Related Experiment Video
Updated: May 26, 2026
Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology
Published on: May 31, 2022
Low-inflammatory macrophage-BMSC crosstalk enhances osteogenesis with attenuation of oxidative stress
Tian Tian1, Changhuan Liu2, Lingnan Zhang3
1Department of Periodontics, Binzhou Medical University Hospital Binzhou 256603, Shandong, P. R. China.
Aim:
Macrophage-mesenchymal stem cell (MSC) interactions critically influence bone regeneration during periodontitis resolution. While macrophage polarization under strong inflammatory conditions has been well studied, the functional role of macrophages exposed to low-grade inflammatory stimulation and their impact on MSC osteogenesis remain poorly understood.
Methods:
RAW264.7 macrophages were stimulated with reduced doses of classical M1 inducers to generate a low-inflammatory transitional phenotype (M1semi). Macrophage polarization and inflammatory cytokine expression were assessed by quantitative real-time PCR. M1semi macrophages were co-cultured with bone marrow mesenchymal stem cells (BMSCs) using conditioned medium, Transwell indirect co-culture, or direct cell-cell contact, with or without oxidative stress induction by hydrogen peroxide. Intracellular reactive oxygen species (ROS) levels in BMSCs were evaluated by fluorescence staining, and osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining, ALP activity, and osteogenic gene expression.
Results:
Compared with classically activated M1 macrophages, M1semi macrophages exhibited reduced expression of pro-inflammatory cytokines and a polarization profile intermediate between M1 and M2 phenotypes. Among the co-culture systems tested, only direct contact with M1semi macrophages significantly enhanced osteogenic and angiogenic marker expression and increased ALP activity in BMSCs. This osteogenic enhancement was accompanied by a marked reduction in intracellular ROS levels. Importantly, exogenous induction of oxidative stress attenuated both ROS suppression and the osteogenic effects observed in direct co-culture.
Conclusion:
Macrophages exposed to low-grade inflammatory stimulation acquire a transitional phenotype with distinct immunomodulatory properties. Direct interaction with these macrophages enhances BMSC osteogenic differentiation, which is associated with reduced intracellular ROS accumulation. These findings suggest that modulation of oxidative stress contributes to macrophage-mediated osteogenesis in low-inflammatory environments, providing insight into immune-regulated bone regeneration during periodontal healing.
