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Scientific dossier : New discoveries on infant skin from the first days of life
1. Formulated under
dermatological control
and tested by paediatrician
The expert on baby’s skin
From birth
Mustela®. The skincare expert for babies and mothers-to-be for over 60 years.
Major new discoveries on infant skin
from the first days of life
Published in
the Journal of
Dermatology
S C I E N T I F I C D O S S I E R
2.
3. Dear Colleagues,
Researchers at the Innovation, Research and Development (IRD) Division of Laboratoires
Expanscience, the baby skin experts for more than three generations, are proud to present you the
EV.E.I.L.S (EValution of Early Infant Life: Skin) research programme.
After reviewing the current state of children’s skincare, Laboratoires Expanscience decided to
deepen their understanding of the physiology of newborn skin, from the very first days of life. For
this purpose, the Innovation, Research and Development Division has brought together researchers
of international recognition.
This research programme, ongoing for the past ten years and the only one of its kind in the world, is
centered on innovative and non-invasive technologies. It has resulted in major discoveries about the
skin barrier and skin hydration as well as epidermal stem cells in infants from the earliest moments
of life.
Acting upon these new findings and in an endeavour to provide baby's skin with what it requires
specifically to protect it and promote its development, the R&D centre has developed an exclusive,
natural and biomimetic active ingredient, derived from the avocado: Avocado Perseose®.
Avocado Perseose® has proven its activity in vitro from the surface of the skin right down to the
epidermal stem cells. It strengthens the skin barrier, helps maintain the level of epidermal hydration
and preserves the irreplaceable lifetime supply of epidermal stem cells.
Mustela® is now able to offer a new generation of clinically-tested skincare products, which act in
perfect harmony with a baby's skin, providing both maximum tolerance and targeted efficacy.
Best regards
Philippe Msika
Director of Innovation
Research and Development
at Laboratoires Expanscience
4.
5. C O N T E N T S
Introduction to the skin page 6
Published knowledge about baby’s skin page 11
EV.E.I.L.S (Evaluation of the Early Infant life: the Skin):
an internationally unprecedented fundamental research programme page 15
A. Innovative and ethical new investigative technologies
a. Scanning Electron Microscopy
b. Raman spectroscopy
c. In vitro epidermal skin models and genomic analysis
B. Three major discoveries about the skin of infants from birth to 2 years
a. On the organisation of the Stratum corneum
b. On the distribution of moisturising factors in the Stratum granulosum
c. On the stem cells of the Stratum germinativum
A new generation active ingredient: Avocado Perseose® page 29
A. Its actions extend from the Stratum corneum through to the Stratum germinativum
B. Unique, patented extraction processes
C. An ethical and responsible supply chain
The formulation: a new technological challenge page 41
A. To incorporate Avocado Perseose® in all formulas
B. To maximise the safety, innocuity and tolerance of the products
C. To ensure a high sensory quality
D. To be consistent with a strategy of environmental responsibility
Conclusion page 47
References page 50
1
2
3
4
5
6
6. 6
The skin is a vital organ that covers and protects the body.
Its principal functions include protection against environmental aggression (chemical,
mechanical, thermal, microbial infections and UV radiation) and control of water loss.
This protective "barrier function" is provided by the epidermis and more particularly by the
cornified layer known as the Stratum corneum.
Introduction to the skin
7. 7
The epidermis is a stratified epithelium, composed of 4 main layers:
• Stratum corneum:
the most superficial layer arranged in a “brick-and-mortar” structure. Composed of corneocytes without
nuclei (representing the bricks) and lipids (forming the mortar), the Stratum corneum fulfills principally
the barrier function.
• Stratum granulosum
and
• Stratum spinosum:
in these two layers, the keratinocytes produce lamellar granules and keratohyalin granules, necessary
for producing lipid cement and for skin hydration, respectively.
The lamellar granules provide, among other functions, the constituents of the lipid cement of the Stratum
corneum.
The keratohyalin granules give rise to NMF (Natural Moisturising Factors).
• Stratum germinativum:
this is the deepest layer of the epidermis, composed of a single layer of proliferating keratinocytes and
basal stem cells responsible for the integrity of the epidermis throughout and individual's lifetime.
8. 8
FOCUS ON THE STRATUM CORNEUM WHICH FULFILS THE BARRIER FUNCTION
Representative schema of the Stratum corneum
It is organised according to a model conventionally called brick and mortar and composed of:
• Corneocytes, anucleate cells of which the cornified envelope is a structure formed by a protein envelope and a
lipid envelope.
• Lipid cement, equimolar mixture of ceramides (40 to 50% of total weight), cholesterol (20-25%) and free fatty
acids (10-20%), playing a role in holding cells together. It has now been clearly established that the structure
of the corneocytes and the organisation of the lipid bilayer constitute a dynamic barrier more complex than a
simple brick wall.
• Corneodesmosomes which also play a primordial role in the cohesion of the Stratum corneum. The corneodes-
mosomes are intercellular junctions, specific to the Stratum corneum, the role of which is to link corneocytes to
one another.
Corneodesmosomesarecomposedmainlyofproteins,suchasdesmoglein-1,desmocollin-1andcorneodesmosin.
FOCUSONTHESTRATUMGRANULOSUMANDSTRATUMSPINOSUMWHICHPLAYANESSENTIALROLEINHYDRATION
The keratinocytes of this layer contain keratohyalin and lamellar granules.
Lamellar granules are rich in lipids and contribute to the production of the intercellular cement.
The keratohyalin granules are hydrolysed into filaggrin, which participates in the consolidation of the network of
keratin formation, a component of corneocytes.
Thefilaggrinisproteolysedintofreepolaraminoacids,makingupthenaturalmoisturisingfactors(orNMF),some
of which are water-trapping molecules.
The NMF (Natural Moisturising Factors) thus play a fundamental role in skin hydration.
