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The University of Manchester Research
Silver Oxysalts Promote Cutaneous Wound HealingIndependent of InfectionDOI:10.1111/wrr.12627
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Citation for published version (APA):Thomason, H., Lovett, J. M., Spina, C. J., Stephenson, C., Mcbain, A., & Hardman, M. J. (2018). Silver OxysaltsPromote Cutaneous Wound Healing Independent of Infection. Wound Repair and Regeneration, 26(2), 144-152.https://doi.org/10.1111/wrr.12627
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Silver Oxysalts Promote Cutaneous Wound Healing Independent of Infection
Helen A. Thomason (Ph.D.)1,2*, Jodie M. Lovett (B.Sc.)1, Carla J. Spina (Ph.D.)3, Christian
Stephenson(B.Sc.)1, Andrew J. McBain (Ph.D.)2*, Matthew J. Hardman (Ph.D.)4.
1 Crawford Healthcare Ltd, King Edward Court, King Edward Road, Knutsford, Cheshire,
WA16 0BE, United Kingdom.
2 Faculty of Biology, Medicine and Health, Stopford Building, The University of Manchester,
Oxford Road, Manchester, M13 9PT, United Kingdom.
3 Exciton Technologies Inc, 10230 Jasper Ave, Edmonton, Alberta, T5J 4P6, Canada.
4 School of Life Sciences, Hardy Building, University of Hull, Cottingham Road, Hull, HU6 7RX,
United Kingdom.
*Correspondence authors:
Dr Helen Thomason Professor Andrew McBain
Crawford Healthcare University of Manchester
King Edward Court Stopford Building
King Edward Road Oxford Road
Knutsford Manchester
WA16 0BE M13 9PT
United Kingdom United Kingdom
[email protected] [email protected]
Running Title: Silver Oxysalts Promote Healing
This article is protected by copyright. All rights reserved.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/wrr.12627
2
Key words: Silver oxysalts, Silver, Reactive oxygen species, Hydrogen peroxide, Oxygen.
Source of funding
This work was supported by a Knowledge Transfer Partnership (KTP 9006) between the
University of Manchester and Crawford Healthcare.
Abstract
Chronic wounds often exist in a heightened state of inflammation whereby excessive
inflammatory cells release high levels of proteases and reactive oxygen species (ROS). While
low levels of ROS play a fundamental role in the regulation of normal wound healing, their
levels need to be tightly regulated to prevent a hostile wound environment resulting from
excessive levels of ROS. Infection amplifies the inflammatory response, augmenting levels of
ROS which creates additional tissue damage that supports microbial growth. Antimicrobial
dressings are used to combat infection; however, the effects of these dressing on the
wound environment and healing independent of infection are rarely assessed. Cytotoxic or
adverse effects on healing may exacerbate the hostile wound environment and prolong
healing. Here we assessed the effect on healing independent of infection of silver oxysalts
which produce higher oxidative states of silver (Ag2+/Ag3+). Silver oxysalts had no adverse
effect on fibroblast scratch wound closure whilst significantly promoting closure of
keratinocyte scratch wounds (34% increase compared to control). Furthermore, dressings
containing silver oxysalts accelerated healing of full-thickness incisional wounds in wild-type
mice, reducing wound area, promoting re-epithelialisation and dampening inflammation.
We explored the mechanisms by which silver oxysalts promote healing and found that
unlike other silver dressings tested, silver oxysalt dressings catalyse the breakdown of
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3
hydrogen peroxide to water and oxygen. In addition, we found that silver oxysalts directly
released oxygen when exposed to water. Collectively, these data provide the first indication
that silver oxysalts promote healing independent of infection and may regulate oxidative
stress within a wound through catalysis of hydrogen peroxide.
Introduction
Chronic wounds, which include diabetic, venous and pressure ulcers, are a significant global
problem, causing patient morbidity and a substantial financial burden on health services
worldwide. The incidence of chronic wounds is currently rising because those populations
most susceptible, the elderly and diabetic, are rapidly expanding1. This in turn puts
increasing financial strain on health services. To put this in perspective, while the UK
population is estimated to rise just 3.8% in the 5 years from 2014 to 2019; the
disproportionate increase in the elderly and diabetic means the incidence of chronic
wounds is expected to rise by more than twice as much (9.8%) in the same period1. In 2014,
the annual NHS spend on wound care was estimated at £2 billion2, while in the U.S. an
estimated US$25 billion is spent on their treatment3. Despite the significant global economic
and social impact of chronic wounds, mechanistic understanding of delayed healing remains
poor.
Wound infection is a major contributing factor to the development and persistence of
chronic non-healing wounds, with wound bioburden (a combination of total bacterial load,
species diversity and presence of pathogenic species) linked to healing outcome4. Most
chronic wounds are stalled in the inflammatory phase of healing whereby excessive tissue
damage results in prolonged and heightened inflammation. Infection amplifies and
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4
perpetuates this inflammation by recruiting more neutrophils and macrophages5.
Neutrophils and macrophages release high levels of proteases which cause excessive tissue
breakdown. In addition, these inflammatory cells are a major source of reactive oxygen
species (ROS; superoxide anion, hydroxyl radicals, H2O2, singlet oxygen). ROS play a
fundamental role in the orchestration of normal wound healing. Tissue wounding induces a
rapid concentration gradient of H2O2, produced by injured epithelia, which acts as a
chemoattractant to guide neutrophils towards injury. The short-lived highly cytotoxic nature
of H2O2 is a key mechanism of the innate immune response in the control of infection.
However, H2O2 production must be tightly regulated as excess levels damage structural
proteins in the extracellular matrix, adversely alter the regulation of signalling pathways and
pro-inflammatory cytokines and causes cell death and tissue necrosis6. It has been
suggested that regulating excessive ROS levels, such as H2O2, in chronic wounds may help
reduce the hostile environment and stimulate healing7.
The harmful nature of the recruited inflammatory cells and their secreted products causes
tissue damage which propagates infection. This cycle leads to a destructive wound
environment. Despite the high levels of inflammatory cells present in infected chronic
wounds, their phagocytic and bactericidal functions are diminished8,9,10,11. As a result the
wound becomes more susceptible to an increase in bacterial bioburden.
Antimicrobial therapies are often used to compensate for the inability of the body’s innate
immune response to control infection in chronic wounds. Dressings incorporating
antimicrobial agents such as silver, iodine, honey and polyhexamethylene biguanide
(PHMB), are commonly favoured over antibiotics for the treatment of infected wounds,
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particularly in the earlier stages of infection12,13. While the effectiveness of antimicrobial
wound dressings on bioburden is paramount, their effects on the wound environment must
also be considered. Dressings containing singly ionic silver (Ag1+) are arguably the most
widely available and frequently used dressing due to their antimicrobial activity and minimal
association with microbial resistance14,15,16. However, concerns remain over potential tissue
cytotoxicity caused by silver dressings. Any detrimental effects of these antimicrobial
dressings on healing may exacerbate the non-healing phenotype observed in chronic
wounds. Thus, the effects of silver dressings on healing subsequent to infection clearance,
and recommendations for their prophylactic use to prevent infection, remain
unclear17,18,19,20,21,22.
New technology can now incorporate silver oxysalts (Ag7NO11) into dressings which, when
exposed to aqueous media such as wound fluid, degrade to produce higher oxidative states
of silver (Ag2+ and Ag3+). In vitro studies have shown these higher oxidative states of silver
exhibit greater antimicrobial activity than singly ionic silver (Ag1+)23,24. Crucially, the potent
antimicrobial activity of Ag2+ and Ag3+ oxysalts significantly reduces the silver concentration
required to achieve bactericidal activity23, reducing the risk of tissue cytotoxicity. However,
the effects of silver oxysalt-containing dressings on healing, independent of infection remain
unknown. In the current investigation, we demonstrate the safety profile of silver oxysalts
using in vitro (cell) and in vivo (murine wound) studies, while also assessing the potential of
silver oxysalts to promote healing independent of infection.
This article is protected by copyright. All rights reserved.
