Epithelial Root Sheath Research Articles

Calcific metamorphosis of pulp after extrusive luxation.

The literature on the pathogenesis of extrusive dental luxation has been focused on periodontal tissue responses, with little attention given to the pulp. The aim of this study was to evaluate the response of dental pulp of teeth following extrusive luxation in a rat model. The maxillary right central incisors of 30 rats were extrusively luxated and repositioned after 5minutes. The animals were euthanized after 7, 15, and 30days to provide three groups: I, II, and III, respectively (n=10). Histological sections were stained with H and E for histomorphometric analysis of the odontoblast layer, reparative dentin deposition, Hertwig's epithelial root sheath, pulp necrosis, and periapical inflammatory infiltrate. In most cases, new vascular formation occured in association with reparative dentin deposition on the root walls and within the pulp. In some cases, dentin deposition occupied the entire pulp space over time, with no other types of non-odontogenic hard tissues being observed. Pulp necrosis with the presence of periapical inflammatory infiltrate was also observed in a few cases. No statistical differences were observed among the studied groups. Following extrusive luxation, calcific metamorphosis of the pulp is very likely to occur.

Taurodontism part 1: history, aetiology and molecular signalling, epidemiology and classification

Abstract: Taurodontism is a developmental morphological anomaly that affects the internal anatomy of the multi-rooted teeth; hence, it is difficult to distinguish from a normal tooth structure based only on external appearance. In this defect, the teeth have elongated and cylindrical pulp cavities, a deep furcation and short roots. This article aims to address an important problem related to the cause of taurodontism. Although it is a well-held belief that taurodontism is caused by delayed or failed invagination of Hertwig's Epithelial Root Sheath (HERS), the molecular mechanism that signals this delay or failure had not been well understood until recently. This article discusses the history, aetiology, prevalence and classification of this morphological disorder, whereas part 2 of this 2-part series will discuss the biomechanics of taurodontic teeth, differential diagnosis and congenital disorders that mimic taurodontism, relevant syndromes, clinical implications and management. CPD/Clinical Relevance: In order to treat cases presenting taurodontism, it is essential to understand this anomaly along with its history, aetiology and associated epidemiology. This will provide clinicians with a deeper understanding when they encounter a relatively unnoticeable anomaly.

A RECENT UPDATE ON HISTOGENESIS OF ADENOMATOID ODONTOGENIC TUMOR: A REVIEW

Adenomatoid odontogenic tumor (AOT) is a relatively rare benign, epithelial tumor of odontogenic origin. There is varying class of thoughts contemplating this lesion to be a hamartoma or neoplastic growth of odontogenic epithelium. Controversy regarding the histogenesis of the lesion is plentiful in earlier literature. The recent advent of immunohistochemical and ultrastructural studies has aided in throwing light on the tissue of origin of this tumor. This review aims at understanding the evolving concepts of histogenesis of AOT to better understand the biological behavior of the lesion. The review of AOT was carried out using PubMed and Google Scholar databases and 39 articles from the year 2001 to 2016 which contributed to the histogenesis of AOT were included for review. Since the origin of the cystic lining is similar to a reduced enamel epithelium and not the dental lamina, we propose the former to be the progenitor of AOT. Furthermore, we consider extra-follicular, as well as peripheral AOTs, originate from the remnants of Hertwig’s epithelial root sheath (epithelial rests of Malassez), which complies with the common histology for all these variants.

Three-dimensional configuration of apical epithelial compartments including stem cell niches in guinea pig cheek teeth

ObjectivesContinuously growing rodent incisors have an apically located epithelial stem cell compartment, known as an “apical bud” (AB). Few studies have described the morphological features of ABs and stem cell niches in continuously growing premolars/molars. We attempted to clarify the relationship between the three-dimensional configuration of ABs and the stem cell niches in guinea pig cheek teeth. MethodsWe perfusion-fixed four-week-old guinea pigs, then decalcified their premolars/molars to produce serial paraffin sections, which we immunostained for Sox2. We reconstructed the serial sections using image processing and analysis software. We processed undecalcified samples for scanning electron microscopy by KOH digestion. ResultsTwo types of epithelia with M and Δ shapes surrounded the S-shaped dental papilla in the apical region of the premolars/molars, and there were three Sox2-positive epithelial bulges above the M- and Δ-shaped epithelia. Sox2-positive epithelial stem cell niches were restricted to the apical side, and cell proliferation and differentiation immediately proceeded in the crown-analogue dentin. The Sox2-positive epithelial stem cell niches were sparsely distributed and extended to the occlusal side. We also detected continuously proliferating cells in the cervical loop and Hertwig’s epithelial root sheath of the root-analogue dentin. ConclusionsOur findings suggest that guinea pig cheek teeth have three ABs, and the complex configuration of these types of teeth may be attributed to the prompt formation of crown-analogue dentin followed by the long-term formation of root-analogue dentin.

Development of immortalized Hertwig\u2019s epithelial root sheath cell lines for cementum and dentin regeneration

BackgroundHertwig’s epithelial root sheath (HERS) is important in guiding tooth root formation by differentiating into cementoblasts through epithelial–mesenchymal transition (EMT) and inducing odontoblastic differentiation of dental papilla through epithelial–mesenchymal interaction (EMI) during the tooth root development. Thus, HERS cells are critical for cementum and dentin formation and might be a potential cell source to achieve tooth root regeneration. However, limited availability and lifespan of primary HERS cells may represent an obstacle for biological investigation and therapeutic use of tooth tissue engineering. Therefore, we constructed, characterized, and tested the functionality of immortalized cell lines in order to produce a more readily available alternative to HERS cells.MethodsPrimary HERS cells were immortalized via infection with lentivirus vector containing the gene encoding simian virus 40 Large T Antigen (SV40LT). Immortalized HERS cell subclones were isolated using a limiting dilution method, and subclones named HERS-H1 and HERS-C2 cells were isolated. The characteristics of HERS-H1 and HERS-C2 cells, including cell proliferation, ability of epithelial–mesenchymal transformation and epithelial–mesenchymal interaction, were determined by CCK-8 assay, immunofluorescence staining, and real-time PCR. The cell differentiation into cementoblast-like cells or periodontal fibroblast-like cells was confirmed in vivo. And the inductive influence of the cell lines on dental papilla cells (DPCs) was also confirmed in vivo.ResultsHERS-H1 and HERS-C2 cells share some common features with primary HERS cells such as epithelial-like morphology, positive expression of CK14, E-Cadherin, and Vimentin, and undergoing EMT in response to TGF-beta. HERS-C2 cells showed the EMT characteristics and could differentiate into cementum-forming cells in vitro and generate cementum-like tissue in vivo. HERS-H1 could induce the differentiation of DPCs into odontoblasts in vitro and generation of dentin-like tissue in vivo.ConclusionsWe successfully isolated and characterized novel cell lines representing two key features of HERS cells during the tooth root development and which were useful substitutes for primary HERS cells, thereby providing a biologically relevant, unlimited cell source for studies on cell biology, developmental biology, and tooth root regeneration.

Insulinーlike growth factorーI stimulates the disintegration of Hertwig's epithelial root sheath and cellular cementogenesis in mouse molars in vitro.

Insulinーlike growth factorーI stimulates the disintegration of Hertwig's epithelial root sheath and cellular cementogenesis in mouse molars in vitro.

Parathyroid hormone-related peptide (1-34) promotes tooth eruption and inhibits osteogenesis of dental follicle cells during tooth development.

Dental follicle cells (DFCs) activate and recruit osteoclasts for tooth development and tooth eruption, whereas DFCs themselves differentiate into osteoblasts to form alveolar bone surrounding tooth roots through the interaction with Hertwig's epithelial root sheath (HERS). Also during tooth development, parathyroid hormone-related peptide (PTHrP) is expressed surrounding the tooth germ. Thus, we aimed to investigate the effect of PTHrP (1-34) on bone resorption and osteogenesis of DFCs in vitro and in vivo. In vitro studies demonstrated that DFCs cocultured with HERS cells expressed higher levels ofBSP and OPN than the DFCs control group, whereas cocultured DFCs treated with PTHrP (1-34) had lower expressions of ALP, RUNX2, BSP, and OPN than the cocultured DFCs control group. Moreover, we found PTHrP (1-34) inhibited osteogenesis of cocultured DFCs by inactivating the Wnt/β-catenin pathway. PTHrP (1-34) also increased the expression of RANKL/OPG ratio in DFCs. Consistently, in vivo study found that PTHrP (1-34) accelerated tooth eruption and inhibited alveolar bone formation. Therefore, these results suggest that PTHrP (1-34) accelerates tooth eruption and inhibits osteogenesis of DFCs by inactivating Wnt/β-catenin pathway.

Are Hertwig’s epithelial root sheath cells necessary for periodontal formation by dental follicle cells?

ObjectiveThe role of Hertwig’s epithelial root sheath (HERS) cells in periodontal formation has been controversial. This study aimed to further clarify whether HERS cells participate in formation of the periodontium, and the necessity of HERS cells in differentiation of dental follicle cells (DFCs) for periodontal regeneration. DesignHERS cells and DFCs were isolated and identified from post-natal 7-day Sprauge-Dawley rats. In vitro, direct co-culture of HERS cells and DFCs as well as the individual culture of HERS and DFCs were performed and followed by alizarin red staining and the quantitative real-time polymerase chain reaction analysis. For in vivo evaluation, the inactivated dentin matrix (iTDM) was fabricated. HERS cells and DFCs were seeded in combination or alone on iTDM and then transplanted into the rat omentum. Scanning electron microscope and further histological analysis were carried out. ResultsIn vitro, mineral-like nodules were found in the culture of HERS cells alone or HERS + DFCs either by alizarin red staining or scanning electronic microscope. The mineralization and fiber-forming relevant mRNA expressions, such as bone sialoprotein, osteopontin, collagen I and collagen III in HERS + DFCs were significantly higher than that of the HERS or DFCs alone group. After transplantation in vivo, cementum and periodontal ligament-like tissues were formed in groups of HERS + DFCs and HERS alone, while no evident hard tissues and attached fibers were found in DFCs alone. ConclusionsHertwig’s epithelial root sheath cells directly participate in the formation of the periodontium, and they are essential for the differentiation of dental follicle cells to form periodontal structures. The combination use of Hertwig’s epithelial root sheath cells and dental follicle cells is a promising approach for periodontal regeneration.

Comparative study on differentiation of cervical-loop cells and Hertwig\u2019s epithelial root sheath cells under the induction of dental follicle cells in rat

Cervical loop cells (CLC) and Hertwig’s epithelial root sheath (HERS) cells are believed to play critical roles in distinct developmental patterns between rodent incisors and molars, respectively. However, the differences in differentiation between CLC and HERS cells, and their response to inductions from dental follicle cells, remain largely unknown. In present study, CLC and HERS cells, as well as incisor dental follicle (IF) cells and molar dental follicle (MF) cells were isolated from post-natal 7-day rats. IF and MF cell derived conditioned medium (CM) was obtained for induction of CLC and HERS cells. In vitro experiments, we found that, under the induction of dental follicle cell derived CM, CLC cells maintained the epithelial polygonal-shapes and formed massive minerals, while part of HERS cells underwent shape transformation and generated granular minerals. CLC cells expressed higher enamel-forming and mineralization related genes, while HERS cells showed opposite expression patterns of BMP2, BMP4, AMBN and AMGN. In vivo, CLC cells generated enamel-like tissues while HERS cells formed cementum-periodontal ligament-like structures. Taken together, CLC and HERS cells present distinct differentiation patterns under the inductions from dental follicle cells.

Malignant tumor of outer root sheath epithelium, trichilemmal carcinoma. Clinical presentations, treatments and outcomes.

Objectives:To emphasize different clinical features of tumor that can be misdiagnosed clinically.Methods:A total of 8 cases operated between September 2009 and 2016 at the Celal Bayar University, Faculty of Medicine were included in the study. Patients’ clinicopathological features, type of surgery and follow up information were evaluated.Results:Six patients were male. The average age was 75.50. The lesions were located on the head and neck, and chest wall. Six patients had a history of the rapid growth of lesion. There was no metastasis at the time of diagnosis. None of the patients needed adjuvant therapy. Mean follow up time was 19.37 months. None of the patients developed recurrence or metastasis.Conclusion:This tumor resembles basal or squamous cell carcinoma. The histopathological evaluation may lead to misdiagnosis. Regional or distant metastasis is very rare. There is no consensus about adjuvant therapy. Screening for metastasis and close follow up are mandatory.

Modulation of microRNAs in Tooth Root and Periodontal Tissue Development.

Tooth root development begins after the completion of tooth crown development. Both the tooth root and crown undergo a series of interactions between the epithelium and adjacent mesenchymal cells. Although many studies have evaluated tooth crown formation, little is known about the regulatory mechanisms of tooth root development. MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein expression through post-transcriptional mechanisms and participate in a broad range of biological processes, from development to tumorigenesis. The functional importance of miRNAs on the development of tooth root and periodontal tissues has been suggested in many studies. To summarize the functions of miRNAs on tooth root and periodontal tissue development. MicroRNAs are important to root odontogenesis, Hertwig's epithelial root sheath and periodontal tissue development, and have functions in stem cells from dental or periodontal tissues. The modulation of miRNAs in tooth root and periodontal tissue development is fine tuning.

Revitalization Teeth: A Prospective Case Series

In addition to caries, dental trauma is one of the most common causes of damage to permanent teeth and pulp. Pulp necrosis or damage to Hertwig’s epithelial root sheath (HERS) leads to arrested tooth root development in immature teeth. Pulp necrosis can be treated by revitalization, a biology-based treatment alternative to apexification. Induction of a blood clot inside the root canal can lead to healing of periapical lesions and increased root length and thickness. Traumatic impact as the cause of pulp necrosis may affect the treatment outcome negatively, depending on the severity of damage to HERS. Revitalization procedures in four teeth with pulp necrosis following dental trauma were performed using a standardized treatment protocol. Three teeth were dislocated, the fourth tooth was avulsed. Each patient exhibited at least two clinical signs of pulp necrosis as well as radiographic evidence of apical periodontitis. X-rays were taken using individualized film holders (IFH) to reliably assess the treatment outcome. Revitalization treatment was performed without instrumentation of the canal walls, but disinfection with sodium hypochlorite and intracanal dressing with triple antibiotic paste (TAP) for three weeks. Provocation of bleeding was induced in a second visit, the blood clot was covered with collagen followed by calcium silicate cement, and teeth were sealed with resin composite. Clinical and radiographic follow-ups were performed after 1, 3, 6 and 12 months. An increase of root length and thickness was evident in the three teeth with dislocation injuries. In one case, formation of mineralized tissue below the calcium silicate cement was observed. The tooth which had been avulsed and replanted showed resorption of the apical root area. The observations made in this study support the assumption that a separation of HERS and the cells that form pulp and dentin during tooth root development may negatively affect the outcome after a standardized revitalization procedure. The consistent implementation of standardized treatment protocols and the use of IFH are helpful receiving a reliable treatment outcome.

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.

Harmine promotes molar root development via SMAD1/5/8 phosphorylation

Tooth and bone are major tissues involved in physiological calcification in the body, and they use similar molecular pathways for development, homeostasis, and regeneration. Harmine (HMN) is a natural small compound that stimulates osteoblast differentiation in vitro and in vivo. Here we examined the biological effect of HMN on the postnatal development of molar tooth roots and periodontal tissues. HMN supported the formation of tooth roots and periodontal tissues in developing tooth germs. In tooth germ organ culture, HMN promoted the elongation of Hertwig's epithelial root sheath (HERS) and stimulated cell proliferation in HERS and dental follicle-derived tissues, including dental papillae and dental follicles. HMN stimulated cell proliferation and cell movement of HERS-derived cells without mesenchymal cells in vitro and directly induced the phosphorylation of SMAD1/5/8 protein in HERS-derived cells. Our results indicated that HMN was the first natural small compound to stimulate postnatal development of tooth germs.

Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae.

During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/β-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted β-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/β-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferationand odontoblastic differentiation.

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.

Localization of RELM-β/FIZZ2 Is Associated with Cementum Formation.

Resistin-like molecule-β/found in inflammatory zone 2 (RELM-β/FIZZ2) is a cysteine-rich secretory protein that is localized in the epithelium of the gastrointestinal tract and lung alveoli. Previous reports have suggested that this protein regulates glucose metabolism and inflammation. In the present study, to analyze the involvement of RELM-β/FIZZ2 in tooth development, we immunohistochemically examined the localization of RELM-β/FIZZ2 in tooth germs of embryonic days (E) 15-20 and postnatal days (P) 7-42 rats. RELM-β/FIZZ2 was hardly detected in the tooth germ at the bud (E15) stage. However, at the cap (E17) and bell (E20) stages, this protein was detectable in the inner enamel epithelium; whereas cells in the other parts of the enamel organ including the outer enamel epithelium and stellate reticulum did not show the immunoreactivity. During the root formation stage (P14-28), cells in Hertwig's epithelial root sheath (HERS) localized RELM-β/FIZZ2. Intense immunoreactivity was also seen in the matrix of the root dentin facing the HERS and the dental follicle. This reactivity was not present on the more upwardly located dentin surface. In contrast, cementum matrix positive for osteopontin and bone sialoprotein was observed on the dentin instead of immunoreactivity for RELM-β/FIZZ2. Osterix-positive cells, indicating cementoblast progenitors, were also detected in the dental follicle near the matrix positive for RELM-β/FIZZ2. These results suggest that RELM-β/FIZZ2 secreted by the inner enamel epithelium was mainly localized in the matrix at the surface of the apical root dentin and might be involved in cementogenesis. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1865-1874, 2017. © 2017 Wiley Periodicals, Inc.

Expression and localization of Yap and Taz during development of the mandibular first molar in rats

Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz) are two downstream factors in the Hippo signaling pathway. Yap and Taz participate in regulating organ size, stem cell self-renewal, proliferation and differentiation. We investigated the spatial-temporal expression and relative expression levels of Yap and Taz using immunohistochemistry and real-time polymerase chain reaction. We found Yap and Taz in the oral epithelium and mesenchyme at embryonic (E) day 14.5 (E14.5) and E16.5. By E18.5, Yap and Taz were detected in the dental papilla and the entire enamel organ. At postnatal (P) day 0 (PN0), PN3 and PN7, Yap and Taz expression was localized in ameloblasts, odontoblasts and stratum intermedium. Yap and Taz were expressed in Hertwig’s epithelial root sheath (HERS) at PN7. At PN3, PN7 and PN14, Yap was detected in the enamel matrix. From PN21 to PN28, Yap and Taz were absent from differentiated ameloblasts, but they were expressed in odontoblasts. From PN0 to PN10, the Yap and Taz mRNA expression increased, then decreased. We found that Yap and Taz may influence the differentiation of ameloblasts and odontoblasts; they also may contribute to enamel mineralization, crown morphogenesis and root formation.

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.

Hertwig's epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by TGF-β.

In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwig's epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-β regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-β was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-β1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-β1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-βsignaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.