Fangzhou Lu (Maastricht / NL), Anna J. L. Lodewijks (Maastricht / NL), Rald Victor Maria Groven (Maastricht / NL), Martijn Poeze (Maastricht / NL), Martijn van Griensven (Maastricht / NL), Taco J. Blokhuis (Maastricht / NL)
Objectives: BMSCs are key to bone regeneration. Neutrophils are the first responders at the fracture site, yet their role in bone healing remains unclear. This study aims to investigate the distinct effects of N1 (inflammatory) and N2 (regenerative) phenotypes on BMSC osteogenic behavior.
Methods: Neutrophils were isolated and polarized into N0 (unstimulated), N1, or N2 phenotypes. N0, N1 and N2 were co-cultured with BMSCs. BMSCs without neutrophils served as controls. After three days, non-adherent neutrophils were removed, and BMSCs were cultured continuously. BMSC viability and ALP activity were assessed on day 7, and ECM mineralization on day 28. Gene and cytokine expression were analyzed by qPCR and ELISA on both days 7 and 28.
Results: On day 7, B/N0 group showed lower ALP activity than both B-only and B/N1 groups, and cell viability in B/N1 group was lower than in B-only group. On day 28, B/N0 group showed reduced mineralization compared to B-only and B/N2 groups. Gene expression analysis revealed no significant differences in COL1A1, but OPN expression significantly increased in all groups from day 7 to day 28. RUNX2 expression increased in B-only, B/N0, and B/N1 groups, remaining unchanged in B/N2 group. OCN expression increased in B-only, B/N1, and B/N2 groups, except in B/N0 group. For cytokine secretion, BMP-2 levels were higher in B/N2 group on day 28 compared to day 7. OPN secretion remained stable across groups, while MMP-13 was undetectable on day 7 but present on day 28, with lower levels in B/N1 group.
Discussion: This study shows that N1 and N2 neutrophil phenotypes differentially influence the osteogenic property of human BMSCs. N1 neutrophils promote early osteogenesis but reduce cell viability, while N2 neutrophils enhance ECM mineralization. These effects seem to occur independently of gene expression changes. The results offer new insights into the roles of neutrophils in bone regeneration.
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