The therapeutic usage of physical stimuli is framed in a highly heterogeneous research area, with variable levels of maturity and of translatability into clinical application. In particular, electrostimulation is deeply studied for its application on the autonomous nervous system, but less is known about the anti- inflammatory effects of such stimuli beyond the inflammatory reflex. Further, reproducibility and meta-analyses are extremely challenging, owing to the limited rationale on dosage and experimental standardization. It is specifically to address the fundamental question on the anti-inflammatory effects of electricity on biological systems, that we propose a series of controlled experiments on the effects of direct and alternate current delivered on a standardized 3D bioconstruct constituted by fibroblasts and keratinocytes in a collagen matrix, in the presence or absence of TNF-$\alpha$ as conventional inflammation inducer. This selected but systematic exploration, with transcriptomics backed by metabolomics at specific time points allows to obtain the first systemic overview of the biological functions at stake, highlighting the differential anti-inflammatory potential of such approaches, with promising results for 5 V direct current stimuli, correlating with the wound healing process. With our results, we wish to set the base for a rigorous systematic approach to the problem, fundamental towards future elucidations of the detailed mechanisms at stake, highlighting both the healing and damaging potential of such approaches.

Electrical stimulation (ES) is widely employed in both clinical therapies and research settings where it has shown promise in promoting tissue regeneration, wound healing, and inflammation control. Research has also highlighted ES as a regulator of DNA demethylation, which plays a critical role in nerve regeneration and cellular repair mechanisms. While the impact of ES on epigenetic processes is recognized, its broader effects on cellular functions, particularly in inflammation and wound healing, are less understood. We recently showed how ES impacts inflammatory states by modulating transcriptomic and metabolomic profiles in a 3Din vitromodel where human fibroblasts and keratinocytes are included in a collagen matrix, i.e., even in the absence of the nervous system. Here, we propose to deepen our exploration on the differential effects on DNA methylation, including an investigation of the correlation with age acceleration using a mitotic clock. These results confirm and caution on the differential effect of DC on inflamed and non-inflamed samples and suggest an involvement of direct current stimuli at 1 V ($DC1$) in the control of senescent processes associated with mitosis and inflammation; the mechanistic details of these will have to be clarified with additional experiments.

The therapeutic usage of physical stimuli is framed in a highly heterogeneous research area, with variable levels of maturity and of translatability into clinical application. In particular, electrostimulation is deeply studied for its application on the autonomous nervous system, but less is known about the anti-inflammatory effects of such stimuli beyond the inflammatory reflex . Further, reproducibility and meta-analyses on existing results are extremely challenging, owing to the limited rationale on dosage and experimental standardization. In this work we propose a series of controlled experiments on the effects of electrical stimuli (in direct and alternate current) delivered on a standardized 3D bioconstruct constituted by fibroblasts and keratinocytes in a collagen matrix. Transcriptomics backed by metabolomics at selected time points allow to obtain a first systematic overview of the biological functions at stake, highlighting the differential anti-inflammatory potential of such approaches, with promising results for 5V direct current stimuli. We hope that our results will trigger an interest and a facilitation in the study of the anti-inflammatory effects of physical stimuli, highlighting not only the potential but also the limitations of such approaches, offering, ultimately, solid evidence for future translation into the clinic.

Electrical stimulation (ES) is widely employed in both clinical therapies and research settings where it has shown promise in promoting tissue regeneration, wound healing and also inflammation control. Research has also highlighted ES as a regulator of DNA demethylation, which plays a critical role in nerve regeneration and cellular repair mechanisms. While the impact of ES on epigenetic processes is recognized, its broader effects on cellular functions, particularly in inflammation and wound healing, are less understood. We recently showed how ES impacts on inflammatory states by modulating transcriptomic and metabolomic profiles in a 3D in vitro model where human fibroblasts and keratinocytes are included in a collagen matrix, i.e even in the absence of nervous system. Here we propose to deepen our exploration on the differential effects on DNA methylation, including investigation of potential age acceleration with the usage of an epigenetic clock. These results support the potential of 5V Direct Current stimuli observed earlier at the transcriptional level to influence inflammatory processes, and suggest an involvement of DC5 in the control of senescent processes associated with inflammation.