SHEDs and BMSCs exhibit distinct lineage preferences in HUVECs dynamic spheroid co-cultures: vascular versus osteogenic commitment.

Stem cells from human exfoliated deciduous teeth (SHEDs) offer a promising alternative to bone marrow-derived mesenchymal stem cells (BMSCs) for bone tissue engineering due to their accessibility, high proliferative potential, and multipotency. In this study, we compared the osteogenic and angiogenic potential of two mesenchymal stem cells subpopulations, SHEDs and BMSCs, when co-cultured with human umbilical vein endothelial cells (HUVECs) into spheroids over a period of 28 days in porous pullulan/dextran scaffolds loaded with hydroxyapatite (HAp) particles as the sole osteoinductive cue. Spheroids were cultured under static and dynamic conditions, with the latter employing a perfusion flow bioreactor to enhance solute transport and oxygenation. Dynamic culture conditions significantly improved cell viability compared to static culture (85% vs. 54% at Day 28), maintained spheroid integrity, and promoted the expression of angiogenic markers, such as the cluster of differentiation 31 (CD31) and von Willebrand factor (vWF), which under static culture were largely confined to the spheroid periphery. Furthermore, alpha-smooth muscle actin/neural-glial-antigen 2 (αSMA/NG2) and CD31/NG2 colocalization reflected close spatial associations between SHEDs and HUVECs, suggesting a supportive perivascular interaction under dynamic culture. In the presence of HUVECs, we found that HAp particles alone were insufficient to induce robust osteogenic differentiation in SHEDs. Weak alkaline phosphatase activity, minimal osteopontin and osteocalcin expression, and incomplete mineralization were observed under both static and dynamic conditions. In contrast, BMSC/HUVEC spheroids exhibited robust osteogenic differentiation and consistent mineral deposition. These results show intrinsic differences in the behavior of SHEDs and BMSCs when co-cultured with endothelial cells; while BMSCs tend to favor osteogenesis, SHEDs appear to adopt a more perivascular or pericytic behavior.