Electrically stimulating wound dressings

Abstract

An activatable dressing of the present disclosure comprises a biocompatible battery including a magnesium-based (Mg) anode; a silver / silver chloride-based (Ag / AgCl) cathode; and a sodium chloride (NaCl) impregnated separator disposed between the anode and cathode in a dry state, the separator comprising an inlet pad configured for introduction of a water-based fluid to the separator for activation of the battery. The activatable dressing further comprises stimulation electrodes electrically coupled to the Mg anode and Ag / AgCl cathode.

Description

ELECTRICALLY STIMULATING WOUND DRESSINGSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to co-pending U.S. provisional application entitled, “Electrically Stimulating Wound Dressings,” having application number 63 / 632,891 , filed April 11 , 2024, which is entirely incorporated herein by reference.BACKGROUND

[0002] Chronic wounds are characterized by impaired healing and affect millions globally. These wounds result from a variety of factors, including pressure ulcers, venous and arterial insufficiencies, peripheral neuropathy, diabetes mellitus, and surgical complications. The United States Centers for Medicare & Medicaid Services alone spends more than $28 billion annually to treat such wounds and the expenditure is expected to grow for the near future due to increasing number of individuals with diabetes, obesity, cancer and other comorbidities.

[0003] Present products for treating chronic wounds include occlusive dressings, hydrogels, skin substitutes, and topical growth factors. While occlusive dressings, gauze, and hydrogels are relatively low-cost and user-friendly, they show limited effectiveness against complex chronic wounds. More advanced treatments such as skin substitutes and topical growth factors are still only moderately effective, at best achieving complete wound healing in ~50% of cases, while often costing well over $1000, and sometimes more than $20,000, per wound closure. Current research into biologics-based systems includes stem cell-loaded hydrogels, growth factor delivery systems, and multilayered scaffolds. However, stem cells and growth factors remain expensive and challenging to work with, and scaffolds can suffer from shrinkage and fail to recreate the heterogeneous skin environment. There is therefore a need to develop low-cost, highly effective treatments.SUMMARY

[0004] The present disclosure presents apparatuses and related methods of electrically stimulating wound dressings. One such apparatus is for an activatable dressing comprised of a biocompatible battery including: a magnesium-based (Mg) anode; a silver / silver chloridebased (Ag / AgCI) cathode; and a sodium chloride (NaCI) impregnated separator disposed between the anode and cathode in a dry state, the separator comprising an inlet pad configured for introduction of a water-based fluid to the separator for activation of the battery. The activatable dressing further comprises stimulation electrodes electrically coupled to the Mg anode and Ag / AgCI cathode.

[0005] Also disclosed herein is a method of treating a wound comprising applying to the wound an activatable dressing, wherein the activatable dressing is comprised of a biocompatible battery including: a magnesium-based (Mg) anode; a silver / silver chloridebased (Ag / AgCI) cathode; and a sodium chloride (NaCI) impregnated separator disposed between the anode and cathode in a dry state, the separator comprising an inlet pad configured for introduction of a water-based fluid to the separator for activation of the battery. The activatable dressing further comprises stimulation electrodes electrically coupled to the Mg anode and Ag / AgCI cathode.

[0006] In one or more aspects of such apparatuses and methods, the battery is packaged within a film; wherein the film is a polyethylene terephthalate (PET) film; the separator comprises a check pad configured to provide an indication of hydration of the separator; the check pad comprises a dry water-soluble dye loaded into a storage membrane; the separator comprises a cellulose-based membrane; the battery is ring shaped allowing visual access to the wound; the stimulation electrodes comprise an outer stimulation electrode that is ring shaped; the battery is on a non-adhesive side of a bandage and the stimulation electrodes are on an adhesive side of a bandage; the stimulation electrodes comprise an spiral shaped inner electrode and a serpentine shaped outer electrode in a ring pattern; the stimulation electrodes comprise a central disk electrode and an outer ring electrode; and / or the separator is loaded with glycerol.

[0007] In one or more aspects, such methods further comprise activating the activatable dressing by droplets of a water to the inlet pad.

[0008] In one or more aspects of such methods, the activatable dressing is activated from wound fluid that is contact with the inlet pad.

[0009] Other apparatuses, methods, systems, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description and be within the scope of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0011] FIG. 1A shows an activatable water powered dressing assembly in accordance with various embodiments of the present disclosure.

[0012] FIG. 1 B shows a schematic illustration of operation of a separator component of the water powered dressing assembly of FIG. 1 A in accordance with various embodiments of the present disclosure.

[0013] FIG. 1 C shows a close-up view of an exemplary water powered dressing assembly in accordance with various embodiments of the present disclosure.

[0014] FIG. 1 D illustrates a schematic representation of components of the dressing assembly of FIG. 1A in accordance with various embodiments of the present disclosure.

[0015] FIG. 2A shows an embodiment of an exemplary water powered dressing assembly having kirigami-based stimulation electrodes in accordance with various embodiments of the present disclosure.

[0016] FIG. 2B shows mechanical finite element analysis (FEA) results from stretching the kirigami-based stimulation electrodes of FIG. 2A.

[0017] FIG. 2C shows a relative change in the electrical resistance of kirigami-based stimulation electrodes (of FIG. 2A) as a function of strain.

[0018] FIG. 2D shows a cross-sectional view of a kirigami-based water powered dressing assembly interfaced with an artificial deep wound with complex shape and contours in accordance with the present disclosure.

[0019] FIG. 3A is a chart showing the battery capacity as a function of volume of water added to the separator of an exemplary water powered dressing assembly in accordance with various embodiments of the present disclosure.

[0020] FIG. 3B is a chart showing the effect of different types of water-based fluids (tap water, deionized water, phosphate-buffered saline, and artificial wound fluid) being used to activate the battery of an exemplary water powered dressing assembly in accordance with various embodiments of the present disclosure.

[0021] FIG. 3C is a chart showing a duration of stimulation for different loads simulating different stages of wound healing for an exemplary water powered dressing assembly of the present disclosure.

[0022] FIG. 3D is a chart showing an effect of temperature on duration of stimulation for different loads simulating different stages of wound healing for an exemplary water powered dressing assembly of the present disclosure.

[0023] FIG. 3E is a chart showing an effect of ambient humidity on duration of stimulation for different loads simulating different stages of wound healing for an exemplary water powered dressing assembly of the present disclosure.

[0024] FIG. 3F is a chart showing an effect of applied pressure on duration of stimulation for different loads simulating different stages of wound healing for an exemplary water powered dressing assembly of the present disclosure.

[0025] FIG. 3G is a chart showing an effect of bending stress on duration of stimulation for different loads simulating different stages of wound healing for an exemplary water powered dressing assembly of the present disclosure.

[0026] FIG. 3H is a chart showing durations of stimulation offered by an exemplary water powered dressing assembly of fixed battery capacity for treating wounds of different diameters.

[0027] FIG. 31 is a chart showing an effect of battery capacity of an exemplary water powered dressing assembly on duration of stimulation for treating wounds of fixed size.

[0028] FIG. 4A shows an electric potential distribution profile between two stimulation electrodes of an exemplary kirigami-based water powered dressing assembly with a battery output voltage of 1 .6 V.

[0029] FIG. 4B shows a top (left) and side (right) view of the electric field distribution at the wound site when treated with a kirigami-based water powered dressing assembly supplying a voltage of 0.5, 1 .0, and 1 .6 V.

[0030] FIGS. 4C-4E show electric field distribution profiles across the wound length for different (C) applied voltages for a wound (diameter: 1 cm), (D) wound shapes, and (E) wound diameters.

[0031] FIG. 5A shows infrared images of a mouse wearing an exemplary water powered dressing assembly before (left) and 5 minutes after (right) activation of the battery for the dressing assembly in accordance with various embodiments of the present disclosure.

[0032] FIG. 5B is a chart showing a relative size of the wounds over time for three groups of mice testing application of an exemplary water powered dressing assembly of the present disclosure.

[0033] FIG. 5C is a chart showing the relative size of the wounds on day 1 1 for the three groups of mice (of FIG. 5B) testing application of the exemplary water powered dressing assembly of the present disclosure.

[0034] FIG. 5D is a chart showing representative movement trajectories for individual ones of the three groups of mice (of FIGS. 5B-5C) over 10 minutes during testing of the exemplary water powered dressing assembly of the present disclosure.

[0035] FIG. 6A shows a representative cross-sectional images of wounds with hematoxylin and eosin stains for the three groups of mice (of FIGS. 5B-5D) after 13 days during testing of the exemplary water powered dressing assembly of the present disclosure.

[0036] FIG. 6B shows representative cross-sectional Masson’s trichrome stains of the center of the wounds for the three groups of mice (of FIGS. 5B-5D and 6A) 13 days after wounding during testing of the exemplary water powered dressing assembly of the present disclosure.

[0037] FIG. 6C is a chart showing quantitative comparison of the epidermal thickness of the three groups of mice (of FIGS. 5B-5D and 6A-6B) during testing of the exemplary water powered dressing assembly of the present disclosure.

[0038] FIG. 6D is a chart showing quantitative comparison of the collagen intensity of the three groups of mice (of FIGS. 5B-5D and 6A-6C) during testing of the exemplary water powered dressing assembly of the present disclosure.

[0039] FIG. 6E is a chart showing levels of immunofluorescence staining for CD31 (red) and DAPI (blue) in tissue from the three groups of mice (of FIGS. 5B-5D and 6A-6D) during testing of the exemplary water powered dressing assembly of the present disclosure.

[0040] FIG. 6F is a chart showing quantification of M1 / M2 macrophage ratio of the three groups of mice (of FIGS. 5B-5D and 6A-6E) during testing of the exemplary water powered dressing assembly of the present disclosure.

[0041] FIG. 7A is a chart showing temporal evolution of wound impedance during testing of the exemplary water powered dressing assembly of the present disclosure.

[0042] FIG. 7B is a chart showing a duration of stimulation as a function of simulated wound impedance during testing of the exemplary water powered dressing assembly of the present disclosure.

[0043] FIG. 70 is a chart showing battery discharge profile as a function of relative humidity (load resistance: 25 kQ) during testing of the exemplary water powered dressing assembly of the present disclosure.

[0044] FIG. 7D is a chart shown the effect of the size of phantom wound on duration of stimulation during testing of the exemplary water powered dressing assembly of the present disclosure.

[0045] FIG. 7E is a chart showing Nyquist plots of phantom wounds of different sizes during testing of the exemplary water powered dressing assembly of the present disclosure.

[0046] FIG. 7F is a chart showing impedance values of phantom wounds of different sizes during testing of the exemplary water powered dressing assembly of the present disclosure.DETAILED DESCRIPTION

[0047] The present disclosure provides examples related to electrically stimulating dressings and their associated methods and applications. In one aspect, as shown in FIG. 1A, an activatable water powered dressing assembly 1 includes an inactive biocompatible battery 2 including: a magnesium-based (Mg) anode 10, supported by a current collector 8; a silver / silver chloride-based (Ag / AgCI) cathode 16 supported by a current collector 14; and a sodium chloride (NaCI) impregnated separator 12 disposed between the anode 10 and cathode 16 in a dry state, with the separator 12 comprising an inlet pad 11 configured for introduction of water to the separator 12 for activation of the battery and a check pad 12 configured to provide an indication of hydration of the separator 12; and stimulation electrodes 22 electrically coupled to the Mg anode 10 and Ag / AgCI cathode 16. In various embodiments, a first adhesive package 6 is applied at a top of the dressing assembly and a second adhesive package 18 is applied at a bottom of the dressing assembly with the occlusive dressing 20 (e.g., transparent film, gauze, etc.) and stimulation electrodes 20 being positioned external to the second adhesive package 18.