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Methods
Scratch wound assays: Conditioned media, KerraContact AgTM (previously Exsalt-T7;
Crawford Healthcare, Knutsford, UK) or control dressing (non-woven Sawabond 4383;
Sandler, Schwarzenbach/Saale, Germany) was prepared by incubating 10cm2 of each
dressing in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM - Sigma-Aldrich, Dorset,
England) containing 10% fetal bovine serum (FBS - Sigma-Aldrich, Dorset, England) for 24 h
with agitation at room temperature. The control dressing, Sawabond 4383, is composed of
the same material as KerraContact Ag, polyethylene and polyester. These materials are
biologically inert and have similar absorptive properties. After 24 h media was collected,
vigorously shaken, diluted 1 in 10 and applied to scratch wounds as follows. Confluent
primary human dermal fibroblasts or human epidermal keratinocytes (HaCaT) cultured in
24-well plates were scratch wounded using the point of a sterile 1000 μl volume pipette tip.
One milliliter of silver oxysalt conditioned media, control dressing conditioned media or
control media was added to each well immediately after wounding, followed by 24 h
incubation at 37 oC under 5% CO2. Cells were washed in PBS, visualized with crystal violet
and imaged using a Nikon Eclipse E600 microscope and SPOT digital camera. Images were
blinded and wound closure was calculated using Image ProPlus version 6.1
(MediaCybernetics Inc, Maryland, USA).
Murine wounding: All animal work was carried out under a UK Home Office project licences
subject to local ethics committee approval and in accordance with the European Guidelines
for Animal Care and Use of Experimental Animals. Eight-week-old female C57BL/6 mice
(Environ) were anaesthetised and wounded following our established protocol25 in
accordance with Home Office regulations. Briefly, two 1 cm full-thickness incisional wounds
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were made through both skin and panniculus carnosus muscle. A 1 cm2 piece of non-woven
Sawabond 4383 control dressing (Sandler, Schwarzenbach/Saale, Germany) or KerraContact
AgTM dressing (previously Exsalt-T7; Crawford Healthcare, Knutsford, UK), pre-moistened in
sterile distilled H2O, was placed over each wound. Dressings were then covered with
Tegaderm (3M, Bracknell, UK) attached via Mastisol liquid adhesive (Eloquest Healthcare,
Michigan, US). Mice were house individually to prevent damage to the wounds and
dressings. Three and 7 d post-wounding mice were sacrificed (schedule 1 method), each
wound was excised, bisected and processed for histology, allowing the midpoint of the
wound to be compared between groups. Ten wounds (five mice) for each treatment group
at each time-point were analysed.
Histological and immunohistochemical analysis: Histological sections were prepared from
wound tissue fixed in 10% buffered formalin saline and embedded in paraffin. Five-
micrometre sections were stained with haematoxylin and eosin or subjected to
immunohistochemistry with rat anti-Ly6G (neutrophils), rat anti-Mac-3 (macrophage) (BD
Biosciences, Pharmingen, Oxford, UK), anti-keratin 14, anti-keratin 1, anti-keratin 6 and anti-
loricrin (Biolegend, San Diego, USA) and the appropriate biotinylated secondary antibody
followed by ABC-peroxidase reagent (Vector Laboratories, UK) with NovaRed substrate or
streptavidin-Cy3 and counterstaining with haematoxylin or DAPI. Slides were blinded before
images captured with a Nikon Eclipse E600 microscope and SPOT digital camera. Total
inflammatory cell numbers, re-epithelialisation and wound area were quantified with Image
Pro Plus software (MediaCybernetics, Maryland, USA) as previously described25. Keratin 14,
keratin 1 and keratin 6 were quantified using Image Pro Plus software (MediaCybernetics,
Maryland, USA) as area of expression within the wound margins. Loricrin expression was
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quantified using Image Pro Plus software (MediaCybernetics, Maryland, USA) as percentage
coverage between the wound margins.
Assessment of oxygen generation from silver compounds: One hundred milligrams of silver
compounds were added to 20ml of foetal bovine serum and oxygen levels in solution
measured using a Hach TQ40D Oxygen probe and meter and levels recorded every minute
for 5 min. This meter detects dissolved oxygen in the range 0.00-22.00 ppm. The operating
principle of the meter is highly specific to oxygen and is not affected by metal ions or
oxidising agents per se.
Assessment of H2O2 breakdown: One hundred milligrams of silver compound were added
to 20ml of foetal bovine serum, vigorously mixed before adding H2O2 to a final
concentration of 0.08% w/w. Oxygen levels were monitored using a Hach TQ40D Oxygen
probe and meter and levels recorded every minute for 5 min. To assess the ability of silver-
containing wound dressings to catalyse the breakdown of H2O2, the oxygen level of
deionised water containing 0.08% w/w H2O2 was recorded immediately prior to the addition
of a single 2x2 cm2 of dressing and at each minute for 5 min following the addition. The
procedure was repeated in triplicate.
Statistical Analysis: Statistical analysis was performed on all quantitative data and power
calculations performed as previously described26. Our statistical power calculations based
on a large experimental data set indicate that an inter-group difference of 20% or greater
can be reliably detected using 8 wounds. All graphs represent mean +/- standard error. In
vitro scratch wound assays, in vivo wound area and inflammatory cell counts were analysed
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with a Mann-Whitney U test. Re-epithelialisation was analysed using an unpaired t-test with
Welch’s correction. For all statistical tests, probability values of P less than 0.05 were
considered statistically significant.
Results
We investigated the effects of silver oxysalts on healing independent of infection in vitro
and in vivo. We first assessed the effects of a dressing containing silver oxysalts on healing
of fibroblast and keratinocyte scratch wounds in vitro. Specifically, confluent layers of
fibroblasts or keratinocytes were scratched and treated for 24 h with control media, media
conditioned with a control dressing or silver oxysalt conditioned media (Figure 1). We found
no difference (P=0.53) in the closure rate of fibroblast scratch wounds treated with silver
oxysalt media versus the controls (Figure 1 A-C). By contrast, keratinocyte scratch wounds
treated with silver oxysalt media showed significantly (P=0.04) accelerated scratch wound
closure when compared to control media (Figure 1 D-F).
Next, we evaluated the effects of silver oxysalts on wound healing in vivo utilising a murine,
full-thickness, incisional wound model. Upon wounding, mice were treated with a control
dressing or a dressing containing silver oxysalts. At both 3 and 7 d post-wounding, silver
oxysalt treated wounds appeared visually smaller than control treated wounds (Figure 2 A-
D). Argyria, a grey-slate discolouration of the skin following topical or systemic
administration of silver compounds was not observed with silver oxysalt treatment.
Histological analysis (Figure 2 E-H) and quantification of wound area from histological
sections (Figure 2 I) confirmed silver oxysalt-treated wounds were smaller than control
treated wounds at 3 and 7 d post-wounding (P=0.03 and P=0.02, respectively). We next
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quantified the extent of re-epithelisation. At 3 d post-wounding silver oxysalt-treated
wounds showed significantly advanced re-epithelialisation compared to controls (P=0.05).
After 7 d both control and silver oxysalt-treated wounds were fully re-epithelialised. To
evaluate restoration of normal epidermal organisation and differentiation27, we profiled
epidermal differentiation markers 7 d post-wounding (Figure 3). Similar expression profiles
of the basal epidermal marker, keratin 14 (Figure 3 A & B), suprabasal epidermal marker,
keratin 1 (Figure 3 C & D), and injury induced keratin 6 (Figure 3 E & F) were observed in
control and silver oxysalt-treated wounds. Quantification of these keratin markers revealed
a non-significant difference in area of expression within the wound margins (P>0.05). We
next examined loricrin expression, a marker of late terminal differentiation. In control
treated wounds, patchy loricrin expression was observed across the newly re-epithelialised
epidermis (Figure 3 G). By contrast, a continuous layer of loricrin expression was observed
covering the newly re-epithelialised epidermis in silver oxysalt treated wounds (Figure 3 H).
Quantification of percentage loricrin expression confirmed significantly greater coverage in
silver oxysalt treated wounds compared to control treated wounds (85% compared to 63%,
respectively, P=0.02).
To determine the effects of silver oxysalts on inflammation in vivo we
immunohistochemically quantified the infiltration of neutrophils and macrophages into
murine granulation tissue (Figure 4). At 3 and 7 d post-wounding silver oxysalt treated
wounds exhibited significantly fewer neutrophils (P=0.015 and P=0.03, respectively; Figure 4
A-E) and macrophages (P=0.03; Figure 4 F-J) compared to control treated wounds.
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When exposed to aqueous media, the breakdown of silver oxysalts to generate higher
oxidative states of silver is proposed to release oxygen28. We therefore tested the ability of
silver compounds to directly generate oxygen when exposed to 100% foetal bovine serum.
Addition of silver oxysalts to foetal bovine serum generated oxygen; a property not
exhibited by any other silver-based compound tested (Figure 5A).
Silver is known to catalyse the breakdown of H2O2 to oxygen and water29. Therefore, we
assessed the ability of silver compounds commonly used in wound dressings to catalyse the
breakdown of H2O2 to oxygen and water by measuring oxygen levels in silver solutions made
up in 100% foetal bovine serum after the addition of H2O2. Oxygen levels increased after the
addition of H2O2 to solutions of silver oxysalts, silver oxides, silver phosphate and silver
metal (Figure 5B). We noted elevated oxygen concentrations in the absence of H2O2 at time
zero with silver oxysalts, confirming that silver oxysalts also evolve oxygen in the absence of
H2O2 (data not shown). We then assessed the ability of commercially available wound
dressings containing various silver compounds to catalyse the breakdown of H2O2 by
measuring oxygen levels in foetal bovine serum (plus dressings) after the addition of H2O2.
Of all the dressings tested, increased oxygen levels compared to the control were only
detected with dressings containing silver oxysalts (KerraContact Ag and KerraCel Ag)
indicating that silver oxysalt-containing dressings alone could catalyse the breakdown of
H2O2 to oxygen and water (Figure 5C). The release of oxygen through the catalytic
breakdown of H2O2 by dressings containing silver oxysalts was visualised as bubbles in a
solution of H2O2, this was unique to silver oxysalt dressings and not observed by dressings
containing other silver compounds (Figure 5D).
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Discussion
There is a wealth of literature assessing the efficacy of antimicrobial wound dressings;
however, few studies determine the impact of these dressings on healing independent of
infection. Adverse effects that may exacerbate the already hostile environment of a chronic
wound need to be determined to ensure antimicrobial treatment is given appropriately. Our
results show that silver oxysalt treatment promotes closure of in vitro keratinocyte scratch
wounds. Furthermore, we found that dressings containing silver oxysalts accelerated healing
of uninfected full-thickness murine wounds, reducing wound area, promoting re-
epithelialisation and dampening inflammation. We explored the possible mechanisms by
which silver oxysalts promote healing and found they release oxygen directly when exposed
to aqueous media and also catalyse the breakdown of hydrogen peroxide to oxygen and
water, a property that was unique to dressings containing silver oxysalts.
Silver is becoming increasingly popular as a topical antimicrobial, and a wide range of silver
dressings are available which incorporate silver in various formulations14. An ongoing
concern with the use of silver dressings to treat infected chronic wounds is potential local or
systemic cytotoxicity, which may be detrimental to healing. Unproven in situ, this concern
arises from extrapolation of in vitro results, where silver induces cell death in culture19,20,21.
In vivo, the effects of silver on healing independent of infection remain controversial; with
studies reporting both adverse17,18 and beneficial outcomes22,31. The higher oxidative states
of silver in silver oxysalts exhibit enhanced antimicrobial action compared to other available
silver technologies23,24. We have assessed the effects of silver oxysalt-containing dressings
on healing independent of infection. Given the potent antimicrobial action of silver oxysalts
we were surprised to demonstrate no adverse effects of silver oxysalts on healing of in vitro
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13
fibroblast scratch wounds and an unexpected acceleration of healing of in vitro keratinocyte
scratch wounds. Accelerated healing was also observed with silver oxysalt treatment of full-
thickness murine incisional wounds, linked to reduced local inflammation and increased re-
epithelialisation. These data contrast with that of Burd and co-workers (2007)17 who
reported impaired re-epithelialisation in an ex vivo porcine and an in vivo murine wound
model when treated with several dressings containing single ionic (Ag1+) silver. These
conflicting observations could simply reflect the differing models employed, however, the
differing effects may be due to the differences in the silver technology.
A number of factors can delay wound healing which can lead to the development of a
chronic wound; these include chronic disease, ageing, diabetes, vascular insufficiency,
pressure, oedema and infection. Despite the underlying cause, many chronic wounds
progress in a similar way, whereby tissue damage results in heightened and prolonged
inflammation. This is characterised by excess neutrophil and macrophage infiltration and
elevated levels of destructive proteinases and reactive oxygen species such as H2O2. We
explored the possible mechanisms by which silver oxysalts promote healing and found that
dressings containing silver oxysalts catalysed the breakdown of H2O2 to water and oxygen.
This catalysis of H2O2 was unique to silver oxysalt dressings and was not observed with any
other silver dressing. During wound healing low levels of H2O2 are essential for effective
repair; however, too much adversely affects healing and prolongs the inflammatory
phase32,33. Tissue wounding induces a rapid concentration gradient of H2O2 from epithelial
cells which signals to recruit inflammatory cells to the site of injury34. Inhibition of this H2O2
signal is sufficient to impair leukocyte recruitment to the wound34. It is hypothesised that
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H2O2 generation by epithelial cells has two essential functions: killing of bacteria and
recruitment of leukocytes. In addition, the recruited inflammatory cells also release H2O2
which is essential for microbial killing; however, at high concentrations H2O2 causes tissue
damage and contributes to chronic inflammation32,33,35. Thus, in chronic wounds excessive
amounts of H2O2 contribute to the delay in healing35,7. This has also been shown in vivo
whereby the addition of low levels of H2O2 (10 mM) to wounds in diabetic mice promotes
healing whereas the addition of higher concentrations of H2O2 (166 mM) retards healing32. It
has therefore been proposed that interventions to alter H2O2 concentrations within a
wound could be a successful therapeutic strategy to reduce the hostile wound environment.
While this does not address the underlying cause of delayed healing it may be sufficient to
promote healing of chronic wounds7.
A hallmark of non-healing chronic wounds is excessive and prolonged inflammation, a direct
causative factor in healing delay. Wounds treated with silver oxysalt-containing dressings
showed reduced numbers of macrophage and neutrophils. Depletion of either or both
neutrophils and macrophage has been shown to enhance the rate of wound repair and
decrease scarring and it has therefore been suggested that modulating leukocyte
recruitment may also therapeutically benefit healing of chronic wounds37. However, in an
infected wound, a reduction in these cells may delay the wounds ability to combat the
infection. The potent antimicrobial action of silver oxysalts is expected to compensate for
the reduction in inflammatory cell recruitment. Furthermore, the ability of the silver
oxysalts to catalyse the breakdown of H2O2 may enhance the innate immune response. The
antimicrobial action of H2O2 released from inflammatory cells within the wound relates not
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15
only to its oxidising properties but also to its breakdown to water, releasing various oxygen
species and free radicals38. These compounds are more reactive than H2O2 but are short
lived and therefore only those radicals generated in the immediate vicinity of microbial cells
are likely to have an antimicrobial effect. The presence of a catalyst has been shown to
dramatically enhance the killing properties of H2O2 against bacterial biofilms39. Moreover,
residual biofilm bacteria are unaffected by antimicrobial treatments unless a catalyst is
included and the strength of bacterial attachment to a test surface is significantly loosened
by the inclusion of a catalyst39. Thus, in an infected chronic wound silver oxysalts acting as a
catalyst at the site of infection may enhance the killing properties of H2O2 through its
breakdown to more reactive antimicrobial species.
Another mechanism by which silver oxysalts could promote healing is its ability to increase
tissue oxygen levels within the wound. We tested the ability of a variety of silver
compounds to directly generate oxygen when added to foetal bovine serum and found that
only silver oxysalts can directly generate oxygen. Oxygen plays a critical role in cell function
and survival and is required in high demand by many of the cells essential for effective
wound repair. Systemic and topical oxygen therapies have been reported to promote
healing; however, the mechanisms by which they do so are still being elucidated and these
mechanisms are likely to differ between systemic and topical treatments. Positive effects on
neo-angiogenesis, collagen formation, re-epithelialisation and antimicrobial activity have
been reported40. Although oxygen is critical for effective wound repair, the main
contraindication to oxygen therapy for treatment of chronic wounds centres on the role of
hypoxia in promoting repair. Upon wounding oxygen levels drop until a state of hypoxia is
reached. Hypoxic conditions within a wound induce a tightly regulated response pathway to
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16
modulate cellular functions and ultimately restore oxygen supply via the upregulation of the
transcription factor hypoxia-inducible factor-1 (HIF-1). In states of prolonged hypoxia, such
as those found in chronic wounds HIF-1 activity is repressed and thus the key signalling
pathways are not stimulated. The optimal oxygen concentration for effective wound repair
is unclear, too much or too little may impede healing. Thus, adequate wound tissue oxygen
is required but may not be sufficient to positively influence healing. Oxygen therapy may
therefore only benefit those cases where hypoxia is the only rate limiting factor and all
other essential factors are functional. The level of oxygen released during silver oxysalts to
silver ion conversion is low; however, direct oxygen release and oxygen produced through
the breakdown of H2O2, together with a reduction in oxygen consuming leukocytes may
sufficiently increase tissue oxygen levels to stimulate healing of silver oxysalt treated
wounds.
