Using thermal spraying technique, we developed a novel titanium material coated with hydroxyapatite (HA) containing silver (Ag). In this study, antibacterial activities of the material were examined both in vitro and in vivo. Two different titanium test pieces were prepared. One is the test piece that was coated by HA containing Ag (HA-Ag) and the other is that was coated by HA only, used as a negative control. Antibacterial activity and efficacy of HA-Ag against Staphylococcus aureus, Escherichia coli, and biofilm-forming methicillin-resistant S. aureus (BF-MRSA) was examined by using the Japanese Industrial Standards test (JIS Z2801). Furthermore, surface area where BF-MRSA was attached and proliferated on the test pieces after 24 hours of incubation was calculated by means of scanning electron microscope (SEM). To investigate the antibacterial activity in vivo, the test pieces were inserted subcutisly into the back of SD rats, and BF-MRSA was inoculated into the inserted pieces. On the 7th day after the inoculation, the numbers of adherent bacteria to the pieces were countered by a sterile cotton-tipped swabs method. By the JIS Z2801 test, HA-Ag showed 104 to 105 times stronger antibacterial activity than HA against all bacteria tested in this study. SEM studies revealed the HA-Ag coated material had 30 to 50 times smaller area of attached bacteria than control. In vivo study showed that viable bacterial numbers on surfaces of HA-Ag were 1000 times less than control. These results indicated that the HA-Ag coated materials have antibacterial activities both in vitro and in vivo. Titanium coated with HA containing Ag has a possibility to be a novel antibacterial biomaterial

Implant-related infection is one of the leading reasons for failure in orthopaedics and trauma, and results in high social and economic costs. Various antibacterial coating technologies have proven to be safe and effective both in preclinical and clinical studies, with post-surgical implant-related infections reduced by 90% in some cases, depending on the type of coating and experimental setup used. Economic assessment may enable the cost-to-benefit profile of any given antibacterial coating to be defined, based on the expected infection rate with and without the coating, the cost of the infection management, and the cost of the coating. After reviewing the latest evidence on the available antibacterial coatings, we quantified the impact caused by delaying their large-scale application. Considering only joint arthroplasties, our calculations indicated that for an antibacterial coating, with a final user’s cost price of €600 and able to reduce post-surgical infection by 80%, each year of delay to its large-scale application would cause an estimated 35 200 new cases of post-surgical infection in Europe, equating to additional hospital costs of approximately €440 million per year. An adequate reimbursement policy for antibacterial coatings may benefit patients, healthcare systems, and related research, as could faster and more affordable regulatory pathways for the technologies still in the pipeline. This could significantly reduce the social and economic burden of implant-related infections in orthopaedics and trauma. Cite this article: C. L. Romanò, H. Tsuchiya, I. Morelli, A. G. Battaglia, L. Drago. Antibacterial coating of implants: are we missing something? Bone Joint Res 2019;8:199–206. DOI: 10.1302/2046-3758.85.BJR-2018-0316

Aim. In the current study we aim to characterize the use of cationic host defense peptides (HDPs) as alternative antibacterial agents to include into novel antibacterial coatings for orthopedic implants. Staphyloccous aureus represent one the most challenging cause of infections to treat by traditional antibacterial therapies. Thanks to their lack of microbial resistance described so far, HDPs represent an attractive therapeutic alternative to antibiotics. Furthermore, HDPs have been showed to control infections via a dual function: direct antimicrobial activity and regulation of immune response. However, HDPs functions characterization and comparison is controversial, as changing test conditions or cell type used might yield different effects from the same peptide. Therefore, before moving towards the development of HDP-based coatings, we need to characterize and compare the immunomodulatory and antibacterial functions under the same conditions in vitro of 3 well-known cathelicidins: human LL-37, chicken CATH-2, and bovine-derived IDR-1018. Method. S. aureus, strain SH1000, was incubated with different concentrations of each HDP and bacterial growth was monitored overnight. Primary human monocytes were isolated from buffy coats using Ficoll-Paque density and CD14 microbeads, and differentiated for 7 days to macrophages. After 24h incubation in presence of LPS and HDPs, macrophages cytokines production was measured by ELISA. Macrophages cultured for 24h in presence of HDPs were infected with serum-opsonized S. aureus. 30 min and 24h after infection, bacterial phagocytosis and intracellular killing by macrophages were measured by flow cytometry and colony forming units (CFU) count respectively. Results. All HDPs efficiently inhibit macrophages LPS-mediated activation, as observed by a reduced production of TNF-α and IL-10. Despite a comparable anti-inflammatory action, only CATH-2 shows direct antibacterial properties at concentrations 10-times lower than those needed to stimulate immune cells. Although stimulation with HDPs fails to improve macrophages ability to kill intracellular S. aureus, IDR-1018 decreases the proportion of cells phagocytosing bacteria. Conclusions. In addition to a strong anti-inflammatory effect provided by all HDPs tested, CATH-2 has direct antibacterial effects while IDR-1018 reduces the proportion of macrophages infected by S. aureus. Use of these HDPs in combination with each other or with other conventional antibacterial agents could lead the way to the design of novel antibacterial coatings for orthopedic implants

Prosthetic joint infections represent complications connected to the implantation of biomedical devices. Bacterial biofilm is one of the main issues causing infections from contaminated orthopaedic prostheses. Biofilm is a structured community of microbial cells that are firmly attached to a surface and have unique metabolic and physiological attributes that induce improved resistance to environmental stresses including toxic compounds like antimicrobial molecules (e.g. antibiotics). Therefore, there is increasing need to develop methods/treatments exerting antibacterial activities not only against planktonic (suspended) cells but also against adherent cells of pathogenic microorganisms forming biofilms. In this context, metal-based coatings with antibacterial activities have been widely investigated and used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing the biofilm formation prevention efficacy. Additionally, standardized and systematic approaches to test antibacterial activity of newly developed coatings are still missing, while standard microbiological tests (e.g. soft-agar assays) are typically used that are limited in terms of simultaneous conditions that can be tested, potentially leading to scarce reproducibility and reliability of the results. In this work, we combined the Calgary Biofilm Device (CBD) as a device for high-throughput screening, together with a novel plasma-assisted technique named Ionized Jet Deposition (IJD), to generate and test new generation of nanostructured silver- and zinc-based films as coatings for biomedical devices with antibacterial and antibiofilm properties. During the experiments we tested both planktonic and biofilm growth of four bacterial strains, two gram-positive and two gram-negative bacterial strains, i.e. Staphylococcus aureus ATCC 6538P, Enterococcus faecalis DP1122 and Escherichia coli ATCC 8739 and Pseudomonas aeruginosa PAO1, respectively. The use of CBD that had the only wells covered with the metal coatings while the biofilm supports (pegs) were not sheltered allowed to selectively define the toxic effect of the metal release (from the coating) against biofilm development in addition to the toxic activity exerted by contact killing mechanism (on biofilms formed on the coating). The results indicated that the antibacterial and antibiofilm effects of the metal coatings was at least partly gram staining dependent. Indeed, Gram negative bacterial strains showed high sensitivity toward silver in both planktonic growth and biofilm formation, whereas zinc coatings provided a significant inhibitory activity against Gram positive bacterial strains. Furthermore, the coatings showed the maximal activity against biofilms directly forming on them, although, Zn coating showed a strong effect against biofilms of gram-positive bacteria also formed on uncoated pegs. We conclude that the metal-based coatings newly developed and screened in this work are efficient against bacterial growth and adherence opening possible future applications for orthopedic protheses manufacturing

The implantation of endoprosthesis is a routine procedure in orthopaedics. Endoprosthesis are mainly manufactured from ceramics, polymers, metals or metal alloys. To ensure longevity of the implants they should be as biocompatible as possible and ideally have antibacterial properties, to avoid periprosthetic joint infections (PJI). Various antibacterial implant materials have been proposed, but have so far only been used sporadically in patients. PJI is one of the main risk factors for revision surgeries. The aim of the study was to identify novel implant coatings that both exhibit antibacterial properties whilst having optimal biocompatibility. Six different novel implant coatings and surface modifications (EBM TiAl6V4, strontium, TiCuN, TiNbN, gentamicin phosphate (GP), gentamicin phosphate+cationic polymer (GP+CP)) were compared to standard CoCrMo-alloy. The coatings were further characterized with regard to the surface roughness. E. coli and S. capitis were cultured on the modified surfaces to investigate the antibacterial properties. To quantify bacterial proliferation the optical density (OD) was measured and viability was determined using colony forming units (CFU). Murine bone marrow derived macrophages (BMMs) were cultured on the surfaces and differentiated into osteoblasts to quantify the mineralisation using the alizarin red assay. All novel coatings showed reduced bacterial proliferation and viability compared to standard CoCrMo-alloy. A significant reduction was observed for GP and GP+CP coated samples compared to CoCrMo (OD. GP,E.coli. = 0.18±0.4; OD. GP+CP,E.coli. = 0.13±0.3; p≤0.0002; N≥7-8). An increase in osteoblast-mediated mineralisation was observed on all surfaces tested compared to CoCrMo. Furthermore, GP and GP+CP coated samples showed a statistically significant increase (M. GP. = 0.21±0.1; M. GP+CP. = 0.25±0.2; p<0.0001; N≥3-6). The preliminary data indicates that the gentamicin containing surfaces have the most effective antibacterial property and the highest osseointegrative capacity. The use of antibiotic coatings on prostheses could reduce the risk of PJI while being applied on osseointegrative implant surfaces

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

Infection in orthopedics is a challenge, since it has high incidence (rates can be up to 15-20%, also depending on the surgical procedure and on comorbidities), interferes with osseointegration and brings severe complications to the patients and high societal burden. In particular, infection rates are high in oncologic surgery, when biomedical devices are used to fill bone gaps created to remove tumors. To increase osseointegration, calcium phosphates coatings are used. To prevent infection, metal- and mainly silver-based coatings are the most diffused option. However, traditional techniques present some drawbacks, including scarce adhesion to the substrate, detachments, and/or poor control over metal ions release, all leading to cytotoxicity and/or interfering with osteointegration. Since important cross-relations exist among infection, osseointegration and tumors, solutions capable of addressing all would be a breakthrough innovation in the field and could improve clinical practice. Here, for the first time, we propose the use antimicrobial silver-based nanostructured thin films to simultaneously discourage infection and bone metastases. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture. These characteristics, in turn, allow tuning silver release and avoid delamination, thus preventing toxicity. In addition, to mitigate interference with osseointegration, here silver composites with bone apatite are explored. Indeed, capability of bone apatite coatings to promote osseointegration had been previously demonstrated in vitro and in vivo. Here, antibacterial efficacy and biocompatibility of silver-based films are tested in vitro and in vivo. Finally, for the first time, a proof-of-concept of antitumor efficacy of the silver-based films is shown in vitro. Coatings are obtained by silver and silver-bone apatite composite targets. Both standard and custom-made (porous) vertebral titanium alloy prostheses are used as substrates. Films composition and morphology depending on the deposition parameters are investigated and optimized. Antibacterial efficacy of silver films is tested in vitro against gram+ and gram- species (E. coli, P. aeruginosa, S. aureus, E. faecalis), to determine the optimal coatings characteristics, by assessing reduction of bacterial viability, adhesion to substrate and biofilm formation. Biocompatibility is tested in vitro on fibroblasts and MSCs and, in vivo on rat models. Efficacy is also tested in an in vivo rabbit model, using a multidrug resistant strain of S. aureus (MRSA, S. aureus USA 300). Absence of nanotoxicity is assessed in vivo by measuring possible presence of Ag in the blood or in target organs (ICP-MS). Then, possible antitumor effect of the films is preliminary assessed in vitro using MDA-MB-231 cells, live/dead assay and scanning electron microscopy (FEG-SEM). Statistical analysis is performed and data are reported as Mean ± standard Deviation at a significance level of p <0.05. Silver and silver-bone apatite films show high efficacy in vitro against all the tested strains (complete inhibition of planktonic growth, reduction of biofilm formation > 50%), without causing cytotoxicity. Biocompatibility is also confirmed in vivo. In vivo, Ag and Ag-bone apatite films can inhibit the MRSA strain (>99% and >86% reduction against ctr, respectively). Residual antibacterial activity is retained after explant (at 1 month). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses

Favoring osseointegration and avoiding bacterial contamination are the key challenges in the design of implantable devices for orthopedic applications. To meet these goals, a promising route is to tune the biointerface of the devices, that can regulate interactions with the host cells and bacteria, by using nanostructured antibacterial and bioactive coatings. Indeed, the selection of adequate metal-based coatings permits to discourage infection while avoiding the development of bacterial resistance and nanostructuring permits to tune the release of the antimicrobial compounds, allowing high efficacy and decreasing possible cytotoxic effects. In addition, metal-doped calcium phosphates-based nanostructured coatings permit to tune both composition and morphology of the biointerfaces, allowing to regulate host cells and bacteria response. To tune the biointerfaces of implantable devices, nanostructured coatings can be used, but their use is challenging when the substrate is heat-sensitive and/or porous. Here, we propose the use of Ionized Jet Deposition (IJD) to deposit metallic and ion-doped calcium phosphates materials onto different polymeric substrates, without heating and damaging the substrate morphology. 3D printed scaffolds in polylactic acid (PLA) and polyurethane (PU), and electrospun matrices in polycaprolactone (PCL) and PLA were used as substrates. Biogenic apatite (HA), ion doped (zinc, copper and iron) tricalcium phosphate (TCP) and silver (Ag) coatings were obtained on porous and custom-made polymeric substrates. Chemical analyses confirmed that coatings composition matches that of the target materials, both in terms of main phase (HA or TCP) and ion doping (presence of Cu, Zn or Fe ion). Deposition parameters, and especially its duration time, influence the coating features (morphology and thickness) and substrate damage. Indeed, SEM/EDS observations show the presence of nanostructured agglomerates on substrates surface. The dimensions of the aggregates and the thickness of the coating films increase increasing the deposition time, without affecting the substrate morphology (no porosity alteration or fibers damaging). The possible substrate damage is influenced by target and substrate material, but it can be avoided modulating deposition time. Once the parameters are optimized, the models show suitable in vitro biological efficacy for applications in bone models, regenerative medicine and infection. Indeed, HA-based coatings favor cells adhesion on printed and electrospun fibers. For antibacterial applications, the ion doped TCP coatings can reduce the bacterial growth and adhesion (E.coli and S.aureus) on electrospun matrices. To conclude, it is possible achieve different properties applying nanostructured coatings with IJD technique on polymeric substrates, modulating deposition conditions to avoid substrate damage

Periprosthetic joint infections (PJI) are one of the most common reasons for orthopedic revision surgeries. In previous studies, it has been shown that silver modification of titanium (Ti-6Al-4V) surfaces by PMEDM (powder mixed electrical discharge machining) has an antibacterial effect on Staphylococcus aureus adhesion. Whether this method also influences the proliferation of bacteria has not been investigated so far. Furthermore, the effect is only limitedly investigated on the ossification processes. Therefore, the aim of this work is to investigate the antibacterial effect as well as the in vitro ossification process of PMEDM machined surfaces modified by integration of silver. In this study, we analyzed adhesion and proliferation of S. aureus in comparison to of surface roughness, silver content and layer thickness of the silver-integrated-PMEDM surfaces (N = 5). To test the in vitro ossification, human osteoblasts (SaOs-2) and osteoclasts (differentiated from murine-bone-marrow-macrophages) were cultured on the silver surfaces (N = 3). We showed that the attachment of S. aureus on the surfaces was significantly lower than on the comparative control surfaces of pure Ti-6Al-4V without incorporated silver, independently of the measured surface properties. Bacterial proliferation, however, was not affected by the silver content. No influence on the in vitro ossification was observed, whereas osteoclast formation was drastically reduced on the silver-modified surfaces. We showed that 1 to 3% of silver in the surface layer significantly reduced the adhesion of S. aureus, but not the proliferation of already attached bacteria. At the same time, no influence on the in vitro ossification was observed, while no osteoclasts were formed on the surface. Therefore, we state that PMEDM with simultaneous silver modification of the machined surfaces represents a promising technology for endoprostheses manufacturing to reduce infections while at the same time optimizing bone ingrowth

Implant-related infections pose a severe economical and societal burden, hence solutions capable of exerting suitable efficacy while not causing toxicity and/or development of resistant bacterial strains are needed. Thus, inorganic antibacterial coatings, and in particular silver coatings, have been extensively studied and used in the clinical practice. However, some drawbacks such as scarce adhesion to the substrate, delamination, or scarce control over silver release have been evidenced. Here, antibacterial nanostructured silver thin films have been developed by a novel plasma-assisted technique. The technique allows deposition on several substrates, including heat sensitive materials and objects of complex shape. Thanks to nanostructured surface, a tuned release can be achieved, preventing citoxicity. Composition (grazing incidence XRD, XPS) and morphology (SEM, AFM, ASTM) of the obtained coatings were characterized, then, their efficacy was validated in vitro against relevant bacterial strains (gram+ S. Aureus and gram– E. Coli). Live/dead kit and confocal microscopy were used to evaluate antibacterial efficacy. Super resolution imaging in the Structured Illumination Microscopy (SIM) setup was used to investigate damage to the bacterial wall. Results indicate that the coatings are composed of nanosized aggregates of metallic silver, indicating a perfect transfer of composition from the deposition target to the coating. Because of the sub-micrometric thickness, they do not alter the micro- and macro- morphology and surface finishing of the implants, developed by the manufacturers to ensure optimal integration in the host bone. Finally, remarkable efficacy was found against both gram+ and gram- bacteria, indicating that the developed coatings are promising for antibacterial applications

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses

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

Bacterial infections related to orthopaedic implants is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. However, antibacterial evaluations of each material are inconsistant, so intercomparison of the antibacterial performance is difficult. This study focused on the Japanese Industrial Standards test (JIS Z2801), which is used for antibacterial evaluation of commodities. The study investigated a suitable evaluation method for in vitro antibacterial activity of biamaterials. In 2007, JIS Z2801 test was approved as international standard ISO 22196. Hydroxyapatite (HA) powder containing 3 wt % of silver oxide (Ag) was sprayed on the surface of titanium disks with the thermal spraying method, using an acetylene torch. This coating has been proved to generate strong antibacterial activity in previous studies. The antibacterial activity was examined with the JIS Z 2801 test and modified JIS Z2801 test. The bacterial strains used in JIS Z2801 test were Escherichia coli (E.coli), Staphylococcus aureus (S.aureus). Bacterial culture medium was instilled onto the surface of the test disks (about 106 cells/ml) and covered with polystyrene films. After cultivation in 1/500 Nutrient Broth for 24 h at 35°C, the bacteria was washed out with the broth. The numbers of viable bacteria in the broth were counted with the agar plate culture method. Additionally, Modified JIS Z2801 test was performed. Modified points were added to the bacterial strain of biofilm-forming methicillin-resistant S.aureus (BF-MRSA), using Fetal Bovine Serum (FBS) as a culture medium, and cultivated at 37°C. In the JIS Z2801 test, Antibacterial activity values of the HA-Ag disk were composed against E.coli 4.1 and S.aureus 5.0. In the modified JIS Z2801 test, antibacterial activity values against E.coli, S.aureus and BF-MRSA were 8.2, 5.5, and 7.1. When this value is greater than 2.0, it shows there is antibacterial activity. The titanium disk coated with HA-Ag showed antibacterial activity in both tests. The JIS Z2801 test is designed to evaluate comodities in poor nutritional environment. However, the environment in the body is eutrophic. It is easy to make bacterial growth. For this reason, it is necessary to consider evaluating for biomaterials with suitable method considered in vivo. In this study, to examine the condition like that found in the body, we cultivated FBS at 37°C. In addition, the antibacterial activity against BF-MRSA was examined to consider the bacterial infection related to orthopaedic implants. The modified JIS Z2801 test showed that it is a suitable evaluation method for in vitro antibacterial activity of biomaterials

Summary Statement. An Implant Disposable Antibacterial Coating (i-DAC®) is described, consisting of a fully resorbable, biocompatible hydrogel, able to release antibacterial and antibiofilm agents. Direct application of the hydrogel on implants prevented infection occurrence in an in vitro model of peri-prosthetic infection. Introduction. Biofilm-related infections are among the main reasons for failure of joint prosthesis with high associated social and economical costs. Bacterial adhesion and subsequent biofilm formation have been shown to develop early after biomaterials implant into the human body, when a “race to the surface” takes place between the host's cells and the colonizing bacteria eventually present at the surgical site. Providing an antibacterial/antibiofilm coating of the implant may then play a strategic role in preventing biofilm related infections. Here we report the results of a series of in vitro and in vivo studies, partially performed under the European 7th Framework Programme (Implant Disposable Antibiotic Coating, IDAC, collaborative research project # 277988), concerning a fully resorbable, biocompatible antibacterial hydrogel coating (DAC®, Novagenit, Italy). The patented hydrogel, a co-polimer comprising of hyaluronic acid and a polylactic acid, has been designed to be mixed with various antibacterial agents and applied directly on the implant at the time of surgery, being fully resorbed within few days. Patients & Methods. The tested hydrogel (DAC®, Novagenit, Italy) is a derivative of a low molecular weight hyaluronan, grafted with poly-D, L-lactic acid and provided in powder form. At the point of care, the powder is hydrated with the antibiotic or antibiofilm solution, thus generating the final compound to be applied onto the implant surface. In vitro studies were conducted using DAC® coating on different biomaterials, including titanium, chrome-cobalt and polyethylene discs. The release of different antibacterial agents, including vancomycin, ciprofloxacin, meropenem, gentamycin, amikacin, tobramycin, clindamycin, doxycyclin, linezolid, NAsalycilate and N-acetylcisteine, adequately mixed with the hydrogel, has been tested by means of gas chromatography and microbiological methods. In vivo studies were then performed on 35 rabbits divided in 7 groups. Animals were implanted with an intramedullary titanium rod in their femur, with a known inoculum of methicillin-resistant Staph. aureus and vancomycin-loaded DAC® at different concentrations (2% and 5%) and compared with controls. Results. Regardless of the tested material, in vitro studies showed the ability of the hydrogel to be loaded and to sustain the release of the following antibacterial/antibiofilm compounds for up to 96 hours: vancomycin, ciprofloxacin, meropenem, gentamycin, amikacin, tobramycin, clindamycin, doxycyclin, linezolid, NAsalycilate, N-acetylcisteine. In vivo studies showed a bacterial load reduction ranging from 94% to 99.9% using vancomycin-loaded DAC®, compared to controls. Discussion/Conclusion. DAC®, a fast-resorbable antibacterial coating, showed the ability to be loaded with various antibacterial compounds and the ability to provide a highly significant reduction of bacterial colonization of implanted biomaterials in an animal model, opening a new pathway to local prevention and treatment of biofilm-/implant-related infections

Aims: A serious problem in orthopedic surgery is the development of infections. The realization of antibacterial and biocompatible/bioactive surfaces represents a challenge. In this study antibacterial behavior has been conferred to surfaces of glasses and glass-caramics, with different degrees of bioactivity, by the introduction of silver through ion exchange. Methods: Materials have been studied both in bulk form, and as coatings. All samples have been analyzed by means of XRD, SEM and EDS before and after the treatment. Coatings’ roughness, porosity and adhesion resistance have been also analyzed. In vitro reactivity and silver release were carried out soaking samples in SBF. Samples have been analyzed by means of SEM/ EDS and XRD; silver has been quantified in solution by GFAAS. Cellular tests have been performed in order to evaluate materials biocompatibility before and after the treatment. Antibacterial behavior has been tested against S.Aureus. Results: Characterization analyses show that glassy or crystalline structure and morphology are maintained after the ion-exchange. As well the coating adhesion resistance is higher then the limit provided by ISO standard for hydroxyapatite coatings. GFAAS analysis determined that silver is gradually released in solution. Cellular tests demonstrate that biocompatibility is generally maintained after treatment but it is closely connected to the amount of silver released. Microbiological tests show antibacterial behavior for silver-doped samples. Conclusions: Ion-exchange technique permits the introduction of controlled silver amount without modifying materials’ structural and morphological properties. Comparing cellular and microbiological tests it is possible to design process parameters to confer, antibacterial properties but not cytotoxic behavior

Periprosthetic joint infections (PJIs) and osteosynthesis-associated infections (OSIs) present significant challenges in trauma and orthopaedic surgery, substantially impacting patient morbidity, mortality, and economic burden. This concern is heightened in patients with pre-existing comorbidities, such as diabetes mellitus, which are not always modifiable at presentation. A novel intraoperative strategy to prevent these infections is the use of Defensive Antibacterial Coating (DAC), a bio-absorbable antibiotic-containing hydrogel applied to implant surfaces at implantation, acting as a physical barrier to prevent infection. The purpose of this study is to assess the use of a commercially available hydrogel (DAC), highlighting its characteristics that make it suitable for managing PJIs and OSIs in orthopaedics and traumatology. Twenty-five patients who underwent complex orthopaedic procedures with intraoperative application of DAC between March 2022 and April 2023 at a single hospital site were included. Post-operative assessment encompassed clinical, laboratory, and radiographic examinations. In this study, 25 patients were included, with a mean age of 70 ± 14.77 years and an average ASA grade of 2.46 ± 0.78. The cohort presented an average Charleston Comorbidity score of 5.45 ± 2.24. The procedures included 8 periprosthetic fractures, 8 foot and ankle surgeries, 5 upper limb surgeries, and 4 elective hip and knee surgeries. Follow-up assessments at 6 weeks and 6 months revealed no evidence of PJI or OSI in any patients, nor were any treatments for PJI or OSI required during the interim period. DAC demonstrated efficacy in preventing infections in high-risk patients undergoing complex orthopaedic procedures. Our findings warrant further investigation into the use of DAC in complex hosts with randomized control trials

Aim. We aimed to compare the in vitro antibacterial activity of Bioactive Glass (BAG) S53P4, which is a compound showing local antibacterial activity, to that of antibiotic-loaded polymethylmethacrylate (PMMA) against multidrug resistant bacteria from osteomyelitis (OM) and prosthetic joint infection (PJI) isolates. Method. We studied convenience samples of multidrug resistant (MDR) microorganisms obtained from patients presenting OM and prosthetic joint infection (PJI). Mixtures containing tryptic soy broth (TSB) and inert glass beads (2mm), BAG-S53P4 granules (0.5–0.8mm and <45 mm) and Gentamicin or Vancomycin-loaded PMMA beads were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MR-CoNS), Pseudomonas aeruginosa or Klebsiella pneumoniae isolates. Glass beads (2.0mm) were used as a control. Antibacterial activity was evaluated by means of time-kill curve, through seeding the strains on blood agar plates, and subsequently performing colony counts after 24, 48, 72, 96, 120 and 168 hours of incubation. Differences between groups were evaluated by means of two-way analysis of variance (ANOVA) and Bonferroni's t test. Results. Inhibition of bacterial growth started soon after 48 hours of incubation, reached zero CFU/ml between 120 and 168 hours of incubation for both antibiotic-loaded PMMA and BAG S53P4 groups, in comparison with inert glass (p< 0.05). No difference regarding time-kill curves between antibiotic-loaded PMMA and BAG S53P4 was observed. Moreover, despite no difference was observed between both Vancomycin - or Gentamicin-loaded PMMA and BAG groups, there was statistical difference between the effectiveness of all treatments (BAG included) against gram-positive cocci and gram-negative bacilli, the latter of which requiring longer time frames for the cultures to yield no bacterial growth. Conclusions. BAG S53P4 presented antibacterial properties as much as antibiotic-loaded PMMA for MDR bacteria producing OM and PJI, although presenting differences between its effectiveness against different bacterial groups

Introduction. The use of narcotic medications to manage postoperative pain after TJA has been associated with impaired mobility, diminished capacity to engage in rehabilitation, and lower patient satisfaction [1]. In addition, side effects including constipation, dizziness, nausea, vomiting and urinary retention can prolong post-operative hospital stays. Intraarticular administration of local anesthetics such as bupivacaine – part of a multimodal postoperative pain management regimen – reduces pain and lowers patients' length of stay [2]. In addition to its anesthetic activity, bupivacaine also has antibacterial activity, particularly against gram-positive bacteria [3]. We have developed a bupivacaine-eluting ultrahigh molecular weight polyethylene (Bupi-PE) formulation; we hypothesized that elution of bupivacaine from polyethylene could have both anesthetic and antibacterial effects in vivo. Methods. In Vivo Antibacterial Efficacy. A total of n=10 male Sprague Dawley rats (250 g) were used in this study. Polyethylene (control) or Bupi-PE plugs (2.5 mm diameter × 5 mm length) were implanted subcutaneously in the rat dorsum. After incision site closure, 5 × 10. 7. cfu of bioluminescent S. aureus were injected around the implants. Bioluminescent signal (photos/second) was measured daily. All rats were euthanized after one week. In Vivo Anesthetic Efficacy. A total of n=10 male Sprague Dawley rats (250 g) were used in this study. Polyethylene (control) and Bupi-PE plugs (2.5 mm diameter × 5 mm length) were implanted into rat knees via a lateral transcondylar approach (Figure 1a). Efficacy was determined by performing a walking track analysis using a highly sensitive Tekscan. ®. sensor (VHR, 5101) (Figure 1b). Walking tracks were performed at baseline (pre-surgery) and every 24 hours for two weeks. All rats were euthanized after two weeks. Results. In Vivo Antibacterial Efficacy. One control rat expired at day 3 and another one expired in day 7. None of the Bupi-PE rats expired during the study. Significantly less bacterial load was observed in rats receiving Bupi-PE, starting at 24 hr post implantation, continuing until the end of study (day 7) (Figure 2). In Vivo Anesthetic Efficacy. 24 hr post surgery, rats in the control group loaded their unoperated hindlimb significantly more than their operated hindlimb. Rats with the Bupi-PE implant loaded both their hindlimbs similarly (Figure 1c). Discussion. The antibiotic activity of the Bupi-PE against an acute S. aureus infection in the subcutaneous dorsum determined that bupivacaine elution from UHMWPE effectively eradicated bacteria within the implant perimeter. In the joint, the release of bupivacaine allowed prompt weightbearing and joint mobilization compared to controls. Conclusion. Bupivacaine-eluting UHMWPE effectively reduced bacterial load in murine subcutaneous dorsum and reduced postsurgical pain in a murine intra-articular model. This material can be promising for use as infection prophylaxis and pain management after TJA. For any figures or tables, please contact authors directly (see Info & Metrics tab above).

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

Summary Statement. The problem facing this research is to promote rapid osteointegration of titanium implants and to minimise the risks of infections by the functionalization with different agents, each designed for a specific action. A patented process gives a multifunctional titanium surface. Introduction. A patented process of surface modification is described. It gives a multifunctional surface with a multiscale roughness (micro and nano topography), that is excellent for osteoblast adhesion and differentiation. It has a high degree of hydroxylation, that is relevant for inorganic bioactivity (apatite-HA precipitation) and it is ready for a functionalization with biological factors. A direct grafting of ALP has been obtained. Moreover, the growth of an antibacterial agent within the surface oxide layer can be useful in order to combine the osteoinduction ability to antimicrobial effects. The selection of an inorganic agent (metal nanoparticles) has the advantage to avoid an eventual development of antibiotic resistance by bacteria. Experimental Methods. Ti-cp and Ti6Al4V samples were polished or blasted, etched in diluted hydrofluoric acid (step 1a), oxidised in hydrogen peroxide (step 1b), incubated in Tresyl chloride (step 2a) and Alkaline phosphatase (ALP) enzyme (step 2b) [1, 2]. A water solution, containing a salt of the metal to be added to the surface as an inorganic antibacterial agent, can be introduced during the oxidation in hydrogen peroxide. Surface morphology and chemical composition were investigated by Scanning Electron Microscopy (SEM) and Field Emission Scanning Electron Microscopy (FESEM) equipped with Energy Dispersive Spectroscopy (EDS). The composition of the outermost surface layer and the chemical state of elements were analyzed by X-Ray Photoelectron Spectroscopy (XPS). The activity of grafted enzyme was studied by an enzymatic activity test. In vitro bioactivity was evaluated by soaking the samples in simulated body fluid and SEM observation to verify hydroxyapatite (HA) precipitation. Antibacterial activity has been determined by inhibition halo test against S aureus. Results and Discussion. A peculiar multi-scale topography, with spongy-like nanometric features, was obtained after the inorganic treatment (step 1a-1b). This morphology can be superimposed on the micro-or macro roughness deriving from acid etching or blasting, by properly optimizing the process parameters. Moreover, the treated surfaces present a high density of hydroxyl groups (XPS data) and they are bioactive (HA precipitation after soaking in SBF for 15 days). Metal (Ag, Cu, Zn) nanoparticles can be grown within the surface oxide layer and they are effective as antimicrobial inorganic agents. The amount of the metal nanoparticles can be tailored in order to have an antibacterial or a bacteriostatic surface. The effective grafting of ALP (step 2a-2b) has been shown by XPS because of the appearance of characteristic peaks in the carbon region. Moreover, it has been observed that ALP maintains its activity after grafting by an enzymatic activity test. ALP grafting improves HA precipitation kinetics. Conclusions. An innovative process was applied to titanium surfaces in order to obtain a better bone integration ability and antibacterial activity. A multi scale surface topography (micro and nano features) was successfully obtained together with an high hydroxylation degree. Modified surfaces are able to induce hydroxyapatite precipitation in vitro and to graft ALP, maintaining its activity and improving bioactivity. Metal nanoparticles embedded in the surface oxide layer have an antibacterial effect

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

Orthopedic metallic medical devices are essential in the treatment of a wide range of skeletal diseases and disabilities. However, they are often related with surgery complications due to acute prosthetic joint infections (PJI) causing devastating complications. Gallium (Ga) antibacterial activity has been recently demonstrated: in aqueous solutions, Ga ionize in a trivalent form Ga. 3+. that can replace Fe. 3+. in bacterial metabolism thus leading to bacteria death. However, it is not yet clear whether such effect is typical to Ga. 3+. release, and how this would affect longer term performance. Here we investigated Ga addition into titanium alloys using metallurgical methods. The study has confirmed that metallurgical addition of gallium even in small amounts (1–2% wt.) to titanium alloys have highly efficient antibacterial function without any visible cytostatic or cytotoxic effects. The presence of gallium within the metal matrix might ensure that antibacterial effect will persist for a long time towards multi-drug resistant S. aureus, which might not be possible if gallium or other metal are only in thin degradable coatings or similar formulations. A 5-logs decrease in CFU number was detected for alloys with 2% Ga and more after 72 h (alamar blue and CFU count assays). The alloys also show to be in vitro cytocompatible with both mature U2OS osteoblasts and progenitor pre-osteoblasts hFOB. Since gallium is metallurgically analogous to aluminium in titanium alloys, it might be used without affecting other alloy properties

Previous studies showed that a fast-resorbable antibacterial hydrogel coating (DAC®, Novagenit Srl, Mezzolombardo, TN, Italy) composed of covalently linked hyaluronan and poly-D, L-lactide, is able to reduce early post-surgical infection both after joint replacement and osteosynthesis. Aim of the present report is to investigate medium-term safety and efficacy of the coating in patients undergoing primary and revision cementless total hip replacement (THR). We designed a two-phases study. In both phases, DAC was prepared according to manufacturer's instructions. In brief, the syringe prefilled with 300 mg of sterile DAC powder was mixed, at the time of surgery, with a solution of 5 mL of sterile water and with the tailored antibiotics, at a concentration ranging from 25 mg/mL to 50 mg/mL. The resulting antibacterial hydrogel was then spread on the outer surface of the prosthesis just before implantation. In the first phase, safety was assessed. Forty-six patients (13 primary and 33 revision THR) were treated with DAC between 2013 and 2015 and evaluated at a 2.8 ± 0.7 years follow up (FU). Antibiotics used for DAC reconstruction were Vancomycin in 33 cases, Vancomycin + Meropenem in 10, Vancomycin + Rifampicin, Teicoplanin or Ceftazidime in 1 case, respectively. Patients were evaluated at 3, 6, 12 months and yearly after with a clinical and radiographic FU. No evidence of infection and no failure/loosening of the prosthesis were observed. No adverse events were reported. The second phase was designed to evaluate efficacy of DAC in preventing infection recurrences after a two stage revision for infected THR. Twenty-seven patients, treated with DAC coating, were compared with a control group of 32, treated in the same time period, without the coating. Demographics, host type and and identified bacteria were similar in the two groups (18.6% of MRSA in DAC group vs 18.5% MRSA in no-DAC group). Patients were evaluated clinically and radiographically at 3, 6, 12 months and yearly thereafter. At a minimum 2 years FU (mean 2.7), we observed 1 dislocation in each group and 2 cases of loosening in the no-DAC group. 4 cases (11%) of recurrence of infection in the no-DAC group (1 MRSA and 3 St. Epidermidis) and no infection recurrences in the DAC group. Due to the small cohort of patients this difference is not statistically significant (Fisher's exact test, p=0.18). This is, to our knowledge, the longest observation concerning the safety and efficacy of the DAC antibacterial coating, applied to hip replacement. The results are in line with those previously reported and point out the absence of side effects of the antibacterial coating in this application and the tendency to reduce re-infection in second stage. This finding needs to be confirmed by a larger dataset

Bone infections due to fractures or implants are a big medical problem. In experimental medicine, many experimental models have been created on different animal species to simulate the disease condition and to do experience treatments. The aim of this paper was to present an antibacterial efficacy of using a bone allograft developed according to the Marburg system of bone bank on a model of chronic osteomyelitis induced in rabbits. In research was used 54 rabbits. Osteomyelitis was induced in rabbits by a human strain of St. aureus ATCC 43300, in the rabbit femur. There have been created 3 groups of animals. In 1. st. group used antibiotic impregnated biodegradable material “PerOssal”. In 2. nd. group used antibiotic impregnated whole bone allograft. In 3. rd. group used antibiotic impregnated perforated bone allograft. Evaluation of installation and evolution of the disease was done by microbiological. A separate study of microbiological data is presented here. This study showed, in the 1. st. and 3. rd. groups there is a persistent decrease in CFU by 14 knocks to 120.4 in the 1. st. group and to 3.5 in the 3. rd. group, and in the 2. nd. group, on the contrary, there is an increase in CFU to 237.33. This shows the lack of effectiveness of using a whole bone allograft. The results showed, after 7 days there was no statistically significant difference between the groups. After 14 days the perforated bone allograft impregnated with antibiotic was better than the biodegradable material “PerOssal”

Orthopaedic infection with bacteria leads to high societal cost and is detrimental to the life quality. Particularly, deep bone infection leading to osteomyelitis results in an inflammatory response whereby localized bone destruction occurs. Current treatments like antibiotic-containing polymethymethacrylate (PMMA) still has the high risk of bacterial resistance. Taking advantages of silver which has antibacterial and anti-inflammatory effect and bioactive collagen, we fabricated a silver nanoparticle (AgNP)-coated collagen membrane by sonication and sputtering. SEM showed good deposition of AgNPs on collagen membrane by both coating methods. The optimal coating concentration was finalized by assessing optimal antibacterial effect against cytotoxicity and finally collagen membrane coated with 1mg/mL AgNPs solution was selected. We also found that the coated collagen membrane demonstrating short-term cytotoxicity within 24 hours with damage to the cell membrane, which was evidenced by MTS and LDH release test, but had no significant influence (p > 0.05) thereafter. The amount of released AgNPs from coated collagen membrane had negligible cytotoxicity (p > 0.05). Confocal laser scanning microscope displayed similar cell morphology in both coated and uncoated collagen membrane. ELISA and qPCR presented the decreased secretion and expression (p < 0.001) of IL-6 and TNF-alpha. Upregulated expression (p < 0.001) of osteogenesis markers (RUNX2, ALP and OPN) could be found and this might be attributed to the modified collagen fibre surface coated by AgNPs. Collectively, the osteogenesis induced by AgNPs demonstrates a promising application in orthopaedic surgery for its use both as an antimicrobial agent, and to enhance bone regeneration

Bacterial infection related to orthopaedic implants is a significant complication today. One of the ways to reduce the incidence of implant-associated infections is assumed to give antibacterial activity to surface of implant itself. We focused attention on Ag, because it has a broad antibacterial spectrum, strong antimicrobial activity and low toxicity. In the previous works, sputtering, electrochemically deposition and sol-gel coating of Ag-containing hydroxyapatite (HA) have been reported. However, since practical technique of HA coating widely used for medical and dental implants has been the “thermal spraying” technique over the last two decades, we aimed at developing the novel thermal spraying technology for Ag-HA coating with antibacterial activity. In this study, physical and chemical properties, in vitro antibacterial activity, inhibition activity of bacterial attachment, HA-forming ability, cytotoxicity and release of Ag ions of the thermal-sprayed Ag-HA coating were evaluated. HA powder containing 3wt % of silver oxide (Ag2O) was sprayed on surface of titanium disks by the thermal spraying method using acetylene torch. SEM images showed a typical structure of the thermal-sprayed coating and the X-ray diffraction (XRD) pattern of the coating showed an amorphous structure. Ag residue in the coating was determined by the elementary analysis. The coating showed strong antibacterial activity and inhabitation activity of bacterial attachment to the methicillin-resistant Staphylococcus aureus (MRSA) in fetal bovine serum (FBS). On the other hand, the coating showed fast HA-forming ability in simulated body fluid (SBF) and no cytotoxicity related to Ag contained in the coating. Therefore, it is expected that the thermal-sprayed Ag-HA coating provides antibacterial and bone-bonding ability on the surface of the implant itself. In addition, though the HA coating is generally liable to adhere bacteria, the thermal-sprayed Ag-HA coating overcomes this problem. Pre-evaluation of release of Ag ions from the Ag-containing ceramic powders indicated that the releasing behavior of Ag ions in SBFs is dependent on the existing form of Ag in the Ag-containing material. It is assumed that most of Ag components in the Ag-HA coating are not retained as metallic Ag but as Ag2O in the amorphous layer. Time-course release tests of Ag ions from the coating in FBS showed a large release rate of Ag ions until 24 h after the immersion. It is expected that the Ag-HA coating could show strong antibacterial activity at the early post-operative stage. In the repeated release testing, the amount of released Ag ions was about 6500 ppb for the first release test, after which it gradually decreased. However, a significant release amount of Ag ions was observed even after the sixth repeat test. Therefore, it was assumed that the thermal-sprayed Ag-HA coating has a slow-release property of Ag ions in FBS

Prior work in the setting of MRSA (clinical isolate), showed that enhancement of Ti6Al4V with anodized nanotubes apparently disrupts the formation and adhesion of MRSA biofilm. The greater amount of cultured MRSA using effluent released from in vitro nanotube surfaces by sonication, compared with thermal plasma sprayed (TPS), indicated probable disruption of biofilm formation and adhesion. The use of nanosilver nanotubes in vivo in a rabbit model showed that after 1 week of infection followed by 1 week of vancomycin treatment, the nanotube MRSA level was 30% that of TPS, and the nanosilver nanotube MRSA level was only 5% of TPS. The implementation of the technology will enhance the remodeled bone locking ability of rough TPS, with surface nanotubes that provide antibacterial properties and increased bone adhesion. Lap shear tests of the nanotubes were performed according to ASTM F1044. In multiple tests, circular adhesive films bonded Ti6Al4V bars containing nanotubes with plain Ti6Al4V. The assemblies were suitably arranged in a tensile tester and pulled to shear failure. There were three modes of failure; shear failure within the adhesive, failure of the adhesive from the plain titanium, and shear failure of the nanotubes from the bar. Tests determined the shear strength of the adhesive and its bonding strength to bare titanium. ImageJ software determined the area of each of the three failure modes. From this analysis, the shear strength of the nanotubes of each sample was calculated. The analyses showed the shear strength of the nanotubes to be as high as 65MPa (9,500psi) with a more typical shear strength of 55MPa (8,000 psi), and several surfaces with 45MPa (6,000 psi). The literature presents models predicting the shear stress in bonded hip stems. Assuming the TPS with nanotubes performs similar to a bonded hip stem, owing to the locking of the bone with the TPS, a typical shear stress prediction for physiological loads is approximately 10 MPa. The nanotube shear strengths were 4–6 times higher than the expected stress during use. For any figures or tables, please contact authors directly

