Please cite this paper as: Silver-loaded seaweed-based cellulosic fibre improves epidermal skin physiology in atopic dermatitis: safety assessment, mode of action and controlled, randomized single-blinded exploratory in vivo study. Experimental Dermatology 2010; 19: e9–e15.
Background: The epidermal part of the skin is the major interface between the internal body and the external environment. The skin has a specific physiology and is to different degrees adapted for protection against multiple exogenous stress factors. Clothing is the material with the longest and most intensive contact to human skin. It plays a critical role especially in inflammatory dermatoses or skin conditions with an increased susceptibility of bacterial and fungal infections like atopic dermatitis. Previously, we have shown a dose-dependent antibacterial and antifungal activity of silver-loaded seaweed-based cellulosic fibres.
Aim of the study: We studied the mode of action of silver-loaded seaweed-based cellulosic fiber and performed a broad safety assessment. The principal aim was to analyse the effects of wearing the textile on epidermal skin physiology in 37 patients with atopic dermatitis in a controlled, randomized single-blinded in vivo study. Furthermore, the sensitization potential was tested in a patch test in 111 panellists.
Results: We could demonstrate in vitro a dose-dependent scavenging of induced reactive oxygen species by silver-loaded seaweed-based cellulosic fibers. Safety assessment of these fibres showed no detectable release of silver ions. Furthermore, ex vivo assessment after 24 h application both in healthy volunteers and patients with atopic dermatitis by sequential tape stripping and subsequently raster electron microscopy and energy dispersive microanalysis analysis revealed no detectable amounts of silver in any of stratum corneum layers. Serum analysis of silver showed no detectable levels. The in vivo patch testing of 111 volunteers revealed no sensitization against different SeaCell® Active (SeaCell GmbH, Rudolstadt, Germany) containing fabrics. The in vivo study on 37 patients with known atopic dermatitis and mild-to-moderate eczema on their arms were randomly assigned to either silver-loaded seaweed fibre T-shirts or to cotton T-shirts for 8 weeks. A significant reduction in Staphylococcus aureus colonization was detectable for the silver T-shirts compared with cotton T-shirts without any changes in non-pathogenic surface bacteria colonization. Furthermore, a more pronounced improvement in barrier function (transepidermal water loss) was observed in mildly involved eczema areas during the first 4 weeks of the study. Stratum corneum hydration and surface pH improved in both treatment groups over time.
Conclusion: The tested silver-loaded seaweed fibre can be regarded as safe and seams to be suited for application in bio-active textiles in atopic dermatitis based on its positive in vivo activity.
The epidermal part of the skin is the major interface between the internal body and the external environment (1–4). Each skin type has a specific skin physiology and is adapted to a different degree for protection against multiple exogenous stress factors. Clothing is the material with the longest and most intensive contact to human skin (5). It plays a critical role especially in inflammatory dermatoses or skin conditions with an increased susceptibility of bacterial and fungal infections like atopic dermatitis (6–9).
A new fibre called SeaCell® Active (SCA) was developed in response to the increasing demand for bio-active textiles (10). The fibre can be manufactured by means of the so-called Lyocell process. This process has been established as an environment-friendly, economically viable, product-enhancing and highly flexible alternative for the manufacture of man-made cellulose fibres. In the Lyocell process, cellulose is dissolved directly without formation of derivatives (11). The non-toxic, aqueous N-methylmorpholine-N-oxide is used as a solvent. The SeaCell® fibres are manufactured by adding grounded seaweed, mainly from the family of brown, red, green and blue algae. Especially the brown algae, Ascophyllum nodosum and/or the red algae Lithothamnium Calcareum are added to form the spinning solution (12–16).
The algae are added either as a powder or as a suspension in one of the process steps preceding the spinning of the cellulose solution. Seaweed has the capability of absorbing the minerals contained in the sea water. According to this variety of active ingredients, seaweed and/or seaweed extracts are used in cosmetics as well as in the pharmaceutical industry (17–21). The natural, cellulose and seaweed-based SeaCell® fibres serves as a functional carrier for the active compound silver, which is known for more than one century to exert antifungal and antibacterial activity (22). Additionally, the seaweed-based, silver-loaded Lyocell fiber SCA contains the minerals calcium, magnesium and sodium, which are known to play a key role in epidermal barrier homeostasis (23). Previously, we have shown a dose-dependent antifungal activity of silver-loaded seaweed-based cellulosic fibres on Candida albicans (DSM 11225), C. tropicalis (ATCC 1169) and C. krusei (ATCC 6258). Furthermore, we were able to show dose-dependent antibacterial activity against two different Staphylococcus aureus strains (ATCC 22926 and ATCC 25923) and one Escherichia coli strain (ATCC 35218) (24).
The present studies were intended to (i) provide insight in the mode of action of silver-loaded and seaweed fibres, (ii) assess the safety profile of the in vivo use of silver-loaded seacell fibres, (iii) show the in vivo antibacterial activity of silver-loaded seaweed-based cellulosic fibre T-shirts compared with cotton wool T-shirts in patient with atopic dermatitis and (iv) assess the effect of cellulosic fibre T-shirts compared with cotton wool T-shirts in patient with atopic dermatitis on skin physiology and clinical parameters.
Materials and methods
Mode of action experiments
Determination of antioxidant capacity
The capability of the tested fibres and fabrics to scavenge reactive oxygen species (ROS) was assessed using chemiluminescent ABEL® Antioxidant Test Kits specific for peroxynitrite and ROS, such as superoxide radical species. Both test kits contain Pholasin® and were purchased from Knight Scientific Limited (Plymouth, UK). Pholasin® is a photoprotein, isolated from the mollusc Pholas dactylus, which emits light in the presence of certain oxidants. As the tests are based on cell-independent systems, the necessary solutions to create reactive molecules that activate the Pholasin® are provided. Peroxynitrite is formed by reaction of superoxide and nitric oxide, released from a 2.5 mm solution of SIN-1 (3-morpholino-sydnomine HCl). Measurements are performed in 96-well microplates (Nuncbrand, Roskilde, Denmark) and carried out as recommended in the kit instructions in a total reaction volume of 200 μl. Samples were placed into the microplate. To each sample, 100 μl assay buffer and 50 μl Pholasin® were added. At the start of the measurement, 50 μl SIN-1 solution were injected to each well with an automatic dispenser. The luminescence was measured for up to 2 h, depending on the peak time of the different samples, at room temperature using the LUMIstar Galaxy plate reader (BMG LABTECH GmbH, Offenburg, Germany). A control without sample was run with each assay. An antioxidant activity delays the appearance of the luminescence peak and lowers its light intensity. The antioxidant capacity of a sample was expressed as percent reduction of peak luminescence as follows:
The chemiluminescent test for superoxide was carried out according to the kit protocol in 96-well microplates, at room temperature. The samples were added to 25 μl assay buffer, 50 μl Pholasin® and 100 μl Solution A. While the well was in the light measuring position, 25 μl Solution B were injected to generate a superoxide flux. As the peak of the light intensity was reached within 5 s, the LUMIstar Galaxy plate reader was programmed to measure each well for 30 s, in short intervals after the injection of Solution B. The antioxidant capacities of the samples were calculated similar to the peroxynitrite test.
In the in vivo part 37 (18 cotton; 19 silver) patients diagnosed with atopic dermatitis according to the criteria of the ‘Erlangen Atopy Score’ (25) were included. The sex ratio was f17:m20 (cotton 9f:9m, silver 8f:11m). The mean age was 25 years. (cotton 25.5, silver 25.4) with a range of 13–51 years. (cotton 13–51, silver 14–47). The studies were approved by the ethics committee of the Friedrich Schiller University under the number 1456–12/04. The patients signed a written informed consent prior to the inclusion into the study. The in vivo study on atopic dermatitis patients was conducted from December 2004 through April 2006 with a break during the summer season in 2005 because of a possible interference of sun exposure on the natural course of atopic dermatitis. The inclusion criteria were age 12–60, atopic dermatitis with a score-value equal or higher than 10 according the ‘Erlangen Atopy Score’ (25), mild eczema on the volar forearm, no other known dermatological diseases. Pregnant or lactating women, patients with a history of malignant diseases, systemic medication for atopic dermatitis 4 weeks prior to the start of the study, any other systemic medication and any known systemic diseases were excluded.
The patch testing was performed in 111 panellists, aged 20–82 years (mean 51.8 years) with 83 females and 28 males.
All data sets were complete regarding clinical and biophysical assessments. Bacterial colonization was positive for all subjects for non-pathogenic bacteria but only in four patients in the cotton group and 10 patients in the silver group. Thus, the obtained data regarding antibacterial properties can only be seen as exploratory.
No dropouts were recorded in the human studies.
The ex vivo safety assessment of potential silver penetration into the blood and the stratum corneum was performed on a subset of four patients with atopic dermatitis of the in vivo study and an age-matched control group of four healthy volunteers.
Skin physiology parameters
Baseline values were obtained from the volar forearms using non-invasive biophysical devices. Assessment of the surface pH (skin-pH-meter PH 900; Courage and Khazaka Electronics, Cologne, Germany), transepidermal water loss (TEWL; Tewameter TM 300; Courage and Khazaka) and capacitance-based skin hydration (Corneometer CM 825, Courage and Khazaka) was performed. All measurements were conducted under standard ambient conditions (room temperature between 20 and 22°C and relative humidity between 30% and 40%) after adequate acclimatization of the participants in the measuring room and according to the published guidelines (26–30). The data obtained were expressed as the mean value of three recordings. Skin surface pH, TEWL and, skin hydration were measured, respectively, on day 0, after 6 and 12 weeks. The parameters were assessed on mildly and severely involved areas.
Testing of antibacterial activity from atopic dermatitis patients
The surface bacterial colonization was assessed using a classical standardized washing method (31) with a glass cylinder (7.069 cm2 surface) and 1 ml of Tween 80 PBS washing solution than further diluted in five dilution steps. The solution was subsequently plated on Columbia agar plates containing 5% sheep blood (bioMérieux, Nürtingen, Germany). For identification of S. aureus and further classification of the bacteria, dedicated commercial kits (Slidex® and ID 32 Staph; both from bioMérieux) were used. Bacterial colonization was assessed at baseline and after 8 weeks of wearing the T-shirts.
The following compositions were tested in the in vivo study: SCA 100% (SCA 100) versus cotton 100% both as long sleeve T-shirts. In the patch test, SCA 100, SCA 100% non-woven (SCA 100NW), SCA 5–20% (SCA 5–20), Lyocell (LC 100), SeaCell® 100% (SC 100) and washed fibre (W). In the silver release experiment, SCA 100 was assessed. In the mode of action studies, LC100, SC 100 and SCA active 100NW were compared with a seacell-free control tissue. The biophysical properties of the tested fibres are listed in Table 1 according to recently published data (32) [alternative sentence: The biophysical properties are discussed in detailed in a recent publication (32)].
|Physcial properties||Cotton||Lyocell||Sea cell||Sea cell active|
|Titer (typical), dtex||1.5||1.3||1.4||1.4|
|Tenacity cond. (cN/tex)||20–24||36.5||35.9||34.4|
|Tenacity wet (cN/tex)||26–30||31.4||31.1||28.0|
|Elongation cond. (%)||7–9||12.1||11.9||9.3|
|Elongation wet (%)||25–20||15.3||13.4||14.2|
Safety testing: silver ion release ex vivo and serum levels
Ten sequential tape strippings were performed with 22 mm D-Squame® discs (CuDerm, Dallas, TX, USA). The ex vivo assessment was carried out after 24 h application both in four healthy volunteers and four patients with atopic dermatitis by sequential tape stripping and subsequently by scanning electron microscopy (SEM, S440i, Leica, Wetzlar, Germany) and energy dispersive microanalysis (EDX, Oxford Instruments Link ISIS, Abingdon, UK).
The tapes were fixed on a conductive carbon pad which was placed on an aluminium sample holder. The samples were coated with a thin layer of carbon for electrical conductivity. The samples were placed in the SEM and magnified (5000 × ). The areas measured by EDX was kept constant to 360 μm × 250 μm. Areas fully covered by the skin cells were selected for these measurements at all times. Three different measurements were made for each of the 10 samples per person. The acceleration voltage was 20 kV, the penetration depth around 5 μm. Prior to each quantitative analysis, a spectrum was taken and the peaks identified. All samples were measured for the presence of the elements sodium, potassium, calcium, silicon, sulphur chlorine, and silver. Measurements on uncovered areas of the sample showed also silicon, along side the expected carbon and oxygen. It could be that the silicon contaminant is most likely to be from the glue on the tape.
The high acceleration voltage was chosen to ensure that at least partially the surface of the keratinocytes – which is glued on the tape by the procedure of tape striping – is included in the measurement. In this way, however, the values measured quantitatively can not really be considered as quantitative compositions of the keratinocytes. This is because, the penetration depth might be deeper than the thickness of the cell, the high organic content causes significant errors for real quantitative data and finally the amount found is so low that principle measure errors are in the range of 50%. Therefore, the data given should be regarded as qualitative and for comparison purposes only.
Serum levels of silver ions were assessed in EDTA-blood of a subset of four atopic patients after wearing the silver T-shirt for 12 weeks via ICP-MS. The detection level was 0.2 μg/l.
Mean values and standard deviations were calculated with Microsoft® Excel software and the statistical comparison with Prism 3.02 (GraphPad, San Diego, CA, USA). P-values were set to be significant at P < 0.05. A baseline adjustment was performed for each group in the in vivo part of the study for the parameters of skin physiology.
Mode of action: antioxidant properties of fibres in vitro
In vitro studies could show a significant peroxynitrite scavenging by SeaCell (without silver loading; 87.4%), SCA (silver-loaded; 91.0%) and the Lyocell fibres (68.8%) compared with the control in the pholasin test induced ROS liberation in cell model (ANOVA P < 0.001; t-test comparing all groups versus control P < 0.01; data not shown). Additionally, the present data revealed a significant prevention of superoxide radical formation with a scavenging of 47.4% for Lyocell, 84.5% for SeaCell and 92.2% for SCA (ANOVA P < 0.001; t-test comparing all groups versus control P < 0.01; data not shown). Figure 1a,b shows the influence of the three test materials on peroxynitrite formation and superoxide radical scavenging compared with pholasin control (ANOVA P < 0.001; t-test comparing all groups versus control P < 0.01).
Antibacterial activity in vivo
Comparing the two groups of patients with atopic dermatitis a significant reduction of S. aureus was detectable for baseline adjusted bacterial counts (Fig. 2a). Apathogenic bacterial colonization showed no change in the cotton group and a slight but not significant decrease was observed in the silver T-shirt group (Fig. 2b) with no significant difference between the two groups.
Skin physiology and clinical in vivo data
Epidermal barrier function
Epidermal barrier function assessed by measuring TEWL decreased significantly in the first part of the study on mildly involved skin in the silver group compared with cotton (Fig. 3a). On the severely involved areas both groups had an improvement of barrier function without being significant when comparing the silver to cotton (Fig. 4b).
Stratum corneum hydration
Stratum corneum hydration measured by capacitance-based corneometry revealed a slight, but not significant increase of stratum hydration in the second part of the study, more prominent in severely involved skin in the silver group compared with cotton (Fig. 4a,b).
In both groups, a decrease of surface pH was detectable without showing a significant difference between silver and cotton both on mildly and on severely affected skin areas (data not shown). In Silver, the reduction in pH-values was faster in the early phase of the study, but still not significantly different compared with cotton.
Silver release below detectable threshold
Silver release from SCA in the serum of atopic patients wearing the T-shirts for 12 weeks was below the detection threshold (data not shown). No silver ions were detectable by raster electron microscopy and EDX. Figure 5 shows a typical SEM micrograph of keratinocytes on D-Squame® used for EDX investigation.
No sensitization against all the tested textiles was detected in the 111 panellists.
Atopic dermatitis is chronic disease with an impaired epidermal barrier, inflammatory processes, decreased SC hydration and high psycho-social impact for the patients and their family members (33). The properties of textiles and their interaction with skin have been subject of several publications (5,32,34–38) The use of silver in dermatology and especially in atopic dermatitis is gaining rapid importance (22,34,38–40). The antibacterial effect of silver is known for approximately 4000 years. The mode of action studies have revealed that silver sulphadiazine dissociates reacting with cellular DNA and that only silver ions bind DNA and inhibit its replication (41,42). Furthermore, silver ions also interfere with the respiratory chain at the cytochromes and in the reduced nicotinamide adenine dinucleotide-succinate dehydrogenase region (43). As silver does not have any negative side effects like skin irritation, this metal is a good candidate for application as an antimicrobial and inflammatory agent in textiles (44). Based on the previous shown antimicrobioal activity of the silver-loaded seaweed-based cellulosic fibre in vitro (45), we tested whether this effect would have additional benefits for patients with atopic dermatitis.
Initially, we were interested to provide insight in the so far not known mode of action: We could demonstrate a dose-dependent scavenging of induced ROS by the silver-loaded seaweed-based cellulosic fibres in vitro with the highest radical scavenging properties for the silver-loaded seaweed-based cellulosic fibre. As free radical formation plays an important role in inflammatory skin diseases (like AD or psoriasis) antioxidative properties, as shown in vitro for the tested fibre, have a clinical rational might potentially beneficial for these patients. In contrast, the formation of free radicals, e.g. by environmental factors (46).
The in vivo study on 37 patients with known atopic dermatitis and mild-to-moderate eczema on their arms were randomly assigned to either silver-loaded seaweed fibre T-shirts or to cotton T-shirts for 8 weeks. A reduction in S. aureus colonization was detectable for the silver T-shirts compared with cotton T-shirts without relevant changes in non-pathogenic surface bacteria colonization. However, the number of S. aureus carriers was very low in both groups. Thus the data have to be regarded as explorative. Gauger and co-workers have shown antibacterial effect of silver-coated textiles in a shorter application than in our study (22). Furthermore, an improvement in barrier Function (TEWL) was observed in mildly involved eczema areas at the beginning especially during the first 4 weeks of the study. Part of this barrier improvement effect was lost in the second part of the study both in mildly and severely involved areas. The positive influence on TEWL in the silver group might be due to the positive effect on the antioxidative network (as shown in the mode of action part of this study). In the second part of the study, this effect might be partially lost because of adaptive processes of the skin to the shirt regarding barrier function. In contrast, stratum corneum hydration and surface pH improved in both treatment groups over time.
The safety assessment of the silver-loaded fibre showed no detectable release of silver ions in any of the performed in vitro or ex vivo tests. The in vivo patch testing of 111 volunteers revealed no sensitization against different SCA containing fabrics.
In conclusion, the silver T-shirt-treated group showed antimicrobial activity against S. aureus without any negative effects on apathogenic bacteria. Clinical signs and as well as physiological parameters showed a slightly positive effect but did only occasionally reach statistical significance for the silver group compared with cotton. The antibacterial, clinical and skin physiology assessment should be regarded as explorative. The mode of action can be attributed, at least partially to antioxidant properties shown in in vitro cell assays. The present results demonstrate for the tested silver-loaded seaweed fibre an excellent safety profile. We conclude that at this stage the silver-loaded seaweed fibre T-shirt can be regarded as safe and seams to be suited as bio-active textile in atopic dermatitis.
This study was funded by SeaCell GmbH Schwarza, Rudolstadt, Germany.