It is reported that more than 10 million Japanese suffer from arthrosis. To cure these cartilage defects, total joint replacements, which are the most popular treatment methods for severe disease situation, have been operated as about two hundred thousand cases a year in Japan. Although the implants made of either ceramics, metals or plastics have high wear resistance quality, it becomes apparent that the endurance life of the artificial joints in considerable cases is limited by aseptic loosening to between 10–15 years. Here we focused on a poly(vinyl alcohol) (PVA) hydrogel as an artificial cartilage tissue to make an improvement of friction surface of the artificial joints. In this paper, we observed morphology of wear particles and assessed immune responses of wear particles from the hydrogel for confirming the validity of the gel as a biotribological material. We prepared 20 w/w% of PVA hydrogel by repeated freezing-thawing method. The number of the freezing-thawing cycles was five times. Polymerization degree and saponification degree of PVA (Kishida Chemical Co. Ltd., Japan) were 2000 and 98.4–99.8 mol%, respectively. To collect the wear particles of PVA hydrogel, we processed wear testing by using a purpose-build wear test machine of reciprocating pin-on-plate tribometer as shown in figure 1. We installed a Co-Cr-Mo ball of 26 mm in diameter as a stationary upper specimen and a PVA hydrogel plate of 2 mm in thickness as reciprocating lower specimen in a water bath. The lubricant was a distilled water containing eluted PVA which PVA-FT gel had been soaked in, filtered by 0.22 μm and autoclaved, subsequently. Siding speed was 50 mm/s and the total sliding distance was 3 km. We observed the wear particles which had been dried in a desiccator, by scanning electron microscope (SEM; SU8000, Hitachi High-Technologies). Additionally, to investigate the effect of the wear particles on response of phagocytosis of macrophages, here we used THP-1 cell line from Human acute monocytic leukemia as a macrophage, which was purchased from JCRB Cell Bank, and attached the macrophages on a dish after stimulating THP-1 by phorbol 12-myristate 13-acetate (PMA; Wako). After the wear test, we harvested the lubricant in aseptic clean hood and applied the particles to the macrophages to clarify the effect of wear particles of PVA hydrogel on immune response of the cell. To assess cytokine biosynthesis as immune responses, we assayed IL-1β and TNF-α biosynthesis in culture medium by ELISA (Thermo scientific), respectively. Figure 2 shows an SEM image of PVA hydrogel after wear test under 4.9 N loading. We observed the wear particles of varied sizes. When applying the wear particles to the macrophages in RPMI-1640 supplement with 10 v/v% fetal bovine serum, it seemed that there were not enough change on cytokine synthesis in culture medium between with/without the particles

Background. Polyethylene (PE) as a bearing material for total joint replacements (TJR) represents the golden standard for the past forty years. However, over the past decade it becomes apparent that PE wear and the biological response to wear products are the limiting factor for the longevity of TJRs. For this reason research has focused onto PE wear particle analysis. A particle analysis highly depends on the methodological work and results often show discrepancies between different research groups. From there, our hypothesis was, that an often unattended influencing factor is the optical magnification which has been used for particle analyses. Material and Methods. In the present study samples of a previous conducted knee wear simulator test were used. Wear particles were isolated from the bovine serum using an established method. 1. Briefly the serum was digested with hydrochloric acid and a continuous stirring and heating. Particles were filtered onto 20nm alumina filters and analyzed using high resolution field emission gun scanning electron microscopy (FEG-SEM). Filters were analyzed on the same points using three different magnifications: 5000, 15000 and 30000. To describe the size and morphology of the particles the equivalent circle diameter (ECD), aspect ratio (AR), roundness (R) and form factor (FF) were specified according to ASTM F 1877-05. The estimated total number (ETN) of particles was calculated based on the number of particles recovered on the filter, the analyzed area, the dilution, evaporation and the total serum volume. Results. The results showed significant differences between the different magnifications. Examples of the analyzed pictures are depicted in Fig. 1. The results are summarized in Tab. 1. In particular the size of the particles highly depends on the choosen optical magnification which becomes apparent in a more than twofold increase when comparing wear particles at magnification of 5k or 30k (p≤0.001). The mean particle diameter distribution (Fig. 2) also shows a shift in the distribution of wear particles: A higher magnification results in a higher fraction of smallest particles (e.g. over 50 percent between 0–0.2µm with magnification 30.000 compared to only 3 percent with a magnification of 5.000) and nearly no particles above 1µm could be verified. The results regarding the particle morphology show smaller but significant differences. The ETN of particles quadruplicates when comparing results with magnifications of 5.000 and 30.000. Conclusion. This study shows great differences in particle size, which can be directly attributed to the differences in optical magnification. According to ASTM F1877-05 a magnification of 10.000 for the analysis of wear particles between 0.1–1µm is recommended. However, results show that this magnification is not sufficient especially for particle sizes below 0.2µm, which account for the greatest number of particles. To the authors opinion a more detailed recommendation concerning the magnification is needed. Additional, a standardized evaluation system which takes the magnification into account is necessary to allow comparison of different research groups

