Aim. To describe the histopathology of the first and last debrided bone tissue in chronic osteomyelitis and answer the following research question; is the last debrided bone tissue viable and without signs of inflammation?. Method. In total, 15 patients with chronic osteomyelitis were allocated to surgical treatment using a one stage protocol including extensive debridement. Suspected infected bone tissue eradicated early in the debridement procedure was collected as a clearly infected sample (S1). Likewise, the last eradicated bone tissue was collected as a suspected non-infected sample (S2), representing the status of the bone void. In all cases, the surgeon debrided the bone until visual confirmation of healthy bleeding bone. The samples were processed for histology, i.e. decalcification and paraffin embedding, followed by cutting and staining with Haematoxylin and Eosin. Immunohistochemistry with MAC-387 antibodies towards the calprotectin of neutrophil granulocytes (NGs) was also performed and used for estimation of a neutrophil granulocyte (NG) score (0, 1, 2 or 3), by the method described for fracture related infections (1). Results. For the S1 samples the median NG score was 3 which is considered confirmatory for infection. However, following debridement the median NG score was significantly (p = 0.032) reduced to 2. Often NGs were seen as single cells, but in seven S1 samples and in one S2 sample massive NG accumulations were observed. The S1 samples showed a mix of granulation tissue, fibrosis, viable bone, and bone necrosis. The S2 samples contained viable bone tissue and occasionally (10/15) small fragments of necrotic bone or bone debris were seen. Furthermore, a large number of erythrocytes were observed in most S2 samples. Conclusions. The present study shows that the inflammatory response still existents after debridement, although the response fades from the center of infection. Therefore, sampling of debrided bone tissue for histology must be performed initially during surgery, to avoid underestimation of the inflammatory response, i.e. the NG score. The last debrided bone tissue cannot by definition be considered completely viable and caution should be made to remove blood (rinse) before intraoperative evaluation of the viability of debrided cancellous bone. Remnant necrotic bone fragments or debris could represent low-vascular hiding places for leftover bacteria. Application of local antibiotics might have a central role in clearing of these small non-viable bone pieces at the bone void interface

Aim. A reason for treatment failure, in cases of periprosthetic bone infections and osteomyelitis, may be incomplete or heterogeneous tissue distribution of antimicrobials to the affected bone. Decreased bioavailability has been demonstrated in healthy bones but never in pathological bone tissue. Therefore, the aim was to obtain pharmacokinetic parameters of cefuroxime in infected bone tissue by means of microdialysis in a porcine model of implant associated osteomyelitis. Method. An implant cavity of 4 mm in diameter was drilled 25 mm into the right tibial bone of ten pigs (30 kg/BW). Subsequently, a small steel implant (K-wire 2 × 2 mm) and 10. 4. CFU of Staphylococcus aureus was inserted and injected into the implant cavity. Five days after inoculation, two additional drill holes of 2 × 25 mm were drilled into the trabecular bone tissue adjacent to the implant cavity and into the left uninfected tibia. After intravenous administration of 1500 mg of cefuroxime, the concentration was measured in plasma and in the three tibial drill holes for 8 hours. All measurements were performed with microdialysis. Post mortem, the presence of bone infection was assessed by computed tomography (CT) scans and cultures of swabs. Results. Destruction of bone tissue was seen on CT scans around all implant cavities but not in the adjacent trabecular bone tissue of the right leg or in the left leg. All swabs from the implant cavity and 8/10 swabs from the adjacent trabecular tissue were positive for S. aureus. Conversely, all swabs from the left tibia were negative. The area under the concentration-time curves differed significantly, with the lowest found in the implant cavity (P<0.001). Although not significant, cefuroxime penetration into the adjacent bone tissue was incomplete. Conclusions. This is the first study to show, by microdialysis, that the destructive bone processes associated with implant associated osteomyelitis significantly impair cefuroxime penetration. Our results support the clinical conception of fast diagnosis and initiation of antibiotic treatment if surgery is to be avoided. It is of crucial importance to know the exact level of antibiotics, which actually reaches a pathological bone focus in order to obtain more targeted and effective antibiotic treatments of bone infections

