BACKGROUND: Mineral metabolism is critical for proper development of hard tissues of the skeleton and dentition. The dentoalveolar complex includes the following 4 mineralized tissues: enamel, dentin, cementum, and alveolar bone. Developmental processes of these tissues are affected by inherited disorders that disrupt phosphate and pyrophosphate homeostasis, although manifestations are distinct from those in the skeleton.
METHODS: The authors discuss original data from experiments and comparative analyses and review articles describing effects of inherited phosphate and pyrophosphate disorders on dental tissues. A particular emphasis is placed on how new therapeutic approaches for these conditions may affect oral health and dental treatments of affected patients.
RESULTS: Disorders of phosphate and pyrophosphate metabolism can lead to reduced mineralization (hypomineralization) or inappropriate (ectopic) calcification of soft tissues. Disruptions in phosphate levels in X-linked hypophosphatemia and hyperphosphatemic familial tumoral calcinosis and disruptions in pyrophosphate levels in hypophosphatasia and generalized arterial calcification of infancy contribute to dental mineralization defects. Traditionally, there have been few options to ameliorate dental health problems arising from these conditions. New antibody and enzyme replacement therapies bring possibilities to improve oral health in affected patients.
CONCLUSIONS: Research over the past 2 decades has exponentially expanded the understanding of mineral metabolism, and has led to novel treatments for mineralization disorders. Newly implemented and emerging therapeutic strategies affect the dentoalveolar complex and interact with aspects of oral health care that must be considered for dental treatment, clinical trial design, and coordination of multidisciplinary care teams.

Schwann cells (SCs), a type of glial cell in the peripheral nervous system, can serve as a source of mesenchymal stem cells (MSCs) to repair injured pulp. This study aimed to investigate the role of SCs in tooth germ development and repair of pulp injury. We performed RNA-seq and immunofluorescent staining on tooth germs at different developmental stages. The effect of L-type calcium channel (LTCC) blocker nimodipine on SCs odontogenic differentiation was analyzed by real-time polymerase chain reaction and Alizarin Red S staining. We used the PLP1-CreERT2/ Rosa26-GFP tracing mice model to examine the role of SCs and Cav1.2 in self-repair after pulp injury. SC-specific markers expressed in rat tooth germs at different developmental stages. Nimodipine treatment enhanced mRNA levels of osteogenic markers (DSPP, DMP1, and Runx2) but decreased calcium nodule formation. SCs-derived cells increased following pulp injury and Cav1.2 showed a similar response pattern as SCs. The different SCs phenotypes are coordinated in the whole process to ensure tooth development. Blocking the LTCC with nimodipine promoted SCs odontogenic differentiation. Moreover, SCs participate in the process of injured dental pulp repair as a source of MSCs, and Cav1.2 may regulate this process.

Thanks to their exceptional diversity, teeth are among the most distinctive features of vertebrates. Parameters such as tooth size, shape, number, identity, and implantation can have substantial implications for the ecology and certain social behaviors of toothed species. Despite decades of research primarily focused on mammalian dentition, particularly using the laboratory mouse model, squamate reptiles (\"lizards\" and snakes) offer a wide array of tooth types and dentition variations. This diversity, which includes differences in size, shape, function, and replacement capacity, provides invaluable opportunities for investigating these fundamental properties. The central bearded dragon (Pogona vitticeps), a popular pet species with well-established husbandry practices, is of particular interest. It features a broad spectrum of morphs and spontaneous mutants and exhibits a wide range of heterodont phenotypes, including variation in the size, shape, number, implantation, and renewal of teeth at both posterior and anterior positions. These characteristics position the species as a crucial model organism for developmental studies in tooth research and for gaining deeper insights into evolutionary patterns of vertebrate dentitions. In this article, we provide an overview of the current understanding of squamate dentition, its diversity, development, and replacement. Furthermore, we discuss the significant advantages offered by squamate species as model organisms for investigating the evolutionary and developmental aspects of vertebrate dentition.

