Time-dependent modulation of MC3T3-E1 pre-osteoblasts by a helium cold plasma: a preliminary study on its potential relevance for alveolar bone regeneration

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.