The Translational Medicine Regarding Ozone in Saline Solutions. Comment on Armeli et al. Ozone Saline Solution Polarizes Microglial Cells Towards an Anti-Inflammatory Phenotype. Molecules 2025, 30, 3932.

This commentary critically evaluates the translational relevance of a recent study investigating the effects of ozonated saline solution (O3SS) on microglial and endothelial cell models. While the original research proposes potential antioxidant and anti-inflammatory benefits of low-dose ozone exposure, we identify significant methodological and conceptual flaws that undermine its conclusions. Key concerns include the unjustified assumption that ozone behaves similarly in microwell cultures and clinical infusion settings, despite known physicochemical differences affecting ozone stability and reactivity. The use of immortalized BV2 and HUVEC cells, which lack the complexity of in vivo systems, further limits the study's applicability. The absence of accurate ozone quantification, proper controls, protein-level validation, and kinetic modeling exacerbates these weaknesses. Our analysis also demonstrates, through differential equation modeling, that ozone rapidly decays in saline solutions, making systemic delivery via infusion chemically implausible as a therapeutic approach. Moreover, the extrapolation of in vitro gene expression data to systemic therapeutic claims lacks scientific justification. We conclude that while the observed cellular responses in vitro are of academic interest, they do not support the efficacy or safety of O3SS in clinical settings. A more rigorous approach is necessary to substantiate the biomedical potential of ozonated solutions.