Aims. The mechanism by which synovial fluid (SF) kills bacteria has not yet been elucidated, and a better understanding is needed. We sought to analyze the antimicrobial properties of exogenous copper in human SF against Staphylococcus aureus. Methods. We performed in vitro growth and viability assays to determine the capability of S. aureus to survive in SF with the addition of 10 µM of copper. We determined the minimum bactericidal concentration of copper (MBC-Cu) and evaluated its sensitivity to killing, comparing wild type (WT) and CopAZB-deficient USA300 strains. Results. UAMS-1 demonstrated a greater sensitivity to SF compared to USA300 WT at 12 hours (p = 0.001) and 24 hours (p = 0.027). UAMS-1 died in statistically significant quantities at 24 hours (p = 0.017), and USA300 WT survived at 24 hours. UAMS-1 was more susceptible to the addition of copper at four (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 four (p = 0.011), 12 (p = 0.011), and 24 hours (p = 0.011). WT and CopAZB-deficient USA300 strains significantly died in SF, demonstrating a MBC-Cu of 50 µM against USA300 WT (p = 0.011). Conclusion. SF has antimicrobial properties against S. aureus, and UAMS-1 was more sensitive than USA300 WT. Adding 10 µM of copper was highly toxic, confirming its bactericidal effect. We found CopAZB proteins to be involved in copper effluxion by demonstrating the high sensitivity of mutant strains to lower copper concentrations. Thus, we propose CopAZB proteins as potential targets and use exogenous copper as a treatment alternative against S. aureus

Over the last decades, biodegradable metals emerged as promising materials for various biomedical implant applications, aiming to reduce the use of permanent metallic implants and, therefore, to avoid additional surgeries for implant removal. However, among the important issue to be solved is their fast corrosion - too high to match the healing rate of the bone tissue. The most effective way to improve this characteristic is to coat biodegradable metals with substituted calcium phosphates. Tricalcium phosphate (β-TCP) is a resorbable bioceramic widely used as synthetic bone graft. In order to modulate and enhance its biological performance, the substitution of Ca2+ by various metal ions, such as strontium (Sr2+), magnesium (Mg2+), iron (Fe2+) etc., can be carried out. Among them, copper (Cu2+), manganese (Mn2+), zinc (Zn2+) etc. could add antimicrobial properties against implant-related infections. Double substitutions of TCP containing couples of Cu2+/Sr2+ or Mn2+/Sr2+ ions are considered to be the most perspective based on the results of our study. We established that single phase Ca3−2x(MˊMˊˊ)x(PO4)2 solid solutions are formed only at x ≤ 0.286, where Mˊ and Mˊˊ—divalent metal ions, such as Zn2+, Mg2+, Cu2+, Mn2+, and that in case of double substitutions, the incorporation of Sr2+ ions allows one to extend the limit of solid solution due to the enlargement of the unit cell structure. We also reported that antimicrobial properties depend on the substitution ion occupation of Ca2+ crystal sites in the β-TCP structure. The combination of two different ions in the Ca5 position, on one side, and in the Ca1, Ca2, Ca3, and Ca4 positions, on another side, significantly boosts antimicrobial properties. In the present work, zinc-lithium (Zn-Li) biodegradable alloys were coated with double substituted Mn2+/Sr2+ β-TCP and double substituted Cu2+/ Sr2+ β-TCP, with the scope to promote osteoinductive effect (due to the Sr2+ presence) and to impart antimicrobial properties (thanks to Cu2+ or Mn2+ ions). The Pulsed Laser Deposition (PLD) method was applied as the coating's preparation technique. It was shown that films deposited using PLD present good adhesion strength and hardness and are characterized by a nanostructured background with random microparticles on the surface. For coatings characterization, Fourier Transform Infrared Spectroscopy, X-ray Diffraction, and Scanning Electron Microscopy coupled with Energy Dispersive X-ray and X-ray Photoelectron Spectroscopy were applied. The microbiology tests on the prepared coated Zn-Li alloys were performed with the Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Salmonella typhimurium, Escherichia coli) bacteria strains and Candida albicans fungus. The antimicrobial activity tests showed that Mn2+/Sr2+ β-TCP -coated and Cu2+/Sr2+ β-TCP coated Zn-Li alloys were able to inhibit the growth of all five microorganisms. The prepared coatings are promising in improving the degradation behavior and biological properties of Zn-Li alloys, and further studies are necessary before a possible clinical translation

