British Jour11al of Dermatology (1974) 91, 27.
`
`Percutaneous absorption and the surface area of
`occluded skin
`
`A SCANNING ELECTRON MICROSCOPIC STUDY
`
`DAVID R.HAR RIS*, C H RISTOPHER M.PAPA AND
`RAYMOND ST ANTON
`
`Letterman Army Institute of Research, Presidio of San Francisco, and t Dermat0log1cal Division, Johnson and
`Johnson, New Brunswick, New Jersey, and Rutgers Medical School, Piscataway, New Jersey, U.S.A.
`
`Accepted for publication 12 October 1973
`
`S UM.MARY
`
`Scanning electron microscopy of samples of human skin occluded for 72 h, revealed that the hydrated
`stratum corneum not only swells, but develops multiple folds. Surface area estimations of such
`stratum corneum, utilizing stereo pairs of the photomicrographs, indicated a 37° 11 increase over the
`normal, non-occluded horny layer values. It is speculated that rhe increase in absorptive area contri(cid:173)
`butes to the increa ed , kin permeability following ocdu!>ion.
`
`The most widely used method of increasing percutaneous absorption of topically applied therapeutic
`agents is by the use of occlusive plastic films (Garb, 1960; Hall-Smith, 1962; Scholtz, 1961; Sulz(cid:173)
`berger & Witten, 1961). With this technique, absorption of some substances can be increa~ed by as
`much as 100 times, in addition co the formation of a long-lasting reservoir within the stratum cor(cid:173)
`neum (Bettley, 1970). Fritsch & Stoughcon (1963) have shown Lhat, in occlusive environments
`approaching 10011
`11 relative humidity, increa~ed penetrability is due almost enLirely to the hydration of
`the stratum corneum.
`ln the course of srudie~ in which skin wac; occluded for 72 h and prepared for examination with the
`scanning electron microscope (SEM), the present authors noted remarkable altcratiom. in the appear(cid:173)
`ance of the horny layer of the occluded skin. The possible influence of such changes on percutaneous
`absorption seemed obvious, since the area of the absorptive surface was apparently increased. The
`potential increment was assessed using SEM photomicrographs and mathematical estimation tech(cid:173)
`niques.
`
`• Present address : Chief of Dermatology Service, Veterans Administration Hospital, Palo Alto, California, and
`Assistant Professor of Dermatology, Stanford University School of Medkioe, U.S.A.
`Reprint requests to : Christopher M. Papa, M .D ., 501 George Street, New Bruni;wick, Ne\\ Jersey 08903,
`U.S.A.
`
`

`

`David R.Harris, Clzrisropher M.Papa and Raymond Seamon
`
`a
`FIG u RE r. Diagrammatic representation of the principle of phorogrammctric cstunation, where two
`photographs of the same object taken at different angles are used to calculate measurements of
`height.
`
`- - -H-
`---
`F I Gu RE 2. Simplified diagram or a plammetric technique where the photographic series is arranged
`in such a way that the intersections or perpendicular lines through known points completes a
`topographical profile.
`
`

`

`Percuraneous absorption and skin surface area
`
`29
`
`Subjects
`Ten healthy male volunteers were included, none of whom had previous skin disease.
`
`METHODS
`
`Histological preparations
`Two 3 mm dermal punch biopsies were taken from each subject; one from a site occluded for 72 h
`with firmly affixed polyethylene film, and one from an un-occluded r:.ite. The specimens were dehy-
`
`PIG URE 3. Skin occluded for 72 h. The horny layer is swollen and thrown into folds. Jndividual,
`desquamating celJs are difficult ro visualize. The oval structure in the centre of the field is an eccrinc
`sweat pore ( x 500).
`
`drated in graded alcohols, air dried and fixed to mounting blocks with the horny layer surfaces
`facing upward. The tissues were coated with gold-palladium in a vacuum evaporator and examined
`in a Jelco JSM-2 Scanning Electron Microscope. Photographs of the resulting images were taken of
`the samples at angles of o-, 5 , ro and r5 .
`
`Estimation of swf ace area
`The basic approach was to use two photographs of the same object taken from different angles (Fig. I)
`
`

`

`30
`
`David R.Harris, Christopher M.Papa and Raymond Stanton
`
`and geometrically calculate measurem ents of height from them. When making topographical maps,
`the photographs are taken from two different Jocarjons, usually from an aeroplane (Thompson, 1966).
`In our case, the camera was fixed and the prepared sample was rotated to obtain different views.
`
`F 1 Gu RE 4. Non-occluded skin. The relatively flat surface and shedding of stratum corneum cells
`is the normal picture. The central oval structure is an eccrine pore ( x 500).
`
`Resulting photographs were then used to plot a series of profiles or cross-sections taken through the
`sample perpendicular to the main plane of the surface. A line was drawn in the same place on each
`photograph through which a profile was to be made. These are shown as lines A 1-E 1 and A2-E2 • The
`photographs were laid out on a drawing board as shown in a simplified profile (Fig. 2), such that
`angles</> and Oare the same as cf, and O in Fig. I. Lines A 1-A 1
`1
`1 were
`1 to E2-E2
`1 to E 1-E 1
`, and Ai-A 2
`drawn perpendicular to the lines A 1- E 1 and A2-E2 • The point a on the profile is determined by the
`1
`1 and point bis determined by the intersection oflines B 1- B 1
`1 and A 2- A 2
`intersection oflines A 1-A1
`and B2-B2
`, ere. In this way, a series of known points on the profile were located. Using the photo-
`1
`
`

