Fracture haematoma proteomics.

Aims: The aim of this study was to determine the fracture haematoma (fxH) proteome after multiple trauma using label-free proteomics, comparing two different fracture treatment strategies. Methods: A porcine multiple trauma model was used in which two fracture treatment strategies were compared: early total care (ETC) and damage control orthopaedics (DCO). fxH was harvested and analyzed using liquid chromatography-tandem mass spectrometry. Per group, discriminating proteins were identified and protein interaction analyses were performed to further elucidate key biomolecular pathways in the early fracture healing phase. Results: The early fxH proteome was characterized by immunomodulatory and osteogenic proteins, and proteins involved in the coagulation cascade. Treatment-specific proteome alterations were observed. The fxH proteome of the ETC group showed increased expression of pro-inflammatory proteins related to, among others, activation of the complement system, neutrophil functioning, and macrophage activation, while showing decreased expression of proteins related to osteogenesis and tissue remodelling. Conversely, the fxH proteome of the DCO group contained various upregulated or exclusively detected proteins related to tissue regeneration and remodelling, and proteins related to anti-inflammatory and osteogenic processes. Conclusion: The early fxH proteome of the ETC group was characterized by the expression of immunomodulatory, mainly pro-inflammatory, proteins, whereas the early fxH proteome of the DCO group was more regenerative and osteogenic in nature. These findings match clinical observations, in which enhanced surgical trauma after multiple trauma causes dysbalanced inflammation, potentially leading to reduced tissue regeneration, and gained insights into regulatory mechanisms of fracture healing after severe trauma.

Porcine Mandibular Bone Marrow-Derived Mesenchymal Stem Cell (BMSC)-Derived Extracellular Vesicles Can Promote the Osteogenic Differentiation Capacity of Porcine Tibial-Derived BMSCs.

OBJECTIVE: Existing research suggests that bone marrow-derived mesenchymal stem cells (BMSCs) may promote endogenous bone repair. This may be through the secretion of factors that stimulate repair processes or directly through differentiation into osteoblast-progenitor cells. However, the osteogenic potential of BMSCs varies among different tissue sources (e.g., mandibular versus long BMSCs). The main aim of this study was to investigate the difference in osteogenic differentiation capacity between mandibular BMSCs (mBMSCs) and tibial BMSCs (tBMSCs). MATERIALS AND METHODS: Bioinformatics analysis of the GSE81430 dataset taken from the Gene Expression Omnibus (GEO) database was performed using GEO2R. BMSCs were isolated from mandibular and tibial bone marrow tissue samples. Healthy pigs (n = 3) (registered at the State Office for Nature, Environment, and Consumer Protection, North Rhine-Westphalia (LANUV) 81-02.04.2020.A215) were used for this purpose. Cell morphology and osteogenic differentiation were evaluated in mBMSCs and tBMSCs. The expression levels of toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively. In addition, mBMSC-derived extracellular vesicles (mBMSC-EVs) were gained and used as osteogenic stimuli for tBMSCs. Cell morphology and osteogenic differentiation capacity were assessed after mBMSC-EV stimulation. RESULTS: Bioinformatic analysis indicated that the difference in the activation of the TLR4/NF-κB pathway was more pronounced compared to all other examined genes. Specifically, this demonstrated significant downregulation, whereas only 5-7 upregulated genes displayed significant variances. The mBMSC group showed stronger osteogenic differentiation capacity compared to the tBMSC group, confirmed via ALP, ARS, and von Kossa staining. Furthermore, qPCR and WB analysis revealed a significant decrease in the expression of the TLR4/NF-κB pathway in the mBMSC group compared to the tBMSC group (TLR4 fold changes: mBMSCs vs. tBMSCs p < 0.05; NF-κB fold changes: mBMSCs vs. tBMSCs p < 0.05). The osteogenic differentiation capacity was enhanced, and qPCR and WB analysis revealed a significant decrease in the expression of TLR4 and NF-κB in the tBMSC group with mBMSC-EVs added compared to tBMSCs alone (TLR4 fold changes: p < 0.05; NF-κB fold changes: p < 0.05). CONCLUSION: Our results indicate that mBMSC-EVs can promote the osteogenic differentiation of tBMSCs in vitro. The results also provide insights into the osteogenic mechanism of mBMSCs via TLR4/NF-κB signaling pathway activation. This discovery promises a fresh perspective on the treatment of bone fractures or malunions, potentially offering a novel therapeutic method.

Liquid Biopsy in Organ Damage: small extracellular vesicle chip-based assessment of polytrauma.

