Enhancement of Osteoblast Differentiation Using No-Ozone Cold Plasma on Human Periodontal Ligament Cells

Periodontitis is an inflammatory disease that leads to periodontal tissue destruction and bone resorption. Proliferation and differentiation of cells capable of differentiating into osteoblasts is important for reconstructing periodontal tissues destroyed by periodontitis. In this study, the effects of the nozone (no-ozone) cold plasma (NCP) treatment on osteoblastic differentiation in periodontal ligament (PDL) cells were investigated. To test the toxicity of NCP on PDL cells, various NCP treatment methods and durations were tested, and time-dependent cell proliferation was analyzed using a water-soluble tetrazolium salts-1 assay. To determine the effect of NCP on PDL cell differentiation, the cells were provided with osteogenic media immediately after an NCP treatment to induce differentiation; the cells were then analyzed using alkaline phosphatase (ALP) staining, an ALP activity assay, real time PCR, and Alizarin Red S staining. The NCP treatment without toxicity on PDL cells was the condition of 1-min NCP treatment immediately followed by the replacement with fresh media. NCP increased ALP, osteocalcin, osteonectin, and osteopontin expression, as well as mineralization nodule formation. NCP treatment promotes osteoblastic differentiation of PDL cells; therefore, it may be beneficial for treating periodontitis.