Merkel-like cells in Malassez epithelium in the periodontal ligament of cats: an immunohistochemical, confocal-laser scanning and immuno electron-microscopic investigation.

The cellular heterogeneity of Malassez epithelium (ME) residing in the periodontal ligament has recently been reported, and the presence and coexistence of the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) in single cells in ME has been shown (1). However, the identity of these neuroendocrine cells has so far not been verified. This study was undertaken in order to elucidate the identity of the neuroendocrine cells in ME by means of transmission electron microscopy, confocal scanning microscopy and immunohistochemistry using antibodies to protein gene product (PGP) 9.5 and cytokeratin 20 (CK). Gingival tissue was included in the study as a positive control for identification of Merkel-like cells in oral epithelium. CK 20 immunopositive cells were present in both Malassez epithelium and in basal cell layers of gingival epithelium showing a distribution consistent with PGP 9.5 labelled cells in both epithelia. The results from PGP 9.5 immuno electron microscopy clearly evidenced the presence of single, intensely labelled cells and some nerve fibres invested between the Malassez epithelial cells. The conformity of the immunopositive cells in Malassez and gingival epithelium verified by double immunolabelling with PGP 9.5 and CK 20, indicates that the labelled neuroendocrine cells are identical in ME and in gingival epithelium. This demonstrates that Malassez epithelium not only exhibits neuroendocrine cells, but additionally that the neuroendocrine cells represent Merkel-like cells.

High-resolution electron microscopy of the crystallites of fossil enamels obtained from various geological ages.

To elucidate the stability of the central dark line (CDL) in biologically induced hydroxyapatite crystals, we examined the diagenetic changes on the microstructures of the crystallites during the course of fossilization. Using transmission electron microscopy, we investigated the enamel crystallites of fossil animals of various geological ages ranging from Pleistocene to Cretaceous. Electron micrographs indicated that the microstructures and lattice images of each crystallite in fossil enamels were well-preserved regardless of the thickness of the enamel layer, and the presence of CDLs in fossil enamel crystals was also confirmed. The results indicated that the microstructure of hydroxyapatite crystals containing lattice images of CDLs appear stable during long geological periods. In addition, we conclude that the existence of lattice images in apatite with CDLs may be an indicator for the assessment of the evolution of dental enamel from fossil remains.

Apoptosis in regressive deciduous tooth germs of Suncus murinus evaluated by the the TUNEL method and electron microscopy.

Apoptosis in regressive primary (deciduous) dental primordia was examined in the embryos of Suncus murinus, which is a monophyodont. The primary tooth germs of S. murinus are temporarily formed and disappear during the embryonic period before they are calcified. Most primary tooth germs reach the bell stage and degenerate by embryonic day 22 (E22). Light microscopy on haematoxylin-eosin-stained sections revealed that intensely labelled granular substances are frequently present in the epithelial portion (enamel organs) of the deciduous tooth germs during the period from E18 to E20. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling method, computer-assisted three-dimensional reconstructions, and electron microscopy confirmed that these variable-sized granular substances are similar to apoptotic cells or bodies. Apoptotic structures were mainly found in the primary tooth germ located on the buccal surface of the secondary (successional) tooth germ. These results lead to the conclusion that apoptosis is closely associated with the involution and disappearance of the deciduous tooth germ in S. murinus. A primary tooth germ was observed on the buccal side of all the corresponding successional tooth germs, although the buccal surface of the secondary tooth germ of third upper molar teeth developed only to epithelial thickening without mesenchymal condensation. The findings, therefore, suggest that apoptosis is responsible for disappearance of the primary dental primordia during tooth development in S. murinus.

Synthesis of carbapenam skeletons using a ruthenium-catalyzed cyclization.

Carbapenam is a very important skeleton of beta-lactam antibiotics, and it has a highly strained structure. When enynes 9 were treated with RuH(2)CO(PPh(3))(3) (10 mol %) in toluene upon heating, carbapenams 10 were obtained in good yields.

