Abstract
There is a growing interest in bioelectric wound treatment and electrotaxis, the process by which cells detect an electric field and orient their migration along its direction, has emerged as a potential cornerstone of the endogenous wound healing response. Despite recognition of the importance of electrotaxis in wound healing, no experimental system to date demonstrates that the actual closing of a wound can be accelerated solely by the electrotaxis response itself, and in vivo systems are too complex to resolve cell migration from other healing stages such as proliferation and inflammation. This uncertainty has led to a lack of standardization between stimulation methods, model systems, and electrode technology required for device development. In this paper, we present a ‘healing-on-chip’ approach that is a standardized, low-cost, model for investigating electrically accelerated wound healing. Our device provides the first convergent field geometry used in a stimulation device. We validate this device by using electrical stimulation to close a 1.5 mm gap between two large (30 mm2) primary skin keratinocyte layers to double the rate of healing over an unstimulated tissue. This proves that convergent electrotaxis is both possible and can accelerate healing, and offers a new ‘healing-on-a-chip’ platform to explore future bioelectric interfaces.
Introduction
Since du Bois-Reymond first characterized the naturally occurring ‘wound current’ nearly two centuries ago (1), there has been significant interest in applying external electrical stimulation to improve wound healing (2–4). The potential for this approach is becoming increasingly apparent— for instance, numerous, recent, in vivo studies show some improvement in skin healing in animal models upon electric field stimulation (5–10), while in vitro assays have demonstrated control of cells and simple tissues using spatially programmed electric cues (11–13). Further, given the increasing prevalence and healthcare burden of wound treatments (14,15), new technologies to expedite and improve wound care are sorely needed. However, despite these and other studies over the past several decades, the few extant commercial products have demonstrated mixed results (16–19), and bioelectric wound therapy is far from the standard of care. This discrepancy is due to broad gaps in both technology development and biological knowledge describing how electrical stimulation may act to improve wound healing. Technologically, optimum stimulation parameters for field strength, biointerface design, and current delivery mode remain unclear (20,21). Biologically, there is uncertainty about how the key wound healing mechanisms--cell migration, proliferation, and inflammation—are affected by electric stimulation (2). This uncertainty has resulted in a lack of standardization in stimulation schemes, model systems, and technology that can all lead to issues of reproducibility and long design iterations time that have slowed progress (22–24).
Here, we begin to address this problem by integrating a popular technical approach used in other branches of biotechnology—’organ-on-a-chip’ systems—to reduce the complexity of biomedical problems to something both tractable and eventually translatable. Organ-on-a-chip (OoC) platforms are in vitro model systems that capture a specific and critical physiological behavior of the in vivo system in a standardized, rapid, lower-cost in vitro model. To date, OoCs have clearly proven their value in other fields by aiding discoveries and treatments for lung, gut, and vascular pathologies (25–27). Here, we use an OoC approach to integrate a ‘healing-on-a-chip’ platform with a custom electrobioreactor designed from the ground up to investigate electrically accelerated wound healing.
While there are many effects that applied electrical stimulation may have on tissue growth and healing, the best-characterized is electrotaxis—the directed motion of cells in response to an electric current. Electrotaxis is seen in over 20 cell types across multiple organisms where cells sense and track electrochemical potential gradients (∼1 V/cm) that emerge during development and injury healing (28–30). The mechanism of detection is thought to be electrophoresis of charged membrane-bound receptors in the presence of an electric field, resulting in an asymmetric distribution of these proteins that triggers downstream signaling of the cell migration machinery (31). In vivo, these fields result in the center of a skin wound being negatively polarized relative to the periphery of the wound (32,33). Direct current fields are analogous to fields in vivo (34), in contrast to the pulsed DC or AC stimulation used in many in vivo studies (5), and are sufficient to induce the electrotaxis response. However, electrotaxis has primarily been studied in isolated single cells to elucidate the molecular biology of the process, and at present there is no study, either in vitro or in vivo that conclusively indicates that electrotaxis itself can accelerate wound closure. This gap stems from the technological limitations of current devices used to study electrotaxis.
Nearly all devices use a single electrode pair to apply a uniform, unidirectional field across tissues—such a field would cause one side of a wound to close and the other side to worsen. In vivo, the wound field converges on the center of a wound, so a new device design is required to capture this characteristic. In addition to this stimulation limitation, most studies and devices do not generalize well to macroscale tissues and wounds since precise tissues with reproducible, millimetric wounds must be grown inside the electrobioreactor. Finally, macroscale cell migration requires stable electrical stimulation over many hours, and the common, bleach-based electrode preparation process is insufficient for long-term stimulation (>4 hours). An ideal bioelectric ‘wound-on-a-chip’ platform should address these issues.
