Editorial - Infectious-disease research during a pandemic: the importance of global unity.

The orthopaedic and trauma community have faced the threat of infection since the introduction of operative fracture fixation many decades ago. The parallel emergence and spread of antimicrobial resistance in clinically relevant pathogens has the potential to significantly complicate patient care. This editorial serves to provide a global context to the issue of antimicrobial resistance and how infectious disease research in general plays a crucial role both on a global scale as evidenced by the current pandemic, but also on a more personal scale for the daily management of orthopaedic trauma patients. The special issue on Orthopaedic Infection in the eCM journal provides a snapshot of the clinically relevant basic research that is being performed in this field.

Gut microbial-derived short-chain fatty acids and bone: a potential role in fracture healing.

Bone healing complications such as delayed healing or non-union affect 5-10 % of patients with a long-bone fracture and lead to reduced quality of life and increased health-care costs. The gut microbiota and the metabolites they produce, mainly short-chain fatty acids (SCFAs), have been shown to impact nearly all organs of the human body including bone. SCFAs show broad activity in positively influencing bone healing outcomes either by acting directly on cell types involved in fracture healing, such as osteoblasts, osteoclasts, chondrocytes and fibroblasts, or indirectly, by shaping an appropriate anti-inflammatory and immune regulatory response. Due to the ability of SCFAs to influence osteoblast and osteoclast differentiation, SCFAs may also affect the integration of orthopaedic implants in bone. In addition, SCFA-derivatives have already been used in a variety of tissue engineering constructs to reduce inflammation and induce bone tissue production. The present review summarises the current knowledge on the role of the gut microbiota, in particular through the action of SCFAs, in the individual stages of bone healing and provides insights into how SCFAs may be utilised in a manner beneficial for fracture healing and surgical reconstruction.

Helical plating - a novel technique to increase stiffness in defect fractures.

Single-plate fixation bridging bone defects provokes nonunion and risks plate-fatigue failure due to under- dimensioned implants. Adding a helical plate to bridge the fracture increases stiffness and balances load sharing. This study compares the stiffness and plate surface strain of different constructs in a transverse contact and gap femoral shaft fracture model. Eight groups of six synthetic femora each were formed: intact femora; intact femora with lateral locking plate; contact and gap transverse shaft osteotomies each with lateral locking plate, lateral locking plate and helical locking plate, and long proximal femoral nail. Constructs underwent non-destructive quasi-static axial and torsional loading. Plate surface strain evaluation was performed under 200 N axial loading. Constructs with both lateral and helical plates demonstrated similar axial and torsional stiffness- independent of the contact or gap situations - being significantly higher compared to lateral plating (p < 0.01). Torsional stiffness of the constructs, with both lateral and helical plates in the gap situation, was significantly higher compared to this situation stabilised by a nail (p < 0.01). Plate surface strain dropped from 0.3 % in the gap situation with a lateral plate to < 0.1 % in this situation with both a lateral and a helical plate. Additional helical plating increases axial and torsional construct stiffness in synthetic bone and seems to provide well-balanced load sharing. Its use should be considered in very demanding situations for gap or defect fractures, where single-plate osteosynthesis provides inadequate stiffness for fracture healing and induces nonunion.

A murine Staphylococcus aureus fracture-related infection model characterised by fracture non-union, staphylococcal abscess communities and myeloid-derived suppressor cells.

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 non-steroidal anti-inflammatory drug carprofen negatively impacts new bone formation and antibiotic efficacy in a rat model of orthopaedic-device-related infection.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for pain management during recovery from orthopaedic surgery. NSAID use is associated with increased risk of bone healing complications but it is currently unknown whether NSAIDs increase the risk of developing an orthopaedic-device-related infection (ODRI) and/or affects its response to antibiotic therapy. The present study aimed to determine if administration of the NSAID carprofen [a preferential cyclooxygenase-2 (COX-2) inhibitor] negatively affected Staphylococcus epidermidis (S. epidermidis) bone infection, or its subsequent treatment with antibiotics, in a rodent ODRI model. Sterile or S. epidermidis-contaminated screws (~ 1.5 x 106 CFU) were implanted into the proximal tibia of skeletally mature female Wistar rats, in the absence or presence of daily carprofen administration. A subset of infected animals received antibiotics (rifampicin plus cefazolin) from day 7 to 21, to determine if carprofen affected antibiotic efficacy. Bone changes were monitored using in vivo µCT scanning and histological analysis. The risk of developing an infection with carprofen administration was assessed in separate animals at day 9 using a screw contaminated with 10² CFU S. epidermidis. Quantitative bacteriological analysis assessed bacterial load at euthanasia. In the 28-day antibiotic treatment study, carprofen reduced osteolysis but markedly diminished reparative bone formation, although total bacterial load was not affected at euthanasia. Antibiotic efficacy was negatively affected by carprofen (carprofen: 8/8 infected; control: 2/9 infected). Finally, carprofen increased bacterial load and diminished bone formation following reduced S. epidermidis inoculum (10² CFU) at day 9. This study suggests that NSAIDs with COX-2 selectivity reduce antibiotic efficacy and diminish reparative responses to S. epidermidis ODRI.

