Loss of Autophagy Disrupts Stemness of Ameloblast-Lineage Cells in Aging.

Autophagy is one of the intracellular degradation pathways and maintains cellular homeostasis, regulating the stress response, cell proliferation, and signal transduction. To elucidate the role of autophagy in the maintenance of dental epithelial stem cells and the subsequent enamel formation, we analyzed autophagy-deficient mice in epithelial cells (Atg7f/f;KRT14-Cre mice), focusing on the influence of aging and stress environments. We also performed in vitro cell and organ culture experiments with an autophagy inhibitor. In young Atg7f/f;KRT14-Cre mice, morphological change was not obvious in maxillary incisors, except for the remarkable cell death in the stratum intermedium of the transitional stage. However, under stress conditions of hyperglycemia, the incisor color changed to white in diabetes Atg7f/f;KRT14-Cre mice. Regarding dental epithelial stem cells, the shape of the apical bud region of the incisor became irregular with age, and odontoma was formed in aged Atg7f/f;KRT14-Cre mice. In addition, the shape of apical bud culture cells of Atg7f/f;KRT14-Cre mice became irregular and enlarged atypically, with epigenetic changes during culture, suggesting that autophagy deficiency may induce tumorigenesis in dental epithelial cells. The epigenetic change and upregulation of p21 expression were induced by autophagy inhibition in vivo and in vitro. These findings suggest that autophagy is important for the regulation of stem cell maintenance, proliferation, and differentiation of ameloblast-lineage cells, and an autophagy disorder may induce tumorigenesis in odontogenic epithelial cells.

A novel GJA1 mutation in oculodentodigital dysplasia with extensive loss of enamel.

OBJECTIVE: To characterize clinical features and identify genetic causes of a patient with oculodentodigital dysplasia (ODDD). SUBJECTS AND METHODS: Clinical, dental, radiological features were obtained. DNA was collected from an affected Thai family. Whole-exome sequencing was employed to identify the disease-causing mutation causing ODDD. The presence of the identified variant was confirmed by Sanger sequencing. RESULTS: The proband suffered with extensive enamel hypoplasia, polysyndactyly and clinodactyly of the 3rd-5th fingers, microphthalmia, and unique facial characteristics of ODDD. Mutation analysis revealed a novel missense mutation, c. 31C>A, p.L11I, in the GJA1 gene which encodes gap junction channel protein connexin 43. Bioinformatics and structural modeling suggested the mutation to be pathogenic. The parents did not harbor the mutation. CONCLUSIONS: This study identified a novel de novo mutation in the GJA1 gene associated with severe tooth defects. These results expand the mutation spectrum and understanding of pathologic dental phenotypes related to ODDD.

GREMLIN 2 Mutations and Dental Anomalies.

Isolated or nonsyndromic tooth agenesis or hypodontia is the most common human malformation. It has been associated with mutations in MSX1, PAX9, EDA, AXIN2, EDAR, EDARADD, and WNT10A. GREMLIN 2 (GREM2) is a strong bone morphogenetic protein (BMP) antagonist that is known to regulate BMPs in embryogenesis and tissue development. Bmp4 has been shown to have a role in tooth development. Grem2(-/-) mice have small, malformed maxillary and mandibular incisors, indicating that Grem2 has important roles in normal tooth development. Here, we demonstrate for the first time that GREM2 mutations are associated with human malformations, which include isolated tooth agenesis, microdontia, short tooth roots, taurodontism, sparse and slow-growing hair, and dry and itchy skin. We sequenced WNT10A, WNT10B, MSX1, EDA, EDAR, EDARADD, AXIN2, and PAX9 in all 7 patients to rule out the effects of other ectodermal dysplasias and other tooth-related genes and did not find mutations in any of them. GREM2 mutations exhibit variable expressivity even within the same families. The inheritance is autosomal dominant with incomplete penetrance. The expression of Grem2 during the early development of mouse teeth and hair follicles and the evaluation of the likely effects of the mutations on the protein structure substantiate these new findings.

Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

A mathematical model for mechanotransduction at the early steps of suture formation.

Growth and patterning of craniofacial sutures is subjected to the effects of mechanical stress. Mechanotransduction processes occurring at the margins of the sutures are not precisely understood. Here, we propose a simple theoretical model based on the orientation of collagen fibres within the suture in response to local stress. We demonstrate that fibre alignment generates an instability leading to the emergence of interdigitations. We confirm the appearance of this instability both analytically and numerically. To support our model, we use histology and synchrotron X-ray microtomography and reveal the fine structure of fibres within the sutural mesenchyme and their insertion into the bone. Furthermore, using a mouse model with impaired mechanotransduction, we show that the architecture of sutures is disturbed when forces are not interpreted properly. Finally, by studying the structure of sutures in the mouse, the rat, an actinopterygian (Polypterus bichir) and a placoderm (Compagopiscis croucheri), we show that bone deposition patterns during dermal bone growth are conserved within jawed vertebrates. In total, these results support the role of mechanical constraints in the growth and patterning of craniofacial sutures, a process that was probably effective at the emergence of gnathostomes, and provide new directions for the understanding of normal and pathological suture fusion.

Oral lining mucosa development depends on mesenchymal microRNAs.

The oral mucosa plays critical roles in protection, sensation, and secretion and can be classified into masticatory, lining, and specialized mucosa that are known to be functionally, histologically, and clinically distinct. Each type of oral mucosa is believed to develop through discrete molecular mechanisms, which remain unclear. MicroRNAs (miRNAs) are 19 to 25nt non-coding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs. miRNAs are crucial for fine-tuning of molecular mechanisms. To investigate the role of miRNAs in oral mucosa development, we examined mice with mesenchymal (Wnt1Cre;Dicer(fl/fl)) conditional deletion of Dicer. Wnt1Cre;Dicer(fl/fl) mice showed trans-differentiation of lining mucosa into an epithelium with masticatory mucosa/ skin-specific characteristics. Up-regulation of Fgf signaling was found in mutant lining mucosal epithelium that was accompanied by an increase in Fgf7 expression in mutant mesenchyme. Mesenchyme miRNAs thus have an indirect effect on lining mucosal epithelial cell growth/differentiation.

Organized tooth-specific cellular differentiation stimulated by BMP4.

Mammalian teeth develop on the oral surface of the first pharyngeal arch by a series of reciprocal interactions between epithelial and mesenchymal cells. The embryonic first pharyngeal arch oral epithelium is able to induce tooth formation when combined with mesenchymal cells from the second pharyngeal arch, a region devoid of tooth development. Second pharyngeal arch mesenchyme is thus competent to form teeth if provided with the correct signals. First-arch oral epithelium expresses several signaling molecules that could be potential inducers of tooth development, including BMP4. The addition of BMP4 to intact second-arch explants resulted in the development of organized structures containing layers of cells that express marker genes of tooth-specific cells, odontoblasts and ameloblasts. Thus, although overt tooth development did not occur, BMP4 has the ability to stimulate organized differentiation of epithelial- and mesenchymal-derived dental-specific cells from non-dental primordia.

Stem-cell-based tissue engineering of murine teeth.

Teeth develop from reciprocal interactions between mesenchyme cells and epithelium, where the epithelium provides the instructive information for initiation. Based on these initial tissue interactions, we have replaced the mesenchyme cells with mesenchyme created by aggregation of cultured non-dental stem cells in mice. Recombinations between non-dental cell-derived mesenchyme and embryonic oral epithelium stimulate an odontogenic response in the stem cells. Embryonic stem cells, neural stem cells, and adult bone-marrow-derived cells all responded by expressing odontogenic genes. Transfer of recombinations into adult renal capsules resulted in the development of tooth structures and associated bone. Moreover, transfer of embryonic tooth primordia into the adult jaw resulted in development of tooth structures, showing that an embryonic primordium can develop in its adult environment. These results thus provide a significant advance toward the creation of artificial embryonic tooth primordia from cultured cells that can be used to replace missing teeth following transplantation into the adult mouth.

Opg, Rank, and Rankl in tooth development: co-ordination of odontogenesis and osteogenesis.

Osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB (RANK), and RANK ligand (RANKL) are mediators of various cellular interactions, including bone metabolism. We analyzed expression of these three genes during murine odontogenesis from epithelial thickening to cytodifferentiation stages. Opg showed expression in the thickening and bud epithelium. Expression of Opg and Rank was observed in both the internal and the external enamel epithelium as well as in the dental papilla mesenchyme. Although Rankl expression was not detected in tooth epithelium or mesenchyme, it was expressed in pre-osteogenic mesenchymal cells close to developing tooth germs. All three genes were detected in developing dentary bone at P0. The addition of exogenous OPG to explant cultures of tooth primordia produced a delay in tooth development that resulted in reduced mineralization. We propose that the spatiotemporal expression of these molecules in early tooth and bone primordia cells has a role in co-ordinating bone and tooth development.

Participation of periodontal ligament cells with regeneration of alveolar bone.

BACKGROUND: It is important to clarify the participation of periodontal ligament (PDL) cells in the regeneration of alveolar bone to establish a reliable approach for obtaining periodontal regeneration. The aim of this study was to determine whether PDL cells play an important role in alveolar bone repair during the course of periodontal regeneration. METHODS: In an in vitro study, the expression of the osteoblast phenotype, such as alkaline phosphatase activity and parathyroid hormone-dependent 3',5'-cyclic adenosine monophosphate accumulation, was investigated in dog PDL cells (DPLC) and dog bone cells isolated from mandibles (DBC). In a related study, the roots of mandibular third premolars extracted from aged dogs were divided into a PDL(+) group, in which the PDL was preserved, and a PDL(-) group, in which the PDL was removed. These roots were respectively transplanted into surgically created bone cavities with buccal and interproximal bone defects in an edentulous area, prepared in advance by extraction of mandibular fourth premolars. These bone defects with the transplanted roots were completely covered with submerged physical barrier membranes. New bone formation and new connective tissue attachment, which require new cementum and insertion of functionally oriented new collagen fibers of periodontal ligament, were histomorphometrically assessed, and were compared between the PDL(+) and PDL(-) groups 6 weeks after transplantation. RESULTS: Both cultured DPLC and DBC exhibited the osteoblast phenotype. New connective tissue attachment was observed only in the PDL(+) group. However, alveolar bone was almost completely regenerated to the original bone height in both the PDL(+) and PDL(-) groups, and the amount of newly formed bone was not significantly different between the 2 groups. CONCLUSIONS: DPLC retain the capability to differentiate into an osteoblast lineage and may act in the regeneration of periodontal ligament with new cementum formation, whereas these cells may have a limited influence on alveolar bone formation during the course of periodontal regeneration.