Quantitative assessment of metastatic involvement of the bony spine is important for assessing disease progression and treatment response. Quantification of metastatic involvement is challenging as tumours may appear as osteolytic (bone resorbing), osteoblastic (bone forming) or mixed. This investigation aimed to develop an automated method to accurately segment osteoblastic lesions in a animal model of metastatically involved vertebrae, imaged with micro computed tomography (μCT). Radiomics seeks to apply standardized features extracted from medical images for the purpose of decision-support as well as diagnosis and treatment planning. Here we investigate the application of radiomic-based features for the delineation of osteoblastic vertebral metastases. Osteoblastic lesions affect bone deposition and bone quality, resulting in a change in the texture of bony material physically seen through μCT imaging. We hypothesize that radiomics based features will be sensitive to changes in osteoblastic lesion bone texture and that these changes will be useful for automating segmentation. Osteoblastic metastases were generated via intracardiac injection of human ZR-75-1 breast cancer cells into a preclinical athymic rat model (n=3). Four months post inoculation, ex-vivo μCT images (µCT100, Scanco) were acquired of each rodent spine focused on the metastatically involved third lumbar vertebra (L3) at 7µm/voxel and resampled to 34µm/voxel. The trabecular bone within each vertebra was isolated using an atlas and level-set based segmentation approach previously developed by our group. Pyradiomics, an open source Radiomics library written in python, was used to calculate 3D image features at each voxel location within the vertebral bone. Thresholding of each radiomic feature map was used to isolate the osteoblastic lesions. The utility of radiomic feature-based segmentation of osteoblastic bone tissue was evaluated on randomly selected 2D sagittal and axial slices of the μCT volume. Feature segmentations were compared to ground truth osteoblastic lesion segmentations by calculating the Dice Similarity Coefficient (DSC). Manually defined ground truth osteoblastic tumor segmentations on the μCT slices were informed by histological confirmation of the lesions. The radiomic based features that best segmented osteoblastic tissue while optimizing computational time were derived from the Neighbouring Gray Tone Difference Matrix (NGTDM). Measures of coarseness yielded the best agreement with the manual segmentations (DSC=707%) followed by contrast, strength and complexity (DSC=6513%, 5428%, and 4826%, respectively). This pilot study using a radiomic based approach demonstrates the utility of the NGTDM features for segmentation of vertebral osteoblastic lesions. This investigation looked at the utility of isolated features to segment osteoblastic lesions and found modest performance in isolation. In future work we will explore combining these features using machine learning based classifiers (i.e. decision forests, support vector machines, etc.) to improve segmentation performance

Aim. The reconstruction of bone critical size defects of the tibia is one of the most complex therapeutic challenges in the orthopedic field. This study aims to describe and evaluate our three-staged surgical protocol of reconstruction of infected defects of the tibia emphasizing in limb salvage rate, resolution of infection, functional outcome and patient satisfaction. Method. A retrospective review was performed in all cases of complex infected tibia fracture with combined soft and bone tissue loss treated in a specialized limb reconstruction center between 2010 and 2018. In all cases, a three-stage protocol was performed: 1) Infected-limb damage control with radical debridement, 2) Soft tissue coverage with vascularized or local flap 3) Bone reconstruction procedure. The minimum follow-up required was 12 months after external fixator removal. Results. Twenty-eight patients with a mean age of 42 years were included. The mean soft tissue defect was 91.7 cm2 and the mean bone defect was 5.8 cm. 67.85% of the cases were classified as a type IV B-local osteomyelitis. The median global treatment time was 456 days. The External Fixator Time (EFT) was 419, 284, 235 for bone transport, shortening-lengthening and acute shortening groups respectively. The median Bone Healing Index (BHI) was 1.82 months/cm in bone transport group and 2.15 months/cm in shortening-lengthening group. The limb salvage rate was 92.85%. Infection resolution rate was 96,42%. We achieved bone union in 92,85% of the cases. Regarding ASAMI bone score, 92.8% of the cases were “good-or-excellent”. Two patients underwent a delayed amputation. Eight cases of non-progressive Docking Site (DS) healing were observed. Nineteen non-expected reinterventions were performed. Functional data: the mean VAS score was 1.0. The mean LEFS score was 55.88 (55.88/80). Regarding ASAMI functional score, 78,6% of the cases were “good-or-excellent”. More than 80% of the patients could return to work. 100% of the patients were “very satisfied” or “moderately satisfied” (75% and 25% respectively). Conclusions. Our results demonstrate that our three-stage surgical approach in infected tibial bone defects with soft tissue damage can result in high infection resolution, good functional outcome, good patient satisfaction and an acceptable limb salvage rate despite the large time of treatment and unexpected reinterventions