Infected non-healing chronic wounds are clinically challenging, requiring antimicrobial
treatment to combat infection and methods to reduce the hostile wound environment.
There are an increasing number of antimicrobial therapies available for the treatment of
infected wounds and whilst many of these are effective at combatting infection, their
effects on healing independent of infection are often unknown. Delineating the effects of
antimicrobial agents on healing independent of infection is therefore essential to guide
clinical protocols for the treatment of infected wounds. The ability of silver oxysalts to
combat infection whilst promoting multiple aspects of healing in vivo offer clinicians a novel
alternative to currently available antimicrobial dressings.
This article is protected by copyright. All rights reserved.
17
Acknowledgements
The authors gratefully acknowledge the support of Bryan Greener in the preparation of this
manuscript. This work was supported by a Knowledge Transfer Partnership (KTP 9006)
between the University of Manchester and Crawford Healthcare.
Conflict of Interest
Helen Thomason, Jodie Lovett and Christian Stephenson are employees of Crawford
Healthcare. Andrew McBain and Matthew Hardman received KTP funding from Innovate UK
and Crawford Healthcare.
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Figure legends
Figure 1. Silver Oxysalts influence human fibroblast and keratinocyte scratch wound
closure. (A & B) Representative images of human fibroblast scratch wounds treated with
control (A) or silver oxysalt-containing (B) media for 24 h. (C) Quantification revealed no
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22
difference in wound closure between scratch wounds treated with control media or control
dressing conditioned media and those incubated with silver oxysalt media. (D & E)
Representative images of human keratinocyte (HaCaT) scratch wounds treated with control
(D) or silver oxysalt (E) media. (F) After 24 h scratch wounds treated with media incubated
with silver oxysalt media showed a significant increase in healing compared to control media
or control dressing conditioned media treated wounds (n=3). *, P <0.05. Scale bars = 500 µm
(A, B, D, E).
Figure 2. Silver oxysalts promote healing of non-infected mouse wounds. Representative
macroscopic (A-D) and histological (E-H) images of incisional wounds treated for 3 d (A-B, E-
F) or 7 d (C-D, G-H) with control dressing (A, C, E, G) or dressing containing silver oxysalts (B,
D, F, H). (I-J) Quantification revealed significantly reduced wound area following silver
oxysalt treatment compared to control treated wounds at 3 d and 7 d post-wounding (I) and
increased re-epithelialisation at 3 d post wounding (J). n=5 mice (10 wounds). Arrows in E-H
indicate wound margins. * indicates P= <0.05. Scale bars = 200 µm (A-H).
Figure 3. Silver oxysalts promote restoration of epidermal differentiation.
Immunofluorescence staining (red) for keratin 14 (A & B), keratin 1 (C & D), loricrin (E & F)
and keratin 6 (G & H) in murine wounds treated with control or silver oxysalt dressings for 7
d. Silver oxysalt treated wounds exhibited an advanced re-establishment of normal terminal
differentiation compared to control treated wounds. Similar expression profiles of K14 (A &
B) and K1 (C & D) were observed with control (A & C) and silver oxysalt treatment (B & D).
Keratin 6 expression, which is up-regulated in the epidermis upon wounding, was reduced in
silver oxysalt treated wounds (F) compared to control treated wounds (E). In control treated
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23
wounds loricrin expression was patchy (G) whereas a continuous layer was observed in silver
oxysalt treated wounds (H). n=5 mice (10 wounds). Scale bars = 50 µm (A-H).