Corneocytes
Corneodesmosomes
Lipid matrix (ceramides)
Cornified envelope (involucrin, transglutaminase) NMF
9. 9
FOCUS ON THE STRATUM GERMINATIVUM, STOREHOUSE OF EPIDERMAL STEM CELLS
The different layers of the epidermis
The Stratum germinativum is the deepest layer of the epidermis: this is where the epidermal
cells are created.
So-called adult stem cells are undifferentiated yet specialised cells which may be defined by
two main and distinct properties:
• their ability to renew themselves and remain in place for long periods of time,
• their ability to generate different types of cells.
The skin, an organ subject to rapid and continuous renewal, belongs to the group of tissues
containing a significant number of stem cells. Accordingly, each compartment of the skin
has stem cells that can develop into melanocytes, adipocytes, nerve cells, endothelial cells,
mesenchymal cells or epidermal cells.
Epidermal stem cells are found in the region of the hair follicle bulb (follicle stem cells), in
sebaceous glands (sebaceous stem cells) and in the Stratum germinativum (interfollicular
epidermal stem cells or basal stem cells).
The basal stem cells (or epidermal stem cells) constitute the main supply of epidermal stem
cells and ensure keratinocyte renewal.
Thus, they contribute to maintaining the skin’s homeostasis as well as to the repair and
regeneration of the epidermis, thereby ensuring its integrity throughout life.
Granular layer
(Stratum granulosum)
Spinous layer
(Stratum spinosum)
Basal layer
(Stratum basale)
Basal stem cell
Transit
amplifying cell
Cornified layer
(Stratum corneum)
12. 12
Current knowledge indicates a difference in skin functions between babies and adults.
Birth and the early months of life are decisive for adaptation from intra-uterine life to a gaseous and relatively
dry environment. The events responsible for these adjustments are characterised by changes in physiological
functions demonstrated by certain biophysical parameters.
1) The pH on the surface of the skin
The pH of the cornified layer plays an essential role in the equilibrium of the skin barrier, through its action on the
formation of the Stratum corneum and on the constitution of the antimicrobial barrier.
The elevated (alkaline) pH levels present at birth, drop to values found in adults between the first and fourth
weeks after birth.
2) Barrier function expressed as IWL (insensible water loss)
In full-term new-borns, a slight increase in insensible water loss (measured in g/m²/h) is observed during the first
hours after birth. IWL values then stabilise quickly, to levels comparable with those observed in adults.
3) Hydration of the Stratum corneum
Skin hydration is one of the key factors in skin resistance against mechanical aggressions.
Minimal at birth, hydration of the Stratum corneum increases during the first six months of life. It then stabilises
at a level comparable to that of an adult.
4) Sebum secretion
At birth, the sebaceous glands are present but functionally immature.
It has been shown that sebum secretion increases after birth to attain a level observed in adults during the first
months of postnatal life.
At six months of age, the values are lower than in adults and remain identical until puberty.
5) Sweat gland secretion
Sweat gland secretion (sweating or perspiration) triggered by emotions or temperature is present in full-term
new-borns.
Birth accelerates the development of sweat gland secretion in response to temperature rather than to emotions.
This represents a major distinction between new-borns and older children.
Published knowledge
about baby's skin
1
13. 13
6) Skin vascularisation
Immediatelyafterbirth,skinmicrovascularisationconsistsinadensehorizontalplexuswithadisorganisedcapil-
lary network.
A looped capillary network can be observed within all skin sites only at the age of 14-17 weeks.
VARIABLES
Functional
parameters
Gestationalage Postnatal age Sex
Anatomical
site
Ambient
temperature
Ambient
humidity
IWL + + 0 + + +
SC hydration + + 0 + + +
Surface pH 0 + +/- + ? ?
Sebum
secretion
? + 0 + ? ?
Sweat gland
secretion
(perspiration)
+ + ? + + +
Skin
vascularisation
+/- + ? + + ?
Variables influencing functional parameters of the skin
« + » : influence present - « 0 » : no influence - « ? » : no data - « +/- » : conflicting data
IWL : insensible water loss SC - SC : Stratum corneum
The literature tells us that, at birth, the skin’s development is incomplete, which makes it highly vulnerable to
aggression.
However, there are no published data about the evolution of the epidermis between birth and 2 years with
regard to:
• the organisation of the Stratum corneum
• the analysis of the biochemical composition of the Stratum corneum and the Stratum granulosum
• changes in gene markers, in particular those of the Stratum germinativum
There thus remain many unanswered questions, especially about how the maturation of the epidermis is
organised between birth and 2 years, from the Stratum corneum to the epidermal stem cells.
This is why Laboratoires Expanscience decided to conduct a programme of fundamental research, which
began in 2003 and for which the initial findings are presented in the following chapters.
T O S U M U P
1
16. 16
The EV.E.I.L.S research programme (Evaluation of the Early Infant Life: the Skin) is a series of
studies, unprecedented in the world, conducted over the past ten years with the support of
international researchers,
J. FLUHR, assistant professor, Department of Dermato-surgery, Berlin,
J.P. HACHEM, professor of dermatology, University of Brussels.
The aim of this research programme is to gain a better understanding of and analyse the maturation of healthy
infant skin from birth and over the course of the first years of life.
Using innovative and non-invasive technology, the IRD centre of Laboratoires Expanscience has carried out
unprecedented fundamental research on the physiology of baby’s skin from the surface down to the stem cell
layer of the epidermis, starting in the earliest days of life.
In vivo study conducted in 66 subjects,
divided into 6 age groups.
In vivo study conducted in 30 subjects,
divided into 5 age groups.
In vivo study conducted in 108 subjects,
divided into 6 age groups.
In vivo study, reconstructed epidermis of
babies models.
1 month, 4 months, 6 months, 1 year, 2 years,
3 years, 7 years, 12 years, Adult
e
d
c
b
a
From the surface of the skin
to the epidermal stem cells
SCANNING ELECTRON
MICROSCOPY
Viewing of the cutaneous
barrier from skin cell
samples (D-Squame®)
RAMAN SPECTROSCOPY
Evaluation of the dynamic
change in skin hydration
EPIDERMAL SKIN MODELS
AND GENOMIC ANALYSIS
Genomic study using
real-time quantitative PCR
of reconstructed epidermis
of babies models
Theinitialresults,whichatthetimeofwritinghavebeenthesubjectof9patentapplicationsand18international
publications - including the British Journal of Dermatology -, are presented to you in this scientific dossier.
PROTOCOLS AND TECHNOLOGIES OF THE EV.E.I.L.S STUDY
The EV.E.I.L.S research programme
EValuation of the Early Infant Life: the Skin
2
17. 17
A. Innovative and ethical new investigative technologies
3 unprecedented studies have been conducted using technologies that are innovative and non-invasive.
a. Scanning Electron Microscopy
Scanning Electron Microscopy is an electron microscope technique capable of producing high resolution images
of the surface of a skin sample.
The technique involves a beam of electrons that sweeps over the surface of the skin
which, in response, emits certain particles. These particles are analysed by different
detectors, thereby enabling the reconstruction of a three-dimensional image.
Scientists at Laboratoires Expanscience have analysed, for the first time in the world,
skin cell samples taken from new-borns using non-invasive D-Squame® sampling discs.
These samples were collected from the forearm then observed with Scanning Electron
Microscopy, enabling changes in the cornified layer to be visualised, at the cellular level.
This in vivo study, divided into 2 phases has enabled researchers to observe and analyse the organisation of the
skin barrier.
A first preliminary phase was conducted in 66 subjects, divided into 6 age groups:
• full-term new-borns (1-15 days)
• 5 weeks (+/- 1 week)
• 6 months (+/- 1 month)
• 1-2 years
• 4-5 years
• adult (20-35 years)
A second validation phase was conducted in 30 subjects, divided into 5 age groups:
• 5 weeks (+/- 1 week)
• 6 months (+/- 1 month)
• 1-2 years
• 4-5 years
• adult (20-35 years)
This innovative approach has enabled the development of the first skin barrier maturity score.
2
18. 18
b. Raman spectroscopy
This technology, uses a probe placed on the skin, and the emission of a laser beam,
to measure the distribution of water in the different layers of the epidermis and the
identification and quantification of NMF (Natural Moisturising Factors).
(Max. depth: 50µm - measurement pitch: 5µm)
Due to the use of this non-invasive technology, scientists at Laboratoires Expanscience
have been able to measure the distribution of water and Natural Moisturising Factors in the various layers of the
Stratum corneum in the skin of babies from the first days of life.
This study was conducted in vivo in 108 subjects, divided into 6 age groups:
• full-term new-borns (1-15 days)
• 5 weeks (+/- 1 week)
• 6 months (+/- 1 month)
• 1-2 years
• 4-5 years
• adult (20-35 years)
Conventional measurements such as insensible water loss (indicating the permeability of the skin barrier), skin
capacitance (measure of hydration of the superficial layers of the epidermis) and the pH of the surface of the skin
were also carried out.
This study has allowed an evaluation of the dynamic changes in hydration not only on the surface of the skin
but also in the deeper layers of the Stratum corneum.
The results have been published in the British Journal of Dermatology.
19. 19
The scientists at
Laboratoires Expans-
cience have, for the
firsttimeintheworld,
used keratinocytes
to create models and analyse the epidermis of
babies of varying ages and obtained specific
models for each age group:
•1 month / 4 month / 6 month / 1 year / 2 years /
3years/7years/12years/Adult
To create these epidermal skin models, an
innovative approach has been developed.
Using samples of skin of different ages, the
keratinocytes of the epidermal cells were
isolated and put into suspension. It was
then possible to reconstruct, using cell
engineering techniques, the epidermis of
infants and children of different ages. A histo-
logical examination then enabled the quality of
the reconstructed epidermis to be confirmed.
This study has enabled the analysis of gene expression, from the skin surface to the stem cells, in
reconstructed epidermal models from babies of different ages.
To get to the core of
the cell and gain a
better understanding
of the changes it
undergoes, Laboratoires Expanscience decided
to analyse thousands of genes.
The expression profiles of marker genes were
analysed using real-time quantitative PCR
(Polymerase Chain Reaction) assay, which
enables the selected genes to be detected easily
and precisely quantified.
c. In vitro epidermal skin models and genomic analysis
2
20. 20
B. Three major discoveries about the skin of infants from birth to 2 years
e
d
c
b
a
From the surface of the skin
to the epidermal stem cells
Cutaneous barrier organisation
Development of a maturity score for the
cutaneous barrier (E.M.I. Score)
Subject of research
Quantification of water distribution
in epidermis’ depth
Quantification of Natural
Moisturising Factors (NMF)
Analysis of the profile of stem cells
gene / biomarker expression
Major discoveries
0-2 years: the skin surface is immature
at birth and reaches a first stage of
maturity from the second year of life
0-2 years: period of vulnerability during
which hydration regulation mechanisms
are perturbed
0-2 years: Maximum stem cells
resources at birth which decrease
significantly between 0 and 2 years
under the effect of outside aggression
These different studies on the skin of babies of varying ages, starting at birth, from the surface of the
epidermis to the stem cells, has enabled us to make 3 major discoveries.
21. 21
a. On the organisation of the Stratum corneum
First discovery and determination of the 1st
skin barrier maturation score: an immature
barrier function that evolves over the course of the first years of life.
• The existence of changes in the organisation of the Stratum corneum with age has been
demonstrated through the observation and description of 300 images obtained by
Scanning Electron Microscopy, in the course of the preliminary study phase.
The images (X 500 magnification) obtained by Scanning Electron Microscopy show that the
Stratum corneum:
is heterogeneous (disorganised) in the newborn,
then organised networks start to appear around the age of 2 years
finally, in adulthood, the organisation is homogeneous.
1-2 years
x 500
4-5 years Adults
Newborns 5-6 weeks 6 months
Skin surface heterogeneous
and disorganised in infants
during the first months of life:
anisotropic organisation
Appearance of organised, non
oriented, homogeneous networks:
isotropicorganisation
2
22. 22
• Due to the analysis of these images, for the first time in the world, researchers at Laboratoires Expanscience
have developed a maturity score for the Stratum corneum: the E.M.I (Electron Microscopy Isotropy) score,
based on 4 criteria:
Cell density,
Cell clustering,
Cohesion between cells,
The shapes of the cells and ability to distinguish between them.
IMAGES OBTAINED BY SEM
(X30 MAGNIFICATION)
IMAGES OBTAINED BY SEM
(X500 MAGNIFICATION)
Cell density Cell clusters Cell adhesion/shape
Ability to distinguish between
cells
0 Cells separated Irregular clusters
Asymmetric cells
Irregular shapes
Cells not distinguishable
1 Cells grouped/scattered
Mixture of regular and
irregular clusters
Cells partially
symmetrical
Shapes partially irregular
Cells partially
distinguishable
2 Cells evenly scattered Mostly regular clusters
Cells mostly
symmetrical
Shapes mostly irregular
Cells mostly
distinguishable
3 Cells linked Regular clusters
Symmetrical cells
Regular shapes
identical in all geometric
directions to an octogon
with regular surface
Cells easily
distinguishable
Basic criteria of the EMI score
23. 23
ISOTROPY SCORE MATURITY
Anisotropy 0-4 Immature
Intermediate isotropy 5-7 Intermediate
Complete isotropy 8-12 Mature
The 3 stages of maturity derived from the EMI score
This study has confirmed that surface of the skin undergoes significant change, and has enabled us to
establish the existence of a first stage of maturity from the second year of life.
Between birth and 2 years, the skin barrier does not fully accomplish its protective role. It is therefore
necessary to support it during its development.
It has been established thereby that the Stratum corneum is:
immature in newborns
at an intermediate stage after 5-6 weeks
in a first stage of maturity from the age of 1-2 years
The EMI score, developed and patented by Laboratoires Expanscience, constitutes an effective tool for analysing
the topography of the surface of baby’s skin throughout the years.
Using the EMI score, the IRD centre has defined three stages of maturity of Stratum corneum organisation:
2
24. 24
b. On the distribution of moisturising factors in the Stratum granulosum
Second discovery: skin dehydrated at birth and mechanisms regulating water balance
disturbed.
Measurements taken with Raman spectroscopy (NMF + water content) have provided
evidence for three phenomena never before demonstrated and which have been reported in
an article published in the British Journal of Dermatology.
• In newborns, a dual phenomenon has been discovered:
water content is low, in correlation with low hydration of the surface measured by
corneometry.
the NMF concentration is high (contrary to expectations).
This phenomenon may be explained by the stress caused at birth by the change from a liquid to a gaseous
environment.
This stress in the newborn triggers compensatory mechanisms (release of high levels of NMF), a phenomenon
that helps to regulate the pH and water content (too high and too low, respectively) and thereby enable the skin
to adapt to its new environment.
• Between 0 and 6 months, an abrupt drop in NMF to regulate pH and hydration is observed
• Between 6 months and 2 years: Gradual return to an NMF level comparable with that of an adult.
18
16
14
12
10
8
6
4
2
0
NB
(1 to 15 d)
5
weeks
6
months
1-2
years
4-5
years
Adults
(20-25
years)
Hydration (corneometry)
Water content (RAMAN)
NMF (RAMAN)
pH (pH meter)
Return to hydration level comparable with adults
Hydration
Water
pH
NMF
25. 25
Changes in NMF concentration with age.
Values calculated from area under the curve of Raman profiles for each age group at depths between 0 and 25 µm (mean +/- SE)
20
15
10
5
0
1-15 days 5-6 weeks 1-2 years 4-5 years 20-35 years6 +/- 1 months
NMFProfile(bulk)AUC(a.u.)
17.2 12.0 12.211.7
6.4 9.7
ResearchatLaboratoiresExpansciencehasrevealedforthefirsttimethemolecularcompositionoftheskinin
relation to its dynamic functional adaptation during the first months of life.
We have shown that the mechanisms of water balance regulation are disturbed between birth and 2 years,
which corresponds to a period of vulnerability.
Thisiswhyitisimportanttomoisturisetheskinofinfantsdaily,frombirthandduringthefirsttwoyearsoflife.
2
26. 26
c. On the stem cells of the Stratum germinativum
Recentadvancesinstem
cell research are extre-
mely relevant to our
study.
The latest scientific
advances have taught
us that these stem cells
reside in a specialised
microenvironment, called
a niche, made up of an extracellular
matrix, rich in proteins, which helps to
maintain the stem phenotype of these
cells.
This microenvironment contributes
to maintaining the stem cells in good
condition, influences cell division and
differentiation on one hand and ensures
toacertainextenttheprotectionofstem
cells against outside aggression on the
other.
Basal stem cells contribute to the
maintenance of skin homeostasis. Their
protection is therefore essential for
limiting the harmful effects of outside
aggression.
Third discovery: stem cells, an essential and
vulnerable supply.
Forthefirsttimeintheworld,wehaveanalysed
the expression of stem cells genes in recons-
tructed epidermis, from babies of varying
ages, both under normal conditions and under
aggression.
To help identify stem cells, we chose to ana-
lyse the expression of the most commonly
cited gene markers in the scientific literature:
• Keratin 19: marker of stem cell proliferation
• Integrin a6, integrin β4, integrin β1: cell
receptors implicated in intercellular commu-
nication, in the adhesion of cells in the extra-
cellular matrix, in the regulation of stem cell
proliferation and differentiation
• Notch Homolog 1: gene that regulates the
correct functioning of stem cell activity
27. 27
100 % 100 %
20%
Integrin alpha-6
(green)
20%
Integrin beta-1
(pink)
Stress conditions Stress conditions
Stemcells,theskin’sguaranteeofsurvival,calledintoactionfortheconstructionoftheepidermisthroughout
anindividual’slifetime,mustbeconservedandprotectedagainstenvironmentalaggressionregardlessofage.
This has revealed 2 major discoveries:
• Under normal conditions, the expression of stem cell marker genes are highest at birth, diminishing
spontaneously in the first few months of life.
• En conditions normales, l’expression des marqueurs des gènes des cellules souches est plus fortement
exprimée dès la naissance puis, diminue spontanément dès les premiers mois de vie.
• Under the effect of outside aggression, the expression of certain basal stem cell marker genes decrease
significantly.
We attached immunofluorescent labels to alpha 6 and beta 1 integrins in the epidermal stem cells of the
basal layer of a skin explant before and after irradiation with UVA (10J/cm² )+ UVB (200J/cm²)
We observed a very marked decrease in the expression of these stem cell genes, confirming the very high
vulnerability of stem cells to environmental aggression.
12 ans
Adulte
120
100
80
60
40
20
0
Stem cells genes change
1 month
4 months
6 months
1 year
2 years
3 years
Normalizedrelativeexpression
(%tothecontrol)
KRT19 ITGB1 NID1
7 years
12 years
Adults
2
28. 28
Laboratoires Expanscience have carried out a programme of research on children’s skin, from birth to
2 yearsofage,fromthesurfacetothestemcellsoftheepidermis,andhaveestablishedthattheperiod
between 0 and 2 years is critical for children’s skin.
From 0 to 2 years:
the Stratum corneum, which guarantees
the barrier function of the skin, is under-
going a process of maturation,
the skin is dehydrated and water
balance is disturbed,
the supply of stem cells is extremely
vulnerable to outside aggression.
This is why it is essential to protect and moisturise the skin daily between the ages of 0
and 2 years.
DISCOVERIES OF THE EV.E.I.L.S PROGRAMME
TO SUM UP
32. 32
A new generation active ingredient:
Avocado Perseose®
3
e
d
c
b
a
From the skin surface
to the epidermal stem cells
AVOCADO PERSEOSE®
REINFORCES THE SKIN BARRIER
AVOCADO PERSEOSE®
MAINTAINS THE HYDRATION LEVEL
OF THE EPIDERMIS
AVOCADO PERSEOSE®
PROTECTS THE SKIN'S STEM CELL
SUPPLY
Having become aware of the need to support the development of the skin and protect the fragile
skin cell supply of infants between 0 and 2 years, Laboratoires Expanscience have pursued their
research programme.
Due to its dual know-how in plant extraction and cell biology, the IRD centre has developed an exclusive, natural
and patented active ingredient: Avocado Perseose®.
Avocado Perseose® is a unique patented active ingredient composed of mannoheptulose and perseitol, sugars
with seven carbon atoms, rare in the plant kingdom and present in very few species.
Avocado Perseose® belongs to the new generation of biomimetic active ingredients: active ingredients of natural
origin, which are able to act in perfect affinity with baby’s skin while combining targeted efficacy and maximum
tolerance.
The researchers at the IRD centre present to you, in this section, the biological activities targeted by Avocado
Perseose® while respecting the skin’s integrity, the unique, patented extraction process for Avocado Perseose®,
as well as the Laboratoires Expanscience pharmaceutical supply chain from which the avocado is sourced.
A. ItsactionsextendfromtheStratumcorneumthroughtotheStratumgerminativum
33. 33
a. Action on the skin barrier
• Avocado Perseose® acts on the cohesion of the Stratum corneum
Protocol: A thin layer chromatography analysis enabled the isolation of ceramides from other major
lipids in the extracellular matrix and precise quantification after treatment with a 0.005% solution of
Avocado Perseose® over 72 hours.
Result:Avocado Perseose® increased the synthesis of ceramides in reconstructed epidermis by 32%.
• Avocado Perseose® acts on the consolidation of the microarchitecture of the Stratum corneum
Protocol: The ELISA technique was used to measure the quantity of involucrin (constituent of the
cornified envelope around cells) and desmoglein-1 (constituent of corneodesmosomes, structures
linking cells together) produced by keratinocytes after treatment with a 0.005% solution of Avocado
Perseose® over 48 hours.
Result: Avocado Perseose® significantly stimulated the expression of involucrin and desmoglein-1 in
keratinocytes: + 49% (p 0.001) and + 41% (p 0.001) respectively.
Production of involucrin and desmoglein-1 in keratinocytes
Neosynthesis of ceramides in reconstructed epidermis
QUANTITY OF CERAMIDES
Relative intensity (mean) % of control
Control epidermis 14.2 100
Differentiated epidermis 19.7 138
Avocado Perseose 0.005% 18.0 132
10
8
6
4
2
0
Untreated
cells
Avocado
Perseose®
Relativequantityofdesmoglein-1/
numberofcells
***
+ 41 %4
3.5
3
2.5
2
1.5
1
0,5
0
Untreated
cells
Avocado
Perseose®
Relativequantityofinvolucrin/
numberofcells
***
+ 49 %
*** p0.001, Dunnett test*** p0.001, Dunnett test
Avocado Perseose® reinforces the skin barrier.
3
34. 34
b. Action on skin hydration
• Avocado Perseose® acts on the retention of water in the epidermis
Protocol: The ELISA technique was used to measure the quantity of hyaluronic
acid produced by keratinocytes after treatment with a 0.005% solution of Avocado
Perseose® over 24 hours.
Result: Avocado Perseose® significantly increased the quantity of hyaluronic acid
produced by keratinocytes: + 47% (p0.05).
Production of hyaluronic acid by keratinocytes
• Avocado Perseose® acts on the semi-permeability of the Stratum corneum
1st
step: detection by immunolabelling
Protocol: Immunolabelling enabled the detection of transglutaminase, an enzyme that promotes the
adhesion of the cornified envelope around cells to maintain water within, produced by keratinocytes
after delipidation of the skin (explant mimicking dry skin) and treatment with Avocado Perseose® for
3 hours.
Result:TopicaltreatmentwithAvocadoPerseose®ofdelipidatedskinexplantsstimulatedtransgluta-
minase expression.
700
600
500
400
300
200
100
0
Untreated
cells
Retinoic acid
(ref)
Avocado
Perseose®
Relativequantityofhyaluronicacid/
numberofcells
***
*
+ 119 %
+ 47 %
* p0.05, *** p0.001, Dunnett test
versus untreated cells
35. 35
Expression of transglutaminase in delipidated skin explants
Immunofluorescent labelling: green = transglutaminase (FITC) - red = cell nuclei (propidium iodide)
2nd
step: activity assay
Protocol: The activity of the transglutaminase produced by keratinocytes was assayed using the
Transglutaminase Colorimetric MicroAssay kit after treatment with a 0.005% solution of Avocado
Perseose® for 72 hours.
Result: Avocado Perseose® significantly stimulated the activity of the enzyme transglutaminase in
keratinocytes: + 175% (p0.05).
Transglutaminase activity in keratinocytes
Dry skin (delipidated) control Dry skin (delipidated) + Avocado Perseose®
0.00005
0.00004
0.00003
0.00002
0.00001
0.00000
Untreated
cells
Reference (Ca) Avocado
Perseose®
Transglutaminaseactivity
(D0)/totalproteins
*
*+ 146 %
+ 175 %
* p0.05, Dunnett test
versus untreated cells
Avocado Perseose® maintains the level of hydration in the epidermis
3
36. 36
c. Action on stem cells
The effect of Avocado Perseose® on basal stem cell markers was studied both under baseline
conditions(withoutaggression)andinconditionsofseriousaggressionsuchasUVirradiation.
• Under normal physiological conditions, Avocado Perseose® has a completely neutral effect
on stem cells.
Protocol: Skin samples were taken from donors aged 1 month, 3 months, 3 years,
6 years and 11 years, as well as from adults. From these skin samples, keratinocytes
wereextractedandculturedfor24hourswithandwithout(control)a0.005%solution
of Avocado Perseose®.
The expression of stem cells marker genes (KRT19, ITGB1, ITGA6, NOTCH1) was
evaluated using the real-time quantitative PCR (Polymerase Chain Reaction) assay
method.
The genomic analysis demonstrated that the expression of basal stem cells genes
was high at birth and decreased with age.
Result: This expression profile is maintained in the presence of Avocado Perseose®,
indicating that the latter is well tolerated by cells and that it maintains the balance
of basal stem cells and the epidermis.
Changes in basal stem cell genes/markers by age in the presence of Avocado Perseose®
120
100
80
60
40
20
0
KRT19 ITGB1 ITGA6 NOTCH1
1 month
3 months
3 years
6 years
11 years
Adult
Relativequantityexpressedin%
37. 37
• Inthefaceofseriousenvironmentalaggression(UVAandUVB),AvocadoPerseose®guaranteestheprotection
of basal epidermal stem cells
Protocol: Skin explants were irradiated with UV (UVA: 10 J/cm2
and UVB: 200 mJ/cm2
) and treated with
or without Avocado Perseose®, 24 hours before and 24 hours after irradiation. Protein immunolabelling
enabled the measurement of expression of alpha-6 and beta-1 integrins, basal stem cell markers,
under different conditions.
Result: In the face of serious environmental aggression (UVA and UVB) and in the presence of
Avocado Perseose®, 80% of stem cell markers were conserved (compared with 20%)
Immunolabelling of basal stem cell markers. Effect of Avocado Perseose® in presence of UV irradiation
INTEGRIN alpha-6 INTEGRIN beta-1
20% 20%
80% 80%
Stress
conditions
Stress
conditions
+ PERSEOSE + PERSEOSE
INTEGRIN alpha-6
(green)
INTEGRIN beta-1
(pink)
Avocado Perseose® helps protect the potential of basal stem cells in the face of serious aggression such as
UV exposure.
Avocado Perseose® hence has a positive effect on the protection of basal stem cell potency and contributes
to the maintenance of the integrity and balance of the epidermis.
3
38. 38
B. Unique, patented extraction processes
Avocado Perseose® is a natural active ingredient from Laboratoires Expanscience produced by its subsidiaries in
South Africa and Peru.
On the strength of knowledge and know-how regarding the chemistry of the avocado developed over the course
of the last thirty years, our RD centre, in its Lipid Chemistry and Plant Extraction Laboratory, has used a unique
and patented process to extract the desired active ingredient from the plant.
Working with a C7 sugar concentration of less than 1% in raw avocado, it proved a real technological challenge to
purify, conserve and concentrate the molecular rarity of Avocado Perseose® from raw avocado, while taking into
consideration the volume restrictions.
39. 39
Selection of raw avocado
Slicing and drying under patented and optimised
conditions to preserve molecular integrity
Extraction by pressing to obtain oil and press cake
Pharmaceutical
grade oil used in
certain Laboratoires
Expanscience
products
Selective extraction of sugars using extraction media
that respect the environment and mankind
Purification and Concentration by specific filtration
systems and stabilisation of the active principle
by lyophilisation
Press cake: mixture of sugars and proteins
Mannoheptulose and Perseitol
Avocado
Perseose
To achieve this, the selected avocado must undergo a process comprising over 8 steps and calling on specific
technological means.
3
40. 40
C. An ethical and responsible supply chain
Avocado Perseose® is the product of a controlled pharmaceutical process, governed by our guidelines for
corporate social responsibility, and subject to evaluation by the UEBT (Union for Ethical Bio Trade).
An organisation inspired by a United Nations initiative, the UEBT is an international not-for-profit association,
which promotes ethical practices for the supply of ingredients sourced from biodiversity.
Joining the UEBT is a pioneering initiative, especially in the pharmaceutical sector.
Since December 2011, Laboratoires Expanscience is the first French pharmaceutical company to become an
official member of the Union for Ethical Bio Trade (UEBT) and through its membership affirms its commitment to
promoting biodiversity.
In addition, Laboratoires Expanscience has willingly adopted a strategy of Corporate Social Responsibility
This commitment is demonstrated in particular by the incorporation of eco-design principles during the
development of pharmaceutical and cosmetic active ingredients.
Specialists in the use of products of natural origin, and due to the extensive knowledge and know-how regarding
the chemistry of the avocado, which has been developed over the course of the last thirty years, Laboratoires
Expanscience is now able to use every part of the fruit to produce a dozen raw materials or cosmetic active
ingredients, including Avocado Perseose®
We present to you here the avocado processing cycle.
41. 41
SLICING + DRYING
Grinding
Exfoliating
avocado
power
Refined
avocado
oil
Unrefined oilMeal
Avocado
oil concentrate
Avocado
oil unsaponifiables
5a Avocuta®
Enz. hydrolysisAvocado peptides
Extraction
Refining
Esterification
Mol. distillation
(MD)
MD + extraction
ACOCADO
SUGARS
(C7)
Free fatty acids
100% of the avocado recovered
EXTRACTION FROM AVOCADO TREE
The RD centre has developed, due to its dual know-how in plant extraction and cell biology, an exclusive,
natural and patented active ingredient: Avocado Perseose®.
AvocadoPerseose®belongstothenewgenerationofbiomimeticactiveingredients,activeingredientsofnatu-
ral origin which are able to act in perfect affinity with skin while combining targeted efficacy and maximum
tolerance, from birth.
The Avocado Perseose® fits the specific needs of the baby skin from 0 to 2 years, and it :
• reinforces the skin barrier
• maintains hydration and regulating water balance
• and protects the skin’s supply of cells
T O S U M U P
3
Pressure
44. 44
The formulation: a new technological challenge
4
Following this programme of unprecedented research, these exciting discoveries and the
development of Avocado Perseose®, Laboratoires Expanscience has completely reviewed the
Mustela® Bébé skincare products.
The global reformulation of Mustela® Bébé line was a fantastic technological challenge, revolved around
4 objectives:
A. To incorporate Avocado Perseose® in all formulas
The formulation of new products incorporating Avocado Perseose® had to conform to a highly ambitious internal
charter reflecting our very strict safety requirements and criteria, while maintaining the galenic properties that
satisfy the demands of our consumers in terms of texture and fragrance, efficacy and high degree of naturalness.
The challenge was to maintain the stability of the formulas, as the addition of Avocado Perseose® upset the
equilibrium of the existing formulas particularly during the incorporation - in certain lipophilic formulas - of a
hydrophilic active ingredient.
To guarantee the beneficial effects of Avocado Perseose® in the products, the efficacy of all the new Mustela® Bébé
products have been tested on babies and children - via in vivo studies in observational clinical tests under real
conditionsofuseandunderdermatologicalandpaediatricsupervision,andinvitro,onreconstructedbabyepidermis.
B. To maximise the safety and tolerance of the products
while guaranteeing a high degree of naturalness and
excludingquestionableingredientsinallproductformulas
D. To be consistent with a volunteer strategy of social
and environmental responsibility
A. To incorporate Avocado Perseose® in all formulas and
guaranteeallitsbeneficialeffectsinthefinishedproducts
C. To ensure a high sensory quality in the products for
unequalled pleasure of use
45. 45
B. Tomaximisethesafety,innocuityandtoleranceoftheproducts
In addition to the stringent regulatory framework of the ISO 22716 standard and adherence to good manufactu-
ringpractices,LaboratoiresExpansciencemaintainsahighlevelofsurveillanceforalltheingredientsselected,at
every stage of development and before releasing the product on the market.
SAFETY – INNOCUITY
• Rigorous selection of raw materials
An in-depth file is assembled and submitted to an independent expert toxicologist. The documents included a
reviewofpublished data foreachrawmaterial,ananalytical study oftherawmaterialaswellasinformationfrom
suppliers on the source, stability and safety of the raw materials.
Concerning fragrances or our fragrance compositions, we ensure their safety is subject to a toxicology assess-
ment. We choose them pursuant to specifications that meet the prevailing cosmetics and perfumery regulations
(Cosmetics Directive 76/768/EC/Regulation (EC) No 1223/2009 of the European Parliament and of the Council
on cosmetic products, and current IFRA regulations), and also require the removal from label-
ling of the 26 allergenic substances (identified as entailing a potential allergen in Cosmetics
Directive 76/768/EC - 2003 revision).
• Priority given to ingredients of natural origin and substitution of a maximum of synthetic raw materials
Today, Mustela® Bébé products contain on average 92% ingredients of natural origin. Priority is given to natural
ingredients or ingredients of natural origin, as long as they meet our demands in terms of safety, efficacy and
pleasure of use.
However,whenthereisnonaturalalternative,weauthorisetherecoursetosyntheticingredients.Theselection
is then made uniquely from ingredients permitted by European regulations, and which meet all our safety and
efficacy requirements.
We limit the use of certain synthetic ingredients, such as synthetic waxes and mineral oils,
silicones, synthetic colouring agents,...
• Acute assessment of the safety and innocuity of the finished product
A rigorous procedure enables us to evaluate the safety of the finished product throughout its life cycle.
EveryMustela®formulaundergoesnumerousteststoestablishthatitisperfectlysafe:toleranceistestedinvitroand
invivoundermaximisingconditions,testsofhypoallergenicityand/orphototoxicityareconducted.
To evaluate product safety in an even more reliable and comprehensive manner, our researchers have pioneered
the reconstruction of baby’s and children’s epidermis from birth (Expanscience Laboratories exclusivity).
ON AV E R A GE
EN MOYENNE
Paraben / Phenoxyethanol / Phtalate /
Chlorphenesin / Alcohol / SLS / EDTA...
4
46. 46
TOLERANCE
All Mustela® products are developed to minimise the risks of allergy.
Theyallpossessalevelofhightolerance,whichhavebeentestedunderworst-caseconditionsinvitroandinvivo,with
proceduresthatareuniqueandexclusivetoExpanscience,includingtestingonreconstructedbabyskinepidermis.
Product tolerance is also evaluated in observational clinical tests, under real use conditions and under
dermatological and paediatric supervision on groups of babies of whom 25% are aged less than 1 month.
in vitro
on reconstructed epidermal
models of baby’s skin
in vitro
These epidermal models of 1-month-old
baby skin constitute a tolerance evaluation
model (histological measurements and cell
viability) for products designed for skincare
and hygiene in babies. These models are now
systematically used in evaluation procedures
for all Laboratoires Expanscience cosmetic
products for babies.
in vivo
specific Mustela stinging test
conducted under worst-case
conditions
The aim of this test is to measure non-visible
objective signs, generally not detected in other
tolerance tests.
We conduct the test under “worst case” conditions
by changing parameters such as ambient
temperature, humidity level, and using kinetic
measurement techniques.
WORLD EXCLUSIVITY
47. 47
C. To ensure a high sensory quality
The formulation challenge was successfully met by the Cosmetic Development Division of the RD Centre of
Laboratoires Expanscience.
With a high sensory quality, and an unequalled comfort and pleasure of use, the new patented formulas of
Mustela® Bébé line contribute to the well-being and sensory awakening of newborns and babies due to the
textures adapted to each of the products without changing the emblematic Mustela® Bébé fragrance.
D. To be consistent with a strategy of environmental responsibility
• From ethical and responsible sourcing
A voluntary and transparent approach taken by Laboratoires Expanscience on the selection of ingredients
details the company’s position on this important issue.
Information on raw materials (The life cycle of Mustela® products) can be found on the Mustela® website:
http://www.mustela.com as well as on the website: www.expanscience.com/en/sustainable-development
• To eco-designed products
Asforpackaging,aneco-designstrategycalled3R(reduce,replace,recycle)isfollowed.Thereductioninweight
of certain bottles and the partial incorporation of recycled plastic in tubes, bottle and lids of certain items have
led to a 9-tonne decrease in the annual consumption of plastic and an additional reduction of 4.4 tonnes in the
amount of virgin plastic used
Use of recycled plastic in tubes
Use of cardboard sourced from sustainably-managed forests
Use of plant-based inks on boxes
No superfluous over-packaging, no paper package inserts
• To the end of the product’s life cycle, in order to reduce the environnemental impact
All our liquid cleansing formulas are biodegradable, in accordance with the guidelines (OCDE 302B)
And Mustela® bottles are 100% recyclable according to French criteria of recyclability (excluding samples).
4
50. 50
Conclusion
5
This research programme, ongoing for the past ten years and unique in the world, is founded on ethical and
innovative investigative technology.
It has enabled us to deepen our understanding of the physiology of baby’s skin from the Stratum corneum
to the epidermal stem cells, from the very first days of life.
Major discoveries about the skin barrier, hydration and epidermal stem cells have enabled us to establish
the existence of a critical period for children’s skin between birth and 2 years.
To provide baby's skin with what it requires specifically during this period of vulnerability between 0 to
2 years, the Expanscience Laboratories RD centre has rethought all the Mustela® Bébé formulas and
enriched them with an exclusive, natural and biomimetic active ingredient, derived from the avocado:
Avocado Perseose®.
Mustela® offers a new generation of skincare products, clinically tested, which act in perfect harmony with
a baby’s skin and combine targeted efficacy, a high level of safety and maximum tolerance, with no less
than 92% ingredients of natural origin on average.
Expanscience
Laboratories
RD centre,
Epernon, France.
51. 51
New Mustela® Bébé line developed thanks to Expanscience Laboratories expertises and manufactured
on the industrial site located in Epernon - France. 5
54. 54
• Direction Innovation, Research and Development (IRD) - Biologie Cellulaire - Laboratoires Expanscience. Dossier
technique: perséose d’avocat et fonction barrière. July 2013.
• Fluhr JW, Darlenski R, Lachmann N et al. Infant epidermal skin physiology: adaptation after birth. British Journal
of Dermatology 2012; 166(3):483-90.
• R D Center Laboratoires Expanscience. Fluhr JW, Darlenski R, Hachem JP et al. Skin adaptation from birth to
childhood: practical considerations.
• Innovation,RDLaboratoiresExpanscience.Belilovsky(de)C,FluhrJW,BaudouinCetal.E.V.E.I.L.SPROGRAMME:
inside infants’ skin: new spectroscopic, microscopic, genomic data leading to cutting edge and patented genera-
tion of baby skin research and care.
• Innovation, R D Laboratoires Expanscience. Fluhr JW, Msika Ph, Baudouin C et al. Surface isotropy as a marker
for epidermal maturation in infancy: development and validation of the patented Electron Microscopy Isotropy
(E.M.I) score.
• R D Center Laboratoires Expanscience. Fluhr JW, Lachmann N, Baudouin C et al. Comparison of the skin
physiology of neonates and young children with that of adults: a randomised clinical trial.
• Innovation, R D Laboratoires Expanscience. Baudouin C, Brédif S, Pedretti N et al. Modeling and genomic
signature of age-specific infants and children epidermis: new data and innovative discoveries.
References
6
55. 55
• Direction Innovation, Research and Development (IRD) - Biologie Cellulaire - Laboratoires Expanscience. Dossier
technique: perséose d’avocat et cellules souches. July 2013.
• Direction Innovation, Research and Development (IRD) - Laboratoires Expanscience. Baudouin C. Point sur les
cellules souches épidermiques. January 2013.
• Innovation, R D Laboratoires Expanscience. Leclère-Bienfait S, Brédif S, Rocheteau J et al. Avocado Perseose®,
a biomimetic patented active ingredient targeted to the needs of infant’s skin.
• R D Center Laboratoires Expanscience.Cochet P, Menu F, Msika Ph. Safety evaluation and formulation of
cosmetic products designed for children under age of 3 years.
• Innovation, R D Center Laboratoires Expanscience. Baudouin C, Belivovsky (de) C, Lachmann N et al. Tolerance
assessmentofcosmeticproductsdedicatedtobaby’sskin:innovativeapproachesusingstingingtestandinfant
epidermis model.
• Fluhr JW, Darlenski R, Taieb A, Hachem JP, Baudouin C, Msika P, Belilovsky (de) C, Berardesca E. Functional skin
adaptation in infancy - almost complete but not fully competent. Experimental dermatology 2010; 19(6): 483-92.
• Mustela® website: www.mustela.fr
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