The Role Of Stratum Intermedium In Differentiation And Secretion Stage Of Ameloblasts

Tooth is a typical epithelium-mesenchyme and epithelium-epithelium organ, which involves a complicated process of cell and molecular interaction. It has been well known that stratum intermedium is an integral part of developing tooth germs. It is a transient and subtle epithelial structure closely associated with inner dental epithelium Stratum intermedium undergoing marked and transient changes during amelogenesis .At the initial stage of ameloblasts differentiation,it has two or three cells layers,then with the differentiation process, it underwent thinning and involution. It is conspicuous that its formation and subsequent behavior are closely related to the cytodifferentiation events of inner dental epithelium and secretion of the ameloblasts. The stratum intermedium represents an important player in the progression of tooth germs, especially in the normal morphopoiesis of amelogenesis.Objective:This study aims to establish a model of developmental stratum intermedium, to identify the morphological changes of the structure and study expression of mineralization-related signaling molecules in stratum intermedium and ameloblasts during the amelogenesis. So as to provide with theoretical basis for further studies of stratum intermedium and their interaction with ameloblasts in the secretion stage.Methods:1. BALB/c postnatal mice were applied to study the development of stratum intermedium by HE.Then an electron microscope was carried out to elucidate the ultrastructural changes of stratum intermedium during the secretion of enamel.2. Tdt-mediated Dutp nick end labeling (TUNEL) technique and immunohistochemical method were apply to study the expression of PCNA and cell apoptosis in stratum intermedium and ameloblasts during the adamantine formation..3. Immunohistochemistry method then was used to localize the distribution of TNSALP, E-cad, OC, BSP, OPN, BGN,DCN and FM in the enemal organ of day 7 and day 13 old mice.Results:1. Stratum intermedium was two or three cells layers during the late bell stage. It was observed slight rough endoplasmic reticulum and mitochondrion in cytoplasm. At this stage, the inner enamal epithelium was columnar, and the rough endoplasmic reticulum and mitochondria increased in number. Desmosomes junctions between the inner enamel epithelium and the stratum intermedium cells were also studied, which increased greatly with advanced secretion from the beginning of differentiation to the formation of enamel.Then with the completion of the process, stratum intermedium underwent thinning and involution.The intercellular space became porous and organeleas were also little than before.2. The positive expression of PCNA was mainly located in stratum intermedium cells and amleoblasts in the early stage, and weakened gradually during amelogenesis; Staining of apoptosis cells were observed in every stage of ameloblasts, especially in the maturestage. TUNEL positive cells were also seen in the late stratum intermedium cells.3. ALP, E-cad, OC, BSP, OPN, DMP1 and FM were positively expressed in stratum intermedium and ameloblasts. The expression of ALP and E-cad in stratum intermedium was earlier than that in ameloblasts, and a spatio-temporal expression pattern could be observed during enamel formation. BSP, OPN, DMP1 and FM were positively expressed in stratum intermedium and ameloblasts in late secretion stage of ameloblasts, but negatively in early stage.Conclusions:1. The number of desmosomes junctions and organelles in the stratum intermedium increased greatly with advanced secretion,as well as the ameloblasts. These morphologic changes indicated that the stratum intermedium may have transport and supporting functions during the amelification.2. Cell proliferation and apoptosis play an important role in plasticity of tooth development. There was a relationship between stratum intermedium and ameloblast for the expression of PCNA and TUNEL. It shows that stratum intermedium plays an important regulating role during the differentiation of ameloblast and the normal formation of enamel morphous .3. There was a temporospatial expression of mineralization-related signaling molecules in stratum intermedium and ameloblasts.These findings raise the possibility that stratum intermedium cells may played an important regulating role during the formation of enamel.