Biomaterials used in regenerative medicine should be able to support and promote the growth and repair of natural tissues. Bioactive glasses (BGs) have a great potential for applications in bone tissue engineering [1, 2]. As it is well known BGs can bond to host bone and stimulate bone cells toward osteogenesis. Silicate BGs, e.g. 45S5 Bioglass® (composition in wt.%: 45 SiO. 2. , 6 P. 2. O. 5. , 24, 5 Na. 2. O and 24.5 CaO), exhibit positive characteristics for bone engineering applications considering that reactions on the material surface induce the release of critical concentrations of soluble Si, Ca, P and Na ions, which can lead to the up regulation of different genes in osteoblastic cells, which in turn promote rapid bone formation. BGs are also increasingly investigated for their angiogenic properties. This presentation is focused on cell behavior of osteoblast-like cells and osteoclast-like cells on BGs with varying sample geometry (including dense discs for material evaluation and coatings of highly porous Al. 2. O. 3. -scaffolds as an example of load-bearing implants). To obtain mechanically competent porous samples with trabecular architecture analogous to those of cancellous bone, in this study Al. 2. O. 3. scaffolds were fabricated by the well-known foam replication method and coated with Bioglass® by dip coating. The resulted geometry and porosity were proven by SEM and μCT. Originating from peripheral blood mononuclear cells formed multinucleated giant cells, i.e. osteoclast-like cells, after 3 weeks of stimulation with RANKL and M-CSF. Thus, the bioactive glass surface can be considered a promising material for bone healing, providing a surface for bone remodeling. Osteoblast-like cells and bone marrow stromal cells were seeded on dense bioactive glass substrates and coatings showing an initial inhibited cell attachment but later a strong osteogenic differentiation. Additionally, cell attachment and differentiation studies were carried out by staining cytoskeleton and measuring specific alkaline phosphatase activity. In this context, 45S5 bioactive glass surfaces can be considered a highly promising material for bone tissue regeneration, providing very fast kinetics for bone-like hydroxyapatite formation (mineralization). Our examinations revealed good results in vitro for cell seeding efficacy, cell attachment, viability, proliferation and cell penetration onto dense and porous Bioglass®-coated scaffolds. Recent in vivo investigations [3] have revealed also the angiogenic potential of bioactive glass both in particulate form and as 3D scaffolds confirming the high potential of BGs for bone regeneration strategies at different scales. Implant surfaces based on bioactive glasses offer new opportunities to develop these advanced biomaterials for the next generation of implantable devices and tissue scaffolds with desired tissue-implant interaction

Introduction. Glenoid loosening, still a main complication in shoulder arthroplasty, could be related to glenohumeral orientation and conformity, cementing techniques, fixation design and periprosthetic bone quality [1,2]. While past numerical analyses were conducted to understand the relative role of these factors, so far none used realistic representations of bone microstructure, which has an impact on structural bone properties [3]. This study aims at using refined microFE models including accurate cortical bone geometry and internal porosity, to evaluate the effects of fixation design, glenohumeral conformity, and bone quality on internal bone tissue and cement stresses under physiological and pathological loads. Methods. Four cadaveric scapulae were scanned at 82µm resolution with a high resolution peripheral quantitative computer tomography (XtremeCT Scanco). Images were processed and virtually implantated with two anatomical glenoid replacements (UHMWPE Keeled and Pegged designs, Exactech). These images were converted to microFE models consisting of nearly 43 million elements, with detailed geometries of compact and trabecular bone, implant, and a thin layer of penetrating cement through the porous bone. Bone tissue, implant and cement layer were assigned material properties based on literature. These models were loaded with a central load at the glenohumeral surface, with the opposite bone surface fully constrained. Effects of glenohumeral conformity were simulated with increases of the applied load area from 5mm-radius to a fully conformed case with the entire glenoid surface loaded. The models were additionally subjected to a superiorly shifted load mimicking torn rotator cuff conditions. These models were solved and compared for internal stresses within the structures (Figure 1) with a parallel solver (parFE, ETH Zurich) on a computation cluster, and peak stresses in each region compared by design and related to apparent bone density. Results. Peak cement stresses were generally located at the interface with bone rather than implant (p<0.05), and peak bone stresses occurred around the cemented region. A larger trabecular load share was predicted with the Pegged compared to the Keeled design (Figure 2a). Superior load shift reduced this ratio but resulted in slight stress increase in the cement and implant, with the Keeled design less sensitive to this shift (Figure 2b). These effects were more pronounced with decreased overall bone density (Figure 2c). Increasing conformity significantly affected peak stresses in the cement and implant for both Keeled and Pegged designs (Figure 3) (p<0.041), but only significantly changed bone stresses for the Keeled design (p<0.047). Generally higher peak cement and trabecular bone stresses were predicted for the Pegged design. Discussion. Our detailed microFE analyses suggest that implant fixation design affects the sensitivity of internal stresses to glenohumeral load shifts, in particular within the cement region and through alterations in load sharing in the periprosthetic bone. Future steps including reverse replacements and more physiological loading conditions, combined with experimental validation tests in dynamic loading, will provide improved insights into the clinical incidences of glenoid loosening

