Epigenetic reprogramming periosteum promotes aging critical segmental bone defect repair via methylation remodeling

Critical segmental bone defects (CSBDs) heal poorly in the aging microenvironment, leading to high complication rates. This study developed a novel periosteum with epigenetic reprogramming capability to address this challenge. The material combines a three-layer structure with bone fracture-derived extracellular vesicles (BFVs) from juvenile mice. It demonstrated stable BFV release over 14 days and favorable tensile properties (modulus ∼0.22 MPa, elongation ∼200%). In vitro , extracts from BFV-loaded periosteum significantly reduced senescence markers (β-gal, γH2A.x, p16, p21) in aging bone marrow stromal cells (BMSCs) while enhancing osteogenesis. The material also modulated macrophage polarization toward the anti-inflammatory M2 phenotype and inhibited osteoclast formation. In senior mice with femoral CSBDs, the BFV-loaded periosteum combined with 3D-printed scaffolds maintained structural integrity and substantially enhanced bone regeneration, achieving repair levels comparable to mature mice. Reduced representation bisulfite sequencing revealed that the material reprogrammed the aging bone microenvironment by promoting DNA methylation in genebody regions. The Forkhead Box O3 ( Foxo3 ) gene emerged as a key regulator in this process. This periosteum effectively bridges mechanical compatibility between 3D-printed scaffolds and host bone while rejuvenating the aged bone microenvironment through epigenetic regulation, offering a promising strategy for treating skeletal injuries in the elderly. We developed a periosteum loaded with the optimized extracellular vesicles derived from the juvenile mice with bone fracture (named BFVs). This material possesses the ability of epigenetic reprogramming. And can improve the poor bone microenvironment and inflammatory microenvironment during the repair of critical segmental bone defects (CSBDs) in the aged by regulating the DNA methylation level. • A bioactive periosteum restores repair capacity of critical segmental bone defects in aged mice to mature levels. • This bioactive periosteum improves the aged bone microenvironment by regulating stem cells and macrophages. • Mechanistically, this bioactive periosteum epigenetically reprograms via genebody DNA methylation remodeling.