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Background: Hypoxia-inducible factor-1α (HIF-1α) and NF-κB play important roles in the inflammatory response after hemorrhagic shock and resuscitation (H/R). Here, the role of myeloid HIF-1α in liver hypoxia, injury, and inflammation after H/R with special regard to NF-κB activation was studied.
Methods: Mice with a conditional HIF-1α knockout (KO) in myeloid cell-line and wild-type (WT) controls were hemorrhaged for 90 min ( mm Hg) and resuscitated. Controls underwent only surgical procedures.
Results: After six hours, H/R enhanced the expression of HIF-1α-induced genes vascular endothelial growth factor (VEGF) and adrenomedullin (ADM). In KO mice, this was not observed. H/R-induced liver injury in HIF-1α KO was comparable to WT. Elevated plasma interleukin-6 (IL-6) levels after H/R were not reduced by HIF-1α KO. Local hepatic hypoxia was not significantly reduced in HIF-1α KO compared to controls after H/R. H/R-induced NF-κB phosphorylation in liver did not significantly differ between WT and KO.
Conclusions: Here, deleting HIF-1α in myeloid cells and thereby in Kupffer cells was not protective after H/R. This data indicates that other factors, such as NF-κB, due to its upregulated phosphorylation in WT and KO mice, contrary to HIF-1α, are rather key modulators of inflammation after H/R in our model.
Highlights
• CD62p + exosomes were significantly increased in septic polytrauma-patients, while CD40+, as well as CD49e + exosomes were diminished.
• Exosomal IL-6 concentration in septic patients reflects the systemic IL-6.
• Exosomal IL-10 concentration seemed to be constant in patients and healthy controls.
• Decrease of miR-21 in exosomes was associated with the development of sepsis, while exosomal miR-93, miR-155 and miR-92a were not specifically altered.
Abstract
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.
Due to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.
The creation of entirely synthetically derived bone substitute materials which are as effective as autologous bone grafts is desirable. Osteogenesis involves the concerted action of several proteins within a signaling cascade. Hedgehog proteins act upstream of this cascade, inducing the expression of various bone morphogenetic proteins (BMPs) and promoting physiological bone healing. Therefore, the hypothesis that hedgehog signaling in bone defects improves bone healing more than BMP signaling alone was tested. Recombinant N-terminal sonic hedgehog protein (N-SHh), BMP-2 or a combination of the two was added to β-tricalcium phosphate (β-TCP) and 5-mm femoral midshaft defects in nude rats were filled with these composites. The defects were stabilized with mini-plates. After eight weeks, the animals were sacrificed and the femora were explanted. The radiological evaluation was followed by a three-point bending test and histological examination. BMP-2/β-TCP composites showed a trend of increased stiffness compared with the controls (β-TCP without protein). N-SHh/β-TCP composites had lower stiffness compared with the control group and the N-SHh/BMP-2/β-TCP composites also had lower average stiffness compared with the controls (all not significant). Histomorphometry, however, revealed abundant cartilage and bone core formation in the N-SHh-composite groups. The sum of the new cartilage and bone was highest in the combination group N-SHh/BMP-2 (not significant). The addition of N-SHh to bone substitute materials appears to delay bone healing at the applied concentration and observation time but also showed a trend for higher amounts of ossifying cartilage.
The Masquelet technique is used to treat large bone defects; it is a two-stage procedure based on an induced membrane. To improve the induced membrane process, demineralized bone matrix in granular (GDBM) and fibrous form (f-DBM) was tested with and without bone marrow mononuclear cells (BMC) as filling of the membrane against the gold standard filling with syngeneic cancellous bone (SCB). A total of 65 male Sprague–Dawley rats obtained a 5 mm femoral defect. These defects were treated with the induced membrane technique and filled with SCB, GDBM, or f-DBM, with or without BMC. After a healing period of eight weeks, the femurs were harvested and submitted for histological, radiological, and biomechanical analyses. The fracture load in the defect zone was lower compared to SCB in all groups. However, histological analysis showed comparable new bone formation, bone mineral density, and cartilage proportions and vascularization. The results suggest that f-DBM in combination with BMC and the induced membrane technique cannot reproduce the very good results of this material in large, non-membrane coated bone defects, nevertheless it supports the maturation of new bone tissue locally. It can be concluded that BMC should be applied in lower doses and inflammatory cells should be removed from the cell preparation before implantation.
Objectives: Reconstruction of long segmental bone defects is demanding for patients and surgeons, and associated with long-term treatment periods and substantial complication rates in addition to high costs. While defects up to 4–5 cm length might be filled up with autologous bone graft, heterologous bone from cadavers, or artificial bone graft substitutes, current options to reconstruct bone defects greater than 5 cm consist of either vascularized free bone transfers, the Masquelet technique or the Ilizarov distraction osteogenesis. Alternatively, autologous cell transplantation is an encouraging treatment option for large bone defects as it eliminates problems such as limited autologous bone availability, allogenic bone immunogenicity, and donor-site morbidity, and might be used for stabilizing loose alloplastic implants.
Methods: The authors show different cell therapies without expansion in culture, with ex vivo expansion and cell therapy in local bone defects, bone healing and osteonecrosis. Different kinds of cells and scaffolds investigated in our group as well as in vivo transfer studies and BMC used in clinical phase I and IIa clinical trials of our group are shown.
Results: Our research history demonstrated the great potential of various stem cell species to support bone defect healing. It was clearly shown that the combination of different cell types is superior to approaches using single cell types. We further demonstrate that it is feasible to translate preclinically developed protocols from in vitro to in vivo experiments and follow positive convincing results into a clinical setting to use autologous stem cells to support bone healing.
Introduction: The induced membrane technique for the treatment of large bone defects is a two-step procedure. In the first operation, a foreign body membrane is induced around a spacer, then, in the second step, several weeks or months later, the spacer is removed and the Membrane pocket is filled with autologous bone material. Induction of a functional biological membrane might be avoided by initially using a biological membrane. In this study, the effect of a human acellular dermis (hADM, Epiflex, DIZG gGmbH) was evaluated for the treatment of a large (5 mm), plate-stabilised femoral bone defect.
Material and Methods: In an established rat model, hADM was compared to the two-stage induced membrane technique and a bone defect without membrane cover. Syngeneous spongiosa from donor animals was used for defect filling in all groups. The group size in each case was n = 5, the induction time of the membrane was 3–4 weeks and the healing time after filling of the defect was 8 weeks.
Results: The ultimate loads were increased to levels comparable with native bone in both membrane groups (hADM: 63.2% ± 29.6% of the reference bone, p < 0.05 vs. no membrane, induced membrane: 52.1% ± 25.8% of the reference bone, p < 0.05 vs. no membrane) and were significantly higher than the control group without membrane (21.5%). The membrane groups were radiologically and histologically almost completely bridged by new bone formation, in contrast to the control Group where no closed osseous bridging could be observed.
Conclusion: The use of the human acellular dermis leads to equivalent healing results in comparison to the two-stage induced membrane technique. This could lead to a shortened therapy duration of large bone defects.
Regeneration of large bone defects is a major objective in trauma surgery. Bone marrow mononuclear cell (BMC)-supported bone healing was shown to be efficient after immobilization on a scaffold. We hypothesized that fibrous demineralized bone matrix (DBM) in various forms with BMCs is superior to granular DBM. A total of 65 male SD rats were assigned to five treatment groups: syngenic cancellous bone (SCB), fibrous demineralized bone matrix (f-DBM), fibrous demineralized bone matrix densely packed (f-DBM 120%), DBM granules (GDBM) and DBM granules 5% calcium phosphate (GDBM5%Ca2+). BMCs from donor rats were combined with different scaffolds and placed into 5 mm femoral bone defects. After 8 weeks, bone mineral density (BMD), biomechanical stability and histology were assessed. Similar biomechanical properties of f-DBM and SCB defects were observed. Similar bone and cartilage formation was found in all groups, but a significantly bigger residual defect size was found in GDBM. High bone healing scores were found in f-DBM (25) and SCB (25). The application of DBM in fiber form combined with the application of BMCs shows promising results comparable to the gold standard, syngenic cancellous bone. Denser packing of fibers or higher amount of calcium phosphate has no positive effect.
The design of novel biomaterials should directly influence the host-immune system and steer it towards high biocompatibility. To date, new implants/materials have been tested for biocompatibility in vitro in cell cultures and in vivo in animal models. The current methods do not reflect reality (cell cultures) or are very time-consuming and deliver results only after weeks (animal model). In this proof-of-concept study, the suitability of a Whole Blood Stimulation Assay (WBSA) in combination with a Protein Profiler Array (PPA), as a readily available and cost-effective screening tool, was investigated. Three different biomaterials based on poly(lactic-co-glycolic acid (PLGA), calcium sulphate/-carbonate (CS) and poly(methyl methacrylate) (PMMA) were exposed to native whole blood from three volunteers and subsequently screened with a PPA. Individual reproducible protein profiles could be detected for all three materials after 24 h of incubation. The most intense reaction resulted from the use of PLGA, followed by CS. If even marginal differences in implants can be reflected in protein profiles, the combination of WBSA and PPA could serve as an early biocompatibility screening tool in the development of novel biomaterials. This may also lead to a reduction in costs and the amount of animal testing required.
In Bone Tissue Engineering (BTE), autologous bone-regenerative cells are combined with a scaffold for large bone defect treatment (LBDT). Microporous, polylactic acid (PLA) scaffolds showed good healing results in small animals. However, transfer to large animal models is not easily achieved simply by upscaling the design. Increasing diffusion distances have a negative impact on cell survival and nutrition supply, leading to cell death and ultimately implant failure. Here, a novel scaffold architecture was designed to meet all requirements for an advanced bone substitute. Biofunctional, porous subunits in a load-bearing, compression-resistant frame structure characterize this approach. An open, macro- and microporous internal architecture (100 µm–2 mm pores) optimizes conditions for oxygen and nutrient supply to the implant’s inner areas by diffusion. A prototype was 3D-printed applying Fused Filament Fabrication using PLA. After incubation with Saos-2 (Sarcoma osteogenic) cells for 14 days, cell morphology, cell distribution, cell survival (fluorescence microscopy and LDH-based cytotoxicity assay), metabolic activity (MTT test), and osteogenic gene expression were determined. The adherent cells showed colonization properties, proliferation potential, and osteogenic differentiation. The innovative design, with its porous structure, is a promising matrix for cell settlement and proliferation. The modular design allows easy upscaling and offers a solution for LBDT.
Treatment of large bone defects is one of the great challenges in contemporary orthopedic and traumatic surgery. Grafts are necessary to support bone healing. A well-established allograft is demineralized bone matrix (DBM) prepared from donated human bone tissue. In this study, a fibrous demineralized bone matrix (f-DBM) with a high surface-to-volume ratio has been analyzed for toxicity and immunogenicity. f-DBM was transplanted to a 5-mm, plate-stabilized, femoral critical-size-bone-defect in Sprague-Dawley (SD)-rats. Healthy animals were used as controls. After two months histology, hematological analyses, immunogenicity as well as serum biochemistry were performed. Evaluation of free radical release and hematological and biochemical analyses showed no significant differences between the control group and recipients of f-DBM. Histologically, there was no evidence of damage to liver and kidney and good bone healing was observed in the f-DBM group. Reactivity against human HLA class I and class II antigens was detected with mostly low fluorescence values both in the serum of untreated and treated animals, reflecting rather a background reaction. Taken together, these results provide evidence for no systemic toxicity and the first proof of no basic immunogenic reaction to bone allograft and no sensitization of the recipient.