Background. External fixation is a method of osteosynthesis currently required in traumatology and orthopaedic surgery. Pin tract infection is a common problem in clinical practice. Infection occurs after a bacterial colonisation of the pin due to its contact with skin and local environment. To prevent such local contamination, one way to handle this issue is to create a specific coating using method which could be applied in the medical field. In this work we develop a surface coating for external fixator pins based on photocatalytic TiOα properties, producing a bactericidal effect with sufficient mechanical strength to be compatible with surgical use. Method. The morphology and structure of the sol-gel coating layers were characterised using, respectively, scanning electron microscopy and X-ray diffraction. Resistance properties of the coating were investigated by mechanical testing. Photo-degradation of acid orange 7 in aqueous solution was used as a probe, to assess the photo-catalytic activity of titanium dioxide layers under UV irradiation. The bactericidal effect induced by the process was evaluated against 2 strains: a Staphylococcus aureus and a multiresistant Staphylococcus epidermidis. Results. The coated pins showed good mechanical strength and efficient antibacterial effect after 1 hour of UV irradiation. Conclusion. Our study allowed to develop an antibacterial coating for stainless steel commonly used in surgical practice. The process using photoactive TiO2 exposed to UV irradiation is actually well known and applied in many disinfection fields, and exhibited efficiency against the two main bactericidal strains involved in pin tract infections. Mechanical tests confirmed the coating's ability to resist to important stresses. Moreover, this kind of coating created by sol-gel dip-coating techniques is not expensive and quite easy to do. As a consequence, we can hope that this new option would treat preventively pin tract infection, even if there is an important optimisation task to be done in order to amplify bactericidal properties. Level of evidence. II

Aim. The aim of this study was to establish an implant-associated osteomyelitis model in rats with the ability to quantify biofilm formation on implants for prospective evaluation of antibacterial effects on micro-structured implant surfaces. Method. Staphylococcus aureus (strain 36/07) suspension with infection concentrations of 106, 105, 104 and 10. 3. CFU/10µl, respectively was injected in the tibia of 32 rats (n=8 per group). Afterwards a titanium implant (0.8×0.8×12 mm) was inserted. 8 rats were implanted with a preincubated implant (107 CFU/ml, 12 h) and 8 rats served as a control (injection of 0.9% NaCl). During the follow up, clinical, radiographic and µ-CT examinations were conducted. On day 21 post op, all rats were sacrificed. Implant and tibia were explanted under sterile conditions. The implant was stained with green and red fluorescent nucleic acid dye (live/ dead) and analyzed by confocal microscopy. The amount of vivid and dead biomass as well as vivid bacteria on the implant surface was calculated with an image software*. Results. In all groups with artificial infection, local bacterial colonization could be detected without systemic infection. While clinical signs of infections (lameness, subcutaneous abscesses) decreased, the volume of bacterial colonization increased on the implant surface with decreasing initial infection CFU. Preincubated implants showed a similar bacterial colonialization of the surface as implants which were infected with 106 CFU as well as a similar bone disintegration due to ongoing osteomyelitis. Conclusions. Establishment of the implant-associated infection model in rats with subsequent quantification of the vivid bacterial volume via confocal microscopy was successful and is now applicable for the evaluation of micro-structured antibacterial implant surfaces. Pre incubation of implants with initiating biofilm formation was established as alternative onset of infection. This work was part of BIOFABRICATION for NIFE and funded by Volkswagen Foundation and MWK. * Imaris® ×64 6.2.1

Bioactive glasses (BAGs) are bone substitutes with bone bonding, angiogenesis promoting and antibacterial properties. The bioactive process leading to bone bonding has been described as a sequence of reactions in the glass and at its surface. Implantation of the glass is followed by a rapid exchange of Na+ in the glass with H+ and H3O+ from the surrounding tissue, leading to the formation of silanol (SiOH) groups at the glass surface. Due to migration of Ca2+ and PO43− groups to the surface and cystallization, a CaO-P2O5 hydroxyapatite (HA) layer is formed on top of the Si-rich layer. Finally, cell interactions with the HA layer subsequently initiate the bone forming pathway. The rapid increase in pH and the subsequent osmotic effect caused by dissolution of the glass have been suggested to partly explain the antibacterial properties observed for BAGs. Comparing bactericidal effects of different BAGs, BAG-S53P4 has been shown to be the most effective, with the fastest killing or growth inhibitory effect. This antibacterial effect has been observed in vitro for all pathogens tested, including the most important aerobic and anaerobic pathogens, as well as very resistant bacteria. In a multicentre study in 2007–2009, BAG-S53P4 was used as bone graft substitute in treatment of osteomyelitis. Eleven patients (nine males, two females) with a radiologically diagnosed osteomyelitis in the lower extremity (N=10) and in the spine (N-1) participated. In the operation, the infected bone and the soft tissue were removed, and the cavitary bone defects were filled with BAG-S53P4 (BonAlive™, Bonalive Biomaterials Ltd., Finland). In four patients, muscle flaps were used as part of the treatment. Eight patients were treated in a one-stage procedure. Kanamycin granules were used in one patient and Garamycin granules (Septocol ®) in two patients. Patient data were obtained from hospital patient' records until August 2010, resulting in a mean follow-up period of 29 months (range 15–43). BAG-S53P4 was well tolerated; no BAG-related adverse effects were seen in any patient. The use of BAG-S53P4 as a bone graft substitute resulted in a fast recovery. Long-term clinical outcome was good or excellent in ten of eleven patients. These primary results indicate that BAG-S53P4 can be considered as a good and usable material in treatment of osteomyelitis. After this study BAG-S53P4 has been used in several other patients with very promising results

Introduction: Since the establishment of osteosynthesis as the treatment of choice for bone fractures, the issues relating to complications and their prophylaxis have become a major topic of scientific discussion and research in the field of traumatology. Infection of the bone and soft tissue represents one major complication that arises after the implantation of osteosynthetic material at the fracture site. The treatment of these infections is often time-consuming and involves repeated, extensive surgical interventions. The aim of this study was to acquire information about the effect of an antibacterial and biodegradable poly-L-lactide (PLLA) coated titanium plate osteosynthesis on local infection resistance. Material and Methods: We compared infection rates in white New Zealand rabbits after titanium plate osteosynthesis of the tibia with or without antibacterial coating after local percutaneous bacterial inoculations at different concentrations (2x10. 5. –2xlO. 8. ):. group I (n=12):uncoated titanium plate,. group II (n=12): PLLA coated titanium plate,. group III (n=12): titanium plate coated with PLLA + 3% Rifampicin and 7% Fusidic acid, group IV (n= 12): titanium plate coated with PLLA + 2% Octenidin und 8% Irgasan. The plate, the contaminated soft tissues and the underlying bone were removed under sterile conditions after 28 days and quantitatively evaluated for bacterial growth. A stepwise experimental design with an “up-and-down” dosage technique was used to adjust the bacterial challenge in the area of the ID50 (50% infection dose). Statistical evaluation of the differences between the infection rates of both groups was performed using the two-sided Fisher exact test (p< 0.05). Results: The overall infection rate was 50%. For group I and II the infection rate was both 83% (10 of 12 animals). In group III and IV with antibacterial coating the infection rate was both 17% (2 of 12 animals). The ID50 in the antibacterial coated groups III and IV was recorded as lxl10. 8. CFU, whereas the ID50 values in the groups I and II without antibacterial coating were a hundred times lower at lxl10. 6. CFU, respectively. The difference between the groups with and without antibacterial coating was statistically significant (p=0.033). Conclusions: Using an antibacterial biodegradable PLLA coating on titanium plates, a significant reduction of infection rate in a canine infection model could be demonstrated. For the first time we were able to show, under standardized and reproducable conditions, that an antiseptic coating leads to the same reduction in infection rate as an antibiotic coating. Taking the problem of antibiotic-induced bacterial resistance into consideration, we thus regard the antiseptic coating, which shows the same level of effectiveness, as advantageous

Infection remains among the first reasons for failure of joint prosthesis. Currently, the golden standard for treating prosthetic joint infections (PJIs) is two-stage revision. However, two-stage procedures have been reported to be associated with higher costs and possible higher morbidity and mortality, compared to one-stage. Furthermore, recent studies showed the ability of a fast-resorbable, antibacterial-loaded hydrogel coating to reduce surgical site infections after joint replacement, by preventing bacterial colonization of implants. Aim of this study was then to compare the infection recurrence rate after a one-stage, cemenless exchange, performed with an antibacterial coated implant versus a standardized two-stage revision procedure. In this two-center prospective study, 22 patients, candidate to revision surgery for PJI, were enrolled to undergo a one-stage revision surgery with cementless implants, coated intra-operatively with a fast-resorbable, antibiotic-loaded hyaluronan and poly-D,L-lactide based hydrogel coating (“Defensive Antibacterial Coating”, DAC, Novagenit, Italy). DAC was reconstructed according to manufacturer indications and loaded with Vancomycin or Vancomycin + Meropenem, according to cultural examinations, and directly spread onto the implant before insertion. This prospective cohort was compared with a retrospective series of 22 consecutive patients, matched for age, sex, host type, site of surgery, that underwent a two stage procedure, using a preformed, antibiotic-loaded spacer (Tecres, Italy) and a cementless implant. The second surgery, for definitive implant placing, was performed only after CRP normalization and no clinical sign of infection. Clinical, laboratory and radiographic evaluation were performed at 3, 6 and 12 months, and every 6 months thereafter. Infection recurrence was defined by the presence of a sinus tract communicating with the joint, or at least two among the following criteria: clinical signs of infections; elevated CRP and ESR; elevated synovial fluid WBC count; elevated synovial fluid leukocyte esterase; a positive cultural examination from synovial fluid; radiographic signs of stem loosening. The two groups did not differ significantly for age, sex, host type and site of surgery (18 knees and 4 hips, respectively). The DAC hydrogel was loaded intra-operatively, according to cultural examination, with vancomycin (14 patients) or vancomycin and meropenem (8 cases). At a mean follow-up of 20.2 ± 6.3 months, 2 patients (9.1%) in the DAC group showed an infection recurrence, compared to 3 patients (13.6%) in the two-stage group. No adverse events associated with the use of DAC or radiographic loosening of the stem were observed at the latest follow-up months. This is the first report on one-stage cementless revision surgery for PJI, performed with a fast-resorbable antibacterial hydrogel coating. Our data, although in a limited series of patients and at a relatively short follow-up, show similar infection recurrence rate after one-stage exchange with cementless, coated implants, compared to two-stage revision. These findings warrant further studies in the possible applications of antibacterial coating technologies to treat implant-related infections

Aim. The treatment of osteomyelitis often requires extensive surgical debridement and removal of all infected tissues and foreign bodies. Resulting bone loss can then eventually be managed with antibacterial bone substitutes, that may also serve as a regenerative scaffold. Aim of the present study is to report the clinical results of a continuous series of patients, treated at our centre with an antibacterial bioglass*. Method. From November 2010 to May 2016, a total of 106 patients, affected by osteomyelitis, were included in this prospective, single centre, observational study. Inclusion criteria were the presence of osteomyelitis with a contained bone defect or segmental defects < 10 mm, with adequate soft tissue coverage. All patients underwent a one-stage procedure, including surgical debridement and bone void filling with the bioactive glass*, with systemic antibiotic therapy and no local antibiotics. Clinical, radiographic and laboratory examinations were performed at 3, 6 and 12 months and yearly thereafter. Results. Two patients were lost to follow-up, hence a total of 104 patients (65 males, 39 females; mean age: 46 ± 17 years, min 6 – max 81) were available at an average follow-up of 38 ± 26 months (range: 12 – 68); forty-eight patients (46.1%) were classified as Type A, 48 (46.1%) as Type B and 8 (7.7%) as Type C hosts, according to McPherson classification. Tibia (N=61) and femur (N=33) were the most common involved bones. On average patients had undergone 2.1 ± 1.3 (min 0 – max 7) previous surgical operations, with a mean infection duration of 18.7 ± 16.6 months (min 2 – max 120). Infection recurrence was observed in 10 patients (9.6%), most often within one year from surgery (8/10). Negative prognostic factors included infection duration > 2 years, Gram negative or mixed flora or negative cultural examination, Type B or C hosts and soft tissue defect. No side effects or complications related to bioglass were noted. Conclusions. This is to our knowledge the longest and the largest single centre consecutive series of patients, affected by bone infections of the long bones, treated according to a one-stage procedure using bioactive glass. Our results confirm, on a larger population and at a longer follow-up, previous reports. Early treatment, pathogen identification and adequate management of soft tissues should be considered to further reduce infection recurrence rate. *BonAlive®

The aim of the study is to evaluate the effect of acrylic cement CMW1 (DePuy) containing 2,5% of gentamicin and addition of 5 % and 10 % of respective vancomycin, meropeneme and ceftriaxone on growth inhibition of reference strains of MRSA, E. faecalis, S. aureus, P. aeruginosa and E. coli. From every portion of investigated acrylic cement CMW1 discs were cut with a diameter of 15mm and a thickness of 5mm, average weight 1.365 g (+/− 0,257g). Inoculum was prepared with the reference strains: MR3 S. aureus methicillin-resistant (MRSA), ATCC 29219 E. faecalis, ATCC 25923 S. ureus, ATCC 27853 P. aeruginosa and ATCC 25922 E. coli. A colonies of bacteria taken from a 18-hour culture on solid medium were addend to tubes with sterile physiological saline solution to obtain a density of 0.5 McFarland (5 × 105 CFU / ml). The suspension was distributed evenly over the Mueller-Hinton (MH) medium (Biomerieux, France). Prepared discs of CMW1 cement were put with a sterile forceps on the plate with a dry medium. The plates were incubated aerobically at 24 hr and the temp. 37°C. After 24 hours the diameter of zone of inhibition of bacterial growth on a plate was measured (in mm) and average size of the inhibition zone was calculated. The CMW1 cement inhibited to a comparable degree growth of reference strains with the exception of E. faecalis. The addition of vancomycin increased by 1/5 inhibitory potential of CMW1 cement on growth of MRSA, S. aureus, P. aeruginosa and E. coli. and significantly for E. faecalis. Changing the concentration of vancomycin, meropeneme and ceftriaxone from 5% to 10% do not increased the inhibitory potential of CMW1 cement on the growth of MRSA, S. aureus, P. aeruginosa, E. coli and E. faecalis. Addition of meropeneme increased inhibitory potential of CMW1 cement against MRSA by 1/3, P. aeruginosa and E. coli by ½, E. faecalis by 3/4 and against S. aureus by 100%. Addition of ceftriaxone to CMW1 cement increased the inhibiting of the growth of MRSA similiarly to 5% and 10% of vancomycin, E. faecalis as meropeneme 5% and 10 %, while the growth of S. aureus and P. aeruginosa, less than meropeneme. Addition of antibiotics to acrylic cement increased its antibacterial properties. Increase if vancomycine concentrations from 5 to 10% had no stronger antibacterial effect

Platelet-rich plasma is a new inductive therapy which is being increasingly used for the treatment of the complications of bone healing, such as infection and nonunion. The activator for platelet-rich plasma is a mixture of thrombin and calcium chloride which produces a platelet-rich gel. We analysed the antibacterial effect of platelet-rich gel in vitro by using the platelet-rich plasma samples of 20 volunteers. In vitro laboratory susceptibility to platelet-rich gel was determined by the Kirby-Bauer disc-diffusion method. Baseline antimicrobial activity was assessed by measuring the zones of inhibition on agar plates coated with selected bacterial strains. Zones of inhibition produced by platelet-rich gel ranged between 6 mm and 24 mm (mean 9.83 mm) in diameter. Platelet-rich gel inhibited the growth of Staphylococcus aureus and was also active against Escherichia coli. There was no activity against Klebsiella pneumoniae, Enterococcus faecalis, and Pseudomonas aeruginosa. Moreover, platelet-rich gel seemed to induce the in vitro growth of Ps. aeruginosa, suggesting that it may cause an exacerbation of infections with this organism. We believe that a combination of the inductive and antimicrobial properties of platelet-rich gel can improve the treatment of infected delayed healing and nonunion

The release of various penicillins and other antibiotics from two brands of polymerised bone cement has been studied in vitro and in vivo in mice. Bone cement plugs containing antibiotics demonstrated antibacterial activity as a result of diffusion of antibiotic from the plugs into the surrounding medium. With all antibiotics tested, from 2-5 to 10 per cent of the antibiotic in the cement was released in vitro in active form within twenty-four hours. Most of the activity appeared within three hours of the start of the test, but in some cases low levels of activity were detected after four days. Antibiotic cement plugs implanted in mice and rats produced low concentrations of antibiotic in the blood up to two hours after implantation, but activity was seldom detected subsequently. In general, penicillins and non-penicillin antibiotics showed similar diffusion characteristics, and the pattern of release in vitro and in vivo was consistent with the leaching of antibiotic from, or near, the surface of the bone cement

Introduction: Extracorporeal shock wave therapy (ESWT) covers a multitude of different indications in modern orthopedics, however, bacterial infections are still considered as contraindications. The goal of the present study was to determine the effect of ESWT on growth of clinically relevant bacteria in orthopedic and trauma surgery. Methods: Standardised suspensions of a methicillin sensitive and a methicillin resistant strain of Staphylococcus aureus, and reference strains of Staphylococcus epidermidis, Pseudomonas aeruginosa and Enterococ-cus faecalis were subjected to 4000 impulses of high-energy shock waves with an energy flux density (EFD) of 0.96 mJ/mm2 and a frequency of 2 Hz. Furthermore, corresponding suspensions of S. aureus ATCC 25923 were exposed to different impulse rates of shock waves (1000 to 6000 impulses) and to different EFDs up to a maximum of 0.96 mJ/mm2 (2 Hz) to evaluate the influence of shock wave parameters. Subsequently, viable bacteria were quantified by culture and compared with an untreated control. Results: A highly significant antibacterial effect of the ESWT was demonstrated for all bacterial strains with a reduction of growth to values between 1,1% and 29,7% (P < 0.01). Reference strains of S. aureus and S. epidermidis reacted most sensitive whereas E. faecium demonstrated highest resistance against high-energy shock waves. After applying different energy levels to S. aureus, a significant bactericidal effect was observed only with a minimum threshold EFD of 0.59 mJ/mm2 (P < 0.05). A threshold impulse rate of more than 1000 impulses could be defined to reduce bacterial growth of S. aureus (P < 0.05). Further elevation of energy and impulse rate exponentially increased bacterial killing. Conclusions: ESWT proved to exert significant antibacterial effect in an energy-dependent manner. The results suggest that infections are not necessarily contraindications to shock wave therapy and could even represent a new indication for ESWT. However, clinical relevance should be assessed in vivo in an animal model

Introduction: Over 75% of hospital-acquired infections are methicillin-resistant staphyloccoal (MRSA) infections. There is an urgent need to find alternatives to treat such infections. We report our experience with the use of a topical antibacterial agent, Polytoxinol, PT (TM), combined with debridement, for the treatment of wound and bone infections where antibiotics had failed. PT is a complex formulation of eucalyptic plant extracts, shown to be strongly bactericidal in vitro against a broad range of aerobic bacteria. Methods: Staphylococcal infections were diagnosed in 6 cases by culture; 4 of these were confirmed as involving MRSA. In 8/9 patients, infection was localised at the site of ligament and/or bone surgery for repair of traumatic injury, or for prosthetic joint replacement. Results: Prior to this series, PT was applied as a biological wound sealant to 180 orthoapedic patients with two instances of localised sensitivity. Eight of the current 9 cases of wound infection, included 4 verified cases involving methicillin-resistant Staphylococcus aureus, were successfully treated by topical application of Polytoxinol, either without (6 patients), or in combination with systemic antibiotics (3 patients). In 8 patients, Polytoxinol application was followed by reduced inflammation, rapid granulation and healing even where infection was of > 2 years standing. Adverse local tissue reaction shown by 1 patient quickly subsided on withdrawal of Polytoxinol. Conclusions: Polytoxinol antimicrobial liquid applied topically to infected wounds and bone is an effective broad spectrum bactericide. It has the potential to supplement, or in many instances replace, antibiotics in the treatment of such infections

Aims

Biofilm formation is intrinsic to prosthetic joint infection (PJI). In the current study, we evaluated the effects of silver-containing hydroxyapatite (Ag-HA) coating and vancomycin (VCM) on methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation.

This study aimed to verify the hypothesis that an antibiotic loaded hydrogel, defensive antimicrobial coating (DAC), reduces overall complication and infection rates when used for high-risk primary and revision total hip arthroplasty (THA).

This was a retrospective study matched cohort study of 238 patients, treated with cementless implants with and without DAC. A sub-group analysis of patients undergoing 2nd stage revision THA for prosthetic joint infection (PJI) was also conducted. Re-infection rates within 2 years, complications necessitating surgical intervention and radiographic analysis for aseptic loosening was assessed.

The mean age was 68.3±11.5 years, with 39 (32.8%) Macpherson class A, 64 (53.8%) class B and 16 (13.4%) class C patients. 4 (3.4%) patients in the DAC group developed complications including 1 PJI and 1 delayed wound healing, while 13 (10.9%) patients in the control group developed complications including 5 PJIs and 3 delayed wound healing (p=0.032). PJI rates (p=0.136) and delayed wound healing rates (p=0.337) were not statistically significant. For 2nd stage revision THA for PJI there were 86 patients in the DAC group and 45 in the control group. 1 (1.2%) patient in the DAC group developed complications with no recurrences of infection or delayed wound healing, while 10 (22.2%) patients in the control group developed complications including 4 recurrent PJI and 1 delayed wound healing (p=0.003). Recurrent PJI rates were statistically significant (p=0.005) while delayed wound healing rates were not (p=0.165). Patients treated with DAC also had lower rates of aseptic loosening (0% vs 6.7%; p=0.015).

