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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

Published in:Wound Repair and Regeneration

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Download date:16. May. 2021

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

Helen.thomason@crawfordpharma.com Andrew.mcbain@manchester.ac.uk

Helen.thomason@manchester.ac.uk

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

This article is protected by copyright. All rights reserved.

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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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

This article is protected by copyright. All rights reserved.

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

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d C

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ControlMedia

ControlDressing

AgOxysalts

Wo

un

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losu

re (

%)

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50

60

70

80

90

100

3 7% R

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hel

ialis

atio

n

Days post-wounding

0.0

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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

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e (m

m2)

Days post-wounding

0

100

200

300

400

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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

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Oxy

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(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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