Background. Gram stain microscopy is a routinely requested investigation in the evaluation of septic arthritis in both paediatric and adult patients. Recent evidence suggests that gram stain microscopy has poor diagnostic accuracy in adults with a sensitivity of only 45%, however the diagnostic accuracy remains unknown in children. We sought to establish the diagnostic utility of gram stain microscopy in the diagnosis of septic arthritis in children. Methods. We conducted a retrospective review of all patients of 16 years and under that underwent aspiration and washout of suspected septic joints in theatre from March 2005 to February 2011. Theatre data were cross referenced with microbiology results and analysed by simple descriptive methods in Excel. Results. We identified 23 paediatric patients undergoing washout or aspiration of a suspected septic joint during the time period studied. 17 (74%) of the patients were female and the average age was 2 years (Range 1 month–16 years). The most commonly affected joints were Knees (12/23, 52%) and Hips (7/23, 30%), with the remainder of infections occurring sporadically. There were 9 cases of culture confirmed septic arthritis (39% of all washouts), and these occurred in 5 knees (56%), 3 Hips (33%) and 1 ankle (11%). Organisms were Staphylococcus Aureus (2/8), Coagulase Negative Staphylococcus (2/9), Streptococcus pneumoniae (2/9), Group B Streptococcus (2/9) and Group A Streptococcus (1/9). Gram stain microscopy identified organisms in 3 aspirates both of which were confirmed on extended culture (Sensitivity 33%, Specificity 100%). Conclusion. Gram stain microscopy identified only 33% of culture positive cases of septic arthritis within this study. Our results suggest that gram stain microscopy lacks the diagnostic accuracy to be used in the exclusion of septic arthritis in the paediatric population

The detailed biomechanical mechanism of annulus fibrosus under abnormal loading is still ambiguous, especially at the micro and nano scales. This study aims to characterize the alterations of modulus at the nano scale of individual collagen fibrils in annulus fibrosus after in-situ immobilization, and the corresponding micro-biomechanics of annulus fibrosus. An immobilization model was used on the rat tail with an external fixation device. Twenty one fully grown 12-week-old male Sprague-Dawley rats were used in this study. The rats were assigned to one of three groups randomly. One group was selected to be the baseline control group with intact intervertebral discs (n=7). In the other two groups, the vertebrae were immobilized with an external fixation device that fixed four caudal vertebrae (C7-C10) for 4 and 8 weeks, respectively. Four K-wires were fixed in parallel using two aluminum alloy cuboids which do not compress or stretch the target discs. The immobilized discs were harvested and then stained with hematoxylin/eosin, scanned using atomic force microscopy to obtain the modulus at both nano and micro scales, and analyzed the gene expression with real-time quantitative polymerase chain reaction. Significance of differences between the study groups was obtained using a two-way analysis of variance (ANOVA) with Fisher's Partial Least-Squares Difference (PLSD) to analyze the combined influence of immobilization time and scanning region. Statistical significance was set at P≤0.05. Compared to the control group, the inner layer of annulus fibrosus presented significant disorder and hyperplasia after immobilization for 8 weeks, but not in the 4 week group. The fibrils in inner layer showed an alteration in elastic modulus from 91.38±20.19MPa in the intact annulus fibrosus to 110.64±15.58MPa (P<0.001) at the nano scale after immobilization for 8 weeks, while the corresponding modulus at the micro scale also underwent a change from 0.33±0.04MPa to 0.47±0.04MPa (P<0.001). The upregulation of collagen II from 1±0.03 in control to 1.22±0.03 in 8w group (P = 0.003) was induced after immobilization, while other genes expression showed no significant alteration after immobilization for both 4 and 8 weeks compared to the control group (P>0.05). The biomechanical properties at both nano and micro scales altered in different degrees between inner and outer layers in annulus fibrosus after immobilization for different times. Meanwhile, the fibril arrangement disorder and the upregulation of collagen II in annulus fibrosus were observed using hematoxylin/eosin staining and real-time RT-PCR, respectively. These results indicate that immobilization not only influenced the individual collagen fibril at the nano scale, but also suggested alterations of micro-biomechanics and cell response. This work provides a better understanding of IVD degeneration after immobilization and benefits to the clinical treatment related to disc immobilization