Figure 4. Silver oxysalts reduce wound neutrophil and macrophage infiltration. (A-D)
Representative neutrophil immunohistochemistry images of control (A & C) and silver
oxysalt treated (B & D) wounds for 3 d (A & B) and 7 d (C & D). (E) Quantification revealed
significantly fewer neutrophils in wounds treated with silver oxysalts after 3 and 7 d versus
control. (F-I) Representative macrophage immunohistochemistry images of control (F & H)
and silver oxysalt treated (G & I) wounds for 3 d (F & G) and 7 d (H & I). (J) Quantification
revealed significantly fewer macrophages in wounds treated with silver oxysalts after 3 d
and 7 d versus control. n=5 mice (10 wounds). * indicates P <0.05. Scale bar = 50 µm (A-D, F-
I).
Figure 5. Oxygen generation and catalytic oxygen release from H2O2 breakdown by silver
oxysalts. (A) Oxygen evolution from silver salt powders in foetal bovine serum was
monitored over 5 minutes. Dissolved oxygen concentrations increased immediately after
addition of silver oxysalts. This behaviour was not observed for any other silver material
tested, including silver oxides. (B) Catalytic breakdown of H2O2 solution to oxygen by a range
of silver salts. Oxygen levels are shown one minute after the addition of H2O2. Catalytic
breakdown of H2O2 to oxygen in foetal bovine serum is achieved by a subset of silver
compounds uncommon in wound care and silver oxysalts. Silver compounds commonly used
in wound care, including chloride (Aquacel Ag), sulfate (Mepilex Ag) and sulfadiazine
(Allevyn Ag) are inactive. (C) Catalytic breakdown of H2O2 solution to oxygen by a range of
silver wound dressings in foetal bovine serum. Only dressings containing silver oxysalts
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24
(KerraContact Ag and KerraCel Ag) were found to catalyse the breakdown of H2O2 to oxygen
and water. (D) Oxygen released through catalytic breakdown of H2O2 by silver oxysalt
dressings (KerraCel Ag) was visualised as bubbles in a solution of H2O2. Bubbles were not
observed by dressings containing other silver compounds.
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Figure 1. – Colour image
*
F
D E A B
C
40
60
80
100
ControlMedia
ControlDressing
AgOxysalts
Wo
un
d C
losu
re (
%)
40
60
80
100
ControlMedia
ControlDressing
AgOxysalts
Wo
un
d C
losu
re (
%)
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50
60
70
80
90
100
3 7% R
e-e
pit
hel
ialis
atio
n
Days post-wounding
0.0
0.2
0.4
0.6
0.8
1.0
3 7
Wo
un
d A
rea
(mm
2)
Days post-wounding
Figure 2. Colour image
A B C D
E F G H
I J
Control Ag Oxysalts Control Ag Oxysalts
3 days post-wounding 7 days post-wounding
*
Ag Oxysalts Control
*
*
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Lor Lor
K6 K6
Figure 3. Colour image
Control Ag Oxysalts K14 K14
K1 K1
A B
C D
E F
G H
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0
400
800
1200
3 7
Mac
rop
hag
e (m
m2)
Days post-wounding
0
100
200
300
400
500
3 7N
eu
tro
ph
ils (
mm
2)
Days post-wounding
Control Ag Oxysalts A B
C D
E
F G
H I
3 d
ays
7
day
s
3 d
ays
7
day
s
Ne
utr
op
hils
M
acro
ph
age
J
Figure 4.
Control Ag Oxysalts
Control Ag Oxysalts
*
*
*
*
Control Ag Oxysalts
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A
C
B
D KerraCel Ag Aquacel Ag Extra
Figure 5.
5
10
15
20
25
Co
ntr
ol
Ag
Sulf
ate
SSD
Ag
Ch
lori
de
Ag
Cit
rate
Ag
Nit
rate
Ag
Oxy
salt
s
Ag(
I,II
I) O
xid
e
Ag(
I) O
xid
e
Ag
Me
tal
Ag
Ph
osp
hat
e
Oxy
gen
(p
pm
)
5
10
15
20
25
0 1 2 3 4 5
Oxy
gen
(p
pm
)
Time (minutes)
Silver Oxysalt Ag(I,III) Oxide
Ag(I) Oxide Ag Metal
Ag Sulfate SSD
Ag Chloride Ag Citrate
Ag Phosphate Ag Nitrate
8
10
12
14
16
0 1 2 3 4 5
Oxy
gen
(p
pm
)
Time (minutes)
Kerrcel Ag Kerracontact Ag
Aquacel Ag Aquacel Ag Extra
Durafiber Ag Control
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