Here we build on our prior work (11,12) to create a new electrobioreactor to study healing in a macroscale skin-on-a-chip model using primary mouse skin monolayers which migrate toward the cathode when stimulated (Fig. 1), and use electrical stimulation to accelerate closure of 1.5 mm large model skin wounds by at least 2X over unstimulated skin layers (Figs. 2, 3). To accomplish this, we developed new electrotaxis infrastructure specifically designed for the constraints of wound healing, delivering a sustained converging electric field to a tissue (Fig. 1). With this device, we were able to engineer and stimulate the largest tissues yet tested with electrotaxis (30 mm2) for 12 hours, while also exploring the consequences of overstimulation.
Results
Our new electrobioreactor significantly departs from extant electrotaxis systems by generating an electric field that converges at the center of model wounds, and functions as follows. The device consists of an acrylic insert clamped to a standard tissue culture dish, holding electrodes and agarose salt bridges in position (Fig. 1A, see ESI Methods for fabrication details). Three chloridized silver stimulation electrodes (anodes at left and right, cathode at center) are isolated from each other in separate saline reservoirs and electrical contact with the culture media is provided by 4% agarose w/v salt bridges cast inside the insert, one per reservoir (Fig. 1B). The ∼500 µm thin, laser-milled, agarose bridge serves as a central cathode and is aligned directly over the wound site (Fig. 1C, see ‘*’). The result is a stable, uniform field that converges upon the central electrode as confirmed by simulation (Fig. 1D,E). To reliably generate reproducible tissues and linear wounds, we use a silicone stencil templating method (12,35) to prepare confluent monolayers the evening before an experiment. We then assemble the electrobioreactor over these tissues prior to imaging. For these experiments, we use layers of keratinocytes from primary skin cultured under basal conditions optimized for electrotaxis (11). After the tissues have grown, the stencils are removed, and the device is clamped over the cells, aligned such that that the central slit electrode is in the gap between cells (Fig. 1E). Then, a computer-controlled source meter (Keithley 2450) is connected to each pair of electrodes (left-center and right-center), with both sources sharing the central cathode, to supply an electric current. The field within the chamber is continuously monitored by a digital oscilloscope and the current output of each source meter is adjusted via closed-loop control to maintain a constant 2 V/cm field strength directed toward the central cathode. We specifically chose this field strength as it has previously been validated and was chosen to amplify the approximate field strength experienced in vivo (28). To extend cathode lifetime, only one source was active at a time, alternating between left-center and right-center stimulation every 30 seconds. Oxygen delivery and waste management are handled by perfusing fresh media through the bioreactor at 2 mL/h, turning over the chamber volume ∼11 times per hour. The resulting system provides a robust convergent field to viable cells.
Stable DC stimulation and cell viability require that the electrodes remain intact throughout an entire experiment, so optimization of electrode chemistry is an important consideration. Virtually all DC electrotaxis chambers use an anode and a cathode to inject Faradaic current through a sample, using combinations of salt bridges, media perfusion, and heavy buffering to prevent the buildup of toxic electrochemical byproducts or harmful pH changes due to electrolysis at the electrodes (20,36,37). Because the current used in our device is moderate (∼6-10 mA) and the 1.5 mm gap between tissues is relatively large, the central cathode must be able to sink current for an extended period to induce tissue convergence, ideally 12 hours or more. To support this, our system uses electrically chloridized silver foil as electrodes, which degrades at the cathode into ionic silver and chloride during stimulation. This reaction is more favorable than the hydrolysis cathodal half-reaction, which evolves hydrogen gas from the solution and increases pH (37). This allows for safe stimulation until AgCl is depleted at the cathode, when evolution of H2 then becomes favorable and pH increases rapidly, which can cause cytotoxicity. Therefore, sufficient chloridization of the silver foil is paramount for extended electrode lifetime. We compared our chloridization method with bleach immersion, another technique commonly used to chloridize silver. We performed repeated cyclic voltammetry to compare electrode preparations and found that our method of electroplating silver chloride resulted in more stable cathodes (see ESI, Fig. S1). Our combined approach of robust silver chloridization, agarose diffusion barriers to prevent ionic silver reaching the tissue, and media perfusion integrates numerous best practices to maximize cell viability during stimulation in our device, allowing for extended wound healing experiments.
To evaluate this platform for in vitro healing, we patterned two 10 x 3 mm tissues spaced 1.5 mm apart with the central cathode aligned over the wound center (Fig. 1F). The acrylic outline of the central cathode slit fluoresces weakly when imaged using a standard DAPI filter set, so the alignment between the central cathode and the tissues could be tuned and verified. We then applied convergent electrical stimulation over 12 hours following a 30-minute control period without field, with striking results (Fig. 2, Video S1). In the non-stimulated control case, cell proliferation and migration lead to the slow expansion of tissues and gradual, but incomplete closure of the wound over 12 hours (∼50% closure, N=3). However, convergent bioelectric stimulation led to complete closure between 11-12 h (N=3). More specifically, the edge migration speed was twice as fast in the stimulated case as in the control, measuring 29.4 ± 3.3 µm/h and 62.2 ± 8.1 µm/h for the control and stimulated cases, respectively. To conclusively attribute this effect to electrical stimulation rather than temperature effects (Joule heating has been linked to increased migration speeds in prior studies (31,38)) we monitored the device temperature during stimulation (Fig. S2). The steady state temperature rose from 37 °C to 38 °C, and this 3% increase is unlikely to account for the 100+% increase in migration speed during stimulation. We hypothesize that perfusion and media turn over helps to exchange heat and mitigate any effects from Joule heating. Taken together, this is the first demonstration of convergent field stimulation accelerating in vitro wound healing, and the results prove that electrotaxis alone is sufficient for this closure.
To better characterize device performance and its effects on large scale tissue growth and motion, we performed particle image velocimetry (PIV) on each tissue. Representative horizontal velocity kymographs for both the control and convergent stimulation cases are shown in Fig. 3 (compare with Video S1). To provide context of spatial dynamics within a given tissue, we show representative heatmaps of horizontal velocity and line integral convolution (LIC) migration maps to visualize the overall flow of cellular motion at 4 hours after the onset of stimulation (steady state). Throughout the control tissue (Figs. 3A-C), there is little net outwards motion, except for slow expansion at the edges. Disorder is apparent in the velocity and migration maps of the control tissues, which lack large regions of coordinated movement, as expected for non-stimulated tissues (Figs. 3B,C). In contrast, bioelectric stimulation resulted in nearly uniformly high-speed motion throughout the tissue, converging on the gap within 15 minutes of the field turning on, as visualized in the velocity and migration maps (Figs. 3D-F).
The large number of parallel streaklines along the stimulation direction in the migration map demonstrates highly coordinated motion across the tissue in alignment with the stimulus (Figs. 3F). These visualizations reveal that the electric field acts a global migration cue across a large area, confirming that cells experience a highly uniform field as predicted by simulation (Fig. 1D,E).
Having demonstrated that the in vitro healing process can be electrically accelerated overall, we next characterized cellular responses specifically during the final stages of wound closure. Unlike traditional electrotaxis chambers where the electrodes are significantly distal to the tissue to ensure a uniform field, our healing-on-a-chip device requires a central electrode to focus cell migration into the wound zone. Since the central electrode has a finite width (∼500 µm here) that is smaller than the wound, this means that tissues will eventually pass underneath the electrode and enter the ‘electrode shadow’ during the final stages of healing and convergence. Any discrete electrode produces field non-uniformities close to its surface, so as cells enter the electrode shadow, they will experience a very different field than out in the fully developed zones far from the center. Our simulation predicts a sharp decrease in electric field strength that begins about 500 µm on either side of the central cathode above the convergence region (Fig. 4A). We quantified the actual effects of the central field singularity by stimulating closed tissues for 6 hours and using a live nuclear dye to track cells in that central zone (Video S2). We averaged PIV across the region surrounding the closure zone over the stimulation period (Fig. 4B, asterisks and error bars) and fit a sigmoid function to the data (Fig. 4B, inset) showing that there is a strong, steady-state response far from the central electrode that steadily weakens as cells approach the central electrode and enter the electrode shadow (Fig. 4, dashed blue line; magenta zone shows electrode shadow). While this local weakening of the electrotactic response closely resembled the trend in our simulations, cells nonetheless continued to directionally migrate deep into the electrode shadow zone, only to dropping to <50% of the steady state velocity once cells were ∼100 µm off the electrode midline. These data show that the effective electrode size is smaller than its physical, 500 µm width (Fig. 4B, compare dotted black boundaries to electrode boundaries). This means that even relatively large electrodes can still promote last-mile healing.
Critically, we also observed potential consequences to continued electrical stimulation after a wound had closed. As has been noted previously (39), electrotaxis appears to override basic cellular safety mechanisms, such as contact inhibition, meaning that cells will continue trying to directionally migrate as long as stimulation is active. In our wound healing model, this meant that stimulating after a tissue had closed would continue to drive cell migration towards where the center of the wound had previously been. This inevitably caused an increase in local cell density, and we measured a >2X increase (from 750 to 1600 cells/mm2) in cell density under the electrode shadow relative to density distal to the central electrode (Fig. 4C-E), showing there is potential for over-densification of cells driven by post-healing stimulation. Comparing individual cell trajectories within the central zone confirmed that cells within the electrode shadow translated horizontally a lower distance than those that were farther away (Fig. 4F,G). This reduction in overall translation extended, in a graded fashion, outwards 500 µm from the center in either direction, consistent with the reduction of speed cells experience as they enter the electrode shadow. Nevertheless, there is net migration towards the center, even for cells that were initially positioned under the electrode, suggesting again that the electrode’s influence extends underneath its width despite significant weakening of the effective field strength.
We suggest two reasons that the ‘effective electrode’ size would be smaller than its physical size. First, the threshold field strength that elicits an electrotaxis response is lower than the 2 V/cm we target in stimulation. As the field strength rolls off, it is still ‘therapeutic’ for some time, given that physiological field strengths are on the order of 1 V/cm (28). Second, the monolayers carry some memory of the electrotaxis response that continues to influence their responses after the stimulus changes (11). Keratinocytes polarize in response to the field stimulus, and this polarization takes time to decay once a stimulus is no longer detected. This could lead to cells effectively coasting, unguided, during the last gap before tissue closure.
Discussion & Conclusion
Overall, we present a bioelectric, healing-on-a-chip (HoC) platform designed specifically to study the role of electrotaxis and other electrical phenomena in wound healing. Unique for electrical stimulation bioreactors, our approach creates a field stimulation pattern that mimics that found in wounds in vivo, with the field converging at the center of the wound gap. This capability allows us to directly explore the actual healing process, rather than purely uni-directional cell migration. Therefore, our platform allows study of the in vitro healing process spanning initial injury, ‘first contact’ as the sides of the wound meet and, critically, post-closure behavior after the wound has healed. Using this platform and unoptimized stimulation parameters, we demonstrate ∼2X acceleration of wound closure in an in vitro skin layer model due solely to electrotactic effects. To our knowledge, this is the first demonstration and visualization of electrotaxis itself accelerating a healing process. The stability, reproducibility, and programmability of the platform make it suitable to deeply explore key technological and biological questions, and we have taken care to ensure the device is easily replicable and accessible to a broad audience.
That even naïve stimulation had a strong, positive effect on in vitro healing is encouraging, and establishes a clear baseline against which future parameter optimization studies can be compared. This approach could be critical to the field as standardization and optimization of stimulation approaches remains an open question. To hint at this, we explored the effects of stimulating beyond initial closure of the wound by electrically stimulating a closed tissue, and the resulting cellular pile-up indicates both the potency of electrotaxis to drive migration and the importance of being able to fine-tune and intelligently adjust stimulation in practice to avoid detrimental effects of overstimulation. Such cellular pile-ups also speak more fundamentally to the role of electrotaxis as a tool to modulate and explore interactions at the boundaries between tissues.
While we specifically investigated healing in monolayers of primary skin cells here, wound healing in vivo clearly involves complex coordination across multiple cell types (e.g. macrophages and immune cells, fibroblasts, and vascular cells, and epidermal cells) and phases, (e.g inflammation, granulation, and re-epithelialization) (2). That our platform supports pre-engineering tissue configurations means that co-cultures or more complex tissue models can be grown first and then incorporated into the bioreactor to allow more complex studies on healing. When linked to stimulation optimization approaches, it may be possible to determine modalities that preferentially target a given cell type, or process such as proliferation vs. migration during healing. Again, these questions benefit from a field geometry that enables a healing phenotype.
Finally, our bioelectric ‘Healing-on-a-Chip’ approach is fully open and intended to be modified and tailored for a variety of applications. We provide complete design files, computational models, and stimulation code (Provided via a GitHub repository: https://github.com/CohenLabPrinceton/SCHEEPDOG), and the basic approach lends itself to easy customization. For instance, electrode shape, size, number, and location can easily be adjusted without additional cost or significant complexity. Field stimulation strategies can be tested by attaching any desired power supplies or running arbitrary stimulation code to activate electrode sequences. Our autofluorescence alignment approach makes it possible to accurately align a given electrode configuration to a given wound and removes much of the ambiguity and difficulty this process would normally introduce. We hope the demonstrations here and flexibility of the device can help accelerate healing-on-a-chip research, improve translation for future in vivo applications, and even support new, research on general interactions between colliding tissues.
Acknowledgements
We gratefully acknowledge Prof. Danelle Devenport and Katie Little at Princeton University for providing primary keratinocytes and culture support. Research reported in this publication was supported by the National Center for Advancing Translational Sciences (NCATS), a component of the National Institute of Health (NIH) under award number TL1TR003019 (TJZ). Further support was provided by National Institutes of Health grant R35GM13357401 (DJC, GS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.