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Odontoblasts Enhance the Maturation of Enamel Crystals by Secreting EMSP1 at the Enamel-Dentin Junction

¹ Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan;
² Department of Periodontology, School of Dental Medicine, Tsurumi University, Yokohama, Japan;
³ Department of Physical Therapy, School of Health Science, Niigata University of Health and Welfare, 3198 Shimami-cho, Niigata 950-3198, Japan; and
⁴ University of Texas Health Science Center at San Antonio, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA;

View larger version (47K): [in this window] [in a new window]   Figure 1. Schematic divisions of the ESEF and cell layer samples prepared from the labial side of a porcine incisor crown-stage tooth germ (a) and RT-PCR products of these samples, using the primers for EMSP1 (b) and enamelysin (c). 1, ESEF; 2, secretory ameloblast cell layer; 3, maturation-stage ameloblast cell layer; 4, odontoblast cell layer at the matrix formation stage; and 5, odontoblast cell layer at the maturation stage. Key for abbreviations: HME, highly mineralized enamel; EDJ, enamel-dentin junction; S, molecular standard.

View larger version (93K): [in this window] [in a new window]   Figure 2. Light micrograph of the ESEF, HME, and cell layer samples prepared according to the divisions shown in Fig. 1a. The length of the ESEF sample is approximately 1 mm. (a) The ESEF sample contained cells from the inner enamel epithelium, stratum intermedium, odontoblasts, and dental papilla cells. (b) The secretory ameloblast sample contained no outer enamel epithelium, which is not shown in the magnified micrograph. (c) HME sample was scraped approximately 30 µm by means of a sharp dental excavator. Approximately 22% of the sample weight was HME, based upon the assumption that the collagen in the sample was derived from dentin. (d) Odontoblast cell layer sample was prepared by scraping with a blunt, small spatula from the cells on predentin as shown in the Fig. Key for panels: a, ESEF; b, secretory ameloblast cell layer; c, HME; d, odontoblast cell layer. Key for abbreviations: IE, immature enamel; PD, predentin; OB, odontoblast; D, dentin; AB, ameloblast; EDJ, enamel-dentin junction.

View larger version (70K): [in this window] [in a new window]   Figure 3. Gelatin (a) and casein (b) zymograms showing the relative proteolytic activities in equal volumes of porcine secretory enamel samples obtained at different depths: outer enamel (lane 1), outer-inner enamel (lane 2), and inner enamel (lane 3). The gel of casein zymogram was incubated in 2 mM calcium. The volumes for 100 mg of outer, outer-inner, and inner layer enamel samples were calculated to be 75 µL, 68 µL, and 64 µL, respectively. A 10-µL quantity of each enamel sample was extracted sequentially with 180 µL of Sorensen buffer (pH 7.4) and 0.05 M carbonate buffer (pH 10.8). We prepared samples for electrophoresis by adding the equal volume of 4% SDS/2% sucrose solution to each extracted solution. Equal proportions of each sample volume were applied to the zymograms. For the gelatin and casein zymograms, 20 µL of sample solution prepared from the neutral-soluble fraction and 10 µL of sample solution from the alkaline-soluble fraction, respectively, were applied to each well.

View larger version (80K): [in this window] [in a new window]   Figure 4. Gelatin (a) and casein (b) zymograms for ESEF (1), predentin (2), dentin (3), and HME (4) samples prepared from the porcine permanent tooth germs. The electrophoresed gels were incubated with 2 mM Ca (+) or 2 mM EDTA (-). The EDTA chelates metal ions and inactivates matrix metalloproteinases. The volumes of these samples applied to wells were almost the same as that of the neutral-soluble fraction of the inner layer enamel sample as shown in Fig. 3.

Journal of Dental Research, Vol. 81, No. 10, 668-672 (2002)
DOI: 10.1177/154405910208101003


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