Zappia, J. | Search bioRxiv

bioRxiv2026

NSAIDs impair fracture healing by disrupting neutrophil-mediated repair

Neutrophils are among the first immune cells recruited to fractures, yet their contribution to bone repair remains poorly understood. Using live imaging and neutrophil perturbation in adult zebrafish, we show that neutrophils establish the early fracture microenvironment. Neutrophils rapidly accumulated at fractures, underwent NETosis, and exhibited transcriptional programmes associated with matrix remodelling. Compromising neutrophil recruitment, via ibuprofen treatment or orthogonal perturbations, increased fracture non-union, delayed osteoblast differentiation, impaired mineralisation, and altered callus architecture. Mechanistically, neutrophils actively interacted with extracellular matrix proteins, including laminin, fibronectin, and collagen I, through uptake, trafficking and secretion-associated pathways. Together, our findings reveal that neutrophils establish a provisional emergency fracture matrix that templates subsequent bone repair, providing a mechanistic link between early non-steroidal anti-inflammatory drug (NSAID) exposure and impaired skeletal regeneration.

immunology↗

bioRxiv2026

Nanovibrational stimulation preferentially enhances osteogenic responses in zebrafish

Mechanical cues are key regulators of bone formation, yet their potential as therapeutic stimuli remains incompletely explored in vivo. Nanovibrational stimulation, which delivers low-amplitude, high-frequency mechanical input, has been shown to promote osteogenic differentiation in vitro, and rodent studies have similarly demonstrated osteogenic effects. However, its impact on other cellular systems at the whole-organism level remains poorly understood. Here, we demonstrate that nanovibrational stimulation enhances myogenic differentiation in vitro before investigating its effects on skeletal development and tissue specificity in zebrafish. Larval zebrafish exposed to nanovibrational stimulation exhibited increased osteoblast numbers and enhanced bone formation relative to controls. In adult zebrafish, nanovibration increased the osteoblast response to a fracture-like injury, indicating enhanced osteogenic activity during repair. To assess tissue specificity, we examined additional cell types and systems relevant to skeletal regeneration, including cartilage, muscle, vasculature and innate immune cells. Although nanovibrational stimulation promoted myogenic differentiation of C2C12 cells in vitro, its effects on muscle and other non-skeletal tissues in zebrafish larvae were comparatively limited. These findings suggest that nanovibrational stimulation exerts a preferential effect on osteogenic processes in vivo. These findings demonstrate that nanovibrational stimulation preferentially enhances osteoblast-mediated bone formation and skeletal injury responses in zebrafish, without causing significant perturbations in other tissues. Our results establish zebrafish as a tractable in vivo model for investigating vibration-induced mechanobiological processes, providing a more physiologically relevant representation of tissue-level mechanotransduction than conventional two-dimensional culture systems. Furthermore, these findings highlight the potential of nanovibrational stimulation as a non-invasive strategy to promote bone regeneration and fracture repair.

physiology↗