Fig. 2: Effect of Bio-iES on cell development and maturation via eliminating neural stimulus-inertia.

a iES induced the opening of VGCC to allow an influx of extracellular Ca²⁺ through the activation of membrane potential. Nevertheless, under the long-term electrically polarization process, commonly used constant iES signals posed the accumulation of external charges on the cell membrane, resulting in the activation of VGCC. Inversely, Bio-iES signals have the ability to retain the activation of membrane potential by dissipating the accumulation of exogenous charges. b Schematic diagram of Bio-iES, Sw-iES, and Tw-iES stimulating motor neurons in vitro. The waveform of Bio-iES obtained from SD rat at active (red line) and resting state (blue line), while Sw-iES and Tw-iES were provided by an electrochemical workstation. c Typical electrophysiological action potential response (yellow and orange areas) from the cell membranes of Bio-iES, Tw-iES and iES groups before and after electrostimulation. d Relative Ca²⁺ level through different stimulation times. e Triple fluorescence staining images of motor neurons after 2 days culture. (Red: neuronal marker protein of III β-tubulin (Tuj 1) for filopodia, green: dendrite marker protein of the microtubule-associated protein-2 (Map) for neurites, blue: neuronal nuclei). Mean axon length f and relative Map expression level g and relative Tuj 1 expression level h were measured from Tuj 1/Map/DAPI triple fluorescence staining. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). All error bars indicate ±S.D.