NRF2-mediated osteoblast anti-ferroptosis effect promotes induced membrane osteogenesis

Induced membrane technique (IMT) is a new method for repairing segmental bone defects. However, the mechanism of its defect repair is not clear. In recent years, several studies have gradually indicated that ferroptosis is closely related to bone remodeling. Therefore, this study mainly explored the impact of NRF2-mediated osteoblast anti-ferroptosis on bone mineralization within the induced membrane. Male Sprague-Dawley rats aged 12-14 weeks were randomly divided into four groups (n = 12): Model group, DMF (NRF2 agonist) group, ML385 (NRF2 inhibitor) group and Sham group. Except for Sham group, an IMT model of the right femur was established in all other groups. After 4 weeks and 8 weeks of treatment with DMF and ML385, compared to Model group, DMF group showed significantly higher levels of bone volume fraction (BV/TV), osteogenic factors and NRF2/ARE pathway-related factors (NRF2, GPX4, HO-1 and SLC7A11), while ferroptosis-related indicators (total iron, 4-HNE and MDA) were significantly lower. Conversely, ML385 group exhibited significantly higher ferroptosis-related indicators and lower levels of NRF2/ARE pathway-related factors and osteogenesis. In vitro, erastin could induce ferroptosis in osteoblasts. Compared to Erastin group, Erastin+oe-NRF2 (NRF2 overexpression) group showed significantly increased cell viability, mineralization ability, and levels of NRF2/ARE pathway-related factors, along with reduced ferroptosis effects. However, Erastin+si-NRF2 (NRF2 small interfering) group displayed enhanced ferroptosis effects and significantly reduced cell viability, mineralization ability, and levels of NRF2/ARE pathway-related factors. In conclusion, in the bone grafting area of the induced membrane, there existed ferroptosis caused by iron overload. Activating the anti-ferroptosis effect of osteoblasts mediated by the NRF2/ARE signaling cascade could promote growth and mineralization of bone grafts within the induced membrane.