Infection around implanted biomaterials in humans is a major healthcare issue and current ability to effectively prevent and treat such infections using antibiotics is limited. The hypothesis of the study was that surface charge could be manipulated to a positive state and thus moderate bacterial adhesion to the implant. The surface charge was manipulated by creating a galvanic cell using a zinc strip in a standard suction drain. Adhesion of Staph. aureus and Staph. Epidermidis to stainless steel and titanium implants in vitro and in vivo was quantified by sonication and log dilution technique. The response to this surface manipulation of charge varied for both the bacterial species and the type of metallic implant. In vitro studies produced an 88% reduction in Staph. aureus adhesion to stainless steel and a 36% reduction in adhesion to titanium. However Staph. epidermidis showed an increased adhesion to stainless steel (Log 1.81 ± 1.12 in vitro) and to titanium (log 1.80 ± 0.12). Staph aureus demonstrated a log increase of 1.56± 0.09 in adhesion to titanium in vivo while Staph. epidermidis generated a log increase of 3.97± 0.10 in adherent bacteria. In this experiment we have shown that alteration of the electrochemical environment around an implant influences bacterial adhesion. While our technique is not therapeutically viable, further manipulation of surface charge of an implant is possible using other electroactive materials. This may be explored in the prophylactic treatment of implant infection

Although there is some clinical evidence of ceramic bearings being associated with a lower infection rate after total hip arthroplasty (THA), available data remains controversial since this surface is usually reserved for young, healthy patients. Therefore, we investigated the influence of five commonly-used biomaterials on the adhesion potential of four biofilm-producing bacteria usually detected in infected THAs. In this in-vitro research, we evaluated the ability of S. aureus, S. epidermidis ATCC 35984, E. coli ATCC 25922 and P. aeruginosa to adhere to the surface of solid biomaterials, including a 28mm cobalt-chromium metal head, a 28mm fourth-generation ceramic head, a 48mm fourth-generation ceramic insert, a 48mm highly-crossed linked polyethylene insert and a 52mm titanium porous-coated acetabular component. After an initial vortex step, a bacterial separation from the surface of each specimen was done until no remaining attached bacteria were observed by digital optical microscope. The colony-forming units were counted to determine the number of viable adherent bacteria and the bacterial density. We found no differences on global bacterial adhesion between the different surfaces. E. coli presented the least adherence potential among the analysed pathogens (p<0.001). The combination of E. coli and S. epidermidis generated an antagonist effect over the adherence potential of S. epidermidis individually (58±4% vs. 48±5%; p=0.007). The combination of P. aeruginosa and S. aureus presented a trend to an increased adherence of P. aeruginosa independently, suggesting an agonist effect (71% vs. 62%; p=0.07). In this study, ceramic bearings appeared not to be related to a lower bacterial adhesion than other biomaterials. However, different adhesive potentials among bacteria may play a major role on infection's inception

Aim. To evaluate bacterial adhesion and biofilm formation to metallic cerclage wire versus polymer cerclage system (SuperCable®). Methods. Experimental in vitro study to evaluate quantitative bacterial adherence to different cerclage wire materials. Two types of cerclage wires were compared: a metallic versus a polymer based wire (SuperCable®). A two-centimeter cerclage wire piece of each material was included in 2 mL of tryptic soy broth (TSB) culture media, inoculated with 10 microliters of a 0.5 McFarland of a Staphylococcus epidermidis strain and cultivated at 37°C during 2h for adhesion and 48h for biofilm formation. After this time, the cerclages were washed using a 1% phosphate buffered saline (PBS) and sonicated in new culture medium. After sonication, dilutions of each culture were spread in TSB agar and incubated 37°C during 24h. The number of colonies were counted and the cfu/cm2 was calculated. Results. There were no differences in the number of colonies counted at 2 hours. At 48 hours, the polymer cerclage system showed a clinically and statistically reduction of 95.2% in the biofilm formation of S. epidermidis. The highest bacterial counts were observed in metallic cerclages after 48h. Conclusion. In in vitro conditions, the polymer cerclage system may offer decreased biofilm formation compared with metallic cerclage wires. However, there are many other factors in in vivo conditions that could play a role in bacterial adhesion to cerclage wires. Further research is needed in order to recommend the use of polymer cerclage systems for septic revision surgery

