We revised seven alumina-blasted cementless hip prostheses (Ti-alloy stems, cp Ti threaded sockets) with low- or high-carbon Co-alloy bearings at a mean of 20.1 months after implantation because of pain and loosening. Histological examination of the retrieved periprosthetic tissues from two cases in which the implant was stable and three in which the socket was loose showed macrophages with basophilic granules containing metal and alumina wear particles and lymph-cell infiltrates. In one of the two cases of stem loosening the thickened neocapsule also contained definite lymphatic follicles and gross lymphocyte/plasma-cell infiltrates. Spectrometric determination of the concentration of elements in periprosthetic tissues from six cases was compared with that of joint capsules from five control patients undergoing primary hip surgery. In the revisions the mean concentration of implant-relevant elements was 693.85 μg/g dry tissue. In addition to Cr (15.2%), Co (4.3%), and Ti (10.3%), Al was predominant (68.1%) and all concentrations were significantly higher (p < 0.001) than those in the control tissues. The annual rates of linear wear were calculated for six implants. The mean value was 11.1 μm (heads 6.25 μm, inserts 4.82 μm). SEM/EDXA showed numerous fine scratches and deep furrows containing alumina particles in loosened sockets, and stems showed contamination with adhering or impacted alumina particles of between 2 and 50 μm in size

In an attempt to increase the life of cementless prostheses, an hydroxyapatite-coated implant which releases a bisphosphonate has been suggested as a drug-delivery system. Our in vitro study was designed to determine the maximum dose to which osteoblasts could be safely exposed. Our findings demonstrated that zoledronate did not impair the proliferation of human osteoblasts when used at concentrations below 1 μ. m. Murine cells can be exposed to concentrations as high as 10 μ. m. . A concentration of 0.01% of titanium particles did not impair the proliferation of either cell line. Zoledronate affected the alkaline phosphatase activity of murine osteoblasts through a chelation phenomenon. The presence of titanium particles strongly decreased the alkaline phosphatase activity of murine osteoblasts. We did not detect any synergic effect of zoledronate and titanium particles on the behaviour of both human and murine osteoblasts

Nanometre-sized particles of ultra-high molecular weight polyethylene have been identified in the lubricants retrieved from hip simulators. Tissue samples were taken from seven failed Charnley total hip replacements, digested using strong alkali and analysed using high-resolution field emission gun-scanning electron microscopy to determine whether nanometre-sized particles of polyethylene debris were generated in vivo. A randomised method of analysis was used to quantify and characterise all the polyethylene particles isolated. We isolated nanometre-sized particles from the retrieved tissue samples. The smallest identified was 30 nm and the majority were in the 0.1 μm to 0.99 μm size range. Particles in the 1.0 μm to 9.99 μm size range represented the highest proportion of the wear volume of the tissue samples, with 35% to 98% of the total wear volume comprised of particles of this size. The number of nanometre-sized particles isolated from the tissues accounted for only a small proportion of the total wear volume. Further work is required to assess the biological response to nanometre-sized polyethylene particles

