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

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

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

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

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

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

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

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

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

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

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.

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