Tooth germ injury can lead to abnormal tooth development and even tooth loss, affecting various aspects of the stomatognathic system including form, function, and appearance. However, the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited. Therefore, it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model. Here, we constructed a Tg(dlx2b:Dendra2-NTR) transgenic line that labeled tooth germ specifically. Taking advantage of the NTR/Mtz system, the dlx2b⁺ tooth germ cells were depleted by Mtz effectively. The process of tooth germ repair was evaluated by antibody staining, in situ hybridization, EdU staining and alizarin red staining. The severely injured tooth germ was repaired in several days after Mtz treatment was stopped. In the early stage of tooth germ repair, the expression of phosphorylated 4E-BP1 was increased, indicating that mTORC1 is activated. Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ. Normally, mouse incisors were repaired after damage, but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress. Overall, we are the first to construct a stable and repeatable repair model of severe tooth germ injury, and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair, providing a potential target for clinical treatment of tooth germ injury.
Animals ; Mice ; Mechanistic Target of Rapamycin Complex 1/pharmacology* ; Signal Transduction ; Tooth/metabolism* ; Tooth Germ/metabolism* ; Odontogenesis

Multiple signaling pathways are involved in the regulation of cell proliferation and differentiation in odontogenesis and dental tissue renewal, but the details of these mechanisms remain unknown. Here, we investigated the expression patterns of a transcription factor, Krüppel-like factor 6 (KLF6), during the development of murine tooth germ and its function in odontoblastic differentiation. KLF6 was almost ubiquitously expressed in odontoblasts at various stages, and it was co-expressed with P21 (to varying degrees) in mouse dental germ. To determine the function of Klf6, overexpression and knockdown experiments were performed in a mouse dental papilla cell line (iMDP-3). Klf6 functioned as a promoter of odontoblastic differentiation and inhibited the proliferation and cell cycle progression of iMDP-3 through p21 upregulation. Dual-luciferase reporter assay and chromatin immunoprecipitation showed that Klf6 directly activates p21 transcription. Additionally, the in vivo study showed that KLF6 and P21 were also co-expressed in odontoblasts around the reparative dentin. In conclusion, Klf6 regulates the transcriptional activity of p21, thus promoting the cell proliferation to odontoblastic differentiation transition in vitro. This study provides a theoretical basis for odontoblast differentiation and the formation of reparative dentine regeneration.
Animals ; Cell Differentiation/physiology* ; Cell Proliferation ; Mice ; Odontoblasts/metabolism* ; Odontogenesis ; Tooth Germ

OBJECTIVETo observe the transcriptional expression of enamelin in developing postnatal rat first mandibular molar germs, for further studies of functions of enamelin in enamel development and mineralization.

METHODSTissue slices of first mandibular molar germ of rat 1, 3, 7, 10, 14 days after birth were prepared. The enamelin mRNA expression was identified by in situ hybridization.

RESULTSEnamelin mRNA was observed in both ameloblast and odontoblast in 1-10 day old rat postnatal first mandibular molar germs. Enamelin mRNA appeared very weakly at 1st day, and increased through 3rd day, reached the maximum at 7th day, and reduced at 10th day and became negative at 14th day postnatally; while the expression of enamelin mRNA in odontoblast maintained lower from 1st to 10th day and negative at 14th day postnatally.

CONCLUSIONEnamelin gene transcriptional expression lasts from preameloblast to maturation ameloblast, which suggests that enamelin may participate in the development of enamel and mantle dentin.

Ameloblasts ; metabolism ; Animals ; Dental Enamel Proteins ; biosynthesis ; genetics ; Gene Expression Regulation ; In Situ Hybridization ; Molar ; embryology ; Odontoblasts ; metabolism ; RNA, Messenger ; analysis ; Rats ; Tooth Germ ; growth & development ; metabolism ; Transcription, Genetic

OBJECTIVE: To observe the expression and distribution of transforming growth factor β3 (TGF-β3) in the mouse tooth germ after advanced bell stage, and to discuss the role of TGF-β3 during the development of tooth germs. METHODS: BALB/C's mouse tooth germs at 4, 11, and 18 days postnatal (4dpn,11dpn,and 18dpn) were collected and processed for routine fixation, decalcification, embedding, and slicing. The expression of TGF-β3 was detected by immunohistochemisty. RESULTS: As to 4dpn tooth germ: Positive expression of TGF-β3 was found in enameloblasts, odontoblasts, ambitus of dental pupilla, with weak positive expression in the intermedial of dental papilla. As to 11dpn tooth germ: Positive expression was seen in enameloblasts, with negative expression in odontoblasts and dental papilla. As to 18dpn tooth, positive expression of TGF-β3 was showed in the vessel wall and its surrounding, with negative expression in other areas. CONCLUSION The distribution of TGF-β3 expression showed a time-space characteristic during the mouse tooth germ development after advanced bell stage, which may exert a regulatory effect on tooth development and this effect is gradually getting weak with the development of tooth germs.
Animals ; Animals, Newborn ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Molar ; metabolism ; Tissue Distribution ; Tooth Germ ; growth & development ; metabolism ; Transforming Growth Factor beta3 ; metabolism

OBJECTIVETo investigate the expression and biological effect of Shh during late bell stage by morphological and semi-quantitative analysis.

METHODSTooth germs were selected from new born Bal b/c mouse (P1, P2, P3, P5, P7). Semi-quality of Shh was measured by Western Blot and the expression place and strength of Shh were observed by in situ hybridization.

RESULTSShh was expressed in the ameloblast layer during late bell stage; the expression strength was high in secretive period and decreased with development; the active N-section was detectable before P3.

CONCLUSIONShh expresses specially in the ameloblast layer in late bell stage, and expression quality is related to the function of ameloblasts.

Ameloblasts ; metabolism ; Animals ; Blotting, Western ; Gene Expression ; Hedgehog Proteins ; In Situ Hybridization ; In Vitro Techniques ; Mice ; Mice, Inbred BALB C ; Tooth Germ ; cytology ; metabolism ; Trans-Activators ; biosynthesis ; genetics

OBJECTIVETo investigate the expression of Shh and its receptors Ptc1 and Ptc2 mRNA in the cap stage of mouse molar and discuss its role in early tooth morphogenesis.

METHODSThe embryonic mouse heads of early tooth development (E10.5 - E15.5) were obtained and 5 micro m serial sections were made. Immunohistochemical staining of PCNA was carried out by SP method. The expression pattern of Shh, Ptc1, and Ptc2 mRNA was analysed by in situ hybridization.

RESULTSE14.5, outer dental epithelium, inner dental epithelium, stellate reticulum and underlying dental mesenchyme were PCNA positive. Most of the enamel knot cells were PCNA negative. A few of the enamel knot cells were PCNA positive. Shh, Ptc1, and Ptc2 mRNA were strongly expressed in outer dental epithelium, inner dental epithelium, stellate reticulum and the enamel knot.

CONCLUSIONIn the cap stage, Shh as a paracrine and autocrine signaling molecule might stimulate epithelium and mesenchyme proliferation.

Animals ; Hedgehog Proteins ; Mice ; Molar ; metabolism ; Patched Receptors ; Patched-1 Receptor ; RNA, Messenger ; biosynthesis ; Receptors, Cell Surface ; biosynthesis ; genetics ; Tooth Germ ; growth & development ; metabolism ; Trans-Activators ; biosynthesis

OBJECTIVETo invesitgate the expression patterns of amelogenin and enamelin in the developing tooth germs.

METHODSMandible sections of postnatal day 1, 3, 7 and 14 mouse were prepared, immunohistochemical analysis and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect the expression patterns of amelogenin and enamelin in mandibular first molars.

RESULTSAmelogenin was observed in the cytoplasm of secretory ameloblasts and the whole enamel matrix layer. It was also transiently expressed in the odontoblasts of postnatal day 1 molars. Enamelin proteins were observed in the enamel layer deposited by secretory ameloblasts, especially intense beneath the ameloblast process and dentino-enamel junction. The mRNA levels of both amelogenin and enamelin were highest on postnatal day 7 (the ratio to glyceraldehyde phosphate dehydrogenase of amelogenin and enamelin: 0.813 ± 0.085 and 0.799 ± 0.064, respectively, P < 0.05).

CONCLUSIONSAmelogenin and enamelin were enamel matrix proteins predominately expressed by secretory ameloblasts. The temporal-spatial expression patterns of amelogenin and enamelin indicate the important roles they played in amelogenesis and biomineralization.

Ameloblasts ; metabolism ; Amelogenesis ; Amelogenin ; genetics ; metabolism ; Animals ; Dental Enamel ; metabolism ; Dental Enamel Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Molar ; metabolism ; Odontoblasts ; metabolism ; RNA, Messenger ; metabolism ; Time Factors ; Tooth Germ ; growth & development ; metabolism

Epithelial-mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA. Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development. Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses. Robust differences in gene expression,as well as genes not previously associated with root formation,were identified. Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction (RT-PCR) supported our findings. These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family. In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.
Animals ; Dental Cementum ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; physiology ; Gene Expression Regulation, Developmental ; Laser Capture Microdissection ; Mice ; Mice, Inbred Strains ; Odontoblasts ; metabolism ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Tooth Germ ; metabolism ; Tooth Root ; growth & development

OBJECTIVETo observe the temporal and spatial expression of Smad7 during human tooth germ development and evaluate the effect of Smad7 on tooth germ development.

METHODSThe expression of Smad7 and its changes at different stages of human tooth germ were detected by using immunohistochemical staining.

RESULTSSmad7 was expressed at all stages of tooth germ, but the distribution patterns at various stages were different. It indicated that temporal and spatial expressing mode of Smad7 during human tooth germ development was specific, which was similar to that of TGF-beta its signal transducer Smad2/3.

CONCLUSIONSmad7 might play an important role in TGF-beta intracellular signaling for modulating the differentiation of ameloblasts and odontoblasts.

Ameloblasts ; cytology ; Cell Differentiation ; DNA-Binding Proteins ; genetics ; metabolism ; physiology ; Fetus ; Humans ; Immunohistochemistry ; Odontoblasts ; cytology ; Odontogenesis ; Signal Transduction ; Smad7 Protein ; Tooth ; growth & development ; Tooth Germ ; embryology ; Trans-Activators ; genetics ; metabolism ; physiology ; Transforming Growth Factor beta ; genetics ; metabolism ; physiology

Generation of bio-engineered teeth by using stem cells will be a major approach for bioengineered implantation. Previous studies have demonstrated that dissociated tooth germ cells are capable of generating a tooth after reaggregation in vitro. However, the cellular and molecular mechanisms underlying this tooth regeneration are not clear. In this study, we dispersed E13.5 molar germ into single cells, immediately reaggregated them into cell pellet, then grafted the reaggregates under mouse kidney capsule for various times of culture. We investigated the morphogenesis and the expression of several developmental genes in dental epithelial cells in reaggregates of tooth germ cells. We found that dissociated tooth germ cells, after reaggregation, recapitulated normal tooth developmental process. In addition, dissociated dental epithelial cells retained the expression of Fgf8, Noggin, and Shh during reaggregation and tooth regeneration processes. Our results demonstrated that, despite of under dissociated status, dental epithelial cells maintained their odontogenic fate after re-aggregation with dental mesenchymal cells. These results provided important information for future in vitro generation of bio-engineered teeth from stem cells.
Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Embryo, Mammalian ; Epithelial Cells ; cytology ; metabolism ; Female ; Gene Expression Regulation, Developmental ; genetics ; Male ; Mice ; Odontogenesis ; genetics ; Tooth Germ ; cytology ; physiology