Antibiotic impregnated hydrogel coatings on cementless implants showed decreased complication rates after complex primary or revision THA. In 2nd stage revision THA for PJI, it was associated with reduced risk of re-infection and aseptic loosening.

We developed a novel silorane-based biomaterial (SBB) for use as an orthopedic cement. SBB is comprised of non-toxic silicon-based monomers, undergoes non-exothermic polymerization, and has weight-bearing strength required of orthopedic cements. We sought to compare the antibiotic release kinetics of this new cement to that of commercially available PMMA bone cement. We also evaluated each material's inherent propensity to support the attachment of bacteria under both static and dynamic conditions.

One gram of either rifampin or vancomycin was added to 40g batches of PMMA and SBB. Pellets were individually soaked in PBS. Eluate was collected and tested daily for 14 days using HPLC. Compressive strength and modulus were tested over 21 days. Bioassays were used to confirm the bioactivity of the antibiotics eluted.

We measured the growth and maturation of staphylococcus aureus (SA) biofilm on the surface of both PMMA and SBB disks over the course of 72 hours in a static well plate and in a dynamic biofilm reactor (CDC Biofilm Reactor). N=4 at 24, 48, and 72 hours. A luminescent strain of SA (Xen 29) was employed allowing imaging of bacteria on the discs.

SBB eluted higher concentrations of vancomycin than did PMMA over the course of 14 days (p<0.001). A significant 55.1% greater day 1 elution was observed from SBB. Silorane cement was able to deliver rifampin in clinically favorable concentrations over 14 days. On the contrary, PMMA was unable to deliver rifampin past day 1. The incorporation of rifampin into PMMA severely reduced its mechanical strength (p<0.001) and modulus (p<0.001).

Surface bacterial radiance of PMMA specimens was significantly greater than that of SBB specimens at all time points (p<0.05).

The novel silorane-based cement demonstrated superior antibiotic release and, even without antibiotic incorporation, demonstrated an innate inhabitation to bacterial attachment and biofilm.

Musculoskeletal disorders is one of most important health problems human population is facing includes. Approximately 310 thousand of hip protheses have been used in 45 years and older patients in total according to the recent studies have been done. [1, 2]. Many factors, including poor osseointegration or relaxation of the implant due to stress, limit the life of the load-bearing implants [3]. To overcome these difficulties and to protect metal implants inside the body, the surfaces of the implants were coated with silver ion doped hydroxyapatite/bioglass. In this study, silver doped hydroxyapatite ceramic powder and 6P57 bioglass were synthesized. Two different coating suspensions, 100% bioglass and 70% Ag-HAp / 30% bioglass, were prepared in methyl alcohol with a solid content of 1% by weight.

Two layers were coated on the external fixator nails by using electrospray method with the bioglass and Ag-Hap/Bioglass suspensions respectively. The coated implants were cut with an equal surface area and kept in human blood plasma for different time. The scanning electron microscopy (SEM, Zeiss Supra 50VP and Zeiss Evo 50EP) and stereo microscope (Zeiss Axiocam Stemi 2000-C) were used to characterize microstructure and thickness of coated surface. Energy dispersive X-ray Spectroscopy was used characterized of chemical composition of coating. Changing of pH value of plasma was measured by pH meter (Hanna HI83414). In addition, the ICP method was used to determine the elements contained in the plasma fluid after dissolution. As a result of this study, physical and chemical changes occurring on the coating surface in different time periods are presented in detail

Aims

Surgical site infection (SSI) is a common complication of surgery with an incidence of about 1% in the United Kingdom. Sutures can lead to the development of a SSI, as micro-organisms can colonize the suture as it is implanted. Triclosan-coated sutures, being antimicrobical, were developed to reduce the rate of SSI. Our aim was to assess whether triclosan-coated sutures cause a reduction in SSIs following arthroplasty of the hip and knee.

With an ever-increasing aging population, total hip and knee arthroplasty is projected to increase by 137% and 601%, respectively, between the period; 2005–2030. Prosthetic Join Infection (PJI) occurs in approximately 2% of total joint replacements (TJRs) in the U.S. PJI is primarily caused by adherence of bacteria to the surface of the prosthesis, ultimately forming an irreversibly attached community of sessile bacteria, known as a biofilm, highly tolerant to antibiotic treatment. Often the only resolution if the ensuing chronic infection is surgical removal of the implant – at high cost for the patient (increased morbidity), and for healthcare resources. Strategies to prevent bacterial adherence have significant potential for medical impact. Laser surface treatment using an automated continuous wave (CW) fiber laser system has shown promise in producing anti-adherent and bactericidal surfaces. Work presented here aims to investigate the effect of this approach on orthopaedic metals as a proof of concept, specifically Ti-6Al-4V (kindly supplied by Stryker Orthopaedics, Limerick). A coupon was surface treated using a laser (MLS-4030; Micro Lasersystems BV, Driel). Samples were incubated in Müller Hinton Broth (MHB) inoculated with methicillin resistant Staphylococcus aureus (MRSA; ATCC 43300) for 24h before Live/Dead staining (BacLight™ solution; Molecular Probes) and inspection by fluorescence microscopy (GXM-L3201 LED; GX Optical). Images were analysed using ImageJ software (NIH) and a significant reduction (p > 0.05, n=24) in total biofilm coverage and Live/Dead ratio was observed between the laser treated and as received surfaces. This data demonstrates the anti-adherent, and indeed bactericidal, effect of Laser-surface treatment.

The development and pre-clinical evaluation of nano-texturised, biomimetic, surfaces of titanium (Ti) implants treated with titanium dioxide (TiO₂) nanotube arrays is reviewed. In vitro and in vivo evaluations show that TiO₂ nanotubes on Ti surfaces positively affect the osseointegration, cell differentiation, mineralisation, and anti-microbial properties. This surface treatment can be superimposed onto existing macro and micro porous Ti implants creating a surface texture that also interacts with cells at the nano level. Histology and mechanical pull-out testing of specimens in rabbits indicate that TiO₂ nanotubes improves bone bonding nine-fold (p = 0.008). The rate of mineralisation associated with TiO₂ nanotube surfaces is about three times that of non-treated Ti surfaces. In addition to improved osseointegration properties, TiO₂ nanotubes reduce the initial adhesion and colonisation of Staphylococcus epidermidis. Collectively, the properties of Ti implant surfaces enhanced with TiO₂ nanotubes show great promise.

Cite this article: Bone Joint J 2018;100-B(1 Supple A):9–16.

Bacterial contamination of endoprostheses especially in revision surgery is an upcoming problem according to increasing number of joint replacements. Early adherence of bacteria producing a biofilm is difficult to treat. Silver coating of implants offers the opportunity to avoid bacterial adhesions acting against all relevant bacteria causing infections on the implant. We developed a new technique of nano-silver coating using elemental silver covered with SiOxCy whose thickness can be varied determing duration of the coating on the implant. The SiOxCy and silver is completely soluble at least at 3 months. The silver coatings used so far are measuring at least 10um and they are not soluble making a cementless implantation of the endoprostheses impossible. The aim of this study was to test the compatibility of the new combined coating with human osteoblastic cells.

The test was carried out with fHOB 1.19 (ATCCR CRL-11372TM). The cells were cultivated in 1:1 mixture of DMEM/Ham's F12 with usual supplements. The protein content was measured colourimetrically using BCA reagents and staining of the cells was done with XTT-reagent (Roche). The cells were incubated on Titanium and PEEK with and without coating for 2,6,16 and 48 hours.

No adverse effects of the silver coating on the early cell adhesion at 2 and 6 hours and the further proliferation at 16 and 48 hours were observed. The adhesion on Titanium showed no significant difference against coated Titanium but an improvement of cell adhesion was seen on coated PEEK.

This soluble silver coating did not negatively influence human osteoblastic cells. As the complete surfacing is soluble it might be possible to combine early protection against bacteria and osseous integration. An animal study is in progress verifying the in vitro results. It should investigate the maximum duration of the coating on the implant not disturbing osseous integration.

Copal bone cement loaded with gentamicin and clindamicin was developed recently as a response to the emerging occurrence of gentamicin-resistant strains in periprothetic infections. The objective of this study was to compare the in vitro antibiotic release and antimicrobial efficacy of gentamicin/clindamicin-loaded Copal bone cement and gentamicin-loaded Palacos R-G bone cement, as well as biofilm formation on these cements.

In order to determine antibiotic release, cement blocks were placed in phosphate buffer and aliquots were taken at designated times for measurement of antibiotic release. In addition, the bone cement discs were pressed on agar to study the effects of antibiotic release on bacterial growth. Biofilm formation on the different bone cements was also investigated after 1 and 7 days using plate counting and confocal laser scanning microscopy (CLSM). Experiments were done with a gentamicin-sensitive S. aureus and a gentamicin-resistant CNS.

Antibiotic release after 672 h from Copal bone cement was more extensive (65% of the clindamycin and 41% of the gentamicin incorporated) than from Palacos R-G (4% of the gentamicin incorporated). The higher antibiotic release from Copal resulted in a stronger and more prolonged inhibition of bacterial growth on agar. Plate counting and CLSM of biofilms grown on the bone cements showed that antibiotic release reduced bacterial viability, most notably close to the cement surface. Moreover, the gentamicin-sensitive S. aureus formed gentamicin-resistant small colony variants on Palacos R-G, and therefore, Copal was much more effective in decreasing biofilm formation than Palacos R-G.

Biofilm formation on bone cement could be more effectively reduced by incorporation of a second antibiotic, next to gentamicin. Antibiotic release from the cements had a stronger effect on bacteria close to the cement than on bacteria at the outer surface of the bio-film. Clinically, bone cement with two antibiotics may be more effective than cement loaded with only gentamicin. The clinical efficacy of antibiotic loaded bone cements in combination with systemic antibiotics can be explained because antibiotics released from cements kill predominantly the bacteria in the bottom of the biofilm, whereas systemic antibiotics can only deal with bacteria at the outer surface of the biofilm.

Aims. There is a lack of biomaterial-based carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotics for bone infections. RIF is also known for causing rapid development of antibiotic resistance when given as monotherapy. This in vitro study evaluated a clinically used biphasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). Methods. The CaS/HA composites containing RIF/GEN/VAN, either alone or in combination, were first prepared and their injectability, setting time, and antibiotic elution profiles were assessed. Using a continuous disk diffusion assay, the antibacterial behaviour of the material was tested on both planktonic and biofilm-embedded forms of standard and clinical strains of Staphylococcus aureus for 28 days. Development of bacterial resistance to RIF was determined by exposing the biofilm-embedded bacteria continuously to released fractions of antibiotics from CaS/HA-antibiotic composites. Results. Following the addition of RIF to CaS/HA-VAN/GEN, adequate injectability and setting of the CaS/HA composites were noted. Sustained release of RIF above the minimum inhibitory concentrations of S. aureus was observed until study endpoint (day 35). Only combinations of CaS/HA-VAN/GEN + RIF exhibited antibacterial and antibiofilm effects yielding no viable bacteria at study endpoint. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with CaS/HA-VAN/GEN + RIF. Conclusion. Our in vitro results indicate that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for local delivery in clinically demanding bone infections. Cite this article: Bone Joint Res 2022;11(11):787–802

Aims. The optimum type of antibiotics and their administration route for treating Gram-negative (GN) periprosthetic joint infection (PJI) remain controversial. This study aimed to determine the GN bacterial species and antibacterial resistance rates related to clinical GN-PJI, and to determine the efficacy and safety of intra-articular (IA) antibiotic injection after one-stage revision in a GN pathogen-induced PJI rat model of total knee arthroplasty. Methods. A total of 36 consecutive PJI patients who had been infected with GN bacteria between February 2015 and December 2021 were retrospectively recruited in order to analyze the GN bacterial species involvement and antibacterial resistance rates. Antibiotic susceptibility assays of the GN bacterial species were performed to screen for the most sensitive antibiotic, which was then used to treat the most common GN pathogen-induced PJI rat model. The rats were randomized either to a PJI control group or to three meropenem groups (intraperitoneal (IP), IA, and IP + IA groups). After two weeks of treatment, infection control level, the side effects, and the volume of antibiotic use were evaluated. Results. Escherichia coli was the most common pathogen in GN-PJI, and meropenem was the most sensitive antibiotic. Serum inflammatory markers, weightbearing activity, and Rissing score were significantly improved by meropenem, especially in the IA and IP + IA groups ( p < 0.05). Meropenem in the IA group eradicated E. coli from soft-tissue, bone, and prosthetic surfaces, with the same effect as in the IP + IA group. Radiological results revealed that IA and IP + IA meropenem were effective at relieving bone damage. Haematoxylin and eosin staining also showed that IA and IP + IA meropenem improved synovial inflammation and bone destruction. No pathological changes in the main organs or abnormal serum markers were observed in any of the meropenem-treated rats. The IA group required the lowest amount of meropenem, followed by the IP and IP + IA groups. Conclusion. IA-only meropenem with a two-week treatment course was effective and safe for PJI control following one-stage revision in a rat model, with less meropenem use. Cite this article: Bone Joint Res 2024;13(10):546–558

Decreasing the chance of local relapse or infection after surgical excision of bone metastases is a main goals in orthopedic oncology. Indeed, bone metastases have high incidence rate (up to 75%) and important cross-relations with infection and bone regeneration. Even in patients with advanced cancer, bone gaps resulting from tumor excision must be filled with bone substitutes. Functionalization of these substitutes with antitumor and antibacterial compounds could constitute a promising approach to overcome infection and tumor at one same time. Here, for the first time, we propose the use of nanostructured zinc-bone apatite coatings having antitumor and antimicrobial efficacy. The coatings are obtained by Ionized Jet Deposition from composite targets of zinc and bovine-derived bone apatite. Antibacterial and antibiofilm efficacy of the coatings is demonstrated in vitro against S. Aureus and E. Coli. Anti-tumor efficacy is investigated against MDA- MB-231 cells and biocompatibility is assessed on L929 and MSCs. A microfluidic based approach is used to select the optimal concentration of zinc to be used to obtain antitumor efficacy and avoid cytotoxicity, exploiting a custom gradient generator microfluidic device, specifically designed for the experiments. Then, coatings capable of releasing the desired amount of active compounds are manufactured. Films morphology, composition and ion-release are studies by FEG- SEM/EDS, XRD and ICP. Efficacy and biocompatibility of the coatings are verified by investigating MDA, MSCs and L929 viability and morphology by Alamar Blue, Live/Dead Assay and FEG-SEM at different timepoints. Statistical analysis is performed by SPSS/PC + Statistics TM 25.0 software, one-way ANOVA and post-hoc Sheffe? test. Data are reported as Mean ± standard Deviation at a significance level of p <0.05. Results and Discussion. Coatings have a nanostructured surface morphology and a composition mimicking the target. They permit sustained zinc release for over 14 days in medium. Thanks to these characteristics, they show high antibacterial ability (inhibition of bacteria viability and adhesion to substrate) against both the gram + and gram – strain. The gradient generator microfluidic device permits a fine selection of the concentration of zinc to be used, with many potential perspectives for the design of biomaterials. For the first time, we show that zinc and zinc-based coatings have a selective efficacy against MDA cells. Upon mixing with bone apatite, the efficacy is maintained and cytotoxicity is avoided. For the first time, new antibacterial metal-based films are proposed for addressing bone metastases and infection at one same time. At the same time, a new approach is proposed for the design of the coatings, based on a microfluidic approach. We demonstrated the efficacy of Zn against the MDA-MB-231 cells, characterized for their ability to form bone metastases in vivo, and the possibility to use nanostructured metallic coatings against bone tumors. At the same time, we show that the gradient-generator approach is promising for the design of antitumor biomaterials. Efficacy of Zn films must be verified in vivo, but the dual-efficacy coatings appear promising for orthopedic applications

Aims. We propose a state-of-the-art temporary spacer, consisting of a cobalt-chrome (CoCr) femoral component and a gentamicin-eluting ultra-high molecular weight polyethylene (UHMWPE) tibial insert, which can provide therapeutic delivery of gentamicin, while retaining excellent mechanical properties. The proposed implant is designed to replace conventional spacers made from bone cement. Methods. Gentamicin-loaded UHMWPE was prepared using phase-separated compression moulding, and its drug elution kinetics, antibacterial, mechanical, and wear properties were compared with those of conventional gentamicin-loaded bone cement. Results. Gentamicin-loaded UHMWPE tibial components not only eradicated planktonic Staphylococcus aureus, but also prevented colonization of both femoral and tibial components. The proposed spacer possesses far superior mechanical and wear properties when compared with conventional bone cement spacers. Conclusion. The proposed gentamicin-eluting UHMWPE spacer can provide antibacterial efficacy comparable with currently used bone cement spacers, while overcoming their drawbacks. The novel spacer proposed here has the potential to drastically reduce complications associated with currently used bone cement spacers and substantially improve patients’ quality of life during the treatment. Cite this article: Bone Joint J 2020;102-B(6 Supple A):151–157

Aims. Biofilm infections are among the most challenging complications in orthopaedics, as bacteria within the biofilms are protected from the host immune system and many antibiotics. Halicin exhibits broad-spectrum activity against many planktonic bacteria, and previous studies have demonstrated that halicin is also effective against Staphylococcus aureus biofilms grown on polystyrene or polypropylene substrates. However, the effectiveness of many antibiotics can be substantially altered depending on which orthopaedically relevant substrates the biofilms grow. This study, therefore, evaluated the activity of halicin against less mature and more mature S. aureus biofilms grown on titanium alloy, cobalt-chrome, ultra-high molecular weight polyethylene (UHMWPE), devitalized muscle, or devitalized bone. Methods. S. aureus-Xen36 biofilms were grown on the various substrates for 24 hours or seven days. Biofilms were incubated with various concentrations of halicin or vancomycin and then allowed to recover without antibiotics. Minimal biofilm eradication concentrations (MBECs) were defined by CFU counting and resazurin reduction assays, and were compared with the planktonic minimal inhibitory concentrations (MICs). Results. Halicin continued to exert significantly (p < 0.01) more antibacterial activity against biofilms grown on all tested orthopaedically relevant substrates than vancomycin, an antibiotic known to be affected by biofilm maturity. For example, halicin MBECs against both less mature and more mature biofilms were ten-fold to 40-fold higher than its MIC. In contrast, vancomycin MBECs against the less mature biofilms were 50-fold to 200-fold higher than its MIC, and 100-fold to 400-fold higher against the more mature biofilms. Conclusion. Halicin is a promising antibiotic that should be tested in animal models of orthopaedic infection. Cite this article: Bone Joint Res 2024;13(3):101–109

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses

Orthopedic Device-Related Infections (ODRIs) are a major medical challenge, particularly due to the involvement of biofilm-encased and multidrug-resistant bacteria. Current treatments, based on antibiotic administration, have proven to be ineffective. Consequently, there is a need for antibiotic-free alternatives. Antimicrobial peptides (AMPs) are a promising solution due to their broad-spectrum of activity, high efficacy at very low concentrations, and low propensity to induce resistance. We aim to develop a new AMP-based chitosan nanogel to be injected during orthopedic device implantation to prevent ODRIs. Chitosan was functionalized with norbornenes (NorChit) through the reaction with carbic anhydride and then, a cysteine-modified AMP, Dhvar5, a peptide with potent antibacterial activity, even against methicillin-resistant Staphylococcus aureus (MRSA), was covalently conjugated to NorChit (NorChit- Dhvar5), through a thiol-norbornene photoclick chemistry (UV= 365 nm). For NorChit-Dhvar5 nanogels production, the NorChit-Dhvar5 solution (0.15% w/v) and Milli-Q water were injected separately into microfluidic system. The nanogels were characterized regarding size, concentration, and shape, using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and Dynamic light scattering (DLS). The nanogels antibacterial properties were assessed in Phosphate Buffer (PBS) for 6 h, against four relevant microorganisms (Pseudomonas aeruginosa, S. aureus and MRSA, and in Muller- Hinton Broth (MHB), 50% (v/v) in PBS, supplemented with human plasma (1% (v/v)), for 6 and 24 h against MRSA. The obtained NorChit-Dhvar5 nanogels, presented a round-shaped and ∼100 nm. NorChit- Dhvar5 nanogels in a concentration of 10. 10. nanogels/mL in PBS were capable of reducing the initial inoculum of P. aeruginosa by 99%, S. aureus by 99%, and MRSA by 90%. These results were corroborated by a 99% MRSA reduction, after 24 h in medium. Furthermore, NorChit-Dhvar5 nanogels do not demonstrate signs of cytotoxicity against MC3T3-E1 cells (a pre-osteoblast cell line) after 14 days, having high potential to prevent antibiotic-resistant infection in the context of ODRIs

Infections are among the main complications connected to implantation of biomedical devices, having high incidence rate and severe outcome. Since their treatment is challenging, prevention must be preferred. For this reason, solutions capable of exerting suitable efficacy while not causing toxicity and/or development of resistant bacterial strains are needed. To address infection, inorganic antibacterial coatings, and in particular silver coatings, have been extensively studied and used in the clinical practice, but some drawbacks have been evidenced, such as scarce adhesion to the substrate, delamination, or scarce control over silver release. Here, antibacterial nanostructured silver-based thin films are proposed, obtained by a novel plasma-assisted technique, Ionized Jet Deposition (IJD). Coatings are obtained by deposition of metallic silver targets. Films thickness is selected based on previous results aimed at measuring extent and duration of silver release and at evaluating toxicity to host cells (fibroblasts). Here, composition (grazing incidence XRD) and morphology (SEM) of the obtained coatings are characterized for deposition onto different substrates, both metallic and polymeric. For heat sensitive substrates, possible alterations caused by coatings deposition in terms of morphology (SEM) and composition (FT-IR) is assessed. Then, a proof-of-concept study of the capability of these films to inhibit microbial biofilm formation is performed by using two different supports i.e., the Calgary Biofilm Device and the microplates. To the best of the Authors knowledge, this is the first study describing the application of specific anti-biofilm analyses to nanostructured coatings. In particular, anti-biofilm activities are tested against the following pathogenic strains: Escherichia (E.) coli NCTC12923, Staphylococcus (S.) aureus ATCC29213 and S. aureus 86. Among these, the strain 86 is not only pathogen but it also possesses several antibiotic resistance genes, allowing the evaluation of the utilization of nanostructured coatings as an alternative anti-microbial system to face the global threat of antibiotic resistance. Results indicate that films deposited from silver targets are composed of nanosized aggregates of metallic silver, indicating a perfect transfer of composition from the deposition target to the coatings. Results obtained here indicate that the films have significant antibacterial and antibiofilm activity. In addition, they prove that the system can be successfully applied for evaluation of coatings antibacterial efficacy for biomedical applications

Introduction. Ultra-high molecular weight polyethylene (UHMWPE) can provide local sustained delivery of therapeutics. 1,2. For example, it can deliver analgesics to address post-arthroplasty pain. 2. Given that several analgesics, such as bupivacaine (anesthetic) and tolfenamic acid (NSAID), were shown to possess antibacterial activity against Staphylococci, we hypothesize that analgesic-loaded UHMWPE can also yield antimicrobial effects, preventing the development of periprosthetic joint infections. Methods. Bupivacaine and tolfenamic acid were incorporated into UHMWPE via phase-separated compression molding. Drug release from the prepared samples was measured using high-performance liquid chromatography. Antibacterial studies of the obtained materials were conducted against methicillin-sensitive, and methicillin-resistant S. aureus, as well as S. epidermidis. Time-kill curves were obtained to characterize antimicrobial activity against planktonic bacteria. The dynamics of bacterial adhesion were assessed to characterize antibiofilm activity. Scanning electron microscopy (SEM) was used to visualize adherent bacteria. Anticolonizing activity of the tested materials was characterized using the “daughter cell” method as outlined elsewhere. 3. Cytotoxicity profile of drug-loaded UHMWPEs was evaluated using MG-63 osteoblast cell line. Results. The bupivacaine release rate generally increased with increasing drug loading (e.g. a model knee implant loaded with bupivacaine would release ca. 15–500 mg over 24 hours). While also proportional, drug release from UHMWPE loaded with tolfenamic acid was much lower. The bacterial viability curves showed that bupivacaine-loaded UHMWPE possessed moderate antibacterial activity against planktonic MSSA, MRSA, and S. epidermidis, slowing bacteria proliferation by up to 70%. Bupivacaine-loaded UHMWPE also mitigated biofilm formation and development during the initial culture period. SEM images confirmed the observed antibiofilm effect (Fig. 1). Tolfenamic acid-loaded UHMWPE allowed proliferation of planktonic bacteria. At the same time, these materials showed pronounced dose-dependent anticolonizing activity against tested strains, providing 3-log reduction of “daughter” cells. Bupivacaine- and tolfenamic acid-loaded UHMWPEs showed little-to-no cytotoxicity against osteoblasts. Discussion & Conclusions. We demonstrated for the first time that bupivacaine-loaded UHMWPE possesses dose-dependent antibacterial properties against planktonic and adherent MSSA, MRSA, and S. epidermidis – pathogens commonly associated with periprosthetic joint infections. Pronounced anticolonizing activity was evident for tolfenamic acid-loaded UHMWPE. Due to the low solubility of tolfenamic acid, the material's antibacterial effect against planktonic bacteria was lower. These results demonstrate that analgesic-loaded UHMWPE, used as a tool in multimodal pain management, can also yield antibacterial effects, opening an entirely new avenue for providing post-arthroplasty antibacterial prophylaxis. This pioneering approach has a potential to reduce patients' morbidity and mortality after arthroplasty. For any tables or figures, please contact the authors directly

Aims. Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. Methods. Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology. Results. Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway. Conclusion. The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells. Cite this article: Bone Joint Res 2023;12(2):91–102

Introduction and Objective. Alveolar bone resorption following tooth extraction or periodontal disease compromises the bone volume required to ensure the stability of an implant. Guided bone regeneration (GBR) is one of the most attractive technique for restoring oral bone defects, where an occlusive membrane is positioned over the bone graft material, providing space maintenance required to seclude soft tissue infiltration and to promote bone regeneration. However, bone regeneration is in many cases impeded by a lack of an adequate tissue vascularization and/or by bacterial contamination. Using simultaneous spray coating of interacting species (SSCIS) process, a bone inspired coating made of calcium phosphate-chitosan-hyaluronic acid was built on one side of a nanofibrous GBR collagen membrane in order to improve its biological properties. Materials and Methods. First, the physicochemical characterizations of the resulting hybrid coating were performed by scanning electron microscopy, X-ray photoelectron, infrared spectroscopies and high-resolution transmission electron microscopy. Then human mesenchymal stem cells (MSCs) and human monocytes were cultured on those membranes. Biocompatibility and bioactivity of the hybrid coated membrane were respectively evaluated through MSCs proliferation (WST-1 and DNA quantification) and visualization; and cytokine release by MSCs and monocytes (ELISA and endothelial cells recruitment). Antibacterial properties of the hybrid coating were then tested against S. aureus and P. aeruginosa, and through MSCs/bacteria interactions. Finally, a preclinical in vivo study was conducted on rat calvaria bone defect. The newly formed bone was characterized 8 weeks post implantation through μCT reconstructions, histological characterizations (Masson's Trichrome and Von Kossa stain), immunohistochemistry analysis and second harmonic generation. Biomechanical features of newly formed bone were determined. Results. The resulting hybrid coating of about 1 μm in thickness is composed of amorphous calcium phosphate and carbonated poorly crystalline hydroxyapatite, wrapped within chitosan/hyaluronic acid polysaccharide complex. Hybrid coated membrane possesses excellent bioactivity and capability of inducing an overwhelmingly positive response of MSCs and monocytes in favor of bone regeneration. Furthermore, the antibacterial experiments showed that the hybrid coating provides contact-killing properties by disturbing the cell wall integrity of Gram-positive and Gram-negative bacteria. Its combination with MSCs, able to release antibacterial agents and mediators of the innate immune response, constitutes an excellent strategy for fighting bacteria. A preclinical in vivo study was therefore conducted in rat calvaria bone defect. μCT reconstructions showed that hybrid coated membrane favored bone regeneration, as we observed a two-fold increase in bone volume / total volume ratios vs. uncoated membrane. The histological characterizations revealed the presence of mineralized collagen (Masson's Trichrome and Von Kossa stain), and immunohistochemistry analysis highlighted a bone vascularization at 8 weeks post-implantation. However, second harmonic generation analysis showed that the newly formed collagen was not fully organized. Despite a significant increase in the elastic modulus of the newly formed bone with hybrid coated membrane (vs. uncoated membrane), the obtained values were lower than those for native bone (approximately 3 times less). Conclusions. These significant data shed light on the regenerative potential of such bioinspired hybrid coating, providing a suitable environment for bone regeneration and vascularization, as well as an ideal strategy to prevent bone implant-associated infections

Objectives . The objective of this study is to determine an optimal antibiotic-loaded bone cement (ALBC) for infection prophylaxis in total joint arthroplasty (TJA). Methods. We evaluated the antibacterial effects of polymethylmethacrylate (PMMA) bone cements loaded with vancomycin, teicoplanin, ceftazidime, imipenem, piperacillin, gentamicin, and tobramycin against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staph. aureus (MRSA), coagulase-negative staphylococci (CoNS), Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Standardised cement specimens made from 40 g PMMA loaded with 1 g antibiotics were tested for elution characteristics, antibacterial activities, and compressive strength in vitro. . Results. The ALBC containing gentamicin provided a much longer duration of antibiotic release than those containing other antibiotic. Imipenem-loading on the cement had a significant adverse effect on the compressive strength of the ALBC, which made it insufficient for use in prosthesis fixation. All of the tested antibiotics maintained their antibacterial properties after being mixed with PMMA. The gentamicin-loaded ALBC provided a broad antibacterial spectrum against all the test organisms and had the greatest duration of antibacterial activity against MSSA, CoNS, P. aeruginosa and E. coli. . Conclusion. When considering the use of ALBC as infection prophylaxis in TJA, gentamicin-loaded ALBC may be a very effective choice. Cite this article: Bone Joint Res 2013;2:220–6

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

Aims. Treatment outcomes for methicillin-resistant Staphylococcus aureus (MRSA) periprosthetic joint infection (PJI) using systemic vancomycin and antibacterial cement spacers during two-stage revision arthroplasty remain unsatisfactory. This study explored the efficacy and safety of intra-articular vancomycin injections for PJI control after debridement and cement spacer implantation in a rat model. Methods. Total knee arthroplasty (TKA), MRSA inoculation, debridement, and vancomycin-spacer implantation were performed successively in rats to mimic first-stage PJI during the two-stage revision arthroplasty procedure. Vancomycin was administered intraperitoneally or intra-articularly for two weeks to control the infection after debridement and spacer implantation. Results. Rats receiving intra-articular vancomycin showed the best outcomes among the four treatment groups, with negative bacterial cultures, increased weight gain, increased capacity for weightbearing activities, increased residual bone volume preservation, and reduced inflammatory reactions in the joint tissues, indicating MRSA eradication in the knee. The vancomycin-spacer and/or systemic vancomycin failed to eliminate the MRSA infections following a two-week antibiotic course. Serum vancomycin levels did not reach nephrotoxic levels in any group. Mild renal histopathological changes, without changes in serum creatinine levels, were observed in the intraperitoneal vancomycin group compared with the intra-articular vancomycin group, but no changes in hepatic structure or serum alanine aminotransferase or aspartate aminotransferase levels were observed. No local complications were observed, such as sinus tract or non-healing surgical incisions. Conclusion. Intra-articular vancomycin injection was effective and safe for PJI control following debridement and spacer implantation in a rat model during two-stage revision arthroplasties, with better outcomes than systemic vancomycin administration. Cite this article: Bone Joint Res 2022;11(6):371–385

Periprosthetic joint infection (PJI) is one of the most dreaded complications after arthroplasty surgery; thus numerous approaches have been undertaken to equip metal surfaces with antibacterial properties. Due to its antimicrobial effects, silver is a promising coating for metallic surfaces, and several types of silver-coated arthroplasty implants are in clinical use today. However, silver can also exert toxic effects on eukaryotic cells both in the immediate vicinity of the coated implants and systemically. In most clinically-used implants, silver coatings are applied on bulk components that are not in direct contact with bone, such as in partial or total long bone arthroplasties used in tumour or complex revision surgery. These implants differ considerably in the coating method, total silver content, and silver release rates. Safety issues, such as the occurrence of argyria, have been a cause for concern, and the efficacy of silver coatings in terms of preventing PJI is also controversial. The application of silver coatings is uncommon on parts of implants intended for cementless fixation in host bone, but this option might be highly desirable since the modification of implant surfaces in order to improve osteoconductivity can also increase bacterial adhesion. Therefore, an optimal silver content that inhibits bacterial colonization while maintaining osteoconductivity is crucial if silver were to be applied as a coating on parts intended for bone contact. This review summarizes the different methods used to apply silver coatings to arthroplasty components, with a focus on the amount and duration of silver release from the different coatings; the available experience with silver-coated implants that are in clinical use today; and future strategies to balance the effects of silver on bacteria and eukaryotic cells, and to develop silver-coated titanium components suitable for bone ingrowth. Cite this article: Bone Joint J 2021;103-B(3):423–429

In order to improve fast osseointegration, to modulate inflammatory response and to avoid biofilm formation, several attempts of surface modifications of titanium alloy in term of surface topography and chemistry have been performed over years, but this is still an open issue. In our research work, a patented chemical treatment was developed and tailored to improve fast osseointegration and to allow further surface functionalization in order to get a multifunctional surface. After the chemical treatment, Ti6Al4V shows a micro and nano-textured surface oxide layer with high density of hydroxyls groups, as summarized Figure 1: it is able to induce apatite precipitation (during soaking in Simulated Body Fluid), high wettability by blood, specific protein adsorption, positive osteoblast response and surface mechanical resistance to implantation friction. Hydroxyl groups exposed by the treated surface also allow binding natural biomolecules such as polyphenols, which can further improve the rate and quality of osseointegration by adding anti-inflammatory, antibacterial and antitumoral effects suitable for implants in critical situations. Polyphenols have the further added value of being a low cost and eco-sustainable product, extractable from byproducts of wine and food industry. On the chemically treated and functionalized samples, the surface characterization was performed using Folin&Ciocalteu test, fluorescence microscopy and XPS analysis in order to check the presence and activity of the grafted biomolecules (polyphenols from red grape pomace and green tea leaves). Cell tests were performed with Kusa A-1 cells highlighting the ability of polyphenols to improve osteoblasts differentiation and deposition of mineralized extracellular matrix. Surface functionalization can also be performed with chitin derived biomolecules to reduce inflammation. With the purpose of obtaining the antibacterial effect, during the chemical treatment a silver precursor can also be added to obtain in situ reduced silver nanoparticles embedded in the nano-structured oxide layer. The samples containing nanoparticles on the surface were characterized by means of TEM and FESEM observation highlighting the presence of well distributed and small-sized nanoparticles on the surface and through the thickness of the oxide layer. A long-lasting release in water was observed up to 14 days and antibacterial tests on Staphylococcus aureus showed the ability of the surface to reduce bacteria viability avoiding biofilm formation. The results showed that the patented chemical treatment can improve the response of osteoblasts to titanium alloy implants, but is also a promising way to obtain multifunctional surfaces with antibacterial, antioxidant, anti-inflammatory and antitumoral properties that can be the future of orthopedic implants

Objectives. Thermal stability is a key property in determining the suitability of an antibiotic agent for local application in the treatment of orthopaedic infections. Despite the fact that long-term therapy is a stated goal of novel local delivery carriers, data describing thermal stability over a long period are scarce, and studies that avoid interference from specific carrier materials are absent from the orthopaedic literature. Methods. In this study, a total of 38 frequently used antibiotic agents were maintained at 37°C in saline solution, and degradation and antibacterial activity assessed over six weeks. The impact of an initial supplementary heat exposure mimicking exothermically curing bone cement was also tested as this material is commonly used as a local delivery vehicle. Antibiotic degradation was assessed by liquid chromatography coupled to mass spectrometry, or by immunoassays, as appropriate. Antibacterial activity over time was determined by the Kirby-Bauer disk diffusion assay. Results. The heat exposure mimicking curing bone cement had minimal effect on stability for most antibiotics, except for gentamicin which experienced approximately 25% degradation as measured by immunoassay. Beta-lactam antibiotics were found to degrade quite rapidly at 37°C regardless of whether there was an initial heat exposure. Excellent long-term stability was observed for aminoglycosides, glycopeptides, tetracyclines and quinolones under both conditions. Conclusions. This study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics, and for material scientists looking to develop next-generation controlled or extended-release antibiotic carriers. Cite this article: E. Samara, T. F. Moriarty, L. A. Decosterd, R. G. Richards, E. Gautier, P. Wahl. Antibiotic stability over six weeks in aqueous solution at body temperature with and without heat treatment that mimics the curing of bone cement. Bone Joint J 2017;6:296–306. DOI: 10.1302/2046-3758.65.BJR-2017-0276.R1

Device-associated bacterial infections are a major and costly clinical challenge. This project aimed to develop a smart new biomaterial for implants that helps to protect against infection and inflammation, promote bone growth, and is biodegradable. Gallium (Ga) doped strontium-phosphate was coated on pure Magnesium (Mg) through a chemical conversion process. Mg was distributed in a graduated manner throughout the strontium-phosphate coating GaSrPO4, with a compact structure and a Ga-rich surface. We tested this sample for its biocompatibility, effects on bone remodeling and antibacterial activities including Staphylococcus aureus, S. epidermidis and E. coli - key strains causing infection and early failure of the surgical implantations in orthopaedics and trauma. Ga was distributed in a gradient way throughout the entire strontium-phosphate coating with a compact structure and a gallium-rich surface. The GaSrPO4 coating protected the underlying Mg from substantial degradation in minimal essential media at physiological conditions over 9 days. The liberated Ga ions from the coatings upon Mg specimens inhibited the growth of bacterial tested. The Ga dopants showed minimal interferences with the SrPO4 based coating, which boosted osteoblasts and undermined osteoclasts in in vitro co-cultures model. The results evidenced this new material may be further translated to preclinical trial in large animal model and towards clinical trial. Acknowledgements: Authors are grateful to the financial support from the Australian Research Council through the Linkage Scheme (ARC LP150100343). The authors acknowledge the facilities, and the scientific and technical assistance of the RMIT University and John Curtin School of Medical Research, Australian National University

This study aimed to determine the optimal formulation of antibiotic-loaded bone cement (ALBC) for periprosthetic joint infection (PJI) using both in vitro and in vivo models incorporating various combinations of gram-positive and gram-negative antibiotics. The in vitro antibiotic release characteristics and antibacterial capacities of ALBCs loaded with either 4 g of vancomycin or teicoplanin and 4 g of ceftazidime, imipenem, or aztreonam were measured against methicillin-susceptible S. aureus, methicillin-resistant S. aureus, coagulase-negative staphylococci, Pseudomonas aeruginosa and Escherichia coli. ALBC spacers with superior in vitro antibacterial capacity were then implanted into ten patients (five females and five males between 29 and 75 years of age) diagnosed with chronic hip/knee PJIs and antibacterial activities within joint fluid were measured. The average duration of ALBC spacer implantation was 80 days (range, 36–155 days). Antibiotic concentrations and antibacterial activities of joint fluid at the site of infection were measured during the initial period as well as several months following spacer implantation. Cement samples loaded with vancomycin/ceftazidime or teicoplanin/ceftazidime exhibited equal or longer antibacterial duration against test bacteria as compared with other ALBCs. Joint fluid samples exhibited antibacterial activity against the test microorganisms including ATCC strains and clinically isolated strains. There were no adverse systemic effects, infection at second stage re-implantation, or recurrent infection at final follow-up. Vancomycin/ceftazidime ALBC provided broad antibacterial capacity both in vitro and in vivo and was shown to be an effective and safe therapeutic measure in the treatment of hip/knee PJIs. We thank H.Y. Hsu for performing bioassay

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

The discussion will focus on new approaches to reduce bacterial adhesion on the surface of polymethylmethacrylate (PMMA) in contact with bone, comparing the clinical and engineering point of view. One possibility is to encourage and speed up direct interaction with the bone, for example by adding a bioactive phase in the cement (hydroxyapatite, glass and bioactive glass ceramic). A widespread strategy is also the addition of different types of antibiotics (gentamicin, tobramycin vancomycin, etc.), although they are known to have some drawbacks: not complete release, resistant strain development. Another strategy could be represented by the PMMA-based composite cements loaded with a completely inorganic filler consisting of a bioactive glass doped with ions whose bioactivity mechanism is well-known and encompasses a chemical and biological interaction with tissues promoting osteoinduction. Bioactive glasses can be doped with antibacterial ions (silver, copper, etc.) preserving their biocompatibility and bioactivity and, at the same time, acquiring antibacterial properties. Thus, it is possible to produce composite cements that combine the properties of the polymer matrix with those of the inorganic filler, overcoming the main problems associated with the use of antibiotics. An additional possibility is the addition of essential oils, vegetable oils with remarkable antibacterial properties

Background. Although described as a commensal bacterium with low pathogenicity, Cutibacterium acnes involvement has been reported in many clinical entities: infections associated with devices, such as shoulder prosthetic joint infections, osteosynthesis, breast implants or cerebrospinal fluid shunts. Various studies show that C. acnes grows as a biofilm, contributing to its persistence by allowing its escape from the action of the immune system and antibiotics. Purpose. Our aim was to assess the activity of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) on eight different well-characterized C. acnes strains after growth in biofilm mode. Methods. Eight susceptible strains of C. acnes were selected for this study, including two reference strains (ATCC6919 and ATCC11827) and six clinical strains. All C. acnes strains were studied using two different methods to study the biofilm production at different time points: the BioFilm Ring Test. ®. technique (early stages of adhesion) and the Crystal Violet (CV) method (mature biofilm). In a second step, the impact of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) was studied. For the CV technique, two types of tests were performed: preventive tests (addition of active substances and bacteria at the same time) and curative challenge tests (addition of active substances on a biofilm already formed after 48h). Transmission electron microscopy was performed to investigate the morphology modifications. Results. C. acnes isolates from phylotypes IA. 1. and IA. 2. , seem to produce more mature biofilm in the first stages of adhesion than other phylotypes. Curative assays were performed to evaluate the efficacy of antibiotics and Myrtacine. ®. on mature biofilm. Significant efficacy of Myrtacine. ®. at 0.03% was observed for C. acnes strains. Moreover, the combination of Myrtacine. ®. and doxycycline appears to decrease the total biofilm biomass. The effect of doxycycline as a preventive measure was minimal. On the contrary, a similar use of Myrtacine. ®. as early as 0.001% showed significant efficacy with a significant decrease in total biofilm biomass for all C. acnes strains. Transmission electron microscopy revealed a significantly decreased biofilm growth in treated bacteria with Myrtacine. ®. compared to untreated bacteria. Moreover, the total number of bacteria decreased as the concentration of Myrtacine. ®. increased suggesting also an antimicrobial effect. Conclusion. These results confirm the difference in biofilm producing ability depending on C. acnes phylotypes. These results suggest that Myrtacine. ®. may be a promising alternative antibacterial and anti-biofilm agent like peroxide de benzoyle to prevent shoulder prosthetic joint infection involving planktonic and biofilm C. acnes

