Stromal vascular fraction self-assembles vascularized osteogenic organoids with immunomodulatory functions

Bone tissue engineering has enormous potential for treating complex orthopedic conditions such as Large Bone Defects (LBDs), delayed union or non-union fractures, and Osteonecrosis (ON). On the other hand, the adipose tissue-derived Stromal Vascular Fraction (SVF) comprises a heterogeneous population of cells, including Adipose-Derived Mesenchymal Stem Cells (ADSCs), Endothelial Progenitor Cells (EPCs), pericytes, and Hematopoietic Stem Cells (HSCs). Owing to its ease of acquisition, abundance, and the intrinsic involvement of its cellular components in bone repair, the SVF is presently considered a viable source for constructing osteogenic organoids. Herein, we leveraged the self-assembly capacity of SVF cells to construct spatially complex organoids with osteogenic, angiogenic, and immunomodulatory properties that mimic native tissues. Compared to spheroids from ADSCs alone, SVF cells exhibited natural tissue-like spatial organization, as well as superior osteogenic, angiogenic, and immunomodulatory potential, after two weeks of 3D spheroid culture. Furthermore, SVF-derived organoids showed distinct spatial architectures and functional profiles under both the growth and osteogenic induction conditions. Proteomic analysis and cytokine array profiling further revealed that SVF organoids were functionally enriched in the cytokine signaling and inflammatory/immune regulation pathways, with greater Extracellular Matrix (ECM) production observed under growth conditions compared to osteogenic induction. Moreover, in vivo experiments revealed that SVF organoids significantly accelerated fracture healing in nude mice. These findings present a simple and effective strategy for constructing multifunctional SVF-derived organoids, offering not only an innovative approach for bone tissue engineering but also a potential therapeutic avenue for clinical applications. A schematic illustration of the experimental workflow. Abdominal adipose tissue was harvested from the same donor and processed to isolate the SVF. Meanwhile, primary ADSCs (P0 ADSCs) were obtained through an initial culture of SVF-derived cells. Both SVF and P0 ADSCs were seeded into ultra-low attachment 96-well U-bottom plates for spontaneous self-assembly into spheroids. After three days of spheroid formation, some spheroids were maintained in the Growth Medium (GM) without induction, while others were switched to the Osteogenic Medium (OM) for osteogenic induction. The spheroids were then cultured until day 21 post-formation. For in vivo transplantation in nude mice, spheroids were collected on day 14 after the initial formation. • Stromal vascular fraction (SVF) cells self-assemble into vascularized organoids with osteogenic and immune properties. • SVF-derived organoids exhibit superior angiogenesis, osteogenesis, and cytokine signaling compared to ADSC spheroids. • SVF organoids form physiologically relevant spatial architectures mimicking native bone tissue microenvironments. • Without prior osteogenic induction, SVF organoids significantly accelerate fracture healing in vivo . • This work provides a simple, scalable, and clinically translatable strategy for bone tissue engineering.