Assessment, Quantification, and Management of Fracture Pain: from Animals to the Clinic

Purpose of review: Fractures are painful and disabling injuries that can occur due to trauma, especially when compounded with pathologic conditions, such as osteoporosis in older adults. It is well documented that acute pain management plays an integral role in the treatment of orthopedic patients. There is no current therapy available to completely control post-fracture pain that does not interfere with bone healing or have major adverse effects. In this review, we focus on recent advances in the understanding of pain behaviors post-fracture. Recent findings: We review animal models of bone fracture and the assays that have been developed to assess and quantify spontaneous and evoked pain behaviors, including the two most commonly used assays: dynamic weight bearing and von Frey testing to assess withdrawal from a cutaneous (hindpaw) stimulus. Additionally, we discuss the assessment and quantification of fracture pain in the clinical setting, including the use of numeric pain rating scales, satisfaction with pain relief, and other biopsychosocial factor measurements. We review how pain behaviors in animal models and clinical cases can change with the use of current pain management therapies. We conclude by discussing the use of pain behavioral analyses in assessing potential therapeutic treatment options for addressing acute and chronic fracture pain without compromising fracture healing. There currently is a lack of effective treatment options for fracture pain that reliably relieve pain without potentially interfering with bone healing. Continued development and verification of reliable measurements of fracture pain in both pre-clinical and clinical settings is an essential aspect of continued research into novel analgesic treatments for fracture pain

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

This work was supported in part by funds from an Oklahoma State Regent Grant for Higher Education (021606), P20RR016478 grant from the National Center for Research Resources (NCRR) a component of National Institute of Health (NIH), and a grant from the University of Central Oklahoma office of Research and Grants to L.C.The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification.Yeshttp://www.plosone.org/static/editorial#pee

[Comment] Redefine statistical significance

The lack of reproducibility of scientific studies has caused growing concern over the credibility of claims of new discoveries based on “statistically significant” findings. There has been much progress toward documenting and addressing several causes of this lack of reproducibility (e.g., multiple testing, P-hacking, publication bias, and under-powered studies). However, we believe that a leading cause of non-reproducibility has not yet been adequately addressed: Statistical standards of evidence for claiming discoveries in many fields of science are simply too low. Associating “statistically significant” findings with P < 0.05 results in a high rate of false positives even in the absence of other experimental, procedural and reporting problems. For fields where the threshold for defining statistical significance is P<0.05, we propose a change to P<0.005. This simple step would immediately improve the reproducibility of scientific research in many fields. Results that would currently be called “significant” but do not meet the new threshold should instead be called “suggestive.” While statisticians have known the relative weakness of using P≈0.05 as a threshold for discovery and the proposal to lower it to 0.005 is not new (1, 2), a critical mass of researchers now endorse this change. We restrict our recommendation to claims of discovery of new effects. We do not address the appropriate threshold for confirmatory or contradictory replications of existing claims. We also do not advocate changes to discovery thresholds in fields that have already adopted more stringent standards (e.g., genomics and high-energy physics research; see Potential Objections below). We also restrict our recommendation to studies that conduct null hypothesis significance tests. We have diverse views about how best to improve reproducibility, and many of us believe that other ways of summarizing the data, such as Bayes factors or other posterior summaries based on clearly articulated model assumptions, are preferable to P-values. However, changing the P-value threshold is simple and might quickly achieve broad acceptance

Assessment, Quantification, and Management of Fracture Pain: from Animals to the Clinic

Purpose of review: Fractures are painful and disabling injuries that can occur due to trauma, especially when compounded with pathologic conditions, such as osteoporosis in older adults. It is well documented that acute pain management plays an integral role in the treatment of orthopedic patients. There is no current therapy available to completely control post-fracture pain that does not interfere with bone healing or have major adverse effects. In this review, we focus on recent advances in the understanding of pain behaviors post-fracture. Recent findings: We review animal models of bone fracture and the assays that have been developed to assess and quantify spontaneous and evoked pain behaviors, including the two most commonly used assays: dynamic weight bearing and von Frey testing to assess withdrawal from a cutaneous (hindpaw) stimulus. Additionally, we discuss the assessment and quantification of fracture pain in the clinical setting, including the use of numeric pain rating scales, satisfaction with pain relief, and other biopsychosocial factor measurements. We review how pain behaviors in animal models and clinical cases can change with the use of current pain management therapies. We conclude by discussing the use of pain behavioral analyses in assessing potential therapeutic treatment options for addressing acute and chronic fracture pain without compromising fracture healing. There currently is a lack of effective treatment options for fracture pain that reliably relieve pain without potentially interfering with bone healing. Continued development and verification of reliable measurements of fracture pain in both pre-clinical and clinical settings is an essential aspect of continued research into novel analgesic treatments for fracture pain

An optimized hepatitis C virus E2 glycoprotein core adopts a functional homodimer that efficiently blocks virus entry

The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects ∼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81

Fracture Healing in Aging: Improving Angiogenesis

BACKGROUND: With an aging world population, there is an increased risk of fracture and impaired fracture healing. Enhancement of angiogenesis is one potential mechanism for improved fracture healing. The question then becomes: How do therapeutic targets improve these angiogenic and vasculitic conditions to facilitate successful fracture union? The current therapy, bone morphogenetic protein 2 (BMP-2), while successful in promoting fracture union, presents with several potential side effects, including excess bone growth and an increased risk of developing cancer. We hypothesize that thrombopoietin (TPO) may serve as a novel therapeutic modality for improved angiogenesis and fracture union. Here we characterize and stimulate endothelial cells (ECs) with TPO in vitro. OBJECTIVE: We hypothesize that thrombopoietin (TPO) may serve as a novel therapeutic modality for improved angiogenesis and fracture union. METHODS: Five young (3 month-old) and 5 old (22-24 month-old) C57BL/6 mice were euthanized and bone marrow was collected from the tibia and humeri for EC isolation. Additionally, five young mice underwent a 2 mm femoral diaphyseal defect surgery. Three weeks post-surgery mice were euthanized and bone marrow from the ipsilateral and contralateral tibia as well as the humeri were collected and ECs isolated. All ECs were assessed for vessel-like formation and proliferation. All in vitro studies were completed at least 3 times with triplicate samples. Statistical analyses were performed using a two-way ANOVA with Tukey’s post-hoc testing. All procedures were approved by the Indiana University IACUC. RESULTS: In vitro characterization of ECs showed impaired vessel-like formation, branch length, number of nodes, mesh area, and proliferation in old ECs compared to young ECs. ECs isolated from the ipsilateral tibia of mice undergoing a SBD surgery had impaired ECs vessel-like parameters and proliferation compared to ECs from the contralateral tibia. Finally, treatment of old ECs with TPO improved vessel-like parameters and proliferation compared to untreated cells. CONCLUSIONS: Although preliminary, our results confirm that EC proliferation and vessel formation are impaired in aging and in the ipsilateral tibia of mice, which underwent a femoral SBD surgery. Although BMP-2 is used by orthopaedic surgeons for bone healing, its use is not without side effects, leaving researchers to investigate alternative treatments. We have previously shown that TPO is effective for bone healing. Here we show that TPO improves vessel formation in aged ECs, and therefore, TPO may improve angiogenesis and fracture healing in old mice, which is currently under investigation

Type 2 Diabetes-Driven Alterations in Bone Healing and Angiogenesis

INTRODUCTION: Two out of five U.S. citizens born after 2014 are expected to develop type 2 diabetes (T2D). T2D patients are at increased risk for fracture and impaired fracture healing, including: increased frequency of partial union, delayed union, or nonunion. These outcomes can in part be explained by T2D-mediated physiological and structural changes in bone; most prominently, compromised microvascular performance, which negatively impacts angiogenesis necessary for bone regeneration. Currently, to enhance bone healing in nonunion, the only FDA-approved biological treatment is the use of bone morphogenetic protein-2 (BMP-2). However, BMP-2 does not always achieve union, has adverse side effects, and may not be optimal for overcoming the T2D-specific impairments to bone healing. OBJECTIVE: In this study, we aimed to characterize the mechanisms by which T2D impairs bone healing and hypothesize that this effect is mediated by impairments in angiogenesis and endothelial cell proliferation and function. METHODS: Using Tie2-CreER;Td-Tomato mice (Tie2CreERT+), we established a high fat diet (HFD)-induced T2D mouse model to compare with low fat diet (LFD)-fed mice. Mice underwent glucose tolerance testing, insulin tolerance testing, and echoMRI to confirm the T2D-like metabolic phenotype. They were then subjected to a femoral critical-size defect surgery and were treated with either saline or BMP-2. 60 mice were allocated to each diet and experimental treatment group. Healing of the defect was assessed by X-rays. Additionally, bone marrow-derived endothelial cells (BMECs) were collected from the surgically treated mice to assess changes in endothelial colony and tube formation in vitro. Statistical analyses were performed with a two-way ANOVA and a Tukey post-hoc test. All procedures completed were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee. RESULTS: Our results showed that after 12 weeks, HFD mice acquired a T2D-like metabolic phenotype. X-ray imaging revealed that fracture healing was impaired in the HFD mice, even with the administration of BMP-2. The isolation of BMECs was confirmed by visualization of fluorescent Tie2+ cells. Unexpectedly, in vitro tube formation assays indicated that HFD improved vessel-like formation properties. BMP-2 treatment appeared to improve some vessel-like formation properties compared to control treatment. CONCLUSIONS: Further data will need to be collected to better characterize differences in bone healing and to analyze angiogenesis in the healing femurs. Still, these data are significant as they reveal the mechanisms by which T2D impairs bone healing and demonstrate the important difference between examining endothelial cells in vitro vs. in vivo. Future investigations will examine if thrombopoietin, which our group has previously shown to improve both fracture healing and angiogenesis, may be a more effective treatment than BMP-2 in this model

The Effects of Bone Morphogenetic Protein 2 and Thrombopoietin Treatment on Angiogenic Properties of Endothelial Cells Derived from the Lung and Bone Marrow of Young and Aged, Male and Female Mice

With an aging world population, there is an increased risk of fracture and impaired healing. One contributing factor may be aging-associated decreases in vascular function; thus, enhancing angiogenesis could improve fracture healing. Both bone morphogenetic protein 2 (BMP-2) and thrombopoietin (TPO) have pro-angiogenic effects. The aim of this study was to investigate the effects of treatment with BMP-2 or TPO on the in vitro angiogenic and proliferative potential of endothelial cells (ECs) isolated from lungs (LECs) or bone marrow (BMECs) of young (3-4 months) and old (22-24 months), male and female, C57BL/6J mice. Cell proliferation, vessel-like structure formation, migration, and gene expression were used to evaluate angiogenic properties. In vitro characterization of ECs generally showed impaired vessel-like structure formation and proliferation in old ECs compared to young ECs, but improved migration characteristics in old BMECs. Differential sex-based angiogenic responses were observed, especially with respect to drug treatments and gene expression. Importantly, these studies suggest that NTN1, ROBO2, and SLIT3, along with angiogenic markers (CD31, FLT-1, ANGPT1, and ANGP2) differentially regulate EC proliferation and functional outcomes based on treatment, sex, and age. Furthermore, treatment of old ECs with TPO typically improved vessel-like structure parameters, but impaired migration. Thus, TPO may serve as an alternative treatment to BMP-2 for fracture healing in aging owing to improved angiogenesis and fracture healing, and the lack of side effects associated with BMP-2