M1 macrophage-conditioned medium and Vitex agnus-castus effects on bone tissue organization in ovariectomized rats: In vivo and in vitro study.

BACKGROUND

Bone repair is a highly coordinated process regulated by the local microenvironment, in which macrophage polarization plays a key role, particularly under estrogen-deficient conditions. Natural immunomodulatory compounds, such as Vitex agnus-castus (monk's pepper; VAC), may influence macrophage-mediated responses and exert osteoprotective effects. This study investigated how M1 macrophage-conditioned medium (CM) and VAC, alone or in combination, influence bone tissue organization and osteogenic activity in ovariectomized (OVX) rats.

METHODS

Ovariectomized and SHAM rats received M1 macrophage-conditioned medium in calvarial defects and/or oral Vitex agnus-castus for 60 days. Bone neoformation, osteocyte density, and collagen organization were evaluated histomorphometrically. In vitro, mesenchymal stem cells were assessed for viability, alkaline phosphatase activity, and mineralization.

RESULTS

Despite systemic osteoporotic changes, OVX and SHAM groups showed similar bone formation patterns in calvarial defects, suggesting site-specific responses to estrogen deficiency. However, CM treatment significantly enhanced osteocyte density and improved the structural organization of newly formed bone in OVX animals. In contrast, VAC alone had limited effects on tissue architecture, while the combined treatment increased osteocyte numbers without improving overall bone organization. Cytokine profiling confirmed a pro-inflammatory macrophage phenotype, supporting a microenvironment-driven mechanism. In vitro, CM enhanced cell viability, alkaline phosphatase activity, and mineralization in OVX-derived cells, whereas VAC showed modest effects. The combined treatment underperformed compared to CM alone.

CONCLUSIONS

These findings indicate that macrophage-derived factors modulate the bone microenvironment, promoting structurally improved tissue repair under osteoporotic conditions and highlighting the complex interplay between immune signaling and bone tissue organization.