Aims

This study investigated vancomycin-microbubbles (Vm-MBs) and meropenem (Mp)-MBs with ultrasound-targeted microbubble destruction (UTMD) to disrupt biofilms and improve bactericidal efficiency, providing a new and promising strategy for the treatment of device-related infections (DRIs).

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

Aims. Biofilm-related infection is a major complication that occurs in orthopaedic surgery. Various treatments are available but efficacy to eradicate infections varies significantly. A systematic review was performed to evaluate therapeutic interventions combating biofilm-related infections on in vivo animal models. Methods. Literature research was performed on PubMed and Embase databases. Keywords used for search criteria were “bone AND biofilm”. Information on the species of the animal model, bacterial strain, evaluation of biofilm and bone infection, complications, key findings on observations, prevention, and treatment of biofilm were extracted. Results. A total of 43 studies were included. Animal models used included fracture-related infections (ten studies), periprosthetic joint infections (five studies), spinal infections (three studies), other implant-associated infections, and osteomyelitis. The most common bacteria were Staphylococcus species. Biofilm was most often observed with scanning electron microscopy. The natural history of biofilm revealed that the process of bacteria attachment, proliferation, maturation, and dispersal would take 14 days. For systemic mono-antibiotic therapy, only two of six studies using vancomycin reported significant biofilm reduction, and none reported eradication. Ten studies showed that combined systemic and topical antibiotics are needed to achieve higher biofilm reduction or eradication, and the effect is decreased with delayed treatment. Overall, 13 studies showed promising therapeutic potential with surface coating and antibiotic loading techniques. Conclusion. Combined topical and systemic application of antimicrobial agents effectively reduces biofilm at early stages. Future studies with sustained release of antimicrobial and biofilm-dispersing agents tailored to specific pathogens are warranted to achieve biofilm eradication. Cite this article: Bone Joint Res 2022;11(10):700–714

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

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

Aims. The purpose of this study was to explore a simple and effective method of preparing human acellular amniotic membrane (HAAM) scaffolds, and explore the effect of HAAM scaffolds with juvenile cartilage fragments (JCFs) on osteochondral defects. Methods. HAAM scaffolds were constructed via trypsinization from fresh human amniotic membrane (HAM). The characteristics of the HAAM scaffolds were evaluated by haematoxylin and eosin (H&E) staining, picrosirius red staining, type II collagen immunostaining, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Human amniotic mesenchymal stem cells (hAMSCs) were isolated, and stemness was verified by multilineage differentiation. Then, third-generation (P3) hAMSCs were seeded on the HAAM scaffolds, and phalloidin staining and SEM were used to detect the growth of hAMSCs on the HAAM scaffolds. Osteochondral defects (diameter: 3.5 mm; depth: 3 mm) were created in the right patellar grooves of 20 New Zealand White rabbits. The rabbits were randomly divided into four groups: the control group (n = 5), the HAAM scaffolds group (n = 5), the JCFs group (n = 5), and the HAAM + JCFs group (n = 5). Macroscopic and histological assessments of the regenerated tissue were evaluated to validate the treatment results at 12 weeks. Results. In vitro, the HAAM scaffolds had a network structure and possessed abundant collagen. The HAAM scaffolds had good cytocompatibility, and hAMSCs grew well on the HAAM scaffolds. In vivo, the macroscopic scores of the HAAM + JCFs group were significantly higher than those of the other groups. In addition, histological assessments demonstrated that large amounts of hyaline-like cartilage formed in the osteochondral defects in the HAAM + JCFs group. Integration with surrounding normal cartilage and regeneration of subchondral bone in the HAAM + JCFs group were better than those in the other groups. Conclusion. HAAM scaffolds combined with JCFs promote the regenerative repair of osteochondral defects. Cite this article: Bone Joint Res 2022;11(6):349–361

