Icariin-Loaded Milk-Derived Extracellular Vesicles: Protective Effect on Inflammatory Bone Defects via HIF-1α

Objective: Icariin (ICA) is an active small molecule extracted from Epimedium, possessing therapeutic potential for inflammatory bone destruction. Small extracellular vesicles (MEVs) derived from bovine milk are safe and efficient drug delivery carriers. We aimed to explore the potential of ICA-loaded bovine milk EVs (ICA-MEVs) to repair inflammatory bone defects in an inflammatory microenvironment and investigated the underlying molecular mechanism, providing new ideas for the treatment of inflammatory bone defects. Methods: We fabricated icariin (ICA)-loaded milk-derived extracellular vesicles (ICA-MEVs) embedded in GelMA hydrogel and systematically evaluated the in vivo repairing efficacy against lipopolysaccharide (LPS)-induced inflammatory calvarial bone defects via micro-CT, HE staining, Masson staining and immunohistochemistry. Subsequent in vitro cellular experiments were carried out to uncover the regulatory mechanism by which ICA-MEVs promotes LPS-inhibited osteoblast proliferation and osteogenic differentiation. Results: ICA-MEVs significantly promoted the repair of inflammatory bone defects, upregulated osteogenic factors such as BMP-2, OCN, and Runx-2, and reduced the levels of IL-1β and TNF-α. ICA-loaded MEVs facilitated the proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts while alleviating cellular inflammatory activation. Mechanistically, ICA-MEVs promoted bone repair by elevating LIM1 expression. Elevated LIM1 bound to the endogenous HIF-1α promoter and triggered subsequent transcriptional activation of HIF-1α. Conclusions: Under inflammatory conditions, ICA-MEVs effectively promoted the proliferation and differentiation of MC3T3-E1 cells and inhibited the expression of inflammatory factors. Mechanistically, ICA-MEVs upregulated HIF-1α transcription and expression by potentiating the LIM1-mediated transcriptional activation of the HIF-1α promoter, thereby facilitating inflammatory bone repair. Although milk-derived EVs exhibited favorable safety profiles in this preclinical study, comprehensive detection of immunogenicity and long-term adverse reactions will be necessary in follow-up research to support clinical transformation.