Endothelial Nitric Oxide Synthase Restores Diabetic Dentin Regeneration via AKT/Endothelial Nitric Oxide Synthase Axis

ABSTRACT: Objective: While diabetes is known to impair dentin regenerative capacity, the specific role of endothelial nitric oxide synthase (eNOS) is poorly defined. This study aimed to elucidate how eNOS regulates the osteo/odontogenic differentiation of human dental pulp stem cells (hDPSCs) under high-glucose conditions and to determine its functional role in dentin regeneration in vivo. Methods: Immunohistochemistry and Western blot were used to analyse eNOS and odontogenic marker expression in diabetic dental pulp. hDPSCs with eNOS knockdown or overexpression were cultured under high glucose and evaluated using ALP activity, alizarin red staining, Western blot, and RT-qPCR. A rabbit tooth extraction model with DPSC implantation assessed in vivo dentin regeneration via micro-CT, histology, and electron microscopy. Results: Diabetic pulp showed reduced eNOS, DSPP, and DMP1 expression. High glucose suppressed p-AKT/eNOS signalling and impaired hDPSC differentiation, which was rescued by eNOS overexpression. In vivo, eNOS-overexpressing cells promoted significantly more dentin-like tissue formation with elevated DSPP/DMP1 expression. Conclusions: eNOS is essential for maintaining hDPSC differentiation under high-glucose conditions. Targeting eNOS signalling may represent a novel strategy to enhance dentin regeneration in diabetic patients. Clinical Relevance: Targeting the AKT/eNOS signalling axis offers a novel therapeutic strategy to enhance dentin regeneration in diabetic patients.