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Supercapacitive Polymer Electrodes for Directing Epithelial Repair

Periodic Reporting for period 2 - SPEEDER (Supercapacitive Polymer Electrodes for Directing Epithelial Repair)

Reporting period: 2019-08-01 to 2021-01-31

Even so the human skin has a remarkable ability to heal after injury this natural healing mechanism occasionally fails and the wound becomes chronic. Such chronic wounds contribute to enormous costs for the European healthcare system and lead to suffering and reduced mobility of affected individuals. Better strategies are therefore urgently needed that can prevent that wounds become chronic in the first place. Every additional day a wound remains open means an increased risk that the tissue enters into a vicious circle of infection and inflammation. Regular wound care aims to mechanically protect and support the wound during closure, but leave the rest up to the natural regenerative process. Far better would be an active wound dressing, that could “fast forward” the natural healing process and quickly restore the protectoral barrier of the epithelium.

The overall goal of SPEEDER is to develop a new concept for accelerated wound healing based on electrical stimulation of the wounded skin. Our hypothesis is that electrical fields, applied over the open wound, act as a guidance signal for skin cells involved in re-epithelialization. In vitro data suggest that such signals could act both to direct cell migration, but also to accelerate the speed at which the cells move. The idea of such a concept is in its own not original to SPEEDER. In fact, numerous studies with electrostimulation of skin have been performed, but with inconsistent outcome. Partially this can be attributed to difficulties in translating the techniques used in a petri dish - where electrical guidance of relevant skin can be demonstrated in a controlled environment - to a functional concept for stimulation on real skin. The key component missing has been an electrode material which makes it possible to sustain direct current stimulation over long periods of time, without corroding or dissolving the stimulating electrodes.

The unique idea in SPEEDER is to use the super-capacitive properties of polymer electrodes, mainly the conducting polymer poly(3-4-ethylene dioxythiophene) (PEDOT), to make direct current stimulation of tissue possible. While metal electrodes corrode under direct current stimulation, a conducting polymer layer in-between the metal and the electrolyte makes it possible to move ions in solution in a completely reversible process. When a voltage is applied over an electrode pair, the polymer electrodes drive current in the form of ions through the solution and an electrical field is generated. If the electrodes have sufficient internal charge storage capability this field can be sustained over many hours.

In SPEEDER we develop this concept, from electrode technology to an active wound dressing. We tailor conducting polymer electrodes which can deliver direct current to cells in a long term sustainable manner. “Long term sustainable” here means the electrodes can be charged and discharged a large number of times, and that each active discharge phase can be sustained for long enough to impact the cells. The established technology is tested on culture models where we mimic the biological environment of a skin wound. This way we can tune our concept using cells isolated from human skin, identifying the threshold stimulation needed to trigger a response. Thanks to scientific exchange with the plastic surgery department we will in the end also have the possibility to test our finished devices on skin biopsies, a so called ex-vivo model. This way, tissue which is left as waste after surgery can be a valuable test platform for our technology.
To validate our idea, we have developed microfluidic test environments (chips) where we seed human skin cells and study their behaviour. The fluidic chips can be seen as “running tracks” for cells where we monitor how the cells move (direction and speed) by TimeLapse imaging (Pic 1). We have analysed response thresholds, meaning the electrical field strengths where the cells first start responding to the applied stimulation. If no field is applied, the cells migrate randomly over the surface which, in the case of a wound, would reduce their ability to close the wound efficiently. When a field of more than 50 mV/mm is applied, keratinocytes align their migration towards the cathode - so called electrotaxis (Pic. 1). These biological experiments have been important, not just for validating that we can influence the migration of relevant cells, but also for verifying that our electrodes do not elute toxic by-products which would interfere with healthy cell proliferation. 50 mV/mm is well within what our technology is capable of and, as long as discharge is mixed with intermittent phases where electrodes are allowed to recharge, they can be relied upon for long treatment times.

The next step has been to set up culture models that are closer to the real skin wounds. Cells cultured in “running tracks” may behave quite different from cells that are allowed to act as a collective in an epithelial layer. A starting point is to let cells form an epithelial layer in a culture dish, which can be “wounded” e.g. by scratching with a scalpel. Far better is however to seed cells in three dimensional constructs involving the two most important cell types involved in wound healing: keratinocytes and fibroblasts (Pic 2). When co-cultured in a collagen matrix these cells form an artificial skin, in which we can punch a hole and study the speed at which the wound closes, with and without applied stimulation. This way we are now analysing if the effect of the stimulation seen with single cells can be reproduced in an environment more similar to a real wound.
To the best of our knowledge, this is the first time a concept for sustainable direct current stimulation of tissue has been demonstrated, in a way that electrodes can be charged and discharged in a fully reversible and cell compatible process. The electrode technology developed for SPEEDER is not exclusively relevant for skin cells, but can also guide other cells in similar manner. This means that direct current stimulation would e.g. also be possible within the body, where the electrodes cannot be removed or replaced that easily. As the stimulation mainly shifts ions that anyways are available in abundance in biological liquids, we do not expect that there is any accumulation of by-products from the stimulation, so that the charge/discharge cycles have no biochemical net effect. If we discharge electrodes above the response threshold, and recharge them again below threshold, the net effect on the cell migration still remains directional.

In the first phase of the project we have verified that the technology in principle works i.e. that the electrodes can generate electrical fields and that these fields are relevant for influencing the cells. We have set up tools which allow us to analyse this in high throughput “running tracks” and advanced culture models where we closely can mimic real skin wounds. Our focus in the next phase will be to use these tools to investigate exactly which stimulation parameters that are the most efficient for wound closure and which other factors (e.g. pH) that might interplay or interfere with the desired function. Thanks to our microfluidic chips, we can approach this rather broadly, performing a large set of experiments in parallel. At project end we expect to be at the point where we are ready to test out the optimized stimulation paradigm and a prototype of the active wound dressing clinically (Pic 3).
Directional migration of cells in "running tracks"
SPEEDER skin stimulation concept
Cross-section of a 3D model of a skin wound with a punched wound (2 mm in diameter)