Silver Deposition and Tissue Staining Associated with Wound Dressings Containing Silver

    Silver has long been recognized for its broad-spectrum antimicrobial activity and compatibility with mammalian tissues. Several reviews have been written on its historical use,^1,2 antimicrobial properties,³ and toxicity.⁴

Silver’s latest resurgence began in the mid-1960s when Moyer and colleagues⁵ revitalized interest in the use of silver salts and silver salt solutions in the treatment of burn patients. Consequently, silver became available in a variety of formulations — eg, as a colloidal silver suspension, a dilute salt solution (0.5% silver nitrate solution), in cream formulations containing silver sulfadiazine (SSD —1% w/w), and more recently in wound dressings that contain silver.

    Many of the earlier colloidal silver products had high silver concentrations (30% or more)⁶ and several reports of facial discoloration (a condition known as argyria) following excessive colloidal ingestion have recently been published.^6,7 Argyria is now considered a rare cutaneous disorder and is thought to be relatively benign apart from causing possible social embarrassment due to permanent skin discoloration.

    Silver nitrate solution (0.5%) has been used extensively in the treatment of burns; however, one of the major drawbacks to using this dilute silver solution is its ability to blacken the skin surface following topical application, due to light exposure.⁴ Although this form of staining may be considered transient, it is still unpleasant and perceived as clinically unacceptable.⁴

    The silver-containing wound dressings presently available have a wide variety of material components, ranging from polyurethane foams, hydrocolloids, and Hydrofiber® to nanocrystalline-coated and charcoal-containing products. Dressing manufacturers have used many technologies to provide ionic silver, from simple ionic silver salt particulate to nanocrystalline high surface area silver coating.

    These dressings contain different amounts of silver and their ability to make ionic silver available is also variable, which has been shown in recent in vitro studies where the antimicrobial properties of several silver-containing dressings were tested.⁸ The results indicated that while a nanocrystalline silver dressing (NSD) produced a rapid antimicrobial effect, evidence of antimicrobial activity using a polyurethane silver-containing foam were not convincing.⁸ Another study using microcorimetry has shown antimicrobial activity equivalence between the silver-containing Hydrofiber® dressing (SHD) and the nanocrystalline dressing.⁹

    Although a great deal of the recent literature speaks to the benefits of topical silver products,^4,10-12 little has been published on the availability of silver, how it may create an environment that supports wound healing,^11,13 its metabolic fate, or its deposition either within the wound or on surrounding skin. Lansdown⁴ recently suggested that silver is taken up by epidermal cells at the wound margin. Others have shown that silver has been found in blood,¹⁴ urine,¹⁴ kidney, and liver.¹⁵ In a porcine wound study,¹³ a gray coloration of the wound tissue was observed following the application of a nanocrystalline dressing. It has been suggested that this is due to silver precipitation associated with the keratinized epidermal layer.

    Clinical evidence suggests that silver deposition onto human skin occurs following the application of silver-containing dressings.^16,17 Although this discoloration appears to produce no negative side effects or have an adverse effect on patient health and safety and may be considered transient, no studies to date have attempted to correlate silver availability rates with skin discoloration. With this in mind, the purpose of the current studies was to analyze the silver content, release profiles, and deposition of two silver-containing dressings using a human skin in vitro model.⁵

Methods

    Test materials. A silver-containing Hydrofiber® dressing (SHD; AQUACEL® Ag, ConvaTec, a Bristol-Myers Squibb Company, Princeton, NJ) and an nanocrystalline silver-containing dressing (NSD; Acticoat™, Smith and Nephew Inc, Largo, Fla) were investigated for their silver content, silver release, and possible human skin deposition. Silver nitrate solutions (0.5% w/v) were prepared in both water and physiological saline (0.9% w/v) and used as positive controls and saline (0.9% w/v) and water were used as the negative controls in human skin studies using tissue from electively amputated (EA) lower limbs. Ethical approval to allow the use of human skin from lower limb amputations was granted by North East Wales Trust, Wrexham Maelor Hospital, Ethics Committee.

    Determination of silver content in silver-containing dressings. One-tenth gram of each dressing was dissolved by the addition of 1 mL of nitric acid (specific gravity [s.g.] 1.42), followed by the addition of 5 mL sulfuric acid (s.g. 1.84, diluted to 66% v/v). This mixture was brought to a boil and when nitrogen dioxide production ceased, 20 mL of distilled water was added. The mixture was re-boiled for 2 to 3 minutes and an additional 10 mL of distilled water was added. When the solution had cooled, it was volumetrically adjusted to yield a solution of between 1 and 4 parts per million (ppm), where 1 ppm = 1µg/mL, and then assayed directly by atomic absorption spectroscopy (AAS). The silver content of each dressing was expressed as a percentage of the dry weight of the dressing (% w/w). A minimum of five determinations was performed for each dressing.

    Availability of silver from silver-containing dressings. Two series of silver availability experiments were conducted. The first was performed under the maximum solubility conditions of an excess volume (120 mL) of constantly stirred distilled water held at a constant 37°C (the use of sterile water for pre-moistening dressings is recommended in the NSD manufacturer’s instructions). The second series of studies used saline (0.9% w/v), chosen to mimic the physiological conditions likely to be encountered in a wound environment (the presence of sodium and chloride ions in wound exudate).

    A fixed test sample area (25 cm²) was used for both dressings. A sample of dissolution liquor (25 mL) was taken after 5 hours and then daily for the next 4 days. Each sample volume removed was replaced with an equal volume of distilled water or saline. Silver concentration in this liquor then was assayed directly by AAS, diluting into the range of the standard calibration curve as necessary. These experiments were performed on five replicate samples for each dressing; the data were expressed as the amount of silver released in µg/mL, taking into account volume corrections and dilution factors.

    In vitro whole human skin staining studies. The skin-staining studies were conducted using an adaptation of a Franz-type horizontal glass diffusion cell, described previously.¹⁸ Briefly, human whole skin samples (1-cm² discs) were punched out using an Osborne 147 punch (Montana Leather Co., Billings, Mont), and placed epidermal side uppermost on the receptor chamber. The donor chamber was placed carefully on top of the receptor chamber. The whole cell was clamped together and placed into a water bath maintained at 32^oC (± 1ºC). The underside of the skin was bathed in distilled water (receptor volume approximately 1.8 mL). The exposed epidermal surface area available for application of silver-containing dressings was 0.64 cm². Individual silver-containing wound dressings (0.64 cm²) were cut and placed onto the exposed human whole skin via the open donor (top) chamber. The SHD and NSD then were hydrated with 200 µL distilled water or saline (0.9%). For positive and negative controls, 200 µl silver nitrate solutions (0.5%) (in distilled water and saline) and 200 µl saline (0.9%), respectively, were used. A minimum of five individual measurements were taken for each topical application. Each donor chamber was covered in Parafilm® (American Can Co., Greenwich, Conn.) to ensure no fluid was lost. Each cell then was left at an ambient temperature (~21°C) for 24 hours and photographs were taken using a Polaroid digital microscope camera (DMC 1e, Polaroid Corp., Waltham, Mass.).

    In vitro determination of the silver content of whole human skin. Once photographed, each piece of skin was removed from the lower chamber and any excess, non-exposed tissue was excised. The remaining exposed area (0.64 cm²) was weighed and dissolved in 5 mL of nitric acid. When the production of nitrogen dioxide gas ceased, 25 mL of distilled water was added, the solution was assayed directly by AAS, and the amount of silver present per gram of wet tissue was noted in micrograms (µg).

    Statistical analyses. Statistical analyses were performed on the skin-staining data using a one-way analysis of variance (ANOVA) for replicate measurements (Stat 100 Statistical package Version 1.26, Biosoft 1995-96). For the silver release data, a two-way ANOVA was used because computations take into account different dressings and time-points with repeated measurements on one factor (ie, silver release). In both instances, Duncan’s Multiple Comparison Test was applied.

Results

    Silver content in silver-containing dressings. Table 1 presents the dry weight percent (w/w) and a calculated weight of silver per unit area. Because the positive control (silver nitrate solution — 0.5% w/v) was already fully solubilized, it was presented as a bolus dose, making it difficult to quantify an estimated dose per unit area. However, assuming the amount of silver nitrate solution applied was between 0.1 and 1 mL/cm² (an estimate of the clinical dosage), this would equate to a silver concentration equivalent to 0.3175 to 3.175 mg silver/cm².

    Availability of silver from silver-containing dressings. The total amount of silver released expressed in µg/mL (allowing for volume removed corrections and dilution factors, as stated in the Methods) was plotted against time for each dressing material in both distilled water and saline (0.9% w/v) (see Figures 1 and 2, respectively). The data for distilled water indicated a significantly higher amount of silver was released from the NSD than the SHD (P <0.005). The silver available from the NSD maximized at approximately 50 µg/mL over 25 to 30 hours; this level of silver release was maintained throughout the remaining time period of the experiment (105 hours), an amount approximately 50 to 60 times higher than the SHD (see Figure 1 inset).

    In the presence of saline (see Figure 2), the silver ion release profiles for the two dressings were low and almost identical. The greatly reduced availability of silver ions from the NSD, compared to the results in water, was a consequence of precipitation of silver ions with chloride ions down to the solubility limit of silver chloride (approximately 1 µg/mL).¹⁹

    In vitro whole human skin staining studies. The application of an aqueous silver nitrate solution (0.5% w/v) blackened the human skin (see control in Figure a versus Figure 3b), a known contraindication for the application of this solution in wound prophylaxis.⁴ When the silver-containing dressings were hydrated with distilled water, markedly less silver was deposited onto human skin by the SHD, compared to the NSD (see Figures 3c and 3d, respectively).

    Although discoloration was still marked in comparison to the control skin (see Figure 4a), skin discoloration was not as evident when silver nitrate solution was prepared in saline compared to when the solution was prepared in water (see Figure 4b versus Figure 3b). However, white deposits were evident on the skin where silver ions had precipitated with chloride ions (see Figure 4b). Following hydration with saline, silver deposition was shown to be similar to SHD (see Figure 4c) and was less evident with the NSD compared to hydration with water (see Figure 4d).

    In vitro determination of the silver content of whole human skin. A marked silver deposition (733 µg/mL ± 84) was noted after the aqueous silver nitrate solution (0.5% w/v) was applied to human skin that, on exposure to light, turned black (see Figure 3b). The silver deposit was shown to be significantly greater (P <0.005) than that seen with the NSD (107 µg/mL ± 44) and approximately 30 times greater than the amount deposited by the SHD (22 µg/mL ± 7) (see Figures 3d, 3c, and 5, respectively). However attempts to solubilize silver nitrate into saline (0.9% w/v) resulted in the formation of a white milky precipitate due to the minimal solubility of silver chloride. Consequently, this milky precipitate helped reduce staining of the skin surface (see Figure 4b) and silver deposition was reduced approximately three-fold (219 µg/mL ± 115) (see Figure 5).

    Solid particles of silver chloride also were visible on the skin surface (see Figure 4b).

    By contrast, when both dressings were hydrated with saline (0.9% w/v), they showed almost identical silver deposition (NSD — 24 µg/mL ± 15 and SHD — 24 µg/mL ± 7, respectively) (see Figure 5).
Both dressings were shown to be significantly different to the positive control (silver nitrate solution 0.5% w/v) (P <0.005), regardless of the hydration medium. However, only the SHD showed no difference relative to both negative controls (ie, distilled water or saline [0.9% w/v]); whereas, following hydration with distilled water, the NSD was shown to have significantly more silver deposition than the control (P >0.005).

Discussion

    Although pure metallic silver is biologically inert, in its ionized form it is a potent antimicrobial agent at concentrations as low as 0.001 to 0.06 ppm (1µg/mL).^3,12 This oligodynamic activity (active with few molecules or ions) was first described by von Naegelli²⁰ in the nineteenth century and relates to the ability of bacteria to accumulate silver preferentially. Hence, the microbicidal activity of ionic silver is likely to be related to the number of silver ions absorbed (10⁵ to 10⁷ ions per cell).⁴

    Although ionic silver is known to be microbicidal at very low concentrations, currently available silver-containing dressings vary widely in silver content and availability of ionic silver (see Table 1). For a dressing to be both effective and non-staining, the ionic silver in a particular dressing needs to be made available in a low and sustained manner. To highlight this point, two dressings were chosen that had notable differences in silver content (see Table 1). The NSD contained approximately 10 times more silver than the SHD. A direct result of this concentration difference was evident in the amount of silver available from the respective dressings.

    The SHD showed a low but sustained availability of silver ions into distilled water; whereas, the availability of silver ions from the NSD was significantly greater (approximately 50 to 60 times). However, water represents a simple dissolution medium, which is not physiologically appropriate because water lacks important counter ions (ie, anions such as chloride ions) commonly present in wound fluid that will precipitate silver ions to form silver salts (eg, silver chloride).

    During the silver availability studies, it was observed that the plastic dissolution vessels used with the NSD developed a silver lining on the surface. The authors believed this was due to silver deposition and suggest that the technology used to create nanocrystalline clusters on the NSD may allow silver displacement caused by mechanical forces (such as applying or removing the dressing). As illustrated in Figure 6, deposits of metallic silver were observed when the NSD was removed from the skin surface.

    It is unlikely that silver ions penetrate through unbroken skin (as used in these studies) due to their high reactivity with skin proteins; hence, leading to silver deposition.^21,22 Histologically, silver granules have been observed on sweat gland membranes, on connective tissue membranes around sebaceous glands, and in collagen below the basement membrane.²³ The fact that silver has a high affinity for sulfated proteins (eg, thiols, sulfides, and selenosulfides), coupled with the increased silver availability from the NSD in distilled water, likely accounts for the increased silver deposition in human skin seen in these in vitro studies (see Figures 3d and Figure 5, respectively). In particular, complexation with selenium, which is thought to have an essential role in skin keratinization,²⁴ forms a highly insoluble silver selenide residue that effectively reduces the availability of monovalent silver to interfere with normal enzymatic activities in tissues.²⁵ It is thought that this property of the selenium-silver complex may explain the permanence and irreversibility of the metallic tinge in the skin of patients with argyria, the lack of significant systemic toxicity, and unsuccessful attempts at chelation to remove silver from the body.²⁵

    Silver displays no known active transport in human skin. Tregear²⁶ suggested that the chloride concentration in normal skin, if distributed to a depth of 10 µ (approximate depth of stratum corneum) should be near isotonicity (ie, 100 meq/L). This equates to a chloride-to-silver ion ratio of approximately 3,000:1. Therefore, the availability of silver ions will be dramatically reduced by their precipitation with chloride ions down to the solubility limit of silver chloride (approximately 1µg/mL). Ionic silver concentrations in excess of the silver chloride solubility limit will be rapidly reduced by the formation of biologically inactive silver chloride as demonstrated when silver nitrate dissolved in saline caused a milky precipitation of silver chloride. This helps explain why similar levels of skin staining were noted for both dressings in the presence of saline respectively (see Figures 4b [silver nitrate], 4c [SHD], and 4d [NSD]).

    Evidence presented suggests that when using the NSD, metallic silver particles may be deposited (see Figure 6). One possible explanation for this may be that hydration of the dressing with water dislodges metallic nanocrystals from the dressing surface. However, Lium¹⁷ has suggested that this deposition and discoloration of the surrounding skin appears to be transient and disappears after a few days. Others have suggested that the level of transient silver staining of the skin with NSD is similar to that of silver nitrate²⁷ (see Figure 7).

Conclusion

    These laboratory studies have shown that the rate of silver release from dressings, as well as subsequent skin deposition and staining, are influenced by the hydrating solution (water or saline). The application of silver nitrate solution (0.5% w/v) leads to skin blackening, possibly due to either photo-oxidation of silver nitrate or the formation of silver selenide. Two forms of silver — ionic and metallic — are available through the NSD. The former, if made available by hydration with water, can lead to silver deposition in skin. The latter may be deposited by the physical detachment of metallic clusters caused by mechanical forces. When saline (0.9% w/v) was used to represent a more physiologically relevant medium, similar amounts of silver were made available from both silver-containing dressings, which resulted in minimal staining.

    Controlling the release of silver ions from wound dressings that contain silver should help reduce the potential for excessive levels of silver to be deposited in wound tissue. Thus, while antimicrobial efficacy is optimized, skin staining will be minimized.

1. Klasen HJ. A historical review of the use of silver in the treatment of burns. I. Early uses. Burns. 2000;26:117–130.

2. Klasen HJ. A historical review of the use of silver in the treatment of burns. II. Renewed interest in silver. Burns. 2000;26:131–138.

3. Russell AD, Hugo WB. Antimicrobial activity and action of silver. In: Ellis GP, Luscombe DK (eds). Progress in Medicinal Chemistry, Vol. 31. Philadelphia, Pa: Elsevier Science; 2004:351-370.

4. Lansdown ABG. The role of silver. ETRS Bulletin. 2002;9:108–111.

5. Moyer C. Brentano L, Gravens, DL, et al. Treatment of large human burns with 0.5% silver nitrate solution. Arch Surg. 1965;90:812–867.

6. Bouts BA. Images in clinical medicine. Argyria. N Engl J Med. 1999;20;340:1554.

7. White JML, Powell AM, Brady K, Russell-Jones R. Severe generalised argyria secondary to ingestion of colloidal protein. Clin Dermatol. 2003;28:254–256.

8. Thomas S McCubbin P. An in vitro analysis of the antimicrobial properties of 10 silver-containing dressings. J Wound Care. 2003;12:1–4.

9. O’Neill MAA, Vine GJ, Beezer AE, et al. Antimicrobial properties of silver-containing wound dressings: a microcalorimetric study. Int J Pharm. 2003;263:61–68.

10. Burrell RE. A scientific perspective on the use of topical silver preparations. Ostomy Wound Manage. 2004;49(5A Suppl):19S–24S.

11. Wright JB, Lam K, Olson ME, Burrell RE. Is antimicrobial efficacy sufficient? A question concerning the benefits of new dressings. WOUNDS. 2003;15:133–142.

12. Demling RH, DeSanti L. Effects of silver on wound management. WOUNDS. 2001:13(suppl A):4–15.

13. Wright JB, Lam K, Buret A, Olson ME, Burrell RE. Early healing events in a porcine model of contaminated wounds: effects of nanocrystalline silver on matrix proteinases, cell apotosis, and healing. Wound Rep Regen. 2002;10:141–151.

14. Wan AT, Conyers RAJ, Coombs CJ, Masterton JP. Determination of silver in blood urine and tissue of volunteers and burn patients. Clin Chem. 1991;37:1683–1687.

15. Wang X-W, Wang NZ, Zhang OZ, Zapata-Sirvent RL, Davies JWL. Tissue deposition of silver following topical use of silver sulfadiazine in extensive burns. Burns. 1985;11:197–201.

16. Clennett S, Hosking G. Management of toxic epidermal necrolysis in a 15-year-old girl. J Wound Care. 2003;12:151–154.

17. Lium A. The use of a silver coated dressing Acticoat® in the treatment of infected wounds following orthopaedic surgery. Six case studies. Presented at European Bone and Joint Infection Society Meeting, Trondheim, 2003.

18. Dugard PH, Walker M, Mawdsley, SJ, Scott, RC. Absorption of some glycol ethers through human skin in vitro. Environ Health Pers. 1984;57:193–197.

19. Lide DR. Handbook of Chemistry and Physics, 72nd edition. Boca Rato, Fla: CRC Press;1991.

20. von Naegeli V. Deut schr Schweiz. Naturforsh Ges. 1893;33:174–182.

21. Skog E, Whalberg JE. A comparative investigation of the percutaneous absorption of metal compounds in the guinea pig by means of the radio isotopes 51Cr, 58Co, 65Zn, 110mAg, 115mCd, 200Hg. J Invest Dermatol. 1964;43:187–192.

22. Hoestynek JJ, Hinz RS, Lorence CR, Price M, Guy RH. Metals and the skin. Critic Rev Toxicol. 1993;23:171–235.

23. Buckley WR, Oster CF, Fasset DW. Localised argyria. II. Chemical nature of the silver containing particles. Arch Dermatol. 1965;92:697–705.

24. Molokhia A, Portnoy B, Dyer A. Neutron activation of trace elements in the skin. VIII. Selenium in normal skin. Br J Dermatol. 1977;101:567–572.

25. Aaseth J, Olsen A, Halse J, Hovig T. Argyria-tissue deposition of silver as selenide. Scand J Clin Lab Invest. 1981;41:247–251.

26. Tregear TR. Physical function of skin. In: Danielli JF (consult ed.) Theoretical and Experimental Biology, Vol. 5. London, UK: Academic Press;1966:46–48.

27. Cantor AJ. Achieving adjunctive success with wound dressings. Podiatry Today. 2003;July:50–63.