Aim. To develop a new system for antibacterial coating of joint prosthesis and osteosynthesis material. The new coating system was designed to release gentamicin immediately after insertion to eradicate surgical contamination. Method. Steel implants (2×15mm) were coated with a solid nanocomposite xerogel made from silica and the dendritic polymer, hyperbranched polyethyleneimine. The xerogel was anchored inside a porous surface made by pre-coating with titanium microspheres. Finally, gentamicin was encapsulated in the xerogel, i.e. no chemical binding. A total of 50 µg gentamicin was captured into each implant. The efficacy of the new coating was evaluated in a porcine model of implant associated osteomyelitis. In total, 30 female pigs were randomized into 3 study groups (n=10). Group A; plain implants + saline, Group B; plain implants + 10. 4. CFU of Staphylococcus aureus, and Group C; coated implants + 10. 4. CFU of S. aureus. Implant + inoculum was placed into a pre-drilled implant cavity of the right tibia and the pig was euthanized 5 days afterwards. Postmortem microbiology and pathology were performed. Two additional pigs were used in a pharmacokinetic study where microdialysis (MD) catheters were placed alongside coated implants. Extracellular fluid was sampled regularly for 24 hours from the MD catheters and analyzed for gentamicin content. Results. Within Groups A and C, all implants were found sterile by sonication and bacteria could not be identified within the surrounding bone tissue. In contrast, all Group B animals had S. aureus positive implant and tissue microbiology. Macroscopic and microscopic pathological examinations confirmed that Group A and C animals were complete identic, i.e. no pus around implants and only minor peri-implant inflammation related to insertion of implants per se. All Group B animals had pus around their implants and a massive peri-implant inflammatory response dominated by neutrophil granulocytes. Maximum gentamicin release (35 µg /mL) was measured in the first obtained MD sample, i.e. after 30 min, and the concentration stayed above the MIC level for the used S. aureus strain for 8 hours. Conclusions. The new xerogel coating prevented development of osteomyelitis. Prevention was due to a fast gentamicin release immediately following insertion and antimicrobial active concentrations were detectable several hours after implantation. This means that the critical time point of most relevant surgical procedures potentially could be protected by the novel coating. The new coating will be investigated on larger scale implants and full-size prosthesis in the future

Biphasic calcium phosphates (BCP) are the most frequently used materials because of their mineral analogy with bio-mineral part of bones. Their chemical synthesis can be modulated by doping, in order to respond to the biological needs. We present here the biological responses induced by copper ions in solution, to characterize its cytotoxicity and antibacterial activity. We also investigate the antibacterial property of Cu-doped BCP (Ca10 Cu0.1 (PO4)6 (OH)1.8 O0.2) on a strain of clinical interest: S. aureus, compared to undoped BCP. The sol-gel route has been used to prepare the BCP ceramics. Human BMC (Bone Marrow Cells) were obtained from metaphysal cancellous bone collected during hip arthroplasty and used for cytotoxicity evaluations. A strain of Staphylococcus aureus isolated from an osteoarticular infection after total knee arthroplasty was used to evaluate antibacterial activities. Results indicate that 3 ppm of copper ions leads to the death of all cultured bacteria in 24 hours and 25 ppm caused the death of all cells in 15 days. Regarding BCP, the undoped bioceramics increased the bacterial growth compared to a control without bioceramic. After 16 hours of contact, the copper ions released by the Cu-doped BCP induced a significant decrease of the bacterial concentration, indeed no viable bacteria were found. These materials seem to be a promising alternative for the preparation of multifunctional bone substitutes

The aim of the work is to develop innovative antibacterial surface modification treatments for titanium capable to limit the bacterial adhesion and proliferation as weel as the biofilm formation while maintaining an high osteointegrative potential. The goal is to contrast the infections which represent a serius complication related to the use of implantable devices. Introduction. Titanium and titanium alloy are considered the golden standard materials for the applications in contact with bone especially for dental and orthopaedic applications. To extend the implantable component lifetime and increase their clinical performance some surface modifications are required, to promote and speed up the osteointegration process increasing the rate of bone bonding. Unfortunately, among the different complications related to the use of titanium implantable devices the infections represent the most serious, often leading to implant failure and revision. The use of surface modification with specific metal ions represents a promising approach to fight implant-related infections. In particular gallium has recently shown efficacy in the treatment of infections: exploiting the chemical similarity of Ga. 3+. with Fe. 3+. , it can interfere in the iron metabolism for a wide range of bacteria. The aim of this work is to develop and characterise new biocompatible biomimetic treatments with anodic spark deposition (ASD) technique on titanium characterised by antibacterial properties maintaining high osteointegrative potential. Experimental Methods. Three surfaces were developed using titanium grade 2 samples (12 mm diam., 0.5 mm thick): i) SiB-Na: ASD treatment performed in an electrolytic solution containing Ca, P, Si and Na. 1. used as control; ii) GaOss: ASD treatment performed in the SiB-Na solution enriched with gallium nitrate and oxalic acid; iii) GaCis: ASD treatment performed in the SiB-Na solution enriched with with gallium nitrate and L-cysteine. The ASD was carried out in galvano-static condition with a current density of 10 mA/cm. 2. reaching 295V (for SiB-Na, GaCis) and 310V for GaOss. Untreated Ti was used as control. The surface morphology and chemistry were analysed using SEM, EDS and XPS. Ga release in D-PBS was studied up to 21 days using ICP/OES analysis. The structure of the titanium oxide was investigated using XRD while the surface wettability was studied using OCA measurements. The coating mechanical stability was evaluated using scratch test and three-point bending test. Human osteoblastic cells (Saos2) indirect citotoxicity was asessed using Alamar Blue assay. Saos2 morphology and adhesion to the treated surfaces were evaluated using SEM and actin staining. Saos2 viability was assessed up to 21 of cell cultured in direct contact with antibacterial surfaces while the Saos2 alkaline phosphatase activity (ALP) was evaluated up to 21 day as a marker of new bone formation. The antibacterial properties were assessed with S. mutans, S. epidermidis and E. coli bacterial strains even after 21 days of the antibacterial agents release to test the long lasting antibacterial activity. Also the effectiveness in limiting biofilm formation was evaluated against S. epidermidis and A. baumanni biofilm producers. Results and discussion. The developed surfaces showed a microporous morphology without the presence of any delamination. The EDS showed the presence of Ga, Si, P and Ca for GaCis and GaOss. Ga-based treatments revealed a similar concentration of the antimicrobial agent although the Ga released from GaOss was extremely higher than on GaCis. XRD analysis revealed the presence of the anatase cristallographic form which is associated with an higher surface wettability than Ti. The coatings showed a good mechanical stability both after three point bending test and scratch test. The antibacterial surfaces did not show any indirect citotoxicity for Saos2. Also the cell morphology and viability were not negatively affected by the presence of the antibacterial agent: GaOss treatment displayed a stimulating effect on ALP activity of osteoblastic cells than controls. A strong reduction of bacterial adhesion and proliferation for both of the Ga-based treatments especially for GaOss (≈ 40% vs Ti) was observed as well as a long-lasting antibaterial activity. Finally, a significative reduction of S. epidermidis and A. baumanni biofilm production than Ti was observed for GaOss and GaCis treatments. Conclusion. The treatments developed in the present study represent a promising class of antibacterial and osteointegrative coatings for titanium in particular for dental and orthopaedic applications

The bone infection osteomyelitis (typically Staphylococcus aureus) requires a multistep treatment process including: surgical debridement, long-term systemic high-dose antibiotics, and often bone grafting. With antibiotic resistance becoming increasingly concerning, alternative approaches are urgently needed. Herein, we develop a one-step treatment for osteomyelitis that combines local, controlled release of non-antibiotic antibacterials (copper) within a proven regenerative scaffold. To maximise efficacy we utilised bioactive glass – an established material with immense osteogenic capacity – as a copper ion delivery reservoir. Copper ions have also been shown to stimulate angiogenesis and induce MSC differentiation down an osteogenic lineage. To eliminate grafting requirements, the copper-doped BG was incorporated into our previously developed collagen scaffolds to produce multifunctional antibacterial, osteogenic, and angiogenic scaffolds. Scaffolds were fabricated by freeze-drying a co-suspension of collagen and bioactive glass particles (+/− copper doping, referred to as CuBG and BG, respectively) at a range of different concentrations (0–300% w/w bioactive glass/collagen). Scaffolds demonstrated a 2.7-fold increase in compressive modulus (300% CuBG vs. 0%; p≤0.01), whilst maintaining >98% porosity. The 300% CuBG scaffolds showed significant antibacterial activity against Staphylococcus aureus (p≤0.001). In terms of osteogenesis, both 100% and 300% CuBG scaffolds increased cell-mediated calcium deposition on the scaffolds at day 14 and 28 (p≤0.05 and p≤0.001), as confirmed by alizarin red staining. 100% CuBG scaffolds significantly enhanced angiogenesis by increased tubule formation (p≤0.01) and VEGF protein production (p≤0.001) (all ≥n=3). In summary, this single-stage, off-the-shelf treatment for osteomyelitis shows potential to minimise bone grafting and antibiotic dependence, while reducing hospital stays and costs

Objectives. Investigate the incorporation of an antibiotic in bone cement using liposomes (a drug delivery system) with the potential to promote osseointegration at the bone cement interface whilst maintaining antibiotic elution, anti-microbiological efficacy and cement mechanical properties. Prosthetic joint infection and aseptic loosening are associated with significant morbidity. Antibiotic loaded bone cement is commonly used and successfully reduces infection rates; however, there is increasing resistance to the commonly used gentamicin. Previous studies have shown gentamicin incorporated into bone cement using liposomes can maintain the cement's mechanical properties and improve antibiotic elution. The phospholipid phosphatidyl-l-serine has been postulated to encourage surface osteoblast attachment and in a liposome could improve osseointegration, thereby reducing aseptic loosening. Preliminary clinical isolate testing showed excellent antimicrobial action with amoxicillin therefore the study aims were to test amoxicillin incorporated into bone cement using liposomes containing phosphatidyl-l-serine in terms of antibiotic elution, microbiological profile and mechanical properties. Methods. Amoxicillin was encapsulated within 100nm liposomes containing phosphatidyl-L-serine and added to PMMA bone cement (Palacos R (Heraeus Medical, Newbury, UK)). Mechanical testing was performed according to Acrylic Cement standards (ISO BS 5833:2002). Elution testing was carried out along with microbiological testing utilising clinical isolates. Results. Liposomal encapsulated amoxicillin PMMA bone cement exceeded minimum ISO BS 5833:2002 standards, had better elution at 12.9% when compared with plain amoxicillin (p=0.036 at 48 hours) or commercial gentamicin cement (Palacos R+G, Heraeus Medical, Newbury, UK – previous studies showed 6% elution over the same time period). Amoxicillin showed superior antimicrobial action when compared with gentamicin of the same concentration. However, liposomal encapsulated amoxicillin in solution and liposomal encapsulated amoxicillin in PMMA were both less effective than free amoxicillin in bacterial growth inhibition. The liposomal amoxicillin also seemed to decrease the cement setting time. Conclusions. Phosphatidyl-l-serine containing liposomes maintained the cement's mechanical properties and seemed to have better antibiotic elution, however, had less effective antibacterial action than plain amoxicillin. This difference in antibacterial action requires further investigation along with investigation of osteoblast attachment to phosphatidyl-l-serine containing liposomes within cement. Plain amoxicillin, for those not penicillin allergic, seems to be a credible alternative to gentamicin for incorporation in PMMA bone cement. It has shown superior antibacterial action, which may improve infection rates, whilst maintaining the cement's mechanical properties

Recent clinical data suggest improvement in the fixation of tibia trays for total knee arthroplasty when the trays are additive manufactured with highly porous bone ingrowth structures. Currently, press-fit TKA is less common than press-fit THA. This is partly because the loads on the relatively flat, porous, bony apposition area of a tibial tray are more demanding than those same porous materials surrounding a hip stem. Even the most advanced additive manufactured (AM) highly porous structures have bone ingrowth limitations clinically as aseptic loosening still remains more common in press-fit TKA vs. THA implants. Osseointegration and antibacterial properties have been shown in vitro and in vivo to improve when implants have modified surfaces that have biomimetic nanostructures designed to mimic and interact with biological structures on the nano-scale. Pre-clinical evaluations show that TiO. 2. nanotubes (TNT), produced by anodization, on Ti6Al4V surfaces positively enhance the rate at which osseointegration occurs and TNT nano-texturization enhances the antibacterial properties of the implant surface. 2. In this in vivo sheep study, identical Direct Metal laser Sintered (DMLS) highly porous Ti6Al4V specimens with and without TNT surface treatment are compared to sintered bead specimens with plasma sprayed hydroxyapatite-coated surface treatment. Identical DMLS specimens made from CoCrMo were also implanted in sheep tibia bi-cortically (3 per tibia) and in the cancellous bone of the distal femur and proximal tibia (1 per site). Animals were injected with fluorochrome labels at weeks 1, 2 and 3 after surgery to assess the rate of bone integration. The cortical specimens were mechanically tested and processed for PMMA histology and histomorphometry after 4 or 12 weeks. The cancellous samples were also processed for PMMA histology and histomorphometry. The three types of bone labels were visualized under UV light to examine the rate of new bony integration. At 4 weeks, a 42% increase in average pull-out shear strength between nanotube treated specimens and non-nanotube treated specimens was shown. A 21% increase in average pull-out shear strength between nanotube treated specimens and hydroxyapatite-coated specimens was shown. At 12 weeks, all specimens had statistically similar pull-out values. Bone labels demonstrated new bone formation into the porous domains on the materials as early as 2 weeks. A separate in vivo study on 8 rabbits infected with methicillin-resistant Staphylococcus aureus showed bacterial colonization reduction on the surface of the implants treated with TNT. In vitro and in vivo evidence suggests that nanoscale surfaces have an antibacterial effect due to surface energy changes that reduce the ability of bacteria to adhere. These in vivo studies show that TNT on highly porous AM specimens made from Ti6Al4V enhances new bone integration and also reduce microbial attachment

Aims. Biofilm formation is one of the primary reasons for the difficulty in treating implant-related infections (IRIs). Focused high-energy extracorporeal shockwave therapy (fhESWT), which is a treatment modality for fracture nonunions, has been shown to have a direct antibacterial effect on planktonic bacteria. The goal of the present study was to investigate the effect of fhESWT on Staphylococcus aureus biofilms in vitro in the presence and absence of antibiotic agents. Methods. S. aureus biofilms were grown on titanium discs (13 mm × 4 mm) in a bioreactor for 48 hours. Shockwaves were applied with either 250, 500, or 1,000 impulses onto the discs surrounded by either phosphate-buffered saline or antibiotic (rifampin alone or in combination with nafcillin). The number of viable bacteria was determined by quantitative culture after sonication. Representative samples were taken for scanning electron microscopy. Results. The application of fhESWT led to a ten-fold reduction in bacterial counts on the metal discs for all impulse numbers compared to the control (p < 0.001). Increasing the number of impulses did not further reduce bacterial counts in the absence of antibiotics (all p > 0.289). Antibiotics alone reduced the number of bacteria on the discs; however, the combined application of the fhESWT and antibiotic administration further reduced the bacterial count compared to the antibiotic treatment only (p = 0.032). Conclusion. The use of fhESWT significantly reduced the colony-forming unit (CFU) count of a S. aureus biofilm in our model independently, and in combination with antibiotics. Therefore, the supplementary application of fhESWT could be a helpful tool in the treatment of IFIs in certain cases, including infected nonunions. Cite this article: Bone Joint Res 2021;10(1):77–84

Antibiotic-laden bone cement is an important strategy of treatment for an established bone infection. It was aimed to find the safe antibiotic dose intervals of the antibiotic cements soaked in Phosphate Buffered Saline solution and to determine whether there was a difference in terms of mechanical strength between the prepared samples. This study was done in our institute Microbiology and Metallurgy laboratories. All samples were prepared using manual mixing technique using 40 g radiopaque Biomet® Bone cement (Zimmer Biomet, Indiana, USA) under sterile conditions at 19 ± 2 ºC. In this study, vancomycin (4 groups − 0.5, 2, 4, 6 g), teicoplanin (4 groups − 0.8, 1.2, 2, 2.4 g), daptomycin (4 groups − 1, 2, 2.5, 3 g), piperacillin-tazobactam (4 groups − 0.125, 0.5, 1, 2 g) and meropenem (4 groups − 0.5, 2, 4, 6 g) were measured in a assay balance and added to the cement powder. Antibiotic levels ranged from the lowest 0.625% to the highest 15%. 80×10×4 mm rectangle prism-shaped sample for mechanical measurements in accordance to ISO 5833 standart and 12×6×1 mm disc-shaped samples for microbiological assesments were used. Four sample for each antibiotic dose and control group was made. Prepared samples were evaluated macroscopically and faulty samples were excluded from the study. Prepared samples were kept in Phosphate Buffered Saline solution renewed every 24 hours at 37 ºC. At the end of 6 weeks, all samples were tested with Instron ® 3369 (Norwood Massachusetts, USA) four point bending test. Staphylococcus aureus (ATCC 29213) strain was used for samples of antibiotics containing vancomycin, teicoplanin and daptomycin after the samples prepared for antibiotic release were maintained under sterile conditions and kept in Phosphate Buffered Saline solution as appropriate. For samples containing meropenem and piperacillin - tazobactam antibiotics, Pseudomonas aeruginosa (ATCC 27853) strain was used. The addition of more than 5% antibiotics to the cement powder was significantly reduced mechanical strength in all groups(p <0.05) however the power of significance was changed depending on the type of antibiotic. In general, adding antibiotics with 2.5% and less for cement amount was not cause significant changes in mechanical measurements. There was a negative correlation between the increase in the amount of antibiotics mixed with cement and the durability of the cement (p: <0.001, r: −0.883 to 0.914). In this study, especially the antibacterial effects of piperacillin-tazobactam, containing 0.25 gr and 0.5 gr antibiotic doses, were found to be low. There was no bacterial growth in all other groups for 21 days. Considering the mechanical properties of groups containing meropenem, vancomycin, daptomycin and teicoplanin, it was observed that all antibiotic cements remained above the limit value of 50 MPa in the bending test at concentrations containing 2.5% and less antibiotics. This was not achieved for the piperacillin-tazobactam group. The findings of the study showed that each antibiotic has different MPa values at different doses. Therefore, it could be concluded that not only the antibiotic dose but also the type oould change the mechanical properties. In the light of these findings, mixing more than 2.5% antibiotics in cement for the antibiotic types included in the study was ineffective in terms of antibacterial effect and mechanically reduces the durability of cement below the standard value of 50 MPa

Introduction. Infection remains as one of the major challenges of total joint surgery. One-stage irrigation, debridement and reimplantation, or two-stage revision surgery with a temporary implantation of antibiotic eluting bone cement spacer followed by reimplantation are two methods often used to treat infected patients with mixed outcomes. Like bone cement, ultra-high molecular weight polyethylene (UHMWPE) can also be used as a carrier for antibiotics. Recently, we demonstrated that vancomycin and rifampin can be delivered from UHMWPE implants at therapeutic levels to eradicate Staphylococcus aureus biofilm in a lupine animal model. There are regulatory challenges in translating these types of combination devices to clinical use. Last year, at this meeting, we presented the preliminary pre-clinical testing for a temporary UHMWPE spacer containing gentamicin sulfate as a first step towards clinical use. Since then, we carried out a survey among the Knee Society membership about their preference for spacer use in two-stage revision surgery and found that 43% prefer to use a CoCr femoral component on an all-poly cemented tibial insert, 22% prefer bone cement spacers molded in the OR, 20% prefer static bone cement spacers, and 14% prefer pre-formed bone cement spacers. We modified our implant design based on the majority's preference for a total knee system, rather than bone cement spacers, in the temporary two-stage approach. In this study, we explored the effect of gentamicin sulfate (GS) elution from UHMWPE/GS tibial inserts on bacterial colonization on CoCr surfaces. Methods. We characterized the gentamicin sulfate (GS) particles with scanning electron microscopy (SEM). We molded UHMWPE/GS powder blends and characterized the morphology using SEM and Energy Dispersive X-Ray Spectroscopy (EDS). We submerged samples of molded UHMWPE/GS in buffered phosphate solution (PBS) at 37°C and quantified the extent of GS elution into PBS with a method described by Gubernator et al. using o-phthaladehyde (OPA) [1]. Under basic conditions, OPA reacts with primary amino groups to form fluorescent complexes. Since gentamicin is the only source of such amino acids in our elution samples, the number of fluorescent complexes formed is directly proportional to the amount of gentamicin in the sample. Using this method, we could quantify gentamicin elution by measuring sample fluorescence post OPA-reaction. We used a plate reader to excite the fluorescent complexes formed in the OPA reaction and measured the resulting emission at wavelengths of 340 nm and 455 nm, respectively. We also quantified the effect of the standard cleaning protocol (heated sonication in alkaline water and alcohol) used to clean UHMWPE implants on subsequent GS elution from UHMWPE/GS samples using the OPA method. We used agar diffusion tests to characterize antibacterial properties of UHMWPE/GS samples after cleaning. For these tests, we collected eluents collected from UHMWPE/GS and gentamicin-impregnated bone cement (BC/GS) following 1, 2, 3, and 4 weeks of elution, and tested against S. aureus (ATCC 12600). We used the “daughter cells” method developed by Bechert et al. to assess anticolonizing properties of UHMWPE/GS [2,3]. We also characterized the colonization of bacteria on CoCr surfaces in the presence of GS eluting from UHMWPE/GS test samples. For this we modified a Pin-on-Disc (PoD) wear tester: An UHMWPE/GS pin and UHMWPE pin (control) articulated against an implant-finish CoCr disc with Tryptic Soy Broth containing S. Aureus as the lubricant. After 18 hrs, we rinsed the articular surfaces of the pin and disc and stamped them onto Agar gel to transfer any adherent bacteria. We incubated the Agar plate overnight such that adherent bacteria proliferated and became visible. Results. SEM characterized the GS particles as hollow spheres (Fig 1a). These formed small groups of agglomerated domains at the virgin resin boundaries of UHMWPE after molding (Fig 1b). Sulfur signature from the EDS analysis identified the agglomerated domains as GS particles (Fig 2). Elution of GS started with an initial burst and was followed by steady elution up to 12 weeks (Fig 3). Cleaning reduced the initial burst GS elution; and the elution remained unchanged after 2 days (Fig 4). The agar diffusion test showed simmilar inhibition zones for the eluents collected from UHMWPE/GS and BC/GS, suggesting that these samples yield similar antibacterial activity against S. aureus (Fig 5). UHMWPE/GS demonstrated pronounced anticolonizing properties, effectively mitigating the proliferation of S. aureus “daughter” cells. Anticolonizing activity of Palacos R+G was not significantly different when compared with UHMWPE/GS. The PoD test showed little-to-no colonization of CoCr surfaces in the presence of UHMWPE/GS pins, indicative of excellent antibacterial properties of UHMWPE/GS against S. aureus. Conclusion. SEM and EDS has allowed us to visualize domains of gentamicin sulfate particles in UHMWPE. Our OPA method has greater precision than traditional agar-well diffusion methods of measuring gentamicin concentration and showed that gentamicin sulfate-loaded UHMWPE elutes at the same rate as Palacos R+G. Pin-on-disc experiments and the daughter cell method both confirmed that these two materials have similar anticolonization abilities. We also found that using the standard cleaning protocol for UHMWPE orthopedic implants decreased the burst of gentamicin eluting from UHMWPE, but after 2 days, it had no effect compared to uncleaned UHMWPE/GS. Finally, we found that UHMWPE/GS can reduce the colonization of bacteria on CoCr. UHMWPE/GS continues to be a promising material for treating PJI. For figures, tables, or references, please contact authors directly