Bone infection: a clinical priority for clinicians, scientists and educators.

Bone infection has received increasing attention in recent years as one of the main outstanding clinical problems in orthopaedic-trauma surgery that has not been successfully addressed. In fact, infection may develop across a spectrum of patient types regardless of the level of perioperative management, including antibiotic prophylaxis. Some of the main unknown factors that may be involved, and the main targets for future intervention, include more accurate and less invasive diagnostic options, more thorough and accurate debridement protocols, and more potent and targeted antimicrobials. The underlying biology dominates the clinical management of bone infections, with features such as biofilm formation, osteolysis and vascularisation being particularly influential. Based on the persistence of this problem, an improved understanding of the basic biology is deemed necessary to enable innovation in the field. Furthermore, from the clinical side, better evidence, documentation and outreach will be required to translate these innovations to the patient. This review presents the findings and progress of the AO Trauma Clinical Priority Program on the topic of bone infection.

The tissue-renin-angiotensin-system of the human intervertebral disc.

Symptomatic intervertebral disc (IVD) degeneration accounts for significant socioeconomic burden. Recently, the expression of the tissue renin-angiotensin system (tRAS) in rat and bovine IVD was demonstrated. The major effector of tRAS is angiotensin II (AngII), which participates in proinflammatory pathways. The present study investigated the expression of tRAS in human IVDs, and the correlation between tRAS, inflammation and IVD degeneration. Human IVD tissue was collected during spine surgery and distributed according to principal diagnosis. Gene expression of tRAS components, proinflammatory and catabolic markers in the IVD tissue was assessed. Hydroxyproline (OHP) and glycosaminoglycan (GAG) content in the IVD tissue were determined. Tissue distribution of tRAS components was investigated by immunohistochemistry. Gene expression of tRAS components such as angiotensin-converting enzyme (ACE), Ang II receptor type 2 (AGTR2), angiotensinogen (AGT) and cathepsin D (CTSD) was confirmed in human IVDs. IVD samples that expressed tRAS components (n = 21) revealed significantly higher expression levels of interleukin 6 (IL-6), tumour necrosis factor α (TNF-α), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and 5 compared to tRAS-negative samples (n = 37). Within tRAS-positive samples, AGT, matrix-metalloproteinases 13 and 3, IL-1, IL-6 and IL-8 were more highly expressed in traumatic compared to degenerated IVDs. Total GAG/DNA content of non-tRAS expressing IVD tissue was significantly higher compared to tRAS positive tissue. Immunohistochemistry confirmed the presence of AngII in the human IVD. The present study identified the existence of tRAS in the human IVD and suggested a correlation between tRAS expression, inflammation and ultimately IVD degeneration.

Preclinical in vivo models of fracture-related infection: a systematic review and critical appraisal.

A fracture-related infection (FRI) is an important complication that can lead to an increase in morbidity, mortality and economic costs. Preclinical in vivo models are critical in the evaluation of novel prevention and treatment strategies, yet it is important that these studies recapitulate the features of an FRI that make it such a clinical challenge. The aim of this systematic review was to survey the available preclinical models of FRIs and assess which of the key FRI-specific parameters are incorporated in these models. A comprehensive search was performed on July 1st 2017 in PubMed, Embase and Web of Science. Overall, 75 preclinical studies were identified, 97.3 % (n = 73) of which use Staphylococcus aureus as the causative microorganism. The most common mode for creation of bone instability is an osteotomy (n = 30; 40 %), followed by the creation of a defect (n = 26; 34.7 %). An actual fracture is created in only 19 studies (25.3 %). 12 (16 %) of the models include a time gap between bacterial inoculation and fixation to mimic the time-to-treatment in clinical open fracture scenarios. This systematic review reveals that animal models used in translational research on prevention and treatment of FRIs rarely incorporate all key clinical features in one model and that there is an over-representation of S. aureus in comparison to actual clinical epidemiology. To improve the relevance of these studies, existing preclinical models should be adapted or new models developed that better recapitulate the clinical condition of FRI.

