolysis in a sex and age dependent manner.

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.

Particulate Debris Released From Breast Implant Surfaces Is Highly Dependent on Implant Type.

BACKGROUND: Although breast implants (BIs) have never been safer, factors such as implant debris may influence complications such as chronic inflammation and illness such as ALCL (anaplastic large cell lymphoma). Do different types of BIs produce differential particulate debris? OBJECTIVES: The aim of this study was to quantify, investigate, and characterize the size, amount, and material type of both loosely bound and adherent surface particles on 5 different surface types of commercial BIs. METHODS: Surface particles from BIs of 5 surface types (n = 5/group), Biocell, Microcell, Siltex, Smooth, SmoothSilk, and Traditional-Smooth, were: (1) removed by a rinsing procedure and (2) removed with ultrapure adhesive carbon tabs. Particles were characterized (ASTM 1877-16) by scanning electron microscopy and energy-dispersive X-ray chemical analysis. RESULTS: Particles rinsed from Biocell, Microcell and Siltex were <1 µm in diameter whereas SmoothSilk and Traditional-Smooth surfaces had median sizes >1 µm (range, 0.4-2.7 µm). The total mass of particles rinsed from the surfaces indicated Biocell had >5-fold more particulate compared with all other implants, and >30-fold more than SmoothSilk or Traditional-Smooth implants (>100-fold more for post-rinse adhesion analysis). Energy-dispersive X-ray analysis indicated that the particulate material for Biocell, Microcell, and Siltex was silicone (>50%), whereas particulates from SmoothSilk and Traditional-Smooth implants were predominantly carbon-based polymers, eg, polycarbonate-urethane, consistent with packaging (and were detected on all implant types). Generally, SmoothSilk and Traditional-Smooth implant groups released >10-fold fewer particles than Biocell, Microcell, and Siltex surfaces. Pilot ex vivo tissue analysis supported these findings. CONCLUSIONS: Particulate debris released from BIs are highly dependent on the type of implant surface and are a likely key determinant of in vivo performance.

Do Battlefield Injury-acquired Indwelling Metal Fragments Induce Metal Immunogenicity?

BACKGROUND: A battlefield-related injury results in increased local and systemic innate immune inflammatory responses, resulting in wound-specific complications and an increased incidence of osteoarthritis. However, little is known about whether severe injuries affect long-term systemic homeostasis, for example, immune function. Moreover, it also remains unknown whether battlefield-acquired metal fragments retained over the long term result in residual systemic effects such as altered immune reactivity to metals. QUESTIONS/PURPOSES: Does a retained metal fragment from a battlefield injury contribute to increased (1) adaptive metal-specific immune responses, (2) systemically elevated metal ion serum levels, and (3) serum immunoglobulin levels compared with combat injuries that did not result in a retained metal fragment? METHODS: In this pilot study, we analyzed metal-immunogenicity in injured military personnel and noninjured control participants using lymphocyte transformation testing (LTT, lymphocyte proliferation responses to cobalt, chromium and nickel challenge at 0.001, 0.01 and 0.1-mM concentrations in triplicate for each participant), serum metal ion analysis (ICP-mass spectroscopy), and serum immunoglobulin analysis (IgE, IgG, IgA, and IgM ). Military personnel with a battlefield-sustained injury self-recruited without any exclusion for sex, age, degree of injury. Those with battlefield injury resulting in retained metal fragments (INJ-FRAG, n = 20 male, mean time since injury ± SD was 12 ± 10 years) were compared with those with a battlefield injury but without retained metal fragments (INJ-NO-FRAG, n = 12 male, mean time since injury ± SD was 13 ± 12 years). A control group comprised of male noninjured participants was used to compare measured immunogenicity metrics (n = 11, males were selected to match battlefield injury group demographics). RESULTS: Military participants with sustained metal fragments had increased levels of metal-induced lymphocyte responses. The lymphocyte stimulation index among military participants with metal fragments was higher than in those with nonretained metal fragments (stimulation index = 4.2 ± 6.0 versus stimulation index = 2.1 ± 1.2 (mean difference 2.1 ± 1.4 [95% confidence interval 5.1 to 0.8]; p = 0.07) and an average stimulation index = 2 ± 1 in noninjured controls. Four of 20 participants injured with retained fragments had a lymphocyte proliferation index greater than 2 to cobalt compared with 0 in the group without a retained metal fragment or 0 in the control participants. However, with the numbers available, military personnel with retained metal fragments did not have higher serum metal ion levels than military participants without retained metal fragment-related injuries or control participants. Military personnel with retained metal fragments had lower serum immunoglobulin levels (IgG, IgA, and IgM) than military personnel without retained metal fragments and noninjured controls, except for IgE. Individuals who were metal-reactive positive (that is, a stimulation index > 2) with retained metal fragments had higher median IgE serum levels than participants who metal-reactive with nonmetal injuries (1198 ± 383 IU/mL versus 171 ± 67 IU/mL, mean difference 1027 ± 477 IU/mL [95% CI 2029 to 25]; p = 0.02). CONCLUSIONS: We found that males with retained metal fragments after a battlefield-related injury had altered adaptive immune responses compared with battlefield-injured military personnel without indwelling metal fragments. Military participants with a retained metal fragment had an increased proportion of group members and increased average lymphocyte reactivity to common implant metals such as nickel and cobalt. Further studies are needed to determine a causal association between exposure to amounts of retained metal fragments, type of injury, personnel demographics and general immune function/reactivity that may affect personal health or future metal implant performance. LEVEL OF EVIDENCE: Level IV, therapeutic study.

The Inflammatory Effects of Breast Implant Particulate Shedding: Comparison With Orthopedic Implants.

Currently, there is a dearth of information regarding the degree of particle shedding from breast implants (BIs) and what are the general biological consequences of BI debris. Thus, it is unclear to what degree BI debris compromises the long-term biological performance of BIs. For orthopedic implants, it is well established that the severity of biological reactivity to implant debris governs long-term clinical performance. Orthopedic implant particulate debris is generally in the range of 0.01 to 100 µm in diameter. Implant debris-induced bioreactivity/inflammation is mostly a peri-implant phenomenon caused by local innate immune cells (eg, macrophages) that produce proinflammatory cytokines such as tumor necrosis factor-α, interleukin-1ß, interleukin-6, and prostaglandin 2 (PGE2). In orthopedics, there have been few systemic concerns associated with polymeric implant debris (like silicone) other than documented dissemination to remote organs (eg, liver, spleen, etc.) with no known associated pathogenicity. This is not true of metal implant debris where normal (well-functioning) implants can induce systemic reactions such as delayed type hypersensitivity. Diagnostic analysis of orthopedic tissues has focused on innate (macrophage mediated) and adaptive (lymphocyte-mediated hypersensitivity) immune responses. Orthopedic implant debris-associated lymphocyte cancers have not been reported in over 40 years of orthopedic literature. Adaptive immune responses such as hypersensitivity reactions to orthopedic implant debris have been dominated by certain implant types that produce specific kinds of debris (eg, metal-on-metal total joint prostheses). Orthopedic hypersensitivity responses and atypical BI bioreactivity such as BI-associated anaplastic large cell lymphoma share crossover markers for diagnosis. Differentiating normal innate immune reactivity to particles from anaplastic large cell lymphoma reactions from delayed type hypersensitivity reactions to BI-associated implant debris remains unclear but vital to patients and surgeons.

Transition from metal-DTH resistance to susceptibility is facilitated by NLRP3 inflammasome signaling induced Th17 reactivity: Implications for orthopedic implants.

Metal hypersensitivity has been recognized as an adverse biologic reaction that can compromise total joint arthroplasty (TJA) performance. However, the etiology of metal hypersensitivity responses in TJAs remains unclear. Metal implant debris is known to act as a danger signal that drives NLRP3 inflammasome activation. It remains unknown if implant debris induced inflammasome activation regulates T cell lineage in TJA metal hypersensitivity responses. In this study, we show both in vivo and in vitro that the pathogenesis of metal hypersensitivity responses to implant debris are largely dependent on activation of the inflammasome/caspase-1 pathway and subsequent production of IL-17A/F by CD4+ T cells. Inhibiting either the inflammasome pathway or IL-17A bioactivity in vivo and in vitro (in vivo using NLRP3 and Caspase-1 deficient mice or in vitro using blocking agents such as Capase-1 inhibitor, IL-1Ra and anti-IL-17A), significantly (p<0.05) mitigated metal-DTH paw inflammation as well as lymphocyte cytokine (IFN-γ and IL-17) and proliferation responses in metal-sensitized mice and primary human PBMCs. This study provides mechanistic insight into how in vivo exposure to orthopedic implant debris, and metals in general, elicits NLRP3 inflammasome activation that mediates the generation of IL-17A/F producing CD4+ T cells, leading to metal-delayed type hypersensitivity reactions.

CoCrMo alloy vs. UHMWPE Particulate Implant Debris Induces Sex Dependent Aseptic Osteolysis Responses In Vivo using a Murine Model.

BACKGROUND: The rate of revision for some designs of total hip replacements due to idiopathic aseptic loosening has been reported as higher for women. However, whether this is environmental or inherently sex-related is not clear. OBJECTIVE: Can particle induced osteolysis be sex dependent? And if so, is this dependent on the type of implant debris (e.g. metal vs polymer)? The objective of this study was to test for material dependent inflammatory osteolysis that may be linked to sex using CoCrMo and implant grade conventional polyethylene (UHMWPE), using an in vivo murine calvaria model. METHODS: Healthy 12 week old female and male C57BL/6J mice were treated with UHMWPE (1.0um ECD) or CoCrMo particles (0.9um ECD) or received sham surgery. Bone resorption was assessed by micro-computed tomography, histology and histomorphometry on day 12 post challenge. RESULTS: Female mice that received CoCrMo particles showed significantly more inflammatory osteolysis and bone destruction compared to the females who received UHMWPE implant debris. Moreover, females challenged with CoCrMo particles exhibited 120% more inflammatory bone loss compared to males (p<0.01) challenged with CoCrMo implant debris (but this was not the case for UHMWPE particles). CONCLUSION: We demonstrated sex-specific differences in the amount of osteolysis resulting from CoCrMo particle challenge. This suggests osteo-immune responses to metal debris are preferentially higher in female compared to male mice, and supports the contention that there may be inherent sex related susceptibility to some types of implant debris.

TLR4 (not TLR2) dominate cognate TLR activity associated with CoCrMo implant particles.

Innate immune reactions to orthopedic implant debris are the primary cause of total joint replacement (TJR) failure over the long term (15-20 years). The role of pathogen associated pattern recognition receptors (i.e., TLRs) in regulating immune reactivity to metal implant particles remains controversial. Do different TLRs (i.e., TLR2 vs. TLR4) activated by their respective ligands in concert with metal implant debris elicit equivalent innate immune responses? In this investigation, our in vitro and in vivo data indicate that Gram-negative PAMPs are more pro-inflammatory than Gram-positive PAMPs. In vitro results indicated TLR4 activation in concert with CoCrMo orthopedic implant debris (CoCrMo/LPS+) challenged primary macrophages resulted in significantly greater inflammatory responses than CoCrMo/PAM3CSK+ (TLR2). Similarly, in vivo results indicated CoCrMo/LPS+ TLR4 challenge induced a twofold increase in inflammation-induced bone resorption (osteolysis) than CoCrMo/PAM3CSK+ (p < 0.01) or CoCrMo (p < 0.03) alone in an established murine calvaria model. This points to a more potent TLR4-based effect of CoCrMo/LPS+ on innate immune responses, that is, IL-1ß, TNF-α, and resulting osteolysis. Differential CoCrMo/LPS+ induced osteolysis compared to CoCrMo/PAM3CSK+, reveals inherent differences in TLR4 versus TLR2 activation which are relevant to (i) how different types of implant debris elicit differential reactivity, (ii) how TLR2 Gram-positive bacteria benefits from less immune activation possibly due to the down-regulation of TLR2 surface expression, that subsequently impacts Gram-positive infections in TJRs, and (iii) how using TLR4 LPS (a Gram-negative agonist) may not accurately model Gram-positive bacteria responses, alone and/or with specific types of implant particles, particularly CoCrMo alloy. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1007-1017, 2017.

Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation.

Cobalt alloy debris has been implicated as causative in the early failure of some designs of current total joint implants. The ability of implant debris to cause excessive inflammation via danger signaling (NLRP3 inflammasome) vs. pathogen associated pattern recognition receptors (e.g. Toll-like receptors; TLRs) remains controversial. Recently, specific non-conserved histidines on human TLR4 have been shown activated by cobalt and nickel ions in solution. However, whether this TLR activation is directly or indirectly an effect of metals or secondary endogenous alarmins (danger-associated molecular patterns, DAMPs) elicited by danger signaling, remains unknown and contentious. Our study indicates that in both a human macrophage cell line (THP-1) and primary human macrophages, as well as an in vivo murine model of inflammatory osteolysis, that Cobalt-alloy particle induced NLRP3 inflammasome danger signaling inflammatory responses were highly dominant relative to TLR4 activation, as measured respectively by IL-1ß or TNF-α, IL-6, IL-10, tissue histology and quantitative bone loss measurement. Despite the lack of metal binding histidines H456 and H458 in murine TLR4, murine calvaria challenge with Cobalt alloy particles induced significant macrophage driven in vivo inflammation and bone loss inflammatory osteolysis, whereas LPS calvaria challenge alone did not. Additionally, no significant increase (p<0.05) in inflammation and inflammatory bone loss by LPS co-challenge with Cobalt vs. Cobalt alone was evident, even at high levels of LPS (i.e. levels commiserate with hematogenous levels in fatal sepsis, >500pg/mL). Therefore, not only do the results of this investigation support Cobalt alloy danger signaling induced inflammation, but under normal homeostasis low levels of hematogenous PAMPs (<2pg/mL) from Gram-negative bacteria, seem to have negligible contribution to the danger signaling responses elicited by Cobalt alloy metal implant debris. This suggests the unique nature of Cobalt alloy particle bioreactivity is strong enough to illicit danger signaling that secondarily activate concomitant TLR activation, and may in part explain Cobalt particulate associated inflammatory and toxicity-like reactions of specific orthopedic implants.

Implant debris particle size affects serum protein adsorption which may contribute to particle size-based bioreactivity differences.

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 (p<0.03), 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-1ß 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.

Osteoclasts lose innate inflammatory reactivity to metal and polymer implant debris compared to monocytes/macrophages.

Long-term aseptic failures of joint replacements are generally attributed to implant debris-induced inflammation and osteolysis. This response is largely mediated by immune and bone cells (monocytes/macrophages and osteoclasts, respectively), that in the presence of implant debris (e.g. metal particles and ions), release pro-inflammatory cytokines such as IL-1ß, TNF-α, and IL-6. The relative degree to which implant debris can illicit inflammatory response(s) from osteoclasts vs monocytes/macrophages is unknown, i.e. are osteoclasts a viable target for anti-inflammatory therapy for implant debris? We investigated relative monocyte versus osteoclast inflammatory responses in a side-by-side comparison using implant debris from the perspective of both danger signaling (IL-1ß) and pathogenic recognition (TNF-α) reactivity (Challenge Agents: Cobalt-alloy, Titanium-alloy, and PMMA particles, 0.9-1.8um-dia ECD and Cobalt, and Nickel-ions 0.01-0.1mM, all with and without LPS priming). Human monocytes/macrophages reacted to implant debris with >100 fold greater production of cytokines compared to osteoclast-like cells. Particulate Co-alloy challenge induced >1000 pg/ml of IL-1ß and TNF-α, in monocytes and <50pg/mL IL-1ß and TNF-α in osteoclasts. Cobalt ions induced >3000pg/mL IL-1ß and TNF-α in monocytes/macrophages and <50pg/mL IL-1ß and TNF-α in osteoclasts. The paracrine effect of supernatants from debris-treated monocytes/macrophages was capable of inducing greater osteoclastogenesis (TRAP+, p<0.06) and inflammation than direct debris challenge on osteoclasts. Our results indicate that as monocytes/macrophages differentiate into osteoclasts, they largely lose their innate immune reactivity to implant debris and thus may not be as relevant a therapeutic target as monocytes/macrophages for mitigating debris-induced inflammation.

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.

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-1ß production, irregular 1 µm CoCrMo-alloy particles induced significant IL-1ß 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-1ß 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-1ß versus similarly sized round/smooth particles (at an equal dose, particles/cell). CoCrMo-alloy particle-size-induced IL-1ß 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.