Abstract
The presence of pre-existing basement membrane (BM) components improves the morphogenesis of epidermis and BM in constructing a human living skin-equivalent (LSE). De-epithelialized amniotic membrane (AM) retains key BM components. We have therefore investigated the usefulness of AM for constructing LSE. De-epithelialized AM was overlaid on type I collagen gel embedded with fibroblasts. Normal human keratinocytes (NHKs) were then seeded onto the epithelial side of the AM to construct an AM-LSE. A conventional LSE was constructed by seeding NHKs on a fibroblast-populated type I collagen gel. When the keratinocytes reached confluence, the LSE was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at various times and investigated morphologically, immunohistochemically, and ultrastructurally. In AM-LSE, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of LSE without AM. A more continuous BM and better-developed hemidesmosomes were found in AM-LSE. The epidermis of AM-LSE outgrew much faster than that of LSE without AM. When transplanted onto nude mice, both LSEs took well; however, the AM-LSE graft showed better morphogenesis of the epidermis, BM, and hemidesmosomes. The better epidermal morphology and better-developed BM in AM-LSE in vitro and in vivo indicates its superiority over LSE without AM for clinical applications.
Similar content being viewed by others
References
Amano S, Akutsu N, Matsunaga Y, Nishiyama T, Champliaud MF, Burgeson RE, Adachi E (2001) Importance of balance between extracellular matrix synthesis and degradation in basement membrane formation. Exp Cell Res 271:249–262
Andriani F, Margulis A, Lin N, Griffey S, Garlick JA (2003) Analysis of microenvironmental factors contributing to basement membrane assembly and normalized epidermal phenotype. J Invest Dermatol 120:923–931
Andriani F, Garfield J, Fusenig NE, Garlick JA (2004) Basement membrane proteins promote progression of intraepithelial neoplasia in 3-dimensional models of human stratified epithelium. Int J Cancer 108:348–357
Bergman R, David R, Ramon Y, Ramon M, Kerner H, Kilim S, Peled I, Friedman-Birnbaum R (1997) Delayed postburn blisters: an immunohistochemical and ultrastructural study. J Cutan Pathol 24:429–433
Blomme EA, Weckmann MT, Capen CC, Rosol TJ (1998) Influence of extracellular matrix macromolecules on normal human keratinocyte phenotype and parathyroid hormone-related protein secretion and expression in vitro. Exp Cell Res 238:204–215
Champliaud MF, Lunstrum GP, Rousselle P, Nishiyama T, Keene DR, Burgeson RE (1996) Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment. J Cell Biol 132:1189–1198
Eaglstein WH, Falanga V (1998) Tissue engineering and the development of Apligraf a human skin equivalent. Adv Wound Care 11:1–8
Espana EM, He H, Kawakita T, Di Pascuale MA, Raju VK, Liu CY, Tseng SC (2003) Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan. Invest Ophthalmol Vis Sci 44:5136–5141
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 11:35–45
Kim JP, Chen JD, Wilke MS, Schall TJ, Woodley DT (1994) Human keratinocyte migration on type IV collagen. Roles of heparin-binding site and alpha 2 beta 1 integrin. Lab Invest 71:401–408
Kim SW, Park KC, Kim HJ, Cho KH, Chung JH, Kim KH, Eun HC, Lee JS, Park KD (2001) Effects of collagen IV and laminin on the reconstruction of human oral mucosa. J Biomed Mater Res 58:108–112
Koizumi N, Fullwood NJ, Bairaktaris G, Inatomi T, Kinoshita S, Quantock AJ (2000) Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Invest Ophthalmol Vis Sci 41:2506–2513
Kubo M, Sonoda Y, Muramatsu R, Usui M (2001) Immunogenicity of human amniotic membrane in experimental xenotransplantation. Invest Ophthalmol Vis Sci 42:1539–1546
Kumar TR, Shanmugasundaram N, Babu M (2003) Biocompatible collagen scaffolds from a human amniotic membrane: physicochemical and in vitro culture characteristics. J Biomater Sci Polym Ed 14:689–706
Llames SG, Del Rio M, Larcher F, Garcia E, Garcia M, Escamez MJ, Jorcano JL, Holguin P, Meana A (2004) Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin. Transplantation 77:350–355
Mahgoub MA, Ammar A, Fayez M, Edris A, Hazem A, Akl M, Hammam O (2004) Neovascularization of the amniotic membrane as a biological immune barrier. Transplant Proc 36:1194–1198
Meana A, Iglesias J, Del Rio M, Larcher F, Madrigal B, Fresno MF, Martin C, San Roman F, Tevar F (1998) Large surface of cultured human epithelium obtained on a dermal matrix based on live fibroblast-containing fibrin gels. Burns 24:621–630
Medalie DA, Eming SA, Collins ME, Tompkins RG, Yarmush ML, Morgan JR (1997) Differences in dermal analogs influence subsequent pigmentation, epidermal differentiation, basement membrane, and rete ridge formation of transplanted composite skin grafts. Transplantation 64:454–465
Nakamura T, Endo K, Cooper LJ, Fullwood NJ, Tanifuji N, Tsuzuki M, Koizumi N, Inatomi T, Sano Y, Kinoshita S (2003a) The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci 44:106–116
Nakamura T, Koizumi N, Tsuzuki M, Inoki K, Sano Y, Sotozono C, Kinoshita S (2003b) Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease. Cornea 22:70–71
Nakamura T, Yoshitani M, Rigby H, Fullwood NJ, Ito W, Inatomi T, Sotozono C, Nakamura T, Shimizu Y, Kinoshita S (2004) Sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction. Invest Ophthalmol Vis Sci 45:93–99
Nishiyama T, Amano S, Tsunenaga M, Kadoya K, Takeda A, Adachi E, Burgeson RE (2000) The importance of laminin 5 in the dermal-epidermal basement membrane. J Dermatol Sci 24:S51–S59
Ojeh NO, Frame JD, Navsaria HA (2001) In vitro characterization of an artificial dermal scaffold. Tissue Eng 7:457–472
Oyama N, Bhogal BS, Carrington P, Gratian MJ, Black MM (2003) Human placental amnion is a novel substrate for detecting autoantibodies in autoimmune bullous diseases by immunoblotting. Br J Dermatol 148:939–944
Park WC, Tseng SC (2000) Modulation of acute inflammation and keratocyte death by suturing, blood, and amniotic membrane in PRK. Invest Ophthalmol Vis Sci 41:2906–2914
Ralston DR, Layton C, Dalley AJ, Boyce SG, Freedlander E, MacNeil S (1999) The requirement for basement membrane antigens in the production of human epidermal/dermal composites in vitro. Br J Dermatol 140:605–615
Ravishanker R, Bath AS, Roy R (2003) “Amnion Bank”—the use of long term glycerol preserved amniotic membranes in the management of superficial and superficial partial thickness burns. Burns 29:369–374
Rejzek A, Weyer F, Eichberger R, Gebhart W (2001) Physical changes of amniotic membranes through glycerolization for the use as an epidermal substitute. Light and electron microscopic studies. Cell Tissue Bank 2:95–102
Rousselle P, Keene DR, Ruggiero F, Champliaud MF, Rest M, Burgeson RE (1997) Laminin 5 binds the NC-1 domain of type VII collagen. J Cell Biol 138:719–728
Sheridan RL, Moreno C (2001) Skin substitutes in burns. Burns 27:92
Shirakata Y, Tokumaru S, Yamasaki K, Sayama K, Hashimoto K (2003) So-called biological dressing effects of cultured epidermal sheets are mediated by the production of EGF family, TGF-beta and VEGF. J Dermatol Sci 32:209–215
Shirakata Y, Ueno H, Hanakawa Y, Kameda K, Yamasaki K, Tokumaru S, Yahata Y, Tohyama M, Sayama K, Hashimoto K (2004) TGF-beta is not involved in early phase growth inhibition of keratinocytes by 1alpha,25(OH)2vitamin D3. J Dermatol Sci 36:41–50
Tseng SC (2001) Amniotic membrane transplantation for ocular surface reconstruction. Biosci Rep 21:481–489
Tsunenaga M, Adachi E, Amano S, Burgeson RE, Nishiyama T (1998) Laminin 5 can promote assembly of the lamina densa in the skin equivalent model. Matrix Biol 17:603–613
von Versen-Hoynck F, Syring C, Bachmann S, Moller DE (2004) The influence of different preservation and sterilisation steps on the histological properties of amnion allografts-light and scanning electron microscopic studies. Cell Tissue Bank 5:45–56
Yang L, Shirakata Y, Tamai K, Dai X, Hanakawa Y, Tokumaru S, Yahata Y, Tohyama M, Shiraishi K, Nagai H, Wang X, Murakami S, Sayama K, Kaneda Y, Hashimoto K (2005) Microbubble-enhanced ultrasound for gene transfer into living skin equivalents. J Dermatol Sci 40:105–114
Acknowledgements
We thank Teruko Tsuda, Eriko Tan, and Wakana Itoh for technical assistance.
Author information
Authors and Affiliations
Corresponding author
Additional information
This work was partly supported by Health Sciences Research Grants for Research on Specific Diseases from the Ministry of Health, Labor, and Welfare of Japan (to K.H.) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K.H. and Y.S.).
L. Yang and Y. Shirakata contributed equally to this work.
Rights and permissions
About this article
Cite this article
Yang, L., Shirakata, Y., Shudou, M. et al. New skin-equivalent model from de-epithelialized amnion membrane. Cell Tissue Res 326, 69–77 (2006). https://doi.org/10.1007/s00441-006-0208-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-006-0208-2