Lokale Inflammation und ossäre Zell-Kommunikation nach Trauma : Untersuchungen im Klein-und Großtiermodell

  • Local inflammation and osseous cell communication after trauma : Studies in small and large animal models

Greven, Johannes; Pradel, Gabriele (Thesis advisor); Müller, Frank (Thesis advisor); Hildebrand, Frank (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Introduction: Based on the results of studies to date, it remains unclear whether concomitant injuries or fracture treatment techniques (osteosynthesis) have a significant effect on local inflammation after a severe trauma and thus cause complications of fracture healing in the form of pseudathroses. Therefore, this dissertation should investigate the specific influence of injuries and osteosynthesis techniques on the local inflammatory response in a clinically relevant polytrauma large animal model. In a second study, the effects of extracellular vesicles (EV) on fracture healing osteoblasts were investigated in a translational rat model, since EVs play a relevant role in the intercellular transport of inflammatory mediators and can thus influence their activity profile. Material and methods:The investigations in the large animal model included three groups: group monotrauma (isolated femur fracture), group polytrauma (femur fracture, thoracic trauma, liver laceration, hemorrhagic shock) and sham group (no trauma, only anesthesia/ventilation and catheterization). Depending on the group, the femur fracture was stabilized with an intramedullary nail or an external fixator. The total test duration was 72 h. Muscle and blood samples were taken at four time points (2, 24, 48 and 72 h post trauma) and cytokine concentration and expression as well as immune cell status were analyzed. Methods used were multiplex ELISAs, qRT-PCR and histological sections.In the rat model, a Krischner wire was first inserted intramedullary into the femur and then a femur fracture was induced by means of a blunt guillotine. Blood samples were collected after 3, 7 and 14 days and extracellular vesicles (EV) were isolated. Subsequently, primary osteoblasts were incubated with them and examined for viability and proliferation. In all approaches, EV-free plasma and plasma as a control were included. The analyses were performed by nanoparticle tracking analysis (NTA), immunohistological staining, newborn counts and a proliferation assay (MTT).Results:The comparison of the two supply strategies showed no significant difference for the local pro-inflammatory cytokine release in the musculature. However, intramedullary nailing in monotrauma compared to external fixation led to a significant IL-10 increase in muscle tissue (48 h after trauma, p< 0.05 and a significantly reduced infiltration of neutrophil granulocytes in adjacent muscle tissue (p = 0.041)). The fracture itself had a significant influence on the pro-inflammatory cytokine concentrations with significantly higher cytokine levels on the traumatized side (T-side) compared to the non-injured side (AT-side) (IL-6: 2 h p = 0.041, 24 h p = 0.0017; IL-8: 2 h p = 0.002, 24 h p = 0.029 and 48 h p = 0.008) in monotrauma. The severity of the overall trauma (mono- vs. polytrauma) also had a significant influence with increased local cytokine concentrations for monotrauma (2 h and 48 h mono- higher than polytrauma p = 0.003; p = 0.032). Furthermore, an increased immigration of neutrophil granulocytes into the muscle (mono vs. polytrauma 15.52 ± 5.39 vs. 8.23 ± 3.36 [neutrophil/visual field]; p = 0.013) after monotrauma was shown. After incubation with EV, primary osteoblasts took them up and processed them in the endoplamatic reticulum (ER). The results of the study shown here show that EV isolated from EV-free plasma contains molecules that promote the metabolism and proliferation of osteoblasts even over a long period of time after fracture. The full plasma used only showed a viability enhancing effect on osteoblasts after 14 days (p = 0.032), whereas EVs isolated from plasma significantly increased the viability of osteoblasts shortly after trauma induction and over a period of several days (3 d p = 0.038, 7 d p = 0.019 and 14 d p = 0.007). Discussion: Local inflammatory processes have a potential influence on fracture healing processes. In our study we were able to show that this local inflammatory reaction proceeds differently after polytrauma and monotrauma. The influence of the fracture treatment strategy also depends on the presence of mono- or polytrauma. Due to the systemic reaction in case of polytrauma, an inflammatory reaction comparable to the fracture side also occurs on the opposite side of the fracture. Furthermore, in polytrauma the supply strategy seems to play a subordinate role in the concentration of cytokines (IL-6, IL-8 and IL-10) in the muscle tissue on both the traumatized and non-traumatized side. Rather, it is influenced in particular by the concomitant injuries. A possible reason for this is the impaired activity of macrophages and neutrophilic granulocytes during trauma. In order to be able to evaluate the significance of care strategies in the context of a polytrauma in more detail, it is therefore necessary to identify more specific markers. The intramedullary nail reduces the number of locally recruited neutrophil granulocytes. In addition, pro-inflammatory (IL-6 and IL-8) cytokine transcription and anti-inflammatory (IL-10) protein concentration in muscle tissue close to the fracture are increased after monotrauma and treatment with an intramedullary nail. It could be postulated that due to the lack of concomitant injuries the recruitment of immune cells after monotrauma could be more targeted. The interaction between the inflammatory response and fracture (healing) could be significantly mediated by systemic EV, since the viability and proliferation of osteoblasts can be modified by EV. Due to this potential relevance of EV a potential use in the treatment of fracture healing disorders (e.g. pseudathrosis) is conceivable.