Yu-Hsin Lin1, Wei-Shan Hsu2, Wan-Yu Chung3, Tse-Hao Ko4 & Jui-Hsiang Lin5
1 Department of Biological Science and Technology, China Medical University, Taichung, Taiwan
2 R&D Center, Bio-medical Carbon Technology Co., Ltd., Taichung, Taiwan
3 R&D Center, Bio-medical Carbon Technology Co., Ltd., Taichung, Taiwan
4 Department of Materials Science and Engineering, Feng Chia University, Taichung, Taiwan
5 R&D Center, Bio-medical Carbon Technology Co., Ltd., Taichung, Taiwan
Open wounds contain a certain degree of bacteria on their surface. States of contamination, colonisation, critical colonisation and invasive infection can be categorised depending on the amount and effects of bacteria in the wound (6,7).
Contamination is defined as the presence of non-replicating bacteria in the wound. Colonisation is defined as the presence of replicating bacteria in the wound without any tissue damage. These two states do not delay the healing processes. However, critical colonisation, in which bacteria do not invade the skin tissue, would delay wound healing. Invasive infection is characterised by bacterial density of 105 microorganisms per gram of the skin tissue (7–9).
Delayed wound healing occurs because of the increased levels of endotoxins produced by bacteria (7). Endotoxins are known to elevate the levels of proinflammatory cytokines, which in turn decrease the production of growth factors and the deposition of collagen in wounds, leading to critical colonisation and invasive infection (7,10,11).
Therefore, it is important to keep the bacterial burden in the wound bed below the level of critical colonisation. All the currently available wound dressings promote wound healing and prevent further harm to the wounded region. An ideal wound dressing maintains moisture in the wound, removes excess exudates, is non-adherent, prevents the entry of microorganisms, controls infection, allows gaseous exchange and leaves no foreign particles in the wound (12,13). Although different dressings have different properties, all the dressings function as a barrier (at various degrees) to prevent the entry of external microorganisms. A recent study by Fujiwara et al. put forth a new concept of bacterial retention in a wound dressing.
They proposed that the ability of a dressing to retain bacteria could be important for reducing bacterial burden in the wound bed. In addition to controlling bacterial burden, a wound dressing actively promotes wound healing, thus accelerating the healing processes (14).
In this study, we examined three silver-containing dressings made of different raw materials and compared their efficiency with that of gauze (control). These three dressings are as follows: (i) KoCarbonAg® (Biomedical Carbon Technology Co., Ltd., Taichung, Taiwan) containing activated carbon fibre (ACF) supported by silver particles, (ii) Acticoat 7 (Smith & Nephew, London, UK) containing alternate layers of rayon and silver-coated polyethylene film and (iii) Aquacel® Ag (ConvaTec Inc., Skillman, NJ) containing hydrofibres and ionic silver.We investigated the ability of these three dressings to block and retain bacteria in vitro and their wound healing efficacy in vivo in a rat model.
The bacteria Pseudomonas aeruginosa (ATCC 27853) were grown on tryptic soy broth (TSB) agar plates under
microaerophilic conditions for 24 hours at 37∘C. A single colony was selected and cultured in TSB medium to an optical density of 1⋅0 at 590 nm (OD590) measured by a visible spectrophotometer (Biochrom, England, UK). The suspension was diluted in TSB medium to achieve a final concentration of 1 × 105 colony-forming unit/ml (CFU/ml) for further use.
Analysis of bacterial blocking activity
For analysing the bacterial blocking activity, 6 ×6 cm2 pieces of the dressings were placed on agar plates with their skin-contacting surface facing downwards. Then, 3 ml suspension of P. aeruginosa (1 × 105 CFU/ml) was inoculated in the centre of the dressing on the exposed upper surface. The plates were incubated at 37∘C for 24 hours. The test pieces of the dressings were then removed aseptically, and the plates were further incubated for 24 hours at 37∘C. The area of bacterial growth in each plate was measured using Image Tool 3.0 (UTHSCSA, San Antonio, TX).
Analysis of bacterial retention activity
In a laminar flow cabinet, 3 ml suspension of P. aeruginosa (1 × 105 CFU/ml)was placed in the centre of the 6 × 6 cm2 piece of each test dressing on the skin-contacting surface. The pieces were incubated for 5minutes to allow the complete absorption of the bacterial suspension. The pieces were then placed on plates, with the skin-contacting surface facing downward, and the plates were incubated at 37∘C for 24 hours. The dressing pieces were then removed aseptically, and the agar plates were further incubated for another 24 hours at 37∘C. The area of bacterial growth in each plate was measured using Image Tool 3.0.
Quantification of bacterial quantity
The areas on the agar plates covered by the dressing pieces were cut and transferred to a new culture plate. Then, 10ml of TSB was added, and the plates were incubated at 37∘C. Next, 100 μL cultures incubated for 1 hour and 6 hours were used for water-soluble tetrazolium salt (WST-1) assay (Roche Diagnostics, Mannheim, Germany).
Wound healing and histologic analysis of wound tissue
Animal care and use complied with the 1996 revision of the ‘guide for the care and use of laboratory animals’ prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press. A total of 20 Sprague–Dawley (SD) rats weighing 250–300 g were individually housed in polycarbonate cages maintained at constant temperature (22±2∘C) and humidity (55%). The rats were subjected to a 12:12 hour light–dark cycle and were provided access to food and water. Their average weight and behaviour did not change significantly during the experiment. Rats were anaesthetised with an intraperitoneal injection of ketamine (90 mg/kg) with xylazine (10mg/kg) (15). The dorsal skin was shaved, application fields were outlined with a marker pen before skin excision and the surgical area was disinfected with 70% ethanol. On the back of each rat,
a full-thickness wound with dimensions of 1 × 1 cm2 was created on each side of the spine by dermo-epidermic excision.
Each of the wounded rats received 100 μl of P. aeruginosa (1⋅5 OD590) on the wound surface. Wound dressings with dimensions of 2 × 2 cm2 were applied onto the wounded rats after 24- hour infection. Each dressing was covered with a sterile compress secured with a hypoallergenic elastic adhesive bandage. Animals were caged individually following identification. The dressings were changed every 3 days; the wounds were photographed and examined to determine wound size reduction. Wound size measurements taken during surgery and at biopsy were used to calculate percentage size reductions of wounds (16).
Wound size reduction (%) = [(A0 –At) ∕A0] × 100
where A0 and At denote initial wound area and wound area after time interval ‘t’, respectively. Wound area was measured from photographs using the Image-Pro Plus (Media Cybernetics, Silver Spring, MD) following calibration. Histologic analysis on day 3 was carried out using light microscopy. Briefly, biopsies were fixed in buffered paraformaldehyde and embedded in paraffin wax (17,18). Sections of 5 μm were stained with Masson’s trichrome. The stained sections of each test sample were then examined under a light microscope for the analysis of collagen synthesis.
Western blotting analysis of wound tissue
Wound tissue extracts were fractionated by performing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene difluoride membrane by using a transfer apparatus (Bio-Rad Laboratories Inc., Hercules, CA), according to the manufacturer’s protocols. After incubation with 5% non-fat milk in TBST (10mM Tris pH 8⋅0, 150mM NaCl, and 0⋅05% Tween-20) for 60minutes, the membrane was washed once with TBST and was incubated with primary antibodies against rabbit anti-collagen-1 antibody (1:500 dilution) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000 dilution) at 4∘C for 16 hours.
The membrane was then washed three times and was incubated with horseradish peroxidase–conjugated anti-rabbit or anti-mouse antibodies (1:5000 dilution) for 1 hour. The blots were washed three times with TBST and the proteins were visualised by enhanced chemiluminescence western blotting detection reagents (GE Healthcare, Little Chalfont, UK) and detected by X-ray films (Kodak, New York, NY). Concentration of collagen-1 was quantified using ImageJ software (National Institute of Health, Bethesda, MD) after normalisation with GAPDH protein.
Ability of the dressings to block the entry of bacteria from external environment
An important function of a wound dressing is to protect the wound area from any contamination from the external environment. In this study, the ability of the three dressings to block the entry of bacteria from the external environment was tested. Percentage bacterial growth on the agar plates after inoculating P. aeruginosa on the exposed surface of the dressings was investigated.
Data obtained from image analysis showed that growth area of P. aeruginosa on plates covered by gauze was 80⋅3%±4⋅9% of the covered area, indicating that gauze barely protected the wound area from the external environment (Figure 1A and B). The gauze blocked only 20% of the inoculated bacteria, and the remaining bacteria penetrated through the gauze and colonised the wound area. The growth area of P. aeruginosa on plates covered by KoCarbonAg® was 0⋅41%±0⋅3% of the covered area, indicating that KoCarbonAg® blocked the entry of >99% bacteria and prevented them from colonising on the wound area.
The growth areas of P. aeruginosa on plates covered by Acticoat 7 and Aquacel® Ag were 18⋅6%±9⋅6% and 30⋅5%±2⋅4%, respectively, of the covered areas. However, this could be because of the interference by brown colour developed on the plate covered by Acticoat 7 or by fibres left on the plate covered by Aquacel® Ag (Figure 1A).
The amount of bacteria on each plate was quantified by performing the WST-1 assay. Bacteria were harvested from
the plates and were cultured in TBS for up to 6 hours. Relative bacterial counts at 1 hour and 6 hours are represented in
Figure 1C. The plate covered with gauze had large amount of bacteria (15- and 96-fold higher than that in the control plate
at 1 and 6 hours, respectively), which was consistent with the results of image analysis.
Plates covered by KoCarbonAg® and Aquacel® Ag had similar amounts of bacteria at both 1 and 6 hours, whereas the plate covered with Acticoat 7 had slightly increased amount of bacteria (1⋅5- and 2-fold higher amount of bacteria than that in the control plate at 1 and 6 hours, respectively). Results of the WST-1 assay indicated that both Aquacel® Ag and KoCarbonAg® had a similar capacity to protect wounds from environmental contamination, indicating that Aquacel® Ag could also block 99% of external bacteria (Figure 1C). The bacterial amount obtained from the plate covered with Acticoat 7 at 1 hour was approximately 1/10th of that obtained from the plate covered by gauze. Bacterial growth area in the plate covered by gauze was 80⋅3%, while it was estimated to be 8% in the plate covered by Acticoat 7.This indicated that Acticoat 7 blocked approximately 92% of external bacteria.
Ability for retaining bacteria in the dressing
Wound dressing is usually applied to the wounded area from a couple of hours to a few days. Besides the surrounding environment, the dressing itself is another source of bacterial contamination. If the dressing cannot retain the absorbed bacteria, then the bacteria would leak into the wound area to induce inflammation or to colonise, thus delaying wound healing. To examine the ability of the wound dressings to retain the absorbed bacteria, the percentage growth of P. aeruginosa inoculated on the skin-contacting surface of the dressings was investigated.
The image analysis data showed that the growth area of P. aeruginosa on the plate covered by gauze was 93⋅2%±9⋅6% of the covered area, indicating that the gauze could not retain the absorbed bacteria (Figure 2A and B). Only approximately 7% of bacteria were retained in the gauze, and the remaining leaked out and colonised the wound area. The growth area of both 1 and 6 hours). The results of the WST-1 assay indicated that Aquacel® Ag and KoCarbonAg® had a similar capacity to retain bacteria, indicating that Aquacel® Ag could also prevent 99% of bacteria from leaking out. The bacterial amount in the Acticoat 7-covered plate at 1 hour was approximately 2⋅2/14 of that in the gauze-covered plate (Figure 2C). Bacterial growth area in the gauze-covered plate was 93⋅2% of the covered area, while that in the Acticoat 7-covered plate was estimated to be 15%, which indicated that Acticoat 7 prevented approximately 85% bacteria from leaking out. The results of blockage and retention tests also represent the antimicrobial activity of these silver-coated dressings. The results of the WST-1 Assay indicated that KoCarbonAg® and Aquacel® Ag eliminated almost all the bacteria that escaped from the dressing. However, Acticoat 7, which contains the highest concentration of silver (1⋅7mg/cm2) among these three dressings, could not eliminate all the bacteria that escaped the dressing.
(Figure 2a, 2b and 2c below)
Wound healing is a complicated process involving many factors that interact reciprocally. Control of bacterial burden is critical in the early phase of wound healing. In the proliferation phase, active promotion of wound healing is a prominent factor. In this study, we showed that the silver-containing dressing KoCarbonAg® not only reduced bacterial burden in the inflammatory phase but also promoted wound healing in the proliferation phase by promoting the production of collagen-1. Although an ideal dressing might not exist, choosing a multifunctional dressing might be cost-effective and helpful to both
patients and clinicians.
The authors would like to thank Enago (http://www.enago.tw) for the English language review.
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