`

`Percutaneous absorption and ski11 surface a,-ea
`
`3r
`
`graphs as a reference, a line was then drawn which connects all the points, thus completing the profile
`(Thompson, 1966; Robinson, 1969).
`In the simplified example shown in Figs. 1 and 2, all profiles taken parallel to a-e would be identical,
`but with the more complicated surfaces of the stratum corncum samples, this was not the case.
`Therefore, !>everal different profiles were made. The length of the line forming each profile was
`measured and divided by the horizontal length (H ), giving a unit profile length (L ). An average L
`value for both the un-occluded and occluded samples was calculated. An approximation of the unit
`surface area was found by squaring the resulting average L values. The ratio of L 2 for occluded
`stratum corneum to L 2 for un-occluded stratum corneum would thus be an approximation of the
`ratio of the two areas.
`
`RESULTS
`
`Skin occluded for 72 h becomes swollen and thrown into undulating folds (Fig. 3). This picture is an
`obvious distortion of the normal, predominantly flat or plate-like, layered texture of the stratum
`corncum seen in SEM images of un-occluded skin (Fig. 4).
`The ratio (L 0 ) of the average surface area calculated for the occluded specimens compared to an
`ideal horizontal line (H) was 1·30. For the un-occluded specimens the ratio (L.,) was I ·II. The ratio of
`the!.e average L values r.302/r·n 2 was found to be 1·37. In.other words, according to these calcu(cid:173)
`lations, the occluded sites had 37° 0 more surface area than the un-occluded.
`
`DISCUSSION
`
`When the stratum corneum becomes hydrated under an occlus ive dressing, che tissue changes from
`one containing little water (5-15° ,,) to one which may contain as much as 50" 0 water (Blank &
`Scheuplein, 1964). For the most part, the water is held in spaces between fibre bundles, swelling the
`interfibrillary structure of the stratum corneum, straightening the polar side-chains, and forming a
`more or Jess continuous aqueous pathway through part of it (Yates, 1971). Virtually all of the resulting
`increased rate of transport of water-soluble substances has been attributed to this internal change in
`the physical structure of the membrane (Fritsch & Stoughton, 1963; Blank & Schcuplein, 1964; Yate!.,
`1971).
`However, even cursory examination of the SEM images (Figs. 3 and 4) reveals that stratum
`corncum hydrated by polyethylene occlusion has a greater surface area than un-occludcd specimens.
`It is reasonable to assume that the increased surface area contributes to increased cutaneous pene(cid:173)
`trability which follows occlusion.
`It is worth noting that the simple, graded alcohol preparation of skin for SEM preserves the swollen,
`convoluted morphology of the occluded samples. The end point of preparation, dryness, does not
`restore their structure to the flat surfaces of non-occluded skin. As has been preYiously noted, the
`fixation which anends gradual dehydration of skin apparently minimizes the production of artifacts
`(Papa & Farber, 1971).
`Our mathematical determination of the relative increase in surface area of occluded skin is, at best,
`a crude estimate. Equipment similar to that used for converting aerial photographs of earth surface
`terrain into line topographical maps (stereo comparagraph) could be used to obtain more precise
`information.
`
`BETTUY, F.R. ( 1970) The epidermal barrier o.nd percuro.ncous absorption. In : An /111roduc1io11 to the Biology of
`the Skin (Ed. by R.H.Charnpion, T.Gillman, A.J.Rook and R.T.Sims), p. 342. Blackwell Scientific
`Publications, Oxford.
`
`REFERENCES
`
`

`

`David R.Harris, Chriszopher M.Papa and Raymond Stamon
`
`32
`BLANK, I.H. & ScHEUPLBIN, R.J. ( 1964) Percutaneous absorption and the epidermal barrier. I n: Progress in the
`Biological Sciences i11 Relation to Dennatolo;:y (Ed. by A.J.Rook and R. H .Champion), vol. 2, p. 247.
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`FRITSCH, W.C. & STOUGHTON, R.B. ( 1963) The effect of temperature and humidity on the penetration of C 14
`acetylsalicylic acid in excised human skin.Journal of lnves1igative Dermatology, 41, 307.
`GARB, J. ( 1960) Nevus verrucosus unilateralis cured with podophyllin ointment. Archives of Den11a10/ogy, 8 1,
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`HALL-SMITH, S.P. (1962) 'Polythene' coverings in dermatological treatment. Brimh Medical Journal, 2, 1233.
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`RoRrNSON, A.H. ( 1969) Elemenrs of Carrography, 3rd cdn, p. 141. John Wiley & Sons, New York.
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`THOMPSON, M.M. (Ed.) (1966) Ma1111al of Phorngrupl11c lwcrpreration, 3rd cdn, p. 19. American Society of
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`YATES, J.R. ( 1971) Mechanism of water uptake by skin. In : Biophysical Properries of the Skin (Ed. by H.R.Elden),
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`
`