[Research progress in methylation modification in tooth root development].

Methylation modification is one of the most common epigenetic modification regulation in eukaryotes, including histone methylation, DNA methylation, RNA methylation, etc., which plays an important regulatory role in physiological processes and pathologic occurrence and development. Tooth root development is carried out by both epithelial and mesenchymal cells and involves a variety of cell-molecular interactions. In recent years, a large number of studies have found that methylation plays a key role in the regulation of tooth root development and expands the mechanism network of tooth root development. In this paper, we review the role and mechanism of methylation modification during root development.

Fibroblast growth factor signalling regulates the development of tooth root.

Fibroblast growth factor (FGF) signalling plays a crucial role in the morphogenesis of multiple tissues including teeth. While the role of the signal has been studied in tooth crown development, little is known about root development. Of several FGF ligands involved in hard tissue formation, we suggest that FGF18 regulates the development of murine tooth roots. We implanted FGF18-soaked heparin beads into the lower first molar tooth buds at postnatal day 6 (P6), followed by transplantation under the kidney capsule. After 3 weeks, FGF18 significantly facilitated root elongation and periodontal tissue formation compared to the control. In situ hybridisation showed that Fgf18 transcripts were initially localised in the dental pulp along Hertwig's epithelial root sheath at P6 and P10 and subsequently in the dental follicle cells at P14. Fgf receptors were expressed in various dental tissues during these stages. In vitro analysis using the dental pulp stem cells revealed that FGF18 inhibited cell proliferation and decreased expression levels of osteogenic markers, Runx2, Alpl and Sp7. Consistently, after 1 week of kidney capsule transplantation, FGF18 application did not induce the expression of Sp7 and Bsp, but upregulated Periostin in the apical region of dental mesenchyme in the grafted molar. These findings suggest that FGF18 facilitates molar root development by regulating the calcification of periodontal tissues.

Application of cryopreservation to tooth germ transplantation for root development and tooth eruption.

We cryopreserved mouse tooth germs with widely open cervical margins of the enamel organ to overcome difficulties in cryoprotectant permeation and tested their efficacy by transplanting them into recipient mice. The upper right first molar germs of 8-day-old donor mice were extracted and categorized into the following four groups according to cryopreservation time: no cryopreservation, 1 week, 1 month, and 3 months. The donor tooth germs were transplanted into the upper right first molar germ sockets of the 8-day-old recipient mice. The upper left first molars of the recipient mice were used as controls. The outcome of the transplantation was assessed at 1, 2, and 3 weeks after transplantation. Stereomicroscopic evaluation revealed that most of the transplanted teeth erupted by 3 weeks after transplantation. Micro-computed tomography analysis revealed root elongation in the transplanted groups as well as in the controls. There was no significant difference between the cryopreserved and non-cryopreserved transplanted teeth, but the roots of the cryopreserved teeth were significantly shorter than those of the control teeth. Histological examination revealed root and periodontal ligament formations in all the transplanted groups. These results suggest that the transplantation of cryopreserved tooth germs facilitates subsequent root elongation and tooth eruption.

Arid1a-Plagl1-Hh signaling is indispensable for differentiation-associated cell cycle arrest of tooth root progenitors.

Chromatin remodelers often show broad expression patterns in multiple cell types yet can elicit cell-specific effects in development and diseases. Arid1a binds DNA and regulates gene expression during tissue development and homeostasis. However, it is unclear how Arid1a achieves its functional specificity in regulating progenitor cells. Using the tooth root as a model, we show that loss of Arid1a impairs the differentiation-associated cell cycle arrest of tooth root progenitors through Hedgehog (Hh) signaling regulation, leading to shortened roots. Our data suggest that Plagl1, as a co-factor, endows Arid1a with its cell-type/spatial functional specificity. Furthermore, we show that loss of Arid1a leads to increased expression of Arid1b, which is also indispensable for odontoblast differentiation but is not involved in regulation of Hh signaling. This study expands our knowledge of the intricate interactions among chromatin remodelers, transcription factors, and signaling molecules during progenitor cell fate determination and lineage commitment.

Lhx6 regulates canonical Wnt signaling to control the fate of mesenchymal progenitor cells during mouse molar root patterning.

