Comparative bone healing with induced membrane technique (IMT) versus empty defects in septic and aseptic conditions in a novel rabbit humerus model.

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

Chronic Periodontal Infection and Not Iatrogenic Interference Is the Trigger of Medication-Related Osteonecrosis of the Jaw: Insights from a Large Animal Study (PerioBRONJ Pig Model).

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

Venous injection of a triphasic calcium-based implant in a sheep model of pulmonary embolism demonstrates minimal acute systemic effects.

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

Influence of screw head diameter on ex vivo fixation of equine lateral condylar fractures with 5.5 mm cortical screws.

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

Continuous Implant Load Monitoring to Assess Bone Healing Status-Evidence from Animal Testing.

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

Differences between auto-fluorescence and tetracycline-fluorescence in medication-related osteonecrosis of the jaw-a preclinical proof of concept study in the mini-pig.

OBJECTIVES: Fluorescence-guided bone surgery is a well-established technique in the treatment of medication-related osteonecrosis of the jaw. No histopathological evidence for bone auto-fluorescence is currently available, and thus, any differences from tetracycline-fluorescence remain unclear. Therefore, the goals of this study were to find out if macroscopic and histological differences occur between the auto- and tetracycline-fluorescence in the delineation of viable and necrotic jawbone in the mini-pig. MATERIALS AND METHODS: According to the proof of concept, osteonecrosis was provoked in eight Göttingen minipigs. Pigs were divided into two groups (AF group: no fluorochrome label; TF group: tetracycline label). Delineation of necrosis and viable bone was evaluated in vivo and in vitro macro-/microscopically, correlated to fluorescence properties and compared between the two study groups. RESULTS: No macroscopic and microscopic clinical differences were seen in fluorescence between the AF and TF groups. Macroscopic and microscopic viable bone fluoresced green, whereas necrotic bone showed no or only pale fluorescence in both groups. The auto-fluorescence was attributable to the arrangements and structure of collagen and the cell-filled bone lacunae. CONCLUSION: Neither in vivo nor in vitro macroscopically differences are apparent between the auto-fluorescence and the tetracycline-fluorescence of bone. The auto-fluorescence is attributable to the arrangements and structure of collagen and the cell-filled bone lacunae. Tetracycline-fluorescence is a mixture of tetracycline (at the bone edges with increased bone formation) and large components of auto-fluorescence. CLINICAL RELEVANCE: Because auto-fluorescence is easy to apply, reproducible, and does not rely on the subjective impression of the surgeon, it promises to be an important standardized alternative to tetracycline-labeled MRONJ therapy.

Subchondral screw abutment: does it harm the joint cartilage? An in vivo study on sheep tibiae.

PURPOSE: Subchondral screw abutment in osteosynthesis of joint fractures is an effective method to achieve sufficient screw grip. In this study we investigated if subchondral screw placement is possible without harming the overlying subchondral plate and joint cartilage iatrogenic. MATERIALS AND METHODS: A 3.5-mm conventional steel screw was placed in the tibia of ten sheep in distances between 1 and 7 mm beneath the joint cartilage. After a follow up of two and four months, evaluation of the subchondral bone and joint cartilage was performed by means of a histological osteoarthritis score, HRpQCT imaging and determination of the glycosaminoglycan content in the cartilage. The control group was the contralateral knee of the same animal. RESULTS: Histomorphometric evaluation of the Mankin osteoarthritis score revealed no significant difference compared to the control after two (p = 0.102) and four months (p = 0.429). No correlation between distance of the screw to the cartilage and histological scoring was found (p = 0.658, R2 = 0.04 after two months and p = 0.171, R2 = 0.18 after four months). HRpQCT measurements of the subchondral thickness between screw and cartilage after two (p = 0.05) and four months (p = 0.424) showed no significant difference. Mean glycosaminoglycan content in the treatment group compared to the control after two months (p = 0.25) and four months (p = 0.523) was not significant different. CONCLUSION: In conclusion subchondral screw abutment did not damage the joint cartilage after a two- and four-month follow up in this sheep model.

Implant retention in a rabbit model of fracture-related infection.

Aims: Fracture-related infection (FRI) is commonly classified based on the time of onset of symptoms. Early infections (< two weeks) are treated with debridement, antibiotics, and implant retention (DAIR). For late infections (> ten weeks), guidelines recommend implant removal due to tolerant biofilms. For delayed infections (two to ten weeks), recommendations are unclear. In this study we compared infection clearance and bone healing in early and delayed FRI treated with DAIR in a rabbit model. Methods: Staphylococcus aureus was inoculated into a humeral osteotomy in 17 rabbits after plate osteosynthesis. Infection developed for one week (early group, n = 6) or four weeks (delayed group, n = 6) before DAIR (systemic antibiotics: two weeks, nafcillin + rifampin; four weeks, levofloxacin + rifampin). A control group (n = 5) received revision surgery after four weeks without antibiotics. Bacteriology of humerus, soft-tissue, and implants was performed seven weeks after revision surgery. Bone healing was assessed using a modified radiological union scale in tibial fractures (mRUST). Results: Greater bacterial burden in the early group compared to the delayed and control groups at revision surgery indicates a retraction of the infection from one to four weeks. Infection was cleared in all animals in the early and delayed groups at euthanasia, but not in the control group. Osteotomies healed in the early group, but bone healing was significantly compromised in the delayed and control groups. Conclusion: The duration of the infection from one to four weeks does not impact the success of infection clearance in this model. Bone healing, however, is impaired as the duration of the infection increases.

Doxycycline-Mediated Inhibition of Snake Venom Phospholipase and Metalloproteinase.

INTRODUCTION: Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline-Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia, and Daboia russelii were tested under inhibitory conditions using doxycycline. MATERIALS AND METHODS: Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii, and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels. RESULTS: Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P < .0001) (Fig. 1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P < .0001) (Fig. 1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus = 20.29 mg/kg (Fig. 2A), N. kaouthia = 0.38 mg/kg (Fig. 2B), and D. russelii = 7.92 mg/kg (Fig. 2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig. 3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P < .0001) (Fig. 4). CONCLUSION: Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species.

Impact of Perioperative Dexamethasone Administration on Infection and Implant Osseointegration in a Preclinical Model of Orthopedic Device-Related Infection.

Glucocorticoids may be given prior to major orthopedic surgery to decrease postoperative nausea, vomiting, and pain. Additionally, many orthopedic patients may be on chronic glucocorticoid therapy. The aim of our study was to investigate whether glucocorticoid administration influences Orthopedic-Device-Related Infection (ODRI) in a rat model. Screws colonized with Staphylococcus epidermidis were implanted in the tibia of skeletally mature female Wistar rats. The treated groups received either a single shot of dexamethasone in a short-term risk study, or a daily dose of dexamethasone in a longer-term interference study. In both phases, bone changes in the vicinity of the implant were monitored with microCT. There were no statistically significant differences in bacteriological outcome with or without dexamethasone. In the interference study, new bone formation was statistically higher in the dexamethasone-treated group (p = 0.0005) as revealed by CT and histopathological analysis, although with relatively low direct osseointegration of the implant. In conclusion, dexamethasone does not increase the risk of developing periprosthetic osteolysis or infection in a pre-clinical model of ODRI. Long-term administration of dexamethasone seemed to offer a benefit in terms of new bone formation around the implant, but with low osseointegration.

Peri-anesthetic hypothermia in rodents: A factor to consider for accurate and reproducible outcomes in orthopedic device-related infection studies.

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

Arginine concentration in arterial vs venous blood in a bleomycin-induced lung inflammation model in mice.

Pneumonia, always a major malady, became the main public health and economic disaster of historical proportions with the COVID-19 pandemic. This study was based on a premise that pathology of lung metabolism in inflammation may have features invariant to the nature of the underlying cause. Amino acid uptake by the lungs was measured from plasma samples collected pre-terminally from a carotid artery and vena cava in mice with bleomycin-induced lung inflammation (N = 10) and compared to controls treated with saline instillation (N = 6). In the control group, the difference in concentrations between the arterial and venous blood of the 19 amino acids measured reached the level of statistical significance only for arginine (-10.7%, p = 0.0372) and phenylalanine (+5.5%, p = 0.0266). In the bleomycin group, 11 amino acids had significantly lower concentrations in the arterial blood. Arginine concentration was decreased by 21.1% (p<0.0001) and only that of citrulline was significantly increased (by 20.1%, p = 0.0002). Global Arginine Bioavailability Ratio was decreased in arterial blood by 19.5% (p = 0.0305) in the saline group and by 30.4% (p<0.0001) in the bleomycin group. Production of nitric oxide (NO) and citrulline from arginine by the inducible nitric oxide synthase (iNOS) is greatly increased in the immune system's response to lung injury. Deprived of arginine, the endothelial cells downstream may fail to provide enough NO to prevent the activation of thrombocytes. Thrombotic-related vascular dysfunction is a defining characteristic of pneumonia, including COVID-19. This experiment lends further support to arginine replacement as adjuvant therapy in pneumonia.

Effects of a Two-Step Silver Diamine Fluoride Varnish on Shear Bond Strength of Restorations, Dentin and Enamel Hardness, and Biofilm Formation.

INTRODUCTION: Dental caries are a limiting factor in maintaining dental and medical readiness in the military. Untreated dental caries can lead to dire health consequences. Consistent and comprehensive access to dental care is often limited due to the intensive operational demands on our nation's warfighters. The standard of care for dental caries is a surgical model where diseased tooth tissue is surgically removed and restored with appropriate restorative materials. While effective, it is not practical in the military operational environment, especially under time constraints. Dental restoratives offer military personnel a simple and preventive treatment of dental caries and are suitable as self-applied first aids. The purpose of this study was to measure the shear bond strengths of two dental restorative materials to human teeth paired with two different fluoride treatments and the hardness and biofilm formation on teeth after applying the fluoride varnishes. MATERIALS AND METHODS: Specimens were made of human molar teeth treated with each of the following four materials: glass ionomer cement GC Fuji II LC Capsules, Filtek Z250, Riva Star steps 1 and 2, or Mark3 NaF varnish. Step 1 of Riva Star consists of silver diamine fluoride and step 2 contains potassium iodide. On human molar slabs, 10 circular specimens of 5 cm in diameter were prepared with restoratives according to manufacturer procedures. Etch-Rite and a proprietary aluminum chloride-based cavity conditioner were used as etchants on tooth surfaces for the Filtek Z250 and glass ionomer cement, respectively. After at least 24 hours underwater, each assembly was removed, and the shear bond strength of the adhesive was measured according to International Organization for Standardization (ISO) 29022.The hardness was measured according to ISO 14233. Hardness measurements were performed before varnish application, then after storage in an incubator at 37 °C for 4 hours in a demineralization solution (pH = 4.5), and after 1 day in a mineralization solution (pH = 7). A crystal violet staining assay was used to measure biofilm formation of Streptococcus mutans bacteria on human molar teeth after the application of fluoride varnish. RESULTS: We report a 16% increase in shear bond strength of the Filtek Z250/Riva Star coupled treatment compared to the Filtek Z250/Mark3 NaF coupled treatment. We also demonstrate a significant 84% decrease in bond strength with a GC Fuji II LC/Mark3 NaF treatment compared to control (P = .0002), while Riva Star remains statistically unchanged. Enamel and dentinal hardness are significantly improved when Riva Star is applied compared to NaF varnish. A 25%-35% (P < .0001) decrease in oral biofilm formation was observed on samples where a Riva Star or NaF varnish was applied. CONCLUSIONS: Mechanical and antimicrobial testing indicated Riva Star, compared favorably with and in some cases, performed better in the laboratory than a Mark3 NaF varnish. Hardness measurements indicated Riva Star is more effective in dentin tubule occlusion compared to NaF varnish. Our findings help provide practical suggestions to dental treatment, particularly to the unique dental environments seen in the military. Riva Star may be used as an adjunctive treatment prior to placing a final restoration. This study supports the use of Riva Star in conjunction with GC Fuji II LC or Filtek Z250 restorative materials, making it a promising treatment in military dental applications.

Genome Sequences of 14 Siphophages That Infect Serratia marcescens.

We announce the complete genome sequences of 14 Serratia bacteriophages isolated from wastewater treatment plants. These phages define two previously undescribed types which we call the Carrot-like phage cluster (phages Carrot, BigDog, LittleDog, Niamh, Opt-148, Opt-169, PhooPhighters, Rovert, Serratianator, Stoker, Swain, and Ulliraptor) and Tlacuache-like phage cluster (Tlacuache and Opt-155).

Genome Sequences of 22 T1-like Bacteriophages That Infect Enterobacteriales.

Here, the full genome sequences of 22 T1-like bacteriophages isolated from wastewater are reported. Eight (BlueShadow, Brooksby, Devorator, ElisaCorrea, Reinasaurus, SorkZaugg, Supreme284, ZeroToHero) were isolated on Citrobacter, six on Klebsiella (Chell, FairDinkum, HazelMika, Opt-817, P528, PeteCarol), and eight on Escherichia (Fulano1, Mishu, Opt-719, PhleaSolo, Punny, Poky, Phunderstruck, Sadiya).

Double-plating of ovine critical sized defects of the tibia: a low morbidity model enabling continuous in vivo monitoring of bone healing.

BACKGROUND: Recent studies using sheep critical sized defect models to test tissue engineered products report high morbidity and complications rates. This study evaluates a large bone defect model in the sheep tibia, stabilized with two, a novel Carbon fibre Poly-ether-ether-ketone (CF-PEEK) and a locking compression plate (LCP) which could sustain duration for up to 6 month with an acceptable low complication rate. METHODS: A large bone defect of 3 cm was performed in the mid diaphysis of the right tibia in 33 sheep. The defect was stabilised with the CF - PEEK plate and an LCP. All sheep were supported with slings for 8 weeks after surgery. The study was carried out for 3 months in 6 and for 6 months in 27 animals. RESULTS: The surgical procedure could easily be performed in all sheep and continuous in vivo radiographic evaluation of the defect was possible. This long bone critical sized defect model shows with 6.1% a low rate of complications compared with numbers mentioned in the literature. CONCLUSIONS: This experimental animal model could serve as a standard model in comparative research. A well defined standard model would reduce the number of experimental animals needed in future studies and would therefore add to ethical considerations.

The relation between fracture activity and bone healing with special reference to the early healing phase - A preclinical study.

BACKGROUND: Fracture healing outcome is to a great extent steered by the mechanical environment. The importance of early phase mechanical fracture stimulation is still controversially discussed, both clinically and scientifically. Furthermore, the role of fracture activity, defined as the number of stimulatory events per time, is particularly for the direct postoperative phase unknown. METHODS: Tibial defects of seven Swiss mountain sheep were stabilized with a dynamizable bone fixator, which allowed for defined interfragmentary motion by limiting the maximum axial displacement. The fixator was further equipped with a telemetric measuring unit to continuously log all occurring displacement events above a predefined amplitude threshold over an 8-weeks observation period. Callus size was measured over time from X-rays. Ultimate torsional strength of the healed defects was assessed after euthanasia. RESULTS: One animal had to be excluded from the experiment due to technical reasons. The remaining six animals exhibited consistently the highest fracture activity in week 1 post-operation with 6'029 displacement events per week for the animal with the lowest activity and 21'866 events per week for the most active animal. Afterwards fracture activity gradually decreased over time. Strong and significant correlations were found for fracture activity in week 1 and 2 with torsional strength of the healed bone (R ≥ 0.881, p ≤ 0.02). No significant correlations were observed at later timepoints. Fracture activity in week 1 and 2 also correlated strongly with the maximum callus area as measured from X-rays (R ≥ 0.846, p ≤ 0.034). CONCLUSIONS: The data demonstrates a positive effect of, within limits, frequent fracture stimulation on bone healing and suggests the importance of the mechanical environment in the direct post-operative healing phase. Clinically, the findings may advocate for the concept of direct post-operative weight bearing. This, however, requires clinical validation and must be considered within the full clinical context including the risk for fixation failure from overloading.

Is a Block of the Femoral and Sciatic Nerves an Alternative to Epidural Analgesia in Sheep Undergoing Orthopaedic Hind Limb Surgery? A Prospective, Randomized, Double Blinded Experimental Trial.

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

Morphology of bony callus growth in healing of a sheep tibial osteotomy.

Long bone fractures typically heal via formation of an external callus, which helps stabilise the bone fragments. Callus composition and morphology influence the mechanical environment, which in turn regulates the progression of healing. Therefore characterising callus development over time is crucial in understanding this mechanobiological regulation. Although bony callus is often assumed to grow towards the fracture from either side, this is not consistent with observations from large animal studies and clinical cases. Therefore, we sought to quantify the morphology of bony callus over time in a large animal model. Sheep tibiae were x-rayed weekly over eight weeks following an osteotomy (n=5), with fixation allowing up to 10% axial displacement under normal weight-bearing. After scaling radiographs by known landmarks and normalising greyscales, bony callus boundaries were defined by manual segmentation. The lateral callus area and coordinates of its centroid were calculated from each image. The external callus initially formed adjacent to the osteotomy site. Over the first four weeks, callus growth from its outer surfaces was characterised by its centre of area moving outwards and away from the osteotomy, on both proximal and distal fragments. Subsequent weeks showed consolidation and resorption from the outer surface of the callus. Our approach allowed bony callus development to be tracked in individuals throughout healing. Contrary to the view that periosteal bone formation originates distant from the fracture, our data showed bony callus adjacent to the defect from early stages, followed by approximately concentric growth. This discrepancy highlights the need for data specific to experimental conditions, and particularly early stages of healing, for evaluating theoretical models of mechanical regulation.

Impact of low bone mass and antiresorptive therapy on antibiotic efficacy in a rat model of orthopedic device-related infection.

A significant proportion of orthopedic devices are implanted in osteoporotic patients, but it is currently unclear how estrogen deficiency and/or exposure to antiresorptive bisphosphonates (BPs) influence orthopedic device-related infection (ODRI), or response to therapy. The aim of this study is to characterize the bone changes resulting from Staphylococcus epidermidis infection in a rodent ODRI model and to determine if ovariectomy (OVX) or BP treatment influences the infection or the success of antibiotic therapy. A sterile or S. epidermidis-contaminated screw was implanted into the proximal tibia of skeletally mature female Wistar rats (n = 6-9 per group). Bone changes were monitored over 28 days using in vivo micro-computed tomography scanning. OVX was performed 12 weeks before screw implantation. The BP zoledronic acid (ZOL) was administered 4 days before screw insertion. A combination antibiotic regimen (rifampin plus cefazolin) was administered from Days 7-21. In skeletally healthy animals, S. epidermidis induced marked changes in bone, with peak osteolysis occurring at Day 9 and woven bone deposition and periosteal mineralization from Day 14 onwards. Antibiotic therapy cleared the infection in the majority of animals (2/9 infected) but did not affect bone responses. OVX did not affect the pattern of infection-induced changes in bone, nor bacterial load, but reduced antibiotic efficacy (5/9 infected). ZOL treatment did not protect from osteolysis in OVX animals, or further affect antibiotic efficacy (5/9 infected) but did significantly increase the bacterial load. This study suggests that both BPs and OVX can influence host responses to bone infections involving S. epidermidis.