Expression of cytokeratins in human enamel organ

Cytokeratin (CK) is a filament which plays a central role in epithelial tissue and, like the polypeptides of intermediate filaments in general, shows a high degree of tissue specificity. The CK expression patterns of odontogenic epithelia are still poorly described. We studied the distribution of individual CK polypeptides in the human enamel organ at bell stage and in remnants of the dental lamina. Our immunohistochemical study showed that epithelial cells stained for CKs 7, 13, 14 and 19 with slight changes in their pattern during the differentiation phase of odontogenesis. There was negative staining for all other CK polypeptides tested (CKs 8, 10, 16, 17 and 18). Most of the CKs in the enamel organ epithelia did not show differences related to the stage-specific state of differentiation, except for CKs 14 and 19 at the inner enamel epithelium. A strong label for CK 14 was present at the inner dental epithelium at early bell stage, and this was substituted by CK 19 at the late bell stage when the ameloblasts were fully differentiated.     

Role of perlecan,a basement membrane-type heparan sulfate proteoglycan,in enamel organ morphogenesis

Perlecan is a multifunctional heparan sulfate proteoglycan that controls cell-signaling events by interacting with several growth factors, cytokines, and other signaling molecules. Perlecan was thought to be localized only in the basement membrane, but recently, intraepithelial localization of perlecan has been demonstrated in some pathophysiological situations. Therefore, perlecan is expected to modulate epithelial cell behavior. Our recent study demonstrated that perlecan accumulates in the stellate reticulum of the enamel organ of murine molar tooth germs in a stage-specific manner. To understand the function of perlecan in the enamel organ, we generated transgenic (Tg) mice that overexpressed perlecan in epithelial cells by using the keratin 5 promoter. Perlecan Tg molars had dull-ended crowns and outward-curved tooth roots, and their enamel was poorly crystallized. The constant overexpression of perlecan and the accompanying disorganized distribution of perlecan-related molecules in the enamel organ resulted in irregular tooth morphology. These results indicate that the time schedule of intraepithelial perlecan expression appears to be critically controlled during enamel organ development. In this brief review, we have described the dynamics of perlecan and its receptors and the timing of cleavage of heparan sulfate chains in odontogenesis, focusing on enamel organ development, and discussed the role of perlecan in enamel organ morphogenesis.     

In vitro effects of calcitonin and/or parathyroid hormone on odontogenesis of mouse embryonic molars

Mandibular first molars of mouse embryos were cultivated for examination of the effects of calcitonin (CT) and/or parathyroid hormone (PTH) on the odontogenesis of the molars, and for determination of whether and how CT, which is a PTH antagonist, has an influence on the effect of PTH on odontogenesis. On the second day, the inner enamel epithelium in the control group had already differentiated into pre-ameloblasts. Typical odontoblasts had secreted a layer of predentin. On the fourth day of culture, the pre-ameloblasts achieved terminal differentiation into secretory ameloblasts, and enamel and dentin had already been deposited. PTH (1 unit/mL) inhibited the odontogenesis of the cultured molars during the designated culture periods (two and four days), while CT (0.5 unit/mL) stimulated odontogenesis. On the second day, the development of the molars in the CT + PTH group showed an intermediate stage between the control and PTH-treated explants, but on day 4 it corresponded to that of the controls. Moreover, when the molars exposed to PTH for two days were untreated and treated with CT for an additional two days, the former produced a small quantity of enamel matrix, while the latter formed a large amount of the matrix. These histological findings were also supported by a morphometric analysis of the enamel matrix in the cultured molars. The present results suggest that CT stimulates, but PTH suppresses, the odontogenesis of the mouse embryonic molars, and that CT is an antagonist to the inhibitory effect of PTH on odontogenesis.     

Dental epithelial histo-morphogenesis in the mouse: positional information versus cell history

Reciprocal epithelial-mesenchymal interactions control odontogenesis and the cap stage tooth germ mesenchyme specifies crown morphogenesis. The aim of this work was to determine whether this mesenchyme could also control epithelial histogenesis. Dental mesenchyme and enamel organ were dissociated from mouse first lower molars at E14. At this early cap stage, the enamel organ consists of four cell types forming the inner dental epithelium (IDE), primary enamel knot (PEK), outer dental epithelium (ODE) and the stellate reticulum (SR). Pelleted trypsin-dissociated single dental epithelial cells, which had lost all positional information, were reassociated to either dental mesenchyme or dissociated mesenchymal cells and cultured in vitro. Although with different timings, teeth developed in both types of experiments showing a characteristic dental epithelial histogenesis, cusp formation, and the differentiation of functional odontoblasts and ameloblasts. The rapid progression of the initial steps of histogenesis suggested that the cell history was not memorized. The dental mesenchyme, as well as dissociated mesenchymal cells, induced the formation of a PEK indicating that no specific organisation in the mesenchyme is required for this step. However, the proportion of well-formed multicusped teeth was much higher when intact mesenchyme was used instead of dissociated mesenchymal cells. The mesenchymal cell dissociation had consequences for the functionality of the newly-formed PEK.     

Immunofluorescent lectin binding patterns and glycoprotein co-localization in the developing murine molar tooth.

Fluorescein-conjugated lectins were used in conjunction with antibodies to laminin, tenascin and amelogenin to investigate saccharide expression in the developing tooth germ. At the bud stage, peanut agglutinin (PNA) binding demonstrated residues that may be D-galactose-(beta 1----3)DGalNAc, and this staining occurred after the expression of tenascin. Only the cap-stage enamel organ suprabasal cells and the enamel knot stained intensely with Ulex europeus agglutinin-I, but not Lotus tetragonolobus agglutinin, implying the transient presence of blood group H type I oligosaccharides. At the late stages of amelogenesis, enamel synthesis is preceded by en bloc loss of inner enamel basement membrane components. Before this, Bandeiraea (Griffonia) simplicifolia--I (BSL-I) staining was lost from postmitotic ameloblasts, suggesting that a glycosylated species is initially removed. Additionally, PNA was co-localized with amelogenin protein, suggesting that it may express beta-D-galactosyl sequences. These results indicate that the glycosylation patterns of matrix components during odontogenesis may be important as they vary in a manner similar to that of the well-known glycoproteins.     

Calbindin-D28 kappa localization in rat molars during odontogenesis

Specific antiserum raised against Calbindin-D28 kappa, a vitamin D-dependent calcium-binding protein (CaBP) isolated from chick intestine, was used for localization of the protein in developing rat molars. Previously, CaBP had been localized in specific cells associated with the continuously erupting rat incisor: late pre-secretory ameloblasts, secretory and maturation zone ameloblasts, stratum intermedium cells adjacent to ameloblasts in the late zone of enamel secretion, and papillary cells underlying maturation zone ameloblasts. In this study, the peroxidase anti-peroxidase technique was used for localization of the peroxidase anti-peroxidase technique was used for localization of CaBP in histological sections of rat mandibles from 18-day-old rat embryos through 20-day-old neonates. CaBP was not detected in any cells of the enamel organ, dental papilla, or dental sac during early odontogenesis from the dental lamina stage through the advanced bell stage. The protein first appeared in secretory ameloblasts which were situated opposite odontoblasts with newly secreted dentin. CaBP was present in the cytoplasm of more mature ameloblasts, but not in less mature ameloblasts. Some stratum intermedium cells subjacent to well-developed secretory and maturation zone ameloblasts also contained CaBP. The protein was not detected in odontoblasts, pulp cells, or other cells associated with the developing molars. It was also absent from the demineralized enamel and dentin matrix. In developing rat molars, the time-course of appearance of CaBP, a protein dependent for its synthesis on the vitamin D endocrine system in other organ systems, suggests a potential direct role of this hormonal system in enamel mineralization.     

Effects of gamma radiation on incisor development of the prenatal albino mouse

Fetuses of pregnant albino mouse exposed to 400 rad of gamma-irradiation, on the 12th gestational day, were compared with unirradiated fetuses to asses the radiation effect on developing incisors. Pregnant animals were sacrificed on day 18 post coitum, and their fetuses were decapitated. Heads were routinely prepared, frontally sectioned, and stained with hematoxylin and eosin. Histologic examination demonstrated that the development of the maxillary and mandibular incisors was retarded in all the experimental fetuses and were in early bell stage, whereas those of the control animals were elaborated their matrices. It was concluded that gamma-irradiation interferes with cytodifferentiation of the enamel organ and dental papilla and subsequently inhibits normal odontogenesis.     

Dynamic expression of E-cadherin in ameloblasts and cementoblasts in mice

During embryonic development, E-cadherin mediates intercellular adhesion in a variety of epithelia in a spatio-temporal pattern. We have analyzed the distribution of this cell adhesion molecule in the mouse during odontogenesis, at both mRNA and protein levels, in the mandibular first molars and incisors. E-cadherin was strongly expressed at the bell stage by the cells of the dental organ, and by the pre-secretory ameloblasts and the cells of stratum intermedium at the early mineralization stage. At the onset of enamel secretion, E-cadherin disappeared from the apical pool of the ameloblasts and was later absent from the post-secretory ameloblasts. E-cadherin was also found in Hertwig's root sheath and later in the cells producing acellular cementum. These findings indicate that E-cadherin may be involved in the polarization of the ameloblasts and in the early stages of cementogenesis.     

Immunohistochemical and immunochemical detection of a similar epitope in enamel proteins and monocyte-macrophage protein recognized by mouse monoclonal antibody MOMA-2.

These studies were made as a first step in elucidating unknown functions of enamel proteins in odontogenesis. The cytoplasm of ameloblasts and the proteins in dentine matrix before mineralization were stained with this monoclonal antibody. SDS-PAGE and Coomassie blue staining of proteins extracted from enamel showed several protein bands. Immunoblotting revealed that proteins recognized by this antibody were situated between 20-30 kDa. These results indicate that enamel proteins, presumably amelogenins, have an epitope resembling monocyte-macrophage protein. The findings suggest that epithelial-mesenchymal interaction in odontogenesis and preosteoblast-preosteoclast interaction in osteogenesis may be similar.     

蕾状期成釉器重建体外模型的建立

目的:研究小鼠下颌第一磨牙胚蕾状期:①牙间充质对牙胚上皮发生的控制作用;②原发釉结节对牙胚间充质的定义作用;③牙胚上皮细胞位置信息在牙齿发育中的作用.材料与方法:使用小鼠蕾状期下颌第一磨牙蕾状期牙胚,在体外利用胰蛋白酶解离后的牙间充质与混合牙胚上皮细胞进行了重组培养至14天.结果与结论:结果显示蕾状期牙胚上皮具有高度的可塑性,在丧失了全部位置信息后牙胚上皮细胞仍能在牙胚间充质的诱导下完成成釉器的重建及一系列的组织发生.这表明胚间充质在原釉结节形成时对牙胚上皮组织学发生具有可能的控制作用,即在原釉结节形成前在牙胚间充质中可能已存在有牙齿发生所需的全部信息.而在成釉器的体外重建过程中,牙胚间充质则起到了支架的作用.     

Immunohistochemical demonstration of enamel proteins in odontogenic tumors

Immunohistochemical localization of two enamel proteins, amelogenin and enamelin, in comparison with that of keratin, was determined in odontogenic tumors and the allied lesions in order to verify functional differentiation of the tumor cells as ameloblasts. Amelogenin and enamelin were demonstrated in small mineralized foci and in the tumor cells surrounding them in adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), and calcifying odontogenic cyst (COC). Hyaline droplets in AOT showed positive staining for both enamel proteins. These mineralized and hyaline materials were not positive for keratin, although tumor cells were positive. On the other hand, no immunoreaction for enamel proteins was obtained in ameloblastoima and odontogenic epithelial cell nests within myxoma and epulis. The results suggest that tumor cells of AOT and CEOT and lining epithelial cells of COC show ameloblastic differentiation in part, but that ameloblastoma cells do not attain functional matauration as secretory phase ameloblasts.     

大鼠成釉器细胞的分离纯化

目的：寻找一种大鼠成釉器细胞分离纯化的简捷方法。方法：分离出生后6dSD大鼠上下颌第一和第二磨牙完整牙胚，剥离牙囊和成釉器，酶消化法原代混合培养。采用多次差别消化法去除成纤维样细胞，纯化上皮样细胞，并进行抗角蛋白染色和RT—PCR检测釉原蛋白、成釉蛋白mRNA表达。结果：原代细胞为混杂细胞，经2～3次差别消化可完全去除成纤维样细胞。上皮样细胞呈多角形，铺路石样生长，抗角蛋白染色阳性，表达釉原蛋白、成釉蛋白mRNA。结论：本研究通过多次差别消化获得了纯化的大鼠成釉器细胞。     

Diffusion of fluoride through the rat enamel organ in vitro

This study investigated the diffusion of fluoride through the enamel organ in vitro. The rat molar explants used were entirely in the secretory stage or predominantly in the maturation stage of enamel formation. The removal of the enamel organ or metabolic inhibition with iodoacetate caused significant increases in enamel fluoride uptake at both stages of enamel formation. Inhibition with dinitrophenol caused a significant increase only in the maturation phase. Uptake of fluoride in enamel was related to the fluoride concentration in the medium, except in the maturation stage explants, where increasing the medium fluoride concentration from 0.05 ppm to 0.08 ppm did not significantly increase fluoride uptake at any of the three observation times. The findings indicate that the enamel organ exists as a diffusion-limiting membrane to the movement of fluoride from the extracellular fluid compartment to the developing enamel.     

Pulpal irritants

The dental pulp is characterized as a connective tissue and as such it is not considered an external tissue, yet its exposure to external stimuli is constant. This is due to a number of factors including the permeability of attrited or disrupted enamel as well as that of physiologic dentin and cementum. The pulp is extraordinarily sensitive to its external environment. Once thought to be a vestigial organ, it is now understood that the dental pulp is an important tissue whose role in the defense of the dentition may be as significant as its role in odontogenesis.     

The ultrastructure of ameloblastoma

The ultrastructural features of five mandibular ameloblastomas were investigated. The morphological comparison between ameloblastoma and enamel organ was made, and the cause of the appearance of granular cells in the tumor was discussed. The peripheral columnar cells were morphologically similar to the inner enamel epithelium of the enamel organ. The central cells were similar to the stellate reticulum cells. There were not stratum intermedium-like cells in the tumors. This implied that the tumors were developed earlier than the enamel organ of bell stage. The squamous cells were sometimes found in the central part of the tumor cell masses. These cells were flat with a few organelles in the plasma, but had a lot of tonofilaments. This suggested that these cells were not active. Upon the ultrastructure, however, it could not be determined whether the appearance of these cells reflected a low differentiation or a degeneracy. Granular cells contained a lot of lysosomes, but no autophage was noticed. We thought that the appearance of these cells was due to accumulation of some unusual substances in the cells, the substances might be related with the glycosaminoglycan.     

The effect of LRAP on enamel organ epithelial cell differentiation

Leucine-rich amelogenin peptide (LRAP) is an alternatively spliced amelogenin found in the developing enamel organ. LRAP functions to regulate the development of mesenchymal-derived cells; however, its effect on cells of the enamel organ remains unclear. The hypothesis tested in this study is that LRAP also regulates human enamel organ epithelial cells. Recombinant human LRAP (rH58) was synthesized in E. coli, purified, and exogenously added to cultures of human primary enamel epithelial cells, which were analyzed for changes in cell proliferation and differentiation. rH58 had no effect on cell proliferation, but altered enamel epithelial cell morphology, resulting in larger, more rounded cells. Immunofluorescence showed that rH58 treatment increased amelogenin synthesis, but down-regulated Notch1 expression in enamel epithelial cells. LAMP-1, a membrane receptor for LRAP in mesenchymal cells, was identified and was up-regulated in the presence of rH58. These results suggest that rH58 promotes differentiation of human enamel organ epithelial cells.     

Epithelium-connective tissue junction in follicular ameloblastoma and ameloblastic fibroma: an ultrastructural analysis

The ameloblastoma and ameloblastic fibroma are tumors of odontogenic origin. During odontogenesis, there is a sequence of inductive stimulations, or interactions, between the epithelium of the enamel organ and the connective tissue of the dental papilla. We review 7 cases of follicular ameloblastoma and 1 case of ameloblastic fibroma under the electron microscope to investigate possible induction-stimulated changes at the epithelium-connective tissue junction. Thickening of the basal lamina by a granulo-filamentous material was a universal finding. Horizontal proliferation and convolutions of this thickened material were found in 2 ameloblastomas. The ameloblastic fibroma evidenced fine aperiodic fibers perpendicular to the basal lamina. These changes are consistent with attempted inductive stimuli directed toward tooth formation.     

人牙齿发育中OSX的表达

目的:通过免疫组织化学方法观察人牙齿发育中OSX的表达规律,探索其功能及意义。方法:取自然流产的人胚胎上下颌骨,分别进行HE染色和免疫组织化学染色,用图像分析仪分析。结果:免疫组化显示,OSX分布于细胞浆中。蕾状期,OSX分布于牙蕾上皮细胞;帽状期,成釉器所有细胞均表达OSX,但在内釉上皮的釉结处表达明显集中;钟状期,成釉细胞、成牙本质细胞及邻近的牙乳头细胞高表达OSX,釉牙本质界、牙本质小管及新形成的釉质有表达。结论:OSX参与成釉细胞、成牙本质细胞的分化、成熟及细胞间信号传导,可能参与调节釉质和牙本质基质的分泌及其生物矿化过程。