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ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2008
Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 381
Skin barrier responses tomoisturizers
IZABELA BURACZEWSKA
ISSN 1651-6206ISBN 978-91-554-7296-2urn:nbn:se:uu:diva-9300
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To my Family
'Well, in OUR country,' said Alice, still panting a little,
'you'd generally get to somewhere else–if you ran very fast
for a long time, as we've been doing.'
'A slow sort of country!' said the Queen. 'Now, HERE, you
see, it takes all the running YOU can do, to keep in the
same place. If you want to get somewhere else, you must
run at least twice as fast as that!'
Lewis Carroll, “Through the Looking-Glass”
PAPERS INCLUDED
This thesis is based on the following papers, which will be referred to in the text by their
Roman numerals:
I. Buraczewska I., Lodén M. Treatment of surfactant-damaged skin in humans with
creams of different pH values. Pharmacology 2005; 73:1–7.
II. Buraczewska I., Berne B., Lindberg M., Törmä, H. and Lodén M. Changes in skin
barrier function following long-term treatment with moisturizers, a randomized
controlled trial. Br J Dermatol 2007; 156:492–8.
III. Buraczewska I., Berne B., Lindberg M., Lodén M. and Törmä, H. Effect of a long
term-treatment with moisturizers on the keratinocyte differentiation and desquamation.
Arch Dermatol Res, in press.
IV. Buraczewska I., Berne B., Lindberg M., Lodén M. and Törmä, H. Moisturizers change
the mRNA expression of enzymes synthesizing skin barrier lipids. Manuscript.
Reprints were made with permission from the publishers.
TABLE OF CONTENTS
Introduction .............................................................................................................................. 11�
The structure and barrier function of the skin ..................................................................... 11�
Skin structure .................................................................................................................. 11�
Stratum corneum as the skin barrier ............................................................................... 13�
Intercellular lipids of the stratum corneum..................................................................... 15�
Enzymes and non-enzymatic regulatory proteins of the skin barrier .................................. 15�
Corneocyte formation ..................................................................................................... 15�
Desquamation ................................................................................................................. 16�
Formation of extracellular lipid matrix of stratum corneum .......................................... 17�
Nuclear hormone receptors and lipoxygenases .............................................................. 18�
Skin pH................................................................................................................................ 19�
Moisturizers and the skin barrier ......................................................................................... 21�
Chemistry of moisturizers ................................................................................................... 22�
Methods used for evaluation of the skin barrier function.................................................... 23�
Aim of the research.............................................................................................................. 24�
Materials and Methods ............................................................................................................. 25�
Volunteers............................................................................................................................ 25�
Test moisturizers ................................................................................................................. 25�
Experimental design ............................................................................................................ 27�
Paper I ............................................................................................................................. 27�
Paper II............................................................................................................................ 27�
Papers III and IV............................................................................................................. 27�
Evaluations .......................................................................................................................... 28�
Calculations and statistics.................................................................................................... 28�
Results ...................................................................................................................................... 30�
Paper I.................................................................................................................................. 30�
Paper II ................................................................................................................................ 30�
Paper III ............................................................................................................................... 31�
Paper IV............................................................................................................................... 33�
Discussion and conclusions...................................................................................................... 37�
Choice of test moisturizers .................................................................................................. 38�
Effect of 7-week use of test preparations on the skin barrier .............................................. 39�
Possible explanations of observed effects ........................................................................... 39�
Lipids .............................................................................................................................. 40�
Water............................................................................................................................... 40�
Emulsifiers and polymers ............................................................................................... 41�
Humectants ..................................................................................................................... 41�
Occlusion ........................................................................................................................ 42�
pH .................................................................................................................................. 43�
Molecular changes induced by Hydrocarbon and Complex creams ................................... 44�
Effect of Hydrocarbon cream on the skin barrier ........................................................... 44�
Nuclear receptors and lipoxygenases.............................................................................. 44�
Enzymes and proteins involved in keratinocyte differentiation and desquamation ....... 45�
Conclusions ......................................................................................................................... 47�
Future perspectives................................................................................................................... 48�
Svensk sammanfattning (summary in Swedish) ...................................................................... 51�
Acknowledgments.................................................................................................................... 52�
References ................................................................................................................................ 54�
ABBREVIATIONS
ACACB acetyl-CoA carboxylase beta
ACSL1 acyl-CoA synthetase long-chain family member 1
ACTB actin, beta (�-actin)
ALOX12B arachidonate 12-lipoxygenase, 12R type
ALOXE3 epidermal arachidonate lipoxygenase 3
ARCI autosomal recessive congenital ichthyosis
CDKN1A cyclin-dependent kinase inhibitor 1A
cDNA complementary deoxyribonucleic acid
CoA Coenzyme A
DAPI 4’-6-diamidino-2-phenylindole
FASN fatty acid synthase
FLG profilaggrin
GBA glucocerebrosidase, beta; acid (�-glucocerebrosidase)
HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase)
HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMG-CoA synthase 1)
IL1A interleukin-1�
IVL involucrin
KLK5 kallikrein 5
KLK7 kallikrein 7
mRNA messenger ribonucleic acid
NMF natural moisturizing factor
o/w oil-in-water
PBS phosphate-buffered saline
PCA pyrrolidone carboxylic acid
PPARA peroxisome proliferator-activated receptor alpha (PPAR-�)
PPARB peroxisome proliferator-activated receptor beta (PPAR-�)
PPARG peroxisome proliferator-activated receptor gamma (PPAR-�)
QRT-PCR quantitative real-time polymerase chain reaction
RT reverse transcription
RXRA retinoid X receptor alpha (RXR-�)
SLS sodium lauryl sulfate
SMPD1 sphingomyelin phosphodiesterase 1, acid lysosomal (acid sphingomyelinase)
SPTLC2 serine palmitoyltransferase, long chain base subunit 2 (serine palmitoyl-
transferase 2)
TEWL transepidermal water loss
TGM1 transglutaminase 1
UGCG UDP-glucose ceramide glucosyltransferase
11
INTRODUCTION
THE STRUCTURE AND BARRIER FUNCTION OF THE SKIN
Terrestrial life would not be possible without protection from dehydration and harmful
factors. The skin gives us such protection, but it is more than an impenetrable shield: it is a
dynamic and complex tissue, mediating a multiplicity of functions. It provides a physical
permeability barrier, hampering excessive water and electrolyte loss from inside-out, and
protects from external chemical, microbial, and mechanical insults. The skin plays an
essential role in thermoregulation, absorbance of ultraviolet radiation, sensation and
sociosexual communication. Since it is our largest organ, its immunological performance as
well as its ability to repair wounds and regenerate itself, is of vital importance for the entire
body.1,2
Skin structure
The skin consists of three distinctive layers, the subcutis, dermis, and epidermis (Figure 1).
The lowest of these layers, the subcutis (hypodermis, subcutaneous fat), is built of adipose
tissue, which helps to cushion and insulate the body. This layer serves as energy storage and
allows for skin mobility over underlying structures.1,2
The dermis constitutes the principle mass of the skin. Its main component, extracellular
matrix (ground substance), attracts and retains water due to presence of strongly hygroscopic
molecules, proteoglycans. Dermis is crossed with nerve and vascular networks and embraces
epidermal appendages such as hair, sweat glands and sebaceous glands. It contains various
cell types, such as fibroblasts, macrophages, mast cells, and transient circulating cells of the
immune system. The dermis is tightly connected to the uppermost layer of the skin, the
epidermis, by the basement membrane, which is the main part of the dermal–epidermal
junction.1,2
The epidermis, a constantly self-renewing stratified structure, is built mostly of keratinocytes,
which account for at least 80% of its total cells. Therefore, the properties and functioning of
keratinocytes determine the condition of the epidermis. The remaining cell types are
melanocytes, Langerhans cells, Merkel cells, and various cells of the immune system. Due to
the differentiation state of keratinocytes, layers of epidermis are classified into the stratum
basale, stratum spinosum, stratum granulosum and stratum corneum (Figure 2).1,2
12
Figure 1 – Skin layers. From: Gilberg S., Tse D. Atlas of Ophthalmology, Edited by Richard
Parrish II, David T. Tse, 2000, Current Medicine Group LLC, with permission.
Figure 2 – Layers of epidermis. From Gilberg S., Tse D. Atlas of Ophthalmology. Edited by
Richard Parrish II, David T. Tse, 2000, Current Medicine Group LLC, with permission.
13
Stratum corneum as the skin barrier
Keratinocytes in the epidermis migrate from stratum basale with a proliferative cell type,
through stratum spinosum, to the stratum granulosum, where cornification, a process of
terminal differentiation, is initiated; transforming keratinocytes into flat anucleated cells
called “corneocytes”. Cornification involves degradation of organelles, organization of keratin
bundles inside the cell, and formation of a cornified envelope around it.1,2 This is
accompanied by secretion of lamellar bodies, organelles containing a mixture of lipids,
mainly polar ones: glucosylceramides, sphingomyelin, and phospholipids, and also
cholesterol, as well as numerous enzymes. After secretion at the stratum granulosum/stratum
corneum interface, polar lipids are enzymatically converted into non-polar products:
ceramides and free fatty acids (reviewed by Feingold3). Lipids derived from lamellar bodies
are assembled into lamellar structures surrounding the corneocytes.1,2
As a result, the stratum corneum consists of layers of corneocytes, which are densely packed
with keratin and embedded in extracellular lipid matrix. Corneocytes are connected to each
other by corneodesmosomes, and are gradually removed from the skin surface by
desquamation, making place for new cells coming from underneath. Process of keratinocyte
turnover in epidermis is highly organized in space and time, so that proliferation and
differentiation of keratinocytes are in balance.1,2
The stratum corneum, though only 10–20 �m thick, is the most essential layer of epidermis
from the perspective of its barrier properties and protection outside-in and inside-out. The
structure of the stratum corneum can therefore be compared to a wall made of bricks
(corneocytes) and mortar (intercellular lipids). This “brick and mortar” model was initially
presented by Michales et al.4 in 1975. The skin barrier models presented later: stacked
monolayer model,5 mosaic domain model,6 single gel-phase model,7 and sandwich model,8
can be regarded as extensions of the brick and mortar model (a concise summary of those
models is presented by Norlén9). They accept the two-compartment structure of the stratum
corneum and focus on explanation of molecular arrangements of intercellular lipids. Although
the organization of the stratum corneum and its components is not completely understood, the
development of new techniques, such as cryo-electron microscopy and cryo-electron
tomography of vitreous skin sections (reviewed by Norlén10), gives new insight into its
structure.
