Epidermal and Dermal Cha

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Epidermal and Dermal Characteristics

in Skin Equivalent after Systemic andTopical Application of Skin CareIngredients

JANA VICANOVA,a CHARBEL BOUEZ,b SOPHIE LACROIX,b

LARS LINDMARK,c AND ODILE DAMOUR b

a DermData s.r.o, Czech Republic

b Banque de Tissus et Cellules, H ̂ opital Edouard Herriot, Hospices Civils de Lyon, Lyon, France

c Imedeen Research, Ferrosan, Denmark

ABSTRACT: Effects of active ingredients from topical and systemic skin-care products on structure and organization of epidermis, dermal– epidermal junction (DEJ), and dermis were examined using an in vitroreconstructed skin equivalent (SE). Imedeen Time Perfection (ITP) in-

gredients (a mixture of BioMarine Complex, grape seed extract, tomatoextract, vitamin C) were supplemented systemically into culture medium.Kinetin, an active ingredient from Imedeen Expression Line ControlSerum, was applied topically. Both treatments were tested separately orcombined. In epidermis, all treatments stimulated keratinocyte prolifer-ation, showing a significant increase of Ki67-positive keratinocytes ( P <0.05). Kinetin showed a twofold increase of Ki67-positive cells, ITP re-sulted in a fivefold, and ITP+kinetin showed a nine-fold increase. Differ-entiation of keratinocytes was influenced only by kinetin since filaggrinwas found only in kinetin and kinetin+ITP samples. At the DEJ, laminin5 was slightly increased by all treatments. In dermis, only ITP increasedthe amount of collagen type I. Both kinetin and ITP stimulated forma-tion of fibrillin-1 and elastin deposition. The effect of kinetin was seen inupper dermis. It stimulated not only the amount of deposited fibrillin-1and 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 treat-ment with ITP had complementary beneficial effects in the formationand development of epidermis and dermis.

K EYWORDS: Imedeen; Mimeskin; skin equivalent; keratinocyte prolifer-

ation; dermal matrix; kinetin


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338 ANNALS NEW YORK ACADEMY OF SCIENCES

Skin aging is associated with changes of skin structure and skin cell activ-ities. The most prominent modifications were found in dermis, but changes

were also observed at the dermal–epidermal 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. Amongavailable testing systems, in vitro reconstructed three-dimensional skin equiv-alent model (SE) Mimeskin proved to be innovative and relevant in testingcosmetic products.2 – 4

This study was aimed to examine the effect of systemic treatment withImedeen Time Perfection active ingredients (ITP) and of topical treatmentwith kinetin, an active ingredient present in Imedeen Expression Line control

serum, using SE. In epidermis, the investigations focused on potential effecton 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 equiva-lents were prepared by adding a suspension of 200,000 fibroblasts/cm2 orig-

inating from healthy 42-year-old female skin on the top of the collagen– glycosaminoglycan–chitosan porous sponge Mimedisk (Coletica, France).All equivalents were cultured for 21 days in fibroblast medium (Dulbecco’sModified Eagle’s Medium (DMEM with Glutamax-1, Invitrogen, CergyPontoise, France) with 10% calf serum (HyClone, Logan, UT), 20 g/mLgentamicin (Panpharma, Fougères, 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. QuentinFallavier, France). The medium was changed daily.

Keratinocytes (250,000 cells/cm2) were seeded on the top of dermal equiv-alent at day 14. After 7 days of submerged culture in the keratinocyte medium[3:1 mixture of DMEM and Ham’s F12 (Invitrogen), with10% calf serum(HyClone), 10 ng/mL epidermal growth factor (EGF) (Austral Biologic, SanRamon, CA), 0.12 IU/mL insulin (Lilly, Saint-Cloud, France), 0.4 g/mLhydrocortisone (UpJohn, St. Quentin en Yvelines, France), 5 g/mL triiodo-L-thyronine (Sigma), 24.3 g/mL adenine (Sigma), and antibiotics as above],the SE were elevated at the air–liquid 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.


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VICANOVA et al.: IMEDEEN IN SKIN EQUIVALENT 339

which was dissolved in tetrahydrofuran (THF). ITP was added from the timeof the first culture medium change until harvesting. Kinetin was obtained from

Senetek (USA). Solution of 0.1% kinetin in ketrol was applied topically fromday 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 topicalketrol, and (v) control without any treatment.

Three samples of material at each condition were fixed in 4% paraformalde-hyde for histologic and immunohistochemical study, dehydrated, and embed-ded in paraffin. Three others were embedded in OCT Tissue-Tek (Miles,Immunotech, Marseille, France). Four histologic vertical 5-m sections were

stained with hematoxylin–phloxin–saffron.For immunohistochemical study, 6-m sections were deparaffinized and

whitened in glycine–HCl (100 mmol/L). The antibody was directed againstKi67 (monoclonal, raised in mouse, dilution 1:50, DAKO, Glostrup, Den-mark). Peroxydase-conjugated goat anti-mouse IgG (1:50 dilution, Santa CruzBiotechnology, Santa Cruz, CA) was used to detect the immune complexes us-ing 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 represen-

tative samples. The number of Ki67-positive cells is expressed as percentageof 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 wereused 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 al- bumin. Antibodies were directed against human elastin (polyclonal, raised inrabbit, 1:150 dilution, Novotec), human type I collagen (polyclonal, raised inrabbit, 1:40 dilution, Tebu Bio, Le Perray, France), human filaggrin (mono-

clonal, raised in mouse; 1:100 dilution, Biomedical Technologies, Stoughton,MA), and human fibrillin-1 (monoclonal, raised in mouse, dilution 1:50, In-terchim, Neomarkers, France). Secondary antibodies, either anti-rabbit IgG(1:50 dilution, Sanofi Diagnostics Pasteur Chaska, MN) or goat anti-mouseIgG (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 antibodiesspecific for human collagen do not cross-react with the bovine collagen thatwas used to prepare the dermal substrate.6


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TABLE 1. Overall grading of epidermal and dermal markers in skin equivalent after

treatment with ITP, kinetin, and ITP+kinetin

Control Kinetin ITP ITP+KinetinEpidermis

Ki67-positive keratinocytes + ++ +++ +++++Filaggrin 0 ++ 0 ++

Basement membraneLaminin + ++ ++ ++

DermisFibrillin-1 + +++ ++ ++++Elastin + ++ ++++ ++++Collagen type I ++ ++ ++++ ++++

NOTE: The individual markers were visualized by immunofluorescence and immunohistochemicaltechniques 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 asignificant 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 fil-

lagrin was found only in kinetin and kinetin+ITP samples (TABLE 1). At thedermal–epidermal junction (DEJ), laminin 5 was slightly increased by all treat-ments. In dermis, only ITP increased the amount of collagen type I. Both kinetinand ITP stimulated formation of fibrillin and elastin deposition. The effect of kinetin was seen in upper dermis. Kinetin stimulates not only the amount butalso the organization of fibrillin-1 and elastin fibers perpendicularly to theDEJ. ITP stimulated formation of fibrillin in deeper dermis.

DISCUSSION

The results of this study confirmed a strong effect of ITP actives on formationand deposition of extracellular matrix components such as collagen type I and fibrillin-1 in reconstructed Skin Equivalent (SE) reported previously.7 In vivohuman studies confirmed that the effect observed in vitro leads to clinicallyvisible improvement of aging skin structure.8,9

In this study, we investigated, for the first time, the effect of ITP on epidermalkeratinocytes, which showed a stimulation of basal cell proliferation. It is

well known that cell proliferation decreases with aging. Such an effect can bemimicked in SE using cells derived from young and aged donors. Stimulationof Ki67 expression in suboptimally proliferating SE generated from aged cells


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VICANOVA et al.: IMEDEEN IN SKIN EQUIVALENT 341

FIGURE 1. Number of Ki67-positive keratinocytes in skin equivalent after treatmentwith ITP, kinetin, and ITP+kinetin. The number of Ki67-positive cells is expressed as

percentage of total cell count in a f ield of 100 cells. Four fields were scored per sample. For statistical analysis, normality test and Mann-Whitney rank sum test were used for multiplecomparison 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 trialon volunteers with moderate signs of skin aging demonstrated objective and statistically significant improvements in several parameters after topical useof kinetin.12 In our study, kinetin alone influenced keratinocyte proliferationand differentiation as well as formation of basement membrane and elasticnetwork 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 mul-

tiple effects on development and organization of epidermis and dermis. ITPand kinetin have complementary beneficial effects on the formation and main-tenance of healthy skin tissue.

ACKNOWLEDGMENTS

We thank Sandrine Vidal for her expert technical assistance.


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4. SCHLOTMANN, K., M. K AETEN, A.F. BLACK , et al . 2001. Cosmetic efficacy claimsin vitro using a 3D human skin model. Int. J. Cosmet. Sci. 23: 309–318.

5. DUPLAN-PERRAT,F.,O.DAMOUR , C . MONTROCHER , etal . 2000. Keratinocytes influ-ence the maturation and organization of the elastin network in a skin equivalent.J. Invest. Dermatol. 114: 365–370.

6. NOBLESSE, E., V. CENIZO, C. BOUEZ, etal . 2004. Lysyl oxidase-like and lysyl oxidaseare present in the dermis and epidermis of a skin equivalent and in human skinand are associated to elastic fibers. J. Invest. Dermatol. 122: 621–630.

7. BOUEZ,C.,O.DAMOUR & J . VICANOVA. 2003. Improvement of dermal extracellular

matrix structure and composition after treatment with Imedeen Time Perfectionin an in vitro skin equivalent. Poster exhibited at meeting of International Societyfor Bioengineering and International Society for Skin Imaging. May, Hamburg,Germany.

8. K IEFFER , M.E. & J. EFSEN. 1998. Imedeen in the treatment of photoaged skin: anefficacy and safety trial over 12 months. J. Eur. Acad. Dermatol. Venereol. 11:129–136.

9. SIGLER , M.L. & P. R ASMUSSEN. 2003. A placebo controlled study of an oral supple-ment (Imedeen Time Perfection) in improving the appearance of photodamaged skin. Poster exhibited at Meeting of European Academy of Dermato Venereology.

October, Barcelona, Spain.10. R ATTAN, S. & B. CLARK . 1994. Kinetin delays the onset of ageing characteristics

in human fibroblasts. Biochem. Biophys. Res. Commun. 201: 665–672.11. BARCISZEWSKI, J., S. R ATTAN, G . SIBOSKA, et al . 1999. Kinetin—45 years on. Plant

Sci. 148: 37–45.12. MCCULLOUGH, J.L. & G.D. WEINSTEIN. 2002. Clinical study of safety and efficacy

of using topical kinetin 0.1% (Kinerase) to treat photodamaged skin. Cosmet.Dermatol. 15: 29–32.


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Epidermal and Dermal Cha