Research Article
Skin Pharmacol Physiol 2019;32:8–21
DOI: 10.1159/000493168
Effect of Physical and Chemical Hair Removal
Methods on Skin Barrier Function in vitro:
Consequences for a Hydrophilic Model Permeant
Astrid Pany a Victoria Klang a, b Marion Brunner a Johanna Ruthofer a
Elisabeth Schwarz a Claudia Valenta a, b
a Department
of Pharmaceutical Technology and Biopharmaceutics, Faculty of Life Sciences, University of
Vienna, Vienna, Austria; b Research Platform “Characterisation of Drug Delivery Systems on Skin and Investigation of
Involved Mechanisms,” University of Vienna, Vienna, Austria
Keywords
Damaged skin · Skin barrier function · Hair removal ·
Skin/material interaction · Shaving
Abstract
Background: Although very common in our society, the ef-
fect of hair removal on physiological skin parameters and on
the ingress of applied chemicals has not been systematically
investigated. Thus, as a first step, the aim of the present
study was to elucidate the effect of hair removal through
epilation (electric epilation, waxing) and depilation (dry and
wet shaving, depilatory cream) on skin properties in vitro us-
ing the porcine ear model. Methods: Attenuated total reflec-
tion Fourier transform infrared spectroscopy, measurement
of the transepidermal water loss (TEWL), visualization by ca-
pacitance-based contact imaging, confocal Raman spectros-
copy (CRS), diffusion cell studies and tape stripping experi-
ments were employed. Results: Increased TEWL and altered
skin permittivity maps were observed. Decreased stratum
corneum thickness was observed after waxing. Diffusion cell
studies showed increased skin permeation especially in case
© 2018 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/spp
Received: May 25, 2018
Accepted after revision: August 22, 2018
Published online: October 19, 2018
of dry shaving, electric epilation and waxing. Conclusion:
Considering CRS and diffusion cell data, a moderate if sig-
nificant decrease in skin barrier function was found after hair
removal by dry shaving (physical skin/material interaction)
and epilation methods (plucking out the entire hair, for ex-
ample, by electrical epilation and waxing). Subsequent ex-
periments will include testing of different permeants cover-
ing a broad range of physicochemical properties in vitro and
confirming our findings in vivo. © 2018 S. Karger AG, Basel
Introduction
Our skin is a strong barrier against the ingress of xe-
nobiotics; it protects us from mechanical or chemical
damage and dehydration [1]. In everyday life, the skin is
subjected to numerous small stresses that may affect its
barrier function. Many of those stresses are caused by cos-
metic interventions, such as hair removal procedures.
The removal of body hair is immensely common in
Western societies; the prevalence and motivations have
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Leiden University Medisch Centrum
Victoria Klang
Department of Pharmaceutical Technology and Biopharmaceutics
Faculty of Life Sciences, University of Vienna
Althanstrasse 14, AT–1090 Vienna (Austria)
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E-Mail victoria.klang @ univie.ac.at
been described for both women [2–4] and men [5, 6]. De-
spite the fact that these cosmetic procedures targeting
hair removal might compromise the skin barrier, there is
lack of data dealing with the impact of such everyday pro-
cedures on physiological skin properties and stratum cor-
neum (SC) barrier function. Individual studies have in-
vestigated the impact of shaving on axillary skin [7],
which is relevant in context with the use of anti-perspi-
rant and the controversial role of aluminium salts [8]. Re-
cent studies have investigated the effect of shaving on skin
barrier function in context with dyes [9] and sunscreen
application [10]. However, a literature research [11] pro-
duced only a small number of articles associated with the
effect of hair removal by shaving on the skin barrier func-
tion; no data on other methods of hair removal were pre-
sented. The authors reported a clear need for further in-
vestigations since little is known about the extent of bar-
rier damage that may be caused by hair removal.
Moreover, very few studies involve the application of
drug-loaded formulations onto shaved skin, left alone
otherwise depilated or epilated skin. Far more data about
the skin penetration of substances with different physico-
chemical properties are required to make an estimation
of barrier damage caused by hair removal methods. Apart
from shaving, further frequently employed hair removal
methods are waxing [3–5], plucking [3] and depilation
creams [3, 5]. However, to the best of our knowledge, the
effect of such cosmetic procedures on physiological skin
parameters associated with skin barrier function and on
the skin penetration of subsequently applied formula-
tions has not been investigated so far.
Thus, as a first step, the aim of this study was to inves-
tigate the effect of different hair removal procedures on
the skin barrier function of excised porcine ear skin in
vitro, the hypothesis being that slight skin damage is like-
ly due to mechanical interaction or effects of chemical
compounds in hair removal products. Since porcine ear
skin is an accepted model for human skin in terms of
morphology and permeability [12–14], different hair re-
moval procedures were adapted and standardised for use
on excised porcine ears. The investigated techniques in-
cluded dry shaving, wet shaving, electric epilation, depil-
atory cream and waxing; shortening hair by scissors was
employed as the control method. Biophysical measure-
ment techniques were selected to investigate skin proper-
ties associated with barrier function, such as measure-
ment of the transepidermal water loss (TEWL) and skin
dielectric permittivity. Attenuated total reflection Fourier
transform infrared (ATR-FTIR) spectroscopy was em-
ployed to investigate the effect of hair removal on SC con-
Hair Removal and Skin Barrier
stituents such as lipid matrix and proteins [15, 16]. The
SC thickness was determined by confocal Raman spec-
troscopy (CRS) [17–19] before and after hair removal. Fi-
nally, the effect of all hair removal methods on the skin
permeation and skin penetration of the hydrophilic mod-
el permeant fluorescein sodium was investigated using
Franz-type diffusion cells and in vitro tape stripping
studies.
Materials and Methods
Materials
Fluorescein sodium (CAS 518-47-8), di-sodium hydrogen
phosphate dodecahydrate (CAS 10039-32-4) and potassium dihy-
drogenphosphate (CAS 7778-77-0) were purchased from Sigma
Aldrich (St. Louis, MO, USA). Sodium chloride (CAS 7647-14-5)
was obtained from Herba Chemosan Apotheker-AG (Vienna,
Austria). For the tape stripping experiments, Corneofix® adhesive
films with an area of 4 cm2 were received from Courage Khazaka
GmbH (Cologne, Germany). Marketed epilation and depilation
products were bought from a local drugstore (dm drogerie markt
GmbH, Austria). Table 1 gives an overview of all employed hair
removal products and devices.
Preparation of Porcine Skin
Untreated intact porcine ears were obtained directly after sac-
rifice from a local abattoir (Johann Gantner GmbH, Hollabrunn,
Austria) and were immediately frozen and stored at –18 ° C for a
maximum of 3 months. For all biophysical measurements (TEWL,
permittivity), spectroscopic analyses (ATR-FTIR and CRS) and
tape stripping experiments the ears were allowed to thaw at room
temperature directly before the measurements; ears were then
cleaned briefly under cold running water and blotted dry with soft
tissue before the respective experiments. Hair removal was per-
formed by the test methods or the control method (i.e., shortening
of the hair with scissors). For the diffusion cell studies, the porcine
ear skin was additionally cut with a dermatome at 0.7 mm (Aescu-
lap GA 630 DBP, Aesculap AG, Germany); the resulting skin sam-
ples were again stored frozen until use.
Depilated Porcine Ear Skin: Razor Shaving
Both dry shaving and shaving with additional shaving foam
were employed using a disposable razor (BIC® Twin Lady Sensitiv,
Société BIC, France). Thus, the mechanical impact of shaving both
with and without introducing potential chemical effects caused by
shaving foam should be determined. For shaving with shaving
foam, 1.0 g of a shaving product (Balea® Rasiergel mit Aloe Vera
und Avocadoöl empfindliche Haut, dm drogerie markt, Austria)
was applied onto the ear directly prior to shaving.
For both dry and wet shaving, shaving was performed with as
little pressure as possible (less than 0.98 N or 100 g, performed on
a balance) by moving the razor 3 times in alternating direction,
that is, in the direction of hair growth, against direction of growth
and again in direction of growth. In cases where shaving foam was
employed, the skin was briefly washed under running water to re-
move any remnants of the product and was then gently blotted dry
with soft tissue.
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DOI: 10.1159/000493168
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 Table 1. Overview of the employed hair removal products and devices
Product Brand and product name
Wax Balea® Warmwachs-RollOn
Shaving gel Balea® Rasiergel mit Aloe Vera
und Avocadoöl empfindliche Haut
Depilatory Balea® Enthaarungscreme mit
cream Aprikosenöl und Mandelöl
empfindliche Haut
Disposable Balea® Twin Lady Sensitiv,
razors Société BIC, France
Electric Philips HP6401 Satinelle Essential,
epilator Philips, Austria
Depilated Porcine Ear Skin: Depilatory Cream
A depilatory cream (Balea® Enthaarungscreme mit Aprikosenöl
und Mandelöl empfindliche Haut, dm drogerie markt, Austria) was
employed according to the producer’s instructions. The hairy skin
surface was covered in a thin layer of the cream and the product
was left on for 10 min. Afterwards, hair and cream were softly re-
moved with the enclosed plastic scraping tool under application of
minor pressure (again, less than 0.98 N or 100 g). The skin was
gently washed under running water to remove any remnants of the
depilation formulation and was blotted dry with soft tissue.
Epilated Porcine Ear Skin: Electrical Device
An electric epilator (Philips HP6401 Satinelle Essential, Philips,
Austria) was employed. Epilation was performed by gently moving
the device across the skin once for every section of interest against
direction of hair growth.
Epilated Porcine Ear Skin: Waxing
A marketed waxing product (Balea® Warmwachs-RollOn, dm
drogerie markt, Austria) was employed for epilation according to
the producer’s instructions. The wax was melted in a microwave.
Care was taken to let the wax cool down appropriately before ap-
plying a thin layer onto the skin in the direction of hair growth.
Thereafter, one of the enclosed fleece stripes was smoothed down