14
The function of the skin as a barrier can be regarded as protective, or even defensive, in
nature, and it is localized mostly in the stratum corneum. Corneocytes and extracellular lipid
matrix fulfill various protective functions, which often co-localize and/or are linked together
(Table 1) (reviewed by Elias11,12). In dermatological research, overall protective properties of
the stratum corneum and epidermis are often referred to as the “skin barrier”, while their
performance is known as the “skin barrier function”. However, it is important to realize that
depending on the research design and the evaluation methods, the exact meaning of the term
“skin barrier” may vary. For example, some studies may focus more on the permeability
barrier to water from inside the skin to the outside, while others investigate selective
absorption of a substance from outside into the epidermis.
Table 1 – Multiple protective function of outer epidermis*
Function Principal compartment Biochemical basis
Permeability Extracellular matrix of SC Ceramides, cholesterol, nonessential fatty acids in proper ratio
Antimicrobial Extracellular matrix of SC Antimicrobial peptides, free fatty acids, sphingosine
Antioxidant Extracellular matrix of SC Cholesterol, free fatty acids, vitamin E, redox gradient
Cohesion (integrity) � desquamation Extracellular matrix of SC Intercellular DSG1/DSC1
homodimers
Mechanical or rheological Corneocyte �-glutamyl isopeptide bonds
Chemical (antigen exclusion) Extracellular matrix of SC Hydrophilic products of
corneodesmosomes
Psychosensory interface Extracellular matrix of SC Barrier lipids
Neurosensory Stratum granulosum Ion channels, neurotransmitters
Hydration Corneocyte Filaggrin proteolytic products, glycerol
Ultraviolet light Corneocyte Trans-urocanic acid (histidase activity)
Initiation of inflammation (first-degree cytokine activity)
Corneocyte Proteolytic activation of pro-interleukin-1�/�
*Modified from Elias12, published with permission; SC = stratum corneum; DSG1 = desmoglein 1; DSC1 = desmocollin 1
15
Intercellular lipids of the stratum corneum
Lipids of the continuous extracellular lipid matrix of the stratum corneum are predominantly
ceramides, cholesterol, cholesteryl esters (cholesterol esters with fatty acids) and free fatty
acids, in an estimated molar ratio 37:32:15:16, respectively.13 Moreover, small quantities of
cholesterol sulfate14 and glucosylceramides15 are present as well. Lipids are organized into
lamellar phases, oriented approximately parallel to the surface of the corneocytes (reviewed
by Bouwstra et al.16). The detailed lamellar organization of intercellular lipids is still not
completely known, as data vary, depending on the method used.17,18 Their lateral organization
is also under investigation, since it determines the permeability of the stratum corneum: lipids
are organized mostly in orthorhombic and hexagonal packing, with the first type being the
least permeable arrangement, while the latter is more permeable.19,20 It has been shown that in
normal skin, lipids of stratum corneum have mostly orthorhombic packing, although
hexagonal packing and blends of hexagonal and orthorhombic packing are also present. By
contrast, patients with atopic dermatitis and lamellar ichthyosis have been found to have
predominantly hexagonal organization of intercellular lipids.20
ENZYMES AND NON-ENZYMATIC REGULATORY PROTEINS OF THE
SKIN BARRIER
The formation of components of stratum corneum, corneocytes and intercellular lipids, as
well as degradation of corneodesmosomes, which results in desquamation, is regulated by
several key enzymes and non-enzymatic proteins. Abnormalities in their expression, structure,
or activity may lead to impairment of the skin barrier, which is often found in various skin
disorders, such as atopic dermatitis, psoriasis and disorders of keratinization.
Corneocyte formation
Among the most important proteins in the process of cornification are transglutaminase 1,
involucrin and filaggrin. Transglutaminase 1, together with transglutaminase 3, crosslinks
various proteins into a mechanically and chemically resistant structure named the “cornified
envelope”, formed around each corneocyte. Involucrin is one of the first proteins linked
during this process and acts as a scaffold for other proteins, e.g., loricrin, trichohyalin, small
proline-rich proteins, cystatin �, and elafin (reviewed by Candi et al.21 and Ishida-Yamamoto
et al.22). Reduced level of involucrin and its altered distribution in epidermis was found in
patients with atopic dermatitis.23 Transglutaminase 1 is also probably involved in the
16
formation of ester bonding between involucrin, possibly also other proteins, and �-
hydroxyceramides, forming a lipid envelope of about 5 nm around the cornified envelope,
which acts as a frame for attaching other intercellular lipids.24 Mutations in gene of
transglutaminase 1 result in enzyme deficiency, and are found in patients with lamellar
ichthyosis and congenital ichthyosiform erythroderma (nonbullous) (reviewed by Richard25).
Keratohyalin granules are small organelles that are visible in stratum granulosum by ordinary
light microscope. They are composed primarily of profilaggrin and loricrin.1 Profilaggrin is
cleaved into filaggrin, which aggregates keratin filaments into tight bundles and causes the
collapse of filament network of keratinocytes, resulting in a flattened cell (reviewed by Candi
et al.21). Later, filaggrin undergoes degradation into free amino acids, pyrrolidone carboxylic
acid (PCA) and urocanic acid.26 Amino acids and PCA are the main components of the bulk
of natural moisturizing factor (NMF), helping to maintain an appropriate hydration level of
the stratum corneum (reviewed by Rawlings et al.27,28). The importance of filaggrin for the
skin barrier function has been demonstrated in recent studies, which link atopic dermatitis,29
chronic irritant contact dermatitis,30 and ichthyosis vulgaris31 to mutations in the gene of
profilaggrin. Individuals with mutation of FLG have been shown to have less NMF, which
may be one of factors predisposing them to dry skin disorders.32 Moreover, FLG mutations
are connected to a higher risk of developing asthma among patients with atopic dermatitis and
severe asthma in individuals without eczema.33
Another essential protein in the cell cycle is cyclin-dependent kinase inhibitor 1A, which has
a broad functionality in skin biology. Together with other inhibitors of the same family, this
protein is involved in cycle progression through the G1 phase into the S phase and in gene
expression regulation. Differentiation of squamous epithelia, including the epidermis, is
associated with increased expression of cyclin-dependent kinase inhibitor 1A (reviewed by
Weinberg et al.34). Increases in both its mRNA and protein levels have been found in psoriatic
plaques, and also after application of irritants and tape stripping.35
Desquamation
Desquamation of corneocytes from the stratum corneum surface, terminating the keratinocyte
life cycle, is mediated by enzymes belonging to the group of proteases, among which
kallikrein 5 and kallikrein 7 (previously known as stratum corneum tryptic enzyme (SCTE)
and stratum corneum chymotryptic enzyme (SCCE), respectively) are currently assumed the
most crucial.36-39 In normal skin, kallikrein 5 and 7 are found in the stratum granulosum and
17
corneum, as well as in the skin appendages.37,38 In transgenic mice with pathologic skin
changes, such as increased epidermal thickness, hyperkeratosis, dermal inflammation, and
severe pruritus, kallikrein 7 was expressed also in the suprabasal epidermal layers.40
Moreover, increased protein expression of kallikrein 5 and 7 was found in stratum corneum of
lesional skin of psoriasis vulgaris, while non-lesional psoriasis skin had no such increase,41
and also in stratum corneum obtained from patients with atopic dermatitis, which had none or
only very mild lichenification.42 These findings suggest an involvement of kallikreins in
various skin disorders, including inflammatory reactions. Kallikrein 7 has also been found to
be affected by the humidity of the environment: the low relative humidity decreased activity
of kallikrein 7 in excised animal skin due to a diminished water concentration in the upper
stratum corneum, reversible by application of a moisturizer or a humectant (glycerin).43
Formation of extracellular lipid matrix of stratum corneum
So far, nine classes of ceramides have been identified in the human stratum corneum, and they
differ from each other with regard to their head group architecture. They can contain
sphingosine, phytosphingosine, or 6-hydroxysphingosine as a base. Ceramides 1, 4, and 9 are
unique, as they contain linoleic acid. The ceramides, especially ceramide 1, have an important
role in the organization of stratum corneum lipids (reviewed by Bouwstra et al.16).
All types of ceramides are synthesized de novo from palmitoyl-CoA and serine, where serine
palmitoyltransferase is a rate-limiting enzyme in this process.1,44 In order to be transported to
stratum corneum in lamellar bodies, ceramides are converted to glucosylceramides and
sphingomyelin, by enzymes UDP-glucosylceramide synthase and sphingomyelin synthase,
respectively.45,46 After extrusions from lamellar bodies, enzymes �-glucocerebrosidase and
serine palmitoyltransferase transform them to ceramides (reviewed by Feingold3).
Deficiency in ceramides or abnormalities in enzymes involved in their formation has been
found in atopic dermatitis, where a significant reduction in the quantity of ceramides has been
observed in both lesional and non-lesional skin.23,47,48 This has been associated with a
decreased activity of acid sphingomyelinase23 and increased activity of glucosylceramide
deacylase,48 but no changes were found in activity of �-glucocerebrosidase.49 The level of
ceramides is also decreased in psoriasis, probably due to decreased levels of serine
palmitoyltransferase, and the severity this disease has been reported to correlate with the level
of this enzyme.50 In psoriasis, mRNA and protein expression of �-glucocerebrosidase has
been decreased in non-lesional skin and increased in lesional skin.51 Except for their structural
18
function, ceramides are also involved in a number of cellular processes including apoptosis,
the cell cycle, and cellular differentiation (reviewed by Ruvolo52). Recently, a role for
ceramides was proposed in the formation of the epidermal pH gradient, as acquiring free fatty
acids from ceramides may contribute to acidification of the stratum corneum.53
HMG-CoA synthase and reductase enzymes are involved in the synthesis of cholesterol in
epidermis, with acetyl-CoA as a substrate.1 The importance of cholesterol synthesis for the
barrier function and recovery has been demonstrated after acute and chronic skin barrier
disruption in mice, by acetone, tape stripping and essential fatty acid-deficiency diet, which
significantly increased the mRNA expression of HMG-CoA reductase and synthase.54 The
same type of barrier damage increased also activity HMG-CoA reductase.55 Topical
application of lovastatin, an inhibitor of HMG-CoA reductase, has been reported to impede
barrier recovery.56 Cholesterol seems to be important for the organization of intercellular
lipids (reviewed by Bouwstra et al.16). Moreover, cholesterol sulfate has a significant role in
desquamation (reviewed by Elias et al.57).
Acetyl-CoA serves as a substrate for synthesis not only of cholesterol, but of fatty acids as
well, involving fatty acid synthase in this process.1 The mRNA expression of fatty acid
synthase was shown to increase after barrier disruption by in mice, as also does the expression
of another lipid-processing enzyme, acetyl-CoA carboxylase beta.54 Interestingly, when
murine skin is occluded just after tape stripping, mRNA expression of acetyl-CoA
carboxylase beta, fatty acid synthase, HMG-CoA reductase and synthase is decreased, which
suggests that their expression is regulated by the skin barrier function itself, not by a non-
specific response to damage.54
Nuclear hormone receptors and lipoxygenases
Recently, the significance of nuclear hormone receptors, namely peroxisome proliferator-
activated receptors PPAR-�, PPAR-� and PPAR-�, and retinoid X receptor alpha (RXR-�),
for the normal skin barrier formation has been demonstrated. PPARs are transcription factors
involved in keratinocyte differentiation and proliferation and lipid synthesis. Their activation
was shown to increase expression of proteins essential in formation of cornified envelope:
involucrin, loricrin, and transglutaminase 1, stimulate synthesis of epidermal lipids and
formation of lamellar bodies, as well as to increase activity of lipid-processing enzymes.