Summary Statement. Antioxidant containing UHMWPE particles induced similar levels of in vitro macrophage proliferation and in vivo inflammation in the mouse air pouch model as UHMWPE particles alone. Benefit of antioxidant in reducing wear particle induced inflammation requires further investigation. Introduction. Wear particles derived from UHMWPE implants can provoke inflammatory reaction and cause osteolysis in the bone, leading to aseptic implant loosening. Antioxidants have been incorporated into UHMWPE implants to improve their long term oxidative stability. However it is unclear if the anti-inflammatory property of the antioxidant could reduce UHMWPE particle induced inflammation. This study evaluated the effect of cyanidin and vitamin E on UHMWPE induced macrophage activation and mouse air pouch inflammation. Methods. Four types of UHMWPE were used: (1) compression molded (CM) conventional GUR1020 (PE); (2) CM GUR1020 blended with 300 ppm cyanidin (C-PE); (3) CM GUR1020 blended with 1000 ppm α-tocopherol (BE-PE); and (4) CM GUR1020, gamma irradiated at 100kGy, diffused with α-tocopherol, and sterilised at 30kGy (DE-PE). Particles were generated by cryomilling. Particle count, size, and aspect ratio were determined using SEM and Image Pro. Each particle group was cultured with RAW264.7 macrophage cells at four concentrations (0.625, 1.25, 2.5, and 5 μg/mL) in a standard medium for 4 days. Cell numbers were quantified using MTT assay. Cytokine expression (IL-1β, TNFα, and IL-6) was measured using RT-PCR and ELISA. Particles were also suspended in PBS at 2 concentrations (0.2 or 1 mg) and injected into subcutaneous air pouches in BALB/c mice. Control animals were injected with PBS alone. Six days post-injection air pouches were harvested, half of which were fixed for histology to measure membrane thickness and inflammatory cell quantity. Remaining air pouches were frozen and analyzed by ELISA for cytokine production. Data were analyzed using one-way ANOVA with post hoc testing. P<0.05 was considered significant. Results. All 4 materials showed similar particle characteristics after cryomilling. Particle size ranged from 1 to 19 μm with 33% of particle population smaller than 2 μm. All particle groups supported macrophage proliferation, showing an inverse correlation between proliferation rate and particle dose. Gene expression of IL-1β and TNFα also showed an inverse correlation with particle dose. Expression of IL-1β, TNFα, and IL-6 appeared lower in cells cultured with C-PE than the other 3 materials. The accumulative protein productions of IL-1β and TNFα were significantly lower while IL-6 production was moderately lower in C-PE, BE-PE and DE-PE when compared to PE. Injection of polyethylene particles increased the air pouch membrane thickness significantly compared to the PBS control in all particle types and doses. Higher particle dose induced thicker membrane in all 4 materials. A similar trend was also observed in the percentage of inflammatory cell infiltration in the pouch membrane. C-PE and DE-PE particles at low dose and C-PE particles at high dose induced lower levels of IL-1β and TNFα than PE. IL-6 production was similar between PE and other 3 groups. Discussion/Conclusion. Antioxidant incorporated in UHMWPE did not alter the level of macrophage proliferation and air pouch inflammation induced by UHMWPE particles, although it reduced cytokine gene expression. Future investigation in a synovial joint environment is desired to evaluate the chronic inflammation response to antioxidant containing UHMWPE wear particles and to verify the effect of antioxidant in UHMWPE properties

Demineralized bone matrix (DBM) is a natural, collagen-based, well-established osteoinductive biomaterial. Nevertheless, there are conflicting reports on the efficacy of this product. The purpose of this study was to evaluate whether DBM collagen structure is affected by particle size and can influence DBM osteoinductivity. Sheep cortical bone was ground and particles were divided in three fractions with different sizes, defined as large (L, 1–2 mm), medium (M, 0.5–1 mm), and small (S, < 0.5 mm). After demineralization, the three DBM samples were characterized by DTA analysis, XRD, ICP-OES, and FTIR. Data clearly showed a particle size-dependent alteration in collagen structure, with DBM-M being altered but not as much as DBM-S. The in vivo study showed that only DBM-M was able to induce new bone formation in a subcutaneous ectopic mouse model. When sheep MSC were seeded onto DBM particles before implantation, all DBM particles were able to induce new bone formation with the best incidence for DBM-M and DBM-S. Gene expression analysis performed on recovered implants supports the histological results and underlines the supportive role of MSC in DBM osteoinduction through the regulation of host cells. In conclusion, our results show a relation between DBM particle size, structural modification of the collagen and in vivo osteoinductivity. The medium particles represent a good compromise between no modification (largest particles) and excessive modification (smallest particles) of collagen structure, yielding highest osteoinduction. We believe that these results can guide researchers to use DBM particles of 0.5–1 mm size range in applications aimed at inducing new bone formation, obtaining results more comparable and reliable among different research groups. Furthermore, we suggest to carefully analyze the structure of the collagen when a collagen-based biomaterial is used alone or in association with cells to induce new bone formation

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic bio-materials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells

The biological significance of cobalt-chromium wear particles from metal-on-metal hip replacements may be different to the effects of the constituent metal ions in solution. Bacteria may be able to discriminate between particulate and ionic forms of these metals because of a transmembrane nickel/cobalt-permease. It is not known whether wear particles are bacteriocidal. We compared the doubling time of coagulase negative staphylococcus, Staphylococcus aureus and methicillin resistant S. aureus when cultured in either wear particles from a metal-on-metal hip simulator, wear particles from a metal-on-polyethylene hip simulator, metal ions in solution or a control. Doubling time halved in metal-on-metal (p = 0.003) and metal-on-polyethylene (p = 0.131) particulate debris compared with the control. Bacterial nickel/cobalt-transporters allow metal ions but not wear particles to cross bacterial membranes. This may be useful for testing the biological characteristics of different wear debris. This experiment also shows that metal-on-metal hip wear debris is not bacteriocidal

We implanted bone harvest chambers (BHCs) bilaterally in ten mature male New Zealand white rabbits. Polyethylene particles (0.3 ± 0.1 −m in diameter, 6.4×10. 12. particles/ml) were implanted for two, four or six weeks bilaterally in the BHCs, with subsequent removal of the ingrown tissue after each treatment. In addition to the particles, one side also received 1.5 −g of recombinant transforming growth factor ß1 (TGFβ1). At two weeks, the bone area as a percentage of total area was less in chambers containing TGFβ compared with those with particles alone (7.8 ± 1.3% v 16.9 ± 2.7% respectively; 95% confidence interval (CI) for difference -14.0 to -4.30; p = 0.002). At four weeks, the percentage area of bone was greater in chambers containing TGFβ compared with those with particles alone (31.2 ± 3.4% v 22.5 ± 2.0% respectively; 95% CI for difference 1.0 to 16.4; p = 0.03). There were no statistical differences at six weeks, despite a higher mean value with TGFβ treatment (38.2 ± 3.9% v 28.8 ± 3.5%; 95% CI for difference -4.6 to 23.3; p = 0.16). The number of vitronectin-receptor-positive cells (osteoclast-like cells) was greater in the treatment group with TGFβ compared with that with particles alone; most of these positive cells were located in the interstitium, rather than adjacent to bone. TGFβ1 is a pleotropic growth factor which can modulate cellular events in the musculoskeletal system in a time- and concentration-dependent manner. Our data suggest that there is an early window at between two and six weeks, in which TGFβ may favourably affect bone ingrowth in the BHC model. Exogenous growth factors such as TGFβ may be a useful adjunct in obtaining osseointegration and bone ingrowth, especially in revisions when there is compromised bone stock and residual particulate debris

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

We used a rat model in vivo to study the effects of the concentration of polyethylene particles on the bone-implant interface around stable implants in the proximal tibia. Intra-articular injections of 10. 4. , 10. 6. or 10. 8. high-density polyethylene (HDPE) particles per joint were given 8, 10 and 12 weeks after surgery. The animals were killed after 14 and 26 weeks and the response at the interface determined. Fibrous tissue was seen at the bone-implant interface when the head of the implant was flush with the top of the tibia but not when it was sunk below the tibial plateau. In the latter case the implant was completely surrounded by a shell of bone. The area of fibrous tissue and that of the gap between the implant and bone was related to the concentration of particles in the 14-week group (p < 0.05). Foreign-body granulomas containing HDPE particles were seen at the bone-implant interface in animals given 10. 8. particles. The pathology resembles that seen around prostheses with aseptic loosening and we suggest that this is a useful model by which to study this process

Summary Statement. Using the latest Next Generation Sequencing technologies, we have investigated miRNA expression profiles in human trabecular bone from total hip replacement (THR) revision surgery where wear particle associated osteolysis was evident. Introduction. A major problem in orthopaedic surgery is aseptic loosening of prosthetic implants caused by wear particle associated osteolysis. Wear debris is known to impact on a variety of cellular responses and genes in multiple pathways associated with the development of the periprosthetic osteolysis. MicroRNAs (miRNAs) act as negative regulators of gene expression and the importance of miRNAs in joint pathologies has only recently been addressed. However, miRNA profiles in osteolytic bone are largely unknown. Using the latest Next Generation Sequencing technologies, we have investigated miRNA expression profiles in human trabecular bone sourced from bone discarded during total hip replacement (THR) revision surgery where wear particle associated osteolysis was evident. Patients and Methods. Three groups of gender and age-matched patients (n=9 per group) were recruited for this study including patients undergoing revision surgery, primary THR patients and healthy subjects. Total RNAs were prepared from trabecular bone specimens. The cDNA libraries were constructed using a TruSeq Small RNA Sample Preparation kit, and then sequenced on an Illumina HiSeq2000 sequencer. All good quality tags were aligned against the reference sequences containing human chromosomal sequences and 18s and 28s rRNA sequences were analysed using Bowtie software. We used miRBase v19 to identify the start positions of all mature miRNA and the edgeR package to analyse differential expression. Osteogenesis pathway-related gene expression was also investigated using RT-qPCR Array assay. Results. We observed a significant difference in expressed miRNAs between revision and primary THR groups, including upexpressed miR127, miR-409, miR-211 and miR-146a. Importantly, the miR-127 (3.1 fold, p=0.005) and miR-146a (3.5 fold, p=0.001) were not only upexpressed in the revision group vs primary group, but also upexpressed in the revision group vs the healthy group. Thus, miR-127 and miR-146a may have potential as both biomarkers to predict osteolysis and as therapeutic targets. The miR-127 and miR-146a are critical in bone diseases because some of their target genes play an important role in osteogenesis. We have thus studied osteogenic genes and confirmed that SMAD4, RUNX2, FGFR1, TGFβ1, COL1A1 and WNT4 were downregulated. Our data also revealed that miR-93 and miR-204a were downexpressed (−3.7 fold, p=0.023; −2.5, p=0.003 respectively) and t IL-6 and IL-6R, which had been reported as miR-204 target genes, were upexpressed. Discussion and Conclusion. Our results showed that upexpressed miR-127, miR-146a, miR-204a and miR-93 in trabecular bone from revision THR may be the key negative regulators in either osteogenic genes involved in osteogenic differentiation of bone formation or inflammatory genes involved in osteoclastogenesis. Aberrant miRNA expressions identified in the revision THR group may also suggest the existence of genetic risk factors favouring the development of osteolysis in certain specific subgroups of patients. An in-depth understanding of the roles of these regulatory miRNAs in the skeleton warrants further investigation

We analysed revised Mathys isoelastic polyacetal femoral stems with stainless-steel heads and polyethylene acetabular cups from eight patients in order to differentiate various types of particle of wear debris. Loosening of isoelastic femoral stems is associated with the formation of polyacetal wear particles as well as those of polyethylene and metal. All three types of particle were isolated simultaneously by tissue digestion followed by sucrose gradient centrifugation. Polyacetal particles were either elongated, ranging from 10 to 150 μm in size, or shred-like and up to 100 μm in size. Polyethylene particles were elongated or granules, and were typically submicron or micronsized. Polyacetal and polyethylene polymer particles were differentiated by the presence of BaSO. 4. , which is added as a radiopaque agent to polyacetal but not to polyethylene. This was easily detectable by back-scattered SEM analysis and verified by energy dispersive x-ray analysis. Two types of foreign-body giant cell (FBGC) were recognised in the histological specimens. Extremely large FBGCs with irregular polygonal particles showing an uneven, spotty birefringence in polarised light were ascribed to polyacetal debris. Smaller FBGCs with slender elongated particles shining uniformly brightly in polarisation were related to polyethylene. Mononucleated histiocytes containing both types of particle were also present. Our findings offer a better understanding of the processes involved in the loosening of polyacetal stems and indicate why the idea of ‘isoelasticity’ proved to be unsuccessful in clinical practice

