Degenerate Wave and Capacitive Coupling Increase Human MSC Invasion and Proliferation While Reducing Cytotoxicity in an In Vitro Wound Healing Model

Non-unions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing; however, the effect of different clinical ES waveforms on BMMSCs cellular activities remains unknown. We compared the effects of direct current (DC), capacitive coupling (CC), pulsed electromagnetic field (PEMF) and degenerate wave (DW) on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G2/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay. All above findings were shown to be statistically significant (p<0.05). We conclude that ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. Application of DW or CC to the fracture site may help in the recruitment of BMMSCs to the wound that may enhance rate of bone healing at the fracture site

Enhanced neurogenic biomarker expression and reinnervation in human acute skin wounds treated by electrical stimulation

Electrical stimulation (ES) is known to promote cutaneous healing; however, its ability to regulate reinnervation remains unclear. First, we show that ES treatment of human acute cutaneous wounds (n = 40) increased reinnervation. Next, to define neurophysiologic mechanisms through which ES affects repair, microarray analysis of wound biopsy samples was performed on days 3, 7, 10, and 14 after wounding. This identified neural differentiation biomarkers TUBB3 (melanocyte development and neuronal marker) and its upstream molecule FIG4 (phosphatidylinositol (3,5)-bisphosphate 5-phosphatase) as significantly up-regulated after ES treatment. To demonstrate a functional ES-TUBB3 axis in cutaneous healing, we showed increased TUBB3 melanocytes and melanogenesis plus FIG4 and nerve growth factor expression, suggesting higher cellular differentiation. In support of this role of ES to regulate neural crest-derived cell fate and differentiation in vivo, knockdown of FIG4 in neuroblastoma cells resulted in vacuologenesis and cell degeneration, whereas ES treatment after FIG4-small interfering RNA transfection enhanced neural differentiation, survival, and integrity. Further characterization showed increased TUBB3 and protein gene product 9.5 Merkel cells during in vivo repair, after ES. We demonstrate that ES contributes to increased expression of neural differentiation biomarkers, reinnervation, and expansion of melanocyte and Merkel cell pool during repair. Targeted ES-assisted acceleration of healing has significant clinical implications

Enhancement of Differentiation and Mineralisation of Osteoblast-like Cells by Degenerate Electrical Waveform in an <i>In Vitro</i> Electrical Stimulation Model Compared to Capacitive Coupling

<div><p>Electrical stimulation (ES) is effective in enhancing bone healing, however the best electrical waveform, mode of application and mechanisms remains unclear. We recently reported the <i>in vitro</i> differential healing response of a novel electrical waveform called degenerate sine wave (DW) compared to other forms of ES. This study further explores this original observation on osteoblast cells. Here, we electrically stimulated SaOS-2 osteoblast-like cells with DW in an <i>in vitro</i> ES chamber (referred to as ‘DW stimulation’) and compared the intracellular effects to capacitive coupling (CC) stimulation. ES lasted for 4 h, followed by an incubation period of 20 h and subsequent ES for 4 additional hours. Cytotoxicity, proliferation, differentiation and mineralisation of the osteoblast-like cells were evaluated to determine the cell maturation process. DW significantly enhanced the differentiation of cells when compared to CC stimulation with increased alkaline phosphatase and collagen I gene expression by quantitative real time- polymerase chain reaction analysis (p<0.01). Moreover, DW significantly increased the mineralisation of cells compared to CC stimulation. Furthermore the transcription of osteocalcin, osteonectin, osteopontin and bone sialoprotein (p<0.05) was also up regulated by DW. However, ES did not augment the proliferation of cells. Translational analysis by immunocytochemistry and Western blotting showed increased collagen I, osteocalcin and osteonectin expression after DW than CC stimulation. In summary, we have demonstrated for the first time that DW stimulation in an <i>in vitro</i> ES chamber has a significant effect on maturation of osteoblast-like cells compared to CC stimulation of the same magnitude.</p></div

Cytotoxicity by LDH assay (2A).

<p>The cytotoxicity was greater for the osteoblast-like cells exposed to CC stimulation compared to DW stimulation. Cell proliferation by WST-1 assay (2B). Cell proliferation was greater for non-stimulated cells compared to cells exposed to electrical stimulation by 28 h. * p <0.05; ** p <0.01; *** p <0.001. OD  =  optical density.</p

Mineral nodule formation (400x magnification) after Alizarin Red S (ARS) staining (4A).

<p>Few mineral nodules could be observed for cells without ES. A greater number of mineral nodules were observed after DW and CC by 28 h with more intense staining for cells exposed to DW than CC. Semi-quantitative analysis of intracellular ARS by CPC extraction method (4B). ARS staining was greater for cells exposed to DW than CC stimulation by 28 h. *** p <0.001; $ p<0.0001.</p

Alkaline phosphatase (ALP) activity in the cell growth medium (3A).

<p>ALP activity was greater after DW stimulation compared to CC stimulation by 28 h. Intracellular ALP activity (3B). ALP activity was greater after DW stimulation compared to CC stimulation. * p <0.05; ** p <0.01; *** p <0.001.</p

Degenerate waveform (1A) when applied to the customized <i>in vitro</i> ES apparatus with ES chamber (1B) or Capacitive Coupling stimulation apparatus (1C).

<p>Degenerate waveform (1A) when applied to the customized <i>in vitro</i> ES apparatus with ES chamber (1B) or Capacitive Coupling stimulation apparatus (1C).</p