Background: Despite major advances in medicine, blood-borne biomarkers are urgently needed to support decision-making, including polytrauma. Here, we assessed serum-derived extracellular vesicles (EVs) as potential markers of decision-making in polytrauma. Objective: Our Liquid Biopsy in Organ Damage (LiBOD) study aimed to differentiate polytrauma with organ injury from polytrauma without organ injury. We analysed of blood-borne small EVs at the individual level using a combination of immunocapture and high-resolution imaging. Methods: To this end, we isolated, purified, and characterized small EVs according to the latest Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from human blood collected within 24 h post-trauma and validated our results using a porcine polytrauma model. Results: We found that small EVs derived from monocytes CD14+ and CD14+CD61+ were significantly elevated in polytrauma with organ damage. To be precise, our findings revealed that CD9+CD14+ and CD14+CD61+ small EVs exhibited superior performance compared to CD9+CD61+ small EVs in accurately indicating polytrauma with organ damage, reaching a sensitivity and a specificity of 0.81% and 0.97%, respectively. The results in humans were confirmed in an independent porcine model of polytrauma. Conclusion: These findings suggest that these specific types of small EVs may serve as valuable, non-invasive, and objective biomarkers for assessing and monitoring the severity of polytrauma and associated organ damage.

Circulating miRNA expression in extracellular vesicles is associated with specific injuries after multiple trauma and surgical invasiveness.

Introduction: Two trauma treatment principles are Early Total Care (ETC), and Damage Control Orthopedics (DCO). Cellular mechanisms that underlie the connection between treatment type, its systemic effects, and tissue regeneration are not fully known. Therefore, this study aimed to: 1) profile microRNA (miRNA) expression in plasma derived Extracellular Vesicles (EVs) from a porcine multiple trauma model at different timepoints, comparing two surgical treatments; and 2) determine and validate the miRNA's messengerRNA (mRNA) targets. Methods: The porcine multiple trauma model consisted of blunt chest trauma, liver laceration, bilateral femur fractures, and controlled haemorrhagic shock. Two treatment groups were defined, ETC (n=8), and DCO (n=8). Animals were monitored under Intensive Care Unit-standards, blood was sampled at 1.5, 2.5, 24, and 72 hours after trauma, and EVs were harvested from plasma. MiRNAs were analysed using quantitative Polymerase Chain Reaction arrays. MRNA targets were identified in silico and validated in vivo in lung and liver tissue. Results: The arrays showed distinct treatment specific miRNA expression patterns throughout all timepoints, and miRNAs related to the multiple trauma and its individual injuries. EV-packed miRNA expression in the ETC group was more pro-inflammatory, indicating potentially decreased tissue regenerative capacities in the acute post-traumatic phase. In silico target prediction revealed several overlapping mRNA targets among the identified miRNAs, related to inflammation, (pulmonary) fibrosis, and Wnt-signalling. These were, among others, A Disintegrin and Metalloproteinase domain-containing protein 10, Collagen Type 1 Alpha 1 Chain, Catenin Beta Interacting Protein 1, and Signal Transducers and Activators of Transcription 3. Validation of these mRNA targets in the lung showed significant, treatment specific deregulations which matched the expression of their upstream miRNAs. No significant mRNA deregulations were observed in the liver. Discussion: This study showed treatment specific, EV-packed miRNA expression patterns after trauma that correlated with mRNA expressions in the lungs, target organs over distance. A systemic response to the increased surgical trauma in the ETC group was identified, with various miRNAs associated with injuries from the trauma model, and involved in (systemic) inflammation, tissue regeneration. EV-transported miRNAs demonstrated a clear role in multiple trauma, warranting further research into tissue-tissue talk and therapeutic applications of EVs after trauma.

Simultaneous C5 and CD14 inhibition limits inflammation and organ dysfunction in pig polytrauma.

Dysfunctional complement activation and Toll-like receptor signaling immediately after trauma are associated with development of trauma-induced coagulopathy and multiple organ dysfunction syndrome. We assessed the efficacy of the combined inhibition therapy of complement factor C5 and the TLR co-receptor CD14 on thrombo-inflammation and organ damage in an exploratory 72-h polytrauma porcine model, conducted under standard surgical and intensive care management procedures. Twelve male pigs were subjected to polytrauma, followed by resuscitation (ATLS® guidelines) and operation of the femur fracture (intramedullary nailing technique). The pigs were allocated to combined C5 and CD14 inhibition therapy group (n=4) and control group (n=8). The therapy group received intravenously C5 inhibitor (RA101295) and anti-CD14 antibody (rMil2) 30 min post-trauma. Controls received saline. Combined C5 and CD14 inhibition reduced the blood levels of the terminal complement complex (TCC) by 70% (p=0.004), CRP by 28% (p=0.004), and IL-6 by 52% (p=0.048). The inhibition therapy prevented the platelet consumption by 18% and TAT formation by 77% (p=0.008). Moreover, the norepinephrine requirements in the treated group were reduced by 88%. The inhibition therapy limited the organ damage, thereby reducing the blood lipase values by 50% (p=0.028), LDH by 30% (p=0.004), AST by 33%, and NGAL by 30%. Immunofluorescent analysis of the lung tissue revealed C5b-9 deposition on blood vessels in five from the untreated, and in none of the treated animals. In kidney and liver, the C5b-9 deposition was similarly detected mainly the untreated as compared to the treated animals. Combined C5 and CD14 inhibition limited the inflammatory response, the organ damage, and reduced the catecholamine requirements after experimental polytrauma and might be a promising therapeutic approach.

Fracture fixation strategy and specific muscle tissue availability of neutrophilic granulocytes following mono- and polytrauma: intramedullary nailing vs. external fixation of femoral fractures.

BACKGROUND: In the stabilization of femoral fractures in mono- and polytrauma, clinical practice has shown better care through intramedullary nailing. However, the reason why this is the case is not fully understood. In addition to concomitant injuries, the immunological aspect is increasingly coming to the fore. Neutrophil granulocytes (PMNL), in particular next to other immunological cell types, seem to be associated with the fracture healing processes. For this reason, the early phase after fracture (up to 72 h after trauma) near the fracture zone in muscle tissue was investigated in a pig model. MATERIAL AND METHODS: A mono- and polytrauma pig model (sole femur fracture or blunt thoracic trauma, hemorrhagic shock, liver laceration, and femur fracture) was used to demonstrate the immunological situation through muscle biopsies and their analysis by histology and qRT-PCR during a 72 h follow-up phase. Two stabilization methods were used (intramedullary nail vs. external fixator) and compared with a nontraumatized sham group. RESULTS: Monotrauma shows higher PMNL numbers in muscle tissue compared with polytrauma (15.52 ± 5.39 mono vs. 8.23 ± 3.36 poly; p = 0.013), regardless of the treatment strategy. In contrast, polytrauma shows a longer lasting invasion of PMNL (24 h vs. 72 h). At 24 h in the case of monotrauma, the fracture treated with external fixation shows more PMNL than the fracture treated with intramedullary nailing (p = 0.026). This difference cannot be determined in polytrauma probably caused by a generalized immune response. Both monotrauma and polytrauma show a delayed PMNL increase in the muscle tissue of the uninjured side. The use of intramedullary nailing in monotrauma resulted in a significant increase in IL-6 (2 h after trauma) and IL-8 (24 and 48 h after trauma) transcription. CONCLUSION: The reduction of PMNL invasion into the nearby muscle tissue of a monotrauma femur fracture stabilized by intramedullary nailing supports the advantages found in everyday clinical practice and therefore underlines the usage of nailing. For the polytrauma situation, the fixation seems to play a minor role, possibly due to a generalized immune reaction.

Validation of the mTICCS Score as a Useful Tool for the Early Prediction of a Massive Transfusion in Patients with a Traumatic Hemorrhage.

The modified Trauma-Induced Coagulopathy Clinical Score (mTICCS) presents a new scoring system for the early detection of the need for a massive transfusion (MT). While validated in a large trauma cohort, the comparison of mTICCS to established scoring systems is missing. This study therefore validated the ability of six scoring systems to stratify patients at risk for an MT at an early stage after trauma. A dataset of severely injured patients (ISS ≥ 16) derived from the database of a level I trauma center (2010-2015) was used. Scoring systems assessed were Trauma-Associated Severe Hemorrhage (TASH) score, Prince of Wales Hospital (PWH) score, Larson score, Assessment of Blood Consumption (ABC) score, Emergency Transfusion Score (ETS), and mTICCS. Demographics, diagnostic data, mechanism of injury, injury pattern (graded by AIS), and outcome (length of stay, mortality) were analyzed. Scores were calculated, and the area under the receiver operating characteristic curves (AUCs) were evaluated. From the AUCs, the cut-off point with the best relationship of sensitivity-to-specificity was used to recalculate sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV). A total of 479 patients were included; of those, blunt trauma occurred in 92.3% of patients. The mean age of patients was 49 ± 22 years with a mean ISS of 25 ± 29. The overall MT rate was 8.4% (n = 40). The TASH score had the highest overall accuracy as reflected by an AUC of 0.782 followed by the mTICCS (0.776). The ETS was the most sensitive (80%), whereas the TASH score had the highest specificity (82%) and the PWH score had the lowest (51.83%). At a cut-off > 5 points, the mTICCS score showed a sensitivity of 77.5% and a specificity of 74.03%. Compared to sophisticated systems, using a higher number of weighted variables, the newly developed mTICCS presents a useful tool to predict the need for an MT in a prehospital situation. This might accelerate the diagnosis of an MT in emergency situations. However, prospective validations are needed to improve the development process and use of scoring systems in the future.

A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.