dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilladerived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of 1 x 10(6) cells/dish in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.]]>
Alkaline Phosphatase ; Ascorbic Acid ; Bone Matrix ; Dental Papilla ; Dental Sac ; Dentin ; Dexamethasone ; Enamel Organ ; Humans ; Mandible ; Molar, Third ; Osteoblasts ; Osteocalcin ; RNA, Messenger ; Stem Cells ; Tooth ; Tooth Germ ; Trypsin ; Young Adult

OBJECTIVETo study role of dental follicle in tooth root development.

METHODSSixteen mandibular first molar dental germs from eight five-day postnatal Balb/c mice were divided into two groups randomly. Dental follicle of germs in one group was undetached and that of another group was removed. Subsequently, each of the germs was separately transplanted to back-muscles of adult nude mice. At seventh and fourteenth day after transplanting, the germs were collected, fixed, demineralized, dehydrated, and embedded in wax in sequence. Serial sections of 5 microm thick were made following the routine methods, stained with haematoxylin-eosin dying solution, and observed under a light microscope.

RESULTSAll implantations were located in the back-muscles with abundant capillary vasculature. Under microscope, although all tooth germs could further develop after grafting, tooth germs without dental follicle developed slowly with small size and low calcification compared to those with dental follicle. Although position of Hertwig's epithelial root sheath of all germs seemed no changing, roots of the group with dental follicle could further develop and the roots develop toward the apical direction; this tendency couldn't be seen in the germs of another group. Inflammatory cells could be seen in and out of the pulp cavity of the two groups at 7th day after grafting, while no obvious inflammatory cell was observed at 14th day after grafting.

CONCLUSIONDental follicle play an important role in tooth root development. It probably can lead tooth root to develop in normal direction.

Animals ; Dental Pulp Cavity ; Dental Sac ; Enamel Organ ; Mice ; Mice, Nude ; Molar ; Odontogenesis ; Tooth ; Tooth Germ ; Tooth Root

PURPOSE: To investigate the expression of bone morphogenetic protein(BMP)-2/4 during eary tooth development after irradiation and calcium-deficient diet. MATERIALS AND METHODS: The pregnant three-week-old Sprague-Dawley rats were used for the study. The control group was non-irradiation/normal diet group(Group 1), and the experimental groups were irradiation/normal diet group(Group 2) and irradiation/calcium-diet group(Group 3). The abdomen of the rats at the 9th day of pregnancy were irradiated with single dose of 350 cGy. The rat pups were sacrificed at embryonic 18 days, 3 days and 14 days after delivery and the maxillae tooth germs were taken. The tissue sections of specimen were stained immunohistochemically with anti-BMP-2/4 antibody. RESULTS: At embryo-18 days, immunoreacivity for BMP-2/4 of the Group 1 was modetate in stratum intermedium of dental organ and weak in dental papilla and dental follicle, but that of Group 2 was weak in cell layer of dental organ, and no immunoreacivity was shown in dental papilla and dental follice of Group 2 and in all tissue components of the Group 3. At postnatal-3 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in cell layer of dental organ, odontoblasts and developing alveolar bone, but that of Group of 2 and Group 3 was weak in odontoblasts and developing alveolar bone. At postnatal-14 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in newly formed cementum, alveolar bone and odontoblasts, but that of Group 2 was weaker than that of Group 1. In the Group 3, tooth forming cell layer showed weak immunoreactivity, but other cell layers showed no immunoreactivity. CONCLUSION: The expression of bone morphogenetic protein(BMP)-2/4 during early tooth development was disturbed after irradiation and calcium-deficient diet.
Abdomen ; Animals ; Bone Morphogenetic Proteins ; Dental Cementum ; Dental Papilla ; Dental Sac ; Diet* ; Maxilla ; Odontoblasts ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Tooth Germ ; Tooth*

Osteocalcin (OC) is the most abundant noncollagenous protein of extracellular matrix in the bone. In an OC deficient mouse, bone formation rates are increased in cancellous and cortical bones. OC is known as a negative regulator of mineral apposition. OC is also expressed in the tooth of the rat, bovine, and human. However, little is known about OC during tooth development in Xenopus. The purpose of this study is to compare the expression of OC with mineralization in the developing tooth of Xenopus, by using von Kossa staining and in situ hybridization. At stage 56, the developmental stage of tooth germ corresponds to the cap stage, and an acellular zone was apparent between the dental papilla and the enamel organ. From stage 57, calcium deposition was revealed by von Kossa staining prior to OC expression, and the differentiated odontoblasts forming predentin were located at adjoining predentin. At stage 58, OC transcripts were detected in the differentiated odontoblasts. At stage 66, OC mRNA was expressed in the odontoblasts, which was aligned in a single layer at the periphery of the pulp. These findings suggest that OC may play a role in mineralization and odontogenesis of tooth development in Xenopus.
Animals ; Calcium ; Dental Papilla ; Enamel Organ ; Extracellular Matrix ; Humans ; In Situ Hybridization ; Mice ; Odontoblasts ; Odontogenesis ; Osteocalcin* ; Osteogenesis ; Rats ; RNA, Messenger ; Tooth Germ ; Tooth* ; Xenopus ; Xenopus laevis*

Differentiation and activation of osteoclasts are crucial in bone resorption. RANKL released from osteoblasts, also known as ODF, and proteins such as OPG/OCIF, decoy receptor of RANKL for inhibition of osteoclast formation play an important role. Teeth exhibit various movement patterns in mandible before eruption, inevitably accompaning peripheral bone resorption. Therefore, it is very meaningful to study osteoclasts differentiation and activation-related various factors near developing teeth for prediction of tooth movement patterns and understanding of mechanism of pre-and post-eruptive movement. This study examined distribution of RANKL and OPG in the mandible of postnatal 1~24 day rats containing developing teeth using immunohistochemical methods. After birth, the mandibular pre-eruptive first and second molars showed tooth germ morphology from bell stage to crown stage. In postnatal day 17, the mandibular first molar crown was completed and continuous eruption was in process along with root formation. On the twenty fourth day after birth, it was exposed to the oral cavity. RANKL exhibited strong positive immunohistochemical reactions in dental follicles and stromal cells around the mandibular first molar. On the third day after birth, strong positive reaction to RANKL was observed in the alvelolar bone above the mandibular first molar along with appearance of numerous osteoclasts. The reactivity was slightly attenuated on the seventh day and increased again on the tenth day after birth. The positive immunoreactivities were found not only in alveolar bone above the developing first molar, but also in stromal cells of the cervical area in contact with HERS. The alveolar bone below developing roots was also strongly stained. OPG displayed a similar patterns in the RANKL expression and locations of posively reacting cells, but relatively weakly stained on the third and tenth day after birth, when numerous osteoclasts made their appearnace. Above results suggest that RANKL and OPG have a close relation to the teeth movement in mandible such as eruption during teeth developing process. They also imply that RANKL and OPG play an important role in osteoclasts formation after synthesized in dental follicular cells and alveolar stromal cells.
Animals ; Bone Resorption ; Crowns ; Dental Sac ; Mandible ; Molar ; Mouth ; Osteoblasts ; Osteoclasts ; Parturition ; Rats* ; Stromal Cells ; Tooth Germ ; Tooth Movement ; Tooth*

Cholinesterase (ChE) is one of the most ubiquitous enzymes and in addition to its well characterized catalytic function, the morphogenetic involvement of ChE has also been demonstrated in neuronal tissues and in non-neuronal tissues such as bone and cartilage. We have previously reported that during mouse tooth development, acetylcholinesterase (AChE) activity is dynamically localized in the dental epithelium and its derivatives whereas butyrylcholinesterase (BuChE) activity is localized in the dental follicles. To test the functional conservation of ChE in tooth morphogenesis among different species, we performed cholinesterase histochemistry following the use of specific inhibitors of developing molar and incisors in the hamster from embryonic day 11 (E11) to postnatal day 1 (P1). In the developing molar in hamster, the localization of ChE activity was found to be very similar to that of the mouse. At the bud stage, no ChE activity was found in the tooth buds, but was first detectable in the dental epithelium and dental follicles at the cap and bell stages. AChE activity was found to be principally localized in the dental epithelium whereas BuChE activity was observed in the dental follicle. In contrast to the ChE activity in the molars, BuChE activity was specifically observed in the secretory ameloblasts of the incisors, whilst no AChE activity was found in the dental epithelium of incisors. The subtype and localization of ChE activity in the dental epithelium of the incisor thus differed from those of the molar in hamster. In addition, these patterns also differed from the ChE activity in the mouse incisor. These results strongly suggest that ChE may play roles in the differentiation of the dental epithelium and dental follicle in hamster, and that morphogenetic subtypes of ChE may be variable among species and tooth types.
Acetylcholinesterase ; Ameloblasts ; Animals ; Butyrylcholinesterase ; Cartilage ; Cholinesterases ; Cricetinae ; Dental Sac ; Epithelium ; Incisor ; Mice ; Molar ; Morphogenesis ; Neurons ; Tooth ; Tooth Germ

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.
Ameloblasts ; Animals ; Basement Membrane ; Collagenases ; Crowns ; Dental Enamel ; Dental Sac ; Epithelium ; Matrix Metalloproteinases ; Molar ; Odontoblasts ; Odontogenesis ; Rats ; Tooth Germ

PURPOSE: To elucidate the effects of the irradiation and calcium-deficient diet on expression of interleukin(IL)-1 during tooth formation of rat molar MATERIALS AND METHODS: The pregnant three-week-old Spague-Dawley rats were used for the study. The control group was non-irradiation/normal diet group, and the experimental groups were irradiation/normal diet group and irradiation/calcium-diet group. The abdomen of the rats on the 9th day of pregnancy were irradiated with single dose of 350 cGy. The rat pups were sacrificed on the 14th day after delivery and the maxillae tooth germs were taken. The specimen were prepared to make sections for light microscopy, and some of tissue sections were stained immunohistochemically with anti-IL-1 antibody. RESULTS: In the irradiation/normal diet group, dental follicle showed fewer blood vessels, mononuclear cells, and fusions of mononuclear cells than in non-irradiation/normal diet group. Alveolar bone showed a few osteoblasts and osteoclasts. Periodontal ligament showed collagen fibers and fibroblasts with irregularity. Weak immunoreactivity for IL-1 was shown in dental follicle, alveolar bone, and periodontal ligament. In the irradiation/calcium-deficient diet group, dental follicle showed sparse cellularity. Alveolar bone showed diminished number of osteoblasts. Periodontal ligament showed irregular collagen fibers and atrophy of cementoblasts and fibroblasts. No immunoreactivity for IL-1 was shown in dental follicle, alveolar bone, and periodontal ligament. CONCLUSION: Irradiation and calcium-deficient diet seems to cause disturbance of the expression of interleukin-1 during tooth formation of rat molar.
Abdomen ; Animals ; Atrophy ; Blood Vessels ; Collagen ; Dental Cementum ; Dental Sac ; Diet* ; Fibroblasts ; Interleukin-1* ; Maxilla ; Microscopy ; Molar* ; Osteoblasts ; Osteoclasts ; Periodontal Ligament ; Pregnancy ; Rats* ; Tooth Germ ; Tooth*

Odontogenic cells express many genes spatiotemporally through complex and intricate processes during tooth formation. Therefore, investigating them during the tooth development has been an important subject for the better understanding of tooth morphogenesis. The present study was performed to identify the genetic profiles which are involved in the morphological changes during the different stages of rat tooth development using the Agilent Rat Oligonucleotide Microarrays. Morphologically, the maxillary 3rd molar germ at 10 days post-partum (dpp) was at the cap/bell stage. In contrast, the maxillary 2nd molar germ showed the root development stage. After microarray analysis, there were a considerable number of up- or down-regulated genes in the 3rd and the 2nd molar germ cells during tooth morphogenesis. Several differentially expressed genes for nerve supply were further studied. Among them, neuroligin 1 (Nlgn 1) was gradually downregulated during tooth development both at the transcription and the translation level. Also, Nlgn 1 was mostly localized in the dental sac, which is an important component yielding the nerve supply. This genetic profiling study proposed that many genes may be implicated in the biological processes for the dental hard tissue formation and, furthermore, may allow the identification of the key genes involved in the nerve supply to the dental sac.
Animals ; Biological Processes ; Dental Sac ; Gene Expression Profiling* ; Gene Expression* ; Germ Cells ; Microarray Analysis ; Molar ; Morphogenesis ; Oligonucleotide Array Sequence Analysis ; Rats* ; Tooth*

Tooth is formed by the reciprocal interactions between the ectoderm and ectomesenchyme derived from neural crest. It has not been clear that neuronal factors involved in the morphogenesis and differentiation of tooth. To identify the roles of neuronal factors during the tooth development, the expression patterns and localization of Uchl1 were investigated in the developing mouse tooth germ by in situ hybridization and immunohistochemistry. Uchl1 transcripts were weakly expressed in the oral epithelium and dental lamina at bud stage. However, expression of Uchl1 was not found in the oral epithelium from cap stage and observed in the inner enamel epithelium, stellate reticulum and dental papilla. From the bell stage, Uchl1 was expressed in the inner enamel epithelium and ameloblasts. Uchl1, was appeared to be localized in the inner enamel epithelium and differentiating ameloblasts of molar and incisors at neonates. Uchl1 was localized strongly in the fully differentiated ameloblasts and adjacent papillary layer whereas localized weakly in the odontoblasts of the molar at postnatal day 5. From these results, Uchl1 was expressed and localized in the differentiating dental epithelium and ameloblasts during tooth development. The results suggest that neuronal protein, Uchl1 may play roles in the histo- and cyto-differentiation of non-neuronal dental epithelium.
Ameloblasts ; Animals ; Dental Enamel ; Dental Papilla ; Ectoderm ; Epithelium* ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Incisor ; Infant, Newborn ; Mice* ; Molar ; Morphogenesis ; Neural Crest ; Neurons ; Odontoblasts ; Reticulum ; Tooth Germ ; Tooth*