Abstract

Scaffold-free, assembloid-like SHED-HUVEC microspheroids promote odontogenic-angiogenic coupling. • Microspheroids promote robust vascularized dental pulp formation in vivo . • 3D assembly activates a novel CX26/CFOS signaling axis in dental pulp stem cells. • CX26/CFOS drives key odontogenic (DMP1/DSPP) and angiogenic (VEGFA) factors. • Platform offers a translatable strategy for functional dental pulp regeneration. Conventional two-dimensional (2D) dental pulp culture models are limited by their inability to recapitulate native tissue complexity and their frequent dependence on exogenous scaffolds. Here, we engineer scaffold-free, assembloid-like microspheroids that integrate stem cells from human exfoliated deciduous teeth (SHED) with human umbilical vein endothelial cells (HUVECs) to model physiological odontogenic-angiogenic coupling. Three-dimensional (3D) SHED micro-spheroids significantly upregulate key odontogenic markers (DMP1, DSPP) and the angiogenic factor VEGFA compared to 2D cultures. When co-assembled with HUVECs, the resulting microspheroids further amplify the expression of angiogenic markers (CD31, EMCN) and the osteo/odontogenic transcription factor RUNX2. Subcutaneous transplantation in nude mice reveals that these SHED–HUVEC microspheroids generate robust H-type vascular networks and extensive mineralized dentin-like structures, outperforming SHED-only constructs. Mechanistically, 3D self-assembly activates the gap junction protein connexin 26 (CX26) and the immediate-early transcription factor CFOS in SHED. CX26/CFOS signaling collectively promote the upregulation of VEGFA, DMP1, and DSPP, a regulatory axis validated through loss-of-function studies. Our findings establish a potent, scaffold-free micro-spheroid platform for functional dental pulp regeneration and uncover the CX26/CFOS pathway as a master regulator of vascularized dental tissue formation, offering a translatable strategy for clinical pulp engineering. Download: Download hig