The increasing N2/N1 neutrophil ratio over time in human fracture hematoma indicates a transition from inflammation to regeneration

OBJECTIVES: Fracture hematoma (FH) initiates fracture healing and is key in the inflammatory phase. Neutrophils, the first infiltrating cells, play a dual role in this process. The discovery of pro-inflammatory N1 and regenerative N2 neutrophil phenotypes provides new insights into their functions. This study aims to investigate the presence, dynamics, and osteogenic effects of N1 and N2 neutrophils in human FH.

METHODS: Human neutrophils were polarized into N1 and N2 phenotypes in vitro. CD15 and CD16 served as general markers, while CD54, CD95, and CD182 expression and TNF-α, IL-8, MCP-1, and SDF-1α secretion characterized N1/N2 polarization. After validation, 15 FH samples (4-14 days post-trauma) were analyzed by flow cytometry and immunofluorescence to determine the N2/N1 ratio and its temporal trend. Functional assays assessed their influence on human BMSC osteogenesis in co-culture.

RESULTS: In vitro, N1 neutrophils expressed high CD54 and CD95 and secreted elevated TNF-α, IL-8, MCP-1, and SDF-1α, whereas N2 cells showed increased CD182 and SDF-1α. Both phenotypes were detected in human FH, with a rising N2/N1 ratio over time, indicating a shift from inflammation to regeneration. Functionally, N1 neutrophils counteracted N0-induced inhibition of early osteogenesis but reduced cell metabolism, while N2 neutrophils alleviated N0-mediated suppression of late mineralization.

CONCLUSIONS: Our findings provide the first evidence of the coexistence and temporal evolution of N1 and N2 neutrophils within human fracture hematoma, consistent with their polarization characteristics observed in vitro. Moreover, these phenotypes exert distinct effects on osteogenesis, in alignment with the progressive increase of the N2/N1 ratio in vivo, supporting the gradual transition from inflammation to regeneration during the human fracture-healing cascade. Collectively, this study advances the understanding of neutrophil heterogeneity in bone regeneration and bridges fundamental immunobiological mechanisms with potential clinical translation.