Superoxide dismutase 2 scavenges ROS to promote osteogenic differentiation of human periodontal ligament stem cells by regulating Smad3 in alveolar bone-defective rats.

Abstract

Osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is an essential event in alveolar bone regeneration. Oxidative stress may be the main inhibiting factor of hPDLSC osteogenesis. Superoxide dismutase 2 (SOD2) is a key antioxidant enzyme, but its effect on hPDLSC osteogenic differentiation is unclear. Several surface markers were detected by flow cytometry, and the differentiation potential of hPDLSCs was validated by alkaline phosphatase (ALP), Alizarin Red S, and Oil Red O staining. Osteogenic indicators of hPDLSCs were detected by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and ALP staining. Furthermore, alveolar bone defect rat models were analyzed through micro-CT, hematoxylin and eosin, and Masson staining. The intracellular reactive oxygen species (ROS) level was evaluated by a ROS assay kit. Finally, the expression of SOD2, Smad3, and p-Smad3 in hPDLSCs was detected by RT-qPCR and Western blotting (WB). SOD2 positively regulated the gene and protein expressions of ALP, BMP6, and RUNX2 in hPDLSCs (p<0.05). Ideal bone formation and continuous cortical bone were obtained by transplanting LV-SOD2 hPDLSCs (lentivirus vector for overexpressing SOD2 in hPDLSCs) in vivo. Exogenous H2 O2 downregulated osteogenic indicators (ALP, BMP6, RUNX2) in hPDLSCs (p<0.05); this was reversed by overexpression of SOD2. WB results showed that the Smad3 and p-Smad3 signaling pathways participated in the osteogenic process of SOD2 in hPDLSCs. SOD2 positively regulated hPDLSC osteogenic differentiation in vitro and in vivo. Mechanistically, SOD2 promotes hPDLSC osteogenic differentiation by regulating the phosphorylation of Smad3 to scavenge ROS. This work provides a theoretical basis for the treatment of alveolar bone regeneration.