Aims. Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella. Methods. For the haematogenous infection, G. mellonella larvae were implanted with a Kirschner wire (K-wire), infected with S. aureus, and subsequently phages and/or gentamicin were administered. For the early-stage biofilm implant infection, the K-wires were pre-incubated with S. aureus suspension before implantation. After 24 hours, the larvae received phages and/or gentamicin. In both models, the larvae also received daily doses of phages and/or gentamicin for up to five days. The effect was determined by survival analysis for five days and quantitative culture of bacteria after two days of repetitive doses. Results. In the haematogenous infection, a single combined dose of phages and gentamicin, and repetitive injections with gentamicin or in combination with phages, resulted in significantly improved survival rates. In the early-stage biofilm infection, only repetitive combined administration of phages and gentamicin led to a significantly increased survival. Additionally, a significant reduction in number of bacteria was observed in the larvae after receiving repetitive doses of phages and/or gentamicin in both infection models. Conclusion. Based on our results, a single dose of the combination of phages and gentamicin is sufficient to prevent a haematogenous S. aureus implant-related infection, whereas gentamicin needs to be administered daily for the same effect. To treat early-stage S. aureus implant-related infection, repetitive doses of the combination of phages and gentamicin are required. Cite this article: Bone Joint Res 2024;13(8):383–391

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Serum inflammatory parameters are widely used to aid in diagnosing a periprosthetic joint infection (PJI). Due to their limited performances in the literature, novel and more accurate biomarkers are needed. Serum albumin-to-globulin ratio (AGR) and serum CRP-to-albumin ratio (CAR) have previously been proposed as potential new parameters, but results were mixed. The aim of this study was to assess the diagnostic accuracy of AGR and CAR in diagnosing PJI and to compare them to the established and widely used marker CRP.

Aims. Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI). Methods. A total of 56 male Sprague-Dawley rats were established in acute PJI models by intra-articular injection of bacteria. The rats were divided into four groups: a Control group, a 0.35% PI group, a LIPUS and saline group, and a LIPUS and 0.35% PI group. All rats underwent DAIR, except for Control, which underwent a sham procedure. General status, serum biochemical markers, weightbearing analysis, radiographs, micro-CT analysis, scanning electron microscopy of the prostheses, microbiological analysis, macroscope, and histopathology evaluation were performed 14 days after DAIR. Results. The group with LIPUS and 0.35% PI exhibited decreased levels of serum biochemical markers, improved weightbearing scores, reduced reactive bone changes, absence of viable bacteria, and decreased inflammation compared to the Control group. Despite the greater antibiofilm activity observed in the PI group compared to the LIPUS and saline group, none of the monotherapies were successful in preventing reactive bone changes or eliminating the infection. Conclusion. In the rat model of PJI treated with DAIR, LIPUS combined with 0.35% PI demonstrated stronger antibiofilm potential than monotherapy, without impairing any local soft-tissue. Cite this article: Bone Joint Res 2024;13(7):332–341

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This study aimed to investigate the clinical characteristics and outcomes associated with culture-negative limb osteomyelitis patients.

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To explore the clinical efficacy of using two different types of articulating spacers in two-stage revision for chronic knee periprosthetic joint infection (kPJI).

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The aim of the present study was to assess the outcomes of the induced membrane technique (IMT) for the management of infected segmental bone defects, and to analyze predictive factors associated with unfavourable outcomes.

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Delayed postoperative inoculation of orthopaedic implants with persistent wound drainage or bacterial seeding of a haematoma can result in periprosthetic joint infection (PJI). The aim of this in vivo study was to compare the efficacy of vancomycin powder with vancomycin-eluting calcium sulphate beads in preventing PJI due to delayed inoculation.

Introduction. Major trauma during military conflicts involve heavily contaminated open fractures. Staphylococcus aureus (S. aureus) commonly causes infection within a protective biofilm. Lactoferrin (Lf), a natural milk glycoprotein, chelates iron and releases bacteria from biofilms, complimenting antibiotics. This research developed a periprosthetic biofilm infection model in rodents to test an Lf based lavage/sustained local release formulation embedded in Stimulin beads. Method. Surgery was performed on adult rats and received systemic Flucloxacillin (Flu). The craniomedial tibia was exposed, drilled, then inoculated with S. aureus biofilm. A metal pin was placed within the medullary cavity and treatments conducted. Lf in lavage solutions: The defect was subject to 2× 50 mL lavage with 4 treatment groups (saline only, Lf only, Bactisure with Lf, Bactisure with saline). Lf embedded in Stimulin beads: 4 bead types were introduced (Stimulin only, Lf only, Flu only, Lf with Flu). At day 7, rats are processed for bioluminescent and X-ray imaging, and tibial explants/pins collected for bacterial enumeration (CFU). Results. Rats without treatments established a mean infection of 2×106 CFU/tibia. 4 treatment groups with a day 0, one-off lavage demonstrated >95% reduction in bacterial load 7 days post-op, with a reduction in CFU from 1×106/tibia down to 1×104/tibia. There was no statistically significant difference between each group (p = 0.55 with one way ANOVA). The stimulin bead experiments are ongoing and complete results will be obtained in the end of July. Conclusions. This research demonstrated a clinically relevant animal model of implanted metalware that establishes infection. No additional benefit was observed with a one-off, adjuvant Lf lavage during the initial decontamination of the surgical wound, compared with saline alone, and in combination with the antiseptic Bactisure. This animal model provides the foundation for future antibiofilm therapies

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Accurate diagnosis of chronic periprosthetic joint infection (PJI) presents a significant challenge for hip surgeons. Preoperative diagnosis is not always easy to establish, making the intraoperative decision-making process crucial in deciding between one- and two-stage revision total hip arthroplasty (THA). Calprotectin is a promising point-of-care novel biomarker that has displayed high accuracy in detecting PJI. We aimed to evaluate the utility of intraoperative calprotectin lateral flow immunoassay (LFI) in THA patients with suspected chronic PJI.

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The mean age of patients undergoing total knee arthroplasty (TKA) has reduced with time. Younger patients have increased expectations following TKA. Aseptic loosening of the tibial component is the most common cause of failure of TKA in the UK. Interest in cementless TKA has re-emerged due to its encouraging results in the younger patient population. We review a large series of tantalum trabecular metal cementless implants in patients who are at the highest risk of revision surgery.

Aims. Fracture-related infection (FRI) is commonly classified based on the time of onset of symptoms. Early infections (< two weeks) are treated with debridement, antibiotics, and implant retention (DAIR). For late infections (> ten weeks), guidelines recommend implant removal due to tolerant biofilms. For delayed infections (two to ten weeks), recommendations are unclear. In this study we compared infection clearance and bone healing in early and delayed FRI treated with DAIR in a rabbit model. Methods. Staphylococcus aureus was inoculated into a humeral osteotomy in 17 rabbits after plate osteosynthesis. Infection developed for one week (early group, n = 6) or four weeks (delayed group, n = 6) before DAIR (systemic antibiotics: two weeks, nafcillin + rifampin; four weeks, levofloxacin + rifampin). A control group (n = 5) received revision surgery after four weeks without antibiotics. Bacteriology of humerus, soft-tissue, and implants was performed seven weeks after revision surgery. Bone healing was assessed using a modified radiological union scale in tibial fractures (mRUST). Results. Greater bacterial burden in the early group compared to the delayed and control groups at revision surgery indicates a retraction of the infection from one to four weeks. Infection was cleared in all animals in the early and delayed groups at euthanasia, but not in the control group. Osteotomies healed in the early group, but bone healing was significantly compromised in the delayed and control groups. Conclusion. The duration of the infection from one to four weeks does not impact the success of infection clearance in this model. Bone healing, however, is impaired as the duration of the infection increases. Cite this article: Bone Joint Res 2024;13(3):127–135

Aims. Biofilm infections are among the most challenging complications in orthopaedics, as bacteria within the biofilms are protected from the host immune system and many antibiotics. Halicin exhibits broad-spectrum activity against many planktonic bacteria, and previous studies have demonstrated that halicin is also effective against Staphylococcus aureus biofilms grown on polystyrene or polypropylene substrates. However, the effectiveness of many antibiotics can be substantially altered depending on which orthopaedically relevant substrates the biofilms grow. This study, therefore, evaluated the activity of halicin against less mature and more mature S. aureus biofilms grown on titanium alloy, cobalt-chrome, ultra-high molecular weight polyethylene (UHMWPE), devitalized muscle, or devitalized bone. Methods. S. aureus-Xen36 biofilms were grown on the various substrates for 24 hours or seven days. Biofilms were incubated with various concentrations of halicin or vancomycin and then allowed to recover without antibiotics. Minimal biofilm eradication concentrations (MBECs) were defined by CFU counting and resazurin reduction assays, and were compared with the planktonic minimal inhibitory concentrations (MICs). Results. Halicin continued to exert significantly (p < 0.01) more antibacterial activity against biofilms grown on all tested orthopaedically relevant substrates than vancomycin, an antibiotic known to be affected by biofilm maturity. For example, halicin MBECs against both less mature and more mature biofilms were ten-fold to 40-fold higher than its MIC. In contrast, vancomycin MBECs against the less mature biofilms were 50-fold to 200-fold higher than its MIC, and 100-fold to 400-fold higher against the more mature biofilms. Conclusion. Halicin is a promising antibiotic that should be tested in animal models of orthopaedic infection. Cite this article: Bone Joint Res 2024;13(3):101–109

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Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells.