We have studied the beneficial effects of a hydroxyapatite (HA) coating on the prevention of the migration of wear debris along the implant-bone interface. We implanted a loaded HA-coated implant and a non-coated grit-blasted titanium alloy (Ti) implant in each distal femoral condyle of eight Labrador dogs. The test implant was surrounded by a gap communicating with the joint space and allowing access of joint fluid to the implant-bone interface. We injected polyethylene (PE) particles into the right knee three weeks after surgery and repeated this weekly for the following five weeks. The left knee received sham injections. The animals were killed eight weeks after surgery. Specimens from the implant-bone interface were examined under plain and polarised light. Only a few particles were found around HA-coated implants, but around Ti implants there was a large amount of particles. HA-coated implants had approximately 35% bone ingrowth, whereas Ti implants had virtually no bone ingrowth and were surrounded by a fibrous membrane. Our findings suggest that HA coating of implants is able to inhibit peri-implant migration of PE particles by creating a seal of tightly-bonded bone on the surface of the implant

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

Summary. Particulate wear debris with different chemical composition induced similar periprosthetic tissue reactions in patients with loosened uncemented and cemented titanium hip implants, which suggests that osteolysis can develop independent of particle composition. Introduction. Periprosthetic osteolysis is a serious long-term complication in total hip replacements (THR). Wear debris-induced inflammation is thought to be the main cause for periprosthetic bone loss and implant loosening. The aim of the present study was to compare the tissue reactions and wear debris characteristics in periprosthetic tissues from patients with failed uncemented (UC) and cemented (C) titanium alloy hip prostheses. We hypothesised that implant wear products around two different hip designs induced periprosthetic inflammation leading to osteolysis. Patients & Methods. Thirty THR-patients undergoing revision surgery were included: Fifteen patients had loose cemented titanium stems (Titan. ®. , DePuy) and 15 had well-fixed uncemented titanium stems (Profile, DePuy), but loose or worn uncemented metal-backed cups with conventional UHMWPE liners (Gemini, Tropic and Tri-Lock Plus, DePuy; Harris/Galante and Trilogy, Zimmer). A semi-quantitative histological evaluation was performed in 59 sections of periprosthetic tissues using light microscopy. Wear particles were counted by polarised light and high resolution dark-field microscopy. Additionally, particle composition was determined by SEM-EDXA following particle isolation using an enzymatic digestion method. Blood metal ions were determined with high resolution-ICP-MS. Results. The implants in the uncemented group were revised after a median of 15.7 years (range: 7.25–19.3) due to osteolysis and high wear of the polyethylene liner and metal backing resulting in gross metallosis, and/or cup loosening. The average lifetime of implants in the cemented group was only 6.5 years (range: 1.5–11.75) due to early stem loosening with large osteolysis pockets in the femur close to the cement mantle. Tissue examination revealed similar results for both groups: numerous mononuclear histiocytes and chronic inflammatory cells, a few neutrophils and multinucleated giant cells, and to some extent necrosis. The amount of metal particles per histiocyte positively correlated with the tissue reactions in the cemented, but not in the uncemented group. A higher particle load (medians: C: 14727 vs. UC: 1382 particles/mm. 2. , p<0.0001) was found in tissues adjacent to cemented stems, which contained mainly submicron ZrO. 2. particles. Particles containing pure Ti or Ti alloy elements (size range: 0.21 to 6.46 µm) were most abundant in tissues from the uncemented group. Here, also PE was more frequent, but accounted only for a small portion of total particles (2.8 PE/mm. 2. ). The blood concentrations of titanium (range: 3.8–138.5 microgram/L) and zirconium (cemented cases, range: 0.6–3.5 microgram/L) were highly elevated in cases with high abrasive wear and metallosis. Discussion/Conclusion. Phagocytosis of different wear particles by histiocytes induced a similar chronic inflammatory reaction in the periprosthetic tissues in both groups. ZrO. 2. particles, originating from bone cement degradation, dominated in the cemented group, while in the uncemented group the high abundance of pure Ti and Ti alloy particles of various sizes indicates wear of the metal-backed cups. The low density of polyethylene particles in the tissues suggests that they are not solely responsible for the tissue reactions and accompanying osteolysis. Our findings suggest that the chemical composition of wear particles plays a minor role in the mechanism of osteolysis. Particle size, load and ionic exposure might be more important

Wear particles commonly used for experiments may carry adherent endotoxin on their surfaces, which may be responsible for the observed effects. In this study, we attached titanium plates to the tibiae of 20 rats. After osseointegration, endotoxin-contaminated or uncontaminated high-density-polyethylene (HDPE) particles were applied. Contaminated specimens showed a dramatic resorption of bone after seven days but new bone filled the site again at 21 days. Uncontaminated specimens showed no resorption. In 18 rats we implanted intramuscularly discs of ultra-high-molecular-weight polyethylene (UHMWPE) with baseline or excess contamination of endotoxin. Excess endotoxin disappeared within 24 hours and the amount of endotoxin remained at baseline level (contamination from production). Uncontaminated titanium discs did not adsorb endotoxin in vivo. The endotoxin was measured by analytical chemistry. Locally-applied endotoxin stimulated bone resorption similarly to that in experiments with wear particles. Endotoxin on the surface of implants and particles appeared to be inactivated in situ. A clean implant surface did not adsorb endotoxin. Our results suggest that endotoxin adhering to orthopaedic implants is not a major cause for concern

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

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

Increased failure rates due to metallic wear particle-associated adverse local tissue reactions (ALTR) is a significant clinical problem in resurfacing and total hip arthroplasty. Histological analysis and particle characterization are important elements for understanding the biological mechanisms of the reaction and different histological subtypes may have unique needs for longitudinal clinical follow-up and complication rates after revision arthroplasty. Consecutive patients (N=285 cases) presenting with ALTR from three major hip implant classes, metal-on-metal resurfacing and total hip arthroplasty (THA) and non-metal-on-metal THA with dual modular neck were identified from our prospective Osteolysis Tissue Database and Repository and 53 cases were selected for wear particle nano-analysis. Conventional histology: Tissue samples taken from multiple regions around the hip with extensive sampling performed at macroscopic examination were examined by light microscopy. Particle analysis: Tissue samples selected after frozen section evaluation for cellularity and particle content were examined by scanning electron microscopy (SEM), backscatter scanning electron microscopy (BSEM), BSEM-energy-dispersive X-ray spectroscopy (EDS) element mapping examination, transmission electron microscopy (TEM), TEM-EDS element mapping, and X-ray diffraction spectrometry (XRD) examination. ALTR encompasses three main histological patterns: 1) macrophage predominant, 2) mixed lymphocytic and macrophagic, and 3) predominant sarcoid-like granulomas. Duration of implantation and composition of periprosthetic cellular infiltrates was significantly different among the three implant types examined. Distinct differences in the size, shape, and element composition of the metallic particulate material were detected in each implant class, with correlation of the severity of the adverse reaction with element complexity of the particles. ALTR encompasses a diverse range of histological patterns, which are reflective of both the implant configuration independent of manufacturer and clinical features such as duration of implantation. Distinct differences in the metallic particulate material can contribute to explain the histological features of the ALTR and variability of performance of the implants. ALTR exhibits different histological patterns and is dependent on the characteristics of the wear particulate material of each implant class and host immunological reaction

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