Aims. This study aimed to determine if macrophages can attach and directly affect the oxide layers of 316L stainless steel, titanium alloy (Ti6Al4V), and cobalt-chromium-molybdenum alloy (CoCrMo) by releasing components of these alloys. Methods. Murine peritoneal macrophages were cultured and placed on stainless steel, CoCrMo, and Ti6Al4V discs into a 96-well plate. Cells were activated with interferon gamma and lipopolysaccharide. Macrophages on stainless steel discs produced significantly more nitric oxide (NO) compared to their control counterparts after eight to ten days and remained elevated for the duration of the experiment. Results. On stainless steel, both nonactivated and activated cell groups were shown to have a significant increase in metal ion release for Cr, Fe, and Ni (p < 0.001, p = 0.002, and p = 0.020 respectively) compared with medium only and showed macrophage-sized corrosive pits on the stainless steel surface. On titanium alloy discs there was a significant increase in aluminum (p < 0.001) among all groups compared with medium only. Conclusion. These results indicated that macrophages were able to attach to and affect the oxide surface of stainless steel and titanium alloy discs. Cite this article: Bone Joint J 2020;102-B(7 Supple B):116–121

Aseptic loosening and osteolysis around prosthetic joints are the principal causes of failure and consequent revision. During this process activated macrophages produce cytokines which are thought to promote osteolysis by osteoclasts. Changes in pressure within the space around implants have been proposed as a cause of loosening and osteolysis. We therefore studied the effect of two different regimes of cyclic pressure on the production of interleukin-1β (IL-1β), IL-6 and tumour necrosis factor-α (TNF-α) by cultured human monocyte-derived (M-D) macrophages. There was a wide variation in the expression of cytokines in non-stimulated M-D macrophages from different donors and therefore cells from the same donor were compared under control and pressurised conditions. Both regimes of cyclic pressure were found to increase expression of IL-6 and TNF-α. Expression of IL-1β was increased by a higher-frequency regime only. Our findings suggest that M-D macrophages are activated by cyclic pressure. Further work will be required to understand the relative roles of frequency, amplitude and duration of applied pressure in the cellular effects of cyclic pressure in this system

Particulate wear debris is associated with periprosthetic inflammation and loosening in total joint arthroplasty. We tested the effects of titanium alloy (Ti-alloy) and PMMA particles on monocyte/macrophage expression of the C-C chemokines, monocyte chemoattractant protein-1 (MCP-1), monocyte inflammatory protein-1 alpha (MIP-1α), and regulated upon activation normal T expressed and secreted protein (RANTES). Periprosthetic granulomatous tissue was analysed for expression of macrophage chemokines by immunohistochemistry. Chemokine expression in human monocytes/macrophages exposed to Ti-alloy and PMMA particles in vitro was determined by RT-PCR, ELISA and monocyte migration. We observed MCP-1 and MIP-1α expression in all tissue samples from failed arthroplasties. Ti-alloy and PMMA particles increased expression of MCP-1 and MIP-1α in macrophages in vitro in a dose- and time-dependent manner whereas RANTES was not detected. mRNA signal levels for MCP-1 and MIP-1α were also observed in cells after exposure to particles. Monocyte migration was stimulated by culture medium collected from macrophages exposed to Ti-alloy and PMMA particles. Antibodies to MCP-1 and MIP-1α inhibited chemotactic activity of the culture medium samples. Release of C-C chemokines by macrophages in response to wear particles may contribute to chronic inflammation at the bone-implant interface in total joint arthroplasty

The cellular mechanisms which account for the formation of osteoclasts and bone resorption associated with enlarging benign and malignant mesenchymal tumours of bone are uncertain. Osteoclasts are marrow-derived, multinucleated, bone-resorbing cells which express a macrophage phenotype. We have determined whether tumour-associated macrophages (TAMs) isolated from benign and malignant mesenchymal tumours are capable of differentiating into osteoclasts. Macrophages were cultured on both coverslips and dentine slices for up to 21 days with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D. 3. (1,25(OH). 2. D. 3. ) and human macrophage colony-stimulating factor (M-CSF) or, in the absence of UMR 106 cells, with M-CSF and RANK ligand. In all tumours, the formation of osteoclasts from CD14-positive macrophages was shown by the formation of tartrate-resistant-acid-phosphatase and vitronectin-receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. These results indicate that the tumour osteolysis associated with the growth of mesenchymal tumours in bone is likely to be due in part to the differentiation of mononuclear phagocyte osteoclast precursors which are present in the TAM population of these lesions

Periprosthetic osteolysis is a major cause of aseptic loosening in artificial joint replacement. It is assumed to occur in conjunction with the activation of macrophages. We have shown in vitro that human osteoblast-like cells, isolated from bone specimens obtained from patients undergoing hip replacement, phagocytose fine particles of titanium alloy (TiAlV). The human osteoblast-like cells were identified immunocytochemically by the presence of bone-specific alkaline phosphatase (BAP). With increasing duration of culture, a variable number of the osteoblastic cells became positive for the macrophage marker CD68, independent of the phagocytosis of particles, with a fine granular cytoplasmic staining which was coexpressed with BAP as revealed by immunodoublestaining. The metal particles were not toxic to the osteoblastic cells since even in culture for up to four weeks massively laden cells were vital and had a characteristic morphology. Cells of the human osteosarcoma cell line (HOS 58) were also able to phagocytose metal particles but had only a low expression of the CD68 antigen. Fluorescence-activated cell scanning confirmed our immunocytochemical results. Additionally, the cells were found to be negative for the major histocompatibility complex-II (MHC-II) which is a marker for macrophages and other antigen-presenting cells. Negative results of histochemical tests for tartrate-resistant acid phosphatase excluded the contamination by osteoclasts or macrophages in culture. Our observations suggest that the osteoblast can either change to a phagocytosing cell or that the phagocytosis is an underestimated property of the osteoblast. The detection of the CD68 antigen is insufficient to prove the monocytic lineage. In order to discriminate between macrophages and osteoblasts additional markers should be used. To our knowledge, this is the first demonstration of cells of an osteoblastic origin which have acquired a mixed phenotype of both osteoblasts and macrophages

Macrophages and their fused products are commonly found at the polymethylmethacrylate cement-bone interface, but it is not known if they contribute directly to the osteolysis associated with loosening of the cemented prosthesis. We isolated mononuclear phagocytes from granulomas formed by subcutaneous implantation of polymethylmethacrylate into mice and incubated them on bone slices in which they formed resorption lacunae after co-culture for seven to 14 days with both marrow stromal cells and osteoblast-like cells (in the presence of 1 alpha,25-dihydroxyvitamin D3 and dexamethasone). Increased numbers of tartrate-resistant acid phosphatase-positive mononuclear and multinucleated cells formed in these cultures. Both in the presence and absence of stromal cells, macrophages produced extensive superficial roughening of the bone surface. Polymethylmethacrylate-induced macrophages are thus capable of low-grade surface and high-grade lacunar osteolysis, the latter requiring the presence of specific hormonal and stromal cell elements. These two forms of bone resorption could account for the pathogenesis and clinical patterns associated with loosening of the cemented prosthesis

We exposed human macrophages isolated from the peripheral blood of healthy donors to metal and bone-cement particles from 0.2 to 10 μm in size. Zymography showed that macrophages exposed to titanium alloy and polymethylmethacrylate (PMMA) particles released a 92- and 72-kDa gelatinase in a dose- and time-dependent manner. Western immunoblotting confirmed that the 92- and 72-kDa gelatinolytic activities corresponded to matrix metalloproteinase-9 and matrix metalloproteinase-2 (MMP-9, MMP-2), respectively. Western immunoblotting also indicated that titanium alloy and PMMA particles increased the release of MMP-1. Northern blotting showed elevated mRNA signal levels for MMP-1, MMP-2, and MMP-9 after exposure to both types of particle. Collagenolytic activity also increased in the macrophage culture medium in response to both types of particle. Our findings support the hypothesis that macrophages release MMPs in proportion to the amount of particulate debris within periprosthetic tissues

Although the response of macrophages to polyethylene debris has been widely studied, it has never been compared with the cellular response to ceramic debris. Our aim was to investigate the cytotoxicity of ceramic particles (Al. 2. O. 3. and ZrO. 2. ) and to analyse their ability to stimulate the release of inflammatory mediators compared with that of high-density polyethylene particles (HDP). We analysed the effects of particle size, concentration and composition using an in vitro model. The J774 mouse macrophage cell line was exposed to commercial particles in the phagocytosable range (up to 4.5 μm). Al. 2. O. 3. was compared with ZrO. 2. at 0.6 μm and with HDP at 4.5 μm. Cytotoxicity tests were performed using flow cytometry and macrophage cytokine release was measured by ELISA. Cell mortality increased with the size and concentration of Al. 2. O. 3. particles. When comparing Al. 2. O. 3. and ZrO. 2. at 0.6 μm, we did not detect any significant difference at the concentrations analysed (up to 2500 particles per macrophage), and mortality remained very low (less than 10%). Release of TNF-α also increased with the size and concentration of Al. 2. O. 3. particles, reaching 195% of control (165 pg/ml v 84 pg/ml) at 2.4 μm and 350 particles per cell (p < 0.05). Release of TNF-α was higher with HDP than with Al. 2. O. 3. particles at 4.5 μm. However, we did not detect any significant difference in the release of TNF-α between Al. 2. O. 3. and ZrO. 2. at 0.6 μm (p > 0.05). We saw no evidence of release of interleukin-1α or interleukin-1ß after exposure to ceramic or HDP particles

