Abstract
Bone organoids (BOs) demonstrate excellent therapeutic effects in bone defect transplantation, but their high cultivation difficulty and cost limit widespread adoption, prompting us to focus on their derivatives. Whether BO-derived extracellular vesicles (BOEVs) can promote bone regeneration and whether they offer greater advantages than stem cell-derived extracellular vesicles (EVs) remains unclear. In this study, we isolated rat bone marrow mesenchymal stem cells (BMSCs) and induced them into BOs. As culture duration increased, BO size gradually enlarged to 203.71 ± 45.30 μm, but the number of spheroids progressively decreased. Messenger RNA (mRNA) sequencing revealed that after seven days of culture, BOs exhibited hypoxic alterations, leading to increased expression of cytokines, including hypoxia-inducible factor-1α (HIF-1α). Subsequently, BOEVs were isolated from BO culture medium at corresponding time points. Both BOEVs cultured for seven days (BOEV-7d) and 14 days (BOEV-14d) were enriched with bone morphogenetic protein 2 (BMP-2), transforming growth factor beta 1 (TGF-β1), HIF-1α, and vascular endothelial growth factor (VEGF). Co-culturing BOEVs with human umbilical vein endothelial cells and hFOB1.19 cells revealed that BOEV-7d significantly promoted tube length and branching while upregulating VEGF and CCN2 mRNA, demonstrating superior effects compared to BOEV-1d and BOEV-14d. Concurrently, BOEV-7d enhanced alizarin red S staining levels in hFOB1.19 cells, while the expression of osteogenesis-related genes Runx2, Col1a1, and Ocn was positively correlated with culture duration. Notably, BOEVs demonstrated significantly greater osteogenic and angiogenic effects than BMSC-derived EVs in 2D culture. This study demonstrates, in vitro, that BOEVs play dual roles in bone and vascular regeneration and have the potential to modulate the vascularization–osteogenesis pathway in bone defects. It offers insights into bone defect treatment and inspires research on organoid-derived EVs.