Mouse Molar Research Articles

Tracing PRX1+ cells during molar formation and periodontal ligament reconstruction

Neural crest-derived mesenchymal stem cells (MSCs) are known to play an essential function during tooth and skeletal development. PRX1+ cells constitute an important MSC subtype that is implicated in osteogenesis. However, their potential function in tooth development and regeneration remains elusive. In the present study, we first assessed the cell fate of PRX1+ cells during molar development and periodontal ligament (PDL) formation in mice. Furthermore, single-cell RNA sequencing analysis was performed to study the distribution of PRX1+ cells in PDL cells. The behavior of PRX1+ cells during PDL reconstruction was investigated using an allogeneic transplanted tooth model. Although PRX1+ cells are spatial specific and can differentiate into almost all types of mesenchymal cells in first molars, their distribution in third molars is highly limited. The PDL formation is associated with a high number of PRX1+ cells; during transplanted teeth PDL reconstruction, PRX1+ cells from the recipient alveolar bone participate in angiogenesis as pericytes. Overall, PRX1+ cells are a key subtype of dental MSCs involved in the formation of mouse molar and PDL and participate in angiogenesis as pericytes during PDL reconstruction after tooth transplantation.

In vitro and in vivo study of the pathogenic role of PPARα in experimental periodontitis.

Objective The purpose of this study is to investigate the pathogenic role of PPARα in periodontal antigen treated gingival cells in vitro and in experimental periodontitis in vivo .Methodology Gingival fibroblasts, gingival epithelial cells and splenocytes were isolated from C57BL/6J wild type (WT) mice and treated with fixed P. gingivalis at for 48 hours. The mRNA levels of PPARs, TNFα, IL-1β and IL-10 were detected by Real-time quantitative PCR. Silk ligatures after being soaked in the P.gingivalis suspension were tied around both maxillary second molars of WT mice or PPARα knock-out (KO) mice for two weeks. PPARα agonist fenofibrate and vehicle control were injected into the different side of the palatal gingiva on days 3, 6, and 9. At day 14, bone resorption and gingival mRNA expression levels of PPARs, TNFα, IL-1β and IL-10 were measured by micro-computed tomography and RT-qPCR respectively.ResultsP. gingivalis treatment downregulated the expression of PPARα, but not PPARβ or PPARγ, and increased the expression of TNF-α and IL-1β in Gingival fibroblasts, gingival epithelial cells and splenocytes from WT mice. Gingival mRNA levels of PPARα were significantly decreased in experimental periodontitis in WT mice. The bone loss of PPARα KO mice in experimental periodontitis was significantly higher than WT mice and was not reduced by fenofibrate treatment. Gingival TNFα protein expressions were significantly increased by P. gingivalis associated ligation and decreased by fenofibrate treatment in WT mice but not in PPARα KO mice.Conclusion This study suggests that PPARα plays an essential role in periodontitis.

Deficiency of Mineralization-Regulating Transcription Factor Trps1 Compromises Quality of Dental Tissues and Increases Susceptibility to Dental Caries.

Dental caries is the most common chronic disease in children and adults worldwide. The complex etiology of dental caries includes environmental factors as well as host genetics, which together contribute to inter-individual variation in susceptibility. The goal of this study was to provide insights into the molecular pathology underlying increased predisposition to dental caries in trichorhinophalangeal syndrome (TRPS). This rare inherited skeletal dysplasia is caused by mutations in the TRPS1 gene coding for the TRPS1 transcription factor. Considering Trps1 expression in odontoblasts, where Trps1 supports expression of multiple mineralization-related genes, we focused on determining the consequences of odontoblast-specific Trps1 deficiency on the quality of dental tissues. We generated a conditional Trps1Col1a1 knockout mouse, in which Trps1 is deleted in differentiated odontoblasts using 2.3kbCol1a1-CreERT2 driver. Mandibular first molars of 4wk old male and female mice were analyzed by micro-computed tomography (μCT) and histology. Mechanical properties of dentin and enamel were analyzed by Vickers microhardness test. The susceptibility to acid demineralization was compared between WT and Trps1Col1a1cKO molars using an ex vivo artificial caries procedure. μCT analyses demonstrated that odontoblast-specific deletion of Trps1 results in decreased dentin volume in male and female mice, while no significant differences were detected in dentin mineral density. However, histology revealed a wider predentin layer and the presence of globular dentin, which are indicative of disturbed mineralization. The secondary effect on enamel was also detected, with both dentin and enamel of Trps1Col1a1cKO mice being more susceptible to demineralization than WT tissues. The quality of dental tissues was particularly impaired in molar pits, which are sites highly susceptible to dental caries in human teeth. Interestingly, Trps1Col1a1cKO males demonstrated a stronger phenotype than females, which calls for attention to genetically-driven sex differences in predisposition to dental caries. In conclusion, the analyses of Trps1Col1a1cKO mice suggest that compromised quality of dental tissues contributes to the high prevalence of dental caries in TRPS patients. Furthermore, our results suggest that TRPS patients will benefit particularly from improved dental caries prevention strategies tailored for individuals genetically predisposed due to developmental defects in tooth mineralization.

Single-cell transcriptomics defines Dot1L interacting partners and downstream target genes in the mouse molar dental pulp.

Although histone methyltransferases are implicated in many key developmental processes, the contribution of individual chromatin modifiers in dental tissues is not well understood. Using single-cell RNA sequencing, we examined the expression profiles of the disruptor of telomeric silencing 1-like (Dot1L) gene in the postnatal day 5 mouse molar dental pulp. Dot1L is the only known enzyme that methylates histone 3 on lysine 79, a modification associated with gene expression. Our research revealed 15 distinct clusters representing different populations of mesenchymal stromal cells (MSCs), immune cells, pericytes, ameloblasts and endothelial cells. We documented heterogeneity in gene expression across different subpopulations of MSCs, a good indicator that these stromal progenitors undergo different phases of osteogenic differentiation. Interestingly, although Dot1L was broadly expressed across all cell clusters within the molar dental pulp, our analyses indicated specific enrichment of Dot1L within two clusters of MSCs, as well as cell clusters characterized as ameloblasts and endothelial cells. Moreover, we detected Dot1L co-expression with protein interactors involved in epigenetic activation such as Setd2, Sirt1, Brd4, Isw1, Bptf and Suv39h1. In addition, Dot1L was co-expressed with Eed2, Cbx3 and Dnmt1, which encode epigenetic factors associated with gene silencing and heterochromatin formation. Dot1l was co-expressed with downstream targets of the insulin growth factor and WNT signaling pathways, as well as genes involved in cell cycle progression. Collectively, our results suggest that Dot1L may play key roles in orchestrating lineage-specific gene expression during MSC differentiation.

Deletion of epithelial cell-specific p130Cas impairs the maturation stage of amelogenesis

Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130CasΔepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130CasΔepi- mice in comparison to p130Casflox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130CasΔepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130CasΔepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na+/Ca2++K+-exchanger in the maturation stage were reduced in p130CasΔepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).

Wound healing in periodontal disease induces macrophage polarization characterized by different arginine-metabolizing enzymes.

Macrophages play important roles from the initiation of inflammation to wound healing. Two phenotypes of macrophages, namely pro-inflammatory type macrophages (M1-MΦ) and anti-inflammatory type macrophages (M2-MΦ), have been reported. Two contrasting metabolic enzymes that use arginine as a substrate, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1), have been identified as M1-MΦ and M2-MΦ markers, respectively. The purpose of this study was to elucidate the temporal dynamics of the macrophage phenotype during the progression and healing phases of experimental periodontitis in mice. A total of 63 C57BL/6J mice were divided into the following 3groups: control (C), periodontitis (P), and healing (H). To induce periodontitis, a silk ligature was placed around the maxillary bilateral second molars of mice in the periodontitis and healing groups. In the healing group, the ligature was removed 3days after ligation to induce tissue healing. Maxillary tissue was collected on day 0 for the control group, days 1, 3, 5, and 7 for the periodontitis group (P1, P3, P5, and P7), and days 5 and 7 for the healing group (H5 and H7: 3days with the ligation + 2days or 4days following ligature removal). The left side of the maxilla was subjected to bone structure analysis using micro-computed tomography and gene expression analysis using polymerase chain reaction. On the right side, immunohistochemistry was performed to histopathologically evaluate the localization of macrophages by phenotype in the periodontal tissue. In the alveolar bone structure analysis, the linear distance of bone height increased significantly in the P5 and P7groups, whereas bone volume fraction and bone mineral density decreased over time after ligature placement; in the healing group (H5 and H7), these parameters improved significantly compared with the periodontitis group (P5 and P7). Expression of genes encoding pro-inflammatory cytokines and iNOS increased in the periodontitis group, and expression of anti-inflammatory cytokine genes and Arg-1 increased in the healing group. Furthermore, the iNOS/Arg-1 expression ratio increased with ligation, whereas the ratio in the healing groups (H5 and H7) significantly decreased compared with the periodontitis groups (P5 and P7). Immunofluorescence staining revealed a significant increase in the number of iNOS-positive macrophages in the periodontitis group and decrease in the healing group. In contrast, the number of Arg-1-positive macrophages decreased in the periodontitis group and increased in the healing group. The results of the present study suggest that wound healing in periodontal disease induces macrophage polarization from M1-MΦ to M2-MΦ characterized by iNOS and Arg-1.

Facilitating Reparative Dentin Formation Using Apigenin Local Delivery in the Exposed Pulp Cavity.

Apigenin, a natural product belonging to the flavone class, affects various cell physiologies, such as cell signaling, inflammation, proliferation, migration, and protease production. In this study, apigenin was applied to mouse molar pulp after mechanically pulpal exposure to examine the detailed function of apigenin in regulating pulpal inflammation and tertiary dentin formation. In vitro cell cultivation using human dental pulp stem cells (hDPSCs) and in vivo mice model experiments were employed to examine the effect of apigenin in the pulp and dentin regeneration. In vitro cultivation of hDPSCs with apigenin treatment upregulated bone morphogenetic protein (BMP)- and osteogenesis-related signaling molecules such as BMP2, BMP4, BMP7, bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN) after 14 days. After apigenin local delivery in the mice pulpal cavity, histology and cellular physiology, such as the modulation of inflammation and differentiation, were examined using histology and immunostainings. Apigenin-treated specimens showed period-altered immunolocalization patterns of tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), NESTIN, and transforming growth factor (TGF)-β1 at 3 and 5 days. Moreover, the apigenin-treated group showed a facilitated dentin-bridge formation with few irregular tubules after 42 days from pulpal cavity preparation. Micro-CT images confirmed obvious dentin-bridge structures in the apigenin-treated specimens compared with the control. Apigenin facilitated the reparative dentin formation through the modulation of inflammation and the activation of signaling regulations. Therefore, apigenin would be a potential therapeutic agent for regenerating dentin in exposed pulp caused by dental caries and traumatic injury.

Enhancement of orthodontic tooth movement and root resorption in ovariectomized mice

Background/purposeAs the number of patients with osteoporosis requiring orthodontic treatment is increasing with the aging of society, it is necessary to evaluate the relations between bone metabolism in old age and orthodontic tooth movement (OTM). However, the effects of changes in bone metabolism due to osteoporosis on OTM and root resorption are still unclear. Therefore, we investigated the effects of OTM and root resorption in a mouse ovariectomy (OVX)-induced osteoporosis model. Materials and methodsEight-week-old female wild-type mice underwent OVX or sham surgery (Sham) as controls. One month after treatment, a nickel titanium coil spring was used to apply a mesial force to the maxillary left first molars of OVX or Sham mice for 12 days. The distance between the maxillary first molar and the second molar changed due to OTM and osteoclast formation was evaluated. The odontoclast formation and root resorption along the root surface of the distobuccal root of the first molar was also evaluated by histological analysis and scanning electron microscopy. ResultsDistance of tooth movement and osteoclast formation were significantly increased in OVX mice compared to Sham controls. Furthermore, root resorption in the mesial surface of the distal molars induced by orthodontic force was significantly increased in OVX mice. ConclusionThe amount of OTM was significantly increased, and the accompanying root resorption was also increased in OVX mice. Therefore, attention should be paid to the risk of root resorption associated with orthodontic treatment in patients with osteoporosis.

The Receptor AT1 Appears to Be Important for the Maintenance of Bone Mass and AT2 Receptor Function in Periodontal Bone Loss Appears to Be Regulated by AT1 Receptor.

A large number of experimental studies has demonstrated that angiotensin II (Ang II) is involved in key events of the inflammatory process. This study aimed to evaluate the role of Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors on periodontitis. Methods: Experimental periodontitis was induced by placing a 5.0 nylon thread ligature around the second upper left molar of AT1 mice, no-ligature or ligature (AT1-NL and AT1-L), AT2 (AT2-NL or AT2-L) and wild type (WT-NL or L). Alveolar bone loss was scanned using Micro-CT. Cytokines, peptides and enzymes were analyzed from gingival tissues by Elisa and RT-PCR. Results: The blockade of AT1 receptor resulted in bone loss, even in healthy animals. Ang II receptor blockades did not prevent linear bone loss. Ang II and Ang 1-7 levels were significantly increased in the AT2-L (p < 0.01) group compared to AT2-NL and AT1-L. The genic expression of the Mas receptor was significantly increased in WT-L and AT2-L compared to (WT-NL and AT2-NL, respectively) and in AT1-L. Conclusions: Our data suggest that the receptor AT1 appears to be important for the maintenance of bone mass. AT2 receptor molecular function in periodontitis appears to be regulated by AT1.

The tooth-specific basic helix-loop-helix factor AmeloD promotes differentiation of ameloblasts.

Tissue-specific basic helix-loop-helix (bHLH) transcription factors play an important role in cellular differentiation. We recently identified AmeloD as a tooth-specific bHLH transcription factor. However, the role of AmeloD in cellular differentiation has not been investigated. The aim of this study was to elucidate the role of AmeloD in dental epithelial cell differentiation. We found that AmeloD-knockout (AmeloD-KO) mice developed an abnormal structure and altered ion composition of enamel in molars, suggesting that AmeloD-KO mice developed enamel hypoplasia. In molars of AmeloD-KO mice, the transcription factor Sox21 encoding SRY-Box transcription factor 21 and ameloblast differentiation marker genes were significantly downregulated. Furthermore, overexpression of AmeloD in the dental epithelial cell line M3H1 upregulated Sox21 and ameloblast differentiation marker genes, indicating that AmeloD is critical for ameloblast differentiation. Our study demonstrated that AmeloD is an important transcription factor in amelogenesis for promoting ameloblast differentiation. This study provides new insights into the mechanisms of amelogenesis.

Integration of Single-Cell RNA- and CAGE-seq Reveals Tooth-Enriched Genes

Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5′-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type–specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type–specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future.

Genome-wide distribution of 5hmC in the dental pulp of mouse molars and incisors

The dental pulp is critical for the production of odontoblasts to create reparative dentin. In recent years, dental pulp has become a promising source of mesenchymal stem cells that are capable of differentiating into multiple cell types. To elucidate the transcriptional control mechanisms specifying the early phases of odontoblast differentiation, we analysed the DNA demethylation pattern associated with 5-hydroxymethylcytosine (5hmC) in the primary murine dental pulp. 5hmC plays an important role in chromatin accessibility and transcriptional control by modelling a dynamic equilibrium between DNA methylation and demethylation. Our research revealed 5hmC enrichment along genes and non-coding regulatory regions associated with specific developmental pathways in the genome of mouse incisor and molar dental pulp. Although the overall distribution of 5hmC is similar, the intensity and location of the 5hmC peaks significantly differs between the incisor and molar pulp genome, indicating cell type-specific epigenetic variations. Our study suggests that the differential DNA demethylation pattern could account for the distinct regulatory mechanisms underlying the tooth-specific ontogenetic programs.

Chromosomal neighbourhoods allow identification of organ specific changes in gene expression.

Although most genes share their chromosomal neighbourhood with other genes, distribution of genes has not been explored in the context of individual organ development; the common focus of developmental biology studies. Because developmental processes are often associated with initially subtle changes in gene expression, here we explored whether neighbouring genes are informative in the identification of differentially expressed genes. First, we quantified the chromosomal neighbourhood patterns of genes having related functional roles in the mammalian genome. Although the majority of protein coding genes have at least five neighbours within 1 Mb window around each gene, very few of these neighbours regulate development of the same organ. Analyses of transcriptomes of developing mouse molar teeth revealed that whereas expression of genes regulating tooth development changes, their neighbouring genes show no marked changes, irrespective of their level of expression. Finally, we test whether inclusion of gene neighbourhood in the analyses of differential expression could provide additional benefits. For the analyses, we developed an algorithm, called DELocal that identifies differentially expressed genes by comparing their expression changes to changes in adjacent genes in their chromosomal regions. Our results show that DELocal removes detection bias towards large changes in expression, thereby allowing identification of even subtle changes in development. Future studies, including the detection of differential expression, may benefit from, and further characterize the significance of gene-gene neighbour relationships.

Application of Ligature-Induced Periodontitis in Mice to Explore the Molecular Mechanism of Periodontal Disease

Periodontitis is an inflammatory disease characterized by the destruction of the periodontium. In the last decade, a new murine model of periodontitis has been widely used to simulate alveolar bone resorption and periodontal soft tissue destruction by ligation. Typically, 3-0 to 9-0 silks are selected for ligation around the molars in mice, and significant bone loss and inflammatory infiltration are observed within a week. The ligature-maintained period can vary according to specific aims. We reviewed the findings on the interaction of systemic diseases with periodontitis, periodontal tissue destruction, the immunological and bacteriological responses, and new treatments. In these studies, the activation of osteoclasts, upregulation of pro-inflammatory factors, and excessive immune response have been considered as major factors in periodontal disruption. Multiple genes identified in periodontal tissues partly reflect the complexity of the pathogenesis of periodontitis. The effects of novel treatment methods on periodontitis have also been evaluated in a ligature-induced periodontitis model in mice. This model cannot completely represent all aspects of periodontitis in humans but is considered an effective method for the exploration of its mechanisms. Through this review, we aimed to provide evidence and enlightenment for future studies planning to use this model.

Quantitative morphometric analysis of molar teeth and alveolar bone using micro-computed tomography in aged mice

ObjectivesIrreversible morphological regressions of the teeth or related structures in older people can diminish their overall health. However, research on human aging in dentistry is complicated by several confounding factors. In this study, we conducted a morphometric analysis of the mandibular second molars and surrounding alveolar bone in C57BL/6 mice to evaluate age-related changes in the oral cavity. MethodsThe animals were divided into five groups based on their age: 4 weeks (juvenile mice; n = 5); 20 weeks (n = 7), 50 weeks (n = 5), 77 weeks (n = 7), and 100 weeks (n = 5); changes were evaluated using micro-computed tomography. ResultsThe molars of juvenile mice had sharp and pointed cusps and presented maximum heights. With age and occlusal wear, the cusp heights demonstrated a significant decrease (up to 75%) until the last stage of life. Conversely, apparent lesions were not observed on the basal portion of the crown, even in the most heavily worn teeth. The roots of the molars continued to grow in length at 4 weeks of age. Alveolar bone resorption begins to occur in middle age and continues throughout life. The proportion of vertical bone loss reached approximately 40% of the entire root length, demonstrating a remarkable increase between weeks 77 and 100. ConclusionsOverall, these morphological changes were similar to those observed in humans. Therefore, it might be appropriate to use aged mice as an experimental model for basic and clinical research in geriatric dentistry.

Identification of novel candidate genes implicated in odontogenic potential in the developing mouse tooth germ using transcriptome analysis.

In tooth bioengineering for replacement therapy of missing teeth, the utilized cells must possess an inductive signal-forming ability to initiate odontogenesis. This ability is called odontogenic potential. In mice, the odontogenic potential signal is known to be translocated from the epithelium to the mesenchyme at the early bud stage in the developing molar tooth germ. However, the identity of the molecular constituents of this process remains unclear. The purpose of this study is to determine the molecular identity of odontogenic potential and to provide a new perspective in the field of tooth development research. In this study, whole transcriptome profiles of the mouse molar tooth germ epithelium and mesenchyme were investigated using the RNA sequencing (RNA-seq) technique. The analyzed transcriptomes corresponded to two developmental stages, embryonic day 11.5 (E11.5) and 14.5 (E14.5), which represent the odontogenic potential shifts. We identified differentially expressed genes (DEGs), which were specifically overexpressed in both the E11.5 epithelium and E14.5 mesenchyme, but not expressed in their respective counterparts. Of the 55 DEGs identified, the top three most expressed transcription factor genes (transcription factor AP-2 beta isoform 3 [TFAP2B], developing brain homeobox protein 2 [DBX2], and insulin gene enhancer protein ISL-1 [ISL1]) and three tooth development-related genes (transcription factor HES-5 [HES5], platelet-derived growth factor D precursor [PDGFD], semaphrin-3A precursor [SEMA3A]) were selected and validated by quantitative RT-PCR. Using immunofluorescence staining, the TFAP2B protein expression was found to be localized only at the E11.5 epithelium and E14.5 mesenchyme. Thus, our empirical findings in the present study may provide a new perspective into the characterization of the molecules responsible for the odontogenic potential and may have an implication in the cell-based whole tooth regeneration strategy.

A modified glass ionomer cement to mediate dentine repair

ObjectivesGlass ionomer cements (GIC) can be used to protect dentine following caries removal. However, GIC have little biological activity on biological repair processes, which means that neo-dentine formation remains reliant on limited endogenous regenerative processes. Wnt/β-catenin signalling is known to play a central role in stimulating tertiary dentine formation following tooth damage and can be stimulated by a range of glycogen synthase kinase (GSK3) antagonists, including lithium ions. MethodsHere, we created lithium-containing bioactive glass (BG) by substituting lithium for sodium ions in 45S5 BG. We then replaced between 10 and 40% of the powder phase of a commercial GIC with the lithium-substituted BG to create a range of formulations of ‘LithGlassGIC’. In vitro physical properties of the resulting glasses were characterised and their ability to stimulate reactionary dentine formation in mouse molars in vivo was tested. ResultsLithium release from LithGlassGIC increased with increasing lithium content in the cement. In common with unmodified commercial GIC, all formations of LithGlassGIC showed in vitro toxicity when measured using an indirect cell culture assay based on ISO10993:5, precluding direct pulp contact. However, in a murine non-exposed pulp model of tooth damage, LithGlassGIC quickly released lithium ions, which could be transiently detected in the saliva and blood. LithGlassGIC also enhanced the formation of tertiary dentine, resulting in a thickening of the dentine at the damage site that restored lost dentine volume. Dentine regeneration was likely mediated by upregulation of Wnt/β-catenin activity, as LithGlassGIC placed in TCF/Lef:H2B-GFP reporter mice showed enhanced GFP activity. SignificanceWe conclude that LithGlassGIC acts as a biological restorative material that promotes tertiary dentine formation and restores tooth structure.

Expression pattern of transcriptional enhanced associate domain family member 1 (Tead1) in developing mouse molar tooth

The Hippo pathway is essential for determining organ size by regulating cell proliferation. Previous reports have shown that impairing this pathway causes abnormal tooth development. However, the precise expression profile of the members of the transcriptional enhanced associate domain family (Tead), which are key transcription factors mediating Yap function, during tooth development is unclear. In this study, among the four isoforms of Tead (Tead1 - 4), only the expression of Tead1 mRNA was observed using semiquantitative RT- PCR in murine developing tooth germ at E16.5. The expression level of Tead1 mRNA in the excised murine mandibular molar tooth germ was significantly higher at E16.5 than at other developmental stages, as determined using quantitative PCR. We found that the mRNA expression of connective tissue growth factor (Ctgf), which is one of the Yap target genes directly controlling cell growth, changed consistently with that of Tead1 in developing molars. Fluorescent immunostaining revealed that Tead1 protein was expressed in both epithelial cells and mesenchymal cells of the dental lamina and dental epithelium, including the primary enamel knot during the cap stage. During the early bell stage (E16.5), Tead1 was expressed intensely in the inner and outer enamel epithelium, including the secondary enamel knot and the neighboring mesenchymal cells. Tead1 then specifically localized to the inner and outer enamel epithelium, which is responsible for enamel formation during the bell stage. These expression patterns were consistent with those of Yap, Taz, and Ctgf protein in developing molars. These results suggest that Tead1 acts as a mediator of the biological functions of Yap, such as the morphogenesis of cusp formation, during tooth development.

In vivo cell proliferation analysis and cell-tracing reveal the global cellular dynamics of periodontal ligament cells under mechanical-loading

Periodontal ligament (PDL) is a uniquely differentiated tissue that anchors the tooth to the alveolar bone socket and plays key roles in oral function. PDL cells can respond rapidly to mechanical stimuli, resulting in accelerated tissue remodeling. Cell proliferation is an initial event in tissue remodeling and participates in maintaining the cell supply; therefore, analyzing cell-proliferative activity might provide a comprehensive view of cellular dynamics at the tissue level. In this study, we investigated proliferating cells in mouse molar PDL during orthodontic tooth movement (OTM)-induced tissue remodeling. Our results demonstrated that the mechanical stimuli evoked a dynamic change in the proliferative-cell profile at the entire PDL. Additionally, cell-tracing analysis revealed that the proliferated cells underwent further division and subsequently contributed to tissue remodeling. Moreover, OTM-induced proliferating cells expressed various molecular markers that most likely arise from a wide range of cell types, indicating the lineage plasticity of PDL cells in vivo. Although further studies are required, these findings partially elucidated the global views of the cell trajectory in mouse molar PDL under mechanical-loading conditions, which is vital for understanding the cellular dynamics of the PDL and beneficial for dental treatment in humans.

Recent, Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity.

Recent, Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity.