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.

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.

Medication-related osteonecrosis of the jaw in a minipig model: Parameters for developing a macroscopic, radiological, and microscopic grading scheme.

OBJECTIVES: To devise a macroscopic, radiological, and histological scale for assessing pathological changes associated with medication-related osteonecrosis of the jaw in a minipig model. MATERIALS AND METHODS: Medication-related osteonecrosis of the jaw was induced in Göttingen minipigs by weekly intravenous administration of bisphosphonate (zoledronic acid) combined with a tooth extraction procedure. Controls either did not receive zoledronic acid or did not undergo tooth extraction. After 20 weeks, minipigs were euthanized and underwent computed tomography and micro-computed tomography scanning. The mandible underwent additional histological examination. RESULTS: The most consistent macroscopic findings in animals that had developed bisphosphonate-related osteonecrosis of the jaw (BRONJ) were necrotic, denuded bone, and formation of fistula and pus. Under radiological examination, impaired extraction socket healing, decrease in attenuation of bone beneath the extraction site, and periosteal reaction were observed. Under histological examination, demineralization of the extracellular bone matrix, denuding of bone, and osteonecrosis were recorded. CONCLUSION: These parameters were used to develop a scoring system for grading BRONJ.

In vivo study on scaffolds based on chitosan, collagen, and hyaluronic acid with hydroxyapatite.

Scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with nano-hydroxyapatite were obtained with the use of the freeze-drying method. Composites swelling behavior was assessed by the liquid uptake test. The adhesion and proliferation of human osteosarcoma SaOS-2 cells on the scaffolds were examined in 4-day culture. The biocompatibility of the chosen scaffolds was further studied by in vivo implantation into subcutaneous tissue of rabbits. The results showed low stability of the scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with hydroxyapatite. The addition of hydroxyapatite delayed the degradation process of the obtained scaffolds. The X-ray images of the tissues surrounding the scaffolds showed that both, the control scaffold without hydroxyapatite (HAp) and those with addition of 50% wt. HAp underwent degradation after 6 months. However, the scaffolds supplemented with 80% wt. HAp premained in the implanted place. The results showed satisfactory tissue response on the implanted scaffolds.

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.

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.

Histamine receptor 2 modifies iNKT cell activity within the inflamed lung.

BACKGROUND: Histamine is a key immunoregulatory mediator and can dampen proinflammatory responses via activation of histamine receptor 2 (H2 R). The aim of this study was to determine the role of H2 R in modulating lung inflammatory responses. METHODS: H2 R was blocked using famotidine or activated using dimaprit in both the ovalbumin (OVA) and house dust mite extract (HDM) murine models of respiratory inflammation. H2 R-deficient animals and CD1d/H2 R-deficient animals were utilized to examine the CD1d presentation of lipid antigens (αGalCer or OCH) to invariant natural killer T (iNKT) cells. RESULTS: Famotidine treatment resulted in more severe airway disease in the OVA model, while dimaprit treatment significantly reduced disease severity. Both OVA and HDM-induced airway diseases were more severe in H2 R-deficient animals. Flow cytometric analysis of lung tissue from H2 R-deficient animals revealed increased numbers of CD1d+ dendritic cells and increased numbers of iNKT cells. In vitro, αGalCer-stimulated iNKT cells from H2 R-deficient mice secreted higher levels of IL-4, IL-5, and GM-CSF. In vivo, αGalCer or OCH administration to the lung resulted in enhanced mucus secretion, inflammatory cell recruitment, and cytokine production in H2 R-deficient or famotidine-treated animals, while dimaprit dampened the lung iNKT cell response to αGalCer. Removal of iNKT cells in H2 R-deficient (CD1d-/- H2 R-/- ) animals normalized the lung response to HDM. CONCLUSION: The deliberate activation of H2 R, or its downstream signaling molecules, may represent a novel therapeutic target for chronic lung inflammatory diseases, especially when CD1d-mediated presentation of lipid antigens to iNKT cells is contributing to the pathology.

Healing pattern of reamed bone following bone harvesting by a RIA device.

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.

Spontaneous bilateral avulsion fracture of the tuberositas tibiae in a New Zealand White rabbit - a counterpart to Osgood-Schlatter disease in humans?

The first reported case describing a spontaneous bilateral avulsion fracture of the tuberositas tibiae in a New Zealand White rabbit is presented. So far in animals, this condition has been only described in dogs and horses. In humans, this condition is also called Osgood-Schlatter disease (OSD) or syndrome, traction apophysitis of the tibial tubercle (ATT) or patellar tendon enthesopathy of the tibial tuberosity respectively. It is mainly seen in young adolescents coinciding with periods of growth spurts. In humans, its pathogenesis is believed to be caused by repetitive tendon/muscle strain at the insertion of the patellar tendon to the immature tibial tuberosity, which has its own secondary ossification center. Morphologically this case is characterized by bilateral chronic avulsion with incomplete separation of the tuberositas tibae, and proximal dislocation of the patella (patella alta). Despite these marked pathological changes, the animal was clinically without findings. Nevertheless, this case emphasizes the need for thorough clinical and radiological examination of rabbits intended for preclinical research studies prior to study begin, especially in orthopedic research.

Significance of hepatic preneoplasia for cancer chemoprevention.

Hepatic preneoplasia represents an early stage in neoplastic development, preceding both benign and malignant neoplasia. This applies particularly to foci of altered hepatocytes (FAH), that precede the manifestation of hepatocellular adenomas and carcinomas in all species investigated. Morphological, microbiochemical and molecular biological approaches in situ have provided evidence for striking similarities in specific changes of the cellular phenotype of preneoplastic FAH emerging in experimental and human hepatocarcinogenesis, irrespective of whether this was elicited by chemicals, hormones, radiation, viruses or, in animal models, by transgenic oncogenes or Helicobacter hepaticus. Different types of FAH have been distinguished and related to three main preneoplastic hepatocellular lineages: (1) the glycogenotic-basophilic cell lineage, (2) its xenomorphic-tigroid cell variant, and (3) the amphophilic-basophilic cell lineage. The predominant glycogenotic-basophilic and tigroid cell lineages develop especially after exposure to DNA-reactive chemicals, radiation, hepadnaviridae, transgenic oncogenes and local hyperinsulinism, their phenotype indicating initiation by insulin or insulinomimetic effects of the oncogenic agents. In contrast, the amphophilic cell lineage of hepatocarcinogenesis has been observed mainly after exposure of rodents to peroxisome proliferators that are not directly DNA-reactive or to hepadnaviridae, the biochemical pattern mimicking an effect of thyroid hormone, including mitochondrial proliferation and activation of mitochondrial enzymes. Hepatic preneoplastic lesions are increasingly used as end-points in carcinogenicity testing, particularly in medium-term carcinogenesis bioassays. This has been complemented more recently by the use of FAH as indicators of chemoprevention, although possible pitfalls of this approach have to be considered carefully. Our ever-increasing knowledge on the metabolic and molecular changes that characterize preneoplastic lesions and their progression to neoplasia provides a new basis for rational approaches to chemoprevention by drugs, hormones or components of the diet.

Insulin receptor substrate-1 is over-expressed in glycogenotic but not in amphophilic preneoplastic hepatic foci induced in rats by N-nitrosomorpholine and dehydroepiandrosterone.

Insulin receptor substrate-1 (IRS-1) is over-expressed in preneoplastic glycogenotic hepatic foci (GSF) and is gradually down-regulated during progression of these lesions, via mixed cell foci (MCF), to the basophilic neoplastic phenotype. The aim of the present study was to investigate the effect of dehydroepiandrosterone (DHEA), a weak hepatocarcinogen and tumour enhancer, on IRS-1 expression. Hepatocellular lesions were induced by N-nitrosomorpholine followed by DHEA. Under these conditions, many glycogen-poor amphophilic (APF) and intermediate cell foci (ICF) appear, in addition to GSF and MCF. IRS-1 was over-expressed in 215 out of 295 GSF, in 50 out of 53 MCF and in a glycogen-rich mixed cell adenoma. IRS-1 expression was not shown in 147 APF, 51 ICF and 5 amphophilic hepatocellular adenomas, and 3 out of 5 hepatocellular carcinomas showed a weak IRS-1 expression. The results suggest a close association of IRS-1 over-expression with the glycogenotic hepatocellular phenotype. The modulation and enhancement of tumour progression by DHEA is associated with a shift from glycogenosis to amphophilia and basophilia, and a down-regulation of IRS-1 expression.

Overexpression of insulin receptor substrate-1 emerges early in hepatocarcinogenesis and elicits preneoplastic hepatic glycogenosis.

Insulin receptor substrate-1 (IRS-1) is a multisite docking protein occupying a central position in signaling cascades stimulated by a number of growth factors including insulin. Using Western blotting and immunohistochemistry, we investigated the expression of IRS-1 in more than 400 preneoplastic foci of altered hepatocytes and in 12 hepatocellular carcinomas induced in rats by oral administration of N-nitrosomorpholine. In both N-nitrosomorpholine-treated and untreated rat livers, IRS-1 was demonstrable by Western blotting, but with the exception of a few single hepatocytes it was not detectable in the normal parenchyma by immunohistochemistry. In contrast, immunohistochemistry revealed that IRS-1 was strongly expressed in the majority of foci of altered hepatocytes particularly in approximately 97% of the clear/acidophilic and mixed cell foci showing excessive storage of glycogen (glycogenosis). In glycogen-poor basophilic foci of altered hepatocytes and hepatocellular carcinomas, IRS-1 was not detected by immunohistochemistry, but a weak expression was observed in small subpopulations of three hepatocellular carcinomas containing remnants of glycogen. These results indicate that the focal overexpression of IRS-1 is an early event in hepatocarcinogenesis, which is closely correlated with preneoplastic hepatic glycogenosis. During progression from glycogenotic foci to hepatocellular carcinomas, IRS-1-overexpression is gradually down-regulated, and this late event is associated with a fundamental metabolic shift leading to the malignant neoplastic phenotype.