|
Sign In to gain access to subscriptions and/or personal tools.
|
Odontoblasts Enhance the Maturation of Enamel Crystals by Secreting EMSP1 at the Enamel-Dentin Junction
M. Fukae1,*,
T. Tanabe1,
T. Nagano2,
H. Ando1,
Y. Yamakoshi1,
M. Yamada3,
J.P. Simmer4 and
S. Oida1
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;
Correspondence: * corresponding author, fukae-m{at}tsurumi-u.ac.jp
 |
ABSTRACT
|
|---|
The temporal expression patterns and activity distributions of enamelysin and EMSP1, which are the major proteinases in immature enamel, were characterized. Extracellular matrix fractions from developing porcine incisors, individually comprised of predentin, dentin, and four secretory-stage enamel samples, including the highly mineralized enamel (HME) at the enamel-dentin junction (EDJ), were isolated, and their resident proteinases were identified by zymography. Soft-tissue fractions, which included cells from the extension site of enamel formation (ESEF), secretory- and maturation-stage ameloblasts, and odontoblasts, were characterized histologically and by RT-PCR for their expression of enamelysin and EMSP1. A significant finding was that EMSP1, expressed by odontoblasts, concentrates in the HME, but is not detected in predentin or dentin. We conclude that odontoblasts deposit EMSP1 via their cell processes into the deepest enamel layer, which facilitates the hardening of this layer and contributes significantly to the functional properties of the EDJ.
Key Words: amelogenesis • highly mineralized enamel at EDJ • odontoblasts • enamelysin • EMSP1 • KLK4
|
INTRODUCTION
|
|---|
During the secretory stage of amelogenesis, ameloblasts secrete an enamel matrix containing a large quantity of structural proteins, including amelogenin, enamelin (Hu et al., 1997b) and sheathlin (ameloblastin/amelin) (Cerny et al., 1996; Krebsbach et al., 1996; Hu et al., 1997a), and small amounts of proteinases, such as enamelysin (MMP-20) (Fukae et al., 1998) and enamel matrix serine proteinase 1 (EMSP1) (Simmer et al., 1998). One apparent function of proteolysis during the secretory stage is to degrade matrix proteins and produce the space for crystal growth. Thus, some loss of protein matrix occurs during the secretory stage (Fukae and Shimizu, 1974), but most occurs during the transition and early maturation stages (Burgess and Maclaren, 1965; Fukae and Shimizu, 1974; Robinson et al., 1988; Uchida et al., 1991).
During crown formation, enamel and dentin form in an extracellular space lined by ameloblasts on one side and odontoblasts on the other. After the mineralization of enamel initiates on the mineralized dentin, secretory ameloblasts continue to secrete matrix as they move away from the enamel-dentin junction (EDJ). At the secretory-stage enamel, there is a decrease in protein content, and a corresponding increase in mineral going from the surface to the EDJ (Fukae and Shimizu, 1974), which is manifested by a progressive thickening of the crystallites with depth (Miake et al., 1993). In addition, highly mineralized enamel (HME) exists as a narrow layer along the EDJ (Suga, 1983). Ameloblasts secrete the structural matrix into the space that becomes the HME, including amelogenin (Uchida et al., 1989), enamelin (Uchida et al., 1991), sheathlin (Uchida et al., 1995), and tuft protein (Robinson et al., 1975). EMSP1 is not detected in the early secretory enamel corresponding to the future HME (Fukae et al., 2001), but EMSP1 is expressed by odontoblasts prior to its onset of expression in transition-stage ameloblasts in the mouse enamel organ (Hu et al., 2000). It is unclear how the HME becomes so hard, positioned as it is in contact with the dentin surface and away from the ameloblast layer.
To better understand the stage-specific expression and distribution of proteinase activities in developing teeth, we have isolated specific soft and hard tissues from crown-stage pig incisors and assayed them for the presence of enamelysin and EMSP1 mRNA by RT-PCR, and for their gelatinolytic and caseinolytic proteinase activities, respectively. A surprising finding is that EMSP1 is specifically deposited by odontoblasts, presumably by secretion through their odontoblastic processes, into the newly deposited enamel layer covering mantle dentin.
|
MATERIALS & METHODS
|
|---|
All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Care Program at the Tsurumi University.
Tissue Preparation
Unless otherwise stated, all preparation and extraction procedures were carried out at 4°C or under ice-cold conditions. The fresh mandibles of six-month-old pigs (an edible hybrid of Landrace, Large White, and Duroc) were purchased from the slaughterhouse and carried to the laboratory under ice-cold conditions. Permanent incisor tooth germs were dissected carefully from the mandible, and secretory-, transition-, and early-maturation-stage enamel samples were separately scraped from the smooth labial surface (Fukae and Shimizu, 1974). Outer, outer-inner, and inner layer enamel samples, which corresponded to approximately 0 30 µm, 3060 µm, and more than 60 µm, respectively, from the surface of secretory-stage enamel, were also prepared as described elsewhere (Tanabe et al., 1992; Fukae and Tanabe, 1998). After scraping the inner layer enamel, we used a dental excavator to scrape the thin, harder enamel layer associated with the enamel-dentin junction (EDJ). We refer to this layer, which has a thickness of approximately 20 µm, as highly mineralized enamel (HME). Since some dentin was possibly removed along with the HME, dentin under the HME was also scraped as a control. Predentin samples were prepared by a method described previously (Fukae et al., 1994).
Various cell layer samples were carefully dissected from the labial side of porcine developing tooth germs according to the divisions shown in Fig. 1a . The extension sites of enamel formation (ESEF) samples were obtained from the cervical margin (approximately 1 mm) of crown-stage incisor tooth germs as described elsewhere (Fukae et al., 2001). The ESEF refers to the point where more functional ameloblasts are being recruited.
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.
|
|
Several cell layer samples were prepared from the permanent incisor tooth germs. We carefully removed the outer enamel epithelium covering the labial side, to avoid injury to the underlying ameloblast cell layer, which we then divided into two pieces by cutting transversely at the beginning of the transition stage using a surgical blade. Two separate ameloblast samples were obtained (Oida et al., 2002). The enamel organ epithelia covering (1) secretory-stage enamel and (2) transition- and early-maturation-stage enamel were collected, and designated as the secretory- and maturation-stage ameloblast cell layers, respectively (Fig. 1a ).
The odontoblast cell layers were prepared in the following manner. After the removal of surrounding soft tissues, the dental papilla was pulled out carefully, and the cervical margin of the tooth was cut off with a surgical blade. The tooth was then cut transversely at the transition-stage margin into two pieces. We separately collected the odontoblast cell layers (Schiess et al., 1966; Munksgaard et al., 1978), which remained on a predentin surface of the two halves, by scraping with a dental spatula.
Extraction of Proteins
The soluble proteins of HME samples were extracted by gentle agitation in 0.5 M acetic acid for 2 hrs at 4°C to minimize contamination from dentin. The soluble proteins of the ESEF were extracted in the same way. The predentin and dentin samples were extracted for total soluble protein with 4 M guanidine solution (0.05 M Tris-HCl, pH 7.4), respectively, although the dentin sample was demineralized in 0.5 M acetic acid before the extraction (Fukae et al., 1994). These extracts were de-salted by ultrafiltration through a YM-3 membrane, and lyophilized.
RNA Preparation and Reverse-transcription/ Polymerase Chain-reaction (RT-PCR)
Total RNA was isolated from each tissue sample by the guanidinium thiocyanate-phenolchloroform method (Chomczynski and Sacchi, 1987). Primer sets were designed to anneal to the porcine EMSP1 and enamelysin cDNA sequences. The EMSP1 sense and antisense primers were 5'-ATAAACGGCGAGGACTGCAA(162-181) and 5'-GTTAACTGGCCTGGATGGTCG(834-854). The enamelysin sense and antisense primers were 5'-ATACGTGCAGCGAATAGATGC(1180-1200) and 5'-CTATTTAGCAACCAATCCAGG(1469-1489). A GAPDH primer set (Clontech Laboratories, Inc., Palo Alto, CA, USA) was used as a control. RT-PCR was carried out with the use of a GENEAmp RNA PCR Kit and protocol (Perkin Elmer, Norwalk, CT, USA).
Histochemistry
Each tissue sample was immersed in a mixture of 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) or 10% formalin for 3 days. After fixation, the specimens were embedded in Epon or paraffin. The mineralized tissues were demineralized before being embedded. Sections (4 µm thick) were mounted on glass slides and stained with toluidine blue or Mayer’s hematoxylin and eosin. The HME and odontoblast cell layer samples, before being scraped, were examined in the same way.
Analytical Methods
Nucleotide sequences of PCR products were determined by means of an automated double-stranded dideoxy sequencing system (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England) and a thermostable DNA polymerase (BcaBEST, Takara. Otsu, Shiga, Japan). For chemical composition analyses, the samples’ fresh wet weights were measured in 100% humidity, and then dried to a constant weight. The dried samples were hydrolyzed in 6 N HCl in an evacuated sealed glass tube at 110°C for 24 hrs. The hydrolysate was analyzed for calcium, phosphorus, and amino acid composition. The total protein content was calculated based upon the amino acid compositions. Collagen content was calculated bsaed on the hydroxyproline content. The volume of each sample was calculated using the densities of water, protein, and mineral, which are 1, 1.35, and 3.0 gm/cm3, respectively. The density of enamel protein was obtained from the specific volume of its amino acid contents (McMeekin and Marshall, 1952).
Zymography
Gelatin and  -casein zymograms were carried out for the detection of EMSP1 and enamelysin, respectively. Each enamel sample was extracted for EMSP1 activity in Sorensen buffer (pH 7.4), by homogenization with Polytron homogenizer for 60 sec at a speed of 6000 rpm. After the supernatant was separated, the pellet was re-suspended in the same volume of 0.05 M carbonate buffer (pH 10.8) and processed in the same way to yield the alkaline-soluble fraction, in which almost all enamelysin activities were recovered (Fukae and Tanabe, 1998).
|
RESULTS
|
|---|
Reverse transcription (RT) was performed on each cell layer sample, and amplified by primers for GAPDH. The GAPDH amplification products were used to normalize the amount of RT product from each cell layer sample. These samples were then amplified for 30 cycles using primers specific for EMSP1 and enamelysin cDNA, and then analyzed by agarose gel electrophoresis (Figs. 1b, 1c). The EMSP1 amplification product was found in the maturation-stage ameloblast and odontoblast samples, but not in the ESEF and secretory ameloblast samples. In the 35-cycle amplifications, a small amount of the EMSP1 PCR product was detected in the secretory ameloblast sample, but not in the ESEF sample. In contrast, the enamelysin PCR product could be detected in all samples, although the amount was low in the ESEF and maturation-stage ameloblast samples. The EMSP1 and enamelysin PCR products from the ameloblast and odontoblast samples were the same by the determination of their DNA sequencing.
Each tissue sample was examined histologically (Fig. 2). The ESEF sample contained predentin, dentin, and a small amount of immature enamel. It also contained numerous cells, but no differentiated ameloblasts (Fig. 2a). The secretory ameloblast sample contained not only secretory ameloblasts, but also from 4 to 6 cell layers of stratum-intermedium-like cells along with a thick, loose connective tissue (Fig. 2b). In contrast, the odontoblast samples contained mostly odontoblasts (Fig. 2d). The HME was ca. 10 to 20 µm deep on the uneven surface of the dentin (Fig. 2c). The chemical composition of the HME was determined to be 9.2% protein, 33% calcium, and 12.5% phosphorus per dry weight, based upon the assumption that the collagen in the sample was derived from dentin. The protein content is quite different from the 28.8% protein of the inner layer enamel sample (Fukae and Tanabe, 1998). The HME contained mainly serum-like proteins, along with a trace amount of amelogenins (data not shown).