Introduction. The complex process of inflammation and osteolysis due to wear particles still is not understood in detail. So far, Ultra-high-molecular-weight-polyethylene (UHMWPE) is the bearing material of choice in knee arthroplasty and revision knee arthroplasty, but there is a growing demand for alternative bearing materials with improved wear properties. Lately, increasing interest developed in the use of natural and carbon-fiber-reinforced-poly-ether-ether-ketones (CFR-PEEK). While there is a lack of data concerning the effects of CFR-PEEK particles on human tissue, the effects of such wear debris in vitro and in animal studies is controversially discussed. The aim of this study was to analyze human tissue containing CFR-PEEK as well as UHMWPE wear debris. The authors hypothesized no difference between the used biomaterials because of similar size parameters of the wear particles in a prior knee simulator study of this implant. Methods and Materials. Synovial tissue samples of 10 patients while knee revision surgery of a rotating hinge knee implant design (Enduro®, Aesculap, Germany) were achieved. The tibial inserts of this design were made from UHMWPE (GUR 1020), whereas the bushings and flanges are made of CFR-PEEK containing 30% polyacrylonitrile (PAN) based carbon fibers (PEEK-Optima LT1, Invibio Ltd. Thornton-Cleveleys, UK). In a prior in vitro test most of the released CFR-PEEK particles were in a size range between 0.1 and 2μm. The implant survival until revision surgery was 22 (2.5–48 min.-max.) months. As a control synovial tissue out of a patient also got knee revision surgery without any PEEK components. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sliced into 2 µm thick sections. stained with hematoxylin and eosin in a standard process. A modified panoptical staining (preincubation in propylenglycol; >3h; 35°C) was also done which stained the UHMWPE particles turquoise. The study was approved by the ethics committee of the local university. Results. Overall, histologically a “wear-type” reaction was seen in the testing and the control group similar as described for other materials in the common literature. In all samples of the testing group the UHMWPE particles were scattered in the tissue similar to the control. Larger UHWMPE particles were incorporated in giant cells. In contrast to these findings, CFR-PEEK particles were not scattered in tissue but located only as conglomerates. In addition, these conglomerates have been found exclusively near to or in vessels. Furthermore, CFR-PEEK particles were collected in macrophages, no CFR-PEEK particles were seen in giant cells. In conclusion, the hypothesis has to be rejected. Interestingely, different behaviour of UHMWPE and PEEK particles has been found in human synovial tissue. This aspect needs further investigation concerning the cytokine expression and also the surface texture of particles. Acknowledgement. This study was supported by Aesculap, Germany

INTRODUCTION. Loosening is concerned to be the major cause of revision in the artificial prosthesis. Wear debris of UHMWPE dispersed into the implant-bone interface are phagocytosed by macrophages releasing inflammatory cytokines such as TNF-α which leads to osteolysis and loosening eventually. It is known that the size and structure [1] as well as attached substances on particle surface such as endotoxin could affect the amount of cytokines released [2]. An in vivo study using rat femurs showed that the presence of polyethylene particles around implants could result in accumulation of lipopolysaccharide (LPS) from exogenous sources that may affect bone remodeling around implants [3]. It is also reported that LPS is transported throughout the body with lipoproteins or LPS binding proteins [4] and Circulating LPS may originate from local sites of infection or via bloodborne bacteria [5]. In this study, we evaluated the effects of LPS that attached to UHMWPE particle surface by measuring TNF-α released from macrophages. MATERIALS AND METHODS. We cultured mouse macrophage cell line RAW 264 with spherical UHMWPE particles (8.7µm and 23µm diameter in average, Mitsui chemicals Co., LTD.) and LDPE particles (3.6µm and 5.8µm diameter in average, Sumitomo Seika Chemicals Co., LTD.) using the Inverse Culture Method for 24 hours before estimating the TNF-α generation by TNF- ALPHA QUANTIKINE ELISA KIT (R&D). Spherical UHMWPE particles (10µm diameter in average, Mitsui chemicals Co., LTD.) with E.coli original LPS (Enzo Life Sciences) attached to them were incubated with cells to see the effects of LPS on the bio-reactivity tests. REAULTS AND DISCUSSIONS. Figure 1 shows the TNF-α concentration of different materials and sizes of polyethylene particles. TNF-α concentration was shown to be dose-dependent to the total surface area of particles added regardless of the materials and sizes. Figure 2 shows TNF-α concentration relative to the particle surface area inverse and non-inverse cultured. No significant difference was observed in TNF-α concentration between particles that were attached to LPS and virgin particles in non-inverse culture method. However, when cultured inversely, the effect of LPS became more significant in higher surface area range in which dose-dependent relationship was not observed. The results suggest that saturation may occur caused by size exclusion, production limitation, etc. However, LPS attached to particle surface may alter the production limitation due to increased presence of particles around macrophages. For any figures or tables, please contact the authors directly

Today's aging society is seeing an increase of patients with rheumatoid arthritis and osteoarthritis, as well as an increase in joint replacement surgery. The artificial joints used in this surgery frequently uses ultra-high molecular weight polyethylene (UHMWPE) as a bearing material. However, UHMWPE wear particles are considered to be a major factor in long-term osteolysis, and implant loosening. Many researchers have reported that the volume and size of particles are critical factors in macrophage activation, with particles in the size range of 0.1 – 1.0 μm being the most biologically active. The micro slurry-jet erosion (MSE) apparatus was introduced to minimize the amount of wear, and increase the size of UHMWPE wear particles by texturing the surfaces of Co-Cr-Mo alloy implants. The MSE apparatus uses a slurry of alumina particles (WA#8000: average diameter 1.2 μm) mixed with water. The slurry and compressed air are mixed within an injection nozzle, which is then applied to the Co-Cr-Mo alloy at high speed to achieve a desired nano-textured surface. In this study, four Co-Cr-Mo alloy surface profiles were prepared. The MSE injection nozzle was fed 40.0 mm in alternating directions across each surface with an orthogonal step of 0.5 mm. The surface M-1 was processed with an injection nozzle feed rate of 1.0 mm/s, and obtained a surface roughness of 5.7 nm. M-2 was processed with a feed rate of 2.0 mm/s, and had a surface roughness of 2.3 nm. The M-4 surface used a 40.0 mm alternating directions surface feed, but with a 1.0 mm orthogonal step, and an injection nozzle feed rate of 0.5 mm/s. It obtained a surface roughness of 4.0 nm. The G-1 surface, with a roughness of 10.0 nm, was processed with the typical lapping method, which is used in conventional artificial joints [Fig. 1]. A pin-on-disk wear tester, capable of multidirectional motion, was used to assess which surface was the most appropriate for artificial joints. The UHMWPE pins were flat ended cylinders, 12.0 mm in diameter, and were placed on the disk with a contact pressure of 6.0 MPa. Tests were carried out in 25% (v/v) fetal calf serum with sodium azide to retard bacterial growth. A sliding speed of 12.1 mm/s, and a total sliding distance of 15.0 km were applied. The wear weight of the MSE textured surface M-1 was significantly lower than the wear weight of the conventional surface. Moreover, the percentages of various wear particle sizes obtained from MSE surface texturing was significantly different from those obtained from the traditional surface