onto the wax layer and removed in a single movement in the op-
posite direction to hair growth. Every skin site of interest was treat-
ed in this manner once. The skin was then rinsed with water to
remove any wax remnants and blotted dry with soft tissue.
10 Skin Pharmacol Physiol 2019;32:8–21
DOI: 10.1159/000493168
Composition (INCI)
Glucose, Glycerin, Citric Acid, Aloe Barbadensis Leaf
Juice, Parfum, Aqua, Propylene Glycol, Chamomilla
Recutita Flower Extract, Potassium Sorbate, Sodium
Benzoate, Sodium Chloride, CI 14720
Aqua, Palmitic Acid, Triethanolamine, Oleth-20,
Isopentane, Sorbitol, Laureth-23, Aloe Barbadensis
Leaf Juice, Cocamide MIPA, Isobutane, Hydrogenated
Polyisobutene, Hydroxyethylcellulose,
Phenoxyethanol, Parfum, Linalool, Persea Gratissima
Oil, Sodium Sulfate, CI 45100
Aqua, Urea, Cetearyl Alcohol, Potassium
Thioglycolate, Ceteareth 20, Calcium Hydroxide,
Bentonite, Magnesium Trisilicate, Ppg-15 Stearyl
Ether, Titanium Dioxide, Propylene Glycol, Parfum,
Acrylates Copolymer, Paraffinum Liquidum, Prunus
Armeniaca Kernel Oil, Prunus Amygdalus Dulcis Oil,
Sodium Gluconate, BHT, Salicylic acid
–
–
Transepidermal Water Loss
The TEWL represents the amount of water that is lost through
the skin per m2 of skin area per hour (g/m2/h) and is frequently
employed to assess the state of the skin barrier function in vivo [20,
21] or in vitro [22]. While measurement of TEWL in vivo provides
a simple biophysical measurement of the SC function, determina-
tion of TEWL in vitro on porcine skin can be employed to assess
the initial state of the skin barrier and its change during skin dam-
age such as tape stripping [23, 24]. In the present study, the TEWL
of thawed porcine skin was generally measured as means of qual-
ity control. As previously reported, skin with TEWL values up to
20 g/m2/h was considered representative for an intact skin barrier
[25–27]. The TEWL of porcine ear skin was measured using a
closed-chamber device (Aquaflux®, Biox Ltd., UK). Since the pres-
ence of hair may affect the determined TEWL due to adsorbed
moisture [28] all control measurements were performed on skin
after shortening the hair with scissors.
As first step, the most appropriate measurement time after hair
removal was determined in a separate set of experiments. After
measuring the initial TEWL on marked skin areas, the hair was
completely removed with scissors or by electric epilation. Subse-
quent TEWL measurements were performed immediately and af-
ter 30, 60, and 90 min; during these time periods, the skin was left
at ambient conditions in a non-occluded fashion. In accordance
with the results of these tests, all further TEWL measurements
were performed immediately after hair removal.
For the main experiments, the TEWL was measured before
(control) and after hair removal on the exact same skin area to
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evaluate potential barrier damage. For each type of epilated/depil-
ated skin at least 18 skin areas from 4 to 5 different ears were em-
ployed (3–4 experiments per ear).
Capacitive Contact Imaging (Skin Permittivity)
The mean permittivity of the porcine SC was analysed using
capacitive contact imaging (Epsilon® Biox Ltd., UK). Since chang-
es in skin barrier function caused by tape stripping had been suc-
cessfully monitored with a precursor system [24], the present in-
vestigation should reveal whether small changes in skin barrier
function caused by hair removal could be detected. The latter of-
fers a calibrated, linear response to near-surface dielectric permit-
tivity (Ԑ), which is linearly related to SC hydration. Thus, dielectric
permittivity images of the investigated area (12.8 × 15 mm, 50 μm
image resolution, approximately 20 μm depth resolution) are ob-
tained. Measurements were performed using an event trigger to
delay the start of the measurement until contact with the skin sam-
ple was established. A region of interest was selected and range
control was applied (3≤ Ɛ ≤85) to exclude areas of poor contact or
remaining surface water.
For preliminary experiments, capacitance-based contact imag-
ing was performed in parallel to TEWL measurements to deter-
mine the most appropriate measurement time after hair removal.
In accordance with the results of these tests, all further permittiv-
ity measurements were performed immediately after hair removal.
For the main experiments, the effect of different hair removal
procedures on skin permittivity was evaluated. Control measure-
ments could inherently be performed only on separate skin sites
that were freed from hair completely with scissors. For each type
of epilated/depilated skin, at least 10 skin areas from at least 3 dif-
ferent ears were employed (3–4 experiments per ear).
ATR-FTIR Spectroscopy
ATR-FTIR spectroscopic analysis of the skin surface was per-
formed to analyse the effect of hair removal on SC lipids and proteins.
Hair removal was performed on defrosted porcine ears (section 2.2.);
porcine ear skin was prepared for ATR-FTIR analysis as reported
[16]: skin samples of 2 × 7 cm were cut and the skin was removed
from the cartilage with a scalpel. For each type of epilated/depilated
skin, at least 10 skin areas from 4 different ears were employed (2–3
experiments per ear). The skin was stretched onto Styrofoam plates,
fixed with needles and incubated at 32 ° C in a petri dish with satu-
    
