Overexpression of transforming growth factor-β1 in teeth results in detachment of ameloblasts and enamel defects

Transforming growth factor- β 1 (TGF- β 1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF- β 1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF- β 1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF- β 1 in teeth impacts the adhesion process of ameloblasts to dentin.     

Enamel organ proteins as targets for antibodies in celiac disease: implications for oral health

Enamel defects in permanent and deciduous teeth may be oral manifestations of celiac disease. Sometimes they are the only sign that points to this underdiagnosed autoimmune pathology. However, the etiology of these specific enamel defects remains unknown. Based on previously reported cross‐reactivity of antibodies to gliadin with the enamel proteins, amelogenin and ameloblastin, we analyzed (using immunohistochemistry) the ability of anti‐gliadin IgG, produced during untreated disease, to recognize enamel organ structures. We used swine germ teeth as a tissue model because they are highly homologous to human teeth in terms of proteins and development biology. Strong staining of the enamel matrix and of the layer of ameloblasts was observed with serum samples from women with celiac disease; high IgG reactivity was found against both gliadin peptides and enamel matrix protein extract, but there was no IgG reactivity against tissue antigens. In line with these findings, the gamma globulin fraction from gliadin‐immunized BALB/c mice showed a similar staining pattern to that of amelogenin‐specific staining. These results strongly suggest a pathological role for antibodies to gliadin in enamel defect dentition for both deciduous and permanent teeth, considering that IgG can be transported through the placenta during fetal tooth development.     

Epithelial-specific knockout of the Rac1 gene leads to enamel defects

The Ras-related C3 botulinum toxin substrate 1 (Rac1) gene encodes a 21-kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction, activities with consequences for cell growth, adhesion, migration, and differentiation. To examine the role(s) played by RAC1 protein in cell-matrix interactions and enamel matrix biomineralization, we used the Cre/loxP binary recombination system to characterize the expression of enamel matrix proteins and enamel formation in Rac1 knockout mice (Rac1(-/-)). Mating between mice bearing the floxed Rac1 allele and mice bearing a cytokeratin 14-Cre transgene generated mice in which Rac1 was absent from epithelial organs. Enamel of the Rac1 conditional knockout mouse was characterized by light microscopy, backscattered electron imaging in the scanning electron microscope, microcomputed tomography, and histochemistry. Enamel matrix protein expression was analyzed by western blotting. Major findings showed that the Tomes' processes of Rac1(-/-) ameloblasts lose contact with the forming enamel matrix in unerupted teeth, the amounts of amelogenin and ameloblastin are reduced in Rac1(-/-) ameloblasts, and after eruption, the enamel from Rac1(-/-) mice displays severe structural defects with a complete loss of enamel. These results support an essential role for RAC1 in the dental epithelium involving cell-matrix interactions and matrix biomineralization.     

A comparison of enamelin and amelogenin expression in developing mouse molars

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.     

Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino-enamel junction, and a decussating enamel rod pattern

Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.     

Elevated TGF-beta2 signaling in dentin results in sex related enamel defects

Initiation of enamel formation requires reciprocal signaling between epithelially and mesenchymally derived cells. OBJECTIVE: In this study, we used a transgenic mouse model which drives overexpression of an activated form of TGF-beta2 under control of the osteocalcin promoter, to investigate the role of TGF-beta2 in the dental mesenchyme, on enamel formation. DESIGN: Dentin and enamel were imaged by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Dentin mechanical properties were characterized for hardness and elasticity, following nanoindentation with a modified AFM. Pores found in enamel were quantified and compared using image analysis software (Scion Imagetrade mark). RESULTS: The elastic modulus of dentin was significantly reduced in the male TGF-beta2 overexpressor mice as compared to male wildtype mice, with no significant differences between female mice. Similarly, there were significantly more pores in enamel of the male transgenic mice as compared to male wildtype mice, with no significant differences between female mice. In situ hybridization of the continuously erupting incisor confirmed that osteocalcin expression was limited to the odontoblast cell layer at all stages of tooth formation. CONCLUSION: TGF-beta2 overexpression in the dentin matrix, results in sex-linked differences in dentin and enamel formation.     

Abnormal tooth tissue in human odontodysplasia.

The affected teeth in this case of odontodysplasia exhibited abnormal hypoplastic enamel, abnormal dentin containing extensive interglobular regions and completely lacking a peritubular matrix, and an abnormal irregular tissue consisting of highly calcified and partially fused granules located within the dentin of the tooth tips. Electron micrographs of the irregular tissue showed that the granules consisted of crystallites densely and radically packed in a noncollagenous amorphous matrix. Granules were surrounded by a slightly calcified, irregularly arranged, collagenous matrix. The irregular tissue formed in the pulp of the tooth tip befofe and independently of the dentin. It was at least partially formed by pulpal calcification. Abnormal dentin matrix was formed by odontoblasts which were less differentiated than normal. Odontodysplastic teeth showed abnormal differentiation of odontoblasts and ameloblasts resulting in defective enamel and dentin and, in extreme cases, in extensive pulpal calcification.     

An amelogenin mutation leads to disruption of the odontogenic apparatus and aberrant expression of Notch1

J Oral Pathol Med (2011) 40 : 235–242 Background: Amelogenins are highly conserved proteins secreted by ameloblasts in the dental organ of developing teeth. These proteins regulate dental enamel thickness and structure in humans and mice. Mice that express an amelogenin transgene with a P70T mutation (TgP70T) develop abnormal epithelial proliferation in an amelogenin null (KO) background. Some of these cellular masses have the appearance of proliferating stratum intermedium, which is the layer adjacent to the ameloblasts in unerupted teeth. As Notch proteins are thought to constitute the developmental switch that separates ameloblasts from stratum intermedium, these signaling proteins were evaluated in normal and proliferating tissues. Methods: Mandibles were dissected for histology and immunohistochemistry using Notch1 antibodies. Molar teeth were dissected for western blotting and RT‐PCR for evaluation of Notch levels through imaging and statistical analyses. Results: Notch1 was immunolocalized to ameloblasts of TgP70TKO mice, KO ameloblasts stained, but less strongly, and wild‐type teeth had minimal staining. Cells within the proliferating epithelial cell masses were positive for Notch1 and had an appearance reminiscent of calcifying epithelial odontogenic tumor with amyloid‐like deposits. Notch1 protein and mRNA were elevated in molar teeth from TgP70TKO mice. Conclusion: Expression of TgP70T leads to abnormal structures in mandibles and maxillae of mice with the KO genetic background and these mice have elevated levels of Notch 1 in developing molars. As cells within the masses also express transgenic amelogenins, development of the abnormal proliferations suggests communication between amelogenin producing cells and the proliferating cells, dependent on the presence of the mutated amelogenin protein.     

Cell proliferation and apoptosis in enamelin null mice

Enamelin is a secreted glycoprotein that is critical for dental enamel formation. Ameloblasts in enamelin (Enam) null mice develop atypical features that include the absence of a Tomes' process, expanded endoplasmic reticulum, apparent loss of polarity, and pooling of extracellular matrix in all directions, including between ameloblasts and the stratum intermedium. We hypothesized that ameloblast pathological changes may be associated with increased cell apoptosis. Our objective was to assess apoptotic activity in maxillary first molars of wild-type, Enam(+/-), and Enam(-/-) mice at postnatal days 5, 7, 9, 14, and 17. Mouse maxillae were characterized by light microscopy after terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labelling (TUNEL) or 5-bromo-2'-deoxyuridine (BrdU) staining. Following the initial deposition of dentin matrix, ameloblasts became highly dysplastic and no enamel crystal ribbons were deposited. Ameloblast apoptosis was observed in the Enam null mice starting in the secretory stage and with no apparent alteration in cell proliferation. We conclude that in the absence of enamelin and subsequent shutdown of enamel formation, ameloblasts undergo pathological changes early in the secretory stage that are evident as radically altered cell morphology, detachment from the tooth surface, apoptosis, and formation of ectopic calcifications both outside and inside the dystrophic enamel organ.     

Traumatic injuries to enamel formation in rat incisors

abstract – Twenty-three fullgrown white male rats were fitted with an inclined plane on the mandibular incisors with consequent raising of the bite by 2.5 mm. After an observation period of between 1 and 30 d the animals were killed. In all cases traumatic injuries to the enamel organs were seen. The defects were mainly of four different types. Type 1 occurred in the early maturation zone of the enamel as a local defect with dedifferentiated ameloblasts. Vascular injury was moderate and deformation of the dentin was seen. After 30 d there was still a local defect of the enamel organ. Type 2 occurred apically to the maturation zone and caused a two-layered ameloblastoma resembling a sandwich. Hemorrhages were common in the surrounding connective tissue, with accompanying appearance of phospholipids in the tissue. After 30 d some restitution of the enamel formation was seen. Type 3 was characterized by fractures of the dentin associated with total ameloblastic injury. Vascular necrosis was seen in the surrounding tissue. After 30 d a new ameloblastoma had developed in some areas. These three types appeared separately or combined. Type 4 occurred as a folding of newly formed apical dentin in areas without differentiated ameloblastoma and was seen in all of the experimental animals.     

Identification of the DSPP mutation in a new kindred and phenotype-genotype correlation

Oral Diseases (2011) 17 , 314–319 Objective: Hereditary dentin defects can be grouped into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia. Tooth enamel is considered normal in patients with hereditary dentin defects, but is easily worn down and fractured due to DSPP mutation‐induced altered dentin properties. The purposes of this study were to identify genetic cause of a family with type II DGI and enamel defects. Materials and methods: We identified a family with type II DGI and a unique form of hypoplastic enamel defect affecting occlusal third of the crown. Family members were recruited for the genetic analysis and DNA was obtained from peripheral whole blood. Results: Mutational analysis revealed a T to A transversion in exon 3 of the DSPP (c.53T>A, p.V18D). Haplotype analysis showed that the same mutation arose separately in two different families having DGI with similar enamel defects, indicating that this phenotype is associated with this specific DSPP mutation. Clinical features suggest that enamel formation was affected in the affected individuals during early amelogenesis, in addition to the dentin defect. Conclusions: We observed that a DSPP gene mutation not only influences dentinogenesis but also affects early stage amelogenesis.     

Overexpression of constitutively active MAP3K7 in ameloblasts causes enamel defects of mouse teeth

ObjectiveCompelling evidence suggests that mitogen-activated protein kinases (Mapks) play an important role in amelogenesis. However, the role of transforming growth factor (TGF)-β-activating kinase 1 (Tak1, Map3k7), which is a known upstream kinase of Mapks, during amelogenesis remains to be determined. The aim of this study was to investigate the possible involvement of Map3k7 in amelogenesis.DesignWe generated transgenic mice that produced constitutively active human MAP3K7 (caMAP3K7) under the control of amelogenin (Amelx) gene promoter. Radiography and micro-computed tomography (μCT) analysis was used to detect the radio-opacity and density of the teeth. The enamel microstructure was observed with a scanning electron microscope. Histological analysis was used to observe the adhesion between ameloblasts and residual organic matrix of the enamel. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression of enamel matrix protein.ResultsThe enamel of mandibular molars in caMAP3K7-overexpressing mice displayed pigmentation and a highly irregular structure compared with the wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. The gross histological appearances of ameloblasts and supporting cellular structures, as well as the expression of the enamel protein amelotin (Amtn) were altered by the overexpression of caMAP3K7.ConclusionsOur data demonstrated that protein expression, processing and secretion occurred abnormally in transgenic mice overexpressing caMAP3K7. The overexpression of caMAP3K7 had a profound effect on enamel structure by disrupting the orderly growth of enamel prisms.     

Quantitative image analysis of hyaluronan expression in human tooth germs

The expression of hyaluronan in human tooth germs was studied by using a biotinylated hyaluronan-binding complex and quantitative digital image analysis. At the cap stage, dental papilla exhibited a moderate staining, while intense reaction was observed in the apical portion of presecretory ameloblasts, stellate reticulum, and in dental basement membrane. When the enamel and dentine matrices started to develop, a strong hyaluronan reaction was evident in the young enamel and the apical portion of secretory ameloblasts. No hyaluronan could be detected in the secretory ameloblasts and enamel matrix of the early (9-wk-old) post-natal stage. It is concluded that hyaluronan may play a transitory role in the early phase of the development of the enamel matrix organization. A very weak signal was observed in the wall of dentin tubules, whereas the rest of the dentine matrix was not stained. The odontoblasts and the pulp were also moderately stained, and these reactions gradually decreased with age, suggesting that hyaluronan may also contribute to the development of dentine matrix and pulp.     

Proteolysis on maturing enamel surface, as shown by gel-coating methods

Degradation of enamel matrix proteins, and their removal during early maturation, is critical for the growth of large enamel crystals in the subsequent processes of enamel maturation. In this study, we sought to demonstrate, using in vivo zymography, the exact sites of proteolysis in maturing enamel and its relationship to the overlying ameloblasts. The maturing enamel surfaces of rat and bovine incisors were exposed and painted either with pre-exposed autoradiographic emulsion or with densely fluorescein-conjugated (DQ) gelatin. After a few hours, photographic development of the emulsion revealed alternate black and white banding patterns over the maturing enamel surface. DQ gelatin also revealed similar banding patterns of fluorescent and non-fluorescent regions. White, powdery areas of emulsion and fluorescent bands of DQ gelatin both corresponded to the areas of ruffle-ended ameloblasts, at least up to the mid stages of enamel maturation, implicating a predominant contribution of ruffle-ended ameloblasts in the degradation of enamel matrix proteins. Powdery white bands in autoradiographic emulsion shifted from the areas of ruffle-ended to smooth-ended ameloblasts in late maturation in both bovine and rat incisors and were not influenced by proteinase inhibitors or heat inactivation, implicating non-enzymatic interactions. DQ gelatin, in fact, did not generate any fluorescence in such smooth-ended ameloblast regions.     

LPA6-RhoA signals regulate junctional complexes for polarity and morphology establishment of maturation stage ameloblasts

ObjectivesLysophosphatidic acid (LPA) is a potent bioactive phospholipid that exerts various functions upon binding to six known G protein-coupled receptors (LPA_1–6); however; its role in a tooth remains unclear. This study aimed to explore the impact of the LPA/LPA receptor 6 (LPA₆)/RhoA signaling axis on maturation stage ameloblasts (M-ABs), which are responsible for enamel mineralization.MethodsThe expression of LPA₆ and LPA-producing synthetic enzymes during ameloblast differentiation was explored through immunobiological analysis of mouse incisors and molars. To elucidate the role of LPA₆ in ameloblasts, incisors of LPA₆ KO mice were analyzed. In vitro experiments using ameloblast cell lines were performed to validate the function of LPA-LPA₆-RhoA signaling in ameloblasts.ResultsLPA₆ and LPA-producing enzymes were strongly expressed in M-ABs. In LPA₆ knockout mice, M-ABs exhibited abnormal morphology with the loss of cell polarity, and an abnormal enamel epithelium containing cyst-like structures was formed. Moreover, the expression of E-cadherin and zonula occludens-1 (ZO-1) significantly decreased in M-ABs. In vitro experiments demonstrated that LPA upregulated the expression of E-cadherin, ZO-1, and filamentous actin (F-actin) at the cellular membrane, whereas LPA₆ knockdown decreased their expression and changed cell morphology. Furthermore, we showed that RhoA signaling mediates LPA-LPA₆-induced junctional complexes.ConclusionsThis study demonstrated that LPA-LPA₆-RhoA signaling is essential for establishing proper cell morphology and polarity, via cell–cell junction and actin cytoskeleton expression and stability, of M-ABs. These results highlight the biological significance of bioactive lipids in a tooth, providing a novel molecular regulatory mechanism of ameloblasts.     

