Epithelial Root Sheath Research Articles

Dental pulp stem cell‑derived extracellular vesicles loaded with hydrogels promote osteogenesis in rats with alveolar bone defects.

Alveolar bone defects caused by inflammation, trauma and tumors adversely affect periodontal health, causing tooth loosening or dentition defects, thus affecting denture or implant repair. Advancements in tissue engineering technology and stem cell biology have significantly improved the regenerative reconstruction of alveolar bone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells play important roles in exerting their therapeutic effects. Several studies have reported the role of dental pulp stem cells (DPSCs) in bone regeneration, but the regenerative effects of DPSC‑EVs on alveolar bone defects are unclear. In the present study, the osteogenic effects of DPSC‑EVs on Hertwig's epithelial root sheath (HERS) cells in vitro and their osteoinductive effects in an alveolar bone defect rat model were investigated. The results showed that DPSC‑EVs significantly promoted the expression of osteogenic genes, such as runt‑related transcription factor 2 and alkaline phosphatase, and increased the osteogenic differentiation capability of HERS. These findings suggested that transforming growth factor β1 inhibition decreased DPSC‑EV‑induced Smad, MAPK and ERK phosphorylation in HERS. In vivo, DPSC‑EV‑loaded hydrogels were transplanted into the alveolar sockets of Sprague‑Dawley rats and observed for eight weeks. The new bone grew concentrically in the DPSC‑EV or DPSC‑EV‑loaded hydrogel group, with greater bone mass than that in the control group, and the bone volume/total volume increased notably. The results confirmed the osteogenic and osteoinductive effects of DPSC‑EVs and DPSC‑Exo‑loaded hydrogels on alveolar bone defects. Due to their low immunogenicity, high stability, good biocompatibility and osteogenic propensity, DPSC‑EV‑loaded hydrogels are a safe cell‑free therapeutic approach for defective alveolar bone regeneration.

Immense potential for self-healing with conservative management of a large radicular cyst associated with pulpectomized primary tooth: A 40-month follow-up

Radicular cysts are one of the most common cysts of inflammatory origin involving the maxillofacial region that arise from the remnants of Herwig’s epithelial root sheath. These are often diagnosed as incidental findings on radiographs, which are seldom symptomatic and are slowly progressive. Decompression could be used as a conservative treatment approach for the management of large radicular cysts in children to have satisfactory healing and minimum damage to adjacent vital structures, simultaneously allowing the eruption of succedaneous tooth. This paper discusses the management of a large radicular cyst in a 12 year old, causing displacement of the mandibular canal and developing tooth germ using decompression that resulted in bony healing and spontaneous eruption of displaced succedaneous premolar, with a periodic follow-up for 40 months. Although decompression is a well-known approach, the present case with long-term follow-up emphasizes the potential of spontaneous healing in pediatric radicular cyst cases restoring the anatomy to normal without any functional impairment. This paper also highlights the importance of periodic clinical and radiographic follow-up in pulpally treated nonvital primary teeth.

Non-surgical treatment of maxillary lateral incisor double dens invaginatus type Ⅲ with apical periodontitis.

Dental invagination is an abnormality of the crown or root development induced during tooth germ development when the enamel-forming apparatus or epithelial root sheath overpopulates and folds into the papilla. In severe cases, the invaginated channels are connected to the pulp and periodontal tissues, often causing endodontic and periapical diseases. The complex anatomical pattern of this disease adds difficulty in its preoperative diagnosis and clinical operation. In this paper, we report a case of non-surgical treatment assisted by cone beam CT and microscopy for maxillary lateral incisor double dens invaginatus type Ⅲ (Ⅲa and Ⅲb) with apical periapical infection. After 1-year follow-up, the affected tooth was asymptomatic and the periapical lesion was significantly reduced.

Genetic Variants in KCTD1 Are Associated with Isolated Dental Anomalies.

KCTD1 plays crucial roles in regulating both the SHH and WNT/β-catenin signaling pathways, which are essential for tooth development. The objective of this study was to investigate if genetic variants in KCTD1 might also be associated with isolated dental anomalies. We clinically and radiographically investigated 362 patients affected with isolated dental anomalies. Whole exome sequencing identified two unrelated families with rare (p.Arg241Gln) or novel (p.Pro243Ser) variants in KCTD1. The variants segregated with the dental anomalies in all nine patients from the two families. Clinical findings of the patients included taurodontism, unseparated roots, long roots, tooth agenesis, a supernumerary tooth, torus palatinus, and torus mandibularis. The role of Kctd1 in root development is supported by our immunohistochemical study showing high expression of Kctd1 in Hertwig epithelial root sheath. The KCTD1 variants in our patients are the first variants found to be located in the C-terminal domain, which might disrupt protein-protein interactions and/or SUMOylation and subsequently result in aberrant WNT-SHH-BMP signaling and isolated dental anomalies. Functional studies on the p.Arg241Gln variant are consistent with an impact on β-catenin levels and canonical WNT signaling. This is the first report of the association of KCTD1 variants and isolated dental anomalies.

A Wnt10a-Notch signaling axis controls Hertwig’s epithelial root sheath cell behaviors during root furcation patterning

Human with bi-allelic WNT10A mutations and epithelial Wnt10a knockout mice present enlarged pulp chamber and apical displacement of the root furcation of multi-rooted teeth, known as taurodontism; thus, indicating the critical role of Wnt10a in tooth root morphogenesis. However, the endogenous mechanism by which epithelial Wnt10a regulates Hertwig’s epithelial root sheath (HERS) cellular behaviors and contributes to root furcation patterning remains unclear. In this study, we found that HERS in the presumptive root furcating region failed to elongate at an appropriate horizontal level in K14-Cre;Wnt10afl/fl mice from post-natal day 0.5 (PN0.5) to PN4.5. EdU assays and immunofluorescent staining of cyclin D1 revealed significantly decreased proliferation activity of inner enamel epithelial (IEE) cells of HERS in K14-Cre;Wnt10afl/fl mice at PN2.5 and PN3.5. Immunofluorescent staining of E-Cadherin and acetyl-α-Tubulin demonstrated that the IEE cells of HERS tended to divide perpendicularly to the horizontal plane, which impaired the horizontal extension of HERS in the presumptive root furcating region of K14-Cre;Wnt10afl/fl mice. RNA-seq and immunofluorescence showed that the expressions of Jag1 and Notch2 were downregulated in IEE cells of HERS in K14-Cre;Wnt10afl/fl mice. Furthermore, after activation of Notch signaling in K14-Cre;Wnt10afl/fl molars by Notch2 adenovirus and kidney capsule grafts, the root furcation defect was partially rescued. Taken together, our study demonstrates that an epithelial Wnt10a-Notch signaling axis is crucial for modulating HERS cell proper proliferation and horizontal-oriented division during tooth root furcation morphogenesis.

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. Insitu 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. Invitro 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.

The IL-1β-p65 axis stimulates quiescent odontogenic epithelial cell rests via TGF-β signalling to promote cell proliferation of the lining epithelia in radicular cysts: A laboratory investigation.