rated atmosphere for 2 h to ensure good contact with the ATR crys-
tal. Subsequently, infrared spectra were obtained using an FTIR spec-
trophotometer (Tensor 27, Bio-ATR I tool, Bruker Optics, Vienna,
Austria) thermostatted at 32 ° C. Skin samples were placed on the
    
ZnSe crystal with the SC facing downwards and were weighted down
with a weight of 100.00 g during analysis to achieve spectra of con-
stant intensity. Data analysis including determination of peak posi-
tions and calculation of peak areas were performed using the OPUS
5.5 software (Bruker Optics, Germany). The amide II absorbance was
employed as internal standard to account for differences in contact
between the skin and the ATR crystal. Thus, the obtained data were
compared in the form of absorbance ratios instead of absolute values.
To assess the amount of skin lipids, the symmetric CH2/amide
II absorbance ratio was compared between control (scissors) and
samples treated with the investigated hair removal methods [29,
30]. The wavenumber limits for integration were 2,861–2,837
cm–1 for the symmetric CH2 absorbance and 1,585–1,487 cm–1 for
the amide II absorbance.
Hair Removal and Skin Barrier
For evaluation of changes in the amide I absorbance, caused by
applied substances or treatments, the amide I/amide II absorbance
ratio was analysed [29, 30]. The wavenumber limits for integration
were 1,707–1,590 cm–1 for the amide I absorbance and 1,585–1,487
cm–1 for the amide II absorbance.
CRS: SC Thickness
CRS was employed to investigate the effect of different hair re-
moval methods on SC thickness by performing before/after mea-
surements on the same skin areas. Control measurements were
conducted after trimming hair with scissors; final measurements
were conducted on the same skin areas after the subsequent hair
removal method.
Hair removal was performed on defrosted porcine ears (section
2.2.). Skin areas of 2 × 2 cm2 were marked and analysed before and
after hair removal with a confocal Raman microspectrometer
(gen2 Skin Composition Analyzer, River Diagnostics, the Nether-
lands). The obtained spectra were evaluated with SkinTools® soft-
ware version 2.0. Measurements were performed in the high wave-
number region (2,000–4,000 cm–1) with the 671 nm laser in 2 µm
depth increments up to a depth of 40 µm at a signal collection time
of 2 s per spectrum on 8 different locations of each area before and
after hair removal. The SC thickness was calculated after con-
structing mean water concentration profiles [17–19]. The water
content within the SC increases gradually from the outside to the
inside until it reaches an almost constant value in the viable tissue
[31]. Using the obtained profiles, 2 straight lines were applied to
the 2 reaches of the curve and SC thickness was obtained by calcu-
lating the intersection of the lines. For each type of epilated/depil-
ated skin, at least 15 skin areas from at least 4 different ears were
employed (3–4 experiments per ear).
Skin Permeation Experiments: Diffusion Cell Studies
Diffusion cell studies were conducted to investigate the effect
of different hair removal procedures on skin permeation of the
hydrophilic model drug fluorescein sodium. Static Franz-type dif-
fusion cells (Permegear, PA, USA) were employed. Thawed skin
patches were cut to appropriate size and mounted between donor
and receptor chamber resulting in a permeation area of 0.95 cm2.
The receptor compartment was filled with 2 mL of phosphate buff-
ered saline (pH 7.4, 0.238% w/w sodium monohydrogen phos-
phate, 0.019% w/w potassium dihydrogen phosphate, 0.8% w/w
sodium chloride) to provide sink conditions. The donor compart-
ment was filled with 200 mg of an aqueous solution of fluorescein
sodium at 0.05% w/w to achieve infinite dose conditions. Donor
compartment and sampling arm were occluded to prevent evapo-
ration. The diffusion cells were kept at 32 ° C and stirred with mag-
netic bars for 24 h. The entire receptor fluid was removed at de-
fined intervals of 2, 4, 6, 8, and 24 h for analysis and replaced with
fresh receptor medium. Consecutive sets of experiments were per-
formed with test samples (n = 20 samples from 4 ears) and control
samples (n = 30 samples from 6 ears).
The receptor fluid samples were analysed for their fluorescein
sodium content by fluorescence spectroscopy to calculate the cu-
mulative permeated drug amounts in percentage of applied dose.
Skin Penetration Experiments: in vitro Tape Stripping
To investigate the effect of hair removal procedures on fluores-
cein sodium skin penetration under finite-dose application, in vi-
tro tape stripping experiments were performed. A finite dose (5
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 30
* *
25 * 30
*
20
TEWL, g/m2/h
15
10
5 5
Electric epilation
Control
0 0
0 30 60 90
a Time, min
Fig. 1. Development of transepidermal water loss (a) and mean
stratum corneum permittivity (b) during prolonged experiment
times in vitro after hair removal by electric epilation (black lines)
or scissors (control, grey lines). Values are means of n = 13–14 ex-
periments ± SD performed on 5 porcine ears respectively. Signifi-
μL/cm2) of an aqueous fluorescein sodium solution (0.05% w/w)
was applied onto a marked skin area and gently spread for one
minute with a fingerstall that had been pre-saturated with aqueous
fluorescein sodium solution. If the fluorescein sodium solution
had not completely been absorbed after spreading and an exposure
time of one hour, as observed after waxing and application of de-
pilatory cream, residues were carefully dabbed off with soft tissue
before proceeding. Adhesive films (Corneofix®, Courage Khazaka
GmbH, Cologne, Germany) were employed to remove the upper-
most SC layers. The amount of corneocytes on each strip was de-
termined by IR-densitometry (Squame ScanTM 850A, Heiland
Electronics, Wetzlar, Germany) as described [32, 33]. The fluores-
cein sodium content of the adhesive tapes was analysed by fluores-
cence spectroscopy.
In the course of the tape stripping experiments, the total SC
thickness of the employed porcine ear skin was determined by IR-
densitometry. Since inter-individual variability in SC thickness has
to be expected [14, 34], the SC thickness was determined on every
employed ear. To this end, adhesive films were removed until the
pseudo absorption was approaching zero. Previously determined
calibration data were employed to calculate the mass of corneo-
cytes on each adhesive film by applying the equation m = Acorr/0.224
(in µg/cm2) [35]. Assuming the mean protein density to be around
1 g/cm3 [12], the mean cumulative amount of removed corneo-
cytes was employed to calculate the total SC thickness and the pen-
etration depth of fluorescein sodium. The latter was expressed as
a per cent of the corresponding individual SC thickness. For each
type of epilated/depilated skin, 12 skin areas from at least 3 differ-
ent ears were employed (3–4 experiments per ear).
Drug Quantification: Fluorescence Spectroscopy
After measuring the pseudo-absorption of the corneocytes on
the adhesive tapes by IR-densitometry, fluorescein sodium was ex-
tracted from the adhesive films with phosphate buffered saline in
12 Skin Pharmacol Physiol 2019;32:8–21
DOI: 10.1159/000493168
35
*
Mean permittivity Ɛ, arbitrary units
25
20
15
10
0 30 60 90
b Time, min
cant differences between electric epilation and control are marked
with asterisks (* p < 0.05, paired t test for comparison of subse-
quent measurements on the same area, unpaired t test or Mann-
Whitney test for comparison between scissors/electric epilation).
an ultrasonic bath for 12 min. Care was taken to protect the sam-
ples from light to avoid photo-bleaching. The samples were cen-
trifuged and the supernatant was analysed by fluorescence spec-
troscopy. To this end, 100 μL of the samples were pipetted into a
microtiter plate in triplicate and analysed against phosphate buff-
ered saline as control. A calibration curve of fluorescein sodium in
phosphate buffered saline ranging from 0.002 to 1 µg/mL was ap-
plied onto every microtiter plate. The correlation coefficients of
the calibration curves obtained by Pearson correlation ranged
from 0.9999 to 1. Samples with fluorescein sodium contents high-
er than 1 µg/mL were diluted prior to analysis. Quantification was
conducted employing a microplate reader (TecanTM infinite 200,
Tecan Ltd., Maennedorf, Switzerland) at an excitation wavelength
of 485 nm and an emission wavelength of 535 nm.
Statistical Data Analysis
For statistical data analysis, GraphPad Prism 3.0 (GraphPad
Software, CA, USA) was employed. One-way analysis of variance
or Student t test (paired t test for CRS data, otherwise unpaired t
test) was performed for the analysis of parametric data. Non-para-
metric data were analysed employing the Kruskal-Wallis test fol-
lowed by Dunn’s Multiple Comparison test or Mann-Whitney/
Wilcoxon signed-rank test (the latter for paired TEWL data). p <
0.05 was considered statistically significant for all tests.
Results
Transepidermal Water Loss
The mean TEWL of untreated porcine ear skin in vitro
after shortening of the hair with scissors was 14.42 ± 2.67
g/m2/h (n = 155).
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 50 ■ Before hair removal
■ After hair removal **
40
*
**
Fig. 2. Box plots of transepidermal water