Sterilisation by gamma irradiation in the presence of air causes free radicals generated in polyethylene (PE) to react with oxygen, which could lead to loss of physical properties and reduction in fatigue strength. Tissue retrieved from failed total hip replacements often has large quantities of particulate PE and most particles associated with peri-implant osteolysis are oxidised. Consequently, an understanding of the cellular responses of oxidised PE particles may lead to clarification of the pathogenesis of osteolysis and aseptic loosening. We have used the agarose system to demonstrate the differential effects of oxidised and non-oxidised PE particles on the release of proinflammatory products such as interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α) from monocytes/ macrophages (M/M). Oxidised PE particles were shown to stimulate human M/M to phagocytose and to release cytokines. Oxidation may alter the surface chemistry of the particles and enhance the response to specific membrane receptors on macrophages, such as scavenger-type receptors

We have investigated whether the particle-stimulated release of inflammatory cytokines from human primary macrophages in vitro was dependent upon the type of bone cement used. Particles of clinically relevant size were produced from Palacos R without radio-opacifier, Palacos R with BaSO. 4. , Palacos R with ZrO. 2. and from CMW3 which contains BaSO. 4. All four preparations produced significantly greater release of tumour necrosis factor alpha, interleukin-6 and interleukin-1 beta than a negative control but there were no significant differences between them. The differences in the ability to stimulate bone resorption and in clinical performance between proprietary bone cements previously recorded are not explained by the release of the cytokines most commonly implicated in osteolysis

Particulate prosthetic materials are often studied by adding them to monocytic cells in vitro and measuring the release of cytokines as an indicator of their inflammatory potential. Endotoxin is known to be a contaminant of particle preparations and also stimulates the release of cytokines. It is usual to use a proprietary endotoxin test to avoid erroneous results. Four different formulations of cement were found to be free from endotoxin using standard, gelclot tests but stimulated different levels of release of cytokines from macrophages. These differences were explained when a more sensitive, kinetic endotoxin assay showed that release correlated with minor contamination with endotoxin. In a repeat experiment using cement particles with low or undetectable levels of endotoxin by kinetic assay, differences in the ability of the formulations to stimulate the release of cytokines were not seen. Endotoxin is adsorbed on to the surface of particles and it is this combination which stimulates increased release of cytokines. In both the above methods for determination of endotoxin, the water in which the particles had been soaked was examined rather than the particles directly. Further investigations showed that a kinetic assay directly on a particle suspension is the most sensitive method to measure contamination with endotoxin

Aims. To investigate metallosis in patients with magnetically controlled growing rods (MCGRs) and characterize the metal particle profile of the tissues surrounding the rod. Methods. This was a prospective observational study of patients with early onset scoliosis (EOS) treated with MCGRs and undergoing rod exchange who were consecutively recruited between February 2019 and January 2020. Ten patients were recruited (mean age 12 years (SD 1.3); 2 M:8 F). The configurations of the MCGR were studied to reveal the distraction mechanisms, with crucial rod parts being the distractable piston rod and the magnetically driven rotor inside the barrel of the MCGR. Metal-on-metal contact in the form of ring-like wear marks on the piston was found on the distracted portion of the piston immediately outside the barrel opening (BO) through which the piston rod distracts. Biopsies of paraspinal muscles and control tissue samples were taken over and away from the wear marks, respectively. Spectral analyses of the rod alloy and biopsies were performed to reveal the metal constituents and concentrations. Histological analyses of the biopsies were performed with haematoxylin and eosin staining. Results. Titanium (Ti), vanadium (V), and neodymium (Nd) concentrations in the biopsies taken near the wear marks were found to be significantly higher than those in the control tissue samples. Significantly increased Nd concentrations were also found in the tissues near the barrel of the MCGR. Chronic inflammation was revealed by the histological studies with fibrosis and macrophage infiltration. Black particles were present within the macrophages in the fibrotic tissues. Conclusion. Ti and V were generated mainly at the BO due to metal-on-metal contact, whereas the Nd from the rotor of the MCGR is likely released from the BO during distraction sessions. Phagocytotic immune cells with black particles inside raise concern regarding the long-term implications of metallosis. Cite this article: Bone Joint J 2020;102-B(10):1375–1383