With its high wear and corrosion resistance, CoCrMo alloy has been widely used for metal-on-metal total hip replacements (THRs). However, the use of the metal-on-metal implants has dropped substantially as a result of several alerts issued by the Medicines and Healthcare products Regulatory Agency (MHRA) due to concern on metal ion release [1]. However, some of the first generation of metal-on-metal THRs have lasted for more than 20 years [2]. It is far from clear why some MoM joints have survived, while other failed. It is known that dynamic changes occur at the metal surface during articulation. For example, a nanocrystalline layer has been reported on the topmost surface of both in vivo and in vitro CoCrMo THRs [3, 4] but it is not known whether this layer is beneficial or detrimental. The current work focuses on the sub-surface damage evolution of explanted MoM hips, which is compared to in vitro tested CoCrMo hip prostheses. Site-specific TEM cross-section of both in vivo and in vitro CoCrMo samples were prepared by focused ion beam (FIB) in situ lift-out method (Quanta 200 3D with Omniprobe, FEI, the Netherlands). TEM of the FIB specimens was performed on various microscopes. Routine bright field imaging was performed on a Tecnai 20 (FEI, the Netherland) operating at 200 kV, while high resolution transmission electron microscopy (HRTEM) of the nanocrystalline layer and other surface species was undertaken on a Jeol 2010F (Jeol, Japan) operating at 200 kV. A nanocrystalline layer (which was not present on the starting surfaces) was observed on both explanted in vivo and in vitro tested materials. For the explanted joints, the nanocrystalline layer was thin (a few 100 nm) and the extent did not appear to correlate with the local wear rate. For in vitro samples, the nanocrystalline layer was thicker (up to micron). HRTEM from this layer are shown in Fig. 1 and Fig. 2. The nanocrystallite size was ∼5 nm and appeared to be a mixture of face centred cubic and hexagonal close packed phases. The formation of the nanocrystalline layer and its correlation with wear behaviour are discussed

Aim. Staphylococcus aureus (SA) can cause various infections and is associated with high morbidity and mortality rates of up to 40%. Antibiotic treatment often fails to eradicate SA infections even if the causative strain has been tested susceptible in vitro. The mechanisms leading to this persistence is still largely unknown. In our work, we to reveal SA interactions with host cells that allow SA to persist at the site of infection. Method. We established a sampling workflow to receive tissue samples from patients requiring surgical debridement due to SA bone-and joint or soft-tissue infections. We developed a multiplex immunofluorescent staining protocol which allowed us to stain for SA, leukocytes, neutrophils, macrophages, B-cells, T-cells, DAPI and cytoplasmatic marker on the same sample slide. Further, distance of SA to cell nuclei was measured. Interaction of immune cells and SA on a single cell level was investigated with high-resolution 3D microscopy. We then validated our findings applying fluorescence-activated cell sorting (FACS) on digested patient samples. Finally, we aimed to reproduce our ex vivo patient results in an in vitro co-culture model of primary macrophages and clinical SA strains, where we used live cell microscopy and high-resolution microscopy to visualize SA-immune cell interactions and a gentamicin protection assay to assess viability of SA. Results. Here, we revealed that CD68+ macrophages were the immune cells closest to SA with a mean distance of 56μm (SD=36.4μm). Counting the amount of SA, we found in total >7000 single SA in nine patients. Two-thirds of SA were located intracellularly. Two-thirds of the affected immune cells with intracellular SA were macrophages. The distribution of intra- to extracellular SA was independent of ongoing antibiotic therapy and underlying infection type. FACS confirmed these findings. In our co-culture model, intracellular SA remained alive for the whole observation period of eight hours and resided in RAB5+ early phagosomes. Conclusions. Our study suggests an essential role of intracellular survival in macrophages in SA infections. These findings may have major implication for future treatment strategies