Delayed wound repair in sepsis is associated with reduced local pro-inflammatory cytokine expression
(2013)
Sepsis is one of the main causes for morbidity and mortality in hospitalized patients. Moreover, sepsis associated complications involving impaired wound healing are common. Septic patients often require surgical interventions that in-turn may lead to further complications caused by impaired wound healing. We established a mouse model to the study delayed wound healing during sepsis distant to the septic focus point. For this reason cecal ligation and puncture (CLP) was combined with the creation of a superficial wound on the mouse ear. Control animals received the same procedure without CPL. Epithelialization was measured every second day by direct microscopic visualization up to complete closure of the wound. As interplay of TNF-α, TGF-β, matrix metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) is important in wound healing in general, TNF-α, TGF-β, MMP7, and TIMP1 were assessed immunohistochemical in samples of wounded ears harvested on days 2, 6, 10 and 16 after wounding. After induction of sepsis, animals showed a significant delay in wound epithelialization from day 2 to 12 compared to control animals. Complete wound healing was attained after mean 12.2± standard deviation (SD) 3.0 days in septic animals compared to 8.7± SD 1.7 days in the control group. Septic animals showed a significant reduction in local pro-inflammatory cytokine level of TNF-α on day 2 and day 6 as well as a reduced expression of TGF-β on day 2 in wounds. A significant lower expression of MMP7 as well as TIMP1 was also observed on day 2 after wounding. The induction of sepsis impairs wound healing distant to the septic focus point. We could demonstrate that expression of important cytokines for wound repair is deregulated after induction of sepsis. Thus restoring normal cytokine response locally in wounds could be a good strategy to enhance wound repair in sepsis.
Epoxyeicosatrienoic acids (EET) facilitate regeneration in different tissues, and their benefit in dermal wound healing has been proven under normal conditions. In this study, we investigated the effect of 11,12 EET on dermal wound healing in diabetes. We induced diabetes by i.p. injection of streptozotocin 2 weeks prior to wound creation on the dorsal side of the mouse ear. 11,12 EET was applied every second day on the wound, whereas the control groups received only solvent. Epithelialization was monitored every second day intravitally up to wound closure. Wounds were stained for VEGF, CD31, TGF-β, TNF-α, SDF-1α, NF-κB, and Ki-67, and fibroblasts were counted after hematoxylin-eosin stain on days 3, 6, 9, and 16 after wounding. After induction of diabetes, wounds closed on day 13.00 ± 2.20 standard deviation (SD). Local 11,12 ETT application improved wound closure significantly to day 8.40 ± 1.39 SD. EET treatment enhanced VEGF and CD31 expression in wounds on day 3. It also seemed to raise TNF-α level on all days investigated as well as TGF-β level on days 3 and 6. A decrease in NF-κB could be observed on days 9 and 16 after EET application. The latter findings were not significant. SDF-1α expression was not influenced by EET application, and Ki-67 was significantly less in the EET group on day 9 after EET application. The number of fibroblasts was significantly increased on day 9 after the 11,12 EET application. 11,12 EET improve deteriorated wound healing in diabetes by enhancing neoangiogenesis, especially in the early phase of wound healing. Furthermore, they contribute to the dissolution of the initial inflammatory reaction, allowing the crucial transition from the inflammatory to proliferative phase in wound healing.
Introduction: Stem cell transplantation is one of the most promising strategies to improve healing in chronic wounds as systemic administration of endothelial progenitor cells (EPC) enhances healing by promoting neovascularization and homing though a high amount of cells is needed. In the following study, we analysed whether local application can reduce the number of EPC needed achieving the same beneficial effect on wound healing.
Material and Methods: Wound healing after local or systemic treatment with EPC was monitored in vivo by creating standardized wounds on the dorsum of hairless mice measuring wound closure every second day. Systemic group received 2 × 106 EPC i.v. and locally treated group 2 × 105 EPC, locally injected. As control PBS injection was performed the same way. Expression of CD31, VEGF, CD90 and, SDF-1α was analysed immunohistochemically for evaluation of neovascularisation and amelioration of homing.
Results: Local (7.1 ± 0.45 SD) as well as systemic (6.1 ± 0.23 SD) EPC transplantation led to a significant acceleration of wound closure compared to controls (PBS local: 9.7 ± 0.5 SD, PBS systemic 10.9 ± 0.38 SD). Systemic application enhanced CD31 expression on day 6 after wounding and local EPC on 6 and 9 in comparison to control. VEGF expression was not significantly affected. Systemic and local EPC treatment resulted in a significantly enhanced SDF-1α and CD90 expression on all days investigated.
Conclusion: Local as well as systemic EPC treatment enhances wound healing. Moreover, beneficial effects are obtained with a tenfold decrease number of EPC when applied locally. Thus, local EPC treatment might be more convenient way to enhance wound healing as number of progenitor cells is limited.
Introduction: Epoxyeicosatrienoic acids (EETs) are able to enhance angiogenesis and regulate inflammation that is especially important in wound healing under ischemic conditions. Thus, we evaluated the effect of local EET application on ischemic wounds in mice.
Methods: Ischemia was induced by cautherization of two of the three supplying vessels to the mouse ear. Wounding was performed on the ear three days later. Wounds were treated either with 11,12 or 14,15 EET and compared to untreated control and normal wounds. Epithelialization was measured every second day. VEGF, TNF-α, TGF-β, matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), Ki67, and SDF-1α were evaluated immunohistochemically in wounds on day 3, 6, and 9.
Results: Ischemia delayed wound closure (12.8 days ± 1.9 standard deviation (SD) for ischemia and 8.0 days ± 0.94 SD for control). 11,12 and14,15 EET application ameliorated deteriorated wound healing on ischemic ears (7.6 ± 1.3 SD for 11,12 EET and 9.2 ± 1.4 SD for 14,15 EET). Ischemia did not change VEGF, TNF-α, TGF-β, SDF-1α, TIMP, MMP7 or MMP9 level significantly compared to control. Local application of 11,12 as well as 14,15 EET induced a significant elevation of VEGF, TGF-β, and SDF-1α expression as well as proliferation during the whole phase of wound healing compared to control and ischemia alone.
Conclusion: In summary, EET improve impaired wound healing caused by ischemia as they enhance neovascularization and alter inflammatory response in wounds. Thus elevating lipid mediator level as 11,12 and 14,15 EET in wounds might be a successful strategy for amelioration of deranged wound healing under ischemia.
BACKGROUND: Local implantation of ex vivo concentrated, washed and filtrated human bone marrow-derived mononuclear cells (BMC) seeded onto β-tricalciumphosphate (TCP) significantly enhanced bone healing in a preclinical segmental defect model. Based on these results, we evaluated in a first clinical phase-I trial safety and feasibility of augmentation with preoperatively isolated autologous BMC seeded onto β-TCP in combination with angle stable plate fixation for the therapy of proximal humeral fractures as a potential alternative to autologous bone graft from the iliac crest.
METHODS: 10 patients were enrolled to assess whether cell therapy with 1.3 × 106 autologous BMC/ml/ml β-TCP, collected on the day preceding the definitive surgery, is safe and feasible when seeded onto β-TCP in patients with a proximal humeral fracture. 5 follow-up visits for clinical and radiological controls up to 12 weeks were performed.
RESULTS: β-tricalciumphosphate fortification with BMC was feasible and safe; specifically, neither morbidity at the harvest site nor at the surgical wound site were observed. Neither local nor systemic inflammation was noted. All fractures healed within the observation time without secondary dislocation. Three adverse events were reported: one case each of abdominal wall shingles, tendon loosening and initial screw perforation, none of which presumed related to the IND.
CONCLUSIONS: Cell therapy with autologous BMC for bone regeneration appeared to be safe and feasible with no drug-related adverse reactions being described to date. The impression of efficacy was given, although the study was not powered nor controlled to detect such. A clinical trial phase-II will be forthcoming in order to formally test the clinical benefit of BMC-laden β-TCP for PHF patients. Trial registration The study was registered in the European Clinical Trial Register as EudraCT No. 2012-004037-17. Date of registration 30th of August 2012. Informed consent was signed from all patients enrolled.
Fragestellung: In einem ausgedehnten Knochendefekt kann das Einwachsen von knochenbildenden Zellen limitiert sein, da ohne Gefässe die Ernährung der regenerativen Zellen im Knochenkonstrukt insuffizient ist. Endotheliale Progenitorzellen (EPC) sind wichtig bei der Neovaskularisierung. Die frühe Vaskularisierung von grossen Knochendefekten kann für das Überleben und die Funktion von mesenchymalen Stammzellen (MSC) und knochenbildenden Zellen entscheidend sein. Kann die Implantation von EPC und MSC auf osteokonduktiven beta-Tricalciumphosphat (beta-TCP) in einem "critical-size" Knochendefekt des Femur von athymischen Ratten die frühe Vaskularisierung und die Knochenheilung in vivo verbessern?
