Due to its antimicrobial, anti-inflammatory and pro-healing properties, the application of cold atmospheric plasma (CAP) has emerged as a new and promising therapeutic strategy in various fields of medicine, including general medicine and dentistry. In this light, the aim of the present study was to investigate the effects of a homemade plasma jet on the cellular behaviour of two important cell types involved in gingivitis, namely gingival fibroblasts (HGF-1 cell line) and macrophages (RAW 264.7 cell line), by the direct application of CAP in different experimental conditions. The cellular behaviour of the HGF-1 cells was investigated in terms of viability/proliferation (LIVE/DEAD and CCK-8 assays), morphological features (immunofluorescent staining of the actin cytoskeleton) and fibronectin expression (immunocytochemical staining of the fibronectin network), while the macrophages’ response was evaluated through the assessment of the cellular survival/proliferation rate (LIVE/DEAD and CCK-8 assays), morphological behaviour (immunofluorescent staining of the actin cytoskeleton) and inflammatory activity (pro-inflammatory cytokine secretion profile (ELISA assay) and foreign body giant cells (FBGCs) formation (immunofluorescent staining of the actin cytoskeleton and multinuclearity index determination)). The in vitro biological assessment revealed an upward trend dependent on treatment time and number of CAP applications, in terms of fibroblasts proliferation (p < 0.0001) and fibronectin expression (p < 0.0001). On the other hand, the macrophages exposed to five consecutive CAP applications for longer treatment times (over 120 s) exhibited a strong pro-inflammatory activity, as evinced by their altered morphology, pro-inflammatory cytokine profile (p < 0.0001) and FBGCs formation. Overall, our results demonstrate that CAP exposure, when used with appropriate operating parameters, has a beneficial effect on the cellular response of HGF-1 and RAW 264.7 cells, thus paving the way for further in vitro and in vivo investigations that will allow the translation of CAP treatment from research to clinic as an alternative therapy for gingivitis.

While atmospheric pressure plasma sources are emerging as potentially innovative instruments in medicine, some aspects of the interaction between plasma and biological substrates remain unclear. The high diversity in both sources and applications in the literature, and the lack of a systematic testing protocol, has resulted in a wide variety of devices that cannot be efficiently compared with one another. In this work, an integrated benchmark involving physical, chemical, and biological diagnostics is proposed. The setup is designed to be stable and fixed, while remaining adaptable to different sources. Three different sources, for a total of five configurations, are compared, demonstrating the possibility of obtaining multimodal data. Comparing the biological effects in terms of E. coli abatement between direct and indirect treatments allowed for the exclusion of short-timescale species and phenomena to have a key role in the abatement. The chemical characterisation describes the equilibrium of reactive oxygen and nitrogen species in treated samples, whose presence in the water has been found to be coherent with the plasma operating gas and the nitrogen vibrational temperatures. Nitrate, nitrite and peroxide are excluded from having an autonomous role in the inactivation biochemistry, suggesting the presence of a synergistic effect.

Cold atmospheric plasma (CAP) has demonstrated promising antimicrobial activity and potential to promote tissue regeneration. However, most existing research has focused on its effects on soft tissue cells, such as fibroblasts, while its influence on osteoblasts, the main cells responsible for alveolar bone formation, is rather limited. As a first step towards addressing this gap, the present study assessed the biological effects of a newly custom-designed CAP source on the pre-osteoblastic MC3T3-E1 cell line, with a primary focus on the cellular survival and osteogenic differentiation. Through the use of diverse in vitro methods, the cytotoxicity (e.g. Live&Dead and CCK-8 assays; morphological characterisation) and osteogenic differentiation potential (e.g. alkaline phosphates activity and Alizarin Red S staining) of a helium-based CAP were assessed. The cytotoxicity results indicated a treatment duration- and exposure number-dependent response, i.e. repeated CAP exposures within the 30 s–60 s range yielded the highest cell survival and metabolic activity, while extended exposures (180 s–240 s) significantly reduced cell viability by approx. 30%. On the contrary, an opposite trend was observed in terms of osteogenic differentiation, where repeated, prolonged exposures (90 s−120 s), resulted in an enhanced matrix mineralisation, suggesting an increased level of late-stage osteogenic differentiation. Overall, these findings indicate that CAP treatment can modulate the MC3T3-E1 pre-osteoblasts’ behaviour in a dose-dependent manner, suggesting a potential relevance for bone regeneration applications, and possibly for alveolar bone regeneration. Importantly, this study represents an initial effort towards the exploration of the effects of CAP exposure on osteoblasts using a pre-osteoblastic cell line and highlights the need for further research through more in-depth studies (e.g. periodontal-specific cell models and in vivo studies) prior to clinical translation.

The detection and quantification of reactive oxygen and nitrogen species (RONS) in plasma-treated water (PTW) are essential for advancing plasma applications in biomedical and agricultural fields. However, RONS characterization remains challenging, as conventional techniques often require chemical reagents that can alter the sample. Electrochemical impedance spectroscopy (EIS) offers a non-destructive alternative by probing the electrical response of aqueous systems and providing information on ionic concentration, charge transfer, and diffusion processes. This study investigates the feasibility of EIS as a diagnostic tool for characterizing physicochemical changes in PTW. Calibration experiments were performed using saline solutions with different ionic concentrations to evaluate the sensitivity of impedance measurements. Impedance spectra were recorded over a frequency range of 0.1 Hz to 10 kHz and analyzed using Nyquist and Bode plots with equivalent circuit modeling. Deionized water was treated with cold atmospheric plasma at different discharge powers (3.53–10.15 W) and treatment times (5–30 min) to generate RONS. The results show that EIS can monitor plasma-induced changes in conductivity and interfacial properties associated with variations in ionic content. In particular, systematic changes in solution resistance and admittance were observed and were correlated with plasma-induced changes in ionic composition. These findings demonstrate that EIS is a sensitive and non-invasive diagnostic method for PTW analysis.