Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.
Mice ; Animals ; Durapatite/metabolism* ; Dental Enamel/metabolism* ; Ameloblasts/metabolism* ; Amelogenesis ; Stem Cells ; Organoids

Tooth development is a complex process that involves precise and time-dependent orchestration of multiple genetic, molecular, and cellular interactions. Ameloblastin (AMBN, also named "amelin" or "sheathlin") is the second most abundant enamel matrix protein known to have a key role in amelogenesis. Amelogenesis imperfecta (AI [MIM: 104500]) refers to a genetically and phenotypically heterogeneous group of conditions characterized by inherited developmental enamel defects. The hereditary dentin disorders comprise a variety of autosomal-dominant genetic symptoms characterized by abnormal dentin structure affecting either the primary or both the primary and secondary teeth. The vital role of Ambn in amelogenesis has been confirmed experimentally using mouse models. Only two cases have been reported of mutations of AMBN associated with non-syndromic human AI. However, no AMBN missense mutations have been reported to be associated with both human AI and dentin disorders. We recruited one kindred with autosomal-dominant amelogenesis imperfecta (ADAI) and dentinogenesis imperfecta/dysplasia characterized by generalized severe enamel and dentin defects. Whole exome sequencing of the proband identified a novel heterozygous C-T point mutation at nucleotide position 1069 of the AMBN gene, causing a Pro to Ser mutation at the conserved amino acid position 357 of the protein. Exfoliated third molar teeth from the affected family members were found to have enamel and dentin of lower mineral density than control teeth, with thinner and easily fractured enamel, short and thick roots, and pulp obliteration. This study demonstrates, for the first time, that an AMBN missense mutation causes non-syndromic human AI and dentin disorders.
Adult ; Amelogenesis Imperfecta ; genetics ; Cells, Cultured ; China ; Codon ; Dentin ; abnormalities ; ultrastructure ; Female ; Humans ; Male ; Microsatellite Repeats ; Microscopy, Electron, Scanning ; Middle Aged ; Mutation, Missense ; Pedigree ; RNA ; analysis ; Transfection ; Whole Exome Sequencing

The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary 2(nd) molar germs of rats. We used the maxillary 2(nd) molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary 2(nd) molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.
Ameloblasts ; Amelogenesis ; Amelogenin ; Animals ; Blotting, Western ; Dental Enamel ; Fluorescent Antibody Technique ; Molar ; Polymerase Chain Reaction ; Rats ; Reverse Transcription ; RNA, Messenger

OBJECTIVE: To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoA^{Dominant Negative} (EGFP-RhoA^DN) transgenic mice model, from the aspect of adherens junctions, and to provide a theory basis for mechanism of enamel development defects. METHODS: The enamel thickness of mandibular first molars of EGFP-RhoA^DN transgenic mice and wild type (WT) mice were observed by scanning electronic microscopy at 20 kV, and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by ImageJ (Rasband, 1997-2009). The enamel organs from mandibular first molars from postnatal-4-day (P4) EGFP-RhoA^DN mice and wild type mice were isolated, and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoA^DN transgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay. RESULTS: The EGFP-RhoA^DN transgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group (n=20), and enamel thickness was (84.60±0.20) μm vs. (106.24±0.24) μm, P<0.05. The protein expressions of E-cadherin, α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoA^DN transgenic mice molars were down-regulated, and the protein level of β-catenin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was down-regulated versus that of WT mice, and the gene expression of E-cadherin was 0.93±0.01 vs. 1.00±0.02, P<0.05. The mRNA level of β-catenin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was up-regulated versus that of WT mice, and the gene expression of β-catenin was 1.23±0.03 vs. 1.00±0.05, P<0.05. CONCLUSION In the mandibular first molars of EGFP-RhoA^DN transgenic mice, the enamel formation was disrupted and the adherens junctions of EGFP-RhoA^DN transgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.
Adherens Junctions ; Ameloblasts ; Amelogenesis ; Animals ; Antigens, CD ; Cadherins/metabolism* ; Dental Enamel/metabolism* ; Enamel Organ ; Humans ; Mice ; Mice, Transgenic ; Molar ; Signal Transduction ; alpha Catenin ; beta Catenin ; rhoA GTP-Binding Protein/physiology*

Amelogenesis imperfecta characterized as abnormally formed enamel is caused by a defect of unique group of genes. Patients affected by this disease might have difficulties in social and psychological aspects due to non-esthetic teeth as well as functional problems caused by enamel detachment and tooth wear from their early ages. Adult patients with amelogenesis imperfecta can be treated with full-mouth restorations, which make functional and esthetic rehabilitations of severely worn tooth. However, the anterior open bite and lack of occlusal clearance for posterior teeth restorations due to compensatory extrusion are the intervening factors in the prosthetic treatment. Therefore, the determination of anterior tooth lengths, vertical dimension, and anterior guidance should be set carefully. Recently, computer-aided design and computer-aided manufacturing (CAD/CAM) techniques help systematic approaches and enable dentists to reduce time-consuming procedures in the diagnosis and treatment of full-mouth rehabilitation. This case report demonstrates the successful full mouth rehabilitation using a CAD/CAM system in a young adult patient with amelogenesis imperfecta and anterior open bite.
Adult ; Amelogenesis Imperfecta* ; Amelogenesis* ; Computer-Aided Design ; Dental Enamel ; Dentists ; Diagnosis ; Humans ; Mouth Rehabilitation ; Open Bite* ; Rehabilitation* ; Tooth ; Tooth Wear ; Vertical Dimension ; Young Adult

Numerous cases of enamel renal syndrome have been previously reported. Various terms, such as enamel renal syndrome, amelogenesis imperfecta and gingival fibromatosis syndrome, and enamel-renal-gingival syndrome, have been used for patients presenting with the dental phenotype characteristic of this condition, nephrocalcinosis or nephrolithiasis, and gingival findings. This report describes a case of amelogenesis imperfecta of the enamel agenesis variety with nephrolithiasis in a 21-year-old male patient who complained of small teeth. The imaging modalities employed were conventional radiography, cone-beam computed tomography, and renal sonography. Such cases are first encountered by dentists, as other organ or metabolic diseases are generally hidden. Hence, cases of amelogenesis imperfecta should be subjected to advanced diagnostic modalities, incorporating both dental and medical criteria, in order to facilitate comprehensive long-term management.
Amelogenesis Imperfecta* ; Amelogenesis* ; Cone-Beam Computed Tomography ; Dental Enamel Hypoplasia ; Dental Enamel* ; Dentists ; Fibromatosis, Gingival ; Humans ; Kidney Diseases ; Male ; Metabolic Diseases ; Nephrocalcinosis ; Nephrolithiasis* ; Phenotype ; Radiography ; Tooth ; Young Adult

PURPOSE: This study was performed to evaluate the prevalence of all types and subtypes of dental anomalies among 7- to 35-year-old patients by using panoramic radiographs. MATERIALS AND METHODS: This cross-sectional study was conducted on 1649 people in Hamadan City, in 2012-2013. The prevalence of four types and 12 subtypes of dental anomalies was evaluated by two observers separately by using panoramic radiography. Dental anomalies were divided into four types: (a) shape (including fusion, taurodontism, and dens invagination); (b) number (including hypodontia, oligodontia, and hyperdontia); (c) structure (including amelogenesis imperfecta, dentinogenesis imperfecta, and dentin dysplasia); and (d) position (including displacement, impaction, and dilacerations). RESULTS: The reliability between the two observers was 79.56% according to the Kappa statistics. The prevalence of dental anomalies diagnosed by panoramic radiographs was 29%. Anomalies of position and number were the most common types of abnormalities, and anomalies of shape and structure were the least in both genders. Anomalies of impaction (44.76%), dilacerations (21.11%), hypodontia (15.88%), taurodontism (9.29%), and hyperdontia (6.76%) were the most common subtypes of dental anomalies. The anomalies of shape and number were more common in the age groups of 7-12 years and 13-15 years, respectively, while the anomalies of structure and position were more common among the other age groups. CONCLUSION: Anomalies of tooth position were the most common type of dental anomalies, and structure anomalies were the least in this Iranian population. The frequency and type of dental anomalies vary within and between populations, confirming the role of racial factors in the prevalence of dental anomalies.
Adult* ; Amelogenesis Imperfecta ; Anodontia ; Cross-Sectional Studies ; Dentin ; Dentinogenesis Imperfecta ; Dentition ; Humans ; Iran* ; Prevalence* ; Radiography, Panoramic ; Tooth

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.
Actins ; analysis ; Activating Transcription Factor 2 ; genetics ; Age Factors ; Ameloblasts ; pathology ; Amelogenesis ; genetics ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; Cell Adhesion Molecules ; analysis ; Cell Differentiation ; genetics ; Cementogenesis ; genetics ; Dental Cementum ; pathology ; Dental Pulp ; blood supply ; Fluorescent Dyes ; Green Fluorescent Proteins ; Male ; Mice ; Mice, Knockout ; Microvessels ; pathology ; Molar ; growth & development ; Molar, Third ; growth & development ; NFI Transcription Factors ; analysis ; Odontoblasts ; pathology ; Odontogenesis ; genetics ; Periodontal Ligament ; growth & development ; Sp7 Transcription Factor ; Stem Cells ; physiology ; Tooth Root ; growth & development ; Transcription Factors ; genetics ; Vascular Endothelial Growth Factor A ; analysis ; Zinc Fingers ; genetics

This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.
Ameloblasts ; physiology ; Amelogenesis ; genetics ; Amelogenin ; analysis ; Bone Morphogenetic Protein 4 ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Cell Lineage ; Embryonic Stem Cells ; drug effects ; physiology ; Epithelial Cells ; drug effects ; physiology ; Fibroblast Growth Factor 8 ; analysis ; Hedgehog Proteins ; analysis ; Homeodomain Proteins ; analysis ; Humans ; Keratins ; analysis ; classification ; Lithium Chloride ; pharmacology ; MSX1 Transcription Factor ; analysis ; Mouth Mucosa ; cytology ; Phenotype ; Regeneration ; physiology ; Skin ; cytology ; Transcription Factors ; analysis ; Tretinoin ; pharmacology

Deletion or mutation of dentin matrix protein 1 (DMP1) leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate (Pi) or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmp1-null (Dmp1(-/-)), Klotho-deficient (kl/kl), Dmp1/Klotho-double-deficient (Dmp1(-/-)/kl/kl) and wild-type (WT) mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography (μCT), scanning electron microscopy (SEM), histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmp1(-/-) (a low Pi level) or kl/kl (a high Pi level) mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice (a high Pi level) displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment (a high Pi level).
Ameloblasts ; pathology ; Amelogenesis ; physiology ; Animals ; Apoptosis ; physiology ; Cell Differentiation ; physiology ; Dental Enamel ; pathology ; Dental Pulp ; pathology ; physiology ; Dental Pulp Cavity ; pathology ; Dentin ; abnormalities ; pathology ; Dentinogenesis ; physiology ; Extracellular Matrix Proteins ; genetics ; physiology ; Glucuronidase ; genetics ; Homeostasis ; physiology ; Hyperphosphatemia ; physiopathology ; Immunohistochemistry ; Mice ; Mice, Knockout ; Microscopy, Electron, Scanning ; Odontoblasts ; pathology ; Odontogenesis ; physiology ; Ossification, Heterotopic ; genetics ; pathology ; Phosphates ; physiology ; Tooth Calcification ; physiology ; X-Ray Microtomography