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Abstract—We report on an innovative, fabric-based
conformable, and easily fabricated electroceutical wound dressing
that inhibits bacterial biofilm infections and shows significant
promise for healing chronic wounds. Cyclic voltammetry
demonstrates the ability of the electroceutical to produce reactive
oxygen species, primarily HOCl that is responsible for bacterial
inhibition. In vitro investigation with the lawn biofilm grown on a
soft tissue mimic assay shows the efficacy of the dressing against
both gram-positive and gram-negative bacteria in the biofilm
form. In vivo, the printed electroceutical dressing was utilized as
an intervention treatment for a canine subject with a non-healing
wound due to a year-long persistent polymicrobial infection. The
clinical case study with the canine subject exhibited the
applicability in a clinical setting with the results showing infection
inhibition within 11 days of initial treatment. This printed
electroceutical dressing was integrated with a Bluetooth® enabled
circuit allowing remote monitoring of the current flow within the
wound bed. The potential to monitor wounds remotely in real-time
with a Bluetooth® enabled circuit proposes a new physical
biomarker for management of infected, chronic wounds.
Index Terms — Electroceuticals, Dressing, Wound, Chronic,
Biofilm, Treatment
I. INTRODUCTION
HRONIC or non-healing wounds present a major healthcare
burden on the US with an estimated $25 billion in cost for
nearly 6.5 million human patients per year [1]. While there is
limited data available for animal populations, chronic wound
management in domestic and livestock animals are estimated to
cost an additional $1.1 billion by 2021 with an expected 7%
increase per year [2]. In human populations, 60% of chronic
wounds have biofilm infections making them recalcitrant to
state-of-art treatment, including antibiotics [3, 4, 5]. In the
biofilm form, bacteria form ‘protective shields’ comprising of
extracellular polymeric substances (EPS) generated by the
bacterial cells which subsequently encapsulate the bacteria [6].
The EPS matrix promotes adhesion to both the wound surface
and between bacterial cells, forming a 3D structure that protects
the bacteria from antimicrobials and host immune defenses [6].
Submitted for review on May 3, 2020. Partial personnel support is
acknowledged from the National Institute of Health for grants R01HL141941
(SP) and R01GM124436 (PS).
1
Rachel Heald, Molly Bennett, Vish V. Subramaniam, Varun Lochab,
Prashanth Mohana Sundaram, J. D. West, and *Shaurya Prakash
(prakash.31@osu.edu) are all with the Department of Mechanical and
Aerospace Engineering, The Ohio State University, Columbus, OH, USA.
2
Devendra Dusane was previously with the Department of Microbial
Infection and Immunity, The Ohio State University and is now at the
Nationwide Children’s Hospital, Columbus, OH, USA.
The EPS matrix also alters the nutrient and chemical gradients
within the wound environment, providing additional defenses
that make eradicating biofilms a difficult challenge, even for the
current state-of-art treatment methods [7,8].
Recently, electroceutical dressings that apply potential
gradients to induce electric fields and may permit electrical
current flow have become an emerging method for inhibition of
bacterial biofilms [9, 10]. However, the commercially available
electroceutical dressings lack a mechanistic understanding of
the treatment outcomes. Commercially available and approved
by the food and drug administration (FDA), passive dressings
such as Arthrex and Procellera apply electric fields to the
wound but have no current flow [9]. These dressings use the
silver (Ag)-zinc (Zn) redox couple organized as electrically
discontinuous dots. The redox couple is activated when a
conducting fluid (e.g., saline or wound fluid) contacts the metal
pattern on a polyester fabric to generate a measurable open
circuit potential. In the presence of an adequate redox potential,
reactive oxygen species (ROS) such as hydrogen peroxide
(H
2
O
2
) may be generated and act as antimicrobials for shallow
or thin infection layers (< 0.25 mm) that may occur in wound
beds [9].
On the other hand, direct current (DC) has proven to have
inhibitory action against planktonic bacteria in systems that are
both static and flowing [10, 11]. There have also been displays
of the treatment efficacy of DC against bacterial biofilms of
Pseudomonas aeruginosa [16] and Staphylococcus epidermidis
[12], but many of these past studies used liquid media to study
biofilms that is not representative of wounds, containing a soft
tissue bed over which the biofilm forms. Our lawn biofilm
grown on a soft tissue mimic assay provided a significant
methodological advance in developing tools for analyzing the
mechanistic actions of our printed electroceutical dressing
(PED) [13]. In the present study we report the bacterial
inhibitory mechanisms and efficacy of our device both in vitro
and in vivo with a new physical biomarker proposed with the
potential to integrate real-time, remote wound monitoring.
3
Sarah Salyer is with the Department of Veterinary Clinical Sciences, The
Ohio State University, Columbus, OH, USA.
4
Paul Stoodley is with the Department of Microbial Infection and Immunity
and the Department of Orthopedics, The Ohio State University, Columbus, OH,
USA. He is also affiliated with the National Centre for Advanced Tribiology at
Southampton and the National Biofilm Innovation Centre, Dept. Mechanical
Engineering, University of Southampton, UK.
Printed Electroceutical Dressings for the Inhibition
of Biofilms and Treatment of Chronic Wounds
Rachel Heald
1
, Molly Bennett
1
, Vish V. Subramaniam
1
, Devendra Dusane
2
, Varun Lochab
1
, Prashanth
Mohana Sundaram
1
, Sarah Salyer
3
, J.D. West
1
, Paul Stoodley
4
, and Shaurya Prakash
1
*
C
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II. FABRICATION
Our novel, printed electroceutical dressing was made by a
facile silk-screen printing process for use on flexible substrates
like fabric. The pattern screen was fabricated by coating a 196
amber polymer mesh with a photoactive polymer (Speedball
Diazo Photo Emulsion). The screen was flood exposed using a
200 W clear incandescent bulb for 70 minutes showing the
relatively simple infrastructure needed for PED fabrication. The
photo-emulsion was then rinsed using regular, laboratory-grade
filtered water, revealing the visible PED pattern (Fig. 1A). The
nominal dimensions of the anode and cathode were 5 mm and
3 mm width with a 1 mm separation arranged in an
interdigitated pattern to maximize electrode coverage over the
7.5 cm x 7.5 cm substrate. Next, the PED pattern was screen-
printed by using a medically compatible Ag/AgCl ink (Creative
Materials #113-09) onto habotai silk substrates (Fig. 1B). Silk-
based materials are commonly used for wound dressings [14]
and the habotai weave provides a breathable, lightweight fabric
with accepted biocompatibility [15]. The screen-printing
process is fast and enables high throughput [16] but also leads
to differences in nominal vs. actual electrode sizes. The
absorption of the ink onto a porous substrate combined with
dispersion along the substrate lead to measured anode and
cathode widths to be 5.5 mm and 4.0 mm (Fig. 1C) with an
uneven (< 1 mm) spacing between the electrodes. Each
electrode set is manually checked with a handheld multimeter
before use to confirm that the entire electrical pattern is
continuous without electrical shorts. The printed electrodes
were then epoxied to the battery pack with two 3V batteries in
series and a current limiting 1 kΩ resistor (Fig 1D). Medical
tape was then used to cover the circuit connections and also
provide a fluid and electrical isolation layer leaving the battery
pack switch and the electrodes accessible for use. The
microstructure of the printed Ag/AgCl ink is seen in the
scanning electron microscopy (SEM) images in figures 1E and
1F.
III. RESULTS
A. Soft Tissue Mimic Assay
In vitro investigation displays biofilm inhibition as a result
of PED operation using our lawn biofilm soft tissue assay [13].
For direct analysis of the antimicrobial activity of the PED, two
geometrically simplified electrodes (3 mm wide x 8 cm long,
separated by 3 cm) were embedded within agar on which 24
Fig. 2
. Two geometrically simplified silver electrodes
depicting the anodic and
cathodic PED
response are e
mbedded in an agar gel with a 24 h PA lawn. The
electrodes are connected to a 6V battery and
1 kΩ ballast resistor for 24 hr.
Fig. 1. (a) The screen with developed pattern for screen printing
the PED electrodes. (b) The Ag/AgCl ink was spread over the
screen mesh and manually printed using a squeegee. (c) The
resultant printed design on the silk substrate with actual
dimensions as marked due to ink spreading on porous substrate.
(d) The printed electrodes were connected to the battery pack
with two 3V batteries in series and a current limiting resistor. The
open electrical connections were isolated by medical tape.
Overlaid is a schematic for the working circuit diagram. (e) An
scanning electron microscopy (SEM) image of the Ag/AgCl ink
printed onto the silk fabric. (f) SEM image of the fabric-electrode
cross-section shows the ink is absorbed within the fabric with a
~50 μm ink layer on the silk fabric.