Aim. Treatment of prosthetic joint infection (PJI) by systemic administration of high doses of long-term antibiotics often proves ineffective, causing severe side effects. Thus, we presented the phage Sb-1, which coding extracellular polymeric substances (EPS) degradation depolymerases, conjugated with rifampicin-loaded liposomes (Lip-RIF@Phage) by bio-orthogonal functionalization strategy to target biofilm (Figure1). Method. Methicillin-resistant Staphylococcus aureus (MRSA) biofilm was grown on porous glass beads for 24 h in vitro. After the biofilm formation, beads were exposed to 0.9% saline, then sonication. Quantitative and qualitative biofilm analyses were performed by colony counting, scanning electron microscopy and isothermal microcalorimetry. A rat model of total knee arthroplasty infected with the bioluminescent MRSA strain was developed as the PJI model to evaluate the efficacy of Lip-RIF@Phage anti-biofilm therapy in vivo, then the creatinine, alanine transaminase, and aspartate transaminase values were evaluated throughout the entire treatment process. Results. After treatment with Lip-RIF@Phage, no bacterial colonies were observed, consistent with findings from scanning electron microscopy. Similarly, isothermal microcalorimetry revealed no detectable heat following Lip-RIF@Phage treatment, aligning with these observations. In vivo experiments demonstrated a significant reduction in biofilm cell load compared to all other tested conditions, with no evidence of systemic toxicity on renal and liver functions attributed to Lip-RIF@Phage. Conclusions. The innovative depolymerase-phagobot nanosystem (Lip-RIF@Phage) exhibits remarkable efficacy in completely eliminating biofilm cells in vitro. It serves as an excellent carrier for antibiotic delivery, enhancing antibiotic penetration through biofilms and improving biofilm eradication efficacy. Furthermore, it enables personalized treatment strategies against biofilm-associated multidrug-resistant (MDR) infections by maximizing the effectiveness of any remaining sensitive antibiotics. For any tables or figures, please contact the authors directly

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

Title. Longitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine Model. Aim. To assess S. aureus vs. host cell colonization of contaminated implants vis intravital multiphoton laser scanning microscopy (IV-MLSM) in a murine model. Method. All animal experiments were approved by IACUC. A flat stainless steel or titanium L-shaped pin was contaminated with 10. 5. CFU of a red fluorescent protein (RFP) expressing strain of USA300LAC, and surgically implanted through the femur of global GFP-transgenic mice. IV-MLSM was performed at 2, 4, and 6 hours post-op. Parallel cross-sectional CFU studies were performed to quantify the bacteria load on the implant at 2,4,6,12,18 and 24 hours. Results. 1) We developed a high-fidelity reproducible IV-MLSM system to quantify S. aureus and host cell colonization of a bone implant in the mouse femur. Proper placement of all implants were confirmed with in vivo X-rays, and ex vivo photos. We empirically derive the ROI during each imaging session by aggregating the imaged volume which ranges from (636.4um × 636.4um × 151um) = 0.625 +/- 0.014 mm. 3. of bone marrow in a global GFP-transgenic mouse. 2) IV-MLSM imaging acquisition of the “race for the surface”.In vitro MPLSM images of implants partially coated with USA300LAC (RFP-MRSA) were verified by SEM image. Results from IV-MLSM of RFP-MRSA and GFP. +. host cell colonization of the contaminated implants illustrated the mutually exclusive surface coating at 3hrs, which to our knowledge is the first demonstration of “the race for the surface” between bacteria and host cells via intravital microscopy. 3) Quantifying the “race for the surface” with CFU verification of S. aureus on the implant. 3D volumetric rendering of the GFP. +. voxels and RFP+ voxels within the ROI were generated in Imaris. The voxel numbers suggeste that the fight for the surface concludes ∼3hrs post-infection, and then transitions to an aggressive MRSA proliferation phase. The results of WT control demonstrate a significant increase in CFU by 12hrs post-op for both stainless steel (P<0.01) and titanium (P<0.01). Conclusions. We developed IV-MLSM to quantify the “Race for the Surface” between host cells and contaminating S. aureus in a murine femur implant model. This race is remarkably fast, as the implant surface is completely covered with 3hrs, peak bacterial growth on the implant occurs between 2 and 12 hours and is complete by 12hrs

Aim. Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria. Method. 19 Human bone tissue samples from patients with osteomyelitis were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their respective mitochondria were histomorphometrically analyzed. The results were compared to the control group of 5 non-infectious human bone tissue samples. Results. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infectious samples as a common finding in both cell types. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number could be found as a sign for increased mitochondrial fission. Conclusions. The results show that mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This suggests that manipulation of mitochondrial dynamics in a way of inhibiting mitochondrial fission may improve bone cell survival and exploit bone cells regenerative potential to aid in the treatment of osteomyelitis

Aim. Multispecies biofilms are associated with difficult periprosthetic joint infections (PJI), particularly if they have different antibiotic sensitivities. We aimed to determine if we could generate and kill a multispecies biofilm consisting of a Gram negative and Gram positive pathogen in-vitro with antibiotic loaded calcium sulfate beads containing single or combination antibiotics. Methods. To establish whether we could co-culture mixed species biofilms various combinations of Pseudomonas aeruginosa (PA), Enterococcus faecalis (EF), Staphylococcus aureus (SA) and Enterobacter faecalis (EF) were grown together on 316L stainless steel coupons and agar plates. Based on this screen we focused on PA + EF and challenged them with high purity calcium sulfate beads (Stimulan Rapid Cure) loaded with vancomycin (V), alone tobramycin (T) alone or vancomycin and tobramycin in combination (V+T). Bioluminescence, light imaging, plate count, confocal microscopy and scanning electron microscopy were used to quantify growth. Results. On 316LSS the V loaded bead reduced both EF and PA by approximately 2 logs compared to unloaded control beads. A T alone loaded bead eliminated PA from the dual species biofilm and caused a 2-log reduction in EF. The V+T-beads reduced PA by 9-logs and EF by 8.3 logs. In terms of total CFUs V+T beads reduced the bioburden by 8.4 logs compared to V or T alone. which resulted in 2.1 and 2.6 log reductions respectively. (* P<0.05, *** P<0.001). On agar PA dominated the culture for the unloaded and V loaded beads. However, when challenged with a T loaded bead both species were able to coexist and a zone of killing was generated in both species in the multispecies biofilms. However, this zone was smaller and included more tolerant variants than the zone generated by V+T-loaded beads. Conclusions. There were species proportion differences between biofilms grown on agar and 316LSS demonstrating the importance of growth conditions on species interactions. Antibiotics against strains with differing sensitivities can shift species interactions. High purity calcium sulfate beads containing tobramycin a broad-spectrum Gram positive and negative antibiotic vancomycin, a Gram-positive targeted antibiotic killed a larger percentage of a multispecies in an in-vitro biofilm than either single gram-specific antibiotic alone, demonstrating the advantage of using combination antibiotics for treating multispecies biofilms

Aims. This study aims to enhance understanding of clinical and radiological consequences and involved mechanisms that led to corrosion of the Precice Stryde (Stryde) intramedullary lengthening nail in the post market surveillance era of the device. Between 2018 and 2021 more than 2,000 Stryde nails have been implanted worldwide. However, the outcome of treatment with the Stryde system is insufficiently reported. Methods. This is a retrospective single-centre study analyzing outcome of 57 consecutive lengthening procedures performed with the Stryde nail at the authors’ institution from February 2019 until November 2020. Macro- and microscopic metallographic analysis of four retrieved nails was conducted. To investigate observed corrosion at telescoping junction, scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDX) were performed. Results. Adjacent to the nail’s telescoping junction, osteolytic changes were observed in bi-planar radiographs of 20/57 segments (35%) after a mean of 9.5 months (95% confidence interval 7.2 to 11.9) after surgery. A total of 8/20 patients with osseous alterations (40%) reported rest and ambulation pain of the lengthened segment during consolidation. So far, 24 Stryde nails were retrieved and in 20 (83%) macroscopic corrosion was observed at the nail’s telescoping junction. Before implant removal 11/20 radiographs (55%) of lengthened segments with these 20 nails revealed osteolysis. Implant retrieval analysis by means of SEM showed pitting and crevice corrosion. EDX detected chromium as the main metallic element of corrosion. Conclusion. Patients are exposed to the risk of implant-related osteolysis of unclear short- and long-term clinical consequences. The authors advocate in favour of an early implant removal after osseous consolidation. Cite this article: Bone Joint Res 2021;10(7):425–436

