Abstract Tape stripping of human stratum corneum is widely used as a method for studying the kinetics and penetration depth of drugs. Several factors can in-.
Arch Dermatol Res (1997) 289 : 514–518
© Springer-Verlag 1997
O R I G I N A L PA P E R
R. G. van der Molen · F. Spies · J. M. van ‘t Noordende · E. Boelsma · A. M. Mommaas · H. K. Koerten
Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin
Received: 21 November 1996
Abstract Tape stripping of human stratum corneum is widely used as a method for studying the kinetics and penetration depth of drugs. Several factors can influence the quantity of stratum corneum that is removed by a piece of tape, such as the manner of tape stripping, the hydration of the skin, cohesion between cells, body site and interindividual differences. However, few data are available about the influence of furrows in the human epidermis on the tape-stripping technique. In this study, we investigated the efficacy of tape stripping in removing complete cell layers from the superficial part of the human stratum corneum. A histological section of skin that was tape-stripped 20 times clearly showed nonstripped skin in the furrows, indicating persistent incomplete tape stripping. Replicas of tape-stripped skin surface demonstrated that even after removing 40 tape strips the furrows were still present. We validated the tape-stripping method further with X-ray microanalysis in the mapping mode by scanning electron microscopy, using a TiO2-containing compound as a marker. TiO2 applied to the skin before the tape-stripping procedures was still present after the tenth tape strip, and was specifically located on the rims of the furrows. We emphasize that results from studies using the tape-stripping method have to be viewed from the perspective that cells on one tape strip of the stratum corneum may be derived from different layers, depending on the position of the tape strip in relation to the slope of the furrow, and such results should be interpreted with considerable caution.
R. G. van der Molen (Y) · F. Spies · J. M. van ‘t Noordende · A. M. Mommaas · H. K. Koerten Laboratory for Electron Microscopy, University Hospital Leiden, PO Box 9503, Building 3, 2300 RA Leiden, The Netherlands Tel. +31 71 5276457; Fax +31 71 5276440 E. Boelsma Department of Dermatology, University Hospital Leiden, PO Box 9600, Building 1-P4Q, 2300 RC Leiden, The Netherlands
Key words Tape stripping · Human · Stratum corneum · Penetration studies · Skin furrows
Introduction The stratum corneum consists of corneocytes embedded in lipid bilayers, and is often described using the analogy of the bricks and mortar of a brick wall (Elias 1983). This wall possesses macroscopic furrows, which run parallel to the surface of the skin (Fiedler et al. 1995). To examine the localization and distribution of substances within the stratum corneum, skin surface tape stripping with adhesive tape is a widely accepted and used method (Bommannan et al. 1990; Higo et al. 1993; Lotte et al. 1993; Pershing et al. 1992; Rougier et al. 1986; Tojo and Lee 1989). The method is also used to provide information about the kinetics of transdermal drug delivery. Adhesive tape removes a sheet of corneocytes. It has been found that on the flexor surface of the forearm about 30 tape strips are needed to strip off most of the horny layer (Pinkus 1951). Usually the quantity of stratum corneum removed by tape stripping is not linearly proportional to the number of tape strips removed, and about two-thirds of each tape strip is estimated to be covered by cells (Pinkus 1951). Several factors can influence the quantity of stratum corneum removed by a piece of tape, and these include the manner of tape stripping, the hydration of the skin, cohesion between cells which increases with depth in the stratum corneum, body site and interindividual differences (King et al. 1979; Marttin et al. 1996). No reports are available, however, on the possible influence of the macroscopic furrows on the tape-stripping technique. These furrows may cause problems in the interpretation of tape-strip results. This is especially important in studies on the effects of various topically applied agents on the ultrastructure of corneocytes particularly in relation to penetration depth. Moreover, the influence of furrows has to be taken into consideration if absorption into the cells of certain cell layers has to be established. In the present study, we investigated the effects of furrows on the removal of cell layers by the tape-stripping
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method. Therefore, we compared histological sections of control and tape-stripped skin by light microscopy (LM). We also used a replication technique in combination with scanning electron microscopy (SEM) to screen large areas of skin surface in situ before and after tape stripping. To establish possible changes in furrows after prolonged tape stripping we continued to 40 tape strips. In addition, we examined the ultrastructural morphology of these tape strips by SEM. Finally, we investigated the influence of the presence of furrows on the efficacy of tape stripping using topical application of a compound containing platelet-shaped TiO2 particles. Because of their size these particles are unlikely to penetrate the skin, and X-ray microanalysis (XRMA) can detect the presence of a titanium signal. The combination of XRMA with SEM allows the direct visualization of the titanium signal on the tapestripped stratum corneum.
Materials and methods Tape stripping The tape-stripping experiments were performed on the flexor forearm of normal healthy volunteers (n = 3) after obtaining informed consent. Tesafilm 4206 PV1 polypropylene adhesive tape (Beiersdorf, Hamburg, Germany), 2 cm wide and 5 cm long, was used. The tape was applied to the skin, rubbed lightly to assure adhesion, and then pulled off with one fluent and decisive movement. Light microscopic examination of tape-stripped skin Freshly excised skin obtained by plastic surgery was placed on polystyrene foam and tape-stripped as described above. Next, samples of excised skin were taken of areas that had been tapestripped and of control (non-tape-stripped) areas. The samples were fixed in buffered 4% formaldehyde, and processed for embedding in paraffin. Sections (5 µm) were cut perpendicular to the surface and stained with haematoxylin and eosin (H/E) for LM examination.
Fig. 1 a, b Light micrographs of 5-µm cross-sections of (a) control skin and (b) skin tape-stripped 20 times. After tape stripping nonstripped areas are still present in the furrows (arrows). Bar 10 µm
Replication technique Replicas of the skin surface were made with Provil-L, a polyvinyl siloxane impression rubber (Bayer Dental, Leverkusen, Germany). Provil-L is obtained by mixing two monomers, a catalyst and a base, at a ratio of 1 : 1. The mix was applied to the skin of a subject with a metal spatula, and allowed to polymerize for about 5 min. After removal from the skin, the replicas were sputter coated with gold (Balzers, MED 010) and examined with a Philips 525M SEM. Replicas made before tape stripping and after tape strip 10, 20, 30 and 40 were compared. The tape strips obtained were sputter coated with gold and examined by SEM. Preparation of tape strips for X-ray microanalysis XRMA (Voyager, Noran Instruments) in combination with SEM was applied to an oil-in-water formulation containing plateletshaped TiO2 (kindly provided by Merck, Darmstadt, Germany). The morphology of the compound was first examined by SEM. XRMA in combination with SEM was used to determine the presence of titanium in the compound. The ointment was applied on two SEM holders, allowed to dry overnight and one sample was sputter coated with gold for SEM and the other was coated with carbon (Balzers, BAF 400) for XRMA. To investigate the efficacy of the tape-stripping technique, 2 mg/cm2 of the TiO2-containing compound was applied with a metal spatula over a marked rectangular area of 18 cm2 to the skin surface of a subject. As a negative control, the TiO2-free vehicle of the TiO2-containing compound was applied. Immediately after application, these areas were tape-stripped 15 times. Each tape strip was cut to size, mounted on a SEM holder and coated with carbon. The tape strips were examined in the mapping mode of the XRMA equipment in the SEM. In the mapping mode every image point is measured for the presence of an element, in this case titanium. The titanium count-rate above a prefixed threshold results in a white dot on the image (Koerten et al. 1990). After the XRMA procedures were completed, these tape strips were sputter-coated with gold for morphological imaging with the SEM.
Results For the morphological investigation, we compared untreated skin with tape-stripped skin, obtained from plastic surgery. On H/E-stained sections of control skin, the various regions of the skin could easily be recognized, i.e. poorly stained dermis and darker stained epidermis. In the epidermis we observed the various strata with in the upper part the horny layers of the stratum corneum with char-
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Fig. 2 a, b Scanning electron micrographs of Provil-L replicas of the surface of (a) control skin and (b) skin tape-stripped 40 times. Furrows (here seen as ridges, arrows) do not disappear after tape stripping. Bar 250 µm
acteristic loosely connected flattened cells (Fig. 1 a). Figure 1 a clearly shows the presence of furrows at the surface of the skin. Layers of loose cells, as observed on top of the superficial area, were continuous in these furrows. The skin samples obtained after tape stripping 20 times showed the same morphology, except for the stratum corneum, which was largely removed. Accurate inspection of the whole surface, however, showed that the loose layers in the furrows were almost untouched by the tapestripping procedure and layers of horny cells were still present there (Fig. 1 b). To visualize large areas of the surface of the skin in situ we applied a replication technique in combination with SEM. The results of this part of the study are illustrated in
Fig. 3 Scanning electron micrograph of the first tape strip, showing domains of corneocytes with empty spaces at the places where furrows could be expected. Note the similarity of the morphology with the replicas shown in Fig. 2 a, b. Bar 250 µm
Fig. 2. The replication technique resulted in furrows being represented as ridges by SEM. Figure 2 a shows a replica of non-tape-stripped skin with the regular presence of furrows (ridges), which cross each other and divide the skin surface into corneocyte-containing domains. Examination of the first tape strip by SEM also showed corneocyte domains separated by empty spaces at sites where furrows could be expected (Fig. 3). After tape stripping 40 times (Fig. 2 b) the ridges were still present. We used SEM in combination with XRMA to study the efficacy of tape stripping after application of a compound containing TiO2 particles. The morphology of the compound itself was examined by SEM, demonstrating platelet-shaped TiO2 particles with a diameter ranging between 5 and 50 µm (Fig. 4). The chemical characteristics were determined by XRMA, which revealed clear peaks for titanium upon irradiation of the TiO2 particles with the focused electron beam (data not shown). To establish the distribution of the compound over the tape strips, XRMA
Fig. 4 The TiO2-containing compound examined by scanning electron microscopy, showing the platelet-shaped particles. Bar 30 µm
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Fig. 5 a, b X-ray microanalysis analogue maps of tape strips taken immediately after application of a TiO2-containing compound. a The first tape strip, showing the presence of titanium signals (white dots) all over the skin domains. b The tenth tape strip: titanium signals are present only on the rims of the furrows (arrows). Bar 250 µm
in the mapping mode was used (Fig. 5). Each white dot on the image represents titanium. As Fig. 5 a shows, corneocyte domains were recognizable on the first tape strip and the titanium signal was abundantly present and evenly distributed over these domains. After ten tape strips, the titanium signal could only be demonstrated on the rims of the furrows and not on the corneocyte domains originating from deeper layers of the stratum corneum (Fig. 5 b). Further tape stripping up to tape strip 40 did not change this distribution pattern (data not shown). Titanium was not detectable on tape strips taken of areas to which the vehicle of the TiO2-containing compound had been applied.
Discussion Although tape stripping is widely used to examine the stratum corneum barrier function (Bommannan et al. 1990; Higo et al. 1993; Lotte et al. 1993; Pershing et al. Fig. 6 Schematic drawing of the tape-strip technique. Superficial corneocytes still present in the furrows after tape stripping, are removed with later tape strips. Note that in this way, tape strips are obtained containing corneocytes which originate from different layers of the stratum corneum
1992; Rougier et al. 1996; Tojo and Lee 1989), several factors can influence the actual technique (Marttin et al. 1996). Until now, no attention has been paid to furrows in the skin, which are commonly present. Therefore, we investigated the effect that furrows can have on the results of tape stripping. We investigated cross-sections of control and tape-stripped skin to determine whether tape strips removed superficial corneocytes from the furrows, and found that these cells were not removed by tape stripping. To establish the effect of furrows on the accuracy of sampling after repeated tape stripping, we applied a replication technique in combination with SEM, which allowed visualization of large areas of the surface of human skin in situ. The advantage of this technique was the possibility of obtaining microscopic information about the surface of the stratum corneum without the need for taking biopsies. Replicas of control and tape-stripped skin showed that furrows were still evident even after removing 40 tape strips. This observation was confirmed by investigation of the removed tape strips by SEM, which revealed empty spaces at the sites of the furrows. Given our doubts about the accuracy of tape stripping, we also investigated a typical application of the tapestripping method. A TiO2-containing compound was applied to skin which was subsequently tape-stripped. The
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tape strips were examined using XRMA with SEM. After repeated tape stripping, we found the persistent presence of the titanium signal, which was restricted to the rims of the furrows, enabling us to confirm that different layers of the stratum corneum are removed with one tape strip (Fig. 6). Usually in penetration studies it is important to ascertain the concentration of certain compounds detected on tape strips, that are interpreted as having penetrated into or across the stratum corneum (Dupuis et al. 1984; Higo et al. 1993; Tojo and Lee 1989). However, our present results indicate that such an interpretation should be made with caution. A second consideration is that residual material of the compound under investigation could accumulate in furrows, thus disturbing the interpretation of the tape-stripping results. In the normal situation residual material is removed before tape stripping by washing with ethanol (Dupuis et al. 1984; Higo et al. 1993; Lotte et al. 1993; Rougier et al. 1986) or methanol (Tojo and Lee 1989) followed by distilled water and drying with cotton wool. Pershing et al. (1992) removed residual material only with a Teflon spatula and gently wiped the skin with three separate dry cotton applicators, allowing it to dry before tape stripping. However, these methods do not ensure complete removal of all residual material, because we found by visual inspection that wiping the skin with a tissue or washing the skin with water failed to remove all residual TiO2-containing compound. This white compound remained clearly visible, marking the furrows as thin white lines (data not shown). Furthermore, cleaning procedures might even damage the skin or enhance penetration of the applied compounds. In order to calculate concentration profiles for compounds penetrating the skin, it is essential to take the influence of variable amounts of stratum corneum on each tape strip into consideration. Usually, the exact number of corneocytes that are removed by one tape strip is determined by weighing the strips before and after tape stripping (Bommannan et al. 1990; Higo et al. 1993). The concentration of the penetrated compound in the tape strips is then determined. Subsequently, the concentration of the compound is plotted as a function of the cumulative stratum corneum weight (Higo et al. 1993). However, the interpretation of the results thus obtained may be misleading owing to residual material in the furrows. Even if all residual material has been removed completely before tape stripping, we still remain with the problem that, depending on the position of the tape strip in relation to the slope of the furrow, deeper layers and superficial layers are present in the same sample (Fig. 6). As a result of this, it may seem that some compounds have penetrated into the deeper layers of the stratum corneum, while in fact they have only penetrated into the top layers. A possible procedure to overcome the problems caused by furrows involves stretching the skin in a symmetrical manner thereby reducing the furrow depth (Dr. F. Spies, personal communication). However, this method may have the disadvantage that the force of stretching to flatten the surface of the skin can influence the architecture of the
skin and thus the penetration of compounds. Whether such a technique does indeed improve the quality of tape stripping requires further investigation. In summary, we conclude that furrows in the skin can present difficulties when performing depth-penetration studies. Although the largest part of the skin surface will be stripped properly, it has to be realized that small areas, represented by the furrows, may still contain high concentrations of the applied substance. In the case of compounds with very poor penetration characteristics, the contribution of such compound remnants in the furrows will be relatively large. In addition to this, we showed that corneocytes from different layers were present on one tape strip, and this may also contribute to misinterpretation of tape-strip results. Acknowledgements The authors wish to thank L.D.C. Verschragen and J van der Meulen for their support in preparation of the micrographs. This work was supported by grant 28-1739-1 of the Praeventiefonds, The Netherlands.
References Bommannan D, Potts RO, Guy RH (1990) Examination of stratum corneum barrier function in vivo by infrared spectroscopy. J Invest Dermatol 95 : 403–408 Dupuis D, Rougier A, Roguet R, Lotte C, Kalopissis G (1984) In vivo relationship between horny layer reservoir effect and percutaneous absorption in human and rat. J Invest Dermatol 82 : 353–356 Elias PM (1983) Epidermal lipids, barrier function, and desquamation. J Invest Dermatol 80 : 44s–49s Fiedler M, Meier WD, Hoppe U (1995) Texture analysis of the surface of the human skin. Skin Pharmacol 8 : 252–265 Higo N, Naik A, Bommannan DB, Potts RO, Guy RH (1993) Validation of reflectance infrared spectroscopy as a quantitative method to measure percutaneous absorption in vivo. Pharm Res 10 : 1500–1505 King CS, Barton SP, Nicholls S, Marks R (1979) The change in properties of the stratum corneum as a function of depth. Br J Dermatol 100 : 165–172 Koerten HK, Hazekamp J, Kroon M, Daems WTh (1990) Asbestos body formation and iron accumulation in mouse peritoneal granulomas after the introduction of crocidolite asbestos fibers. Am J Pathol 136 : 141–157 Lotte C, Wester RC, Rougier A, Maibach HI (1993) Racial differences in the in vivo percutaneous absorption of some organic compounds: a comparison between black, Caucasian and Asian subjets. Arch Dermatol Res 284 : 456–459 Marttin E, Neelissen-Subnel MTA, De Haan FHN, Boddé HE (1996) A critical comparison of methods to quantify stratum corneum removed by tape-stripping. Skin Pharmacol 9 : 69–77 Pershing LK, Silver BS, Krueger GG, Shah VP, Skelley JP (1992) Feasibility of measuring the bioavailability of topical betamethasone dipropionate in commercial formulations using drug content in skin and a skin blanching bioassay. Pharm Res 9 : 45–51 Pinkus H (1951) Examination of the epidermis by the strip method of removing horny layers. I. Observation on thickness of the horny layer, and on mitotic activity after stripping. J Invest Dermatol 16 : 383–386 Rougier A, Dupuis D, Lotte C, Roguet R, Wester RC, Maibach HI (1986) Regional variation in percutaneous absorption in man: measurement by the stripping method. Arch Dermatol Res 278 : 465–469 Tojo K, Lee AC (1989) A method for prediction steady-state of skin penetration in vivo. J Invest Dermatol 92 : 105–108