TEWL, g/m2/h
30
loss before (white boxes) and directly after
hair removal (grey boxes) with different
*
procedures. The line in the middle indi- 20
*
cates the median value, the extent of the
lines the lowest and highest values among
n = 18–21 experiments performed on 4–5 10
ears respectively. The lower and upper
parts of the rectangles depict the first and
third quartile. Significant differences are 0
Depilatory cream Wet shaved Dry shaved Epilated Waxed
marked with asterisks (*  p < 0.05, **  p <
0.01, Wilcoxon signed-rank test).

**
**
50 ***
45
Mean permitivity, arbitrary units

40
35
30
25
20
Fig. 3. Mean skin permittivity values of 15
porcine ear skin directly after hair removal.
10
Values are means of n = 10–17 experiments
± SD performed on 3–4 ears respectively. 5
Significant differences to the control are 0
marked with asterisks (** p < 0.01, *** p < Control Dep. cream Wet shaved Dry shaved Epilated Waxed
0.001, unpaired t test).

No changes in TEWL during prolonged experiment
times after hair removal were found in preliminary ex-
periments (Fig. 1a, control and electric epilation as repre-
sentative example of hair removal, n = 13–14, p > 0.05
over time for both). Therefore, the main TEWL measure-
ments were conducted directly after hair removal. Sig-
nificantly higher TEWL values were observed for epilated
skin versus the control at all measurement points (Fig. 1a,
n = 13–14, p < 0.05). This is in agreement with the results
of the main TEWL experiments (Fig. 2). When measuring
TEWL on shortened hair and again on the same area after
hair removal significantly higher values were observed for
all investigated hair removal methods (n = 18–21, p <
0.05). When comparing the percentage change in TEWL
related to the respective initial values, no differences be-
tween the individual hair removal methods were ob-
served (n = 18–21, p > 0.05).
Electrical Permittivity
In preliminary studies, the mean SC permittivity of
untreated porcine ear skin in vitro after removing the hair
with scissors was 30.45 ± 4.93 arbitrary units (n = 47). No
changes in mean SC permittivity during prolonged ex-
periment times after hair removal were found (Fig.  1b,
control and electric epilation as representative example of
hair removal, n = 13–14, p > 0.05 over time). Therefore,
the main permittivity experiments were conducted di-
rectly after hair removal. Significantly higher mean SC
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Hair Removal and Skin Barrier Skin Pharmacol Physiol 2019;32:8–21 13

DOI: 10.1159/000493168
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 Color version available online
a b

c d

Fig. 4. Effect of hair removal on skin condition visualised by capacitance-based sensor imaging (Epsilon® E100,
Biox Ltd., UK). Bright areas indicate high permittivity/high stratum corneum (SC) hydration. Images were taken
before (left-hand side) and after 60 s of occlusion (right-hand side) following hair removal by dry shaving (a),
waxing (b), depilatory cream (c), electric epilation (d).

permittivity values were observed for epilated skin versus
the control directly after epilation (Fig. 1b, n = 13–14, p <
0.05). No differences between epilated skin and control
skin were observed during the following measurements
points after 30, 60 and 90 min (n = 13–14, p > 0.05). The
initial difference in SC permittivity after hair removal is
reflected in the results of the main permittivity experi-
ments (Fig.  3). Significantly lower permittivity values
were obtained for skin after dry shaving and electric epi-
lation, while significantly higher values were observed af-
ter the depilatory cream (n = 10–17, all p < 0.05 versus
control).
The corresponding skin permittivity maps revealed ir-
regular skin surface properties after some of the investi-
gated hair removal methods (Fig.  4). Uniformly higher
skin permittivity was observed after waxing and particu-
larly after the application of the depilatory cream (Fig. 4c,
brighter image due to occlusion effects), while an uneven
skin status due to irregular skin lesions was observed es-
pecially after dry shaving (Fig. 4a, white circles).
ATR-FTIR Spectroscopy: Skin Lipid Matrix
The absorbance bands caused by intercellular lipids or
keratin within the SC were evaluated in terms of peak po-
sitions and peak areas. In regard to the conformation
of the intercellular lipids, the CH2 asymmetric (∼2,917
cm–1) and symmetric (∼2,850 cm–1) stretching vibrations
and the CH2 scissoring mode (∼1,466 to 1,455 cm–1) were
analysed [15]. No statistically significant shifts of the
spectral bands were observed (n = 10–13, p > 0.05 vs. con-
trol). None of the hair removal methods or formulations
caused transitions of the skin lipid order.
Regarding the protein peaks, the positions of the am-
ide I (∼1,642 cm–1) as well as amide II (∼1,541 cm–1) vi-
brations were only slightly affected by the different hair
removal methods (n = 10–13, p > 0.05 in all cases). No
structural changes to the keratin within the corneocytes
were observed.
The symmetric CH2/amide II absorbance ratio is rep-
resentative for the amount of SC lipids. A trend towards
higher values was observed for dry shaving > depilatory
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 1,642 cm–1
CH2 asymmetric vibrations (~2,917 cm–1)
CH2 symmetric vibrations (~2,850 cm–1)
1,541 cm–1
CH2 scissoring mode (~1,466–1,455 cm–1)

Amide I (~1,642 cm–1)

Amide II cm–1 (~1,541 cm–1)

Control
Wet shaved
0.8 1,466–1,455 cm–1
2,917 cm–1
Absorbance units

2,850 cm–1
0.6

0.4

0.2

3,500 3,000 2,500 2,000 1,500 1,000

a Wavenumber, cm–1

Waxed ***
Epilated

Dry shaved *
Wet shaved **
Depilatory
cream ** ***
Control

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0 0.5 1.0 1.5 2.0
b Symmetric CH2/amide II peak area ratio c Amide I/amide II peak area ratio

Fig. 5. a Representative attenuated total reflection Fourier trans-
form infrared (ATR-FTIR) spectrum of skin after hair removal
with scissors (control, dashed line) and after wet shaving (solid
line). b Comparison of the symmetric CH2/amide II absorbance
ratio measured after hair removal by scissors (control) and differ-
ent hair removal methods. c Comparison of the amide I/amide II
absorbance ratio measured after hair removal by scissors (control)
and different hair removal methods. Values in 5b/5c are means of
n = 10–13 experiments ± SD performed on 4 ears respectively. Sig-
nificant differences to the control are marked with asterisks (* p <
0.05, ** p < 0.01, *** p < 0.001, t test or Mann-Whitney test).

cream > wet shaving > waxing > electric epilation > con- tained after waxing and the depilatory cream (n = 10–13,
trol (Fig. 5b). Dry shaving, wet shaving and the depila- p < 0.001 vs. control).
tory cream led to significantly higher lipid values (n =
10–13, p < 0.05 vs. control). CRS: SC Thickness
Regarding the amide I/amide II absorbance ratio at the The results of SC thickness determination are given in
skin surface (Fig. 5c), significantly higher values were ob- Figure 6. A statistically significant decrease of mean SC
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 20 ■ Before hair removal
18 ■ After hair removal

Stratum corneum thickness, μm
16
14
**
12
10
Fig. 6. Stratum corneum thickness calcu- 8
lated from water concentration profiles ob-
tained by confocal Raman spectroscopy be- 6
fore (white bars) and after (grey bars) hair 4
removal. Values are means of n = 15–16 ex- 2
periments ± SD performed on 4–5 ears re-
spectively. Significant differences to the 0
Depilatory cream Wet shaved Dry shaved Epilated Waxed
control are marked with asterisks (**  p <
0.01, paired t test).

100

10 ***
***
Cumulated permeation, %

*** ***
1 ***
■ Control
** ■ Depilatory cream
■ Wet shaved
0.1
■ Dry shaved
■ Epilated
■ Waxed
0.01

0.001
2h 4h

100

*** *** *
10 *** *** *
*** ***
Cumulated permeation, %

*** ***
1

0.1

0.01

0.001
6h 8h 24 h

Fig. 7. Box plot of the mean cumulated permeation of fluorescein
sodium expressed in percentage of the applied dose after 2, 4, 6, 8,
and 24 h (logarithmic scale). The line in the middle indicates the
median value, the extent of the lines the lowest and highest values
among n = 20–30 diffusion cells with skin samples obtained from
4 to 6 ears, respectively. The lower and upper parts of the rectangles
depict the first and third quartile. Significant differences to the
control are marked with asterisks (* p < 0.05, ** p < 0.01, *** p <
0.001, Kruskal-Wallis test, Dunn’s Multiple Comparison test).
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 70
60
Skin penetration depth, % SC
50
40
30
Fig. 8. Skin penetration depth of fluores-
cein sodium in percent of total stratum cor- 20
neum (SC) thickness by IR-densitometry
(dark grey bars) and corrected for the esti- 10
mated decrease of SC thickness determined
by confocal Raman spectroscopy (CRS; 0
light grey bars). Values are means of n = 12
experiments ± SD performed on 3–4 ears,
respectively. Significant differences to the
control (white bar) are marked with aster-
isks (** p < 0.01, unpaired t test).
thickness by 8% could be observed after waxing (n = 15–
16, p < 0.01); a trend towards slightly lower values was
observed after dry and wet shaving (mean decrease of
6–7% of SC thickness, n = 15–16). Correction factors
were calculated and employed to account for lost SC
thickness in the evaluation of the tape stripping experi-
ments.
Diffusion Cell Studies
Skin permeation experiments were conducted under
occluded infinite-dose conditions (Fig.  7). Significantly
higher permeated amounts of fluorescein sodium were
found at all sampling times (2, 4, 6, 8, and 24 h) for skin
after dry shaving, electric epilation and waxing (n = 20–
30, p < 0.05). Trends towards higher values were observed
for skin after wet shaving and depilatory cream.
Tape Stripping
Tape stripping experiments under finite-dose condi-
tions were performed; the observed penetration depths
of fluorescein sodium into the SC after hair removal are
given in Figure 8. Conventional evaluation of the per-
centage penetration depth (dark grey bars) by IR-densi-
tometry was corrected for corneocyte layers potentially
lost in the hair removal process due to skin/material in-
teraction (light grey bars); correction factors were deter-
mined by CRS analysis. The skin penetration depth of
fluorescein sodium was only moderately affected in this
experimental setup; significantly higher penetration
Hair Removal and Skin Barrier
**
Control Depilatory Wet shaved Dry shaved Epilated Waxed
cream
■ Corrected for the potential decrease of SC thickness as determined by CRS
■ As calculated with the obtained NIR values
depths were observed only after dry shaving (n = 12, p <
0.01 vs. control).
Regarding the total penetrated drug amounts 1.3 ± 0.2
µg/cm2 of fluorescein sodium were found in the first few
micrometres of the SC of control skin (n = 12). Compa-
rable drug amounts were found for skin after dry shaving,
electric epilation and waxing (n = 12), slightly lower drug
amounts around 0.9 ± 0.4 µg/cm2 after depilatory cream
and wet shaving (n = 12, p < 0.05).
Representative in vitro skin penetration profiles of flu-
orescein sodium into porcine ear skin are depicted in Fig-
ure 9 for control skin and skin after dry shaving consider-
ing the potential loss of corneocyte layers as determined
by CRS (compare Fig. 8, light grey bars).
Discussion
Transepidermal Water Loss, Electrical Permittivity
and Barrier Function
The correlation between TEWL and skin barrier func-
tion following physical damage in vitro has been subject
to debate [36, 37]; in our experience, the measurement of
TEWL in vitro is a simple and useful tool to exclude dam-
aged skin areas. In the present study, measurement of the
TEWL before and after hair removal on the same skin
area was employed to investigate potentially small barrier
defects caused by the procedures. Care was taken to en-
sure stable environmental conditions [38]; a closed-
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 0
1
10

20 5
30

Horny layer thickness, %

40

Tape number
10
50

60

70

80

90
■ Concentration of fluorescein sodium, µg/cm2
100
a 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

0
Potential decrease of SC thickness as determined by CRS
10
1
20

30 5
Horny layer thickness, %

40

Tape number
50 10
60

Fig. 9. Representative in vitro skin penetra- 70

tion profiles of fluorescein sodium into
80
porcine ear skin for control skin (scissors
[a]) and skin after dry shaving, considering 90
the potential loss of corneocyte layers as ■ Concentration of fluorescein sodium, µg/cm2
100
determined by confocal Raman spectros-
b 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
copy (CRS; compare Fig. 8, light grey bars
[b]).

chamber device was employed. Due to the in vitro nature
of the experimental setup, no sweat gland activity could
affect the results.
The mean TEWL of untreated porcine ears after short-
ening the hair with scissors exhibited comparatively small
fluctuations; values were in agreement with literature
[14]. All investigated hair removal methods led to in-
creased TEWL values; no differences between individual
methods were observed. In general, TEWL values after
hair removal showed higher variability implying uneven
skin damage, which is in agreement with the visualisation
by capacitive contact imaging. This can be expected even
under standardised conditions of hair removal, since skin
is not a perfectly uniform barrier but shows inter-individ-
ual differences in superficial morphology and sensitivity
to stress. We conclude that measurement of the TEWL
can detect small changes in skin barrier function in vitro
if the exact same measurement areas are used for com-
parative studies; otherwise, the high inter-individual vari-
ations of TEWL values will mask potential effects. Future
in vivo studies might reveal stronger differences between
individual hair removal procedures regarding barrier
damage and recovery time. The effect of regular long-
term hair removal on skin barrier function is also an as-
pect worth investigating in vivo.
Visualisation of skin permittivity by the employed sen-
sor technology allowed an interesting insight into the way
the different procedures affected the skin surface. Like for
TEWL measurements, the technique provides more in-
formation when applied in vivo, but can serve as simple
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biophysical tool for rapid assessment of skin status in vi-
tro. The lower values observed after electric epilation and
dry shaving might be ascribed to water loss of the tissue,
while treatment with the depilatory cream caused in-
creased SC hydration due to occlusion. Skin lesions were
particularly visible in case of dry shaving and – to a small-
er extent – for the other methods.
ATR-FTIR Spectroscopy and SC Compounds
ATR-FTIR spectroscopy of the skin surface was em-
ployed to investigate whether the different hair removal
methods, in particular those including dermal rinse-off
formulations such as shaving foam or depilatory cream,
would affect the SC on a molecular level. No shifting of
the lipid- or protein- associated spectral bands were ob-
served after different hair removal methods; it can there-
fore be assumed that none of the hair removal methods
or formulations caused transitions of the skin lipid order
or structural changes to the keratin.
When considering the symmetric CH2/amide II absor-
bance ratio, it is evident that higher values were obtained
after hair removal, suggesting the removal of superficial
corneocytes (e.g., in case of dry shaving). The remaining
skin surface exhibited higher relative amounts of intercel-
lular lipids. In case of wet shaving and the depilatory
cream, the higher absorbance of the analysed alkyl chain
signal might also have been caused by penetration of ap-
plied compounds into the SC. This is particularly likely in
case of the depilatory cream, which remained on the skin
surface for 10 min during application.
Regarding the amide I/amide II absorbance ratio at the
skin surface, significantly higher values were obtained af-
ter waxing and the depilatory cream; previous groups
have associated such changes with an increased moisture
content of the SC [29, 39]. For our data, this observation
would be in good agreement with the capacitive contact
imaging data, where a visual tendency towards uniformly
higher mean permittivity values was observed after wax-
ing and the depilatory cream.
CRS and SC Thickness
All hair removal techniques that involve some kind of
physical skin/material interaction may remove corneo-
cytes from the outermost SC layers, thus reducing the to-
tal thickness of the SC barrier. On the one hand, the ex-
tent of this potential reduction of barrier thickness by hair
removal is an interesting observation in itself. On the oth-
er hand, knowledge of the reduction of SC thickness
through hair removal is an important asset for evaluation
of the obtained tape stripping data [40].
Hair Removal and Skin Barrier
CRS measurements were performed to investigate the
influence of the different hair removal techniques on SC
thickness. The CRS technique was chosen due to its non-
destructive nature and the possibility to determine the SC
thickness via skin hydration profiling on the same mea-
surement area before and after hair removal.
Hair removal methods with more pronounced physi-
cal skin/material interaction (waxing, but also trends for
dry/wet shaving) removed a higher amount of corneo-
cytes from the SC surface, resulting in a decrease of SC
thickness, than plucking out the hair individually, which
did not affect SC thickness (electric epilation). In case of
the depilatory cream, which is scraped off the skin surface
after 10 min of application, the effect of the physical in-
teraction by scraping was presumably masked by the al-
tered skin hydration profile due to the lipid replenishing
formula of the employed product and occlusion effects.
In vitro Skin Permeation and Penetration Studies
Skin permeation experiments employing Franz type
diffusion cells and an infinite dose were performed to in-
vestigate whether and to what extent skin permeability of
fluorescein sodium would be affected by the different hair
removal methods. As a general trend, higher skin perme-
ation of fluorescein sodium was observed for all investi-
gated hair removal methods when compared to the con-
trol. This is in agreement with the results of a previous
study that showed higher skin permeation through shaved
skin in context with dyes [9]. It seems evident that meth-
ods of epilation, where the entire hair including the root
is ripped out by force (epilation, waxing), led to signifi-
cant increases in skin permeability at all times of the ex-
periment. However, even for dry shaving, significantly
higher permeability could be observed and this might be
related to a decrease of SC thickness due to physical skin/
material interaction. Smaller effects could be observed af-
ter the other depilation methods (depilatory cream, wet
shaving), which is in agreement with the total drug
amounts found by tape stripping and presumably associ-
ated with the employed formulations (depilatory cream,
shaving foam).
Interestingly, the observed differences in skin perme-
ation were more accentuated during the first 8 h of the
experiments, which is presumably related to the experi-
mental setup. Swelling of the skin after prolonged exper-
iment times might have reduced potential differences in
barrier function caused by the different hair removal
techniques. Due to the hydrophilic nature of the model
substance, diffusion along the hydrophilic head groups
within the SC lipid double layer is strongly enhanced after
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DOI: 10.1159/000493168
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prolonged swelling irrespective of small differences in
skin properties.
To confirm the diffusion cell data, tape stripping ex-
periments with a finite dose were performed. Again, the
influence of the different hair removal methods on skin
penetration of fluorescein sodium from an aqueous solu-
tion was evaluated. The obtained penetration depth in
micrometres was related to the respective determined to-
tal SC thickness on each porcine ear. The obtained SC
thicknesses varied between 6.7 and 19.9 µm. Convention-
al evaluation of the percentage penetration depth by IR-
densitometry was corrected for corneocyte layers poten-
tially lost in the hair removal process due to skin/mate-
rial interaction with correction factors determined by
CRS. The skin penetration depth of fluorescein sodium
was only significantly increased in case of dry shaving.
The total penetrated drug amounts were comparable for
most hair removal methods and the control. In general,
very low amounts of fluorescein were found. Only in case
of methods including rinse-off products (depilatory
cream, wet shaving with foam), slightly lower penetrated
drug amounts were found. This can be ascribed to lipid
replenishing effects protecting the skin surface against
the ingress of chemicals. These results are in agreement
with the diffusion cell experiments, where generally high-
er drug amounts were involved due to the set-up, but no
significant differences were observed between the control
and these 2 methods except after 24 h.
Summarising Remarks
The results suggest that hair removal by dry shaving
without suitable dermal formulations has the strongest
effect on skin barrier function of all depilation methods.
In general, epilation methods such as electric epilation or
waxing appear to have an equally strong effect due to the
fact that the entire hair is pulled out. Follow-up in vivo
studies will bring more information on the practical rel-
evance of these findings, since long-term effects are also
related to the frequency of the treatment, which is of
course very different for depilation and epilation meth-
ods.
Conclusions
Mild changes in SC barrier properties caused by differ-
ent hair removal methods were found, confirming the ex-
istence of small barrier alterations introduced through
mechanical interaction or chemical compounds in hair
removal products. Significantly decreased SC thickness
was found by CRS in case of waxing. Diffusion cell studies
showed enhanced skin permeation of the hydrophilic
model drug fluorescein sodium especially in case of dry
shaving, electric epilation and waxing. Tape stripping ex-
periments confirmed this trend for dry shaving. Subse-
quent experiments will include testing of different per-
meants covering a broad range of physicochemical prop-
erties in vitro and confirming our findings in vivo, where
a representative assessment of barrier impairment and
recovery time on human skin can be undertaken.
Statement of Ethics
Pig ears were received from a local abattoir (EU Schlachthof
Gantner GmbH, Gewerbering 19, 2020 Hollabrunn, Austria). No
living animals were involved in any of the studies or preparations
thereof; the employed porcine ears were obtained as waste from
routine animal sacrifices.
Disclosure Statement
The authors report no conflicts of interest. No external funding
was received and the authors alone are responsible for the content
and writing of the paper.

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