393 www.ecmjournal.org C Nishio et al. Formation/regeneration of junctional epithelium European Cells and Materials Vol. 20 2010 (pages 393-402) ISSN 1473-2262 Abstract The junctional epithelium (JE) adheres to the tooth surface, and seals off periodontal tissues from the oral environment. This incompletely differentiated epithelium is formed initially by the fusion of the reduced enamel organ with the oral epithelium (OE). Two proteins, odontogenic ameloblast-associated (ODAM) and amelotin (AMTN), have been identified in the JE. The objective of this study was to evaluate their expression pattern during formation and regeneration of the JE. Cytokeratin 14 was used as a differentiation marker for oral epithelial cells, and Ki67 for cell proliferation. Immunohistochemistry was carried out on erupting rat molars, and in regenerating JE following gingivectomy. In the reducing enamel organ and in established JE, ODAM and AMTN were present at the cell- tooth interface while only ODAM and CK14 were found throughout the JE. Both were also conspicuously present in cell clusters situated between the erupting tooth and OE. During JE regeneration, ODAM was detected first at the leading wound edge and then in the regenerating JE. Some cell clusters in the subjacent connective tissue were also positive for ODAM. AMTN appeared later and both AMTN and ODAM accumulated at the interface with the tooth. Cytokeratin 14 gradually appeared in the regenerating JE but the cell clusters showed variable labeling. Cells associated with JE formation and regeneration exhibited higher division activity than adjacent epithelial cells. These findings suggest that ODAM and AMTN have a role at the cell-tooth interface, and that ODAM is likely also implicated in cellular events during formation and regeneration of the JE. Keywords: Apin, amelotin, cytokeratin 14, development, gingival, junctional epithelium, ODAM, regeneration. * Address for correspondence: Antonio Nanci Laboratory for the Study of Calcified Tissues and Biomaterials Department of Stomatology, Faculty of Dentistry, Université de Montréal 2900 Edouard-Montpetit, Pavillon Roger-Gaudry, Room A-212, H3T1J4 Montréal, Québec, Canada Telephone Number: +1514.3435846 FAX Number: +1514.3432233 E-mail: antonio.nanci@umontreal.ca Introduction The junctional epithelium (JE) is a specialized epithelial structure that seals off the supporting tissues of the tooth from the aggressive oral environment, and represents the first line of defense against periodontal diseases (Takata et al., 1986; Schroeder and Listgarten, 1997; Schroeder and Listgarten, 2003). This stratified squamous, non- keratinizing epithelium is considered to be incompletely differentiated, producing components for tooth attachment instead of progressing along a keratinization pathway (Schroeder and Listgarten, 2003; Shimono et al., 2003). The attachment of the gingiva to the enamel surface is provided by a structural complex called the epithelial attachment consisting of an inner basal lamina (BL) and hemidesmosomes. The BL is formed and maintained by the superficial JE cells to which they are attached by hemidesmosomes (Hormia et al., 1992; Hormia et al., 2001; Masaoka et al., 2009; Tsuchiya et al., 2009). The BL of the JE is atypical because it contains laminin-5 but not other typical components, such as γ1 chain-containing laminins, and type IV and VII collagens (Hormia et al., 2001). However, the exact composition and attachment mechanism of the BL to the tooth surface are still not completely understood. It is believed that during tooth development, the primary JE forms by the fusion of the reduced enamel organ with the oral epithelium (OE), and will be replaced in time by a secondary JE derived solely from cells of the OE (Sabag et al., 1984; Abe et al., 1995; Schroeder, 1996; Shimono et al., 2003; Bosshardt and Lang, 2005). Although the structure and the function of the JE are influenced by the underlying connective tissue (Mackenzie, 1987) and by contact with a solid substratum, such as enamel, dentin or cementum (Abe et al., 1995; Schroeder, 1996), the exact mechanisms that lead to the formation and regeneration of the JE remain unclear. Efforts to identify the secretome of the epithelial cells responsible for creating tooth enamel (Moffatt et al., 2006a) have led to the identification of two genes encoding for secreted proteins called amelotin (AMTN) (Moffatt et al., 2006b) and APIN (Moffatt et al., 2008), APIN now being called odontogenic ameloblast-associated (ODAM). ODAM (Moffatt et al., 2006a; Moffatt et al., 2008) and AMTN (Iwasaki et al., 2005; Moffatt et al., 2006a; Moffatt et al., 2006b) are encoded by two different genes classified on the basis of their genomic location and architecture as part of the secretory calcium-binding phosphoprotein (SCPP) gene cluster (Kawasaki and Weiss, 2003; Huq et al ., 2005; Kawasaki and Weiss, 2006). This cluster contains a number of genes coding for proteins that EXPRESSION PATTERN OF ODONTOGENIC AMELOBLAST-ASSOCIATED AND AMELOTIN DURING FORMATION AND REGENERATION OF THE JUNCTIONAL EPITHELIUM Clarice Nishio 1 , Rima Wazen 1 , Shingo Kuroda 1 , Pierre Moffatt 2 , and Antonio Nanci 1* 1 Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Montréal, Québec, Canada 2 Shriners Hospital for Children, Montréal, Québec, Canada