Original Contribution

Dermal penetration of creatine from a face-care formulation

containing creatine, guarana and glycerol is linked to effective
antiwrinkle and antisagging efficacy in male subjects
Reto I Peirano, PhD,* Volker Achterberg, PhD,* Hans-Jürgen Düsing, Mehdi Akhiani, Urte Koop,
Sören Jaspers, Andrea Krüger, Helge Schwengler, Tina Hamann, MSc, Horst Wenck, PhD, Franz
Stäb, PhD, Stefan Gallinat, PhD & Thomas Blatt, PhD
Beiersdorf AG, Research and Development, Skin Research, Hamburg, Germany

Summary Background The dermal extracellular matrix provides stability and structure to the skin.
With increasing age, however, its major component collagen is subject to degeneration,
resulting in a gradual decline in skin elasticity and progression of wrinkle formation.
Previous studies suggest that the reduction in cellular energy contributes to the
diminished synthesis of cutaneous collagen during aging.
Aims To investigate the potential of topically applied creatine to improve the clinical
signs of skin aging by stimulating dermal collagen synthesis in vitro and in vivo.
Patients ⁄ Methods Penetration experiments were performed with a pig skin ex vivo model.
Effects of creatine on dermal collagen gene expression and procollagen synthesis were
studied in vitro using cultured fibroblast-populated collagen gels. In a single-center,
controlled study, 43 male Caucasians applied a face-care formulation containing
creatine, guarana extract, and glycerol to determine its influence on facial topometric
features.
Results Cultured human dermal fibroblasts supplemented with creatine displayed a
stimulation of collagen synthesis relative to untreated control cells both on the gene
expression and at the protein level. In skin penetration experiments, topically applied
creatine rapidly reached the dermis. In addition, topical in vivo application of a creatine-
containing formulation for 6 weeks significantly reduced the sagging cheek intensity in
the jowl area as compared to baseline. This result was confirmed by clinical live scoring,
which also demonstrated a significant reduction in crow’s feet wrinkles and wrinkles
under the eyes.
Conclusions In summary, creatine represents a beneficial active ingredient for topical use
in the prevention and treatment of human skin aging.
Keywords: creatine, rapid penetration, collagen, skin aging, topical application

Correspondence: V Achterberg, PhD, Department of Skin Biology and Skin
Structure, Beiersdorf AG, Research and Development, Bf. 510, Unnastrasse
48, 20245 Hamburg, Germany. E-mail: volker.achterberg@beiersdorf.com
*Both authors contributed equally to this work.
Accepted for publication August 18, 2011
Introduction
Like all other organs, human skin is subject to
physiological and morphological changes during aging.
In the dermis, age-associated changes are characterized
by alterations to the dermal extracellular matrix (ECM).

 2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 273–281 273
Anti-aging effects of creatine • R I Peirano et al.

These alterations are in part attributed to the degra- Material and methods
dation of the major ECM component collagen.1 The
cellular balance between collagen synthesis and colla- In vitro studies
gen breakdown is modified by changing cellular redox
Determination of penetration kinetics using pig skin as a
homeostasis and ⁄ or decreasing cutaneous energy
model substrate
metabolism. Ultraviolet (UV) exposition, for example,
Absorption and percutaneous penetration experiments
activates collagen-degrading matrix metalloproteinases
were performed as previously described.11 In brief,
via generation of reactive oxygen species (ROS).2 As a
unboiled back skin of female pigs (approximately
result, detrimental modifications of dermal structures
130 days old, weighing approximately 100 kg, delivered
are observed which contribute to the development of
by a local, commercial butcher) was cleaned using tap
the well-known clinical signs of skin aging: facial
water, gently dry-shaven, and full-thickness skin disks
wrinkles, fine and coarse lines, and sagging of
were obtained (thickness: 3–4 mm; diameter: 5 cm). The
cheeks.3,4
disks were mounted on glass penetration cells (Franz-type
To improve the status of the ECM and to combat
cell with an application area of 4.9 cm2) and kept at 32 C
visible skin alterations, collagen synthesis needs to be
with a thermostat. For experiments, 20 lL of an aqueous
activated in aging cells. Cutaneous collagen synthesis
solution of 14C-radiolabeled creatine (activity of
requires large amounts of energy, but with progressing
1 mCi ⁄ mL; Biotrend, Cologne, Germany) was added to
age, cellular energy levels are declining.5,6 In this
approximately 500 mg of a commercially available,
context, creatine, a naturally occurring substance in
creatine-containing cosmetic face-care formulation
all vertebrates including humans, is brought into
(Beiersdorf, Hamburg, Germany). To guarantee a homo-
focus.
geneous distribution of radioactivity within the formula-
Creatine is known to play a crucial role in the body’s
tion, the cream was intensively stirred with a spatula and
energy supply. It serves as an energy store, which can,
kept for at least 20 h at room temperature prior to
in the case of high cellular energy demand, rapidly
application (approximately 450 Bq ⁄ g activity concentra-
phosphorylate ADP to generate ATP.7 Sufficient energy
tion). The formulation was applied to the surface of the pig
levels in the form of ATP are necessary to maintain vital
skin disk mounted on the penetration cell at a dose of
function and health of human skin cells. ATP is required
about 4 mg ⁄ cm2. The receptor fluid, suitable to dissolve
for all cellular mechanisms involved in repair and
hydrophilic and lipophilic test substances, was composed
defense processes, and also for synthesis of important
of 0.9% sodium chloride, 1% bovine serum albumin and
cutaneous bio-molecules such as collagen. Cells attain
0.02% gentamycin sulfate in water.
their physiological levels of creatine by biosynthesis from
After 1 and 3 h of exposure, respectively, dermal
the amino acids such as arginine, glycine, and methi-
absorption and distribution of creatine between skin
onine. As endogenous creatine synthesis in humans is
surface (nonabsorbed), horny layer, epidermis, dermis
not sufficient, creatine needs to be taken up with the diet
and receptor fluid were determined as follows: excessive
especially by ingestion of meat and fish. In human skin,
test sample was removed by using cotton swabs, the
both the highly specific creatine transport protein (CRT)
horny layer was removed by stripping with adhesive
and creatine kinase (CK) are expressed.8 Creatine
tape, epidermis and dermis were heat-separated on a
synthesis in human cells declines with age, and from
80 C hot plate for 45 s and dissolved in SOLUENE-350
the age of approximately 30 years on, a decrease in
(Packard Instrument Company, Meriden, CT, USA).
cutaneous cellular concentration and also in the activity
Radioactivity in analytical samples was measured using
of CK can be determined.9,10
the liquid scintillation counter LS 6500 (Beckman
The objective of this study was to investigate whether
Coulter Inc., Brea, CA, USA) with automatic quench-
topically applied creatine reaches dermal skin layers and
correction and Hionic-Fluor (Packard Instrument
can facilitate collagen synthesis in vitro and in vivo.
Company) as scintillation cocktail. Nonlabeled sub-
Here, we demonstrate that application of creatine in
stances were analyzed with either HPLC or gas
vitro leads to substantial stimulation of collagen syn-
chromatography. Experiments were performed in
thesis and to an effective improvement in facial skin
triplicate (three skin disks).
texture in vivo, as assessed by clinical live scoring.
Overall, our findings underline that creatine can support
Human primary dermal fibroblast cell culture
dermal cells energetically, making this compound a
Human primary dermal fibroblasts (HDF) were cultured
beneficial active ingredient for topical treatment of
by outgrowth from skin biopsies obtained from different
human skin aging.

274  2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 273–281

donors (43–69 years) as previously described.12 All
donors provided written informed consent. Briefly, cells
were cultivated in Dulbecco’s modified Eagle medium
(DMEM; Invitrogen, Karlsruhe, Germany) supplemented
with 10% fetal calf serum (FCS; PAA, Linz, Austria),
50 U ⁄ mL penicillin, 50 lg ⁄ mL streptomycin, and 1·
glutamine (all obtained from Invitrogen) by incubation
in a humidified atmosphere of 7% CO2 at 37 C. After
having reached almost confluency, fibroblast cultures
were routinely passaged by trypsination and used for
experiments up to passage 6. For each experiment,
fibroblasts obtained from different donors were used.
Generation of fibroblast-populated collagen lattices
To establish fibroblast-populated collagen lattices
(FPCLs), used as a dermal equivalent, 100 mg of
collagen type I isolated from rat tails (Sigma-Aldrich,
Munich, Germany) was dissolved in 0.1% sterile acetic
acid (25 mL) at 4 C. After preparation of a homogenous
solution, 9.5 mL of the solution was mixed in an ice bath
with 11.7 mL DMEM and 1 mL 10· Hank’s salt solution
(Sigma-Aldrich) and then neutralized with 0.8 mL 0.2 N
NaOH solution.
Human dermal fibroblasts (6 · 106) suspended in
1 mL DMEM were added, and 2 mL of the resulting
fibroblast–collagen solution was cast into each tissue
culture dish (35 mm diameter). After an hour of
incubation at 37 C, FPCLs were solidified and contained
1.6 mg ⁄ mL collagen and 2.7 · 105 fibroblasts ⁄ mL.
Next, gels were covered with 1 mL DMEM containing
20% FCS (final concentration in the gel 6.7%) either
supplemented with creatine (final concentration
240 mg ⁄ L; Goldschmidt, Essen, Germany) or not. Every
2–3 days, 1 mL of the medium was replaced with the
respective fresh medium. During the entire incubation
time, gels were kept attached. As positive control,
10 ng ⁄ mL of TGF-b1 (human, recombinant; Sigma-
Aldrich Chemie GmbH, Taufkirchen, Germany) was used.
Isolation of total RNA and determination of collagen 1A1
gene expression
To determine collagen 1A1 gene expression, FPCLs were
dissolved after 21 days of culture using collagenase: Gels
were rinsed with phosphate-buffered saline (PBS; PAA,
Linz, Austria) and incubated for 45 min at 37 C in
0.5 mL collagenase type 1 (3.3 mg ⁄ mL, isolated from
Clostridium histolyticum; Sigma-Aldrich) dissolved in
collagenase buffer (130 mm NaCl, 10 mm Na-acetate,
10 mm Ca-acetate, 20 mm HEPES, pH 7.2). Following
two washing steps with PBS and a subsequent centri-
fugation for 5 min at 300 g, RNA was isolated from the
resulting cell pellet using the RNeasy Mini Kit (Qiagen
 2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 273–281
Anti-aging effects of creatine • R I Peirano et al.
GmbH, Hilden, Germany) according to the manufac-
turer’s instructions. Quantitative RT-PCR was carried
out utilizing a TaqMan low-density custom array
(Applied Biosystems, Foster City, CA, USA).
The analysis of mRNA expression was carried out with
the real-time TaqMan-PCR using a 7900HT Fast-Real-
Time-PCR System (Applied Biosystems). FAM-labeled
primers for RT-PCR (Applied Biosystems) were as follows:
Inventoried TaqMan assays for the internal control 18S
ribosomal ribonucleic acid (rRNA) (Hs99999901_s1)
and the target RNA collagen 1A1 (Hs00164004_m1).
PCR conditions were as follows: 50 C for 2 min, 94.5 C
for 10 min followed by 40 cycles at 97 C for 30 s and
59.7 C for 1 min. Real-time PCR data were analyzed
using the Sequence Detector Version 2.2.2 (Applied
Biosystems) supplied with the 7900 HT System. The
threshold cycle (Ct) for collagen 1A1 gene was normalized
to the housekeeper 18S rRNA, resulting in delta Ct-values
(DCt). To analyze relative changes in gene expression
between the treated sample and the untreated control, the
relative quantification (RQ) parameter was determined
(RQ ¼ 2ðDCt treatedDCt controlÞ ) according to the method
described by Livak and Schmittgen.13
Determination of procollagen concentration
After 18 days of incubation, gel supernatants were
collected and subsequently analyzed for procollagen
levels using the Procollagen Type I C-Peptide EIA Kit
(Takara Bio Inc., Otsu, Japan) according to the manu-
facturer’s instructions.
In vivo study
Measurements of facial topometry features after application
of a creatine-containing face-care formulation
To determine a decrease in sagging and wrinkle inten-
sity, 43 male volunteers (Caucasians, aged 32–60 years)
were enrolled in a single-center, controlled (compared to
baseline t0) study. The participants applied a formulation
containing 0.2% creatine ⁄ 0.05% creatinine for 6 weeks
to their faces, especially in the cheek and periorbital
regions. The formulation consists of a creme gel with
approximately 8% glycerol and 0.4% guarana extract
(Paullinia cupana) stabilized by a polyacrylate-based
thickener.
During the last 10 days prior to the baseline visit and
during the entire study, the volunteers were required to
desist from using special face-care and self-tanning
products in the facial area, as well as any systemic or
topical medical treatments such as retinoic acid or
corticosteroids. Volunteers were also asked to prevent a
significant weight gain or loss. During the last 5 days
275
Anti-aging effects of creatine • R I Peirano et al.
prior to the first scheduled measurement, volunteers
were required to refrain from using any skin care
products in the face including sun protecting products;
only cleansing was allowed. Visits to saunas and
swimming-pools as well as very demanding exercise
were prohibited for 1 day prior to the scheduled baseline
visit. On the days of measurements, subjects were
required to refrain from using the test samples and to
shave 2–3 h prior to measurement. Intensive sun
exposure was prohibited as well as visits to solariums.
Volunteers were asked to apply the same sample
quantity used in everyday practice (2 mg ⁄ cm2) twice
daily. After baseline measurements were taken, the
investigator demonstrated correct application of the test
formulation and trained volunteers in self-application.
Measurements were performed by trained and experi-
enced personnel after acclimatization for at least 30 min
under standard atmospheric conditions (21.5 ± 1.0 C
and 45 ± 5% relative humidity). During the visit in the
test center, the consumption of caffeine was prohibited.
The effects of treatment especially with regard to
antisagging and antiwinkling were assessed by using the
following methods:
• Phase shift rapid in vivo measuring of human skin
(PRIMOS): At baseline visit and after 4 and 6 weeks
of treatment, the volume in the jowl area was
evaluated by the enhanced PRIMOS body system (Gf
Messtechnik, Berlin, Germany) with a measuring
field of 300 · 200 lm2 as described earlier.14 The
PRIMOS system represents an established and
objective method to quantify changes in not only
skin surface roughness but also skin contours.
• Expert assessment: At the baseline visit, after 4 and
6 weeks, clinical live scoring was performed by two
clinical study experts to evaluate the skin condition
and skin textural changes such as sagging cheeks,
crow’s feet wrinkles, wrinkles under the eyes and
intensity of eye bags in the face using an 11-step
scale (scores from 0 to 5.0 in steps of 0.5). As we
focused on visible changes in eye bag intensity, we
did not use PRIMOS or other quantitative methods15
for measuring this parameter.
• Volunteer self-assessment: Volunteers completed a
questionnaire after 4 and 6 weeks of treatment
with the help of a score. The score, based upon a
five-tiered scale (from ‘‘strongly disagree’’: 1 to
‘‘strongly agree’’: 5), established the extent of
agreement with the following statements: (A) ‘‘The
product treatment noticeably improves my skin
structure,’’ (B) ‘‘The product has a noticeably
tightening effect,’’ (C) ‘‘Overall, the product
improves facial contours.’’
276
In this in vivo study, the recommendations of the
current version of the Declaration of Helsinki and the
guideline of the International Conference on Harmoni-
zation Good Clinical Practice (ICH GCP) were observed
as applicable to a nondrug study. All volunteers provided
written informed consent.
Statistics
Statistical analysis was performed with nonparametric
methods based on two-sided hypotheses using a signif-
icance level of 0.05 (alpha).
Determination of collagen 1A1 gene expression and
procollagen concentration in fibroblast-populated collagen
gels
Paired comparisons were carried out using Wilcoxon’s
signed rank test. Software MS Office Excel (2007),
Statistica V9.2 (Stat Soft Inc., Tulsa, OK, USA), was
used.
In vivo study
Comparisons regarding sagging cheek intensity, clinical
live scoring, and self-assessment were carried out using
Wilcoxon’s signed rank test. Test for relevance of the
inquired statements in self-assessment was based on the
comparison with the neutral score 3. Software MS Office
Excel (2007), sas Software Package for Windows V9.2
(SAS Institute Inc., Cary, NC, USA), was used.
Results
In vitro studies
Determination of penetration kinetics using pig skin as a
model substrate
Radioactively labeled creatine was incorporated into a
commercially available cosmetic formulation, and pene-
tration kinetics was investigated after topical application
to excised pig skin. Sixty minutes after application, the
amount of creatine absorbed through pig skin reached a
value of 52%. After 3 h of exposure, this amount
remained the same. As demonstrated in Figure 1, the
distribution of radioactively labeled creatine after an
exposure of 60 min was determined as follows: 86.5%
stratum corneum, 9% epidermis, and 4.5% dermis.
Within 3 h after application, radioactively labeled
creatine was still enriched in the stratum corneum to
79.6%, whereas the other fractions rose to 13.2%
(epidermis) and 7.1% (dermis), respectively. A small
quantity of label (0.1%) could be detected in the receptor
fluid.
 2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 273–281

Figure 1 Penetration depth of topically applied 14C-creatine
using pig skin as model substrate. Results are shown in percent
(n = 3).
Determination of collagen 1A1 gene and procollagen
expression in fibroblast-populated collagen gels following
treatment with creatine
To determine whether creatine exerts beneficial effects
on the connective tissue, collagen 1A1 mRNA expres-
sion of fibroblasts cultivated in FPCLs was assessed by
Real-Time TaqMan-PCR. Compared with untreated
controls, the resulting RQ value of 1.28 ± 0.45
(n = 13; P = 0.0107, Figure 2) revealed a significant
increase after stimulation with creatine. This enhanced
gene expression resulted in an elevated protein level as
well. As shown in Figure 3, compared with the
Figure 2 Relative collagen 1A1 gene expression (RQ) of cells
cultivated in fibroblast-populated collagen gels after supplemen-
tation with creatine relative to nontreated cells. Results are
shown as mean ± SD (n = 13). Significant differences are marked
with an asterisk (*P £ 0.05).
 2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 273–281
Anti-aging effects of creatine • R I Peirano et al.
untreated control (set as 100%), procollagen secretion
by fibroblasts cultivated in FPCL was significantly
enhanced following treatment with creatine
(109.7 ± 12.0%; n = 13; P = 0.0128). With the posi-
tive control, we found the expected strong stimulation of
collagen expression and procollagen secretion, namely
RQ = 4.6 ± 2.0% and 449 ± 204%, respectively.
In vivo study
Improvement in facial topometry features after application of
a creatine-containing face-care formulation
The PRIMOS measurements showed that treatment with
the creatine-containing test formulation resulted in a
significantly decreased volume in the jowl area com-
pared to that of baseline values. After 4 weeks of
treatment, a significant decrease in volume in the
treated jowl area by )0.21 ± 0.37 mL (P = 0.0000,
n = 40) was detected compared to the volume deter-
mined at the baseline visit (Fig. 4). After 6 weeks of
treatment, this significant volume reduction was
determined as )0.27 ± 0.46 mL (P = 0.0000, n = 39)
compared to that at baseline.
As demonstrated in Figure 5, after 6 weeks of treat-
ment with the creatine-containing formulation, clinical
live scoring revealed a statistically significant improve-
ment in all parameters investigated compared to those at
baseline, such as wrinkles under the eye, crow’s feet
wrinkles, as well as sagging cheek, and eye bag intensity
(Table 1).
As the study was conducted to determine improve-
ments in facial skin contours volunteers carried out a
Figure 3 Determination of procollagen protein concentration in
fibroblast-populated collagen lattice gel supernatants after treat-
ment with creatine. Results are depicted as mean ± SD (n = 13)
relative to the untreated control (set as 100%). Significant dif-
ferences are marked with an asterisk (*P £ 0.05).
277
Anti-aging effects of creatine • R I Peirano et al.

Table 1 Clinical live scoring after treatment with a creatine-

containing formulation (P-value determined relative to t0; only
depicted, when P < 0.05)

Criteria t0 baseline t1 4 weeks t2 6 weeks

Sagging cheek
n 43 43 42
mean ± SD 2.26 ± 0.54 2.08 ± 0.55 1.93 ± 0.54
P = 0.0057 P = 0.0000
Crow’s feet winkles
n 40 40 40
mean ± SD 2.06 ± 1.02 1.86 ± 0.91 1.63 ± 0.95
P = 0.0000
Wrinkles under the eye
n 42 42 41
Figure 4 Relative change in volume in the jowl area after mean ± SD 3.05 ± 1.10 2.93 ± 1.03 2.77 ± 1.12
treatment with a creatine-containing formulation. Results are P = 0.0244
depicted as mean topometrical volume difference to t0 mL ±SD Intensity of eye bags
(n = 40 and 39, respectively). Significant differences ti ) t0 are n 33 33 33
marked with an asterisk (*P £ 0.05). mean ± SD 1.94 ± 1.35 1.81 ± 1.21 1.31 ± 0.99
P = 0.0005

Figure 5 Clinical live scoring performed by two experts after

treatment with a creatine-containing formulation regarding
changes in sagging cheeks, crow’s feet wrinkles, wrinkles under
the eyes and intensity of eye bags in the face. Results are shown
as mean score ±SD (n = 33–43). Significant differences ti ) t0 are
marked with an asterisk (*P £ 0.05).
self-assessment after 4 (t1) and 6 (t2) weeks of treatment.
As shown in Figure 6, the mean score achieved by
statement (A) ‘‘The product treatment noticeably
improves my skin structure’’ was 3.63 ± 1.05
(P = 0.0009, n = 43) after 4 weeks and 3.76 ± 1.09
(P = 0.0001, n = 41) after 6 weeks of treatment. State-
ment (B) ‘‘The product has a noticeably tightening
effect’’ received the following mean scores: 3.65 ± 0.90
(t1, P = 0.0014, n = 43) and 3.71 ± 1.02 (t2,
P = 0.014, n = 42), whereas the mean score regarding
statement (C) ‘‘Overall, the product improves facial
contours’’ improved from 3.67 ± 0.97 (t1, P = 0.0001,
n = 43) to 3.74 ± 1.08 (t2, P = 0.0028, n = 42). All
Figure 6 Self-assessment of volunteers with respect to efficacy of
the creatine-containing formulation after 4 and 6 weeks of
treatment. Shown are approval rates as mean ± SD based upon a
five-tiered scale with regard to statements (A) ‘‘The product
treatment noticeably improves my skin structure,’’ (B) ‘‘The
product has a noticeably tightening effect’’ and (C) ‘‘Overall, the
product improves facial contours.’’
three statements were confirmed with statistical evi-
dence after 4 and 6 weeks of use.
Results also demonstrated that the creatine-contain-
ing formulation was tolerated very well by the study
population over the entire duration of usage as no
incompatibility reactions were observed.
Discussion
Despite the existence of many scientific aging theories,
the exact mechanisms leading to age-related skin

278  2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 273–281

alterations are not comprehensively understood. One
prominent hypothesis focuses on the deterioration of the
dermal ECM because of extrinsic influences such as UV
irradiation. The amount of inflicted damage is dependent
on the incidence and intensity of UV exposition as well
as the efficiency of intrinsic repair processes. The ability
to repair UV-induced damage of cellular and extracellu-
lar components, and in this course to combat structural
dermal degeneration, is highly dependent on cellular
energy levels. If, owing to an insufficient energy supply,
repair mechanisms cannot keep pace, detrimental
changes in skin structure can occur, leading to the
visible signs of aging. Imperfect repair processes accel-
erate skin aging, which consequently leads to the further
decrease in cutaneous cellular energy levels. Out of this
reason, it is believed that declining mitochondrial
function and energy production can be considered both
a cause and effect of aging.16–19 The maintenance of a
sustained supply of cutaneous cellular energy in the
form of creatine may be a strategy to break this vicious
circle and decelerate, stop, or even reverse skin aging.20
Creatine is known to play a crucial role in the body’s
energy supply and influences the energy metabolism of
cutaneous cells. But skin cells may show signs of reduced
substrate levels for creatine, presumably caused by a
stress and age-related decrease of dermal vasculariza-
tion.21 Lenz et al. 10 demonstrated that the epidermal
creatine system deteriorates under oxidative stress
conditions as well as during skin aging. A reactivation
of the creatine system was achieved by the addition of
creatine to human keratinocytes in vitro. Clinical trials
investigating the exogenous supplementation of human
epidermal cells with creatine in a topical formulation
revealed protective effects against UV-induced dam-
age.10 Also, after 4 weeks of topical application, skin
sites treated with creatine exhibited a significant
increase in papillary density.22
Based on these previous findings, we speculated that a
creatine supplementation might be able to balance
energy deficits in human dermal cells and consequently
improve the structure of the ECM by increasing collagen
synthesis. The overall objective of the present study was
to stimulate collagen neo-synthesis, counteract wrinkle
formation and the loss of skin elasticity and resilience,
by reinforcing the endogenous energy levels with
topically supplied creatine. To put this approach into
practice, we investigated the effects of a creatine-
induced stimulation of dermal collagen synthesis
in vitro with a special focus on the improvement of skin
firmness in vivo.
To be able to affect the cellular energy metabolism and
induce collagen synthesis, creatine needs to penetrate
 2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 273–281
Anti-aging effects of creatine • R I Peirano et al.
the skin to reach dermal fibroblasts. As our data showed
that topically applied radioactively labeled creatine
efficiently and rapidly penetrated pig skin, leading to
elevated creatine levels in the dermis already 60 min
after application. The ex vivo ⁄ in vitro pig skin model is a
standard assay for the estimation of percutaneous
penetration of topically applied cosmetic and pharma-
ceutical ingredients showing a good agreement with
skin penetration data on human skin.23 As previously
demonstrated, once in the dermis, creatine is immedi-
ately (within minutes) taken up by fibroblasts via a
cutaneous creatine transport system.22
To elucidate the beneficial effects of creatine on
collagen synthesis in more detail, we investigated
creatine-induced cell-matrix interactions in a three-
dimensional context utilizing the attached FPCL as a
dermal tissue equivalent.24 As in this experimental
approach, fibroblasts are present in the synthetically
active form,25 the attached FPCL is a tool to investigate
the modulation of ECM synthesis and remodeling.
Human dermal fibroblasts cultured in this gel matrix
in the presence of creatine showed a significant increase
in collagen 1A1 gene expression. Considering the age-
dependent reduction in collagen gene expression,26,27
these data suggest that creatine stimulates collagen
synthesis in the dermal ECM. It has to be considered,
however, that collagen synthesis represents a multistage
process and may be influenced at different regulatory
levels. Out of this reason, we supplemented FPCLs with
creatine and determined the levels of procollagen
protein. In this experimental setting, creatine signifi-
cantly increased also collagen neo-genesis, underlining
the fact that a stabilized energy supply elicits beneficial
effects on collagen gene expression and protein levels as
well. In comparison with creatine, the collagen stimu-
lation by TGF-b was much more pronounced. However,
the in vitro effects of creatine were statistically signifi-
cant. And furthermore, when creatine is topically
applied for an extended period of time (4 weeks), we
found a tendency toward elevated procollagen levels in
suction blister fluids, as shown in an ex vivo study
performed with 24 elderly volunteers (increase of 36%,
data not shown).
It is also of interest to note that Berneburg et al. 28
showed that in cultured fibroblasts, creatine counter-
acted the enzymatic degradation of collagen by matrix
metalloproteinase-1, further hinting to a role of creatine
in the dermal collagen metabolism. With respect to the
in vivo efficacy, our next aim was to address the question
whether creatine improves the firmness of the connec-
tive tissue, ideally by stimulating and regenerating the
collagen network.
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Anti-aging effects of creatine • R I Peirano et al.
After conducting in vitro studies using fibroblasts
obtained from female donors, we now selected a male
population of Caucasians, investigating the role of
creatine in the improvement of antisagging and anti-
wrinkle efficacy in male skin in vivo. It is well established
that men’s and women’s skin physiology can differ in
hormone metabolism, hair growth, sweat rate, sebum
production, surface pH and fat accumulation, to name a
few characteristics.29
To ensure the stability of pure creatine in a cosmetic
formulation, a special technology was used, because
creatine undergoes transformation to creatinine, if not
stabilized properly in an aqueous system. To maintain a
chemically balanced quantity of highly reactive
creatine, it is crucial to sustain an optimized crea-
tine ⁄ creatinine ratio in this formulation. Out of this
reason, traces of its decomposition product creatinine
were added, mimicking the natural balance occurring in
the human body. Additionally, to generate an optimal
formulation especially designed for male skin care, a
natural guarana extract (Paullinia cupana) was incorpo-
rated into the formulation, as this extract has been
shown to exert vitalizing effects upon male skin fibro-
blasts (23% higher cellular esterase activity, data not
shown).
This special creatine-containing formulation was
tested in a controlled study in which male volunteers
applied the formulation twice daily for 6 weeks on the
face. As our results obtained by in vivo topometry
demonstrated that the sagging cheek intensity in the
jowl area was significantly reduced by the creatine-
containing formulation, as compared to baseline. These
data are supported by results from clinical live scoring
indicating significant reduction in the sagging cheek
intensity in the jowl area, and also in textural changes
such as a reduced intensity of crow’s feet wrinkles,
wrinkles under the eyes and eye bags. The investigation
of eye bag formation was chosen as a parameter, as skin
laxity is one of the factors contributing to eye bag
formation.30 To assess the extent of agreement to
various efficacy statements, volunteers completed a
self-assessment questionnaire that further corroborated
these findings. Furthermore, the antiwrinkle data are in
line with the result of an earlier vehicle-controlled study
that showed a wrinkle-reducing effect of a creatine-
containing formulation after 2 months of application
tested on 30 female volunteers.22
As the focus of this in vivo study was to detect and
ensure optimal consumer perception of benefits achieved
by our treatment, the study was controlled with com-
parison to baseline t0. To further prove that the effects of
our creatine-containing formulation can be attributed to
280
creatine itself, guarana was also tested for collagen
induction in vitro, but no stimulation was found.
Moreover, glycerol as a humectant might have an acute
swelling effect that could influence the measurements on
wrinkle intensity. However, this is unlikely, because the
volunteers did not apply the formulation on the day of
the measurement. Furthermore, all of our in vivo
parameters, such as wrinkle intensity, are not subject
to seasonal variations as shown recently by Qiu et al. 31
and therefore should not change over the study treat-
ment time of 6 weeks. To summarize, the only ingredi-
ent in the cosmetic formulation used showing a proven
collagen stimulation is creatine, which makes it likely,
that the observed antiwrinkle and firming effects are
based on this action. A recent publication also provided
new data on the interrelationship between elastic fibers
and wrinkle formation.32 However, we could not find a
stimulation of tropoelastin gene expression by creatine
in our in vitro test system (data not shown).
In summary, the results presented here demonstrate
that application of creatine led to substantial stimulation
of collagen synthesis, ultimately resulting in a noticeable
reduction in skin textural changes in vivo. Our data
underline that creatine is a beneficial active ingredient
for topical use and helps to diminish the signs of human
skin aging.
Acknowledgments
We kindly thank Mrs Marie-Christine Leneveu-
Duchemin for conducting expert statistical analysis
and Mrs Heike Diekmann for excellent technical
assistance.
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