Odontoblast processes in human dentin revealed by fluorescence labeling and transmission electron microscopy

In the present undertaking, the distribution of odontoblast processes in human dentin was determined through the DiI carbocyanine dye fluorescent staining of the cell membrane, while F-actin was identified by rhodamine-phalloidin. Confocal laser scanning microscopy revealed intense labeling for both agents in inner dentin, while transmission electron microscopy (TEM) identified dentinal tubules including odontoblast processes in this area, each process being surrounded by a cell membrane and containing an abundance of filamentous structures. Electron-dense "lamina limitans" lined the dentinal tubules. Individual cell processes became narrower toward the middle area, and their overall numbers decreased as well under TEM. Labeling for F-actin was absent in both middle and outer dentin, while faint labeling for DiI was visible along the dentinal tubules as far as the dentino-enamel junction (DEJ), where it was also recognized within the tubules themselves. Under TEM, the dentinal tubules lined with electron-dense structures were, in fact, empty in the middle and outer dentin. Immediately below the DEJ, however, the tubules manifested dense concentrations of fine granular material. Our study, therefore, appears to suggest that odontoblast processes do not extend beyond the inner dentin of fully erupted human premolars.     

Oxalate desensitising treatment of dentinal surface

It is well known that a typical painful feeling is caused by impact of different agents and by thermodynamic conditions upon the dentine layer of the tooth. Therefore the action by artificial solutions should be tested to study how the induced modifications might inhibit the pain. The aim of the present study is to evaluate by scanning electron microscopy (SEM) the morphology of dentine surface after different chemical treatments. Oxalate solutions are able to produce a layer of large crystals, while acid solutions remove the smear layer and open the dentinal tubules.     

Electron-microscope study of the dentine-enamel junction of kangaroo (Macropus giganteus) teeth using selected-area argon-ion-beam thinning

Summary Transmission electron microscopy of selected-area argon-ion-beam thinned kangaroo (Macropus giganteus) enamel revealed a complex ultrastructure in the region of the dentine-enamel junction (DEJ). Characteristic features were multiple branching of dentinal tubules, rejoining of enamel tubules, elongated defects, extended protrusions of dentine into enamel, two types (A and B) of hypomineralized enamel and a continuity between dentinal and enamel tubules. In the intertubular regions of the DEJ a complex intermingling of finer enamel and dentine crystals, similar to that found in human enamel, was observed. The varicosities observed in the light microscope were a combined optical effect caused by the hypomineralized (type A) enamel and the branching and rejoining of the enamel tubules.     

Visualization of initial bacterial colonization on dentine and enamel in situ

Bacterial colonization of dentine is of high relevance in cariology, endodontology and periodontology. The aim of the present in situ study was to establish recent methods for visualization and quantification of initial bacterial adherence to dentine in comparison to enamel. For this purpose, bovine enamel and dentine slabs were fixed on buccal sites of individual upper jaw splints worn by 6 subjects for 30 min, 120 min and 360 min, respectively. Adherent bacteria on the slabs were visualized and quantified with DAPI-staining (4′,6-diamidino-2-phenylindole) and fluorescence in situ hybridization (FISH) of streptococci and eubacteria using the CLSM (confocal laser scanning microscopy) as well as an epifluorescence microscope. In addition, the number of colony forming units was quantified after desorption. Representative samples were processed for SEM (scanning electron microscopy) and TEM (transmission electron microscopy). All methods clearly indicated that a significantly higher number of bacteria adhered to dentine than to enamel. Furthermore, the amount of bacteria on the dentine increased with increasing oral exposure time, but remained rather constant on the enamel. The CLSM allowed visualization of bacteria in the dentinal tubules. Bacteria were found preferentially at the openings of the dentine tubules, but were distributed randomly on the enamel.In conclusion, the adopted methods are suitable for visualization and quantification of bacterial adhesion to dentine. Even the initial bacterial colonization of dentine is much more pronounced than bacterial adherence to the enamel.     

Structure of ivory

Profiles with all orientations have been used to visualize the 3D structure of ivory from tusks of elephant, mammoth, walrus, hippopotamus, pig (bush, boar, and warthog), sperm whale, killer whale, and narwhal. Polished, forming, fractured, aged, and stained surfaces were prepared for microscopy using epi-illumination. Tusks have a minor peripheral component, the cementum, a soft derivative of the enamel layer, and a main core of dentine=ivory. The dentine is composed of a matrix of particles 5-20 microm in diameter in a ground substance containing dentinal tubules about 5 microm in diameter with a center to center spacing of 10-20 microm. Dentinal tubules may be straight (most) or curly (pigs). The main findings relate to the way that dentinal tubules align in sheets to form microlaminae in the length of the tusk. Microlaminae are sheets of laterally aligned dentinal tubules. They are axial but may be radial (most), angled to the forming face (pigs and hippopotamus canines), or radial but helical (narwhals). Within the microlaminae the dentinal tubules may be radial, angled to the axis (whales, walrus, and pigs), or may change their orientation from one microlamina to the next in helicoids (canines of hippopotamuses, incisors of proboscidea). In the nonbanded, featureless ivories from the hippopotamus incisors, the dentinal tubules form radial microlamina from which the arrangements in other ivories can be derived. In the canines of hippopotamuses and incisors of proboscidea, the dentinal tubule orientation changes incrementally from one microlamina to the next in a helicoid, a stack of dentinal tubules that change their orientation by 180 degrees anticlockwise. Dentinal tubules having different orientations are laid down concurrently, not layer by layer as in most examples of helicoidal architecture (e.g., insect cuticle). In proboscidean ivory, the microlaminae are radial, normal to the banding of growth layers marking the plane of deposition. They form radial segments with each 180 degrees turn in the orientation of their constituent dentinal tubules. Below the cementum they are almost complete 180 degrees helicoids, but nearer to the core they become narrower with the loss of radially oriented dentinal tubules. These truncated helicoidal patterns appear in longitudinal profile as VVVV feather patterns rather than intersection intersection intersection intersection, each V or intersection being the side view of a partial or complete helicoid. The Schreger pattern in proboscidean ivory consists of these helicoids divided tangentially into columns in the length of the tusk. Narwhals have the most abundant matrix particles with their radial/helical dentinal tubules having a twist opposite to that in the cementum.     

A method for studying histochemical reactions in intact human dentine with a polarizing incident light microscope

Localization and distribution of non-specific esterases has been studied in intact human dentine, by reflected light microscopy. The method of specimen preparation described here permits the visualization of optical sections in depth within the specimen at high optical resolution. Non-specific esterase was found deposited as discrete bands across the tubules. or as droplets, or as a diffuse microsomal variety in the dentinal tubules and in the interglobular spaces. It was possible to distinguish the droplet variety from the microsomal variety, of esterase within the same tubule, by means of a novel optical method using antiflex and differential interference contrast systems of reflected light microscopy. It was found that the coefficient of reflection of dentine diminished gradually from the enamel to the pre-dentine and was inversely related to the scattering of light in dentine. This scattering plays an important role in the formation of the image with reflected light microscopy. The reflected light microscope offers an economically attractive alternative or a supplementary mode of microscopy to the confocal scanning microscopes for studying intact dentine at varying depths.     

Enamel and dentine ultrastructure in the early Jurassic therian Kuehneotherium

Three fragmentary molars of Kuehneotherium from Wales were progressively abraded by grinding and studied by scanning electron microscopy. A fourth molar was reduced to small blocks, thin sections of which were examined by transmission electron microscopy. Enamel showed a 'preprismatic' pattern, consisting of columns of crystals disposed as pinnae but not separated by an interprismatic substance. No enamel tubules were observed. Incremental lines were present. Inner dentine was permeated by numerous tubules not surrounded by peritubular dentine but a peripheral dentine layer adjacent to the enamel-dentine junction was atubular. Calcified deposits were occasionally observed in the lumen of dentine tubules, some of which were interpreted here as dentinal sclerosis consecutive to masticatory wear.     

Cytodifferentiation and degeneration of odontoclasts in physiologic root resorption of kitten deciduous teeth

To investigate the cytodifferentiation and degeneration of odontoclasts in physiologic root resorption, we studied deciduous incisors undergoing resorption in 6-month-old kittens by electron microscopy of ultrathin sections. The endogenous peroxidase activity within the cells was also examined by incubating the tissue slices in diaminobenzidine-H2O2 medium. The resorbing tissues, consisting of multinucleated giant cells, macrophages, granular leukocytes, fibroblasts and many blood vessels, were observed at the resorbing surface of the root dentine. Macrophages and granular leukocytes exhibited endogenous peroxidase activity, but mononuclear and multinucleated preodontoclasts and multinucleated odontoclasts did not. These preodontoclasts contained abundant mitochondria, a moderate amount of rough endoplasmic reticulum, stacks of Golgi membranes, lysosomes and numerous polyribosomes scattered throughout the cytoplasm. Many cellular processes extended from their cell surfaces by which the preodontoclasts appeared to fuse to one another during their multinucleation. Concomitant with the multinucleation process, the preodontoclasts developed numerous pale vacuoles throughout the cytoplasm. These vacuoles seemed to arise from some smooth endoplasmic reticula, perhaps representing Golgi-endoplasmic reticulum-lysosome, and the Golgi saccules. However, the preodontoclasts did not yet form a ruffled border and clear zones. When these preodontoclasts came into direct contact with resorbing dentine surfaces, they began to form the clear zones against dentine surfaces. Characteristically, numerous pale vacuoles were accumulated in the cytoplasm adjacent to the clear zone, then they penetrated into the cytoplasm of the clear zone, and with this, ruffles of the plasma membranes appeared. Through a further movement of more pale vacuoles towards the ruffled plasma membranes, the odontoclasts developed typical ruffled borders against the resorbing dentine surfaces. At this differential phase, little pale vacuoles appeared in the Golgi area, but the cisterns of the Golgi apparatus themselves reached their greatest extent during cellular differentiation. Fully differentiated odontoclasts frequently extended long broad cellular processes into the dentinal tubules exposed to the resorption lacunae. Although some odontoclastic processes penetrating the dentinal tubules contained vacuoles and lysosomal structures, most processes lacked any cytoplasmic organelles, and their cytoplasm resembled that of the clear zone. But these processes never exhibited ruffled-border-like structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

The microstructure of dentine in taxonomic and phylogenetic studies

Traditionally, physical anthropologists have focused their dental studies on features of teeth that can be observed with the unaided eye, measured with calipers, or observed with light microscopes. With the advent of the scanning electron microscope, there has been renewed interest in the use of the microstructure of enamel in phylogenetic reconstruction and taxonomic classification. The microstructure of dentine has received far less research attention than has enamel although several investigators have proposed that dentine has taxon specificity. We report the first results of a study in which we investigated the taxon specificity of dentine. In our study, we exposed the dentinal tubules on the mesial root surfaces of 12 Canis, 11 Papio, and 12 Homo mandibular first molars and compared tubule density and pattern among the three taxa. Based on these parameters, 91.43% of the teeth were correctly identified as being dog, baboon, or human, respectively. We conclude that dentine has taxon-specific structural characteristics, which can be used in anthropological investigations. To our knowledge, this is the first comparative study of the micro-structure of teeth utilizing statistical analysis.     

蜥臀目霸王龙及有鳞目沧龙牙齿组织的微细结构

本文是利用扫描电子显微镜对陆栖恐龙tvrannosaurid与海栖渐增mosasaurid”牙齿结构进行的比较解剖学研究。化石采自加拿大RedDeerhiverValley上白里统Horse－shoeCanyon组。通过研究地层中出现的生物化石,特别是动物牙齿的组织结构,可以了解动物为了适应生活环境而发生的进化过程,也可以推测它们的系统发育关系。tyrannosaund与mosas。id都拥有锥状的同形齿,牙齿侧向扁平,且略向后弯曲。研究结果确认了tyrannosaurid的牙齿由于薄层的无柱釉质bPrismaticenamel）向齿质的侵人而造成许多的凹凸构造,此锯齿状构造沿着牙齿的前后缘,由牙齿的顶端分布至基部。因此tyrannosaurid的牙齿呈现着锐利的切缘;在这些凹凸状切缘的沟与小窝的深部可观察到有机物的沉积。但是类似的锯齿状构造只能在齿冠呈钝圆状的mosasaurid牙齿的基部附近观察到。我们以扫描电子显微镜（SEM）检索,确认两爬行类的齿质皆是属于中间型的真性齿质（intermediatetypeorthodentine）;所谓orthodentine即是细管齿质（tubulardentine）。tyrannosaurid的真性齿质的齿质小管只在齿质一釉质相接处Uentino—enamaljunction）附近放散出规则性的分歧与末枝。但mosasaurid的真性齿质的齿质J。管,在齿质的中间层与表层中,呈现着由复杂     

Structure of comblike teeth of the ayu sweetfish,Plecoglossus altivelis (Teleostei: Isospondyli): I. Denticles and tooth attachment

The structure and tooth attachment of the comblike teeth and denticles of the ayu sweetfish, Plecoglossus altivelis, were examined by light and scanning electron microscopy. The denticle is composed of a spoonlike crown with a spine pointed anteriorly, a triangular plate in the cervical region, and a root that curves laterally and tapers off to a point. The root apex is fused with a long thin pedicle that turns abruptly anteriad toward the jaw bone. Planes of the spine, the spoonlike crown, the triangle plate and the root of the denticle are varied, and the denticle is twisted in the region of the triangle plane. The superficial layer of the dentine is homogeneously calcified and is considered to be enameloid, because some of the inner dentinal epithelial cells in the tooth germ are columnar and possess cellular processes at their apical ends. The dentine is fibrous and fine dentinal tubules are visible in dentine treated with sodium hydroxide and observed by scanning electron microscopy. The upper half of the root is surrounded by a dense layer of collagen fibers running parallel to the tooth axis, and the lower half is encompassed by interlaced collagen fibers. The lower part of the root is open on its lingual side. The pedicle is a long rod which is homogeneously calcified and enmeshed by interlaced collagen fibers, and it curves mediad as it nears the jaw bone. The pedicles are interposed between a layer of gelatinous connective tissue and the jaw bone and terminate on the periosteum. Comparative aspects of ayu tooth morphology are discussed. © 1993 Wiley-Liss, Inc.     

The presence of open dentinal tubules affects the biological properties of dental pulp cells <Emphasis Type="Italic">Ex Vivo</Emphasis>

To investigate the effects of open dentinal tubules on the morphological and functional characteristics of dental pulp cells. Morphological changes in human dental pulp cells that were seeded onto dentin discs with open dentinal tubules were investigated on days 1, 2, 4, and 10 of culture using scanning electron microscopy and fluorescence microscopy. Samples collected on days 1, 3, 6, 8, and 10 of culture were evaluated for cell proliferation rate and alkaline phosphatase activity. Cultured human dental pulp cells developed a columnar or polygonal morphology and monopolar cytoplasmic processes that extended into the dentinal tubules. The cells formed a multilayer and secreted an extracellular matrix onto the cell surface. Scanning electron microscopy and fluorescence microscopy revealed polarized organization of odontoblasts. Cells seeded onto dentin discs proliferated minimally but showed high levels of ALP activity. Dental pulp cells seeded onto treated dentin discs develop an odontoblastlike phenotype, which may be a potential alternative for use in experimental research on dentinogenesis.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Calcium oxalate smear layer: mineralogical and crystallographic study.

Different methods to reduce dentine hypersensitivity have been tested by means of oxalate treatments. Natural material (20 teeth) and commercial oxalates have been employed. As a result the dentinal tubules are filled by small crystals preventing dental disease. Permeability measurements have been performed in order to quantify results. The composition of the synthetic smear layer produced after oxalate treatment have been investigated by powder diffraction method suggesting calcium oxalate formation.     

Er,Cr:YSGG激光照射对牙周病牙根面的影响

目的:观察不同能量输出的Er,C r:YSGG激光照射对牙周病牙根面的影响,探讨Er,C r:YSGG激光用于牙周治疗的可行性和有效性。方法:选择因牙周病松动Ⅲo而拔除牙12颗,随机分3组,每组4颗。分别以1.5 W、2.0 W、2.5 W激光照射。扫描电镜(SEM)观察各组牙根表面和纵剖面。结果:低倍镜下各组根面改变无明显差异,表面干净,粗糙程度均匀,未见明显弹坑状痕迹;高倍镜下1.5 W激光照射组显示根面粗糙程度更均匀一致,无弹坑状改变,无熔融、炭化迹象,未见牙本质小管暴露。结论:Er,C r:YSGG激光能有效去除牙周病牙根面玷污层及病变牙骨质,根面无熔融、炭化等改变。1.5 W激光照射组优于2.0 W组和2.5 W组。     

Cryopreservation and image enhancement of juvenile and adult dentine mineral

The inorganic component of bone and related hard tissues is generally described as sheets of uniform needle- and plate-like crystals. However, cryofixation has become the method of choice for ultrastructural studies of bone mineral when ladder-like arrangements of filaments contained within deformable microspheres about 1µm in diameter are apparently the prime structural feature and are consistent with the optical image. The same methodology has now been applied to mature human dentine in caries-free juvenile and adult teeth. These were fixed, sliced, stained for mineral and examined optically or were snap frozen, fragmented under liquid nitrogen, freeze-substituted with methanol or acetone and embedded without thawing in Lowicryl K4M for electron microscopy. Others were processed by traditional transmission electron microscopy methods. To obtain maximum resolution, the electron micrographs were photographically printed as negatives and image-enhanced by digitisation using a Polaroid Sprint Scan 45 and laser printer. In both optical and cryopreparations of juvenile and adult dentine, mineral microspheres up to 1µm in diameter, were present in the dentinal tubules and peritubular dentine. Within these objects, the mineral was primarily in the form of sinuous electron dense filaments, 5nm thick, which had a characteristic periodicity. In these preparations needle-like and plate-like structures were rare. In contrast, after traditional transmission electron microscopy preparation although similar filamentous structures remained, the mineral more generally had the familiar form of needles measuring approximately 50nm in the long axis. The cryopreserved calcified filaments were apparently particularly densely distributed in the intertubular dentine where their parallel ladder-like arrays often formed highly orientated struts and stays. It was concluded that early dentine mineral has the form of filamentous microspheres and as in bone (and other calcifying tissues and cells) has no specific association with collagen. It was also concluded that these structures compact and deform with maturity into a sub-structural framework which may relate to powerful biomechanical forces transmitted through the tissue. Needle- or plate-like mineral is probably rare in vivo in dentine, only becoming commonplace after extensive chemical processing.     

Microscopy of the junctional region between human coronal primary and secondary dentine.

The juction between human primary dentine and regular and irregular secondary dentine was examined with a number of different light and electron microscopic techniques. In decalcified material, a narrow band along the innermost surface of the primary dentine stained intensely. The walls of the tubules within the band stained intensely, whereas the tubular walls within the bulk of the primary dentine were not stained. Generally, the walls of the tubules in both types of secondary dentine were also preferentially stained. Although not readily apparent in ground sections, observations of thin sections revealed a dramatic reduction in the number of tubules in regular secondary dentine. Generally, the radiodensity of the intertubular matrix was the same in primary and secondary dentine and the intensely stained band was not seen radiographically. The pulpal ends of the tubules in primary dentine were often occluded with a material having the same radiodensity as peritubular matrix. Both patent and occluded tubules were seen in irregular secondary dentine. Scanning electron microscopy of acid-etched specimens of secondary dentine revealed that some tubules had irregular walls of highly mineralized matrix which was less acid-soluble then the peritubular matrix of primary dentine.     

Use of Poly (Amidoamine) Dendrimer for Dentinal Tubule Occlusion: A Preliminary Study

The occlusion of dentinal tubules is an effective method to alleviate the symptoms caused by dentin hypersensitivity, a significant health problem in dentistry and daily life. The in situ mineralization within dentinal tubules is a promising treatment for dentin hypersensitivity as it induces the formation of mineral on the sensitive regions and occludes the dentinal tubules. This study was carried out to evaluate the in vitro effect of a whole generation poly(amidoamine) (PAMAM) dendrimer (G3.0) on dentinal tubule occlusion by inducing mineralization within dentinal tubules. Dentin discs were treated with PAMAM dendrimers using two methods, followed by the in vitro characterization using Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), Field emission scanning electron microscopy (FE-SEM) and Energy-Dispersive X-ray Spectroscopy (EDS). These results showed that G3.0 PAMAM dendrimers coated on dentin surface and infiltrated in dentinal tubules could induce hydroxyapatite formation and resulted in effective dentinal tubule occlusion. Moreover, crosslinked PAMAM dendrimers could induce the remineralization of demineralized dentin and thus had the potential in dentinal tubule occlusion. In this in vitro study, dentinal tubules occlusion could be achieved by using PAMAM dendrimers. This could lead to the development of a new therapeutic technique for the treatment of dentin hypersensitivity.     

Impact of high-energy laser irradiation on pulmonic fibroblast in human embryo

This impact was attained by medicinal laser application to alexandrite, dye, erbium and gallium arsenid in various conditions on cultures of pulmonic fibroblasts of human embryo. Obtained results were estimated by transmission and scanning microscopy. The highest cell destruction was observed in dye and alexandrite, being less expressed in gallium arsenid under laser irradiation. Impulsive action of erbium laser did not cause any substantional cell destruction. So, laser application to dye alexandrite and gallium arsenide for healing scar and contracture damages is completely justified for skin pathogeny.