Osteomyelitis is an infection of bone or bone marrow with a concomitant inflammation involving the bone marrow and the surrounding tissues. Chronic osteomyelitis is historically treated in a two-stage fashion with antibiotic-loaded polymethylmethacrylate as local antibacterial therapy. Two-stage surgeries are associated with high morbidity, long hospitalization and high treatment costs. Next to antibiotic releasing biomaterials, S53P4 bioactive glass is a biomaterial that enables one-stage surgery in local treatment of chronic osteomyelitis. S53P4 bioactive glass is gaining interests in recent years in clinical treatment of chronic osteomyelitis in a one-stage fashion due to its antibacterial and bone regenerating capacities. By changing local pH and osmotic pressure S53P4 bioactive glass attack bacteria in a different way as compared to antibiotics. In this presentation, we will present current clinical treatment options for osteomyelitis, clinical results and level of evidence of various biomaterials used in osteomyelitis treatment. In addition, the clinical results and health-economic results of S53P4 bioactive glass will be detailed. Thereafter a summary of the current standing across the board in osteomyelitis treatment will be provided

The enhancement of current bone cement properties is a challenging issue that has been the focus of much research. Developing bone composites with high level of cytocompatibility, mechanical and antibacterial properties is a challenging task. We overcome this challenge by designing a nanocomposite that contain two-dimensional (2D) nanosheets. To develop our novel bone cement nanocomposite, 2D nanosheets were synthesized, mixed in different ratios, and then added to the PMMA matrix. The results reveal that the incorporation of 2D nanosheets into the PMMA matrix leads to increase in the antibacterial properties of the bone cement composite against E. coli bacteria. In addition, the 2D nanosheets improve the compression strength of the bone cement nanocomposite significantly. We also show that nanosheets increased the bioactivity of the bone cements. Finally, MTT assay results indicate that PMMA as a control sample has the lowest cytocompatibility, however, our novel nanocomposites have the highest amount of cytocompatibility. Thus, the current study suggests that 2D nanosheets are potential filler components for the next generation of PMMA bone cement nanocomposites. The findings of this work reveal that the excellent performance of the proposed bone composite can result in a paradigm shift in design of state-of-the art bone cement composites

Aim. Bacteriophages, viruses specific of bacteria, are receiving substantial attention as alternative antibacterial agents to treat bacteria frequently multi-resistant to antibiotics and/or able to form biofilms, such as staphylococci. The latter are responsible for very difficult to treat bone and joint infections (BJIs). In this context, our consortium aims to develop a production of therapeutic phages in accordance with the will of ANSM (French National Agency for the Safety of Medicines and Health Products) to encourage the development of a national academic platform for phage therapy. We report the isolation and characterization of new anti-Staphylococcus phages as well as the evaluation of their activity on a collection of clinical strains of S. aureus (SA) and coagulase-negative staphylococci (CNS) in order to assess their therapeutic potential. Method. Seventeen phages were isolated from wastewater samples. Their identification was obtained by Illumina whole genome sequencing. To evaluate their spectrum of activity, 30 genetically characterized SA strains representative of the main genetic backgrounds as well as 32 strains belonging to 7 CNS species responsible for BJIs were included. The spot test technique, based on the determination of the Efficiency Of Plating ratio, was used (EOP, ratio between the phage titer obtained on a tested strain/titer on a reference strain, close to 1 if high sensitivity to the phage). Results. All isolated phages belonged to the Myoviridae family: 14/17 and 3/17 to the Kayvirus and Silviavirus genera respectively. Silviavirus phages were more active on SA strains (EOP>0.001 for 73–90% of strains) than Kayvirus phages (EOP>0.001 for 13–70% of strains, except for V1SA21: 80%). In total, 83% of strains were susceptible to the phage with the broadest spectrum in each genus, their combination representing a promising opportunity to prevent the emergence of resistance. Kayvirus phages had polyvalent activity on several CNS species (maximum 47% of tested strains), mainly S. lugdunensis, S. capitis and S. caprae, whereas Silviavirus phages were only active on 6–12% of the tested strains. Conclusions. We report the characterization of a large collection of novel phages with complementary spectra against a collection of SA and CNS strains. Further work is currently focused on i) the isolation of anti-S. epidermidis phages, bacterial species against which the present collection of phages was insufficiently active, while it is a major pathogen in this context, ii) the development of production and purification protocols in order to meet the requirements of ANSM for human use

Aim. Staphylococcus epidermidis (S. epidermidis) is one of the main pathogens responsible for bone and joint infections especially those involving prosthetic materials (PJI). Although less virulent than S. aureus, S. epidermidis is involved in chronic infections notably due to its ability to form biofilm. Moreover, it is frequently multiresistant to antibiotics. In this context, the development of additional or alternative antibacterial therapies targeting the biofilm is a priority. Method. The aim of this study was to evaluate in vitro the activity of phage lysin exebacase (CF-301) against biofilms formed by 19 S. epidermidis clinical strains responsible for PJI. We determined the remaining viable bacteria inside the biofilm (counting after serial dilution and plating) and the biomass (bacteria and extracellular matrix, using crystal violet staining) after 24h of exposition to exebacase at different concentrations, alone (0.05; 0.5; 5; 50 and 150 mg/L) or in combination (5, 50 and 150 mg/L) with antibiotics commonly used to treat multi-resistant S. epidermidis PJI (rifampin (1 mg/L), vancomycin (10mg/L) and daptomycin (10mg/L)). In this study, synergy was defined as a significantly higher effect of the association in comparison to the sum of the effect of each molecule. Results. Exebacase showed a dose-dependent reduction of biomass, ranging from 11 % at 0.5 mg/L to 66 % at 150 mg/L. Exebacase showed a significant bactericidal activity at 50 and 150 mg/l, with a mean decrease of the inoculum of 0.94 and 1.7 log, respectively. In addition, synergistic effects were observed in association with i) rifampin (1 mg/L) showing a mean decrease up to 84% of the biomass and 3.5 log CFU at 150 mg/L of exebacase, ii) vancomycin (10 mg/L) showing a mean decrease up to 81% of the biomass and 2.82 log CFU at 150 mg/L of exebacase, iii) and daptomycin (10 mg/L) showing a mean decrease up to 85% of the biomass and 3.1 log CFU at 150 mg/L of exebacase. Conclusions. Exebacase showed, in vitro, synergistic activity with antibiotics against S. epidermidis biofilms. It is a promising adjuvant therapy to rifampin, vancomycin and daptomycin in the context of PJI. Further studies are needed, in vitro to understand the mechanism of action on S. epidermidis biofilm and the heterogeneity of strain behaviour and in vivo to confirm the present data

Introduction: Various antibiotic coatings have been proposed to prevent bacteria colonization and infection of orthopaedic implants. While most of the available technologies seem to provide an effective implant protection from infection, unknown long-term effects of antibiotic coatings raise some concerns for extensive application. Aim of the present study was to develop and test a new fast-resorbable antibacterial carrier to be used as a temporary coating to prevent early bacteria colonization of metallic implants. Methods: The patented tested hydrogel is a co-polimer comprising hyaluronic acid (HA) and a biocompatible polyester (poly-lactic acid) with or without polyethylene glycol chains to further modulate hydrophilicity and anti-fouling characteristics of the compound. The HA derivative is then added to water and mixed, just before its use, with the chosen antibacterial agent. For the purpose of this study, different HA-PLA derivatives have been tested, with two vancomycin and tobramycin concentrations and manually spread to uniformly cover the surface of a titanium specimen. To evaluate the release of vancomycin or tobramycin, high performance chromatographic analysis (HPLC) was carried out. Results: Antibacterial hydrogels provided vancomycin release ranging from 47 % to 80 % in two hours to 100 % (complete release) in 24 to 72 hours, with antibiotic concentrations up to 400 times the minimum inhibiting concentration. The combined release of the two antibiotics (1 % w/v) showed 26.8 % release of vancomycin and 35.8 % of tobramycin at 2 hours and complete release at 72 hours. Doubling antibiotic concentration (2 % w/v), yielded 56.6 % and 76.6 % antibiotic release, respectively for vancomycin and tobramycin at 2 hours and complete release at 48 hours. Discussion and Conclusion: HA chemical derivatization with polyesters leads to the formation of copolymers which can be used to produce antibacterial hydrogels with promising applications in the orthopedic field. These antibacterial hydrogels are in fact easily prepared and spread over a surface, showing the ability of releasing high concentrations of antibiotics for a desired, limited, period of time. Adding antibiotics to the hydrogel just before its use, allows customized antibiotic choice and dosing, avoiding shelf-life problems

Bacterial infection related to prosthetic replacement is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. In order to reduce the incidence of replacement-associated infections, we developed a novel coating technology of Hydroxyapatite (HA) containing silver (Ag). We reported the Ag-HA coating showed high antibacterial activity against E. coli, S. aureus and methicillin-resistant S. aureus (MRSA) under static condition. However, human bodies have a circulating body fluid, which is not a static condition. And the growth and the maturation of biofilm, which is said that a common course of persistent infections at a surgical site, are enhanced by the flow of broth in culture environment. Therefore, we evaluated whether the Ag-HA coating inhibits the biofilm formation on its surface or not by a biofilm-forming test under flow condition in this study. Ag-HA or HA powder was sprayed onto the commercial pure titanium disks using a flame spraying system. The HA coating disks were used as negative control. The biofilm-forming methicillin sensitive S. aureus (BF-MSSA; Seattle 1945) strain and the BF-MRSA (UOEH6) strain were used. The pre-culture bacterial suspension (about 10. 5. colony forming units; CFU) was inoculated onto the Ag-HA and HA coating disks. After cultivation at 37 °C for 1 h, the disks were rinsed twice with 500 μL sterile PBS (-) to eliminate the non-adherent bacteria. The number of the adherent bacteria on these disks was counted using culture method. After rinsing, the disks were transferred into petri-dish containing Trypto–Soy Broth (TSB) + 0.25% glucose with a stirring bar on the magnetic stirrer and they were cultured at 37°C for 7 days. In the meantime, the stirring bar was spun at 60 rounds per minute. Then, the disks were immersed in a fluorescent reagent to stain the biofilm. Finally, the biofilm on each disk was observed by a fluorescence microscope and the biofilm-covered rate on the surfaces of them was calculated using the NIH image software. The number of the bacteria on these disks was not so different between Ag-HA and HA coating after rinsing. After biofilm-forming test, the coverage of the biofilm of BF-MSSA was 2.1% and 81.0% on the Ag-HA and HA coatings, respectively. Similarly, in the case of BF-MRSA, it was 7.7% and 72.0% on the Ag-HA and HA coatings, respectively. Though bacteria slightly adhered, biofilm was hardly observed on the Ag-HA coating. The biofilm on the HA coating was extensive and mature. The inhibition effect of biofilm formation on the Ag-HA coating might be ascribed to the antibacterial effect by Ag ions released from the coating. Because Ag ions have a broad spectrum of antibacterial activity against pathogens, including biofilm forming bacteria, they inhibited the biofilm formation on the Ag-HA coating by killing adherent bacteria. Even in a flow condition, it was suggested that the AgHA shows the antibacterial activity, though the conditions in this work are different from those in living body

Development of antibacterial surfaces or coatings to prevent bacterial adhesion and hence colonization of implants and biofilm formation is an attractive option, in order to reduce the tremendous impact of implant-related infections associated with modern surgery. To overcome the lack of in vivo and clinical models, able to evaluate the performance of anti-adhesive coatings, we designed an in vitro experimental setting that allows to quantitatively evaluate the ability of a coating to reduce bacterial adhesion on a given surface; this model may efficiently serve as a surrogate endpoint to validate anti-adhesive medical devices and compounds. Here we report the results the evaluation of the anti-adhesive properties of a patented, fast-resorbable hydrogel coating, (“Defensive Antibacterial Coating”, DAC). Sterile sandblasted titanium discs of approximately 5cm. 2. surface area were used as substrates for bacterial adhesion. The gel was prepared as follows: syringes prefilled with 300 mg of DAC powder (Novagenit Srl) were reconstituted with 5 ml of sterile water to obtain a hydrogel with a DAC concentration of 6%. Two experiments were conducted. In the first, 200 mg of hydrogel were homogenously spread on the surface of titanium disc, with the spreading device provided by the manufacturer. Both coated and uncoated substrates (controls) were overlaid with a standardized inoculum (10. 8. CFU/ml) of a wild methicillin-resistant Staphylococcus aureus strain, previously isolated from a peri-prosthetic joint infection, for 15, 30, 60 and 120 minutes. Afterwards, non-adherent bacteria were removed by rinsing with sterile saline. The remaining adhered cells were seeded on agar plates for CFU count. In the second experiment, the discs were first inoculated with bacterial cells followed by a treatment with the hydrogel and bacterial count as described above. Ten discs were used for each condition and each time interval (total 160 discs). The adhesion density of S. aureus on titanium discs pre-treated with DAC was significantly lower than that observed on untreated controls at each time point. In particular, the average number of adherent bacteria at 15, 30, 60 and 120 minutes of incubation, was respectively reduced by 86.8%, 80.4%, 74.6% and 66.7%, compared to controls (p<0.001). DAC treatment of discs with previously adhered S. aureus reduced bacterial adhesion, at 15, 30, 60 and 120 minutes of incubation, by, respectively, 84.0% (p<0.05), 72.8%, 72.3% and 64.3% (p<0.001), compared to untreated controls. Our results shows that DAC, “Defensive Antibacterial Coating”, has anti-adhesive properties that allow to reduce bacterial adhesion on a sanded titanium surface by more than 80%, even in the presence of remarkably high bacterial loads (10. 8. CFU/ml), of multi-resistant bacteria (MRSA) and even in the case of previous contamination. Providing anti-adhesive properties to a surface with a fast-resorbable coating may be a safe option to protect inorganic and organic surfaces and biomaterials. Those observation could be the pre-requisite for its in vivo application

Infection remains as one of the major challenges of total joint surgery. One-stage irrigation, debridement and reimplantation or two-stage revision surgery with a temporary implantation of antibiotic eluting bone cement spacer followed by reimplantation are two methods often used to treat infected patients with mixed outcomes. Like bone cement, ultra-high molecular weight polyethylene (UHMWPE) can also be used as a carrier for antibiotics. Recently, we demonstrated that vancomycin and rifampin can be successfully delivered from UHMWPE implants at therapeutic levels to eradicate Staphylococcus aureus biofilm in a lupine animal model. There are regulatory challenges in translating these types of combination devices in to clinical use. One approach is to follow a stepwise strategy, with the first step of seeking clearance for a temporary UHMWPE spacer containing gentamicin sulfate. In this study, we explored the effect of gentamicin sulfate (GS) content in UHMWPE on GS elution rate and antimicrobial activity against methicillin-sensitive S. aureus(MSSA). We also assessed the effect of spacer fabrication on the activity of gentamicin sulfate. We prepared and consolidated UHMWPE/GS blends in varying concentrations. After consolidation, we fabricated test samples with surface area (350mm2) to volume (300mm3) ratio of 1.2 for elution in 1.5ml phosphate buffered saline at body temperature for up to six months and quantified eluted GS content using liquid chromatography – mass spectrometry (LCMS). We assessed the antibacterial activity of the obtained samples in vitro against various concentrations of MSSA (103–106 CFU/ml). Furthermore, we quantified the probability of bacterial colonization of UHMWPE impregnated with GS compared to GS containing bone cement. We assessed any detectable changes in activity of eluted GS caused by spacer fabrication by screening m/z peaks of GS isomers in mass spectra obtained from LC-MS. Gentamicin sulfate activity was not compromised by the elevated temperature and pressure used during spacer fabrication. Elution rate of GS increased with increasing GS content in the blends studied. At comparable elution rates, the GS-loaded UHMWPE was either equivalent or better in terms of antibacterial and anticolonization properties when compared with gentamicin containing bone cement. GS-impregnated UHMWPE is a promising material for temporary spacers

Periprosthetic infection remains a clinical challenge that may lead to revision surgeries, increased spending, disability, and mortality. The cost for treating hip and knee total joint infections is anticipated to be $1.62 billion by 2020. There is a need for implant surface modifications that simultaneously resist bacterial biofilm formation and adhesion, while promoting periprosthetic bone formation and osseointegration. In vitro research has shown that nanotextured titanium promotes osteoblast differentiation, and upregulates metabolic markers of osteoblast activity and osteoblast proliferation. In vivo rat studies confirmed increased bone-implant contact area, enhanced de novo bone formation on and adjacent to the implant, and higher pull-out forces compared to non-textured titanium. The authors have advanced a benign electrochemical anodization process based on ammonium fluoride that creates a nanotube surface in as little as 10 minutes (Fig. 1), which can also integrate antibacterial nanosilver (Fig. 2). The work reported here summarizes in vitro post-inoculation and in vivo post-implantation studies, showing inherent inhibition of methicillin-resistant Staphylococcus aureus (MRSA) by titanium surfaces with nanotubes (TiNT), nanotubes with nanosilver (TiNT+Ag), plain (Ti), and thermal plasma sprayed (TPS) titanium. Ti6Al4V was the base material for all surfaces. In vitro studies evaluated Ti, TPS, four TiNT groups with varying nanotube diameters (60nm, 80nm, 110nm, 150nm), and TiNT+Ag. After seeding with MRSA (10. 5. , 10. 6. , and 10. 8. CFU/mL), the 110nm diameter nanotubes showed MRSA inhibition up to three-orders of magnitude lower than the Ti and TPS surfaces at 2, 6, and 48 hours. Following on the in vitro results, New Zealand White rabbits underwent a bilateral implantation of intramedullary tibial implants of the four material groups (4 mm outside diameter; 110nm NT diameter on TiNT and TiNT+Ag implants). One intramedullary canal was inoculated with clinically-derived MRSA (10. 5. CFU in broth) at the time of implantation; one canal had only culture media introduced (control). At a 2-week endpoint, limbs were harvested for analysis, including implant sonication with sonicant bacterial cultured, histology, and microcomputed chromatography. In the sonicant analysis cohort, TPS showed the lowest average MRSA count, while TiNT and TiNT+Ag were the highest. There was one sample each of TPS, TiNT and TiNT+Ag that showed no MRSA. After an additional 24-hour implant incubation, the TiNT and TiNT+Ag samples had no bacteria, but the TPS grew bacteria; therefore, the authors hypothesize that MRSA more readily releases from the TiNT and TiNT+Ag implants during sonication, indicating weaker biofilm adhesion and development. Histologic analysis is currently underway. In a therapeutic experiment, rabbits underwent bilateral implantation, followed by 1 week of infection development, and then 1 week of vancomycin treatment. At the endpoint, implants were sonicated and bacteria was quantified from the sonicant. TiNT showed viable MRSA at only 30% that of TPS-coated levels, while TiNT+Ag implants showed viable MRSA at only 5% that of TPS-coated levels (Fig. 3). These early results indicate that the TiNT and TiNT+Ag surfaces have some inherent antibacterial activity against MRSA, which may increase the efficacy of systemic antibiotic treatments in the setting of periprosthetic joint infections

Currently, no clinical options are available to prevent infections on uncemented orthopedic implants. Therefore we investigated the efficacy of DAC-hydrogel (disposable antibacterial coating(1), Novagenit, Italy) as carrier for various agents to prevent infections in an in vivo implant-model. Titanium rods were implanted in the left tibiae in New Zealand White rabbits. Prior to implantation, the implant bed was contaminated with 10∧5 colony forming units S. aureus. In the experimental groups, the hydrogel was loaded prior to be coated on the rods with: 2%(w/v) vancomycin (Van2 group, N=6), 5%(w/v) vancomycin (Van5 group, N=6), 10%(w/v) bioactive glass (BonAlive, Finland) (BAG group, N=6), which is antibacterial(2) and osteoconductive(3), or 0.5%(w/v) N-acetyl cysteine (NAC group, N=6), which inhibits bacterial growth and decreases biofilm formation(4). In the control group, empty hydrogel was applied (Gel group, N=12). Blood values were measured weekly. Following explantation on day 28, the anterior tibia was processed for bacterial culture. The posterior tibia and rod were used for measuring bone-implant contact using micro-CT and for histopathology. Results of the experimental groups were compared to the Gel group results. The blood values in the Van2 and Van5 groups were lower on day 7. Moreover, culture results demonstrated less animals with an infection in both groups at day 28. In accordance, these groups showed lower grades for infection. Further, the Van2 group demonstrated more bone-implant contact. These results suggest that infection was reduced in the Van2 and Van5 groups. In contrast, blood values, histological grades, and bone-implant contact of the BAG and NAC groups were comparable with the Gel group. These results suggest that infection was not prevented in the BAG and NAC groups. Local application of vancomycin-loaded DAC-hydrogel successfully reduced implant-related infections. Loading of the hydrogel with BAG or NAC did not prevent infection. It is possible that BAG in powder form, as used in the present study, dissolved before the antibacterial effect could take place. Instead, BAG granules may be a viable alternative. Next, it is possible that the NAC concentration was too low to prevent infections in an in vivo environment, although this concentration was proven effective in vitro for its antibacterial properties

