Pre‐Vascularized hMSC and hPDC Spheroids as Building Block Units for Bone Tissue Engineering

ABSTRACT Spheroids have emerged as valuable tools in bone tissue engineering, mimicking the cellular interactions in native tissues. However, the application of small and low‐cell‐number spheroids for simultaneous bone regeneration and vascularization remains underexplored. In this study, small pre‐vascularized spheroids (250 cells each) were developed, using human mesenchymal stem cells (hMSCs) or human periosteum‐derived stem cells (hPDCs), co‐cultured with human umbilical vein endothelial cells (HUVECs). Spheroids were evaluated for stability, osteogenic differentiation, and angiogenic potential. Results indicated that hMSC and hPDC spheroids formed stable structures, while HUVEC monocultures failed to achieve spheroid stability. Co‐cultures showed HUVEC localization patterns mimicking native vascular structures. Gene and protein analyses revealed distinct osteogenic potential between hMSC and hPDC spheroids, with the latter demonstrating superior and earlier differentiation. Additionally, vascular endothelial growth factor expression was higher in co‐cultures, suggesting enhanced angiogenic potential, particularly in hPDC spheroids. Using small‐diameter spheroids addresses limitations of conventional large spheroids, such as necrotic core formation and heterogeneous differentiation. These findings emphasize the promise of pre‐vascularized spheroids for scaffold‐free and scaffold‐based tissue engineering applications. Furthermore, their small size enables the exploration of their potential applications in 3D bioprinting, paving the way for the future development of more biomimetic vascularized bone constructs.