Epidermal and Dermal Characteristics

in Skin Equivalent after Systemic and

Topical Application of Skin Care
Ingredients
JANA VICANOVA,a CHARBEL BOUEZ,b SOPHIE LACROIX,b
LARS LINDMARK,c AND ODILE DAMOURb
a DermData

s.r.o, Czech Republic

b Banque

de Tissus et Cellules, Hopital Edouard Herriot,

Hospices Civils de Lyon, Lyon, France

c Imedeen

Research, Ferrosan, Denmark

ABSTRACT: Effects of active ingredients from topical and systemic skincare products on structure and organization of epidermis, dermal
epidermal junction (DEJ), and dermis were examined using an in vitro
reconstructed skin equivalent (SE). Imedeen Time Perfection (ITP) ingredients (a mixture of BioMarine Complex, grape seed extract, tomato
extract, vitamin C) were supplemented systemically into culture medium.
Kinetin, an active ingredient from Imedeen Expression Line Control
Serum, was applied topically. Both treatments were tested separately or
combined. In epidermis, all treatments stimulated keratinocyte proliferation, showing a significant increase of Ki67-positive keratinocytes (P <
0.05). Kinetin showed a twofold increase of Ki67-positive cells, ITP resulted in a fivefold, and ITP+kinetin showed a nine-fold increase. Differentiation of keratinocytes was influenced only by kinetin since filaggrin
was found only in kinetin and kinetin+ITP samples. At the DEJ, laminin
5 was slightly increased by all treatments. In dermis, only ITP increased
the amount of collagen type I. Both kinetin and ITP stimulated formation of fibrillin-1 and elastin deposition. The effect of kinetin was seen in
upper dermis. It stimulated not only the amount of deposited fibrillin-1
and elastin fibers but also their organization perpendicularly to the DEJ.
ITP stimulated formation of fibrillin-1 in deeper dermis. In summary,
the combination of topical treatment with kinetin and systemic treatment with ITP had complementary beneficial effects in the formation
and development of epidermis and dermis.
KEYWORDS: Imedeen; Mimeskin; skin equivalent; keratinocyte proliferation; dermal matrix; kinetin
Address for correspondence: Jana Vicanova, Ph.D., Osvobozeni 920, 273 51 Unhost, Czech Republic.
Voice: +420 603 468 949; fax: +420 312 699 236.
e-mail: javi@ferrosan.com
C 2006 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1067: 337342 (2006). 
doi: 10.1196/annals.1354.046

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Skin aging is associated with changes of skin structure and skin cell activities. The most prominent modifications were found in dermis, but changes
were also observed at the dermalepidermal junction (DEJ) and in epidermis.1
In recent years, a search for active ingredients that would prevent, counteract,
or reverse degenerative changes in skin aging has been increasing. Among
available testing systems, in vitro reconstructed three-dimensional skin equivalent model (SE) Mimeskin proved to be innovative and relevant in testing
cosmetic products.24
This study was aimed to examine the effect of systemic treatment with
Imedeen Time Perfection active ingredients (ITP) and of topical treatment
with kinetin, an active ingredient present in Imedeen Expression Line control
serum, using SE. In epidermis, the investigations focused on potential effect
on keratinocyte proliferation (Ki67) and differentiation (filaggrin). In dermis,
amount and localization of deposited dermal components such as fibrillin-1,
elastin, and collagen type I were examined.

EXPERIMENTAL METHODS
The SE Mimeskin was prepared as described previously.5 Dermal equivalents were prepared by adding a suspension of 200,000 fibroblasts/cm2 originating from healthy 42-year-old female skin on the top of the collagen
glycosaminoglycanchitosan porous sponge Mimedisk (Coletica, France).
All equivalents were cultured for 21 days in fibroblast medium (Dulbeccos
Modified Eagles Medium (DMEM with Glutamax-1, Invitrogen, Cergy
Pontoise, France) with 10% calf serum (HyClone, Logan, UT), 20 g/mL
gentamicin (Panpharma, Foug`eres, France), 100 IU/mL penicillin (Sarbach,
Suresnes, France), 1 g/mL amphotericin B (Bristol Myers Squibb, Puteaux,
France), and 10 g/mL L-ascorbic acid 2-phosphate (Sigma, St. Quentin
Fallavier, France). The medium was changed daily.
Keratinocytes (250,000 cells/cm2 ) were seeded on the top of dermal equivalent at day 14. After 7 days of submerged culture in the keratinocyte medium
[3:1 mixture of DMEM and Hams F12 (Invitrogen), with10% calf serum
(HyClone), 10 ng/mL epidermal growth factor (EGF) (Austral Biologic, San
Ramon, CA), 0.12 IU/mL insulin (Lilly, Saint-Cloud, France), 0.4 g/mL
hydrocortisone (UpJohn, St. Quentin en Yvelines, France), 5 g/mL triiodoL-thyronine (Sigma), 24.3 g/mL adenine (Sigma), and antibiotics as above],
the SE were elevated at the airliquid interface and cultured in a simplified
keratinocyte medium containing DMEM supplemented with 10% calf serum,
10 ng/mL EGF, 0.12 IU/mL insulin, 0.4 g/mL hydrocortisone, antibiotics,
and 10 g/mL L-ascorbic acid, and were changed daily.
ITP ingredients were from Ferrosan A/S (Denmark). The final concentrations in medium were: sodium ascorbate (5 g/mL), grape seed extract
(GE; 2.5 g/mL), tomato extract (TE; 5 g/mL), and BioMarine ComplexTM
(BMC; 35 g/mL). All ingredients were water-soluble except tomato extract,

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which was dissolved in tetrahydrofuran (THF). ITP was added from the time
of the first culture medium change until harvesting. Kinetin was obtained from
Senetek (USA). Solution of 0.1% kinetin in ketrol was applied topically from
day 28 (after 1 week of air-exposed epidermis) and reapplied every second
day. Final samples were collected at day 49. The culture conditions were: (i)
kinetin, (ii) ITP, (iii) ITP+kinetin, (iv) control with systemic THF and topical
ketrol, and (v) control without any treatment.
Three samples of material at each condition were fixed in 4% paraformaldehyde for histologic and immunohistochemical study, dehydrated, and embedded in paraffin. Three others were embedded in OCT Tissue-Tek (Miles,
Immunotech, Marseille, France). Four histologic vertical 5-m sections were
stained with hematoxylinphloxinsaffron.
For immunohistochemical study, 6-m sections were deparaffinized and
whitened in glycineHCl (100 mmol/L). The antibody was directed against
Ki67 (monoclonal, raised in mouse, dilution 1:50, DAKO, Glostrup, Denmark). Peroxydase-conjugated goat anti-mouse IgG (1:50 dilution, Santa Cruz
Biotechnology, Santa Cruz, CA) was used to detect the immune complexes using diaminobenzidine as substrate (DAKO). Counterstaining was performed
using Harris hematoxylin. For controls, the primary antibody was omitted.
Multiple serial sections of each specimen were processed to ensure representative samples. The number of Ki67-positive cells is expressed as percentage
of total cell count in a field of 100 cells. Four fields were scored per sample.
For statistical analysis, normality test and Mann-Whitney rank sum test were
used for multiple comparison versus control group.
For immunofluorescence, 6-m frozen sections were air dried, blocked in
phosphate buffered saline solution containing 1% (wt/vol) bovine serum albumin. Antibodies were directed against human elastin (polyclonal, raised in
rabbit, 1:150 dilution, Novotec), human type I collagen (polyclonal, raised in
rabbit, 1:40 dilution, Tebu Bio, Le Perray, France), human filaggrin (monoclonal, raised in mouse; 1:100 dilution, Biomedical Technologies, Stoughton,
MA), and human fibrillin-1 (monoclonal, raised in mouse, dilution 1:50, Interchim, Neomarkers, France). Secondary antibodies, either anti-rabbit IgG
(1:50 dilution, Sanofi Diagnostics Pasteur Chaska, MN) or goat anti-mouse
IgG (1:50 dilution, Santa Cruz Biotechnology), labeled with FITC, were mixed
with 0.1 % Evans Blue to reduce non-specific staining of the sponge network.
For controls, the primary antibody was omitted. The type I collagen antibodies
specific for human collagen do not cross-react with the bovine collagen that
was used to prepare the dermal substrate.6

RESULTS
Effects of ITP, kinetin, and their combination on selected epidermal and
dermal markers are summarized in TABLE 1.

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TABLE 1. Overall grading of epidermal and dermal markers in skin equivalent after
treatment with ITP, kinetin, and ITP+kinetin

Epidermis
Ki67-positive keratinocytes
Filaggrin
Basement membrane
Laminin
Dermis
Fibrillin-1
Elastin
Collagen type I

Control

Kinetin

ITP

ITP+Kinetin

+
0

++
++

+++
0

+++++
++

++

++

++

+
+
++

+++
++
++

++
++++
++++

++++
++++
++++

NOTE: The individual markers were visualized by immunofluorescence and immunohistochemical

techniques and evaluated according to semiquantitative scale. 0 = no effect, absent; + = very low;
++ = low; +++ = moderate; ++++ = high; +++++ = very high.

In epidermis, all treatments stimulated keratinocyte proliferation, showing a

significant increase of Ki67-positive keratinocytes (P < 0.05). Kinetin showed
a twofold increase of Ki67-positive cells, ITP resulted in a fivefold increase,
and ITP+kinetin showed a ninefold increase (FIG. 1).
Differentiation of keratinocytes was influenced only by kinetin since fillagrin was found only in kinetin and kinetin+ITP samples (TABLE 1). At the
dermalepidermal junction (DEJ), laminin 5 was slightly increased by all treatments. In dermis, only ITP increased the amount of collagen type I. Both kinetin
and ITP stimulated formation of fibrillin and elastin deposition. The effect of
kinetin was seen in upper dermis. Kinetin stimulates not only the amount but
also the organization of fibrillin-1 and elastin fibers perpendicularly to the
DEJ. ITP stimulated formation of fibrillin in deeper dermis.

DISCUSSION
The results of this study confirmed a strong effect of ITP actives on formation
and deposition of extracellular matrix components such as collagen type I and
fibrillin-1 in reconstructed Skin Equivalent (SE) reported previously.7 In vivo
human studies confirmed that the effect observed in vitro leads to clinically
visible improvement of aging skin structure.8,9
In this study, we investigated, for the first time, the effect of ITP on epidermal
keratinocytes, which showed a stimulation of basal cell proliferation. It is
well known that cell proliferation decreases with aging. Such an effect can be
mimicked in SE using cells derived from young and aged donors. Stimulation
of Ki67 expression in suboptimally proliferating SE generated from aged cells
suggests a potential for the tested ingredients to optimize epidermal turnover
in vivo.
To further enhance positive effects on aging skin tissue, another active,
kinetin, was applied topically in combination with systemic supplementation

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FIGURE 1. Number of Ki67-positive keratinocytes in skin equivalent after treatment

with ITP, kinetin, and ITP+kinetin. The number of Ki67-positive cells is expressed as
percentage of total cell count in a field of 100 cells. Four fields were scored per sample. For
statistical analysis, normality test and Mann-Whitney rank sum test were used for multiple
comparison versus control group.

of ITP. Kinetin is a cytokinin that displays a variety of biological effects,

including those on cell proliferation and anti-aging effects.10,11 Human trial
on volunteers with moderate signs of skin aging demonstrated objective and
statistically significant improvements in several parameters after topical use
of kinetin.12 In our study, kinetin alone influenced keratinocyte proliferation
and differentiation as well as formation of basement membrane and elastic
network in the upper dermis. Combined treatment of kinetin and ITP seemed
to reinforce the effects of each other regarding keratinocyte proliferation and
elastic network formation.
In conclusion, treatment with topical kinetin and systemic ITP showed multiple effects on development and organization of epidermis and dermis. ITP
and kinetin have complementary beneficial effects on the formation and maintenance of healthy skin tissue.

ACKNOWLEDGMENTS
We thank Sandrine Vidal for her expert technical assistance.
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