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hour biofilm lawns of bioluminescent strains of Pseudomonas
aeruginosa Xen-41 (PA) or Staphylococcus aureus SAP231
(SA) (two common pathogens found in infected wounds
representing both gram-negative and gram-positive bacteria
respectively) and connected to a PED equivalent circuit. The
6V battery with a 1 kΩ resistor had an active connection with
the electrodes embedded in the agar for 24 h (Fig 2). In Vivo
Imaging System (IVIS) images were used to evaluate the
inactivation of the biofilm bacteria over the agar gel in which
the electrodes were embedded. The bioluminescent biofilms of
Pseudomonas aeruginosa (Fig. 3A) and Staphylococcus aureus
(Fig. 3D) are indicated by red within the IVIS images where the
bacteria are present and metabolically active. The blue (and
other darker colors) indicate the absence of bacteria or
metabolically inactive bacteria. For both biofilm species, the
anode and cathode displayed different antimicrobial activity,
with bacterial inhibition occurring primarily over the anode
(Fig 3). Electrochemical reactions caused by the powered
electrodes produce ROS, specifically HOCl [11] that is
responsible for the damage to the bacterial cells over the anode.
SEM images show the EPS present in the bacteria on areas
away from the electrodes (Fig 3B) while the EPS is absent the
regions over the anode (Fig 3C), indicating the breakdown of
the biofilm. The bacteria over the cathode displayed less
inhibition than over the anode. While the area of inhibition for
Staphylococcus aureus appears fairly large, its inhibitory
effects along the length of the cathode towards the center of the
petri dish remain limited in comparison to the anode. The
bacterial inhibition across both biofilms indicates the efficacy
of the PED against both gram-positive and gram-negative
bacteria with the specific differences in the treatment response
being the subject of continued research.
B. Real-Time Wound Monitoring
State of art clinical practice requires frequent visual
inspections of the wound in order to determine healing
outcomes including estimating infection clearance. The visual
inspections are generally carried out by trained medical
professionals adding significant work burden to the practice of
wound management, especially for chronic wounds. In this
work, we report on a potentially new way to assist clinicians by
allowing remote monitoring of the wound site. Since the PED
is an electroceutical dressing with active electrodes, the PED
provides an opportunity to reduce the need for visual
inspections and provide quantitative wound healing metrics.
Therefore, we propose a new physical biomarker for healing.
An initial in vitro test with the soft tissue assay demonstrated
Bluetooth® integration of the PED with an Adafruit Feather
Fig. 4
.
The capability of the PED to be connected to a Bluetooth circuit for
wound monitoring. The PED was embedded in the agar gel to measure the
current flow via the Bluetooth circuit with the data read out displayed on the
electronic device
(cellular phone).
Fig. 5
. The Bluetooth circuit and picoammeter
current measurements recorded
over time while the PED was embedded into the agar gel.
Fig. 3
. (a) The IVIS image of post-
24 h treatment of the powered electrodes to
PA
24 hr lawn. Red represents
metabolically active/live and blue/black
represents
metabolically inactive or killed
bacteria. (b) The SEM image of the
PA biofilm on the area of the gel not directly above the electrodes
acts as control
for untreated area
. (c) The SEM image of the PA biof
ilm on an area directly
above the anode that was powered for 24 hr. The breakdown of EPS is visible.
(d) The IVIS image of post
-24 h treatment of the powered electrodes to SA 24
h
lawn.
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32u4 Bluefruit that records and transmits up to 20 ft real-time
current monitoring (Fig 4). The internal resistance of the
Bluetooth® enabled monitoring circuit is lower than the
resistance of the agar assay during a direct measurement leading
to a higher recorded current than when measured directly
(Fig 5) using an ammeter due to the direct connections with
shorter, silver wires for the bluetooth enabled circuit. However,
the trend over time is the same and may be sufficient for real-
time wound monitoring.
C. Cyclic Voltammetry
To further analyze the electrochemical reactions and ROS
produced by the PED, cyclic voltammetry (CV) of the PED
within the soft tissue agar assay was performed (Fig 6). The
electric potential sweep started at -2 V and switched sweeping
direction upon reaching +2 V at a scan rate of 200 mV/s. It has
been previously reported that at an applied electrode potential
of 1.38 V, HOCl may be generated as the primary ROS [11]. At
an applied potential of -0.6 V, H
2
O
2
is produced [15]. The flow
of current present at these two potentials with our system shows
that by altering the electrode potential, we can control the ROS
produced.
