Fracture-healing is a highly complex and timely orchestrated process. Non-healing fractures are still a major clinical problem and treatment remains difficult. A 16 Hz extremely low-frequency pulsed electromagnetic field (ELF-PEMF) was identified as non-invasive adjunct therapy supporting bone-healing by inducing reactive oxygen species (ROS) and Ca²⁺-influx. However, ROS and Ca²⁺-influx may stimulate neutrophils, the first cells arriving at the wounded site, to excessively form neutrophil extracellular traps (NETs), which negatively affects the healing process. Thus, this study aimed to evaluate the effect of this 16 Hz ELF-PEMF on NET formation. Neutrophils were isolated from healthy volunteers and exposed to different NET-stimuli and the 16 Hz ELF-PEMF. NETs were quantified using Sytox Green Assay and immunofluorescence, Ca²⁺-influx and ROS with fluorescence probes. In contrast to mesenchymal cells, ELF-PEMF exposure did not induce ROS and Ca²⁺-influx in neutrophils. ELF-PEMF exposure did not result in basal or enhanced PMA-induced NET formation but did reduce the amount of DNA released. Similarly, NET formation induced by LPS and H₂O₂ was reduced through exposure to ELF-PEMF. As ELF-PEMF exposure did not induce NET release or negatively affect neutrophils, the ELF-PEMF exposure can be started immediately after fracture treatment.

Type 2 diabetes mellitus (T2DM) significantly impairs fracture healing, with neutrophils playing a crucial role in this process. In T2DM, these immune cells are over-activated, leading to the excessive release of neutrophil extracellular traps (NETs), increasing inflammation and hindering recovery. Thus, a need for markers to assess patients in the risk group arises. This study demonstrates that circulating cell-free DNA (cfDNA) can be efficiently quantified from serum samples by a single-step qPCR and be used as a marker for NETosis. Our results revealed that trauma patients with T2DM have the highest cfDNA levels, followed by trauma patients, and the healthy group has the lowest. The method shows strong correlations between cfDNA and neutrophil-specific markers such as MPO, citH3, AZU1, and α-defensin, highlighting its potential as a rapid indicator of NETosis. This approach could allow the timely interference for high-risk patients, ultimately improving healing outcomes and reducing complications such as chronic inflammation, non-union fractures, and diabetic foot ulcers.

Diabetes is a worldwide evolving disease with many associated complications, one of which is delayed or impaired wound healing. Appropriate wound healing strongly relies on the inflammatory reaction directly after injury, which is often altered in diabetic wound healing. After an injury, neutrophils are the first cells to enter the wound site. They have a special defense mechanism, neutrophil extracellular traps (NETs), consisting of released DNA coated with antimicrobial proteins and histones. Despite being a powerful weapon against pathogens, NETs were shown to contribute to impaired wound healing in diabetic mice and are associated with amputations in diabetic foot ulcer patients. The anti-diabetic drugs metformin and liraglutide have already been shown to regulate NET formation. In this study, the effect of insulin was investigated. NET formation after stimulation with PMA (phorbol myristate acetate), LPS (lipopolysaccharide), or calcium ionophore (CI) in the presence/absence of insulin was analyzed. Insulin led to a robust delay of LPS- and PMA-induced NET formation but had no effect on CI-induced NET formation. Mechanistically, insulin induced reactive oxygen species, phosphorylated p38, and ERK, but reduced citrullination of histone H3. Instead, bacterial killing was induced. Insulin might therefore be a new tool for the regulation of NET formation during diabetic wound healing, either in a systemic or topical application.

Chronic wounds represent an increasing global health burden for both patients and healthcare systems. These non-healing lesions frequently result in amputation and are particularly prevalent among individuals with diabetes. Hallmarks of chronic wounds include a prolonged inflammatory phase characterized by sustained neutrophil infiltration, excessive formation of neutrophil extracellular traps (NETs), and an elevated alkaline tissue pH. Emerging evidence suggests a link between pH and wound healing, with acidic conditions being more favorable for tissue repair. Here, we investigated the pH-dependent modulation of primary neutrophil functions, including viability, reactive oxygen species (ROS) production, NET formation, and exocytosis. Neutrophils cultured in media with pH values ranging from 6.1 to 8.4 exhibited no significant differences in viability. In contrast, acidic conditions suppressed ROS production and NET formation in a pH-dependent manner, whereas alkaline conditions caused responses comparable to those observed at physiological pH (7.4). Furthermore, levels of antioxidant enzymes were markedly increased under acidic conditions following phorbol 12‑myristate 13‑acetate (PMA) stimulation. Notably, alkaline pH promoted granule polarization in neutrophils, thereby impairing exocytosis. In contrast, acidic conditions (pH < 7.0) reduced granule polarization and enhanced the release of granule-associated proteins upon PMA stimulation. Pharmacological inhibition of degranulation confirmed that these effects were mediated by neutrophil exocytosis. In summary, extracellular pH critically modulates neutrophil effector functions and may contribute to the distinct healing dynamics observed in acute versus chronic wounds.