A Ruys, School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney, Sydney. The effects of bone anabolics can be maximised by systemic co-treatment with an anti-catabolic. Local treatment may reduce the total drug required and produce superior outcomes, although high dose local bisphosphonate has been reported to impair bone formation. We have explored local co-delivery of anabolic/anti- catabolic bone drugs at different doses. We manufactured biodegradable poly-D,L-lactic acid (PDLLA) polymer pellets containing 25g BMP-7 as an anabolic with or without 0.002mg-2mg Pamidronate (PAM) as an anti-catabolic. Polymer pellets were surgically implanted into the hind limb muscle of female C57BL6 mice. Animals were sacrificed at three weeks post- implantation and bone formation was assessed by radiography, microcomputed tomography (microCT) and histology. Histological staining on five Âm paraffin sections included haematoxylin/eosin, alcian blue/picrosirius red, and tartrate- resistant acid phosphatase (TRAP). Radiographic and microCT data confirmed that 0.02mg and 0.2mg local PAM doses significantly augmented BMP-7 induced bone formation. In contrast, 2mg local PAM dramatically reduced the amount of bone present. This dose was comparable to that used by Choi et al who also reported impaired bone formation in a skull defect model.2 three-dimensional microCT and histological analyses of the ectopic bone and surrounding muscle showed a cortical shell covering the polymer pellet, which had not completely resorbed. Histological analysis at the pellet/bone interface showed tissue granulation and no inflammation, suggesting a high biocompatibility of the PDLLA polymer. The presence of bisphosphonate also decreased the amount of fatty marrow tissue seen within between the cortical shell and the unresorbed polymer. For the first time we can demonstrate synergy with local BMP/bisphosphonate. This study confirms that high local PAM doses can have negative effects, indicating a need to avoid overdosing. The lack of implant degradation suggests a need to optimise polymer degradation for bone tissue engineering application

Gene-activated scaffolds have shown potential in localised gene delivery resulting in bone tissue regeneration. In this study, the ability of two gene delivery vectors, polyethyleneimine (PEI) and nano-hydroxyapatite (nHA), to act as carriers for the delivery of therapeutic genes when combined with our collagen-nHA (coll-nHA) scaffolds to produce gene-activated scaffolds [1, 2], was determined. In addition, coll-nHA-dual gene scaffolds containing both an angiogenic gene and an osteogenic gene were assessed for bone healing in an in vivo Wistar rat calvarial defect model. When cells were applied to the coll-nHA scaffolds under osteogenic conditions in vitro, the dual scaffolds exhibited significantly superior osteogenic potential when analysed using microCT, calcium quantification and histology compared to single-gene scaffolds and gene-free controls. When the dual scaffolds were assessed in vivo, the nHA dual scaffold outperformed all other groups as early as 4 weeks post-implantation as determined using X-ray, microCT, quantification of new bone volume, histology and vessel formation. This research has demonstrated the potential of using novel coll-nHA scaffolds for therapeutic gene therapy while also being capable of simultaneously delivering numerous genes. This study underlines the effect of specifically tailoring gene-activated scaffolds for bone regeneration applications

Aim. The current antibiotic treatment of periprosthetic joint infection (PJI) is optimized by measuring concentrations in plasma. However, it remains unclear whether effective concentrations of the antibiotics are reached at the site of PJI. Nonetheless, adequate target site concentrations are important to achieve effective eradication of the micro-organism. In order to determine the efficacy of cefuroxime and flucloxacillin in synovial fluid, synovial tissue and bone tissue in relation to the minimal inhibitory concentration (MIC) of the pathogen causing the PJI, we perform a pharmacokinetic/pharmacodynamic (PK/PD) study. Therefore, we aimed to develop validated analytical methods for analysis of cefuroxime and flucloxacillin in synovial fluid, synovial tissue and bone tissue. Method. Blank samples of synovial fluid, synovial tissue and bone tissue were obtained by orthopedic surgeons during surgery. For validation the samples of each matrix were spiked with both cefuroxime and flucloxacillin. Synovial tissue and bone tissue was pulverized with a mikro-dismembrator. Samples were kept frozen at −20°C until analysis. After a sample preparation quantification of cefuroxime and flucloxacillin in each matrix was performed on the ultra-performance convergence chromatography-tandem mass spectrometry (UPC2-MS/MS). Stable-isotope-labeled meropenem-d6 served as internal standard. The linearity, limits of quantification, accuracy and precision and carry-over were determined for all methods separately. The methods were validated according to the European Medicine Agency (EMA) and Food and Drug Administration (FDA) guidelines on bioanalytical method validation. Results. These methods were successfully validated for cefuroxime and flucloxacillin quantification in all matrices according to the EMA and FDA guidelines. The limits of quantification were adequate to cover potential cefuroxime and flucloxacillin concentration in synovial fluid, synovial tissue and bone tissue as described in literature, with a range of 1–100mg/L for synovial fluid and 1–20 µg/g for synovial tissue and bone tissue (r >0.995). Accuracy and within-run precision were validated according to acceptance values (RSD <15%). Carry over was less than 20%. Matrix effects and recovery were investigated for synovial fluid. The results were within the range of 80–120%. Conclusions. The results of the validation fall within the limits of quantification according to the EMA and FDA guidelines. Therefore, these methods can be applied during a PK/PD study to discover the exposure of antibiotics in synovial fluid, synovial tissue and bone tissue at the site of infection in patients with a PJI

