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

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

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

Summary Statement. Wear of total knee replacement (TKR) is a clinical concern. This study demonstrated low-conformity moderately cross-linked-polyethylene fixed bearing TKRs showed lower volumetric wear than conventional-polyethylene curved fixed bearing TKRs highlighting potential improvement in TKR performance through design and material selection. Introduction. Wear of total knee replacement (TKR) continues to be a significant factor in the clinical performance of the implants. Historically, failure due to delamination and fatigue directed implant design towards more conforming implants to reduce contact stress. However, the new generations of more oxidatively-stable polyethylene have improved the long-term mechanical properties of the material, and therefore allowed more flexibility in the bearing design. The purpose of this study was to investigate the effect of insert conformity and material on the wear performance of a fixed bearing total knee replacement through experimental simulation. Methods. The wear of TKR bearings were investigated using a physiological six station Prosim knee wear simulator (Simulator Solutions, UK). Six samples of each test configuration (Sigma CR fixed bearing knees (DePuy Synthes, UK) were studied, and compared with previously reported data, tested under identical conditions (1, 2). The central axis of the implant was offset from the aligned axes of applied load and tibial rotation to replicate a right knee. High kinematics, under anterior-posterior displacement control was used for this study (3). The lubricant was 25% (v/v) calf serum supplemented with 0.03% (v/v) sodium azide solution in deionised water, as an antibacterial agent, and was changed approximately every 0.33Mc. Wear was assessed gravimetrically and moisture uptake accounted for using unloaded soak controls. Results. The wear rates for the moderately cross-linked inserts (XLK) were significantly lower than the conventional polyethylene (GVF) for all geometries (ANOVA, p<0.05). There was a significant reduction in wear rate as the insert geometry became less conforming for both materials (ANOVA, p<0.05). The wear scars areas were comparable in size and shape between materials, within a geometry group. The size of the wear scar changed with conformity, with the curved inserts showing the largest scars in both anterior-posterior and medial-lateral dimensions, and the flat inserts showing the smallest wear scars. Discussion/Conclusion. The introduction of a moderately cross-linked polyethylene insert was shown to significantly reduce the wear of a fixed bearing total knee replacement compared with a conventional material. There was a trend for reducing wear rate with reducing conformity for both materials, suggesting that reduced conformity results in higher contact pressures and reduced contact area, leading to a reduced surface for wear to occur. Both material and conformity were shown to have a significant impact on the wear of a fixed bearing TKR, and therefore provide opportunity for enhancing wear performance through material and design selection