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 (10 µm) including incisors were used from Wistar rats ages 10-12 weeks. 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 24 h, 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.

Deletion of Slc26a1 and Slc26a7 Delays Enamel Mineralization in Mice.

Amelogenesis features two major developmental stages-secretory and maturation. During maturation stage, hydroxyapatite deposition and matrix turnover require delicate pH regulatory mechanisms mediated by multiple ion transporters. Several members of the Slc26 gene family (Slc26a1, Slc26a3, Slc26a4, Slc26a6, and Slc26a7), which exhibit bicarbonate transport activities, have been suggested by previous studies to be involved in maturation-stage amelogenesis, especially the key process of pH regulation. However, details regarding the functional role of these genes in enamel formation are yet to be clarified, as none of the separate mutant animal lines demonstrates any discernible enamel defects. Continuing with our previous investigation of Slc26a1-/- and Slc26a7-/- animal models, we generated a double-mutant animal line with the absence of both Slc26a1 and Slc26a7. We showed in the present study that the double-mutant enamel density was significantly lower in the regions that represent late maturation-, maturation- and secretory-stage enamel development in wild-type mandibular incisors. However, the "maturation" and "secretory" enamel microstructures in double-mutant animals resembled those observed in wild-type secretory and/or pre-secretory stages. Elemental composition analysis revealed a lack of mineral deposition and an accumulation of carbon and chloride in double-mutant enamel. Deletion of Slc26a1 and Slc26a7 did not affect the stage-specific morphology of the enamel organ. Finally, compensatory expression of pH regulator genes and ion transporters was detected in maturation-stage enamel organs of double-mutant animals when compared to wild-type. Combined with the findings from our previous study, these data indicate the involvement of SLC26A1and SLC26A7 as key ion transporters in the pH regulatory network during enamel maturation.

Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.

Critical roles for WDR72 in calcium transport and matrix protein removal during enamel maturation.

Defects in WDR72 (WD repeat-containing protein 72) cause autosomal recessive hypomaturation amelogenesis imperfecta. We generated and characterized Wdr72-knockout/lacZ-knockin mice to investigate the role of WDR72 in enamel formation. In all analyses, enamel formed by Wdr72 heterozygous mice was indistinguishable from wild-type enamel. Without WDR72, enamel mineral density increased early during the maturation stage but soon arrested. The null enamel layer was only a tenth as hard as wild-type enamel and underwent rapid attrition following eruption. Despite the failure to further mineralize enamel deposited during the secretory stage, ectopic mineral formed on the enamel surface and penetrated into the overlying soft tissue. While the proteins in the enamel matrix were successfully degraded, the digestion products remained inside the enamel. Interactome analysis of WDR72 protein revealed potential interactions with clathrin-associated proteins and involvement in ameloblastic endocytosis. The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts. Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane. Fewer blood vessels were observed in the papillary layer supporting ameloblasts. Specific WDR72 expression by maturation stage ameloblasts explained the observation that enamel thickness and rod decussation (established during the secretory stage) are normal in the Wdr72 null mice. We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization.

Enamel maturation: a brief background with implications for some enamel dysplasias.

The maturation stage of enamel development begins once the final tissue thickness has been laid down. Maturation includes an initial transitional pre-stage during which morphology and function of the enamel organ cells change. When this is complete, maturation proper begins. Fully functional maturation stage cells are concerned with final proteolytic degradation and removal of secretory matrix components which are replaced by tissue fluid. Crystals, initiated during the secretory stage, then grow replacing the tissue fluid. Crystals grow in both width and thickness until crystals abut each other occupying most of the tissue volume i.e. full maturation. If this is not complete at eruption, a further post eruptive maturation can occur via mineral ions from the saliva. During maturation calcium and phosphate enter the tissue to facilitate crystal growth. Whether transport is entirely active or not is unclear. Ion transport is also not unidirectional and phosphate, for example, can diffuse out again especially during transition and early maturation. Fluoride and magnesium, selectively taken up at this stage can also diffuse both in an out of the tissue. Crystal growth can be compromised by excessive fluoride and by ingress of other exogenous molecules such as albumin and tetracycline. This may be exacerbated by the relatively long duration of this stage, 10 days or so in a rat incisor and up to several years in human teeth rendering this stage particularly vulnerable to ingress of foreign materials, incompletely mature enamel being the result.

Análise da maturação em profundidade do esmalte de dentes com diferentes idades eruptivas por meio da microdureza longitudinal / In-depth analysis of the enamel maturation in teeth with different eruptive ages by the longitudinal microhardness

Estudos epidemiológicos e experimentos com animais têm demonstrado que a susceptibilidade à doença cárie é maior logo após o período pós-eruptivo dos dentes, decrescendo posteriormente. Na literatura, várias pesquisas ressaltam a importância do conhecimento das características do esmalte durante e após o processo de erupção, assim como dos mecanismos envolvidos na desmineralização dentária para o estabelecimento de medidas preventivas eficazes. Variações idade dependentes na susceptibilidade à doença cárie são de grande importância clínica. Entretanto, a literatura é escassa em estudos que avaliem as alterações no esmalte em dentes inclusos e após a erupção na cavidade bucal. O objetivo deste estudo in vitro foi avaliar a maturação do esmalte em profundidade, de dentes hígidos com diferentes idades eruptivas (antes da erupção na cavidade bucal; 2 a 3 anos após a erupção; 4 a 10 anos após a erupção; e com mais de 10 anos após a erupção) por meio da microdureza longitudinal. Foram utilizados 12 espécimes de esmalte de dentes hígidos de humano para cada idade eruptiva. A comparação entre a microdureza longitudinal das diferentes idades e entre as diferentes profundidades foi realizada por meio da Análise de Variância a dois critérios (ANOVA), adotando-se um nível de significância de 5%. De acordo com a metodologia adotada na presente pesquisa, foi possível concluir que houve diferença entre a microdureza longitudinal dos espécimes com diferentes idades eruptivas em todas as profundidades estudadas, evidenciando-se um comportamento crescente da mineralização quanto maior a idade eruptiva. Pôde-se observar ainda uma tendência crescente dos valores da microdureza quanto maior a profundidade do esmalte, em todos os grupos de idade.

Epidemiologic studies and caries experiments with animals have suggested that caries susceptibility of teeth decreases with age. Many researches in literature, stands out the importance of the enamel characteristics knowledge during and after the eruption process, as well as of the involved mechanisms in dental demineralization in order to establish efficient methods of prevention. Age-dependent variations in the susceptibility to caries lesions are of great clinical importance. However, literature is scarce in studies that evaluate the alterations that occur in the unerupted teeth enamel and after its eruption in the oral cavity. The aim of this study was to evaluate, in vitro, the maturation in depth of sound teeth enamel with different eruptive ages (before the eruption in the mouth, 2 to 3 years after the eruption; 4 to10 years after the eruption; and over 10 years after the eruption) through the longitudinal microhardness. We used 12 specimens of sound teeth enamel for each eruptive age. The comparison between the longitudinal microhardness of the different eruptive ages and in different depths was performed by analysis of variance with two criteria (ANOVA), adopting a significance level of 5%. According to the methodology adopted in this research, it was possible to conclude that there was a difference between the longitudinal microhardness of specimens with different eruptive ages at all depths, revealing an increase of the mineralization with age. It was observed also an increasing trend of the values of microhardness as the enamel depth increased, in all age groups studied.