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

Aim: To investigate the effect of manipulation of the electrochemical environment around metallic implants on bacterial biofilm formation. Background: The inability to prevent and treat prosthetic bacterial infection is a significant orthopaedic problem. Current antimicrobials are ineffective against bacterial biofilm communities. It is hypothesised that the alteration of the micro-environment could inhibit bacterial adhesion sufficiently to prevent biofilm formation allowing normal tissue integration to occur. Previous work by this group using zinc caused increased bacterial biofilm formation. Platinum being at the opposite end of the galvanic spectrum should cause the opposite effect. Materials and Methods: Titanium 2mm Kirschner (K) wires (N=14) and Stainless Steel K wires (N=14) were cut into 50mm segments and sterilised. These were inoculated with either Staphylococcus Epidermitis (NC011047) or Staphylococcus Aureus (NC012973) suspensions. Superficial, non-adherent bacteria were removed by serial rinsing in phosphate buffered solution (PBS). The K wires were added to either the culture media alone or the culture media containing platinum and incubated at 37 degrees for 24 hours. The wires were then removed from the media and rinsed in PBS. Samples were subjected to sonication, to fragment biofilms thereby releasing the bacteria, which were then quantified by serial log dilution technique and manual counting. The presence of platinum reduced the adhesion of both Staph Aureus and Staph Epidermidis to stainless steel. This reduction was statistically significant using paired t-test (SPSS version 6.0). There was a significant reduction of adhesion with platinum in the Staph Aureus and titanium group while the reduction in the Staph Epidermidis and titanium group did not reach statistical significance. Conclusion: The use of platinum to manipulate the microcurrent around metallic implants reduces bacterial biofilm formation in vitro. This has obvious clinical implications in prevention of implant infections

We have assessed the different adhesive properties of some of the most common bacteria associated with periprosthetic joint infection on various types of ultra high molecular Weight Polyethylene (UHMWPE). Quantitative in vitro analysis of the adhesion of biofilm producing strains of Staphylococcus aureus and Escherichia coli to physically and chemically characterised standard UHMWPE (PE), vitamin E blended UHMWPE (VE-PE) and oxidised UHMWPE (OX-PE) was performed using a sonication protocol. A significant decreased bacterial adhesion was registered for both strains on VE-PE, in comparison with that observed on PE, within 48 hours of observation (S. aureus p = 0.024 and E. coli p = 0.008). Since Vitamin E reduces bacterial adhesive ability, VE-stabilised UHMWPE could be valuable in joint replacement by presenting excellent mechanical properties, while reducing bacterial adhesiveness.

Cite this article: Bone Joint J 2014;96-B:497–501.

Summary

Prosthetic UHMWPE added with vitamin E and crosslinked UHMWPE are able to decrease significantly the adhesion of various bacterial and fungal strains limiting biomaterial associated infection and consequent implant failure.

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

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

One of the most common bacteria in orthopaedic prosthetic infections is Staphylococcus Aureus. Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion on nanofabricated materials. We hypothesise that surface nanotopography impacts the differential ability of staphylococci species to adhere via altered metabolomics and may reduce orthopaedic implant infection rate. Bacteria were grown and growth conditions optimised. Polystyrene and titanium (Ti) nanosurfaces were studied. The polystyrene surfaces had different nanopit arrays, while the Ti surfaces expressed different nanowire structures. Adhesion analysis was performed using fluorescence imaging, quantitative PCR and bacterial percentage coverage calculations. Further substitution with ‘heavy’ labelled glucose into growth medium allowed for bacterial metabolomic analysis and identification of any up-regulated metabolites and pathways. Our data demonstrates reduced bacterial adhesion on specific nanopit polystyrene arrays, while nanowired titanium showed increased bacterial adhesion following qPCR (P<0.05) and percentage coverage calculations (P<0.001). Further metabolomic analysis identified significantly increased intensity counts of specific metabolites (Pyruvate, Aspartate, Alanine and Carbamoyl aspartate). Our study shows that by altering nanotopography, bacterial adhesion and therefore biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. The identification of metabolic pathways involved in adhesion may allow for a targeted approach to biofilm eradication in S. aureus. This is of significant benefit to both the patient and the surgeon, and may well extend far beyond the realms of orthopaedics

Aim. Biofilm-related infections represent a recurrent problem in the orthopaedic setting. In recent years, great interest was directed towards the identification of novel molecules capable to interfere with pathogens adhesion and biofilm formation on implant surfaces. In this study, two stable forms of α-tocopherol, the hydrophobic acetate ester and the water-soluble phosphate ester, were tested in vitro as coating for titanium prostheses. Method. Antimicrobial activity against microorganisms responsible of prosthetic and joints infections was assessed by broth microdilution method. In addition, α-tocopherol esters were evaluated for both their ability to hamper bacterial adhesion and biofilm formation on sandblasted titanium surfaces. Results. Only α-tocopheryl phosphate displayed antimicrobial activity against the tested strains. Both esters were able to significantly interfere with bacterial adhesion and to prevent biofilm formation, especially by Staphylococcus aureus and Staphylococcus epidermidis. The activity of α-tocopheryl phosphate was greater than that of α-tocopheryl acetate. Alterations at membrane levels have been reported in literature1 and may be likely responsible for the interference on bacterial adhesion and biofilm formation shown by α-tocopherol esters. Conclusions. Although further studies are needed to better investigate the mechanisms of action and the spectrum of activity of α-tocopherol esters, these characteristics, together with the positive effect on wound healing and immune response, make these molecules promising candidate for coating in order to prevent implant-associated infections

Summary Statement. Description of an original in vitro protocol for assessing combined bacteria and cell competitive adherence on the surface of biomaterials of medical interest. Objectives. Biomaterial-related infections are a major clinical problem. The pathogenesis of this syndrome has been described as a competitive adherence between bacteria and human cells in the so-called “race for the surface” theory. The aim of this study is to develop an in vitro protocol method that can be used to investigate this competence and its implications in the development of materials aimed to be used in orthopedic surgery. Methods. An in vitro study was performed using as a substrate a surface of chemically polished Ti6Al4V (CP) alloy ELI grade, according to the standard ASTM F136-02. For bacterial adhesion studies we used S. aureus 15981 and S. epidermidis ATCC 35984 strains. Bacteria were inoculated in tryptic soy broth with 5% CO2. After overnight culture, bacteria were centrifuged for 10 min at 3500g at 22°C. Supernatant was discarded and the pellet was washed × 3 with sterile PBS. Bacteria were then suspended and diluted in PBS to obtain a 10. 8. CFU/mL concentration. Serial 1:10 dilutions were then made in order to obtain different bacterial concentrations (10. 7. , 10. 6,. 10. 5. , 10. 4. , 10. 3. CFU/mL). Osteoblastic MC3T3-E1 cells at of 100.000 cells/mL were mixed (1:1, v/v) with the different bacterial dilutions in a total volume of 2 ml of α-MEM. A bacterial control (1 mL of 10. 8. CFU/mL of bacteria + 1 mL of PBS), a cellular control (1 mL of 100.000 cells/mL + 1 mL of PBS) and a negative control (2 mL of PBS) were also included. A total of 9 CP specimens were covered with the different dilutions tested, and incubated for 6 h at 37°C to allow adhesion in a static model. After incubation, samples were washed three times with PBS, and fixed to the CP surface with MicroFIX spray during 1 minute. After that, they were washed again with sterile water, stained for 2 min with acridine orange, and rinsed with sterile water. Eight photographs were made for each sample at 40 × magnification to study the bacterial adhesion, and at 20X magnification to study the cellular adhesion, by using an ultraviolet microscope. Results and Discussion. Bacterial adhesion was observed in the specimens inoculated with 10. 8. , 10. 7. , 10. 6. CFU/mL, but not at lower dilutions. Cellular adhesion was observed for all samples. Differences in the cellular morphology were observed when the bacterial concentration decrease, probably because bacterial adhesion has a negative influence in the cellular adhesion to the surface. This protocol can be useful for the evaluation of the bacterial and cellular adherence process on modified biomaterials. Conclusion. These results demonstrate that bacteria cannot colonise cell-covered material at a certain breakpoint

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

The most common bacteria in orthopaedic prosthetic infections are Staphylococcus, namely Staphylococcus Epidermidis (SE) and Staphylococcus Aureus (SA). Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion and biofilm formation on nanofabricated materials. Bacteria studied were clinically relevant from an orthopaedic perspective, SA and SE. We hypothesise that that nanosurfaces can modulate bacterial adherence and biofilm formation and may reduce orthopaedic implant infection rate. Isolated bacteria were grown and growth conditions optimised. Bacterial concentrations were calculated by using qPCR. Statistical analysis allowed identification of optimal biofilm growth conditions. These were refined on standard, non-nanopatterned surfaces, and then control and nanopatterned polystyrene (nanopits) and titanium plates (nanowires). Adhesion analysis was performed using fluorescence imaging and quantitative PCR. 4 bacterial strains were isolated and cultured. Growth kinetics based on 24hr cultures allowed isolation of optimal media for biofilm conditions (Dulbecco's Modified Eagle Medium with additional supplements). Highest bacterial concentrations were found following 2hrs incubation with Lysozyme during qPCR. Bacterial concentration significantly increased between 30, 60 and 90 minutes incubation. Differences in percentage coverage on different polysyrene nanosurfaces (nanopits) were noted varying. This was confirmed by qPCR extractions that showed different bacterial concentrations on different nanopatterns. Titanium nanowire surfaces significantly increased bacterial adhesion (P<0.05). Our study cultured and quantified bacterial biofilm and suggests that by altering nanotopography, bacterial adhesion and therefore biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. Clearly this is of significant benefit to the patient, the surgeon and the NHS, and may well extend far beyond the realms of orthopaedics

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

Aims. Periprosthetic joint infection (PJI) is a debilitating condition with a substantial socioeconomic burden. A novel autologous blood glue (ABG) has been developed, which can be prepared during surgery and sprayed onto prostheses at the time of implantation. The ABG can potentially provide an antimicrobial coating which will be effective in preventing PJI, not only by providing a physical barrier but also by eluting a well-known antibiotic. Hence, this study aimed to assess the antimicrobial effectiveness of ABG when impregnated with gentamicin and stem cells. Methods. Gentamicin elution from the ABG matrix was analyzed and quantified in a time-dependent manner. The combined efficiency of gentamicin and ABG as an anti-biofilm coating was investigated on titanium disks. Results. ABG-gentamicin was bactericidal from 10 μg/ml and could release bactericidal concentrations over seven days, preventing biofilm formation. A concentration of 75 μg/ml of gentamicin in ABG showed the highest bactericidal effect up to day 7. On titanium disks, a significant bacterial reduction on ABG-gentamicin coated disks was observed when compared to both uncoated (mean 2-log reduction) and ABG-coated (mean 3-log reduction) disks, at days 3 and 7. ABG alone exhibited no antimicrobial or anti-biofilm properties. However, a concentration of 75 μg/ml gentamicin in ABG sustains release over seven days and significantly reduced biofilm formation. Its use as an implant coating in patients with a high risk of infection may prevent bacterial adhesion perioperatively and in the early postoperative period. Conclusion. ABG’s use as a carrier for stem cells was effective, as it supported cell growth. It has the potential to co-deliver compatible cells, drugs, and growth factors. However, ABG-gentamicin’s potential needs to be further justified using in vivo studies. Cite this article: Bone Joint Res 2020;9(12):848–856

Objectives. Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied. Methods. We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis. Results. Our results show that clinical strains adhere to the material surface at lower concentrations than collection strains. A destructive effect of bacteria on preosteoblastic cells was also detected, especially with higher concentrations of bacteria. Conclusions. The method described herein can be used to evaluate the effect of surface modifications on bacterial adherence more accurately than conventional monoculture studies. Clinical strains behave differently than collection strains with respect to bacterial adherence. Cite this article: M. Martinez-Perez, C. Perez-Jorge, D. Lozano, S. Portal-Nuñez, R. Perez-Tanoira, A. Conde, M. A. Arenas, J. M. Hernandez-Lopez, J. J. de Damborenea, E. Gomez-Barrena, P. Esbrit, J. Esteban. Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the “race for the surface” theory. Bone Joint Res 2017;6:315–322. DOI: 10.1302/2046-3758.65.BJR-2016-0226.R2

