• International Journal of Oral Biology
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• v.43 no.4
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• pp.177-183
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• 2018

The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary $2^{nd}$ molar germs of rats. We used the maxillary $2^{nd}$ molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary $2^{nd}$ molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.

• Applied Microscopy
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• v.46 no.1
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• pp.58-66
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• 2016

Thymosin ${\beta}4$ ($T{\beta}4$) has been recently reported to play a role in dentinogenesis by regulating the expression of dentin matrix proteins. Based on previous studies, it is hypothesized that $T{\beta}4$ is associated with the formation of the enamel matrix and thus plays an important role in ameloblast. However, there is no report on the function of $T{\beta}4$ during tooth development so far. Therefore, in this study, we aimed to investigate the expression of $T{\beta}4$ and its function in ameloblasts during mouse tooth development. $T{\beta}4$ was expressed strongly in the tooth bud at the bud stage and in the dental lamina and oral epithelium at the cap stage. In advanced bell stage at postnatal day 4, large elongated ameloblasts were observed and the expression of the $T{\beta}4$ protein was the highest, with the enamel being was thicker than that in the early bell stage. The length of ameloblasts increased from the presecretory to the secretory stage and decreased from the maturation to the protective stage. These results suggest that $T{\beta}4$ participates not only in the proliferation of oral epithelial cells during the early stage of tooth development but also regulates enamel protein secretion in ameloblasts and enamel mineralization.

• Applied Microscopy
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• v.34 no.2
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• pp.145-157
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• 2004

The aim of the present study was to examine in detail, both at light and electron microscopical levels, the morphological variations in ameloblast of the fetal rat incisor enamel organ. Rats were started on distilled water at the beginning of pregnancy. The pups were sacrificed 11 days after delivery and animals were perfused intravascularly with glutaraldehyde and the incisors were removed. To examine on the ultrastructure of the ameloblast, the study employed primary light microscopy but electron microscopy was used to clarify some of the light microscopic finding. Longitudinal sections through the incisors of the rat show a continuous layer of ameloblasts on the labial surface of the tooth. This layer contains the entire sequence of developmental stages in enamel production. The ameloblast layer was divided into three main zones: 1) Presecretory zone, region of ameloblasts facing pulp. 2) Secretory zone, region of inner and outer enamel secretion. 3) Maturation zone, region of reduced ameloblasts. In particularly, the present study has shown that two distinctively different types of ameloblasts appear in the enamel organ during enamel maturation in the rat incisor. These two types have been designated ruffle-ended ameloblasts (rAB) and smooth-ended ameloblasts (sAB). The fluoride produces marked alteration in the fine structure of ameloblast from teeth of young rats, such as large confluent distensions of the endoplasmic reticulum and swelling of isolated mitochondria. This experimental data suggested that exposure prolonged of animal to high level of fluoride appears to induce a few dramatic changes in the normal appositional growth and initial mineralization of enamel created during amelogenesis.

• International Journal of Oral Biology
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• v.41 no.2
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• pp.89-96
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• 2016

Tooth development shows dynamic morphological changes from the stages of cap to hard tissue formation and is strictly regulated during development. In the present study, we compared expression and localization of 3 major enamel matrix proteins in rats: amelogenin, enamel and ameloblastin. DD-PCR and RT-PCR revealed differential expression of the major proteins from the cap stage to root stage. Immunofluorescence staining results indicated that amelogenin was not detected in either inner enamel epithelium or reduced enamel epithelium, but highly immunoreactive in preameloblasts and ameloblasts; in addition, it was sporadically expressed in preodontoblasts abutting preameloblasts. Ameloblastin expression was also observed in not only differentiated ameloblasts but also osteoblasts. Immunoreactivity to ameloblastin in ameloblasts was strong in Tomes' processes. Enamelin was exclusively localized along the entire newly formed and maturing enamel. Enamelin was largely localized in near Tomes' processes and enamel rods in maturing enamel. Alendronate treatment resulted in down-regulation of amelogenin and ameloblastin at both transcription and translation levels; whereas, enamelin expression was unchanged in response to the treatment. These results suggested that amelogenin, ameloblastin and enamelin might be implicated in cell differentiation, adhesion of ameloblasts to enamel and enamel crystallization during enamel matrix formation, respectively.

• International Journal of Oral Biology
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• v.36 no.4
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• pp.195-201
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• 2011

Matrix metalloproteinases (MMPs) have been implicated in tissue development and re-modeling. Dynamic morphological changes of tooth germs reflect involvement of these enzymes during odontogenesis. The present study was performed to investigate expression and localization of MMP-2 and MMP-9, which have been known to have type IV collagenase activities, in rat tooth germs at different developmental stages. MMP-2 expression was increased gradually in the tooth germs from cap to crown staged germs at both transcription and translation levels. The localization of this molecule was detected in secretory ameloblasts and preameloblasts. The strong immunoreactivities were occasionally seen along the basement membrane between ameloblasts (or preameloblasts) and odontoblasts (preodontoblasts). However, weak reactivity was detected in odontoblasts and reduced enamel epithelium. The level of MMP-9 expression in the tooth germs was higher in cap stage than in crown staged germs at both transcription and translation levels. They were strongly expressed in both ameloblasts and odontoblasts. Even though reduced enamel epithelium after enamel formation and inner enamel epithelium at the cap stage exhibited weak reactivity, strong reactivity was detected in dental follicles and perifollicular tissues surrounding cap staged germs. These results suggested that MMP-2 may involve degradation of the basement membrane during hard tissue formation, whereas MMP-9 might be involved in remodeling of follicular tissues.

• Journal of the korean academy of Pediatric Dentistry
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• v.11 no.1
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• pp.131-143
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• 1984

150 rats weighting about 150gm were devided into control group of 80 and experimental group of 70. Control group was subdivided into the irradiated vitamin D injection group and X-ray irradiated group. Experimental group was given 2.0mg ergocalciferol by four intramuscular injection prior to X-ray irradiation with single 800 rads and 1,500 rads respectively. Experimental animals from each group was sacrificed after 1, 3, 7, 14, and 28 days and their incisors were investigated by histopathological examination. The results were as follows; 1. In the irradiated groups, it showed dentin hypoplasia and formation of dentinoid substance caused by degeneration of odontoblast at the early stage. Especially, 1,500 rads group which was severely effected showed formation of osteoid dentin at the apical portion and severe injuries of dental papilla at the first week. 2. In the vitamin D2 administration group, it showed thinned dentin layer at the early stage but, taking time, predentin and dentin layer was thickened. At the fourth week, dentin was chiefly composed of interglobular dentin, especially in the lingual portion. 3. Using in combination of overdose vitamin D2 administration and X-ray irradiation, it effected severely odontoblast, undifferentiated mesenchymal cells around tooth germ and pulp tissue. At the early stage, dentin layer was thinned but, taking time, it was thickened and composed of interglobular dentin caused by calcification of predentin layer. 4. In 800 rads irradiation after the overdose vitamin D2 administration, it showed formation of osteoid dentin in the lingual portion at the first week. In the 1,500 rads irradiation after the overdose vitamin D2 administration, it showed formation of osteoid dentin and degeneration of ameloblast in both buccal and lingual portion at the first week, and enamel hypoplasia caused by edema and loss of polarity of ameloblasts at the second week. 5. By the entire experiment, the overdose vitamin D2 administration and X-ray irradiation effected severely odontoblasts, undifferentiated mesenchymal cells of dental papilla, and primitive cells of tooth germ among the dental tissue. Especially using combination of overdose vitamin D2 administration and X-ray irradiation also effected ameloblasts, resulting in enamel hypoplasia.

• Journal of the korean academy of Pediatric Dentistry
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• v.31 no.4
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• pp.651-658
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• 2004

Runx2 is a transcription factor in homologous with Drosophila runt gene and it is essential for bone formation during embryogenesis and a critical gene for osteoblast differentiation and osteoblast function. Runx2-haploinsufficency causes cleidocranial dysplasia (CCD). CCD is an autosomal-dominant inherited disorder characterized by hypoplastic clevicle and delayed ossification in fontanelles and wormian bones. Dental defects are possibly shown to CCD patients : multiple supernumerary teeth, irregular and compressed permanent tooth crowns, hypoplastic and hypomineralized defects in enamel and dentin, an excess of epithelial root remnants, the absence of cellular cementum, and abnormally shaped roots. In addition, delayed eruption of the secondary dentition is a constant finding. The aim of this study is to evaluate the role of Runx2 in the tooth development and eruption through analyzing the expression pattern of Runx2 by in situ hybridization during crown (late bell stage) and root formation of tooth, using postnatal day 1, 4, 7, 14 and 21 mice mandibular molar teeth. mRNA of Runx2-full length is expressed in dental follicle and surrounding tissue at postnatal day1 and 4. At postnatal day 7, it is expressed in ameloblasts of occlusal surface of enamel and bone area surrounding the tooth. In comparison with previous stage, at postnatal day 14, it is expressed in ameloblasts of proximal surface of enamel. At postnatal day 21 it's expression is observed only in bone area. mRNA of Runx2-typeII is not expressed. At postnatal day 1 and 7. At postnatal day 14 and 21, it's expression is observed in the bone area. In this study, we suggest that Runx2 have a relation of ameloblasts differentiation and an important role to tooth eruption made by dental follicle during intraosseous eruption stage. Also we can confirm that Runx2 has a role to bone formation.

• Journal of dental hygiene science
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• v.8 no.2
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• pp.89-96
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• 2008

Effects of sodium fluoride exposure on the amelogenesis during fetal formation were investigated using 11 days rat incisor of control group and two experimental groups. According to results of morphological analysis using an electron microscope, enamel organ in the rat incisor consisted of presecretory, secretory, and maturation zone, especially maturation zone had ruffle-ended ameloblasts (rAB) that additionally supply inorganic ions and smooth-ended ameloblasts (sAB) that remove water and organic compounds. Such a histological composition was same in fetal and adult rats. According to experimental results using calcein (green fluorescence) in order to reveal the modulation cycle of ameloblast, modulation cycle of experimental group decreased on an average one time than control group, as increase of density of sodium fluoride indicated that thickness of smooth-ended ameloblast decreased. Also ratio of thickness on sagittal total length of sAB increased than rAB in experimental groups than control group. In total length of teeth, an injected 100 ppm sodium fluoride group was similar control group but as injected 200 ppm group became short. In experimental group, thickness of sAB and rAB became narrow to the tip of cutting edge. According to concentration of sodium fluoride grows, the modulation cycle and total length of teeth were decreased, finally it prevented teeth growth.

• Development and Reproduction
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• v.17 no.3
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• pp.215-220
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• 2013

Recently, the RNA/DNA-binding protein FUS, Fused in sarcoma, was shown to play a role in growth, differentiation, and morphogenesis in vertebrates. Because little is known about Fus, we investigated its expression pattern in murine tooth development. In situ hybridization of mouse mandibles at specific developmental stages was performed with a DIG-labeled RNA probe. During early tooth development, Fus was detected in the dental epithelium and dental mesenchyme at 11 days postcoitum (dpc) and 12 dpc. From 14 dpc, Fus was strongly expressed in the dental papilla and the cervical loop of the dental epithelium. At postnatal day 4 (PN4), Fus expression was observed in the odontoblasts, ameloblasts, the proliferation zone of the pulp, and the cervical loop. At PN14, the expression pattern of Fus was found to be maintained in the odontoblasts and the proliferation zone of the pulp. Furthermore, Fus expression was especially strong in the Hertwig's epithelial root sheath (HERS). Therefore, this study suggests that Fus may play a role in the HERS during root development.

• International Journal of Oral Biology
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• v.34 no.3
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• pp.143-150
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• 2009

Amino acid transporters play important roles in supplying nutrients to cells. In our current study, we investigated the expression of LAT1 and measured the amino acid uptake in ameloblast cultures to further elucidate the roles of this transporter during the differentiation of these cells. RT-PCR, observations of cell morphology, Alizaline red-S staining, and uptake analyses were performed following the experimental induction of differentiation in the cultures. LAT1 mRNA was detectable and found to gradually increase over time whereas LAT2 mRNA was not evident in the ameloblast cultures. Transcripts of 4F2hc, a cofactor of LAT1 and LAT2, were also found to be expressed in ameloblast cultures and increase with time. Amelogenin mRNA was expressed in the early stage ameloblast cultures. L-leucine uptake was observed to increase over 14 days of growth in culture. Our data suggest that LAT1 has a key role in the differentiation of ameloblasts and in providing these cells with neutral amino acids, including several essential amino acids.