Periodic Reporting for period 4 - SPEEDER (Supercapacitive Polymer Electrodes for Directing Epithelial Repair)
Periodo di rendicontazione: 2022-06-01 al 2023-07-31
Even though human skin has a remarkable ability to heal after injury, this natural healing mechanism occasionally fails, resulting in a chronic wound. Such chronic wounds contribute to enormous costs for the European healthcare system and heavily diminish the quality of life of affected individuals. Improved strategies to tackle this problem are urgently needed. Here, SPEEDER technology can step in as a preventive or corrective measure. By using electrical stimulation to drive the migration of skin cells, the epithelial cell layer can cover the wound area faster, reducing the time window for infection to occur. Thus, while regular wound care focuses on mechanically protecting and supporting the wound during closure while leaving the rest up to the natural regenerative process, SPEEDER is meant to actively “fast forward” the natural healing process and restore the epithelium’s protectoral barrier.
The working hypothesis of SPEEDER has been that electrical fields (EFs), applied over an open wound, act as a guidance signal for skin cells involved in re-epithelialization. In other words, we exploit skin cells’ “electrotactic” ability to manipulate and force them to move faster into the wound area. The idea of such a concept is not original to SPEEDER and has, in fact, been explored in numerous studies but with inconsistent outcomes. A key component has been missing for successfully exploiting electrotaxis, mainly an electrode material capable of sustaining direct current (DC) stimulation over long periods without dissolving the stimulating electrodes and damaging surrounding tissue. SPEEDER has managed to close this gap by working in parallel on biological models, materials science, and electrochemistry.
The unique idea we have followed is to use the super-capacitive properties of polymer electrodes, mainly the conducting polymer poly(3-4-ethylene dioxythiophene (PEDOT), to make DC stimulation of tissue possible. While metal electrodes corrode under DC, a conducting polymer layer makes it possible to move ions in solution through a reversible process.
In SPEEDER, we have deep-dived into the fundamentals of skin cell electrotaxis and, in parallel, studied how the electrode material plays a crucial role in enabling such stimulation in a biocompatible manner. We have developed novel electrode material compositions, which led us to step away from metals and instead introduce combinations of laser-induced graphene (LIG) and conducting polymers to make cheap, scalable, and sustainable versions of the technology possible. We have demonstrated our concept on healthy epithelial cell layers, as well as cell layers modified to mimic impaired wound healing in diabetes. Finally, we propose how this concept can best be shaped using methods and materials that would allow scaling to large-area wound dressings that could be sufficiently cheap to be used as consumables in medical care.
The working hypothesis of SPEEDER has been that electrical fields (EFs), applied over an open wound, act as a guidance signal for skin cells involved in re-epithelialization. In other words, we exploit skin cells’ “electrotactic” ability to manipulate and force them to move faster into the wound area. The idea of such a concept is not original to SPEEDER and has, in fact, been explored in numerous studies but with inconsistent outcomes. A key component has been missing for successfully exploiting electrotaxis, mainly an electrode material capable of sustaining direct current (DC) stimulation over long periods without dissolving the stimulating electrodes and damaging surrounding tissue. SPEEDER has managed to close this gap by working in parallel on biological models, materials science, and electrochemistry.
The unique idea we have followed is to use the super-capacitive properties of polymer electrodes, mainly the conducting polymer poly(3-4-ethylene dioxythiophene (PEDOT), to make DC stimulation of tissue possible. While metal electrodes corrode under DC, a conducting polymer layer makes it possible to move ions in solution through a reversible process.
In SPEEDER, we have deep-dived into the fundamentals of skin cell electrotaxis and, in parallel, studied how the electrode material plays a crucial role in enabling such stimulation in a biocompatible manner. We have developed novel electrode material compositions, which led us to step away from metals and instead introduce combinations of laser-induced graphene (LIG) and conducting polymers to make cheap, scalable, and sustainable versions of the technology possible. We have demonstrated our concept on healthy epithelial cell layers, as well as cell layers modified to mimic impaired wound healing in diabetes. Finally, we propose how this concept can best be shaped using methods and materials that would allow scaling to large-area wound dressings that could be sufficiently cheap to be used as consumables in medical care.
Comment: Ref-nr. refer to publications in the report.
The first action point of SPEEDER was to explore candidate electrode materials for DC. As starting point we used the conducting polymer PEDOT on top of sputtered iridium oxide (SIROF). We showed that our electrodes indeed could drive electrotaxis in cells [6]. We soon realized that not only the PEDOT layer had an important role, but furthermore the underlying substrate electrode. Development of the optimal polymer coating needed to be matched with efforts to make substrate electrodes as good as SIROF, but easier and cheaper to fabricate. We came across laser pyrolysis allowing to carbonize common polymer materials into a graphene like conducting carbon layer - LIG. We developed methods for binding hydrogel-like PEDOT to the LIG electrodes and showed how this resulted in a stable and low impedance skin-electrode interface [7]. We performed extensive mapping to validate the broad range of materials and developed and define the boundaries for safe, bio-tolerable vs unsafe stimulation protocols in the DC regime [10, 13].
The second point was to explore how DC stimulation act on cells and tissue, specifically skin cells active in wound healing. We first explored under which conditions the cells exhibited electrotaxis [6]. The next step was to explore analyse how electrotactic behavior changed when going from cells moving as solitaires to a collective cell layer more representative of real skin [7]. Finally, we studied how the two dominant cell types keratinocytes and fibroblasts, react to the stimulation when merged in the same culture dish [18]. We could confirm a field dependent and consistent electrotactic behavior of keratinocytes and fibroblasts [6,18] and the co-cultures revealed that there is a window of opportunity to activate both cells simultaneously. However, they are directed in opposite directions, implying that the optimal wound closing strategy should have radially oriented stimulation acting to drive keratinocytes inwards in the upper layer of the skin, with a return path in the deeper layer where the fibroblasts are present. This nevertheless could be revised, based on our work on epithelial layers with scratch wounds [9]. There we instead saw that when cells move as a collective, the direction of the electrical field is less important. It’s the combination of interaction with other cells and the field that result in more efficient wound closure. Stimulation across the scratch made it close up to three times faster [9] and the positive effect of stimulation furthermore persisted when investigating modified cells mimicking impaired wound closure in diabetes.
To summarize, we were able to identify a that the stimulation paradigm most relevant for bioelectronic wound dressings may be a field across the wound, and develop materials suitable for driving this stimulation. This will have great impact on how we proceed with stimulation at a real wound, to make the best possible use of electrotactic guiding mechanisms.
The first action point of SPEEDER was to explore candidate electrode materials for DC. As starting point we used the conducting polymer PEDOT on top of sputtered iridium oxide (SIROF). We showed that our electrodes indeed could drive electrotaxis in cells [6]. We soon realized that not only the PEDOT layer had an important role, but furthermore the underlying substrate electrode. Development of the optimal polymer coating needed to be matched with efforts to make substrate electrodes as good as SIROF, but easier and cheaper to fabricate. We came across laser pyrolysis allowing to carbonize common polymer materials into a graphene like conducting carbon layer - LIG. We developed methods for binding hydrogel-like PEDOT to the LIG electrodes and showed how this resulted in a stable and low impedance skin-electrode interface [7]. We performed extensive mapping to validate the broad range of materials and developed and define the boundaries for safe, bio-tolerable vs unsafe stimulation protocols in the DC regime [10, 13].
The second point was to explore how DC stimulation act on cells and tissue, specifically skin cells active in wound healing. We first explored under which conditions the cells exhibited electrotaxis [6]. The next step was to explore analyse how electrotactic behavior changed when going from cells moving as solitaires to a collective cell layer more representative of real skin [7]. Finally, we studied how the two dominant cell types keratinocytes and fibroblasts, react to the stimulation when merged in the same culture dish [18]. We could confirm a field dependent and consistent electrotactic behavior of keratinocytes and fibroblasts [6,18] and the co-cultures revealed that there is a window of opportunity to activate both cells simultaneously. However, they are directed in opposite directions, implying that the optimal wound closing strategy should have radially oriented stimulation acting to drive keratinocytes inwards in the upper layer of the skin, with a return path in the deeper layer where the fibroblasts are present. This nevertheless could be revised, based on our work on epithelial layers with scratch wounds [9]. There we instead saw that when cells move as a collective, the direction of the electrical field is less important. It’s the combination of interaction with other cells and the field that result in more efficient wound closure. Stimulation across the scratch made it close up to three times faster [9] and the positive effect of stimulation furthermore persisted when investigating modified cells mimicking impaired wound closure in diabetes.
To summarize, we were able to identify a that the stimulation paradigm most relevant for bioelectronic wound dressings may be a field across the wound, and develop materials suitable for driving this stimulation. This will have great impact on how we proceed with stimulation at a real wound, to make the best possible use of electrotactic guiding mechanisms.
To the best of our knowledge, SPEEDER 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 unlocks a multitude of possibilities, with the possibility most extensively explored in SPEEDER being the use of field guided migration of cells at wounds, to reduce the risk that wounds becomes chronic. We were able to make substantial progress towards this goal, most importantly to identify that the stimulation paradigm where an electrical field is acting across the wound is more efficient to promote fast closure, than the more complex concept driving cells as solitary units [9]. In culture models of wounds we could see and effect with up to three times faster closure, something that generated enormous public interest at the time of publication [9, press release dissem. 30]. Our developments gives hopes that patients with wounds at risk, in the future could be offered preventive stimulation that supports faster re-epithelialization before the wound becomes chronic. More work remains to prepare for clinical translation which is now be made possible by the follow-up proof-of-concept grant ProBER.
The electrode technology developed for SPEEDER is not exclusively relevant for skin cells, but can also guide other cells in similar manner. We see that SPEEDER will allow us to tap into a broader future application range of regenerative bioelectronics.
The electrode technology developed for SPEEDER is not exclusively relevant for skin cells, but can also guide other cells in similar manner. We see that SPEEDER will allow us to tap into a broader future application range of regenerative bioelectronics.