Mammalian tooth crown formation has long served as a model for investigating how patterning and morphogenesis are orchestrated during development. However, the mechanism underlying root patterning and morphogenesis remains poorly understood. In this study, we find that Lhx6 labels a subpopulation of root progenitor cells in the apical dental mesenchyme, which is closely associated with furcation development. Loss of Lhx6 leads to furcation and root number defects, indicating that Lhx6 is a key root patterning regulator. Among the multiple cellular events regulated by Lhx6 is the odontoblast fate commitment of progenitor cells, which it controls in a cell-autonomous manner. Specifically, Lhx6 loss leads to elevated expression of the Wnt antagonist Sfrp2 and down-regulation of Wnt signaling in the furcation region, while overactivation of Wnt signaling in Lhx6+ progenitor cells partially restore the furcation defects in Lhx6-/- mice. Collectively, our findings have important implications for understanding organ morphogenesis and future strategies for tooth root regeneration.

Cell dynamics in Hertwig's epithelial root sheath are regulated by ß-catenin activity during tooth root development.

ß-catenin, a key mediator of Wnt signaling, plays multiple roles in tooth development. However, the role of ß-catenin in Hertwig's epithelial root sheath (HERS) during root formation remains unclear. In this study, we generated inducible tissue-specific ß-catenin conditional knockout mice (Ctnnb1i∆shh ) to investigate how ß-catenin in HERS affects tooth root development. The inactivation of ß-catenin in HERS led to interrupted root elongation due to premature disruption of HERS. This phenotype was accompanied by reduced cell-cell adhesion and decreased expression of junctional proteins, as well as increased epithelial-to-mesenchymal transition of HERS cells upon ß-catenin depletion. Accordingly, stabilization of ß-catenin in HERS (Catnbi∆shh ) led to the formation of unfragmented HERS and resulted in the failure of HERS dissociation, with increased expression of junctional proteins. Our results suggest that fine control of ß-catenin is important for HERS to guide root formation through regulating its structural integrity.

Therapeutic potential of HERS spheroids in tooth regeneration.

Hertwig's epithelial root sheath (HERS) plays indispensable roles in tooth root development, including controlling the shape and number of roots, dentin formation, and helping generate the cementum. Based on these characteristics, HERS cell is a potential seed cell type for tooth-related tissue regeneration. However, the application is severely limited by a lack of appropriate culture methods and small cell numbers. Methods: Here, we constructed a 3D culture method to expand functional HERS cells into spheroids, and investigated characteristics and application of dental tissue regeneration of these spheroids. HERS spheroids and HERS cells (2D monolayer culture) were compared in terms of biological characteristics (such as proliferation, self-renewal capacity, and stemness) in vitro and functions (including differentiation potential and inductive ability of dentin formation) both in vitro and in vivo. Further, transcriptome analysis was utilized to reveal the molecular mechanisms of their obvious differences. Results: HERS spheroids showed obvious superiority in biological characteristics and functions compared to 2D monolayers of HERS cells in vitro. In vivo, HERS spheroids generated more mineralized tissue; when combined with dental papilla cells (DPCs), HERS spheroids contributed to dentin-like tissue formation. Moreover, the generation and expansion of HERS spheroids rely to some degree on the HIF-1 pathway. Conclusion: HERS spheroid generation is beneficial for functional HERS cell expansion and can provide a useful cell source for further tooth regeneration and mechanistic research. Notably, HIF-1 pathway plays a critical role in HERS spheroid formation and function.

Runx2 Regulates Mouse Tooth Root Development Via Activation of WNT Inhibitor NOTUM.

Progenitor cells are crucial in controlling organ morphogenesis. Tooth development is a well-established model for investigating the molecular and cellular mechanisms that regulate organogenesis. Despite advances in our understanding of how tooth crown formation is regulated, we have limited understanding of tooth root development. Runt-related transcription factor 2 (RUNX2) is a well-known transcription factor in osteogenic differentiation and early tooth development. However, the function of RUNX2 during tooth root formation remains unknown. We revealed in this study that RUNX2 is expressed in a subpopulation of GLI1+ root progenitor cells, and that loss of Runx2 in these GLI1+ progenitor cells and their progeny results in root developmental defects. Our results provide in vivo evidence that Runx2 plays a crucial role in tooth root development and in regulating the differentiation of root progenitor cells. Furthermore, we identified that Gli1, Pcp4, NOTUM, and Sfrp2 are downstream targets of Runx2 by integrating bulk and single-cell RNA sequencing analyses. Specifically, ablation of Runx2 results in downregulation of WNT inhibitor NOTUM and upregulation of canonical WNT signaling in the odontoblastic site, which disturbs normal odontoblastic differentiation. Significantly, exogenous NOTUM partially rescues the impaired root development in Runx2 mutant molars. Collectively, our studies elucidate how Runx2 achieves functional specificity in regulating the development of diverse organs and yields new insights into the network that regulates tooth root development. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Origins of Alterations to Rankl Null Mutant Mouse Dental Root Development.

