Search

A fracture-related infection (FRI) is a serious complication that can occur after surgical fixation of bone fractures. Affected patients may encounter delayed healing and functional limitations. Although it is well established that Staphylococcus aureus (S. aureus) is the main causative pathogen of an FRI, the pathophysiology of an S. aureus-induced FRI is not well characterised over time. Therefore, an experimental study in mice comparing S. aureus-inoculated and non-inoculated groups was performed that particularly focused on staphylococcal abscess communities (SACs) and host cellular response. C57Bl/6N female mice received a double osteotomy of the femur, which was stabilised using a titanium 6-hole MouseFix locking plate and four screws. Animals were either S. aureus-inoculated or non-inoculated and euthanised between 1 and 28 d post-surgery. Histopathological evaluation showed normal bone healing for non-inoculated mice, whereas inoculated mice had no fracture consolidation and severe osteolysis. Within the bone marrow of inoculated mice, SACs were observed from 7 d, which increased in size and number over time. A fibrin pseudocapsule enclosed the SACs, which were surrounded by many Ly6G+ neutrophils with some Ly6C+ monocytes and F4/80+ macrophages, the majority of which were viable. The abscesses were encapsulated by fibrin(ogen), collagen and myofibroblasts, with regulatory T cells and M2 macrophages at the periphery. Only bone marrow monocytes and neutrophils of inoculated mice displayed functional suppression of T cells, indicative of myeloid-derived suppressor cells. The present study revealed that an FRI in mice is persistent over time and associated with osteolysis, SAC formation and an immunosuppressive environment.

Staphylococcus aureus (S. aureus) osteomyelitis remains a major clinical problem. Anti-glucosaminidase (Gmd) antibodies (1C11) are efficacious in prophylactic and therapeutic murine models. Feasibility, safety and pharmacokinetics of 1C11 passive immunisation in sheep and endogenous anti-Gmd levels were quantified in osteomyelitis patients. 3 sheep received a 500 mg intravenous (i.v.) bolus of 1C11 and its levels in sera were determined by enzyme-linked immunosorbent assay (ELISA) over 52 d. A humanised anti-Gmd monoclonal antibody, made by grafting the antigen-binding fragment (Fab) portion of 1C11 onto the fragment crystallisable region (Fc) of human IgG1, was used to make a standard curve of mean fluorescent intensity versus concentration of anti-Gmd. Anti-Gmd serum levels were determined in 297 patients with culture-confirmed S. aureus osteomyelitis and 40 healthy controls. No complications or adverse events were associated with the sheep 1C11 i.v. infusion and the estimated circulating half-life of 1C11 was 23.7 d. Endogenous anti-Gmd antibody levels in sera of osteomyelitis patients ranged from < 1 ng/mL to 300 µg/mL, with a mean concentration of 21.7 µg/mL. The estimated circulating half-life of endogenous anti-Gmd antibodies in sera of 12 patients with cured osteomyelitis was 120.4 d. A clinically relevant administration of anti-Gmd (500 mg i.v. = 7 mg/kg/70 kg human) was safe in sheep. This dose was 8 times more than the endogenous anti-Gmd levels observed in osteomyelitis patients and was predicted to have a half-life of > 3 weeks. Anti-Gmd passive immunisation has potential to prevent and treat S. aureus osteomyelitis. Further clinical development is warranted.

Fracture-related infection (FRI) is a major complication in surgically fixed fractures. Instability of the fracture after fixation is considered a risk factor for infection; however, few experimental data are available confirming this belief. To study whether stable fractures led to higher infection clearance, mouse femoral osteotomies were fixed with either stable or unstable fixation and the surgical site was contaminated with either Staphylococcus epidermidis (S. epidermidis)or Staphylococcus aureus (S. aureus)clinical isolates. Infection progression was assessed at different time points by quantitative bacteriology, total cell counts in spleen and lymph node and histological analysis. Operated, non-inoculated mice were used as controls. Two inbred mouse strains (C57BL/6 and BALB/c) were included in the study to determine the influence of different host background in the outcome. Stable fixation allowed a higher proportion of C57BL/6 mice to clear S. epidermidis inoculation in comparison to unstable fixation. No difference associated with fixation type was observed for BALB/c mice. Inoculation with S. aureus resulted in a more severe infection for both stable and unstable fractures in both mouse strains; however, significant osteolysis around the screws rendered the stable group functionally unstable. Our results suggested that fracture stability could have an influence on S. epidermidis infection, although host factors also played a role. No differences were observed when using S. aureus, due to a more severe infection, leading to osteolysis and loss of stability in both groups. Further studies are required in order to address the biological features underlying the differences observed.

The treatment of chronic orthopaedic device-associated infection (ODRI) often requires multiple surgeries and prolonged antibiotic therapy. Despite this extensive treatment protocol, the procedure is associated with significant failure rates. Currently, no large animal model is available that recapitulates a failed revision. Therefore, our aim was to establish a large animal model for failed treatment of an ODRI in order to serve as a testbed for future interventional strategies. Adult Swiss Alpine sheep received an intramedullary nail in the tibia and a localised inoculum of either a methicillin-sensitive or methicillin-resistant Staphylococcus aureus (MSSA, MRSA respectively). After 8 weeks, when chronic infection had been established, the animals underwent a staged revision with debridement and temporary placement of an antibiotic-loaded cement spacer. Antibiotics were delivered systemically in a standard or pathogen-adapted manner. Debridement and implant exchange alone failed to treat the MSSA infection. Neither local therapy alone nor systemic therapy alone were effective in resolving infection with MSSA, but a combination of local and systemic therapy was effective against it. MRSA infection was not resolved by the combination of local and systemic antibiotics (standard or pathogen-adapted). A model for failed revision of MRSA infection is described despite the use of local and systemic antibiotics. Novel interventions may be assessed using this model, including antibiotic and non-antibiotic interventions.

The local mechanical environment at a fracture is known to influence biological factors such as callus formation, immune cell recruitment and susceptibility to infection. Infection models incorporating a fracture are therefore required to evaluate prevention and treatment of infection after osteosynthesis. The aim of this study was to create humane, standardised and repeatable preclinical models of implant-related bone infection after osteosynthesis in the rabbit humerus. Custom-designed interlocked intramedullary nails and commercially available locking plates were subjected to biomechanical evaluation in cadaveric rabbit humeri; a 10-week in vivo healing study; a dose response study with Staphylococcus aureus over 4 weeks; and finally, a long-term infection of 10 weeks in the plate model.Outcome measures included biomechanical testing, radiography, histology, haematology and quantitative bacteriology. Both implants offered similar biomechanical stability in cadaveric bones, and when applied in the in vivo study, resulted in complete radiographic and histological healing and osteotomy closure within 10-weeks. As expected in the infection study, higher bacterial doses led to an increasing infection rate. In both infected groups, there was a complete lack of osteotomy closure at 4 weeks. C-reactive protein (CRP), lymphocyte: granulocyte ratio and weight loss were increased in infected animals receiving IM nails in comparison with non-inoculated equivalents, although this was less evident in the plate group. In the 10-week infection group, healing does not occur in the plated rabbits. We have successfully developed a rabbit model that is suitable for further studies, particularly those looking into preventative strategies for post-traumatic implant-related osteomyelitis.

Intramedullary nailing has been used for decades to treat fractures of the long bones. However, complications related to the increase in medullary pressure culminated in the development of the Reamer Irrigator Aspirator (RIA). Since its first clinical use, the RIA has moved from a reaming device to a cell and autologous bone-harvesting tool. This increase in use brings with it further clinical questions; namely, does the endosteal bone regenerate sufficiently to allow subsequent reaming procedures. In the current study, endosteal bone regeneration post reaming was assessed in an ovine model. The study included six animals that had one tibia reamed, while the contralateral tibia acted as an intact control. Animals were administered fluorochrome labels in vivo, and bone regeneration was assessed using radiographical analysis. The endpoint of the study was 12 weeks post-surgery, at which time ex vivo analysis consisted of computed tomography and histological assessments. In vivo radiographs indicated limited healing of the reamed bone. However, ex vivo computer tomographical analysis indicated no significant differences in terms of bone volume between the reamed bone and the intact bone. Histological assessment of these regions indicated new bone formation. Fluorescent labelling indicates strong bone formation from 9 weeks post-surgery and as such, the bone formed at 12 weeks was immature in nature and was actively undergoing remodelling. These results indicate that bone regeneration post-reaming was continuing at three months. Therefore, given more time it may have sufficiently healed to allow a surgeon to use the intramedullary canal for a re-reaming procedure.

