Embryonic Tooth Germs Research Articles

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

Bimodal expression of Wnt5a in the tooth germ: A comparative study using in situ hybridization and immunohistochemistry

BackgroundDuring tooth development, Wnt5a, a member of the noncanonical Wnt ligand, is expressed prominently in the dental mesenchyme. However, the spatiotemporal profiles of Wnt5a protein production and distribution in tooth germs are largely unknown, which impairs elucidation of the Wnt5a-mediated regulatory mechanism of tooth development. MethodsWe performed analyzes of the spatiotemporal expression of Wnt5a in embryonic tooth germs (E11.5–E18.5) by using in situ hybridization and immunohistochemistry in parallel. The developmental stages of the embryonic tooth germs were determined by HE staining. In order to compare the spatiotemporal distribution patterns of Wnt5a mRNA-expressing cells and those of Wnt5a protein-expressing cells, serial frontal sections of paraffinized mouse embryo heads were used for the analyzes. When needed, the immunohistochemistry images were subjected to digital detection analysis of Wnt5a immunostaining signal using the WinROOF 2018 Ver. 4.19.0 image processing software program. ResultsThroughout the developmental process, cells expressing Wnt5a mRNA were found in various tissues including the dental follicle, dental papilla, inner and outer enamel epithelium, stratum intermediate, and stellate reticulum. However, odontoblasts differentiating and polarizing at E18.5 were the only cells representing an accumulation of Wnt5a protein in the apical region of the odontoblast process. The Wnt5a protein was undetectable in undifferentiated mesenchymal cells as well as any other cells positive for Wnt5a mRNA. ConclusionDifferentiating odontoblasts execute Wnt5a expression, the mode of which is distinct from that executed by the other cells constituting tooth germ. Change of the mode of Wnt5a expression begins to take place in the mesenchymal cells by E18.5, starting the elongation of the cytoplasmic process.

Mouse Embryonic Tooth Germ Dissection and Ex vivo Culture Protocol.

A tooth germ ex vivo organ culture allows visualization of its development in different stages, thus enabling investigation of the molecular mechanisms of regulatory factors. Tooth germs can be rapidly dissected from E13 mouse embryos and placed on cell culture inserts for observation of subsequent tooth germ development in a three-dimensional situation in real time. This method is also suitable for other organs that develop by epithelial-mesenchymal interactions, including salivary gland, hair, lung, and kidney. In addition, siRNAs or growth factors can be easily added to ex vivo tooth germ cultures to investigate the detailed molecular function of specific genes. The present protocol provides an efficient and practical method for isolation and ex vivo culture of embryonic tooth germs.

Effects of oleanolic acid acetate on bone formation in an experimental periodontitis model in mice.

We evaluated the role of oleanolic acid acetate (OAA), a triterpenoid commonly used in the treatment of liver disorders, inflammatory diseases, and metastasis, in bone formation after tooth loss by periodontitis. Periodontitis causes the sequential degradation of the alveolar bone and associated structures, resulting in tooth loss. Several studies have attempted to regenerate the bone for implantation following tooth loss. Maxillary left second molar was extracted from 8-week-old male mice following induction of periodontitis by ligature for 5days. The extraction socket was treated with 50ng/µL OAA for 1, 2, and 3weeks. Detailed morphological changes were examined using Masson's trichrome staining, and the precise localization patterns of various signaling molecules, including CD31, F4/80, interleukin (IL)-6, and osteocalcin, were observed. The volume of bone formation was examined by Micro-CT. Osteoclasts were enumerated using tartrate-resistant acid phosphatase (TRAP) staining. For molecular dissection of signaling molecules, we employed the hanging-drop in vitro cultivation method at E14 for 1day and examined the expression pattern of transforming growth factor (TGF)-β superfamily and Wnt signaling genes. Histomorphometrical examinations showed facilitated bone formation in the extraction socket following OAA treatment. In addition, OAA-treated specimens showed the altered localization patterns of inflammatory and bone formation-related signaling molecules including CD31, F4/80, IL-6, and osteocalcin. Also, embryonic tooth germ mesenchymal tissue cultivation with OAA treatment showed the significant altered expression patterns of signaling molecules such as transforming growth factor (TGF)-β superfamily and Wnt signaling. Oleanolic acid acetate induces bone formation and remodeling through proper modulation of osteoblast, osteoclast, and inflammation with regulations of TGF-β and Wnt signaling.

Compound odontoma in the primary dentition

Odontomas are odontogenic tumors with a time-limited growth. They are divided clinically and histopathologically into compound odontomas and complex odontomas. A malformation with local tissue excess is caused by a disturbance in embryonic tooth germ development.

Isolation and multiple differentiation potential assessment of human gingival mesenchymal stem cells.

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.

Microfluidics co-culture systems for studying tooth innervation.

Innervation plays a key role in the development and homeostasis of organs and tissues of the orofacial complex. Among these structures, teeth are peculiar organs as they are not innervated until later stages of development. Furthermore, the implication of neurons in tooth initiation, morphogenesis and differentiation is still controversial. Co-cultures constitute a valuable method to investigate and manipulate the interactions of nerve fibers with their target organs in a controlled and isolated environment. Conventional co-cultures between neurons and their target tissues have already been performed, but these cultures do not offer optimal conditions that are closely mimicking the in vivo situation. Indeed, specific cell populations require different culture media in order to preserve their physiological properties. In this study we evaluate the usefulness of a microfluidics system for co-culturing mouse trigeminal ganglia and developing teeth. This device allows the application of specific media for the appropriate development of both neuronal and dental tissues. The results show that mouse trigeminal ganglia and teeth survive for long culture periods in this microfluidics system, and that teeth maintain the attractive or repulsive effect on trigeminal neurites that has been observed in vivo. Neurites are repealed when co-cultured with embryonic tooth germs, while postnatal teeth exert an attractive effect to trigeminal ganglia-derived neurons. In conclusion, microfluidics system devices provide a valuable tool for studying the behavior of neurons during the development of orofacial tissues and organs, faithfully imitating the in vivo situation.

Genome-wide analysis of gene expression in human embryonic tooth germ.

While numerous genes that play important regulatory roles during tooth development in mice have been identified, little is known about gene expression profile and their function during human odontogenesis. To unveil expression profile of odontogenic genes in humans, we conducted genome-wide gene expression analysis by microarray assays to analyze differential gene expression between tooth germ and lip tissue from 11-week old human fetuses. We identified 167 genes that are strongly expressed in the cap stage tooth germ as compared to the lip tissue. Among them, 145 genes were further identified by gene ontology enrichment analysis that are highly represented in multiple gene ontology classes, include extracellular components, sequence-specific DNA binding proteins, Wnt-protein binding molecules, system development, organogenesis, and cell differentiation. Sixty-seven genes that are known to be associated with mammalian tooth development and tooth abnormalities were identified. Real-time PCR was further employed to validate microarray data. Moreover, in situ hybridization assay demonstrated tooth type specific expression of ISL1 and BARX1 in the incisor, canine, and molar respectively, consistent with microarray results. Our results represent a set of reliable data that could provide a solid base for future elaboration of molecular mechanisms underlying human tooth development.

Expression of DPP6 in Meckel's cartilage and tooth germs during mouse facial development

Dipeptidyl peptidase-like protein 6 (DPP6), a member of the dipeptidyl aminopeptidase family, plays distinct roles in brain development, but its expression in embryonic Meckel's cartilage and tooth germs development is unknown. We analyzed the expression pattern of DPP6 in Meckel's cartilage and tooth germs development using in situ hybridization. DPP6 was detected in different patterns in Meckel's cartilage and tooth germs during mouse facial development from 11.5 to 13.5 days post-coitus (dpc) embryos. The expression pattern of DPP6 suggests that it may be involved in mandible and tooth development.

Expression of SHH signaling molecules in the developing human primary dentition

BackgroundOur current knowledge on tooth development derives primarily from studies in mice. Very little is known about gene expression and function during human odontogenesis. Sonic Hedgehog (SHH) signaling has been demonstrated to play crucial roles in the development of multiple organs in mice, including the tooth. However, if SHH signaling molecules are expressed and function in the developing human embryonic tooth remain unknown.ResultsWe conducted microarray assay to reveal the expression profile of SHH signaling pathway molecules. We then used in situ hybridization to validate and reveal spatial and temporal expression patterns of a number of selected molecules, including SHH, PTC1, SMO, GLI1, GLI2, and GLI3, in the developing human embryonic tooth germs, and compared them with that in mice. We found that all these genes exhibit similar but slightly distinct expression patterns in the human and mouse tooth germ at the cap and bell stages.ConclusionsOur results demonstrate the operation of active SHH signaling in the developing human tooth and suggest a conserved function of SHH signaling pathway during human odontogenesis.

An Immune-compromised Method for Tooth Transplantation Using Adult Bone Marrow Stromal Cells and Embryonic Tooth Germ

An Immune-compromised Method for Tooth Transplantation Using Adult Bone Marrow Stromal Cells and Embryonic Tooth Germ

Differentiation of Dermal Multipotent Cells Into Odontogenic Lineage Induced by Embryonic and Neonatal Tooth Germ Cell–Conditioned Medium

Stem cell-based therapy represents a novel and more advantageous modality of treatment for tooth defect or loss. However, this strategy is challenged in the circumstances where tooth-derived stem cells are not readily accessible. In present study we sought to explore the possibility of utilizing dermal multipotent cells (DMCs) easily available from skin tissue for odontogenic induction. Using the limiting dilution technique, colony-forming cell population was isolated and characterized by proliferative activity and multilineage differentiation potential. By exposure to conditioned medium of embryonic and neonatal tooth germ cells in culture, the proliferation and mineralization activity of DMCs was elevated, while the embryonic tooth germ cell-conditioned medium (ETGC-CM) produced more significant effects. Meanwhile, ETGC-CM-treated DMCs phenocopied the odontoblasts in vitro as indicated by specific lineage markers. Following in vivo transplantation as cell pellet, ETGC-CM-treated DMCs were capable of producing blocks of mineralized tissues, which resembled those of dental pulp stem cell (DPSC) explants in the same subcutaneous pockets environment. These observations suggest that although more sufficient and continuous inductive microenvironment may be needed for undifferentiated DMCs to perform as odontoblasts, ETGC-CM-treated DMCs indeed acquire properties as those of DPSCs. Our work highlights the potential utility of DMCs as an alternative candidate cell source in hopes of developing more practical strategy of tooth regeneration research and offering promising opportunities for therapeutic approach.

Amelogenin in cranio‐facial development: the tooth as a model to study the role of amelogenin during embryogenesis

The amelogenins comprise 90% of the developing extracellular enamel matrix proteins and play a major role in the biomineralization and structural organization of enamel. Amelogenins were also detected, in smaller amounts, in postnatal calcifying mesenchymal tissues, and in several nonmineralizing tissues including brain. Low molecular mass amelogenin isoforms were suggested to have signaling activity; to produce ectopically chondrogenic and osteogenic-like tissue and to affect mouse tooth germ differentiation in vitro. Recently, some amelogenin isoforms were found to bind to the cell surface receptors; LAMP-1, LAMP-2 and CD63, and subsequently localize to the perinuclear region of the cell. The recombinant amelogenin protein (rHAM(+)) alone brought about regeneration of the tooth supporting tissues: cementum, periodontal ligament and alveolar bone, in the dog model, through recruitment of progenitor cells and mesenchymal stem cells. We show that amelogenin is expressed in various tissues of the developing mouse embryonic cranio-facial complex such as brain, eye, ganglia, peripheral nerve trunks, cartilage and bone, and is already expressed at E10.5 in the brain and eye, long before the initiation of tooth formation. Amelogenin protein expression was detected in the tooth germ (dental lamina) already at E13.5, much earlier than previously reported (E19). Application of amelogenin (rHAM(+)) beads together with DiI, on E13.5 and E14.5 embryonic mandibular mesenchyme and on embryonic tooth germ, revealed recruitment of mesenchymal cells. The present results indicate that amelogenin has an important role in many tissues of the cranio-facial complex during mouse embryonic development and differentiation, and might be a multifunctional protein.

Expression survey of genes critical for tooth development in the human embryonic tooth germ

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks.

Cell proliferation in teeth reconstructed from dispersed cells of embryonic tooth germs in a three‐dimensional scaffold

Tissue engineering can now reproduce tooth from postnatal tooth cells. However, crown formation is not accurately reconstituted, even when the complex structure of the enamel dentin is reproduced. Here, we showed that a tissue-engineered (TE) tooth, exhibiting morphogenesis according to regular crown-cusp pattern formation, was produced by embryonic tooth germ cells in a three-dimensional scaffold. Heterogeneous cells dissociated from embryonic day 14 (E14) mice tooth germs were seeded on a scaffold and implanted under a kidney capsule in adult mice. The developmental process of the implants was examined for up to 14 d. At 5 d, the cells had formed initial tooth germ, followed by enamel-covered dentin tissue formed symmetrically. To study the developmental process, we examined the growth pattern using 5-bromo-2'-deoxyuridine (BrdU)-labeling analysis. The initial cell-proliferation patterns of the TE teeth were similar to that at the cap and early bell stages in natural teeth. This was particularly true in the cervical loop, which showed a similar distribution pattern of BrdU-positive cells in TE- and natural teeth. These results suggested that even when embryonic tooth germs are dissociated, the single cells can reconstitute tooth, and that enamel organ morphogenesis proceeds as in natural teeth.

Inositol hexasulphate, a casein kinase inhibitor, alters the distribution of dentin matrix protein 1 in cultured embryonic mouse tooth germs.

Immunohistochemical studies using a polyclonal antibody, raised against the recombinant form of dentin matrix protein 1 (DMP1), show that DMP1 was detected mainly in odontoblasts in cultured mouse embryonic tooth germs. However, in restricted areas, DMP1 staining was also observed in secretory ameloblasts, in the stratum intermedium and stellate reticulum, but only when the odontoblasts located in front of them were unstained. When the embryonic tooth germs were cultured in the presence of inositol hexasulfate, a casein kinase I and II inhibitor, staining of odontoblasts was weak or nil, whereas, in contrast, ameloblasts and enamel organ were strongly immunolabelled, suggesting an enhanced translocation of DMP1 after secretion to the secretory ameloblasts and/or stratum intermedium and stellate reticulum. Moreover, DMP1--was shown to be a good substrate for gelatinase A (MMP-2), but not to gelatinase B (MMP- 9). We hypothesized that DMP1--or the sub-fractions cleaved by the MMP--could behave as diffusible signaling molecule (s) rather than as a true dentin extracellular matrix component.

Inositol Hexasulphate, a Casein Kinase Inhibitor, Alters Enamel Formation in Cultured Embryonic Mouse Tooth Germs

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

Developmentally regulated changes in phospholipid composition in murine molar tooth.

In order to explore the possibility that phospholipids are differently expressed during the cascade of events leading to tooth formation, we decided to carry out simultaneous biochemical, histological and electron histochemical studies. High performance thin-layer chromatography and gas-liquid chromatography were used to compare the composition of embryonic mouse first molar tooth germs at day 18 of gestation (E18) and at birth (D1), erupting teeth at day 7 (D7) and erupted molars at day 21 (D21). For the latter, non-demineralized and EDTA-demineralized lipid extracts were analysed separately. Moreover, an ultrahistochemical study was carried out using the iodoplatinate reaction which retains and visualizes phospholipids. Developmentally regulated changes occurred and were closely correlated with an increase in cell membrane phospholipids. Gradual accumulation of phospholipids was identified in the extracellular matrix, at an early stage of tooth germ development within the basement membrane and later, as predentine/dentine and enamel components participating in mineralization processes. Matrix vesicles transiently present in dentine were partly responsible for the lipids that were detected. A first group of phospholipids including phosphatidylcholine as the major membrane-associated phospholipid and phosphatidylinositol as the intracellular second messenger increased by a factor of 2.3 between E18 and D21. This increase is probably associated with cell lengthening and was relatively modest compared with the higher increase detected for a second group of phospholipids, namely phosphatidylethanolamine (x4.8), phosphatidylserine (x 5.9) and sphingomyelin (x5.4). This second group of extracellular matrix-associated phospholipids constituted 68% of the demineralized lipid extract and, therefore, contributes to the mineralization of dental tissues.

Localization of nerve cells in the developing rat tooth.

Earlier studies have shown that mammalian tooth formation can take place in the absence of peripheral nerve fibers. This has been taken to indicate that neurons are not needed for mammalian tooth development. However, our recent localization of peripherin, which is a neuronal cell marker, has suggested that neuronal cell bodies may be associated with developing teeth. In this study, we have analyzed in vivo and in vitro the presence of neuronal cells in developing rat tooth germs. When E14 and E16 rat first molars (thickening of presumptive dental epithelium and bud-stage tooth germ, respectively) were cultured in vitro, peripheral trigeminal axons degenerated. However, with antibodies against peripherin and L1 neural cell adhesion protein, we detected neuronal cell bodies and their axons in the explants. Next, the expression of neurofilament light-chain (NF-L) mRNAs was studied by in situ hybridization of embryonic E12 first branchial arches and tooth germs from initiation to completion of crown morphogenesis (E13, five-day post-natal teeth). NF-L transcripts were first seen at the bud stage (E15) next to the dental epithelium at the buccal side of the tooth germ. At the cap stage (E18), NF-L mRNAs were located under the oral epithelium at some distance from dental epithelium. These expression patterns correlate to the previous localization of peripherin-positive cells and suggest that NF-L expression also revealed neuronal cells. Taken together, these results demonstrate that, in addition to projections of peripheral neurons, neuronal cells are associated with the developing teeth. Hence, it is possible that neuronal cells may participate in the regulation of mammalian tooth formation.

Inhibition of in vitro mineralization in osteoblastic cells and in mouse tooth germ by phosphatidylinositol-specific phospholipase C

We have examined the effect of phosphatidylinositol-specific phospholipase C (PIPLC) on the in vitro mineralization during cultivation of both osteoblast-like MC3T3-E1 cells and the lower molar tooth germ from mouse embryo. PIPLC not only caused the release of alkaline phosphatase (ALP) from cell membranes into the culture medium but also produced a disturbance of in vitro mineralization in MC3T3-E1 cells and in mouse embryonic tooth germ. These findings strongly suggest that ALP, functioning as an ectoenzyme, is involved in the physiological mineralization process of both bone and teeth.