Angiogenesis is a critical component for processes in wound healing and is defined as the formation of new capillaries from pre-existing blood vessels [1, 2]. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation [4–8]. Electrical stimulation (ES) in its various forms has been shown to enhance wound healing by promoting the migration of keratinocytes and macrophages, enhancing angiogenesis, stimulating fibroblasts, and influencing protein synthesis throughout the inflammatory, proliferative and remodelling phases of healing [9–11]. Electrical signals have been shown to stimulate angiogenesis and organise blood vessel formation [3]. There is limited information on the influence of ES on angiogenesis after acute wounding in human skin, as most research is restricted to animal models. Animal studies have shown that angiogenesis is induced by ES in ischemic and non-ischemic rat limbs, and is facilitated by the increased expression of VEGF in muscle cells [12,13]. Endogenous electric fields are able to direct the migration of epithelial cells during wound healing and may contribute to regulation of angiogenesis [14]. Three dimensional imaging analysis demonstrated a statistically significant reduction in would diameter and improved healing after electrical stimulation with ES microcurrent. This form of treatment modality is currently in use by facilities such as the Cleveland Clinic in Ohio, a multispecialty academic center that integrates clinical and applied research [47-51].
Previous studies have shown that certain electrical currents, such as direct and alternating currents, are useful in treating; diabetic foot ulcers, skin ulcers and chronic wounds [15,16]. We have investigated the in vitro effect of different types of ES on the expression of collagen in skin fibroblasts. Importantly, we highlighted the role of a novel waveform termed degenerate wave (DW is a degenerating sine wave, which deteriorates over time) and demonstrated its beneficial effects compared to other known waveforms such as direct and alternating currents [17]. In a recent in vivo study, we also demonstrated the application of an ES biofeedback device, which produces DW in a human volunteer study (n = 20) involving only one time point (day 14) punch biopsy [18]. The results showed increased blood flow in acute cutaneous wounds following the biopsy compared to controls that had not received ES. Additional experimental gene and protein studies corroborated these findings by demonstrating up-regulation of angiogenesis and down-regulation of inflammation in ES treated wounds [19]. However, this study [18,19] only evaluated the effects of ES at one time point (day 14), with 20 participants and using only two objective measures.

In view of the above scientifically interesting and clinically relevant findings, 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 to identify if the enhanced effect occurred earlier than day 14. In addition to multiple time points, we also used an increased number of sequential skin biopsies, increased number of participants (n = 40) with randomisation verified using additional non-invasive objective devices such as a three-dimensional camera and a Dermacorder (BioElectromed Corporation, USA).
Healthy participants were enrolled into the study at the University Hospital of South Manchester NHS Foundation Trust, England, UK. South Manchester Research Ethics Committee and Trust Research and Development department approval were granted for the study (Ethics number: 09/H1012/3). If suitable (inclusion and exclusion criteria outlined in S1 Table), participants were asked to provide written consent to take part and were then enrolled in the study. All study participants provided written consent.

Demographics - Cohort 1. Twenty participants were recruited onto cohort 1, the majority of which were female (n = 14, 70%). Most participants were between the ages of 19–24 years and all were of Caucasian ethnicity (n = 40, 100%). Most participants had a Fitzpatrick skin type of II (fair skin, tans poorly, burns easily) (n = 9, 45%) or skin type III (fair skin, tans moderately, sometimes burns) (n = 9, 45%). Furthermore, all participants were right handed.

Demographics - Cohort 2. Twenty participants were recruited onto cohort 2, the majority of which were female (n = 15, 75%). Most participants were between the ages of 25–30 years and all were of Caucasian ethnicity (n = 40, 100%). Most participants had a Fitzpatrick skin type III (fair skin, tans moderately, sometimes burns) (n = 10, 50%). Furthermore, all participants were right handed. Demographic data for both cohorts are outlined in S2 Table.

Forty healthy volunteers were split equally into two groups: Cohort 1 and Cohort 2 (Fig 1). The rationale for including two cohorts was to look at multiple time points. This would not have been possible in one cohort to look at day 0, 3, 7, 10 and 14 due to the large number of punch biopsies to be performed in each participant.

Due to the variability and skewness of the data distributions, non-parametric summaries of median and range (minimum, maximum) were utilised. Additionally, measurements for the control arm and post-ES arm were compared on all wound days using pair-wise Wilcoxon signed ranks tests. All analyses used a two-sided 1% significance level; adjusting for repeated comparisons on multiple study days using Bonferroni adjustment. For cohort 1, wound days 30, 60 and 90 were considered to correspond to post-ES arm wound days 23, 53 and 83. At these follow-up time points, there was no control arm. The data were analysed to identify any trends or assess any effects that had been sustained in the treated arm. The randomisation was conducted by the statisticians in nQuery Advisor 7.0 using a computer generated permuted block design with mixed block sizes and random seed. All summaries and analyses were performed using SPSS (version 20; IBM Corporation, Armonk, NY, USA). For histological experiments, data is presented as mean +/- standard deviation from three independent experiments performed in triplicates (n = 3). Statistical analysis was calculated using one way ANOVA for comparison between three groups with Tukey post hoc test, and student’s t test for comparison between two groups. Confidence intervals of 95% with corresponding p value of 0.05 was chosen throughout analysis. Data collection and analysis were not blinded as a single researcher performed all aspects of the study except statistical analysis, which was performed by independent statisticians. Additionally, the histological analysis was performed by a scientist at the laboratory. Three dimensional imaging analysis showed a significant reduction in wound volume on days 7, 10 and 14 after electrical stimulation.

Wound volume was lower in the post-ES arm compared to the control arm at all time points (Fig 3A and S7 Table). Control arm measurements for wound volume on day 7 were generally higher with a median of 1.98 (quartiles 1.50 and 2.44); whereas post-ES volume measurements were significantly lower with a median of 1.21 (quartiles 0.56 and 1.95) (p = 0.003). There was a 38.9% decrease in wound volume post-ES on day 7. On day 10, the volume in the control arm was higher with a median of 2.48 (quartiles 1.78 and 3.81); whereas post-ES, the volume was significantly lower with a median of 1.54 (quartiles 1.26 and 2.77) (p = 0.002). There was a 37.9% decrease in wound volume post-ES on day 10. On day 14, wound volume was higher with a median of 2.04 (quartiles 1.45 and 3.31); whereas post-ES wound volume was significantly lower with a median of 1.14 (quartiles 0.69 and 1.34) (p<0.001). There was a 44.1% decrease in wound volume post-ES on day 14.

Three dimensional imaging analysis demonstrated a statistically significant reduction in wound diameter on days 10, 14 and 90 after electrical stimulation. Wound diameter was less in the post-ES arm compared to the control arm at all time points (Fig 3A and S8 Table). Although, there were no statistically significant differences in diameter size on days 3, 7, 30 and 60 when comparing the control to the post-ES arm. However, wound diameter was significantly less on days 10, 14 and 90. On day 10, control arm measurements for wound diameter were higher with a median of 4.32 (quartiles 3.98 and 4.81); whereas post-ES the diameter was significantly less with a median of 3.84 (quartiles 3.21 and 4.45) (p = 0.009). There was an 11.1% decrease in wound diameter post-ES on day 10. On day 14, control measurements for wound diameter were increased with a median of 3.75 (quartiles 3.39 and 4.45); whereas post-ES measurements were significantly reduced with a median of 3.15 (quartiles 2.82 and 3.44) (p = 0.002). There was a 16% decrease in wound diameter post-ES at day 14. On day 90, wound diameter was significantly reduced post-ES compared to controls (p = 0.007). There was a 16.2% decrease in diameter post-ES on day 90.

Wound depth measurements were less in the post-ES arm compared to the control arm at all time points although not significantly (Fig 3A and S9 Table). Wound depth measurements remained approximately similar across all time points for both arms (day 3; p = 0.260, day 7; p = 0.048, day 10; p = 0.390, day 14; p = 0.341, day 30; p = 0.732, day 60; p = 0.040, day 90; p = 0.014).

Electrical field (EF) measurements were higher after electrical stimulation.
EF measurements at the wound edges were mostly increased in the post-ES arm on days 3 (p = 0.050), 10 (p = 0.028), 14 (p = 0.012) and 30 (p = 0.169) compared to the control arm and were reduced in the post-ES arm on days 60 and 90 (Table 1). Although, there were no statistically significant differences noted. At day 3, EF measurements demonstrated a 62% increase in the post-ES arm. At day 10, EF measurements showed a 47% increase in the post-ES arm compared to the control. At day 14, there was a 79% increase in the EF post-ES compared to the control arm.

This multiple sequential biopsy study has demonstrated significant beneficial effects of ES in enhancing acute cutaneous wound healing. We have shown that wound volume was significantly reduced following ES on day 7. Additionally, volume, surface area and diameter of the wounds were significantly reduced post-ES on days 10 and 14. Furthermore, we noted a statistically significant increase in blood flow on days 10 and 14 post-ES. This particular finding was further verified with a simultaneous up regulation of angiogenic biomarkers in wound biopsies. The EF present at the wound edges was increased following ES in the initial days of healing albeit decreased in the later stages, compared to the control. To our knowledge, this detailed study, is unique in investigating the role of ES in enhancing cutaneous wound healing in human skin. Multiple sequential time points were correlated with a number of objective parameters including histological markers to demonstrate the role of an exogenous asymmetric biphasic current in positively impacting the rate of progression of cutaneous wound healing.

The benefits of this study were the relatively larger sample size of 40 compared to our previously published study of twenty subjects [18], the inclusion of sequential temporal biopsies, randomisation to account for handedness and the utilisation of multiple non-invasive measuring devices to monitor the progression of wound healing. In the previous study, we did not utilise a device to measure the size of the wounds, therefore in the current study the use of a 3D camera proved invaluable as it enabled quantitative measurements of wound volume, surface area and diameter before and after application of ES. Additionally, for the first time, we used Dermacorder to measure the EF at the wound edges. The homogeneity of the cohorts was a strength of the study as all subjects were of a similar Fitzpatrick skin type, all were of same ethnic origin (Caucasian), all were right handed and all were of a similar age group (under 30 years of age). The participant arms were randomised to account for handedness as their dominant hand may have affected the healing of the wound, as this arm would be most active arm and in constant use. Additionally, the Fitzpatrick score gave a range of similar skin types for the participants in relation to having a homogenous cohort of subjects for melanin level measurement pre and post ES treatment. Furthermore, a single researcher conducted the treatments and data collections; therefore, inter-observer biases and errors were reduced. Evidence of wound crusting did not cause a problem when measuring the biopsy wounds. By day 7, the majority of crusting was not present with contracting 5mm biopsy wounds, therefore there was either none or minimal crusting present.

We demonstrated that the wound dimensions reduced following ES treatment compared to the controls, which was verified objectively and quantitatively by 3D imaging that calculated the surface area, volume, diameter and depth of the wound sites. This technique has also been used in other studies [2831]. Contraction of the wound begins soon after wounding and peaks at 2 weeks [32]. The degree of wound contraction varies with the depth of the wound. For full-thickness wounds as in our study, contraction is an important part of healing and accounts for up to a 40% decrease in the size of the wound [32]. In this study, the greatest reductions following ES were for wound volume on days 7, 10 and 14 and this reduced by 38.9%, 37.9% and 44.1% respectively.

We used the Dermacorder to measure the EF at the wound edges and demonstrated that the EF was increased following the application of ES. We noted that the potential difference between the wound and the healthy skin was increased on the initial days of healing post-ES treatment. EF = Potential difference / Distance. On day 3, there was a significant reduction in the diameter of the wounds post-ES, while the EF was increased. This shows that the potential difference was increased post-ES treatment. However, on days 10 and 14 the combined effect of both reduction in wound diameter and increase in potential difference may have resulted in a greater EF post-ES treatment. It is thought that current of injury decreases with subsequent time points in wound healing [33]. On day 60, EF was reduced following ES, while the wound diameter remained similar compared to the normal healing process. This demonstrates that there is a greater reduction in potential difference following ES application in the later stages of wound healing, which indicates accelerated repair. This also indicates that a non-leaky epidermal barrier had been established faster post-ES treatment.

Nuccitelli et al [26] developed this new approach, which does not require any electrode contact at the wound site therefore making it possible to quantitate the EF near skin wounds [25,26]. The endogenous EF generated near skin wounds is of interest because keratinocyte migration and wound healing are strongly influenced by EF [26]. A previous study on the subject of galvanotaxis involving human keratinocytes indicated that the mean translocation velocity of cells were proportional to the EF [34]. Additionally, during the maturation and remodeling phase of wound healing, the epidermis re-established its structure and resistance [26]. Thus, the wound current and EF would approach to normal or control levels. The epidermal lateral EF decreases gradually with stratification in re-epithelialisation [26]. This correlates with the results observed here.

In this study, we also showed that there may be a direct effect of ES current through the skin on blood flow. We demonstrated that blood flow was significantly increased on days 10 and 14 following the application of ES. Our results are further corroborated by the findings from our previous study [18], where we showed that blood flow was significantly increased at day 14 (p = 0.027). These results were further supported by the previous gene and protein studies [19], which showed that ES significantly up-regulated angiogenesis and down-regulated inflammation on day 14. However, to validate the results in the current study, we further investigated VEGF-A and PLGF mitogens. VEGF-A is involved in the normal physiological development of blood vessels [35] and its biological effects on maturing endothelium usually takes place over the course of minutes to days during angiogenesis in wound healing. This phenomenon includes VEGF receptor phosphorylation, existing vascular dilation and permeability, and activation of endothelial cell precursors [3639]. PLGF is also a member of the VEGF family [40] and has been shown to have strong angiogenic properties in mice [41]. However, it has been shown to be up-regulated in the active angiogenic phase of wound healing in both migrating keratinocytes and endothelial cells of blood vessels within the human wound bed [42].

The increasing vascularisation over time, associated with multiple signs of active neo-angiogenesis in the dermal microvasculature indicated that the quiescent endothelium was responsive to ES, which activated potent angiogenic mitogens [43,44]. However, the developing vascular phenotype of human dermis following ES and controls, had no distinct features, other than an increase in capillary/vessel number when observed in ES treated tissues. This striking angiogenic phenotype induced by up-regulated VEGF-A and PLGF raises the possibility that these cytokines could be further modulated exogenously when ES is given to enhance neo-angiogenesis for therapeutic purposes.

An association was noted between blood flow and wound diameter, volume and surface area post-ES on days 10 and 14, compared to the control. Blood flow was increased whilst wound dimensions decreased following treatment with ES. It has been shown in a previous study using transcutaneous electrical nerve stimulation in patients with peripheral arterial disease that capillary density was increased following treatment at 3 and 6 weeks [45]. Additionally, an increase in transcutaneous oxygen measurements were reported at the same time points. Therefore, it may be possible that ES could enhance the formation of capillaries. Follow-up time points on days 60 and 90 were used in both the current study (Cohort 1) and the previous trial [18]. The results from both studies were compared, looking at the data produced from the common measurement device, FLPI, measuring blood flow. There were some similarities between the results from both studies. However, there was a reduction in blood flow by day 90 that was shown in both studies. The ES device used forms part of a biofeedback link with the individual’s normal physiological repair. This modality follows the theory that the normal electrical potential of skin forms a global electrical network, which reflects the underlying neurological activity through changes in skin impedance. The treatment was well tolerated by all participants and was relatively easy to use in clinical practice.

Limitations of this study include, the total number of time points employed for evaluation and performing sequential biopsies. In addition, the potential use of additional tools and objective devices for monitoring the process of repair non-invasively may add further detail and value to this study. Future studies are required to note the long-term effect of delivering ES to acute wounds beyond the day 14 time point and whether there is a need for additional treatments to be given beyond the time points discussed in this study. This study indicates that ES accelerates acute cutaneous wound healing evidenced by a reduction in wound volume, diameter and surface area and an increase in blood flow. There is clear evidence from invasive and non-invasive modalities that treatment with ES resulted in increased angiogenesis. This study further substantiates the role of ES in enhancing cutaneous wound repair evidenced by quantifiable objective measures and histological analysis observed in multiple time points. Furthermore, this treatment may have potential application for treatment of delayed and chronic wounds.

Fig 1. Images from the 3-dimensional camera are shown which demonstrate the size of the biopsy wound sites are smaller post electrical stimulation (ES) compared to the control. The normal wound image is displayed with the difference in heights images corresponding shown below. The difference in heights images show the highest points are indicated in red and yellow, whilst the lowest points are displayed as green and blue. B. Images from the full-field laser perfusion imager at each time point demonstrating the blood flow for the control and post-ES arm. The differences in blood flow (flux) in the biopsy sites are depicted by the change of colour intensity in the images.  Image credit:


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