Osteomyelitis (OM) is a progressive, inflammatory infection of bone caused predominately by Staphylococcus aureus. Herein, we engineered an antibiotic-eluting collagen-hydroxyapatite scaffold capable of eliminating infection and facilitating bone healing. An iterative freeze-drying and chemical crosslinking approach was leveraged to modify antibiotic release kinetics, resulting in a layered dual-release system whereby an initial rapid release of antibiotic to clear infection was followed by a sustained controlled release to prevent reoccurrence of infection. We observed that the presence of microbial collagenase accelerated antibiotic release from the crosslinked layer of the scaffold, indicating that the material is responsive to microbial activity. As exemplar drugs, vancomycin and gentamicin-eluting scaffolds were demonstrated to be bactericidal, and supported osteogenesis in vitro. In a pilot murine model of OM, vancomycin-eluting scaffolds were observed to reduce S. aureus infection within the tibia. Finally, in a rabbit model of chronic OM, gentamicin-eluting scaffolds both facilitated radial bone defect healing and eliminated S. aureus infection. These results show that antibiotic-eluting collagen-hydroxyapatite scaffolds are a one-stage therapy for OM, which when implanted into infected bone defects simultaneously eradicate infection and facilitate bone tissue healing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Eamon J Sheehy has patent #17773914 pending to The Royal College of Surgeons in Ireland. Amro Widaa has patent #17773914 pending to The Royal College of Surgeons in Ireland. Alan Ryan has patent #17773914 pending to The Royal College of Surgeons in Ireland. Adolfo Lopez-Noriega has patent #17773914 pending to The Royal College of Surgeons in Ireland. Fergal O’ Brien has patent #17773914 pending to The Royal College of Surgeons in Ireland. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: With the introduction and continuous improvement in operative fracture fixation, even the most severe bone fractures can be treated with a high rate of successful healing. However, healing complications can occur and when healing fails over prolonged time, the outcome is termed a fracture non-union. Non-union is generally believed to develop due to inadequate fixation, underlying host-related factors, or infection. Despite the advancements in fracture fixation and infection management, there is still a clear need for earlier diagnosis, improved prediction of healing outcomes and innovation in the treatment of non-union., Areas Covered: This review provides a detailed description of non-union from a clinical perspective, including the state of the art in diagnosis, treatment, and currently available biomaterials and orthobiologics.Subsequently, recent translational development from the biological, mechanical, and infection research fields are presented, including the latest in smart implants, osteoinductive materials, and in silico modeling., Expert Opinion: The first challenge for future innovations is to refine and to identify new clinical factors for the proper definition, diagnosis, and treatment of non-union. However, integration of in vitro , in vivo , and in silico research will enable a comprehensive understanding of non-union causes and correlations, leading to the development of more effective treatments.

Fracture-related infections (FRIs), particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA), are challenging to treat. This study designed and evaluated a hydrogel loaded with a cocktail of bacteriophages and vancomycin (1.2 mg/mL). The co-delivery hydrogel showed 99.72% reduction in MRSA biofilm in vitro. The hydrogel released 54% of phages and 82% of vancomycin within 72 h and maintained activity for eight days, in vivo the co-delivery hydrogel with systemic antibiotic significantly reduced bacterial load by 0.99 log10 CFU compared to controls, with active phages detected in tissues at euthanasia (2 × 10 3 PFU/mL). No phage resistance was detected in the phage treatment groups, and serum neutralization resulted in only a 20% reduction in phage count. In this work, we show that a phage-antibiotic co-delivery system via CMC hydrogel is a promising adjunct to systemic antibiotic therapy for MRSA-induced FRI, highlighting its potential for localized, sustained delivery and improved treatment outcomes., (© 2024. The Author(s).)

Non-steroidal anti-inflammatory drugs (NSAIDs) are crucial components of multimodal analgesia for musculoskeletal injuries, targeting cyclooxygenase (COX) enzymes (COX-1 and/or COX-2 isoenzymes). Concerns exist regarding their potential interference with bone healing and orthopaedic device-related infections (ODRI), where data are limited. This study aimed to investigate whether the COX-selectivity of NSAIDs interfered with antibiotic efficacy and bone changes in the setting of an ODRI. In vitro testing demonstrated that combining celecoxib (a COX-2 inhibitor) with cefazolin significantly enhanced antibacterial efficacy compared to cefazolin alone ( p < 0.0001). In vivo experiments were performed using Staphylococcus epidermidis in the rat proximal tibia of an ODRI model. Long and short durations of celecoxib treatment in combination with antibiotics were compared to a control group receiving an antibiotic only. The long celecoxib treatment group showed impaired infection clearance, while the short celecoxib treatment showed increased bone formation (day 6, p < 0.0001), lower bone resorption (day 6, p < 0.0001), and lower osteolysis (day 6, BV/TV: p < 0.0001; BIC: p = 0.0005) compared to the control group, without impairing antibiotic efficacy ( p > 0.9999). Given the use of NSAIDs as part of multimodal analgesia, and considering these findings, short-term use of COX-2 selective NSAIDs like celecoxib not only aids pain management but also promotes favorable bone changes during ODRI.

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., Competing Interests: A. Vallejo Diaz, D. Arens, S. Zeiter, C. Siverino, R. G. Richards, and T. F. Moriarty report institutional funding (paid to AO Research Institute Davos) from AOTrauma as part of the Clinical Priority, (© 2024 Puetzler et al.)