Keratins are typical intermediate filament proteins of the epithelium that exhibit highly specific expression patterns related to the epithelial type and stage of cellular differentiation. They are important for cytoplasmic stability and epithelial integrity and are involved in various intracellular signaling pathways. Several keratins are associated with enamel formation. However, information on their expression patterns during tooth development remains lacking. In this study, we analyzed the spatiotemporal expression of keratin family members during tooth development using single-cell RNA-sequencing (scRNA-seq) and microarray analysis. scRNA-seq datasets from postnatal Day 1 mouse molars revealed that several keratins are highly expressed in the dental epithelium, indicating the involvement of keratin family members in cellular functions. Among various keratins, keratin 5 (Krt5), keratin 14 (Krt14), and keratin 17 (Krt17) are highly expressed in the tooth germ; KRT17 is specifically expressed in the stratum intermedium (SI) and stellate reticulum (SR). Depletion of Krt17 did not affect cell proliferation in the dental epithelial cell line SF2 but suppressed their differentiation ability. These results suggest that Krt17 is essential for SI cell differentiation. Furthermore, scRNA-seq results indicated that Krt5, Krt14, and Krt17 exhibited distinct expression patterns in ameloblast, SI, and SR cells. Our findings contribute to the elucidation of novel mechanisms underlying tooth development.

BACKGROUND: Cellular cementum (CC) includes cementocytes, cells suspected to regulate CC formation or resorption as osteocytes do in bone. Sclerostin (SOST) is a secreted negative regulator of Wnt/β-catenin signaling expressed by osteocytes and cementocytes. Osteocyte SOST expression reduces bone formation. We investigated the functional importance of SOST in CC compared with alveolar bone (AB) using a Sost knockout (Sost-/-) mouse model to better understand the role of cementocytes in CC.
METHODS: Mandibles and femurs of Sost-/- and wild-type (WT) mice were analyzed at 42 and 120 days postnatal (dpn). Maxillary first molars were bilaterally extracted at 42 dpn and both AB healing (maxillary molar sockets) and CC apposition (mandibular first molars) were examined at 21 days post-procedure. Analyses included micro-computed tomography, histology, and immunohistochemistry.
RESULTS: Femur cortical and trabecular bone and mandibular bone volumes were similarly increased in Sost-/- versus WT mice at 42 and/or 120 dpn. In contrast to previous reports, CC was not increased by Sost-/- at either age. We conducted challenge experiments on AB and CC to explore tissue-specific responses. Post-extraction AB healing was improved by Sost deletion. In contrast, experimentally-induced apposition in molars failed to stimulate increased CC formation in Sost-/- versus WT mice. Wnt pathway markers AXIN2 and DKK1, which were increased in Sost-/- versus WT AB osteocytes, were unchanged in cementocytes.
CONCLUSIONS: These data indicate CC is less responsive than AB to SOST deletion. Within the study limitations, these results do not support cementocytes as critical for directing increased CC formation.
CONCLUSIONS: Sclerostin is a protein known to inhibit bone formation, and removing sclerostin leads to more bone formation. Cementum is the thin layer that covers the surface of the tooth\'s root. Previous studies suggest that inhibiting sclerostin can similarly increase the amount of cementum. We wanted to compare the response of cementum and bone when sclerostin is absent to understand similarities and differences between these two tissues. In this study, we removed the Sost gene (the gene which produces sclerostin) in mice. We found that mice without sclerostin have more bone in their legs and jaws. Moreover, mice without sclerostin also healed better after tooth removal compared with normal mice. Surprisingly, unlike previous studies, we found that the amount of cementum was not different in mice without sclerostin compared with normal mice. Additionally, we challenged the cementum by taking out the opposing tooth to cause the first mandibular molar to move up by building more cementum. Even with this challenge, we found no difference in the amount of cementum in mice lacking sclerostin compared with normal mice. Therefore, we conclude here that cementum is less sensitive to the absence of sclerostin compared with bone.

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

To investigate the cell linkage between tooth dentin and bones, we studied TGF-β roles during postnatal dentin development using TGF-β receptor 2 (Tgfβr2) cKO models and cell lineage tracing approaches. Micro-CT showed that the early Tgfβr2 cKO exhibit short roots and thin root dentin (n = 4; p<0.01), a switch from multilayer pre-odontoblasts/odontoblasts to a single-layer of bone-like cells with a significant loss of ~85% of dentinal tubules (n = 4; p<0.01), and a matrix shift from dentin to bone. Mechanistic studies revealed a statistically significant decrease in odontogenic markers, and a sharp increase in bone markers. The late Tgfβr2 cKO teeth displayed losses of odontoblast polarity, a significant reduction in crown dentin volume, and the onset of massive bone-like structures in the crown pulp with high expression levels of bone markers and low levels of dentin markers. We thus concluded that bones and tooth dentin are in the same evolutionary linkage in which TGF-β signaling defines the odontogenic fate of dental mesenchymal cells and odontoblasts. This finding also raises the possibility of switching the pulp odontogenic to the osteogenic feature of pulp cells via a local manipulation of gene programs in future treatment of tooth fractures.

Primary cultured odontoblasts rapidly lose their tissue-specific phenotype. To identify transcription factors (TF) that are important for the maintenance of the odontoblast phenotype, primary cultures of C57BL/6 J mouse dental mesenchymal cells (DMC) were isolated, and expression of TF and odontoblast marker genes in cells immediately after isolation and 2 days after culture were comprehensively evaluated and compared using RNA-sequencing (RNA-seq). The expression of odontoblast markers in mouse dental mesenchymal cells decreased rapidly after isolation. In addition, the expression of Hedgehog-related, Notch-related, and immediate- early gene (IEG)-related transcription factors significantly decreased. Forced expression of these genes in lentiviral vectors, together with fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), and the Wnt pathway activator CHIR99021, significantly induced the expression of odontogenic marker genes. These results indicate, for the first time, that Notch signaling and early genes may be important for maintaining odontoblast cultures. Furthermore, simultaneous stimulation of FGF, Wnt, Hedgehog, Notch pathways, and IEG transcription factors cooperatively promoted the maintenance of the odontoblast phenotype. These results suggest that the Hedgehog and Notch signaling pathways may play an important role in maintaining odontoblast phenotypes, in addition to FGF and Wnt signaling.

OBJECTIVE: The aim of this study was to investigate the impact of birth weight on tooth development in children aged 7-8 years.
METHODS: This retrospective cohort study comprised 75 children born at Bint Al-Huda Hospital, Bojnurd, in 2013-2014. The children were categorized into three groups based on their birth weight: Normal Birth Weight (NBW), Low Birth Weight (LBW), and Very Low Birth Weight (VLBW). Panoramic radiographs were taken for orthodontic examination, and Demirjian\'s 8-teeth method was employed to determine dental age. The study compared dental and chronological age within each group. Data analysis utilized SPSS software version 26, employing One-way ANOVA and chi-square tests. Statistical significance was set at P ≤ 0.05.
RESULTS: The mean difference in dental and chronological age for Very Low Birth Weight (VLBW) children was 0.22 ± 0.44 years, for Low Birth Weight (LBW) children it was 0.19 ± 0.45 years, and for Normal Birth Weight (NBW) children, it was 0.08 ± 0.46 years. Although the mean difference decreased with increasing birth weight, this trend did not achieve statistical significance (P = 0.55). Furthermore, no significant differences were observed between the weight groups (P = 0.529) or genders (P = 0.191).

OBJECTIVE: This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process.
METHODS: We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1.
RESULTS: scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes.
CONCLUSIONS: Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.