Endoprosthetic reconstruction for pathologic acetabular fractures is associated with a high risk of periprosthetic joint infection. In this setting, bone defect reconstruction utilising co-delivery of a synthetic bone substitute with an antibiotic, is an attractive treatment option from both, therapeutic and prophylactic perspective. We wished to address some concerns that remain regarding the possible presence of potentially wear inducing particles in the periprosthetic joint space subsequent to this procedure. We analysed a drain fluid sample from an endoprosthetic reconstruction of a pathologic acetabular fracture with implantation of a gentamicin eluting, biphasic bone graft substitute, consisting of 40% hydroxyapatite (HA) and 60% calcium sulphate (CERAMENT G), into the residual peri-acetabular bone defect. This sample was divided into two 1.5ml subsamples, to one of which 100mg HA particles were added as control before burning off all organic substance at very high temperature. These heat treated samples were then examined with scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDAX) and compared to a reference sample consisting of HA particles only. On SEM, hydroxyapatite particles were readily recognisable in the control and reference samples, whereas only very few particles over 2μm were apparent in the ”pure” drain sample. EDAX revealed that very large amounts of salts were present in both drainage samples. The pure drainage sample however, contained markedly lower amounts of calcium and phosphate compared to reference and control samples. No HA particles as such, were seen in the pure sample, however their presence cannot be excluded with absolute certainty, as some particles might have been hidden within the large salt conglomerates. We could not find clear evidence that the drain fluid really contained HA particles. More thorough investigations are needed and future analyses with prior removal of the high salt content would likely yield more conclusive results

Introduction:. The earliest evidence of particle-induced response is found in the synovium, leading to osteolytic defect. The degree of synovitis can be quantified by magnetic resonance imaging (MRI). This is the first long-term, prospective, matched-pair study using MRI to analyze wear-induced synovitis and osteolysis between rotating-platform posterior-stabilized (RP-PS), fixed-bearing metal-back (FB-MB), and all-polyethylene tibial (APT) designs in active patients with identical femoral components and polyethylene. Methods:. From September 1999 to October 2001, a matched-pair analysis of 24 TKAs (18 patients, 3 groups: 8 RP-PS, 8 FB-MB, and 8 APT) was performed. TKAs were matched for age, sex, body mass index (BMI), and University of California Los Angeles (UCLA) activity scores. All patients underwent MRI using MAVRIC (multi-acquisition variable-resonance image combination) knee protocol designed to reduce metal susceptibility artifact. Images were evaluated for volumetric measure of synovitis and/or osteolysis and presence of fibrous membrane formation at the cement-bone interface. Results:. The mean age was 64 ± 5 years (59–72). The mean follow-up was 11.6 ± 0.7 years (10–13). The mean UCLA score at the time of surgery was 8.5 ± 2.6 (5–10). Reactive synovitis was observed in 6 RP-PS (75%), all 8 FB-MB (100%), and 6 APT (75%) knees. There was a significant difference between the volumetric synovitis in RP-PS (4046 mm3 ± 4502 mm3), and FB-MB knees (24498 mm3 ± 22248 mm3), p < 0.001. Osteolysis with bone loss more than 4 mm was seen in 3 FB-MB, 2 APT, but in none of the RP-PS knees. There was no statistical difference for osteolysis between the three designs. Conclusion:. Based of this study, it appears that particle induced synovitis is evident in all 3 types of bearing surfaces, however, it is significantly less in the RP-PS group. This is in contradiction to the report of retrieval studies

Aim. Biomaterial-associated infections (BAI) present a formidable clinical challenge. Bioactive glasses (BG) have proven highly successful in diverse clinical applications, especially in dentistry and orthopaedics. In this study, we aimed to determine the effect of three commonly used BG composition and particle sizes on cell and bacterial attachment and growth. Our focus is on understanding the changes in pH and osmotic pressure in the surrounding environment during glass degradation. Method. First, three different melt-derived glasses were characterized by analyzing particle size and glass network structure using Raman and NMR. The different glasses were then tested in vitro by seeding 4x 10. 4. cells/well (SaOS Cell line) in a 48 well plate. After a pre-incubation period of 72 hours, the different BGs and particle sizes were added to the cells and the pH value, ion release and live/dead staining was measured every hour. The effect of BG against bacteria (S. epidermidis) was analyzed after 24 and 72 hours of treatment by using XTT viability assay and CFU counting by plating out the treated aliquot agar to estimate the viable bacteria cells. Results. All three BG compositions tested showed a significant increase in pH, which was highest in BG composition 45S5 with a value of 11 compared to the other BG compositions 10 and 9 in S53P4 and 13-93 respectively. This strong increase in the pH in all BG samples tested results in a strongly reduced cell viability rate of more than 75% compared to the untreated control and 6-fold reduction in bacterial viability compared to the untreated control. The live/ dead assay also showed an increased cell viability with increasing glass particle size (i. e smallest glass particle < 25% viable cell and largest glass particle> 65% viable cell). The ion release concentration over 50 h showed an increase in sodium ions to 0.25 mol/L, calcium to 0.003 mol/L and a decrease in phosphorus. Conclusions. These results show that the composition of the bioactive glass and the choice of particle size have a major influence on subsequent applications. In addition to the different compositions of the BG, particle size and additional medium change also influence the pH and ion release, and therefore also on cells or bacteria viability. The sizes of the bioactive glass particle are inversely proportional to it. Further tests are necessary to develop custom design BG compositions, which simultaneously stimulate osteoblasts proliferation and prevent microbial adhesion