Aims. With the ageing population, fragility fractures have become one of the most common conditions. The objective of this study was to investigate whether microbiological outcomes and fracture-healing in osteoporotic bone is worse than normal bone with fracture-related infection (FRI). Methods. A total of 120 six-month-old Sprague-Dawley (SD) rats were randomized to six groups: Sham, sham + infection (Sham-Inf), sham with infection + antibiotics (Sham-Inf-A), ovariectomized (OVX), OVX + infection (OVX-Inf), and OVX + infection + antibiotics (OVX-Inf-A). Open femoral diaphysis fractures with Kirschner wire fixation were performed. Staphylococcus aureus at 4 × 10. 4. colony-forming units (CFU)/ml was inoculated. Rats were euthanized at four and eight weeks post-surgery. Radiography, micro-CT, haematoxylin-eosin, mechanical testing, immunohistochemistry (IHC), gram staining, agar plating, crystal violet staining, and scanning electron microscopy were performed. Results. Agar plating analysis revealed a higher bacterial load in bone (p = 0.002), and gram staining showed higher cortical bone colonization (p = 0.039) in OVX-Inf compared to Sham-Inf. OVX-Inf showed significantly increased callus area (p = 0.013), but decreased high-density bone volume (p = 0.023) compared to Sham-Inf. IHC staining showed a significantly increased expression of TNF-α in OVX-Inf compared to OVX (p = 0.049). Significantly reduced bacterial load on bone (p = 0.001), enhanced ultimate load (p = 0.001), and energy to failure were observed in Sham-Inf-A compared to Sham-Inf (p = 0.028), but not in OVX-Inf-A compared to OVX-Inf. Conclusion. In osteoporotic bone with FRI, infection was more severe with more bone lysis and higher bacterial load, and fracture-healing was further delayed. Systemic antibiotics significantly reduced bacterial load and enhanced callus quality and strength in normal bone with FRI, but not in osteoporotic bone. Cite this article: Bone Joint Res 2022;11(2):49–60

Aims. Biofilm formation is intrinsic to prosthetic joint infection (PJI). In the current study, we evaluated the effects of silver-containing hydroxyapatite (Ag-HA) coating and vancomycin (VCM) on methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation. Methods. Pure titanium discs (Ti discs), Ti discs coated with HA (HA discs), and 3% Ag-HA discs developed using a thermal spraying were inoculated with MRSA suspensions containing a mean in vitro 4.3 (SD 0.8) x 10. 6. or 43.0 (SD 8.4) x 10. 5. colony-forming units (CFUs). Immediately after MRSA inoculation, sterile phosphate-buffered saline or VCM (20 µg/ml) was added, and the discs were incubated for 24 hours at 37°C. Viable cell counting, 3D confocal laser scanning microscopy with Airyscan, and scanning electron microscopy were then performed. HA discs and Ag HA discs were implanted subcutaneously in vivo in the dorsum of rats, and MRSA suspensions containing a mean in vivo 7.2 (SD 0.4) x 10. 6.   or 72.0 (SD 4.2) x 10. 5.   CFUs were inoculated on the discs. VCM was injected subcutaneously daily every 12 hours followed by viable cell counting. Results. Biofilms that formed on HA discs were thicker and larger than those on Ti discs, whereas those on Ag-HA discs were thinner and smaller than those on Ti discs. Viable bacterial counts in vivo revealed that Ag-HA combined with VCM was the most effective treatment. Conclusion. Ag-HA with VCM has a potential synergistic effect in reducing MRSA biofilm formation and can thus be useful for preventing and treating PJI. Cite this article:Bone Joint Res. 2020;9(5):211–218

