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.

     

Toll‐like receptors and their adaptors are regulated in macrophages after phagocytosis of lipopolysaccharide‐coated titanium particles

Macrophages phagocytose metallic wear particles and produce mediators, which can induce cellular host response and aseptic implant loosening. Lipopolysaccharide (LPS) on the wear debris can stimulate macrophages via Toll‐like receptor 4 (TLR4) and enhance the response. However, the precise functional role and interaction of TLRs and their adaptor molecules is still unclear. Rat bone marrow macrophages were stimulated with titanium particle (Ti) coated by LPS (Ti/LPS+) and LPS‐free Ti (Ti/LPS?). mRNA levels of cytokines, TLRs and their adaptor molecules were measured using real time PCR. mRNA levels of TNF‐α, IL‐1β, and IL‐6 increased in Ti/LPS+ than Ti/LPS?. In contrast, mRNA levels of TLR4, TLR5, and TLR9 decreased in Ti/LPS+ compared to Ti/LPS?. mRNA levels of MyD88, IRAK1, IRAK4 decreased gradually, and TRAF6 underwent an initial transient increase, followed by suppression in Ti/LPS+. However, mRNA levels of TLR2 and IRAK2 increased after phagocytosis of Ti/LPS+ than Ti/LPS?. The increased expressions of proinflammatory cytokines found in Ti/LPS+ indicated that their productions cytokines could be enhanced by phagocytosis of LPS‐coated particles. Subsequent down‐regulation of TLR4, TLR5, TLR9, MyD88, IRAK1, and IRAK4 suggests that self‐protective mechanisms to regulate excessive host responses are activated in macrophages. Increase of TLR2 and IRAK2 and a transient increase of TRAF6 in Ti/LPS+ suggest that another possible pathway to modulate TLR‐mediated cellular response to prolong inflammatory response in foreign body reaction of aseptic loosening. This down‐ and/or up‐regulation of the potential TLR‐mediated responses to LPS‐coated particles reflects the proactive behavior of effector cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 984–992, 2011     

Inhibition of the PI3K-Akt signaling pathway reduces tumor necrosis factor-alpha production in response to titanium particles in vitro

BACKGROUND: Wear debris contributes to implant loosening after total joint arthroplasty, and few advances have been made in our ability to inhibit the biological response to wear particles. Bacterial endotoxins augment the effects of wear particles in vitro and in vivo. The cytokine, tumor necrosis factor-alpha (TNF-alpha), is produced by macrophages in response to bacterial endotoxins and wear particles, and it increases osteoclast activity resulting in bone resorption and implant loosening. The phosphoinositol-3-kinase (PI3K)-Akt intracellular signal transduction pathway contributes to cytokine production in response to soluble endotoxin. We investigated the role of the PI3K-Akt pathway in the production of TNF-alpha in response to wear particles with adherent endotoxin and so-called endotoxin-free wear particles. METHODS: Cultured RAW264.7 murine macrophages were incubated with titanium particles with adherent endotoxin or with endotoxin-free titanium particles in the presence and absence of specific inhibitors of PI3K (LY294002) or Akt (SH-5). Akt activation was assessed with use of Western blot. TNF-alpha production was measured with use of enzyme-linked immunosorbent assay. Cytotoxicity was determined by measuring lactic dehydrogenase release. RESULTS: Titanium particles with adherent endotoxin increased Akt activation, whereas endotoxin-free titanium particles did not. The PI3K inhibitor reduced TNF-alpha production by 70% in response to titanium with adherent endotoxin without increasing cytotoxicity. Similarly, the Akt inhibitor reduced TNF-alpha production by 83% in response to titanium particles with adherent endotoxin without increasing cytotoxicity. High concentrations of endotoxin-free titanium particles resulted in a small delayed increase in TNF-alpha production that was completely blocked by the PI3K inhibitor. CONCLUSIONS: Inhibition of the PI3K-Akt pathway reduces macrophage TNF-alpha production in response to titanium particles with adherent endotoxin and endotoxin-free particles in vitro.     

Inflammatory Response to Implant Particulates in a Macrophage/Osteoblast Coculture Model

The purpose of this study was to further define the cellular response to titanium and polymethylmethacrylate (PMMA) particles in aseptic loosening, and to determine if the use of pamidronate may be effective in inhibiting bone resorption associated with this response. Macrophages and osteoblasts were cocultured to simulate the environment around an aseptically loose prosthesis. Macrophages were plated on the bottom of six well plates and osteoblasts were plated on culture dish inserts, and placed into the wells with the macrophages. Incubation of macrophages with PMMA in this system led to release of prostaglandin E (PGE₂), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin-6 (IL-6). Incubation with titanium led to release of tumor necrosis factor (TNF) and IL-6. Exposure of calvaria to media from cells exposed to either PMMA or titanium led to release of calcium 45. Incubation of calvaria with pamidronate was able to inhibit release of calcium 45 associated with exposure to the macrophage/osteoblast/particle conditioned medium. Bone resorption at the interface between implant and bone is a consistent feature leading to loosening of orthopedic implants. By inhibiting bone resorption associated with the inflammatory response to implant particulates, pamidronate or other bisphosphonates may have clinical utility in the treatment or prevention or aseptic loosening. Received: 22 December 1995 / Accepted: 3 May 1996     

Inhibition of the PI3K/AKT Pathway Reduces Tumor Necrosis Factor‐Alpha Production in the Cellular Response to Wear Particles In Vitro

Joint replacement is the most effective treatment for end‐stage osteoarticular disease. However, macrophage‐mediated aseptic loosening of joint prosthesis severely hampers the clinical effects of joint replacement. Until now, the mechanism by which macrophages regulate the secretion of inflammatory cytokines after particle stimulation is not clear. It is well known that the PI3K/AKT pathway participates in multiple cellular processes, including cell growth, survival, and inflammation. However, whether the PI3K/AKT pathway participates in the proinflammatory response of macrophages after particle stimulation and secondary aseptic loosening is still unknown. In this study, ceramic and titanium particles of different sizes were prepared to stimulate macrophages. LY294002, a specific inhibitor of PI3K, was pretreated prior to particle stimulation. The expression of tumor necrosis factor‐alpha (TNF‐α) and all the subunits of PI3K and AKT were detected by real‐time polymerase chain reaction, enzyme‐linked immunosorbent assay, and Western blot. The result showed that LY294002 could suppress the RNA and protein expression of TNF‐α in RAW264.7 cells after stimulation of different particles. The subunits of PI3K (p110β and p85β), followed by activation of phosphor‐AKT (Ser473), participated in the regulation of activating macrophages by wear particles, ultimately resulting in the secretion of TNF‐α.     

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. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 847–854, 2009     

Adherent endotoxin on orthopedic wear particles stimulates cytokine production and osteoclast differentiation.

Aseptic loosening of orthopedic implants is thought to be caused primarily by osteoclast differentiation induced by bone resorptive cytokines produced in response to phagocytosis of implant-derived wear particles. This study examined whether adherent endotoxin on the wear particles is responsible for inducing osteoclast differentiation as well as production of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor a (TNF-alpha). Removal of adherent endotoxin almost completely inhibited the responses to titanium (Ti) particles by both murine marrow cells and human peripheral blood monocytes. In vivo experiments showed that endotoxin removal reduced particle-induced osteolysis by 50-70%. Addition of lipopolysaccharide (LPS) to the "endotoxin-free" particles restored their ability to induce cytokine production and osteoclast differentiation in vitro. Moreover, marrow cells from mice that are hyporesponsive to endotoxin because of mutation of Toll-like receptor 4 induced significantly less cytokine production and osteoclast differentiation in response to Ti particles with adherent endotoxin than did marrow cells from normoresponsive mice. This mutation also resulted in significantly less particle-induced osteolysis in vivo. Taken together, these results show that adherent endotoxin is involved in many of the biological responses induced by orthopedic wear particles and should stimulate development of new approaches designed to reduce the activity of adherent endotoxin in patients with orthopedic implants.     

Adherent lipopolysaccharide inhibits the osseointegration of orthopedic implants by impairing osteoblast differentiation

Osseointegration is the process by which an orthopedic implant makes direct bone-to-implant contact and is crucial for the long-term function of the implant. Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopedic implants after sterilization and impair osseointegration. For example, specific lots of implants that were associated with impaired osseointegration and high failure rates were discovered to have contaminants including bacterial debris. Therefore, the goals of this study were to determine if bacterial debris exists on sterile orthopedic implants and if adherent bacterial debris inhibits the osseointegration of orthopedic implants. We found that debris containing lipopolysaccharide (LPS) from Gram-negative bacteria exists on both sterile craniofacial implants and wrist implants. Levels of bacterial debris vary not only between different lots of implants but within an individual lot. Using our murine model of osseointegration, we found that ultrapure LPS adherent to the implants inhibited bone-to-implant contact and biomechanical pullout measures. Analysis of osseointegration in knock-out mice demonstrated that adherent LPS inhibited osseointegration by signaling through its primary receptor, Toll-like receptor 4, and not by signaling through Toll-like receptor 2. Ultrapure LPS adherent to titanium alloy discs had no detectable effect on early stages of MC3T3-E1 osteogenesis in vitro such as attachment, spreading or growth. However, later stages of osteogenic differentiation and mineralization were inhibited by adherent LPS. Thus, LPS may inhibit osseointegration in part through cell autonomous effects on osteoblasts. These results highlight bacterial debris as a type of surface contaminant that can impair the osseointegration of orthopedic implants.     

Toll‐like receptors and aseptic loosening of hip endoprosthesis—a potential to respond against danger signals?

Bacterial remnants and subclinical biofilms residing on prosthesis surfaces have been speculated to play a role in hip implant loosening by opsonizing otherwise relatively inert wear particles. The innate immune system recognizes these microbial pathogen‐associated molecular patterns (PAMPs) using Toll‐like receptors (TLRs). Our objective was to evaluate the possible presence of TLRs in aseptic synovial membrane‐like interface tissue. Bacterial culture‐negative, aseptic (n = 4) periprosthetic synovial membrane‐like tissue was compared to osteoarthritis synovial membrane (n = 5) for the presence of cells positive for all known human functional TLRs, stained using specific antibodies by immunohistochemistry, and evaluated using morphometry. In comparison to osteoarthtritic synovium, the number of TLR‐positive cells was found to be increased in the aseptic setting, reflecting the considerable macrophage infiltration to the tissues investigated. Thus aseptic periprosthetic tissue seems to be very reactive to PAMPs. It has been recently recognized that TLR do not only respond to traditional PAMPs, but also to endogenous alarmings or danger signals released from necrotic and activated cells. Alarming‐TLR interaction in the periprosthetic tissue might be a novel mechanism of aseptic loosening of endoprosthesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:184–190, 2010     

Propofol has anti‐inflammatory effects on alveolar type II epithelial cells

Background: We investigated whether lipopolysaccharide (LPS) induced inflammation in alveolar epithelial type II (ATII) cells is through cluster of differentiation 14 (CD14) and Toll‐like receptor 4 (TLR4) and the effect of different dosages of propofol on the inflammation in primary cultured rat ATII cells. Methods: Cultured ATII cells were randomly assigned to one of the following five groups: Group C: untreated group (control) cultured in the absence of propofol and LPS; Group LPS: treated with 1 μg/ml LPS; Group P1: treated with 1 μg/ml LPS and 25 μM propofol; Group P2: treated with 1 μg/ml LPS and 50 μM propofol; Group P3: treated with 1 μg/ml LPS and 100 μM propofol. ATII cells in all groups were cultured at 37 °C for 3 h. CD14 and TLR4 mRNA was detected using real‐time polymerase chain reaction. Western blot was used to detect CD14 and TLR4 protein expression. CD14 and TLR4 expression on the ATII cells was imaged using immunofluorescence. Tumor necrosis factor‐α (TNF‐α) production was determined using an ELISA kit. Results: LPS stimulation resulted in an increased CD14 and TLR4 expression and increased TNF‐α production in ATII cells. Propofol, at concentrations ≥50 μM, significantly (P<0.05) and dose‐dependently decreased CD14 and TLR4 mRNA expression and protein expression in ATII cells. This was accompanied by a decrease in TNF‐α production (P<0.05). Conclusion: These results suggest that propofol, at clinically relevant concentrations, can reduce inflammatory responses in LPS‐induced ATII cells injury through downregulation of CD14 and TLR4 expression.     

Severe gunshot injuries in a porcine model: impact on central markers of innate immunity

Background: The mechanisms behind lipopolysaccharide (LPS) tolerance remain obscure. LPS signals through Toll‐like receptor 4 (TLR4) and severe trauma/haemorrhage may influence binding and signalling through this receptor, e.g. by changing membrane expression or by releasing endogenous ligands like High Mobility Group Box 1 (HMGB1). The aim of this study was to examine these relations further in a porcine model with standardized trauma. Methods: Nine anaesthetized pigs sustained one gunshot through the femur and one pistol shot through the upper abdomen. Blood was sampled before and 90 min after shooting. The samples were stimulated for 4 h with LPS 10 ng/ml or an equivalent amount of normal saline. The leucocyte response was evaluated by measuring the tumour necrosis factor‐α (TNF‐α) and CXC ligand 8 (CXCL8) in the supernatant. Flow cytometry was used to measure the surface expression of TLR4 on CD14+ monocytes. HMGB1 concentrations were measured in the plasma. Results: Trauma and treatment caused a significant decline in the LPS‐stimulated concentrations of TNF‐α [4.53 ± 0.24 pg/ml (ln) at 0 min, 3.54 ± 0.35 pg/ml (ln) at 90 min, P=0.026], but did not modify the release of CXCL8. Monocyte TLR4 expression was unchanged. Plasma HMGB1 increased significantly [<0.92 vs. 3.02 ± 0.19 ng/ml (ln), P<0.001]. The concentrations of TNF‐α and CXCL8 did not correlate with TLR4 expression or HMGB1 concentrations. Conclusion: The results suggest that trauma‐induced LPS tolerance is not primarily regulated by TLR4 expression on circulating CD14+ monocytes or by the release of HMGB1 from damaged tissues.     

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. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1867–1873, 2011     

Adherent endotoxin mediates biological responses of titanium particles without stimulating their phagocytosis.

Aseptic loosening of orthopaedic implants is thought to be primarily due to stimulation of cytokine production by wear particles from the implants. The cytokines increase osteoclast differentiation, leading to osteolysis and implant loosening. Accumulating evidence indicates that adherent endotoxin mediates the biological responses induced by the wear particles. One mechanism by which adherent endotoxin may act is by increasing phagocytosis of the wear particles. To test this hypothesis, the effect of adherent endotoxin on phagocytosis of titanium particles was determined. First, we developed reliable confocal and fluorescence microscopy methods to examine both the attachment and internalization steps of phagocytosis. Use of these methods showed that adherent endotoxin does not detectably alter the rate or the extent of phagocytosis of titanium particles by RAW 264.7 cells. Despite this lack of an effect on phagocytosis, adherent endotoxin dramatically increases the ability of RAW 264.7 cells to produce TNF-alpha and induce osteoclast differentiation. Thus, adherent endotoxin mediates these biological responses by a mechanism that does not rely on increased phagocytosis. These results also demonstrate that phagocytosis is not sufficient to induce cytokine production and osteoclast differentiation but do not rule out the possibility that phagocytosis is required for induction of these responses by titanium particles with adherent endotoxin.     

The balance between endotoxin accumulation and clearance during particle-induced osteolysis in murine calvaria.

Bacterial endotoxin may contribute to aseptic loosening of orthopedic implants even in the absence of clinical or microbiological evidence of infection. One potential source of endotoxin during aseptic loosening is systemically circulating endotoxin, derived from intestinal flora, minor infections, or dental procedures, that may bind to wear particles. The current study demonstrates that systemically derived endotoxin accumulates when 'endotoxin-free' titanium and polyethylene particles are implanted on murine calvaria. Time-course experiments and experiments using germ-free mice rule out the possibility that the observed endotoxin accumulation may be due to bacterial contamination. In contrast, endotoxin is cleared from titanium particles that originally carry high amounts of adherent endotoxin. The mechanism of endotoxin clearance is not dependent on induction of a respiratory burst. Taken together, these results indicate that a balance between endotoxin accumulation and endotoxin clearance controls the steady-state level of endotoxin surrounding orthopedic wear particles implanted on murine calvaria. This balance may regulate the rate of osteolysis in the murine calvaria model as well as in patients with aseptic loosening.     

Osteoclastic differentiation by mononuclear phagocytes containing biomaterial particles

Aseptic loosening of implant components is a common and important complication of both cemented and uncemented prosthetic joint replacements. Wear particles derived from organic polymer and metal implant biomaterials are commonly found within macrophages and macrophage polykaryons in the fibrous membrane between loose implant components and the host bone undergoing resorption. In order to determine whether biomaterial particle-containing, foreign-body macrophages may contribute to periprosthetic bone resorption, we cultured murine monocytes that had phagocytosed particles of biomaterials commonly employed in bone implant surgery [polymethylmethacrylate (PMMA), ultra-high molecular weight polyethylene (PE), titanium and chromium-cobalt] on bone slices and glass coverslips with UMR 106 osteoblast-like stromal cells in the presence of 1,25-dihydroxy-vitamin D₃. Under these conditions, all biomaterial particle-containing, foreign-body macrophages differentiated into osteoclastic cells, i.e. tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells capable of extensive lacunar bone resorption. This study shows that particle phagocytosis by macrophages does not abrogate the ability of these cells to undergo osteoclast differentiation. These findings emphasise the importance of the foreign-body macrophage response to biomaterial wear particles in the pathogenesis of aseptic loosening.     

PKC-zeta is essential for endotoxin-induced macrophage activation

BACKGROUND: A critical element in sepsis-induced tissue injury is the release of pro-inflammatory mediators from LPS-activated macrophages. The cellular mechanisms involved in this process remain incompletely understood. The aim of the current study was to further clarify the mechanism of LPS activation through the TLR4 receptor complex by examining the roles of the various isoforms of PKC. MATERIALS AND METHODS: Differentiated THP-1 cells were subjected to LPS stimulation. Selected cells were pretreated with various concentrations of G?6983 to inhibit conventional, novel, and atypical PKC isoforms. Lipid raft, cellular, and nuclear proteins were then extracted and analyzed by Western blot and EMSA for components of the TLR4 pathway. Supernatants harvested under the various conditions were analyzed by ELISA for the production of TNF-alpha. RESULTS: LPS stimulation led to the mobilization of TLR4 to lipid rafts followed by phosphorylation and activation of IRAK, ERK 1/2, p38, and JNK/SAPK. Subsequently, LPS induced the activation of NF-kappaB and AP-1. Activation of these TLR4-signaling components resulted in the production of TNF-alpha. Inhibition of conventional and novel PKC isoforms had no significant effect on macrophage activation. Inhibition of the atypical PKC, PKC-zeta, was associated with significant attenuation in the mobilization of TLR4 to lipid rafts, the activation of all TLR4-signaling components, and the production of TNF-alpha. CONCLUSION: This study demonstrates that the atypical PKC isoform, PKC-zeta, is critical to regulation of LPS-induced TLR4 lipid raft mobilization within macrophages, TLR4-signaling, and TNF-alpha production. Although the mechanism of its activation remains unresolved, it appears that modulation of PKC-zeta activity during Gram-negative infections may limit associated inflammatory-induced morbidity.     

Measurement and removal of adherent endotoxin from titanium particles and implant surfaces.

Aseptic loosening is thought to be due primarily to osteolysis induced by cytokines and prostaglandins that are produced in response to implant-derived wear particles. Because endotoxin has many of the same effects as have been reported for wear particles, we hypothesized that adherent endotoxin may be responsible for the biological responses induced by wear particles. We demonstrated the presence of significant levels of adherent endotoxin on commonly used preparations of titanium particles as well as on titanium and titanium-alloy implant surfaces. In contrast, supernatants obtained by centrifugation of particle suspensions contained approximately 1% as much endotoxin as did the particles. Therefore, it is erroneous to assume that particles do not contain endotoxin on the basis of data that it cannot be detected in their supernatants or filtrates. These results emphasize the importance of considering the potential role of adherent endotoxin when examining the in vitro effects of wear particles and the in vivo performance of orthopaedic implants. We also developed a protocol that removed more than 99.94% of the adherent endotoxin from the titanium particles without detectably affecting their size or shape. The removal of adherent endotoxin will allow comparison of the biological responses induced by particles with or without adherent endotoxin.     

Antisense oligonucleotide targeting TNF‐α can suppress co‐cr‐mo particle‐induced osteolysis

The most common cause of implant failure in joint replacement is aseptic loosening due to particle‐induced osteolysis. TNF‐α has been shown to be one of the key factors in the process of osteoclastogenesis. Anti‐TNF agents are useful in the treatment of joint inflammation related to osteolysis. This study investigated the effect of a single subcutaneous dose of an antisense oligonucleotide (ASO) on particle‐induced osteolysis. We utilized the murine calvaria osteolysis model in C57BL/J6 mice. Bone resorption was measured by the toluidine blue staining. Osteoclasts were detected by tartrate resistant acid phosphatase (TRAP) staining assay and were quantified by a TRAP quantification kit. Results show that bone resorption is 0.347 ± 0.09 mm² in mice with particle implantation, and decreased to 0.123 ± 0.05 mm² and 0.052 ± 0.02 mm² after ASO treatment with low and high doses, respectively. The number of osteoclasts in animal calvaria treated with ASO is reduced compared with that of untreated animals, and the quantification results indicate that about 90% of osteoclastogenesis is suppressed by the ASO. In addition, the osteoclastogenesis can be reestablished by the addition of TNF‐α. In conclusion, we demonstrate that the antisense oligonucleotide targeting to TNF‐α can suppress osteolysis induced by metal particles in a murine calvaria model. This new finding may be of value in the search for novel therapeutic methods for implant loosening. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1114–1120, 2008     

Chemiluminescent Responses of Macrophages Exposed to Biomaterials: A Biocompatibility Screening Test

Chemiluminescence (CL) response of alveolar macrophages (AM) exposed to either phagocytosable or nonphagocytosable particles of polymethylmethacrylate (PMMA) or titanium in vitro was monitored. The results were correlated to known in vivo bone tissue compatibility of the materials. Morever, the quantity and morphology of adherent AM on titanium and PMMA disks were assessed using scanning electron microscopy (SEM). Our results with PMMA indicated that a high CL response to phagocytosable as well as nonphagocytosable particles of the biomaterial in question, correlated with poor bone tissue compatibility. By contrast, a low CL response to phagocytosable particles and no response to nonphagocytosable particles correlated with a satisfactory bone tissue compatibility, as in the case of titanium. SEM showed 48% more AM adherent to PMMA than titanium per unit area. Also, the percentage of cells with membrane damage was three times higher on PMMA than on titanium. It appears that the CL response of AM exposed to biomaterials in vitro thus can be used as an easy to do screening test for potential orthopaedic implant materials before in vivo tests are considered