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Microbial growth in wounds often manifests itself as biofilms, which interfere with healing and are difficult to eradicate. New silver dressings claim to combat wound infections, but their antibiofilm efficacy and infection-independent healing effects are generally unknown. Using in vitro and in vivo biofilm models of Staphylococcus aureus and Pseudomonas aeruginosa, we report the effectiveness of Ag1+ ion-generating dressings; Ag1+ dressings containing ethylenediaminetetraacetic acid and benzethonium chloride (Ag1+/EDTA/BC), and dressings containing silver nitrate (Ag oxysalts). , which produce Ag1+, Ag2+ and Ag3+ ions to combat wound biofilm and its effect on healing. Ag1+ dressings had minimal effects on wound biofilm in vitro and in mice (C57BL/6j). In contrast, oxygenated Ag salts and Ag1+/EDTA/BC dressings significantly reduced the number of viable bacteria in biofilms in vitro and demonstrated a significant reduction in bacterial and EPS components in mouse wound biofilms. These dressings had different effects on the healing of biofilm-infected and non-biofilm-infected wounds, with oxygenated salt dressings having more beneficial effects on reepithelialization, wound size, and inflammation compared to control treatments and other silver dressings. The different physicochemical properties of silver dressings may have different effects on wound biofilm and healing, and this should be considered when selecting a dressing for the treatment of biofilm-infected wounds.
Chronic wounds are defined as “wounds that fail to progress through the normal stages of healing in an orderly and timely manner” 1 . Chronic wounds create a psychological, social and economic burden for patients and the healthcare system. Annual NHS spending on treating wounds and associated comorbidities is estimated at £8.3 billion in 2017–182. Chronic wounds are also currently a pressing problem in the United States, with Medicare estimating the annual cost of treating patients with wounds at $28.1–$96.8 billion3.
Infection is a major factor preventing wound healing. Infections often manifest as biofilms, which are present in 78% of non-healing chronic wounds. Biofilms form when microorganisms become irreversibly attached to surfaces, such as wound surfaces, and can aggregate to form extracellular polymer (EPS)-producing communities. Wound biofilm is associated with an increased inflammatory response leading to tissue damage, which can delay or prevent healing4. The increase in tissue damage may be due in part to increased activity of matrix metalloproteinases, collagenase, elastase and reactive oxygen species5. Moreover, inflammatory cells and biofilms are themselves high consumers of oxygen and can therefore cause local tissue hypoxia, depleting cells of the vital oxygen needed for effective tissue repair6.
Mature biofilms are highly resistant to antimicrobial agents, requiring aggressive strategies to control biofilm infections, such as mechanical treatment followed by effective antimicrobial treatment. Because biofilms can rapidly regenerate, effective antimicrobials can reduce the risk of re-formation after surgical debridement7.
Silver is increasingly used in antimicrobial dressings and is often used as a first-line treatment for chronic infected wounds. There are many commercially available silver dressings, each containing a different silver composition, concentration, and base matrix. Advances in silver armbands have led to the development of new silver armbands. The metallic form of silver (Ag0) is inert; To achieve antimicrobial effectiveness, it must lose an electron to form ionic silver (Ag1+). Traditional silver dressings contain silver compounds or metallic silver which, when exposed to liquid, decompose to form Ag1+ ions. These Ag1+ ions react with the bacterial cell, removing electrons from structural components or critical processes necessary for survival. Patented technology has led to the development of a new silver compound, Ag oxysalts (silver nitrate, Ag7NO11), which is included in wound dressings. Unlike traditional silver, the decomposition of oxygen-containing salts produces states of silver with higher valence (Ag1+, Ag2+ and Ag3+). In vitro studies have shown that low concentrations of oxygenated silver salts are more effective than single ion silver (Ag1+) against pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli8,9. Another new type of silver dressing includes additional ingredients, namely ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride (BC), which are reported to target biofilm EPS and thereby increase the penetration of silver into the biofilm. These new silver technologies offer new ways to target wound biofilms. However, the impact of these antimicrobials on the wound environment and infection-independent healing is important to ensure that they do not create an unfavorable wound environment or delay healing. Concerns about in vitro silver cytotoxicity have been reported with several silver dressings10,11. However, in vitro cytotoxicity has not yet translated into in vivo toxicity, and several Ag1+ dressings have demonstrated a good safety profile12.
Here, we investigated the effectiveness of carboxymethylcellulose dressings containing novel silver formulations against wound biofilm in vitro and in vivo. In addition, the effects of these dressings on immune responses and healing independent of infection were assessed.
