<scp>SSUH2</scp> Promotes Odontogenic Differentiation of <scp>SCAPs</scp> via the <scp>FOXM1</scp> / <scp>PDK1</scp> ‐Mediated Regulation of Mitochondrial Function

AIM: Regenerative endodontics focuses on the restoration of the pulp-dentine complex by promoting odontogenic differentiation of stem cells of the apical papilla (SCAPs). Although SSUH2 has been implicated in developmental processes and dentine dysplasia type I (DD-I), the specific role of SSUH2 in regulating SCAPs differentiation remains unclear. In this study, we identified SSUH2 as a novel biomarker for SCAPs odontogenic differentiation and revealed its critical regulatory mechanism. METHODOLOGY: Immunohistochemical staining of mouse mandibular first molar sections and immunofluorescence staining of human dental pulp tissues were performed to investigate the expression characteristics of SSUH2 during root development. SCAPs were treated with siRNA-mediated knockdown or lentivirus-mediated overexpression of SSUH2, followed by assessment of proliferation and odontogenic differentiation. Subcutaneous implantation of hydrogel-scaffold-root fragments loaded with SSUH2-overexpressing SCAPs in nude mice was performed to evaluate in vivo odontogenic capacity. Mechanistically, transcriptome sequencing and bioinformatic analysis identified downstream signalling pathways. Co-immunoprecipitation, nuclear-cytoplasmic fractionation, and immunofluorescence confirmed the direct interaction between SSUH2 and FOXM1, promoting FOXM1 nuclear translocation and subsequent transcriptional upregulation of PDK1. Moreover, transmission electron microscopy, mitochondrial membrane potential assays and ROS detection collectively demonstrated that SSUH2 regulates mitochondrial function in SCAPs via the FOXM1/PDK1 axis. RESULTS: During the development of the first molar in the mouse's lower jaw, SSUH2 is enriched in the root tip papilla region and it is also enriched in the root tip papilla region of humans. SSUH2 positively regulated the odontogenic differentiation of SCAPs in vitro. In vivo transplantation models showed that SSUH2 overexpression enhanced the odontogenic differentiation capacity of SCAPs and promoted dentine-pulp-like structure formation. Transcriptomic and functional analyses indicated that SSUH2 maintained mitochondrial function. SSUH2 directly interacted with FOXM1, promoted its nuclear translocation, and upregulated PDK1 expression. FOXM1 silencing abrogated SSUH2-mediated enhancement of both mitochondrial function and odontogenic differentiation in SCAPs. CONCLUSIONS: Our study is the first to reveal that SSUH2 promotes the odontogenic differentiation of SCAPs through the FOXM1/PDK1 axis by regulating mitochondrial function. These findings suggest that SSUH2 could serve as a potential therapeutic target for enhancing the odontogenic differentiation of SCAPs and provide a novel strategic direction for pulp-dentine complex regeneration.