Enamel Maturation Research Articles

The phosphorylation of serine55 in enamelin is essential for murine amelogenesis

Amelogenesis imperfecta (AI) is an inherited developmental enamel defect affecting tooth masticatory function, esthetic appearance, and the well-being of patients. As one of the major enamel matrix proteins (EMPs), enamelin (ENAM) has three serines located in Ser-x-Glu (S-x-E) motifs, which are potential phosphorylation sites for the Golgi casein kinase FAM20C. Defects in FAM20C have similarly been associated with AI. In our previous study of EnamRgsc514 mice, the Glu57 in the S55-X56-E57 motif was mutated into Gly, which was expected to cause a phosphorylation failure of Ser55 because Ser55 cannot be recognized by FAM20C. The severe enamel defects in ENAMRgsc514 mice reminiscent of Enam-knockout mouse enamel suggested a potentially important role of Ser55 phosphorylation in ENAM function. However, the enamel defects and ENAM dysfunction may also be attributed to distinct physicochemical differences between Glu57 and Gly57. To clarify the significance of Ser55 phosphorylation to ENAM function, we generated two lines of Enam knock-in mice using CRISPR-Cas9 method to eliminate or mimic the phosphorylation state of Ser55 by substituting it with Ala55 or Asp55 (designated as S55A or S55D), respectively. The teeth of 6-day or 4-week-old mice were subjected to histology, micro-CT, SEM, TEM, immunohistochemistry, and mass spectrometry analyses to characterize the morphological, microstructural and proteomic changes in ameloblasts, enamel matrix and enamel rods. Our results showed that the enamel formation and EMP expression in S55D heterozygotes (Het) were less disturbed than those in S55A heterozygotes, while both homozygotes (Homo) had no mature enamel formation. Proteomic analysis revealed alterations of enamel matrix biosynthetic and mineralization processes in S55A Hets. Our present findings indicate that Asp55 substitution partially mimics the phosphorylation state of Ser55 in ENAM. Ser55 phosphorylation is essential for ENAM function during amelogenesis.

Loss of BMP2 and BMP4 Signaling in the Dental Epithelium Causes Defective Enamel Maturation and Aberrant Development of Ameloblasts.

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme’s activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation.

FULL MOUTH REHABILITATION OF DILAPIDATED DENTITION DUE TO AMELOGENESIS IMPERFECTA- A CASE REPORT

Amelogenesis imperfecta is a congenital disor¬der which affects deposition, calcification and/or maturation of dental enamel. This genetic disorder affects teeth in both deciduous and succedaneous dentition leading to functional as well as esthetic in adequacies. This condition exerts radical reper¬cussions on stomatognathicsyste mandintangible effects on the confidence of the patient. This clinical report elaborates on the multidisciplinary approach adopted for full mouth rehabilitation of a dentition affected by this genetic disorder in young adult patient. The treatment plan aimed at upheaving esthetics, restoring the vertical dimension and re¬instating the masticatory function. Pankey–Mann– Schuyler philosophy of full mouth rehabilitation was adhered to achieve these treatment objectives. Full mouth rehabilitation albeit restoring esthetics and functional so imparts a substantial positive psychological upliftment and therefore revamps the patient confidence.

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods’ axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.

The synergistic effects of TGF-β1 and RUNX2 on enamel mineralization through regulating ODAPH expression during the maturation stage.

Transforming growth factor β1 (TGF-β1) and Runt-related transcription factor 2 (RUNX2) are critical factors promoting enamel development and maturation. Our previous studies reported that absence of TGF-β1 or RUNX2 resulted in abnormal secretion and absorption of enamel matrix proteins. However, the mechanism remained enigmatic. In this study, TGF-β1-/-Runx2-/- and TGF-β1+/-Runx2+/- mice were successfully generated to clarify the relationship between TGF-β1 and RUNX2 during amelogenesis. Lower mineralization was observed in TGF-β1-/-Runx2-/- and TGF-β1+/-Runx2+/- mice than single gene deficient mice. Micro-computed tomography (μCT) revealed a lower ratio of enamel to dentin density in TGF-β1-/-Runx2-/- mice. Although μCT elucidated a relatively constant enamel thickness, variation was identified by scanning electron microscopy, which revealed that TGF-β1-/-Runx2-/- mice were more vulnerable to acid etching with lower degree of enamel mineralization. Furthermore, the double gene knock-out mice exhibited more serious enamel dysplasia than the single gene deficient mice. Hematoxylin-eosin staining revealed abnormalities in ameloblast morphology and arrangement in TGF-β1-/-Runx2-/- mice, which was accompanied by the absence of atypical basal lamina (BL) and the ectopic of enamel matrix. Odontogenesis-associated phosphoprotein (ODAPH) has been identified as a component of an atypical BL. The protein and mRNA expression of ODAPH were down-regulated. In summary, TGF-β1 and RUNX2 might synergistically regulate enamel mineralization through the downstream target gene Odaph. However, the specific mechanism by which TGF-β1 and RUNX2 promote mineralization remains to be further studied.

Permanent signatures of birth and nursing initiation are chemically recorded in teeth

In 2013 we presented a model for identifying nursing behavior from primate teeth based on rapid postnatal concentration changes in the non-essential trace element barium. Here we leverage the permanent neonatal (birth) line in the enamel of several dozen primate M1 cusps to compare pre- and postnatal trends in barium, zinc, strontium, and oxygen, as each element is believed to evince developmental patterning. Barium and zinc are the most consistent biomarkers of nursing initiation; a majority of M1 cusps shows concentration increases from prenatal to postnatal enamel, whereas strontium shows decreases or no change with similar frequency. Exceptions to the pattern of barium increase occurred in cusps that had been mineralizing for less than three weeks, suggesting that subsequent enamel maturation has only a minor impact on detecting real time events. Oxygen isotope compositions (δ18O) show rapid and marked fluctuations (∼1–2‰) within two weeks of birth in 93% of M1 cusps (n = 27/29). This is likely due to measurements of hypomineralized perinatal enamel and physiological changes in the body water of newborn infants. Ongoing work integrating elemental concentration gradients with isotopic variation will help establish the degree to which milk intake may cause elevated δ18O in teeth. We show that chemical identification of pre-to postnatal transitions may be robust to slight planar deviations that commonly obscure growth increments under light microscopy, and could help validate the identification of potential neonatal lines, making this approach a useful complement to bioarchaeological studies and public health investigations.

Advances in biomimetic mineralization of tooth enamel based on cell-free strategies

Tooth enamel is a highly-mineralized hard tissue covering the outermost layer of the dental crown, and amelogenesis is inseparable from the participation of necessary components such as ameloblasts, organic matrix proteins, and mineral ions, such as Ca2+ and PO43-. However, mature enamel is an acellular tissue and it is difficult to self-repair once damaged. The current treatment methods for enamel damage are filling or repairing with alloys, ceramics, or composite resins. However, the mechanical properties of these materials are quite different from the natural enamel and they can’t ensure a completely closed interface with the remaining enamel surface, which usually causes a series of post-repair problems. At present, the biomimetic mineralization of tooth enamel is a research hotspot in the field of prosthodontics, and has great clinical application needs and prospects, especially the researches on cell-free strategies have made significant accomplishment. Here, based on the cell-free strategies, we review the recent knowledge from ex situ and in situ two dimensions in the remineralization of tooth ename.

СТОМАТОЛОГИЧЕСКИЙ СТАТУС И МИНЕРАЛЬНЫЙ СОСТАВ ТВЕРДЫХ ТКАНЕЙ ЗУБОВ У ДЕТЕЙ В ЗАВИСИМОСТИ ОТ УРОВНЯ АНТРОПОГЕННОЙ НАГРУЗКИ

Anthropogenic load significantly affects the human body. It used to think that children, due to the high metabolism, are most susceptible to the effects of the urban ecosystem. Unfavorable environmental factors affect children's health, including dental health. Heavy metals tend to accumulate in the human body, namely in bones, hair, nails, and teeth. This can serve as an indicator of the technogenic load on the body. The process of maturation of tooth enamel is closely related to the intake of mineral components into the child's body. The aim of the work is a comparative analysis of the dental status and mineral composition of hard dental tissues in children at different levels of anthropogenic load. Material and methods. With the help of basic and additional examination methods, a comparative analysis of the dental status of primary school children was carried out and described. Using atomic emission spectrometry, the mineral composition of the hard tissues of the teeth of the children under study was studied. Results. It was found that in children in an area with a high anthropogenic load, the prevalence of dental caries and the presence of systemic hypoplasia of the enamel of permanent teeth are higher. Also, these children showed a relatively low caries resistance of the enamel and the content of micro-macroelements in the hard tissues of the teeth. Conclusions. To study dental morbidity in children of primary school age at different anthropogenic loads, it is necessary to carry out a complete examination, including basic and additional research methods. Also, laboratory methods of examination are recommended, namely, the determination of the mineral composition of the hard tissues of the teeth.

Molar-incisor hypomineralisation: narrative review on etiology, epidemiology, diagnostics and treatment decision.

Molar-incisor hypomineralisation (MIH) is clinically defined as demarcated structural enamel defects affecting at least one first permanent molar with or without the involvement of incisors. It is foremost a qualitative developmental defect of systemic origin. The prevalence for MIH is estimated at 12.9% with significant differences between countries. Its etiology and pathogenesis are still not completely understood. Several environmental and medical causes have been suggested to alter enamel maturation. The hypomineralised enamel may collapse shortly after eruption and as a consequence caries lesions seem more likely to develop. Besides cavitation, hypersensitivity and/or pain are the hallmarks of clinical symptoms. Both are associated with increased dental anxiety and fear of children suffering from MIH. Consequently, patients' care and management are challenging and necessitates a large range of non-, micro- and invasive strategies. MIH might be mixed up with three different other types of developmental defects in the enamel: fluorosis, enamel hypoplasia, and amelogenesis imperfecta. Careful diagnostic differentiation should be made before starting any dental treatment. A recent published classification system links the severity of the lesion to a treatment need index. This index is based on four values regarding two key symptoms: hypersensitivity and post-eruptiv enamel breakdown (PEB). Without PEB sealing is strongly recommended in order to prevent caries. For hypersensitive teeth as well as those with PEB use of glass ionomer cement as an intermediate cover, but mainly composite resins are materials of choice. For improvement of aesthetically compromised MIH-incisors, the resin infiltration technique has been proposed.

Expression and localization of amelotin, laminin γ2 and odontogenesis-associated phosphoprotein (ODAPH) on the basal lamina and junctional epithelium.

At maturation stage of enamel development, a specialized basal lamina (sBL) was built between ameloblasts and enamel. After the teeth eruption, the ameloblasts transform into the inner cell layer of junctional epithelium. The inner cell layer forms the internal basal lamina of junctional epithelium. However, the composition of the sBL and internal basal lamina was not clarified. The objective of our study was to make a description of the localization of amelotin (AMTN), laminin γ2 (LAMC2) and Odontogenesis-associated phosphoprotein (ODAPH) on the sBL and internal basal lamina. In immunohistochemical study, AMTN, LAMC2 and ODAPH were detected on the sBL at maturation stage. AMTN was also detected in ameloblasts at maturation stage. The expression of AMTN decreased from early-to-late maturation stage. In contrast, the expression of LAMC2 and ODAPH was stable. Immunofluorescence double-staining showed the localization of AMTN was close to enamel surface. However, the localization of ODAPH was close to ameloblasts. LAMC2 and ODAPH were observed on internal basal lamina of junctional epithelium. In contrast, no expression of AMTN was detected on internal basal lamina of junctional epithelium. Our results suggested that ODAPH might participate in enamel maturation and periodontal health, which might provide a better understanding of enamel defects and periodontal disease in clinic.

Effect of Amelotin on Bone Growth in the Murine Calvarial Defect Model.

Amelotin (AMTN) is a protein that is expressed during the maturation of dental enamel and has important role in enamel hydroxyapatite mineralization. However, it is not well understood whether AMTN has a strong mineral-promoting ability in bone. In this study, the effect of AMTN on bone healing was investigated using mice calvarial defect model in vivo, and the expression of bone marker genes and cell proliferation were investigated to clarify the role of AMTN in bone mineralization using mouse osteogenic cells (MC3T3-E1) in vitro. Collagen membranes, with or without recombinant human (rh) AMTN, were applied to calvarial defects created on the parietal bones of C57BL/6N mice. Microcomputed tomography and histological observation revealed that the defect largely filled with mineralized tissue by the rhAMTN-containing membrane in eight weeks. Moreover, CD31 positive cells were observed in the newly formed mineralized tissue and around the rhAMTN-containing membrane. In the presence of rhAMTN, the expression of the Spp1gene in MC3T3-E1 cells significantly increased within ten days in an osteoinductive medium. Moreover, rhAMTN significantly enhanced MC3T3-E1 cell proliferation. These findings indicate that AMTN positively influences bone repair by promoting hydroxyapatite mineralization.

Comparison of successful outcome of pit and fissure sealants bonded with etch-and-rinse adhesive and self-etch adhesive system.

Dental caries is one of the most common diseases around the world. The newly erupted permanent posterior teeth are at great risk due to the complex occlusal morphology of pits and fissures, infraocclusion and incomplete maturation of enamel. Pit and fissure sealants are the treatment of choice in such conditions. To compare the successful outcome of pit and fissure sealants bonded with etch-and-rinse adhesive system and self-etch adhesive systems in newly erupted permanent first molars. This randomized clinical trial was conducted at the Fatimah Jinnah Dental Hospital from June 2017 to May 2019. Children aged between 6 to 9 years who fulfilled the inclusion criteria were recruited for this study after parents' consent. Patients were randomly divided into two groups by the lottery method. Thirty patients with 60 first molar teeth in Group A were treated with etch-and-rinse adhesive system, while the other thirty patients with 60 first molar teeth in Group B were treated with self-etch adhesive system. After adhesive placement, all children received the same fissure sealants in newly erupted maxillary and mandibular permanent first molar. Each group was clinically evaluated at 6 and 12 months follow-up visit. Modified United States Public Health Service (USPHS) criteria were used for direct clinical evaluation of successful outcomes such as sealant retention and marginal discoloration. Successful outcome was statistically significant in Group B compared to Group A (60% vs. 30% p= 0.020) at 12months follow-up. Stratification analysis was performed with respect to age in which a successful outcome was not significant (p= 0.43) between groups for 6 to 7 years of age, while it was significant (p= 0.012) between groups in 8 to 9 years of age children. Self-etch adhesive system is better than etch-and-rinse adhesive system in terms of long-term sealant retention. In addition, the bonding procedure is simple with relatively short clinical application time therefore it would offer great benefit to clinicians in treating pediatric patients.

Signaling Modulation by miRNA-221-3p During Tooth Morphogenesis in Mice.

miRNAs are conserved short non-coding RNAs that play a role in the modulation of various biological pathways during tissue and organ morphogenesis. In this study, the function of miRNA-221-3p in tooth development, through its loss or gain in function was evaluated. A variety of techniques were utilized to evaluate detailed functional roles of miRNA-221-3p during odontogenesis, including in vitro tooth cultivation, renal capsule transplantation, in situ hybridization, real-time PCR, and immunohistochemistry. Two-day in vitro tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of miRNA-221-3p. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including β-catenin, Bmp2, Bmp4, Fgf4, Ptch1, and Shh, when inhibited with miRNA-221-3p and mimic. Also, the inhibition of miRNA-221-3p demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of Shh and Fgf4 within inner enamel epithelium (IEE) in E13 + 2 days in vitro cultivated teeth. Moreover, 1-week renal transplantation of in vitro cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and Shh expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of miRNA-221-3p did not affect tooth phenotype, while the loss of function resulted in long and slender teeth with short mesiodistal length. This study provides evidence that a suitable level of miRNA-221-3p is required for the modulation of major signaling pathways, including Wnt, Bmp, and Shh, during tooth morphogenesis.

Structural and functional relations between the connective tissue and epithelium of enamel organ and their role during enamel maturation.

The morphological and possible functional interactions between the connective tissue and enamel organ cells were examined during the maturation phase of enamel formation, using immunohistochemical techniques. Decalcified mandibular sections(10µm) including incisors were used from Wistar rats ages 10-12weeks. Sections were incubated with one or two primary antibodies targeting cell cytoskeleton (vimentin, α-actin, α-tubulin), dendritic marker (OX6), gap junctions (cx-43), enzymes (nitric-oxide synthase (nos1) and cyclooxygenase (cox1)), and the ion transporters (Na+/H+ exchanger (NHE1) and Na+/Ca2+ exchanger (NCX)) for 24h, before incubation with the appropriate conjugated fluorescent secondary antibodies. Sections were examined by fluorescence microscopy. Haematoxylin-eosin slides were also employed. Cellular heterogeneity and morphological modulations were identified within enamel organ cells and connective tissue covering suggesting complex cellular interactions and indicating a new functional concept and possible complementary role during enamel maturation. Also, some ion transportation activity, and nos1 and cox1 signalling pathways have been identified, indicating intercellular communication between these regions. A hypothesis is suggested, to explain the morphological modulation of ameloblasts and papillary cells during enamel maturation which functions to increase the transporting membrane surface area to accomplish faster and bulker ion transportation to achieve controlled pH and to direct Ca2+ towards enamel.

Molar-incisor hypomineralisation: narrative review on etiology, epidemiology, diagnostics and treatment decision.

Molar-incisor hypomineralisation (MIH) is clinically defined as demarcated structural enamel defects affecting at least one first permanent molar with or without the involvement of incisors. It is foremost a qualitative developmental defect of systemic origin. The prevalence for MIH is estimated at 12.9% with significant differences between countries. Its etiology and pathogenesis are still not completely understood. Several environmental and medical causes have been suggested to alter enamel maturation. The hypomineralised enamel may collapse shortly after eruption and as a consequence caries lesions seem more likely to develop. Besides cavitation, hypersensitivity and/or pain are the hallmarks of clinical symptoms. Both are associated with increased dental anxiety and fear of children suffering from MIH. Consequently, patients' care and management are challenging and necessitates a large range of non-, micro- and invasive strategies. MIH might be mixed up with three different other types of developmental defects in the enamel: fluorosis, enamel hypoplasia, and amelogenesis imperfecta. Careful diagnostic differentiation should be made before starting any dental treatment. A recent published classification system links the severity of the lesion to a treatment need index. This index is based on four values regarding two key symptoms: hypersensitivity and post-eruptiv enamel breakdown (PEB). Without PEB sealing is strongly recommended in order to prevent caries. For hypersensitive teeth as well as those with PEB use of glass ionomer cement as an intermediate cover, but mainly composite resins are materials of choice. For improvement of aesthetically compromised MIH-incisors, the resin infiltration technique has been proposed.

Investigating early husbandry strategies in the southern Caucasus: intra-tooth sequential carbon and oxygen isotope analysis of Neolithic goats, sheep, and cattle from Göytepe and Hacı Elamxanlı Tepe

The beginning of agriculture marks major changes in food acquisition technology in human history. In the southern Caucasus, agricultural villages emerged around 6000 cal. BC, 2000 to 3000 years later than in the Fertile Crescent despite their geographical closeness to each other. This study aims to provide isotopic evidence indicative of animal herding strategies in the early farming communities in the southern Caucasus. To analyze intra-individual sequential carbon and oxygen isotope variations, we used tooth enamel of five goats, four sheep, and three cattle from the Neolithic settlements, Göytepe and Hacı Elamxanlı Tepe (Azerbaijan). The analyses also included reference samples of modern domestic goat and sheep that are known to have grazed in the vicinity of the sites. As a result, the sequential carbon and oxygen isotope variations of the modern goat and sheep samples showed large amplitude and a synchronous pattern during the whole enamel maturation period. The same pattern was observed in some of the Neolithic goat and sheep samples, suggesting they may have grazed in the vicinity of the lowland site throughout the year. Other Neolithic goat and cattle samples exhibited relatively small amplitudes and inverse cyclical variation patterns. While these patterns may have been caused by multiple factors, such as drinking water and food/fodder, vertical transhumance has also been proposed for the similar isotope patterns. Despite the difficulty in specifying exact causes for the observed isotopic patterns, their diversity within/among the species implies multiple pastoral strategies that may have had adaptive advantages in new agricultural frontiers in the southern Caucasus.

Results of Application of Clinpro White Varnish® and Tooth Mousse® during the Enamel Mineral Maturation and its Focal Demineralization

According to some authors, the final maturation of tooth enamel occurs in 1- 2 years after eruption, and then for 2-3 years this process continues in just the fissure area. Full-fledged mineralization during this period is carried out due to the absorption of minerals from saliva, especially in respect to fluorine, calcium and phosphorus ions. As a result of the course of prophylactic treatment with Clin Pro White Varnish® and Tooth Mooth®, an activation of remineralization process was observed ended with the stabilization of the process or complete remineralization in the junior (6-7 years old) and the middle school group children (12-13 years old), which was confirmed by the obtained indices of the "DiagnodentKaVo" laser fluorometry test and the colorimetric test. The use of these preparations is a convenient and effective method in these age groups of children during the preventive treatment provision.

In the absence of a basal lamina, ameloblasts absorb enamel in a serumless and chemically defined organ culture system

ObjectivesTooth organ development was examined in a serumless, chemically defined organ culture system to determine whether morphological and functional development was identical to that in in vivo and serum-supplemented organ cultures. MethodsMouse mandibular first molar tooth organs at 16 days of gestation were cultured for up to 28 days in a Tronwell culture system using a serum-supplemented or serumless, chemically defined medium. After culture, specimens were processed for assessing tooth development using ultrastructural, immunohistochemical, and mRNA expression analyses. ResultsIn serum-supplemented conditions, inner enamel epithelial cells differentiated into secretory-stage ameloblasts, which formed enamel and reached the maturation stage after 14 and 21 days of culture, respectively. Ameloblasts deposited a basal lamina on immature enamel. Conversely, in serumless conditions, ameloblasts formed enamel on mineralized dentin after 21 days. Moreover, maturation-stage ameloblasts did not form basal lamina and directly absorbed mineralized enamel after 28 days of culture. RT-PCR analysis indicated that tooth organs, cultured in serumless conditions for 28 days, had significantly reduced expression levels of ODAM, amelotin, and laminin-322. ConclusionsThese results indicate that several differences were detected compared to the development in serum-supplemented conditions, such as delayed enamel and dentin formation and the failure of maturation-stage ameloblasts to form basal laminae. Therefore, our results suggest that some factors might be required for the steady formation of mineralized dentin, enamel, and a basal lamina. Additionally, our results indicate that a basal lamina is necessary for enamel maturation.

Odontogenesis-associated phosphoprotein truncation blocks ameloblast transition into maturation in OdaphC41*/C41* mice

Mutations of Odontogenesis-Associated Phosphoprotein (ODAPH, OMIM *614829) cause autosomal recessive amelogenesis imperfecta, however, the function of ODAPH during amelogenesis is unknown. Here we characterized normal Odaph expression by in situ hybridization, generated Odaph truncation mice using CRISPR/Cas9 to replace the TGC codon encoding Cys41 into a TGA translation termination codon, and characterized and compared molar and incisor tooth formation in Odaph+/+, Odaph+/C41*, and OdaphC41*/C41* mice. We also searched genomes to determine when Odaph first appeared phylogenetically. We determined that tooth development in Odaph+/+ and Odaph+/C41* mice was indistinguishable in all respects, so the condition in mice is inherited in a recessive pattern, as it is in humans. Odaph is specifically expressed by ameloblasts starting with the onset of post-secretory transition and continues until mid-maturation. Based upon histological and ultrastructural analyses, we determined that the secretory stage of amelogenesis is not affected in OdaphC41*/C41* mice. The enamel layer achieves a normal shape and contour, normal thickness, and normal rod decussation. The fundamental problem in OdaphC41*/C41* mice starts during post-secretory transition, which fails to generate maturation stage ameloblasts. At the onset of what should be enamel maturation, a cyst forms that separates flattened ameloblasts from the enamel surface. The maturation stage fails completely.

Calmodulin binds and modulates K+-dependent Na+/Ca2+-exchanger isoform 4, NCKX4

The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1–5, each being the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4 and confirmed the interaction using glutathione-S-transferase fusion pull-down. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4 and was abolished by the double-mutant I328D/F334D. When cotransfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a ∼2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, coexpression of wild-type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin prepositioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.