Development of a Therapeutic Device for Wound Healing with Enzymatic Biofuel Cells
Authors: Tsubota Ayaka , Ogawa Yudai , Yamauchi Takeshi , Kai Hiroyuki , Yamasaki Kenshi , Nishizawa Matsuhiko 
Published: February 2020

Galvanotaxis, cell migration in response to ion current, is suggested to play an important role in wound healing. In an uninjured skin, ion channels of the cells transport K+ ion and Na+ ion toward a dermal layer (the interior of skin), and Cl-ion toward a cortical layer (the surface of skin). This ion transport leads to ion concentration gradient and electric potential difference between the dermal layer and the cortical layer. When the skin is wounded, this balance is disturbed and horizontal ion transport toward the wound occurs at the epidermis, where cells move along ionic flow by galvanotaxis and gather to close the wound [1]. In the previous studies, artificial electric current was applied to a wounds by an external power source and wound healing was accelerated [2]. Miniaturization of such devices will make electrical wound healing more readily accessible.
In this work, we develop a wound healing device with enzymatic biofuel cells, which makes a small ion current at the wound and induces galvanotaxis of cells. Enzymatic biofuel cells (EBFCs) generate electricity by redox reactions of enzymes in mild, ambient conditions, which is suited for wearable devices. The EBFC in this work has a carbon fabric modified with carbon nanotubes as an electrode. Fructose dehydrogenase (FDH) that oxidizes fructose was used for an anode, and bilirubin oxidase (BOD) that reduces oxygen for a cathode.
First, we conducted in vitro experiment to examine whether cell migration can be driven by the EBFC. We used a microchannel having 500 µm height to test how much current density is necessary to induce galvanotaxis (Fig.1(a)). The microchannel device for monitoring cell migration consists of two parts, a cell culture chamber with a microchannel and an EBFC chamber, which are separated by a Nafion membrane as an ion exchange membrane. Separation of the two parts by the Nafion membrane prevents contamination of the cell culture chamber with ingredients of the EBFC chamber, thus suppressing bad influence on the cells while ions pass through this membrane in this device to form a closed circuit to generate current. We seeded epidermal keratinocytes in a microchannel, applied electric current in the microchannel for 12 hours. Directional cell migration was observed with the EBFC generating 0.4 mA/cm2current density in a microchannel.
Since in vitro cell migration by the EBFC was confirmed, we tested the effectiveness of the EBFC for wound healing in vivo. We fabricated an electric plaster device with the same EBFC as above, and applied it to a wound on the back skin of a mouse (Fig.1 (b)). The electric plaster device is composed of an EBFC, a double-network gel (DN gel) that supplies fuel, and medical tape that fixes the plaster on a mouse. The DN gel is soaked in 200 mM fructose as fuel and 400 mM citrate buffer as electrolyte solution. The EBFC has two anodes with FDH, one cathode with BOD and a composite film of poly(3,4-ethylenedioxythiohene) and polyurethane as a flexible resistor. The two anodes were placed outside a wound, and the cathode was placed on the center of the wound, which causes ionic flow toward the center of the wound. This plaster device stably adhered to the back of the mouse for one day, and it did not affect activity of the mouse. The current density of the EBFC of the plaster device on a wound remained 0.4 mA/cm2 for 12 hours in vivo, which is sufficient for cell migration as confirmed in the in vitroexperiment above. Next, we observed wound healing process while the plaster was applied several days. We made a circular wound of 4 mm in diameter on a mouse skin and placed the plaster on the wound. The plaster was replaced every 12 hours, and the wound was observed for 6 days. Wound healing was faster with the plaster with the EBFC than a negative control with a DN gel with fructose and a buffer.
[1] McCaig, C. D., Rajnicek, A. M., Song, B., and Zhao, M. (2005). Physiol. Rev., 85(3), 943–978.
[2] Lee, R. C., Canaday, D. J., and Doong, H. (1993). J. Burn Care Rehabil., 14(3), 319–335.

Figure 1