accelerated wound healing on skin by electrical

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1700465 (1 of 5) © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Accelerated Wound Healing on Skin by Electrical Stimulation with a Bioelectric Plaster

Hiroyuki Kai, Takeshi Yamauchi, Yudai Ogawa, Ayaka Tsubota, Takahiro Magome, Takeo Miyake, Kenshi Yamasaki, and Matsuhiko Nishizawa*

DOI: 10.1002/adhm.201700465

in vitro have been achieved by externally applied electric current.[8,9]

The discovery of wound current led to the use of external electrical stimula-tion to cure chronic wounds and ulcers, for example, in patients with diabetes,[10] which was approved by the US govern-ment in 2002.[11] In addition, treatment of acute wounds by electrical stimulation has been tested using model animals.[12,13] Various modalities such as direct current ranging from a few µA to a few mA[14] and pulsed current[15] have been used for electrical stimulation on a wound. How-ever, there is still debate on the efficacy and mechanism of electrical stimulation in wound healing in vivo,[16] and there is no established standard conditions for electrical stimulation of wound healing.[17] Despite the variation in electrical stimula-tion protocols, most treatments on human skin have applied electric current only for

a few hours per day, because it is ethically inappropriate to bind a patient to the large equipment for a long time.

As with the recent progress in wearable electronics that may make electronic devices ubiquitous in our lives,[18–20] minia-turization of equipment for wound healing as a wearable patch will expand the use of electrical stimulation of wound healing for medical applications. A lightweight patch that fits to skin does not hinder motion of a living body thus more suitable to practical applications than conventional wired electric devices. Enzymatic biofuel cells (EBFCs) generate low-intensity direct current by enzymatic reactions using biomolecules such as sugars as fuel.[21] They have advantages as a wearable power source such as mild operating conditions and a simple device structure by eliminating a separator membrane. EBFCs can generate electricity on and in the living body using preloaded sugars or even biological fluid as fuel,[22–27] and implant-able applications of EBFCs to drive electric devices were also demon strated.[28–30] Nonetheless, the application of ionic cur-rent by EBFCs to wound healing on a living skin has never been reported. A major hindrance to application of EBFCs to wound healing on skin is the difficulty of stable physical con-tact with a living body with constant movement and stretching as well as maintaining the ionic current above the wound for a long time. To realize wearable applications of EFBCs, we previ-ously developed flexible and stretchable EBFCs by combining textile-based enzymatic electrodes, hydrogel, and an elastic resistor.[31–33]

Wound healing on skin involves cell migration and proliferation in response to endogenous electric current. External electrical stimulation by electrical equipment is used to promote these biological processes for the treatment of chronic wounds and ulcers. Miniaturization of the electrical stimulation device for wound healing on skin will make this technology more widely avail-able. Using flexible enzymatic electrodes and stretchable hydrogel, a stretch-able bioelectric plaster is fabricated with a built-in enzymatic biofuel cell (EBFC) that fits to skin and generates ionic current along the surface of the skin by enzymatic electrochemical reactions for more than 12 h. To investigate the efficacy of the fabricated bioelectric plaster, an artificial wound is made on the back skin of a live mouse and the wound healing is observed for 7 d in the presence and absence of the ionic current of the bioelectric plaster. The time course of the wound size as well as the hematoxylin and eosin staining of the skin section reveals that the ionic current of the plaster leads to faster and smoother wound healing. The present work demonstrates a proof of concept for the electrical manipulation of biological functions by EBFCs.

Dr. H. Kai, Dr. Y. Ogawa, A. Tsubota, T. Magome, Dr. T. Miyake, Prof. M. NishizawaDepartment of FinemechanicsGraduate School of EngineeringTohoku University6-6-01 Aramaki, Aoba-ku, Sendai 980-8579, JapanE-mail: [email protected]. T. Yamauchi, Prof. K. YamasakiDepartment of DermatologyGraduate School of MedicineTohoku University1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adhm.201700465.