Local application of a gentamicin-loaded thermo-responsive hydrogel allows for fracture healing upon clearance of a high Staphylococcus aureus load in a rabbit model.

Antibiotic-loaded biomaterials (ALBs) have emerged as a potential useful adjunctive antimicrobial measure for the prevention of infection in open fracture care. A biodegradable thermo-responsive poly(N-isopropylacrylamide) grafted hyaluronic acid (HApN) hydrogel loaded with gentamicin has recently been shown to prevent implant-related infection in a rabbit osteosynthesis model. The primary aim of this study was to determine the influence of this HApN hydrogel on bone healing at an early stage (4 weeks). A rabbit humeral osteotomy model with plating osteosynthesis was used to compare fracture healing in rabbits receiving the hydrogel as compared with control animals. The secondary aim was to observe fracture healing in groups treated with and without antibiotic-loaded hydrogel in the presence of bacterial contamination. In all groups, outcome measures were mechanical stability and histological score, with additional quantitative bacteriology in the inoculated groups. Application of the HApN hydrogel in non-inoculated rabbits did not significantly influence humeral stiffness or histological scores for fracture healing in comparison to controls. In the inoculated groups, animals receiving the bacterial inoculum without hydrogel were culture-positive at euthanasia and found to display lower humeral stiffness values and higher histopathological scores for bacterial presence in comparison with equivalents receiving the gentamicin-loaded HApN hydrogel, which were also infection-free. In summary, our data showed that HApN was an effective antibiotic carrier that did not affect fracture healing. This data supported its suitability for application in fracture care. Addition of osteopromotive compounds could provide further support for accelerating fracture healing in addition to successful infection prophylaxis.

Transcriptional activation of ENPP1 by osterix in osteoblasts and osteocytes.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the main source of extracellular pyrophosphate. Along with tissue-nonspecific alkaline phosphatase (TNAP), ENPP1 plays an important role in balancing bone mineralisation. Although well established in pre-osteoblasts, the regulating mechanisms of ENPP1 in osteoblasts and osteocytes remain largely unknown. Using bioinformatic methods, osterix (Osx), an essential transcription factor in osteoblast differentiation and osteocyte function, was found to have five predicted binding sites on the ENPP1 promoter. ENPP1 and Osx showed a similar expression profile both in vitro and in vivo. Over-expression of Osx in MC3T3-E1 and MLO-Y4 cells significantly up-regulated the expression of ENPP1 (p < 0.05). The consensus Sp1 sequences, located in the proximal ENPP1 promoter, were identified as Osx-regulating sites using promoter truncation experiments and chromatin immunoprecipitation (ChIP) assays. The p38-mitogen-activated protein kinase (MAPK) signalling pathway was demonstrated to be responsible for ENPP1 promoter activation by Osx. Runt-related transcription factor 2 (Runx2) was confirmed to have synergistic effects with Osx in activating ENPP1 promoter. Taken together, these results provided evidence of the regulating mechanisms of ENPP1 transcription in osteoblasts and osteocytes.

A large animal model for a failed two-stage revision of intramedullary nail-related infection by methicillin-resistant Staphylococcus aureus.

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.

Influence of fracture stability on Staphylococcus epidermidis and Staphylococcus aureus infection in a murine femoral fracture model.

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.

Reamed locked intramedullary nailing for studying femur fracture and its complications.

Morbidity associated with femur fractures in polytrauma patients is known to be high. The many unsolved clinical questions include the immunological effect of the fracture and its fixation, timing of fracture fixation, management of fracture non-union, effect of infection and critical size of bone defects. The aim of this study was to establish a clinically-relevant and reproducible animal model with regards to histological, biomechanical and radiological changes during bone healing. A custom-designed intramedullary nail with interlocking system (RabbitNail, RISystem AG, Davos Platz, Switzerland) was used for fixation, following femur fracture. New Zealand White rabbits were assigned to two groups: 1. closed fracture model (CF; non-survival model: n = 6, survival model: n = 3) with unilateral mid-shaft femur fracture created by blunt force; 2. osteotomy model (OT; survival model: n = 14) with unilateral transverse osteotomy creating femur fracture. There were no intraoperative complications and full-weight bearing was achieved in all survival rabbits. Significant periosteal reaction and callus formation were confirmed from 2 weeks postoperatively, with a significant volume formation (739.59 ± 62.14 mm3) at 8 weeks confirmed by micro-computed tomography (µ-CT). 2 months after fixation, there was no difference between the osteotomised and contralateral control femora in respect to the maximum torque (3.47 ± 0.35 N m vs. 3.26 ± 0.37 N m) and total energy (21.11 ± 3.09 N m × degree vs. 20.89 ± 2.63 N m × degree) required to break the femur. The data confirmed that a standardised internal fixation technique with an intramedullary nail for closed fracture or osteotomy produced satisfactory bone healing. It was concluded that important clinically-relevant studies can be conducted using this rabbit model.

Innovating in the medical device industry - challenges & opportunities ESB 2015 translational research symposium.

The European Society for Biomaterials 2015 Translational Research Symposium focused on 'Innovating in the Medical Device Industry - Challenges & Opportunities' from different perspectives, i.e., from a non-profit research organisation to a syndicate of small and medium-sized companies and large companies. Lecturers from regulatory consultants, industry and research institutions described the innovation process and regulatory processes (e.g., 510K, PMA, combination product) towards market approval. The aim of the present article is to summarise and explain the main statements made during the symposium, in terms of challenges and opportunities for medical device industries, in a constantly changing customer and regulatory environment.

A rabbit humerus model of plating and nailing osteosynthesis with and without Staphylococcus aureus osteomyelitis.

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.

Progressing innovation in biomaterials. From the bench to the bed of patients.

A Translational Research Symposium was organized at the 2014 annual meeting of the European society for biomaterials. This brought together leading Tier one companies in clinical biomaterials and medical device markets, small and medium enterprises and entrepreneurial academics who shared their experiences on taking biomaterials technologies to commercial endpoints, in the clinics. The symposium focused on "Progressing Innovation in Biomaterials. From the Bench to the Bed of Patients". The aim of the present document is to illustrate the content of the symposium and to highlight the key lessons from selected lectures.

Challenges in linking preclinical anti-microbial research strategies with clinical outcomes for device-associated infections.

Infections related to implanted medical devices have become a significant health care issue in recent decades. Increasing numbers of medical devices are in use, often in an aging population, and these devices are implanted against a background of increasing antibiotic-resistant bacterial populations. Progressively more antibiotic resistant infections, requiring ever more refined treatment options, are therefore predicted to emerge with greater frequency in the coming decades. Improvements in the prevention, diagnosis and treatment of these device-associated infections will remain priority targets both for clinicians and the translational research community charged with addressing these challenges. Preclinical strategies, predictive of ultimate clinical efficacy, should serve as a control point for effective translation of new technologies to clinical applications. The development of new anti-infective medical devices requires a validated preclinical testing protocol; however, reliable validation of experimental and preclinical antimicrobial methodologies currently suffers from a variety of technical limitations. These include the lack of agreement or standardisation of experimental protocols, a general lack of correlation between in vitro and in vivo preclinical results and lack of validation between in vivo preclinical implant infection models and clinical (human) results. Device-associated infections pose additional challenges to practicing clinicians concerning diagnosis and treatment, both of which are complicated by the biofilms formed on the medical device. The critical challenges facing both preclinical research and clinical laboratories in improving both diagnosis and treatment of medical device-associated infections are the focus of this review.

Role and regulation of RUNX2 in osteogenesis.

Runt-related transcription factor 2 (RUNX2) is a transcription factor closely associated with the osteoblast phenotype. While frequently referred to, the complexity of its regulation and its interactions within the osteoblast differentiation pathway are often overlooked. This review aims to summarise the knowledge of its regulation at the transcriptional, translational and post-translational level. In addition, the regulation of RUNX2 by factors commonly used during osteogenic studies will be discussed.

Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production.

PURPOSE: Platelet-rich plasma (PRP) contains growth factors and creates a 3D structure upon clotting; PRP or platelet lysate (PL) might be considered for annulus fibrosus (AF) repair. METHODS: Bovine AF cells were cultured with 25% PRP, 50% PRP, 25% PL, 50% PL, or 10% FBS. After 2 and 4 days, DNA, glycosaminoglycan (GAG), and mRNA levels were analyzed. Histology was performed after injection of PRP into an AF defect in a whole disc ex vivo. RESULTS: By day 4, significant increases in DNA content were observed in all treatment groups. All groups also showed elevated GAG synthesis, with highest amounts at 50% PL. Collagen I and II expression was similar between groups; aggrecan, decorin, and versican expression was highest at 25% PL. Injection of PRP into the AF defect resulted in an increased matrix synthesis. CONCLUSIONS: Platelet-rich preparations increased the matrix production and cell number and may therefore be considered to promote AF repair.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days.

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.