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