Aims. Pin-site infection remains a significant problem for patients treated by external fixation. A randomized trial was undertaken to compare the weekly use of alcoholic chlorhexidine (CHX) for pin-site care with an emollient skin preparation in patients with a tibial fracture treated with a circular frame. Methods. Patients were randomized to use either 0.5% CHX or Dermol (DML) 500 emollient pin-site care. A skin biopsy was taken from the tibia during surgery to measure the dermal and epidermal thickness and capillary, macrophage, and T-cell counts per high-powered field. The pH and hydration of the skin were measured preoperatively, at follow-up, and if pin-site infection occurred. Pin-site infection was defined using a validated clinical system. Results. Out of 116 patients who were enrolled in the study, 23 patients (40%) in the CHX group and 26 (44%) in the DML group had at least one bad or ugly pin-site infection. This difference was not statistically significant (p = 0.71). There was no significant relationship between pH or hydration of the skin and pin-site infection. The epidermal thickness was found to be significantly greater in patients who had a pin-site infection compared with those who did not (p = 0.01). Skin irritation requiring a change of treatment occurred in four patients (7%) using CHX, and none using DML. Conclusion. We found no significant difference in the incidence of pin-site infection between the CHX and DML treatment groups. Dermol appeared to offer a small but significant advantage in terms of tolerability. We did not find a significant association between patient or treatment related factors and pin-site infection. It is therefore difficult to make specific recommendations based upon these results. The use of either cleaning agent appears to be appropriate. Cite this article: Bone Joint J 2021;103-B(2):279–285

Aims. Patients with flexion instability after total knee arthroplasty (TKA) often present with a recurrent effusion, which may be a haemarthrosis. While the radiographic factors contributing to flexion instability have been elucidated, the clinical diagnosis remains challenging. Our aim, in this study, was to determine the mean white cell count and differential profile in pre-operative aspirations of synovial fluid in a consecutive series of patients undergoing revision TKA for flexion instability. Patients and Methods. Between 2000 and 2010, 60 patients undergoing aseptic revision TKA for flexion instability were identified. The results of the pre-operative aspiration of synovial fluid were available for 53 patients (88%). These patients were 1:2 matched to 106 patients who underwent aseptic TKA for indications other than flexion instability. The mean age of the patients at revision TKA was 65 years (44 to 82) and 55% were women. The mean follow-up was 4.3 years (2 to 10.2). Results. In the flexion instability group, the median total cell count was 312 cells/µL (interquartile range (IQR) 104 to 624), with a mean distribution of 45% macrophages (2% to 90%), 30% lymphocytes (1% to 69%), 18% neutrophils (0% to 80%), 0.5% eosinophils (0% to 6%) and 7% other cells (0% to 42%; mainly synovial cells). There was no significant difference in the median total cell count (p = 0.14) or mean distribution of macrophages (p = 0.42), lymphocytes (p = 0.38), neutrophils (p = 0.19) and eosinophils (p = 0.89) between the flexion instability and control groups. There was a significant difference in the percentage of bloody serosanguineous aspirations which was 58% in the flexion instability group and 18% in the control group (odds ratio = 6.5; p = 0.0001). Conclusions. In the group of patients who underwent revision TKA for flexion instability, most had a mean cell count and differential similar to those who underwent revision for other aseptic indications. However, bloody serosanguineous aspirations were 6.5 times more common in those with flexion instability confirming that many of the recurrent effusions seen in this condition are haemarthroses. Cite this article: Bone Joint J 2017;99-B:1477–81

The most frequent cause of failure after total hip replacement in all reported arthroplasty registries is peri-prosthetic osteolysis. Osteolysis is an active biological process initiated in response to wear debris. The eventual response to this process is the activation of macrophages and loss of bone. Activation of macrophages initiates a complex biological cascade resulting in the final common pathway of an increase in osteolytic activity. The biological initiators, mechanisms for and regulation of this process are beginning to be understood. This article explores current concepts in the causes of, and underlying biological mechanism resulting in peri-prosthetic osteolysis, reviewing the current basic science and clinical literature surrounding the topic

Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis. In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition. Cite this article: Bone Joint J 2013;95-B:1021–5

