Engineering Osteogenic Spheroids: The Impact of Endothelial Cell Localization on Vascularization and Differentiation

Abstract Bone tissue is highly vascularized, and robust blood flow is critical for successful bone regeneration. However, the intricate architecture of bone vascular networks and the mechanisms governing their development remain poorly understood, necessitating the development of in vitro models that replicate the native bone microenvironment. In this study, we investigated the influence of endothelial cell positioning within co‐cultured spheroids on vascularization and osteogenic differentiation. Human umbilical vein endothelial cells (HUVECs) and human bone marrow‐derived mesenchymal stem cells (hBMSCs) are employed to generate spheroids using two distinct co‐culture strategies: core‐shell and mixed configurations. Core‐shell spheroids are fabricated in two arrangements: hBMSCs‐core/HUVECs‐outer layer (M2H) and HUVECs‐core/hBMSCs‐outer layer (H2M). Mixed spheroids are created by co‐aggregation of HUVECs and hBMSCs. In vitro analyses revealed that endothelial cell localization significantly impacted spheroid morphology and function. Notably, M2H spheroids exhibited the highest VE‐cadherin levels, suggesting enhanced endothelial cell‐cell interactions. In Matrigel assays, M2H spheroids demonstrated superior angiogenic potential, evidenced by vascular network formation. Furthermore, we evaluated osteoblast differentiation within the spheroids to elucidate the interplay between endothelial cell positioning, vascularization, and bone development. This study provides valuable insights into the processes governing bone vascularization and offers a foundation for developing advanced tissue engineering strategies for regeneration.