Understanding the niche interactions between blood and bone through the in vitro co-culture of osteo-competent cells and endothelial cells is a key factor in unraveling therapeutic potentials in bone regeneration. This can be additionally supported by employing numerical simulation techniques to assess local physical factors, such as oxygen concentration, and mechanical stimuli, such as shear stress, that can mediate cellular communication. In this study, we developed a Mesenchymal Stem Cell line (MSC) and a Human Umbilical Vein Endothelial Cell line (HUVEC), which were co-cultured under flow conditions in a three-dimensional, porous, natural pullulan/dextran scaffold that was supplemented with hydroxyapatite crystals that allowed for the spontaneous formation of spheroids. After 2 weeks, their viability was higher under the dynamic conditions (>94%) than the static conditions (<75%), with dead cells central in the spheroids. Mineralization and collagen IV production increased under the dynamic conditions, correlating with osteogenesis and vasculogenesis. The endothelial cells clustered at the spheroidal core by day 7. Proliferation doubled in the dynamic conditions, especially at the scaffold peripheries. Lattice Boltzmann simulations showed negligible wall shear stress in the hydrogel pores but highlighted highly oxygenated zones coinciding with cell proliferation. A strong oxygen gradient likely influenced endothelial migration and cell distribution. Hypoxia was minimal, explaining high viability and spheroid maturation in the dynamic conditions. © 2024 by the authors.< Réduire