釉原蛋白、釉蛋白及成釉蛋白在小鼠牙胚组织发育过程中的表达

目的:比较小鼠釉原蛋白(amelogenin,AMELX)、成釉蛋白(ameloblastin,AMBN)和釉蛋白(enamelin,ENAM)在牙胚组织发育过程中表达的差异,从而揭示其不同的功能。方法:选择牙胚发育期分别位于钟状期、钟状晚期、釉质成熟期的昆明小鼠头颅切片,使用抗鼠AMELX、AMBN和ENAM多克隆抗体(由美国德州大学健康科学中心Dr.Sim-mer赠送),行免疫组织化学EnVision二步法染色,观察三种蛋白在处于三个不同发育阶段小鼠头颅切片中的表达情况。结果:免疫组化结果显示,三种蛋白在三种小鼠牙胚的成釉细胞、釉质、牙本质中的表达存在时空顺序差异,随着牙胚发育和釉质成熟,AMELX呈递减表达,而AMBN和ENAM则较恒定表达。AMELX在周围发育中的编织状骨组织中也有微弱表达。结论:AMELX、AMBN和ENAM为主要的釉质基质蛋白,在牙胚发育的不同阶段有不同的调节矿化的作用,AMELX在釉质矿化早期促进晶体生长,而其在骨中的表达则进一步表明其在骨修复中可能的作用;AMBN促进釉质基质矿化,并维持晶体形状、大小;ENAM促进和维持晶体生长,并与再矿化有关。     

人牙胚体外器官培养模型的建立

目的 :建立人牙胚体外器官培养模型 .方法 :分泌前期人牙胚采用Trowell型支架培养法 ,RPMI 16 4 0培养基(含 2 0 0 g·L-1FBS ,2 0 0mg·L-1谷氨酰胺 ,2 0 0mg·L-1L 抗坏血酸 ,1× 10 -7mol·L-1维甲酸 ,10 0kU·L-1青霉素 ,10 0kU·L-1链霉素 ) ,在 37℃ ,5 0mL·L-1CO2 +95 0mL·L-1空气条件下进行培养 ,每 4 8h更换一次培养基 .体外培养 .2 ,4 ,6和 8d随机抽取牙胚进行组织学观察 .结果 :培养的牙胚组织结构关系与对照组一致 ,牙尖部形态进一步突出 ,细胞形态典型 .在 8d时 ,牙尖之间部分造釉器细胞和成牙本质细胞开始出现坏死 .结论 :为体外研究人类牙齿正常发育及各种因素对牙齿发育的影响提供了一个实用的模型     

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.     

Dental and Cranial Pathologies in Mice Lacking the Cl−/H+‐Exchanger ClC‐7

ClC‐7 is a 2Cl^?/1H⁺‐exchanger expressed at late endosomes and lysosomes, as well as the ruffled border of osteoclasts. ClC‐7 deficiencies in mice and humans lead to impaired osteoclast function and therefore osteopetrosis. Failure of tooth eruption is also apparent in ClC‐7 mutant animals, and this has been attributed to the osteoclast dysfunction and the subsequent defect in alveolar bone resorptive activity surrounding tooth roots. Ameloblasts also express ClC‐7, and this study aims to determine the significance of ClC‐7 in enamel formation by examining the dentitions of ClC‐7 mutant mice. Micro‐CT analysis revealed that the molar teeth of 3‐week old ClC‐7 mutant mice had no roots, and the incisors were smaller than their age‐matched controls. Despite these notable developmental differences, the enamel and dentin densities of the mutant mice were comparable to those of the wild‐type littermates. Scanning electron microscopy showed normal enamel crystallite and prismatic organization in the ClC‐7 mutant mice, although the enamel was thinner (hypoplastic) than in controls. These results suggested that ClC‐7 was not critical to enamel and dentin formation, and the observed tooth defects may be related more to a resulting alveolar bone phenotype. Micro‐CT analysis also revealed abnormal features in the calvarial bones of the mutant mice. The cranial sutures in ClC‐7 mutant mice remained open compared to the closed sutures seen in the control mice at 3 weeks. These data demonstrate that ClC‐7 deficiency impacts the development of the dentition and calvaria, but does not significantly disrupt amelogenesis. Anat Rec, 298:1502–1508, 2015. © 2015 Wiley Periodicals, Inc.     

Biophysical characterization of synthetic amelogenin C-terminal peptides

Amelogenin plays a key role in the formation of the highly mineralized structure of tooth enamel. During the secretory stage, amelogenin is cleaved gradually by a protease, matrix metalloproteinase-20 (MMP-20), releasing hydrophilic C-terminal peptides. In this study, the biophysical properties of synthetic C-terminal peptides (of 28, 17, and 11 residues), mimicking native peptides, were explored in vitro. A sudden decrease was observed in the zeta (ζ)-potential upon the addition of calcium or phosphates, which was also accompanied by an increased aggregation propensity of the peptides. Under most of the experimental conditions, the particle size increased at a pH 2-3 units higher than the isoelectric point (pI) of the peptides, while the peptides existed as smaller particles (<2 nm) near their pI values and in the acidic range. They showed poor affinity for calcium and phosphates, comparable to full-length amelogenin and variants. The secondary structure determination showed that the 11-amino-acid peptide contained defined secondary structure comprising beta-sheets and turns. Atomic force microscopy analysis revealed the presence of thin, disk-like nanostructures of 54.4 nm diameter for the 28-amino-acid peptide and 54.9 nm diameter for the 11-amino acid peptide, whereas no definite structures were observed for the 17-amino-acid peptide. It is concluded that the amelogenin C-terminal peptides are capable of interacting with calcium and phosphate ions, of self-assembly into nanostructures, and may have some secondary structure, and hence may have some role in enamel synthesis.     

人胚成釉细胞体外培养的实验研究

目的 :探索成釉细胞离体培养方法 ,建立成釉细胞体外模型。方法 :运用组织细胞培养技术分离人胚成釉细胞进行原代培养 ,倒置显微镜下观察培养细胞生长特点 ,免疫组织化学染色检测培养细胞釉原蛋白。结果 :培养细胞成片生长 ,细胞形态结构清楚。免疫组化染色 ,多数细胞抗釉原蛋白抗体染色阳性。结论 :用消化培养法 ,胶原做基质饲养层 ,含 5 %小牛血清的HAMF -12作培养基并加入EGF、INS、HC ,可用于人成釉细胞的体外培养     

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.     

Distribution of Amelotin in Mouse Tooth Development

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT‐PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.