Xeno-Free Biomimetic-Scaffold Supports hiPSC-Derived Dental Organoid Formation and Tooth Regeneration

Tooth regeneration remains an unmet clinical challenge due to the structural complexity of dental tissues and the requirement for precise epithelial–mesenchymal interactions during development. Here, we present a xeno-free, biomimetic strategy for dental organoid engineering by integrating human induced pluripotent stem cell (hiPSC)–derived oral epithelial cells (OECs) and neural crest cells (NCCs) within a biodegradable chitosan–alginate (CA) scaffold. The CA scaffold provides a mechanically compliant and highly porous 3D microenvironment that supports hiPSC maintenance, lineage-specific differentiation, and spatial organization. Recombined OECs and NCCs self-organized into dental organoids that recapitulated key features of early odontogenesis, including coordinated epithelial–mesenchymal interactions, progressive expression of odontogenic markers (DSPP and AMBN), and time-dependent mineral deposition. Transcriptomic profiling revealed activation of gene programs associated with odontogenesis, amelogenesis, and craniofacial morphogenesis. Upon orthotopic implantation into a rat maxillary molar extraction site, cell-laden scaffolds supported cell survival, early tooth-like tissue organization, and localized mineralized matrix formation in vivo. Together, these results establish a xeno-free, scaffold-based platform for dental organoid formation and early tooth regeneration, providing a translational framework for stem cell–based dental tissue engineering and regenerative therapies. Overall design: Bulk RNA-seq analysis of hiPSC and the hiPSC-derived dental organoid at week 5