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

OBJECTIVE: 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.

METHODS: 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.

RESULTS: 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.

CONCLUSIONS: 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.