Neuroendocrine cells in Malassez epithelium and gingiva of the cat.

Malassez epithelium has been designated as epithelial cell rests, the biological significance of which is still under debate. This study was designed to analyze Malassez epithelium for the presence of neuroendocrine cells. Gingival tissue was included as a positive control. Using immunohistochemistry, confocal and light microscopy, Malassez epithelium and gingival epithelium from mature cats (n = 5) were examined for cells containing the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Both Malassez epithelium and the basal epithelial cell layers in gingival rete pegs regularly displayed cells immunoreactive to CGRP, SP, and VIP. The immunopositive cells were most frequently present in the epithelial cell clusters and strands of Malassez located in the cervical half of thc periodontal ligament. Double immunolabeling revealed cellular co-expression of CGRP or SP with VIP, and the neuropeptides were co-localized in the cellular compartments. Labeled cells in both epithelia were occasionally supported by immunoreactive nerve fibers. This study shows that cells immunoreactive to CGRP, SP, and VIP arc located within the cat Malassez epithelium. The localization of neuroendocrine cells verifies the diversity of this epithelium and confirms that Malassez epithelium is composed of different cell types, in common with epithelia from other locations. The presence of neuroendocrine cells in Malassez epithelium strongly suggests biological functions of this tissue, and the neuropeptide content may thus indicate endocrine functions of the cells.

Application of high resolution microfocus X-ray CT for the observation of human tooth.

The calcification degree of extracted human teeth was observed by using high resolution microfocus X-ray CT. As samples, upper and lower first premolars extracted from a 21-year-old female were used. The computed tomograms were produced by high resolution microfocus X-ray CT with a open vacuum X-ray source, rotating sample stage, and image sensor. The distinction between enamel and dentin was very clear, and the shape of the pulp cavity was also clearly identified. The secondary dentin was visible in the circumpulpal dentin. The color map displays showed the heterogeneity of the calcification degree not only in the dentin but also in the enamel. The enamel was divided into three layers according to the calcification degree. High resolution microfocus X-ray CT was very useful for the observation of the internal structure of human teeth without destroying the samples.

Visualization of calcium channels involved in transmitter release from neuronal growth cones.

Immunocytological localization of omega-agatoxin IVA (omega-aga IVA)-sensitive Ca2+ channels involved in glutamate release from growth cones of cultured rat dorsal root ganglion (DRG) neurons was studied with field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). The omega-aga IVA-sensitive Ca2+ channels were visualized by labeling with immuno-gold particles (30 nm). FE-SEM and TEM images showed that immuno-gold particles were present in the area of growth cones as well as somata, and generally absent on neurite stem and fibroblasts. TEM images of vertical ultra-thin sections showed that the immuno-gold particles were present on the surface of the plasma membrane. Since the gold particles indicate the immunological presence of omega-aga IVA-sensitive Ca2+ channels, the Ca2+ channels involved in transmitter release are present on growth cones before making synapse formation.

SEM and EDS analysis of calcospherites in human teeth.

The present study was designed to characterize the morphology and composition of calcospherites in the coronal and root predentin of human permanent teeth by scanning electron microscopy (SEM) and scanning electron microscopy energy dispersive spectroscopy (SEM-EDS). Human incisors, premolars, and molars were used. The calcospherites in the coronal predentin were globular and 10-20 microm in diameter. The calcospherites in the root predentin were smaller and their shape was different. Polygonal calcospherites and stellate calcospherites were observed in the intermediate region of the root predentin. Calcospherites were rarely present in the apical region of the root predentin. Calcified matrix fibers were observed in the apical region of the root predentin. The Ca/P molar ratio in crown calcospherites (1.63 +/- 0.27) differed significantly from that in root calcospherites (1.46 +/- 0.28). Sulfur was detected from the cervical region to the root region, but not in the horn region. Odontoblast activity and the local environment of the predentin are thought to determine the shape, size, and composition of calcospherites.

Degeneration of tooth germ in the developing dentition of the gray short-tailed opossum (Monodelphis domestica).

The aim of this study was to examine the developmental aspects of the dental lamina and the tooth germ of the marsupial opossum (Monodelphis domestica), and to clarify the dental formula of this animal. Specimens were 12-, 16-, and 18-d-old opossums. 3-D reconstructions were constructed from frontal serial sections. In these animals, the tooth germs of the deciduous maxillary and mandibular canine, deciduous third premolar and first molar, and the deciduous maxillary first incisor and second molar had a successional dental lamina and a replacement tooth germ. The tooth germ of the deciduous maxillary fourth incisor and the mandibular first incisor were reduced. The dental lamina was continuous in each jaw except for the deciduous maxillary first incisor. The first dentition (deciduous dentition) remained as the permanent dentition on the deciduous maxillary first incisor, and the deciduous maxillary and mandibular canine and first molar. The maxillary fourth incisor and the mandibular first incisor were the second dentition (successional dentition). Only the deciduous third premolars were replaced. These results showed monophyodonty caused by both deciduous and replacement tooth germ degeneration.

Innervation of the periodontal ligament in the alligatorid Caiman crocodilius.

The mode of development and structure of crocodilian teeth and periodontium parallels that of mammals, but the teeth are continuously replaced throughout the lifetime of those animals. In this report, the innervation and fibres of the crocodilian periodontal ligament were examined using histology, immunohistochemistry for S-100 protein and transmission electron microscopy. Crocodilian periodontal ligaments had the following characteristics: (1) horizontal fibres, which connect the alveolar bone to the root cementum and (2) longitudinal fibres, which ran parallel to the tooth axis, with nerves and blood vessels in the middle layer of the ligament. The apex of root and tooth germs were both embedded in thick circular fibres. S-100 protein was detected in neural elements including terminal portions which were densely distributed in the periodontal ligament and dental follicle. The S-100 positive neural elements formed a periodontal plexus. We found two types of nerve endings; free endings and simple encapsulated corpuscles as described in mammals. The presence of such nerve endings in caiman suggests that these teeth, in addition to having a biting function, may also act as highly sensitive sensory organs.

The function and structure of the marsupial enamel.

The aims of this study are to clarify the structure of tubular enamel and the function of enamel tubules on the marsupial of opossum (Monodelphis domestica). Almost all enamel prisms, surrounded by interprismatic enamel, ran obliquely from the dentinoenamel junction (DEJ), and bent near the enamel surface. The enamel tubules are distributed in both enamel prisms and the interprismatic enamel near the DEJ. From the middle to the surface of the enamel, one enamel tubule ran within a single enamel prism. Most of enamel tubules continued from the DEJ to near the enamel surface. It is suggested that each enamel tubule developed in relation to one ameloblast. The fibers of odontoblastic process penetrated the DEJ from the dentinal tubules into the enamel tubules, and some branched across the enamel prisms. The odontoblastic process may be actively cross into the ameloblastic layer and may be involved in the formation of enamel tubules. After in vivo injection of tetracycline, tetracycline labeling showed that the odontoblastic tubules continued to enamel tubules. And strontium was detected in enamel tubules from the DEJ to the enamel surface, as was the dentinal tubules. In conclusion, there was active transport by the odontoblast and it's process through the enamel tubules.

Helix structure of ribbon-like crystals in bovine enamel.

In the early mineralized enamel crystals, ribbon-like crystals appear near the ameloblasts. Some ribbon-like crystals showed helical or spiral structure within restricted environment during the preparation of embryonic bovine specimens for electron microscope. These specimens did not suffer from the cutting damages nor staining effects. The main cause of the helix structure is considered a result of the dehydration during preparation. The periodic structure must reflect the regularity of initial enamel crystals. If dehydration caused the ribbon-like crystal to induce the periodic helix, it is one possibility that the earliest enamel crystal is OCP which has been proposed as a precursor of HA. Because it is considered that OCP is more sensitive to dehydration and more symmetric structure than biological HA. The periodicity of the helical ribbon-like structure was about 25 to 55 nm long and could be compared to the periodicity of organic helices which had observed in an immature rat enamel.

Scanning electron microscopy and energy dispersive spectroscopy analysis of calciotraumatic lines in rat labial dentin after acute exposure to strontium chloride.

Rats were given strontium chloride (SrCl2) intraperitoneally at a dose of 500 mg/kg. The upper incisors were removed after injection of strontium. These incisors were studied by scanning electron microscopy-energy dispersive spectroscopy analysis (SEM-EDS) and light microscopy to examine the calciotraumatic lines of strontium in the rat incisor labial dentin. At 24 hours after injection of strontium, the calciotraumatic response was observed in the predentin using hematoxylin and eosin (H-E) staining. At 5 days, three layers of calciotraumatic lines were present in the labial dentin using an H-E staining and backscattered electron imaging in the SEM. The external layer consisted of unmineralized dentin, the intermediate layer of relatively unmineralized dentin, ane the internal layer of unmineralized dentin. By SEM-EDS analysis, strontium was detected in these layers. The elemental dot map showed that the external and internal unmineralized layers had a low calcium content. The magnesium concentration was higher in the internal unmineralized layer than the external unmineralized layer.

Evaluation of jawbone calcification in osteopetrotic (op/op) mice using computed tomography.

The study was conducted to evaluate the degree of calcification in the jawbones of 3 pairs of 4-week-old male op/op mice and littermates using computed tomography (CT) numbers. The mean CT numbers for op/op mice (n = 3) were 251.3 HU (range, 226 to 278) in the maxillary bone and 353.7 HU (range, 320 to 398) in the mandibular bone. The mean CT numbers for littermates were 336.7 HU (range, 324 to 350) and 596.6 HU (range, 580 to 604), respectively. The CT numbers of littermates were higher than in op/op mice for both maxillary and mandibular bones. These results indicated that the degree of bony calcification in littermates was higher than that in 4-week-old op/op mice in both the maxillary and mandibular bones. CT is able to demonstrate the degree of bony calcification in the jawbones of op/op mice using CT numbers, and it is suggested that CT is an extremely effective tool for examining the conditions of bone in vivo. This study also shows that op/op mice are useful for investigation of bone calcification or maturation.

Thermal stability of mineralized and demineralized dentin: a differential scanning calorimetric study.

The purpose of this study was to determine the difference, if any, in the thermal stability of collagen in mineralized and demineralized dentine compared to that in unmineralized tissues, using differential scanning calorimetry, DSC. Human tooth dentin blocks, about 1 x 1 x 2 mm in size, were used in this study. Some dentin blocks were demineralized using a Plank Rychlo solution; others, using EDTA solution. The mineralized dentin showed an exothermic peak at about 310 degrees C and the combustion of organic materials was completed at about 450 degrees C. For the demineralized dentin, the combustion was completed at higher temperature range and showed a strong exothermic peak at about 470 degrees C. An exotherm at the temperature between 450 degrees C and 470 degrees C was also observed in DSC pattern of native type I collagen from calf skin and rat tail tendon. DSC pattern of rat tail collagen showed a close similarity to that of the demineralized dentin. Statistically, the same heat flow value was obtained both from the mineralized dentin and the demineralized dentin and from the native type I collagen. These findings indicated that the thermal stability of collagen in dentin is lower than collagen in uncalcified connective tissue. It is suggested that in calcified collagen, the apatite crystallites may have intruded into spaces of the crosslinks of intra- and inter-fibrils, and in so doing, destroyed the crosslinks.

Calcospherites in rabbit incisor predentin.

Calcospherites from the lower incisor dentin of rabbits were investigated by scanning and transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS) and electron diffraction analyses. In the labial predentin, globular calcospherites of 8-31 microns were present at the root apex, decreasing in size toward the incisal region. The calcospherites at the intermediate region were of mulberry- as well as of spindle-shape of 1.5-4 microns diameter. The incisal pulp horn contained micro-calcospherites of 0.3-0.6 micron in diameter. In the lingual predentin, small granular calcospherites of 1.8-3 microns were present at the root apex, increasing in size toward the intermediate region. Ultrathin sections of globular calcospherites showed bundles of collagen fibrils at the root apex of the labial predentin. The diameters of individual bundles ranged from 1.2-3.4 microns. The width of the fibrils in the bundles was approximately 120-170 nm. Bundles of collagen fibrils were not found in the lingual predentin. Crystals of calcospherites were identified as apatite by electron diffraction. Those at the intermediate region showed preferred orientation of the c-axis. TEM-EDS analyses indicated that Ca and P were the major elements, with small amounts of Mg. The Mg/Ca molar ratios decreased from the root apex to the incisal pulp horn. Ca peak intensities increased from the root apex to the incisal region.

Morphological study of calcospherites in rat and rabbit incisor dentin.

Calcospherites from incisors of rats and rabbits were studied, by means of secondary and backscattered electron images, using scanning electron microscopy. Part of each incisor specimen was made anorganic to allow observation of the surface of the mineralization front by scanning electron microscopy; the other part was ground on one side for observation by scanning electron microscopy with a backscattered electron image detector. In both species the labial mineralization front was wavy and the image showed fused calcospherites, whereas the lingual mineralization front appeared to consist of a combination of linear and globular forms. In rat labial dentin the calcospherites were large and globular form, but they did not develop in the lingual dentin and were small and oval. The shape and size of rabbit incisor calcospherites varied from the pulp horn to the root apex. There were great differences between rats and rabbits with respect to the size and shape of the calcospherites at the mineralization front. This may be due to species differences and possibly the differing rates of dentinogenesis in rats and rabbits.

Microprobe analyses of the potassium-calcium distribution relationship in predentine.

Apex regions of continuously growing incisors of Wistar rats were quickly dissected, shock-frozen in liquid nitrogen-cooled propane, freeze-dried at -80 degrees C and infiltrated with Spurr's resin. 400nm thick dry sections were cut with a diamond knife on an ultramicrotome. Relatively flat sections were transferred with an eye lash onto collodium coated aluminum grids. They were flattened with a glass stick and by placing another collodium coated aluminum grid just on top of the first one, exerting a uniform pressure. After carbon coating the sections were observed using the backscattered and secondary electron signals in a scanning microscope. The predentine was analyzed for calcium and potassium with an energy dispersive x-ray analysis system. The x-ray spectra revealed in the predentine regions with beginning dentine formation, near the apex, an uneven K-distribution with very low as well as more prominent x-ray peaks. The K peaks were always lower than those of calcium. In areas with advanced dentine formation, prominent K-peaks were always observed. They were normally higher than the Ca-peaks up to a distance of 5-10 microns from the dentine border. Closer to the dentine border the K concentration decreased while the Ca-peak increased. This might indicate that (besides Na) K is used to balance the negative charges of the macromolecules till K is replaced by Ca at the onset of apatite crystal formation.

[The relationship between the reduced form tooth and the structures. The conical shaped upper lateral incisor].

The tooth structure of conical shaped incisor is observed with ground sections by light microscope. The Hunter-Schreger bands runs slightly convexed to the cervical from the dentino-enamel junction to the enamel surface. Enamel prisms arrange alternately and form almost arched type. Enamel spindles are observed remarkably. Many globular and interglobular structures are observed in the dentine. These structures are almost similar that of molar teeth expect some special types of enamel prisms. It is concluded that the conical shape did not cause of the reduced pattern of teeth structures.