Staphylococcus aureus is the most frequently isolated organism in periprosthetic joint infections. The mechanism by which synovial fluid (SF) kills bacteria has not yet been elucidated, and a better understanding of its antibacterial characteristics is needed. We sought to analyze the antimicrobial properties of exogenous copper in human SF against S. aureus. SF samples were collected from patients undergoing total elective knee or hip arthroplasty. Different S. aureus strains previously found to be sensitive and resistant, UAMS-1 and USA300 WT, respectively, were used. We performed in-vitro growth and viability assays to determine the capability of S. aureus to survive in SF with the addition of 10µM of copper. We determined the minimum bactericidal concentration of copper (MBC-Cu) and evaluated the sensitivity to killing, comparing WT and CopAZB-deficient USA300 strains. UAMS-1 evidenced a greater sensitivity to SF when compared to USA300 WT, at 12 (p=0.001) and 24 hours (p=0.027). UAMS-1 significantly died at 24 hours (p=0.017), and USA300 WT survived at 24 hours. UAMS-1 was more susceptible to the addition of copper at 4 (p=0.001), 12 (p=0.005) and 24-hours (p=0.006). We confirmed a high sensitivity to killing with the addition of exogenous copper on both strains at 4 (p=0.011), 12 (p=0.011), and 24 hours (p=0.011). Both WT and CopAZB-deficient USA300 strains significantly died in SF, evidencing a MBC-Cu of 50µM against USA300 WT (p=0.011). SF has antimicrobial properties against S. aureus, and UAMS-1 was more sensitive than USA300 WT. The addition of 10µM of copper was highly toxic for both strains, confirming its bactericidal effect. We evidenced CopAZB-proteins involvement in copper effluxion by demonstrating the high sensitivity of the mutant strain to lower copper concentrations. Thus, we propose CopAZB-proteins as potential targets and the use of exogenous copper as possible treatment alternatives against S. aureus

Aim. Antibacterial activity of coatings based on metal and metal oxide nanoparticles (NPs) often depends on materials and biotic targets resulting in a material-specific killing activity of selected Gram-positive and Gram-negative bacteria, including drug-resistant strains. In this perspective, the NPs loading amount, the relative elemental concentration inside the nanogranular building blocks and the deposition method are of paramount importance when the goal is to widen the antimicrobial spectrum, but at the same time to avoid high levels of metal content to limit undesired toxic effects. Aim of the present study was evaluation of the antimicrobial properties of two multielement nanogranular coatings composed of Titanium-Silver and Copper and of Magnesium-Silver and Copper. Method. Ti-Ag-Cu and Mg-Ag-Cu NPs were deposited on circular cover glasses (VWR) by Supersonic Cluster Beam Deposition. Biofilm-producer strains of Staphylococcus aureus (methicillin susceptible and resistant), Staphylococcus epidermidis (methicillin susceptible and resistant), Escherichia coli (fully susceptible and producer of extended spectrum beta lactamases), and Pseudomonas aeruginosa (susceptible and multidrug-resistant) were selected. The abilities of the selected strains to adhere, colonize and produce biofilm on the discs coated with Ti-Ag-Cu or Mg-Ag-Cu NPs were compared to uncoated circular cover glasses which were used as growth control. Cytotoxicity was also evaluated in order to assess the biocompatibility of the newly synthesized NPs. Results. In comparison to uncoated controls, both coatings showed significant anti-adhesive properties against S. aureus, S. epidermidis, and E. coli. Reduction in adhesion to Mg-Ag-Cu coated discs was observed also for P. aeruginosa isolates, although differences vs uncoated controls did not reach statistical significance. Biofilm formation was reduced on discs coated with Mg-Ag-Cu compared to Ti-Ag-Cu and, again, coatings had a milder effect on P. aeruginosa, probably due to its exceptional capability of attachment and matrix production. These results were confirmed by the evaluation of bacterial colonization on nanoparticles-coated discs by means of confocal laser scanning microscopy. A viability of 95.8% and 89.4% of cells cultured in the presence of Ti-Ag-Cu and Mg-Ag-Cu discs, respectively, when compared to negative controls was observed, thus excluding cytotoxic effects on eukaryotic cells. Conclusions. The newly synthesized Ti-Ag-Cu and Mg-Ag-Cu coatings are able to limit bacterial adhesion colonization and biofilm production, thus highlighting the safe use of multi-element families of NPs as new strategies against bacterial attachment to the surface of biomedical implants. However, further studies addressing activity against P. aeruginosa and including a wide number of isolates are warranted

