Modulation of Osteogenesis by Conditioned Media of Differently Activated Macrophages: A Comparative In Vitro Study

Macrophages play an important role in bone regeneration by releasing bioactive factors that regulate the behavior of osteoprogenitor cells. In this in vitro study, we investigated how conditioned media (CM) derived from RAW 264.7 macrophages treated with lipopolysaccharide (LPS; 1 or 100 ng/mL), dexamethasone (DEX; 10^-6 M), or left untreated, modulate the proliferation and osteogenic differentiation of MC3T3-E1 preosteoblasts. Exposure to LPS and DEX produced different patterns of macrophage activation, assessed by microscopic morphology, nitroblue tetrazolium (NBT) reduction, and measurement of TNF-α secretion from macrophages. In the L929 bioassay, no CM was cytotoxic, while effects on MC3T3-E1 proliferation and differentiation varied with macrophage treatment and CM dilution. CM obtained from DEX-treated macrophages elicited the most pronounced pattern of osteogenic activity that included reduced cell proliferation, acquisition of osteoblast-like morphology, enhanced von Kossa staining, and pronounced osteocalcin and osteopontin immunoexpression. CM from untreated macrophages also supported osteogenesis, although to a lesser extent. At 100%, CMs from LPS-treated macrophages supported greater preosteoblast proliferation than other CMs, while their effects on differentiation depended on LPS dose and CM dilution. These findings indicate that macrophage-derived signals differentially modulate preosteoblast proliferation and differentiation depending on their functional state. This study provides a valuable in vitro cell model for the development of immunomodulatory and regenerative strategies in bone tissue engineering and broader applications in wound healing.