Cell and Tissue Research

, Volume 319, Issue 2, pp 279–287 | Cite as

Transplantation of reconstructed human skin on nude mice: a model system to study expression of human tenascin-X and elastic fiber components

  • Manon C. Zweers
  • Joost Schalkwijk
  • Toin H. van Kuppevelt
  • Ivonne M. van Vlijmen-Willems
  • Mieke Bergers
  • Claire Lethias
  • Evert N. Lamme
Regular Article


Tenascin-X is a large extracellular matrix protein that is widely expressed in connective tissues during development and in the adult. Genetically determined deficiency of tenascin-X causes the connective tissue disease Ehlers–Danlos syndrome. These patients show reduced collagen density and fragmentation of elastic fibers in their skin. In vitro studies on the role of tenascin-X in elastic fiber biology are hampered because monolayers of fibroblasts do not deposit tenascin-X and elastic fibers into the extracellular matrix. Here, we applied an organotypic culture model of fibroblasts and keratinocytes to address this issue. We investigated the deposition of tenascin-X and elastin into skin-equivalent in vitro and also in vivo after transplantation onto immunodeficient mice. Whereas tenascin-C and fibrillin-1 were readily expressed in the skin-equivalents before transplantation, tenascin-X and elastin were not present. Three weeks post-grafting, a network of elastin was observed that coincided with the appearance of tenascin-X. At the ultrastructural level, microfibrils were observed, some of which were associated with elastin. Transplanted skin-equivalents containing tenascin-X-deficient fibroblasts showed deposition of immunoreactive elastin in similar quantities and distribution as those containing control fibroblasts. This suggests that tenascin-X is important for the stability and maintenance of established elastin fibers, rather than for the initial phase of elastogenesis. Thus, the transplantation of reconstructed skin on nude mice allows the study of tenascin-X and elastin expression and could be used as a model system to study the potential role of tenascin-X in matrix assembly and stability.


Tenascin-X Connective tissue Organotypic culture Skin-equivalent Transplantation Human Mouse (male athymic nude BALB/c) 


  1. Berthod F, Germain L, Li H, Xu W, Damour O, Auger FA (2001) Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice. Matrix Biol 20:463–473CrossRefPubMedGoogle Scholar
  2. Burch GH, Bedolli MA, McDonough S, Rosenthal SM, Bristow J (1995) Embryonic expression of tenascin-X suggests a role in limb, muscle, and heart development. Dev Dyn 203:491–504PubMedGoogle Scholar
  3. Christiano AM, Uitto J (1994) Molecular pathology of the elastic fibers. J Invest Dermatol 103:53S–57SCrossRefPubMedGoogle Scholar
  4. Debelle L, Tamburro AM (1999) Elastin: molecular description and function. Int J Biochem Cell Biol 31:261–272CrossRefPubMedGoogle Scholar
  5. Deckner M, Lindholm T, Cullheim S, Risling M (2000) Differential expression of tenascin-C, tenascin-R, tenascin/J1, and tenascin-X in spinal cord scar tissue and in the olfactory system. Exp Neurol 166:350–362CrossRefPubMedGoogle Scholar
  6. Duplan-Perrat F, Damour O, Montrocher C, Peyrol S, Grenier G, Jacob MP, Braye F (2000) Keratinocytes influence the maturation and organization of the elastin network in a skin equivalent. J Invest Dermatol 114:365–370CrossRefPubMedGoogle Scholar
  7. Fleischmajer R, MacDonald ED, Contard P, Perlish JS (1993) Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin. J Histochem Cytochem 41:1359–1366Google Scholar
  8. Geffrotin C, Garrido JJ, Tremet L, Vaiman M (1995) Distinct tissue distribution in pigs of tenascin-X and tenascin-C transcripts. Eur J Biochem 231:83–92PubMedGoogle Scholar
  9. Geffrotin C, Tricaud Y, Crechet F, Castelli M, Lefaix JL, Vaiman M (1998) Unlike tenascin-X, tenascin-C is highly up-regulated in pig cutaneous and underlying muscle tissue developing fibrosis after necrosis induced by very high-dose gamma radiation. Radiat Res 149:472–481PubMedGoogle Scholar
  10. Geffrotin C, Horak V, Crechet F, Tricaud Y, Lethias C, Vincent-Naulleau S, Vielh P (2000) Opposite regulation of tenascin-C and tenascin-X in MeLiM swine heritable cutaneous malignant melanoma. Biochim Biophys Acta 1524:196–202PubMedGoogle Scholar
  11. Greenlee TK Jr, Ross R, Hartman JL (1966) The fine structure of elastic fibers. J Cell Biol 30:59–71CrossRefPubMedGoogle Scholar
  12. Guerret S, Govignon E, Hartmann DJ, Ronfard V (2003) Long-term remodeling of a bilayered living human skin equivalent (Apligraf) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix. Wound Repair Regen 11:35–45CrossRefPubMedGoogle Scholar
  13. Kielty CM, Sherratt MJ, Shuttleworth CA (2002) Elastic fibres. J Cell Sci 115:2817–2828PubMedGoogle Scholar
  14. Kozel BA, Ciliberto CH, Mecham RP (2004) Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells. Matrix Biol 23:23–34CrossRefPubMedGoogle Scholar
  15. Latijnhouwers M, Bergers M, Ponec M, Dijkman H, Andriessen M, Schalkwijk J (1997) Human epidermal keratinocytes are a source of tenascin-C during wound healing. J Invest Dermatol 108:776–783CrossRefPubMedGoogle Scholar
  16. Lethias C, Descollonges Y, Boutillon MM, Garrone R (1996) Flexilin: a new extracellular matrix glycoprotein localized on collagen fibrils. Matrix Biol 15:11–19CrossRefPubMedGoogle Scholar
  17. Mao JR, Bristow J (2001) The Ehlers–Danlos syndrome: on beyond collagens. J Clin Invest 107:1063–1069PubMedGoogle Scholar
  18. Mao JR, Taylor G, Dean WB, Wagner DR, Afzal V, Lotz JC, Rubin EM, Bristow J (2002) Tenascin-X deficiency mimics Ehlers–Danlos syndrome in mice through alteration of collagen deposition. Nat Genet 30:421–425CrossRefPubMedGoogle Scholar
  19. Matsumoto K, Saga Y, Ikemura T, Sakakura T, Chiquet ER (1994) The distribution of tenascin-X is distinct and often reciprocal to that of tenascin-C. J Cell Biol 125:483–493CrossRefPubMedGoogle Scholar
  20. Matsumoto K, Takayama N, Ohnishi J, Ohnishi E, Shirayoshi Y, Nakatsuji N, Ariga H (2001) Tumour invasion and metastasis are promoted in mice deficient in tenascin-X. Genes Cells 6:1101–1111CrossRefPubMedGoogle Scholar
  21. Mecham RP (1991) Elastin synthesis and fiber assembly. Ann N Y Acad Sci 624:137–146PubMedGoogle Scholar
  22. Milewicz DM, Urban Z, Boyd C (2000) Genetic disorders of the elastic fiber system. Matrix Biol 19:471–480CrossRefPubMedGoogle Scholar
  23. Minamitani T, Ariga H, Matsumoto K (2002) Adhesive defect in extracellular matrix tenascin-X-null fibroblasts: a possible mechanism of tumor invasion. Biol Pharm Bull 25:1472–1475CrossRefPubMedGoogle Scholar
  24. Murphy GaRJJ (2002) Extracellular matrix degradation. In: Royce PM, Steinmann B (eds) Connective tissue and its heritable disorders. Molecular, genetic, and medical aspects. Wiley-Liss, New York, pp 343–384Google Scholar
  25. Partridge SM (1969) Elastin, biosynthesis and structure. Gerontologia 15:85–100PubMedGoogle Scholar
  26. Pasquali-Ronchetti I, Baccarani-Contri M (1997) Elastic fiber during development and aging. Microsc Res Tech 38:428–435CrossRefPubMedGoogle Scholar
  27. Ponec M, Weerheim A, Kempenaar J, Mulder A, Gooris GS, Bouwstra J, Mommaas AM (1997) The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C. J Invest Dermatol 109:348–355CrossRefPubMedGoogle Scholar
  28. Pouliot R, Larouche D, Auger FA, Juhasz J, Xu W, Li H, Germain L (2002) Reconstructed human skin produced in vitro and grafted on athymic mice. Transplantation 73:1751–1757CrossRefPubMedGoogle Scholar
  29. Raghunath M, Bachi T, Meuli M, Altermatt S, Gobet R, Bruckner-Tuderman L, Steinmann B (1996) Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autografts: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation. J Invest Dermatol 106:1090–1095CrossRefPubMedGoogle Scholar
  30. Ramirez F (2000) Pathophysiology of the microfibril/elastic fiber system: introduction. Matrix Biol 19:455–456CrossRefPubMedGoogle Scholar
  31. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343CrossRefPubMedGoogle Scholar
  32. Rosenbloom J (1984) Elastin: relation of protein and gene structure to disease. Lab Invest 51:605–623PubMedGoogle Scholar
  33. Rosenbloom J, Abrams WR (2002) Elastin and the microfibrillar apparatus. In: Royce PM, Steinmann B (eds) Connective tissue and its heritable disorders. Molecular, genetic, and medical aspects. Wiley-Liss, New York, pp 249–269Google Scholar
  34. Sakai LY, Keene DR, Engvall E (1986) Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol 103:2499–2509CrossRefPubMedGoogle Scholar
  35. Sakai T, Furukawa Y, Chiquet ER, Nakamura M, Kitagawa S, Ikemura T, Matsumoto K (1996) Tenascin-X expression in tumor cells and fibroblasts: glucocorticoids as negative regulators in fibroblasts. J Cell Sci 109:2069–2077PubMedGoogle Scholar
  36. Schalkwijk J, Zweers MC, Steijlen PM, Dean WB, Taylor G, Van Vlijmen IM, Haren B van, Miller WL, Bristow J (2001) A recessive form of the Ehlers–Danlos syndrome caused by tenascin-X deficiency. N Engl J Med 345:1167–1175CrossRefPubMedGoogle Scholar
  37. Schwartz E, Fleischmajer R (1986) Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts. J Histochem Cytochem 34:1063–1068PubMedGoogle Scholar
  38. Smola H, Thiekotter G, Fusenig NE (1993) Mutual induction of growth factor gene expression by epidermal–dermal cell interaction. J Cell Biol 122:417–429CrossRefPubMedGoogle Scholar
  39. Toselli P, Salcedo LL, Oliver P, Franzblau C (1981) Formation of elastic fibers and elastin in rabbit aortic smooth muscle cell cultures. Connect Tissue Res 8:231–239PubMedGoogle Scholar
  40. Zhang H, Apfelroth SD, Hu W, Davis EC, Sanguineti C, Bonadio J, Mecham RP, Ramirez F (1994) Structure and expression of fibrillin-2, a novel microfibrillar component preferentially located in elastic matrices. J Cell Biol 124:855–863CrossRefPubMedGoogle Scholar
  41. Zweers MC, Vlijmen-Willems IM, Van Kuppevelt TH, Mecham RP, Steijlen PM, Bristow J, Schalkwijk J (2004) Deficiency of tenascin-X causes abnormalities in dermal elastic fiber morphology. J Invest Dermatol 122:885–891CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Manon C. Zweers
    • 1
  • Joost Schalkwijk
    • 1
  • Toin H. van Kuppevelt
    • 2
  • Ivonne M. van Vlijmen-Willems
    • 1
  • Mieke Bergers
    • 1
  • Claire Lethias
    • 3
  • Evert N. Lamme
    • 1
  1. 1.Department of DermatologyUniversity Medical Centre NijmegenNijmegenThe Netherlands
  2. 2.Department of BiochemistryUniversity Medical Centre NijmegenNijmegenThe Netherlands
  3. 3.Institut de Biologie et Chimie des ProteinesCNRSLyonFrance

Personalised recommendations