Background:

Dental follicle stem cells (DFSCs) show mesenchymal stem cell properties with the potential for oral bone regeneration. Stem cell properties can be impaired by reactive oxygen species (ROS), prompting us to examine the importance of scavenging ROS for stem cell-based tissue regeneration. This study aimed to investigate the effect and mechanism of N-acetylcysteine (NAC), a promising antioxidant, on the properties of DFSCs and DFSC-based oral bone regeneration. Methods DFSCs were cultured in media supplemented with different concentrations of NAC (0–10 mM). Cytologic experiments, RNA-sequencing and antioxidant assays were performed in vitro. Rat maxillary first molar extraction models were constructed, histological and radiological examinations were performed at day 7 post-surgery to investigate oral bone regeneration in tooth extraction sockets after local transplantation of NAC, DFSCs or NAC-treated DFSCs. Results 5 mM NAC-treated DFSCs exhibited better proliferation, less senescent rate, higher stem cell-specific marker and immune-related factor expression with the strongest osteogenic differentiation; 10 mM NAC was not beneficial for maintaining stem cell properties. RNA-sequencing identified 803 differentially expressed genes between DFSCs with and without 5 mM NAC. “Developmental process (GO:0032502)” was prominent, bioinformatic analysis of 394 involved genes revealed functional and pathway enrichment of ossification and PI3K/AKT pathway, respectively. Furthermore, after NAC treatment, the reduction of ROS levels (ROS, superoxide, hydrogen peroxide), the induction of antioxidant levels (glutathione, catalase, superoxide dismutase), the upregulation of PI3K/AKT signaling (PI3K-p110, PI3K-p85, AKT, phosphorylated-PI3K-p85, phosphorylated-AKT) and the rebound of ROS level upon PI3K/AKT inhibition were showed. Local transplantation of NAC, DFSCs or NAC-treated DFSCs were safe and promoted oral socket bone formation after tooth extraction, with application of NAC-treated DFSCs possessing the best effect. Conclusions The proper concentration of NAC enhances DFSC properties, especially osteogenesis, via PI3K/AKT/ROS signaling, and offers clinical potential for stem cell-based oral bone regeneration.