Aims. To investigate the efficacy of ethylenediaminetetraacetic acid-normal saline (EDTA-NS) in dispersing biofilms and reducing bacterial infections. Methods. EDTA-NS solutions were irrigated at different durations (1, 5, 10, and 30 minutes) and concentrations (1, 2, 5, 10, and 50 mM) to disrupt Staphylococcus aureus biofilms on Matrigel-coated glass and two materials widely used in orthopaedic implants (Ti-6Al-4V and highly cross-linked polyethylene (HXLPE)). To assess the efficacy of biofilm dispersion, crystal violet staining biofilm assay and colony counting after sonification and culturing were performed. The results were further confirmed and visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). We then investigated the efficacies of EDTA-NS irrigation in vivo in rat and pig models of biofilm-associated infection. Results. When 10 mM or higher EDTA-NS concentrations were used for ten minutes, over 99% of S. aureus biofilm formed on all three types of materials was eradicated in terms of absorbance measured at 595 nm and colony-forming units (CFUs) after culturing. Consistently, SEM and CSLM scanning demonstrated that less adherence of S. aureus could be observed on all three types of materials after 10 mM EDTA-NS irrigation for ten minutes. In the rat model, compared with NS irrigation combined with rifampin (Ti-6Al-4V wire-implanted rats: 60% bacteria survived; HXLPE particle-implanted rats: 63.3% bacteria survived), EDTA-NS irrigation combined with rifampin produced the highest removal rate (Ti-6Al-4V wire-implanted rats: 3.33% bacteria survived; HXLPE particle-implanted rats: 6.67% bacteria survived). In the pig model, compared with NS irrigation combined with rifampin (Ti-6Al-4V plates: 75% bacteria survived; HXLPE bearings: 87.5% bacteria survived), we observed a similar level of biofilm disruption on Ti-6Al-4V plates (25% bacteria survived) and HXLPE bearings (37.5% bacteria survived) after EDTA-NS irrigation combined with rifampin. The in vivo study revealed that the biomass of S. aureus biofilm was significantly reduced when treated with rifampin following irrigation and debridement, as indicated by both the biofilm bacterial burden and crystal violet staining. EDTA-NS irrigation (10 mM/10 min) combined with rifampin effectively removes S. aureus biofilm-associated infections both in vitro and in vivo. Conclusion. EDTA-NS irrigation with or without antibiotics is effective in eradicating S. aureus biofilm-associated infection both ex and in vivo. Cite this article: Bone Joint Res 2024;13(1):40–51

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This study aimed to evaluate the clinical application of the PJI-TNM classification for periprosthetic joint infection (PJI) by determining intraobserver and interobserver reliability. To facilitate its use in clinical practice, an educational app was subsequently developed and evaluated.

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses

Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. 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). Medical grade titanium implants were dip-coated in subsequent solutions of hyperbranched polymer, polyethyleneimine and 10 mM sodium iodide, and 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. 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. 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. Acknowledgements: This research was financially supported by the Health∼Holland/LSH-TKI call 2021–2022, project 25687, NACQAC: ‘Novel antimicrobial coatings with stable non-antibiotic Quaternary Ammonium Compounds and photosensitizer technology'

The Global Burden of Disease Study 2019 showed a 33.4% increase in fractures and a 65.3% increase in Years lived with disability (YLD) since 1990. Although the overall rate of fracture related infection (FRI) is low, it increases to 30% in complex fractures. In addition, the implantation of foreign materials, such as fracture stabilizing implants, decreases the number of bacteria needed to cause an infection. Then, when infections do occur, they are difficult to treat and often require multiple surgeries to heal. The bacteria can persist in the canaliculi of the bony tissue, in cells, in a biofilm on material or necrotic bone or in abscess communities. In the last decades, different approaches have been pursued to modify biomaterials as well as implant surface and to develop antimicrobial surfaces or local drug release strategies. This talk will give an introduction to the problem of bony and implant associated infections and presents the development and preclinical (as well as clinical) studies of two approaches for local drug delivery

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses