The aim of this study was to create and characterize a tissue-engineered mucosal equivalent (TEM) that closely resembles native mucosa. TEM consists of human primary keratinocytes and fibroblasts isolated from biopsies taken from healthy donors and seeded onto a de-epidermized dermis and cultured for 14 days at the air/liquid interface. The structure of TEM was examined and compared with native nonkeratinizing oral mucosa (NNOM). The various components of the newly formed epidermal layer, basement membrane and underlying connective tissue were analyzed using immunohistochemistry. The mucosal substitute presented in this study showed a mature stratified squamous epithelium that was similar to that of native oral mucosa, as demonstrated by K19, desmoglein-3 and involucrin staining. In addition, the expression of basement membrane components collagen type IV, laminin-5 and integrin α6 and β4 in TEM proved to be consistent with native oral mucosa. The expression of PAS, Ki67, K10 and K13, however, appeared to be different in TEM compared to NNOM. Nevertheless, the similarities with native oral mucosa makes TEM a promising tool for studying the biology of mucosal pathologies such as oral mucositis or fibrosis as well as the development of new therapies.

Keywords:
Tissue engineering, Oral mucosa, Keratinocyte, Fibroblast
1.
Alaminos, M., I. Garzon, M.C. Sanchez-Quevedo, G. Moreu, M. Gonzalez-Andrades, A. Fernandez-Montoya, A. Campos (2007) Time-course study of histological and genetic patterns of differentiation in human engineered oral mucosa. J Tissue Eng Regen Med 1: 350–359.
2.
Bechetoille, N., C. Dezutter-Dambuyant, O. Damour, V. Andre, I. Orly, E. Perrier (2007) Effects of solar ultraviolet radiation on engineered human skin equivalent containing both Langerhans cells and dermal dendritic cells. Tissue Eng 13: 2667–2679.
3.
Boyce, S.T., G.D. Warden (2002) Principles and practices for treatment of cutaneous wounds with cultured skin substitutes. Am J Surg 183: 445–456.
4.
Chung, J.H., K.H. Cho, D.Y. Lee, O.S. Kwon, M.W. Sung, K.H. Kim, H.C. Eun (1997) Human oral buccal mucosa reconstructed on dermal substrates: a model for oral epithelial differentiation. Arch Dermatol Res 289: 677–685.
5.
El Ghalbzouri, A., S. Gibbs, E. Lamme, C.A. Van Blitterswijk, M. Ponec (2002a) Effect of fibroblasts on epidermal regeneration. Br J Dermatol 147: 230–243.
6.
El Ghalbzouri, A., E. Lamme, M. Ponec (2002b) Crucial role of fibroblasts in regulating epidermal morphogenesis. Cell Tissue Res 310: 189–199.
7.
El Ghalbzouri, A., M. Ponec (2004) Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components. Wound Repair Regen 12: 359–367.
8.
Garzon, I., M.C. Sanchez-Quevedo, G. Moreu, M. Gonzalez-Jaranay, M. Gonzalez-Andrades, A. Montalvo, A. Campos, M. Alaminos (2009) In vitro and in vivo cytokeratin patterns of expression in bioengineered human periodontal mucosa. J Periodontal Res 44: 588–597.
9.
Green, K.J., J.C. Jones (1996) Desmosomes and hemidesmosomes: structure and function of molecular components. FASEB J 10: 871–881.
10.
Izumi, K., S.E. Feinberg, A. Iida, M. Yoshizawa (2003) Intraoral grafting of an ex vivo produced oral mucosa equivalent: a preliminary report. Int J Oral Maxillofac Surg 32: 188–197.
11.
Izumi, K., J. Song, S.E. Feinberg (2004) Development of a tissue-engineered human oral mucosa: from the bench to the bed side. Cells Tissues Organs 176: 134–152.
12.
Karring, T., N.P. Lang, H. Loe (1975) The role of gingival connective tissue in determining epithelial differentiation. J Periodontal Res 10: 1–11.
13.
Kautsky, M.B., P. Fleckman, B.A. Dale (1995) Retinoic acid regulates oral epithelial differentiation by two mechanisms. J Invest Dermatol 104: 224–230.
14.
Kinikoglu, B., C. Auxenfans, P. Pierrillas, V. Justin, P. Breton, C. Burillon, V. Hasirci, O. Damour (2009) Reconstruction of a full-thickness collagen-based human oral mucosal equivalent. Biomaterials 30: 6418–6425.
15.
Klausner, M., S. Ayehunie, B.A. Breyfogle, P.W. Wertz, L. Bacca, J. Kubilus (2007) Organotypic human oral tissue models for toxicological studies. Toxicol In Vitro 21: 938–949.
16.
Lauer, G., R. Schimming (2001) Tissue-engineered mucosa graft for reconstruction of the intraoral lining after freeing of the tongue: a clinical and immunohistologic study. J Oral Maxillofac Surg 59: 169–175, discussion 175–167.
17.
Mackenzie, I.C., M.W. Hill (1984) Connective tissue influences on patterns of epithelial architecture and keratinization in skin and oral mucosa of the adult mouse. Cell Tissue Res 235: 551–559.
18.
Moharamzadeh, K., I.M. Brook, R. Van Noort, A.M. Scutt, K.G. Smith, M.H. Thornhill (2008) Development, optimization and characterization of a full-thickness tissue engineered human oral mucosal model for biological assessment of dental biomaterials. J Mater Sci Mater Med 19: 1793–1801.
19.
Pena, I., L.M. Junquera, A. Meana, E. Garcia, V. Garcia, J.C. De Vicente (2010) In vitro engineering of complete autologous oral mucosa equivalents: characterization of a novel scaffold. J Periodontal Res 45: 375–380.
20.
Presland, R.B., R.J. Jurevic (2002) Making sense of the epithelial barrier: what molecular biology and genetics tell us about the functions of oral mucosal and epidermal tissues. J Dent Educ 66: 564–574.
21.
Rakhorst, H.A., S.J. Posthumus-Van Sluijs, W.M. Tra, J.W. Van Neck, G.J. Van Osch, S.E. Hovius, A. El Ghalbzouri, S.O. Hofer (2006) Fibroblasts accelerate culturing of mucosal substitutes. Tissue Eng 12: 2321–2331.
22.
Rheinwald, J.G., H. Green (1975a) Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell 6: 317–330.
23.
Rheinwald, J.G., H. Green (1975b) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331–343.
24.
Tjabringa, G., M. Bergers, D. van Rens, R. de Boer, E. Lamme, J. Schalkwijk (2008) Development and validation of human psoriatic skin equivalents. Am J Pathol 173: 815–823.
25.
Tomakidi, P., D. Breitkreutz, N.E. Fusenig, J. Zoller, A. Kohl, G. Komposch (1998) Establishment of oral mucosa phenotype in vitro in correlation to epithelial anchorage. Cell Tissue Res 292: 355–366.
26.
van Oijen, M.G., M.M. Gilsing, G. Rijksen, G.J. Hordijk, P.J. Slootweg (1998) Increased number of proliferating cells in oral epithelium from smokers and ex-smokers. Oral Oncol 34: 297–303.
27.
Wang, X., Y. Liu, Z. Deng, R. Dong, S. Hu, Y. Li, Y. Jin (2009) Inhibition of dermal fibrosis in self-assembled skin equivalents by undifferentiated keratinocytes. J Dermatol Sci 53: 103–111.
28.
Winning, T.A., G.C. Townsend (2000) Oral mucosal embryology and histology. Clin Dermatol 18: 499–511.
29.
Xu, Q., K. Izumi, T. Tobita, Y. Nakanishi, S.E. Feinberg (2009) Constitutive release of cytokines by human oral keratinocytes in an organotypic culture. J Oral Maxillofac Surg 67: 1256–1264.
© 2011 S. Karger AG, Basel
2011
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