The maximum and minimum current peaks present in
Figure 6 are asymmetrical in their magnitudes and applied
potential with respect to the zero-applied potential. This
asymmetric shape to the CV profile indicates that the
electroceutical treatment of biofilms grown on agar is
electrochemically irreversible i.e., non-Nernstian [18]. The
irreversibility indicates that the concentration of species needed
to produce ROS are the limiting steps for the electrochemical
reaction rates and not the transport of the species to and from
the electrodes. Due to this non-Nernstian response, cyclic
voltammetry is not an ideal technique to quantitatively evaluate
HOCl concentrations even though the actively flowing current
measured indicates the production of the ROS at their
respective potentials [18].
D. Canine Case Study
In vivo, the PED was used in a clinical case study as an
experimental treatment for the therapeutic intervention on a
non-healing and chronically infected wound. The wound was
on the left front leg of a canine subject. Originally, the wound
presented with a polymicrobial infection that was treated with
open wound management for fifteen days followed by a skin
graft and a negative pressure dressing as part of the state-of-art
wound care at Ohio State’s small animal clinic in the College
of Veterinary Medicine. Significant topical antibiotics were
used to treat a persistent infection with the overall treatment and
wound management lasting over a year. The wound was found
to be non-healing with a continued polymicrobial infection. A
tissue culture from punch biopsy revealed the infection
contained primarily Staphylococcus pseudintermedius and
Streptococcus canis. At this stage, as an experimental
treatment, the PED (Fig. 1) was connected to a 6V battery with
a ballast resistor limiting the peak current to 0.6 mA. After
11 days of PED treatment, a punch biopsy revealed no
pathologically detected wound infection. The canine was
released to in-home care with no additional treatment other than
standard open wound care recommended with no topical or
systemic antimicrobials. The next clinical inspection was
possible (since the subject’s home city was hours away) only
67 days later when the canine subject was brought back to the
clinic for visual wound inspection showing complete healing of
the wound. (Fig 7).
IV. D
ISCUSSION
The in vitro investigations of the PED reveal bacterial
inhibition to be primarily over the anode for both gram-positive
and gram-negative bacteria. This localized inhibition motivates
the design of the electrodes to favor a large anode (Fig. 1). The
positive potential held at the anode favors the production of
HOCl [11] responsible for the bacterial clearance displayed in
both Staphylococcus aureus and Pseudomonas aeruginosa
biofilms [13]. The limited bacterial inhibition observed at the
cathode may be due to the antimicrobial action of H
2
O
2
, a ROS
that is favored at negative potentials [17], and also likely to be
produced within our system. H
2
O
2
has also been shown to be an
Fig. 7 Day
0, before the PED was applied with the polymicrobial infection.
Day 67, following the beginning of PED treatment with a fully closed wound.
Fig. 6
.
Cyclic voltammogram of the PED embedded in the soft tissue agar gel
to evaluate the products of the electrochemical products. The cycle had a
starting potential of
-2V and a switching potenti
al of +2V at a scan rate of
200
mV/s.
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effective agent against Pseudomonas aeruginosa [19].
However, Staphylococcus aureus recovers from the initial
oxidative stress that is induced by H
2
O
2
and the cells continue
to grow as if they were untreated [20].
Persistent infection creates clinical complications in wound
healing. For example, the polymicrobial infection present in a
canine wound was not removed to allow natural healing
processes to induce wound closure despite state-of-art wound
care through open wound management, topical and systemic
antimicrobial medications, a skin graft, and a negative pressure
dressing. Following initial treatment and with mounting
veterinary care costs, the canine was released to in-home care
with the recommendation of open wound management.
However, the presence of continued infection opposed the
effectiveness of these treatments, evident in the non-healing
state of the wound one year later. As noted in the results section,
a punch biopsy revealed continuation of the polymicrobial
infection with gram positive bacteria Staphylococcus
pseudintermedius and Streptococcus canis present. The PED,
which now is known to be safe for use in humans [21],
demonstrated efficacy in removing persistent infection and
leading to complete wound closure presents a new clinically
viable method for infected, chronic wound management.
