Characterization and ex vivo evaluation of excised skin samples as substitutes for human dermal barrier in pharmaceutical and dermatological studies

Abstract

Background: Excised animal and human skins are frequently used in permeability testing in pharmaceutical research. Several factors exist that may have influence on the results. In the current study some of the skin parameters that may affect drug permeability were analysed for human, mouse, rat and pig skin.

Materials and methods: Classic biophysical skin parameters were measured (e.g. pH, hydration, permittivity, transepidermal water loss). Physiological characteristics of the skins were also analysed by confocal Raman spectroscopy, scanning electron microscopy and two-photon microscopy.

Results: Based on biophysical testing, skin barrier function was damaged in psoriatic mouse skin and in marketed pig skin. Hydration and pH values were similar among the species, but freezing and thawing reduced the water content of the skins and shifted the surface pH to acidic. Aging reduced hydration and permittivity, resulting in impaired barrier function. Mechanical sensitization used in permeability studies resulted in proportional thinning of dead epidermis.

Discussion: Results indicate that depending on the scientific question it should be considered whether fresh or frozen tissue is used, and for certain purposes rodent skins are well usable. The structure of the skin tissue (ceramide, cholesterol, keratin, natural moisturizing factor or urea) is similar in rats and mice, but due to the higher skin thickness the lipid distribution is different in porcine skin. Psoriasis led to irregular chemical composition of the skin.

Conclusion: A comprehensive evaluation of skin samples of four species was performed. The biophysical and microscopic observations should be considered when selecting drug penetration models and experimental conditions.

Keywords: TEWL; aging; confocal Raman spectroscopy; dermal barrier; excised skins; freezing/thawing; hydration; scanning electron microscopy; tape stripping; two-photon microscopy.

FIGURE 1

Comparison of biophysical parameters of excised, frozen native skin samples (0TS: no Tape Strippings, i.e., untreated) of four different species (human, rat, mouse, pig). (A) Transepidermal water loss (TEWL) representing barrier function, (B) stratum corneum surface pH, (C) permittivity by Epsilon sensor, (D) hydration by Corneometer. The number of experiments was n = 5/group. Pig skin was purchased from a local market, contrary to the other animal tissues which were collected from laboratory animals. Statistical significance is marked with *p < 0.05; **p < 0.01; ***p < 0.005

FIGURE 2

Comparison of biophysical parameters of excised, frozen and sensitized skin samples (10TS: 10 Tape Strippings) from young and old rats. (A) Transepidermal water loss (TEWL) representing barrier function, (B) stratum corneum surface pH, (C) permittivity by Epsilon sensor, (D) hydration by Corneometer. The number of experiments was n = 5/group. Statistical significance is marked with *p < 0.05; **p < 0.01; ***p < 0.005

FIGURE 3

Comparison of biophysical parameters of excised, fresh, and frozen native (0TS: no Tape Strippings, i.e., untreated) skin samples from rat abdomen. (A) Transepidermal water loss (TEWL) representing barrier function, (B) stratum corneum surface pH, (C) permittivity by Epsilon sensor, (D) hydration by Corneometer. The number of experiments was n = 5/group. Statistical significance is marked with *p < 0.05; **p < 0.01; ***p < 0.005

FIGURE 4

Comparison of biophysical parameters of excised, fresh native skin samples (0TS: no Tape Strippings, i.e., untreated) from different species. (A) Transepidermal water loss (TEWL) representing barrier function, (B) stratum corneum surface pH, (C) permittivity by Epsilon sensor and (D) hydration by Corneometer. The animal skins (abdominal) were measured ex vivo, while the human skin was tested in vivo on the inner forearm as a control. the number of experiments was n = 3/group of mice, rats and pigs and n = 1 for psoriatic mouse skin and in vivo human control. *p < 0.05; **p < 0.01; ***p < 0.005

FIGURE 5

Confocal Raman spectroscopy (CRS) depth profiles of physiological skin parameters measured in normal and diseased (psoriatic) fresh mouse skins, rat, and laboratory pig skins. (A) Ceramide content, (B) cholesterol content, (C) keratin content, (D) natural moisturizing factor content, (E) urea content, (F) water content of the skin at different measurement depths from skin surface (0 micron) and stratum corneum until the dermis (28 micron). The number of experiments was n = 3 samples/group except for psoriatic mouse skin (n = 1 sample); all measurements were performed with individual measurements of n = 5–8 in the fingerprint region and n = 4–6 in the HVN region; results are given as means +/‐ SD

FIGURE 6

Comparative scanning electron microscopy to study the effect of tape stripping on skin surface morphology. Rat abdominal skin after shaving and epilation without mechanical sensitization (0TS), with 10x (10TS) and 20x sensitizations (20TS)

FIGURE 7

Comparative scanning electron microscopy of the skin surface of two species. Upper panels: human abdominal skin, lower panels: mouse abdominal skin after shaving and epilation, all without mechanical sensitization (0TS)

FIGURE 8

Two‐photon microscopy of excised rat abdominal skin. Left: intact skin without mechanical sensitization (0TS), Right: with 10x mechanical sensitizations (10TS). The skin samples were not labelled, they show autofluorescence at the given laser wavelengths

FIGURE 9

Two‐photon microscopy of mouse (A and B) and human (C and D) abdominal skin samples. Left: laser pictures, right: camera pictures. ‘A’ is a cross sectional view, ‘C’ is an air interface surface view. Red arrow indicates the skin surface stratum corneum. Blue arrow indicates the subcutaneous adipocytes