Hip and knee arthroplasty (HKA) are two of the most successful orthopaedic procedures. However, one major complication necessitating revision surgery is osteolysis causing aseptic loosening of the prosthesis. JAK-STAT has been demonstrated to influence bone metabolism and can be regulated by microRNA (miRNA). Adult patients with osteolysis or aseptic loosening undergoing revision HKA were recruited. Age and gender matched patients undergoing primary hip or knee arthroplasty were our controls. Samples of bone, tissue and blood were collected and RNA isolation was performed. The best quality samples were used for RNA-sequencing. Data analysis was performed using RStudio and Galaxy to identify differentially expressed genes. Western blotting of IL6 was used to confirm protein expression. Five circulating miRNA were identified which had 10 differentially expressed genes in bone and 11 differentially expressed genes in tissue related to the JAK-STAT pathway. IL6 in bone and EpoR in bone were highly significant and IL6 in tissue, MPL in bone, SOCS3 in tissue, JAK3 in bone and SPRED1 in bone were borderline significant. Western blot results demonstrated up-expression of IL6 in bone tissue of revision patients. Periprosthetic osteolysis and aseptic loosening can be attributed to miRNA regulation of the JAK-STAT pathway in osteoblasts and osteoclasts, leading to increased bone resorption. These findings can be used for further experiments to determine utility in the clinical setting for identifying diagnostic markers or therapeutic targets

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

Aim. Osteomyelitis (OM) is a debilitating infection of the bone that originates from hematogenous spreading of microbes or contamination after surgery/fracture. OM is mainly caused by the opportunistic bacterium Staphylococcus aureus (SA), which can evade the host immune response, acquire antibiotic resistance and chronically colonize the musculoskeletal tissue . 1,2. , yet the underlying molecular and cellular processes are largely unclear. This study aimed to characterize the pathogenetic mechanisms of SA-OM with a focus on the long pentraxin 3 (PTX3), a soluble pattern recognition molecule and bone tissue component that is emerging as a new player in osteoimmunology . 3. and a diagnostic marker of periprosthetic joint infections, a common form of OM. 4. . Method. A murine model of OM based on intra-bone injection of SA was developed that closely mimicked surgery/trauma-related OM in humans and allowed addressing the role of PTX3 in gene-modified (Ptx3-/-) animals. Local and systemic infection and inflammation were assessed via microbiology, flow cytometry, histochemistry and microCT techniques. Results. SA-injected mice developed chronic infection with measurable levels of viable bone-resident bacteria up until 30 days from microbial challenge. The infection was confined to the treated limbs only and accompanied by extensive tissue remodelling. The bacterial load was higher in WT than Ptx3. -/-. animals at 6 and 14 days from SA injection. Accordingly, WT mice had enhanced systemic inflammation with expanded innate immune compartment in the spleen and increased serum levels of inflammatory cytokines and chemokines. PTX3 levels were higher in SA- than vehicle (PBS)-injected WT animals both in the serum and bone tissue. Furthermore, administration of a PTX3-targeting antibody reduced the bacterial burden in the bones of SA-injected WT mice. Conclusions. In a mouse model of SA-OM, genetic deficiency of PTX3 protected from infection and inflammation, pointing to this pentraxin as a crucial player in OM pathogenesis and a novel therapeutic target in bone infections. The study was approved by the Italian Ministry of Health (approval n. 520/2019-PR issued on 19/07/2019) and supported by Fondazione Beppe and Nuccy Angiolini

Aim. Adequate debridement of necrotic bone is of paramount importance for eradication of infection in chronic osteomyelitis. Currently, no tools are available to detect the exact amount of necrotic bone in order to optimize surgical resection. The aim of the present study was to evaluate the feasibility of an intraoperative illumination method (VELscope. ®. ) and the correlation between intraoperative and pathohistological findings in surgically treated chronic fracture related infection patients. Method. Ten consecutive patients with chronic fracture related infections of the lower extremity were included into this prospectively performed case series. All patients had to be treated surgically for fracture related infections requiring bony debridement. An intraoperative illumination method (VELscope®) was used to intraoperatively differentiate between viable and necrotic bone. Tissue samples from the identified viable and necrotic bone areas were histopathologically examined and compared to intraoperative findings. Results. In all included patients, the intraoperative illumination was deemed helpful to differentiate between necrotic and viable bone tissues during bony debridement. The histopathological examination of the samples showed good correlation of the intraoperative illumination findings with histopathological signs of necrosis for areas deemed dead and histopathological signs of intact bone for areas deemed vital during illumination. Conclusions. The fluorescence-assisted, intraoperative detection of necrotic and viable bone using the VELscope. ®. is an easy-to-use procedure that can help surgeons to optimize intraoperative bone resection in chronic fracture related infections by unmasking viable from necrotic bone tissue. This may help to improve resection techniques and eventually treatment outcome in patients in the future

Aim. Flucloxacillin is conventionally administered intravenously for perioperative prophylaxis, while oral administration is typical for bacterial inoculation prophylaxis following smaller traumatic wounds. We aimed to assess the time, for which the free flucloxacillin concentration was maintained above the minimum inhibitory concentration (fT>MIC) for meticillin-susceptible Staphylococcus aureus in soft and bone tissue, after intravenous and oral administration, using microdialysis in a porcine model. Method. 16 pigs were randomly allocated to either intravenous (Group IV) or oral (Group PO) flucloxacillin 1 g every 6 h during 24 h. Microdialysis was used for sampling in cancellous and cortical bone, subcutaneous tissue, and the knee joint. In addition, plasma was sampled. The flucloxacillin fT>MIC was evaluated using a low MIC target (0.5 μg/mL) and a high MIC target (2.0 μg/mL). Results. Intravenous administration resulted in longer fT>MIC (0.5 μg/mL) compared to oral administration, except for cortical bone. In Group IV all pigs reached a concentration of 0.5 μg/mL in all compartments. The mean fT>MIC (0.5 μg/mL) was 149 min in subcutaneous tissue and 61–106 min in bone tissue. In Group PO 0/8 pigs reached a concentration of 0.5 μg/mL in all compartments. For the high MIC target (2.0 μg/mL), fT>MIC was close to 0 min in both groups across compartments. Conclusions. Although intravenous administration of flucloxacillin 1g provided higher fT>MIC for the low MIC target compared to oral administration, concentrations were surprisingly low, particularly for bone tissue. Achievement of sufficient bone and soft tissue flucloxacillin concentrations may require a dose increase or continuous administration. Acknowledgement. The study was supported by the following grants: Sofus Carl Emil Friis Foundation, Aase & Ejnar Danielsens Foundation, the Augustinus Foundation, Direkt⊘r Emil Hertz og hustru Inger Hertz Foundation, and the Novo Nordisk Foundation

Aim. To study the antimicrobial effect of a gentamicin loaded bio-composite bone void filler in relation to a limited or extensive debridement of osteomyelitis lesions, respectively. Methods. Nine pigs were inoculated into the right proximal tibial bone with a high virulent gentamicin sensitive strain of Staphylococcus aureus (10. 4. CFU). Seven days after inoculation, Group A pigs (n=3) were exposed to a limited debridement of the bone lesion, whereas Group B pigs (n=3) were exposed to an extensive debridement. The bone defects of Groups A and B were filled with (2–5 ml) of an absorbable gentamicin (175 mg/10 mL) loaded bio-composite. The animals of Group A and B were euthanized 12 days after revision surgery. Group C animals did not undergo revision surgery and were euthanized seven (n=1) or nineteen (n=2) days post inoculation in order to follow the development of the untreated infection. None of the animals were treated with systemic antimicrobials. All bones were exposed to a post mortem CT scan and rigours pathological examinations. The surrounding bone tissue and the bio-composite were sampled for microbiology. Results. All animals developed a substantial purulent bone infection in the inoculated leg prior to revision surgery. In the cases of limited debridement, the bone lesions surrounding the bio-composite bone void filler had clearly expanded since revision surgery, and contained extensive amounts of pus, necrotic bone tissue and oedematous fibrotic tissue. In the cases of extensive debridement, the bio-composite bone void filler was surrounded by only a few mm of fibrosis and sclerotic bone tissue i.e. the bone lesions were not expanding. However, in one pig the bio-composite bone void filler was communicating with a small purulent osteolytic lesion without a sclerotic border indicating appearance after revision surgery. In all pigs, S. aureus bacteria were post mortem cultured from the adjacent bone tissue and the bio-composite surface. Conclusions. The gentamicin concentrations within the bio-composite could not eradicate the residual infection after debridement. However, extensive debridement and filling of the bone void with gentamicin loaded bio-composite contained the lesion formed by revision surgery, which are important complementing roles as adjuvant to systemic antimicrobial therapy and the immune system in eradication of the infection. The present study emphasizes that extensive debridement is fundamental for successful treatment of bone infections and that antimicrobial loaded bone void fillers or bone substitutes should not be used as an alternative to extensive debridement

Aim. To develop a new system for antibacterial coating of joint prosthesis and osteosynthesis material. The new coating system was designed to release gentamicin immediately after insertion to eradicate surgical contamination. Method. Steel implants (2×15mm) were coated with a solid nanocomposite xerogel made from silica and the dendritic polymer, hyperbranched polyethyleneimine. The xerogel was anchored inside a porous surface made by pre-coating with titanium microspheres. Finally, gentamicin was encapsulated in the xerogel, i.e. no chemical binding. A total of 50 µg gentamicin was captured into each implant. The efficacy of the new coating was evaluated in a porcine model of implant associated osteomyelitis. In total, 30 female pigs were randomized into 3 study groups (n=10). Group A; plain implants + saline, Group B; plain implants + 10. 4. CFU of Staphylococcus aureus, and Group C; coated implants + 10. 4. CFU of S. aureus. Implant + inoculum was placed into a pre-drilled implant cavity of the right tibia and the pig was euthanized 5 days afterwards. Postmortem microbiology and pathology were performed. Two additional pigs were used in a pharmacokinetic study where microdialysis (MD) catheters were placed alongside coated implants. Extracellular fluid was sampled regularly for 24 hours from the MD catheters and analyzed for gentamicin content. Results. Within Groups A and C, all implants were found sterile by sonication and bacteria could not be identified within the surrounding bone tissue. In contrast, all Group B animals had S. aureus positive implant and tissue microbiology. Macroscopic and microscopic pathological examinations confirmed that Group A and C animals were complete identic, i.e. no pus around implants and only minor peri-implant inflammation related to insertion of implants per se. All Group B animals had pus around their implants and a massive peri-implant inflammatory response dominated by neutrophil granulocytes. Maximum gentamicin release (35 µg /mL) was measured in the first obtained MD sample, i.e. after 30 min, and the concentration stayed above the MIC level for the used S. aureus strain for 8 hours. Conclusions. The new xerogel coating prevented development of osteomyelitis. Prevention was due to a fast gentamicin release immediately following insertion and antimicrobial active concentrations were detectable several hours after implantation. This means that the critical time point of most relevant surgical procedures potentially could be protected by the novel coating. The new coating will be investigated on larger scale implants and full-size prosthesis in the future

Aim. Staphylococcus aureus (SA) chronic bone and joint infections (BJI) are characterized by a progressive destruction of bone tissue associated to SA persistence which results in a large number of relapses (10–20%). The main factors proposed for these failures are: i) a weak diffusion of antibiotics in bone tissue, ii) formation of biofilm, iii) the bacterial internalization by the cells responsible for bone mineralization, namely the osteoblasts (OB). Our in vitro and in vivo work aimed at providing new information on the impact of SA, more specifically of internalized SA, on bone homeostasis. Method. Effect of SA infection (8325–4/FnBP+; DU5883/FnBP-) on the viability, differentiation and mineralization of an OB cell line was measured in vitro by MTT and Phosphatase Alcaline (PAL) activity assays and quantification of calcium deposits using Alizarin red, respectively. A gentamicin protection assay (GPA) confirmed that the effects observed are due solely to the internalized SA. In vivo, X-ray microtomography (μCT) and 3D reconstruction was used to evaluate the impact of SA infection on bone formation and bone resorption in a mouse model of femur infection. Results. In vitro, the infection of pre-OB decreases their capacity of differentiation into mature OB displaying a PAL activity. This effect depends on both the multiplicity of infection and invasion capacities of the strains used (8325–4 (invasion competent) vs DU5883 (invasion incompetent)). The infection delays mineralization after 5 days (p <0.0001), likely due to a cytotoxic effect. Indeed, after bacterial clearance at J21, this delay is made up (no difference between infected and uninfected cells). These results are consistent with the preliminary in vivo observations (μCT) showing a significant decrease in the thickness of trabecular of infected femurs with 8325–4 compared to DU5883 and non-infected femurs (p< 0, 0041). Conclusions. These results suggest that the internalization of SA leads to an imbalance of bone remodeling, in particular by a cytotoxic effect on the pre-OB and a slowed-down formation of bone tissue by OB, leading to a significant bone loss. The ongoing study of the cellular and bacterial mechanisms involved in this internalization should allow a better management of chronic BJI

Background. Residual stress remains in bone tissues after press-fit-fixation of a joint prosthesis, recently employed for joint arthroplasty. The response of bone tissues to the residual stress is, however, unknown because it is not physiological. This unnatural stimulus may have adverse effects on bone tissues, including causing thigh pain or bone resorption. In the present study, we designed an experimental method to apply a stationary load from inside an animal femur using a loop spring of titanium alloy with super elasticity. The femoral response was assessed based on the migration of the wire into bone twelve weeks after implantation. As the results, wire migration was noted in 10 of 11 cases. Methods. We developed a method using a loop spring made of super elastic titanium alloy, which can maintain sufficient stress in a rat femur for a prolonged period. This titanium alloy, which contains 43.94% titanium and 56.06% nickel, was supplied as a wire (WDL1, Actment Co., Ltd., Kasukabe, Japan). In the present study, an experimental method was designed to apply a stationary load from inside a rat femur by inserting a loop spring made of super elastic wire. Results. Ten weeks after implantation, migration of the spring wire into the cortical bone was noted in 10 of 11 cases. To assess spring migration in cortical bone, we measured the distance from the endocortical surface to the tip of springs on micro-CT images. The line of the endocortical surface was extrapolated from the adjacent to the wire contact area. The estimated load was distributed from 1.19 to 3.28 N. The migration depth on anterior and posterior sides was not associated with the estimated load. Discussion. In the present study, we developed a method of generating a stationary stress field in a rat femur using a loop spring made from Ni-Ti alloy with super ermore, implantation of the pin was presumed to be able to interpretation by the thelasticity. The load that originated from elastic deformation was large enough to apply mechanical stimulation to bone tissue. The estimated load was distributed from 1.19 to 3.28 N. Migration of the implanted loop spring in the femur was observed in ten of the eleven. The migration depth apparently did not increase with the increase in estimated load. Therefore, regardless of the load, that is stationary load is applied has been suggested to be involved in the migration of the pin. Furtheory of bone remodeling. n a state where the pin load of is applied, the bone implanted pin to which was destroyed by osteoclasts, was presumed to occur is bone formation in the implanted state. Interpretation. The present findings suggest that an excessive stationary load at the implant surface induces endosteal bone resorption together with the migration or protrusion of a prosthesis

Background. Synthetic interbody spinal fusion devices are used to restore and maintain disc height and ensure proper vertebral alignment. These devices are often filled with autograft bone to facilitate bone bridging through the device while providing mechanical stability. Nonporous polyetheretherketone (PEEK) devices are widely used clinically for such procedures. 1. Trabecular Metal devices are an alternative, fabricated from porous tantalum. It was hypothesized that the porous Trabecular Metal device would better maintain autograft viability through the center of the device, the ‘graft hole’ (GH). Methods. Twenty-five goats underwent anterior cervical discectomy and fusion using a Trabecular Metal or PEEK device for 6, 12 or 26 weeks. The GH of each device was filled with autograft bone morsels harvested from the animal at implantation. Fluorochrome labeling oxytetracycline was administered to the animals and used to determine bone viability in the device regions. Following necropsy, the vertebral segments were embedded in poly(methyl methacrylate) sectioned and analyzed using fluorescence and backscatter electron (BSE) imaging. The percent of bone tissue present within the GH was measured as a volume percent using BSE images (Fig. 1). Results. Bone percent analysis demonstrated that there was no significant difference (p<0.05) in volume of bone tissue within the GH of the two devices at 6 and 26 weeks (Fig. 2). At 12 weeks the animals implanted with the Trabecular Metal device had significantly greater volumes of bone within the GH region. Viable bone was observed in the host bone region and periprosthetic to the implant of all PEEK (n=12) and Trabecular Metal (n=12) animals within the study, determined by the presence of fluorescent labels (Fig. 3). Viable bone was also observed in the GH region of all animals with a Trabecular Metal device. However, only 5 of 12 PEEK animals showed bone viability within the GH (2 at 12 weeks and 3 at 26 weeks). A Fisher's exact comparison of the number of animals with viable bone in the GH showed a significant difference between the two devices, p<0.05. Conclusion. Autograft viability was better maintained within the GH for the porous Trabecular Metal device compared to the PEEK device. Although the amount of bone tissue within the GH of the PEEK devices was determined to have no significant difference compared to the Trabecular Metal devices at 6 and 26 weeks, the GH bone tissue was not viable in a number of the PEEK animals at each time point. The interconnected network and high volume porosity of the Trabecular Metal device may have allowed for fluid exchange, angiogenesis and increased blood supply to the autograft morsels. The viability of the autograft morsels also played an important role in the success of bone bridging through the GH between the vertebral endplates. In this animal model it was demonstrated that the autograft bone placed within the PEEK spinal fusion device did not always remain viable after implantation, but sometimes only filled the GH and did not necessarily facilitate fusion between the vertebrae as intended

Aim. Despite the expanding research focusing on bacterial biofilm formation, specific histochemical biofilm stains have not been developed for light microscopy. Therefore, pathologists are often not aware of the presence of biofilm formation when examining slides for diagnosing bacterial infections, including orthopaedic infections. The aim of the present study was to develop a combined histochemical and immunohistochemical biofilm stain for simultaneous visualization of Staphylococcus aureus bacteria and extracellular matrix in different colours using light microscopy. Methods. Infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with the biofilm forming S. aureus strain S54F9. The infection time was 5 and 15 days, respectively. First, 25 common histochemical protocols were used in order to find stains that could identify extracellular biofilm matrix. Hereafter, the histochemical protocols for Alcian Blue pH3, Luna and Methyl-pyronin green were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. Finally, the three new combined protocols were applied to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. For all combined protocols applied on all types of tissue (porcine and human) the number of double stained bacterial aggregates were counted. On the same sections the percentage of extracellular matrix of representative bacterial aggregates was calculated by image analysis. Results. Simultaneous visualization of bacterial cells and extracellular matrix in different colours was detected in both porcine and human tissue sections with all three combined protocols. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain i.e. if it was Alcian blue pH3 (colouring polysaccharides), Luna or Methyl green-pyronin (both colouring extracellular DNA), respectively. In the porcine models, 10 percent of the bacterial aggregates in a 10× magnification field revealed both the extracellular matrix and bacteria simultaneously in two different colours. For the human case, this was seen in 90 percent of the bacterial aggregates. The percentage of extracellular matrix of representative bacterial aggregates was 60 and 20 percent in the human and porcine tissues, respectively. Conclusions. The amount of S. aureus biofilm extracellular matrix increased with infection time. A combination of histochemical and immunohistochemical staining is a practical method for identification and evaluation of S. aureus biofilm in orthopaedic infections