Wound Healing

Living bodies have ability to heal cutaneous wounds to maintain homeostasis in the skin. Wound healing and closure are elabo-rately organized biological processes in which cells migrate and proliferate at and around the wound site.[1] It has long been known that an endogenous electric current arises at the wound site.[2] This “wound current,” together with other biological signals such as secreted cytokines, is suggested to enhance cell migration[3] and proliferation[4] in wound healing. In the last decade, the molecular biological mechanisms of electri-cally guided wound healing in vivo have been studied in more detail,[5–7] as well as directional cell migration and proliferation

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We here, for the first time to the best of our knowledge, report the accelerated skin wound healing by a wearable patch with an integrated stretchable EBFC. Taking advantage of our totally stretchable EBFC, we constructed a “bioelectric plaster” that fits tightly to the skin to promote wound healing by gener-ating ionic current above the wound on the skin (Figure 1). The bioelectric plaster consists of enzymatic electrodes, an elastic conductive resistor, a hydrogel, and medical adhesive tape. The electrode is fabricated according to the procedure we previously reported.[33,34] It is made of a piece of carbon fiber fabric coated with carbon nanotubes, on which a redox enzyme (fructose dehydrogenase for an anode, bilirubin oxidase for a cathode) was immobilized by spontaneous physical adsorption in buffer solution. The large specific surface area and high conductivity of the carbon nanotube-coated carbon fiber fabric enable the fabrication of enzymatic electrodes with high current density. Once the enzymes were immobilized, the electrodes main-tained the electrical output in buffer solution for more than 12 h (Figure S1, Supporting Information). The elastic conduc-tive resistor was made of a composite of poly(3,4-dioxyethyl-enethiophene) and polyurethane[35] and cut into a narrow strip, resulting in the resistance of 10 kΩ for a 6 mm gap between the electrodes. As a hydrogel, a double-network hydrogel made of gellan gum and poly(acrylamide)[36] was adopted. Double-network hydrogel is a tough and stretchable hydrogel made of an interpenetrating network of a hard brittle polymer and a soft ductile polymer.[37,38] The hydrogel works as a reservoir of a buffer and fuel, an ion transport path, and a stretchable con-tact material with skin. The dimensions of the hydrogel were 10 mm × 50 mm × 0.5 mm, whose total volume was 0.25 mL and contained fructose was 17 mg (94 µmol). All these compo-nents are assembled and fixed on the skin by wrapping them in stretchable medical adhesive tape. Overall, the assembled bio-electric plaster maintains stretchability and good ionic contact with the skin of a living body.

Two factors are required to utilize a bioelectric plaster prac-tically for effective wound healing; (1) the bioelectric plaster must be biologically safe, as it makes direct physical contact with a skin and a wound, and (2) it should maintain the electric current at least for a few hours to days until wound closure. Previous in vitro studies showed that the migration speeds of keratinocytes (the predominant cells in the epidermis, the out-ermost layer of skin)[39] and fibroblasts (the predominant cells

in the dermis, a skin layer below the epidermis)[40] increase with increasing direct current voltage that is externally applied. Even though these studies reported an electric field instead of current density as a parameter, current density is positively correlated with the applied voltage in theory. Therefore, their results give the design principles for the bioelectric plaster; it should generate sustained electric current on skin to maximize the degree of cell migration. In addition, the bioelectric plaster becomes more practical if the electric current lasts longer and a user replaces the plaster less frequently.

To find optimal buffer solution for hydrogel as an electrolyte reservoir, we first examined the safety of hydrogel with buffer solution against skin using living mice (Figure 2a–d). The effect of the hydrogel on the skin was tested by placing hydro-gels with different buffer solutions on skin for 7 d followed by observation by eye. A hydrogel with buffer solution of high concentration (1 m; Figure 2d) or high pH (pH 7; Figure 2b) caused rash on the skin, while a hydrogel with 200 × 10−3 m cit-rate buffer solution of pH 5 (Figure 2c) did not have any visible detrimental effect on the skin, and the skin appeared normal like the skin without any treatment (Figure 2a).

Next, we investigated the stability of output current of the bio-electric plaster for 12 h under the conditions with varied buffer concentrations and external resistances (Figure 2e). The thick-ness of hydrogel was fixed at 0.5 mm, which is the minimum possible thickness for sufficient mechanical strength, good fitting to skin, and a wet environment for the electrodes. The thinnest hydrogel gives the highest electric current density in the hydrogel provided that the same electrodes and the resistor are used and total current is constant. Fructose concentration (400 × 10−3 m) and enzyme loading (≈1.5 mg bilirubin oxidase per cathode, ≈1 mg fructose dehydrogenase per anode) were fixed for all the following experiments. Bioelectric plasters had better stability for higher concentration of electrolyte, because diffusion is limited in hydrogel and higher concentration of electrolyte facilitates more effective buffer action. Note that this lower stability for lower resistance contrasts with the fact that the stability of the EBFCs in solution is identically excellent (>98% current was retained after 12 h) for varied buffer con-centrations (Figure S1, Supporting Information). The lower sta-bility of bioelectric plasters can be attributed to a limited volume of electrolyte solution resulting in lower buffer capability that leads to deactivation of the enzyme, as well as drying of the