PPARs are also involved in anti-inflammatory processes and cutaneous carcinogenesis.
Therefore, their performance is essential for the skin barrier formation and function. They
19
may play an important role as drug targets for such skin diseases as psoriasis and atopic
dermatitis, and also in skin cancer (reviewed by Feingold,3 Schmuth et al.,58 and Sertznig et
al.59). RXR-� is a receptor for the vitamin A metabolite, 9-cis retinoic acid, and is the most
abundant retinoid X receptor in the epidermis.60 It heterodimerizes with PPARs, and it has
been shown that these heterodimers act as signal transducers in different signaling pathways.61
The exact function of RXR-� for the skin barrier formation and function remains not
completely understood, but studies on mice show that mutation of RXR-� induces
hyperproliferation and abnormal differentiation of epidermal keratinocytes.60,61
The formation of the skin barrier lipids can be regulated at the transcriptional level by
substances acting as ligands to the PPARs. Enzymes believed to generate endogenous ligands
for these receptors have recently been described.62 Mutations in the coding regions of two of
these enzymes, lipoxygenases arachidonate 12-lipoxygenase, 12R type and epidermal
arachidonate lipoxygenase 3, were discovered in patients with autosomal recessive congenital
ichthyosis (ARCI), characterized by a defect water diffusion barrier in the skin.63,64 The exact
functions of those two lipoxygenases still remain unknown, but it seems that they may be
connected to lipid metabolism of the lamellar granule contents or intercellular lipid layers, as
well as formation of cornified envelope (reviewed by Akiyama et al.65).
SKIN PH
The pH value of the skin has been investigated since the end of 19th century. The acidic nature
of the skin surface was first mentioned by Heuss66 in 1892. In 1928, Schade and
Marchionini67 coined the term “acid mantle” of the skin (“Säuremantel”). Since then, many
studies have been carried out aiming to explain the mechanism of pH gradient formation and
its importance for the skin and its barrier function, but to this day, this issue is not completely
understood.
It is important to realize that the term “skin pH” is not completely correct, as pH values
should refer only to diluted aqueous solutions (�0.1 mol/kg),68 while the epidermis is a dense
structure containing only about 20–30% of water in the stratum corneum and about 70%
water in deeper layers.69,70 Moreover, various residues located on the skin surface may
influence the readings. Therefore, what is actually measured is pH of the “(extractable) water-
soluble constituents of skin”, and that measured pH of the skin is not the pH in a precise
analytical–chemical sense.71 Consequently, instead of “pH of the skin”, terms such as “pH on
the skin” or “apparent pH” have been proposed to be more appropriate.71 However, despite
20
the mentioned considerations, it is widely accepted to use the terms “skin pH” or “pH of the
skin” and these expressions are also used in this thesis.
Several studies show that the pH value on the surface of healthy, undamaged skin of adults is
slightly acidic, about 5, varying between 4 and 6. A mixture of various substances secreted on
the skin surface with sweat, sebum and NMF, such as lactic acid, butyric acid, PCA, amino
acids, and free fatty acids, helps to shift the surface pH towards acidic values. In addition,
ingredients of exogenous origin, such as metabolites of cutaneous microflora (e.g., free fatty
acids) and cosmetic products may be present. However, it seems that pH on the skin surface
depends mainly on processes taking place in deeper layers of the epidermis (reviewed by
Parra et al.,72 Rippke et al.,73 and Fluhr et al.74).
Below the skin surface, in the epidermis, pH increases from acidic values in the upper layers
of the stratum corneum to near-neutral values of around 7.4 in viable epidermis, forming a
gradient through the stratum corneum.75-77 The exact course of this gradient is still uncertain.
Measurement of tape-stripped human skin with a glass electrode has shown a gradual
decrease in pH towards the skin surface.75-77 Visualization of hydrogen ions in human skin
biopsies demonstrated that pH decreases sharply at the stratum corneum/granulosum
interface, but later slightly increases within stratum corneum, and then decreases again
towards the skin surface.78 However, recent investigation with fluorescence lifetime imaging
microscopy (FLIM) suggests that the observed pH is in fact an average from two distinct pH
gradients formed by two types of acidic microdomains, each of them localized in one
compartment of stratum corneum: corneocytes or extracellular lipid matrix.79 As the result,
the average pH of the stratum corneum decreases towards the surface, due to an increase in
the ratio of acidic to neutral regions.79
Protons forming the pH gradient are generated most likely by several mechanisms and
perhaps not all of them have been identified yet. Two endogenous mechanisms are currently
believed to be the most important for acidification of the epidermis: formation of free fatty
acids from phospholipids through the action of secretory phospholipase A2 (PLA2) and
exchange of protons for sodium ions by non-energy-dependent sodium-proton exchangers
(Na+/H+ exchanger isoform 1, NHE1) in the membranes of keratinocytes at the stratum
corneum/stratum granulosum interface.80-84 The latter mechanism explains the decrease in pH
at the border between the stratum corneum and the stratum granulosum, as NHE1 is expressed
in the same place. Recently, a new pathway was proposed; in which epidermal ceramidase
contributes to endogenous skin pH by generating free fatty acids from ceramides.53
21
The acidic pH on the skin surface is assumed to inhibit the growth of pathogenic
microorganisms and keep the skin microflora in balance (reviewed by Fluhr et al.74). If the
skin surface pH is elevated, e.g., after usage of alkaline soaps, prolonged occlusion, or in skin
disorders like atopic dermatitis, the growth of pathogens increases.85-88 However, recent
studies reveal another important role of skin pH. The pH gradient through the epidermis
seems to be essential for several epidermal enzymes involved in the formation and function of
the skin barrier, as their activity is pH-dependent, e.g., �-glucocerebrosidase has an optimum
activity at pH 5.6, phospholipase A2 at pH 7–8, acid sphingomyelinase at pH 4.5, and
cholesterol sulfatase at pH 8 (reviewed by Redoules et al.89). Kallikrein 5 and 7 has been
shown to exhibit maximum activity at pH 8, but have a considerable activity also at pH 5.5.37
The importance of pH for activity of the epidermal enzymes, and therefore for the skin
barrier, was shown in a recent study on mice. Perturbed skin barrier recovered normally when
the skin was exposed to solutions buffered to an acidic pH, while initiation of the recovery
was delayed when the damaged skin was exposed to neutral or alkaline pH. This delay in
barrier recovery was suggested to be a consequence of a lower activity of �-
glucocerebrosidases.90
MOISTURIZERS AND THE SKIN BARRIER
The primary function of moisturizers is to smoothen the skin surface and to increase water
content in the stratum corneum, i.e., to moisture the skin. After application, water and other
volatile ingredients gradually evaporate; leaving a deposit of remaining ingredients, which
may stay on the skin surface or penetrate into the epidermis and be removed from the skin
surface by washing, friction and evaporation.
The increase in water content in the epidermis is achieved by water-binding properties of
humectants, e.g., glycerin, and by formation of a semi-occlusive layer on the skin surface,
which hampers water evaporation and increases water content in the upper epidermis.91
Moreover, an immediate increase in hydration of stratum corneum may be caused by an
uptake of water from the applied product.91 The increase in water content and the
simultaneous filling of the fractures on the skin surface, makes the skin more elastic, and
visibly and tactilely smoother, as well as decreases itch and brings relief (reviewed by
Lodén92,93)
If moisturizers are used repeatedly, as for example in the case of patients with various dry
skin disorders, who require treatment over a long period, or even over a lifetime, it may be
22
speculated what consequences this may have for the skin and its barrier function. Recurring
application of various substances of exogenous origin on the skin, followed by such
physicochemical changes as increase in water content in the stratum corneum or change in
skin surface pH, may influence epidermis, and therefore, the skin barrier function. Few
studies about long-term treatment with moisturizers on normal and diseased human skin have
shown both increases and decreases in skin barrier function, as measured by non-invasive
techniques. Two, 3 or 4-week treatments of normal or atopic skin with moisturizers
containing urea decreased transepidermal water loss (TEWL) and susceptibility to sodium
lauryl sulfate (SLS).94-98 Treatment with a moisturizer containing another humectant, glycerin,
seems to have a less pronounced impact on the skin barrier.98,99 On the other hand, in studies
by Held et al.100,101, a moisturizer containing high lipid content (70%) impaired the barrier of
normal skin after 5-day and 4-week treatments, measured as increased skin susceptibility to
SLS, although no change in TEWL of undamaged skin was found. The same cream also
increased susceptibility to nickel in nickel-allergic volunteers after 7-day treatment.102 In a
study on patients with lamellar ichthyosis, an 8-week treatment with moisturizers containing
high amount of lactic acid significantly increased TEWL, as well as dryness and scaling
decreased.103 Moisturizers have also been shown to influence skin barrier recovery after
exposure to a skin irritant.95,104 These studies demonstrate that prolonged application of
moisturizers may have a substantial impact on parameters used for evaluation of the skin
barrier. However, the factors responsible for the observed effects are unknown, especially
since the studies were performed using moisturizers containing several ingredients.
CHEMISTRY OF MOISTURIZERS
Moisturizers are formulated predominantly as oil-in-water (o/w) emulsions, where oil droplets
are dispersed in water and stabilized by emulsifiers. Reversed, water-in-oil (w/o) emulsions
are used less frequently due to their poor spreadability and the greasier feeling they leave on
the skin; however, they can offer other attributes, e.g., water resistance. Emulsions are
categorized into creams or lotions, depending on their viscosity. Moisturizers may also be gels
containing only hydrophilic material or ointments with only lipophilic ingredients. Other
forms of moisturizers exist, but they are much less common, e.g., multiple emulsions,
silicone-in-water emulsions, or suspensions. The choice of the form of a moisturizer depends
on its desired effect and the ingredients that are supposed to be incorporated.
23
Moisturizers may either have a simple composition and contain only a few ingredients, or be a
complex mixture of many substances. In the case of o/w and w/o emulsions, the simplest
possible moisturizer must contain three ingredients, namely, water, a lipid (oil), and an
emulsifier. Many ingredients used in moisturizers are the same as those found in the
epidermis or on the skin surface: fatty acids, ceramides, vitamins, urea, lactic acid, PCA, etc.
Lipids can be of vegetable, animal or mineral origin. Emulsifiers, which stabilize the lipid
droplets in an emulsion, can be either low-molecular substances, e.g., PEG-100 stearate, or
long-chained polymers of large size, such as acrylates/C10–30 alkyl acrylate crosspolymer.
However, it is rare that emulsions contain only three ingredients, and usually they are
mixtures of at least 15–20 substances. Those additional ingredients allow achieving desired
properties, efficacy, and stability of the product. Moisturizers usually contain humectants,
such as polyols: glycerin, propylene glycol, butylene glycol, sorbitol; alpha-hydroxy acids
(AHAs) and their salts e.g., sodium lactate; low-molecular substances: urea, betaine, amino
acids; and high-molecular polymers with water-binding capacity: sodium hyaluronate. To
increase stability of the emulsion and to adjust its rheology to either cream or lotion form,
viscosity-increasing agents must be added into the formulation, such as polymers: carbomer,
acrylates copolymer, xanthan gum; and high-melting waxes: glyceryl stearate, cetearyl
alcohol. Sensory properties may be modified with silicones, such as dimethicone and
cyclohexasiloxane. Additionally, moisturizers may contain several other ingredients, such as
antioxidants, vitamins, herbal extracts, salts, and UV filters. Depending on the composition of
moisturizers, their pH is adjusted to between slightly acidic and slightly alkaline and usually
ranges from 4 to 7, but in case of formulations containing stearic acid and zinc oxide, pH is
slightly alkaline.
Since the majority of moisturizers contain several ingredients, identification of the parameters
responsible for their effects on the skin barrier is difficult. Consequently, factors such as the
concentration and type of lipids, humectants, and other ingredients, as well as pH adjustment,
should be taken into account.
METHODS USED FOR EVALUATION OF THE SKIN BARRIER FUNCTION
Methods used for evaluation of the skin and its barrier function are numerous. Skin condition
may be assessed visually, e.g., for dryness, scaling and redness. There are several instruments
available for non-invasive assessments of the skin, where no skin sampling is necessary, as
they measure the functional changes on the skin surface or within a defined skin depth, e.g.,
24
TEWL, skin capacitance, skin impedance, blood flow, pH, surface topography, and elasticity.
Such equipment is often portable and easy to use, and consequently, non-invasive
measurements are a common tool in dermatological research. Today, assessment of TEWL is
the most common method for evaluation of the skin barrier function. TEWL is increased
when the skin barrier is impaired, e.g., in dry skin disorders or after damage with an irritant,
but also when the skin is hydrated.
However, in order to investigate processes in the skin in greater detail, at the molecular and
cellular level, skin samples are required. They may be punch or shave biopsies or samples
obtained by tape stripping. Studies utilizing such invasive methods are more complicated to
perform, require more resources and assessment from ethical perspective. Although they may
be common in basic dermatological research, they have rarely been used in research about
moisturizers and their effect on the skin barrier. It is also possible to perform studies in vivo
on mice or in vitro on keratinocyte cultures or skin equivalents. However, results obtained
from experiments performed using animal models or in vitro systems do not always correlate
to the in vivo situation in humans. Analyses of human skin biopsies may give a lot of
information about changes in epidermal structure, and gene and protein expression, as well as
allowing for staining with antibodies against various proteins.
AIM OF THE RESEARCH
The objective of the present work was to increase the understanding of the mechanism by
which long-term treatment with moisturizers influences the skin and its barrier function. The
impact of formulation variables such as pH, lipid type and humectants was assessed in vivo in
healthy human volunteers. Functional changes in the skin were explored using non-invasive
techniques. Moreover, the impact of moisturizers on the skin barrier function was assessed
also at molecular and cellular level, investigating gene and protein expression as well as
histology of epidermis.
25
MATERIALS AND METHODS
VOLUNTEERS
All studies were performed on healthy adult human volunteers. Exclusion criteria were skin
diseases, pregnancy, and allergy to ingredients used in the test preparations. Informed consent
and health declarations were obtained from volunteers before commencement of the studies.
The studies were approved by the Regional Ethical Review Board at Uppsala University,
Uppsala, Sweden. The number and age of the volunteers participating in each study are given
in Table 2.
Table 2 � Number and age of volunteers participating in each study.
Study Number of volunteers Women Men Age
(years)
Paper I 18 15 3 21–54
Paper II Treatment with test moisturizers Irritancy patch test
78 11
58 8
20 3
25–60 27–46
Papers III and IV 20 15 5 23–59
TEST MOISTURIZERS
Altogether, five test preparations were used in the investigations: one ordinary cream,
hereafter called “Complex cream”; three simplified creams emulsified with a long-chained
polymer, with the hydrophobic lipid phase consisting of either the hydrocarbons
isohexadecane and paraffin (“Hydrocarbon cream”) or a vegetable triglyceride oil, canola oil
(“Canola cream” and “Canola/urea cream”); and one lipid-free gel consisting of the polymer
used in simplified creams (“Polymer gel”). All test moisturizers, except for the Polymer gel,
were oil-in-water (o/w) emulsions. Table 3 shows the detailed compositions of the test
moisturizers, their pH, the number of volunteers testing each preparation, and the papers in
which the results are presented.
Tabl
e 3 �
Com
posit
ion
of th
e te
st m
oist
uriz
ers,
thei
r in
gred
ient
s and
pH
, and
the
pape
rs in
whi
ch th
e re
sults
are
pre
sent
ed.
Num
ber
of v
olun
teer
s T
est
prep
arat
ion
Lip
ids
Em
ulsi
fiers
O
ther
ingr
edie
nts
Ure
a pH
Pa
per
I Pa
per
II
Pape
rs II
I an
d IV
4.0
7.5
18
Com
plex
cr
eam
a
20%
ca
pric
/cap
rylic
tri
glyc
erid
e, c
anol
a oi
l, ce
tear
yl
alco
hol,
para
ffin
, gl
ycer
yl st
eara
te
1.3%
PE
G-1
00 st
eara
te,
carb
omer
, po
lyso
rbat
e 60
wat
er, p
ropy
lene
gl
ycol
, gly
cery
l po
lym
etha
cryl
ate,
di
met
hico
ne,
sodi
um la
ctat
e,
met
hylp
arab
en,
prop
ylpa
rabe
n,
lact
ic a
cid,
citr
ic
acid
5%
5
15
10
Hyd
roca
rbon
cr
eam
40%
is
ohex
adec
aneb
(20%
), pa
raffi
nc (2
0%)
0.4%
ac
ryla
tes/
C10
–30
alky
l acr
ylat
e cr
ossp
olym
erd
wat
er
0%
5
16
10
Can
ola
crea
m
40%
ca
nola
oile
0.4%
ac
ryla
tes/
C10
–30
alky
l acr
ylat
e cr
ossp
olym
erd
wat
er
0%
5
15
Can
ola/
urea
cr
eam
40
%
cano
la o
ile
0.4%
ac
ryla
tes/
C10
–30
alky
l acr
ylat
e cr
ossp
olym
erd
wat
er
5%
5
16
Poly
mer
gel
0%
0.4%
ac
ryla
tes/
C10
–30
alky
l acr
ylat
e cr
ossp
olym
erd
wat
er,
met
hylp
arab
enf
0%
5
16
a Can
oder
m
krä
m 5
%, A
CO
HU
D N
OR
DIC
AB
, Sto
ckho
lm, S
wed
en; b A
rlam
ol H
D, U
niqe
ma,
Gou
da, T
he N
ethe
rland
s; c M
erku
r W
hite
Oil
Phar
ma,
Mer
kur
Vas
elin
e,
Ham
burg
, Ger
man
y; d Pe
mul
en T
R-2
, Nov
eon
Inc.
, Cle
vela
nd, O
H, U
SA; e A
kore
x L,
Kar
lsha
mns
AB
, Kar
lsha
mn,
Sw
eden
; f Nip
agin
M, C
laria
nt In
tern
atio
nal,
Pont
yprid
d,
Uni
ted
Kin
gdom
.
27
EXPERIMENTAL DESIGN
The studies were double-blinded and randomized. Test moisturizers were distributed to
volunteers in white coded tubes. Volunteers were allowed to wash normally, but not to use
any skin care products on the test areas (forearms and gluteal skin) at least three days before,
and during, the test period.
Paper I
Volunteers treated each volar forearm with one of two test preparations, twice daily for 8
days. The test preparations were identical creams, except for pH, which was adjusted to either
4.0 or 7.5 (Table 3; Complex cream). Before the first application of the creams, one area of
each forearm was exposed to the skin irritant SLS for 24 hours, which induced irritancy.
Assessments of TEWL, blood flow, and skin capacitance, as well as visual scoring of
irritancy, were performed repeatedly during the subsequent days. On day 8, the examined
areas were exposed once again to SLS for 7 hours and evaluated finally on day 9.
Paper II
This study consisted of two parts: a long-term treatment study with the test preparations, and a
test of their irritancy potential. In the long-term treatment study, volunteers treated one volar
forearm twice daily for 7 weeks with one test preparation (Table 3), leaving the other forearm
to serve as the untreated control. After 7 weeks, on day -1, both volar forearms, treated and
control, were exposed to SLS for 24 hours. TEWL and blood flow were assessed on SLS-
exposed and undamaged skin on each forearm on day 1. Skin capacitance was also measured
on undamaged skin. Moreover, test preparations were assessed for their acute irritancy
potential using a 24-hour patch test, evaluated by visual scoring and TEWL measurements.
Papers III and IV
The volunteers applied one of the test preparations (Table 3; Complex cream or Hydrocarbon
cream) on one volar forearm and one buttock twice daily for 7 weeks, leaving the other
forearm and buttock untreated to serve as control sites. The side of treatment was randomized.
After 7 weeks, one shave and one punch biopsy were taken from each buttock, preceded by
TEWL measurements of the biopsy area. The shave biopsies were used for gene expression
analysis and the punch biopsies for histological and other molecular evaluations. Moreover,
the skin of the forearms was patch-tested with SLS for 24 hours. Non-invasive evaluations
were performed on undamaged and SLS-exposed skin.
28
EVALUATIONS
A list of evaluation techniques used in all presented studies is given in Table 4. The
techniques are described in detail in the “Materials and methods” section of each paper.
Table 4 – Summary of analyses performed in Papers I–IV
Analyses performed Paper I Paper II Paper III Paper IV
Non-invasive in vivo evaluations of the skin
TEWL x x x
Blood flow x x x
Skin capacitance x x x
Visual scoring x x
Molecular analyses of skin biopsies RNA isolation and cDNA synthesis x x
QRT-PCR x x
Histological evaluations x
Immunofluorescence x
Immunohistochemistry x
Lipid staining x TEWL = transepidermal water loss; RNA = ribonucleic acid; cDNA = complementary deoxyribonucleic acid;
QRT-PCR = quantitative real-time polymerase chain reaction
CALCULATIONS AND STATISTICS
The results are expressed either as percentage ratio to the corresponding values obtained from
control (untreated) skin areas, which are given as 100%, or as absolute values. They are
presented in graphs as box plots with the median value as a line across the box and the first
quartile value at the bottom and the third at the top. The whiskers are lines that extend from
the top and bottom of the box to the lowest and the highest observation within a defined
region, with outliers plotted as asterisks outside this region. For results presented in a table or
in the text, the median value is given, followed by the lower (Q1) and upper (Q3) quartiles in
brackets.
To analyze differences between results of non-invasive measurements and molecular
analyses, a Wilcoxon signed rank test on paired data was used. Possible linear relationships
between two variables were analyzed using the Pearson product moment correlation
29
coefficient. A Kruskal-Wallis test of equality of medians was used to investigate the
differences between various moisturizers.
Minitab® statistical software (Minitab Inc., State College, PA, USA), was used for
calculations and plots. The level of significance was set at p < 0.05.
30
RESULTS
PAPER I
Treatment of surfactant-damaged skin in humans with creams of different pH values
After the initial exposure to SLS, there was no difference in the skin barrier recovery between
skin treated with the pH 4.0 cream and skin treated with the pH 7.5 cream, evaluated as
TEWL, blood flow, and skin capacitance, and by visual scoring. After 8 days of cream
application, there was no difference between treatment groups in susceptibility to SLS at the
second SLS exposure.
PAPER II
Changes in skin barrier function following long-term treatment with moisturizers, a
randomized controlled trial
Treatment of normal skin for 7 weeks with Hydrocarbon cream, Canola cream, Canola/urea
cream, and Polymer gel increased TEWL in comparison to the untreated areas. The opposite
effect was found for Complex cream, where TEWL was reduced. Skin capacitance decreased
following treatment with Hydrocarbon cream, but no difference was found for the other test
preparations. Treatment with all test moisturizers did not change blood flow in comparison to
control areas.
After SLS-exposure, the TEWL of skin treated with Hydrocarbon cream, Canola cream,
Canola/urea cream, and Polymer gel was increased in comparison with the corresponding
SLS-exposed skin on the untreated forearm, while after treatment with Complex cream, it was
reduced. However, only Canola/urea cream resulted in a higher relative increase in TEWL
compared with the untreated control skin after SLS exposure.
There were no differences between the simplified creams, Hydrocarbon cream, Canola cream,
and Canola/urea cream, in their effects on TEWL of undamaged skin, TEWL of SLS-exposed
skin, or blood flow of SLS-exposed skin. However, there was a difference in impact on skin
capacitance.
The irritancy patch test showed that the five test preparations were not more irritant than
water.
A summary of the effects of the test preparations on the skin barrier is presented in Table 5.
31
Table 5 – Summary of the effects of test moisturizers on the skin barrier
after 7 weeks of exposure.
Undamaged skin SLS-exposed skin Moisturizer
TEWL Capaci-tance
Blood flow TEWL Blood
flow
Complex cream – –
Hydrocarbon cream � – � �
Canola cream � – – � �
Canola/urea cream � – – � �
Polymer gel � – – � – � = increased in comparison with untreated skin; = decreased; – = no difference;
SLS = sodium lauryl sulfate; TEWL = transepidermal water loss
PAPER III
Long-term treatment with moisturizers affects the mRNA levels of genes involved in
keratinocyte differentiation and desquamation
After 7 weeks of twice-daily treatment of buttock and forearm skin, TEWL was increased on
the Hydrocarbon cream-treated sites, but decreased on the Complex cream-treated sites, as
compared with untreated skin. TEWL measured on untreated areas of gluteal skin was
significantly higher than on untreated forearms: 10.8 (8.7–14.3) vs 7.3 (6.5–12.3) gm-2h-1,
respectively (p<0.001). None of the creams induced changes in superficial skin blood flow or
influenced the messenger ribonucleic acid (mRNA) expression of interleukin-1� (IL1A),
indicating absence of inflammation.
After SLS-exposure of forearms, a higher degree of irritation was found in skin treated with
Hydrocarbon cream compared with its corresponding untreated control skin site, while skin
treated with Complex cream showed less irritation than control skin. Skin capacitance
decreased after exposure to Hydrocarbon cream, while no difference was found for Complex
cream.
Histological analysis of sectioned skin biopsies showed no differences in the thickness of
epidermis or stratum corneum after treatment with either of the creams compared with
controls. In addition, the corneocyte size was not significantly influenced by any of the
treatments.
32
Treatment with Complex cream decreased the expression of cyclin-dependent kinase inhibitor
1A (CDKN1A), while the remaining genes were unaffected. Hydrocarbon cream significantly
increased the gene expression of involucrin (IVL), transglutaminase 1 (TGM1), kallikrein 7
(KLK7), and kallikrein 5 (KLK5) compared with untreated controls, while no changes were
found in the levels of profilaggrin (FLG) and cyclin-dependent kinase inhibitor 1A
(CDKN1A). In an attempt to identify genes important for the normal barrier function we
examined possible correlations between TEWL and mRNA expressions, thickness of the
stratum corneum, and size of corneocytes of untreated buttock skin. However, no linear
correlation was found between TEWL and gene expressions of analyzed genes, thickness of
stratum corneum, and size of corneocytes. A summary of gene expressions analyses presented
in Papers III and IV is given in Table 6.
The immunofluorescence staining did not reveal significant changes at the protein level of
involucrin, transglutaminase 1, and filaggrin (Figure 3). An example of photographs used for
semi-quantitative analyses of protein expression is given in Figure 4.
Perc
enta
ge ra
tio [%
]
Filaggrin Transglutaminase 1 Involucrin
350
300
250
200
150
100
50
p=0.
415
p=0.
083
p=0.
683
p=0.
083
p=0.
067
p=0.
919
Complex creamHydrocarbon cream
Figure 3 – Protein expression of involucrin, transglutaminase 1 and filaggrin in skin treated
with test moisturizers (n=10), assessed using the semi-quantitative method. For an
explanation of the boxplots, see the “Calculations and statistics” section. The untreated
control site is given as 100%. P-values relate to differences between treated and control areas.
33
Figure 4– Examples of photographs used for semi-quantitative analyses of protein expression
of transglutaminase 1: untreated skin of a volunteer using Complex cream (a); treated skin of
a volunteer using Complex cream (b); untreated skin of a volunteer using Hydrocarbon cream
(c); and, treated skin of a volunteer using Hydrocarbon cream (d). The dashed line represents
the dermal–epidermal border. Bar = 50 �m.
PAPER IV
Moisturizers change the mRNA expression of enzymes synthesizing skin barrier
lipids
After a 7-week exposure of the skin to Hydrocarbon cream, an increased mRNA expression of
the ceramide synthesizing enzymes �-glucocerebrosidase (GBA), serine palmitoyltransferase
2, (SPTLC2) and acid sphingomyelinase (SMPD1), but not of UDP-glucose ceramide
glucosyltransferase (UGCG) was observed. Identical treatment with Complex cream did not
affect the expression of any of these enzymes. Regarding the two analyzed enzymes involved
in cholesterol synthesis, treatment with Hydrocarbon cream increased the expression of
HMG-CoA synthase 1 (HMGCS1), but not HMG-CoA reductase (HMGCR), while Complex
cream had no effect on either. None of test moisturizers significantly influenced the mRNA
expression of enzymes involved in free fatty acid metabolism: acetyl-CoA carboxylase beta
(ACACB), fatty acid synthase (FASN) and acyl-CoA synthetase long-chain family member 1
34
(ACSL1). Treatment with Complex cream increased the mRNA expression of one nuclear
receptor, PPAR-� (PPARG), while exposure to Hydrocarbon cream decreased it. There was
no effect of any of the treatments on the expression of PPAR-� (PPARA), PPAR-� (PPARB)
and RXR-� (RXRA). Moreover, treatment with Hydrocarbon cream increased expression of
both analyzed lipoxygenases arachidonate 12-lipoxygenase 12R type (ALOX12B) and
epidermal arachidonate lipoxygenase 3 (ALOXE3), while Complex cream did not (a
summary of gene expression analyzed in Papers III and IV, is presented in Table 6).
In biopsies from the untreated skin, we examined whether there was a correlation between the
mRNA expression of any of the analyzed genes and TEWL (TEWL results were reported in
Paper III). The mRNA expression of two of the 15 examined genes, PPARG and ACACB,
exhibited an inversed linear correlation to TEWL, and high expression of these genes was
associated with low TEWL.
The amount and organization of non-polar lipids, examined by Nile Red in situ staining,
revealed no changes induced by either of the two treatments, in comparison with control
areas.
Since treatment with the two test moisturizers altered mRNA expression of PPARG in
opposite directions, the protein expression of PPAR-� was examined in the biopsies. Two
patterns of nuclear staining were observed: one was the staining of the entire viable epidermis
and the other was more restricted to the rete ridges. These patterns were equally distributed
between the volunteers, irrespective of the type of moisturizer used. In most cases, every
volunteer exhibited the same pattern, both in treated and in control sites, and when performing
semi-quantitative analysis of the staining intensity, the cream-exposed areas were no different
from control areas. An example of photographs used for semi-quantitative analysis of PPAR-
��is presented in Figure 5.
35
Figure 5 – Examples of photographs used for semi-quantitative analyses of protein expression
of PPAR-�: untreated skin of a volunteer using Complex cream (a); treated skin of a volunteer
using Complex cream (b); untreated skin of a volunteer using Hydrocarbon cream (c); and,
treated skin of a volunteer using Hydrocarbon cream (d). Bar = 50 �m.
36
Table 6 - Summary of the gene expression analysis presented in Papers III and IV.
Function Gene Complex cream
Hydrocarbon cream
IVL – �
TGM1 – � FLG – –
Proteins involved in keratinocyte differentiation
CDKN1A –
KLK5 – � Enzymes involved in the process of desquamation KLK7 – �
GBA – �
SPTLC2 – �
SMPD1 – � Enzymes involved in ceramide synthesis
UGCG – –
HMGCS1 – � Enzymes involved in cholesterol synthesis HMGCR – –
ACACB – –
FASN – – Enzymes involved in fatty acid metabolism
ACSL1 – –
PPARA – –
PPARB – –
PPARG � Nuclear hormone receptors
RXRA – –
ALOX12B – � Lipoxygenases
ALOXE3 – � Interleukin IL1A – –
� = increased messenger ribonucleic acid (mRNA) expression in comparison with untreated skin; = decreased mRNA expression; – = no difference; ACACB = acetyl-CoA carboxylase beta; ACSL1 = acyl-CoA synthetase long-chain family member 1; ALOX12B = arachidonate 12-lipoxygenase, 12R type; ALOXE3 = epidermal arachidonate lipoxygenase 3; CDKN1A = cyclin-dependent kinase inhibitor 1A; FASN = fatty acid synthase; FLG = profilaggrin; GBA = �-glucocerebrosidase; HMGCR = HMG-CoA reductase; HMGCS1 = HMG-CoA synthase 1; IL1A = interleukin-1�; IVL = involucrin; KLK5 = kallikrein 5; KLK7 = kallikrein 7; PPARA = PPAR-�; PPARB = PPAR-�; PPARG = PPAR-�; RXRA = RXR-�; SMPD1 = acid sphingomyelinase; SPTLC2 = serine palmitoyltransferase 2; TGM1 = transglutaminase 1; UGCG = UDP-glucose ceramide glucosyltransferase.
37
DISCUSSION AND CONCLUSIONS
Moisturizers are often used as supplements to topical and/or systemic anti-inflammatory drugs
in various types of skin conditions and disorders, such as contact dermatitis, atopic dermatitis,
psoriasis, and ichthyosis, in order to bring relief and break a dry skin cycle (reviewed by
Lodén93,105 and Proksch et al.106). Such skin conditions usually require long-lasting treatment
with moisturizers, and in the case of atopic dermatitis, their use is recommended even when
the eczema is cleared.107 Vehicles of many topical drugs are moisturizes as well. Use of
moisturizer is also widespread among people that self-perceive their skin as dry or rough, e.g.,
in elderly, due to a dry climate or frequent contact with cleaning agents, and they use
moisturizers to obtain relief and for smoothening of the skin. Moreover, skin protection
creams (also called “barrier creams”) are widespread at various workplaces to minimize the
percutaneous penetration of chemicals. Use of moisturizers is therefore common and is
practiced by a significant percentage of the population. Although the importance of using
moisturizers often is overlooked, and they are not perceived as an “active” treatment, they
have been shown to influence skin properties and the barrier function in both healthy and
diseased skin.94-98,100-103,108
Studies on the impact of moisturizers on the skin barrier have mostly focused on short-term
effects, showing that moisturizers are able to increase skin hydration, decrease roughness and
scaling, and improve the condition of dry skin (reviewed by Lodén et al.92,93,105,109). However,
little is known about the effects of their long-term use, lasting weeks, months, or even years,
which better reflects the real-life situation. The few studies that have looked into such effects,
have shown that the moisturizers studied influenced the skin barrier function and recovery, as
measured by non-invasive techniques, such as TEWL, skin capacitance and susceptibility to
an irritant, e.g., SLS and nickel salts.94-98,100-103,108 Therefore, the aim of present research was
to gain further understanding of the mechanism by which long-term treatment with
moisturizers influences the skin in vivo in healthy human volunteers, using not only non-
invasive techniques, but also other tools, such as quantitative real-time polymerase chain
reaction (QRT-PCR), immunofluorescence, immunohistochemistry, and histological
evaluations, allowing investigating the epidermis at molecular and cellular level.
Few available studies demonstrate that moisturizers may have an impact on epidermis at the
molecular level, when used on normal skin. Short et al.110 showed that a moisturizer
containing high amounts of glycerin and silicones increased maximum epidermal thickness,
38
decreased epidermal melanin content, and altered protein expressions of keratins 6, 10, and
16, as assessed with immunohistochemistry after a 4-week treatment. Moreover, all these
changes were accompanied by decreased TEWL. Another study revealed that a 6-week
treatment with a moisturizer containing glycerin and erythritol increased the number of
keratinocytes with a well-matured cornified envelope; also, the interleukin-1 receptor
agonist/interleukin-1� (IL-1ra/IL-1�) ratio in the epidermis was altered.111 However, it may
well be individual ingredients that affect the skin at a molecular level, i.e., not the moisturizer
as a whole. Linoleic acid, found in certain vegetable oils commonly used in topical
preparations, is a ligand of peroxisome proliferator activated receptors (PPAR) which has
been found to have effects comparable to a potent topical glucocorticoid in animal models.112
Another substance, nicotinamide, was shown to increase levels of ceramides and free fatty
acids and to decrease TEWL, as compared with placebo-treated skin.113
CHOICE OF TEST MOISTURIZERS
Three test moisturizers used in the present studies were formulated in such way as to
minimize their complexity, in order to diminish the number of possible confounding factors.
Therefore, they contained only few ingredients, in contrast to the majority of commercially
available topical preparations that contain over a dozen substances. In addition, one
moisturizer, Complex cream, containing several ingredients and previously shown to
influence the skin barrier in normal and atopic skin, was chosen as a reference for this
investigation.96,108 The following factors were examined: the impact of a humectant (urea);
difference between creams of minimum complexity (Hydrocarbon, Canola and Canola/urea
creams) and a cream containing more ingredients (Complex cream); and different types of
lipids, which were either pure hydrophobic hydrocarbons derived from mineral oil (paraffin
and isohexadecane) or a more polar vegetable oil consisting of triglycerides and sterols
(canola oil). The simple creams were stabilized with a polymeric emulsifier, which was
expected not penetrate and influence skin barrier function due to its large molecular size. A
gel, consisting of water and this polymer, was also investigated (Polymer gel). By contrast,
Complex cream contained a mixture of lipids of various origins and was emulsified with a
combination of polymers and low-molecular emulsifiers. To evaluate the effect of pH on the
skin barrier, Complex cream was adjusted to pH 4.0 or 7.5, as certain epidermal enzymes
have optimum activity in either acidic or alkaline pH. To investigate the effect of long-term
39
use of moisturizers, treatment time was chosen to be 7 weeks, as the average turnover time of
epidermis is reported to be 40–45 days.114,115
EFFECT OF 7-WEEK USE OF TEST PREPARATIONS ON THE SKIN
BARRIER
Twice-daily treatment of normal skin for 7 weeks with all test preparations examined in this
research, induced changes in the skin barrier function, as evaluated with non-invasive
methods. Hydrocarbon cream, Canola cream and Canola/urea cream appear to have a negative
effect on the skin barrier, as their application resulted in elevated TEWL and increased skin
susceptibility to SLS. Polymer gel, consisting of polymer and water, had similar effects.
Moreover, Hydrocarbon cream decreased skin capacitance. By contrast, treatment with
Complex cream decreased TEWL and susceptibility to SLS. Molecular analyses of skin
biopsies after use of Hydrocarbon cream and Complex cream on normal skin for 7 weeks
revealed that these two creams induced different effects on the mRNA expression of genes
involved in the keratinocyte differentiation, corneocyte formation and desquamation as well
as lipid metabolism. Treatment with Hydrocarbon cream changed the expression of 11 out of
22 analyzed genes, while exposure to Complex cream affected expression of only two of
them. At the same time, the moisturizers had no effect on protein expression of four analyzed
proteins. Stratum corneum thickness, epidermal thickness, the size of corneocytes, and non-
polar lipid staining, were also unaffected by the treatments.
The studies therefore revealed that moisturizers had different effects on the barrier function of
normal skin and that these changes were dependent on the composition of the moisturizer.
The test preparations had an impact on the normal function and/or structure of the skin. The
observed changes may be caused either by the ingredients of the test preparations (e.g., lipids,
humectants, emulsifiers, or water), which have a direct or indirect effect on skin barrier
components, or by the physical effects of moisturizers on the skin, such as occlusion. The
impact of pH of moisturizers was also investigated, and it was assessed to have no
significance for their effects on the skin barrier.
POSSIBLE EXPLANATIONS OF OBSERVED EFFECTS
Despite the apparent relationship between gene expression and the skin barrier function, it
was not possible to ascertain whether the observed functional changes, such as
40
increased/decreased TEWL, susceptibility to SLS or skin capacitance, were the effect of
molecular changes in the epidermis, or vice versa (“hen-and-egg” situation). The first
possibility is that functional changes in the skin barrier, induced by moisturizers, could trigger
epidermal keratinocytes to alter their gene expression. An example of such functional change
may be the delivery of exogenous lipids from the moisturizers into the intercellular lipids of
the stratum corneum, resulting in altered barrier function, or the impact of emulsifiers,
humectants or exposure to water.
Lipids
Lipids in moisturizers may remain on the skin surface or enter the skin116, and more
physiological lipids may penetrate into the epidermis and affect skin barrier structure and
recovery.117-119 Changes in lateral packing of stratum corneum lipids were observed in
patients with atopic dermatitis and psoriasis after 3-week treatment with a moisturizer
containing 10% petrolatum.20 Therefore, in our study, the possible penetration of lipids from
test moisturizers could alter skin barrier properties.
The three simplified creams investigated in our study, all of which had a negative effect on
the skin barrier, contained high proportions of lipids, 40%, but the type of lipid (hydrocarbons
or triglycerides) was of no importance for the effect. At the same time, Complex cream,
containing 20% lipids, did not deteriorate the skin barrier. Similar effects as for simplified
creams have been obtained also with a moisturizer containing 70% lipids, which made the
skin more susceptible to SLS.100,101 This suggests that differences in the lipid content or
uptake rate may be important for the effect of moisturizers on the skin barrier function. As
exogenous lipids may change the highly organized structure of intercellular lipid layers,
TEWL or the ion flux may also be altered. Such changes may be recognized as barrier
impairment by epidermal keratinocytes, initiating a repair process including altered gene
expression.
Water
Penetration of lipids, however, cannot explain the impairment of the skin barrier obtained
after treatment with Polymer gel, as it contains no lipids but much more water compared with
other moisturizers, 99%. After application of a moisturizer, water, which is one of the main
ingredients, evaporates within a short time.120,121 The effect on the skin barrier of twice-daily
exposure to water for a few weeks is unknown. It has been shown that prolonged contact with
water disrupts the intercellular lipid lamellar structure in the stratum corneum, and may
41
contribute to dryness and increased TEWL.122 Moreover, changes in gene expression of
epidermal enzymes and non-enzymatic proteins were found after exposure of skin only to
water under occlusion.123 It has also been suggested that higher TEWL of the dorsal surface of
hands, compared with the forearm and back, may be due to more frequent contact with
water.124 Increased TEWL and dryness of the skin could also be caused by contact with
irritants, but the test preparations, including Polymer gel, were shown not to be irritant, by an
acute irritancy test and by blood flow measurements. Moreover, treatment with Hydrocarbon
cream and Complex cream did not alter expression of IL1A, indicating lack of inflammation.
Furthermore, though all simple creams and Complex cream contain similar amounts of water,
around 60%, they induced different changes in the skin barrier.
Emulsifiers and polymers
Emulsifiers are essential in moisturizers, as they stabilize the emulsion. Emulsifiers
commonly used in o/w emulsions have been shown to influence the skin barrier function in
normal and SLS-exposed skin.125 Although the emulsifier used in the simple creams and
Polymer gel, acrylates/C10–30 alkyl acrylate crosspolymer, was expected not to penetrate into
the epidermis due to its large molecular size, its negative effect could have been caused by
monomers, which may be present at low concentrations. However, the Complex cream also
contained a polymeric emulsifier, carbomer, which is similar in structure to acrylates/C10–30
alkyl acrylate crosspolymer, lacking a negative impact on the skin barrier. Interestingly, it has
been suggested that polymers themselves may have an effect on the skin barrier, as tested on
mice, accelerating or delaying the barrier recovery.126 Although this phenomenon is not fully
understood, it is possible that polymers together with their counter-ions form an electric
double layer on the skin surface, influencing the skin barrier.126
Humectants
Humectants that are added to moisturizers, such as urea, glycerin, and propylene glycol, may
penetrate into the skin.127,128 It has been suggested that the decreased TEWL and lower
response to SLS after treatment with Complex cream was due to its urea content.96,97,108
Moreover, it could be expected that the addition of urea to a moisturizer would be beneficial,
as it has been reported that urea replaces water in the skin, leaving the physical properties of
the stratum corneum intact.129 However, not only did the addition of urea to Canola/urea
cream not improve the skin barrier, but it also made the skin more susceptible to SLS in
comparison to Canola cream without urea. Urea may have favoured the absorption of
42
potentially damaging ingredients in this formulation, or allowed for increased penetration of
SLS during the 24-hour patch test exposure. It might be that in Canola/urea cream,
penetration of urea was different than in case of Complex cream, due to another emulsion
composition.
Urea has been reported to act as a keratolytic agent;130 however, absence of a measurable
thinning of the stratum corneum by the 5% urea in Complex cream in the present
investigation, does not support keratolytic properties at a concentration commonly found in
moisturizing creams.
Although treatment with Complex cream containing 5% urea significantly decreased TEWL
and susceptibility to SLS, it had only minor effects on the mRNA expression of the analyzed
genes. Therefore, the effect of urea on the barrier function may depend on the whole
composition of the moisturizers, including the lipid content. Urea has also been found to
diminish epidermal proliferation in psoriasis, measured as a decreased expression of
involucrin and an increased expression of cytokeratins.131 However, no difference in
expression of proteins or the genes involved in keratinocyte differentiation and desquamation,
apart from CDKN1A, was detected in the present study. Decreased expression of CDKN1A
suggests influences in cell cycle progression after treatment with the urea-containing Complex
cream, which could be interpreted as decrease in cell differentiation,34,132 though not
detectable by histological evaluations, since the thickness of the epidermis and the stratum
corneum remained unchanged, as did corneocyte size.
Occlusion
As an alternative explanation, gene expression may be influenced by altered activity of
various signaling pathways, resulting in a changed skin barrier function. However, it is still
not clear what type of signal triggers changes in gene expression: the changes could be due to
ingredients included in the moisturizers, or could have been indirectly induced by, e.g.,
changes in ion or water flux. One possible signal may be a reduction in TEWL due to
occlusion by the topically applied moisturizer, hypothetically resulting in water flux changes.
We have previously shown that occlusion of the skin with a semi-permeable membrane,
mimicking the occlusion effect by a moisturizer, decreased TEWL and susceptibility to an
irritant, in a group wearing the membrane 23 hours a day for 3 weeks, suggesting changes in
skin barrier function.133 However, there is no evidence of a correlation between occlusive
properties of creams and their effects on the skin barrier, determined as degree of irritation
43
after SLS-exposure, since Complex cream and a lipid-rich cream show similar occlusive
capacity133, but opposite effects on the skin barrier function.96,97,100,101,108 Therefore, the
detected difference in the effect on the skin barrier is more likely to have causes other than
difference in occlusion between the creams. It is still not known whether a prolonged
occlusion per se influences mRNA expression, since no investigation of this aspect has yet
been performed. It is also worth noting that the semi-occlusive layer formed by a moisturizer
is usually removed from the skin within a few hours.
pH
The impact of the pH of topical preparations on the skin barrier is still not fully understood.
The results presented in Paper I show that the pH of moisturizers seems not to be of major
importance for their effects on the skin barrier. We found no difference in the impact on skin
barrier recovery or susceptibility to an irritant between moisturizers of pH 4 and 7.5. The lack
of difference in effect on skin barrier recovery of the moisturizers of acidic or alkaline pH
values in our study disagrees with a previous study in mice, where barrier recovery was
delayed after exposure to slightly alkaline pH.90 However, the endogenous mechanisms
involved in the formation of the pH gradient in stratum corneum,80-84 as well as continuous
exogenous excretion of sweat and sebum and NMF, could be expected to counteract the
change in the skin surface pH induced by a topical application. It has been shown that after
use of an alkaline soap, initially elevated skin surface pH decreases back towards acidic
values,87 and therefore the same effect can be expected after a moisturizer.
Moisturizers usually contain only small quantities of buffering ingredients, which make them
unable to produce persistent changes in the skin surface pH, while the mentioned skin barrier
recovery study on mice was performed using strong buffer systems.90 Moreover, the majority
of moisturizers have pH in a range of 4 to 6, which is in the range of the skin surface pH.
However, it cannot be excluded that some ingredients of moisturizers may penetrate into the
epidermis, and influence the pH gradient there, which would have an effect on the skin barrier
function.
44
MOLECULAR CHANGES INDUCED BY HYDROCARBON AND
COMPLEX CREAMS
Regardless of the mechanisms involved, Hydrocarbon cream and Complex cream influenced
the skin barrier in a way detectable by non-invasive measurements and molecular analyses of
mRNA expression: the latter, however, were not confirmed at the protein level.
Effect of Hydrocarbon cream on the skin barrier
Hydrocarbon cream appears to have a negative effect on the skin barrier, since it elevates the
TEWL and makes the skin more susceptible to SLS. In the present study, Hydrocarbon cream
increased the mRNA expression of genes responsible for the synthesis of ceramides and
cholesterol: GBA, SPTLC2, SMPD1, and HMGCS1, but not the expression of free fatty acid
metabolizing enzymes. These results can be interpreted as an epidermal response to barrier
damage. Our results support such a hypothesis, since Hydrocarbon cream increased also
mRNA expression of IVL and TGM1, both being key proteins in formation of cornified
envelope. Other studies of skin barrier damage induced in mice by acetone, surfactant, tape
stripping, or an essential fatty acid-deficient diet, also describe increased mRNA expressions
or activities of lipid-processing enzymes.54,55,134,135
However, in a recent study on healthy human volunteers, two creams containing 5% urea, the
same amount as in Complex cream, increased mRNA expression of IVL, FLG, loricrin, and
TGM1 after a 2-week treatment, which was also accompanied by a significant decrease in
TEWL.136 This gene expression profile is more similar to the effect of Hydrocarbon cream,
although the effect on TEWL is like that of Complex cream. This suggests that the
relationship between mRNA expression and TEWL is not so straightforward, and also that the
gene expression profile may change during treatment time.
Nuclear receptors and lipoxygenases
Hydrocarbon cream decreased the mRNA expression of PPARG, as well as influenced
mRNA expression of the two lipoxygenases, ALOX12B and ALOXE3. PPAR-� and other
PPARs are involved in regulation of keratinocyte differentiation and expression of several of
the lipid-processing enzymes. In skin equivalent models, activation of PPAR-� receptors by
synthetic ligands resulted in an increase in the mRNA expression of lipid-metabolizing
enzymes, three of which were the same as in our present study (GBA, SPTLC2 and
HMGCS1).137 Although Hydrocarbon cream does not contain any known PPAR�agonists, the
45
elevated expressions of ALOX12B and ALOXE3, genes of lipoxygenases that produce
derivatives acting as PPAR-� agonists,62 may suggest an endogenous formation of PPAR-�
agonists by the Hydrocarbon cream treatment.
While Hydrocarbon cream decreased the mRNA expression of PPARG, Complex cream
increased it. However, no difference in protein expression of PPAR-� was found after use of
any of the test preparations. Nevertheless, the opposite effect on the expression of PPARG
and TEWL induced by Hydrocarbon and Complex cream, as well as a linear correlation
between these two parameters in untreated skin, suggests an importance of PPAR-��for the
skin barrier function. Usually, PPAR-���activation affects cell proliferation, cell
differentiation, immune responses, and apoptosis in the skin. PPAR�� signaling is triggered by
various types of ligands, including linoleic acid (reviewed by Feingold,3 Schmuth et al.,58 and
Sertznig et al.59). Although Complex cream contains a vegetable oil, which may contain a
small fraction of free fatty acids, e.g., linoleic acid, our present results do not suggest any
activation of PPARs.
A linear correlation between TEWL and mRNA expression in untreated skin was also found
for ACACB. Enzyme encoded by this gene is involved in synthesis of fatty acids and its
importance for the skin barrier has been shown after barrier disruption in mice, which
increased mRNA expression of this enzyme.54 The regulation of ACACB in keratinocytes is
unknown, but in mouse hepatocytes, it has been shown that the PPAR-� agonist WY-16,643
increases the expression of ACACB.138
Enzymes and proteins involved in keratinocyte differentiation and
desquamation
The treatment with Hydrocarbon cream increased the mRNA level of INV and TGM1, but it
was not accompanied by a corresponding increase in protein expression. This may indicate
that the epidermis was preparing itself for possible repair of the impaired skin barrier, or that
the repair phase had already been completed, but there was still ongoing transcription.
Another reason for the lack of correlation between mRNA and protein expression after
treatment may be that the mRNA had not been translated into protein, or that the protein
turnover had increased for unknown reasons. The inhibition of mRNA translation has been
shown to be caused by micro RNA. For example, certain genes in the epidermis of psoriasis
patients exhibit diminished translation due to the presence of certain micro RNAs.139
Furthermore, since protein and mRNA expressions were analyzed on a single occasion, after 7
46
weeks of treatment, it is possible that changes in protein levels occurred at another time,
perhaps after a few days, but became “normalized” after a few weeks due to some adaptation
mechanism. As already mentioned before, a recent study with two creams containing 5%
urea, showed another mRNA expression profile of IVL, FLG and TGM1 than in our study,
but analyzed after a 2-week treatment.136
It is also possible that the analytical method used was not sufficiently sensitive to detect
differences in protein expression. The mRNA expression increased by about 55% in the case
of IVL and 120% in the case of TGM1, which may not be enough to show in detectable
differences using an immunofluorescence technique. Other techniques, e.g. western blot, may
have been more powerful, but would have necessitated obtaining additional biopsies from the
volunteers, which was not possible for ethical reasons. However, another study describes
changes in mRNA expression of a magnitude similar to that seen in the present study, resulted
in visible increased protein levels after 1–7 days after exposure to SLS.123 It suggests also that
the effect of moisturizers at the protein level may also have occurred earlier.
Exposure to Hydrocarbon cream also increased the gene expression of KLK5 and KLK7,
which may suggest an excessive desquamation, since these two proteases are involved in this
process.36-39 However, since the thickness of the stratum corneum remained unchanged, it
could be possible that treatment with Hydrocarbon cream initially induced thickening of
stratum corneum, but increased performance of kallikreins counteracted it. However, the
altered expression of kallikreins may also be due to other, not well known, functions in the
skin barrier, since it has been shown that kallikrein 7 degrade some lipid-processing
enzymes.140
The altered expression of kallikreins may also be linked in some way to decreased skin
capacitance after use of Hydrocarbon cream, indicating low skin hydration levels,141 since
these enzymes have been found to have increased protein expression in and psoriasis41 and
atopic dermatitis.42 However, the protein expression of kallikreins was not assessed in the
present study. Interestingly, decreased skin capacitance was not accompanied by any change
in the mRNA expression of FLG or the protein expression of filaggrin, which degrades into
free amino acids and PCA, the main components of NMF (reviewed by Rawlings et al.27,28).
47
CONCLUSIONS
In the present investigation, moisturizers were examined for their effect on the skin barrier
after long-term treatment, with regard to such factors as pH, lipid type, and presence of a
humectant, as well as complexity of the product. In conclusion, moisturizers are able to
modify the skin barrier function, detected as changes in TEWL, skin capacitance, and
susceptibility to an irritant, and also to change the mRNA expression of certain genes
involved in the assembly, differentiation and desquamation of the stratum corneum, as well as
lipid metabolism. Therefore, moisturizers should not be perceived simply as inert topical
preparations, even if they do not contain any ingredients commonly perceived as “active”.
However, the mechanisms behind the observed effects are still not fully understood. The
observed outcome is most likely the combination of interplay and effects of several factors,
rather than only one, which makes it more difficult to draw any firm conclusions. More
research of this kind is needed to understand better the mechanism of action of moisturizers.
Such research would aid in the treatment of various skin disorders through improved design
of moisturizers, which can target specific skin problems and conditions both more efficiently
and safely.
48
FUTURE PERSPECTIVES
In the present thesis, we used traditional, non-invasive methods to investigate the skin barrier
function, combined with invasive techniques allowing us to study changes in the epidermis
after use of moisturizers at the molecular and cellular level. Analysis of gene and protein
expressions and histological evaluations lead to new hypotheses and answer more questions
than non-invasive methods alone.
Our investigation showed that treatment with moisturizers might change gene expression of a
number of epidermal proteins, including structural proteins and enzymes. The study was
performed on selected genes known to be important for the skin barrier, and since their
number was not very high, QRT-PCR was used for analyses. However, further investigations
could include also analyses using cDNA microarrays, which allow for screening several
thousands of genes at the same time. This would give a broader perspective over changes in
gene expression in epidermis and help to identify additional genes and proteins that should be
investigated more in details.
Since our research investigated the effects of moisturizers after 7 weeks of treatment, one may
consider exploring also their impact on the skin barrier at earlier time points. The course of
gene and protein expression and histological changes may vary between shortly after
application to after few-week use. It would also be interesting to continue the investigation for
some time after use of a moisturizer is terminated, following the return of the skin barrier to
the state from before the treatment.
Although molecular analyses provide plenty of information about the skin barrier, non-
invasive evaluations should be utilized as well, as they do not require skin sampling and
provide a different type of data, i.e. skin barrier function. The new types of non-invasive
methods developed during recent years help us acquire detailed facts about the skin structure
and function in vivo and may be used in studies investigating the effects of moisturizers. For
example, Raman spectroscopy helps to evaluate the moisturizing effect by giving a detailed
water profile in the epidermis and assess stratum corneum and epidermal thickness,69,70,142 as
well as to follow drug penetration (reviewed by Wartewig et al.143). Multiphoton laser
tomography144 and confocal laser scanning microscopy (reviewed by Branzan et al.145), allow
us to “look” inside the skin and may be exploited in various ways.
49
The results presented in this thesis as well as other studies demonstrate that the composition
of moisturizers plays an essential role in their effects on the skin and its barrier function.
Therefore, it would seem reasonable at first to systematically test various ingredients,
combinations of ingredients and complete moisturizers for their effects, in order to find those
giving the desired efficacy, both in healthy and in diseased skin. However, taking into
consideration that over 500 ingredients classified as humectants and about 1,500 emollients
are available,146 the number of possible combinations is endless, even if the number of
ingredients were limited to only those used more frequently. Therefore, it is more realistic to
continue the research about these few ingredients, which are common in many moisturizers
and for which some data is already available, regarding the effect of glycerin, urea, vegetable
oils, and hydrocarbons.
As the knowledge about the effect of various ingredients and their combinations on the skin
barrier is scarce, testing of complete moisturizers is currently the best way to assure benefits
for the consumers. However, although clinical trials are mandatory for pharmaceuticals (over-
the-counter or prescription drugs) to prove their efficacy, moisturizers available as cosmetic
products are rarely evaluated for their effect on the skin barrier. Therefore, in theory, some
commercially available moisturizers may have a negative impact on the skin, and their use
could worsen the skin condition, facilitate penetration of irritants, and even lead to eczema.
Consequently, the composition of a moisturizer should be an important issue when
recommending it to a patient with skin problems, since this choice may have an impact on the
skin status and therefore on quality of life. Treatment with corticosteroids and other drugs
improves the condition of the skin with eczema, but a relapse may only be a question of time.
However, while the use of a moisturizer with the skin barrier-improving effect after
corticosteroid therapy may increase an eczema-free period in comparison with no
treatment147, it is not known if use of a moisturizer with barrier-impairing properties causes an
earlier relapse of eczema. This issue should be investigated further, as it may have a major
impact on the approach to the treatment of dry skin disorders.
Improving or maintaining the skin status and quality of life may also involve more than
choosing a proper moisturizer, since compliance to treatment is also required. If a moisturizer
is not attractive to a patient from a cosmetic perspective, e.g., because it is too greasy and
tacky, difficult to apply, has an unpleasant odour, or its package is impractical, there is a high
probability that the patient will not use it according to guidelines, and as a result will require
more medical attention. The same problem may exist in the case of barrier creams, which are
50
supposed to inhibit penetration of irritant substances. Therefore, the cosmetic properties of
moisturizers must be taken into consideration during the early stages of the development,
which is a challenge for researchers and formulating chemists, as various ingredients added to
make a product more stable and attractive may weaken its performance or cause adverse
reactions.
In conclusion, it is important to remember that moisturizers differ in their composition, which
determines their impact on the skin barrier. A better understanding of the influence of
moisturizers on the skin barrier, obtained by combination of non-invasive methods and
molecular analyses, would facilitate designing skin care products adjusted to specific skin
problems. It would also provide more efficient management of dry skin disorders, and,
hopefully, help to influence the development of moisturizers so that fewer products will have
negative effects on the skin.
51
SVENSK SAMMANFATTNING (SUMMARY IN SWEDISH)
Mjukgörande krämer (s.k. mjukgörare) används vid behandling av olika hudsjukdomar, men
också av personer med frisk hud. Det är inte ovanligt att mjukgörare används kontinuerligt i
veckor, månader eller till och med under flera år. Studier har visat att vissa mjukgörare
förstärker hudens barriärfunktion medan andra har en negativ påverkan, av orsaker som inte
är helt klarlagda. En bättre förståelse kring mekanismer hur långtidsbehandling med
mjukgörande krämer påverkar hudbarriären är av stor klinisk betydelse. Krämer som
försvagar hudens barriärfunktion kan leda till ökad penetration av allergener eller irriterande
ämnen vilket i sin tur kan ge upphov till torr hud och eksem. Mjukgörare som stärker
hudbarriären skulle dock kunna motverka många av dessa problem.
I den här avhandlingen har icke-invasiva tekniker kombinerats med analyser av hudbiopsier
vilket möjliggjort studier av epidermis på cellulär och molekylär nivå. Olika mjukgörande
formuleringar och deras effekt på hudbarriären har studerats på friska försökspersoner.
Parametrar såsom mjukgörarens pH, lipidtyp, innehåll av fuktbindande ämne samt
komplexiteten av formuleringar har studerats.
Efter sju veckors behandling med de olika mjukgörarna detekterades förändringar i hudens
vattenavgivande förmåga, hudens kapacitans samt dess känslighet för irritation, vilket
indikerar förändringar i hudens barriärfunktion. Dessutom, mRNA-uttryck av flera gener som
är involverade i differentieringen och avfjällningen av stratum corneum, liksom
lipidmetabolismen, var förändrad i hud behandlad med en av mjukgörarna medan en annan
inducerade färre förändringar.
Långtidsanvändning med mjukgörare kan förstärka hudens barriärfunktion eller försämra den
och ge upphov till torrhet. Mjukgörare har en signifikant påverkan på hudens biokemi, vilket
kan mätas på molekylär nivå. Genom att noggrant val av innehållsämnen skulle det vara
möjligt att styra mjukgörarnas effekt på huden till en önskad klinisk effekt.
52
ACKNOWLEDGMENTS
I wish to express my sincere gratitude to:
Hans Törmä, my supervisor, for taking me under your wings and being incredibly patient and
understanding, in all situations. Your guidance helped me to look at the skin from a broader
perspective and ignited my enthusiasm about investigating the skin barrier at molecular level.
Thank you for our vigorous discussions.
Marie Lodén, my co-supervisor, for introducing me into a field of the skin barrier and giving
the opportunity to perform research in this area. Your vision and knowledge about
moisturizers, countless ideas and passion for research are sincerely inspiring. I am deeply
grateful for your guidance and persuading me to challenge the barriers, no matter what they
are.
Berit Berne, my co-supervisor, for excellent discussions, creative brainstormings, and being
very thorough in checking the manuscripts, as well as for all “clinical” help. Your inner
calmness was a great counterbalance to my nervousness.
Magnus Lindberg, my co-supervisor, for sharing your knowledge and experience, teaching
me to be cautious about interpretation of obtained results and to express my ideas as concise
as possible (I still need to master that). I am grateful for making me to understand that stratum
corneum should be treated as a ‘black box’.
Hans, Marie, Berit and Magnus–it was an honour to work with you.
Annika Grindborg, managing director of ACO HUD, for believing in me and making this
research possible.
Anders Vahlquist, head of Department of Dermatology, for providing excellent work
conditions.
Tove Agner, Marie Virtanen and Lars Norlén, for valuable comments and interesting
discussion during my “half-time control”.
Jacek Arct, the president of the Polish Society of Cosmetic Chemists, who significantly
influenced my life and showed me that cosmetics are fascinating from all perspectives.
All volunteers, for sacrificing their skin for science, although sometimes it was painful.
My parents, Janina and Krzysztof, and my brother Adam, for their love and support.
53
My dear Tomas, for patience and being by my side.
Ragna Lindeke-Strömqvist, Emil Schwan and Jouni Frid, for moral support, encouragement
in time of despair and discussions about meaning of life.
All my colleagues at ACO HUD, for help and being enthusiastic about my research.
My colleagues at Uppsala, for all support, scientific and not only (otherwise I would have
gone crazy in my dark room), and especially to Inger Pihl-Lundin, for skilful technical
assistance.
Proper English AB, for help with language corrections.
This research was financially supported by ACO HUD NORDIC AB, Uppsala University and
the Welander and Finsen Foundation.
54
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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 381
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)
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