¹BG Klinik Tübingen, Eberhard Karls Universität Tübingen, Tübingen, Deutschland
²Klinisch-Experimentelle Chirurgie, Universität des Saarlandes, Homburg, Deutschland
³BG Trauma Center, Eberhard Karls Universität Tübingen, Siegfried Weller Institute for Trauma Research, Tübingen, Deutschland
⁴Robert Bosch Center for Tumor Diseases, Stuttgart, Deutschland

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Objectives and questions: Non-unions represent a major complication in trauma and orthopedic surgery. Notably, inflammation plays a centrale role during bone regeneration. Inflammatory cells and mediators lead to the migration of stem cells into the callus tissue, which forms the basis for tissue growth. An overshooting and prolonged inflammatory response, on the other hand, may deteriorate fracture healing. In recent years, inflammasomes, i.e. multi-protein complexes that play a crucial role in controlling the inflammatory response, have gained increasing interest in preclinical research. However, there is no information on their expression during fracture healing and non-union formation, and their potential role in fracture healing failure.

Material and methods: Twelve- to 16-week-old CD1-mice were used in the present study. An intramedullary screw was applied in a closed femoral fracture model to simulate fracture healing (union group). To induce non-union formation, a 1.8 mm segmental defect was created and fixed with a pin-clip device, guaranteeing rotational and axial stability (non-union group). The callus tissue was analyzed after 7, 10 and 14 days and after 10 weeks (n = 5 each group) by means of X-ray, µCT, biomechanics, histology, immunohistochemistry and Western blotting. Comparison between the two groups was performed using the unpaired Student's t-test. Statistical significance was accepted for p < 0.05.

Results: The X-ray, µCT and histomorphometric analysis showed a significantly lower amount of bone tissue in the non-union group when compared to mice of the union group. This was associated with a significantly reduced bending stiffness. Additional immunohistochemical analyses demonstrated a higher number of granulocytes and macrophages within the callus tissue of non-unions. Finally, Western blot analyses revealed different expression patterns of inflammasomes between the two study groups. The expression of the NLR family pyrin domain containing (NLRP)1 and NLRP3 inflammasomes was significantly higher within the callus tissue of the animals of the non-union-group. Accordingly, the expression of interleukin-1-beta (IL-1β) was also enhanced when compared to mice of the union group.

Discussion and conclusions: Our results demonstrate an increased expression of the NLRP1 and NLRP3 inflammasome within the callus tissue of non-unions when compared to unions. This is associated with a prolonged inflammatory response, as indicated by enhanced pro-inflammatory cell infiltration and an elevated expression of IL-1β. Hence, novel treatment approaches may target NLRP1 and NLRP3 inflammasome expression to attenuate the overshooting inflammatory response and, thus, to stimulate bone regeneration and to overcome non-union formation.