Biogerontology

, 8:97 | Cite as

Supplementation with a complex of active nutrients improved dermal and epidermal characteristics in skin equivalents generated from fibroblasts from young or aged donors

  • Sophie Lacroix
  • Charbel Bouez
  • Sandrine Vidal
  • Valérie Cenizo
  • Corinne Reymermier
  • Virginie Justin
  • Jana Vičanová
  • Odile Damour
Research Article

Abstract

Cultured skin equivalent (SE, Mimeskin®) was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan dermal substrate. In order to examine donor age effect, fibroblasts from 19- (young) or 49- (aged) year-old females were used. Culture medium was supplemented with nutrients complex containing soy extract, tomato extract, grape seed extract, white tea extract, sodium ascorbate, tocopherol acetate, zinc gluconate and BioMarine™ complex. Epidermal and dermal structure and composition were examined after 42 and 60 days of culture. In untreated samples, SE generated from young fibroblasts was superior to SE from aged fibroblasts in all characteristics. Those include number and regularity of keratinocyte layers, number of keratinocytes expressing proliferation marker Ki67, content of collagen type I, fibrillin-1, elastin, and SE lifespan. Effects of nutritional supplementation were observed in SE from both young and aged fibroblasts, however, those effects were more pronounced in SE from aged fibroblasts. In epidermis, the treatment increased number of keratinocyte layers and delayed epidermal senescence. The number of cells expressing Ki67 was nine folds higher than those of controls, and was similar to that of young cell SE. In dermis, the treatment increased mRNA synthesis of collagen I, fibrillin-1 and elastin. In conclusion, skin cell donor age had major important effect on formation of reconstructed SE. Imperfections in epidermal and dermal structure and composition as well as life span in SE from aged cells can be improved by supplementation with active nutrients.

Keywords

Ageing Dermis Fibroblast Imedeen In vitro Mimeskin Keratinocyte Skin equivalent 

Abbreviations

ECM

Extracellular matrix

EGF

Epidermal growth factor

DMEM

Dulbecco’s modified Eagle’s medium

GAG

Glycosaminoglycan

IPRC

Complex of active ingredients from Imedeen Prime Renewal™

RT-PCR

Reverse transcription polymerase chain reaction

SE

Skin equivalent

References

  1. Ahmad N, Katiyar SK, Mukhtar H (2001) Antioxidants in chemoprevention of skin cancer. Curr Probl Dermatol 29:128–139PubMedCrossRefGoogle Scholar
  2. Augustin C, Frei V, Perrier E, Huc A, Damour O (1997) A skin equivalent model for cosmetological trials: an in vitro efficacy study of a new biopeptide. Skin Pharmacol 10:63–70PubMedCrossRefGoogle Scholar
  3. Augustin C, Frei V, Perrier E, Huc A, Damour O (1997) An in vitro selection of new cosmetic active compounds from screening test on monolayer fibroblast culture to efficiency on 3-D dermal equivalent. J Appl Cosmetol 15:1–11Google Scholar
  4. Augustin C, Collombel C, Damour O (1997) Use of in vitro dermal equivalent and skin equivalent kits for evaluating cutaneous toxicity of cosmetic products. In vitro Tox 10:21–29Google Scholar
  5. Augustin C, Collombel C, Damour O (1997) Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro. Photodermatol Photoimmunol Photomed 13:27–36PubMedGoogle Scholar
  6. Augustin C, Collombel C, Damour O (1997) Measurement of the protective effect of topically applied sunscreens using in vitro dermal and skin equivalents. Photochem Photobiol 66(6):853–859PubMedGoogle Scholar
  7. Augustin C, Collombel C, Damour O (1998) Use of dermal equivalent and skin equivalent models for in vitro cutaneous irritation testing of cosmetic products: comparison with in vivo human data. J Toxicol Cut Ocular Toxicol 17:5–17Google Scholar
  8. Batisse D, Bazin R, Baldeweck T, Querleux B, Leveque JL (2002) Influence of age on the wrinkling capacities of skin. Skin Res Technol 8:148–154PubMedCrossRefGoogle Scholar
  9. Bernerd F, Asselineau D, Vioux C, Chevallier-Lagente O, Bouadjar B, Sarasin A, Magnaldo T (2001) Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro. Proc Natl Acad Sci USA 98:7817–7822PubMedCrossRefGoogle Scholar
  10. Berthod F, Saintiguy G, Chrétien F, Hayek D, Collombel C, Damour O (1994) Optimization of thickness, pore size and mechanical properties of a biomaterial designed for deep burn coverage. Clin Mater 15:259–265PubMedCrossRefGoogle Scholar
  11. Berthod F, Sahuc F, Hayek D, Damour O, Collombel C (1996) Deposition of collagen fibril bundles by long-term culture of fibroblasts in a collagen sponge. J Biomed Mat Res 32:87–93CrossRefGoogle Scholar
  12. Berthod F, Germain L, Lethias C, Garrone R, Damour O, van der Rest M, Auger FA (1997) Differential expression of collagens XII and XIV in human skin and in reconstructed skin. J Invest Dermatol 108:37–42CrossRefGoogle Scholar
  13. Bouez C, Damour O, Vičanová J (2003) Improvement of dermal extracellular matrix structure and composition after treatment with Imedeen Time Perfection in an in vitro skin equivalent. Paper presented at the meeting of International Society for Bioengineering and International Society for Skin Imaging, Hamburg, 21–24 May 2003Google Scholar
  14. Bouez C, Reynaud C, Noblesse E, Thepot A, Gleyzal C, Kanitakis J, Perrier E, Damour O, Sommer P (2006) The lysyl oxidase LOX is absent in basal and squamous cell carcinomas and its knockdown induces an invading phenotype in a skin equivalent model. Clin Cancer Res 12(5):1463–1469PubMedCrossRefGoogle Scholar
  15. Coulomb B, Lebreton C, Dubertret L (1993) Influence of human dermal fibroblasts on epidermalization. J Invest Dermatol 92:122–125CrossRefGoogle Scholar
  16. Damour O, Augustin C, Black AF (1998) Applications of reconstructed skin models in pharmaco-toxicological trials. Med Biol Eng Comput 6:1–8Google Scholar
  17. Damour O, Gueugniaud PY, Berthin-Maghit M, Rousselle P, Berthod F, Sahuc F, Collombel C (1994) A dermal substrate made of collagen–GAG–chitosan for deep burn coverage: first clinical uses. Clin Mater 15:273–276PubMedCrossRefGoogle Scholar
  18. Davis EC, Mecham RP (1998) Intracellular trafficking of tropoelastin. Matrix Biol 17:245–254PubMedCrossRefGoogle Scholar
  19. Debelle L, Tamburro AM (1999) Elastin: molecular description and function. Int J Biochem Cell Biol 31:261–272PubMedCrossRefGoogle Scholar
  20. Duplan-Perrat F, Damour O, Montrocher C, Peyrol S, Grenier G, Jacob M-P, Braye F (2000) Keratinocytes influence the maturation and organization of the elastin network in a skin equivalent. J Invest Dermatol 114:365–370PubMedCrossRefGoogle Scholar
  21. El Ghalbzouri A, Ponec M (2004) Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components. Wound Repair Regen 12(3):359–367PubMedCrossRefGoogle Scholar
  22. Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ (1997) Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med 13(20):1419–1428CrossRefGoogle Scholar
  23. Frances C, Robert L (1984) Elastin and elastic fibers in normal and pathologic skin. Int J Dermatol 23(3):166–179PubMedGoogle Scholar
  24. Fulop T Jr, Jacob MP, Varga Z, Foris G, Leovey A, Robert L (1986) Effect of elastin peptides on human monocytes: Ca2+ mobilization, stimulation of respiratory burst and enzyme secretion. Biochem Biophys Res Commun 141(1):92–98PubMedCrossRefGoogle Scholar
  25. Furth JJ (1991) The steady-state levels of type I collagen mRNA are reduced in senescent fibroblasts. J Gerontol 46:B122–B124PubMedGoogle Scholar
  26. Gilchrest BA, Yaar M (1992) Ageing and photoageing of the skin: observations at the cellular and molecular level. Br J Dermatol 127(Suppl 41):25–30PubMedGoogle Scholar
  27. Glaser DA (2004) Anti-aging products and cosmeceuticals. Facial Plast Surg Clin North Am 12(3):363–372, viiPubMedCrossRefGoogle Scholar
  28. Gregory C, Sephel BZ, Davidson JM (1986) Elastin production in human skin fibroblasts culture and its decline with age. J Invest Dermatol 86:279–295CrossRefGoogle Scholar
  29. Jacob MP, Sauvage M, Osborne-Pellegrin M (2001) Regulation of elastin synthesis. J Soc Biol 195:131–141PubMedGoogle Scholar
  30. Jacob MP, Fulop TJR, Foris G, Robert L (1987) Effect of elastin peptides on ion fluxes inmononuclear cells, fibroblasts, and smooth muscle cells. Proc Natl Acad Sci USA 84:995–999PubMedCrossRefGoogle Scholar
  31. Jenkins G (2002) Molecular mechanisms of skin aging. Mech Ageing Develop 123:801–810CrossRefGoogle Scholar
  32. Kieffer ME, Efsen J (1998) Imedeen® in the treatment of photoaged skin: an efficacy and safety trial over 12 months. J Eur Acad Dermatol Venereol 11:129–136PubMedGoogle Scholar
  33. Kligman AM (1997) Topical treatments for photoaged skin. Separating the reality from the hype. Postgrad Med 102:115–118, 123–126PubMedCrossRefGoogle Scholar
  34. Martin M, Nabout RE, Lafuma C, Crechet F, Remy J (1990) Fibronectin and collagen gene expression during in vitro aging of pig skin fibroblasts. Exp Cell Res 191:8–13PubMedCrossRefGoogle Scholar
  35. Noblesse E, Cenizo V, Bouez C, Borel A, Gleyzal C, Peyrol S, Jacob MP, Sommer P, Damour O (2004) Lysyl oxidase-like and lysyl oxidase are present in the dermis and epidermis of a skin equivalent and in human skin and are associated to elastic fibers. J Invest Dermatol 122(3):621–630PubMedCrossRefGoogle Scholar
  36. Pasquali-Ronchetti I, Baccarani-Contri M (1997) Elastic fiber during development and aging. Microsc Res Tech 38:428–435PubMedCrossRefGoogle Scholar
  37. Peterkofsky B, Chojkier M, Bateman J (1982) Determination of collagen synthetis in tissue and cell culture system. In: Furthmayr (ed) Immunochemistry of the extracellular matrix, vol 2H. CRC Press, Boca Raton, FL, pp 19–47Google Scholar
  38. Peterkofsky B (1972) The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch Biochem Biophys 152(1):318–328PubMedCrossRefGoogle Scholar
  39. Ricciarelli R, Maroni P, Ozer N, Zingg JM, Azzi A (1999) Age-dependent increase of collagenase expression can be reduced by alpha-tocopherol via protein kinase C inhibition. Free Radic Biol Med (7–8):729–737CrossRefGoogle Scholar
  40. Rittie L, Fisher GJ (2002) UV light induced signal cascades and skin ageing. Ageing Res Rev 1(4):705–720PubMedCrossRefGoogle Scholar
  41. Sahuc F, Nakazawa K, Berthod F, Damour O, Collombel C (1996) Mesenchymal–epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermal–epidermal junction formation in a skin equivalent model. Wound Rep Reg 4:93–102CrossRefGoogle Scholar
  42. Scharffetter-Kochanek K, Brenneisen P, Wenk J, Herrmann G, Ma W, Kuhr L, Meewes C, Wlaschek M (2000) Photoaging of the skin from phenotype to mechanisms. Exp Gerontol 35:307–316PubMedCrossRefGoogle Scholar
  43. Schlotmann K, Kaeten M, Black Af, Damour O, Forster T (2001) Cosmetic efficacy claims in vitro using a 3D human skin model. Int J Cosmet Sci 23:309–318CrossRefPubMedGoogle Scholar
  44. Sigler ML, Rasmussen P (2003) A placebo controlled study of an oral supplement (Imedeen Time Perfection) in improving the appearance of photodamaged skin. Paper presented at the meeting of European Academy of Dermato Venereology, Barcelona, 16–18 October 2003Google Scholar
  45. Skovgaard G, Jensen AS (2006) Effect of novel dietary supplement on skin aging in post-menopausal women. Eur J Clin Nutr 1–6Google Scholar
  46. Smit N, Vičanová J, Cramers P, Vrolijk H, Pavel S (2004) The combined effects of extracts containing carotenoids and vitamins E and C on growth and pigmentation of cultured human melanocytes. Skin Pharmacol Physiol 17(5):238–245PubMedCrossRefGoogle Scholar
  47. Uitto J (1979) Biochemistry of the elastic fibers in normal connective tissues and its alterations in diseases. J Invest Dermatol 72(1):1–10Google Scholar
  48. Vaughan MB, Ramirez RD, Brown SA, Yang JC, Wright WE, Shay JW (2004). A reproducible laser-wounded skin equivalent model to study the effects of aging in vitro. Rejuvenation Res 7(2):99–110PubMedCrossRefGoogle Scholar
  49. Vičanová J, Bouez C, Lacroix S, Lindmark L, Damour O (2006) Epidermal and dermal characteristics in skin equivalent after systemic and topical application of skin care ingredients. Ann NY Acad Sci 1067:337–342PubMedCrossRefGoogle Scholar
  50. Wei H, Bowen R, Zhang X, Lebwohl M (1998) Isoflavone genistein inhibits the irrradiation and promotion of two-stage skin carcinogenesis in mice. Carcinogenesis 19:1509–1514PubMedCrossRefGoogle Scholar
  51. West MD, Pereira-Smith OM, Smith JR (1989) Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity. Exp Cell Res 184(1):138–147PubMedCrossRefGoogle Scholar
  52. Yaar M, Eller M, Gilchrest B (2002) Fifty years of skin aging. J Invest Dermatol Symp Proc 7:51–58CrossRefGoogle Scholar
  53. Zhao J, Wang J, Chen Y, Agarwal R (1999) Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5-3'-gallate as the most effective antioxidant constituent. Carcinogenesis 20(9):1737–1745CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Sophie Lacroix
    • 1
  • Charbel Bouez
    • 1
  • Sandrine Vidal
    • 1
  • Valérie Cenizo
    • 2
  • Corinne Reymermier
    • 3
  • Virginie Justin
    • 1
  • Jana Vičanová
    • 4
    • 5
  • Odile Damour
    • 1
    • 2
  1. 1.Banque de Tissus et Cellules, Hôpital Edouard HerriotHospices Civils de LyonLyonFrance
  2. 2.Institut de Biologie et Chimie des ProtéinesLyonFrance
  3. 3.EngelhardLyonFrance
  4. 4.DermDataTrutnovCzech Republic
  5. 5.UnhostCzech Republic

Personalised recommendations