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

Abstract. 3D printing of synthetic scaffolds mimicking natural bone chemical composition, structure, and mechanical properties is a promising approach for repairing bone injuries. Direct ink writing (DIW), a type of 3D printing, confers compatibility with a wide range of materials without exposing these materials to extreme heat. Optimizing ink properties such as filament formation capabilities, shear-thinning, and high storage modulus recovery would improve DIW fabrication characteristics. In this study, composite inks based on biodegradable polycaprolactone (PCL), reinforced with nano-hydroxyapatite (HAp), and loaded with vancomycin were designed and evaluated for their rheological properties, wettability, mechanical properties, and antimicrobial properties. The formulated composite inks displayed a shear-thinning behaviour exhibited storage modulus recovery percentages above 80% for all formulations, which is essential for extrusion deposition by DIW at room temperature. Ink formulations were able to form fully interconnected lattice scaffolds with porosities ranging from 42% to 65%. Increasing the HAp concentrations from 55% to 85% w/w increased the shear thinning behaviour and reduced the printed filament width to more closely match the nozzle diameter; this indicates higher HAp proportion reduces ink shrinkage. The scaffold had high wettability at HAp proportions above 65% w/w and the compressive elastic modulus of DIW printed scaffolds exhibited within the range of trabecular bone. Antimicrobial activity was apparent from the agar diffusion assay; zones of inhibition ranging from 15.82 ± 0.25 mm and 20.06 ± 0.25 mm were observed after 24 hr for composite scaffolds loaded with 3% and 9% w/w vancomycin respectively. Vancomycin-loaded PCL/HAp composite inks were developed, displaying good printability, wettability, mechanical properties, and antimicrobial properties, making them an attractive choice for bone repair and regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Uncemented implants combining antimicrobial properties with osteoconductivity would be highly desirable in revision surgery due to periprosthetic joint infection (PJI). Silver coatings convey antibacterial properties, however, at the cost of toxicity towards osteoblasts. On the other hand, topological modifications such as increased surface roughness or porosity support osseointregation but simultaneously lead to enhanced bacterial colonization. In this study, we investigated the antibacterial and osteoconductive properties of silver-coated porous titanium (Ti) alloys manufactured by electron beam melting, rendering a macrostructure that mimics trabecular bone. Trabecular implants with silver coating (TR-Ag) or without coating (TR) were compared to grit-blasted Ti6Al4V (GB) and glass cover slips as internal controls. Physicochemical characterization was performed by X-ray diffraction (XRD) and energy dispersive X-rays (EDX) together with morphological characterization through electron scanning microscopy (SEM). Bacterial adherence after incubation of samples with Staphylococcus (S.) aureus and S. epidermidis strains harvested from PJI patients was quantitatively assessed by viable count after detachment of adherent bacteria by collagenase/dispase treatment. Primary human osteoblasts (hOB) were used to investigate the osteoconductive potential by lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity. Cell morphology was investigated by fluorescence microscopy after staining with carboxifluorescein diacetate succinimidyl ester (CFDA-SE) and 4′,6-diamidino-2-phenylindole (DAPI). The trabecular implants depicted a porosity of 70% with pore sizes of 600µm. The amount of silver analyzed by EDX accounted for 35%wt in TR-Ag but nil in TR. Silver-coated TR-Ag implants had 24% lower S. aureus viable counts compared to non-coated TR analogues, and 9% lower compared to GB controls. Despite trabecular implants, both with and without silver, had higher viable counts than GB, the viable count of S. epidermidis was 42% lower on TR-Ag compared to TR. The percentage of viable hOB, measured by LDH and normalized to controls and area at 1 day, was lower on both TR-Ag (18%) and on TR (13%) when compared with GB (89%). However, after 1 week, cell proliferation increased more markedly on trabecular implants, with a 5-fold increase on TR-Ag, a 3.4-fold increase on TR, and a 1.7-fold increase on GB. Furthermore, after 2 weeks of hOB culture, proliferation increased 20-fold on TR-Ag, 29-fold on TR, and 3.9-fold for GB, compared to 1 day. The osteoconductive potential measured by ALP illustrated slightly higher values for TR-Ag compared to TR at 1 day and 2 weeks, however below those of GB samples. Cell morphology assessed by microscopy showed abundant growth of osteoblast-like cells confined to the pores of TR-Ag and TR. Overall, our findings indicate that the silver coating of trabecular titanium exerts limited cytotoxic effects on osteoblasts and confers antimicrobial effects on two PJI-relevant bacterial strains. We conclude that improving material design by mimicking the porosity and architecture of cancellous bone can enhance osteoconductivity while the deposition of silver confers potent antimicrobial properties

