Macrophages enhance mesenchymal stem cell osteogenesis via down-regulation of reactive oxygen species.

OBJECTIVES

The role played by macrophages in regulating the differentiation of mesenchymal stem cells (MSCs) during wound healing and bone regeneration is increasingly being recognized. The present study compared the pro-osteogenic effects of three co-culture methods, conditioned medium generated by macrophages (CM), indirect culture (IC) or direct culture (DC) with macrophages, on bone marrow MSCs (BMMSCs).

METHODS

Primary BMMSCs were isolated, characterized and co-cultured with RAW264.7 mouse macrophages. Cell morphology and intracellular reactive oxygen species (ROS) levels were determined by scanning electron microscopy (SEM) and flow cytometry, respectively. Alkaline phosphatase (ALP) staining and assay, Alizarin red staining (ARS) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to evaluate osteogenic differentiation.

RESULTS

Inclusion of macrophages in any of the three co-culture methods resulted in improvement in osteogenic differentiation and mineralization of BMMSCs (DC > IC > CM), as measured by ALP staining and activity, ARS and osteoblastic gene expression (Runx2, Alp, Ocn and Bmp2). The enhanced osteogenesis was reversed with hydrogen peroxide. Macrophages reduced the increased levels of intracellular ROS generated by BMMSCs during osteogenic differentiation in a manner similar to the use of an antioxidant, N-acetyl cysteine.

CONCLUSIONS

Macrophages exert an osteogenesis-enhancing effect to accelerate BMMSC osteogenesis via ROS downregulation.

CLINICAL SIGNIFICANCE

The present findings suggest that targeting MSC-macrophage interaction is an effective strategy for regulating stem cell fate and facilitating bone regeneration.