Osteoporosis poses a major public health challenge, and it is characterized by low bone mass, deterioration of the microarchitecture of bone tissue, causing a consequent increase in bone fragility and susceptibility to fractures and complicating bone fixation, particularly screw implantation. In the present study, our aim was to improve implant stability in osteoporotic bone using a thermoresponsive hyaluronan hydrogel (HA-pNIPAM) to locally deliver the bisphosphonate zoledronic acid (ZOL) to prevent bone resorption and bone morphogenetic protein 2 (BMP2) to induce bone formation. Adult female Wistar rats (n = 36) were divided into 2 treatment groups: one group of SHAM-operated animals and another group that received an ovariectomy (OVX) to induce an osteoporotic state. All animals received a polyetheretherketone (PEEK) screw in the proximal tibia. In addition, subgroups of SHAM or OVX animals received either the HA-pNIPAM hydrogel without or with ZOL/BMP2, placed into the defect site prior to screw implantation. Periprosthetic bone and implant fixation were monitored using longitudinal in vivo microCT scanning post-operatively and at 3, 6, 9, 14, 20 and 28 days. Histological assessment was performed post-mortem. Our data showed that pure hydrogel has no impact of implant fixation The ZOL/BMP2-hydrogel significantly increased bone-implant contact and peri-implant bone fraction, primarily through reduced resorption. STATEMENT OF CLINICAL SIGNIFICANCE: Local delivery of ZOL and BMP2 using a biocompatible hydrogel improved implant stability in osteoporotic bone. This approach could constitute a potent alternative to systemic drug administration and may be useful in avoiding implant loosening in clinical settings., Competing Interests: Declaration of competing interest The authors are not compensated, and no other institutional subsidies, corporate affiliations, or funding sources support this work unless clearly documented and disclosed. No external funds were received in support of this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2 -/- Il2rg -/- Sirpa NOD Flk2 -/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR + monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice., Competing Interests: The authors have no conflict of interest to declare., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Bacteriophage (phage) therapy has shown promise in treating fracture-related infection (FRI); however, questions remain regarding phage efficacy against biofilms, phage-antibiotic interaction, administration routes and dosing, and the development of phage resistance. The goal of this study was to develop a dual antibiotic-phage delivery system containing hydrogel and alginate microbeads loaded with a phage cocktail plus meropenem and evaluate efficacy against muti-drug resistant Pseudomonas aeruginosa. Two phages (FJK.R9-30 and MK.R3-15) displayed enhanced antibiotic activity against P. aeruginosa biofilms when tested in combination with meropenem. The antimicrobial activity of both antibiotic and phage was retained for eight days at 37 °C in dual phage and antibiotic loaded hydrogel with microbeads (PA-HM). In a mouse FRI model, phages were recovered from all tissues within all treatment groups receiving dual PA-HM. Moreover, animals that received the dual PA-HM either with or without systemic antibiotics had less incidence of phage resistance and less serum neutralization compared to phages in saline. The dual PA-HM could reduce bacterial load in soft tissue when combined with systemic antibiotics, although the infection was not eradicated. The use of alginate microbeads and injectable hydrogel for controlled release of phages and antibiotics, leads to the reduced development of phage resistance and lower exposure to the adaptive immune system, which highlights the translational potential of the dual PA-HM. However, further optimization of phage therapy and its delivery system is necessary to achieve higher bacterial killing activity in vivo in the future., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Intervertebral disc (IVD) disorders (e.g., herniation) directly contribute to back pain, which is a leading cause of global disability. Next-generation treatments for IVD herniation need advanced preclinical testing to evaluate their ability to repair large defects, prevent reherniation, and limit progressive degeneration. This study tested whether experimental, injectable, and nonbioactive biomaterials could slow IVD degeneration in an ovine discectomy model., Methods: Ten skeletally mature sheep (4-5.5 years) experienced partial discectomy injury with cruciate-style annulus fibrosus (AF) defects and 0.1 g nucleus pulposus (NP) removal in the L1-L2, L2-L3, and L3-L4 lumbar IVDs. L4-L5 IVDs were Intact controls. IVD injury levels received: (1) no treatment (Injury), (2) poly (ethylene glycol) diacrylate (PEGDA), (3) genipin-crosslinked fibrin (FibGen), (4) carboxymethylcellulose-methylcellulose (C-MC), or (5) C-MC and FibGen (FibGen + C-MC). Animals healed for 12 weeks, then IVDs were assessed using computed tomography (CT), magnetic resonance (MR) imaging, and histopathology., Results: All repaired IVDs retained ~90% of their preoperative disc height and showed minor degenerative changes by Pfirrmann grading. All repairs had similar disc height loss and Pfirrmann grade as Injury IVDs. Adhesive AF sealants (i.e., PEGDA and FibGen) did not herniate, although repair caused local endplate (EP) changes and inflammation. NP repair biomaterials (i.e., C-MC) and combination repair (i.e., FibGen + C-MC) exhibited lower levels of degeneration, less EP damage, and less severe inflammation; however, C-MC showed signs of herniation via biomaterial expulsion., Conclusions: All repair IVDs were noninferior to Injury IVDs by IVD height loss and Pfirrmann grade. C-MC and FibGen + C-MC IVDs had the best outcomes, and may be appropriate for enhancement with bioactive factors (e.g., cells, growth factors, and miRNAs). Such bioactive factors appear to be necessary to prevent injury-induced IVD degeneration. Application of AF sealants alone (i.e., PEGDA and FibGen) resulted in EP damage and inflammation, particularly for PEGDA IVDs, suggesting further material refinements are needed., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Long bone defects resulting from primary trauma or secondary to debridement of fracture-related infection (FRI) remain a major clinical challenge. One approach often used is the induced membrane technique (IMT). The effectiveness of the IMT in infected versus non-infected settings remains to be definitively established. In this study we present a new rabbit humerus model and compare the IMT approach between animals with prior infection and non-infected equivalents., Methods: A 5 mm defect was created in the humerus of New Zealand White rabbits (n = 53) and fixed with a 2.5 mm stainless steel plate. In the non-infected groups, the defect was either left empty (n = 6) or treated using the IMT procedure (PMMA spacer for 3 weeks, n = 6). Additionally, both approaches were applied in animals that were inoculated with Staphylococcus aureus 4 weeks prior to defect creation (n = 5 and n = 6, respectively). At the first and second revision surgeries, infected and necrotic tissues were debrided and processed for bacteriological quantification. In the IMT groups, the PMMA spacer was removed 3 weeks post implantation and replaced with a beta-tricalcium phosphate scaffold and bone healing observed for a further 10 weeks. Infected groups also received systemic antibiotic therapy. The differences in bone healing between the groups were evaluated radiographically using a modification of the radiographic union score for tibial fractures (RUST) and by semiquantitative histopathology on Giemsa-Eosin-stained sections., Results: The presence of S. aureus infection at revision surgery was required for inclusion to the second stage. At the second revision surgery all collected samples were culture negative confirming successful treatment. In the empty defect group, bone healing was increased in the previously infected animals compared with non-infected controls as revealed by radiography with significantly higher RUST values at 6 weeks (p = 0.0281) and at the end of the study (p = 0.0411) and by histopathology with increased cortical bridging (80% and 100% in cis and trans cortical bridging in infected animals compared to 17% and 67% in the non-infected animals). With the IMT approach, both infected and non-infected animals had positive healing assessments., Conclusion: We successfully developed an in vivo model of bone defect healing with IMT with and without infection. Bone defects can heal after an infection with even better outcomes compared to the non-infected setting, although in both cases, the IMT achieved better healing., (© 2023. The Author(s).)

Background: Staphylococcus aureus is the leading pathogen in fracture-related infection. Previous in vitro experiments, in vivo testing in wax moth larvae, and genomic analysis of clinical S. aureu s isolates from fracture-related infection identified low-virulence (Lo-SA5464) and high-virulence (Hi-SA5458) strains. These findings correlated with acute fracture-related infection induced by Hi-SA5458, whereas Lo-SA5464 caused a chronic fracture-related infection in its human host. However, it remains unclear whether and to what extent the causative pathogen is attributable to these disparities in fracture-related infections., Question/purpose: Are there differences in the course of infection when comparing these two different clinical isolates in a murine fracture-related infection model, as measured by (1) clinical observations of weight loss, (2) quantitative bacteriology, (3) immune response, and (4) radiographic and histopathologic morphology?, Methods: Twenty-five (including one replacement animal) female (no sex-specific influences expected), skeletally mature C57Bl/6N inbred mice between 20 and 28 weeks old underwent femoral osteotomy stabilized by titanium locking plates. Fracture-related infection was established by inoculation of high-virulence S. aureus EDCC 5458 (Hi-SA5458) or low-virulence S. aureus EDCC 5464 (Lo-SA5464) in the fracture gap. Each of these groups consisted of 12 randomly assigned animals. Mice were euthanized 4 and 14 days postsurgery, resulting in six animals per group and timepoint. The severity and progression of infection were assessed in terms of clinical observation of weight loss, quantitative bacteriology, quantitative serum cytokine levels, qualitative analysis of postmortem radiographs, and semiquantitative histopathologic evaluation., Results: For clinical observations of weight change, no differences were seen at Day 4 between Hi-SA5458- and Lo-SA5464-infected animals (mean -0.6 ± 0.1 grams versus -0.8 ± 0.2 grams, mean difference -0.2 grams [95% CI -0.8 to 0.5 grams]; p =0.43), while at 14 days, the Hi-SA5458 group lost more weight than the Lo-SA5464 group (mean -1.55 ± 0.2 grams versus -0.8 ± 0.3 grams; mean difference 0.7 grams [95% CI 0.2 to 1.3 grams]; p = 0.02). Quantitative bacteriological results 4 days postoperatively revealed a higher bacterial load in soft tissue samples in Hi-SA5458-infected animals than in the Lo-SA5464-infected cohort (median 6.8 x 10 7 colony-forming units [CFU]/g, range 2.2 x 10 7 to 2.1 x 10 9 CFU/g versus median 6.0 x 10 6 CFU/g, range 1.8 x 10 5 to 1.3 x 10 8 CFU/g; difference of medians 6.2 x 10 7 CFU/g; p = 0.03). At both timepoints, mice infected with the Hi-SA5458 strain also displayed higher proportions of bacterial dissemination into organs than Lo-SA5464-infected animals (67% [24 of 36 organs] versus 14% [five of 36 organs]; OR 12.0 [95% CI 3.7 to 36]; p < 0.001). This was accompanied by a pronounced proinflammatory response on Day 14, indicated by increased serum cytokine levels of interleukin-1β (mean 9.0 ± 2.2 pg/mL versus 5.3 ± 1.5 pg/mL; mean difference 3.6 pg/mL [95% CI 2.0 to 5.2 pg/mL]; p < 0.001), IL-6 (mean 458.6 ± 370.7 pg/mL versus 201.0 ±89.6 pg/mL; mean difference 257.6 pg/mL [95% CI 68.7 to 446.5 pg/mL]; p = 0.006), IL-10 (mean 15.9 ± 3.5 pg/mL versus 9.9 ± 1.0 pg/mL; mean difference 6.0 pg/mL [95% CI 3.2 to 8.7 pg/mL]; p < 0.001), and interferon-γ (mean 2.7 ± 1.9 pg/mL versus 0.8 ± 0.3 pg/mL; mean difference 1.8 pg/mL [95% CI 0.5 to 3.1 pg/mL]; p = 0.002) in Hi-SA5458-infected compared with Lo-SA5464-infected animals. The semiquantitative histopathologic assessment on Day 4 revealed higher grades of granulocyte infiltration in Hi-SA5458-infected animals (mean grade 2.5 ± 1.0) than in Lo-SA5464-infected animals (mean grade 1.8 ± 1.4; mean difference 0.7 [95% CI 0.001 to 1.4]; p = 0.0498). On Day 14, bone healing at the fracture site was present to a higher extent in Lo-SA5464-infected animals than in Hi-SA5458-infected animals (mean grade 0.2 ± 0.4 versus 1.8 ± 1.2; mean difference -1.6 [95% CI -2.8 to -0.5]; p = 0.008)., Conclusion: Similar to septic infection in a human host, infection with Hi-SA5458 in this murine model was characterized by a higher bacterial load, more-pronounced systemic dissemination, and stronger systemic and local inflammation. Thus, there is strong support for the idea that pathogenic virulence plays a crucial role in fracture-related infections. To confirm our observations, future studies should focus on characterizing S. aureus virulence at the genomic and transcriptomic levels in more clinical isolates and patients. Comparing knockout and wildtype strains in vitro and in vivo, including the S. aureus strains studied, could confirm our findings and identify the genomic features responsible for S. aureus virulence in fracture-related infections., Clinical Relevance: For translational use, virulence profiles of S. aureus may be useful in guiding treatment decisions in the future. Once specific virulence targets are identified, one approach to fracture-related infections with high-virulence strains might be the development of antivirulence agents, particularly to treat or prevent septic dissemination. For fracture-related infections with low virulence, prolonged antimicrobial therapy or exchange of an indwelling implant might be beneficial owing to slower growth and persistence capacity., Competing Interests: Each author certifies that there are no funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article related to the author or any immediate family members. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research ® editors and board members are on file with the publication and can be viewed on request., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Association of Bone and Joint Surgeons.)