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. Periprosthetic joint infection (PJI) remains the main cause of failure in primary and revision total knee arthroplasties (TKAs). Local delivery of antibiotics, mainly antibiotic-loaded bone cement (ALBC), is commonly employed to prevent PJI. Over the past decade, tantalum and porous titanium have been successfully utilized as metaphyseal fixation devices to address bone loss and improve biologic fixation during revision TKA. However, no study has examined the antimicrobial properties compared to bone cement. The purpose of this study was to compare the ability of tantalum, 3D porous titanium, antibiotic-loaded bone cement (ALBC) and smooth titanium alloy (STA) to inhibit Staphylococci bacterial agents in an in vitro medium environment, based on the evaluation of the zone of inhibition (ZOI) and the antibacterial activity duration. Our study hypothesis was that we will found no significant difference between groups to inhibit Methicillin-Sensitive or Methicillin-Resistant Staphylococcus aureus (MSSA/MRSA) agents. Methods. Thirty beads made of 3 different materials (tantalum/ 3D porous titanium/ STA) were bathed during 1hour inside of a solution made of 1g vancomycin with 20-mL of sterile water for injection (bath concentration: 50 mg/mL). Ten 1cm. 3. cylinders were also created mixing standard surgical cement with 1g of Vancomycin in standardized sterile molds (ALBC beads). Finally, thirty beads made of tantalum/ 3D porous titanium/ STA were bathed in phosphate buffered saline solution to act as a control group. Cylinders were then placed on agar plates inoculated with MSSA and MRSA. Inhibition zone diameters were measured each day and cylinders were transferred onto a new inoculated plate. Inhibition zones were measured with a manual Vernier caliper and with automated software. The mean inhibition zones between groups were compared using the Wilcoxon Test. Results. The inter-class coefficient correlation values indicated an optimal intra-observer and inter-observer reproducibility for ZOI measurement (ICC 0.96 and ICC 0.98). For MSSA and MRSA, no inhibitory effect was found in the control group and antibiotic-loaded STA beads exhibited a short inhibitory effect until day 2. For MSSA, both tantalum and 3D porous titanium beads exhibited larger inhibition zones than cement beads (all p<0.01) each day until day 7 for tantalum and until day 3 for 3D porous titanium. After 6 days, ALBC presented larger inhibition zone than the 3D porous titanium, but no difference was found with tantalum. For MRSA, both tantalum and 3D porous titanium beads had significantly larger inhibition zones than ALBC each day until day 6 for tantalum (all p<0.01) and until day 3 for 3D porous titanium (all p<0.04). ALBC presented larger inhibition zone than tantalum and 3D porous titanium from day 7 to 9 (all p<0.04). Conclusion. Our results demonstrate that porous metal implants can deliver local antibiotics over slightly varying time frames based on our in vitro analysis. Antibiotic-impregnated tantalum and 3D porous titanium constructs exhibited superior antimicrobial properties when compared to STA. Future goals include impregnating porous metals with antibiotics for intraoperative use during revision TKA. For figures, tables, or references, please contact authors directly

Objectives. The surface of pure titanium (Ti) shows decreased histocompatibility over time; this phenomenon is known as biological ageing. UV irradiation enables the reversal of biological ageing through photofunctionalisation, a physicochemical alteration of the titanium surface. Ti implants are sterilised by UV irradiation in dental surgery. However, orthopaedic biomaterials are usually composed of the alloy Ti6Al4V, for which the antibacterial effects of UV irradiation are unconfirmed. Here we evaluated the bactericidal and antimicrobial effects of treating Ti and Ti6Al4V with UV irradiation of a lower and briefer dose than previously reported, for applications in implant surgery. Materials and Methods. Ti and Ti6Al4V disks were prepared. To evaluate the bactericidal effect of UV irradiation, Staphylococcus aureus 834 suspension was seeded onto the disks, which were then exposed to UV light for 15 minutes at a dose of 9 J/cm. 2. To evaluate the antimicrobial activity of UV irradiation, bacterial suspensions were seeded onto the disks 0, 0.5, one, six, 24 and 48 hours, and three and seven days after UV irradiation as described above. In both experiments, the bacteria were then harvested, cultured, and the number of colonies were counted. Results. No colonies were observed when UV irradiation was performed after the bacteria were added to the disks. When the bacteria were seeded after UV irradiation, the amount of surviving bacteria on the Ti and Ti6Al4V disks decreased at 0 hours and then gradually increased. However, the antimicrobial activity was maintained for seven days after UV irradiation. Conclusion. Antimicrobial activity was induced for seven days after UV irradiation on both types of disk. Irradiated Ti6Al4V and Ti had similar antimicrobial properties. Cite this article: T. Itabashi, K. Narita, A. Ono, K. Wada, T. Tanaka, G. Kumagai, R. Yamauchi, A. Nakane, Y. Ishibashi. Bactericidal and antimicrobial effects of pure titanium and titanium alloy treated with short-term, low-energy UV irradiation. Bone Joint Res 2017;6:108–112. DOI: 10.1302/2046-3758.62.2000619

Post-surgical infections are still one of the most frequent adverse events in the prosthetic surgery. PMMA-based cements are widely employed in orthopaedic surgery as filler or prosthetic fixing device. The main problems associated with this material are poor bone integration and infection development. Aiming to avoid bacterial adhesion and to extend the longevity of implants, different solutions were proposed, both in terms of operative procedures and new materials development. Regarding the materials advancement, innovative PMMA-based composite bone cements, contemporaneously bioactive and antibacterial (without the use of antibiotics), were developed. The composites are based on a PMMA matrix containing a bioactive glass, doped with antibacterial ions (Ag+ or Cu++); so, the same filler shows at the same time the ability of promoting bone ingrowth and an antibacterial effect. Composite cements were characterized in terms of morphology and composition, curing parameters and mechanical properties; in vitro tests were performed to verify the material ability to release antibacterial ions and to promote the precipitation of hydroxyapatite. Moreover, cytotoxicity and antimicrobial properties were verified. The cements characteristics were tested using different commercial matrix and different viscosities; therefore, the proposed formulations represent an innovative solution for a new family of antibiotic-free, bioactive and antibacterial cements

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. Tantalum trabecular metal components are increasingly used to reconstruct major bone defects in revision arthroplasty surgery. It is known that some metals such as silver have antibacterial properties. Recent reports have raised the question as to whether Tantalum components are protective against infection in revision surgery. This is based on a retrospective, single institution review, of revision cases comparing tantalum with titanium acetabular implants, which reported a lower incidence of subsequent infection in the tantalum group. This laboratory study aimed to establish if tantalum had any intrinsic antibacterial properties against planktonic bacteria or ability to inhibit biofilm formation. Materials and methods. Equal sized pieces of tantalum (Trabecular metal, Zimmer UK) and titanium (Trilogy, Zimmer UK) were sterilised and then incubated with a low dose inoculum of either Staphylococcus aureus or Staphylococcus epidermidis for 24 hours. After serial dilution, colony forming units were quantified on MH agar plates. To establish the ability to inhibit biofilm formation these tantalum and titanium pieces were then washed twice, sonicated and washed again to remove loosely adhered planktonic bacteria. They were then re-incubated for 24 hours prior to quantifying colony forming units. All experiments were performed in triplicate. Results. More than 1×10. 8. cfu/ml were observed in both the titanium and tantalum experiments. After washing and sonication more than 2×10. 7. cfu/ml were observed for both tantalum and titanium groups. The results were the same for both Staph Aureus and Staph Epidermidis. Discussion. Compared with titanium controls tantalum did not demonstrate any intrinsic antibacterial activity or ability to inhibit biofilm formation. The intrinsic properties of tantalum do not account for the previously observed reduction in subsequent infection when tantalum was used in the revision procedure. Conclusion. Tantalum does not have any intrinsic antimicrobial properties or ability to inhibit biofilm formation

The use of platelet-leukocyte gel (PLG), made from platelet rich plasma, to stimulate bone formation and wound healing has been investigated extensively. As leukocytes play an important role in the innate host-defence, we hypothesised that PLG might also have antimicrobial properties. The purpose of this study was to investigate the antimicrobial activity of PLG against Staphylococcus aureus in an in vitro experiment. To determine the contribution of myeloperoxidase (MPO), present in leukocytes, in this process, MPO release was measured. Platelet rich plasma (PRP) was prepared from whole blood of 6 donors. In this process platelet poor plasma (PPP) was obtained as well. PLG was prepared by mixing PRP with either autologous (PLG-AT) or bovine thrombin (PLG-BT). The antimicrobial activity of PLG-AT, PLG-BT, PRP and PPP was determined in a bacterial kill assay, containing 1x106 CFU/ml of Staphylococcus aureus, during a 24-hour period. MPO release was measured by ELISA. Cultures showed a rapid decrease in the number of bacteria in the presence of both PLG-AT and PLG-BT, which was maximal between 4 and 8 hours, to approximately 1% of the bacteria in controls. Also PRP and PPP induced a statistically significant bacterial kill, but the effect of PLG-AT was the largest (p=0.093 vs. PLG-BT; p=0.004 vs. PRP and p< 0.001 vs. PPP). PLG-AT, PLG-BT and PRP showed a comparable, gradually increasing MPO release for 8 to 12 hours. Some MPO was also measured in the PPP samples. No correlation between MPO release and bacterial kill could be found. PLG appears to have potent antimicrobial capacity, but the role of MPO in this activity is questionable. PLG might represent a useful strategy against postoperative infections. Further research should investigate its antimicrobial capacity in the in vivo situation

Summary. The two-step labeling protocol using Lysostaphin and bio-orthogonal click chemistry for staining bacteria is described. The click protocol is efficient in labeling staphylococci and is non-toxic. This protocol promises the efficient of infections that are difficult to assess by conventional imaging. Introduction. Infection diagnostics in clinics is time consuming, invasive and relays on microbiological cultures. New probes and labeling protocols enabling rapid and specific detection of infection in vivo shall improve the situation. We investigated the potential of a new click labeling protocol to detect staphylococci. Azido (N3) - modified Lysostaphin and DIBO (Di-benzocyclooctyne) - dye were used in the two-step bacteria-labeling protocol. N3 and DIBO were the counterparts of the bioorthogonal “click” reaction. In the first step, Lysostaphin-N3 bound to Staphylococcus aureus. In the second step, N3 clicked to DIBO thus achieving S. aureus selective labeling. Methods. S. aureus NCTC 10788 and E. coli NCTC 12241 (from National Collection of Type Cultures), primary sheep osteoblasts and C57BL/6 mice were used for this study. DIBO-Alexa488 (Invitrogen ®), DyeLight488 (Thermofisher ®), NHS-N3 (Lumiprobe ®), Lysostaphin (Sigma-Aldrich ®) were purchased. In vitro we used standard microbiological protocols to assess antimicrobial and labeling activity of the “click” probe (Lysostaphin-N3 plus DIBO-dye), one-step-labeled Lysostaphin-Dye and non-labeled Lysostaphin. Flow cytometry, Fluorescence microscopy, and Spectrophotometry were employed to measure binding of the probes to bacteria. The cytotoxicity of the probes on osteoblasts was performed using Presto Blue Cell Viability test (Invitrogen ®). In vivo we used Fluorescence Intravital Microscopy and mice with dorsal skin-fold chambers (approved by the local governmental animal care committee). Subsequently to anesthesia each mouse received S. aureus strain Cowan I intravenously. This was followed by intravenous injections of the test probes. Results. Lysostaphin-N3 partially lost its antimicrobial property if compare to Lysostaphin alone, but still bound to S. aureus efficiently and clicked DIBO-dye afterwards. There was no significant cytotoxicity of “click” reagents on sheep osteoblasts. In vitro the two-step labeling with Lysostaphin-Click was more efficient than the one-step labeling with Lysostaphin-Dye. In vivo the two-step and one-step labeling differed rather qualitatively. In the two-step protocol the “click” probe labeled bacteria adherent to the blood vessels and bacteria extravasated into the soft tissue around 30 minutes post-injection. In the one-step protocol bacteria were labeled quickly (within minutes) in the blood flow and on the blood vessels but not in the soft tissue. Discussion/Conclusion. Effective labeling bacteria with the click probe could be useful for a quantitative and selective assessment of infection, in particular for staphylococci infections, which are common in orthopedic implants but often difficult to detect. The one and two-step protocols successfully labeled bacteria in mice but the labeling was different. In the one-step protocol bacteria were labeled in the blood flow quickly; in the two-step protocol bacteria adherent to the blood vessels and extravasated into the soft tissue bacteria were labeled with a delay

Introduction: Deep periprosthetic infections are infrequent but devastating situations in total joint arthroplasty. During the last years the total number and the percentage of total joint infections with multiresistant bacteria has increased. The aim of this study was to investigate the antimicrobial activity of a new bone cement loaded with nanoparticulate silver against bacteria with different antibiotic resistance. Material and Methods: An in vitro proliferation test was used to test antimicrobial properties of 1% nanoparticulate silver bone cement, gentamicin-loaded, tobramycin-loaded and plain bone cement. This in vitro testing method consisted of two incubation steps. During the first step the tested bacteria could adhere to the bone cement surface. In the second step bacteria either seeded out of vital daughter cells in case of no antimicrobial effect of the cement or were killed by the antibacterial properties of the cement. Seeding out of daughter cells was detected by a microplate reading system resulting in specific time proliferation curves. Several staphylococci and gram-negative strains with different resistance profiles against methicillin, tobramycin, and gentamicin were tested including MRSA and MRSE. Results: 1% nanoparticulate silver bone cement showed bactericidal effect against all tested strains, including MRSA and MRSE. Gentamicin and tobramycin cement was not effective against bacteria with high resistance level against the respective antibiotic. Plain bone cement was not effective against any strain. Conclusion: 1% nanoparticulate silver bone cement exhibited excellent antibacterial properties that could not be reached by gentamicin or tobramycin-loaded cement. Good activity against MRSA could also already be shown in a first animal trial. If further in vivo investigations confirm these promising results nanoparticulate silver bone cement is a new alternative for prophylaxis in total joint arthroplasty

Aims

Biofilm-related infection is a major complication that occurs in orthopaedic surgery. Various treatments are available but efficacy to eradicate infections varies significantly. A systematic review was performed to evaluate therapeutic interventions combating biofilm-related infections on in vivo animal models.

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

Aims

Hand trauma, consisting of injuries to both the hand and the wrist, are a common injury seen worldwide. The global age-standardized incidence of hand trauma exceeds 179 per 100,000. Hand trauma may require surgical management and therefore result in significant costs to both healthcare systems and society. Surgical site infections (SSIs) are common following all surgical interventions, and within hand surgery the risk of SSI is at least 5%. SSI following hand trauma surgery results in significant costs to healthcare systems with estimations of over £450 per patient. The World Health Organization (WHO) have produced international guidelines to help prevent SSIs. However, it is unclear what variability exists in the adherence to these guidelines within hand trauma. The aim is to assess compliance to the WHO global guidelines in prevention of SSI in hand trauma.

