gms | German Medical Science

Objectives: Impaired fracture healing, which affects up to 15% of patients undergoing surgical treatment, poses a significant clinical challenge and socioeconomic burden. Neutrophil extracellular traps (NETs) are intricate structures formed by neutrophils that play a dual role in host defense and tissue damage. NETosis, mediated by PAD4 and resolved by DNAse1 and DNA1-like3, disrupts tissue integrity and healing processes, but its role in bone regeneration remains unclear. Based on the rapid influx of neutrophils into the fracture hematoma after injury, the aim of this study was to define the role of NETs in physiologic fracture healing and also in nonunion formation by using genetic and pharmacologic means to alter NETosis.

Methods: To assess physiological fracture healing, WT, combined DNAse1- and DNAse1-like3-deficient (DNAse1/3) and combined DNAse1/3;PAD4-deficient mice were subjected to a femoral osteotomy, which was stabilized using an external fixator. To induce pathological healing (nonunion), the periosteum of the fracture ends was additionally cauterized in independent sets of mice. For pharmacologic experiments, fractured WT mice with/without additional periosteal cauterization received systemic injections of FDA-approved recombinant DNase1 to facilitate resolution of NETs. Outcome parameters were assessed using qRT-PCR, ELISA, micro-CT and undecalcified histology.

Results and conclusion: In WT mice, increased expression of the NETs generator PAD4 in callus tissue and elevated serum levels of released NETs components were measured after osteotomy. DNAse1/3-deficient mice showed impaired fracture healing and a high incidence of fracture nonunion, which was completely reversed by additional deletion of PAD4. In the nonunion model, the impaired fracture consolidation observed in DNAse1/3-deficient mice was also rescued and even significantly improved compared to WT mice by additional deletion of PAD4. Finally, pharmacological facilitation of NETs resolution in WT mice by systemic DNAse1-treatment improved fracture bridging in both the physiologic fracture and the nonunion model. Taken together, the data indicate that NETs release impairs fracture union in both physiologic and pathologic healing states and demonstrate that NETosis can be targeted pharmacologically to improve bone regeneration in mice.