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus are the most common infecting agents associated with acute PJI, and also appear in some cases of delayed PJI. 1. S. aureus biofilm development can be divided in two stages: adhesion and proliferation. 2. To avoid PJI bacterial adhesion has to be decreased. Hybrid organo-inorganic sol-gel coatings are proposed as a promising biomaterial improvement. 3. One of these compounds is a mixture of two organopolisiloxanes: 3-methacryloxypropyltrimethoxysilane (MAPTMS) and tetramethylorthosilicate (TMOS). The aim of this work was to evaluate bacterial adhesion on MAPTMS-TMOS coating compared to titanium parts made by powder metallurgy. MAPTMS-TMOS sol-gel coating was produced using a molar ratio of 1:2 (MAPTMS:TMOS) and dispersed in ethanol. The sol-gel was deposited by dip-coating on titanium parts made by powder metallurgy followed by a thermal treatment at 120 ºC for 30 minutes. 4. Titanium parts without sol-gel coating were used as control. S. aureus 15981 strain adherence study was performed using the protocol described by Kinnari et al. 5. with 90 min incubation. After incubation, the samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of total adhered, live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate. The statistical data were analyzed by pairwise comparisons using the nonparametric Mann-Whitney test with a level of statistical significance of p<0.05. Values are cited and represented as medians. S. aureus 15981 adherence was 942-fold lower on MAPTMS-TMOS coating than on uncoated titanium. According with our results, MAPTMS-TMOS sol-gel coating is a promising antiadherent surface for S. aureus. More studies are necessary in order to evaluate this property with other species and strains

The aim of this study was to evaluate whether coating titanium discs with selenium in the form of sodium selenite decreased bacterial adhesion of Staphylococcus aureus and Staph. epidermidis and impeded osteoblastic cell growth. In order to evaluate bacterial adhesion, sterile titanium discs were coated with increasing concentrations of selenium and incubated with bacterial solutions of Staph. aureus (ATCC 29213) and Staph. epidermidis (DSM 3269) and stained with Safranin-O. The effect of selenium on osteoblastic cell growth was also observed. The adherence of MG-63 cells on the coated discs was detected by staining with Safranin-O. The proportion of covered area was calculated with imaging software. The tested Staph. aureus strain showed a significantly reduced attachment on titanium discs with 0.5% (p = 0.011) and 0.2% (p = 0.02) selenium coating. Our test strain from Staph. epidermidis showed a highly significant reduction in bacterial adherence on discs coated with 0.5% (p = 0.0099) and 0.2% (p = 0.002) selenium solution. There was no inhibitory effect of the selenium coating on the osteoblastic cell growth. Selenium coating is a promising method to reduce bacterial attachment on prosthetic material. Cite this article: Bone Joint J 2013;95-B:678–82

In England and Wales in 2012 over 160,000 primary total hip and knee replacements were performed with 57% of hip replacements utilising uncemented prostheses. The main cause of failure, affecting approximately 10% of patients, is aseptic loosening. Previous research has found that functionalising titanium with lysophosphatidic acid (LPA) induces an increase in human osteoblast maturation on the implant surface through co-operation with active metabolites of vitamin D3. This feature, the small size of the LPS molecule and its affinity to readily bind to titanium and hydroxylapatite makes it an especially desirable molecule for bone biomaterials. Nevertheless biomaterials that also demonstrate anti-microbial properties are highly desirable. To test the antimicrobial efficacy of the LPA-functionalised titanium, a clinical isolate of Staphylococcus aureus, obtained from an infected revision surgery, was cultured on the surface of titanium discs functionalised with 0, 0.1. 0.5, 1, 2 and 5μM LPA. Bacterial adhesion was quantified at 1, 2, 6, 12 and 24 hours by live/dead counts and biofilm mass quantified by crystal violet staining after 24, 48, 72 and 96 hours culture. To elucidate the mechanisms of action of LPA, proteomic analysis of adhered bacteria was performed using SDS-PAGE and Western blots. 500nM to 1μM LPA were the optimum concentrations to significantly inhibit bacterial adhesion (ANOVA, p<0.001). These concentrations also reduced biofilm mass on the surface of the titanium. Proteomic analysis highlighted an increase in low molecular weight proteins as a result of optimal LPA surface concentrations. Fatty acid chains as found in LPA have previously been associated with causing leakage of low molecular weight proteins through increased cell membrane permeability. LPA coatings have the potential to enhance implant osseointegration whilst simultaneously reducing bacterial attachment. This technology may reduce both septic and aseptic failure of cementless joint prostheses, ultimately prolonging implant longevity and patient quality of life