[0048] It is noted that chronic wounds affect ~2% of the U.S. population and increase risks of amputation and mortality. Unfortunately, treatments for such wounds are oftenexpensive, complex, and only moderately effective. In this context, electrotherapy, which involves the use of an external electrical field to drive wound closure, shows great promise with recent examples illustrating low-cost, skin-friendly electrodes for efficient delivery of electrical stimulation to wounds. Preclinical and clinical studies show that electrotherapy leads to increased cell migration, venous blood flow, and cell proliferation, among other pro-healing effects resulting in rapid wound closure. However, achieving the best patient outcomes using this method requires daily treatment for several hours typically for weeks at a time. This places an exorbitant strain on the patients, as the stimulation electrodes are connected to bulky benchtop devices, restricting patient mobility for the duration of treatment and limiting the practical use of electrotherapy in wound care.

[0049] Emerging work in the area of battery-free, radio frequency (RF)-powered and battery-powered wearable electronics enables the realization of miniaturized, smart dressings that support electrotherapy. However, battery-powered systems are still quite bulky and pose safety concerns when interfaced with the delicate wound. Battery-free, RF-powered platforms require that patients be in the vicinity of a transmitting antenna to wirelessly power the device, which restricts patient mobility. These systems also require meticulously designed transmission and receiving antennae and rely on expensive and bulky equipment such as RF amplifiers, function generators, and advanced impedance matching units. Moreover, the fragility of both battery-powered and battery-free electronics-based systems limit their use in body regions that bear high pressures and are prone to developing chronic wounds, such as the heel of the foot and the sacrum. Triboelectric nanogenerators offer an unconventional approach to delivering electrotherapy, though they generate an electrical field only when the participant is moving or when interfaced with an external tethered linear motor. Beyond wound healing, electrotherapy systems also show promise in other areas of tissue regeneration, including cell modulation, nerve regeneration, and osteogenesis.

[0050] The present disclosure describes a unique external electronics-free water powered dressing assembly 1 that electrically stimulates the wound, leading to among the fastest rates of wound closure, comparable to those shown with expensive biologies- orelectronics-based approaches at a fraction of the cost (< $1 / dressing). In various embodiments, the assembly device 1 includes an unconventional ultra-lightweight flexible, water powered battery 2 and a pair of thin-film stimulation electrodes 22 seamlessly integrated with occlusive dressing 20. The battery 2 produces an electrical field across the stimulation electrodes 22 when activated with water. Unlike present examples of electronics-based electro-therapy dressings, a water powered dressing assembly 1 of the present disclosure can offer several hours of continuous stimulation with no restriction on patient mobility, robust performance even when subjected to high pressures and bending stressors, and long shelflife owing to carefully selected materials, design architecture, and working principle.

[0051] In various embodiments, an exemplary water powered dressing assembly 1 is characterized as being ultra-lightweight, thin, and flexible and involves a biocompatible magnesium-silver / silver chloride (Mg-Ag / AgCI) battery 2 (area: 0.64 cm2; weight: 47 mg; capacity: 0.4 mAh) affixed to the non-adhesive side of an occlusive bandage 20 (e.g., TEGADERM dressing) and a pair of carbon-based stimulation electrodes 22 on the adhesive side (facing the wound). Table 1 and Table 2 show the specifications and performance metrics for the water powered dressing assembly, respectively.Table 1Table 2

[0052] The complete system (battery 2 + electrodes 22 + occlusive bandage / dressing 20) is light (290 mg, only ~20% higher than TEGADERM dressing) and highly flexible for conformal attachment on curvilinear body parts such as toes or fingers. In various embodiments, the water powered battery 2 can be shaped in the form of a ring (or circle) for easy visual assessment of the wound, and the battery 2 includes an Mg anode 10 and an Ag / AgCI cathode 16 with a dry cellulose separator 12 impregnated with sodium chloride (NaCI). In various embodiments, the battery 2 may be packaged with a polyethylene terephthalate (PET) film and is in an open-circuit state (i.e. , inactive state) when the separator 12 is dry. A degradation test performed by continuously incubating the carbon-based stimulation electrodes 22 in artificial wound fluid (AWF) at 37°C for 7 days shows negligible impact on their electrical properties (resistance increment of <10%) indicating their inert electrical behavior and suitability for interfacing with wounds.

[0053] In various embodiments, as shown in FIG. 1 B, the separator 12 includes an inlet pad 11 where the user introduces water droplets 15 that is absorbed by the separator 12 (e.g., a microfluidic wicking material) to activate the battery and a check pad 13 to visually indicate complete hydration of the separator 12 and successful activation of the battery 2. In various embodiments, the check pad 13 comprises a dry water-soluble dye 16 loaded into a cellulose- based storage membrane of the separator 12 and laminated to the back side of the separator 12. Hydration of the separator 12 results in dissolution and subsequent transport of the dye from the storage membrane to the separator 12 thus providing a visual indication of the status of separator hydration.

