Development and characterization of a predictive microCT-based non-union model in Fischer F344 rats.

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

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.

Deriving a dose and regimen for anti-glucosaminidase antibody passive-immunisation for patients with Staphylococcus aureus osteomyelitis.

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

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.

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.

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.

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.

Deficiency of inducible and endothelial nitric oxide synthase results in diminished bone formation and delayed union and nonunion development.

BACKGROUND: Between 5% and 10% of all fractures fail to heal adequately resulting in nonunion of the fracture fragments. This can significantly decrease a patient's quality of life and create associated psychosocial and socio-economic problems. Nitric oxide (NO) and nitric oxide synthases (NOS) have been found to be involved in fracture healing, but until now it is not known if disturbances in these mechanisms play a role in nonunion and delayed union development. In this study, we explored the role of endothelial and inducible NOS deficiency in a delayed union model in mice. MATERIALS AND METHODS: A 0.45mm femur osteotomy with periosteal cauterization followed by plate-screw osteosynthesis was performed in the left leg of 20-24week old wild type, Nos2(-/-) and Nos3(-/-) mice. Contralateral unfractured legs were used as a control. Callus volume was measured using micro-computed tomography (µCT) after 28 and 42days of fracture healing. Immuno histochemical myeloperoxidase (MPO) staining was performed on paraffin embedded sections to assess neutrophil influx in callus tissue and surrounding proximal and distal marrow cavities of the femur. After 7 and 28days of fracture healing, femurs were collected for amino acid and RNA analysis to study arginine-NO metabolism. RESULTS: With µCT, delayed union was observed in wild type animals, whereas in both Nos2(-/-) and Nos3(-/-) mice nonunion development was evident. Both knock-out strains also showed a significantly increased influx of MPO when compared with wild type mice. Concentrations of amino acids and expression of enzymes related to the arginine-NO metabolism were aberrant in NOS deficient mice when compared to contralateral control femurs and wild type samples. DISCUSSION AND CONCLUSION: In the present study we show for the first time that the absence of nitric oxide synthases results in a disturbed arginine-NO metabolism and inadequate fracture healing with the transition of delayed union into a nonunion in mice after a femur osteotomy. Based on these data we suggest that the arginine-NO metabolism may play a role in the prevention of delayed unions and nonunions.

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.

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.

Semicircular canal dehiscence in HR multislice computed tomography: distribution, frequency, and clinical relevance.

The literature about bony defects in the semicircular canal system is highly inconsistent. Therefore, we analyzed a series of 700 high-resolution multislice CT examinations of the temporal bone for semicircular canal dehiscencies. An unselected group of ENT patients with different clinical symptoms and variable age was chosen. We found semicircular canal dehiscence in 9.6% of temporal bones, superior semicircular canal was affected mostly (8%), less common posterior semicircular canal (1.2%); only in 3 cases (0.4%), lateral semicircular canal showed dehiscence. In 60% of SSC dehiscence, we registered bilateral manifestation. The so-called "third mobile window" in semicircular canal dehiscence causes a great variety of clinical symptoms like vertigo, nystagmus, oscillopsies, hearing loss, tinnitus and autophonia. Comparison with anatomic studies shows that CT examination implies the risk of considerable overestimation; this fact emphasizes the important role of clinical and neurophysiological testing.

Augmentation of bone defect healing using a new biocomposite scaffold: an in vivo study in sheep.

Previous studies support resorbable biocomposites made of poly(L-lactic acid) (PLA) and beta-tricalcium phosphate (TCP) produced by supercritical gas foaming as a suitable scaffold for tissue engineering. The present study was undertaken to demonstrate the biocompatibility and osteoconductive properties of such a scaffold in a large animal cancellous bone model. The biocomposite (PLA/TCP) was compared with a currently used beta-TCP bone substitute (ChronOS, Dr. Robert Mathys Foundation), representing a positive control, and empty defects, representing a negative control. Ten defects were created in sheep cancellous bone, three in the distal femur and two in the proximal tibia of each hind limb, with diameters of 5 mm and depths of 15 mm. New bone in-growth (osteoconductivity) and biocompatibility were evaluated using microcomputed tomography and histology at 2, 4 and 12 months after surgery. The in vivo study was validated by the positive control (good bone formation with ChronOS) and the negative control (no healing with the empty defect). A major finding of this study was incorporation of the biocomposite in bone after 12 months. Bone in-growth was observed in the biocomposite scaffold, including its central part. Despite initial fibrous tissue formation observed at 2 and 4 months, but not at 12 months, this initial fibrous tissue does not preclude long-term application of the biocomposite, as demonstrated by its osteointegration after 12 months, as well as the absence of chronic or long-term inflammation at this time point.