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.
|
|
After normalizing the volumes of each secretory enamel sample, we compared proteolytic activities at progressive depths in developing enamel by zymography (Fig. 3). EMSP1 activity, which corresponds to 30- and 34-kDa bands (Tanabe, 1984; Simmer et al., 1998), was detected in the neutral soluble fraction of the inner layer enamel sample (Fig. 3a, lane 3) (Tanabe et al., 1992), and was not detected in the alkaline-soluble fraction (data not shown). It was strong in the HME at the EDJ (Fig. 4a, lane 4). Enamelysin activity, which corresponds to 41- and 46-kDa bands (Fukae et al., 1998), was detected mostly in the alkaline-soluble fraction and throughout the entire depth of secretory-stage enamel, but the level of activity decreased in the inner-layer enamel.
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.
|
|
Proteinases in the ESEF, dentin, and predentin samples were also characterized by zymography (Fig. 4). No EMSP1 activity was detected in these tissues, although gelatinolytic activities enhanced by Ca ions were detected at 56 and 61 kDa. The ESEF sample showed the 41- and 46-kDa caseinolytic activities of enamelysin enhanced by Ca ions (Fig. 4b, lane 1). In addition to the enamelysin bands, strong 25- and 54-kDa activities were detected in the predentin sample (Fig. 4b, lane 2). The proteinase responsible for the 54-kDa activity is larger than enamelysin, and therefore cannot be generated from it by proteolysis. Since the enamelysin activity in the ESEF is not associated with the 54-kDa activity characteristic of the predentin sample, the enamelysin activity in the ESEF was contributed by the immature enamel itself, and was not derived from the predentin component, although the ESEF sample contained some predentin.
|
DISCUSSION
|
|---|
To gain a better understanding of when and where enamel proteinases are expressed, and where their activities localize in developing teeth, we carefully dissected crown-stage developing porcine permanent teeth into their component tissues, examined the slices histologically, and individually assayed them by RT-PCR and zymography. The results confirm previous findings concerning the temporal and spatial patterns of enamelysin and EMSP1 expression, but more significantly, we have characterized the relative activities of these proteinases at specific sites in the developing teeth. Enamelysin activity was detected throughout the entire thickness of secretory-stage enamel (in the outer, outer-inner, and inner enamel layers, as well as in HME covering mantle dentin), and its activity decreases with increasing depth. The early expression of enamelysin mRNA during murine dental differentiations, first shown by in situ hybridization (Bègue-Kirn et al., 1998), was confirmed by our RT-PCR analysis of the ESEF. Casein zymogram of the ESEF demonstrates the presence of active enamelysin at this early stage. Enamelysin expression increases during the secretory stage and is barely detected after the onset of the transition stage. In contrast, enamelysin mRNA is consistently detected at high levels in odontoblasts beneath secretory- and maturation-stage ameloblasts.
In the enamel organ, EMSP1 mRNA was detected in the maturation-stage ameloblast cell layer after 30 cycles of PCR amplification. After 35-cycle amplification, this mRNA was expressed in the secretory ameloblast cell layer. This supports that the pro-enzyme of EMSP1 is found in the outer layer enamel of the secretory stage (Tanabe et al., 1996). A large quantity of EMSP1 expression was determined by in situ hybridization in transition-stage ameloblasts, with continued expression throughout the maturation stage (Hu et al., 2000). Similarly, we confirm that EMSP1 is expressed by odontoblasts beneath secretory- and maturation-stage ameloblasts. An important new contribution by this study concerns the localization of EMSP1 activity during the secretory stage, and the source of this active proteinase. Active EMSP1 was not detected in the outer or outer-inner enamel layers of secretory-stage enamel. A small amount was detected in the inner enamel, while a reliably strong signal was evident in HME covering mantle dentin. These findings suggest that odontoblasts secrete EMSP1 via dentinal tubules into the EDJ, since EMSP1 activity was not found in either the predentin or dentin matrices. We conclude that EMSP1, by the common mechanism of degrading enamel proteins that control the thickening of enamel crystallites, is secreted by odontoblasts and by maturation-stage ameloblasts to facilitate the hardening of enamel.
|
ACKNOWLEDGMENTS
|
|---|
This investigation was supported in part by a grant-in-aid for Scientific Research, No. 11671859, from the Ministry of Education, Science and Culture of Japan.
Received for publication September 25, 2001.
Revision received June 18, 2002.
Accepted for publication July 24, 2002.
|
REFERENCES
|
|---|
- Bègue-Kirn C, Krebsbach PH, Bartlett JD, Butler WT (1998). Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiations. Eur J Oral Sci 106:963–970.[CrossRef][Medline]
[Order article via Infotrieve]
- Burgess RC, Maclaren CM (1965). Proteins in developing bovine enamel. In: Tooth enamel I. Stack MV, Fearnhead RW, editors. Bristol: John Wright & Sons Ltd., pp. 74-82.
- Cerny R, Slaby I, Hammarström L, Wurtz T (1996). A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains. J Bone Miner Res 11:883–891.[Medline]
[Order article via Infotrieve]
- Chomczynski P, Sacchi N (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.[Medline]
[Order article via Infotrieve]
- Fukae M, Shimizu M (1974). Studies on the proteins of developing bovine enamel. Arch Oral Biol 19:381–386.[CrossRef][Medline]
[Order article via Infotrieve]
- Fukae M, Tanabe T (1998). Degradation of enamel matrix proteins in porcine secretory enamel. Connect Tissue Res 39:123–129.[Medline]
[Order article via Infotrieve]
- Fukae M, Tanabe T, Yamada M (1994). Action of metalloproteinases on porcine dentin mineralization. Calcif Tissue Int 55:426–435.[Medline]
[Order article via Infotrieve]
- Fukae M, Tanabe T, Uchida T, Lee SK, Ryu OH, Murakami C, et al. (1998). Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis. J Dent Res 77:1580–1588.
- Fukae M, Tanabe T, Yamakoshi Y, Yamada M, Ujiie Y, Oida S (2001). Immunoblot detection and expression of enamel proteins at the apical portion of the forming root in porcine permanent incisor tooth germs. J Bone Miner Metab 19:236–243.[CrossRef][Medline]
[Order article via Infotrieve]
- Hu CC, Fukae M, Uchida T, Qian Q, Zhang CH, Ryu OH, et al. (1997a). Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins. J Dent Res 76:648–657.
- Hu CC, Fukae M, Uchida T, Qian Q, Zhang CH, Ryu OH, et al. (1997b). Cloning and characterization of porcine enamelin mRNAs. J Dent Res 76:1720–1729.
- Hu CC, Ryu OH, Chen JJ, Uchida T, Wakida K, Murakami C, et al. (2000). Cloning of the murine EMSP1 cDNA and analysis of expression during tooth development. J Dent Res 79:70–76.
- Krebsbach PH, Lee SK, Matsuki Y, Kozak CA, Yamada K, Yamada Y (1996). Full-length sequence, localization, and chromosomal mapping of ameloblastin: a novel tooth-specific gene. J Biol Chem 271:4431–4435.[Abstract/Free Full Text]
- McMeekin T, Marshall K (1952). Specific volumes of proteins and the relationship to their amino acid contents. Science 116:142–143.[Free Full Text]
- Miake Y, Shimoda S, Fukae M, Aoba T (1993). Epitaxial overgrowth of apatite crystals on the thin-ribbon precursor at early stages of porcine enamel mineralization. Calcif Tissue Int 53:249–256.[Medline]
[Order article via Infotrieve]
- Munksgaard EC, Rhodes M, Mayne R, Butler WT (1978). Collagen synthesis and secretion by rat incisor odontoblasts in organ culture. Eur J Biochem 82:609–617.[Medline]
[Order article via Infotrieve]
- Oida S, Nagano T, Yamakoshi Y, Ando H, Yamada M, Fukae M (2002). Amelogenin gene expression in porcine odontoblasts. J Dent Res 81:103–108.
- Robinson C, Lowe NR, Weatherell JA (1975). Amino acid composition, distribution and origin of "tuft" protein in human and bovine dental enamel. Arch Oral Biol 20:29–42.[CrossRef][Medline]
[Order article via Infotrieve]
- Robinson C, Kirkham J, Weatherell JA, Richards A, Josephsen K, Fejerskov O (1988). Mineral and protein concentrations in enamel of the developing permanent porcine dentition. Caries Res 22:321–326.[Medline]
[Order article via Infotrieve]
- Schiess AV, Okigaki T, Rounds DE (1966). Human odontoblasts in vitro. I. The harvest of homogeneous cell populations. J Dent Res 45:1101–1104.
- Simmer JP, Fukae M, Tanabe T, Yamakoshi Y, Uchida T, Xhu J, et al. (1998). Purification, characterization, and cloning of enamel matrix serine proteinase 1. J Dent Res 77:377–386.
- Suga S (1983). Comparative histology of the progressive mineralization pattern of developing enamel. In: Mechanisms of tooth enamel formation. Suga S, editor. Tokyo: Quintessence Publishing Co. Inc., pp. 167-203.
- Tanabe T (1984). Purification and characterization of proteolytic enzymes in porcine immature enamel. Tsurumi Shigaku 10:443–452.[Medline]
[Order article via Infotrieve]
- Tanabe T, Fukae M, Uchida T, Shimizu M (1992). The localization and characterization of proteinases for the initial cleavage of porcine amelogenin. Calcif Tissue Int 51:213–217.[CrossRef][Medline]
[Order article via Infotrieve]
- Tanabe T, Fukae M, Shimizu M (1996). Possible actions of metalloproteinases found in porcine enamel in an early secretory stage. Adv Dent Res 10:170–172.[Abstract/Free Full Text]
- Uchida T, Tanabe T, Fukae M (1989). Immunocytochemical localization of amelogenins in the deciduous tooth germs of the human fetus. Arch Histol Cytol 52:543–552.[Medline]
[Order article via Infotrieve]
- Uchida T, Tanabe T, Fukae M, Shimizu M (1991). Immunocytochemical and immunochemical detection of a 32 kDa nonamelogenin and related proteins in porcine tooth germs. Arch Histol Cytol 54:527–538.[Medline]
[Order article via Infotrieve]
- Uchida T, Fukae M, Tanabe T, Yamakoshi Y, Satoda T, Murakami C, et al. (1995). Immunochemical and immunocytochemical study of a 15 kDa non-amelogenin and related proteins in the porcine immature enamel: proposal of a new group of enamel proteins sheath proteins. Biomed Res 16:131–140.
Journal of Dental Research, Vol. 81, No. 10,
668-672 (2002)
DOI: 10.1177/154405910208101003
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter What's this?
This article has been cited by other articles:
|
|
|
|
 
J. P. Simmer, Y. Hu, R. Lertlam, Y. Yamakoshi, and J. C.-C. Hu
Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice
J. Biol. Chem.,
July 10, 2009;
284(28):
19110 - 19121.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Yamakoshi, J. C-C. Hu, T. Iwata, K. Kobayashi, M. Fukae, and J. P. Simmer
Dentin Sialophosphoprotein Is Processed by MMP-2 and MMP-20 in Vitro and in Vivo
J. Biol. Chem.,
December 15, 2006;
281(50):
38235 - 38243.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J-W Kim, J P Simmer, T C Hart, P S Hart, M D Ramaswami, J D Bartlett, and J C-C Hu
MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta
J. Med. Genet.,
March 1, 2005;
42(3):
271 - 275.
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION