Titanium particles up-regulate the activity of matrix metalloproteinase-2 in human synovial cells

Purpose

Wear debris particle-induced osteolysis and subsequent aseptic loosening is one of the major causes of failure of total joint replacement. The purpose of this study was to investigate the effect of titanium implant material and inflammatory cytokines on human synovial cells and the development to osteolysis and aseptic loosening.

Methods

This study investigated the effect of titanium implant material on the ECM-degraded MMP-2 in human synovial cells and analyzed the contribution of synovial cells in osteolysis and aseptic loosening.

Results

When human synovial cells are exposed to titanium materials, MMP-2 activity is induced by 1.72 ± 0.14-fold with Ti disc and 3.95 ± 0.10-fold with Ti particles, compared with that of the controls, respectively. Inflammatory cytokines TNFα and IL-1β are also shown to induce MMP-2 activity by 3.65 ± 0.28-fold and 6.76 ± 0.28-fold, respectively. A combination of Ti particles and cytokines induces MMP-2 activities to a higher level (10.54 ± 0.45-fold). Inhibitors of various signal pathways involved in MMP-2 reverse Ti particle-induced MMP-2 activities.

Conclusions

Synovial cells surrounding the bone–prosthesis interface may contribute to production of MMP-2, and NFκB inhibitors may be explored as potential therapeutics to alleviate wear debris-induced osteolysis and aseptic loosening.     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohistochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis. All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohis-tochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis.

All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.     

Modulation of IL-1beta,IL-6, TNF-alpha and PGE(2) by pharmacological agents in explants of membranes from failed total hip replacement

Introduction and goal Proinflammatory cytokines and prostaglandin E(2) (PGE(2)) play an important role in the pathophysiology of osteolysis and implant loosening. The aim of this study was to evaluate the role of pharmacological agents in the inhibition of Interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) and PGE(2) in explants of interface membranes from failed total hip replacements (fTHR).Material and methods Membranes from fTHR were retrieved (N=20) and explants were incubated for 72h in the absence or presence of tenidap at three different concentrations (5, 20 or 50 microg/ml) or diclofenac (125 microg/l). IL-1beta, IL-6, TNF-alpha, and PGE(2) levels were measured in the culture medium using ELISA Capture or EIA kits. Statistical analysis was done using the Mann-Whitney U-test.Results A statistically significant inhibition in IL-1beta synthesis was found at tenidap concentrations of 20 microg/ml (71.3%, P< 0.05) and 50 microg/ml (79.3%,P< 0.02). Tenidap reduced IL-6 levels by 90.4% at 20 microg/ml (P< 0.005) and 96.0% (P< 0.05) at 50 microg/ml. Tenidap also reduced the synthesis of TNF-alpha by 66.9% (P< 0.05) and 77.4% at concentrations of 20 microg/ml and 50 microg/ml. Tenidap had a marked suppressive effect of over 90% (P< 0.0001) on PGE(2) synthesis in all three concentrations. Diclofenac (125 microg/l) decreased PGE(2) production by 95% (P< 0.0001), but had no significant effect in IL-1beta, IL-6, and TNF-alpha levels in the culture medium.Conclusion The ability to simultaneously suppress the release of proinflammatory cytokines and PGE(2) may help control osteolysis and prevent aseptic loosening of THR. This effect could increase implant longevity and lead the way to the pharmacological treatment of this pathology.     

Presence of interleukin-17C in the tissue around aseptic loosened implants

Purpose

The most common long-term complication of joint arthroplasty is aseptic loosening. The proinflammatory cytokines secreted by macrophages are involved in aseptic loosening. Recently, a novel proinflammatory cytokine IL-17C was reported to participate in inflammatory diseases by synergising with proinflammatory cytokines. However, the relationship between IL-17C and the aseptic loosening is unclear.

Methods

The tissues around aseptic loosened implants were collected during revision surgery and handled by formalin fixation and embedded in paraffin. The presence of IL-17C in the tissues around the aseptic loosened implants was investigated in 12 aseptic loosening patients using immunofluorescence.

Results

The presence of IL-17C protein in the tissues around aseptic loosened implants was detected by immunofluorescence. There are no statistical differences between optical density of IL-17C in aseptic loosening samples and in rheumatoid arthritis samples (positive control).

Conclusions

These results suggest the presence of IL-17C in aseptic loosening. Interleukin-17C was related to the inflammation of aseptic loosening, possibly by contributing to the inflammation and osteolysis in the tissues surrounding aseptic loosened implants.     

PGE2 and IL-6 production by fibroblasts in response to titanium wear debris particles is mediated through a Cox-2 dependent pathway.

Aseptic loosening of orthopaedic implants is precipitated by wear debris-induced osteolysis. Central to this process are the pro-inflammatory mediators that are produced in response to wear by the fibroblastic cells, which comprise the majority of periprosthetic membranes. Since this pro-inflammatory cascade is mediated by a plethora of factors with redundant functions, it is imperative to establish a hierarchy. Two well-known fibroblast derived pro-inflammatory factors that stimulate wear debris-induced osteoclastic resorption are prostaglandin E2 (PGE2) and IL-6. However, their relationship to each other in this process is poorly defined. Here we show immunohistochemistry of retrieval membranes indicating that COX-2 is the principal cyclooxygenase responsible for PGE2 production in fibroblasts around failed implants. We also performed in vitro experiments with fibroblasts derived from wild-type (WT), COX-1 (-/-) and COX-2 (-/-) mice, which demonstrated that COX-2 is required for Ti wear debris-induced PGE2 production. Interestingly, COX-2 was also required for IL-6 production in these assays, which could be rescued by the addition of exogenous PGE2 (10(-6) M). Pharmacology studies that utilized the COX-1 selective inhibitor SC 560, the COX-2 selective inhibitor celecoxib, and the nonselective COX inhibitor indomethacin confirmed these results. Taken together, these results indicate that selective inhibition of prostaglandin signaling could favorably impact aseptic loosening beyond its direct effects on PGE2 synthesis, in that it inhibits downstream pro-inflammatory/pro-osteoclastic cytokine production.     

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.     

Quantitative small-animal surrogate to evaluate drug efficacy in preventing wear debris-induced osteolysis.

Individuals who suffer from severe joint destruction caused by the various arthritidies often undergo total joint arthroplasty. A major limitation of this treatment is the development of aseptic loosening of the prosthesis in as many as 20% of patients. The current paradigm to explain aseptic loosening proposes that wear debris generated from the prosthesis initiates a macrophage-mediated inflammatory response by resident macrophages, leading to osteoclast activation and bone resorption at the implant interface. No therapeutic interventions have been proved to prevent or inhibit aseptic loosening. The development of therapeutic strategies is limited due to the absence of a quantitative surrogate in which drugs can be screened rapidly in large numbers of animals. We have previously described a model in which titanium particles implanted on mouse calvaria induce an inflammatory response with osteolysis similar to that observed in clinical aseptic loosening. Here, we present new methods by which the osteolysis in this model can be quantified. We determined that 6-8-week-old mice in normal health have a sagittal suture area of 50 (+/-6) microm2, which contains approximately five osteoclasts. As a result of the titanium-induced inflammation and osteolysis, the sagittal suture area increases to 197 (+/-27) microm2, with approximately 30 osteoclasts, after 10 days of treatment. The sagittal suture area and the number of osteoclasts in the calvaria of sham-treated mice remained unchanged during the 10 days. We also determined the effects of pentoxifylline, a drug that blocks the responses of tumor necrosis factor-alpha to wear debris, and the osteoclast inhibitor alendronate. We found that both drugs effectively block wear debris-induced osteolysis but not osteoclastogenesis. In conclusion, we found the measurements made with this model to be reproducible and to permit quantitative analysis of agents that are to be screened for their potential to prevent aseptic loosening.     

Monitoring of IL-6 levels in patients after total hip joint replacement]

Total hip replacement became a method of choice in treatment of the severe osteoarthritis. Despite the progress in constructing the implants and also the surgical technique, the number of complications rises together with the number of arthroplasties performed. The periprosthetic osteolysis and its consequence--the loosening is the one of the greatest problems of today's joint replacement. It creates the main obstacle for the long-term efficiency of the total hip arthroplasty. It was proved by the numerous research, wear debris of the implant induce the chronic periprosthetic inflammatory process. Many studies emphasize the influence of the proinflammatory cytokines on the bone metabolism. The aim of the study was the evaluation of the inflammatory process in patients with the severe osteoarthritis before the surgery and in subsequent periods after total hip replacements and also in patients with the aseptic loosening of the endoprosthesis, by the monitoring the levels of IL-6 in serum of the peripheral blood. The results suggest, that in patients following THA with the elevated level of IL-6, the inflammatory process was present. This inflammation may lead in future to the aseptic loosening of the implant.     

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.     

Polyethylene and titanium particles induce osteolysis by similar, lymphocyte-independent, mechanisms.

Periprosthetic osteolysis is a major clinical problem that limits the long-term survival of total joint arthroplasties. Osteolysis is induced by implant-derived wear particles, primarily from the polyethylene bearing surfaces. This study examined two hypotheses. First, that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles. Second, that lymphocytes do not play a major role in particle-induced osteolysis. To test these hypotheses, we used the murine calvarial model that we have previously used to examine titanium-induced osteolysis. Polyethylene particles rapidly induced osteolysis in the murine calvaria 5-7 days after implantation. The polyethylene-induced osteolysis was associated with large numbers of osteoclasts as well as the formation of a thick periosteal fibrous tissue layer with numerous macrophages containing phagocytosed polyethylene particles. Polyethylene-induced osteolysis was rapidly repaired and was undetectable by day 21 after implantation. Lymphocytes were noted in the fibrous layer of wild-type mice. However, the amount of osteolysis and cytokine production induced by polyethylene particles was not substantially affected by the lack of lymphocytes in Pfp/Rag2 double knock out mice. All of these findings are similar to our observations of osteolysis induced by titanium particles. These results provide strong support for both of our hypotheses: that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles and that lymphocytes do not play a major role in particle-induced osteolysis.     

The influence of biomaterial on patterns of failure after cemented total hip replacement

We examined aseptic loosening and osteolysis in 77 revised McKee-Arden total hip arthroplasties (THAs) using polyethylene cups and identical femoral stems made from either cobalt chrome alloy or titanium alloy. Time to failure was significantly shorter in the titanium group. Loosening and peri-prosthetic osteolysis occurred with significantly higher frequency in the titanium group compared to the cobalt chrome group.     

Osteolysis in third-generation alumina ceramic-on-ceramic hip bearings with severe impingement and titanium metallosis

The most common cause of long-term failure of total hip arthroplasty is osteolysis and aseptic loosening secondary to wear debris. Combinations of hard materials such as ceramic-on-ceramic generate smaller volumes of particulate wear debris than traditional combinations such as metal-on-polyethylene. We describe 2 cases where osteolysis arose in hips with third-generation alumina ceramic-on-ceramic couplings. Periarticular tissue in both cases contained titanium wear debris due to impingement of the neck of the titanium femoral component against the rim of the titanium shell and ceramic debris from edge loading wear (stripe wear) of the ceramic. It is not clear whether the titanium debris, the ceramic debris, or both caused the osteolysis. These cases illustrate that the risk of osteolysis persists, even with third-generation alumina ceramics.     

Efficacy of etanercept for wear debris-induced osteolysis.

A major limitation of total joint arthroplasty is that up to 20% of patients require revision surgery to correct prosthetic loosening. Aseptic loosening is believed to result from the phagocytosis of wear debris particles by macrophages, which secrete proinflammatory cytokines that stimulate osteolysis. Tumor necrosis factor alpha (TNF-alpha) has been shown to be one of the prominent cytokines in this cascade and to be involved critically in the generation of particle-induced osteolysis. Etanercept is a soluble inhibitor of TNF-alpha, which is widely used for the treatment of rheumatoid arthritis. Here, we show this agent's ability to prevent wear debris-induced osteolysis. In vitro we show that Etanercept can inhibit directly osteoclastic bone resorption in a bone wafer pit assay, as well as cytokine production from titanium (Ti)-stimulated macrophages. Using a quantitative in vivo model of wear debris-induced osteolysis, we show that Etanercept prevents bone resorption and osteoclastogenesis. In mice treated with Etanercept at the time of osteolysis induction, bone resorption and osteoclast numbers were reduced to background levels in both normal and human TNF-alpha (hTNF-alpha) transgenic mice. In an effort to evaluate its effect on established osteolysis, Etanercept was administered 5 days after Ti implantation, and we observed that further osteolysis was prevented. These data support the concept that TNF-alpha is involved critically in osteoclastogenesis and bone resorption during periprosthetic osteolysis and suggest that soluble TNF-alpha inhibitors may be useful as therapeutic agents for the treatment of prosthetic loosening in humans.     

Cytokine levels in synovial fluid from hips with well-functioning or loose prostheses

We analysed synovial fluid from 88 hips, 38 with osteoarthritis and 12 with well-functioning and 38 with loose hip prostheses. The levels of TNF-alpha, IL-1beta (71 hips) and IL-6 (45 hips) were measured using the ELISA technique. Joints with well-functioning or loose prostheses had significantly increased levels of TNF-alpha compared with those with osteoarthritis. Hips with aseptic loosening also had higher levels of IL-1beta but not of IL-6 compared with those without an implant. The levels of TNF-alpha and IL-1beta did not differ between hips with stable and loose prostheses. Higher levels of TNF-alpha were found in hips with bone resorption of type II and type III (Gustilo-Pasternak) compared with those with type-I loosening. The level of cytokines in joint fluid was not influenced by the time in situ of the implants or the age, gender or area of the osteolysis as measured on conventional radiographs. Our findings support the theory that macrophages in the joint capsule increase the production of TNF-alpha at an early phase probably because of particle load and in the absence of clinical loosening. Since TNF-alpha has an important role in the osteolytic process, the interfaces should be protected from penetration of joint fluid.     