All dressings used were commercially available. 3M Kerracel Gel Fiber Dressing (3M, Knutsford, UK) is a non-antimicrobial 100% carboxymethylcellulose (CMC) gel fiber dressing that was used as the control dressing in this study. Three antimicrobial CMC silver dressings were evaluated, namely 3M Kerracel Ag dressing (3M, Knutsford, UK), which contains 1.7 wt%. oxygenated silver salt (Ag7NO11) in higher valence silver ions (Ag1+, Ag2+ and Ag3+). During the decomposition of Ag7NO11, Ag1+, Ag2+ and Ag3+ ions are formed in a ratio of 1:2:4. Aquacel Ag Extra dressing containing 1.2% silver chloride (Ag1+) (ConvaTec, Deeside, UK) 13 and Aquacel Ag + Extra dressing containing 1.2% silver chloride (Ag1+), EDTA and benzethonium chloride (ConvaTec, Deeside, UK) 14.
The strains used in this study were Pseudomonas aeruginosa NCTC 10781 (Public Health England, Salisbury) and Staphylococcus aureus NCTC 6571 (Public Health England, Salisbury).
Bacteria were grown overnight in Muller-Hinton broth (Oxoid, Altrincham, UK). The overnight culture was then diluted 1:100 in Mueller-Hinton broth and 200 µl plated onto sterile 0.2 µm Whatman cyclopore membranes (Whatman plc, Maidstone, UK) onto Mueller-Hinton agar plates (Sigma-Aldrich Company Ltd, Kent , Great Britain). ) Colonial biofilm formation at 37°C for 24 hours. These colonial biofilms were tested for logarithmic shrinkage.
Cut the dressing into 3 cm2 square pieces and pre-moisten with sterile deionized water. Place the bandage over the biofilm of the colony on the agar plate. Every 24 ha of biofilm was removed, and viable bacteria within the biofilm (CFU/ml) were quantified by serial dilution (10−1 to 10−7) in Day-Angle neutralization broth (Merck-Millipore). After 24 hours of incubation at 37°C, standard plate counts were performed on Mueller-Hinton agar plates. Each treatment and time point was performed in triplicate, and plate counts were repeated for each dilution.
Pork belly skin is obtained from female Large White pigs within 15 minutes of slaughter in accordance with European Union export standards. The skin was shaved and cleaned with alcohol wipes, then frozen at -80°C for 24 hours to devitalize the skin. After thawing, 1 cm2 skin pieces were washed three times with PBS, 0.6% sodium hypochlorite, and 70% ethanol for 20 minutes each time. Before removing the epidermis, remove any remaining ethanol by washing 3 times in sterile PBS. The skin was cultured in a 6-well plate with a 0.45-μm-thick nylon membrane (Merck-Millipore) on top and 3 absorbent pads (Merck-Millipore) containing 3 ml fetal bovine serum (Sigma) supplemented with 10% Dulbecco’s modified Eagle. Medium (Dulbecco’s Modified Eagle Medium – Aldrich Ltd.).
Colonial biofilms were grown as described for biofilm exposure studies. After culturing the biofilm on the membrane for 72 hours, the biofilm was applied to the skin surface using a sterile inoculation loop and the membrane was removed. The biofilm was then incubated on the pig’s dermis for an additional 24 hours at 37°C to allow the biofilm to mature and adhere to the pig’s skin. After the biofilm had matured and attached, a 1.5 cm2 dressing, pre-moistened with sterile distilled water, was applied directly to the skin surface and incubated at 37°C for 24 hours. Viable bacteria were visualized by staining by uniformly applying PrestoBlue cell viability reagent (Invitrogen, Life Technologies, Paisley, UK) to the apical surface of each explant and incubating it for 5 minutes. Use the Leica DFC425 digital camera to instantly capture images on the Leica MZ8 microscope. Areas colored pink were quantified using Image Pro software version 10 (Media Cybernetics Inc, Rockville, MD Image-Pro (mediacy.com)). Scanning electron microscopy was performed as described below.
Bacteria grown overnight were diluted 1:100 in Mueller-Hinton broth. 200 μl of culture was added to sterile 0.2 μm Whatman cyclopore membrane (Whatman, Maidstone, UK) and plated on Mueller-Hinton agar. Biofilm plates were incubated at 37°C for 72 hours to allow mature biofilm formation.
After 3 days of biofilm maturation, a 3 cm2 square bandage was placed directly on the biofilm and incubated at 37°C for 24 hours. After removing the bandage from the biofilm surface, 1 ml of PrestoBlue Cell Viability Reagent (Invitrogen, Waltham, MA) was added to the surface of each biofilm for 20 seconds. The surfaces were dried before color changes were recorded using a Nikon D2300 digital camera (Nikon UK Ltd., Kingston, UK).
Prepare overnight cultures on Mueller-Hinton agar, transfer individual colonies to 10 ml Mueller-Hinton broth and incubate on a shaker at 37°C (100 rpm). After overnight incubation, the culture was diluted 1:100 in Mueller-Hinton broth and 300 µl was spotted onto 0.2 µm circular Whatman cyclopore membrane (Whatman International, Maidstone, UK) on Mueller-Hinton agar and incubated at 37°C within 72 hours. . The mature biofilm was applied to the wound as described below.
All work with animals was carried out at the University of Manchester under a project license approved by the Office of Animal Welfare and Ethical Review (P8721BD27) and in accordance with guidelines published by the Home Office under the 2012 revised ASPA. All authors adhered to the arrival guidelines. Eight-week-old C57BL/6j mice (Envigo, Oxon, UK) were used for all in vivo studies. Mice were anesthetized with isoflurane (Piramal Critical Care Ltd, West Drayton, UK) and their dorsal surfaces were shaved and cleaned. Each mouse was then given a 2 × 6 mm excisional wound using a Stiefel biopsy punch (Schuco International, Hertfordshire, UK). For biofilm-infected wounds, apply 72-hour colonial biofilm grown on the membrane as described above to the dermal layer of the wound using a sterile inoculation loop immediately after injury and discard the membrane. One square centimeter of dressing is pre-moistened with sterile water to maintain a moist wound environment. Dressings were applied directly to each wound and covered with 3M Tegaderm film (3M, Bracknell, UK) and Mastisol liquid adhesive (Eloquest Healthcare, Ferndale, MI) applied around the edges to provide additional adhesion. Buprenorphine (Animalcare, York, UK) was administered at a concentration of 0.1 mg/kg as an analgesic. Cull mice three days after injury using the Schedule 1 method and remove, halve, and store the wound area as needed.
Hematoxylin (ThermoFisher Scientific) and eosin (ThermoFisher Scientific) staining was performed according to the manufacturer’s protocol. Wound area and reepithelialization were quantified using Image Pro software version 10 (Media Cybernetics Inc, Rockville, MD).
Tissue sections were dewaxed in xylene (ThermoFisher Scientific, Loughborough, UK), rehydrated with 100–50% graded ethanol, and briefly immersed in deionized water (ThermoFisher Scientific). Immunohistochemistry was performed using the VectaStain Elite ABC PK-6104 kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Primary antibodies to neutrophils NIMP-R14 (Thermofisher Scientific) and macrophages Ms CD107b Pure M3/84 (BD Biosciences, Wokingham, UK) were diluted 1:100 in blocking solution and added to the cut surface, followed by 2 antibodies Anti-, VectaStain ABC and Vector Nova Red Peroxidase (HRP) substrate kit (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. Images were acquired using an Olympus BX43 microscope and an Olympus DP73 digital camera (Olympus, Southend-on-Sea, UK).
Skin samples were fixed in 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M HEPES (pH 7.4) for 24 hours at 4°C. Samples were dehydrated using graded ethanol and dried in CO2 using a Quorum K850 critical point dryer (Quorum Technologies Ltd, Loughton, UK) and sputter coated with gold-palladium alloy using a Quorum SC7620 mini sputterer/glow discharge system. Specimens were imaged using an FEI Quanta 250 scanning electron microscope (ThermoFisher Scientific) to visualize the central point of the wound.
Toto-1 iodide (2 μM) was applied to the excised mouse wound surface and incubated for 5 min at 37 °C (ThermoFisher Scientific) and treated with Syto-60 (10 μM) at 37 °C (ThermoFisher Scientific). 15-minute Z-stack images were created using a Leica TCS SP8.
Biological and technical replicate data were tabulated and analyzed using Graphpad Prism V9 software (GraphPad Software, La Jolla, CA). One-way analysis of variance with multiple comparisons using Dunnett’s post hoc test was used to test for differences between each treatment and the non-antimicrobial control dressing. A p value <0.05 was considered significant.
The effectiveness of silver gel fibrous dressings was first assessed against biofilm colonies of Staphylococcus aureus and Pseudomonas aeruginosa in vitro. Silver dressings contain different formulas of silver: traditional silver dressings produce Ag1+ ions; silver dressings, which can produce Ag1+ ions after the addition of EDTA/BC, can destroy the biofilm matrix and expose bacteria to silver under the antibacterial effect of silver. ions15 and dressings containing oxygenated Ag salts that produce Ag1+, Ag2+ and Ag3+ ions. Its effectiveness was compared with a non-antimicrobial control dressing made from gelled fibers. Remaining viable bacteria within the biofilm were assessed every 24 hours for 8 days (Figure 1). On day 5, the biofilm was reinoculated with 3.85 × 105S. Staphylococcus aureus or 1.22×105P. aeruginosa to assess biofilm recovery. Compared to non-antimicrobial control dressings, Ag1+ dressings had minimal effect on bacterial viability in Staphylococcus aureus and Pseudomonas aeruginosa biofilms over 5 days. In contrast, dressings containing oxygenated Ag and Ag1+ + EDTA/BC salts were effective in killing bacteria within the biofilm within 5 days. After repeated inoculation with planktonic bacteria on day 5, no restoration of the biofilm was observed (Fig. 1).
Quantification of viable bacteria in Staphylococcus aureus and Pseudomonas aeruginosa biofilms after treatment with silver dressings. Biofilm colonies of Staphylococcus aureus and Pseudomonas aeruginosa were treated with silver dressings or non-antimicrobial control dressings, and the number of viable bacteria remaining was determined every 24 hours. After 5 days, the biofilm was reinoculated with 3.85×105S. Staphylococcus aureus or 1.22×105P. Colonies of the bacterioplankton Pseudomonas aeruginosa were formed individually to assess biofilm recovery. Graphs show mean +/- standard error.
To visualize the effect of silver dressings on biofilm viability, dressings were applied to mature biofilms grown on porcine skin ex vivo. After 24 hours, the dressing is removed and the biofilm is stained with a blue reactive dye, which is metabolized by living bacteria to a pink color. Biofilms treated with control dressings were pink, indicating the presence of viable bacteria within the biofilm (Figure 2A). In contrast, the biofilm treated with the Ag oxysols dressing was primarily blue, indicating that the remaining bacteria on the surface of the pig’s skin were nonviable bacteria (Figure 2B). Mixed blue and pink coloration was observed in biofilms treated with Ag1+-containing dressings, indicating the presence of viable and non-viable bacteria within the biofilm (Figure 2C), whereas EDTA/BC dressings containing Ag1+ were predominantly blue with some pink spots. indicating areas not affected by the silver dressing (Figure 2D). Quantification of active (pink) and inactive (blue) areas showed that the control patch was 75% active (Figure 2E). Ag1+ + EDTA/BC dressings performed similarly to oxygenated Ag salt dressings, with survival rates of 13% and 14%, respectively. The Ag1+ dressing also reduced bacterial viability by 21%. These biofilms were then observed using scanning electron microscopy (SEM). After treatment with the control dressing and the Ag1+ dressing, a layer of Pseudomonas aeruginosa was observed covering the porcine skin (Figure 2F,H), whereas after treatment with the Ag1+ dressing, few bacterial cells were found and few bacterial cells were found underneath. Collagen fibers can be considered as the tissue structure of porcine skin (Figure 2G). After treatment with Ag1+ + EDTA/BC dressing, bacterial plaques and underlying collagen fiber plaques were visible (Figure 2I).
Visualization of Pseudomonas aeruginosa biofilm after silver dressing treatment. (A–D) Bacterial viability in Pseudomonas aeruginosa biofilms grown on porcine skin was visualized using PrestoBlue viability dye 24 hours after treatment with silver dressings or non-antimicrobial control dressings. Live bacteria are pink, non-viable bacteria and pig skin are blue. (E) Quantification of Pseudomonas aeruginosa biofilms grown on porcine skin (pink spot) using scanning electron microscopy Image Pro version 10 (FI) and treated with a silver dressing or a non-antimicrobial control dressing for 24 hours. SEM scale bar = 5 µm. (J–M) Colonial biofilms grew on filters and were stained with PrestoBlue reactive dye after 24 h of incubation with silver dressings.
To determine whether close contact between the dressings and the biofilms affected the effectiveness of the dressings, colonial biofilms placed on a flat surface were treated with the dressings for 24 hours and then stained with reactive dyes. The untreated biofilm was dark pink in color (Figure 2J). In contrast to biofilms treated with dressings containing oxygenated Ag salts (Figure 2K), biofilms treated with dressings containing Ag1+ or Ag1+ + EDTA/BC showed bands of pink staining (Figure 2L,M). This pink coloration indicates the presence of viable bacteria and is associated with the suture area within the dressing. These sewn-in areas create dead spaces that allow bacteria within the biofilm to survive.
To evaluate the effectiveness of silver dressings in vivo, full-thickness excised wounds of mice infected with mature S. aureus and P. aeruginosa biofilms were treated with nonantimicrobial control dressings or silver dressings. After 3 days of treatment, macroscopic image analysis showed smaller wound sizes when treated with oxygenated salt dressings compared to non-antimicrobial control dressings and other silver dressings (Figure 3A-H). To confirm these observations, wounds were harvested and wound area and reepithelialization were quantified on hematoxylin and eosin-stained tissue sections using image pro software version 10 (Figure 3I-L).
The effect of silver dressings on the wound surface and re-epithelialization of wounds infected with biofilms. (A–H) Small cells infected with biofilms of Pseudomonas aeruginosa (A–D) and Staphylococcus aureus (E–H) after three days of treatment with a nonantimicrobial control dressing, an oxygenated Ag salt dressing, an Ag1+ dressing, and an Ag1+ dressing. Representative macroscopic images. wounds of mice with Ag1+ + EDTA/BC dressing. (IL) Representative Pseudomonas aeruginosa infection, histological sections stained with hematoxylin and eosin, used to quantify wound area and epithelial regeneration. Quantification of wound area (M, O) and percentage reepithelialization (N, P) of wounds infected with Pseudomonas aeruginosa (M, N) and Staphylococcus aureus (O, P) biofilms (per treatment group n = 12). Graphs show mean +/- standard error. * means p = < 0.05 ** means p = < 0.01; Macroscopic scale = 2.5 mm, histological scale = 500 µm.
Quantification of wound area in wounds infected with Pseudomonas aeruginosa biofilm (Figure 3M) showed that wounds treated with Ag oxysalts had an average wound size of 2.5 mm2, while the non-antimicrobial control dressing had an average wound size of 3.1 mm2 , which is not true. reached statistical significance (Figure 3M). p = 0.423). Wounds treated with Ag1+ or Ag1+ + EDTA/BC showed no reduction in wound area (3.1 mm2 and 3.6 mm2, respectively). Treatment with oxygenated Ag salt dressing promoted re-epithelialization to a greater extent than the non-antimicrobial control dressing (34% and 15%, respectively; p = 0.029) and Ag1+ or Ag1+ + EDTA/BC (10% and 11%) (Figure 3N). . , respectively).
Similar trends in wound area and epithelial regeneration were observed in wounds infected with S. aureus biofilms (Figure 3O). Dressings containing oxygenated silver salts reduced wound area (2.0 mm2) by 23% compared with the control non-antimicrobial dressing (2.6 mm2), although this reduction was not significant (p = 0.304) (Fig. 3O). In addition, the wound area in the Ag1+ treatment group was slightly reduced (2.4 mm2), while the wound treated with Ag1+ + EDTA/BC dressing did not reduce the wound area (2.9 mm2). Oxygen salts of Ag also promoted re-epithelialization of wounds infected with S. aureus biofilm (31%) to a greater extent than those treated with non-antimicrobial control dressings (12%, p = 0.003) (Figure 3P). Ag1+ dressing (16%, p = 0.903) and Ag+1 + EDTA/BC dressing (14%, p = 0.965) showed levels of epithelial regeneration similar to control.
To visualize the effect of silver dressings on the biofilm matrix, Toto 1 and Syto 60 iodide staining was performed (Fig. 4). Toto 1 iodide is a cell-impermeable dye that can be used to accurately visualize extracellular nucleic acids, which are abundant in the EPS of biofilms. Syto 60 is a cell permeable dye used as a counterstain16. Observations of Toto 1 and Syto 60 iodide in wounds inoculated with biofilms of Pseudomonas aeruginosa (Figure 4A-D) and Staphylococcus aureus (Figure 4I-L) showed that after 3 days of dressing treatment, EPS in the biofilm was significantly reduced. containing oxygenated salts Ag and Ag1+ + EDTA/BC. Ag1+ dressings without additional antibiofilm components significantly reduced cell-free DNA in wounds inoculated with Pseudomonas aeruginosa but were less effective in wounds inoculated with Staphylococcus aureus.
In vivo imaging of wound biofilm after 3 days of treatment with control or silver dressings. Confocal images of Pseudomonas aeruginosa (A–D) and Staphylococcus aureus (I–L) stained with Toto 1 (green) to visualize extracellular nucleic acids, a component of extracellular biofilm polymers. To stain intracellular nucleic acids, use Syto 60 (red). acids. P. Scanning electron microscopy of wounds infected with Pseudomonas aeruginosa (E–H) and Staphylococcus aureus (M–P) biofilms after 3 days of treatment with control and silver dressings. SEM scale bar = 5 µm. Confocal imaging scale bar = 50 µm.
Scanning electron microscopy showed that mice inoculated with biofilm colonies of Pseudomonas aeruginosa (Figure 4E-H) and Staphylococcus aureus (Figure 4M-P) had significantly fewer bacteria in their wounds after 3 days of treatment with all silver dressings.
To evaluate the effect of silver dressings on wound inflammation in biofilm-infected mice, sections of biofilm-infected wounds treated with control or silver dressings for 3 days were immunohistochemically stained using antibodies specific for neutrophils and macrophages. Quantitative determination of neutrophils and macrophages internally. granulation tissue. Figure 5). All silver dressings reduced the number of neutrophils and macrophages in wounds infected with Pseudomonas aeruginosa compared with non-antimicrobial control dressings after three days of treatment. However, treatment with the oxygenated silver salt dressing resulted in a greater reduction in neutrophils (p = <0.0001) and macrophages (p = <0.0001) compared to other silver dressings tested (Figure 5I,J). Although Ag1+ + EDTA/BC had a greater effect on wound biofilm, it reduced neutrophil and macrophage levels to a lesser extent than the Ag1+ dressing. Moderate wounds infected with S. aureus biofilm were also observed after dressing with Ag (p = <0.0001), Ag1+ (p = 0.0008) and Ag1++ EDTA/BC (p = 0.0043) oxisols compared to control . Similar trends are observed for neutropenia. bandage (Fig. 5K). However, only the oxygenated Ag salt dressing showed a significant reduction in the number of macrophages in granulation tissue compared with control in wounds infected with S. aureus biofilms (p = 0.0339) (Figure 5L).
Neutrophils and macrophages were quantified in wounds infected with Pseudomonas aeruginosa and Staphylococcus aureus biofilms after 3 days of treatment with non-antimicrobial control or silver dressings. Neutrophils (AD) and macrophages (EH) were quantified in tissue sections stained with antibodies specific for neutrophils or macrophages. Quantification of neutrophils (I and K) and macrophages (J and L) in wounds infected with Pseudomonas aeruginosa (I and J) and Staphylococcus aureus (K & L) biofilms. N = 12 per group. Graphs show mean +/- standard error, significance values compared to non-antibacterial control dressing, * means p = < 0.05, ** means p = < 0.01; *** means p = < 0.001; indicates p = <0.0001).
We then assessed the effect of silver dressings on infection-independent healing. Non-infected excisional wounds were treated with a non-antimicrobial control dressing or a silver dressing for 3 days (Figure 6). Among the silver dressings tested, only wounds treated with the oxygenated salt dressing appeared smaller on macroscopic images than wounds treated with the control (Figure 6A-D). Quantification of wound area using histological analysis showed that the average wound area after treatment with the Ag oxysols dressing was 2.35 mm2 compared to 2.96 mm2 for wounds treated with the control group, but this difference did not reach statistical significance (p = 0.488 ) (Fig. 6I). In contrast, no reduction in wound area was observed after treatment with Ag1+ (3.38 mm2, p = 0.757) or Ag1+ + EDTA/BC (4.18 mm2, p = 0.054) dressings compared to the control group. Increased epithelial regeneration was observed with the Ag oxysol dressing compared to the control group (30% vs. 22%, respectively), although this did not reach significance (p = 0.067), this is quite significant and confirms previous results. A dressing with oxysols promotes re-epithelialization. -Epithelization of uninfected wounds17. In contrast, treatment with Ag1+ or Ag1+ + EDTA/BC dressings had no effect or showed decreased re-epithelialization compared to control.
Effect of silver wound dressing on wound healing in uninfected mice with complete resection. (AD) Representative macroscopic images of wounds after three days of treatment with a non-antimicrobial control dressing and a silver dressing. (EH) Representative wound sections stained with hematoxylin and eosin. Quantification of wound area (I) and percentage of reepithelialization (J) were calculated from histological sections at the midpoint of the wound using image analysis software (n = 11–12 per treatment group). Graphs show mean +/- standard error. * means p = <0.05.
Silver has a long history of use as an antimicrobial therapy in wound healing, but the many different formulations and delivery methods may result in differences in antimicrobial efficacy 18 . Moreover, the antibiofilm properties of specific silver delivery systems are not fully understood. Although the host immune response is relatively effective against planktonic bacteria, it is generally less effective against biofilms19. Planktonic bacteria are readily phagocytosed by macrophages, but within biofilms, aggregated cells pose additional problems by limiting the host response to the extent that immune cells can undergo apoptosis and release proinflammatory factors to enhance the immune response20. It has been observed that some leukocytes can penetrate biofilms21 but are unable to phagocytose bacteria once this defense is compromised22. A holistic approach should be used to support the host immune response against wound biofilm infection. Wound debridement can physically disrupt the biofilm and remove most of the bioburden, but the host immune response may be ineffective against remaining biofilm, especially if the host immune response is compromised. Thus, antimicrobial therapies such as silver dressings can support the host immune response and eliminate biofilm infections. Composition, concentration, solubility, and delivery substrate can influence the antimicrobial effectiveness of silver. In recent years, advances in silver processing technology have made these dressings more effective9,23. As silver dressing technology advances, it is important to understand the effectiveness of these dressings in controlling wound infection and, more importantly, the impact of these potent forms of silver on the wound environment and healing.
In this study, we compared the effectiveness of two advanced silver dressings with conventional silver dressings that produce Ag1+ ions against biofilms using different in vitro and in vivo models. We also assessed the effect of these dressings on the wound environment and infection-independent healing. To minimize the influence of the delivery matrix, all silver dressings tested were composed of carboxymethylcellulose.
Our preliminary evaluation of these silver dressings against colonial biofilms of Pseudomonas aeruginosa and Staphylococcus aureus shows that, unlike traditional Ag1+ dressings, two advanced silver dressings, Ag1+ + EDTA/BC and oxygenated Ag salts, are effective at 5. Effectively killing biofilm bacteria in within a few days. In addition, these dressings prevent re-formation of biofilm upon repeated exposure to planktonic bacteria. The Ag1+ dressing contained silver chloride, the same silver compound and base matrix as Ag1+ + EDTA/BC, and had a limited effect on bacterial viability within the biofilm over the same period. The observation that an Ag1+ + EDTA/BC dressing was more effective against biofilm than an Ag1+ dressing consisting of the same matrix and a silver compound supports the notion that additional ingredients are required to increase the effectiveness of silver chloride against biofilm, as has been reported elsewhere15. These results support the idea that BC and EDTA play an additional role contributing to overall dressing effectiveness and that the absence of this component in Ag1+ dressings may have contributed to the failure to demonstrate in vitro efficacy. We found that oxygenated Ag salt dressings producing Ag2+ and Ag3+ ions exhibited stronger antibacterial efficacy than Ag1+ and at levels similar to Ag1+ + EDTA/BC. However, due to the high redox potential, it is unclear how long Ag3+ ions remain active and effective against wound biofilms and therefore merit further study. Additionally, there are many different dressings that generate Ag1+ ions that were not tested in this study. These dressings are composed of different silver compounds, concentrations, and base matrices, which may influence the delivery of Ag1+ ions and their effectiveness against biofilms. It is also worth noting that there are many different in vitro and in vivo models used to evaluate the effectiveness of wound dressings against biofilms. The type of model used, as well as the salt and protein content of the media used in these models, will influence the effectiveness of the dressing. In our in vivo model, we allowed the biofilm to mature in vitro and then transferred it to the dermal surface of the wound. The host mouse immune response is relatively effective against planktonic bacteria applied to the wound, thereby forming a biofilm as the wound heals. The addition of mature biofilm to a wound limits the effectiveness of the host immune response to biofilm formation by allowing the mature biofilm to establish itself within the wound before healing can begin. Thus, our model allows us to evaluate the effectiveness of antimicrobial dressings on mature biofilms before wounds begin to heal.
We also found that dressing fit influenced the effectiveness of silver dressings on in vitro-grown biofilms and porcine skin. Close contact with the wound is considered critical for the antimicrobial effectiveness of the dressing24,25. Dressings containing oxygenated Ag salts were in close contact with mature biofilms, resulting in a significant reduction in the number of viable bacteria within the biofilm after 24 hours. In contrast, when treated with Ag1+ and Ag1+ + EDTA/BC dressings, significant numbers of viable bacteria remained. These dressings contain sutures along the entire length of the dressing, which creates dead spaces that prevent close contact with the biofilm. In our in vitro studies, these non-contact areas prevented the killing of viable bacteria within the biofilm. We assessed bacterial viability only after 24 hours of treatment; Over time, as the dressing becomes more saturated, there may be less dead space, reducing the area for these viable bacteria. However, this highlights the importance of the composition of the dressing, not just the type of silver in the dressing.
While in vitro studies are useful for comparing the effectiveness of different silver technologies, it is also important to understand the effects of these dressings on biofilms in vivo, where host tissue and immune responses contribute to the effectiveness of the dressings against biofilms. The effect of these dressings on wound biofilms was observed using scanning electron microscopy and EPS staining of the biofilm using intracellular and extracellular DNA dyes. We found that after 3 days of treatment, all dressings were effective in reducing cell-free DNA in biofilm-infected wounds, but the Ag1+ dressing was less effective in Staphylococcus aureus-infected wounds. Scanning electron microscopy also showed that significantly less bacteria were present in wounds treated with the silver dressings, although this was more pronounced with the oxygenated Ag salt dressing and the Ag1+ + EDTA/BC dressing compared to the Ag1+ dressing. These data show that the silver dressings tested had varying degrees of impact on biofilm structure, but none of the silver dressings were able to eradicate the biofilm, supporting the need for a holistic approach to the treatment of wound biofilm infections; use of silver armbands. Treatment is preceded by physical debridement to remove most of the biofilm.
Chronic wounds are often in a state of severe inflammation, with excess inflammatory cells remaining in the wound tissue for an extended period of time, causing tissue damage and depleting the oxygen needed for efficient cellular metabolism and function in the wound26. Biofilms exacerbate this hostile wound environment by negatively affecting healing in a variety of ways, including inhibition of cell proliferation and migration and activation of proinflammatory cytokines27. As silver dressings become more effective, it is important to understand the impact they have on the wound environment and healing.
Interestingly, although all silver dressings affected biofilm composition, only oxygenated silver salt dressings increased re-epithelialization of these infected wounds. These data support our previous findings17 and those of Kalan et al. (2017)28, which demonstrated good safety and toxicity profiles of oxygenated silver salts, as lower concentrations of silver were effective against biofilms.
Our current study highlights the differences in silver technology between antimicrobial silver dressings and the impact of this technology on the wound environment and infection-independent healing. However, these results differ from previous studies showing that Ag1+ + EDTA/BC dressing improved healing parameters of injured rabbit ears in vivo. However, this may be due to differences in animal models, measurement times, and bacterial application methods29. In this case, wound measurements were taken 12 days after injury to allow the active ingredients of the dressing to act on the biofilm over a longer period of time. This is supported by a study that showed that clinically infected leg ulcers treated with Ag1+ + EDTA/BC initially increased in size after one week of treatment, and then over the next 3 weeks of treatment with Ag1+ + EDTA/BC and within 4 weeks of use of non-antimicrobials. drugs. CMC dressings to reduce the size of ulcers30.
Certain forms and concentrations of silver have previously been shown to be cytotoxic in vitro 11 , but these in vitro results do not always translate into adverse effects in vivo. In addition, advances in silver technology and a better understanding of silver compounds and concentrations in dressings have led to the development of many safe and effective silver dressings. However, as silver dressing technology advances, it is important to understand the impact of these dressings on the wound environment31,32,33. It was previously reported that the increased rate of re-epithelialization corresponds to an increased proportion of anti-inflammatory M2 macrophages compared to the pro-inflammatory M1 phenotype. This was noted in a previous mouse model where silver hydrogel wound dressings were compared with silver sulfadiazine and non-antimicrobial hydrogels34.
Chronic wounds may exhibit excessive inflammation and it has been observed that the presence of excess neutrophils may be detrimental to wound healing35. In a study in neutrophil-depleted mice, the presence of neutrophils delayed reepithelialization. The presence of excess neutrophils leads to high levels of proteases and reactive oxygen species, such as superoxide and hydrogen peroxide, which are associated with chronic and slow-healing wounds37,38. Likewise, an increase in macrophage numbers, if uncontrolled, can lead to delayed wound healing39. This increase is particularly important if macrophages are unable to transition from a pro-inflammatory phenotype to a pro-healing phenotype, resulting in wounds failing to exit the inflammatory phase of healing40. We observed a decrease in neutrophils and macrophages in biofilm-infected wounds after 3 days of treatment with all silver dressings, but the decrease was more pronounced with oxygenated salt dressings. This decrease may be a direct result of the immune response to silver, a response to decreased bioburden, or the wound being in a later stage of healing and therefore immune cells in the wound being reduced. Reducing the number of inflammatory cells in the wound may maintain an environment conducive to wound healing. The mechanism of action of how Ag oxysalts promote infection-independent healing is unclear, but the ability of Ag oxysalts to produce oxygen and destroy harmful levels of hydrogen peroxide, a mediator of inflammation, may explain this and requires further study17.
Chronic non-healing infected wounds pose a problem for both doctors and patients. Although many dressings claim antimicrobial effectiveness, research rarely focuses on other key factors influencing the wound microenvironment. This study shows that different silver technologies have different antimicrobial efficacy and, importantly, different effects on the wound environment and healing, independent of infection. Although these in vitro and in vivo studies show the effectiveness of these dressings in treating wound infections and promoting healing, randomized controlled trials are needed to evaluate the effectiveness of these dressings in the clinic.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Post time: Jul-15-2024