Reply to Franzini et al. 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”

In this Reply, we address the criticisms raised by Franzini, Valdenassi, and Chirumbolo concerning our study on the effects of ozonized saline solution (O3SS) on microglial polarization and endothelial responses in vitro. We clarify that the primary aim of the original work was mechanistic, relying on rigorously controlled cellular models that are universally recognized as essential preclinical tools in translational medicine. We reaffirm the validity of our experimental approach, including the preparation and characterization of O3SS based on empirically validated methodologies, direct ozone quantification, and standardized protocols consistent with the existing literature and clinical practice. Concerns regarding ozone chemistry, dose relevance, and hypochlorite formation are addressed through analytical validation, biological threshold considerations, and the use of certified assays. We further justify the choice of BV2 microglia and HUVEC cells as established and widely used models for investigating inflammatory and vascular pathways under reproducible conditions. Statistical analyses, gene expression interpretation, and the absence of comparative pharmacological agents are discussed in the context of the study’s focused objectives. Finally, we place our findings within the established framework of ozone as an indirect pro-oxidant that elicits adaptive redox signaling (“oxidative eustress”), emphasizing the translational relevance of in vitro systems for elucidating early mechanistic events. Overall, we maintain that our study provides a robust, balanced, and evidence-based contribution to the understanding of ozone-derived redox biology.