Aim: Secondary bone healing requires an adequate level of mechanical stimulation expressed by the extent of interfragmentary motion in the fracture. However, there is no consensus about when the mechanical stimulation should be initiated to ensure a timely healing response. Therefore, this study aims to compare the effect of the immediate and delayed application of mechanical stimulation in a large animal model., Methods: Twelve Swiss White Alpine sheep underwent partial osteotomy of a tibia that was stabilised with an active fixator inducing well-controlled mechanical stimulation. Animals were randomly assigned into two groups with different stimulation protocols. The immediate group received daily stimulation (1000 cycles/day) from the first day post-operation, while in the delayed group, stimulation began only on the 22 nd day post-operation. Healing progression was evaluated daily by measuring the in vivo stiffness of the repair tissue and by quantifying callus area on weekly radiographs. All animals were euthanised five weeks post-op. Post-mortem callus volume was determined from high-resolution computer tomography (HRCT)., Results: Fracture stiffness (p < 0.05) and callus area (p < 0.01) were significantly larger for the immediate group compared to the delayed stimulation group. In addition, the callus volume measured on the post-mortem HRCT showed 319 % greater callus volume for the immediate stimulation group (p < 0.01)., Conclusions: This study demonstrates that a delay in the onset of mechanical stimulation retards fracture callus development and that mechanical stimulation already applied in the early post-op phase promotes bone healing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The authors are not compensated and there are no other institutional subsidies, corporate affiliations, or funding sources supporting this work unless clearly documented and disclosed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background and Objectives : Antiresorptive drugs are widely used in osteology and oncology. An important adverse effect of these drugs is medication-induced osteonecrosis of the jaw (MRONJ). There is scientific uncertainty about the underlying pathomechanism of MRONJ. A promising theory suspects infectious stimuli and local acidification with adverse effects on osteoclastic activity as crucial steps of MRONJ etiology. Clinical evidence showing a direct association between MRONJ and oral infections, such as periodontitis, without preceding surgical interventions is limited. Large animal models investigating the relationship between periodontitis and MRONJ have not been implemented. It is unclear whether the presence of infectious processes without surgical manipulation can trigger MRONJ. The following research question was formulated: is there a link between chronic oral infectious processes (periodontitis) and the occurrence of MRONJ in the absence of oral surgical procedures? Materials and Methods: A minipig large animal model for bisphosphonate-related ONJ (BRONJ) using 16 Göttingen minipigs divided into 2 groups (intervention/control) was designed and implemented. The intervention group included animals receiving i.v. bisphosphonates (zoledronate, n = 8, 0.05 mg/kg/week: ZOL group). The control group received no antiresorptive drug (n = 8: NON-ZOL group). Periodontitis lesions were induced by established procedures after 3 months of pretreatment (for the maxilla: the creation of an artificial gingival crevice and placement of a periodontal silk suture; for the mandible: the placement of a periodontal silk suture only). The outcomes were evaluated clinically and radiologically for 3 months postoperatively. After euthanasia a detailed histological evaluation was performed. Results: Periodontitis lesions could be induced successfully in all animals (both ZOL and NON-ZOL animals). MRONJ lesions of various stages developed around all periodontitis induction sites in the ZOL animals. The presence of MRONJ and periodontitis was proven clinically, radiologically and histologically. Conclusions: The results of this study provide further evidence that the infectious processes without prior dentoalveolar surgical interventions can trigger MRONJ. Therefore, iatrogenic disruption of the oral mucosa cannot be the decisive step in the pathogenesis of MRONJ.

Pneumonia, always a major malady, became the main public health and economic disaster of historical proportions with the COVID-19 pandemic. This study was based on a premise that pathology of lung metabolism in inflammation may have features invariant to the nature of the underlying cause. Amino acid uptake by the lungs was measured from plasma samples collected pre-terminally from a carotid artery and vena cava in mice with bleomycin-induced lung inflammation (N = 10) and compared to controls treated with saline instillation (N = 6). In the control group, the difference in concentrations between the arterial and venous blood of the 19 amino acids measured reached the level of statistical significance only for arginine (-10.7%, p = 0.0372) and phenylalanine (+5.5%, p = 0.0266). In the bleomycin group, 11 amino acids had significantly lower concentrations in the arterial blood. Arginine concentration was decreased by 21.1% (p<0.0001) and only that of citrulline was significantly increased (by 20.1%, p = 0.0002). Global Arginine Bioavailability Ratio was decreased in arterial blood by 19.5% (p = 0.0305) in the saline group and by 30.4% (p<0.0001) in the bleomycin group. Production of nitric oxide (NO) and citrulline from arginine by the inducible nitric oxide synthase (iNOS) is greatly increased in the immune system's response to lung injury. Deprived of arginine, the endothelial cells downstream may fail to provide enough NO to prevent the activation of thrombocytes. Thrombotic-related vascular dysfunction is a defining characteristic of pneumonia, including COVID-19. This experiment lends further support to arginine replacement as adjuvant therapy in pneumonia., Competing Interests: Slobodan Tepic is a co-founder and CEO of Kyon Biotech AG, Zurich, the parent company of Hepius Biotech AG. He is also the inventor of a pending European patent application (EP 21200735.5, Treatment of infective pneumonias by arginine), now abandoned. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Tepic et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Autologous cancellous bone graft is the gold standard in large bone defect repair. However, studies using autologous bone grafting in rats are rare. To determine the feasibility of autologous cancellous bone graft harvest from different anatomical donor sites (humerus, ilium, femur, tibia, and tail vertebrae) in rats and compare their suitability as donor sites, a total of 13 freshly euthanized rats were used to describe the surgical technique, determine the cancellous bone volume and microstructure, and compare the cancellous bone collected quantitatively and qualitatively. It was feasible to harvest cancellous bone grafts from all five anatomical sites with the humerus and tail being more surgically challenging. The microstructural analysis using micro-computed tomography showed a significantly lower bone volume fraction, bone mineral density, and trabecular thickness of the humerus and iliac crest compared to the femur, tibia, and tail vertebrae. The harvested weight and volume did not differ between the donor sites. All donor sites apart from the femur yielded primary osteogenic cells confirmed by the presence of alkaline phosphatase and Alizarin Red S stain. Bone samples from the iliac crest showed the most consistent outgrowth of osteoprogenitor cells. In conclusion, the tibia and iliac crest may be the most favorable donor sites considering the surgical approach. However, due to the differences in microstructure of the cancellous bone and the consistency of outgrowth of osteoprogenitor cells, the donor sites may have different healing properties, that need further investigation in an in vivo study., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Orthopedic device-related infection (ODRI) preclinical models are widely used in translational research. Most ODRI models require induction of general anesthesia, which frequently results in hypothermia in rodents. This study aimed to evaluate the impact of peri-anesthetic hypothermia in rodents on outcomes in preclinical ODRI studies. A retrospective analysis of all rodents that underwent surgery under general anesthesia to induce an ODRI model with inoculation of Staphylococcus epidermidis between 2016 and 2020 was conducted. A one-way multivariate analysis of covariance (one-way MANCOVA) was used to determine the fixed effect of peri-anesthetic hypothermia (hypothermic defined as rectal temperature <35°C) on the combined harvested tissue and implant colony-forming unit (CFU) counts, and having controlled for the study groups including treatments received, duration of surgery and anesthesia, and study period. The results showed a significant effect of peri-anesthetic hypothermia on the post-mortem combined CFU counts from the harvested tissue and implant(s) (p = 0.01) when comparing normo- versus hypothermic rodents. Using Wilks' Λ as a criterion to determine the contribution of independent variables to the model, peri-anesthetic hypothermia was the most significant, though still a weak predictor, of increased harvested CFU counts. Altogether, the data corroborate the concept that bacterial colonization is affected by abnormal body temperature during general anesthesia at the time of bacterial inoculation in rodents, which needs to be taken into consideration to decrease infection data variability and improve experimental reproducibility., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non-unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow - derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1 rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies. STATEMENT OF SIGNIFICANCE: Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Orthopedic-device-related infection is one of the most severe complications in orthopedic surgery. To reduce the associated morbidity and healthcare costs, new prevention and treatment modalities are continuously under development. Preclinical in vivo models serve as a control point prior to clinical implementation. This study presents a mouse model of subcutaneously implanted titanium discs, infected with Staphylococcus aureus , to fill a gap in the early-stage testing of antimicrobial biomaterials. Firstly, three different inocula were administered either pre-adhered to the implant or pipetted on top of it following implantation to test their ability to reliably create an infection. Secondly, the efficacy of low-dose (25 mg/kg) and high-dose (250 mg/kg) cefazolin administered systemically in infection prevention was assessed. Lastly, titanium implants were replaced by antibiotic-loaded bone cement (ALBC) discs to investigate the efficacy of local antibiotics in infection prevention. The efficacy in infection prevention of the low-dose perioperative antibiotic prophylaxis (PAP) depended on both the inoculum and inoculation method. Bacterial counts were significantly lower in animals receiving the high dose of PAP. ALBC discs with or without the additional PAP proved highly effective in infection prevention and provide a suitable positive control to test other prevention strategies.

Intervertebral disc degeneration is a common cause of low back pain, the leading cause of disability worldwide. Appropriate preclinical models for intervertebral disc research are essential to achieving a better understanding of underlying pathophysiology and for the development, evaluation, and translation of more effective treatments. To this end, in vivo animal and ex vivo organ culture models are both widely used by spine researchers; however, the relative strengths and weaknesses of these two approaches are a source of ongoing controversy. In this article, members from the Spine and Preclinical Models Sections of the Orthopedic Research Society, including experts in both basic and translational spine research, present contrasting arguments in support of in vivo animal models versus ex vivo organ culture models for studies of the disc, supported by a comprehensive review of the relevant literature. The objective is to provide a deeper understanding of the respective advantages and limitations of these approaches, and advance the field toward a consensus with respect to appropriate model selection and implementation. We conclude that complementary use of several model types and leveraging the unique advantages of each is likely to result in the highest impact research in most instances., Competing Interests: Lachlan J. Smith, Sibylle Grad, Victor Leung, Christine L. Le Maitre, and Devina Purmessur are members of the JOR Spine Advisory Review Board. The authors have no other relevant conflicts to declare., (© 2022 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Introduction: Cell transplantation shows promising results for intervertebral disc (IVD) repair, however, contemporary strategies present concerns regarding needle puncture damage, cell retention, and straining the limited nutrient availability. Mesenchymal stromal cell (MSC) homing is a natural mechanism of long-distance cellular migration to sites of damage and regeneration. Previous ex vivo studies have confirmed the potential of MSC to migrate over the endplate and enhance IVD-matrix production. In this study, we aimed to exploit this mechanism to engender IVD repair in a rat disc degeneration model., Methods: Female Sprague Dawley rats were subjected to coccygeal disc degeneration through nucleus pulposus (NP) aspiration. In part 1; MSC or saline was transplanted into the vertebrae neighboring healthy or degenerative IVD subjected to irradiation or left untouched, and the ability to maintain the IVD integrity for 2 and 4 weeks was assessed by disc height index (DHI) and histology. For part 2, ubiquitously GFP expressing MSC were transplanted either intradiscally or vertebrally, and regenerative outcomes were compared at days 1, 5, and 14 post-transplantation. Moreover, the homing potential from vertebrae to IVD of the GFP + MSC was assessed through cryosection mediated immunohistochemistry., Results: Part 1 of the study revealed significantly improved maintenance of DHI for IVD vertebrally receiving MSC. Moreover, histological observations revealed a trend of IVD integrity maintenance. Part 2 of the study highlighted the enhanced DHI and matrix integrity for discs receiving MSC vertebrally compared with intradiscal injection. Moreover, GFP rates highlighted MSC migration and integration in the IVD at similar rates as the intradiscally treated cohort., Conclusion: Vertebrally transplanted MSC had a beneficial effect on the degenerative cascade in their neighboring IVD, and thus potentially present an alternative administration strategy. Further investigation will be needed to determine the long-term effects, elucidate the role of cellular homing versus paracrine signaling, and validate our observations on a large animal model., Competing Interests: TW is a paid employee of TUNZ Pharma Co. Ltd. (Osaka, Japan). DS is a scientific advisor of TUNZ Pharma Co. Ltd., (© 2022 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Purpose: Implant leakage is the most common complication of vertebral augmentation. Alternative injectable materials must demonstrate intravascular safety comparable to or better than polymethyl methacrylate (PMMA). This study assessed the systemic effects of a triphasic calcium-based implant or PMMA injected directly into the femoral vein in a large animal model designed to mimic severe intravascular implant leakage., Methods: Six skeletally mature female sheep were randomly assigned (n = 3) to either the PMMA or the triphasic implant (AGN1, composition: calcium sulfate, β-tricalcium phosphate, brushite) treatment group. Femoral veins of each sheep were directly injected with 0.5 mL of implant material to mimic leakage volumes reported during PMMA vertebroplasty. To compare acute systemic effects of the materials, cardiovascular parameters, laboratory coagulation markers, and calcium and sulfate serum levels were monitored for 60 min after implant injection. Thrombotic and embolic events were evaluated by radiologic imaging, necropsy, and histopathology., Results: Heart rate, systemic arterial blood pressure, arterial oxygenation, arterial carbon dioxide content, and coagulation markers remained within physiological range after either AGN1 or PMMA injection. No blood flow interruption in the larger pulmonary vessels was observed in either group. Lung histopathology revealed that the severity of thrombotic changes after AGN1 injection was minimal to slight, while changes after PMMA injection were minimal to massive., Conclusion: Acute systemic effects of intravascular AGN1 appeared to be comparable to or less than that of intravascular PMMA. Furthermore, in this preliminary study, the severity and incidence of pulmonary histological changes were lower for AGN1 compared to PMMA., (© 2022. The Author(s).)

Objective: Surgery is an integral part of many experimental studies. Aseptic and minimal invasive surgical technique and optimal perioperative and post-operative care are prerequisites to achieve surgical success and best possible animal welfare outcomes. Good surgical practice cannot only improve the animal's postoperative recovery, but also study outcome and validity. There seems to be a lack of implementation of good surgical practice during rodent surgery. The aim of this systematic review is to identify, critically evaluate and compare the currently recommended standards and underlying guidelines for rodent surgery-and finally to compile a comprehensive guideline of good surgical practice for rodent surgery., Search Strategy: PubMed, Embase and Web of Science were searched to identify guidelines published in peer-reviewed journals. To identify grey literature and unpublished guidelines, we will perform a Google search for published guidelines and search laboratory animal sciences books for relevant book chapters. Additionally, we will conduct a survey among animal researchers enquiring about the guidelines they use., Screening and Study Selection: For publications retrieved by the systematic search, unique references are screened by two reviewers, first for eligibility based on title and abstract and subsequently for final inclusion based on full text. Eligibility of books is based on title and content, final inclusion based on chapter full text. Guidelines are either retrieved by Google searches or a survey. Google searches will be conducted by at least four of the authors. Thereafter, guidelines will be screened by two of the authors., Data Extraction and Synthesis: We will extract data from publications, book chapters and guidelines. Based on the extracted data, we will perform a descriptive synthesis of the bibliographical details, guideline development and endorsement, and the prevalence of individual recommendations, including subgroup analysis of the guidance per continent or country and differences between peer-reviewed versus non-peer-reviewed guidance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