The purpose of the present study was to assess the early stages of development of mouse first molar roots in the osteopetrotic context of RANKL invalidation in order to demonstrate that the radicular phenotype observed resulted not only from defective osteoclasts, but also from loss of cell-to-cell communication among dental, periodontium and alveolar bone cells involving RANKL signaling. Two experimental models were used in this study: Rankl mutants with permanent RANKL invalidation, and C57BL/6J mice injected during the first postnatal week with a RANKL neutralizing antibody corresponding to a transient RANKL invalidation. The dento-alveolar complex was systematically analyzed using micro-CT, and histological and immunohistochemical approaches. These experiments showed that the root elongation alterations observed in the Rankl-/- mice were associated with reduced proliferation of the RANK-expressing HERS cells with a significant decrease in proliferating cell nuclear antigen (PCNA) expression and a significant increase in P21 expression. The phenotypic comparison of the adult first molar root at 35 days between permanent and transitory invalidations of RANKL made it possible to demonstrate that alterations in dental root development have at least two origins, one intrinsic and linked to proliferation/differentiation perturbations in dental-root-forming cells, the other extrinsic and corresponding to disturbances of bone cell differentiation/function.

Sonic Hedgehog Signaling and Tooth Development.

Sonic hedgehog (Shh) is a secreted protein with important roles in mammalian embryogenesis. During tooth development, Shh is primarily expressed in the dental epithelium, from initiation to the root formation stages. A number of studies have analyzed the function of Shh signaling at different stages of tooth development and have revealed that Shh signaling regulates the formation of various tooth components, including enamel, dentin, cementum, and other soft tissues. In addition, dental mesenchymal cells positive for Gli1, a downstream transcription factor of Shh signaling, have been found to have stem cell properties, including multipotency and the ability to self-renew. Indeed, Gli1-positive cells in mature teeth appear to contribute to the regeneration of dental pulp and periodontal tissues. In this review, we provide an overview of recent advances related to the role of Shh signaling in tooth development, as well as the contribution of this pathway to tooth homeostasis and regeneration.

[Effects of post-natal inhibition of RANKL on molar eruption and root formation in C57BL/6 mice]. / Effets de l'inhibition post-natale de RANKL sur l'éruption et la formation radiculaire des molaires de souris C57BL/6.

INTRODUCTION: Recent observations performed in the orthodontic department of La Pitié-Salpêtrière hospital in Paris reported an increase of non-familial eruption defects of permanent molars. Our recent data have evidenced the involvement of osteoclasts (OC) in both the eruption and the dental retention processes through the RANKL/RANK/OPG signaling pathway. These facts are at the origin of the hypothesis of the existence of an environmental etiology for those eruption defects that would correspond to the perturbation of cellular autocrine/paracrine signaling pathways as the RANKL/ RANK/OPG. MATERIALS AND METHODS: C57BL/6 mice were submitted to repeated injections with anti-RANKL neutralizing antibody during the nine days following birth. A phenotypic comparison with transgenic mice overexpressing RANK was performed for the functional characterization of the RANKL/RANK/OPG pathway. The dento-alveolar complex was analyzed using micro-CT for bone density and Masson's trichrome staining for histological examination. RESULTS: The RANKL transient invalidation of RANKL stopped the molar root development and tooth eruption contrary to transgenic mice overexpressing RANK. The recruitment and the OC activity were strongly impacted. DISCUSSION: This research is of direct clinical interest in understanding the pathology of eruption as indirect in establishing orthodontic treatment protocols for particular cases.

New radiological findings and radiculomegaly in oculofaciocardiodental syndrome with a novel BCOR mutation: A case report.

RATIONALE: Oculofaciocardiodental syndrome (OFCD) patients who show radiculomegaly are very rare. We treated a new OFCD patient orthodontically, and performed longitudinal observation for 30 years. New findings, termed calcified-dental-papillae (CDPs) beneath open-apices (OAs) of developing radiculomegalies, pulp-stone-like-calcifications (PSLCs) and the process of radiculomegaly development were observed. A novel mutation of BCL-6 interacting corepressor (BCOR) was identified. Cone-beam-computed-tomography (CBCT) images of the radiculomegalies clarified their morphology. PATIENT CONCERNS: A female patient and her parents were referred to orthodontic clinic for alignment of the teeth. DIAGNOSIS: A CDP that harbored bulbous-round-calcified-tissue in the dental papilla beneath the OA of a developing radiculomegaly was found radiographically. PSLCs were observed in the dental pulp. Genetic analysis revealed a novel mutation c.265G>A on Exon 4 and diagnosed as OFCD. CBCT images confirmed round-calcified-tissue and PSLC and that the length of an affected canine was 38.0 mm and calculated as +14.8SD. These novel findings were not observed in lateral incisors and molars. INTERVENTIONS: Observation was performed for 29 years and 3 months including orthodontic treatment for 2 years and 9 months. OUTCOME: Longitudinal follow-up for 26 years and 7 months after the treatment revealed that the development of radiculomegaly every few months or years, CDPs beneath OAs and PSLCs were observed. CDPs, PSLCs, and OAs were associated with radiculomegaly. The patient and the affected teeth including aligned teeth showed no particular change after the completion of the radiculomegaly. CBCT images showed bulbous-calcified-tissue and PSLCs in the mature dental pulp associated with radiculomegaly. LESSONS: The radiographical findings of CDP, OA and PSLC help early diagnose of OFCD and have importance for initiating orthodontic treatment until radiculomegaly completion.

The role of Rho-GEF Trio in regulating tooth root development through the p38 MAPK pathway.

Trio, the Rho guanine nucleotide exchange factor (Rho-GEF), plays diverse roles in cell migration, cell axon guidance and cytoskeleton reorganization. Conserved during evolution, Trio encodes two guanine nucleotide exchange factor domains (GEFs) and activates small GTPases. The Rho-family small GTPases RhoA and Rac1, which are target molecules of Trio, have been described to engage in craniofacial development and tooth formation. However, the exact role of Trio in tooth development remains elusive. In this study, we generated Wnt1-cre;Triofl/fl mice to address the potential function of Trio in tooth development. Wnt1-cre;Triofl/fl mice showed short root deformity as well as decreased expression of odontogenic makers such as RUNX2, OSX, OCN, and OPN. In vitro, Trio was silenced in human stem cells of dental papilla (SCAPs). Compared with the control group, the proliferation and migration ability in the experimental group was disrupted. After knocking down Trio in SCAPs, the cells showed phenotypes of poor odontogenic differentiation and weak mineralized nodules. To study the underlying mechanism, we investigated the p38 MAPK pathway and found that loss of Trio blocked the cascade transduction of p38 MAPK signaling. In conclusion, we identified Trio as a novel coordinator in regulating root development and clarified its relevant molecular events.

Regenerative endodontics: a comprehensive review.

The European Society of Endodontology and the American Association for Endodontists have released position statements and clinical considerations for regenerative endodontics. There is increasing literature on this field since the initial reports of Iwaya et al. (Dental Traumatology, 17, 2001, 185) and Banchs & Trope (Journal of Endodontics, 30, 2004, 196). Endogenous stem cells from an induced periapical bleeding and scaffolds using blood clot, platelet rich plasma or platelet-rich fibrin have been utilized in regenerative endodontics. This approach has been described as a 'paradigm shift' and considered the first treatment option for immature teeth with pulp necrosis. There are three treatment outcomes of regenerative endodontics; (i) resolution of clinical signs and symptoms; (ii) further root maturation; and (iii) return of neurogenesis. It is known that results are variable for these objectives, and true regeneration of the pulp/dentine complex is not achieved. Repair derived primarily from the periodontal and osseous tissues has been shown histologically. It is hoped that with the concept of tissue engineering, namely stem cells, scaffolds and signalling molecules, that true pulp regeneration is an achievable goal. This review discusses current knowledge as well as future directions for regenerative endodontics. Patient-centred outcomes such as tooth discolouration and possibly more appointments with the potential for adverse effects needs to be discussed with patients and parents. Based on the classification of Cvek (Endodontics and Dental Traumatology, 8, 1992, 45), it is proposed that regenerative endodontics should be considered for teeth with incomplete root formation although teeth with near or complete root formation may be more suited for conventional endodontic therapy or MTA barrier techniques. However, much is still not known about clinical and biological aspects of regenerative endodontics.

