3D-printed dictamni-calcium silicate scaffolds modulate the osteoimmune microenvironment and enhance macrophage-derived exosomal miR-21 signaling in vascularized bone regeneration

Reconstruction of critical-sized bone defects remains a formidable clinical challenge due to a persistent, localized, pro-inflammatory microenvironment that disrupts the coordinated process of osteogenic-angiogenic coupling. To address this, we engineered 3D-printed calcium silicate scaffolds containing Dictamni Radicis Cortex to modulate the osteoimmune niche. In vitro models demonstrated that Dic5 scaffolds effectively attenuated the pro-inflammatory state, promoting the transition of macrophages from an M1 to a proregenerative M2 phenotype, characterized by anti-inflammatory cytokine secretion. In parallel, elevated levels of angiogenesis-related factors were also observed. In vivo assessments using a rabbit bone defect model corroborated these findings, with Dic5 scaffolds implantation was associated with the rapid resolution of local tissue inflammation, facilitation of early vascular network formation, and accelerated highly mineralized bone healing compared to the Dic0 group. To elucidate the molecular mechanisms linking this immunomodulation to the observed therapeutic efficacy, we investigated macrophage-mediated paracrine signaling. Transcriptomic profiling using next-generation sequencing revealed that extracellular vesicles secreted by these modulated macrophages were significantly enriched with miR-21-5p. Subsequent in vitro mechanistic studies demonstrated that upon internalization by human mesenchymal stem cells, exosomal miR-21 acts as an epigenetic regulator, targeting and silencing inhibitory genes, specifically Spry1 and Pten, thereby unlocking downstream signaling pathways to promote the expression of osteogenic effectors, including Runx2 and osteocalcin. Finally, blocking miR-21 with a specific inhibitor significantly abolished Dic5-M2EV-mediated osteogenic capabilities. In summary, this study presents a translatable phytochemically engineered 3D bioscaffold and elucidates the regulatory role of the biomaterial-macrophage-exosomal miR-21 axis, providing a promising cell-free therapeutic strategy for vascularized bone tissue engineering.