¹Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Deutschland

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Objectives and questions: Heterotopic ossification (HO) is a common complication following injury or surgery, particularly in patients with elbow fractures. Although post-traumatic inflammatory processes are thought to be critical for subsequent HO formation, the underlying mechanisms remain poorly understood. Current treatment options are both unspecific and ineffective. Neutrophils are among the first cells recruited to injured tissue, including sites of HO formation. They can extrude a meshwork of DNA covered with granule-derived antimicrobial peptides and enzymes aimed at killing bacteria, which are termed neutrophil extracellular traps (NETs). Given the importance of neutrophils in the early post-traumatic inflammatory response, this study explored the involvement of NETs in the formation of HO.

Material and methods: This study employed both clinical and experimental approaches. Clinically, histological examination of human HO samples after a median of 16 months after upper extremity injury was performed. Bone wedges from otherwise healthy patients undergoing elective femoral or tibial osteotomy were used as controls. Concentrations of NETs markers were measured by ELISA in serum samples from affected patients. Experimentally, traumatic HO was induced in mice via Achilles tendon tenotomy, with radiological and histological analyses conducted at 8 weeks post-operatively. To assess the role of NETs in HO formation, mice with genetic modifications and pharmacological treatment to suppress NETs formation or clearance were used, respectively.

Results: Clinically, HO samples showed indices of excessive bone formation, immature bone structure, and infiltration of leukocytes. Immunohistofluorescence with co-localization of the NETs markers cell-free DNA, myeloperoxidase and neutrophil elastase demonstrated the presence of neutrophils undergoing various stages of NETs shedding in HO lesions. This was associated with increased serum levels of NETs components in HO patients compared to healthy controls. Experimentally, mice with a genetic impairment in NETs clearance showed increased HO lesion size compared to wild-type (WT) controls. Conversely, additional deletion of PAD4, which encodes the key enzyme required for NETs formation, led to a strong reduction in HO formation. Pharmacological administration of a PAD4 inhibitor or recombinant DNAse1, an FDA-approved agent that facilitates the clearance of NETs, resulted in a significant reduction in HO volume in WT mice.

Discussion and conclusions: This study provides a clinical and mechanistic link between NETs and HO formation. Our findings suggest that NETs are key mediators of HO development and that strategies to inhibit NETs release or promote their clearance may provide novel therapeutic approaches for the treatment of HO.