N1 and N2 neutrophil phenotypes may play differential roles in stimulating the endochondral ossification behaviors of ATDC5 cells.

Endochondral ossification is vital for bone healing, with neutrophils playing a crucial role in the osteoimmune system. While N1 (pro-inflammatory) and N2 (regenerative) neutrophils are documented in other contexts, their role in endochondral ossification remains unclear. This study investigates their effects on ATDC5 cells. Neutrophils from five healthy volunteers were isolated and polarized into N0 (unstimulated), N1, or N2 phenotypes. After culturing for 4 h, neutrophil-conditioned media was mixed (20 % v/v) with chondrogenic differentiation media (DM). ATDC5 cells were cultured with mixture or DM alone for 24 h, followed by continued DM culturing. On days 7 and 14, several gene expressions, ALP activity, and TGF-β3 levels were assessed. SOX9 peaked in the ATDC5 (A)/N2 group on day 7, while the A/N1 group showed the highest levels on day 14. COL2A1 was elevated in the A/N0 group on day 7. RUNX2 was higher in A/N1 and A/N2 on day 7, with A/N1 remaining elevated on day 14. MMP-13 was significantly higher in A/N1 on day 7. COL10A1 expression showed no significant changes. COL1A1 and COX2 were continually elevated in the A/N1 group. ALP activity was consistently enhanced in the A/N1 group, and TGF-β3 was lower in both A/N1 and A/N2 on day 14. This study indicates that N1 neutrophils may promote chondrocyte maturation and osteogenic differentiation, while N2 neutrophils may support proliferation, hypertrophy, and maturation, providing a cell reservoir for ossification. These findings highlight their distinct roles in directing chondrogenic progenitors toward bone rather than cartilage formation.