Introduction. Implant associated infections are responsible for over 10 % of recorded orthopaedic revision surgeries across the UK, with higher infection rates commonly observed for other endoprostheses such as cranioplasties. To prevent colonization and biofilm formation on implant surfaces, the use of silver coatings has shown positive results in clinical setting due to its synergistic function with conventional antibiotic prophylaxes. Additive manufacturing allows manufacture of entirely new implant geometries such as lattice structures to enhance osseointegration, however this limits the ability to uniformly coat implants. Direct integration of silver into the powder feedstock for selective laser melting (SLM) may allow manufacture of a biomedical alloy with innate, long lasting antimicrobial properties without compromising possible geometries and with no coating process necessary. Methods. Feedstock powders of 15–45 micron Grade 5 Ti-64 (Renishaw Plc) and Ag-999 powder (CooksonGold) were characterized using laser particle size analysis, ICP-OES, LECO-ONH, and morphological analysis in SEM. A blend of Ti-64 with 3 wt% Ag-999 powder (Ti-643) was produced by tumble blending, and validated by SEM and EDS. Parameters for manufacture were established using a 17 point design of experiment (DoE) exploring a 2D parameter space of applied laser power and laser scanning speed. Samples were manufactured using a ConceptLaser M2 LaserCusing SLM. Density was assessed by He pycnometry, and cross-sections analysed for defects by optical microscopy. Silver distribution was mapped by micro X-Ray Fluoroscopy (µXRF) and energy-dispersive X-ray spectroscopy (EDS). Optimum parameters were identified and used to manufacture all subsequent samples. Cylindrical Ti-643 samples were manufactured for further physical characterization and bacterial investigation, alongside control Ti-64 samples manufactured using existing optimum parameters. Samples were polished using silicon carbide papers to a 4000-grit surface finish. Contact angle measurements were made by goniometry. Silver elution characteristics were assessed by immersion in water refreshed on a daily basis, and sampled over a 14 day period using ICP-OES. Viability of S. aureus was compared to control samples according to the Japanese standard test method, JIS Z 2801:2000. Results. Across the entire parameter space tested, selective laser melting (SLM) of all 17 samples was successful, with no delamination. An increased recoater blade speed was required to achieve uniform spreading in process versus pure Ti-6Al- 4V powder, indicating an increased cohesivity of the Ti-643 blend. The presence of silver in all samples was confirmed by µXRF, indicating that there was no excessive evaporation of silver in-process. Laser parameters were found to alter the defect density and microstructure scale, though sample density was tightly clustered in a range from 4.415 to 4.453 gcm-3, showing relatively low process variation. No significant difference in bacterial survival was found between control and Ti-643 samples, indicating that further microstructural optimization is needed to guarantee efficacy

INTRODUCTION. Biomaterial-related infections are an important complication in orthopaedic surgery [1], and Staphylococcus sp. accounts for more than half of the prosthetic joint infection cases [2]. Adhesion of bacteria to biomaterial surfaces is a key step in pathogenesis of such infections [3]. Titanium alloys are widely used in orthopaedic implants because their biocompatibility [4]. Surface incorporation of ions with antimicrobial properties, like fluorine, is one strategy previously studied with good results [5]. MATERIAL AND METHODS. A 18mm diameter rod of Ti–6Al–4V alloy ELI grade according to the standard ASTMF136-02 supplied by SURGIVAL was cut into 2 mm thick disk specimens, ground through successive grades of SiC paper to 1200 grade, degreased with a conventional detergent and rinsed in tap water followed by deionised water. The specimens were then chemically polished (CP). The disks were anodized only on one side by using a two electrode cell in a suitable electrolyte. TiO. 2. barrier layers, without fluoride (BL), were produced by anodizing in 1 M H. 2. SO. 4. at 15 mA cm-2 to 90 V, reaching 200 nm of thickness. Fluoride barrier layers (FBL) were produced in an electrolyte containing 1 M NH. 4. H. 2. PO. 4. and 0.15 M NH. 4. F, at constant voltage controlled at 20 V for 120 min at 20°C; the thickness of the layer is 140 nm. Laboratory biofilm-forming strains of Staphylococcus aureus 15981 [6] and Staphylococcus epidermidis ATCC 35984 were used in adherence studies, which were performed using the protocol by Kinnari et al [7]. Photographs obtained were studied by ImageJ software. Statistical analysis was performed by EPI-INFO software. The experiments were performed in triplicates. RESULTS. Lower adherence was detected when compared FBL with unmodified controls (CP and BL). A statistical significant difference (p<0.01) was detected in the adhesion to modified material between both species, being the adherence of S. aureus lower than that of S. epidermidis (Figure 1). DISCUSSION & CONCLUSIONS. There is currently a discussion about the actual antibacterial properties of fluorine when incorporated in biomaterial surfaces. In this study we have demonstrated that both S. aureus and S. epidermidis strains showed a decrease of bacterial adhesion to modified surfaces with fluorine, a decrease that cannot be due to other surface modifications. Further studies, including adhesion studies with clinical strains [8], must be performed to confirm these results, which can lead to the development of new materials with a potential use in orthopaedic surgery

Implant-associated infection is a major source of morbidity in orthopaedic surgery. There has been extensive research into the development of materials that prevent biofilm formation, and hence, reduce the risk of infection. Silver nanoparticle technology is receiving much interest in the field of orthopaedics for its antimicrobial properties, and the results of studies to date are encouraging. Antimicrobial effects have been seen when silver nanoparticles are used in trauma implants, tumour prostheses, bone cement, and also when combined with hydroxyapatite coatings. Although there are promising results with in vitro and in vivo studies, the number of clinical studies remains small. Future studies will be required to explore further the possible side effects associated with silver nanoparticles, to ensure their use in an effective and biocompatible manner. Here we present a review of the current literature relating to the production of nanosilver for medical use, and its orthopaedic applications.

Cite this article: Bone Joint J 2015; 97-B:582–9.