Methodik: Humane EPC wurden aus Buffy-Coat und humane MSC aus Knochenmarkaspirat durch Dichtezentrifugation isoliert. 2.5 x 105 kultivierte und differenzierte EPC und MSC wurden in vitro auf beta-TCP geladen. In 145 athymischen, männlichen Ratten wurde das Femur osteotomiert, ein 5 mm Knochendefekt erzeugt und mit Fixateur externe stabilisiert. Die Knochendefekte wurden mit beta-TCP (Gruppe 1), beta-TCP und MSC (Gruppe 2), beta-TCP und EPC (Gruppe 3), beta-TCP und EPC und MSC (Gruppe 4) oder autologem Knochen (Gruppe 5) gefüllt. Nach 1 Woche (n=40), 4 Wochen (n=40), 8 Wochen (n=40) und 12 Wochen (n=25) wurden die Ratten getötet. Bei Pinlockerung wurde die Ratte ausgeschlossen. Die (immun)histologische Analyse (Färbung mit HE, VEGF-R2, vWF) der Vaskularisierung und Knochenneubildung erfolgte mit Image-Analysis-System. Nach 8 und 12 Wochen erfolgte ein µCT und ein 4-Punkte-Biegungstest. Für die statistische Analyse wurde der Kruskal-Wallis-Test verwendet.
Ergebnisse und Schlussfolgerungen: Nach 1 Woche zeigte sich bei der Implantation von EPC/MSC und EPC allein signifikant mehr primitive vaskuläre Plexus (p=0.01;p=0.048) als in Vergleichsgruppen. Im Vergleich zur TCP Gruppe war in allen anderen Versuchsgruppen signifikant mehr Knochenneubildung zu sehen (p<0.01). Ausserdem war in der EPC/MSC-Gruppe signifikant mehr Knochenbildung zu erkennen als in der MSC-Gruppe (p=0.03). Nach 12 Wochen zeigten alle Gruppen eine knöcherne Durchbauung des Defektes, jedoch zeigten bereits 8 Wochen nach Implantation von MSC/EPC 83% der Defekte eine stabile, knöcherne Durchbauung. Bei der Implantation von MSC kam es in 18% der Knochendefekte zum knöchernen Durchbau. Alle anderen experimentellen Gruppen zeigten nach 8 Wochen keine knöcherne Durchbauung. Diese Resultate konnten im µCT, biomechanischen Test und in der Histologie quantifiziert werden. EPC scheinen die frühe Vaskularisierung innerhalb eines Knochenkonstrukt in vivo zu stimulieren und das Einwachsverhalten von MSC zu verbessern, was zu einer beschleunigten Knochenheilung im Knochendefektmodell der Ratte führt.
Influence of antibiotic management on microbial selection and infectious complications after trauma
(2021)
Background: The inflammatory response and post-traumatic complications like infections play an important role in the pathophysiology of severe injuries. This study examines the microbiological aspects in anti-infective treatment of trauma patients and their inflammatory response in post-traumatic infections complications. Patients and Methods: A retrospective analysis of prospectively collected data in trauma patients (ISS ≥ 16) over a 1-year period (01/2018 to 12/2018) is provided. Patient population was stratified into severely injured patients without post-traumatic infection (inf-PT), and severely injured patients who developed an infection (inf+PT).Results: Of 114 trauma patients, 45 suffered from post-traumatic infection during the first 10 days of hospitalization. Severely injured patients with concomitant traumatic brain injury (PT+TBI) showed the highest rate of post-traumatic infection. Pro-inflammatory reaction was tracked by levels of Interleukin (IL-)6 (day 3: inf+T 190.8 ± 359.4 pg/dL > inf-PT 56.2 ± 57.7 pg/mL (mean ± SD); p = 0.008) and C-Reactive-Protein (CRP, day 3: inf+PT 15.3 mg/dL > inf-PT 6.7 mg/dL, p = 0.001) which were significantly higher in trauma patients who develop an infectious complication and showed a significant positive correlation with the occurrence of infection. The leading entity of infection was pneumonia followed by infections of the urinary tract mainly caused by gram-negative Enterobacteriaceae. 67.5% of all trauma patients received single-shot antibiosis during initial care in trauma bay. The development of secondary colonization was not relevant positively correlated with single-shot antibiosis (r = 0.013, p = 0.895) and prophylactically calculated antibiotic administration (r = 0.066, p = 0.500).Conclusion: Severely injured trauma patients have an increased risk for development of infectious complications, which mainly is pneumonia followed by infection of the urinary tract mainly caused by gram-negative Enterobacteriaceae. Based on the data in this study, the one-time antibiotic and prophylactic calculated use of antibiotics, like Cephalosporins must be critically discussed in terms of their role in the development of post-traumatic infections and microbial selection.
miR-142-3p expression is predictive for severe traumatic brain injury (TBI) in trauma patients
(2020)
Background: Predictive biomarkers in biofluids are the most commonly used diagnostic method, but established markers in trauma diagnostics lack accuracy. This study investigates promisingmicroRNAs(miRNA)releasedfromaffectedtissueafterseveretraumathathavepredictive values for the effects of the injury.
Methods: A retrospective analysis of prospectively collected data and blood samples of n = 33 trauma patients (ISS≥16) is provided. Levels of miR-9-5p, -124-3p, -142-3p, -219a-5p, -338-3pand-423-3p inseverelyinjuredpatients (PT)withouttraumatic braininjury (TBI) or with severe TBI (PT + TBI) and patients with isolated TBI (isTBI) were measured within 6 h after trauma.
Results: The highest miR-423-3p expression was detected in patients with severe isTBI, followed by patients with PT + TBI, and lowest levels were found in PT patients without TBI (2−∆∆Ct,p = 0.009). ApositivecorrelationbetweenmiR-423-3plevelandincreasingAIShead (p = 0.001) and risk of mortality (RISC II, p = 0.062) in trauma patients (n = 33) was found. ROC analysis of miR-423-3p levels revealed them as statistically significant to predict the severity of brain injury in trauma patients (p = 0.006). miR-124-3p was only found in patients with severe TBI, miR-338-3p was shown in all trauma groups. miR-9-5p, miR-142-3p and miR-219a-5p could not be detected in any of the four groups. Conclusion: miR-423-3p expression is significantly elevated after isolated traumatic braininjuryandpredictableforsevereTBIinthefirsthoursaftertrauma. miR-423-3pcouldrepresent a promising new biomarker to identify severe isolated TBI.
The inflammatory response plays an important role in the pathophysiology of multiple injuries. This study examines the effects of severe trauma and inflammatory response on markers of neuronal damage. A retrospective analysis of prospectively collected data in 445 trauma patients (Injury Severity Score (ISS) ≥ 16) is provided. Levels of neuronal biomarkers (calcium-binding Protein B (S100b), Enolase2 (NSE), glial fibrillary acidic protein (GFAP)) and Interleukins (IL-6, IL-10) in severely injured patients (with polytrauma (PT)) without traumatic brain injury (TBI) or with severe TBI (PT+TBI) and patients with isolated TBI (isTBI) were measured upon arrival until day 5. S100b, NSE, GFAP levels showed a time-dependent decrease in all cohorts. Their expression was higher after multiple injuries (p = 0.038) comparing isTBI. Positive correlation of marker level after concomitant TBI and isTBI (p = 0.001) was noted, while marker expression after PT appears to be independent. Highest levels of IL-6 and -10 were associated to PT und lowest to isTBI (p < 0.001). In all groups pro-inflammatory response (IL-6/-10 ratio) peaked on day 2 and at a lower level on day 4. Severe TBI modulates kinetic profile of inflammatory response by reducing interleukin expression following trauma. Potential markers for neuronal damage have a limited diagnostic value after severe trauma because undifferentiated increase.