Aims. Biofilm formation is one of the primary reasons for the difficulty in treating implant-related infections (IRIs). Focused high-energy extracorporeal shockwave therapy (fhESWT), which is a treatment modality for fracture nonunions, has been shown to have a direct antibacterial effect on planktonic bacteria. The goal of the present study was to investigate the effect of fhESWT on Staphylococcus aureus biofilms in vitro in the presence and absence of antibiotic agents. Methods. S. aureus biofilms were grown on titanium discs (13 mm × 4 mm) in a bioreactor for 48 hours. Shockwaves were applied with either 250, 500, or 1,000 impulses onto the discs surrounded by either phosphate-buffered saline or antibiotic (rifampin alone or in combination with nafcillin). The number of viable bacteria was determined by quantitative culture after sonication. Representative samples were taken for scanning electron microscopy. Results. The application of fhESWT led to a ten-fold reduction in bacterial counts on the metal discs for all impulse numbers compared to the control (p < 0.001). Increasing the number of impulses did not further reduce bacterial counts in the absence of antibiotics (all p > 0.289). Antibiotics alone reduced the number of bacteria on the discs; however, the combined application of the fhESWT and antibiotic administration further reduced the bacterial count compared to the antibiotic treatment only (p = 0.032). Conclusion. The use of fhESWT significantly reduced the colony-forming unit (CFU) count of a S. aureus biofilm in our model independently, and in combination with antibiotics. Therefore, the supplementary application of fhESWT could be a helpful tool in the treatment of IFIs in certain cases, including infected nonunions. Cite this article: Bone Joint Res 2021;10(1):77–84

Objectives. The Precice nail is the latest intramedullary lengthening nail with excellent early outcomes. Implant complications have led to modification of the nail design. The aim of this study was to perform a retrieval study of Precice nails following lower-limb lengthening and to assess macroscopical and microscopical changes to the implants and evaluate differences following design modification, with the aim of identifying potential surgical, implant, and patient risk factors. Methods. A total of 15 nails were retrieved from 13 patients following lower-limb lengthening. Macroscopical and microscopical surface damage to the nails were identified. Further analysis included radiology and micro-CT prior to sectioning. The internal mechanism was then analyzed with scanning electron microscopy and energy dispersive x-ray spectroscopy to identify corrosion. Results. Seven male and three female patients underwent 12 femoral lengthenings. Three female patients underwent tibial lengthening. All patients obtained the desired length with no implant failure. Surface degradation was noted on the telescopic part of every nail design, less on the latest implants. Microscopical analysis confirmed fretting and pitting corrosion. Following sectioning, black debris was noted in all implants. The early designs were found to have fractured actuator pins and the pin and bearings showed evidence of corrosive debris. The latest designs showed evidence of biological deposits suggestive of fluid ingress within the nail but no corrosion. Conclusion. This study confirms less internal corrosion following modification, but evidence of titanium debris remains. We recommend no change to current clinical practice. However, potential reuse of the Precice nail, for secondary limb lengthening in the same patient, should be undertaken with caution. Cite this article: V. C. Panagiotopoulou, K. Davda, H. S. Hothi, J. Henckel, A. Cerquiglini, W. D. Goodier, J. Skinner, A. Hart, P. R. Calder. A retrieval analysis of the Precice intramedullary limb lengthening system. Bone Joint Res 2018;7:476–484. DOI: 10.1302/2046-3758.77.BJR-2017-0359.R1

In 2020 almost 90% of femoral heads for total hip implants in Germany were made of ceramic. Nevertheless, the cellular interactions and abrasion mechanisms in vivo have not been fully understood until now. Metal transfer from the head-neck taper connection, occurring as smear or large-area deposit, negatively influences the surface quality of the articulating bearing. In order to prevent metal transfer, damage patterns of 40 Biolox delta ceramic retrievals with CoC and CoPE bearings were analysed. A classification of damage type and severity for each component (n=40) was done according to an established scoring system. To investigate the physical properties, the surface quality was measured using confocal microscopy, quantitative analysis of phase composition were performed by Raman spectroscopy and qualitative analysis of metal traces was done by scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDX). The periprosthetic tissue was analysed for abrasion particles with SEM and EDX. Both bearing types show different damage patterns. Dotted/ drizzled metal smears were identified in 82 % of CoC (n=16) and 96 % of CoPE (n=24) bearings. Most traces on the ceramic heads were identified in the proximal area while they were observed predominantly in the distal area for the ceramic inlays. The identified marks are similar to those of metallic bearings. Metallic smears lead to an increase of up to 30 % in the monoclinic crystalline phase of the ceramic. The roughness increases by up to six times to Ra=48 nm. Ceramic and metallic wear particles from the articulating surfaces or head neck taper junctions were found in the periprosthetic tissue. Damage patterns on CoC hip implants seem to be similar to those of metallic implants. More detailed analysis of CoC implants are needed to understand the described damage patterns and provide advice for prevention

Despite advancements, revision rates following total ankle replacement (TAR) are high in comparison to other total joint replacements. This explant analysis study aimed to investigate whether there was appreciable metal particulate debris release from various contemporary TARs by describing patterns of material loss. Twenty-eight explanted TARs (9 designs: 3 fixed and 6 mobile bearing), revised for any reason, were studied. The articulating surfaces of the metal tibial and talar components as well as the polyethylene insert were assessed for damage features using light microscopy. Based on the results of the microscopic analysis, scanning electron microscopy with energy dispersive X-ray spectroscopy was performed to determine the composition of embedded debris identified, as well as non-contacting 3D profilometry. Pitting, indicative of material loss, was identified on the articulating surfaces of 54% of tibial components and 96% of talar components. Bearing constraint was not found to be a factor, with similar proportions of fixed and mobile bearing metal components showing pitting. More cobalt-chromium than titanium alloy tibial components exhibited pitting (63% versus 20%). Significantly higher average surface roughness (Sa) values were measured for pitted areas in comparison to unpitted areas of these metal components (p<0.05). Additionally, metallic embedded debris (cobalt-chromium likely due to pitting of the tibial and talar components or titanium likely from loss of their porous coatings) was identified in 18% of polyethylene inserts. The presence of hard 3. rd. body particles was also indicated by macroscopically visible sliding plane scratching, identified on 79% of talar components. This explant analysis study demonstrates that metal debris is released from the articulating surfaces and the coatings of various contemporary TARs, both fixed and mobile bearing. These findings suggest that metal debris release in TARs may be an under-recognised issue that should be considered in the study of painful or failed TAR moving forwards

Objectives. The paradoxical migration of the femoral neck element (FNE) superomedially against gravity, with respect to the intramedullary component of the cephalomedullary device, is a poorly understood phenomenon increasingly seen in the management of pertrochanteric hip fractures with the intramedullary nail. The aim of this study was to investigate the role of bidirectional loading on the medial migration phenomenon, based on unique wear patterns seen on scanning electron microscopy of retrieved implants suggestive of FNE toggling. Methods. A total of 18 synthetic femurs (Sawbones, Vashon Island, Washington) with comminuted pertrochanteric fractures were divided into three groups (n = 6 per group). Fracture fixation was performed using the Proximal Femoral Nail Antirotation (PFNA) implant (Synthes, Oberdorf, Switzerland; n = 6). Group 1 was subjected to unidirectional compression loading (600 N), with an elastomer (70A durometer) replacing loose fracture fragments to simulate surrounding soft-tissue tensioning. Group 2 was subjected to bidirectional loading (600 N compression loading, 120 N tensile loading), also with the elastomer replacing loose fracture fragments. Group 3 was subjected to bidirectional loading (600 N compression loading, 120 N tensile loading) without the elastomer. All constructs were tested at 2 Hz for 5000 cycles or until cut-out occurred. The medial migration distance (MMD) was recorded at the end of the testing cycles. Results. The MMDs for Groups 1, 2, and 3 were 1.02 mm, 6.27 mm, and 5.44 mm respectively, with reliable reproduction of medial migration seen in all groups. Bidirectional loading groups showed significantly higher MMDs compared with the unidirectional loading group (p < 0.01). Conclusion. Our results demonstrate significant contributions of bidirectional cyclic loading to the medial migration phenomenon in cephalomedullary nail fixation of pertrochanteric hip fractures. Cite this article: G. W. Law, Y. R. Wong, A. K-S. Yew, A. C. T. Choh, J. S. B. Koh, T. S. Howe. Medial migration in cephalomedullary nail fixation of pertrochanteric hip fractures: A biomechanical analysis using a novel bidirectional cyclic loading model. Bone Joint Res 2019;8:313–322. DOI: 10.1302/2046-3758.87.BJR-2018-0271.R1

Meniscal injuries affect over 1.5 million people across Europe and the USA annually. Injury greatly reduces knee joint mobility and quality of life and frequently leads to the development of osteoarthritis. Tissue engineered strategies have emerged in response to a lack of viable treatments for meniscal pathologies. However, to date, constructs mimicking the structural and functional organisation of native tissue, whilst promoting deposition of new extracellular matrix, remains a bottleneck in meniscal repair. 3D bioprinting allows for deposition and patterning of biological materials with high spatial resolution. This project aims to develop a biomimetic 3D bioprinted meniscal substitute. Meniscal tissue was characterised to effectively inform the design of biomaterials for bioprinting constructs with appropriate structural and functional properties. Histology, gene expression and mass spectrometry were performed on native tissue to investigate tissue architecture, matrix components, cell populations and protein expression regionally across the meniscus. 3D laser scanning and magnetic resonance imaging were employed to acquire the external geometrical information prior to fabrication of a 3D printed meniscus. Bioink suitability was investigated through regional meniscal cell encapsulation in blended hydrogels, with the incorporation of growth factors and assessed for their suitability through rheology, scanning electron microscopy, histology and gene expression analysis. Meniscal tissue characterisation revealed regional variations in matrix compositions, cellular populations and protein expression. The process of imaging through to 3D printing highlighted the capability of producing a construct that accurately replicated meniscal geometries. Regional meniscal cell encapsulation into hydrogels revealed a recovery in cell phenotype, with the incorporation of growth factors into the bioink's stimulating cellular re-differentiation and improved zonal functionality. Meniscus biofabrication highlights the potential to print patient specific, customisable meniscal implants. Achieving zonally distinct variations in cell and matrix deposition highlights the ability to fabricate a highly complex tissue engineered construct. Acknowledgements: This work was undertaken as part of the UK Research and Innovation (UKRI)-funded CDT in Advanced Biomedical Materials

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 success of cementless orthopaedic implants relies on bony ingrowth and active bone remodelling. Much research effort is invested to develop implants with controllable surface roughness and internal porous architectures that encourage these biological processes. Evaluation of these implants requires long-term and costly animal studies, which do not always yield the desired outcome requiring iteration. The aim of our study is to develop a cost-effective method to prescreen design parameters prior to animal trials to streamline implant development and reduce live animal testing burden. Ex vivo porcine cancellous bone cylinders (n=6, Ø20×12mm) were extracted from porcine knee joints with a computer-numerically-controlled milling machine under sterile conditions within 4 hours of animal sacrifice. The bone discs were implanted with Ø6×12mm additive manufactured porous titanium implants and were then cultured for 21days. Half underwent static culture in medium (DMEM, 10% FBS, 1% antibiotics) at 37°C and 5% CO. 2. The rest were cultured in novel high-throughput stacked configuration in a bioreactor that simulated physiological conditions after surgery: the fluid flow and cyclic compression force were set at 10ml/min and 10–150 N (1Hz,5000 cycles/day) respectively. Stains were administered at days 7 and 14. Samples were evaluated with widefield microscopy, scanning electron microscopy (SEM) and with histology. More bone remodelling was observed on the samples cultured within the bioreactor: widefield imaging showed more remodelling at the boundaries between the implant-bone interface, while SEM revealed immature bone tissue integration within the pores of the implant. Histological analysis confirmed these results, with many more trabecular struts with new osteoid formation on the samples cultured dynamically compared to static ones. Ex vivo bone can be used to analyse new implant technologies with lower cost and ethical impact than animal trial. Physiological conditions (load and fluid flow) promoted bone ingrowth and remodelling

Aim. A novel anti-infective biopolymer implant coating was developed to prevent bacterial biofilm formation and allow on-demand burst release of anti-infective silver (Ag) into the surrounding of the implant at any time after surgery via focused high-energy extracorporeal shock waves (fhESW). Method. A semi-crystalline Poly-L-lactic acid (PLLA) was loaded with homogeneously dissolved silver (Ag) applied onto Ti6Al4V discs. A fibroblast WST-1 assay was performed to ensure adequate biocompatibility of the Ag concentration at 6%. The prevention of early biofilm formation was investigated in a biofilm model with Staphylococcus epidermidis RP62A after incubation for 24 hours via quantitative bacteriology. In addition, the effect of released Ag after fhESW (Storz DUOLITH SD1: 4000 impulses, 1,24 mJ/mm. 2. , 3Hz, 162J) was assessed via optical density of bacterial cultures (Escherichia coli TG1, Staphylococcus epidermidis RP62A, Staphylococcus aureus 6850) and compared to an established electroplated silver coating. The amount of released Ag after the application of different intensities of fhESW was measured and compared to a control group without fhESW via graphite furnace atomic absorption spectrometry (GF-AAS), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Results. The coating with 6% Ag reduced Staphylococcus epidermidis biofilm formation by 99.7% (mean±SD: 2.1×10^5 ± 3,9×10^5 CFU/µL) compared to uncoated controls (6.8×10^7 ± 4.9×10^7 CFU/µL); (p=0.0001). After applying fhESW the commercially available electroplated silver coating did not prevent the growth of all tested bacterial strains. Bacterial growth is delayed with 4% Ag and completely inhibited with 6% Ag in the novel coating, except for a small increase of S. aureus after 17 hours. SEM and EDS confirmed a local disruption of the coating after fhESW. Conclusions. This novel anti-infective implant coating has the potential to prevent bacterial biofilm formation. The on-demand burst release of silver via fhESW could be an adjunctive in the treatment of implant related infection and is of particular interest in the concept of single stage revision surgery

Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. To date, there are no animal models that can fully recapitulate how a biofilm is challenged in vivo in the setting of GN-PJI. The purpose of this study is to establish a clinically representative GN-PJI in vivo model that can reliably depict biofilm formation on titanium implant surface. We hypothesized that the biofilm formation on the implant surface would affect the ability of the implant to be osseointegrated. The model was developed using a 3D-printed, medical-grade titanium (Ti-6Al-4V), monoblock, cementless hemiarthroplasty hip implant. This implant was used to replace the femoral head of a Sprague-Dawley rat using a posterior surgical approach. To induce PJI, two bioluminescent Pseudomonas aeruginosa (PA) strains were utilized: a reference strain (PA14-lux) and a mutant strain that is defective in biofilm formation (DflgK-lux). PJI development and biofilm formation was quantitatively assessed in vivo using the in vivo imaging system (IVIS), and in vitro using the viable colony count of the bacterial load on implant surface. Magnetic Resonance Imaging (MRI) was acquired to assess the involvement of periprosthetic tissue in vivo, and the field emission scanning electron microscopy (FE-SEM) of the explanted implants was used to visualize the biofilm formation at the bone-implant interface. The implant stability, as an outcome, was directly assessed by quantifying the osseointegration using microCT scans of the extracted femurs with retained implants in vitro, and indirectly assessed by identifying the gait pattern changes using DigiGaitTM system in vivo. A localized prosthetic infection was reliably established within the hip joint and was followed by IVIS in real-time. There was a quantitative and qualitative difference in the bacterial load and biofilm formation between PA14 and DflgK. This difference in the ability to persist in the model between the two strains was reflected on the gait pattern and implant osseointegration. We developed a novel uncemented hip hemiarthroplasty GN-PJI rat model. This model is clinically representative since animals can bear weight on the implant. PJI was detected by various modalities. In addition, biofilm formation correlated with implant function and stability. In conclusion, the proposed in vivo GN-PJI model will allow for more reliable testing of novel biofilm-targeting therapetics

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. 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