Distribution pattern of cholinesterase enzymes in human tooth germs

The two distinct molecular forms of cholinesterase (ChE) are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Our previous studies have reported that ChE is involved in tooth development. However, further experiments are needed to understand the precise action of ChE in tooth development. This study aimed to localise types of ChE in human tooth germs, and identify their distribution pattern.ChE were localised in frozen sections of jaws which were prepared from dead fetuses, neonates and stillborns who were free from visible abnormalities by Karnovsky and Root method.AChE was identified in the inner and outer enamel epithelia including the cervical loop region, stratum intermedium and preameloblasts of tooth germs at bell stage. Secretory ameloblasts were free from staining. The bud and cap stages of permanent tooth germs showed AChE activity on the lingual aspect and top surface of the epithelial ingrowths, respectively. BuChE activity was localised in the degenerating dental lamina.Our study reported the first evidence of localisation of ChE in human tooth development and identified the possible molecular form of ChE in tooth germs as AChE. Also, our results have provided strong evidence to speculate the action of AChE is on the cells of enamel organ during tooth development.     

Ultrastructure and composition of basement membrane separating mature ameloblasts from enamel

At a late stage of amelogenesis, a basement-membrane-like (BML) structure appears between mature ameloblasts and the enamel surface. Although this BML structure is known to contain certain basement membrane components, its detailed nature and role were not well defined. As such, this study examined the BML structure using high-resolution electron microscopy combined with immunohistochemical staining. Mandibular rat incisors were processed for the preparation of Epon sections for ultrastructural observations, and frozen sections were used for immunostaining laminin, heparan sulphate proteoglycan (HSPG) and type IV collagen. The BML structure was characterized by the presence of abundant ribbon-like 'double tracks', 4.5-5.0 nm wide; the form known to be taken by HSPG in basal laminae. The main ultrastructural component of basal laminae, known as 'cords', was replaced by fine filaments of type IV collagen. Immunohistochemical staining of the BML structure showed an intense reaction for HSPG, moderate staining for type IV collagen and negligible staining for laminin. These observations indicate that this structure is an atypical basement membrane in which the cord network is replaced by type IV collagen filaments. However, the BML structure was found to be unusually rich in HSPG, similar to kidney glomerular basement membrane. It is likely that this specialized basement membrane mediates firm attachment of mature ameloblasts to the enamel surface, and filters the influx and efflux of materials to and from enamel during maturation.     

Differential expression of LAR tyrosine phosphatase in the rat developing molar tooth germ

OBJECTIVE: This study examined the molecules implicated in the cytodifferentiation of dental hard tissue cells. METHODS: Rat pups at postnatal days 4, 7 and 10 were used. Differential display-polymerase chain reaction (DD-PCR), Western blot and immunofluorescent localisation were performed to search differentially expressed genes in tooth development. RESULTS: Leukocyte-common antigen-related tyrosine phosphatase (lar-tp) was differentially detected between the rat maxillary 2nd and 3rd molar germs on postnatal day 10, which were at the dental hard tissue formation and cap/early bell developmental stages, respectively. Both the mRNA and protein expression levels of lar-tp were higher in the 3rd molar germs than in the 2nd. In addition, the levels in the 2nd molar germs at postnatal days 4, 7 and 10, which corresponded to the early/late bell, crown and root stages, respectively, decreased in a time dependent manner. The immunoreactivity against intracellular P-subunit of lar-tp was detected in the ameloblasts and odontoblasts as well as in the undifferentiated inner enamel epithelia and dental papilla cells. However, strong immunoreactivity against extracellular E-subunit was observed only in the undifferentiated inner enamel epithelia and dental papilla cells in the 3rd molar germs and in the stratum intermedium in the 2nd molar germs. CONCLUSION: This is the first identification of lar-tp in the molar tooth development and suggests that this molecule may be involved in the cytodifferentiation of dental hard tissue cells.     

Formation of tight junctions in differentiating and secretory ameloblasts of rat molar tooth germs

Forty newborn rats were perfused with Karnovsky fixative and the tight junctions in differentiating and secretory ameloblasts were examined by conventional electron microscopy and freeze-fracture replications. Pre-ameloblasts were divided into types I, II and III based on morphology. Initial indications of tight-junction formation appeared as linear aggregations of particles in type II. The apparent tight junctional strands were observed in type III and in secretory ameloblasts. Though the junctional strands were numerous and long, no complete barrier between pre-ameloblasts at their distal ends was present. Complete zonular tight junctions were first observed at the distal ends of secretory ameloblasts; at this stage, proximal tight junctions incompletely sealed the paracellular spaces around the ameloblasts. Throughout their formative processes, the tight junctional strands were engaged in forming gap junctions. The structural features of tight junctions were considered to be closely associated with the cytodifferentiation of ameloblasts and permeability in the ameloblast layer.     

Notch1在小鼠下颌第一磨牙牙胚发育中的表达

目的探讨Notch1在小鼠下颌第一磨牙胚胎发育过程中的组织学分布。方法制作ICR小鼠下颌第一磨牙不同发育阶段的冰冻组织切片,对小鼠下颌第一磨牙自牙胚发育起始期至出生后2天不同发育阶段组织的Notch1分布情况进行免疫组织化学染色。结果 Notch1在小鼠下颌第一磨牙牙胚发育起始期和蕾状期牙板上皮上方或其包绕的口腔上皮中表达,在牙板上皮中没有表达。自帽状期至钟状期,Notch1在牙胚的中间层表达,而在内釉上皮中无表达。至牙釉质和牙本质分泌期,Notch1仍在颈环部位的中间层表达。此外,Notch1还在牙胚发育不同时期的间充质、牙乳头和早期牙髓中表达。结论 Notch1可能在小鼠下颌第一磨牙发育过程中的牙上皮特别是内釉上皮的细胞分化,以及牙囊和牙乳头细胞分化及分化完成后的牙髓干细胞的稳定性方面有重要作用。     

Notch1蛋白在小鼠下颌切牙发育中的表达

目的探讨Notch1在小鼠下颌切牙牙胚发育过程中的组织学分布。方法制作ICR小鼠下颌切牙不同发育阶段的冰冻组织切片,对小鼠下颌切牙牙胚自牙胚发育起始期至钟状晚期不同发育阶段组织Notch1的分布情况进行免疫组织化学染色。结果 Notch1在小鼠下颌切牙发育蕾状期牙胚的口腔侧上皮中表达,而在和间充质相邻的牙胚上皮中没有表达。从帽状期至钟状期,Notch1在牙胚的中间层表达,而在内釉上皮中没有表达。钟状期的唇侧颈环部位星网状层和部分牙上皮细胞也表达Notch1。此外,Notch1还在牙胚发育不同时期的间充质、牙乳头和早期牙髓中表达。结论 Notch1可能在小鼠下颌切牙发育过程中的牙上皮,特别是内釉上皮的细胞分化,以及牙囊和牙乳头细胞分化及分化完成后的牙髓干细胞的稳定性方面有重要作用。     

骨涎蛋白、骨桥蛋白在小鼠骨组织和牙胚组织发育中的表达

目的:探讨骨涎蛋(bone sialoprotein,BSP)、骨桥蛋白(osteopontin,OPN)在小鼠骨组织和牙胚组织发育过程中的分布特点及两者在骨组织形成和牙矿化过程中的作用.方法:取E16d胎鼠及出生后第10、30天的BALB/c小鼠共9只,拉颈处死,分离解剖下颌骨及其他骨组织,新鲜配制4%多聚甲醛固定过夜,常规石蜡包埋,近、远中向5μm连续切片,采用免疫组化法检测E16d胎鼠不同骨组织及10d、30d小鼠牙胚组织中BSP、OPN的表达及分布情况.结果:BSP、OPN在牙胚及骨组织发育中的分布模式存在差异.BSP在已经矿化成熟的矿化组织基质中呈阳性表达,而OPN主要在矿化的前沿及矿化活性强的矿化组织中强阳性表达.结论:BSP、OPN参与骨组织的发育和牙胚组织的矿化成熟,并在小鼠牙及骨组织的形成和矿化中具有重要作用.     