Introduction. Introduction: Pin site infection is a common complication during treatment with a circular frame external fixator and increases time and support patients require from the limb reconstruction team. Wound swabs were not routinely sent by the clinical nurse specialists prior to this study, with most pin site infections treated as Staphylococcus aureus with flucloxacillin (clindamycin in penicillin allergy). The aim of this study was to ascertain whether routine sending of wound swabs in pin site infection would change antibiotic treatment. Materials & Methods. Materials and Method: Patients presenting at clinic or physiotherapy with clinical signs of pin site infection were assessed using the Maz Oxford Nuffield (MON) Pin Site Infection Grading System© (OUH, 2021). Antibiotics were commenced as per unit guidelines and swabs sent for microscopy, culture and sensitivity. Results. Results: There were forty patients treated with pin site infections. Swab results showed S. aureus in 60% (N=24), other organisms in 15% (N=6) and no growth or mixed skin organisms in 10% (N=10). Flucloxacillin was prescribed to 77.5% (n=31) of patients with the remaining 22.5% (n=9) of patients receiving clindamycin (due to allergy) or rifampicin and ciprofloxacin (due to a previous MRSA diagnosis). Antibiotic management was subsequently changed in only 5% (n=2) of patients due to one patient with a new MRSA diagnosis and one patient growing Enterobacter in three pin sites tested. Conclusions. Conclusion: This study has shown that routine swab testing of pin sites does not usually change antibiotic management but should still be used in multiple, severe or unresolving pin site infections

Aim. Quadrupled hamstring anterior cruciate ligament plasties (4xHp) have been described as having a higher risk of infection than bone patellar tendon bone plasties (BPTBp). There are 2 theories that might explain this phenomenon. One is the presence of sutures in a 4xHp that could act as a foreign body, The other is the more complex preparation of a 4xHp that might lead to higher contamination rates during the process. The objective of the present study was to evaluate the formation of biofilm in these plasties and to compare it between a 4xHp and a BPTBp. The hypothesis was that the presence of sutures in 4xHp would increase the amount of biofilm present in them in comparison to BPTBp. Method. A descriptive in vitro study was conducted. One 4xHp and one BPTBp were prepared. They were subsequently divided into 8 fragments. Three of them were reserved for negative control, and the rest were contaminated with a strain of S. Epidermidis (ATCC 35984) 10–5. Finally, a quantitative analysis was carried out by means of microcalorimetry and sonication with plating. Additionally, a qualitative analysis was carried out by means of electron microscopy. Results. In isothermal microcalorimetry, both contaminated plasties showed the same growth dynamics with a population peak (200uW) at 8h. No significant differences were found between the bacterial growth profiles of 4xHp and BPTBp. The product of sonication was plated and the number of colony forming units per milliliter (CFU/ml) was counted at 24 hours. No significant differences were detected between the 4×Hp (mean +/− sem = 3,5×107 +/− 3450000) and the BPTBp (4,6 ×107 +/− 1,455e+7). With a p value of 0.6667, there were no differences of significance (Mann-Whitney test). In the samples analyzed with electron microscopy, no specific biofilm growth pattern was identified upon comparing BPTBp with 4xHp. Conclusions. There were no significant differences at either the quantitative or qualitative level when comparing bacterial growth in BPTBp and 4xHp. Therefore, the presence of sutures in 4xHp cannot be established as a predisposing factor to higher infection rates. These findings may be justified in the sense that the plasties themselves already behave like foreign bodies. Therefore, the presence of sutures does not increase the possibility of biofilm forming on their surface

Objectives. Several studies have reported elevated blood cobalt (Co) and chromium (Cr) concentrations in patients with total knee replacements (TKRs). Up to 44% of tissue samples taken from patients with failed TKRs exhibit histological evidence of metal sensitivity/ALVAL. In simulated conditions, metal particles contribute approximately 12% of total wear debris in TKR. We carried out this investigation to determine the source and quantity of metal release in TKRs. Design and Methods. We analysed 225 explanted fixed-bearing TKRs (Attune, Genesis II, NexGen, PFC, and Vanguard) revised for any indication. These were analysed using peer-reviewed [coordinate measuring machine (CMM)] methodology to measure the volumetric wear of the polyethylene (PE) bearing surfaces and trays. The trays were analysed using 2D profilometry (surface roughness-Ra) and light microscopy. Histological and blood metal ion concentration analyses were performed in a sub-sample of patients. Results. The median (IQR) PE wear rate was 14 (6 to 20) mm. 3. /year. Microscopic examination of the superior surface of trays exhibited pitting on 132 (59%) of trays. There was a statistically significant (p<0.05) increase in Rvk on the pitted area of trays for each design, indicating material removal from the pits compared to the unpitted area. The inferior surface of 116(51%) of trays displayed polishing, indicative of abrasive wear. The median(range) Co and Cr concentrations were 2.5µg/l (0.2–69.4) and 1.7µg/l (0.5-12.5) respectively in 40 patients. Of the tissue samples examined in 30 patients, 6 had at least “mild”-ALVAL infiltrate. All corresponding “ALVAL” explants were found to be pitted and/or show evidence of loosening of the tray. Conclusion. This study provides further evidence that CoCr release in TKR appears to be an under-appreciated cause of adverse clinical outcomes. The generation of metal particles was predominantly from the metal tray, which may explain elevated metal ions after TKRs, despite no direct metal-on-metal contact

