Importance of bicarbonate transport in pH control during amelogenesis — need for functional studies

Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory product, the enamel matrix, is quite different. Ameloblasts direct the formation of hydroxyapatite crystals, which liberate large quantities of protons that then need to be buffered to allow mineralization to proceed. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. Many investigations have used immunohistochemical and knockout studies to determine the effects of these genes on enamel formation, but up till recently very little functional data were available for mineral ion transport. To address this, we developed a novel 2D in vitro model using HAT‐7 ameloblast cells. HAT‐7 cells can be polarized and develop functional tight junctions. Furthermore, they are able to accumulate bicarbonate ions from the basolateral to the apical fluid spaces. We propose that in the future, the HAT‐7 2D system along with similar cellular models will be useful to functionally model ion transport processes during amelogenesis. Additionally, we also suggest that similar approaches will allow a better understanding of the regulation of the cycling process in maturation‐stage ameloblasts, and the pH sensory mechanisms, which are required to develop sound, healthy enamel.     

Localization of Core Planar Cell Polarity Proteins,PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation

To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.     

Transforming growth factor-β1 expression is up-regulated in maturation-stage enamel organ and may induce ameloblast apoptosis

Transforming growth factor-β1 (TGF-β1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-β1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-β1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-β1-mediated growth arrest and are prone to TGF-β1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-β1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-β1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-β1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development.     

Effects of ascorbate-deficiency on collagen secretion and resorption in cultured mouse incisor germs

The effects of ascorbic acid deficiency on mouse incisors, grown in vitro, has been investigated at the histological and cytological levels. In this model, continuously growing mouse incisors are characterized by the existence of different type of predentin-dentin matrix on its lingual (root-analogue) and labial (crown-analogue) surface and the absence of enamel on the lingual surface. Our observations indicated that ascorbate-deficiency affected the behavior of mouse tooth germs in vitro: odontoblast differentiation was disturbed and morphological evidence for odontoblast-mediated collagen resorption were observed. An abnormal amorphous predentin-dentin matrix existed and the basement membrane was prematurely disrupted. The dentin mineralization, as well as functional differentiation of ameloblasts were strongly hampered. Chronic deficiency led to disorganization of the dental tissues.     

Fluoride down-regulates the expression of matrix metalloproteinase-20 in human fetal tooth ameloblast-lineage cells in vitro

Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10  µ M fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10  µ M NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10  µ M NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel.     

Differentiation of human ameloblast-lineage cells in vitro

Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19–24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5–15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering.     

Towards second-generation proteome analysis of murine enamel-forming cells

Proteome analysis of rat enamel-forming cells, initiated over a decade ago, has provided valuable insights to enamel biology. In preparation for a more comprehensive, second-generation proteomic exploration, we evaluated an updated microsample-profiling strategy that comprises sequential extraction of enamel epithelium, parallel one- and two-dimensional gel electrophoresis, and mass spectrometric sequence analysis. The results indicated that several hundred proteins, representing various cellular compartments (including membranes), are amenable to identification with a starting tissue volume of < 10  µ l. With its increased proteomic depth and breadth, this straightforward approach constitutes a major advance from the first-generation work (10-fold increased proteome coverage), although care was needed to ensure a comparably high stringency of protein identification. Expression proteomics has an exciting potential to elucidate the inner workings of murine enamel epithelial cells, leading to an improved understanding of enamel in health and disease.     

大鼠釉原蛋白抗血清的制备及其特异性研究

目的：制备抗大鼠釉原蛋白的抗血清，并研究其在组织学上的特异性。方法：采用弗氏完全／不完全佐剂，经皮下多点注射免疫；将所得抗血清用ＤＥ－３２纤维素纯化其ＩｇＧ抗体；用微量免疫电泳法测试抗体与釉质基质蛋白之间的免疫反应；用免疫组织化学法测试抗体与大鼠切牙及人牙胚中的组织特异反应。结果：所制备的抗血清与釉原蛋白三种组份Ａ１、Ａ２、Ａ３之间都发生沉淀反应，但沉淀弧形及位置不同，不与釉蛋白Ｅ１发生反应。抗角蛋白单克隆抗体与成釉细胞发生强阳性反应，但不与釉质基质发生反应。抗釉原蛋白抗体与成釉细胞发生阳性反应，但在童氏突与釉质浅层区反应最强，不与牙本质细胞发生交叉反应。结论：抗大鼠釉原蛋白抗体能特异地与成釉细胞外基质———釉原蛋白发生免疫反应。     

Expression of odontogenic ameloblast-associated protein, amelotin, ameloblastin, and amelogenin in odontogenic tumors: immunohistochemical analysis and pathogenetic considerations

Screening for expression of amelogenesis-related proteins represents a powerful molecular approach to characterize odontogenic tumors and investigate their pathogenesis. In this study, we have examined the presence and distribution of odontogenic ameloblast-associated protein (ODAM), amelotin (AMTN), ameloblastin (AMBN), and amelogenin (AMEL) by immunohistochemistry in samples of adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), developing odontoma, ameloblastoma, calcifying cystic odontogenic tumor (CCOT), ameloblastic fibroma (AF), myxoma, odontogenic fibroma (OF), and reduced enamel epithelia (REE). Positive results were obtained in those tumors with epithelial component, except for AF, OF, and ameloblastoma. ODAM was found around mineralized structures (dystrophic calcifications) and CEOT's amyloid, whereas AMTN stained the eosinophilic material of AOTs. The CCOT transitory cells to ghost cells were strongly positive with all proteins except AMEL, and the REE as well as odontomas showed immunoexpression for ODAM, AMTN, AMBN, and AMEL similar to those found in normal rat tooth germs. Based on these results, some histopathogenetic theories were formulated.