Important Role In Tooth Development Research Articles

Research Progress in the Molecular Regulatory Mechanisms of Alveolar Bone Restoration

Alveolar bone, the protruding portion of the maxilla and the mandible that surrounds the roots of teeth, plays an important role in tooth development, eruption, and masticatory performance. In oral inflammatory diseases, including apical periodontitis, periodontitis, and peri-implantitis, alveolar bone defects cause the loosening or loss of teeth, impair the masticatory function, and endanger the physical and mental health of patients. However, alveolar bone restoration is confronted with great clinical challenges due to the the complicated effect of the biological, mechanical, and chemical factors in the oral microenvironment. An in-depth understanding of the underlying molecular regulatory mechanisms will contribute to the exploration of new targets for alveolar bone restoration. Recent studies have shown that Notch, Wnt, Toll-like receptor (TLR), and nuclear factor-κB (NF-κB) signaling pathways regulate the proliferation, differentiation, apoptosis, and autophagy of osteoclasts, osteoblasts, osteocytes, periodontal ligament cells, macrophages, and adaptive immune cells, modulate the expression of inflammatory mediators, affect the balance of the receptor activator for nuclear factor-κB ligand/receptor activator for nuclear factor-κB/osteoprotegerin (RANKL/RANK/OPG) system, and ultimately participate in alveolar bone restoration. Additionally, alveolar bone restoration involves AMP-activated protein kinase (AMPK), phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT), Hippo/YAP, Janus kinase/signal transducer and activator of transcription (JAK/STAT), and transforming growth factor β (TGF-β) signaling pathways. However, current studies have failed to construct mature molecular regulatory networks for alveolar bone restoration. There is an urgent need for further research on the molecular regulatory mechanisms of alveolar bone restoration by using new technologies such as single-cell transcriptome sequencing and spatial transcriptome sequencing.

BMPR2 Variants Underlie Nonsyndromic Oligodontia.

Oligodontia manifests as a congenital reduction in the number of permanent teeth. Despite the major efforts that have been made, the genetic etiology of oligodontia remains largely unknown. Bone morphogenetic protein receptor type 2 (BMPR2) variants have been associated with pulmonary arterial hypertension (PAH). However, the genetic significance of BMPR2 in oligodontia has not been previously reported. In the present study, we identified a novel heterozygous variant (c.814C > T; p.Arg272Cys) of BMPR2 in a family with nonsyndromic oligodontia by performing whole-exome sequencing. In addition, we identified two additional heterozygous variants (c.1042G > A; p.Val348Ile and c.1429A > G; p.Lys477Glu) among a cohort of 130 unrelated individuals with nonsyndromic oligodontia by performing Sanger sequencing. Functional analysis demonstrated that the activities of phospho-SMAD1/5/8 were significantly inhibited in BMPR2-knockout 293T cells transfected with variant-expressing plasmids, and were significantly lower in BMPR2 heterozygosity simulation groups than in the wild-type group, indicating that haploinsufficiency may represent the genetic mechanism. RNAscope in situ hybridization revealed that BMPR2 transcripts were highly expressed in the dental papilla and adjacent inner enamel epithelium in mice tooth germs, suggesting that BMPR2 may play important roles in tooth development. Our findings broaden the genetic spectrum of oligodontia and provide clinical and genetic evidence supporting the importance of BMPR2 in nonsyndromic oligodontia.

Co‐opting Lef‐1 and miR‐26bactivities to regulate dental epithelial stem cells and supporting tissues

ObjectiveStem cells are potent to self‐renew and differentiate which contribute to the regenerative process of various mature tissue types. It is important to understand how stem cell behaviors are regulated to give further insights to the application of stem cell therapies in tissue regeneration and repair. We use the developing murine lower incisor as a model to study stem cell maintenance and differentiation, which are housed in a niche and are fated to only a handful of differentiated cell types. Tooth development is a complex process involving rigid temporal and reciprocal regulation of transcription factors including Sox2 and Lef‐1, and signaling pathways including Wnt. We are particularly interested in the regulation between Lef‐1 and miR‐26b due to their inverse expression profiles during tooth development. Lef1 harbors a miR‐26b binding site in its 3’‐UTR and is negatively regulated by miR‐26b. In this study, we aim to understand the role of Lef‐1 and miR‐26b in dental epithelial stem cell (DESC) differentiation, determine the molecular mechanism of miR‐26b regulated Lef‐1 expression and identify genetic pathways for tooth development and regeneration.MethodsWith our newly developed miR‐26b over‐expression (miR‐26b OE) and Lef‐1 conditional over‐expression (COEL) mouse models, we determined the expression of miR‐26b and Lef‐1 by microarray and qPCR. To evaluate the phenotype of tooth development of these models, we performed H&E and IF staining during embryogenesis. Knowing the regulatory relationship between miR‐26b and Lef‐1, we crossed COEL and miR‐26b OE mice and evaluated their phenotype by microCT, H&E and IF staining to validate that COEL mouse phenotype could be rescued by miR‐26b OE. Because Lef‐1 is a Wnt signaling target, a Wnt signaling array was performed in miR‐26b OE E18.5 mandibles.ResultsThe COEL mouse exhibits tusk‐like incisors, a new stem cell compartment in the LaCL and increased DESC proliferation, whereas the miR‐26b OE mice do not have teeth. The expression of miR‐26b is decreased in the COEL mouse and Lef‐1 is decreased in miR‐26b OE mouse. The incisor phenotype observed in the COEL mouse can be rescued by crossing to the miR‐26b OE mouse. Other Wnt related genes including Wnt5a are downregulated whereas some Wnt related genes including Nanog are upregulated in miR‐26b OE mouse.ConclusionCOEL and miR‐26b OE mice have very different incisor phenotypes and crossing COEL and miR‐26b OE mouse rescues the phenotypes. These results further validate that miR‐26b regulates the dental epithelial stem cell niche by negatively regulating Lef‐1, which also indicate their important roles in tooth development. Wnt signaling participates in this regulatory network.SignificanceWith the first miR‐26b OE mouse model and COEL mouse model, we are the first to demonstrate a molecular mechanism for how stage specific miR‐26b expression acts to compartmentalize the stem cell niche through regulating the known developmental transcription factor Lef‐1. Our future direction is to determine the transcriptome and epigenome profiles of the DESC niche in WT, COEL and COEL x miR‐26b OE mice. We expect to identify new regulatory pathways using Lef‐1 and miR‐26b that can be used in bioengineering to repair or regenerate tissues.

Wnt signalling in oral and maxillofacial diseases.

Wnts include more than 19 types of secreted glycoproteins that are involved in a wide range of pathological processes in oral and maxillofacial diseases. The transmission of Wnt signalling from the extracellular matrix into the nucleus includes canonical pathways and noncanonical pathways, which play an important role in tooth development, alveolar bone regeneration, and related diseases. In recent years, with the in-depth study of Wnt signalling in oral and maxillofacial-related diseases, many new conclusions and perspectives have been reached, and there are also some controversies. This article aims to summarise the roles of Wnt signalling in various oral diseases, including periodontitis, dental pulp disease, jaw disease, cleft palate, and abnormal tooth development, to provide researchers with a better and more comprehensive understanding of Wnts in oral and maxillofacial diseases.

The critical role of nuclear factor I-C in tooth development.

Nuclear factor I-C (NFIC) plays a critical role in regulating epithelial-mesenchymal crosstalk during tooth development. However, it remains largely unknown about how NFIC functions in dentin and enamel formation. In the present review, we aim to summarize the most recent discoveries in the field and gain a better understanding of the roles NFIC performs during tooth formation. Nfic-/- mice exhibit human dentin dysplasia type I (DDI)-like phenotypes signified by enlarged pulp chambers, the presence of short-root anomaly, and failure of odontoblast differentiation. Although loss of NFIC has little effect on molar crown morphology, researchers have detected aberrant microstructures of enamel in the incisors. Recently, accumulating evidence has further uncovered the novel function of NFIC in the process of enamel and dentin formation. During epithelial-mesenchyme crosstalk, the expression of NFIC is under the control of SHH-PTCH-SMO-GLI1 pathway. NFIC is closely involved in odontoblast lineage cells proliferation and differentiation, and the maintenance of NFIC protein level in cytoplasm is negatively regulated by TGF-β signaling pathway. In addition, NFIC has mild effect on ameloblast differentiation, enamel mineralization and cementum formation. NFIC plays an important role in tooth development and is required for the formation of dentin, enamel as well as cementum.

Endogenous Enzymatic Activity of Primary and Permanent Dentine.

Matrix metalloproteinases (MMPs) play an important role in tooth development and influence caries development and hybrid layer degradation. Literature is scant on the differences in the activity of MMPs between primary and permanent dentine. Accordingly, the aim of the present study was to investigate endogenous gelatinolytic activity in primary and permanent dentine. Separate batches of dentine powder were obtained from intact human primary and permanent molars (n = 6). Each batch was divided in two subgroups: (1) mineralised; and (2) demineralised with 10% H3PO4. After protein extraction, gelatine zymography was performed. Furthermore, in situ zymography was performed on dentine sections of the same groups (n = 3). The slices were polished, covered with fluorescein-conjugated gelatine and evaluated using a confocal microscope. In situ zymography data were analysed using two-way analysis of variance and post hoc Holm–Šidák statistics (α = 0.05). Primary dentine showed poorly defined bands in the zymograms that vaguely corresponded to the pro-form and active form of MMP-2 and the pro-form of MMP-9. In permanent dentine, demineralised powder demonstrated stronger gelatinolytic activity than mineralised powder. In situ zymography identified stronger enzymatic activity in primary etched dentine (p < 0.05). Stronger enzymatic activity recorded in primary dentine may be related to the differences in morphology and composition between primary and permanent dentine.

HDAC6 Regulates the Fusion of Autophagosome and Lysosome to Involve in Odontoblast Differentiation.

Odontoblast differentiation is an important process during tooth development in which pre-odontoblasts undergo elongation, polarization, and finally become mature secretory odontoblasts. Many factors have been found to regulate the process, and our previous studies demonstrated that autophagy plays an important role in tooth development and promotes odontoblastic differentiation in an inflammatory environment. However, it remains unclear how autophagy is modulated during odontoblast differentiation. In this study, we found that HDAC6 was involved in odontoblast differentiation. The odontoblastic differentiation capacity of human dental papilla cells was impaired upon HDAC6 inhibition. Moreover, we found that HDAC6 and autophagy exhibited similar expression patterns during odontoblast differentiation both in vivo and in vitro; the expression of HDAC6 and the autophagy related proteins ATG5 and LC3 increased as differentiation progressed. Upon knockdown of HDAC6, LC3 puncta were increased in cytoplasm and the autophagy substrate P62 was also increased, suggesting that autophagic flux was affected in human dental papilla cells. Next, we determined the mechanism during odontoblastic differentiation and found that the HDAC6 substrate acetylated-Tubulin was up-regulated when HDAC6 was knocked down, and LAMP2, LC3, and P62 protein levels were increased; however, the levels of ATG5 and Beclin1 showed no obvious change. Autophagosomes accumulated while the number of autolysosomes was decreased as determined by mRFP-GFP-LC3 plasmid labeling. This suggested that the fusion between autophagosomes and lysosomes was blocked, thus affecting the autophagic process during odontoblast differentiation. In conclusion, HDAC6 regulates the fusion of autophagosomes and lysosomes during odontoblast differentiation. When HDAC6 is inhibited, autophagosomes can't fuse with lysosomes, autophagy activity is decreased, and it leads to down-regulation of odontoblastic differentiation capacity. This provides a new perspective on the role of autophagy in odontoblast differentiation.

Expression of BMP2/4/7 during the odontogenesis of deciduous molars in miniature pig embryos.

Bone morphogenetic proteins (BMPs) play important roles in tooth development. However, their expression has not been studied in miniature pigs, which have many anatomical similarities in oral and maxillofacial region compared to human. This study investigated BMP2/4/7 expression patterns during deciduous molar development in miniature pigs on embryonic days (E) 40, 50, and 60. The mandibles were fixed, decalcified, and embedded before sectioning. H&E staining, immunohistochemistry, in situ hybridization using specific radionuclide-labeled cRNA probes, and real-time PCR were used to detect the BMP expression patterns during morphogenesis of the third deciduous molar. H&E staining showed that for the deciduous third molar, E40 represented the cap stage, E50 represented the early bell stage, and E60 represented the late bell stage or secretory stage. BMP2 was expressed in both the enamel organ and in the dental mesenchyme on E40 and E50 and was expressed mainly in pre-odontoblasts on E60. BMP7 expression was similar to BMP2 expression, but BMP7 was also expressed in the inner enamel epithelium on E60. On E40, BMP4 was expressed mainly in the epithelium, with some weak expression in the mesenchyme. On E50, BMP4 expression was stronger in the mesenchyme but weaker in the epithelium. On E60, BMP4 was expressed mainly in the mesenchyme. These data indicated that BMP2/4/7 showed differential spatial and temporal expression during the morphogenesis and odontogenesis of deciduous molars, suggesting that these molecules were associated with tooth morphogenesis and cell differentiation.

Deletion of epithelial cell-specific Cdc42 leads to enamel hypermaturation in a conditional knockout mouse model

Recent evidence suggests that GTPases Rho family plays an important role in tooth development; however, the role of Cdc42 in tooth development remains unclear. We aimed to investigate the function of Cdc42 in tooth development and amelogenesis. We generated an epithelial cell-specific K5-Cdc42 knockout (KO) mouse to evaluate post-eruption dental phenotypes using a K5-Cre driver line. This model overcomes the previously reported perinatal lethality. Tooth phenotypes were analyzed by micro X-ray, micro-computed tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), wear rate, shear strength, and a microhardness test. Enamel matrix protein expression was determined by immunohistochemistry.KO mice displayed a hypomaturation phenotype, including incisors that lacked yellow pigmentation and were abnormally white, rapid attrition of molars following eruption, and decreased micro-hardness and shearing strength. Micro-CT data revealed that of incisor and molar enamel volumes were smaller in the KO than in wild-type (WT) mice. SEM analysis showed that the enamel prism structure was disordered. In addition, HE staining indicated a remarkable difference in the ameloblast morphology and function between KO and WT mice, and immunohistochemistry showed increased expression of amelogenin, ameloblastin, matrix metallopeptidase 20, kallikrein-related peptidase 4 and amelotin in the KO mice teeth.Our results suggest epithelium cell-specific Cdc42 deletion leads to tooth hypomaturation and transformation of the enamel prism structure that is likely due to altered ameloblast morphology and the secretion of enamel matrix proteins and proteases. This is the first in vivo evidence suggesting that Cdc42 is essential for proper tooth development and amelogenesis.

Al-Awadi-Raas-Rothschild syndrome with dental anomalies and a novel WNT7A mutation

Al-Awadi-Raas-Rothschild syndrome (AARRS; OMIM 276820) is a very rare autosomal recessive limb malformation syndrome caused by WNT7A mutations. AARRS is characterized by various degrees of limb aplasia and hypoplasia. Normal intelligence and malformations of urogenital system are frequent findings. Complete loss of WNT7A function has been shown to cause AARRS, however, its partial loss leads to the milder malformation, Fuhrmann syndrome. An Indian boy affected with AARRS is reported. A novel homozygous base substitution mutation c.550A > C (p.Asn184Asp) is identified in the patient. Parents were heterozygous for the mutation. In addition to the typical features of AARRS, the patient had agenesis of the mandibular left deciduous lateral incisor. The heterozygous parents had microdontia of the maxillary left permanent third molar and taurodontism (enlarged dental pulp chamber at the expense of root) in a number of their permanent molars. Whole exome sequencing of the patient and his parents ruled out mutations in 11 known hypodontia-associated genes including WNT10A, MSX1, EDA, EDAR, EDARADD, PAX9, AXIN2, GREM2, NEMO, KRT17, and TFAP2B. In situ hybridization during tooth development showed Wnt7a expression in wild-type tooth epithelium at E14.5. All lines of evidence suggest that WNT7A has important role in tooth development and its mutation may lead to tooth agenesis, microdontia, and taurodontism. Oral examination of patients with AARRS and Fuhrmann syndromes is highly recommended.

The Impact of the Eda Pathway on Tooth Root Development

The Eda pathway (Eda, Edar, Edaradd) plays an important role in tooth development, determining tooth number, crown shape, and enamel formation. Here we show that the Eda pathway also plays a key role in root development. Edar (the receptor) is expressed in Hertwig’s epithelial root sheath (HERS) during root development, with mutant mice showing a high incidence of taurodontism: large pulp chambers lacking or showing delayed bifurcation or trifurcation of the roots. The mouse upper second molars in the Eda pathway mutants show the highest incidence of taurodontism, this enhanced susceptibility being matched in human patients with mutations in EDA-A1. These taurodont teeth form due to defects in the direction of extension of the HERS from the crown, associated with a more extensive area of proliferation of the neighboring root mesenchyme. In those teeth where the angle at which the HERS extends from the crown is very wide and therefore more vertical, the mutant HERSs fail to reach toward the center of the tooth in the normal furcation region, and taurodont teeth are created. The phenotype is variable, however, with milder changes in angle and proliferation leading to normal or delayed furcation. This is the first analysis of the role of Eda in the root, showing a direct role for this pathway during postnatal mouse development, and it suggests that changes in proliferation and angle of HERS may underlie taurodontism in a range of syndromes.

Structural and Morphometric Comparison of Lower Incisors in PACAP-Deficient and Wild-Type Mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with widespread distribution. PACAP plays an important role in the development of the nervous system, it has a trophic and protective effect, and it is also implicated in the regulation of various physiological functions. Teeth are originated from the mesenchyme of the neural crest and the ectoderm of the first branchial arch, suggesting similarities with the development of the nervous system. Earlier PACAP-immunoreactive fibers have been found in the odontoblastic and subodontoblastic layers of the dental pulp. Our previous examinations have shown that PACAP deficiency causes alterations in the morphology and structure of the developing molars of 7-day-old mice. In our present study, morphometric and structural comparison was performed on the incisors of 1-year-old wild-type and PACAP-deficient mice. Hard tissue density measurements and morphometric comparison were carried out on the mandibles and the lower incisors with micro-CT. For structural examination, Raman microscopy was applied on frontal thin sections of the mandible. With micro-CT morphometrical measurements, the size of the incisors and the relative volume of the pulp to dentin were significantly smaller in the PACAP-deficient group compared to the wild-type animals. The density of calcium hydroxyapatite in the dentin was reduced in the PACAP-deficient mice. No structural differences could be observed in the enamel with Raman microscopy. Significant differences were found in the dentin of PACAP-deficient mice with Raman microscopy, where increased carbonate/phosphate ratio indicates higher intracrystalline disordering. The evaluation of amide III bands in the dentin revealed higher structural diversity in wild-type mice. Based upon our present and previous results, it is obvious that PACAP plays an important role in tooth development with the regulation of morphogenesis, dentin, and enamel mineralization. Further studies are required to clarify the molecular background of the effects of PACAP on tooth development.

FHL2 mediates tooth development and human dental pulp cell differentiation into odontoblasts, partially by interacting with Runx2.

The differentiation of mesenchymal cells in tooth germ and dental pulp cells into odontoblasts is crucial for dentin formation, and the transcription factor runt-related transcription factor (Runx2) is necessary for odontoblast differentiation. Our previous study demonstrated that four and a half LIM domains 2 (FHL2) may play an important role in tooth development and human dental pulp cell differentiation. This study aimed to determine whether FHL2 mediated the mesenchymal cells in tooth development and human dental pulp cell differentiation into odontoblasts by interacting with Runx2. The expression patterns of FHL2 and Runx2 were examined at the early stages of mouse molar development using double immunofluorescence staining. Western blot analysis and co-immunoprecipitation (Co-IP) were conducted for the preliminary study of the relationship between FHL2 and Runx2 in human dental pulp cell differentiation into odontoblasts. Results of double immunofluorescence staining showed that FHL2 and Runx2 exhibited similar expression patterns at the early stages of tooth development. Western blot analysis indicated that the expression patterns of FHL2 and Runx2 were synchronized on day 7 of induction, whereas those on day 14 differed. Co-IP analysis revealed positive bands of protein complexes, revealing the interaction of FHL2 and Runx2 on days 0, 7 and 14 of induction. Our data suggested that FHL2 might interact with Runx2 to mediate mesenchymal cell differentiation at the early stages of tooth development and human dental pulp cell differentiation.

Coordinated expression of H3K9 histone methyltransferases during tooth development in mice.

Tissue-specific gene expression is subjected to epigenetic and genetic regulation. Posttranslational modifications of histone tails alter the accessibility of nuclear proteins to DNA, thus affecting the activity of the regulatory complex of nuclear proteins. Methylation at histone 3 lysine 9 (H3K9) is a crucial modification that affects gene expression and cell differentiation. H3K9 is known to have 0-3 methylation states, and these four methylated states are determined by the expression of sets of histone methyltransferases. During development, teeth are formed through mutual interactions between the mesenchyme and epithelium via a process that is subjected to the epigenetic regulation. In this study, we examined the expression of all H3K9 methyltransferases (H3K9MTases) during mouse tooth development. We found that four H3K9MTases-G9a, Glp, Prdm2, and Suv39h1-were highly expressed in the tooth germ, with expression peaks at around embryonic days 16.5 and 17.5 in mice. Immunohistochemical and in situ hybridization analyses revealed that all four H3K9MTases were enriched in the mesenchyme more than in the epithelium. Substrates of H3K9MTases, H3K9me1, H3K9me2, and H3K9me3 were also enriched in the mesenchyme. Taken together, these data suggested that coordinated expression of four H3K9MTases in the dental mesenchyme might play important roles in tooth development.

Multi-Dimensional Prioritization of Dental Caries Candidate Genes and Its Enriched Dense Network Modules

A number of genetic studies have suggested numerous susceptibility genes for dental caries over the past decade with few definite conclusions. The rapid accumulation of relevant information, along with the complex architecture of the disease, provides a challenging but also unique opportunity to review and integrate the heterogeneous data for follow-up validation and exploration. In this study, we collected and curated candidate genes from four major categories: association studies, linkage scans, gene expression analyses, and literature mining. Candidate genes were prioritized according to the magnitude of evidence related to dental caries. We then searched for dense modules enriched with the prioritized candidate genes through their protein-protein interactions (PPIs). We identified 23 modules comprising of 53 genes. Functional analyses of these 53 genes revealed three major clusters: cytokine network relevant genes, matrix metalloproteinases (MMPs) family, and transforming growth factor-beta (TGF-β) family, all of which have been previously implicated to play important roles in tooth development and carious lesions. Through our extensive data collection and an integrative application of gene prioritization and PPI network analyses, we built a dental caries-specific sub-network for the first time. Our study provided insights into the molecular mechanisms underlying dental caries. The framework we proposed in this work can be applied to other complex diseases.

A case–control study of the association between tooth‐development gene polymorphisms and non‐syndromic hypodontia in the Chinese Han population

Hypodontia is one of the most common anomalies of human dentition. Recent genetic studies provide information on a number of genes related to both syndromic and non-syndromic forms of hypodontia. Fifty putative single nucleotide polymorphisms (SNPs) in 20 genes that play important roles in tooth development were selected, and a case-control study was conducted in 273 subjects with hypodontia (cases) and 200 subjects without hypodontia (controls). DNA was obtained from samples of whole blood or saliva. Genotyping was performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A significant difference was observed, between subjects with non-syndromic hypodontia and controls, in the allele and genotype frequencies of two markers [rs929387 of GLI family zinc finger 3 (GLI3) and rs11001553 of Dickkopf-related protein 1 (DKK1)]. Similar results were observed in a subgroup analysis of test subjects (stratified by gender or missing tooth position). However, this analysis showed no significant difference in the haplotype distribution between the controls and the affected subjects. These data demonstrate an association between some SNPs in tooth development-associated genes and sporadic non-syndromic hypodontia in Chinese Han individuals. This information may provide further understanding of the molecular mechanisms of tooth agenesis. Furthermore, these genes can be regarded as candidates for mutation detection in individuals with tooth agenesis.

Effects of Wnt/β‐catenin signalling on proliferation and differentiation of apical papilla stem cells

The Wnt signalling pathway has been shown to play an important role in tooth development, however its effects with stem cells from the apical papilla (SCAP) have remained unclear. The purpose of this study was to determine effects of Wnt/β-catenin on proliferation and differentiation of SCAP in vitro. SCAP were obtained, identified and cultured. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of mineralization-related genes and mineralized nodule formation were measured in presence or absence of various concentrations of lithium chloride. MTT assay and flow cytometry demonstrated that Wnt/β-catenin activity could promote proliferation of SCAP. Real-time PCR analysis found that Wnt/β-catenin strongly upregulated expression of dentine sialophosphoprotein, osteocalcin and ALP in SCAP after incubation with mineralization induction medium, while ALP and alizarin red staining indicated that Wnt/β-catenin enhanced ALP activity and formation of mineralized nodules. Our results suggest that canonical Wnt/β-catenin signalling promotes proliferation and odonto/osteogenic differentiation of SCAP.

Bmp2b and bmp4 are dispensable for zebrafish tooth development

Bone morphogenetic protein (Bmp) signaling has been shown to play important roles in tooth development at virtually all stages from initiation to hard tissue formation. The specific ligands involved in these processes have not been directly tested by loss-of-function experiments, however. We used morpholino antisense oligonucleotides and mutant analysis in the zebrafish to reduce or eliminate the function of bmp2b and bmp4, two ligands known to be expressed in zebrafish teeth and whose mammalian orthologs are thought to play important roles in tooth development. Surprisingly, we found that elimination of function of these two genes singly and in combination did not prevent the formation of mature, attached teeth. The mostly likely explanation for this result is functional redundancy with other Bmp ligands, which may differ between the zebrafish and the mouse.

Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants

The Msx2-interacting nuclear target protein (MINT) is a nuclear matrix protein that regulates the development of many tissues. However, little is known regarding the role of MINT in tooth development. In this study, we prepared polyclonal antibodies against MINT, and found that that MINT was expressed in different cells at each stage of tooth germ development by immunohistochemistry. The role of MINT in tooth development was further illustrated by the misshapen and severely hypoplastic tooth organ in the cultured mandibular explants of MINT deficient mice. From the initiation to cap stage, the differences between mutants and wild-type molars were more and more distinguished histologically. In the MINT-deficient mandibular explants, the tooth germ was reduced in the overall size and lacked enamel knot, with abnormal dental lamina and collapsed stellate reticulum. Furthermore, the BrdU incorporation experiment showed that the proliferation activity was significantly reduced in MINT-deficient dental epithelium. Our results suggest that MINT plays an important role in tooth development, in particular, epithelial morphogenesis.

Effect of Wnt6 on Human Dental Papilla Cells In Vitro

Introduction The Wnt signaling pathway plays an important role in tissue development by acting on proliferation, differentiation, and cell fate decisions. Because the role of Wnt6 in tooth development was still unknown, the purpose of this study was to investigate the role of Wnt6 in tooth morphogenesis and dental tissue mineralization by elucidating its effect on human dental papilla cells (hDPCs) in vitro. Methods Human dental papilla cells were enzymatically separated from tooth germs. Recombinant adenovirus encoding full-length Wnt6 cDNA was constructed to overexpress Wnt6, and the biologic effects of Wnt6 on hDPCs were investigated. Wnt6-transduced changes in hDPC proliferation were examined by means of a 5-bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. Wnt6-transduced changes in hDPC differentiation were investigated by evaluating alkaline phosphatase (ALPase) activity, by a mineralization assay, and analysis of mineralization-related gene expression including ALP, type I collagen (Col I), osteonectin (ON), osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein-1 (DMP-1). Results Wnt6 overexpression had no significant effect on the proliferation of hDPCs by BrdU incorporation assay and flow cytometric analysis. Wnt6 enhanced differentiation of hDPCs into functional odontoblast-like cells with up-regulated activity of ALPase and the expression of mineralization-related genes such as ALP, Col I, ON, OPN, BSP, and DMP-1. Wnt6 overexpression also promoted the mineralization of hDPCs. Conclusions Our findings verified that Wnt6 plays an important role in tooth development by promoting hDPC differentiation, without significant effects on hDPC proliferation.