A Comprehensive Review of Mouse Diaphyseal Femur Fracture Models

Complications related to treatment of long bone fractures still stand as a major challenge for orthopaedic surgeons. Elucidation of the mechanisms of bone healing and development, and the subsequent alteration of these mechanisms to improve outcomes, typically requires animal models as an intermediary between in vitro and human clinical studies. Murine models are some of the most commonly used in translational research, and mouse fracture models are particularly diverse, offering a wide variety of customization with distinct benefits and limitations depending on the study. This review critically examines three common femur fracture models in the mouse, namely cortical hole, 3-point fracture (Einhorn), and segmental bone defect. We lay out the general procedure for execution of each model, evaluate the practical implications and important advantages/disadvantages of each and describe recent innovations. Furthermore, we explore the applications that each model is best adapted for in the context of the current state of murine orthopaedic research

Analysis of the effects of spaceflight and local administration of thrombopoietin to a femoral defect injury on distal skeletal sites

With increased human presence in space, bone loss and fractures will occur. Thrombopoietin (TPO) is a recently patented bone healing agent. Here, we investigated the systemic effects of TPO on mice subjected to spaceflight and sustaining a bone fracture. Forty, 9-week-old, male, C57BL/6 J were divided into 4 groups: (1) Saline+Earth; (2) TPO + Earth; (3) Saline+Flight; and (4) TPO + Flight (n = 10/group). Saline- and TPO-treated mice underwent a femoral defect surgery, and 20 mice were housed in space ("Flight") and 20 mice on Earth for approximately 4 weeks. With the exception of the calvarium and incisor, positive changes were observed in TPO-treated, spaceflight bones, suggesting TPO may improve osteogenesis in the absence of mechanical loading. Thus, TPO, may serve as a new bone healing agent, and may also improve some skeletal properties of astronauts, which might be extrapolated for patients on Earth with restraint mobilization and/or are incapable of bearing weight on their bones

Fracture Healing and Pain Tolerance in Aging Populations

Introduction: Delayed fracture healing is associated with increased morbidity and mortality, and patients with fractures suffer from injury-associated pain as well as post- operative surgical pain. Age is associated with an increased risk of fracture and delayed/impaired fracture healing. Opioids are commonly prescribed for post-orthopedic pain mitigation. Opioids, beyond eliciting cognitive impairment, are commonly associated with tolerance and addiction. This class of drugs, including buprenorphine, may also be associated with a slower resolution of pain and functional status. Objective: Here we used a mouse femoral segmental bone defect (SBD) bone repair model to examine the impact of opioids and fracture/fracture healing on pain behaviors in old and young mice. Methods: Young (3-4-month-old) and old (22-24-month-old) C57BL/6 male mice underwent a SBD surgery (8-10 mice/group). Animals were treated with either bone morphogenetic protein 2 (BMP-2) or vehicle (saline). Bone healing was examined via longitudinal x-rays and terminal micro-computed tomography (μCT). All mice were subjected to a three-day course of buprenorphine. Stimulus dependent mechanical and thermal pain thresholds as well as stimulus independent locomotion activity and exploration behavior were determined before and after SBD surgery/opioid exposure. Addition-ally, asymmetric directed pain behaviors including flinching, licking, lifting, shaking of the ipsilateral hindlimb were assayed in both treatment groups at post-injury timepoints using 20- minute-long video sessions by blinded observers. All animal procedures were approved by the Indiana University IACUC. Results: The ability of BMP-2 to enhance SBD bone union was evident by 2 weeks post-surgery (x-ray) and the return to pre-injury levels of ambulation/locomotion activity was observed by week 4. Vehicle-treated SBD mice did not meet these milestones even at 8 weeks post-surgery. The relative change in tactile allodynia was determined between baseline and 2 weeks post-surgery for young and old mice treated with saline or BMP-2. Contrary to what would be expected, all 4 groups: old, young, healed and unhealed had a reduction of their pain threshold by ~70% at 2 weeks post-surgery (p\u3e0.05). Stimulus- independent pain behavior was also evident in both vehicle and BMP-2 treated CSD animals as defined by asymmetric directed behaviors including flinching, licking, lifting, and shaking of the ipsilateral hindlimb. Conclusion: The data indicates that post-fracture pain thresholds are similar between both healed and unhealed, young and old mice. Therefore, the effect of musculoskele-tal injury and/or buprenorphine may be responsible for the pain experienced following trauma. Distinguishing between these will be critical for further understanding how to best treat acute and chronic pain associated with musculoskeletal injury, particularly for geriatric patients

High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)).To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology.Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33-100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples.Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the specificity of CTC identification and the accuracy of biomarker evaluation

A Controlled Clinical Trial of E5 Murine Monoclonal IgM Antibody to Endotoxin in the Treatment of Gram-Negative Sepsis

Objective. —To assess the efficacy of adjunctive monoclonal antibody antiendotoxin immunotherapy in patients with gram-negative sepsis. Design. —Double-blind, randomized, placebo-controlled trial. Setting. —Thirty-three university-affiliated centers, including Veterans Affairs, community, and municipal hospitals. Patients. —Hospitalized adults with signs of gram-negative infection and a systemic septic response. Intervention. —Patients were assigned to receive either 2 mg/kg of a murine monoclonal antibody directed against gram-negative endotoxin (E5) or placebo. A second infusion was administered 24 hours later. Main Outcome Measures. —Mortality over the 30-day study period, resolution of organ failures, and safety. Results. —Four hundred eighty-six patients were enrolled. Three hundred sixteen had confirmed gram-negative sepsis (54% bacteremic, 46% nonbacteremic). The survival difference was not statistically significant for all patients. Among patients with gram-negative sepsis who were not in shock at study entry (n = 137), E5 treatment resulted in significantly greater survival (relative risk, 2.3; P =.01). Resolution of individual organ failures was more frequent among these patients, occurring in 19 (54%) of 35 patients in the E5 group vs eight (30%) of 27 in the placebo group (P =.05). Four reversible allergic reactions occurred among 247 patients (1.6%) receiving E5. No other toxicity was identified. Conclusions. —Treatment with E5 antiendotoxin antibody appears safe. It reduces mortality and enhances the resolution of organ failure among patients with gram-negative sepsis who are not in shock when treated.(JAMA. 1991;266:1097-110

A Controlled Clinical Trial of E5 Murine Monoclonal IgM Antibody to Endotoxin in the Treatment of Gram-Negative Sepsis

Objective. —To assess the efficacy of adjunctive monoclonal antibody antiendotoxin immunotherapy in patients with gram-negative sepsis. Design. —Double-blind, randomized, placebo-controlled trial. Setting. —Thirty-three university-affiliated centers, including Veterans Affairs, community, and municipal hospitals. Patients. —Hospitalized adults with signs of gram-negative infection and a systemic septic response. Intervention. —Patients were assigned to receive either 2 mg/kg of a murine monoclonal antibody directed against gram-negative endotoxin (E5) or placebo. A second infusion was administered 24 hours later. Main Outcome Measures. —Mortality over the 30-day study period, resolution of organ failures, and safety. Results. —Four hundred eighty-six patients were enrolled. Three hundred sixteen had confirmed gram-negative sepsis (54% bacteremic, 46% nonbacteremic). The survival difference was not statistically significant for all patients. Among patients with gram-negative sepsis who were not in shock at study entry (n = 137), E5 treatment resulted in significantly greater survival (relative risk, 2.3; P =.01). Resolution of individual organ failures was more frequent among these patients, occurring in 19 (54%) of 35 patients in the E5 group vs eight (30%) of 27 in the placebo group (P =.05). Four reversible allergic reactions occurred among 247 patients (1.6%) receiving E5. No other toxicity was identified. Conclusions. —Treatment with E5 antiendotoxin antibody appears safe. It reduces mortality and enhances the resolution of organ failure among patients with gram-negative sepsis who are not in shock when treated.(JAMA. 1991;266:1097-110