In the clinical treatment of fractures, rhBMP-2 administration is associated with a well-established profile of side-effects, including osteolysis and ectopic bone formation, which are driven by pro-inflammatory processes triggered by the use of high doses. Immunomodulatory strategies could minimize the incidence of side-effects by enabling the use of lower, and safer, rhBMP-2 doses. This study investigated whether interleukin-1 receptor antagonist (IL-1Ra) can enhance the therapeutic efficacy of a low dose of rhBMP-2 in a weight-bearing femoral fracture healing model. Exogenous IL-1Ra, in combination with rhBMP-2, was delivered using a collagen-hydroxyapatite scaffold (CHA) to attenuate IL-1β produced in response to fracture. Femoral defects were treated with CHA scaffolds alone, or loaded with IL-1Ra (2.5 µg), rhBMP-2 (1 µg), IL-1Ra (2.5 µg) in combination with rhBMP-2 (1 µg). Bone healing was assessed over 14 weeks in comparison to control groups, empty defect, and a higher dose of rhBMP-2 (5 µg), which were recently demonstrated to lead to non-union, and successful bridging of the defect, respectively. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, at both week 4 and 6, compared to a low dose of rhBMP-2 alone. By 14 weeks, the combination of IL-1Ra and a rhBMP-2 promoted full bridging of femurs, which were 3-fold more mechanically reliable compared to the femurs treated with a low dose of rhBMP-2 alone. Taken together, this study demonstrates that IL-1Ra can significantly enhance femoral bone healing when used in combination with a low dose of rhBMP-2. STATEMENT OF SIGNIFICANCE: Enabling the use of lower and safer doses of rhBMP-2, a potent inducer of bone formation, is of clinical relevance in orthopaedic medicine. In this study, the immunomodulatory interleukin-1 receptor antagonist (IL-1Ra) was investigated for its capacity to enhance the therapeutic efficacy of rhBMP-2 when used at lower doses in a weight-bearing femoral fracture healing model. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, and resulted in more mechanically reliable healed femurs, compared to a low dose of rhBMP-2 alone. This demonstrates for the first time in a rat long bone healing model that IL-1Ra can significantly enhance bone healing when used in combination with a low dose of rhBMP-2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Background and Objectives : Fracture healing is currently assessed through qualitative evaluation of radiographic images, which is highly subjective in nature. Radiographs can only provide snapshots in time, which are limited due to logistics and radiation exposure. We recently proposed assessing the bone healing status through continuous monitoring of the implant load, utilizing an implanted sensor system, the Fracture Monitor. The device telemetrically transmits statistically derived implant parameters via the patient's mobile phone to assist physicians in diagnostics and treatment decision-making. This preclinical study aims to systematically investigate the device safety and performance in an animal setting. Materials and Methods : Mid-shaft tibial osteotomies of different sizes (0.6-30 mm) were created in eleven Swiss mountain sheep. The bones were stabilized with either a conventional Titanium or stainless-steel locking plate equipped with a Fracture Monitor. Data were continuously collected over the device's lifetime. Conventional radiographs and clinical CT scans were taken longitudinally over the study period. The radiographs were systematically scored and CTs were evaluated for normalized bone volume in the defect. The animals were euthanized after 9 months. The sensor output was correlated with the radiologic parameters. Tissue samples from the device location were histologically examined. Results : The sensors functioned autonomously for 6.5-8.4 months until energy depletion. No macroscopic or microscopic adverse effects from device implantation were observed. The relative implant loads at 4 and 8 weeks post-operation correlated significantly with the radiographic scores and with the normalized bone volume metric. Conclusions : Continuous implant load monitoring appears as a relevant approach to support and objectify fracture healing assessments and carries a strong potential to enable patient-tailored rehabilitation in the future.

Background and Objectives : The objective of this study was to evaluate the effects of bisphosphonate (BP) administration on tooth growth, using CT-data of a minipig animal model investigation. Materials and Methods : Tooth growth was evaluated in minipigs, with eight animals receiving weekly zoledronate (ZOL) and three animals serving as the control group. Tooth growth was evaluated at the right 2nd molar (M2) in the maxilla. A computed tomography-based measuring method was applied to evaluate tooth growth in the coronal-apical, buccal-oral and mesial-distal axis. Results : ZOL-administration was found to impact tooth growth in all evaluated measuring axes, with the highest effect observed in the coronal-apical axis. Conclusions : Detrimental effects of BP administration on growing teeth have been reported by a number of investigators. The results of this investigation demonstrate that intravenous ZOL affects the growth of the whole tooth within a short period of administration. With BPs being administered to a growing number of pediatric patients, further studies should be conducted to qualify and quantify the effects of BPs on developing teeth.

Objective: To determine the influence of screw head diameter on equine condylar fracture fixation with 5.5 mm cortical screws., Study Design: Ex vivo, biomechanical study, blinded, matched-pair design., Sample Population: Fifteen pairs of equine third metacarpal (MC3) bones., Methods: Lateral condylar fractures were simulated by parasagittal osteotomies and repaired pairwise by 2 × 5.5 mm cortical screws of 8 mm (standard) or 10 mm (modified) head diameter. Interfragmentary compression at maximum screw insertion torque was measured. The instrumented specimens were pairwise stratified for biomechanical testing under the following modalities (n = 5): (1) screw insertion torque to failure, (2) quasi-static axial load to failure, and (3) cyclic axial load to 2 mm displacement followed by failure. Tests (1) and (2) were analyzed for yield, maximum, and failure torque/angle and load/displacement, respectively. Number of cycles to 2 mm displacement and failure was assessed from test (3)., Results: Maximum insertion torque was greater, and failure angle smaller, when constructs repaired with modified screws were tested (8.1 ± 0.5 vs. 7.4 ± 0.5 Nm; P = .0047 and 550 ± 104 vs. 1130 ± 230; P = .008). Axial yield (7118 ± 707 vs. 5740 ± 2267 N; P = .043) and failure load (12 347 ± 3359 vs. 8695 ± 2277 N; P = .043) were greater for specimens repaired with modified screws. No difference was detected between constructs in the number of cycles to 2 mm displacement., Conclusion: Condylar MC3 osteotomies repaired with modified 5.5 mm cortical screws sustained greater maximal hand torque insertion, smaller insertion failure angle and 1.4 fold greater quasi-static failure forces than constructs repaired with standard 5.5 mm screws., Clinical Significance: Use of modified screws with larger heads may improve the fixation of condylar fractures in horses. These results provide evidence to justify clinical evaluation in horses undergoing fracture repair., (© 2022 American College of Veterinary Surgeons.)

Local antibiotic therapy is increasingly being recognised for its role in preventing and treating orthopaedic device-related infection (ODRI). A bioresorbable, injectable gentamicin-loaded hydrogel has been developed to deliver local antibiotics at the time of surgery with potential for both prevention and treatment of ODRI. In a prophylaxis model, the antibiotic hydrogel was compared with systemic perioperative antibiotic prophylaxis alone in twelve sheep (six per group) at the time of intramedullary (IM) nail insertion to the tibia, which was inoculated with methicillin-sensitive Staphylococcus aureus (MSSA). In a treatment model of single-stage revision surgery, adjunctive antibiotic-loaded hydrogel was compared with systemic antibiotics alone in a single stage revision of MSSA infection associated with a tibia intramedullary nail in eleven sheep (five/six per group). The primary endpoint was quantitative microbiological results of soft tissue, bone and sonicate fluid from explanted hardware at the time of euthanasia. At euthanasia, the control sheep that received no local antibiotics in the prophylaxis model were all culture-positive (median 1x10 8 , range 7x10 6 -3x10 8 colony forming units, CFU) while only two of six sheep receiving local gentamicin had any culture positive biopsies (median 1x10 1 , range 0 - 1x10 5 CFU). For the treatment model, sheep receiving only systemic antibiotics were all culture-positive (median 8x10 5 , range 2x10 3 - 9x10 6 CFU) while only two of six sheep treated with gentamicin-loaded hydrogel had any culture positive biopsies (median 3x10 2 , range 0 - 7x10 4 CFU). Local gentamicin concentrations measured in extracellular fluid in the tibial canal show a burst release of gentamicin from the hydrogel. Serum gentamicin concentrations peaked in both models at one day post application and were below detection limit thereafter. This study has demonstrated the effective use of a locally delivered antibiotic hydrogel for both the prevention and treatment of ODRI that is superior to that of systemic antibiotics alone. Future studies will endeavour to translate from preclinical to clinical research trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Boot, Foster, Guillaume, Eglin, Schmid, D’Este, Zeiter, Richards and Moriarty.)

Background: The current standard of care for intervertebral disc (IVD) herniation, surgical discectomy, does not repair annulus fibrosus (AF) defects, which is partly due to the lack of effective methods to do so and is why new repair strategies are widely investigated and tested preclinically. There is a need to develop a standardized IVD injury model in large animals to enable comparison and interpretation across preclinical study results. The purpose of this study was to compare in vivo IVD injury models in sheep to determine which annulus fibrosus (AF) defect type combined with partial nucleus pulposus (NP) removal would better mimic degenerative human spinal pathologies., Methods: Six skeletally mature sheep were randomly assigned to one of the two observation periods (1 and 3 months) and underwent creation of 3 different AF defect types (slit, cruciate, and box-cut AF defects) in conjunction with 0.1 g NP removal in three lumbar levels using a lateral retroperitoneal surgical approach. The spine was monitored by clinical CT scans pre- and postoperatively, at 2 weeks and euthanasia, and by magnetic resonance imaging (MRI) and histology after euthanasia to determine the severity of degeneration (disc height loss, Pfirrmann grading, semiquantitative histopathology grading)., Results: All AF defects led to significant degenerative changes detectable on CT and MR images, produced bulging of disc tissue without disc herniation and led to degenerative and inflammatory histopathological changes. However, AF defects were not equal in terms of disc height loss at 3 months postoperatively; the cruciate and box-cut AF defects showed significantly decreased disc height compared to their preoperative height, with the box-cut defect creating the greatest disc height loss, while the slit AF defect showed restoration of normal preoperative disc height., Conclusions: The tested IVD injury models do not all generate comparable disc degeneration but can be considered suitable IVD injury models to investigate new treatments. Results of the current study clearly indicate that slit AF defect should be avoided if disc height is used as one of the main outcomes; additional confirmatory studies may be warranted to generalize this finding., Competing Interests: The authors declare no conflict of interest, (© 2022 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Introduction: Non-unions remain a clinical problem and are characterised by the failure to heal after a defined period of time. Current preclinical non-union models apply a wide variety of techniques to diminish intrinsic healing potential deviating from the clinical situation. The aim of this study was to develop and characterise a non-union model in rats using internal plate fixation without the need for additional healing insults, whereby bone healing can be longitudinally assessed using microCT. It was hypothesized that healing/non-unions can be accurately predicted at early time points by microCT., Materials and Methods: Female, skeletally mature Fischer F344 rats received a 2 mm or 1 mm femoral osteotomy, stabilized with either a 2 mm thick plate or a 1.25 mm thick plate. Healing was monitored by microCT over 14 weeks and histological analysis at euthanasia. The mechanical environment was characterised using finite element (FE) modelling and biomechanical testing., Results: The majority of animals receiving the 2 mm thick plate displayed poor healing responses in both the 2 mm and 1 mm defect size groups. Bone and cartilage formation were markedly improved using the 1.25 mm thick plate. MicroCT could accurately predict bone forming capacity at early time points (3-4 weeks)., Conclusions: The 2 mm thick plating system confers poor healing responses in female Fischer F344 rats, comparable to atrophic non-unions. By reducing plate thickness to increase interfragmentary strain within the defect site healing is improved, leading to borderline healing situations or increased abundance of cartilage tissue present in the defect site with ultimate failure to bridge the defect (hypertrophic non-union). Furthermore, microCT can reliably identify delayed/non-healing animals within 4 weeks, thereby allowing their selective targeting for the testing of novel, clinically relevant treatment strategies in different clinical situations aimed at restoring impaired bone healing., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

Although several studies have shown promising clinical outcomes of phage therapy in patients with orthopedic device-related infections, questions remain regarding the optimal application protocol, systemic effects, and the impact of the immune response. This study provides a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection (FRI) caused by Staphylococcus aureus. In a prevention setting, phage in saline (without any biomaterial-based carrier) was highly effective in the prevention of FRI, compared to systemic antibiotic prophylaxis alone. In the subsequent study involving treatment of established infection, daily administration of phage in saline through a subcutaneous access tube was compared to a single intraoperative application of a phage-loaded hydrogel and a control group receiving antibiotics only. In this setting, although a possible trend of bacterial load reduction on the implant was observed with the phage-loaded hydrogel, no superior effect of phage therapy was found compared to antibiotic treatment alone. The application of phage in saline through a subcutaneous access tube was, however, complicated by superinfection and the development of neutralizing antibodies. The latter was not found in the animals that received the phage-loaded hydrogel, which may indicate that encapsulation of phages into a carrier such as a hydrogel limits their exposure to the adaptive immune system. These studies show phage therapy can be useful in targeting orthopedic device-related infection, however, further research and improvements of these application methods are required for this complex clinical setting. IMPORTANCE Because of the growing spread of antimicrobial resistance, the use of alternative prevention and treatment strategies is gaining interest. Although the therapeutic potential of bacteriophages has been demonstrated in a number of case reports and series over the past decade, many unanswered questions remain regarding the optimal application protocol. Furthermore, a major concern during phage therapy is the induction of phage neutralizing antibodies. This study aimed at providing a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection caused by Staphylococcus aureus. Phage therapy was applied as prophylaxis in a first phase, and as treatment of an established infection in a second phase. The development of phage neutralizing antibodies was evaluated in the treatment study. This study demonstrates that phage therapy can be useful in targeting orthopedic device-related infection, especially as prophylaxis; however, further research and improvements of these application methods are required.

Short-chain fatty acids (SCFAs) produced by the gut microbiota have previously been demonstrated to play a role in numerous chronic inflammatory diseases and to be key mediators in the gut-bone signaling axis. However, the role of SCFAs in bone fracture healing and its impact on systemic inflammation during the regeneration process has not been extensively investigated yet. The aim of this study was to first determine the effects of the SCFA butyrate on key cells involved in fracture healing in vitro , namely, osteoclasts and mesenchymal stromal cells (MSCs), and second, to assess if butyrate supplementation or antibiotic therapy impacts bone healing, systemic immune status, and inflammation levels in a murine osteotomy model. Butyrate significantly reduced osteoclast formation and resorption activity in a dose-dependent manner and displayed a trend for increased calcium deposits in MSC cultures. Numerous genes associated with osteoclast differentiation were differentially expressed in osteoclast precursor cells upon butyrate exposure. In vivo , antibiotic-treated mice showed reduced SCFA levels in the cecum, as well as a distinct gut microbiome composition. Furthermore, circulating proinflammatory TNF α , IL-17a, and IL-17f levels, and bone preserving osteoprotegerin (OPG), were increased in antibiotic-treated mice compared to controls. Antibiotic-treated mice also displayed a trend towards delayed bone healing as revealed by reduced mineral apposition at the defect site and higher circulating levels of the bone turnover marker PINP. Butyrate supplementation resulted in a lower abundance of monocyte/macrophages in the bone marrow, as well as reduced circulating proinflammatory IL-6 levels compared to antibiotic- and control-treated mice. In conclusion, this study supports our hypothesis that SCFAs, in particular butyrate, are important contributors to successful bone healing by modulating key cells involved in fracture healing as well as systemic inflammation and immune responses., Competing Interests: All authors declare that they have no conflicts of interest., (Copyright © 2021 Alexandra Wallimann et al.)

Orthopaedic device-related infection (ODRI) presents a significant challenge to the field of orthopaedic and trauma surgery. Despite extensive treatment involving surgical debridement and prolonged antibiotic therapy, outcomes remain poor. This is largely due to the unique abilities of Staphylococcus aureus , the most common causative agent of ODRI, to establish and protect itself within the host by forming biofilms on implanted devices and staphylococcal abscess communities (SACs). There is a need for novel antimicrobials that can readily target such features. Enzybiotics are a class of antimicrobial enzymes derived from bacteria and bacteriophages, which function by enzymatically degrading bacterial polymers essential to bacterial survival or biofilm formation. Here, we apply an enzybiotic-based combination regimen to a set of in vitro models as well as in a murine ODRI model to evaluate their usefulness in eradicating established S. aureus infection, compared to classical antibiotics. We show that two chimeric endolysins previously selected for their functional efficacy in human serum in combination with a polysaccharide depolymerase reduce bacterial CFU numbers 10,000-fold in a peg model and in an implant model of biofilm. The enzyme combination also completely eradicates S. aureus in a SAC in vitro model where classical antibiotics are ineffective. In an in vivo ODRI model in mice, the antibiofilm effects of this enzyme regimen are further enhanced when combined with a classical gentamicin/vancomycin treatment. In a mouse model of methicillin-resistant S. aureus (MRSA) ODRI following a fracture repair, a combined local enzybiotic/antibiotic treatment regimen showed a significant CFU reduction in the device and the surrounding soft tissue, as well as significant prevention of weight loss. These outcomes were superior to treatment with antibiotics alone. Overall, this study demonstrates that the addition of enzybiotics, which are distinguished by their extremely rapid killing efficacy and antibiofilm activities, can enhance the treatment of severe MRSA ODRI.

Biomechanical stability plays an important role in fracture healing, with unstable fixation being associated with healing disturbances. A lack of stability is also considered a risk factor for fracture-related infection (FRI), although confirmatory studies and an understanding of the underlying mechanisms are lacking. In the present study, we investigate whether biomechanical (in)stability can lead to altered immune responses in mice under sterile or experimentally inoculated conditions. In non-inoculated C57BL/6 mice, instability resulted in an early increase of inflammatory markers such as granulocyte-colony stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin (IL)-6 within the bone. When inoculated with Staphylococcus epidermidis, instability resulted in a further significant increase in G-CSF, IL-6 and KC in bone tissue. Staphylococcus aureus infection led to rapid osteolysis and instability in all animals and was not further studied. Gene expression measurements also showed significant upregulation in CCL2 and G-CSF in these mice. IL-17A was found to be upregulated in all S. epidermidis infected mice, with higher systemic IL-17A cell responses in mice that cleared the infection, which was found to be produced by CD4+ and γδ+ T cells in the bone marrow. IL-17A knock-out (KO) mice displayed a trend of delayed clearance of infection (P=0.22, Fisher's exact test) and an increase in interferon (IFN)-γ production. Biomechanical instability leads to a more pronounced local inflammatory response, which is exaggerated by bacterial infection. This study provides insights into long-held beliefs that biomechanics are crucial not only for fracture healing, but also for control of infection., Competing Interests: Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2021. Published by The Company of Biologists Ltd.)

Peripheral nerve blocks are commonly used in human and veterinary medicine. The aim of the study was to compare the analgesic efficacy of a combined block of the femoral and sciatic nerves with an epidural injection of ropivacaine in experimental sheep undergoing orthopaedic hind limb surgery. Twenty-five sheep were assigned to two groups (peripheral nerve block; sciatic and femoral nerves (P); epidural analgesia (E)). In group P 10 mL ropivacaine 0.5% was injected around the sciatic and the femoral nerves under sonographic guidance and 10 mL NaCl 0.9% into the epidural space while in group E 10 mL ropivacaine 0.5% was injected into the epidural space and 10 mL NaCl 0.9% to the sciatic and the femoral nerves. During surgery, heart rate, respiratory rate and mean blood pressure were used as indicators of nociception. In the postoperative phase, nociception was evaluated every hour by use of a purposefully adapted pain score until the animal showed painful sensation at the surgical site. The mean duration of analgesia at the surgical wound was 6 h in group P and 8 h in group E. Mean time to standing was 4 h in group P and 7 h in group E. In conclusion time to standing was significantly shorter in group P while the duration of nociception was comparable in both groups. The peripheral nerve block can be used as an alternative to epidural analgesia in experimental sheep.

It is well known that mechanical stimulation promotes indirect fracture healing by triggering callus formation. We investigated the short-term response of healing tissue to mechanical stimulation to compare the changes in tissue stiffness during stimulation and resting phases in a preclinical case-series. Four sheep underwent a tibial osteotomy and were instrumented with a custom-made active fixator which applied a mechanical stimulation protocol of 1000 cycles/day, equally distributed over 12 h, followed by 12 h of rest. During each cycle, a surrogate metric for tissue stiffness was measured, enabling a continuous real-time monitoring of the healing progression. A daily stiffness increase during stimulation and an increase during resting were evaluated for each animal. One animal had to be excluded from the evaluation due to technical reasons. For all included animals, the stiffness began to increase within the second week post-op. A characteristic pattern was observed during daily measurements: the stiffness dropped considerably within the first stimulation cycles followed by a steady rise throughout the rest of the stimulation phase. However, for all included animals, the average daily stiffness increase within the first three weeks post operation was larger during resting than during stimulation (Sheep I: 16.9% vs. -5.7%; Sheep II: 14.7% vs. -1.8%; Sheep III: 8.9% vs. 1.6%). A continuous measurement of tissue stiffness together with a controlled fracture stimulation enabled the investigation of the short-term effects of specific stimulatory parameters, such as resting periods. Resting was identified as a potentially determining factor for bone healing progression. Optimizing the ratio between stimulation and resting may contribute to more robust fracture healing in the future.

A fracture-related infection (FRI) is a serious complication that can occur after surgical fixation of bone fractures. Affected patients may encounter delayed healing and functional limitations. Although it is well established that Staphylococcus aureus (S. aureus) is the main causative pathogen of an FRI, the pathophysiology of an S. aureus-induced FRI is not well characterised over time. Therefore, an experimental study in mice comparing S. aureus-inoculated and non-inoculated groups was performed that particularly focused on staphylococcal abscess communities (SACs) and host cellular response. C57Bl/6N female mice received a double osteotomy of the femur, which was stabilised using a titanium 6-hole MouseFix locking plate and four screws. Animals were either S. aureus-inoculated or non-inoculated and euthanised between 1 and 28 d post-surgery. Histopathological evaluation showed normal bone healing for non-inoculated mice, whereas inoculated mice had no fracture consolidation and severe osteolysis. Within the bone marrow of inoculated mice, SACs were observed from 7 d, which increased in size and number over time. A fibrin pseudocapsule enclosed the SACs, which were surrounded by many Ly6G+ neutrophils with some Ly6C+ monocytes and F4/80+ macrophages, the majority of which were viable. The abscesses were encapsulated by fibrin(ogen), collagen and myofibroblasts, with regulatory T cells and M2 macrophages at the periphery. Only bone marrow monocytes and neutrophils of inoculated mice displayed functional suppression of T cells, indicative of myeloid-derived suppressor cells. The present study revealed that an FRI in mice is persistent over time and associated with osteolysis, SAC formation and an immunosuppressive environment.

The probiotic Bifidobacterium longum subsp. longum 35624® (B. longum 35624®), with its surface exopolysaccharide (EPS624), has previously been demonstrated to induce immunoregulatory responses in the host and may, therefore, be a novel approach to prevent bone loss in inflammatory conditions such as post-menopausal osteoporosis (PMO). The aim of this study was to investigate the effect of EPS624 on osteoclast and osteoblast differentiation and to assess the potential of B. longum 35624® to prevent bone loss in vivo. In vitro cell assays were used to assess the impact of EPS624 on osteoclast and osteoblast differentiation. The potential of two probiotic B. longum 35624® strains, including an EPS-deficient strain, for preventing ovariectomy (Ovx)-induced bone loss was assessed in a murine model. EPS624 prevented osteoclast formation from murine bone marrow precursors under both normal and TNFα-induced inflammatory conditions and modestly increased mineralized matrix deposition in osteogenic cell cultures. However, in the presence of an anti-TLR2 blocking antibody, or in MyD88 -/-  osteoclast precursors, the inhibitory effect of EPS624 on osteoclast formation was diminished or completely prevented, respectively. Moreover, EPS624 induced IL-10 production in osteoclast precursors in a TLR2-dependent manner, although IL-10 was dispensable in the EPS624-mediated inhibition of osteoclast formation. In addition, EPS624-producing B. longum 35624® partially prevented bone loss in Ovx mice when administered by oral gavage. This study introduced EPS624 as a potential anti-resorptive therapy, although optimal in vivo delivery of the probiotic strain for treating low-grade inflammatory diseases such as PMO remains to be determined.

Orthopedic device-related infections remain a serious challenge to treat. Central to these infections are bacterial biofilms that form on the orthopedic implant itself. These biofilms shield the bacteria from the host immune system and most common antibiotic drugs, which renders them essentially antibiotic-tolerant. There is an urgent clinical need for novel strategies to prevent these serious infections that do not involve conventional antibiotics. Recently, a novel antibiofilm coating for titanium surfaces was developed based on 5-(4-bromophenyl)- N -cyclopentyl-1-octyl-1 H -imidazol-2-amine as an active biofilm inhibitor. In the current study we present an optimized coating protocol that allowed for a 5-fold higher load of this active compound, whilst shortening the manufacturing process. When applied to titanium disks, the newly optimized coating was resilient to the most common sterilization procedures and it induced a 1 log reduction in biofilm cells of a clinical Staphylococcus aureus isolate (JAR060131) in vitro , without affecting the planktonic phase. Moreover, the antibiofilm effect of the coating in combination with the antibiotic cefuroxime was higher than cefuroxime treatment alone. Furthermore, the coating was successfully applied to a human-scale fracture fixation device resulting in a loading that was comparable to the titanium disk model. Finally, an in vivo biocompatibility and healing study in a rabbit osteotomy model indicated that these coated implants did not negatively affect fracture healing or osteointegration. These findings put our technology one step closer to clinical trials, confirming its potential in fighting orthopedic infections without compromising healing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Coppola, Onsea, Moriarty, Nehrbass, Constant, Zeiter, Aktan, Braem, Van der Eycken, Steenackers and Metsemakers.)