Engineered Dll4-overexpressing osteocyte-derived exosomes enhanced bone regeneration by regulating osteogenesis and angiogenesis

<b>Rationale:</b> Delayed fracture healing often results from impaired osteocyte network reconstruction and inadequate vascularization. Our prior work demonstrated that osteocytes engineered to overexpress Dll4 (Dll4-osteocytes) exert dual pro-osteogenic/angiogenic effects. Thus, this study explores the exosomes derived from Dll4-osteocytes (Dll4-Exo) as a cell-free strategy to coordinate bone-vascular regeneration and accelerate repair. <b>Methods:</b> Dll4-Exo were isolated from lentivirus-transduced Dll4-osteocytes. Mouse bone marrow stromal cells (ST2 cells) and human umbilical vein endothelial cells (HUVECs) were treated with Dll4-Exo to evaluate osteogenesis (ALP staining, mineralization, qRT-PCR) and angiogenesis (scratch/transwell migration, tube formation). Notch dependence was tested with γ-secretase inhibitor DAPT. <i>In vivo</i>, Dll4-Exo was locally administered in a mouse tibial fracture model. Healing was assessed via X-ray imaging, histology, immunohistochemistry, and immunofluorescence staining at days 14, 21, and 28. Exosomal miRNA profiles were analyzed by sequencing, and miR-23a-5p function was validated through mimic/inhibitor transfections. <b>Results:</b> Dll4-Exo activated Notch signaling in ST2 cells, significantly upregulating osteogenic genes (Alpl: 9.4-fold increase; mineralization: 62% increase) and enhancing HUVEC migration (2.6-fold) and tube formation. In the fracture model, Dll4-Exo accelerated callus formation, improved bone remodeling (OCN: 1.52-fold increase), and promoted revascularization (CD31⁺ vessel density: 1.56-fold increase with enhanced maturity). Through miRNA sequencing, miR-23a-5p was identified as the most enriched miRNA in Dll4-Exo, which was functionally transferred to both ST2 cells (3.0-fold increase) and HUVECs (2.7-fold increase). Mechanistic studies demonstrated that the pro-osteogenic effect of Dll4-Exo is exerted by miR-23a-5p via Notch signaling activation in ST2 cells, whereas its pro-angiogenic effect on HUVECs occurs through miR-23a-5p-independent mechanisms. <b>Conclusion:</b> Dll4-Exo carrying miR-23a-5p activates Notch-dependent osteogenesis in ST2 cells, while stimulating angiogenesis in HUVECs through alternative mechanisms, synergistically accelerating fracture healing and osteocyte network reconstruction. This engineered exosome platform represents a clinically viable strategy for bone regeneration.