Bone resorption deficiency affects tooth root development in RANKL mutant mice due to attenuated IGF-1 signaling in radicular odontoblasts.

The tooth root is essential for normal tooth physiological function. Studies on mice with mutations or targeted gene deletions revealed that osteoclasts (OCs) play an important role in tooth root development. However, knowledge on the cellular and molecular mechanism underlying how OCs mediate root formation is limited. During bone formation, growth factors (e.g. Insulin-like growth factor-1, IGF-1) liberated from bone matrix by osteoclastic bone resorption stimulate osteoblast differentiation. Thus, we hypothesize that OC-osteoblast coupling may also apply to OC-odontoblast coupling; therefore OCs may have a direct impact on odontoblast differentiation through the release of growth factor(s) from bone matrix, and consequently regulate tooth root formation. To test this hypothesis, we used a receptor activator of NF-κB ligand (RANKL) knockout mouse model in which OC differentiation and function was entirely blocked. We found that molar root formation and tooth eruption were defective in RANKL-/- mice. Disrupted elongation and disorganization of Hertwig's epithelial root sheath (HERS) was observed in RANKL-/- mice. Reduced expression of nuclear factor I C (NFIC), osterix, and dentin sialoprotein, markers essential for radicular (root) odontogenic cell differentiation indicated that odontoblast differentiation was disrupted in RANKL deficient mice likely contributing to the defect in root formation. Moreover, down-regulation of IGF/AKT/mTOR activity in odontoblast indicated that IGF signaling transduction in odontoblasts of the mutant mice was impaired. Treating odontoblast cells in vitro with conditioned medium from RANKL-/- OCs cultured on bone slices resulted in inhibition of odontoblast differentiation. Moreover, depletion of IGF-1 in bone resorption-conditioned medium (BRCM) from wild-type (WT) OC significantly compromised the ability of WT osteoclastic BRCM to induce odontoblast differentiation while addition of IGF-1 into RANKL-/- osteoclastic BRCM rescued impaired odontoblast differentiation, confirming that root and eruption defect in RANKL deficiency mice may result from failure of releasing of IGF-1 from bone matrix through OC bone resorption. These results suggest that OCs are important for odontoblast differentiation and tooth root formation, possibly through IGF/AKT/mTOR signaling mediated by cell-bone matrix interaction. These findings provide significant insights into regulatory mechanism of tooth root development, and also lay the foundation for root regeneration studies.

BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice.

Signaling pathways are used reiteratively in different developmental processes yet produce distinct cell fates through specific downstream transcription factors. In this study, we used tooth root development as a model with which to investigate how the BMP signaling pathway regulates transcriptional complexes to direct the fate determination of multipotent mesenchymal stem cells (MSCs). We first identified the MSC population supporting mouse molar root growth as Gli1+ cells. Using a Gli1-driven Cre-mediated recombination system, our results provide the first in vivo evidence that BMP signaling activity is required for the odontogenic differentiation of MSCs. Specifically, we identified the transcription factors Pax9, Klf4, Satb2 and Lhx8 as being downstream of BMP signaling and expressed in a spatially restricted pattern that is potentially involved in determining distinct cellular identities within the dental mesenchyme. Finally, we found that overactivation of one key transcription factor, Klf4, which is associated with the odontogenic region, promotes odontogenic differentiation of MSCs. Collectively, our results demonstrate the functional significance of BMP signaling in regulating MSC fate during root development and shed light on how BMP signaling can achieve functional specificity in regulating diverse organ development.

The Sirt6 gene: Does it play a role in tooth development?

Dental Mesenchymal Cells (DMCs) are known to play a role in tooth development as well as in the repair and regeneration of dental tissue. A large number of signaling molecules regulate the proliferation and differentiation of DMC, though the underlying mechanisms are still not fully understood. Sirtuin-6 (SIRT6), a key regulator of aging, can exert an impact on embryonic stem cell (ESC) differentiation. The experimental deletion of Sirt6 in mouse bone marrow cells has been found to have an inhibiting impact on the bone mineral density and the osteogenic differentiation of these cells. The possible role of Sirt6 in tooth development, however, has at present remained largely unexplored. In the present study, we found that SIRT6 had no effect on tooth development before birth. However, Sirt6 gene deletion in knockout mice did have two post-natal impacts: a delay in tooth eruption and sluggishness in the development of dental roots. We propose an explanation of the possible molecular basis of the changes observed in Sirt6-/- mice. SIRT6 is expressed in mouse odontoblasts. Sirt6 deletion enhanced the proliferation of DMCs, as well as their capacity for adipogenic differentiation. On the other hand, it inhibited their capacity for in vitro osteogenic/chondrogenic differentiation. Further studies suggested that other factors may mediate the role of Sirt6 in odontogenesis. These include the nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (p38-MAPK), extracellular regulated MAP kinase (ERK) pathways and the mitochondrial energy. We demonstrated that Sirt6 plays a role in tooth root formation and confirmed that SIRT6 is necessary for DMC differentiation as well as for the development of the tooth root and for eventual tooth eruption. These results establish a new link between SIRT6 and tooth development.

F-spondin negatively regulates dental follicle differentiation through the inhibition of TGF-ß activity.

OBJECTIVE: F-spondin is an extracellular matrix (ECM) protein that belongs to the thrombospondin type I repeat superfamily and is a negative regulator of bone mass. We have previously shown that f-spondin is specifically expressed in the dental follicle (DF), which gives rise to the periodontal ligament (PDL) during the tooth root formation stage. To investigate the molecular mechanism of PDL formation, we investigated the function of f-spondin in DF differentiation. DESIGN: The expression patterning of f-spondin in the developing tooth germ was compared with that of periodontal ligament-related genes, including runx2, type I collagen and periostin, by in situ hybridization analysis. To investigate the function of f-spondin during periodontal ligament formation, an f-spondin adenovirus was infected into the bell stage of the developing tooth germ, and the effect on dental differentiation was analyzed. RESULTS: F-spondin was specifically expressed in the DF of the developing tooth germ; by contrast, type I collagen, runx2 and periostin were expressed in the DF and in the alveolar bone. F-spondin-overexpresssing tooth germ exhibited a reduction in gene expression of periostin and type I collagen in the DF. By contrast, the knockdown of f-spondin in primary DF cells increased the expression of these genes. Treatment with recombinant f-spondin protein functionally inhibited periostin expression induced by transforming growth factor-ß (TGF-ß). CONCLUSION: Our data indicated that f-spondin inhibits the differentiation of DF cells into periodontal ligament cells by inhibiting TGF-ß. These data suggested that f-spondin negatively regulates PDL differentiation which may play an important role in the immature phenotype of DF.

Cellular and molecular mechanisms of tooth root development.

The tooth root is an integral, functionally important part of our dentition. The formation of a functional root depends on epithelial-mesenchymal interactions and integration of the root with the jaw bone, blood supply and nerve innervations. The root development process therefore offers an attractive model for investigating organogenesis. Understanding how roots develop and how they can be bioengineered is also of great interest in the field of regenerative medicine. Here, we discuss recent advances in understanding the cellular and molecular mechanisms underlying tooth root formation. We review the function of cellular structure and components such as Hertwig's epithelial root sheath, cranial neural crest cells and stem cells residing in developing and adult teeth. We also highlight how complex signaling networks together with multiple transcription factors mediate tissue-tissue interactions that guide root development. Finally, we discuss the possible role of stem cells in establishing the crown-to-root transition, and provide an overview of root malformations and diseases in humans.

Increasing Risk of Disturbed Root Development in Permanent Teeth in Childhood Cancer Survivors Undergoing Cancer Treatment at Older Age.

BACKGROUND: Structural anomalies of teeth are observed at high rates in childhood cancer survivors (CCS). Several therapeutic exposures have been shown to be associated with dental developmental disturbances. This study was conducted to analyze the risk factors for dental developmental abnormality (DDA) and investigate the association between age at the time of cancer treatment and DDA in CCS. PATIENTS AND METHODS: Fifty-six CCS were enrolled. Orthopantomography and dental examination were performed in all the patients. We evaluated the prevalence of DDA and analyzed the risk factors for each type of DDA. RESULTS: DDAs were observed in 46.4% of CCS, including hypodontia in 9 (16.1%), abnormal roots in nine (16.1%), enamel defects/hypoplasia in 6 (10.7%), and microdontia in 12 (21.4%) patients. The number of patients with abnormal roots was significantly higher in the group treated with stem cell transplantation or at an age older than 4 years. We observed that the formation period of abnormal teeth coincided with the treatment period in the majority of CCS with DDA. CONCLUSIONS: Particularly regarding the root abnormality, treatment at elder age may be a risk factor for root developmental disturbances. Risk evaluation, appropriate follow-up, and early detection of dental issues are required for all CCS.