Synergistic effect of particles and cyclic pressure on cytokine production in human monocyte/macrophages: proposed role in periprosthetic osteolysis.

Macrophages, activated by particulate wear debris, are important in the process of osteolysis, which occurs during joint implant loosening. We previously found increased levels of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha in cultured macrophages subjected to cyclical pressure of 0.138 MPa, suggesting that cyclic pressure may be another relevant cause of macrophage activation. The current study first investigated the effects of a range of cyclic pressures on cultured macrophages, including an investigation of the time course of cytokine expression. At 0.138 MPa, supernatant levels of TNF-alpha were maximal at 12 h, whereas IL-6 and IL-1beta were maximal at 24 h. All four cyclic pressure levels tested (without particles) resulted in increased production of all three cytokines relative to control. These increases were most marked at 0.069 and 0.035 MPa, and the increase in cytokine production at 0.017 MPa was not statistically significant. Further studies demonstrated that conditioned media from cyclically pressurized macrophages stimulated bone resorption in a neonatal mouse calvarial assay system. There were increased levels of calcium released from calvaria cultured in conditioned media from pressurised monocytes, and an increase in tartate-resistant acid phosphatase-positive osteoclasts was observed microscopically. As particulate wear debris is important in implant loosening, ultra high molecular weight polyethylene particles were also added to the pressurized cell cultures. The experiments compared the effect of atmospheric pressure, cyclic pressure alone, particles alone, and particles and cyclic pressure combined. A combination of ultra high molecular weight polyethylene particles and cyclic pressure at 0.017 MPa resulted in a dramatic synergistic elevation of levels of all three cytokines compared with the levels found with either pressure or particles alone. We propose that monocyte/macrophage activation by cyclic pressure plays a major role in the osteolysis seen in aseptic loosening of implants. The synergistic effect observed between particles and pressure could accelerate implant loosening, and implies that reduction in either cyclic pressure (by improving implant fixation) or wear debris load would reduce osteolysis.     

雷尼酸锶对钛颗粒诱导炎性骨溶解的抑制作用

目的观察雷尼酸锶（strontium ranelate,SR）对磨损颗粒诱导炎性骨溶解的影响。方法 30只雄性C57BL/J6小鼠,随机分为空白组、对照组和药物组,每组10只。采用钛（Ti）颗粒诱导的小鼠颅骨溶解模型,药物组建模当日经灌胃予SR[600mg/（kg·d）],空白组和对照组不予处理;持续至建模后10d,处死取材。HE染色观察颅骨溶解程度及骨膜厚度;抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色检测成熟破骨细胞;酶联免疫吸附试验（ELISA）检测肿瘤坏死因子（TNF-α）、白细胞介素（IL）-1β和IL-6表达水平。结果HE染色结果,对照组骨膜明显增厚,颅骨溶解区域广;图像分析软件测量结果,对照组骨膜厚度（0.27±0.04）mm,骨溶解率为0.47±0.11,与药物组[（0.11±0.02）mm,0.18±0.05]比较,差异有统计学意义（P〈0.05）;TRAP染色结果,对照组颅骨大片紫红色区域,SR治疗后明显减少;ELISA检测结果,SR加入后,TNF-α、IL-1β和IL-6的表达量分别为[（145.6±14.2）ng/L、（130.2±8.2）ng/L和（137.6±8.2）μg/L],与对照组[（210.2±8.9）ng/L、（159.6±9.7）ng/L、（170.8±9.5）μg/L]比较,差异有统计学意义（P〈0.05）。结论 SR能够减轻Ti颗粒引起的炎症反应、减少炎症因子分泌,抑制骨溶解。     

Aseptic loosening in total hip arthroplasty secondary to osteolysis induced by wear debris from titanium-alloy modular femoral heads

Since 1984, we have used components made of titanium alloy for total joint arthroplasty. Recently, two patients needed revision hip arthroplasty, approximately three years after the initial procedure, because of aseptic loosening secondary to severe osteolysis that had been induced by metallic debris. Although implants made of titanium alloy have many favorable qualities--most importantly, superb biocompatibility--the alloy is more susceptible to wear by particles of acrylic cement and tends to generate more polyethylene wear than do components made of stainless steel or chromium-cobalt. A new process of implanting ions has reportedly improved resistance to wear as well as fatigue properties and has enhanced the resistance to corrosion of the implants. Although, to our knowledge, only in vitro studies of this process have been reported to date, we recommend avoiding the use of components made of titanium alloy in which ions have not been implanted. We suggest considering the possibility of osteolysis secondary to appreciable metallic debris in patients who have aseptic loosening of titanium-alloy components that were not implanted with ions.