In this present study, the combination of five scientifically-proven technologies, namely ultrasound (US), iontophoresis currents-also known as impulse microcurrents (MC), electroporation (ELPO) and high-voltage pulsatile currents (HVPC), were applied on fibroblast cells to learn more about the individual and synergic effects on cell proliferation and collagen synthesis. A safety assessment was conducted simultaneously. In addition, we were also seeking to establish a correlation between different stimuli, the production of nitric oxide (NO), and the production of collagen and cell proliferation. Nitric oxide (NO) has been suggested to be a pathophysiological modulator of cell proliferation, cell cycle arrest, and apoptosis. Several studies have indicated that low relative concentrations of NO seem to favor cell proliferation and anti-apoptotic responses and higher levels of NO favor pathways inducing cell cycle arrest, mitochondria respiration, senescence, or apoptosis [18] .

The individual stimulations were: ultrasound at a frequency of 3 MHz, microcurrents with an intensity level of 500 and 600 µA, electroporation, high-voltage pulsatile currents, control (no stimulation) and basic (placebo = stimulation environment).

The combined stimulations were: Zone I and Zone II stimulations as shown in Table 1.

Please see Figure 1 below: The medical device RMD: STRUCTURAL REMODELING SYSTEM, conceived and manufactured by ST. Medic, a division of Silhouet-Tone, Canada.

Murine fibroblast-derived cell line L929 (American Type Culture Collection, NCTC clone 929 of strain L) was used for the experiments. Cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and maintained in 25 cm² polystyrene culture flasks at 37˚C in a humidified atmosphere of 5/95% CO₂/air. Subcultures were obtained after reaching 70% - 80% confluence by trypsinization (0.25% trypsin in citrate saline). In order to test the cytotoxicity of the materials, cell suspensions were plated in a 12 well-plate in a density of 1 × 10⁵ cells/well with EMEM supplemented with 10% FBS and kept overnight at 37˚C in a 5% CO₂ humidified atmosphere. Then, each well was treated with different stimulations for 5 min. After 24 h of incubation, viability was measured using the trypan blue method according to the manufacturer’s protocol. Untreated cells were used as a negative control (C−) for normalization. Experiments were replicated three times.

In order to test the morphology of the cells after the different stimulations, L929 cells (1 × 10⁵ cells/well) were cultured in a single well plate of 35 mm for 24 h. Each plate received a different stimulation for 5 minutes and was incubated at 37˚C and 5% CO₂ continued for another 24 h. Finally, each plate was properly mounted in a microscope. All images were collected using the Leica TCS SP5 inverted Laser Scanning Confocal Microscope (LSCM) with the DM600 fixed stage microscope and the Leica Application Suite software. The settings for the Laser Scanning Confocal Microscope were originally chosen as outlined by the manufacturer’s description.

To determine the number of cells and their viability using trypan blue, 100 ml of trypsinized and re-suspended cells were mixed with 20 ml of 0.4% trypan blue dye solution (Sigma-Aldrich) for 1 min. Cells were immediately counted using a Neubauermicrochamber with a light microscope. All counts were done using four technical duplicates of each sample.

The free radical nature of NO makes it highly reactive and very short-lived in biological systems. Therefore, NO is generally viewed as an autocrine or paracrine messenger, mostly regulating local intracellular processes or acting on cells in its near vicinity. The major metabolic pathway for NO involves its rapid oxidation into the higher nitrogen oxides nitrite and nitrate. While the colorimetric Griess reagent detects nitrite, nitrate needs to be reduced to nitrite for detection. NO produced by fibroblast cells after stimulation was estimated spectrophotometrically as a formed nitrite (NO₂). NO synthesized by fibroblasts dissolves in medium and is then oxidized to yield both nitrate and nitrite. Twenty four hours after stimulation, 500 ml of culture medium was taken from each well. To measure the nitrite content, 100 ml of the culture medium was incubated with 100 ml of Griess reagent (1% sulfanilamide in 0.1 mol/l HCl and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride) at room temperature for 10 min. Then the absorbance was measured at 540 nm using a microplate reader. The nitrite content was calculated based on a standard curve constructed with NaNO₂.

The effects of stimulation on fibroblast collagen synthesis were determined using a collagen assay test kit (Sircol, Biocolor Ltd.). EMEM media, in the absence of a sample, was maintained as negative control. As per the Sircol instructions, the supernatant was collected and mixed with the Sircol dye reagent and gently mixed at room temperature for 30 min, followed by centrifugation at 1500 g for 10 min to collect the collagen-dye pellets. The samples were subsequently dissolved in 750 µL alkali reagent and absorbance was measured at 555 nm. The data was expressed in terms of optical density of collagen absorbance by spectrophotometer.

Our results show that cell density increases after stimulation. The different stimuli do not exhibit the same cellular proliferation. The largest cell densities occur upon stimulation with ultrasound (50%), microcurrent (500 µA) and the zone II combination (Figure 2).

Trypan blue staining analysis revealed that cell proliferation increases with stimulation. Aside from electroporation, all other forms of stimulation cause an increase in the number of living cells 24 hours after stimulation compared to control. Stimulation known as Zone II, which is the combination of all stimuli (ultrasound, microcurrent, electroporation and HVPC), has the highest proliferation rate compared to zone I, which is a combination of two types of stimuli (US and MC) (Figure 3). Microcurrents appear to stimulate proliferation more than ultrasound.

Stimulation has an immediate effect on the production of nitric oxide (NO): The Control and Basic conditions show no nitric oxide before 24 h. Stimulation by ultrasound shows the synthesis of nitrogen monoxide 1 hour after, while the other stimulations show nitric oxide just 5 min after stimulation. As shown in Figure 4, at the cellular level, 24 hours after stimulation, individual stimuli are similar to the Basic condition. However, the synergy of the five technologies, represented by Zone II, shows a greater production of nitric oxide. The amount of nitric oxide increases over time. HVPC presents the maximum production of nitric oxide 5 minutes after stimulation, while it is after 20 minutes for zone I. The production of nitric oxide for Zone II is higher than others at 35 min.

Collagen is a protein in the extracellular matrix that is synthesized by fibroblasts in the dermis, where the

balance between collagen synthesis and degradation is an important element in the wound healing and skin rejuvenation processes. With regard to the wound healing effects of the KRB extract one day and two days post- treatment, our results show that collagen synthesis takes place after individual stimulations such as ultrasound, electrophoresis and also after combined stimuli are applied. However, HVPC does not induce collagenesis, as shown in Figure 5.