Decrease of exosomal miR-21-5p and the increase of CD62p+ exosomes are associated with the development of sepsis in polytraumatized patients.

Sepsis as a severe systemic inflammation leads oftentimes to organ dysfunction and subsequently to death. In polytrauma patients, septic complications represent with 45% the predominant cause of late death and are responsible for extremely high costs in the healthcare system. Therefore, clinicians have to detect as early as possible the begin of sepsis to improve the patient's outcome. One new promising diagnostic tool to diagnose septic complications in polytraumatized patients are exosomes. Plasma samples from polytraumatized patients (Injury Severity Score (ISS) ≥16) which developed sepsis (n = 10) and without sepsis (n = 10), were collected at emergency room (ER), 24h and 5 days after trauma. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with plasma EVs from healthy controls (n = 10). Moreover, exosomal cytokine concentrations were measured via high-sensitive ELISA and were correlated with systemic concentrations. For miRNA cargo analysis, we analysed the miRNAs miR-1298-5p, miR-1262, miR-125b-5p, miR-92a-3p, miR-93-5p, miR-155-5p and miR-21-5p and compared their exosomal concentrations by means of RT-qPCR. CD62p + exosomes were significantly increased in septic polytrauma-patients (p ≤ 0.05), while CD40+exosomes, as well as CD49e + exosomes were diminished (p ≤ 0.05). Furthermore, we observed that the exosomal IL-6 concentration reflects the systemic IL-6 concentration (r2 = 0.63) and did not significantly alter between patients with and without sepsis. The exosomal IL-10 concentration seemed to be constant in all patients and healthy controls. We observed that a decrease of miR-21-5p in exosomes was associated with the development of sepsis (p ≤ 0.05), while exosomal miR-93-5p, miR-155-5p and miR-92a-3p were not specifically altered in septic patients. Taken together, the present study in polytraumatized patients demonstrated that the development of sepsis is associated with an increase of CD62p + exosomes. Furthermore, the exosomal cargo was changed in septic patients: miR-21-5p was diminished.

Human Acellular Collagen Matrices-Clinical Opportunities in Tissue Replacement.

The field of regenerative medicine is increasingly in need of effective and biocompatible materials for tissue engineering. Human acellular dermal matrix (hADM)-derived collagen matrices stand out as a particularly promising candidate. Their ability to preserve structural integrity, coupled with exceptional biocompatibility, positions them as a viable choice for tissue replacement. However, their clinical application has been largely confined to serving as scaffolds. This study aims to expand the horizon of clinical uses for collagen sheets by exploring the diverse cutting-edge clinical demands. This review illustrates the clinical utilizations of collagen sheets beyond traditional roles, such as covering skin defects or acting solely as scaffolds. In particular, the potential of Epiflex®, a commercially available and immediately clinically usable allogeneic membrane, will be evaluated. Collagen sheets have demonstrated efficacy in bone reconstruction, where they can substitute the induced Masquelet membrane in a single-stage procedure, proving to be clinically effective and safe. The application of these membranes allow the reconstruction of substantial tissue defects, without requiring extensive plastic reconstructive surgery. Additionally, they are found to be apt for addressing osteochondritis dissecans lesions and for ligament reconstruction in the carpus. The compelling clinical examples showcased in this study affirm that the applications of human ADM extend significantly beyond its initial use for skin defect treatments. hADM has proven to be highly successful and well-tolerated in managing various etiologies of bone and soft tissue defects, enhancing patient care outcomes. In particular, the application from the shelf reduces the need for additional surgery or donor site defects.

Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)-Establishment of the First EV Biobank for Polytraumatized Patients.

In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing-thawing cycles and different storage conditions (RT, 4/-20/-80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing-thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.

Identification of novel blood-based extracellular vesicles biomarker candidates with potential specificity for traumatic brain injury in polytrauma patients.

