Tonsil‑derived mesenchymal stem cell‑derived extracellular vesicles suppress MAPK‑NF‑κB signaling and restore osteogenic differentiation in LPS‑stimulated periodontal ligament fibroblasts

The present study evaluated and compared the anti‑inflammatory and osteogenic effects of extracellular vesicles (EVs) derived from tonsil‑derived mesenchymal stem cells (T‑MSC‑EVs) in a lipopolysaccharide (LPS)‑induced in vitro model of periodontitis using human periodontal ligament fibroblasts (hPDLFs). hPDLFs were treated with LPS to induce inflammation, followed by treatment with T‑MSC‑EVs. Cell viability was assessed using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. The expression levels of inflammatory cytokines (<i>IL‑1β</i>, <i>IL‑6</i>, <i>IL‑8</i> and I<i>FN‑γ</i>) and osteogenic markers [alkaline phosphatase (ALP), bone sialoprotein, osteopontin, osteocalcin and sclerostin] were evaluated using reverse transcription‑quantitative PCR. Inflammatory signaling proteins (phosphorylated ERK, phosphorylated JNK, c‑Fos, c‑Jun and NF‑κB) were analyzed by western blotting. Osteogenic activity was assessed using an ALP activity assay and alizarin red staining over 21 days. Treatment with T‑MSC‑EVs significantly protected hPDLFs from LPS‑induced growth suppression. T‑MSC‑EVs exhibited selective immunomodulation, reducing IL‑8 and IFN‑γ expression, while preserving IL‑6 and IL‑1β expression, which was accompanied by inhibited MAPK‑activator protein 1 and NF‑κB signaling. Finally, T‑MSC‑EVs restored the osteogenic potential by recovering ALP activity, mineral deposition and expression of osteogenic marker genes repressed by LPS. These findings underscore the therapeutic potential of EVs as next‑generation biologics for periodontitis and emphasize the importance of selecting appropriate EV sources to achieve targeted immune modulation and tissue regeneration.