Cyst formation of the jaws is frequently accompanied by the proliferation of odontogenic epithelial cells located in the periodontal ligament (PDL), which consists of heterozygous cells and includes the most fibroblasts. The lining epithelium of radicular cyst, an odontogenic cyst of inflammatory origin, is derived from the proliferation of the remnants of the Hertwig epithelial root sheath (odontogenic epithelial cell rests of Malassez; ERMs) in the PDL. ERMs are maintained at a lower proliferative state under physiological conditions, but the regulatory mechanisms underlying the inflammation-dependent enhanced-proliferative capabilities of ERMs are not fully understood. The aim of this study was to evaluate the effects of cytokine pathway association between TGF-β signalling and IL-1β signalling on the regulation of odontogenic epithelial cell proliferation using radicular cyst pathological specimens and odontogenic epithelial cell lines. Immunofluorescence analyses were performed to clarify the expression levels of Smad2/3 and Ki-67 in ERMs of 8-week-old mouse molar specimens. In radicular cyst (n = 52) and dentigerous cysts (n = 6) specimens from human patients, the expression of p65 (a main subunit of NF-κB), Smad2/3 and Ki-67 were investigated using immunohistochemical analyses. Odontogenic epithelial cells and PDL fibroblastic cells were co-cultured with or without an inhibitor or siRNAs. Odontogenic epithelial cells were cultured with or without TGF-β1 and IL-1β. The proliferative capabilities and Smad2 phosphorylation levels of odontogenic epithelial cells were examined. Immunohistochemically, Smad2/3-positivity was increased, and p65-positivity and Ki-67-positivity were decreased both in ERMs and in the epithelial cells in dentigerous cysts, a non-inflammatory developmental cyst. In contrast, p65-positive cells, along with the expression of Ki-67, were increased and Smad2/3-positive cells were decreased in the lining epithelia of radicular cysts. Co-culture experiments with odontogenic epithelial cells and PDL fibroblastic cells revealed that PDL cells-derived TGF-β1/2 and their downstream signalling suppressed odontogenic epithelial cell proliferation. Moreover, TGF-β1 stimulation induced Smad2 phosphorylation and suppressed odontogenic epithelial cell proliferation, while IL-1β stimulation reversed these phenotypes through p65 transactivation. These results suggest that IL-1β-p65 signalling promotes odontogenic epithelial cell proliferation through suppressing TGF-β-Smad2 signalling, which would be involved in the pathogenesis of radicular cysts.

Three-dimensional visualization of Hertwig's epithelial root sheath during tooth root development with the miniTESOS tissue clearing method.

Hertwig's epithelial root sheath (HERS) acts as a signaling center that regulates the size, shape, and number of tooth roots. Therefore, understanding the anatomical changes in HERS during development is crucial for investigating its effect on root formation. However, the three-dimensional morphology of HERS and its changes during tooth root development remain largely unknown due to the limitations of traditional histological techniques. We developed an improved tissue clearing method for mouse embryonic and early postnatal mandibles, designated as a mini Transparent Embedding Solvent System (miniTESOS), based on the Transparent Embedding Solvent System (TESOS). We applied this method to the K14-Cre;Ai14 mouse line to systematically investigate the spatiotemporal dynamics of HERS at the cellular level during the development of the roots of mandibular first molars (MM1) and mandibular second molars (MM2). Additionally, using MM1 as a model, we quantitatively investigated the spatiotemporal changes in HERS in the root bifurcation region during the development of two-rooted teeth. In the early stages of root development, differences in growth rates and developmental patterns between MM1 and MM2 were observed in the mesiodistal and buccolingual directions, from the initiation to the fusion of buccal and lingual HERS. In the elongation stage of two-rooted teeth, continuous HERS was found exclusively at the leading edge of the root, gradually decreasing in length as the root extended. In the root bifurcation area, HERS undergoes four developmental stages: initiation, elongation, contact, and complete fragmentation, each characterized by specific morphological features. This study improves the understanding of the alterations in HERS during root development and summarizes its developmental pattern in the root bifurcation region. The miniTESOS tissue clearing method provides a new strategy to investigate tooth development.

Determinants of the Number of Main Canals in a Tooth: Deciphering Potential Mechanisms

Background: Although millions of root canal treatments are performed globally on a daily basis, factors that determine the number of main root canals in a tooth have not yet been elucidated. Variations in the number of root canals in different teeth is of utmost importance in clinical practice. However, clinicians aren´t aware about the determinants of such number, let alone these determinants have been approached in the literature, to the best of our knowledge. Purpose: This narrative review aimed to integrate the potential mechanisms involved in determining the number of main canals in a permanent tooth. Methods: We used the search terms “root canal number,” “root canal morphology,” “tooth morphology,” “root development,” and “root formation” to identify articles from the PubMed and Scopus databases. Results: 57 articles and 2 books were obtained. A multifactorial basis is plausible considering the influence of anthropological, demographic, environmental, genetic, epigenetic, tooth size related mechanisms and the pivotal role of Hertwig’s epithelial root sheath. Live-cell imaging techniques, mathematical models, quantitative genetics and dental phenomics could provide insightful information in the near future. Conclusions: Overall, it seems that the potential mechanisms determining the number of main canals in a tooth have a multifactorial basis. The orchestrating role of the Hertwig's epithelial root sheath seems pivotal, although the specific regulatory signals that induce or repress its diaphragmatic processes remain unknown. However, there is a dire need for molecular studies that help unveil these and other potential mechanisms involved.

Impaired breakdown of Herwig's epithelial root sheath disturbs tooth root development.

Wnt/β-catenin signaling plays a variety of roles in both the dental epithelium and mesenchyme at most stages of tooth development. In this study, we verified the roles of Hertwig's epithelial root sheath (HERS) breakdown in tooth root development. This breakdown results in formation of epithelial cell rests of Malassez (ERM). Following induction of β-catenin stabilization in the epithelium of developing tooth at the moment of HERS breakdown, HERS failed to break down for ERM formation. HERS with stabilized β-catenin was altered into a multicellular layer enveloping elongated root dentin with higher expression of junctional proteins such as Zo-1 and E-cadherin. Importantly, this impairment of HERS breakdown led to arrest of further root elongation. In addition, the portion of root dentin enveloped by the undissociated HERS remained in a hypomineralized state. The odontoblasts showed ectopically higher expression of pyrophosphate regulators including Ank and Npp1, whereas Tnap expression was unchanged. Our data suggest that Wnt/β-catenin signaling is decreased in HERS for ERM formation during root development. Furthermore, ERM formation is important for further elongation and dentin mineralization of the tooth roots. These findings may provide new insight to understand the contribution of ERM to root formation.

CDC42-mediated Wnt signaling facilitates odontogenic differentiation of DPCs during tooth root elongation

BackgroundCDC42 is a member of Rho GTPase family, acting as a molecular switch to regulate cytoskeleton organization and junction maturation of epithelium in organ development. Tooth root pattern is a highly complicated and dynamic process that dependens on interaction of epithelium and mesenchyme. However, there is a lack of understanding of the role of CDC42 during tooth root elongation.MethodsThe dynamic expression of CDC42 was traced during tooth development through immunofluorescence staining. Then we constructed a model of lentivirus or inhibitor mediated Cdc42 knockdown in Herwig’s epithelial root sheath (HERS) cells and dental papilla cells (DPCs), respectively. Long-term influence of CDC42 abnormality was assessed via renal capsule transplantation and in situ injection of alveolar socket.ResultsCDC42 displayed a dynamic spatiotemporal pattern, with abundant expression in HERS cells and apical DPCs in developing root. Lentivirus-mediated Cdc42 knockdown in HERS cells didn’t disrupt cell junctions as well as epithelium-mesenchyme transition. However, inhibition of CDC42 in DPCs undermined cell proliferation, migration and odontogenic differentiation. Wnt/β-catenin signaling as the downstream target of CDC42 modulated DPCs’ odontogenic differentiation. The transplantation and in situ injection experiments verified that loss of CDC42 impeded root extension via inhibiting the proliferation and differentiation of DPCs.ConclusionsWe innovatively revealed that CDC42 was responsible for guiding root elongation in a mesenchyme-specific manner. Furthermore, CDC42-mediated canonical Wnt signaling regulated odontogenic differentiation of DPCs during root formation.

Molar‐incisor‐malformation in a paediatric cancer patient: Case report

AbstractAimTo report a case of molar‐incisor‐malformation (MIM) in a 7‐year‐old boy with a history of congenital infantile fibrosarcoma of the upper chest, which was surgically treated, without adjuvant radio/chemotherapy.Material and MethodsThe boy presented with mobility and pain on percussion of the permanent mandibular first molars. The radiographic features were suggestive of MIM. Both teeth were extracted and processed for conventional light microscopic examination and non‐decalcified ground sections.ResultsThe microscopic features confirmed the diagnosis of MIM. Regular follow‐up was recommended.ConclusionsMedical interventions during hospitalization for cancer treatment, other than chemo/radiotherapy, may cause MIM by adversely affecting the function of Hertwig's epithelial root sheath. Regular dental screening in paediatric cancer patients may reveal more MIM cases and help in elucidating its pathogenesis.

Epithelial-specific deletion of FAM20A leads to short root defects

Short Root Defects defined by a reduced ratio of root to crown, may culminate in root resorption and subsequent tooth loss, in spite of the absence of apparent symptoms. Such defects present considerable impediments to orthodontic treatment and restoration. Recent identification of Fam20a, an emergent pseudokinase, has been associated with enamel development and tooth eruption, yet its definitive role in root formation and eruption remains ambiguous. In this research, we initially ascertained that the targeted knockout of Fam20a within the epithelium led to truncated tooth roots, irregular breaks in the epithelial root sheath initiation of the WNT signaling pathway, and decreased expression of the cell polarity-related transcription factor Cdc42 in murine models. This was concomitant with the participation of the associated epithelial root sheath developmental pathways BMP2, Gli1, and Nfic. Furthermore, we observed that Fam20a predominantly affects the intraosseous eruption phase of tooth emergence. During this phase, the osteoclast peak around the mandibular first molar in cKO mice is delayed, leading to a slower formation of the eruption pathway, ultimately resulting in delayed tooth eruption in mice. The findings of this study enrich the extant knowledge regarding the role of Fam20a, suggesting its potential regulatory function in tooth root development through the WNT/β-catenin/Cdc42 pathway.

Atp6i deficient mouse model uncovers transforming growth factor-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation

The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood. We found that Atp6i deficient mice (Atp6i−/−) arrested tooth root formation, indicated by truncated Hertwig’s epithelial root sheath (HERS) progression. Furthermore, Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation. Atp6i−/− mice had largely decreased expression of odontoblast differentiation marker gene expression profiles (Col1a1, Nfic, Dspp, and Osx) in the alveolar bone. Atp6i−/− mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers. Additionally, there was a significant reduction in Smad2/3 activation, inhibiting transforming growth factor-β (TGF-β) signaling in Atp6i−/− odontoblasts. Through treating pulp precursor cells with Atp6i−/− or wild-type OC bone resorption-conditioned medium, we found the latter medium to promote odontoblast differentiation, as shown by increased odontoblast differentiation marker genes expression (Nfic, Dspp, Osx, and Runx2). This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization, whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i−/− OC conditioned medium. Importantly, ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i−/− mice tooth germs were transplanted under mouse kidney capsules. Collectively, our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation. As such, this study uncovers TGF-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.

A Combinatorial Approach to Regenerate the Periodontal Ligament and Cementum in a Nondental Microenvironment

While treated dentin matrix (TDM) has been used for regeneration of dental tissues, the quality and quantity of regenerated periodontal tissue structure are suboptimal. The present study was undertaken to test whether the combined use of the TDM with dental follicle cells (DFCs) and Hertwig’s epithelial root sheath (HERS) cells enhances the regeneration of periodontal structures in a nondental microenvironment. TDMs were fabricated from 3-month-old Sprague–Dawley (SD) rats. DFCs and HERS cells were isolated from postnatal 7-day SD rats. Purified DFCs and HERS cells, both in combination or alone, were seeded and cultured on TDM in vitro and characterized. The cell-seeded TDMs were subsequently implanted into a 3-month-old rat greater omentum for 6 weeks, and further histological evaluation was performed. The results showed that cells grew well on the surface of TDMs, and mineralized nodules could be seen, especially in the HERS + DFCs group. After transplantation in rat omentum, periodontal ligament-like fibers and cementum-like structures were observed around the TDM in 1/3 of the samples in both the HERS group and the DFCs group and in 2/3 of the samples in the HERS + DFCs group, while almost no attached tissue formation was found in the TDM only group. The formed cementum width and the periodontal ligament length were significantly larger in the HERS + DFCs group. The periodontal ligament-like fibers in the HERS + DFCs group were orderly arranged and attached to the cementum-like tissues, which resembled the cementum-periodontal structure. Therefore, the combined use of DFCs, TDM, and HERS cells may be a promising strategy for the regeneration of the periodontal structures, especially in the nondental microenvironment.

Loss of Stat3 in Osterix+ cells impairs dental hard tissues development

BackgroundMutations in the signal transducers and activators of transcription 3 (STAT3) gene result in hyper-IgE syndrome(HIES), a rare immunodeficiency that causes abnormalities in immune system, bones and teeth. However, the role of Stat3 in development of dental hard tissues was yet to investigate.MethodsIn this study, a transgenic mouse of conditional knockout of Stat3 in dental mesenchymal cells (Osx-Cre; Stat3fl/fl, Stat3 CKO) was made. The differences of postnatal tooth development between control and Stat3 CKO mice were compared by histology, µCT and scanning electron microscopy.ResultCompared with the control, Stat3 CKO mice were presented with remarkable abnormal tooth phenotypes characterized by short root and thin dentin in molars and incisors. The enamel defects were also found on mandibular incisors. showed that Ki67-positive cells significantly decreased in dental mesenchymal of Stat3 CKO mice. In addition, β-catenin signaling was reduced in Hertwig's epithelial root sheath (HERS) and odontoblasts of Stat3 CKO mice.ConclusionsOur results suggested that Stat3 played an important role in dental hard tissues development, and Stat3 may regulate dentin and tooth root development through the β-catenin signaling pathway.

Wnt signaling from Gli1-expressing apical stem/progenitor cells is essential forthe coordination of tooth root development.

Stem cell regulation plays a crucial role during development and homeostasis. Here, an essential source of Wnts from Gli1+ stem/progenitor cells was identified in the murine molar. Loss of Wnt production in Gli1+ apical stem/progenitor cells led to loss of Axin2 at the root apex, mis-regulation of SOX9, loss of BMP and Hh signaling, and truncation of root development. In the absence of Wnt signals, the root epithelium lost its integrity and epithelial identity. This phenotype could be partially mimicked by loss of Sox9 in the Gli1 population. Stabilization of Wnt signaling in the apical papilla led to rapid unordered differentiation of hard tissues and fragmentation of the epithelial root sheath. Wnt signaling from Gli1+ stem/progenitor cells, therefore, orchestrates root development, coordinating mesenchymal and epithelial interactions via SOX9 to regulate stem/progenitor cell expansion and differentiation. Our results demonstrate that disparate stem/progenitor cell populations are unified in their fundamental signaling interactions.

The role of cell division cycle 42 in root development of tooth and the involved mechanisms

Objective: To explore the effect of cell division cycle 42 (CDC42) on root development and its regulation on cell proliferation and migration in Hertwig's epithelial root sheath (HERS). Methods: Trace the spatiotemporal expression of CDC42 in root development process [postnatal day 5 (P5), P7, P14] through immunofluorescence staining. Nine eight-week-old C57BL/6J male mice were randomly divided into 3 groups using a simple random sample method (n=3 in each group). P3 tooth germ was cultured in air-liquid system for 1 day and then transplanted to renal capsule each to observe tooth root development. The control group implanted tooth germ only. The phosphate buffered saline (PBS) group implanted tooth germ and gel beads soaked with PBS, while the ML141 group implanted tooth germ and gel beads soaked with CDC42 inhibitor (ML141). Cdc42 in HERS cells was inhibited via lentivirus transfection. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and scratch assay were performed. The distribution of Golgi apparatus (GM130) and cytoskeleton (F-actin) in migrated cells were mapped via immunofluorescence staining. Results: CDC42 was expressed in epithelial cells of HERS, polarized ameloblasts and odontoblasts, as well as adjacent dental papilla and dental follicle cells during tooth root development. The root length of the ML141 group [(0.61±0.09) mm] was substantially shorter than that of control group [(1.03±0.19) mm, P=0.007] and PBS group [(0.98±0.10) mm, P=0.021] according to the data of renal capsule transplantation. After lentiviral transfection, the relative expression of Cdc42 in knockdown group (0.31±0.33) was significantly lower than that in control group (1.05±0.08) (t=15.38, P<0.001), demonstrating the knockdown efficiency closed to 70%. Cell viabilities were significantly inhibited in knockdown group (0.87±0.04, 0.96±0.10, 0.59±0.06, respectively) compared with those in control group (1.09±0.13, 1.55±0.32, 1.10±0.09, respectively) after 3, 4 and 5 days (t=3.16, P=0.016; t=4.23, P=0.002; t=5.08, P<0.01), and the cell proliferation ability in knockdown group [(1.65±0.64)%] also decreased than that in the control group [(4.02±1.12)%](t=5.21, P<0.001). In addition, the cell migration rates after 24 and 48 h [(45.1±4.2)%, (56.4±8.3)%] in knockdown group were obviously lower than those in the control group [(63.8±7.4)%, (80.2±7.8)%] (t=3.78, P=0.019; t=3.62, P=0.023). After Cdc42 was knocked down, Golgi apparatus distributed along the nucleus while behaved oriented in the control group. Conclusions: CDC42 plays an important role in the regulation of root length during root development, which may mediate root elongation by affecting the migration and proliferation of HERS cells.

A Mutation in CACNA1S Is Associated with Multiple Supernumerary Cusps and Root Maldevelopment

Enamel knots and Hertwig epithelial root sheath (HERS) regulate the growth and folding of the dental epithelium, which subsequently determines the final form of tooth crown and roots. We would like to investigate the genetic etiology of seven patients affected with unique clinical manifestations, including multiple supernumerary cusps, single prominent premolars, and single-rooted molars. Oral and radiographic examination and whole-exome or Sanger sequencing were performed in seven patients. Immunohistochemical study during early tooth development in mice was performed. A heterozygous variant (c. 865A>G; p.Ile289Val) in CACNA1S was identified in all the patients, but not in an unaffected family member and control. Immunohistochemical study showed high expression of Cacna1s in the secondary enamel knot. This CACNA1S variant seemed to cause impaired dental epithelial folding; too much folding in the molars and less folding in the premolars; and delayed folding (invagination) of HERS, which resulted in single-rooted molars or taurodontism. Our observation suggests that the mutation in CACNA1S might disrupt calcium influx, resulting in impaired dental epithelium folding, and subsequent abnormal crown and root morphology.

Difference in apical resorption activity during rat molar root formation in response to mechanical force.

To investigate whether there is a difference in apical resorption activity during the development of roots in response to mechanical force in vivo. Maxillary first molars (M1) from postnatal day (PN) 21 and PN35 male rats were selected as representatives of the root-developing and root-completing groups, respectively. A mechanical force of 3 cN was applied to M1 on PN21 and PN35, and the maxilla was collected on PN28 and PN42. Odontoclastogenesis and root morphology were investigated using micro-focus X-ray computed tomography, followed by immunohistochemistry and quantitative real-time polymerase chain reaction to clarify root resorption activity. Development of the mesiobuccal root (MBR) preceded the mesial root (MR). In the PN28 force application (FA) group, the dentine was bent, but the histology, including Hertwig's epithelial root sheath (HERS), was intact. No odontoclasts and resorption lacunae were found in the apical area of the MRs, and only lateral root resorption was observed. External apical root resorption (EARR) was observed in the MR of PN42 (FA) and in the MBR of both PN28 (FA) and PN42 (FA). The expression of osteopontin changed accordingly. No significant change occurred in osteoprotegerin or receptor activator of nuclear factor-κB ligand expression in the MRs of the PN28 (FA) group. Our animal model did not adequately simulate the clinical process of tooth movement in humans. Force application delayed HERS dissociation on the compression side of the developing roots, leading to inhibitory effects on cementogenesis, which resulted in decreased odontoclast differentiation and prevention of EARR.