Objective: The goal of this study was to identify changes in extracellular vesicles (EV) surface proteins specific to traumatic brain injury (TBI), which could be used as a diagnostic and prognostic tool in polytrauma patients. Summary Background Data: Known serum TBI-specific biomarkers (S100B, NSE, and GFAP), which can predict the severity and outcome of isolated TBI, lose their predictive value in the presence of additional extracranial injuries. Extracellular vesicles (EVs) are released from cells in response to various stimuli and carry specific cargo/surface molecules that could be used for tracking injury-responding cells. Methods: EVs were isolated using size exclusion chromatography (SEC) from the plasma of two groups of patients (with isolated TBI, ISS≥16, AIShead≥4, n=10; and polytraumatized patients without TBI ISS≥16, AIShead=0, n=10) collected in the emergency room and 48 h after trauma. EVs' surface epitope expression was investigated using a neurospecific multiplex flow cytometry assay and compared with healthy controls (n=10). Three enrichments of EV epitopes found to be specific to TBI were validated by western blot. Results: The expression of 10 EV epitopes differed significantly among the patient and control groups, and five of these epitopes (CD13, CD196, MOG, CD133, and MBP) were TBI-specific. The increased expression of CD196, CD13, and MOG-positive EVs was validated by western blot. Conclusion: Our data showed that TBI is characterized by a significant increase of CD13, CD196, MOG, CD133, and MBP-positive EVs in patients' plasma. A high level of MOG-positive EVs negatively correlated with the Glasgow Coma Scale score at admission and could be an indicator of poor neurological status.

Evaluation of New Cardiac Damage Biomarkers in Polytrauma: GDF-15, HFABP and uPAR for Predicting Patient Outcomes.

Background: Polytrauma is one of the leading mortality factors in younger patients, and in particular, the presence of cardiac damage correlates with a poor prognosis. Currently, troponin T is the gold standard, although troponin is limited as a biomarker. Therefore, there is a need for new biomarkers of cardiac damage early after trauma. Methods: Polytraumatized patients (ISS ≥ 16) were divided into two groups: those with cardiac damage (troponin T > 50 pg/mL, n = 37) and those without cardiac damage (troponin T < 12 pg/mL, n = 32) on admission to the hospital. Patients' plasma was collected in the emergency room 24 h after trauma, and plasma from healthy volunteers (n = 10) was sampled. The plasma was analyzed for the expression of HFABP, GDF-15 and uPAR proteins, as well as miR-21, miR-29, miR-34, miR-122, miR-125b, miR-133, miR-194, miR-204, and miR-155. Results were correlated with patients' outcomes. Results: HFABP, uPAR, and GDF-15 were increased in polytraumatized patients with cardiac damage (p < 0.001) with a need for catecholamines. HFABP was increased in non-survivors. Analysis of systemic miRNA concentrations showed a significant increase in miR-133 (p < 0.01) and miR-21 (p < 0.05) in patients with cardiac damage. Conclusion: All tested plasma proteins, miR-133, and miR-21 were found to reflect the cardiac damage in polytrauma patients. GDF-15 and HFABP were shown to strongly correlate with patients' outcomes.

Elevated extracellular particle concentration in plasma predicts in-hospital mortality after severe trauma.

Background: Extracellular particles (EPs), particularly extracellular vesicles, play a crucial role in regulating various pathological mechanisms, including immune dysregulations post-trauma. Their distinctive expression of cell-specific markers and regulatory cargo such as cytokines or micro-ribonucleic acid suggests their potential as early biomarkers for organ-specific damage and for identifying patients at risk for complications and mortality. Given the critical need for reliable and easily assessable makers to identify at-risk patients and guide therapeutic decisions, we evaluated the early diagnostic value of circulating EPs regarding outcomes in severely injured multiple-trauma patients. Methods: Plasma samples were collected from 133 severely injured trauma patients (Injury Severity Score (ISS) ≥16) immediately upon arrival at the emergency department (ED). Patients were categorized into survivors and non-survivors. Injury characteristics and outcomes related to sepsis, pneumonia, or early (<1 day after admission) and late mortality were assessed. Circulating EPs, cytokine profiles, and blood counts of platelets and leukocytes were determined. Receiver operating characteristic analyses were conducted. Results: Despite no significant differences in injury pattern or severity, non-survivors exhibited significantly elevated counts of circulating EPs compared to survivors. The optimal cut-off for EPs <200 nm indicating non-survivors was 17380/µl plasma, with a sensitivity of 77% and a specificity of 61% in predicting in-hospital mortality. Later non-survivors received significantly higher numbers of units of packed red blood cells [8.54 ± 5.45 vs. 1.29 ± 0.36 units], had higher serum lactate [38.00 ± 7.51 vs. 26.98 ± 1.58 mg/dL], significantly lower platelet counts [181.30 ± 18.06 vs. 213.60 ± 5.85 *10³/µL] and lower heart rates [74.50 ± 4.93 vs. 90.18 ± 2.06 beats/minute] upon arrival at the ED compared to survivors. Conclusion: Our results demonstrate the high diagnostic potential of elevated concentrations of circulating EPs <200 nm for identifying patients at risk of mortality after severe trauma. This parameter shows comparable sensitivity to established clinical predictors. Early evaluation of EPs concentration could complement assessment markers in guiding early therapeutic decisions.