Summary Statement. A single, locally-delivered injection of a human placental product containing multipotent stromal cells reduced severity of infection in an immunosuppressed murine osteomyelitis model and eliminated infection in 25% of animals compared with 0% of controls without the use of antibiotics. Introduction. Implant–associated osteomyelitis is a serious orthopaedic condition and is particularly difficult to treat in immunosuppressed individuals. Despite great advancement in the field of biomaterials and pharmaceuticals, emerging patterns of antibiotic resistance, complex biofilm production and penetration of therapeutic concentrations of effective antibiotics into bone continue to represent unmet clinical challenges. The promise of adult multipotent stromal cells (MSCs) for tissue regeneration has been of intense interest in recent years. Among their many potential therapeutic uses, MSCs have also been shown to have direct antimicrobial properties. The objective of this study was to evaluate the efficacy of a locally–delivered human placental-based tissue product containing multipotent stromal cells (hAmSC) to reduce the severity of implant-associated Staphylococcus aureus osteomyelitis in an immunosuppressed murine model. We hypothesised that athymic mice with implant-associated osteomyelitis would have diminished infection following treatment with hAmSC as evidenced by decreased bioluminescence intensity and lower histologic scores for infection and bacterial load when compared to saline-treated controls. Methods. An athymic murine model of chronic implant-associated osteomyelitis was developed using luciferase-transfected Staphylococcus aureus to study the antimicrobial effects of a human placental-based product containing multi-potent stromal cells (hAmSC). Sixteen athymic mice had osteomyelitis established in the right femoral diaphysis. Fifteen days after inducing luc S. aureus osteomyelitis, the mice were randomised to receive a single 0.5 cc injection of hAmSC (n=8) or vehicle (0.9% saline) (n=8) into the soft tissues immediately adjacent to the infected bone. No antibiotics were administered throughout the duration of the study. Mice were imaged with an In Vivo Imaging System (IVIS 1000, PerkinElmer) twice weekly for 30 days to assess change in bioluminescence intensity from baseline immediately prior to treatment with either hAmSC or saline. Radiographs were obtained at days −10, 0, 10, 20 and 30 days post-injection and scored for bone changes secondary to osteomyelitis by a reviewer blinded to treatment group. Mice were sacrificed 30 days after treatment and femurs were examined histologically and scored for bacterial load and degree of inflammation by a pathologist blinded to treatment group. Results. Osteomyelitis was successfully established in all mice as evidenced by baseline bioluminescence imaging and radiographs. Mean bioluminescence intensity decreased from baseline in animals receiving hAmSC and remained below baseline for 28 days, whereas vehicle-treated animals showed an increase in mean bioluminescence intensity throughout the study period. Osteomyelitis resolved in 2/8 hAmSC-treated animals and 0/8 vehicle-treated animals as evidenced by bioluminescence imaging and histological examination for bacteria/inflammation at sacrifice. Radiograph scores for secondary bone changes were lower in mice treated with hAmSC than vehicle at 10, 20 and 30 days post injection. Median inflammatory score was lower in the hAmSC-treated mice than vehicle treated controls. Conclusions. A single injection of hAmSC was effective at reducing the severity of S. aureus infection without the use of antibiotics in this chronic implant associated osteomyelitis immunosuppressed murine model. In addition to reduced bioluminescence intensity below baseline for 28 days during the study period, infection was eliminated in 25% of animals in the hAmSC-treated group

Introduction: Platelets play a central role in hemostasis and healing processes. Upon their activation, platelet alfa-granules release over 30 cytokines including platelet-derived growth factor (PDGF), transforming growth factor-alfa (TGF-alfa), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF) and also active substances like serotonin, catecholamines, von Willebrand factor, proaccelerin, osteonectin and antimicrobial proteins. By concentrating platelets, platelet-rich plasma (PRP) with higher levels of growth factors might be reached, which could stimulate the healing processes. The activator for PRP is a mixture of thrombin and calcium chloride. After connecting these substances platelet-rich gel (PRG) is formed. Aims: In present study, we investigated in vitro antimicrobial activity of PRG after antibiotic administration. Material and Methods: 30 minutes after iv Amoxillin/ clavulanic acid administration 54 ml of whole blood was collected from each of 10 donors. PRPs were prepared with using GPS system from Biomet. In vitro laboratory susceptibility to PRG was determined by the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Becton Dickinson). Baseline antimicrobial activity was assessed by measuring the zones of inhibition. Agar plates were coated with one of the following strain: Staphylococcus aureus ATCC 43300 (MRSA), Staphylococcus aureus ATCC 25923 (MSSA), Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 35218 (ESBL), Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853. Results: We tested 10 samples of PRG. Zones of inhibition produced by PRG ranged between 6 – 23 mm in diameter. PRG inhibited the growth of Staphylococcus aureus. PG also was active against Escherichia coli, Enterococcus faecalis. No activity against Klebsiella pneumoniae and Pseudomonas aeruginosa was detected. Conclusions: Our previous study showed PRG no activity against Enterococcus faecalis without antibiotic administration. In this investigation we observed PRG strong activity against this bacteria after iv Amoxicillin-clavulanic acid administration. In infections during antibiotic treatment, PRG antimicrobial properties are enhanced by antibiotics that are concentrated in plasma

Aims

The aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses.