Hesperetin modulates osteoprogenitor cells and macrophages under zoledronic acid and inflammatory stress

To investigate the osteogenic and immunomodulatory effects of hesperetin (HT) on alveolar bone-derived mesenchymal stem cells (aBMSCs) and macrophages under zoledronic acid (ZA) and inflammatory stress. aBMSCs were exposed to ZA (0-10 µM) for 3 days, followed by HT (0-1000 µM) for 3 days. Cell viability was assessed for 3 days of treatments, and osteogenic activity was evaluated by alizarin red quantification at 14 and 21 days. THP-1-derived macrophages were polarized to M1 using lipopolysaccharides (LPS, 1 µg/mL) and treated with HT (1-50 µM) to evaluate cell viability and synthesis of cytokines (ELISA). A co-culture system of aBMSCs and macrophages (1:1 ratio) was established under inflammatory stimulation (LPS ± 20 µM HT) to assess cell viability, cytokine release and mineralized matrix formation. Data were analyzed by ANOVA/post-hoc tests (α = 5%). ZA significantly reduced aBMSCs viability and mineralization in a dose-dependent manner. HT (5-50 µM) enhanced mineralization in healthy aBMSCs and partially restored it after ZA exposure. In M1 macrophages, HT (5-20 µM) decreased TNF-α, IL-1α, and IL-6 synthesis without affecting viability. In inflammatory co-cultures, HT (20 µM) preserved cell viability, increased mineralized matrix deposition, and reduced cytokine release compared to LPS-only controls. This study provides the first evidence that HT can concurrently stimulate osteogenic differentiation and suppress inflammatory responses under ZA- and LPS-induced stress. HT emerges as a promising osteoimmunomodulatory adjuvant to enhance bone regeneration and mitigate bisphosphonate-related osteonecrosis. • Hesperetin enhanced osteogenic differentiation of bone mesenchymal stem cells. • Zoledronic acid impaired mineralization, partially rescued by hesperetin. • Hesperetin reduced the synthesis of pro-inflammatory cytokines in M1 macrophages. • Hesperetin enhanced mineralization in co-culture of bone cells and M1 macrophages. • Hesperetin shows translational potential for MRONJ prevention and bone repair.