[0054] Next, FIG. 1C shows a close-up view of an exemplary water powered dressing assembly 1 , while Fig. 1 D illustrates a schematic representation of components of the dressing assembly 1 . As shown, the water powered battery 2 is ring-shaped for easy visual assessment of the wound. The battery 2 includes a magnesium (Mg) anode 10 and a silver / silver chloride (Ag / AgCI) cathode 16 with a dry cellulose separator 12 impregnated with sodium chloride (NaCI). The battery 2 is packaged within a polyethylene terephthalate film and is in an opencircuit state (i.e. , inactive state) when the separator 12 is dry.

[0055] Upon activation, the battery 2 produces a voltage (~1.5 V) across the pair of stimulation electrodes 22 which include a central disk electrode 22A connected to the anode 10 (Mg; negative electrode) and an outer ring electrode 22B attached to the cathode 16 (Ag / AgCI; positive electrode). Such a configuration ensures a radial electrical field pointing to the wound center in line with the endogenous electrical field, critical for promoting healing. Attributes of using the water powered battery 2 over conventional thin-film batteries include the ability to offer on-demand electrotherapy, simplicity, and no reliance on toxic electrolytes, as is the case with conventional batteries.

[0056] Eventual evaporation of the water from the separator 12 leads to an increase in the internal resistance of the battery 2 culminating in its deactivation when the separator 12 is nearly dry. In various embodiments, the check pad indicator 13 can include a humidity indicator affixed to the inlet pad 1 1 to provide a real-time visual indication of the moisturecontent of the separator 12. For example, the humidity indicator may change its color from blue to light pink when water is added to the inlet pad 1 1 , where the humidity indicator returns to its blue color when the separator 12 dries due to water evaporation. Alternatively, the check pad 13 may include a dye indicator as shown in FIG. 1 C. In either case, a user can simply look at the color of the check pad 13 to gauge the real-time status of the water powered dressing assembly’s ability to deliver electrical stimulation. In various embodiments, the additional electrochromic displays may be included that provide a colorimetric indication of the instantaneous output voltage of the dressing assembly 1 for a more accurate assessment of its real-time ability to electrically stimulate wounds.

[0057] Given that wound beds can have complex, deep, and irregularly shaped three- dimensional (3D) contours, various embodiments of the water powered dressing assembly of the present disclosure feature stimulation electrodes with mechanical features capable of conformally mounting on such 3D-contoured soft tissues. In this context, certain embodiments of stimulation electrodes 22 feature a kirigami-inspired design having a serpentine-shaped (or S-shaped) outer electrode (OE) (that forms a ring or circular pattern) and a spiral-shaped inner electrode (IE), which offers an ability to assume 3D morphable structures and accommodate large, out-of-plane deformations, as shown in FIGS. 2A-2B.

[0058] In FIG. 2A, the dressing assembly 1 includes a serpentine outer electrode (OE) 22B (in a ring or circular shape) and spiral-shaped inner electrode (IE) 22A. FIG. 2B shows the ability of the kirigami-based stimulation electrodes 22A, 22B to expand in 3D under varying degrees of strains and further shows corresponding simulation results capturing the stress generated within the stimulation electrodes 22. The kirigami-based stimulation electrodes 22 exhibit elastic deformation with uniform stress distribution across the electrode lengths resulting in a stable mechanical deformation. The configurable nature of the kirigami-based stimulation electrodes (of FIGS. 2A-2B) allows the OE 22B to expand laterally for mounting it along the periphery and the spiral-shaped IE 22A to unwind and interface with the center of wounds with various complex shapes, sizes, and depths, as shown in FIGS. 2C and 2D, where FIG. 2C shows a relative change in the electrical resistance of kirigami-based stimulationelectrodes as a function of strain and FIG. 2D shows a cross-sectional view of the kirigami- based water powered dressing assembly 1 interfaced with an artificial deep wound with complex shape and contours.

[0059] Electrical characterization of the stimulation electrodes 22 reveals stable resistance for both electrodes with only ~14 and ~13% increase in resistance for the OE under 100% biaxial strain (ultimate elongation of —130%) and IE under ~2500% uniaxial strain (ultimate elongation of ~3300%), respectively, which is comparable to previous studies. The sharp rise in the resistance beyond these strains could be attributed to microcracks generated in the carbon layer.

[0060] Complete hydration of the separator 12 is important to ensure the battery 2 attains maximum possible capacity which is critical for delivering long durations of electrical stimulation. FIG. 3A shows the effect of the total volume of water applied on the areal capacity of the battery 2 (where the data reported in FIGS. 3A-3G are acquired by interfacing the water powered dressing assembly with a resistor (25 kilohms) to mimic average wound impedance). As shown by the figure, increasing the amount of water introduced into the battery 2 leads to an increase in the ionic conductivity of the separator 12 and a decrease in the battery’s internal resistance resulting in increasing areal capacity. The areal capacity peaks at ~4.5 mAh / cm2for ~20 l / cm2of water followed by a small decrease for batteries exposed to higher quantities of water. This decrement in the battery capacity can be attributed to the accelerated oxide formation on the Mg anode 10 leading to an increase in the battery’s internal resistance. FIG. 3B shows that the capacity of the battery is negligibly affected by the source of the water (e.g., water-based fluids: tap water, deionized water, phosphate-buffered substrate, or artificial wound fluid) and that it can be powered even using the wound exudate, thus opening the possibility for developing a dressing that harvests local wound exudate to power the electrotherapy system.

[0061] The duration of stimulation offered by the water powered dressing assembly 1 also depends on the impedance of the wound which changes as the wound heals. During testing, as shown in FIG. 7A, daily wound impedances (wound size: 1-cm diameter) were measuredin male diabetic (db / db) mice (age: 11 to 13 weeks; n = 6) widely used for studying diabetic foot ulcer, a type of chronic wound. In the subsequent experiments, the wound impedance was measured by applying a pair of carbon electrodes (same as that used for stimulation) to the wound and interfacing it with an electrochemical impedance analyzer. The impedance was observed to gradually increase from 10 to 60 kilohms from the day of injury to full recovery (by day 15), which is consistent with previous studies. Thus, in vitro characterization of water powered dressings involves connecting the stimulation electrodes across a resistor of varying resistance (to simulate wound status at different stages of healing), applying water to power the dressing, and recording the voltage across the resistor. All experiments include dressings composed of a 0.64-cm2battery connected to a central disk electrode (diameter: 2 mm) and outer ring electrode (outer diameter: 9 mm; inner diameter: 7.4 mm) designed to treat a 1-cm diameter wound unless otherwise noted. The results obtained with such stimulation electrodes 22 are applicable to the kirigami-based water powered dressing assembly as well, since the performance of the water powered dressing assembly 1 does not depend on the design of the stimulation electrodes 22.

[0062] Several prior studies show that daily treatment of electrical stimulation ranging from 15 min to 6 hours can expedite wound healing. Moreover, these studies show that a wide range of electrical stimulation parameters (amplitudes, DC / AC, and duration of stimulation) have similar effects on wound healing. Separately, previous studies reveal that a minimum electrical field strength of 100 mV / mm is necessary to support pro-healing processes and expedite wound closure. These studies indicate that even a time-varying electrical field (as is the case in water powered dressings) will promote healing as long as the field strength is greater than 100 mV / mm. For the subsequent experiments, the present disclosure therefore defines the duration of stimulation for water powered dressing assembly as the duration over which the dressing assembly offers an electrical field strength of at least 100 mV / mm.

[0063] Correspondingly, FIG. 3C shows that the duration of stimulation varies from 0.5 to greater than 7 hours depending on the wound impedance. FIG. 7B shows the reproducible performance of the water powered dressing as a function of wound impedance. The dressingassembly 1 attains an initial output voltage of 1 .5 which gradually decreases until it reaches 0 V. The maximum voltage (1.5 V) is consistent with previous reports, where stimulating voltages ranging from 1 to 6 V have been reported. Unlike previous examples of electronicsbased electrotherapy systems that produce a constant electrical field for a predetermined period of time, the exemplary water powered dressing assembly 1 generates a time-varying electrical field with a duration of stimulation dependent on the wound impedance. Certain embodiments of the water powered dressing assembly 1 can include a preprogrammed voltage regulator to stabilize its output voltage. Nevertheless, it delivers long durations of stimulation (at least ~0.5 hours) irrespective of the wound impedance, which is critical for facilitating healing.

[0064] The water powered dressing assembly 1 with a 0.4-mAh battery offers greater than 3.5-hour stimulation at temperatures up to 35°C, as shown in FIG. 3D. Investigation of the dressing’s ability to deliver electrical stimulation at extreme temperatures (<0° and >35°C) reveals that the duration of stimulation decreases only by ~1 hour for elevated temperatures (up to 45°C) due to higher rates of water evaporation. On the other hand, the supersaturated salt electrolyte remains in the liquid state at subzero temperatures, ensuring the functioning of the battery 2 even at such low temperatures. The reduced rates of evaporation at these temperatures facilitate the delivery of a substantially higher duration of stimulation. Accordingly, these results highlight the dressing’s ability to perform even under harsh conditions wherein conventional lithium and alkaline battery-powered dressings face deterioration, thus opening possibilities for offering effective wound treatments in extreme weather conditions. The rate of water evaporation at higher temperatures may be minimized by using separators having hygroscopic polymers that tightly bind water molecules. Under arid conditions (e.g., 30% relative humidity (RH)), the dressing assembly 1 offers stimulation for ~1 hour which increases to ~10.5 hours at 60% RH and then falls to ~4.5 hours at 90% RH, as shown in FIG. 3E. FIG. 7C exhibits typical evolution of voltage as a function of time under different ambient humidity levels. These results are expected as increased humidity levels reduce water evaporation while exceedingly high humidity levels accelerate Mg oxidationleading to a lower duration of stimulation and increased variability in voltage due to excessive generation of hydrogen gas bubbles at the electrode-separator interface. In various embodiments, these issues can be addressed by using Mg alloys, special electrode coatings, and / or additives to the separator 12 to reduce corrosion and hydrogen gas production. The duration of stimulation can be extended by nearly six times by preloading glycerol in the separator 12 which acts as a humectant and reduces water evaporation from the separator 12. Such modifications to the water powered dressing assembly 1 may be beneficial to its performance in low-humidity settings. While the performance of the water powered dressing assembly 1 depends on ambient temperature and humidity, the results reveal that the water powered dressing assembly 1 offers hours-long stimulation irrespective of changes in these factors.

[0065] Next, FIG. 3F shows the performance of the water powered dressing assembly 1 when subjected to external pressures in the range typically experienced by these body parts (0 to 450 kPa). For example, chronic wounds often form at the foot or sacrum, areas that experience high external pressure loads. As indicated by FIG. 3F, the duration of stimulation increases with pressure possibly due to reduced separation between the anode 10 and cathode 16 resulting in lower internal battery resistance and thus lower energy loss. Correspondingly, FIG. 3G illustrates the effect of bending stress on the dressing assembly 1 . The bending radii are selected to emulate conditions when the dressing assembly is applied to curved surfaces (e.g., toes). Atrend similarto FIG. 3F is recorded for such bending stresses. Additional testing data reveal that battery voltage decreases by only ~11 % when the dressing is subjected to a bending strain of 100% (bending radius: 0.5 mm). Results in FIGS. 3F and 3G are quite interesting as they reveal that the dressing performs robustly for several hours even when continuously subjected to extremely high pressures (450 kPa) and bending stress (bending radius: 0.5 mm), unlike the expected performance from wireless electronics-based devices.

[0001] The effect of wound size and battery capacity is investigated by applying the water powered dressing assembly 1 o a phantom wound (chicken breast). FIG. 3H shows that theduration of stimulation reduces by only 30% as the wound size increases fourfold for the three sizes of the phantom wounds studied in this experiment. FIG. 7D exhibits representative voltage versus duration of stimulation plots, while FIG. 7E and FIG. 7F illustrate the Nyquist plots and impedances for the three sizes of the phantom wounds studied in this experiment. Here, the battery (capacity: 0.4 mAh; area: 0.64 cm2) and the central disk stimulation electrode (diameter: 2 mm) are maintained for all wound sizes, while the diameter of the ring electrode is matched with that of the wound. Results obtained while investigating the effect of battery size (and hence its capacity) on stimulation performance appear in Fig. 3I. A clear increase in duration of stimulation for a fixed wound size (diameter: 1 cm) is recorded as a function of battery area and capacity, demonstrating the ability of water powered dressing assemblies of the present disclosure to offer uninterrupted, multiday electrotherapy treatment. Furthermore, the dry nature of the batteries offers long shelf life; when tested after 7 months from the time of fabrication, the batteries show an average discharge capacity of 0.39 mAh which represents a 2.5% reduction in the capacity when compared to that of a freshly fabricated battery (0.4 mAh). The small decrease in the battery capacity can be attributed to the formation of an oxide layer on the Mg surface during storage and can be minimized with tight vacuum-sealed packaging. The systematic experiments described in FIGS. 3A-3I clearly demonstrate the utility of water powered dressing assemblies to offer reliable, wound-specific electrotherapy treatment under conditions simulating real-life scenarios in a completely untethered and unencumbered fashion.

[0002] For the kirigami-based water powered dressing assembly, a 3D finite element analysis (FEA)-based wound-on-skin model is used to allow spatial simulation of the electric field distribution around the wound site. The disk electrode 22A is positioned on the adipose tissue, while the OE 22B is placed on the dermis, replicating in vivo conditions. This setup allows the electric field to permeate the dermis and fortify the endogenous electric field, facilitating wound healing. The directional electric field, originating from the spiral and serpentine shaped electrical stimulation electrodes, creates an inward DC flow from the healthy site toward the center of the wound, mimicking naturally occurring endogenous woundcurrents. FIG. 4A shows the spatial potential distribution for a wound (diameter: 1 cm). A peak electric field strength of ~530 mV / mm is produced near the central disk electrode 22A which attenuates to ~100 mV / mm near the inner edge of the ring electrode 22B, as shown in FIG. 4B. Such electrical field strengths are recognized as sufficient to induce migration of human keratinocyte cells and modulate cell behavior, including cell-cell junctions, cell division orientation, and cell migration trajectories (galvanostatic or electrotactic) necessary to promote wound closure. Moreover, this distribution aligns with the effective DC electric field intensity known to enhance dorsal root ganglion neurite outgrowth, stimulate neurotrophic factors released by Schwann cells, and induce PC12 cell differentiation, as reported in previous studies. The electric field strength and distribution are influenced by the output voltage of the battery (as shown in FIG. 4C), wound shape (as shown in FIG. 4D), and wound size (as shown in FIG. 4E). The water powered dressing assembly can be easily adapted to treat deep wounds with narrow openings (e.g., puncture wounds). For example, the two stimulation electrodes 22 may be mounted on either side of the wound to generate an electrical field across the wound. This type of configuration for placing stimulation electrodes is widely used in several clinical settings. In the case of deep wounds, the electric field substantially diminishes when electrodes are positioned at the center and the periphery of the wound. However, kirigami-based electrodes of the present disclosure may offer enhanced spatial electrical field strength and distribution while enabling improved conformal attachment to treat such wounds.

[0003] Before testing in vivo, the biocompatibility of an exemplary water powered dressing assembly 1 was evaluated using NIH / 3T3 mouse fibroblasts cultured with standard media or standard media conditioned with the dressing for 72 hours. Quantification of live / dead staining at 72 hours reveals no substantial difference between the two groups, confirming the dressing’s biocompatibility. Prior studies reveal that electrical stimulation accelerates migration in many cell types, including keratinocytes, fibroblasts, and macrophages. However, when tested in an in vitro scratch assay, water powered dressing assembly showed the migration of NIH / 3T3 mouse fibroblasts. Studies further show that electrical stimulation cancause NIH / 3T3 mouse fibroblasts to contract and develop larger focal adhesions. Separately, studies reveal that focal adhesion size causes a decrease in cell migration speed above a certain threshold. From microscopic images of cells after receiving electrical stimulation for 24 hours via the water powered dressing assembly 1 (control: no electrical stimulation), a clear contraction in cell morphology is observed in cells that received electrical stimulation which may correlate with an increase in focal adhesion size and thus decreased migration speed. The exact mechanism by which electrical stimulation accelerates healing is still being actively investigated, and in vitro testing does not wholly recreate the in vivo environment. Given the pro-healing results in vivo, it is likely that the effects of electrical stimulation are due to a combination of cellular processes, such as an up-regulation of angiogenic factors and other growth factors that cannot be captured in a vitro scratch assay.

[0004] Correspondingly, investigation of an exemplary water powered dressing assembly in a wound closure diabetic (db / db) mouse model reveals its ability to accelerate wound closure. Wounds in these animals heal through a combination of epithelialization and contraction and several studies leverage this model to study wound healing as it recapitulates wound repair impairment of human diabetic ulcers. Experiments involve creating a fullthickness excisional dermal wound (diameter: 1 cm) on the dorsum of each mouse followed by covering it with the water powered dressing assembly 1 (“Stim”; n = 8), and then, applying water to the battery initiates electrotherapy. The animal studies involve the water powered dressing assembly design of FIGS. 1 B-1C, because of the simple, circular, and shallow nature of the wounds. FIG. 5A shows infrared (IR) images of a mouse before and after activation of the water powered dressing revealing that the electrical field generated by the dressing does not cause any measurable temperature variations in the adjacent tissue which is important to avoid any thermally induced damage to the wound. Daily replacement and activation of the dressing over a period of 11 days after injury ensure sustained electrotherapy treatment. Such diurnal treatment is similar to the recommended treatment for becaplermin (U.S. Food and Drug Administration-approved product for diabetic foot ulcers). Moreover, exchanging the water powered dressing assembly is fast, taking <1 min, which is comparable to replacingconventional dressings. Additional studies involving animals treated with inactive dressing (“Sham”; no activation of the battery with water; n = 8) and occlusive dressing (“Control”; Tegaderm bandage-only; n = 7) enable deconvolution of the effects of electrotherapy on wound closure from those due to material and geometric features of the stimulation electrodes and Tegaderm.

[0005] Representative images of wounds in three randomly selected mice each belonging to the three treatment groups were captured on days 0, 3, 6, and 11 after injury appear, with the three treatment groups comprising a Stim group wearing the activated water powered dressing assembly, the Sham group wearing the non-activated water powered dressing assembly, and the Control group wearing an occlusive dressing. Accordingly, FIG. 5B provides the longitudinal evolution of wound size for all animals in each group. Investigation of these results reveals that the Stim group displays substantially increased rates of wound closure beginning day 4 after injury compared to other groups. More specifically by day 1 1 , 75% of wounds in the Stim group fully closed compared to 12.5 and 0% in the Sham and Control groups, respectively. Extended assessment beyond day 1 1 reveals that 88% of wounds in the Stim group completely close by day 13, while only 50 and 28% of wounds in the Sham and Control groups close at this time point. Overall, the animals that receive water powered dressing treatment show 1 .23 times faster closure of wounds by day 11 as compared to other groups, as depicted in FIG. 5C. The wound closure rate offered by these low-cost, easy-to-use dressings is 1.2 times faster than becaplermin and is comparable to wound closure rates reported in the existing literature that rely on expensive biologies and / or external electronics. The higher wound closure rates observed in the Sham group compared to the Control group can possibly be attributed to the stimulation electrodes serving as anchoring structures for migrating cells to adhere to, similar to the role many scaffolds play in wound healing. FIG. 5D exhibits representative movement profiles and total average distance traveled, respectively, by mice in the three groups, and these results reveal that the water powered device assembly 1 does not impede animal mobility in any way owing to its conformal, lightweight nature.

[0006] FIGS. 6A-6F involve histological analysis of the wounds which further confirm the ability of an exemplary water powered dressing assembly 1 to stimulate wound healing beyond simply accelerating closure. Hematoxylin and eosin (H&E) staining (as shown in FIG. 6A) and Masson’s trichrome staining (MTS) (as shown in FIG. 6B) performed on tissue samples collected 13 days after injury reveal that the epidermis is 43 and 72% thicker (P < 0.0001) in the Stim group compared with the Sham and Control groups, respectively, with no notable difference between the epidermal thickness in the Sham and Control groups (as shown in FIG. 6C). In FIG. 6D, analysis affirms increased collagen intensity in the Stim group as compared with the Sham and Control groups (P < 0.01).

[0007] Epidermal regeneration is a central aspect of re-epithelialization, as the healthy epidermis acts as a barrier against pathogens and prevents fluid loss. Collagen is a main component of the extracellular matrix and contributes to the elasticity and tensile strength of skin. As wounds heal, collagen density, fiber size, and orientation all change to return the skin to a healthy state. The increased rate of wound closure, thicker epidermis, and increased collagen intensity seen in the Stim group highlight the device’s ability to promote effective healing beyond simply accelerating wound closure.

[0008] Immunohistochemical staining results further support improvements in healing for wounds treated with water powered dressing assemblies. Previous studies in human and animal models of wound healing show the ability of electrical stimulation to promote angiogenesis, which facilitates the transport of oxygen and nutrients to the wound site. CD31 (also known as PECAM-1) is a commonly used biomarker to identify blood vessels. CD31 staining is notably higher (P < 0.05) for the Stim group than for the Sham or Control groups, as shown in FIG. 6E. In addition, as a wound progresses through the phases of wound healing, from inflammation to proliferation and remodeling, pro-inflammatory M1 macrophages are replaced with M2 macrophages that promote cell proliferation and tissue remodeling. Staining was performed for F4 / 80 (a pan-macrophage marker) and inducible nitric oxide synthase (iNOS; an M1 marker) or CD206 (an M2 marker). As shown in FIG. 6F, a substantially (P = 0.007) lower ratio of M1 to M2 markers is seen in the Stim group compared to the Sham andControl groups, but no notable difference between the Sham and Control groups is observed. The low M1 / M2 macrophage ratio suggests that the exemplary water powered dressing assembly 1 itself does not trigger an inflammatory response and that electrotherapy offered by water powered dressings reduces inflammation. Chronic wounds often fail to exit the inflammatory phase; as such, the anti-inflammatory characteristics of water powered dressings further render it useful for chronic wound treatment.

[0009] Reported examples of electrical stimulation-based devices offer increases in CD31 staining, epidermal thickness, collagen intensity, and a shift in the ratio of M1 to M2 macrophages. Likewise, other wound-healing technologies show increased CD31 expression, epidermal thickness, and collagen expression. The user-friendly water powered dressing assembly offers similar pro-healing capabilities at a fraction of the cost compared to the abovereferenced technologies, making it preferable for treating wounds.

[0010] An exemplary fabrication process of a water powered dressing assembly for one embodiment, among others, of the present disclosure involves fabrication of the water powered battery by screen printing of carbon ink (E3178, Ercon, USA) on a 75-pm-thick sheet of polyimide (PI; Argon Inc., CA, USA), subsequent baking at 120°C for 20 minutes, and CO2laser (Fusion Edge, Epilog, USA) patterning (speed: 10%; power: 3%; frequency: 7%) formed the current collectors for the anode 10 and cathode 16. In various embodiments, anode fabrication involves applying a thin layer of carbon ink onto the laser patterned Pl / carbon- based anode current collector 8 followed by attaching a 100-pm-thick magnesium (Alibaba) ring to it, and baking at 120°C for 10 min. The cathode fabrication process comprised screenprinting Ag / AgCI ink (E2414, Ercon, USA) onto the cathode current collector 14 and baking at 80° and 120°C each for 20 min. A separator 12 with NaCI salt loadings of 2.4 mg / cm2 may then be fabricated. Insertion of the separator 12 between the anode 10 and cathode 16 and adhesive packaging 6, 18 of the battery assembly 1 may be performed with a laser-patterned waterproof tape (Gorilla Glue Inc., OH).

[0011] Fabrication of the stimulation electrodes 22 can involve laser patterning of a PI sheet, followed by the application of carbon ink on both sides and subsequent baking at 120°Cfor 10 min. Next, encapsulation of interconnects with epoxy resin (Epoxy Marine, Loctite, Henkel Corp., OH) can be performed to define the active electrode areas and contact pads.

[0012] For assembly of the water powered dressing assembly, an exemplary process can begin with punching two 1 .5-mm-diameter holes in a 4.5 cm x 4.5 cm occlusive bandage (Tegaderm, 3M, USA) which can serve as apertures for connecting stimulation electrodes 22 with the battery 2. Next, mounting the battery 2 on top (non-adhesive side) and the stimulation electrode pair 22 on the bottom (adhesive side) can be performed of the occlusive bandage dressing 22 followed by bonding the IE to the contact pad of the anode (Mg electrode) and OE to the contact pad of the cathode (Ag / AgCI electrode) using carbon ink to complete the assembly process for the water powered dressing assembly 1 .

[0013] In summary, the present disclosure provides a low-cost, electronics-free, water powered dressing for delivering electrotherapy that accelerates wound healing at rates comparable to those offered by expensive therapeutics. The present disclosure uses an Mg- Ag / AgCI battery with a cellulose separator. The addition of a small amount of water activates the battery, which provides on-demand electrical stimulation for several hours even when the system is subjected to extreme strains. Benchtop studies further show that tailoring the battery size enables continuous electrical stimulation over several days thus circumventing the need for daily replacement of the dressing. This device is lightweight and highly flexible and can be easily integrated into existing bandages. The manufacturing versatility enables the realization of electrotherapy dressings with advanced morphable stimulation electrodes (e g., 3D morphable kirigami-based stimulating electrodes) that can be readily configured to treat wounds of different shapes, sizes, and contours. In addition, an exemplary water powered dressing assembly requires no external electronics or specialized knowledge, rendering it ideal for point-of-care treatment.

[0014] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methodsbelong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0015] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope of the present disclosure.

[0016] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0017] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) without departing substantially from the spiritand principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

CLAIMSTherefore, at least the following is claimed:1 . An activatable dressing, comprising: a biocompatible battery including: a magnesium-based (Mg) anode; a silver / silver chloride-based (Ag / AgCI) cathode; and a sodium chloride (NaCI) impregnated separator disposed between the anode and cathode in a dry state, the separator comprising an inlet pad configured for introduction of a water-based fluid to the separator for activation of the battery; and stimulation electrodes electrically coupled to the Mg anode and Ag / AgCI cathode.

2. The activatable dressing of claim 1 , wherein the battery is packaged within a film.

3. The activatable dressing of claim 2, wherein the film is a polyethylene terephthalate (PET) film.

4. The activatable dressing of claim 1 , wherein the separator comprises a check pad configured to provide an indication of hydration of the separator.

5. The activatable dressing of claim 4, wherein the check pad comprises a dry water- soluble dye loaded into a storage membrane.

6. The activatable dressing of claim 1 , wherein the separator comprises a cellulose- based membrane.

7. The activatable dressing of claim 1 , wherein the battery is ring shaped allowing visual access to the wound.

8. The activatable dressing of claim 7, wherein the stimulation electrodes comprise an outer stimulation electrode that is ring shaped.

9. The activatable dressing of claim 1 , wherein the battery is on a non-adhesive side of a bandage and the stimulation electrodes are on an adhesive side of a bandage.

10. The activatable dressing of claim 1 , wherein the stimulation electrodes comprise an spiral shaped inner electrode and a serpentine shaped outer electrode in a ring pattern.11 . The activatable dressing of claim 1 , wherein the stimulation electrodes comprise a central disk electrode and an outer ring electrode.

12. The activatable dressing of claim 1 , wherein the separator is loaded with glycerol.

13. A method of treating a wound comprising: applying to the wound the activatable dressing of claims 1 to 11 .

14. The method of claim 13, further comprising: activating the activatable dressing by droplets of a water to the inlet pad.

15. The method of claim 13, wherein the activatable dressing is activated from wound fluid that is contact with the inlet pad.

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