Silver Ion-releasing Dressing (CONTREET) Does Not Disturb Angiogenesis and Microvascularization In Vivo

Disclosure: Coloplast GmbH Hamburg provided financial support and the dressings used in this study.

Disturbed development of new microvasculature may affect the wound healing process. Presently, dressing containing silver are widely used for wound management. Silver-based dressings, which release silver ions into the wound bed, are available in many varieties—particularly for the treatment of burns, chronic leg ulcers, and infected wounds. Some data seems to support the clinical use of these products.¹ However, recent findings indicate that the silver may affect the wound healing process.² Contreet® (Coloplast, Holtedam, Denmark) is one of the new silver-releasing wound dressings designed for restarting the healing process in
infected wounds. The active component of this foam dressing is ionized silver (silver/sodium/hydrogen/zirconium/phosphate). The silver compounds are homogeneously distributed within the polyurethane matrix. A sustained release of silver for up to 7 days is guaranteed.³ The silver-based dressing is supposed to possess antimicrobial activity and reduce infection in wounds. Preliminary clinical trials have found this silver-containing dressing promotes healing in infected chronic venous leg ulcers and diabetic foot ulcers.^1,4–6 In addition, effectiveness against a wide range of microorganisms including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) was reported. An induction of bacterial resistance was not detected in these in-vitro studies.^2,7,8 Cost-effective faster wound healing in bacterially challenged, delayed healing leg ulcers was shown in a 4-week health economic analysis for Contreet as compared to alternative antiseptic dressings.⁹
Aside from the encouraging reports regarding this silver-based dressing in wound management, in-vivo studies concerning the impact of silver on microcirculation have not been performed as of yet. Despite extensive testing and clinical experience, not much is known about the effect of ionic silver on wound angiogenesis, therefore the influence of silver ions rebased from this product has only been explored here.
The aim of this study was to investigate the effects of a silver-based, polyurethane dressing (Contreet) versus silver-free polyurethane (Biatain™, Coloplast, Hotledam, Denmark) and alginate dressings (SeaSorb®, Coloplast, Hotledam, Denmark) on specific wound healing events, such as angiogenesis and vascularization. The investigations were performed in an established microcirculatory model using the dorsal skinfold chamber of BALB/c mice.

Materials and Methods

Animals. The BALB/c mice were 12–14 weeks old and weighed 18–22 g. They were kept at 21˚C in a normal 12/12 hours of light/dark pattern and fed a laboratory diet and water ad libitum. The experiments were conducted in accordance with German law regarding protection and welfare of laboratory animals.
Dorsal skinfold chamber. The dorsal skinfold chamber is a widely used and accepted model in microcirculatory research in hamsters and mice.^10–13 The animals were anesthetized by intramuscular injection of ketamine (100 mg/kg body weight) and xylazine 2% (10 mg/kg body weight). After chemical depilation, an extended double layer of skin from the dorsal skinfold was sandwiched between 2 symmetrical titanium frames (frame to frame distance = 400–450 µm). A circular area 15 mm in diameter from 1 layer of skin was completely removed and the remaining layer (epidermis, subcutaneous tissue, thin striated skin muscle) was covered with a glass coverslip incorporated into 1 of the titanium frames. All surgical procedures were performed under sterile conditions. The animals tolerated the chamber well showing no signs of discomfort or effects on eating or sleeping habits.
Wound dressing implants. The noninfected dorsal skinfold chamber was used to make the microvascular measurements after incorporation of implants. Thirty BALB/c mice were assigned to 3 groups. Group 1: Contreet (silver-containing, porous hydrocolloid polyurethane foam, silver content: 1 mg/cm²); Group 2: Biatain (silver-free, porous hydrocolloid polyurethane foam); Group 3: SeaSorb Soft (silver-free, porous amorphous structure of 85% calcium alginate and 15% CMC hydrofiber). Discoid implants of 2 mm diameter and an average thickness of 300–400 µm were used. Ten implants were studied per group. After preparation of the implants from the sterile wound dressings they were single packed, randomized, labeled, and stored in a dark area prior to implantation. All chambers showed intact microcirculation 48 hours after chamber preparation. The animals were again anesthetized. The cover glass of the chamber preparation was opened and a single implant transferred onto the striated skin muscle of the chamber. The chamber preparation was closed with a new, sterile cover glass. Special care was taken to avoid drying of the skin muscle. The animals were released to their cages and had free access to food and water.
Intravital fluorescence microscopy (IFM). After incorporation of implants animals were allowed a recovery period of 72 hours to exclude surgical effects on the microvasculature. The alert mice were then immobilized in a Plexiglas tube and the chamber was attached to the microscope stage of the IFM setup. The setup allowed for repeat scanning of identical tissue areas containing microvessels. A 40-fold water immersion objective (Zeiss, Axiotech Vario 100 HD, Acroplan 20 x 0.5 W, Zeiss, Oberkochen, Germany) was used to observe the microvessels. The total on-screen magnification was 355-fold under epi-illumination. The images were captured using a charge coupled device video camera and digitalized (Pinnacle MovieBox, Pinnacle Systems, Braunschweig, Germany) on hard disc for later off-line analysis. Epi-illumination was achieved using a 12 V, 100 W halogen lamp in conjunction with a Zeiss filter set (BP 450-490, FT 510, LP 520) for measurements after intravenous (IV) injection of fluorescein-isothiocyanate labeled dextran (FITC-Dextran, MW 500.000, Sigma Chemicals Co, St. Louis, Mo).
Histology. At the end of the protocol (Day 12 post implantation) the animals were sacrificed with an overdose of intraperitoneal pentobarbital. Implants with underlying and surrounding skin tissue were harvested and processed for histological evaluation. The morphological alterations of the vascular development were analyzed with light microscopy. After fixation in a 5.0% formaldehyde solution, the specimens were divided, oriented, and placed in a processing cassette. They were taken through a graded ethanol series and embedded in paraffin for a complete edge-to-edge cross sectional view of the implant disc. Semi-thin (~ 5 µm) tissue sections (along with the implant) were obtained using a microtome. Sections were deparaffinized and standard stained with hematoxylin and eosin. Inflammation, fibrosis, and vascularity were judged qualitatively using computer assistance (analySIS®, Soft Imaging System, Muenster, Germany).
Experimental procedure and study design. For the IFM measurements, 0.10 mL of 5% FITC-Dextran was injected into a tail vein for contrast enhancement prior to each measurement. Observations were performed in 2 different areas: surrounding host tissue (striated skin muscle) and border zone of the implant. In each area, 3 different microvascular regions of interest were selected, recorded, and their X-Y coordinates stored on the computer so that it was possible to re-locate the identical areas within the chamber. Measurements were performed on Day 3, and repeated on Day 7 and Day 12 following implantation. At the end of the observations the animals were released to their cages.
Data acquisition and statistics. Analysis of the digital images was performed offline using the commercially available CapImage® computer program (Zeintl, Heidelberg, Germany). The functional vessel density (the length of perfused vessels per observation area, [mm/mm²]) was assessed.¹
The commercially available program SigmaStat™ (Jandel Scientific, San Rafael, Calif) was used for statistical analysis of the data. Comparison between groups was performed using Kruskal-Wallis 1-way analysis of variance on ranks followed by a pair-wise multiple comparison procedure (Bonferroni t-test) in case of significant differences. A P value < 0.05 was considered statistically significant.

Results

No infection was detected during the examination of all chambers. Erythema, exudate, exuberant granulation tissue, or poor hair growth (signs of inflammation) were not observed. Photographs documented the characteristics of the macroscopical observation during the implantation time (Figure 1 A–C). From about Day 3 of treatment, wounds exposed to silver-releasing dressings showed no significant disturbance in the development of new blood vessels. Intravital fluorescence microscopy revealed a constant development of a well-perfused microvessel network surrounding the implants in all groups. The intensity of new blood vessel development was expressed differently for the various implant types. An increased density of perfused microvessels in the border zone of the implants was already observed on Day 3 post implantation. However, on Day 3 and even more so on Day 7, Group 1 showed stronger morphological alteration of mature microvessel network at the border zone compared to Group 2 and 3 (the “silver-free” groups). Reinforced dilatation, elongation, and torsion of capillaries as well as a progressive growth of already perfused preliminary stages of new blood vessels were observed for silver-based implants (Figure 2A and B). Also the FVD of the border zone of silver-based implants on Day 3 (257.1 ± 33.9 mm/mm²⁾ had significantly increased compared to Group 2 (230.0 ± 31.30) and Group 3 (210.1 ± 35.7) at this observation time. At Day 7, the FVD in Group 1 was still significantly higher than that measured in Group 3 (265.9 ± 30.7 versus 219.7 ± 42.6). However, no significant difference in FVD between Groups 1 and 2 on Day 7 (265.9 ± 30.7 versus 269.5 ± 31.9), and on Day 12 (258.7 ± 38.1 versus 268.3 ± 39) remained (Figure 3). The intravital microscopical observations of border zones of the silver containing implants still showed a well perfused network of new microvessels (Figure 2C). Functional vessel density of the surrounding host tissue (striated skin muscle) did not change significantly over the observation period in all groups and ranged from 110.6 to 120.2 mm/mm² on Day 3 and from 119.5 to 124.1 mm/mm² on Day 12. A normal morphology of parallel-distributed perfused capillaries was observed throughout the implantation time within the surrounding skin muscle tissue (Figure 2D).
The morphological alterations within the border zone of implants were detected by light microscopy. Under light microscopy, a reduced development of vascularized granulation tissue surrounding the hydrofiber alginate implants (Group 3) as well as reduced ingrowth of new developed microvessels into implants was found (Figure 4A). In contrast, the matrix of silver-containing polyurethane foam implants showed vascularized granulation tissue formation at the border zone (Figure 4B).

Discussion

Silver has been used as an antibacterial agent for many years in wound treatment. Today, silver is integrated as an active component in many new wound dressings. Understanding the action of silver in vivo is essential for its clinical use in silver-containing products. The bioactive properties of the dressings imparted by the silver coating and its harmful influence on the microvascular aspects of wound healing and tissue repair cannot be excluded. Recent findings indicate that silver delays the wound-healing process. Topically applied silver sulfadiazine (SSD 2%) was found to be highly cytotoxic in cultured human dermal fibroblast cells.¹⁴ Another in-vitro study¹⁵ showed that silver released from nitrate solution as well as nanocrystalline silver released from a commercially available dressing was highly toxic to keratinocytes and fibroblasts in monolayer culture. The cytotoxity of a polyethylene mesh coated with nanocrystalline silver has also been demonstrated on cultured human keratinocytes cultivated on a hyaluronate-derived membrane using dermal fibroblasts as the feeder layer.¹⁶ The cytotoxicity of silver to fibroblast and keratinocytes reported by the in-vitro tests is important in understanding wound healing pathophysiology. Not much is known about the influence of silver on the microvasculature. A study analyzing the
in-vivo nutritive perfusion and leukocytic response after implantation of commercially available pure silver samples demonstrated a distinct and persistent activation of leukocytes combined with a marked disruption of microvascular endothelial integrity. A massive extravasation of leukocytes and considerable venular dilatation could be detected.¹⁷
The mechanism of action of silver during wound healing is unclear. The investigation of the impact of silver-based wound dressings on angiogenesis is clinically relevant. Disturbed growth of new blood vessels may lead to impairment of microcirculation, and consequently, the wound healing process. This study examined the effect of a silver-releasing wound dressing on angiogenesis and vascularization in noninfected wounds to evaluate whether exposure to silver dressing decreases the growth of new blood vessels. Using IVM, constant development of a sufficient network of well-perfused microvessels surrounding the silver-based implants was demonstrated. The exposure to silver did not affect the growth of new blood vessels. The silver-based implants did not show any anti-angiogenic effects compared to the silver-free implants that were used. On the contrary, a stronger induction of new blood vessel development as well as a significantly higher FVD was observed for silver-releasing implants.

Conclusions

Silver release to a striated skin muscle does not affect angiogenesis and microvascularization as compared to silver-free implants. Moreover, the FVD of the border zone of silver based implants was found to be significantly increased compared to silver-free implants at the first observation time point. Subsequently, no potentially important differences in the microvascular response between the study groups were detected. The silver-releasing implants showed no anti-angiogenic activity. The similarity of the microcirculatory observations of silver-free and silver-based dressings confirms the favorable biocompatibility of the silver ions in the dressings used in this study.

Acknowledgment
We acknowledge Dr. Inge Schmitz from the Department of Pathology, University Hospital Bergmannsheil for the histology work. We thank Coloplast for providing the materials used in this study and for funding this research.