Nonthermal Atmospheric Plasma Induces Osteogenic Activity in Osteoblasts through the ROS/WNT Signaling Axis

Nonthermal plasma (NTP) has garnered attention for its potential in various biological activities. Due to the interactions with proteins and other cellular components, NTPs might stimulate osteoblast activity, but the exact mechanisms are still unknown. This study used two kinds of NTP: dielectric barrier discharge (DBD) and jet NTP (JNTP) to treat serum-free cell culture media at different time intervals. The indirect (plasma-media interfaces of ion exchange) exposure to osteoblasts was analyzed to determine whether it could influence osteoblast behavior and stimulate their osteogenic activity. Among various NTP-ionized media, 25% to 100% of the JNTP medium (JNTPM, treated for 10 min) showed noticeable induction of ALP activity in osteoblasts. 25% JNTPM induced maximum ALP activity and also demonstrated enhanced mRNA expression of osteogenic markers like Runx-2, Osterix, collagen 1α, bone sialoprotein, and osteocalcin. In addition, JNTPM treatment to osteoblasts demonstrated enhanced collagen production and mineralization. The administration of JNTPM to SaOS-2 cells increased Axin-2 reporter activity, along with enhanced stabilization of β-catenin and phosphorylation of GSK3-β, indicating the stimulation of the WNT signaling pathway. Moreover, JNTPM treatment increased osteoblasts' intracellular reactive oxygen species (ROS) levels. Prior exposure of N-acetyl-l-cysteine (NAC) to JNTPM-treated osteoblasts diminished the phosphorylation of GSK3-β, Axin-2 reporter activity, and the stability of β-catenin, highlighting the importance of intracellular ROS in enhancing Wnt signaling and osteogenic activity in osteoblasts in response to JNTPM. Thus, JNTPM treatment induces osteogenic stimulatory properties in osteoblasts through cross-talk between ROS and the Wnt signaling axis, suggesting a potential therapeutic approach for bone formation.