BMP7在小型猪乳磨牙胚不同发育阶段的表达研究

目的：目的：研究与牙齿发育相关的骨形态发生蛋白7（bone morphogenetic proteins 7,BMP7）信号分子在小型猪乳磨牙胚中的特异性时空表达情况。方法：采用小型猪第三乳磨牙胚作为研究对象,获取不同孕期的小型猪胎猪胚胎的下颌骨,分别进行 HE 染色、免疫组织化学染色、放射性核素标记探针的原位杂交及实时定量 RT －PCR 检测 BMP7分子在小型猪乳磨牙不同发育时期的表达情况。结果：HE 染色显示小型猪第三乳磨牙在胚胎 E40、E50、E60分别处于帽状期、钟状期以及钟状晚期（分泌期）。免疫组织化学染色从蛋白水平显示,BMP7在 E40时在成釉器和间充质均有表达,成釉器表达相对强于间充质;在 E50时,BMP7仍然表达在牙胚成釉器和间充质,与 E40表达情况类似;在 E60时则主要集中表达在间充质特别是前成牙本质细胞和前成釉细胞。原位杂交与实时定量 RT －PCR 从 mRNA 水平也验证了其表达趋势与免疫组织化学基本一致。结论：BMP7信号分子在小型猪乳磨牙发育过程中呈特异性的时空表达,提示其与牙齿形态发生和细胞分化相关。     

小鼠牙胚发育过程中釉原蛋白和釉蛋白的表达特征

目的 观察釉原蛋白和釉蛋白在小鼠牙胚发育中的表达特征,进一步揭示釉原蛋白和釉蛋白的生物学特性.方法 分别制备出生后第1、3、7和14天小鼠下颌第一磨牙牙胚切片,采用免疫组织化学和反转录聚合酶链反应法检测釉原蛋白和釉蛋白的组织学定位和基因转录表达水平.结果 釉原蛋白表达于分泌期成釉细胞的胞质和釉质基质全层,在新生小鼠牙胚成牙本质细胞中有一过性表达;釉蛋白表达于分泌期釉质基质中,在成釉质细胞突边缘和釉质基质深层呈强阳性表达,釉原蛋白和釉蛋白在矿化成熟的釉质中均未见表达.釉原蛋白和釉蛋白在出生后第7天mRNA表达量的相对值分别为0.813±0.085和0.799±0.064,显著高于其他时间的表达水平(P＜0.05).结论 釉原蛋白和釉蛋白主要由分泌期成釉细胞合成并分泌到釉质基质中,其表达具有高度的时空特异性,提示它们在釉质形成和生物矿化中有重要的作用.     

釉基质蛋白衍生物对大鼠牙移动后复发和牙根修复的影响

目的:观察釉基质蛋白衍生物对大鼠正畸牙移动后早期复发和牙根吸收的影响.方法:选用20只10周龄雄性SD大鼠,实验组和对照组各10只,在左上第一磨牙施加100 g力,使其近中移动,加力14d后拆除装置.自拆除加力装置起,实验组局部注射釉基质蛋白衍生物,对照组不注射任何药物.分别于拆除装置后当天及第14天分别行Micro-CT活体扫描,分析牙根吸收陷窝以及牙移动距离的变化.采用SPSS19.0软件包对数据进行统计学分析.结果:拆除装置14 d后,实验组与对照组牙根吸收陷窝体积修复量分别为(0.0295±0.0052) ×1 07 μm3、(0.0189±0.0086)×107 μm3;牙移动后复发距离及复发百分率分别为(0.089±0.005)mm、(64.76±3.63)％和(0.127±0.010)mm、(92.28±1.90)％.统计学分析表明,拆除装置14 d后,牙根吸收陷窝体积修复量、牙移动后复发距离及复发百分率均有显著差异(P＜0.05).结论:一定浓度的釉基质蛋白衍生物可在一定程度上加强大鼠正畸移动后牙根吸收后修复效应,减弱牙移动后早期复发.     

Bilateral asymmetry of tooth formation is elevated in children with simple hypodontia

Objective

Tooth formation normally progresses symmetrically between sides; the goal in this study was to test the clinical impression that left–right asymmetry in tooth formation is elevated in children with simple hypodontia.

Materials and methods

Data from panoramic X-rays of American white children (5–14 years of age) with simple hypodontia (n = 158) were compared to a comparable group from the same practises with all teeth present (n = 206). Children with hypodontia were otherwise phenotypically normal, with no cleft or recognized syndrome. Crown–root formation of each tooth (ignoring third molars) was scored using an 11-grade scheme. Analysis relied on chi-square goodness-of-fit tests and odds ratios.

Results

Hypodontia typically occurs unilaterally; it is more common in girls than boys; and it most frequently affects second premolars (omitting third molars). No evidence of a side preference was found as regards absence of the tooth or tooth formation. Tooth formation was decidedly more frequently asymmetric in those with hypodontia, though again the distribution by side was random in the sample. Summed over all tooth types, asymmetric formation occurred in 18.6% of cases with hypodontia compared to 11.9% in controls, and this is significant by chi-square (P = 0.03), with an odds ratio of 1.43 (CL: 1.02, 2.04). All tooth types exhibit elevated developmental asymmetry in the hypodontic sample despite only one or a few teeth being agenic.