Objectives. We identified an unusual pattern of backside deformation on polyethylene (PE) inserts of contemporary total knee replacements (TKRs). The PE backside's margins were inferiorly deformed in TKRs with NexGen central-locking trays. This backside deformation was significantly associated with tray debonding. Furthermore, recent studies have shown high rate of tray debonding in PS NexGen TKRs. Subsequently, a field safety notice was issued regarding the performance of this particular device combination and the Option tray has been withdrawn from use. Therefore, we hypothesised that the backside deformation of PS inserts may be greater than that of CR inserts. Design and Methods. At our national implant retrieval centre, we used peer-reviewed techniques to analyse changes in the bearing wear rate and backside surface deformation of NexGen PE inserts using coordinate measuring machines [N=84 (CR-43 and PS-41) TKRs with non-augmented-trays]. Multiple regression was used to determine which variable had the greatest influence on backside deformation. The amount of cement cover on trays was quantified as a %of the total surface using Image-J software. Results. The median (IQR) bearing wear rate of the PS PEs [14(8-22) mm. 3. /year] was not significantly different(p=0.154) to that of the CR PEs [18(8-27)mm. 3. /year]. The median (IQR) backside deformation of the PS inserts [294(239-361) µm] was significantly greater (p<0.001) than that of the CR inserts [212(158-258)µm]. Multiple regression modelling showed that duration in-vivo (p=0.037), central-clearance between insert and tray (p<0.001) and constraint (p=0.003) were significantly associated with PE backside-deformation. 38(93%) PS and 31(72%) of CR trays exhibited ≤10% of cement cover. Non-contacting profilometry and microscopy revealed marked pitting and abrasive changes to the superior surface of the tray. Conclusion. This explant study showed the PE backside deformation was significantly higher in PS than in CR inserts and this may be one explanation for the unsatisfactory clinical performance reported with this device combination

Aim. Here, we are aimed to evaluate bacteriophage (191219) to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella. Method. The bacteriophages (191219) were provided from D&D Pharma GmbH. These bacteriophages were tested against S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) strains. To assess the activity of bacteriophages against planktonic growth phase, bacteriophages, and S. aureus EDCC 5055(1×10. 7. CFU/ml) were co-cultured in LB media as multiplicity of infection (MOI) of 10, 1, 0.1, and 0.01 for 24 hours at 37. o. C and finally plated out on the LB agar plates to estimate the bacterial growth. The antimicrobial activity of bacteriophages on biofilms in vitro was measured by analysing the incubating the several fold dilutions of bacteriophages in LB media with biofilms formed on 96-well plate. The eradication of biofilm was analysed with crystal violet as well as CFU analysis methods. Later, the effect of bacteriophages on intracellular growth of S. aureus in side osteoblast was tested by treating the S. aureus infected osteoblasts at 2h, 4h and 24h time points of post treatment. In addition, we have analysed synergistic effect with gentamicin and rifampicin antibiotics to clear intracellular S. aureus. Finally, experiments are performed to prove the effect of bacteriophages to clear in vivo biofilm using alternative insect model G. mellonella as well as to detect the presence of bacteriophages inside the osteoblasts through transmission electron microscopy (TEM) analysis. Results. Our results demonstrate the in vitro efficacy of bacteriophages against planktonic S. aureus. Transmission electron microscopy (TEM) experiments revealed severe infection of bacteria by bacteriophages. Bacteriophages also eradicated in a dose-dependent manner in vitro S. aureus biofilm formation and were active against intracellular S. aureus in an osteoblastic cell line. TEM analysis visualized the effect of the bacteriophages on S. aureus inside the osteoblasts with the destruction of the intracellular bacteria and formation of new bacteriophages. For the Galleria infection model, single administration of phages failed to show improvement in survival rates, but exhibited some synergistic effects with gentamicin or rifampicin, which was not statistically significant. Conclusions. In summary, bacteriophages could be a potential adjuvant treatment strategy for patients with implant-associated biofilm infections. Further preclinical and clinical trials are required to establish adequate treatment protocols

Aim. Orthopedic implants play a tremendous role in fixing bone damages due to aging as well as fractures. However, these implants tend to get colonized by bacteria on the surface, leading to infections and subsequently prevention of healing and osteointegration. Recently, Roupie et al. showed that a nisin layer-by-layer based coating applied on biomaterials has both osteogenic and antibacterial properties. The Galleria mellonella larva is a well-known insect infection model that has been used to test the virulence of bacterial and fungal strains as well as for the high throughput screening of antimicrobial compounds against infections. Recently, we have developed an insect infection model with G. mellonella larvae to study implant-associated biofilm infections using Kirschner (K)-wires as implant material. Here, we would like to test the antibacterial capacity of nisin layer-by-layer based coatings on K-wires against Staphylococcus aureus in the G. mellonella larva implant infection model. Method. Prior to the implantation procedure, G. mellonella larvae are maintained at room temperature on wheat germ in an incubator. The larvae received bare titanium K-wires (uncoated), or either control-coated or nisin-coated K-wires. After one hour, the larvae were injected with 5×10. 5. S. aureus bacteria per larva (i.e., hematogenous implant infection model). Next, the larvae were incubated at 37. o. C in an incubator and the survival of the larvae was monitored for five days. Moreover, the number of bacteria on the implant surface and in the surrounding tissue was determined after 24h of incubation. Further, scanning electron microscopy (SEM) analyses were performed to study the effect of nisin on biofilm formation. Results. The larvae receiving the nisin-coated K-wires showed significantly higher survival rates compared to uncoated titanium K-wires, although not when compared to control-coated K-wires. A more than 1-log reduction in number of bacteria on the implant surface and in the surrounding tissue was observed in larvae receiving the nisin-coated K-wires, when compared to uncoated titanium K-wires SEM analysis showed reduced colonization of the bacteria nisin-coated K-wires compared to the controls. Conclusions. In conclusion, the antimicrobial nisin layer-by-layer based coating applied on titanium surfaces is able to prevent implant-related S. aureus biofilm infection in G. mellonella and is a promising antimicrobial strategy to prevent implant-related infections

Introduction. In specific conditions, infection may lead to bone loss and is difficult to treat. 1. Current clinical approaches rely on the introduction of antibiotics. While these may be effective, there are concerns regarding the rise of antimicrobial resistance. There is therefore interest in the development of antimicrobial bone graft substitutes for dental and trauma surgery. Aim & Objectives. The incorporation of zinc into biomaterials has been shown to confer broad spectrum antimicrobial activity, but this has not yet been applied to the development of a commercial bone graft substitute. The aim of this research was therefore to prepare and characterise a series of zinc-substituted nanoscale hydroxyapatite (nHA) materials, including evaluation of antimicrobial activity. Method. Zinc (Zn) substituted nHA materials were prepared (0, 5, 10, 15 & 20 mol.% Zn) using a wet chemical precipitation method with a rapid mixing. (2). The reaction was carried out using zinc hydroxide at pH 10. The suspension formed was washed and dried into both powder & paste forms. The resultant powders were characterized using transmission electron microscopy (TEM) and X-ray diffraction (XRD). The antimicrobial activity was evaluated against Staphylococcus aureus (S8650 strain - isolated from an osteomyelitis case), by two techniques. The Miles and Misra method was applied to determine the number of colony-forming units (CFUs) in bacterial suspensions incubated with pastes. Secondly, a biofilm initialization method was used to evaluate the capacity of the materials to prevent biofilm formation. One-way analysis of variance (ANOVA) was used for the statistical analysis and results with p-value < 0.05 were considered statistically significant. Results. XRD indicated the formation of pure hydroxyapatite with up to 10 mol.% Zn without any side products. However, when Zn was increased to 15 & 20 mol %, zinc oxide (ZnO) peaks were detected. The TEM showed nanoscale needle-like particles when Zn was increased compared to nHA particles. Regarding the antibacterial activity, ZnHA pastes at all concentrations caused a significant reduction in bacterial CFUs in a dose-dependent manner (50, 100 & 200 mg). Additionally, even the lowest zinc substitution (5 mol.%) significantly reduced biofilm formation. Conclusion. The results demonstrated a novel method to produce a Zn-substituted nHA that showed antimicrobial activity against a pathogen isolated from a bone infection

Aim. The use of medical devices has grown significantly over the last decades, and has become a major part of modern medicine and our daily life. Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. For still not well understood reasons, the presence of a foreign body strongly increases susceptibility to infection. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. Formation of biofilms on the biomaterial surface is generally considered the main reason for these persistent infections, although bacteria may also enter the surrounding tissue and become internalized within host cells. To prevent biofilm formation using a non-antibiotic based strategy, we aimed to develop a novel permanently fixed antimicrobial coating for titanium devices based on stable immobilized quaternary ammonium compounds (QACs). Method. Medical grade titanium implants (10×4×1 mm) were dip-coated in a solution of 10% (w/v) hyperbranched polymer, subsequently in a solution of 30% (w/v) polyethyleneimine and 10 mM sodium iodide, using a dip-coater, followed by a washing step for 10 min in ethanol. The QAC-coating was characterized using water contact angle measurements, scanning electron microscopy, FTIR, AFM and XPS. The antimicrobial activity of the coating was evaluated against S. aureus strain JAR060131 and S. epidermidis strain ATCC 12228 using the JIS Z 2801:2000 surface microbicidal assay. Lastly, we assessed the in vivo antimicrobial activity in a mouse subcutaneous implant infection model with S. aureus administered locally on the QAC-coated implants prior to implantation to mimic contamination during surgery. Results. Detailed material characterization of the titanium samples showed the presence of a homogenous and stable coating layer at the titanium surface. Moreover, the coating successfully killed S. aureus and S. epidermidis in vitro. The QAC-coating strongly reduced S. aureus colonization of the implant surface as well as of the surrounding tissue, with no apparent macroscopic signs of toxicity or inflammation in the peri-implant tissue at 1 and 4 days after implantation. Conclusions. An antimicrobial coating with stable quaternary ammonium compounds on titanium has been developed which holds promise to prevent BAI. Non-antibiotic-based antimicrobial coatings have great significance in guiding the design of novel antimicrobial coatings in the present, post-antibiotic era

Aim. Chemical debridement is a fundamental step during Periprosthetic joint infection (PJI) surgery. Antiseptic solutions are commonly used, but evidence on the optimal antiseptic, concentration, and irrigation time is lacking. The aim of this study is to analyze and compare the anti-biofilm capacity of povidone iodine, H. 2. 0. 2. , acetic acid and Bactisure™ after different exposure times, as well as their combinations. Method. Surgical steel discs inoculated with methicillin susceptible (MSSA) and resistant S. aureus (MRSA), P. aeruginosa, and S. epidermidis were exposed to the following antiseptic solutions: 0.3% (PI0.3) and 10% povidone iodine (PI10), H. 2. 0. 2. , 3% Acetic acid (AA3) and Bactisure™. Combinations included AA3, H. 2. 0. 2. , and PI10 in various orders. Exposure time for the antiseptics solutions was 1, 3 and 5 minutes, while combinations had a 9-minute total exposure, 3 minutes per antiseptic sequentially. All experiments were performed in triplicate and with a sterile saline control. nThe reduction in colony-forming units (CFU) was measured after sonication, and biofilm structure was analyzed via scanning electron microscopy. Results. PI showed the highest antibiofilm activity. PI0.3 eradicated bacteria on the discs after 3 and 5 minutes of exposure, but only achieved a 77.1% reduction after 1 minute. After PI10 treatment, we did not recover any bacteria regardless of exposure time. H. 2. 0. 2. , AA3, and Bactisure™ reached a significantly lower bacterial decrease at all exposure times compared to PI0.3 and PI10. AA3 was less effective against MSSA and S. epidermidis. H. 2. 0. 2. showed less activity against MRSA than PI0.3, PI10, and Bactisure™. Combinations of antiseptics starting with AA3 showed the best results in terms of CFU reduction and cell viability. Conclusions. We propose a sequential combination of AA3 + H. 2. 0. 2. + PI10 with an exposure time of 9 minutes for the chemical debridement in PJI surgery. First, AA3 performs debridement and disruption of the biofilm. Then, H. 2. 0. 2. has a bactericidal effect and increases the porosity of the cell wall, and PI10 has a final bactericidal effect. If combinations are unavailable, PI is a cost-effective alternative

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