In a new finding, we developed and reported on the use of
electrical current as a potential parameter to monitor wound
healing. Dry, unbroken skin behaves as an insulator to DC and
therefore for a 6V applied potential leads to a near-zero
recorded current [15]. However, as the wound occurs and the
PED is in contact with the wound bed and biological fluids, a
measurable electric current is likely to occur [15]. The
integration of Bluetooth-enabled current monitoring circuit
with the PED allows additional wound evaluation methods to
be incorporated. Such a method may be complementary to
measurements of trans-epidermal water loss (TEWL) in
quantifying the barrier function of healed skin. TEWL is a
quantitative evaluation of wound closure and the restoration of
skin barrier function [22]. In our initial in vitro tests, we were
able to measure current flow in the gel-plate representative of
tissue up to 20 feet away from the gel-plate. The current profile
was recorded on a mobile phone app and is then easily
transmitted to clinicians. The Adafruit Feather 32u4 Bluefruit
circuit requires the addition of a resistor and battery to transmit
current/voltage data to the mobile device to which it’s paired.
Given such components presently compose the circuit of the
PED, Bluetooth monitoring of the wound is a viable option. If
such a technology can be developed and validated for clinical
use, the use of a simple physical parameter based on direct
measurement of electrical current within the wound bed can be
an easily monitored parameter for state of wound healing
without active visual inspections by trained medical
professionals. Clearly, such validation is beyond the scope of
this paper and is part of our on-going efforts.
V. CONCLUSION
We report on a novel, printed electroceutical dressing that
inhibits biofilms and has a potential to provide physical metrics
for real-time wound monitoring. Screen printing Ag/AgCl ink
onto silk substrates generates a fast and inexpensive fabrication
process for high throughput. Through the connection of a 6V
battery and current limiting resistor, the PED showed efficacy
in the soft tissue biofilm assay due to the production of ROS.
HOCl is the main contributor to the antimicrobial effect we
observe primarily over the anode. The PED was used to treat a
chronic wound in vivo in a canine subject.
A
CKNOWLEDGMENT
We thank The Ohio State University Public Health
Preparedness for Infectious Disease (PHPID) Transdisciplinary
Team Grant, Departments of Microbial Infection and
Immunity, Mechanical and Aerospace Engineering. We
acknowledge supports from the staff at campus microscopy and
imaging facility (CMIF) this facility is supported in part by
grant P30 CA016058, National Cancer Institute, Bethesda, MD,
and center for electron microscopy and analysis (CEMAS) at
The Ohio State University for assistance in various imaging and
experimental aspects of this research effort.
R
EFERENCES
[1] C. K. Sen, G. M. Gordillo, S. Roy, R. Kirsner, L.
Lambert, T. K. Hunt, F. Gottrup, G. C. Gurtner, and M.
T. Longaker, "Human skin wounds: a major and
snowballing threat to public health and the economy."
Wound Repair and Regeneration. vol. 17, no. 6, pp 763-
771, 2009.
[2] https://www.marketsandmarkets.com/Market-
reports/animal-wound-care-market-253831778.html
[3] G. A. James, E. Swogger, R. Wolcott, E. Pulcini, P.
Secor, J. Sestrich, J.W. Costeron, and P. S. Stewart,
"Biofilms in chronic wounds." Wound Repair and
Regeneration. vol. 16, no. 1, pp. 37-44, 2008.
[4] M. C. Ammons, “Anti-biofilm strategies and the need for
innovations in wound care.” Recent Patents on Anti-
Infective Drug Discovery. vol. 5, no. 1, pp. 10-17, 2010.
[5] C. Attinger and R. Wolcott, “Clinically addressing
biofilm in chronic wounds.” Advances in Wound Care.
vol. 1, no. 3, pp. 127-132, 2012.
[7] H. Koo, R. N. Allan, R. P. Howlin, P. Stoodley, and L.
Hall-Stoodley, “Targeting microbial biofilms: current and
prospective therapeutic strategies.” Nat Rev Microbiol.
vol. 15, no. 12, pp. 740–755, 2017.
[8] D. Lebeaux, J. M. Ghigo, and C. Beloin, “Biofilm related
infections: bridging the gap between clinical management
and fundamental aspects of recalcitrance toward
antibiotic.” Microbiology and Molecular Biology
Reviews. vol. 78, no. 3, pp. 510-543, 2014.
[9] J. Banerjee, P. D. Ghatak, S. Roy, S. Khanna, E. K.
Sequin, K. Bellman, B. C. Dickinson, P. Suri, V. V.
Subramaniam, C. J. Chang, and C. K. Sen, “Improvement
of Human Keratinocyte Migration by a Redox Active