Conclusions

Increased asymmetry suggests a breakdown in the rigour of developmental timing in cases with simple hypodontia. In concert with increased frequencies of other growth issues in such cases, such as side differences in size and morphology, hypodontia is best viewed as a symptom of an anatomically broad relaxation of developmental canalization between homologous structures, not an isolated dental feature.     

Graded changes in enamel component volumes resulted from a short tooth bleaching procedure

AimTo test the hypothesis that changes in enamel component volumes (mineral, organic, and water volumes, and permeability) are graded from outer to inner enamel after a short bleaching procedure.Materials and methodsExtracted unerupted human third molars had half of their crowns bleached (single bleaching session, 3 × 15 min), and tooth shade changes in bleached parts were analyzed with a spectrophotometer. Ground sections were prepared, component volumes and permeability were quantified at histological points located at varying distances from the enamel surface (n = 10 points/location), representing conditions before and after bleaching.ResultsTooth shade changes were significant (p < 0.001; 95% CI = −1/−8; power = 99%), and most of the enamel layer was unaffected after bleaching, except at the outer layers. Multiple analysis of covariances revealed that most of the variance of the change in enamel composition after bleaching was explained by the combination of the set of types of component volume (in decreasing order of relevance: mineral loss, organic gain, water gain, and decrease in permeability) with the set of distances from the enamel surface (graded from the enamel surface inward) (canonical R² = 0.97; p < 0.0001; power > 99%).ConclusionsChanges in enamel composition after a short bleaching procedure followed a gradient within component volumes (mineral loss > organic gain > water gain > decrease in permeability) and decreased from the enamel surface inward.     

The dentin sialoprotein (DSP) expression in rat tooth germs following fluoride treatment: an immunohistochemical study

Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications. OBJECTIVE: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride. DESIGN AND METHODS: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry. RESULTS: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (F(L)) but not in the group supplemented with the high fluoride concentration (F(H)). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong. CONCLUSIONS: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs.     

Localization of two distinct acid phosphatases in secretory ameloblasts of rat molar tooth germs

Acid phosphatases were examined histochemically at the light- and electron-microscopic levels using para-nitrophenyl phosphate (pNPP) and beta-glycerophosphate (beta-GP) as substrates. By light microscopy, there was intense activity with pNPP in supranuclear and distal regions of the secretory ameloblast, and moderate or slight activity respectively in those regions with beta-GP. These enzyme activities were less at the late secretory stage of amelogenesis and disappeared at the transitional stage. By electron microscopy, acid phosphatase activity was seen in the trans side cisternae of the Golgi apparatus, in lysosome-like granules, and in small vesicles in the Tomes' processes. The activity with pNPP but not beta-GP was also localized at the plasma membrane (proximal, lateral and distal surface). Activity with beta-GP was completely inhibited by 1 mM sodium tartrate and by 1 mM NaF; activity with pNPP was inhibited by 1 mM NaF and 10 mM sodium tartrate, but not by 1 mM sodium tartrate. Thus there are at least two different acid phosphatases, one tartrate-sensitive and the other 1 mM tartrate-resistant, in the secretory ameloblast; the tartrate-resistant enzyme is plasma-membrane bound.     

Histology, innervation and radiographic appearance of fetal rat tooth germs developing in oculo

Abstract – Rat molar and incisor tooth germs from gestational days 18–21 were homologously grafted to the anterior eye chamber of adult recipients. The fetal tooth transplants survived and grew considerably in the eye, attached to and vascularized from the anterior surface of the host iris. Grafts were examined histologically and compared with in situ controls after up to 6 months in oculo. Radiographic examinations of the grafts showed a normal rate of mineralization and a distinct enamel/dentin border after intraocular development. The size of the tooth grafts was somewhat smaller than in situ but the grafted teeth attained a form much resembling in situ incisors and molars. Light microscopy revealed a regular dentin and predentin, a distinct dentin/enamel border and a well innervated and vascularized pulp. No degenerative changes in dentin or enamel layers could be found in long-term grafts (6 months). Thus, intraocular tooth grafts in rats develop many histologic and anatomic features typical of normal teeth, emphasizing the importance of intrinsic regulatory mechanisms in tooth development.     

Pronamel and tooth mousse: an initial assessment of erosion prevention in vitro

OBJECTIVES: The aim of this study was to examine whether a single topical application of proenamel or tooth mousse would prevent enamel erosion METHODS: Enamel samples were treated with either proenamel or tooth mousse applied for 15 min. The control group was placed in distilled water for 15 min. All specimens were then exposed to an erosive challenge of 0.2% citric acid for 1h. Enamel loss was determined using surfometry. RESULTS: The mean amount of enamel removed in the control group was 5.02 microm (S.D. 1.16). The mean enamel loss in the proenamel group was 2.60 microm (S.D. 0.90) and the mean enamel loss in the tooth mousse group was 3.28 microm (S.D. 1.22). The results for the proenamel group were statistically significantly different from the control group at the p<0.01 level and the results for the tooth mousse group were statistically significantly different from the control group at the p<0.05 level. CONCLUSIONS: Tooth mousse and proenamel may offer a degree of protection from erosion of permanent enamel.     

Expression of planar cell polarity genes during mouse tooth development

ObjectivePlanar cell polarity (PCP) refers to the cell polarity across the tissue plane and controls various cell behaviors and structures. Although the expression of several PCP signaling components has been detected in tooth germs, knowledge of the gene expression patterns of these PCP components during tooth development remains incomplete. The aim of this study is to characterize the temporal and spatial changes in PCP gene expression during tooth development.DesignExpression of Celsr1 and 2, Fzd3 and 6, Vangl1 and 2, and Dvl1-3 genes was analyzed in mouse molar germs from the bud to the bell stage using in situ hybridization.ResultsAt the bud stage, all target genes were expressed in all areas of the tooth bud. In the enamel organ at the cap stage, expression of Fzd3 was suppressed in the enamel knot, whereas Fzd6 was strongly expressed there. Expression of Vangl2 was strongly expressed in the inner dental epithelium from the cap stage onwards. In the inner dental epithelium, strong expression of Fzd3, Dvl2 and Vangl2 was noted at the early bell stage, and of Celsr1, Fzd3, Fzd6, Vangl2 and Dvl2 at the bell stage. Furthermore, differentiated odontoblasts strongly expressed Celsr1, Vangl2, and Dvl2.ConclusionThe gene expression patterns delineated in this study improve our understanding of the role(s) of PCP components during tooth development.     

c-jun原癌基因在鼠牙胚中的表达

目的 通过观察ｃ－ｊｕｎ原癌基因在鼠牙胚中的表达,探讨成牙本质细胞分化的转录调节。方法 利用免疫组化方法检测ｃ－ｊｕｎ原癌基因在鼠牙胚中的表达。结果 ｃ－ｊｕｎ原癌基因在小鼠牙胚的蕾状期、帽状期呈阴性表达,在钟状期牙胚的成牙本质细胞中呈阳性表达。结论 ｃ－ｊｕｎ可能参与了成牙本质细胞分化的转录调节,并且可能成为牙本质细胞的标志分子之一。     

The effect of tooth bleaching on the bond strength of an experimental primer to enamel

This study evaluated the effect of tooth bleaching on the microtensile bond strength (μTBS) of an experimental primer to enamel. Materials used were an experimental tooth manicure system (Shofu) composed of primer and light-cured flowable resin composite. Flattened enamel surfaces of bovine teeth were bleached with Nite White Excel (Discus Dental) or Hi-Lite (Shofu), with nonbleached teeth used as a control group. Each bleaching group was subdivided into three bonding modes. These were group A, application of primer for 3 s, followed by 5 s of air blowing; group B, application of primer for 10 s, followed by 5 s of air blowing; and group C, application of 20% phosphoric acid for 10 s, spraying with water for 5 s, and then air blowing for 5 s. The flowable resin paste was placed and polymerized after each enamel surface treatment. Using a low-speed diamond saw, the specimens were sectioned into beam-shaped samples with a cross-sectional area of approximately 1 mm² at the bonded interface. The samples were subjected to the μTBS test with a 1.0 mm/min crosshead speed. Data were statistically analyzed using two-way analysis of variance (ANOVA) followed by the Bonferroni/Dunn post-hoc test. The two-way ANOVA revealed significant differences in the effects of the bleaching systems and bonding mode, and significant differences were also found for the interaction between them (P < 0.01). There were no statistically significant differences in μTBS values among the specimens in groups A and B regardless of bleaching or nonbleaching (P > 0.05). In contrast, the μTBS value of group C without bleaching was significantly higher than that of all other experimental groups (P < 0.01).