Neuroregenerative Potential of Stem-Cells-from-Apical-Papilla–Derived Neuronal Cell Spheroids Regulated by Stem Cells from Apical Papillae Under Various Microenvironments in a Pulp-On-Chip System

Introduction: Stem cells from apical papillae (SCAPs) display potent neuroregenerative potential by secreting neurotrophic factors and differentiating into neuronal-like cells, which are regulated by local niches. This study aimed to explore the interactions between SCAPderived neuronal cell spheroids and SCAPs under various microenvironments in a pulp-on-achip system. Methods: SCAPs were induced into neuronal cells by a cocktail of chemical molecules. The expression of neuronal markers was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. SCAP-derived neuronal cell spheroids were generated with micromolded agarose gels and embedded in collagen hydrogel. After characterization, the SCAP-derived neuronal cell spheroids were cocultured with SCAPs in a pulp-on-chip system under various microenvironments: a-Minimum Essential Medium (MEM)/neuronal maturation medium (NMM) (neurosphere only); undifferentiated SCAPs with a-MEM/NMM (neurosphere 1 SCAP); osteogenic-induced SCAPs (Oi-SCAPs) with osteogenic medium/NMM containing 0.02% hydroxyapatite nanoparticles (neurosphere 1 Oi-SCAP/hydroxyapatite nanoparticle); and Oi-SCAPs with a-MEM/NMM (neurosphere 1 Oi-SCAP). Neurite outgrowth and MAP2 and TUJ1 expression in SCAPderived neuronal cell spheroids were assessed by immunostaining. Results: SCAPs were efficiently chemically induced into neuronal-like cells. The expression levels of MAP2 and TUJ1 were upregulated in SCAP-derived neuronal cell spheroids when cocultured with undifferentiated SCAPs, followed by Oi-SCAPs with a-MEM and neurospheres cultured alone, while coculturing with Oi-SACPs in osteogenic medium/NMM with hydroxyapatite nanoparticles displayed the lowest expression. Neurite spreading was more prominent in the spheroids cocultured with undifferentiated SCAPs than in those of the other 3 groups. Conclusion: This work revealed that local microenvironments critically regulate the neuroregenerative potential of SCAP-derived neuronal cell spheroids. (J Endod 2022;48:1367-1377.)