Titanium knee, shoulder and hip implants are typically grit-blasted, thermal plasma spray coated, or sintered to provide ingrowth surface features having texture with pore sizes on the order of hundreds of micrometers. This provides macro and micro-mechanical locking upon bone remodeling. However, at the nanoscale and cellular level, these surfaces appear smooth. In vitro and in vivo research shows surfaces with nanoscale features result in enhanced osseointegration, greater bone-implant contact area and pullout force, and the potential to be bactericidal via a simple hybrid anodization surface modification process. Prior processes for creating nanotube nano-textured surfaces via electrochemical anodization relied on hydrofluoric acid electrolyte and platinum cathodes. This novel process uses ammonium fluoride electrolytes and graphite cathodes which are more cost effective and easier to handle during processing. Hybrid electrolytes with differing concentrations of ethylene glycol, water, and ammonium fluoride provide a variety of nanotube morphologies and sizes. Nano-tubular surfaces on knee tibial and femoral implants, hip stems and acetabular cups, bone screws and other 3D printed parts have been enhanced by this method of nano-texturing in as little as 30 minutes. In vivo work in a Sprague Dawley rat model showed bone-implant contact area up to 2.9-times greater, and uniaxial pullout forces up to 6.9-times greater, than implanted smooth titanium controls at 4 and 12-week time points. In these tests, 1.25mm Kirschner wires were implanted in the rat femora to simulate an intramedullary nail. Histomorphometry in the mid-shaft and distal regions showed greater trabecular thickness and bone tissue mineral density than controls. Axial pullout tests often resulted in bone failure before the bone-implant interface. In vitro evidence suggests that nanoscale surfaces may have an antibacterial effect due to surface energy changes that reduce the ability of bacteria to adhere. However, it is recognized that silver is highly antibacterial in appropriate concentrations. It is also recognized that nanosilver, approximately 10–20nm, is especially effective. Ammonium fluoride anodization is modified using a hybrid electrolyte that includes silver fluoride. By substituting some of the ammonium fluoride with silver fluoride, to maintain a constant total fluorine mass, nanosilver is integrated within and among the nanotubes in the same single process that forms the nanotubes. This hybrid process in nano-texturing titanium implants can be integrated into current manufacturing production at low cost

Orthopaedic and trauma implant related infection remains one of the major complications that negatively impact clinical outcome and significantly increase healthcare expenditure. Hydroxyapatite has been used for many years to increase implant osseointegration. Silver has been introduced into hydroxyapatite as an antimicrobial coating for orthopedic implants. This surface coatings can both increase tissue compatibility and prevent implant-related infections. We examined infection markers and blood silver values, liver and kidney function tests of 30 patients with of three groups of orthopedic implants, external fixators, intramedullary nails and hip replacements, coated with Ag + ion doped CaP based ceramic powder to determine safety and effectiveness of this dual-function coating. During 1 year follow-up, the pin sites were observed at the external fixator group, and wound areas for the proximal femoral nail and hip arthroplasty group at regular intervals. In addition, liver and kidney function tests, infection markers and blood silver values were checked in patients. In the external fixator group, only 4 out of 91 pin sites (%4.39) were infected. The wound areas healed without any problem in patients with proximal femoral nails and hip arthroplasty. There was no side effect suggesting silver toxicity such as systemic toxic side effect or argyria in any patient and blood silver level did not increase. Compared to similar patient groups in the literature, much lower infection rates were obtained (p = 0.001), and implant osseointegration was good. In patients with chronic infection, the implants were applied acutely after removing the primary implant and with simple debridement. Unlike other silver coating methods, silver was trapped in hydroxyapatite crystals in the ionic form, which is released from the coating during the process of osseointegration, thus, the silver was released into the systemic circulation gradually that showed antibacterial activity locally. We conclude that the use of orthopedic implants with a silver ion added calcium phosphate-based special coating is a safe method to prevent the implant-related infection. This work was supported by TUBİTAK Project Number 315S101

Aim. Thermal stability is a key property determining the suitability of an antibiotic agent for local application. Long-term data describing thermal stability without interference from carrier materials are scarce. Method. In this study, a total of 38 common antibiotic agents have been maintained at 37 °C in saline solution, and degradation and antibacterial activity assessed over 6 weeks. The impact of an initial supplementary heat exposure mimicking exothermically-curing bone cement has also been tested. Antibiotic degradation was assessed by chromatography coupled to mass spectrometry or immunoassays, as appropriate. Antibacterial activity was determined by Kirby-Bauer disk diffusion assay. Results. The heat exposure mimicking curing bone cement had minimal effect on stability for most antibiotics, except for gentamicin, which experienced approximately 25% degradation as measured by immunoassay. Beta-lactam antibiotics were found to degrade quite rapidly at 37°C regardless of whether there was an initial heat exposure or not. However some of them maintained relevant concentrations and activity for 2–3 weeks, particularly aztreonam. Excellent long-term stability was observed for aminoglycosides, glycopeptides, tetracyclines and quinolones under both conditions. Conclusions. This study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics. For example, tobramycin would be more suitable for application with bone cement than gentamicin, as it was found to be resistant to heat exposure mimicking curing bone cement

Aim. The preparation of antibiotic-containing polymethyl methacrylate (PMMA), as spacers generates a high polymerization heat, which may affect their antibiotic activity; it is desirable to use bone cement with a low polymerization heat. Calcium phosphate cement (CPC) does not generate heat on polymerization, and comparative elution testings are reported that vancomycin (VCM)-containing CPC (VCM-CPC) exceeded the antibiotic elution volume and period of PMMA (VCM-PMMA). Although CPC alone is a weak of mechanical property spacer, the double-layered, PMMA-covered CPC spacer has been created and clinically used in our hospital. In this study, we prepared the double-layered spacers: CPC covered with PMMA and we evaluated its elution concentration, antimicrobial activity and antibacterial capability. Method. We prepared spherical, double-layered, PMMA-coated (CPC+PMMA; 24 g CPC coated with 16 g PMMA and 2 g VCM) and PMMA alone (40 g PMMA with 2 g VCM) spacers (5 each). In order to facilitate VCM elution from the central CPC, we drilled multiple holes into the CPC from the spacer surface. Each spacer was immersed in phosphate buffer (1.5 mL/g of the spacer), and the solvent was changed daily. VCM concentrations were measured on days 1, 3, 7, 14, 28, 56, and 84. Antimicrobial activity against MRSA and MSSA was evaluated by the broth microdilution method. After measuring all the concentration, the spacers were compressed at 5 mm/min and the maximum compressive load up to destruction was measured. Results. The VCM concentration of the CPC+PMMA spacer exceeded that of the PMMA spacer at all-time points; in particular, it was approximately 7.3 times (109.30 vs. 15.03 μg/mL) and approximately 9.1 times (54.47 vs. 6.50 μg/mL) greater on days 14 and 28, respectively. Using the broth microdilution method, we found that the CPC+PMMA spacer had higher antimicrobial activity than the PMMA model. On day 56, the PMMA spacer lost the capability to inhibit bacterial growth, but the CPC+PMMA spacer maintained this ability. The average maximum compressive load for the CPC+PMMA was 7.28 kN, and that of PMMA was 16.21 kN. Conclusions. The CPC+PMMA spacer was superior to PMMA alone in VCM elution volume and duration, so CP- C+PMMA may be effective for the treatment of MRSA and MSSA infection. The double-layered, antibiotic-loaded cement spacer may maintain antibacterial capability and sufficient strength

Background and purpose: Commercial gentamicin-loaded bone cement beads (Septopal. ®. ) constitute an effective delivery system for local antibiotic therapy. However, these beads are not commercially available in all parts of the world, and are too expensive for common use in others. Therefore, orthopedic surgeons worldwide make antibiotic-loaded beads themselves. However, these beads are usually not as effective as the commercial beads because of inadequate release kinetics. The aim of this study was to develop a simple, cheap and effective formulation to prepare gentamicin-loaded beads with release properties and antibacterial efficacy similar to the ones of commercially available beads. Methods: Acrylic beads were first prepared with variable monomer contents: 500 μl/g polymer (100%), 375 μl/g polymer (75%), and 250 μl/g polymer (50%) to increase gentamicin release through the creation of a less dense polymer matrix. After optimal monomer content was defined, different gel-forming polymeric fillers were added to enhance the permeation of fluids into the beads. Polyvinylpyrrolidone (PVP) 17 was selected as a suitable filler, its concentration was varied and the antibiotic release and antibacterial efficacy of the final beads were compared with the ones of Septopal. ®. beads. Results: Gentamicin release rate and extend of release from beads prepared with 50% monomer increased upon increasing the PVP 17 content in the beads. Beads with 15 w/w% PVP 17 released 87% of their antibiotic content within 336 h. Importantly, this is significantly more than the gentamicin-release from Septopal. ®. beads, that appeared confined to only 59% within 336 h. In addition, acrylic beads with 15 w/w% PVP 17 reduced bacterial growth up to 93%, which is a similar reduction as achieved with Septopal. ®. . Interpretation: A simple, cheap and effective formulation and preparation process has been described for hand-made gentamicin-releasing acrylic beads, with release kinetics and antibacterial efficacy similar to the ones of commercially available Septopal. ®. beads

Introduction. Infection of endoprostheses is a serious complication in orthopedic surgery. As silver is known for its antibactierial effects, silver-coated endoprostheses have gained increased attention to decrease infection rates. However, cytotoxic effects of silver on bone cells have not been investigated in detail. We aimed to investigate whether silver nano-/microparticles and ionic silver exert cytotoxic effects on osteoblasts and osteoclasts in vitro and to correlate potential effects with the antibacterial effect on Staph. epidermidis. Methods. Murine osteoclasts (OC) and murine osteoblasts (OB) were treated with silver particles (avg. sizes: 50nm, 3μm, 30μm, 8μg/ml–500μg/ml) and Ag+NO3- (0.5μg/ml–500μg/ml). Silver treatment started on day 3 to prevent interference with cell adhesion. XTT assays were performed to assess cell viability. Tartrate resistant acidic phosphatase (TRAP) activity and alkaline phosphatase (ALP) activity served as measures for OC and OB differentiation, respectively. The release of silver ions from silver particles was quantified with atomic emission spectometry (AES). Titanium particles (avg. sizes: 50nm and 30μm) were used as controls to investigate whether potential silver effects were particle- or ion-mediated. The antimicrobial activity of silver ions and particles was tested with Staph. epidermidis agar inhibition assays. Results. Ionic silver had the strongest impact on cell differentiation and viability of OC and OB (OC differentiation: mean IC50 = 5 μg/ml, OC viability: mean IC50 = 14 μg/ml, OB differentiation: mean IC50 = 1 μg/ml, OB viability: mean IC50 = 1 μg/ml). Silver nanoparticles decreased cell differentiation and viability in a dose dependent manner (OC differentiation: mean IC50 = 5μg/ml, OC viability: mean IC50 = 14μg/ml, OB differentiation: mean IC50 = 1μg/ml, OB viability: mean IC50 = 1μg/ml). Silver microparticles as well as titanium nano- and microparticles had no effect on cell differentiation and viability. AES showed a size and dose dependent release of silver ions from silver nano- and microparticles. Agar inhibition assays showed a dose correlation of the antibacterial effect of silver with the cytotoxic effects on OB and OC. Conclusion. Silver nanoparticles and silver ions exert dose-dependent cytotoxic effects on OB and OC in vitro resulting in a severe alteration of cell differentiation and viability. The effect of silver on OB and OC seems to be mediated primarily by silver ions and correlates with the substance's antibacterial effects. The cytotoxicity of silver nanoparticles is mediated primarily by the size-dependent liberation of silver ions. Disturbance of OB and OC survival may have deleterious effects on the osseointegration of orthopedic implants. Further in vivo studies are needed to investigate the osseointegration of silver coated implants prior to their widespread clinical application

Chronic osteomyelitis is historically treated in a two stage fashion with antibiotic-loaded polymethylmethacrylate (PMMA) as local antibacterial therapy. However, two-stage surgeries are associated with high morbidity, long hospitalization and high treatment costs. In recent years new biomaterials were developed that allow to change this treatment algorithm. S53P4 bioactive glass is such a novel biodegradable antibacterial bone graft substitute that enables a one-stage surgery in local treatment of chronic osteomyelitis. This study aimed to explore the eradication of infection and bone healing capacities of S53P4 bioactive glass in clinical practice. In this prospective longitudinal outcome study, clinical applicability of S53P4 bioactive glass in treatment of patients with chronic osteomyelitis was assessed. All patients with clinically, haematologically and radiologically evident chronic osteomyelitis were included. All patients were treated with an extensive debridement surgery, S53P4 bioactive glass implantation and systemic antibiotic administration. Primary endpoint of this study is eradication of infection. During follow-up eradication was analysed based on clinical outcomes, blood samples (inflammatory parameters) and radiological outcomes. The secondary endpoint, bone healing, is assessed using conventional radiographic images of the treated region. Between 2011 and 2016, 25 patients were included in this study, with a mean follow-up of 23 months (range 4 – 57). Hospital stay was short with a mean of 18 days (range 4 – 40) and patients required an average of 1,4 surgeries (range 1 – 4). The inflammatory parameter C-reactive protein (CRP) showed a normalization after a mean duration of 46 days (range 0 – 211). At the end of follow-up haematological and clinical outcomes showed eradication of infection in 24 (96%) of all patients. Radiologically none of all patients showed persisting signs of infection and bone healing was observed in 22 (88%) patients based on changes on conventional radiographic images. One patient had a persistent infection without any bone healing, this patient had an infected non-union prior to surgery. There were two other patients with an initial infected non-union fracture which was not consolidated at last follow-up, although they had successful infection treatment. Another patient had a femoral fracture after surgery that needed additional surgery which did not interfere with eradication of infection. Four (16%) of all patients had initial wound healing problems related to compromised skin and/or soft tissue prior to surgery. Based on the results of our clinical experience, S53P4 bioactive glass can successfully be used in a one-stage procedure for treatment of chronic osteomyelitis. Eradication of infection was successful in almost all patients and so far no patients required a second surgery due to infection recurrence. Bone healing (incorporation of the bioactive glass) was seen in all patients except for the patients with an initial infected non-union fracture. As a consequence of these results, we changed our institutional protocol for treatment of chronic osteomyelitis to a one-stage approach instead of a two-step approach

Objective. Bacterial infection is a serious complication after joint replacement surgery. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and epidermidis(MRSE) are very difficult to eradicate in infected prosthetic joint. Therefore, the retention rate of initial prosthesis affected with such resistant microorganisms is still low. Gentian violet shows potent antibacterial activity against gram-positive cocci as minimal bactericidal concentration is less than 0.1 %. In the present study, we investigated the efficacy of treatment with gentian violet against MRSA and MRSE infections after THA, TKA, and bipolar hip hemiarthroplasty (BHP). Methods. There were 8 patients in this study; five patients with deep periprosthetic MRSA infection (2 THA, 2 BHP, 1 revision TKA); 3 patients with MRSE infection (1 revision THA, 1 BHP, 1 TKA). When infection was suspected after the surgery, we quickly obtained synovial fluid and periprosthetic soft tissue from the joint and applied to culture and microscopic examinations for detection of microorganisms. After identification of bacterial species, we immediately debrided the affected joint and washed thoroughly twice with 0.1% solution of gentian violet for 3 minutes each, followed by intra-articular multiple injection of arbekacin sulfate solution. Then we inserted an aspiration tube into the joint and administered appropriate antibiotics intravenously. If the inflammatory symptoms persisted in spite of the first treatment, we repeated the treatment until inflammation signs and intra-articular microorganisms could not be detected. Results. At first we examined the bactericidal activity of gentian violet solution against MRSA and MRSE by culture examination with or without the solution. We confirmed gram-negative bacillicould be alive but the both MRSA and MRSE could not be alive by the treatment with 0.01 % solution of gentian violet for 3 minutes. The treatment with gentian violet allowed subsidence of the infection in all patients. Furthermore, we could preserve 4 prostheses with MRSA infection; 1 THA, 1 revision TKA and 2 BHPs; 2 prostheses with MRSE infection; 1 TKA, 1BHP. However, we could not preserve 2 prostheses, 1 THA with MRSA infection and 1 revision THA with MRSE infection. Thus, the prosthetic retention rate of this study for MRSA and MRSE infections was 75 %. We performed two-stage operation for the 2 patients in whom we could not preserve the prostheses as follows: after excision of the infected total hips followed by successful reimplantation. Moreover, all of the patients in this study are able to keep the walking ability after joint replacements. Conclusion. Infection of methicillin-resistant Staphylococcus species after joint replacement surgery has tended to increase. Gentian violet exerts a potent antibacterial activity against such microorganisms at the concentration of less than 0.1 %. We obtained good retention rate by the treatment using gentian violet against MRSA and MRSE infections after the joint surgeries. In addition, both MRSA and MRSE have not showed drug resistance for gentian violet. Therefore, we suggest that gentian violet will become a promising adjunct agent for infection after joint replacement surgery not only methicillin-sensitive but also methicillin–resistant Staphylococcus species

Surgical site infection related to orthopaedic implants is one of the serious complications. In the previous works, we developed a novel thermal spraying technology combined silver with hydroxyapatite (HA) in order to resolve such problems, and reported the property and antibacterial effect of them in vitro. However, no previous reports have investigated in vivo. Therefore, we monitored serum silver level in rats to clarify in vivo kinetics of silver released from the coating. HA loaded with 3 wt % of silver oxide (HA-Ag) and plain HA powder were sprayed on surface of titanium disks (20 mm diameter × 1 mm thick) by the flame spraying, which is a kind of thermal spraying method with acetylene torch. All these test pieces were obtained from Japan Medical Materials Corporation (JMM, Osaka, Japan). Both samples were implanted singly into the back subcutaneous pockets of male Sprague-Dawley rats (150–200 g). Rats were housed individually and given ad libitum access to food and water. After 24 h, 48 h, 7 d, 14 d and 28 d, the rats were sacrificed, and then the blood was drawn from common iliac vein. All procedures were operated under anesthesia. These blood samples were spun down and serum silver levels were measured by an inductively coupled plasma mass spectrometry. The average serum silver level in HA-Ag group had increased to more than 40 ppb until 48 h after implantation, and then decreased rapidly to normal level. There were significant differences (p < 0.05) between HA-Ag and HA group, at each measurement period. This is the first report to elucidate the serum silver level in rats implanted HA-Ag coatings. To date, reported coating technologies have included direct-loading antibacterial agents or heavy metals including silver with prosthesis base. The combine technology HA with silver would be effective in not only antibacterial but also osteoconductive respect. Our experimental results highlight the following 2 features: the serum silver levels peaked relatively early, and the levels reduced immediately to normal level after the peak. Therefore, we speculate that the released silver would not be accumulated generally, which not contribute long-term toxicity, and the coating would be suitable for prevention of early surgical site infections. This study provides novel and important information on in vivo release- property for HA-Ag coating, and suggests this coating is effective against not late but rather early infection related to orthopaedic implants

The June 2014 Research Roundup. 360 . looks at:Intraoperative irrigation a balance of toxicities; Ibandronate effective in bone marrow oedema; Risk stratification in damage control surgery; Osteoblast like cells potentially safe; Better wear and antibacterial?; Assessing outcomes in hip fracture

Aim. Focused high energy extracorporeal shockwave therapy (fhESWT) is used to support fracture healing in non-union cases and has been shown to have antibacterial effects. We trialed fhESWT as an adjunct to conventional treatment in a clinically relevant rabbit model of fracture related infection. Method. A complete humeral osteotomy was performed in 31 rabbits and fixed with a 7-hole-LCP. A fracture-related infection (FRI) was established with Staphylococcus aureus. After two weeks, a revision surgery was performed with debridement, irrigation and implant retention. Rabbits then received: no further treatment (controls); shockwaves (at day 2 and 6 after revision, 4'000 Impulses each time with 23kV); systemic antibiotics (rifampin and nafcillin) over one week in weight adjusted dosages; or the combination of antibiotics and shockwaves. Treatments were applied over one week. Blood cultures were taken before and after shockwave sessions. After an additional week without treatment, rabbits were euthanized, and quantitative bacteriology was performed on implants and tissues to determine infection burden. Indicator organs (brain, heart, liver, lungs, kidneys and spleen) were cultured to assess possible bacteraemia due to fhESWT. Results. All rabbits were infected at revision surgery as determined by bacteriological culture of debrided materials. fhESWT in combination with antibiotic treatment lowered the bacterial burden at euthanasia hundredfold compared to antibiotic treatment alone in all samples (p=0.38). This effect was most prevalent for the implant sample (p=0.08). No significant effect was seen for fhESWT alone compared to untreated controls. No signs of bacteraemia occurred. Conclusions. The additon of systemic antibiotics had the biggest effect on reduction of bacteria. Although further lowering the bacterial burden in our model the effect of fhESWT as an adjunct was not big enough to be statistically secured in this in vivo rabbit model. In certain difficult-to-treat infections the addition of fhESWT might be beneficial. The method appears to be safe in this model of acute FRI as no signs of bacteremia occurred despite the high energy and impulse number. Further investigations are needed to identify the correct indication

Aims

Delayed postoperative inoculation of orthopaedic implants with persistent wound drainage or bacterial seeding of a haematoma can result in periprosthetic joint infection (PJI). The aim of this in vivo study was to compare the efficacy of vancomycin powder with vancomycin-eluting calcium sulphate beads in preventing PJI due to delayed inoculation.