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Amelogenin is a Cell Adhesion Protein

¹ Department of Periodontics,
² Department of Cellular and Structural Biology, and
³ Department of Pediatric Dentistry, University of Texas Health Science Center, San Antonio, TX 78229;

View larger version (67K): [in a new window]   Figure 1. SDS-PAGE analysis of Emdogain® and recombinant porcine amelogenin. Samples of Emdogain® and recombinant porcine amelogenin were analyzed by SDS-PAGE on 15% (w/v) cross-linked polyacrylamide mini-slab gels under reducing conditions and stained with Coomassie Brilliant Blue R-250. The analysis showed that Emdogain® contains several protein species. The molecular masses were clearly detectable from less than 14.3 to over 77 kDa. A major protein band, corresponding to processed 20-kDa amelogenin, was clearly visible in the Emdogain® preparations. In comparison, the native, unprocessed recombinant porcine amelogenin migrated as a single band with a mass of 25 kDa. Mr, molecular-weight markers x 10-3, are indicated.

View larger version (21K): [in a new window]   Figure 2. Amelogenin and Emdogain® promote cell adhesion. Plates were coated with the indicated amounts of amelogenin or Emdogain®. Cell adhesion assays were carried out as described in the text, with MG63 cells, and results are presented as the means and standard deviations of 3 determinations. Amelogenin and Emdogain® have similar dose-response characteristics in their ability to support MG63 cell attachment. The concentrations of amelogenin and Emdogain® stock solutions were checked by the BCA assay (Pierce Chemical Co., Rockford, IL, USA), which indicated that the basis of the small difference between amelogenin and Emdogain® dose-response curves was not due to the amount of protein added.

View larger version (115K): [in a new window]   Figure 3. Amelogenin promotes both cell attachment and cell spreading. Using the cell adhesion assay described in MATERIALS & METHODS, we found MG63 cells to attach to and spread on plastic substrata coated with 100 µg/plate Emdogain® (Panel A), 100 µg/plate amelogenin (Panel B), 100 µg/plate fibronectin (Panel C), and control (heat-inactivated BSA only) (Panel D). Note that the rounded cells in Panels A-C were attached well enough to resist washing of the plate, while the rounded cells in the control (Panel D) would have been removed if the plate had been washed.

View larger version (17K): [in a new window]   Figure 4. Requirement for divalent cations. Using minimal attachment medium lacking divalent cations, we studied the effect of Ca2+ (filled circles) or Mg2+ (squares) on amelogenin-mediated cell attachment. The results presented are the means and standard deviations of 3 determinations. Both Ca2+ and Mg2+ promoted cell attachment in a dose-response fashion at concentrations up to 10 mM, beyond which cell attachment decreased. This result is similar to findings for the divalent cation dependence of fibronectin-mediated cell attachment (Klebe et al., 1977).