Introduction. Orthopedic implants are subject to wear and release ultra-high molecular weight polyethylene (UHMWPE) debris. Analysis of UHMWPE wear particles is critical in determining the safety and effectiveness of novel orthopedic implants. Complete digestion of periprosthetic tissue and wear fluid is necessary to ensure accurate morphological and quantitative particle analysis. Acid digestion methods are more effective than enzymatic and base digestion approaches [Baxter+ 2009]. However, optimal digestion times, quantity, and type of acid are unclear for particle isolation. In addition, imaging and analysis techniques are critical to ensure accurate reporting of particle characteristics. Here, we 1) compared the efficacy of three acid-based digestion methods in isolating particles from a) bovine serum and b) animal/human tissue, and 2) analyzed the effects of imaging location on particle quantity/morphology results. Methods. 1a) UHMWPE (GUR 150) particles were generated by Mode I knee wear testing for 1 million cycles in bovine serum. Serum was digested in one of four solutions: 12.2M HCl, 15.8M HNO. 3. , a 1:1 volume ratio of HNO. 3. :HCl (aqua regia), or filtered H. 2. O (control). The serum:solution volume ratio was 1:5 [Niedzwiecki+ 2001, ISO 17853:2011]. Digestion occurred for 60min on a stir plate at 60°C. Each digest was combined with MeOH at a 1:5 digest:MeOH volume ratio and filtered using a 100 nm polycarbonate membrane. The particle-containing membranes were imaged (12 images/membrane) using scanning electron microscopy (SEM) to determine particle characteristics, including quantity, equivalent circular diameter (ECD) and aspect ratio (AR). 1b) Based on 1a, HNO. 3. was used to digest porcine and human tissue at concentrations of 1:40, 1:60, or 1:80 tissue:HNO. 3. volume ratios for either 1, 12, or 24 hours, followed by SEM analysis. 2) Particle characteristics were compared at nine locations (20 images/location) across a particle-containing membrane to determine the effects of imaging location. Results. 1a) HNO. 3. and aqua regia methods successfully digested the bovine serum, whereas the HCl and H. 2. O methods were unsuccessful (Fig.1A). Comparing HNO. 3. and aqua regia groups, particle characteristics and ECD frequency distribution were nearly identical (Fig.1B). 1b) Nitric acid did not fully digest porcine or human tissues. 2) Similar particle characteristics were observed in all nine locations analyzed across the polycarbonate membrane. The particle quantity, ECD, and AR for a representative center vs. intermediate location were 808 vs. 780 particles, 0.33±0.28 vs. 0.35±0.29 µm, and 1.57±0.56 vs. 1.51±0.4, respectively (Fig.2). Conclusions. Nitric acid and aqua regia are capable of digesting bovine serum using low quantities of acid for short duration, allowing precise analysis of UHMWPE particle debris from orthopedic implants. However, further optimization of digestion techniques for animal/human tissue is warranted. In addition, an accurate representation of particle distribution can be achieved without analyzing hundreds of images, because membrane location does not strongly influence particle results. Finally, ASTM F1877-16 – Standard Practice for Characterization of Particles – could benefit from adding software-based automated particle characterization as an optional method. An automated approach that uses k-means clustering image segmentation to identify particles and computer vision tools to extract relevant morphological features is under development and validation

Complex acetabular reconstruction for oncology and bone loss are challenging for surgeons due to their often hostile biological and mechanical environments. Titrating concentrations of silver ions on implants and alternative modes of delivery allow surgeons to exploit anti-infective properties without compromising bone on growth and thus providing a long-term stable fixation. We present a case series of 12 custom acetabular tri-flange and custom hemipelvis reconstructions (Ossis, Christchurch, New Zealand), with an ultrathin plasma coating of silver particles embedded between layers of siloxane (BioGate HyProtect™, Nuremberg, Germany). At the time of reporting no implant has been revised and no patient has required a hospital admission or debridement for a deep surgical site infection. Routine follow up x-rays were reviewed and found 2 cases with loosening, both at their respective anterior fixation. Radiographs of both cases show remodelling at the ilium indicative of stable fixation posteriorly. Both patients remain asymptomatic. 3 patients were readmitted for dislocations, 1 of whom had 5 dislocations within 3 weeks post-operatively and was immobilised in an abduction brace to address a lack of muscle tone and has not had a revision of their components. Utilising navigation with meticulous implant design and construction; augmented with an ultrathin plasma coating of silver particles embedded between layers of siloxane with controlled and long-term generation of silver ion diffusion has led to outstanding outcomes in this series of 12 custom acetabular and hemipelvis reconstructions. No patients were revised for infection and no patients show signs of failure of bone on growth and incorporation. Hip instability remains a problem in these challenging mechanical environments and we continue to reassess our approach to this multifaceted problem

Introduction. In specific conditions, infection may lead to bone loss and is difficult to treat. 1. Current clinical approaches rely on the introduction of antibiotics. While these may be effective, there are concerns regarding the rise of antimicrobial resistance. There is therefore interest in the development of antimicrobial bone graft substitutes for dental and trauma surgery. Aim & Objectives. The incorporation of zinc into biomaterials has been shown to confer broad spectrum antimicrobial activity, but this has not yet been applied to the development of a commercial bone graft substitute. The aim of this research was therefore to prepare and characterise a series of zinc-substituted nanoscale hydroxyapatite (nHA) materials, including evaluation of antimicrobial activity. Method. Zinc (Zn) substituted nHA materials were prepared (0, 5, 10, 15 & 20 mol.% Zn) using a wet chemical precipitation method with a rapid mixing. (2). The reaction was carried out using zinc hydroxide at pH 10. The suspension formed was washed and dried into both powder & paste forms. The resultant powders were characterized using transmission electron microscopy (TEM) and X-ray diffraction (XRD). The antimicrobial activity was evaluated against Staphylococcus aureus (S8650 strain - isolated from an osteomyelitis case), by two techniques. The Miles and Misra method was applied to determine the number of colony-forming units (CFUs) in bacterial suspensions incubated with pastes. Secondly, a biofilm initialization method was used to evaluate the capacity of the materials to prevent biofilm formation. One-way analysis of variance (ANOVA) was used for the statistical analysis and results with p-value < 0.05 were considered statistically significant. Results. XRD indicated the formation of pure hydroxyapatite with up to 10 mol.% Zn without any side products. However, when Zn was increased to 15 & 20 mol %, zinc oxide (ZnO) peaks were detected. The TEM showed nanoscale needle-like particles when Zn was increased compared to nHA particles. Regarding the antibacterial activity, ZnHA pastes at all concentrations caused a significant reduction in bacterial CFUs in a dose-dependent manner (50, 100 & 200 mg). Additionally, even the lowest zinc substitution (5 mol.%) significantly reduced biofilm formation. Conclusion. The results demonstrated a novel method to produce a Zn-substituted nHA that showed antimicrobial activity against a pathogen isolated from a bone infection

Currently, different techniques to evaluate the biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal/ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. Moreover, cell culture systems evaluate the biological responses of cells to a biomaterial as the combined effect of both particles and ions. We have developed a novel cell culture system suitable for testing the all three type of particles and ions, separately. The method was tested by evaluating the biological responses of human peripheral blood mononuclear cells (PBMNCs) to UHMWPE, cobalt-chromium alloy (CoCr), and Ti64 alloy wear particles. Methods. Clinically relevant sterile UHMWPE, CoCr, and Ti64 wear particles were generated in a pin-on-plate wear simulator. Whole peripheral blood was collected from healthy human donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell, UK) and seeded into the wells of 96-well and 384-well cell culture plates. The plates were then incubated for 24 h in 5% (v/v) CO. 2. at 37°C to allow the attachment of mononuclear phagocytes. Adherent phagocytes were incubated with UHMWPE and CoCr wear debris at volumetric concentrations of 0.5 to 100 µm. 3. particles per cell for 24 h in 5% (v/v) CO. 2. at 37°C. During the incubation of cells with particles, for each assay, two identical plates were set up in two configurations (one upright and one inverted). After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer, UK). Intracellular oxidative stress was measured using the DCFDA-based reactive oxygen species detection assay (Abcam, UK). TNF-α cytokine was measured using sandwich ELISA. DNA damage was measured by alkaline comet assay. The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. Results and Discussion. Cellular uptake of UHMWPE, CoCr and Ti64 particles was confirmed by optical microscopy. PBMNCs incubated with UHMWPE particles did not show any adverse responses except the release of significant levels of TNF-α cytokine at 100 µm. 3. particles per cell, when in contact with particles. PBMNCs incubated with CoCr wear particles showed adverse responses at high particle doses (100 µm. 3. particles per cell) for all the assays. Moreover, cytotoxicity was observed to be a combined effect of both particles and ions, whereas oxidative stress and DNA damage were mostly caused by ions. Ti64 wear particles did not show any adverse responses except cytotoxicity at high particle doses (100 µm. 3. particles per cell). Moreover, this cytotoxicity was mostly found to be a particle effect. In conclusion, the novel cell culture system is suitable for evaluating the biological impact of orthopaedic wear particles and ions, separately

Introduction. Aseptic loosening is a major cause of revision of total joint arthroplasty (TJA). Although crosslinked Ultra-high molecular weight polyethylene (UHMWPE) have improved wear resistance, residual radicals remaining in the material have a possibility to increase bio-reactivity of particles [2]. In this study, we attempt to evaluate the effects of irradiation and residual radicals on bio-reactivity of the material with a new method called the inverse culture method [1]. Material and methods. UHMWPE particles (10µm diameter in average, Mitsui chemicals Co., LTD) along with irradiated particles (RAD, 300kGy electron irradiation) and particles annealed after the irradiation (RAD+ANN, 100°C 72 hours) are co-incubated with mouse macrophage cell line RAW264 using the inverse culture method. The amount of TNF-α was measured with ELISA. Results and discussion. The amount of TNF-α released by macrophages reacting with virgin UHMWPE, RAD and RAD+ANN is shown in Figure 1. The horizontal axis represents the total surface area of the particles. The coefficient of determination and inclination of the approximate curve are calculated to analyze the result. The coefficient of determination suggested that cytokines released from macrophage is dose-dependent to the surface area of polyethylene particles, which was consistent with the result of our former study[1]. We use the inclination of the approximation curve in Figure 1 as an index to evaluate the bio-reactivity. The values of the index of virgin, RAD and RAD+ANN were 21×10. -4. gLm. -2. , 100×10. -4. gLm. -2. and 59×10. -4. gLm. -2. The inclination of the approximation line of RAD is significantly larger than that of virgin (test for the difference of regression line angle). These suggest that the irradiation to UHMWPE particles increases their bio-reactivity possibly due to radicals. The increased reactivity cannot be eliminated by annealing (100°C 72 hours) completely. Conclusion. Although electron irradiation increases the bio-reactivity of UHMWPE particles, annealing after the irradiation can decrease it, but cannot restore to original reactivity. For any figures or tables, please contact authors directly

Polyethylene wear-debris induced inflammatory osteolysis is known as the main cause of aseptic loosening and long term revision total hip arthroplasty. Although recent reports suggest that antioxidant impregnated ultra-high molecular weight polyethylene (UHMWPE) wear-debris have reduce the osteolytic potential in vivo when compared to virgin UHMWPE, little is known about if and/or how PE rate of oxidation affects osteolysis in vivo. We hypothesized that oxidized UHMWPE particles would cause more inflammatory osteolysis in a murine calvarial bone model when compared to virgin UHMWPE. Male C57BL/6 eight weeks old received equal amount of particulate debris overlaying the calvarium of (n=12/group): sham treatment (no particles), 2mg (6,75×107 particles/mg) of endotoxin-free UHMWPE particles (PE) or of endotoxin-free highly oxidized-UHMWPE (OX) particles. In vivo osteolysis was assessed using high resolution micro-CT and inflammation with L-012 probe dependent luminescence. At day 10, calvarial bone was examined using high resolution micro-CT, histomorphometric, immunohistochemistry analyses and qRT-PCR to assess OPG, RANK, RANK-L, IL-10, IL-4, IL-1b and TRAP genes expression using the protocol defined by individual TaqManTM Gene Expression Assays Protocol (Applied Biosystems). In vivo inflammation was significantly higher in the OX (1.60E+06 ± 8.28E+05 photons/s/cm2) versus PE (8.48E+05 ± 3.67E+05) group (p=0.01). Although there was a statistically significant difference between sham (−0.27% ± 2.55%) and implanted (PE: −9.7% ± 1.97%, and OX: − 8.38% ± 1.98%) groups with regards to bone resorption (p=0.02), this difference was not significant between OX and PE (p = 0.14). There was no significant difference between groups regarding PCR analyses for OPG, RANK, RANK-L, IL-10, IL-4, IL-1b and TRAP (p = 0.6, 0.7, 0.1, 0.6, 0.3, 0.4, 0.7 respectively). Bone volume density was significantly decreased in PE (13.3%±1.2%) and OX (12.2%±1.2%) groups when compared to sham (15%±0.9%) (p < 0 .05). Histomorphometric analyses showed a significantly decreased Bone Thickness/Tissue Thickness ratio in the implanted group (0.41±0.01 mm and 0.43±0.01 mm) compared to sham group (0.69± 0.01) (p < 0 .001). However, there were no significant difference between OX and PE (p = 0.2). Our findings suggest that oxidized UHMWPE particles display increased inflammatory potential. Results were not significant regarding in vivo or ex vivo osteolysis. As antioxidant-diffused UHMWPE induce less inflammation activity in vivo, the mechanism by which they cause reduced osteolysis requires further investigation

Introduction. It is well-known that wear debris generated by metal-on-metal hip replacements leads to aseptic loosening. This process starts in the local tissue where an inflammatory reaction is induced, followed by an periprosthetic osteolysis. MOM bearings generate particles as well as ions. The influence of both in human bodies is still the subject of debate. For instance hypersensitivity and high blood metal ion levels are under discussion for systemic reactions or pseudotumors around the hip replacement as a local reaction. The exact biopathologic mechanism is still unknown. The aim of this study was to investigate the impact of local injected metal ions and metal particles. Material and Methods. We used an established murine inflammation model with Balb/c mice and generated three groups. Group PBS (control group, n=10) got an injection of 50µl 0.1 vol% PBS-suspension, Group MI (Metal-ion, n=10) got an injection of 50µl metal ion suspension at a concentration of 200µg/l and Group MP (Metal-particles, n=10) got an injection of 50µl 0.1 vol% metal particle suspension each in the left knee. After incubation for 7 days the mice were euthanized and the extraction of the left knee ensued. Followed by immunhistochemical treatment with markers of inflammation that implied TNFα, IL-6, IL-1β, CD 45, CD 68, CD 3, we counted the positive cells in the synovial layer in the left knees by light microscopy, subdivided into visual fields 200× magnified. The statistical analysis was done with Kruskal-Wallis test and a post hoc Bonferroni correction. Results. The Group with metal particles showed significantly elevated inflammatory markers (TNFα, IL-6, IL-1β, CD 68, CD 45) compared to all other groups. Interestingly, CD 3 as a marker for T-lymphocytes showed no increased levels in all groups. The metal ion group showed significant elevated CD 45 expressions compared to the control group. Conclusion. The results clearly demonstrate that especially metal wear particles lead to an intensive inflammatory reaction. The tissue formations in the metal particle group show an osseous destructive behavior in previously demonstrated results, comparable to pseudotumors. But, in this study, the expression of the immunohistological markers CD 3, CD 45 and CD 68 indicate that the tissue consists of leucocytes and macrophages, whereas lymphocytes could not be detected. This might be due to an acute inflammatory reaction, whereas the adaptive immune response by T-lymphocytes seems not (yet) to be activated. Overall it must be stated that solid metal wear particles are responsible for local inflammatory reactions, whereas it is still unknown whether wear particles corrode in vivo and release a potentially high level of locally toxic metal ions

Background. Increasing evidence suggests a link between the bearing surface used in total hip arthroplasty (THA) and the occurrence of infection. It is postulated that polyethylene has immunomodulatory effects and may influence bacterial function and survival, thereby impacting the development of periprosthetic joint infection (PJI). This study aimed to investigate the association between polyethylene type and revision surgery for PJI in THA using data from the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR). We hypothesized that the use of XLPE would demonstrate a statistically significant reduction in revision rates due to PJI compared to N-XLPE. Methods. Data from the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) spanning September 1, 1999, to December 31, 2021, were used to compare the infection revision rates between THA using N-XLPE and XLPE. We calculated the Cumulative Percentage Revision rate (CPR) and Hazard Ratio (HR) while controlling for factors like age, sex, body mass index (BMI), American Society of Anesthesiologists’ (ASA) grade, and head size. Results. From the total 361,083 primary THAs, 26,827 used N-XLPE and 334,256 used XLPE. Excluding data from the first 6 months post-surgery, 220 revisions occurred in the N-XLPE group and 1,055 in the XLPE group for PJI. The HR for infection revision was significantly higher in N-XLPE compared to XLPE, at 1.64 (95% CI, 1.41–1.90, p<0.001). Conclusions. This analysis provides evidence of an association between N-XLPE and revision for infection in THA. We suspect that polyethylene wear particles contribute to the susceptibility of THA to PJI, resulting in a significantly higher risk of revision for infection in N-XLPE hips compared to those with XLPE. Level of Evidence. Therapeutic Level III

Aim. The prevention of surgical-site infection (SSI) is of great importance. Airborne particulate correlates with microbial load and SSI. There are many potential sources of airborne particulates in theatre and from an experimental point of view impossible to control. We evaluated the effectiveness of a novel air decontamination-recirculation system (ADRS) in reducing airborne particles in a laboratory environment and controlled the introduction of particulate using diathermy. Methods. Airborne particles were measured with and without activation of the ADRS in PC2 laboratory to provide a baseline. Particles were generated in a controlled manner utilising electrocautery ablation of porcine skin tissue. Ablation was performed at 50W power (Cut) for 60 seconds at a constant rate with and without the ADRS operating in the PC2 laboratory. Particles were measured continuously in 30s intervals at two sites 0.5m and 3m from the site of diathermy. Adequate time was allowed for return to baseline between each repetition. Each experiment was repeated 10 times. Results. The ADRS significantly reduced baseline airborne particles in the empty PC2 laboratory. When using electrocauterization (as a source of particle generation), peak particles were significantly higher at 0.5m compared to 3m. Small particles (0.3–0.5 microns) were reduced at 0.5m with ADRS whilst larger particles were not. The ADRS significantly reduced all particles of 0.3–10.0 microns at 3m. Particles also returned to a lower baseline and at a faster rate with the ADRS. Conclusion. Airborne particle counts are a surrogate measure of microbial load. As likelihood of SSI is assumed to increase with the quantity of airborne pathogens present, there is a great deal of interest in methods of reducing airborne particle count in the operating theatre. Distance from the source of particle generation influences particle load and has potential clinical relevance for the operating theatre layout and staff. The ADRS effectively reduced the peaks and baseline of airborne particles and hastened the clearance of generated particles. The use of this technology in the operating theatre is of great interest for further research as suppression of airborne particulate may play a role in reducing SSIs. Diathermy provides a simple means to introduce particles in a controlled manner for such experiments

Wear and corrosion debris generated from total hip replacements (THR) can cause adverse local tissue reactions (ALTR) or osteolysis, often leading to premature implant failure. The tissue response can be best characterized by histopathological analysis, which accurately determines the presence of cell types, but is limited in the characterization of biochemical changes (e.g. protein conformation alteration). Fourier transform infrared micro-spectroscopy imaging (FTIRI) enables rapid analysis of the chemical structure of biological tissue with a high spatial resolution, and minimal additional sample preparation. The data provides the most information through multivariate method carried out by hierarchical clustering analysis (HCA). It is the goal of this study to demonstrate the beneficial use of this multivariate approach in providing pathologist with biochemical information from cellular and subcellular organization within joint capsule tissue retrieved from THR patients. Joint capsule tissue from 2 retrieved THRs was studied. Case 1: a metal-on-polyethylene THR, and Case 2: a dual modular metal-on-metal THR. Prior to FTIRI analysis, tissue samples were formalin-fixed paraffin-embedded and 5μm thick microtome sectioned samples were prepared and mounted on BaF. 2. discs and deparaffinized. FTIRI data were collected using high-definition transmission mode (pixel size: ∼1.1 μm. 2. ). Hyperspectral images were exported to CytoSpec V2.0.06 for processing and reconstruction into pseudo-color maps based on cluster assignments. Case 1 exhibited a strong presence of lymphocytes and macrophages (Fig. 1a). Since the process of taking second derivatives reduces the half width of the spectral peaks, it increases the sensitivity toward detecting shoulders or second peaks that may not be apparent in the raw spectra (Fig. 1b). Thus, areas occupied by lymphocytes and macrophages can be easily distinguished providing a fast tissue screening method. Here, HCA was able to distinguish macrophages and lymphocytes based on the infrared response, even in areas where both occurred intermixed. (Fig. 1c) The tissue in direct proximity to cells had a slightly altered collagenous structure. Case 1 also exhibited multiple glassy, green particles which can typically observed around THRs that underwent taper corrosion (Fig. 2a). HCA image was able to visualize and distinguish large CrPO. 4. particles, embedded within fibrin exudate rich areas, collagenous tissue without inflammatory cells, and a nearby area with a strong macrophage presence and some finer CrPO. 4. particles (Fig. 2d). Moreover, this method can not only locate macrophages, but distinguish particle-laden macrophages depending the type of particles within the cells. In Case 2 (Fig. 3a), clustering results (Fig. 3 b&c) are consistent with the fact that different particle types are associated with MoM bearing surface wear (Co rich particles), corrosion of the CoCrMo taper junctions (Cr-oxides and –phosphate), fretting of Ti-alloy dual modular tapers (Ti-oxides, Ti alloy particles), and even suture debris, which all occurred in this case. Although details of debris types are not available, specifications are possible by coupling other techniques. The results demonstrate that multivariate FTIRI based spectral histopathology is a powerful tool to characterize the chemical structure and foreign body response within periprosthetic tissue, thus providing insights into the biological impact of different types of implant debris. For any figures or tables, please contact the authors directly

