In vitro and in vivo investigation of a thyroid hormone system-specific interaction with triazoles.

Alterations in thyroid hormones (TH) and thyroid-stimulating hormone levels are frequently found following exposure to chemicals of concern. Dysregulation of TH levels can severely perturb physiological growth, metabolism, differentiation, homeostasis in the adult and developmental processes in utero. A frequently identified mode of action for this interaction is the induction of hepatic detoxification mechanisms (e.g. SULTs and UGTs), which lead to TH conjugation and elimination and therefore interfere with hormonal homeostasis, fulfilling the endocrine disruptors (EDs) definition. A short-term study in rats with dietary exposure to cyproconazole, epoxiconazole and prochloraz was conducted and hepatocyte hypertrophy, hepatic UGT activity and Phase 1/2 gene expression inductions were observed together with changes in TH levels and thyroid follicular hypertrophy and hyperplasia. To test for specific interaction with the thyroid hormone system, in vitro assays were conducted covering thyroidal I-uptake (NIS), TH transmembranal transport via MCT8 and thyroid peroxidase (TPO) function. Assays for iodothyronine deiodinases (DIO1-DIO3) and iodotyrosine deiodinase (DEHAL1) were included, and from the animal experiment, Dio1 and Dehal1 activities were measured in kidney and liver as relevant local indicators and endpoints. The fungicides did not affect any TH-specific KEs, in vitro and in vivo, thereby suggesting hepatic conjugation as the dominant MoA.

Matrine and Oxymatrine: evaluating the gene mutation potential using in silico tools and the bacterial reverse mutation assay (Ames test).

The quinolizidine alkaloids matrine and its N-oxide oxymatrine occur in plants of the genus Sophora. Recently, matrine was sporadically detected in liquorice products. Morphological similarity of the liquorice plant Glycyrrhiza glabra with Sophora species and resulting confusion during harvesting may explain this contamination, but use of matrine as pesticide has also been reported. The detection of matrine in liquorice products raised concern as some studies suggested a genotoxic activity of matrine and oxymatrine. However, these studies are fraught with uncertainties, putting the reliability and robustness into question. Another issue was that Sophora root extracts were usually tested instead of pure matrine and oxymatrine. The aim of this work was therefore to determine whether matrine and oxymatrine have potential for causing gene mutations. In a first step and to support a weight-of-evidence analysis, in silico predictions were performed to improve the database using expert and statistical systems by VEGA, Leadscope (Instem®), and Nexus (Lhasa Limited). Unfortunately, the confidence levels of the predictions were insufficient to either identify or exclude a mutagenic potential. Thus, in order to obtain reliable results, the bacterial reverse mutation assay (Ames test) was carried out in accordance with OECD Test Guideline 471. The test set included the plate incorporation and the preincubation assay. It was performed with five different bacterial strains in the presence or absence of metabolic activation. Neither matrine nor oxymatrine induced a significant increase in the number of revertants under any of the selected experimental conditions. Overall, it can be concluded that matrine and oxymatrine are unlikely to have a gene mutation potential. Any positive findings with Sophora extracts in the Ames test may be related to other components. Notably, the results also indicated a need to extend the application domain of respective (Q)SAR tools to secondary plant metabolites.

Penthrox® (Methoxyflurane) as an Analgesic for Removal of Circular External Fixators and Minor Procedures during the COVID-19 Pandemic.

Introduction: Methoxyflurane has excellent analgesic properties and is approved for use in the United Kingdom and Ireland since 2015. It is currently used in emergency departments for analgesia during fracture reductions. During the COVID-19 pandemic, with theatre access severely restricted, Penthrox® had the potential to provide adequate pain relief to aid frame and wire removal in the clinic setting. Materials and methods: Patients presenting to the limb reconstruction service elective clinic and requiring frame removal or minor procedures were included in the study. Patients with renal, cardiac or hepatic disease, a history of sensitivity to fluorinated anaesthetic agents and those on any nephrotoxic or enzyme-inducing drugs were excluded. All procedures were performed in an appropriate isolated room in the clinic. Patient demographics, procedure details, visual analogue score, Richmond Agitation Scale and patient satisfaction were recorded. Results: A total of 39 patients were included in the study of which 17 had Ilizarov frames removed, 10 had hexapod removals, nine had heel rings removed and three had an external fixator removed. Eleven patients received additional pain relief in the form of oral analgesia. All patients were satisfied or very satisfied with the experience. One patient required a general anaesthetic for the removal of a wire that could not be removed in the clinic due to bony overgrowth. Conclusion: Patient satisfaction was very high (>95%), and it was possible to perform frame removals and minor procedures in the clinic environment during the COVID-19 pandemic. We see potential for regular use of Penthrox® in the future for the removal of external fixation outside of the operating theatre. Clinical significance: Penthrox as an analgesic for frame adjustments and removals is safe and has the potential for significant financial savings for the National Health Service (NHS). How to cite this article: Debuka E, Birkenhead P, Shah S, et al. Penthrox® (Methoxyflurane) as an Analgesic for Removal of Circular External Fixators and Minor Procedures during the COVID-19 Pandemic. Strategies Trauma Limb Reconstr 2023;18(2):82-86.

Mechanical parameter assessment of fresh human cancellous bone of the femoral head in atraumatic femoral head necrosis and primary coxarthrosis.

BACKGROUND: Atraumatic femoral head necrosis is a rare pathological change of the femoral head. It is characterized by local necrosis of the cancellous bone as a result of reduced blood supply to the bone. Even today it remains unclear how to assess the hardness of the necrosis, whether it is soft tissue that is easily removed, or hard tissue that is difficult to resect. METHODS: Femoral heads with primary coxarthrosis were selected as a comparison group. For this purpose, 49 femoral heads obtained during total hip arthroplasty surgery with either condition (23 femoral head necrosis, 26 coxarthrosis) were transferred to the testing laboratory in fresh condition. Cylindrical specimens were obtained using a tenon cutter along the main trabecular load direction in the subchondral region of the femoral head. Additionally, thin bone slices were extracted proximal and distal to the specimens for density measurements. Brass plates were glued to the circular surfaces of the specimens. After curing of the adhesive, the specimens were mounted in the testing machine and destructive uniaxial compression tests were conducted. FINDINGS: The recorded mean compressive strengths and elastic moduli were almost identical for both groups, but the necrosis group showed significantly higher data scattering and range regarding the elastic modulus. The mean density of the coxarthrosis specimens was significantly higher than that of the necrotic specimens. INTERPRETATION: The mechanical properties of cancellous bone vary considerably in the presence of femoral head necrosis. The existence of hard necrosis implies a potential challenge regarding the clinical resection of these tissues.

Dynamic biomechanical investigation of a novel sulcus bicipitalis plate in combination with a conventional locking plate for the treatment of complex proximal humerus fractures.

BACKGROUND: Complex proximal humerus fractures place high demands on osteosynthetic treatment. In some cases, double plating has already been used to increase primary stability of the osteosynthesis. This approach was advanced in the present study by developing an additive plate for the sulcus bicipitalis. To demonstrate the superior primary stability of the newly developed plate osteosynthesis, a biomechanical comparison against a conventional locking plate with an additional calcar screw was performed. METHODS: Ten pairs of cadaveric humeri were treated proximally with a locking plate (PENTA plate small fragment, INTERCUS). Each had a two-part fracture model with a fracture gap of 10 mm. All right humeri were treated with an additive novel plate that extends along the bicipital sulcus and encircles the lesser tuberosity proximally. First, the specimens were loaded sinusoidally at 250 N in 20° abduction for 5000 cycles. Afterwards quasi-static loading until failure was applied. FINDINGS: The movement at the fracture gap due to the cyclic loading occurred mainly as rotation around the z-axis, corresponding to a tilt medially and distally. The double plate osteosynthesis reduces the rotation by approximately 39%. For all load cycles observed, except 5000 cycles, medial and distal rotation of the head was significantly reduced by the double plate. The failure loads showed no significant differences between the groups. INTERPRETATION: In the tested scenario under cyclic loading, the novel double plate osteosynthesis showed a significant superiority of primary stability over the conventional treatment with one locking plate. Furthermore, the study showed the advantages of cyclic load application over quasi-static load application until failure.

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor. In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

Mixture effects of co-formulants and two plant protection products in a liver cell line.

Plant protection products (PPPs) consist of one or more active substances and several co-formulants. Active substances provide the functionality of the PPP and are consequently evaluated according to standard test methods set by legal data requirements before approval, whereas co-formulants' toxicity is not as comprehensively assessed. However, in some cases mixture effects of active substances and co-formulants might result in increased or different forms of toxicity. In a proof-of-concept study we hence built on previously published results of Zahn et al. (2018[38]) on the mixture toxicity of Priori Xtra® and Adexar® to specifically investigate the influence of co-formulants on the toxicity of these commonly used fungicides. Products, their respective active substances in combination as well as some co-formulants were applied to human hepatoma cell line (HepaRG) in several dilutions. Cell viability analysis, mRNA expression, abundance of xenobiotic metabolizing enzymes and intracellular concentrations of active substances determined by LC-MS/MS analyses demonstrated that the toxicity of the PPPs is influenced by the presence of co-formulants in vitro. PPPs were more cytotoxic than the mix of their active substances. Gene expression profiles of cells treated with the PPPs were similar to those treated with their respective mixture combinations with marked differences. Co-formulants can cause gene expression changes on their own. LC-MS/MS analyses revealed higher intracellular concentrations of active substances in cells treated with PPPs compared to those treated with the respective active substances' mix. Proteomic data showed co-formulants can induce ABC transporters and CYP enzymes. Co-formulants can contribute to the observed increased toxicity of PPPs compared to their active substances in combination due to kinetic interactions, necessitating a more comprehensive evaluation approach.

Biomechanical testing of different fixation techniques for intraoperative proximal femur fractures: a technical note.

Intraoperative proximal femoral fractures (IPFF) represent a rare but challenging complication of total hip arthroplasties. They usually occur as a longitudinal split. This pilot trial aimed to compare the biomechanical primary stability of different fixation techniques for IPFF. Standardised longitudinal medial split fractures of the proximal femur (type II, Modified Mallory Classification) were created in artificial osteoporotic and non-osteoporotic composite femora after implantation of a cementless femoral stem. Five different fixation techniques were compared: cerclage band, cerclage wiring with one or two wires, and lag screw fixation with one or two lag screws. A quasi-static loading protocol was applied and failure loads were evaluated. The observed median failure loads were 4192N (3982N - 5189N) for one cerclage band, 4450N (3577N - 4927N) for one cerclage wire, 5016N (4175N - 5685N) for two cerclage wires, 6085N (5000N - 8907N) for one lag screw, and 4774N (4509N - 8502N) for two lag screws. Due to the wide range of failure loads within the experimental groups, there were no observable differences between the groups. All fixation techniques provided sufficient primary stability in osteoporotic and non-osteoporotic composite bones. Further cadaveric studies with larger sample sizes may be needed to confirm the results presented here.

The sacrotuberous ligament is preloaded in situ.

The objective of this study was to evaluate the intrinsic preload of the sacrotuberous ligament. Preload measurements and anatomical experiments were performed on 20 specimens from 10 human cadavers and assessed to consider the thesis of the ligamentous tension band system as a possible load distribution. The result was an unexpectedly high preload force with an overall average of 118 N ± 74 N. Age has been significantly different between females and males in the cohort (median 94 vs. 77 years). Nevertheless, there is preliminary evidence of the sex-dependent sacrotuberous ligament preload force with an average value of approximately 65 N for the 10 female cadaver specimens and 172 N for the 10 male cadaver specimens. The assessment of further influencing factors and their statistical evaluation also showed a dependence of the sacrotuberous ligament preload force on body height, age and elastin content. Thus, the sacrotuberous ligament is more preloaded in the cadaver than previously assumed in the literature. Therefore, and contrary to most assumptions, it could possibly also be more preloaded in a living person in an upright position under a muscular load. This leads to the hypothesis that pelvic stability is more dependent on ligamentous preload than previously thought. These considerations should be taken more into account in numerical simulations of sacroiliac joint function.

Scalable expansion of iPSC and their derivatives across multiple lineages.

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for "ready-to-use" cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools.

Atraumatic femoral head necrosis: a biomechanical, histological and radiological examination compared to primary hip osteoarthritis.

INTRODUCTION: Atraumatic necrosis of the femoral head (AFHN) is a common disease with an incidence of 5000-7000 middle-aged adults in Germany. There is no uniform consensus in the literature regarding the configuration of the bone in AFHN. The clinical picture of our patients varies from very hard bone, especially in idiopathic findings, and rather soft bone in cortisone-induced necrosis. A better understanding of the underlying process could be decisive for establishing a morphology-dependent approach. The aim of this study is the closer examination of the condition of the bone in the AFHN compared to the primary hip osteo arthritis (PHOA). MATERIALS AND METHODS: The preparations were obtained as part of elective endoprosthetic treatment of the hip joint. Immediately after sample collection, thin-slice CT of the preserved femoral heads was performed to determine the exact density of the bone in the necrosis zone. Reconstruction was done in 0.8-1 mm layers in two directions, coronary and axial, starting from the femoral neck axis. Density of the femoral heads was determined by grey value analysis. The value in Hounsfield units per sample head was averaged from three individual measurements to minimize fluctuations. For biomechanical and histomorphological evaluation, the samples were extracted in the load bearing zone perpendicular to the surface of the femoral head. Group-dependent statistical evaluation was performed using single factor variance analysis (ANOVA). RESULTS: A total of 41 patients with a mean age of 64.44 years were included. The mean bone density of the AFHN samples, at 1.432 g/cm3, was about 7% higher than in the PHOA group with a mean value of 1.350 g/cm3 (p = 0.040). The biomechanical testing in the AFHN group showed a 22% higher-but not significant-mean compressive strength (20.397 MPa) than in the PHOA group (16.733 MPa). On the basis of histological analysis, no differentiation between AFHN and PHOA samples was possible. CONCLUSIONS: The present study (NCT, evidence level II) shows that AFHN has a very well detectable higher bone density compared to PHOA. However, neither biomechanical stress tests nor histomorphological evaluation did show any significant difference between the groups. The results allow the conclusion that there is no "soft" necrosis at all in the AFHN group.

Effects of co-formulants on the absorption and secretion of active substances in plant protection products in vitro.

Currently, the authorisation process for plant protection products (PPPs) relies on the testing of acute and topological toxicity only. Contrastingly, the evaluation of active substances includes a more comprehensive set of toxicity studies. Nevertheless, mixture effects of active ingredients and co-formulants may result in increased toxicity. Therefore, we investigated effects of surface active co-formulants on the toxicity of two PPPs focussing on qualitative and quantitative toxicokinetic effects on absorption and secretion. The respective products are based on the active substances abamectin and fluroxypyr-meptyl and were tested for cytotoxicity in the presence or absence of the corresponding surfactants and co-formulants using Caco-2 cells. In addition, the effect of co-formulants on increased cellular permeation was quantified using LC-MS/MS, while potential kinetic mixture effects were addressed by fluorescence anisotropy measurements and ATPase assays. The results show that surface active co-formulants significantly increase the cytotoxicity of the investigated PPPs, leading to more than additive mixture effects. Moreover, analytical investigations show higher efflux ratios of both active substances and the metabolite fluroxypyr upon combination with certain concentrations of the surfactants. The results further point to a significant and concentration-dependent inhibition of Pgp transporters by most of the surfactants as well as to increased membrane fluidity. Altogether, these findings strongly support the hypothesis that surfactants contribute to increased cytotoxicity of PPPs and do so by increasing the bioavailability of the respective active substances.

Development of a fully human assay combining NGN2-inducible neurons co-cultured with iPSC-derived astrocytes amenable for electrophysiological studies.

Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a "fully-humanized" set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at https://cells.ebisc.org/.

Monkey V1 epidural field potentials provide detailed information about stimulus location, size, shape, and color.

Brain signal recordings with epidural microarrays constitute a low-invasive approach for recording distributed neuronal signals. Epidural field potentials (EFPs) may serve as a safe and highly beneficial signal source for a variety of research questions arising from both basic and applied neuroscience. A wider use of these signals, however, is constrained by a lack of data on their specific information content. Here, we make use of the high spatial resolution and the columnar organization of macaque primary visual cortex (V1) to investigate whether and to what extent EFP signals preserve information about various visual stimulus features. Two monkeys were presented with different feature combinations of location, size, shape, and color, yielding a total of 375 stimulus conditions. Visual features were chosen to access different spatial levels of functional organization. We found that, besides being highly specific for locational information, EFPs were significantly modulated by small differences in size, shape, and color, allowing for high stimulus classification rates even at the single-trial level. The results support the notion that EFPs constitute a low-invasive, highly beneficial signal source for longer-term recordings for medical and basic research by showing that they convey detailed and reliable information about constituent features of activating stimuli.

Influence of the fixation technique on the mechanical properties of human cancellous bone of the femoral head.

BACKGROUND: The femoral head is of central importance for the force transmission from the suprapelvic body mass to the lower extremity. However, the condition of the subcortical bone and its mechanical properties in case of pathological changes due to coxarthrosis or femoral head necrosis differ from the healthy condition. METHODS: Fresh femoral heads were gathered during hip total endoprosthesis surgeries and cylindrical cancellous bone samples were extracted with a hollow drill. By means of a uniaxial tensile-compression test system, the compressive strength was determined for two different specimen types (fresh and 24 h storage in acetone). Exemplary tests on an exceptionally large femoral head were performed to compare properties of fresh, fresh-deep-frozen and acetone-stored samples. FINDINGS: The deformation behaviour and the material parameters determined were very heterogeneous. For most of the specimens, a destructive material test was successfully carried out, i.e. the compressive strength was determined. The average strength of fresh specimens was slightly higher than that of acetone specimens. On the other hand, the average Young's modulus of the acetone specimens was higher than that of the fresh specimens. INTERPRETATION: The lower Young's moodulus of the fresh samples compared to the acetone samples could indicate a causal effect of the degreasing influence of the acetone. The partly considerable individual differences in compressive strength and failure compression can have patient-specific influencing factors such as constitution and physical fitness as well as causes in the initial pathological condition.

Podcasts as a teaching tool in orthopaedic surgery : Is it beneficial or more an exemption card from attending lectures?

OBJECTIVE: The aim of this study was to evaluate the influence of the introduction of online podcasts as part of the main lecture series in orthopaedics on the number of lecture attendees, the examination results and the assessment of teaching by the students. Additionally, we evaluated the use of other media for examination preparation. METHODOLOGY: At the beginning and end of the lecture series questionnaires were handed out to the students to evaluate their attitudes towards attending lectures, the use of video podcasts and examination preparation. In addition, the number of lecture attendees and podcast usage during the semester were counted and the statements of the students in the evaluation assessments of orthopaedic teaching were evaluated. The examination results were correlated in a statistical analysis with the learning materials provided by the students for examination preparation. RESULTS: At the end of the lecture series, 284 students stated that they used the lecture podcast about twice as often as attending lectures; however, for the majority of the students the provision of a video podcast was no reason not to attend the lecture. For example, 37.2% stated that they never and 26.8% stated that they rarely had not attended the lecture by providing the podcasts. Of the students 91-95% considered the availability of lecture podcasts to be a rather meaningful or very meaningful supplement to the lecture visit. Students increasingly used digital media to prepare for examinations instead of using traditional analogue methods. None of the learning methods or materials examined showed a statistically significant advantage in examination results. CONCLUSION: Students in the age of digitalization use a variety of learning materials and are no longer bound to classical analog teaching methods. The use of online podcasts had no negative impact on examination performance. Most students perceived lecture podcasts as a useful supplement to lecture attendance. The students praised the expansion of the teaching curriculum to include additional digital offers with positive comments in the evaluations, but without achieving an improvement in these student evaluations.

The influence of different sample preparation on mechanical properties of human iliotibial tract.

In the run-up to biomechanical testing, fresh human tissue samples are often frozen in order to inhibit initial decomposition processes and to achieve a temporal independence of tissue acquisition from biomechanical testing. The aim of this study was to compare the mechanical properties of fresh tissue samples of the human iliotibial tract (IT) to fresh-frozen samples taken from the same IT and those modified with different concentrations of Dimethylsulfoxide (DMSO) prior to freezing. All samples were partial plastinated and destructive tensile tests were conducted with a uniaxial tensile test setup. A plastination technique already established in the laboratory was modified to improve the clamping behaviour of the samples. Material failure was caused by a gradual rupture of the load-bearing collagen fibre bundles. Contrary to our expectations, no significant difference was found between the tensile strength of fresh and fresh frozen specimens. The addition of 1 wt% DMSO did not increase the tensile strength compared to fresh-frozen samples; an addition of 10 wt% DMSO even resulted in a decrease. Based on our findings, the use of simple fresh-frozen specimens to determine the tensile strength is viable; however fresh specimens should be used to generate a complete property profile.