The interactions between the different cell types in periprosthetic tissue are still unclear. We used a non-contact coculture model to investigate the effects of polymethylmethacrylate (PMMA) particles and human macrophage-derived soluble mediators on fibroblast activation. Macrophages were either exposed or not exposed to phagocytosable PMMA particles, but fibroblasts were not. Increasing numbers of macrophages were tested in cocultures in which the fibroblast cell number was held constant and cultures of macrophages alone were used for comparison of cytokine release. We used the release of interleukin-1 beta (IL-1β), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α), lysosomal enzyme and metalloproteinase activity to assess the cultivation of macrophages and fibroblasts. In cocultures, IL-6 release was increased 100-fold for both unchallenged and particle-challenged cultures when compared with macrophage cultures alone. Furthermore, particle-challenged cocultures had threefold higher IL-6 levels than unchallenged cocultures. Release of TNF-α was similar in cocultures and in macrophage cultures. IL-1β release in cocultures was independent of the macrophage-fibroblast ratio. Lysosomal enzyme activity and metalloproteinase activity were increased in cocultures. Our data show that macrophages and fibroblasts in coculture significantly increase the release of IL-6 and to a less degree other inflammatory mediators; particle exposure accentuates this effect. This suggests that macrophage accumulation in fibrous tissue may lead to elevated IL-6 levels that are much higher than those caused by particle activation of macrophages alone. This macrophage-fibroblast interaction represents a novel concept for the initiation and maintenance of the inflammatory process in periprosthetic membranes

The role of inflammatory cells in aseptic loosening and failure of cemented joint replacements is unclear. Inflammatory cells from the revision joint capsule of four failed hip arthroplasties were examined to determine their nature and resorptive capacity. The capsules contained numerous macrophages and abundant foreign-body macrophage polykaryons, distinguished from osteoclasts by their antigenic phenotype and lack of response to calcitonin. When cultured on cortical bone slices in vitro, both macrophages and macrophage polykaryons produced small resorption pits and were associated with areas of superficial resorption of the bone surface. These results indicate that foreign-body induced macrophages and macrophage polykaryons are capable of a type of low-grade bone resorption which may be of pathogenic significance in the loosening of cemented joint prosthetic components

Aseptic loosening is a major cause of failure of total hip arthroplasty. The adverse tissue response to prosthetic wear particles, with activation of cytokine and prostanoid production, contributes to bone loss around the implants. We have investigated the possibility that inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) are expressed in macrophages in the pseudomembrane at the bone-implant interface, thereby contributing to the periprosthetic bone resorption. We also assessed whether peroxynitrite, a nitric oxide (NO)-derived oxidant associated with cellular injury, is generated in the membrane. Enzymatic activity of iNOS was measured using the arginine-citrulline assay technique and prostaglandin E. 2. (PGE. 2. ), as an indicator of COX-2 activity, was measured using an enzyme immunoassay. Cellular immunoreactivity for iNOS, nitrotyrosine (a marker of peroxynitrite-induced cellular injury) and COX-2 was assessed by quantitative peroxidase immunocytochemistry while immunofluorescence methods were used for subsequent co-localisation studies with CD68. +. macrophages. The presence of calcium-independent iNOS activity and PGE. 2. production was confirmed in the homogenized interface membrane. Immunocytochemistry showed that periprosthetic CD68. +. wear-debris-laden macrophages were the most prominent cell type immunoreactive for iNOS, nitrotyrosine and COX-2. Other periprosthetic inflammatory and resident cell types were also found to immunolocalise nitrotyrosine thereby suggesting peroxynitrite-induced protein nitrosylation and cellular damage not only in NO-producing CD68. +. macrophages, but also in their neighbouring cells. These data indicate that both iNOS and COX-2 are expressed by CD68. +. macrophages in the interface membrane and peroxynitrite-induced cellular damage is evident in such tissue. If high-output NO and peroxynitrite generation were to cause macrophage cell death, this would result in the release of phagocytosed wear debris into the extracellular matrix. A detrimental cycle of events would then be established with further phagocytosis by newly-recruited inflammatory cells and subsequent NO, peroxynitrite and prostanoid synthesis. Since both NO and have been implicated in the induction and PGE. 2. maintenance of chronic inflammation with resulting loss of bone, and peroxynitrite in the pathogenesis of disease states, they may be central to the pathogenesis of aseptic loosening

We investigated the possibility that the macrophages which are seen around implants may stimulate bone resorption and cause loosening. We found that macrophages release mediators that stimulate bone resorption, and that the amount of resorption increased by between 2.5 and 10 times when the macrophages adhered to a foreign surface. This bone resorption depended on the surface energy and roughness of the foreign surface, varying with these physical properties rather than with the chemical nature of the material. It is concluded that loosening of orthopaedic implants is likely to be influenced by the surface energy and roughness of the implant