Introduction. Periprosthetic joint infection (PJI) and particle-induced osteolysis are closely related to peri-implant local immunity and macrophage function. We previously demonstrated that titanium particles attenuate the immune response of macrophages caused by chronic inflammation [1]. In a separate study, we have determined that UHMWPE wear particles containing vitamin E (VE) induce less osteolysis compared to HXL UHMWPE wear particles in a murine calvarium model [2]. For this study we hypothesized that macrophages exposed to HXL UHMWPE particles containing VE would better maintain their ability to respond to S. aureus compared to HXL UHMWPE without VE. Methods. A gamma-sterilized, HXL UHMWPE tibial bearing containing VE (E1, Biomet, “VE-PE”) and 100kGy irradiated and melted UHMWPE (“CISM 100”) were cryomilled to particles by Bioengineering Solutions (Oak Park, IL). In the first in vitro study, RAW 264.7 mouse macrophages were exposed (inverted co-culture) to either VE-PE particles or CISM100 particles and lipopolysaccharide (LPS) for 1–7 days. Macrophage viability was measured using a cell counting kit (CCK-8). Control group with no particles and a LPS group were also included. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to determine macrophage apoptosis rate in response to particle exposure over time. In the second study, macrophages were exposed to VE-PE or CISM100 particles for 48h, then exposed to LPS for 30 min. Subsequently, reactive oxygen species (ROS) generation and extracellular regulated protein kinase (ERK) phosphorylation were measured. In a third study, after exposure to particles for 48h, fatigued macrophages were co-cultured with bioluminescent S. aureus strain Xen29 for 3h and 6h. Bioluminescence signal was determined to measure the total amount of bacteria. Bacterial live/dead staining and optical density at 600 nm (OD 600) were also performed to determine S. aureus viability. Statistical analysis was performed using one-way or two-way ANOVA with a post hoc examination. *indicates p<0.05. Results. CISM100 particles significantly decreased macrophage viability at day 5 and day 7 (p<0.05, Fig. 1A), while the viability of macrophages exposed to VE-PE particles was similar to controls (macrophages not exposed to particles). After 48h, macrophages exposed to VE-PE particles showed a lower TUNEL-positive rate (less apoptosis) compared to CISM100 particles (Fig. 1B, C). 48h-exposure to VE-PE particles increased ROS generation and ERK phosphorylation in 30 min-LPS-activated macrophages when compared to CISM100 particles (Fig. 2). This immune response caused by VE-PE particles resembles that of macrophages without particles. Furthermore, 48h exposure to E1 particles showed less S. aureus at 6h (Fig. 3). Conclusions. These results suggest that VE-PE particles cause reduced macrophage apoptosis and protect the macrophages' immune response. VE-PE particles also preserved the innate immunity of macrophages, unlike CISM100, as evidenced by the S. aureus co-culture study. Thus, patients with vitamin-E containing implants may be less likely to develop PJI

Aim. Prosthetic joint infections pose a major clinical challenge. Developing novel material surface technologies for orthopedic implants that prevent bacterial adhesion and biofilm formation is essential. Antimicrobial coatings applicable to articulating implant surfaces are limited, due to the articulation mechanics inducing wear, coating degradation, and toxic particle release. Noble metals are known for their antimicrobial activity and high mechanical strength and could be a viable coating alternative for orthopaedic implants [1]. In this study, the potential of thin platinum-based metal alloy coatings was developed, characterized, and tested on cytotoxicity and antibacterial properties. Method. Three platinum-based metal alloy coatings were sputter-coated on medical-grade polished titanium discs. The coatings were characterized using optical topography and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Ion release was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). Cytotoxicity was tested according to ISO10993-5 using mouse fibroblasts (cell lines L929 and 3T3). Antibacterial surface activity, bacterial adhesion, bacterial proliferation, and biofilm formation were tested with gram-positive Staphylococcus aureus ATCC 25923 and gram-negative Escherichia coli ATCC 25922. Colony forming unit (CFU) counts, live-dead fluorescence staining, and SEM-EDS images were used to assess antibacterial activity. Results. Three different platinum-based metal alloys consisting of platinum-iridium, platinum-copper, and platinum-zirconium. The coatings were found 80 nm thick, smooth (roughness average < 60 nm), and non-toxic. The platinum-copper coating showed a CFU reduction larger than one logarithm in adherent bacteria compared to uncoated titanium. The other coatings showed a smaller reduction. This data was confirmed by SEM and live-dead fluorescence images, and accordingly, ICP-OES measurements showed low levels of metal ion release from the coatings. Conclusions. The platinum-copper coating showed low anti-adhesion properties, even with extremely low metal ions released. These platinum-based metal alloy coatings cannot be classified as antimicrobial yet. Further optimization of the coating composition to induce a higher ion release based on the galvanic principle is required and copper looks most promising as the antimicrobial compound of choice. Acknowledgments. This publication is supported by the DARTBAC project (with project number NWA.1292.19.354) of the research program NWA-ORC which is (partly) financed by the Dutch Research Council (NWO); and the AMBITION project (with project number NSP20–1-302), co-funded by the PPP Allowance made available by Health-Holland, Top Sector Life Sciences & Health to ReumaNederland