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.

Angiogenesis is induced and wound size is reduced by electrical stimulation in an acute wound healing model in human skin.

Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.

Electrical stimulation enhances epidermal proliferation in human cutaneous wounds by modulating p53-SIVA1 interaction.

Cutaneous wounds establish endogenous "wound current" upon injury until re-epithelialization is complete. Keratinocyte proliferation, regulated partly by p53, is required for epidermal closure. SIVA1 promotes human double minute 2 homolog (HDM2)-mediated p53 regulation. However, the role of SIVA1 in wound healing is obscure. Here, we report that electrical stimulation (ES) accelerates wound healing by upregulating SIVA1 and its subsequent ability to modulate p53 activities. Cultured donut-shaped human skin explants, subjected to ES, exhibited better epidermal stratification, increased proliferation, and upregulation of gene and protein expression of HDM2/SIVA1, compared with non-ES-treated explants. ES significantly increased in vitro keratinocyte proliferation and phospho-p53-SIVA1 interaction; however, this showed stable expression of phospho-p53, which increased significantly in the absence of SIVA1. Here, HDM2 alone was unable to downregulate nuclear-accumulated phospho-p53, which was evident from decreased proliferation and increased sub-G1 population seen by flow cytometry. Further examination of the epidermis of human cutaneous wounds showed higher p53-SIVA1 coexpression and proliferation 7 days after injury in ES-treated wounds compared with control wounds. In summary, ES-inducible SIVA1 modulates p53 activities in proliferating keratinocytes, and exogenous ES affects p53/HDM2/SIVA1 axis leading to increased proliferation during re-epithelialization. This highlights ES as a potential strategy for enhancing cutaneous repair.

Enhancement of differentiation and mineralisation of osteoblast-like cells by degenerate electrical waveform in an in vitro electrical stimulation model compared to capacitive coupling.

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 in vitro 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 in vitro 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 in vitro ES chamber has a significant effect on maturation of osteoblast-like cells compared to CC stimulation of the same magnitude.

Significant reduction of symptoms of scarring with electrical stimulation: evaluated with subjective and objective assessment tools in a prospective noncontrolled case series.

INTRODUCTION: Fenzian wave (FW) electrical stimulation has been shown to influence cutaneous wound healing. The authors previously published a case series investigating the effect of FW on symptomatic abnormal skin scars (raised dermal scars [RDS]) using spectrophotometric intracutaneous analysis (SIAscopy). In addition, a human volunteer sequential biopsy study in acute cutaneous wounds was conducted, which demonstrated that FW increased vascularity. The aim of this study was to evaluate the effectiveness of FW on symptomatic RDS using full-field laser perfusion imaging (FLPI) to assess changes in dermal blood flow. METHODS: Eighteen patients with RDS and long-term pain and pruritus participated.Time points analyzed were day 0, weeks 1 and 2, and months 1 and 2. Symptoms were monitored using a subjective numerical rating scale. Additionally, a Manchester Scar Scale and digital photographywere used. Objective noninvasive measures captured quantitative data: SIAscopy to measure melanin, hemoglobin and collagen levels, and FLPI to assess the dermal blood flow. RESULTS: There were statistically significant reductions in pain scores (from day 0 to month 1, P = 0.007) and pruritus scores (from day 0 to week 1, P = 0.007; and day 0 to month 1, P = 0.002). The trend for melanin levels demonstrated an increase from day 0 to week 1, hemoglobin levels showed an increase from day 0 to week 2, and hemoglobin flux increased from day 0 to week 2 (not statistically significant). CONCLUSION: This report demonstrates that FW electrical stimulation significantly reduces the symptoms of pain and pruritus in patients with RDS. This unique treatment has the potential for management of symptomatic skin scarring. .

Electrical stimulation increases blood flow and haemoglobin levels in acute cutaneous wounds without affecting wound closure time: evidenced by non-invasive assessment of temporal biopsy wounds in human volunteers.

Recent studies highlighted the beneficial effects of a novel electrical stimulation waveform, the degenerate wave (DW), on skin fibroblasts and symptomatic skin scarring. However, no study to date has investigated the role of DW on acute cutaneous wounds. Therefore, we evaluated this in a trial using a temporal punch biopsy model. Twenty healthy volunteers had a biopsy performed on day 0 (left arm) and day 14 (right arm). On day 14, DW was applied. Participants were randomised into two groups. Objective non-invasive assessments were performed on days 0, 7, 14, 60 and 90 using spectrophotometric intracutaneous analysis and full-field laser perfusion imaging. There were statistically significant increases in mean flux on day 14 (P = 0.027) in the post-DW arm. Haemoglobin levels increased on day 7 for the post-DW arm compared to without DW (P = 0.088). Differences in melanin levels were higher post-DW on the left arm between randomised groups on day 90 (P = 0.033). Haemoglobin levels in the vascular ring increased significantly from day 7 to 90 (P < 0.001 for post-DW and without DW arms). This study, for the first time, shows that DW increases blood flow and haemoglobin levels in acute healing wounds without affecting wound closure time and may have potential application in enhancing acute cutaneous healing.

Acceleration of cutaneous healing by electrical stimulation: degenerate electrical waveform down-regulates inflammation, up-regulates angiogenesis and advances remodeling in temporal punch biopsies in a human volunteer study.

We previously demonstrated the beneficial effect of a novel electrical stimulation (ES) waveform, degenerate wave (DW) on skin fibroblasts, and now hypothesize that DW can enhance cutaneous wound healing in vivo. Therefore, a punch biopsy was taken from the upper arm of 20 volunteers on day 0 and repeated on day 14 (NSD14). A contralateral upper arm biopsy was taken on day 0 and treated with DW for 14 days prior to a repeat biopsy on day 14 (ESD14). A near-completed inflammatory stage of wound healing in ESD14, compared to NSD14 was demonstrated by up-regulation of interleukin-10 and vasoactive intestinal peptide using quantitative real time polymerase chain reaction and down-regulation of CD3 by immunohistochemistry (IHC) (p < 0.05). In addition to up-regulation (p < 0.05) of mRNA transcripts for re-epithelialization and angiogenesis, IHC showed significant overexpression (p < 0.05) of CD31 (15.5%), vascular endothelial growth factor (66%), and Melan A (8.6 cells/0.95 mm²) in ESD14 compared to NSD14 (9.5%, 38% and 4.3 cells/0.95 mm², respectively). Furthermore, granulation tissue formation (by hematoxylin and eosin staining), and myofibroblastic proliferation demonstrated by alpha-smooth muscle actin (62.7%) plus CD3+ T lymphocytes (8.1%) showed significant up-regulation (p < 0.05) in NSD14. In the remodeling stage, mRNA transcripts for fibronectin, collagen IV (by IHC, 14.1%) and mature collagen synthesis (by Herovici staining, 71.44%) were significantly up-regulated (p < 0.05) in ESD14. Apoptotic (TUNEL assay) and proliferative cells (Ki67) were significantly up-regulated (p < 0.05) in NSD14 (5.34 and 11.9 cells/0.95 mm²) while the proliferation index of ESD14 was similar to normal skin. In summary, cutaneous wounds receiving DW electrical stimulation display accelerated healing seen by reduced inflammation, enhanced angiogenesis and advanced remodeling phase.

Addition of novel degenerate electrical waveform stimulation with photodynamic therapy significantly enhances its cytotoxic effect in keloid fibroblasts: first report of a potential combination therapy.

BACKGROUND: We recently reported use of photodynamic therapy (PDT) for treating keloid disease (KD). However, in view of high recurrence rates post any treatment modality, adjuvant therapies should be considered. Additionally, we previously demonstrated the effect of a novel electrical waveform, the degenerate wave (DW) on differential gene expression in keloid fibroblasts. OBJECTIVE: In this study, we evaluated the in vitro cytotoxic effect of PDT at 5J/cm(2) and 10J/cm(2) of red light (633 ± 3nm) using 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) with and without DW, on keloid fibroblasts compared to normal skin fibroblasts. METHODS: The rate of intracellular photosensitizer (protoporphyrin IX, PPIX) generation and disintegration, reactive oxygen species (ROS) generation, LDH cytotoxicity, WST-1 cytoproliferation, apoptosis by Caspase-3 activation, mitochondrial membrane potential assessment by JC-1 aggregates, qRT-PCR, flow cytometry and In-Cell Western Blotting were performed. RESULTS: PPIX accumulation and disintegration rate was higher in keloid than normal fibroblasts after incubation with MAL compared to ALA. Increased cytotoxicity and decreased cytoproliferation were observed for keloid fibroblasts after PDT+DW treatment compared to PDT alone. ROS generation, mitochondrial membrane depolarization, apoptosis (Caspase-3 activation) and collagens I and III gene down-regulation were higher in keloid compared to normal skin fibroblasts after MAL-PDT+DW treatment. An increase in the number of cells entering apoptosis and necrosis was observed after PDT+DW treatment by flow cytometry analysis. All positive findings were statistically significant (P<0.05). CONCLUSION: The cytotoxic effect of PDT on keloid fibroblasts can be enhanced significantly with addition of DW stimulation, indicating for the first time the utility of this potential combinational therapy.

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 G(2)/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.

A novel in vitro assay for electrophysiological research on human skin fibroblasts: degenerate electrical waves downregulate collagen I expression in keloid fibroblasts.

Electrical stimulation (ES) has been used for the treatment of wounds and has been shown to alter gene expression and protein synthesis in skin fibroblasts in vitro. Here, we have developed a new in vitro model system for testing the effects of precisely defined, different types of ES on the collagen expression of normal and keloid human skin fibroblasts. Keloid fibroblasts were studied because they show excessive collagen production. Both types of fibroblasts were electrically stimulated with alternating current (AC), direct current (DC) or degenerate waves (DW). Cells were subjected to 20, 75 and 150mV/mm electric field strengths at 10 and 60Hz frequencies. At lower electric fields, all types of ES upregulated collagen I in both cell types compared to controls. However, at higher electric field strength (150mV/mm) and frequency (60Hz), DW maximally downregulated collagen I in keloid fibroblasts, yet had significantly lower cytotoxic effects on normal fibroblasts than AC and DC. Compared to unstimulated cells, both normal skin and keloid fibroblasts showed a significant decrease in collagen I expression after 12h of DW and AC stimulation. In contrast, increasing amplitude of DC upregulated collagen I and PAI-1 gene transcription in normal and keloid fibroblasts, along with increased cytotoxicity effects. Thus, our new preclinical assay system shows highly differential effects of specific types of ES on human fibroblast collagen expression and cytotoxicity and identifies DW of electrical current (DW) as a promising, novel therapeutic strategy for suppressing excessive collagen I formation in keloid disease.

Effective inspired oxygen concentration measured via transtracheal and oral gas analysis.

BACKGROUND: The fraction of inspired oxygen (F(IO(2))) is quoted for different oxygen delivery systems, but variations in inspiratory flow and tidal volume make precise measurement difficult. We developed a reliable method of measuring the effective F(IO(2)) in patients receiving supplemental oxygen. METHODS: Ten subjects with chronic hypoxemia breathed through a mouthpiece with a sampling probe connected to a mass spectrometer. Four of the 10 subjects had transtracheal catheters that allowed direct sampling of tracheal gas. We used oxygen concentrations of 47% and 97%, and flow rates between 1 L/min and 8 L/min. We also compared oxygen delivery via nasal cannula and transtracheal catheter. Effective F(IO(2)) was derived from plots of the fractional concentrations of carbon dioxide versus oxygen. RESULTS: We found excellent correlation between the effective F(IO(2)) values from tracheal and oral sampling (r = 0.960, P < .001). With 97% oxygen via nasal cannula, effective F(IO(2)) increased by 2.5% per liter of increased flow (P < .001); effective F(IO(2)) reached 32.7% at 5 L/min while P(aO(2)) increased by 12 mm Hg per liter of increased flow. In 4 subjects with a transtracheal catheter, effective F(IO(2)) increased 5.0% (P < .001) per liter of increased flow, and P(aO(2)) increased by 13 mm Hg per liter of increased flow, whereas in the same 4 subjects using nasal cannula for oxygen delivery, P(aO(2)) increased by only 6 mm Hg per liter of increased flow. CONCLUSIONS: Exhaled gas sampled at the mouth accurately reflected the effective F(IO(2)) in the trachea. In relation to inspired oxygen flow, the effective F(IO(2)) was lower than is conventionally thought. Compared to nasal cannula, transtracheal catheter approximately doubled the effective F(IO(2)) at a given flow rate. Accurate knowledge of F(IO(2)) should aid clinicians in managing patients with acute and chronic lung diseases.