CACNA1S mutation-associated dental anomalies: A calcium channelopathy.

OBJECTIVES: To identify the molecular etiology of distinct dental anomalies found in eight Thai patients and explore the mutational effects on cellular functions. MATERIALS AND METHODS: Clinical and radiographic examinations were performed for eight patients. Whole exome sequencing, mutant protein modelling, qPCR, western blot analysis, scratch assays, immunofluorescence, confocal analysis, in situ hybridization, and scanning electron micrography of teeth were done. RESULTS: All patients had molars with multiple supernumerary cusps, single-cusped premolars, and a reduction in root number. Mutation analysis highlighted a heterozygous c.865A>G; p.Ile289Val mutation in CACNA1S in the patients. CACNA1S is a component of the slowly inactivating L-type voltage-dependent calcium channel. Mutant protein modeling suggested that the mutation might allow leakage of Ca2+ or other cations, or a tightening, to restrict calcium flow. Immunohistochemistry analysis showed expression of Cacna1s in the developing murine tooth epithelium during stages of crown and root morphogenesis. In cell culture, the mutation resulted in abnormal cell migration of transfected CHO cells compared to wildtype CACNA1S, with changes to the cytoskeleton and markers of focal adhesion. CONCLUSIONS: The malformations observed in our patients suggest a role for calcium signaling in organization of both cusps and roots, affecting cell dynamics within the dental epithelium.

Impact of scientific and technological advances.

Advancements in research and technology are transforming our world. The dental profession is changing too, in the light of scientific discoveries that are advancing biological technology-from new biomaterials to unravelling the genetic make-up of the human being. As health professionals, we embrace a model of continuous quality improvement and lifelong learning. Our pedagogical approach to incorporating the plethora of scientific-technological advancements calls for us to shift our paradigm from emphasis on skill acquisition to knowledge application. The 2017 ADEE/ADEA workshop provided a forum to explore and discuss strategies to ensure faculty, students and, ultimately, patients are best positioned to exploit the opportunities that arise from integrating new technological advances and research outcomes. Participants discussed methods of incorporating the impact of new technologies and research findings into the education of our dental students. This report serves as a signpost of the way forward and how to promote incorporation of research and technology advances and lifelong learning into the dental education curriculum.

A conserved tooth resorption mechanism in modern and fossil snakes.

Whether snakes evolved their elongated, limbless bodies or their specialized skulls and teeth first is a central question in squamate evolution. Identifying features shared between extant and fossil snakes is therefore key to unraveling the early evolution of this iconic reptile group. One promising candidate is their unusual mode of tooth replacement, whereby teeth are replaced without signs of external tooth resorption. We reveal through histological analysis that the lack of resorption pits in snakes is due to the unusual action of odontoclasts, which resorb dentine from within the pulp of the tooth. Internal tooth resorption is widespread in extant snakes, differs from replacement in other reptiles, and is even detectable via non-destructive µCT scanning, providing a method for identifying fossil snakes. We then detected internal tooth resorption in the fossil snake Yurlunggur, and one of the oldest snake fossils, Portugalophis, suggesting that it is one of the earliest innovations in Pan-Serpentes, likely preceding limb loss.

Coordinated labio-lingual asymmetries in dental and bone development create a symmetrical acrodont dentition.

Organs throughout the body develop both asymmetrically and symmetrically. Here, we assess how symmetrical teeth in reptiles can be created from asymmetrical tooth germs. Teeth of lepidosaurian reptiles are mostly anchored to the jaw bones by pleurodont ankylosis, where the tooth is held in place on the labial side only. Pleurodont teeth are characterized by significantly asymmetrical development of the labial and lingual sides of the cervical loop, which later leads to uneven deposition of hard tissue. On the other hand, acrodont teeth found in lizards of the Acrodonta clade (i.e. agamas, chameleons) are symmetrically ankylosed to the jaw bone. Here, we have focused on the formation of the symmetrical acrodont dentition of the veiled chameleon (Chamaeleo calyptratus). Intriguingly, our results revealed distinct asymmetries in morphology of the labial and lingual sides of the cervical loop during early developmental stages, both at the gross and ultrastructural level, with specific patterns of cell proliferation and stem cell marker expression. Asymmetrical expression of ST14 was also observed, with a positive domain on the lingual side of the cervical loop overlapping with the SOX2 domain. In contrast, micro-CT analysis of hard tissues revealed that deposition of dentin and enamel was largely symmetrical at the mineralization stage, highlighting the difference between cervical loop morphology during early development and differentiation of odontoblasts throughout later odontogenesis. In conclusion, the early asymmetrical development of the enamel organ seems to be a plesiomorphic character for all squamate reptiles, while symmetrical and precisely orchestrated deposition of hard tissue during tooth formation in acrodont dentitions probably represents a novelty in the Acrodonta clade.

Transformation of tooth type induced by inhibition of BMP signaling.

Mammalian dentitions are highly patterned, with different types of teeth positioned in different regions of the jaws. BMP4 is an early oral epithelial protein signal that directs odontogenic gene expression in mesenchyme cells of the developing mandibular arch. BMP4 was shown to inhibit expression of the homeobox gene Barx-1 and to restrict expression to the proximal, presumptive molar mesenchyme of mouse embryos at embryonic day 10. The inhibition of BMP signaling early in mandible development by the action of exogenous Noggin protein resulted in ectopic Barx-1 expression in the distal, presumptive incisor mesenchyme and a transformation of tooth identity from incisor to molar.

High yield expression of biologically active recombinant full length human tuftelin protein in baculovirus-infected insect cells.

Tuftelin is an acidic protein expressed at very early stages of mouse odontogenesis. It was suggested to play a role during epithelial-mesenchymal interactions, and later, when enamel formation commences, to be involved in enamel mineralization. Tuftelin was also detected in several normal soft tissues of different origins and some of their corresponding cancerous tissues. Tuftelin is expressed in low quantities, and undergoes degradation in the enamel extracellular matrix. To investigate the structure and function of tuftelin, the full length recombinant human tuftelin protein was produced. The full length human tuftelin cDNA was cloned using Gateway recombination into the Bac-to-Bac system compatible transfer vector pDest10. This vector adds a hexahistidine tag to the N-terminus of the expressed protein, enabling one-step affinity purification on nickel column. The recombinant human tuftelin protein was transposed into the bacmid and expressed in Spodoptera frugiperda (Sf9) insect cells. The yield of the purified, his-tagged recombinant full length human Tuftelin (rHTuft+) was 5-8 mg/L culture. rHTuft+ was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, restriction mapping and MS/MS sequencing. The availability of the purified, full length recombinant human tuftelin protein opened up the possibility to investigate novel functions of tuftelin. Application of rHTuft+ agarose beads onto embryonic mouse mandibular explants caused changes in the surrounding epithelial cells, including morphology, orientation and spatial organization. Further studies using DiI labeling, revealed that rHTuft+, placed on the tooth germ region, brought about recruitment of adjacent embryonic mesenchymal cells. These findings support the hypothesis that tuftelin plays an important role during embryogenesis.

Molar Bud-to-Cap Transition Is Proliferation Independent.

Tooth germs undergo a series of dynamic morphologic changes through bud, cap, and bell stages, in which odontogenic epithelium continuously extends into the underlying mesenchyme. During the transition from the bud stage to the cap stage, the base of the bud flattens and then bends into a cap shape whose edges are referred to as "cervical loops." Although genetic mechanisms for cap formation have been well described, little is understood about the morphogenetic mechanisms. Computer modeling and cell trajectory tracking have suggested that the epithelial bending is driven purely by differential cell proliferation and adhesion in different parts of the tooth germ. Here, we show that, unexpectedly, inhibition of cell proliferation did not prevent bud-to-cap morphogenesis. We quantified cell shapes and actin and myosin distributions in different parts of the tooth epithelium at the critical stages and found that these are consistent with basal relaxation in the forming cervical loops and basal constriction around enamel knot at the center of the cap. Inhibition of focal adhesion kinase, which is required for basal constriction in other systems, arrested the molar explant morphogenesis at the bud stage. Together, these results show that the bud-to-cap transition is largely proliferation independent, and we propose that it is driven by classic actomyosin-driven cell shape-dependent mechanisms. We discuss how these results can be reconciled with the previous models and data.

Tooth agenesis: from molecular genetics to molecular dentistry.

Tooth agenesis may originate from either genetic or environmental factors. Genetically determined hypodontic disorders appear as isolated features or as part of a syndrome. Msx1, Pax9, and Axin2 are involved in non-syndromic hypodontia, while genes such as Shh, Pitx2, Irf6, and p63 are considered to participate in syndromic genetic disorders, which include tooth agenesis. In dentistry, artificial tooth implants represent a common solution to tooth loss problems; however, molecular dentistry offers promising solutions for the future. In this paper, the genetic and molecular bases of non-syndromic and syndromic hypodontia are reviewed, and the advantages and disadvantages of tissue engineering in the clinical treatment of tooth agenesis are discussed.

Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice.

During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.

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.

Apoptosis in Early Salivary Gland Duct Morphogenesis and Lumen Formation.

Salivary glands are essential for the maintenance of oral health by providing lubrication and antimicrobial protection to the mucosal and tooth surfaces. Saliva is modified and delivered to the oral cavity by a complex multifunctional ductal system. During development, these ducts form as solid tubes, which undergo cavitation to create lumens. Apoptosis has been suggested to play a role in this cavitation process along with changes in cell polarity. Here, we show that apoptosis occurs from the very earliest stages of mouse salivary gland development, much earlier than previously reported. Apoptotic cells were observed in the center of the first epithelial stalk at early-stage embryonic day 12.5 (E12.5) according to both TUNEL staining and cleaved caspase 3 immunofluorescence. The presumptive lumen space was highlighted by the colocalization of a predictive lumen marker, cytokeratin 7. At E14.5, as lumens start to form throughout the glands, apoptotic expression decreased while cytokeratin 7 remained positive. In vitro inhibition of all caspases in E12.5 and E13.5 salivary glands resulted in wider ducts, as compared with the controls, and a defect in lumen formation. In contrast, no such defect in lumen formation was observed at E14.5. Our data indicate that apoptosis is involved during early stages of gland formation (E12.5 onward) and appears important for shaping the forming ducts.

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.

Apoptosis-related factors (Fas receptor, Fas ligand, FADD) in early tooth development of the field vole (Microtus agrestis).

Fas (CD95/APO-1) belongs to the TNF receptor (TNFR) family. Fas ligand binding followed by Fas-receptor oligomerisation leads to formation of a death-inducing signal complex starting with recruitment of the Fas-adapter protein (FADD). Components of this initiation complex (Fas, Fas-L, FADD) were correlated with apoptotic cells, detected by specific DNA fragmentation and morphological criteria. Apoptotic cells can be detected throughout the embryonic development of molar teeth. Restricted temporospatial distribution suggests several important roles for apoptosis in tooth morphogenesis. However, the mechanisms employed in dental apoptosis remain unclear. Frontal sections of the field vole at stage 13.5-15.5 of embryonic development were exploited to investigate and correlate location of Fas, Fas-ligand, FADD molecules and apoptosis in developing first molars by immunohistochemistry. During these stages the primary enamel knot appears and is gradually terminated by apoptosis. Initially, apoptotic cells were demonstrated in the most superficial layer of the dental lamina. The number of TUNEL-positive cells expanded from late bud to cap stages. Restricted areas of apoptotic cells were found in the stalk and primary enamel knot. Fas, Fas-L and FADD were co-localised, particularly in the primary enamel knot, and the stalk, correlating with the occurrence of apoptosis in these areas. Fas-L, however, was also found in proliferating parts of the developing tooth germ, such as in the cervical loops. Interestingly, FADD molecules were also observed in areas, where Fas protein was not detected. According to the immunohistochemical data, Fas-mediated signalling may have a triggering or enhancing role in dental apoptosis. This remains to be functionally confirmed.

Salivary Gland Dysplasia in Fgf10 Heterozygous Mice: A New Mouse Model of Xerostomia.

Xerostomia, or chronic dry mouth, is a common syndrome caused by a lack of saliva that can lead to severe eating difficulties, dental caries and oral candida infections. The prevalence of xerostomia increases with age and affects approximately 30% of people aged 65 or older. Given the large numbers of sufferers, and the potential increase in incidence given our aging population, it is important to understand the complex mechanisms that drive hyposalivation and the consequences for the dentition and oral mucosa. From this study we propose the Fgf10 +/- mouse as a model to investigate xerostomia. By following embryonic salivary gland development, in vivo and in vitro, we show that a reduction in Fgf10 causes a delay in branching of salivary glands. This leads to hypoplasia of the glands, a phenotype that is not rescued postnatally or by adulthood in both male and female Fgf10 +/- mice. Histological analysis of the glands showed no obvious defect in cellular differentiation or acini/ductal arrangements, however there was a significant reduction in their size and weight. Analysis of saliva secretion showed that hypoplasia of the glands led to a significant reduction in saliva production in Fgf10 +/- adults, giving rise to a reduced saliva pellicle in the oral cavity of these mice. Mature mice were shown to drink more and in many cases had severe tooth wear. The Fgf10 +/- mouse is therefore a useful model to explore the causes and effects of xerostomia.

Root and Eruption Defects in c-Fos Mice Are Driven by Loss of Osteoclasts.

c-Fos homozygous mice lack osteoclasts with a failure of the teeth to erupt and with an arrest of root development. Here, we characterize the defects associated with the failure in root development and the loss of the tooth-bone interface, and we investigate the underlying causes. We show that, while homozygous c-Fos mice have no multinucleated osteoclasts, heterozygous mice have a reduction in the number of osteoclasts with a reduction in the tooth-bone interface during development and subtle skeletal defects postnatally. In the homozygous mutants bone is found to penetrate the tooth, particularly at the apical end, physically disrupting the root forming HERS (Hertwig's epithelial root sheath) cells. The cells of the HERS continue to proliferate but cannot extend downward due to the presence of bone, leading to a loss of root formation. Tooth germ culture showed that the developing tooth invaded the static bone in mutant tissue, rather than the bone encroaching on the tooth. Although c-Fos has been shown to be expressed in developing teeth, the defect in maintenance of the tooth-bone interface appears to be driven solely by the lack of osteoclasts, as this defect can be rescued in the presence of donor osteoclasts. The rescue suggests that signals from the tooth recruit osteoclasts to clear the bone from around the tooth, allowing the tooth to grow, form roots, and later erupt.

Stationary intraoral digital tomosynthesis using a carbon nanotube X-ray source array.

OBJECTIVES: Intraoral dental tomosynthesis and closely related tuned-aperture CT (TACT) are low-dose three-dimensional (3D) imaging modalities that have shown improved detection of multiple dental diseases. Clinical interest in implementing these technologies waned owing to their time-consuming nature. Recently developed carbon nanotube (CNT) X-ray sources allow rapid multi-image acquisition without mechanical motion, making tomosynthesis a clinically viable technique. The objective of this investigation was to evaluate the feasibility of and produce high-quality images from a digital tomosynthesis system employing CNT X-ray technology. METHODS: A test-bed stationary intraoral tomosynthesis unit was constructed using a CNT X-ray source array and a digital intraoral sensor. The source-to-image distance was modified to make the system comparable in image resolution to current two-dimensional intraoral radiography imaging systems. Anthropomorphic phantoms containing teeth with simulated and real caries lesions were imaged using a dose comparable to D-speed film dose with a rectangular collimation. Images were reconstructed and analysed. RESULTS: Tomosynthesis images of the phantom and teeth specimen demonstrated perceived image quality equivalent or superior to standard digital images with the added benefit of 3D information. The ability to "scroll" through slices in a buccal-lingual direction significantly improved visualization of anatomical details. In addition, the subjective visibility of dental caries was increased. CONCLUSIONS: Feasibility of the stationary intraoral tomosynthesis is demonstrated. The results show clinical promise and suitability for more robust observer and clinical studies.

Molecular genetics of tooth morphogenesis and patterning: the right shape in the right place.

Development of the mammalian tooth has for many years served as a useful model system for the study of cell-cell interactions in organogenesis. Early development of teeth (tooth buds) shows many morphological and molecular similarities with early development of other organs such as the lung, hair, kidney, etc. There has been much progress toward understanding epithelial/mesenchymal cell signaling in tooth germ formation. Advances in understanding the formation of different shapes of teeth (morphogenesis) at their correct positions in the jaws (patterning) has, until recently, been less forthcoming. We review here the latest ideas on the control of odontogenic patterning and morphogenesis. The stages of early tooth development are well-defined histologically and have been described in numerous textbooks. The progression from localized thickenings of oral epithelium to bud, cap, and bell stages provides an adequate description of the gross morphological changes seen in the epithelial cells of early developing tooth germs. Less obvious are the concomitant changes taking place in the dental (ecto)mesenchymal cells which originate from the cranial neural crest and which condense around the tooth bud epithelium. However, it is very clear that these mesenchymal cells are equal partners with epithelium during the early stages of tooth germ formation and undergo complex changes which, although not obvious histologically, are revealed with molecular (gene) probes. Genes identified as being important for the early communication between the epithelial and ectomesenchymal cells mainly comprise those which code for proteins which act as secreted signals between the cells (ligands) and those that code for nuclear proteins that act to control gene expression in response to the signals. Little is presently known about the changes in structural proteins such as cell adhesion molecules which are involved in mediating the physical interactions between cells and generating the morphological changes.

Death in the life of a tooth.

Programmed cell death (apoptosis) constitutes an important mechanism in embryonic development. Although there is substantial evidence for essential roles of apoptosis in organ shaping and controlling of cell number, the mechanisms of these processes are poorly understood. The regulation of cell proliferation to form tooth buds of the appropriate size and at the correct positions must involve a balance between cell division and cell death. Apoptosis has been suggested to play both passive and active roles in bud formation and morphogenesis and in reduction of the dental lamina, as well as silencing of the enamel knot signaling centers. The location of apoptotic cells during tooth development has been described and suggests their temporospatial roles. Unfortunately, there is little functional evidence on these roles, and the aim of this review is to highlight areas where apoptosis may play key roles in odontogenesis.

Non-apoptotic functions of caspase-7 during osteogenesis.

Caspase-3 and -7 are generally known for their central role in the execution of apoptosis. However, their function is not limited to apoptosis and under specific conditions activation has been linked to proliferation or differentiation of specialised cell types. In the present study, we followed the localisation of the activated form of caspase-7 during intramembranous (alveolar and mandibular bones) and endochondral (long bones of limbs) ossification in mice. In both bone types, the activated form of caspase-7 was detected from the beginning of ossification during embryonic development and persisted postnatally. The bone status was investigated by microCT in both wild-type and caspase-7-deficient adult mice. Intramembranous bone in mutant mice displayed a statistically significant decrease in volume while the mineral density was not altered. Conversely, endochondral bone showed constant volume but a significant decrease in mineral density in caspase-7 knock-out mice. Cleaved caspase-7 was present in a number of cells that did not show signs of apoptosis. PCR array analysis of the mandibular bone of caspase-7-deficient versus wild-type mice pointed to a significant decrease in mRNA levels for Msx1 and Smad1 in early bone formation. These observations might explain the decrease in the alveolar bone volume of adult knock-out mice. In conclusion, this study is the first to report a non-apoptotic function of caspase-7 in osteogenesis and also demonstrates further specificities in endochondral versus intramembranous ossification.

Interactions of the tooth and bone during development.

The tooth works as a functional unit with its surrounding bony socket, the alveolar bone. The growth of the tooth and alveolar bone is co-ordinated so that a studied distance always separates the 2, known as the tooth-bone interface (TBI). Lack of mineralization, a crucial feature of the TBI, creates the space for the developing tooth to grow and the soft tissues of the periodontium to develop. We have investigated the interactions between the tooth and its surrounding bone during development, focusing on the impact of the developing alveolar bone on the development of the mouse first molar (M1). During development, TRAP-positive osteoclasts are found to line the TBI as bone starts to be deposited around the tooth, removing the bone as the tooth expands. An enhancement of osteoclastogenesis through RANK-RANKL signaling results in an expansion of the TBI, showing that osteoclasts are essential for defining the size of this region. Isolation of the M1 from the surrounding mesenchyme and alveolar bone leads to an expansion of the tooth germ, driven by increased proliferation, indicating that, during normal development, the growth of the tooth germ is constrained by the surrounding tissues.