Figure 1. a) Schematic structure of a bioelectric plaster. b) A photograph of the bioelectric plaster applied on a wound of a live mouse skin.



hydrogel resulting in loss of conductivity. In addition to buffer concentration, change in external resistance also modulates the output current and stability. As the resistance is decreased, the initial current becomes higher, while the stability of the current becomes lower. Due to this trade-off, the bioelectric plaster with 10 kΩ external resistance was chosen as it achieves the high amount of accumulated electric current over 12 h. Based on safety to skin and stability of the bioelectric plaster investigated above, we selected a hydrogel with 200 × 10−3 m citrate buffer solution of pH 5 with 400 × 10−3 m fructose and a stretchable resistor of 10 kΩ (Figure 2e, right bottom, orange) for the bio-electric plaster for the further investigation.

With this optimized buffer formulation for the hydrogel, the bioelectric plaster was applied on a live mouse skin to inves-tigate the effect on wound healing and closure. An oval hole of ≈8 mm width (length perpendicular to a mouse body axis)

and ≈4 mm height (length parallel to a body axis) was punched on a back skin of a female ICR mouse to cause artificial wounding. In this wound, both the epidermis and dermis were removed, and this can be considered as a model of delayed wound healing in a large wound. The wound healing process was observed for 7 d after this wounding. Twenty-one mice were divided into three groups of seven mice each (Figure 3a). Group A was without treatment. In Group B, only a hydrogel was placed on the wound. Group C was treated with the bio-electric plaster. Since the wound healing should be affected by both the presence of hydrogel and the ionic current, we tried to separate these two factors. The specific kind of hydrogel used in the bioelectric plaster may significantly affect wound healing, and the comparison between Groups B and C, which use the same kind of hydrogel, isolates the net effect of ionic current by the bioelectric plaster. For Groups B and C, the


Figure 2. Optimization of buffer for the bioelectric plaster. Representative images of mouse skins after 7 d application of hydrogel with different buffer solutions are shown: a) no hydrogel (negative control), b) 100 × 10−3 m phosphate buffer, c) pH 7, 100 × 10−3 m citrate buffer, pH 5, and d) 1 m citrate buffer, pH 5. e) Time-dependent current changes of the bioelectric plaster with different external resistances and citrate buffer solutions with different concentrations are shown. Current density is calculated by dividing current by cross sectional area of the hydrogel. Citrate buffer concentration (pH 5) with 400 × 10−3 m fructose are 1 m (left top), 200 × 10−3 m (left bottom and ×4 magnification in right bottom), and 50 × 10−3 m (right top). Resistances of stretchable resistors are 100 kΩ (blue), 10 kΩ (orange), and 1 kΩ (green).

Figure 3. Wound healing in the presence and absence of a BFC plaster on a mouse skin observed for 7 d. a) Three groups of treatments. b) Representa-tive chronological wound images of each group are shown up to 7 d. c) Changes of wound width and height of Group A (gray), Group B (red), and Group C (blue). Lines and error bars indicate mean and standard error of mean. (n = 3, *: p < 0.05 between Groups B and C)



hydrogel or the bioelectric plaster was replaced every 12 h. Up to 7 d after wounding, a photograph of the wound was taken every day, as well as the wound width and height were meas-ured (Figure 3b). Since there is slight variation in the initial size of a wound, the progress of wound closure was evaluated by the relative size to day 0. Comparison between Groups B and C gives the evaluation of the net effect of ionic current by the bioelectric plaster. Group C showed more significant decrease in the wound width than Group B (Figure 3c,d). Difference of width decrease between Groups B and C at day 6 was 25% (95% confidence interval: 7–43%), and that of day 7 was 22% (95% confidence interval: 2–42%). Although the small sample size and the intrinsic individual difference led to the wide con-fidence intervals, the wound width of Group C was statistically significantly smaller than that of Group B (p < 0.05). In Group B, slight height expansion of the wound by hydrogel until day 1 occurred, as seen in the photographs of the wound (Figure 3b, day 1), indicating that the initial contraction of wound was inhibited by hydrogel. On the other hand, Group C did not show such initial expansion, and the wound size continuously decreased over 7 d. From this comparison between Groups B and C, it can be concluded that electric current of the EBFC effectively promoted closure of the skin wound. Wound closure consists of four processes: coagulation, inflammatory phase, proliferative phase, and remodeling phase. The prolifera-tive phase starts approximately at day 3,[41] in which keratino-cytes and fibroblasts start to proliferate and migrate toward the wound. In the time course of the wound width measured above, Group C after day 3 showed more pronounced decrease than Group B, which suggested that wound closure by the pro-liferation and migration of the cells was effectively promoted by the ionic current of the bioelectric plaster.

In terms of the wound size decrease, almost identical time courses for no treatment (Group A) and treatment with the bio-electric plaster (Group C) were observed. To further investigate wound healing under the application of the bioelectric plaster, the skin section samples of the three groups at day 7 were stained by hematoxylin and eosin (Figure 4). Epidermis (stained in purple) of healed skin tissue in Group A was thin and a basal membrane layer, which is boundary to dermis (stained in red), was flat (Figure 4f, left bottom), which suggested contracture (tightening of skin tissue) during the initial process of wound healing. By contrast, Groups B and C showed thick epidermis with a winding boundary, which is indicative of the lack of con-tracture. This lack of contracture for Groups B and C is charac-teristic of moist healing, a wound healing process in which skin tissue recovers more smoothly with less scarring.[42] From these observations of the time course of wound size and stained skin sections, it can be concluded that application of the bioelectric plaster combines both the advantages of electrical stimulation and moist healing: increased wound closure speed due to elec-trical stimulation (shown by comparison between Groups B and C) and smoother skin healing due to moist healing condi-tions (shown by comparison between Groups A and C), as sum-marized in Table S1 (Supporting Information).

In the experiment above, the bioelectric plaster was applied on an acute wound that was made by cutting an oval hole on the back skin of a mouse. Although the previous examples of elec-trical wound healing in human clinical treatment have mostly been carried out for chronic wounds, there are similarities between the processes of normal healing of acute wounds and electrically treated healing of chronic wounds.[41] Both involve cell migration and proliferation in the healing process, which can be promoted by the bioelectric plaster. Therefore, it can be


Figure 4. Microscopy images of skin sections at the wound at day 7 that were stained by hematoxylin and eosin: Group A (left column), Group B (middle column), and Group C (right column). Images in the bottom row are magnified images of black rectangles in the top row images. Scale bars: 500 µm (top), 200 µm (bottom). Annotations: a) the boundary between normal tissue and healed tissue, b) the area of normal tissue, c) the area of healed tissue, d) dermis, e) fat tissue, and f) epidermis.




concluded that the developed bioelectric plaster may be effective for both the chronic and acute wounds.

In conclusion, we have successfully developed a bioelectric plaster that can accelerate wound healing on skin. The com-parison between conditions with and without EBFCs suggested that ionic current in the hydrogel promotes the wound healing process, although the detailed biological mechanism is still to be investigated. To the best of our knowledge, the present work is the first application of ionic current of EBFCs to direct manip-ulation of biological functions of a living body. The further improvement of bioelectric plasters, such as the better choice of hydrogel that does not slow down the wound closure speed per se, will expand the use of electrical stimulation in wound healing for in vivo studies as well as clinical applications.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsThe authors are grateful to Masanori Fujisawa, Airi Anzai, and Yasuyuki Omura for their technical assistance in the development of an eight-channel battery logger for 12 h stability measurement of bioelectric plasters. All animal experiments were approved by the Animal Care and Experimentation Committee of Tohoku University Graduate School of Medicine. This work was supported in part by Center of Innovation Program (COI-Stream) and Creation of Innovation Centers for Advanced Interdisciplinary Research Area Program from Japan Science and Technology Agency (JST), Regional Innovation Strategy Support Program “Knowledge-based Medical Device Cluster/Miyagi Area,” Grand-in-Aid for Scientific Research A (25246016), and Challenging Exploratory Research (K15K13315) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Conflict of InterestThe authors declare no conflict of interest.

Keywordselectrical stimulation, enzymatic biofuel cells, wearable devices, wound healing

Received: April 10, 2017Revised: July 25, 2017

Published online: September 20, 2017

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