Developmental disturbances of the rat molar induced by two diphosphonates

Very few reports have been published about the effects of diphosphonates on the cells and tissues of developing teeth. The present study was designed to investigate possible morphological changes in ameloblasts and odontoblasts and relate these changes to defects in the enamel surface of erupted teeth. Young rats were injected subcutaneously with single or multiple doses of HEDP or Cl2MDP (10 mg P/kg b.w.). Light microscopic examination of developing maxillary first molars showed that single injections of HEDP or Cl2MDP induced subameloblastic cysts between the secretory ameloblasts and the developing enamel. The ameloblastic lining of the cysts contained numerous calcified deposits. A few days after injection, hypoplasias were seen in the enamel in areas previously occupied by cysts. In the erupted teeth, scanning electron microscopic examination revealed enamel hypoplasias which were mainly localized on the mesial sides of the cusps. In addition to the previously mentioned disturbances, multiple injections resulted in more extensive cysts, some of which contained non-mineralized enamel matrix. Inhibition of the mineralization of dentin and alveolar bone was also noticed.     

The amyloid protein APin is highly expressed during enamel mineralization and maturation in rat incisors

This study investigated the expression and localization of APin (which was previously identified and cloned from a rat odontoblast cDNA library), during ameloblast differentiation in rat incisors, by using in situ hybridization and immunohistochemistry. The subcellular localization of APin varied during ameloblast differentiation, but was stage-specific. APin mRNA was not expressed in pre-ameloblasts, was weakly expressed in secretory ameloblasts, and was strongly expressed in maturation-stage ameloblasts as well as in the junctional epithelium attached to the enamel of erupted molars. In the maturation-stage ameloblasts, APin protein was conspicuous in the supranuclear area (Golgi complex) of smooth-ended ameloblasts as well as in both the supranuclear area and the ruffle end of ruffle-ended ameloblasts. During ameloblast-lineage cell culture, APin was expressed at a low level in the early stages of culture, but at a high level in the late stage of culture, which was equivalent to the maturation stage. APin protein was efficiently secreted from transfected cells in culture. Furthermore, its overexpression and inactivation caused an increase and decrease in matrix metalloproteinase-20 (MMP-20) and tuftelin expression, respectively. These findings indicate a functional role for APin in the mineralization and maturation of enamel that is mediated by the expression of MMP-20 and tuftelin.     

Gene expression and immunolocalization of amelogenin in enamel hypoplasia induced by successive injections of bisphosphonate in rat incisors

Successive injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) in rats induce enamel hypoplasia. To elucidate the pathogenesis of this hypoplasia, male Wistar rats were daily injected with HEBP or physiological saline for 7 days. After the last injection, they were killed under anaesthesia and their maxillary incisors were examined using an in situ hybridization technique and immunohistochemical staining to detect the gene expression and localization of amelogenin protein, respectively. In the HEBP-injected rats, several islets of partially mineralized enamel were present along crown-analogous surface of the incisor in the secretory stage of amelogenesis and enamel-free zones existed between these islets. In situ hybridization demonstrated amelogenin gene expression over the ameloblasts facing the islets of the matrix enamel as well as over those of the enamel-free zones. Immunohistochemical studies using rabbit antiamelogenin antibody revealed positive reaction both in the enamel matrix of the control group and in the islets of enamel matrix of the HEBP-injected group. Some small granules immunoreactive to amelogenin antibody were found in the distal portions of the ameloblasts in the HEBP-injected rats. The results indicate that HEBP does not alter amelogenin gene expression over ameloblasts, or the protein's existence in enamel matrix. There appeared to be some accumulation of amelogenin in the HEBP-treated ameloblasts. It is therefore suggested that the enamel hypoplasia in this experiment may not be due to a disturbance in amelogenin synthesis but to a disturbance in a later process, presumably of protein secretion.