Beneficial effects of non-invasive physical plasma on human periodontal ligament cells in vitro

Abstract

Introduction: Periodontitis is a chronic inflammatory disease of the periodontium that can lead to the loss of affected teeth if left untreated. It is induced by a multifactorial process centered on microbial pathogens such as Fusobacterium nucleatum (F.n.). Non-invasive physical plasma (NIPP), a highly reactive gas, has become a focus of research, not only for its hemostatic, proliferation-enhancing and apoptotic properties, but also for its antimicrobial potential. The objective of this study was to examine the impact of NIPP on human periodontal ligament (PDL) cells that had been induced into a state of periodontal infection in vitro.

Methods: Initially, the solitary effect of NIPP was evaluated by measuring temperature and pH and analyzing reactive oxygen species (ROS). Additionally, DAPI and phalloidin staining were employed to investigate possible cytotoxic effects. The cells were pre-incubated with F.n. and treated with NIPP after 24 hours. Interleukin (IL)-6 and IL-8 were analyzed at mRNA and protein levels, respectively, by real-time PCR and ELISA.

Results: NIPP alone had no significant effect on PDL cells. However, the F.n.-induced upregulation of IL-6 and IL-8 was counteracted by NIPP.

Discussion: Thus, the utilization of NIPP may be regarded as a promising therapeutic strategy for the treatment of periodontal diseases.

Keywords: antimicrobial; cold plasma; human periodontal ligament cells; non-invasive physical plasma; non-thermal plasma; periodontology.

FIGURE 1

Basic characterization of NIPP during treatment: (A) temperature of the NIPP device and treated objects, n = 5. * Statistical significance compared to NIPP 0 s (p < 0.05). (B) Plasma ONE with probe PS30 and counter electrode in discharge mode. (C) Magnification of the probe tip. (D) Thermal image of the probe tip using the Optris PI 400i infrared camera with Optri PIX Connect analysis software (Optris, Berlin, Germany). (E) Thermal image of the bottom view of the probe tip.

FIGURE 2

Basic characterization of NIPP during treatment of medium: (A) pH of medium, n = 5 (B) O₃ concentration in medium, n = 2 (C) H₂O₂ concentration in medium, n = 6 (D) NO₃^– concentration in medium, n = 5 (E) NO₂^– concentration in medium, n = 5. *Statistical significance compared to control (p < 0.05).

FIGURE 3

Immunofluorescence staining of ROS production in PDL cells, 10 min after NIPP treatment. Cells were treated for 0 s (A), 30 s (B), 120 s (C), 180 s (D). The scale bar represents 100 μm. ROS fluorescence was quantified using a fluorescent reader (E). n = 4. *Statistical significance (p < 0.05).

FIGURE 4

Immunofluorescence staining of periodontal ligament (PDL) cells with DAPI (blue staining of the nucleus) and phalloidin (green staining of the cytoskeleton) that were left untreated (A) or treated with NIPP for 30 s at 24 h (B). NIPP treatment showed no cytotoxic effects. The scale bar represents 100 μm.

FIGURE 5

IL-6 and IL-8 levels in PDL cells after pre-incubation with inactivated F.n. at different concentrations (OD = 0.025, 0.05, 0.01). (A) IL-6 mRNA expression at 24 h, n = 9 (B) IL-6 protein level at 48 h, n = 12 (C) IL-8 mRNA expression at 24 h, n = 9 (D) IL-8 protein level at 48 h, n = 12. *Statistical significance (p < 0.05).

FIGURE 6

IL-6 regulation in PDL cells after 24 h pre-incubation with inactivated F.n. at OD 0.025 (+) and after 30 s of NIPP treatment (+). (A) IL-6 mRNA expression at 24 h, n = 6 (B) IL-6 protein level at 48 h, n = 12. * Statistical significance (p < 0.05).

FIGURE 7

IL-8 regulation in PDL cells after 24 h pre-incubation with inactivated F.n. at OD 0.025 (+) and after 30 s of NIPP treatment (+). (A) IL-8 mRNA expression at 24 h, n = 6 (B) IL-8 protein level at 48 h, n = 12. * Statistical significance (p < 0.05).