Immunolocalization of podoplanin/E11/gp38, CD44, and endomucin in the odontoblastic cell layer of murine tooth germs.

In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.

Vascular endothelial growth factor (VEGF) in human tooth germ center.

Many oncogenis and tumour suppressor genes found inside normal and pathological cells are fundamental for the processes of development, proliferation and tissue differentiation. The purpose of our study is to show the presence and a possible relationship of the VEGF protein during different phases of the development of human dental germ centers. After cephalometric investigation in 8 orthodontic patients with a mean age of 13 years, (4 females and 4 males), hyperdivergence of the third molars were extracted. The 40 surgical samples were tested with monoclonal human anti-VEGFs antibodies carrying out a semi-quantitative analysis to look for a positive reaction. Reaction for anti-VEGF antibodies was detected in normal embryological tissues and in microvessels near odontogenic cells. During different phases of embryologic development of the dental bud our search showed intracytoplasmatic positive immunoreactions both in the ameloblastic and odontoblastic cells. Additionally, a positive reaction was observed for the VEGF protein in the cells of the stellate reticulum and in those endothelial tissue surrounding the microvessels in all the samples examined.

Structural features of forming and developing blood capillaries of the enamel organ of rat molar tooth germs observed by light and electron microscopy.

The process of vascularization of the enamel organ, a unique epithelial structure, occurs when the tooth germ is fully developed, i.e., at the onset of dentinogenesis. Although the three-dimensional organization of the capillaries has been previously investigated, the structural features underlying the formation of the new capillaries remains poorly understood. Thus, in the hope of better understanding the mechanism of formation of the stellate reticulum capillaries, upper first molar tooth germs of newborn and 3-day-old rats were fixed in glutaraldehyde-formaldehyde and processed for light and electron microscopy. Our results showed that blood capillaries are initially in close proximity to the outer enamel epithelium. Between and intercalated with the capillaries are round/ovoid clusters of cells, some of which are vacuolated, closely apposed to the outer enamel epithelium. The outer enamel epithelium is not a continuous layer, but exhibits gaps between the cells. This suggests that the capillaries penetrate the enamel organ through these gaps, since no invagination of the epithelium was observed. The presence of a cluster of cells containing vacuoles suggests that vasculogenesis is taking place. Images showing loss of the basal lamina, proliferation of endothelial cells, presence of filopodia and lateral sprouting suggests that angiogenesis is also occurring. Thus, neoformation of capillaries of the molar enamel organ of rat seems to occur simultaneously by mechanisms of vasculogenesis and angiogenesis.

Light microscopy and computer three-dimensional reconstruction of the blood capillaries of the enamel organ of rat molar tooth germs.

We performed a light microscope and a computer three-dimensional reconstruction study of serial sections of the molar enamel organ of 3- and 5-day-old rats perfused with Indian ink through the arterial system. The tooth germs were fixed in Bouin's solution, embedded in paraffin, sectioned and stained with haematoxylin and eosin. For the three-dimensional reconstruction, light micrographs of the serial sections were digitized, and aligned using the serial EM Align software downloaded from http://synapses.bu.edu/tools/. After alignment, the boundaries of the India-ink-filled blood vessels were manually traced with a mouse using the software IGL trace (version 1.26b), also downloaded from the above website. After tracing, a three-dimensional representation of the blood vessel contours was generated in a VRML format and visualized with the help of the software Cortona Web3D viewer (version 4.0) downloaded from http://www.parallelgraphics.com/products/cortona/. Our results showed that in regions where ameloblasts are polarized the capillaries are arranged in three distinct levels: (1) penetrating and leaving capillaries in relation to the outer enamel epithelium; (2) capillaries crossing and branching inside the stellate reticulum; and (3) capillaries branching and anastomosing profusely within the stratum intermedium, thereby forming an extensive capillary plexus intimately associated with the cells of the stratum intermedium. The existence of a conspicuous capillary plexus intermingled with cells of the stratum intermedium, as shown in our results, suggests that some molecules produced by cells of the stratum intermedium could be released into the capillary plexus and thereafter carried to the dental follicle.

Association between vascular endothelial growth factor (VEGF) expression and tumor angiogenesis in ameloblastomas.

BACKGROUND: Expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and microvessel density (MVD), assessed by the use of anti-CD34 antibody, were immunohistochemically examined in benign and malignant ameloblastomas, as well as tooth germs, to clarify the possible role of angiogenesis in epithelial odontogenic tumors. METHODS: Specimens of 5 tooth germs, 35 benign ameloblastomas and 5 malignant ameloblastomas were examined by immunohistochemistry using anti-VEGF and CD34 monoclonal antibodies. RESULTS: Immunoreactivity for VEGF was detected in both normal and neoplastic odontogenic epithelial cells, and weakly in microvessels near odontogenic epithelial cells, suggesting that this angiogenic factor acts on endothelial cells via a paracrine mechanism in odontogenic tissues. Both benign and malignant ameloblastomas showed elevated VEGF expression as compared to tooth germs. VEGF expression was low in keratinizing cells in acanthomatous ameloblastomas and granular cells in granular cell ameloblastomas, and acanthomatous ameloblastomas showed the lowest VEGF reactivity among the subtypes of ameloblastomas. MVD in both benign and malignant ameloblastomas was higher than that in tooth germs, indicating increased demands for blood in the neoplastic tissues. CD34-positive microvessels in follicular ameloblastomas were numerous and small, whereas those in plexiform ameloblastomas were scattered and dilated. MVD tended to depend on VEGF expression levels in both benign and malignant ameloblastomas. CONCLUSIONS: VEGF was considered to be an important mediator of angiogenesis in these epithelial odontogenic tumors, and up-regulation of VEGF might be associated with neoplastic or malignant changes of odontogenic epithelial cells.

Immunohistochemical and electron microscopic demonstration of nerve fibres in relation to gingiva, tooth germs and functional teeth in the lower jaw of the cichlid Tilapia mariae.

Immunohistochemistry revealed the presence of numerous neurofilament (NF)-like immunoreactive axons in relation to gingiva and dental follicles surrounding mineralizing tooth germs. The gingival nerve fibres frequently approached the prospective papilla of early tooth primordia. Electron microscopic (EM) analysis revealed the presence of bundles of unmyelinated axons immediately below the epithelial-proprial junction of the gingiva. Bundles of nerve fibres were also present in the border zone between the prospective papilla of bud-stage tooth germs and surrounding mesenchyme and in close proximity to blood vessels of the follicles surrounding older tooth germs, but no axons were observed within the emerging dental papilla. In the individual functional tooth, a bundle of NF-like immunoreactive nerve fibres entered the apical part of the pulp forming a subodontoblastic plexus at mid-pulpal levels. EM analysis showed that the apical bundle consisted of many unmyelinated and a few myelinated axons invested by Schwann cell processes. The subodontoblastic plexus contained unmyelinated axons only. Thin, axon-like profiles were also seen in predentinal tubules. Nerve fibres were not observed at pulpal horn levels and in the ligamentous attachment. It is concluded that both immature and mature parts of the lower-jaw dentition of the cichlid T. mariae are innervated and that the microscopic anatomy of this innervation is partly similar to the pattern seen in developing and adult mammals.

Nerve fibres and cells immunoreactive to neurochemical markers in developing rat molars and supporting tissues.

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) was compared to the general neurochemical markers for nerves and neuroendocrine cells protein gene product 9.5 (PGP 9.5) and neurone-specific enolase (NSE), by use of the avidin-biotin peroxidase complex method in developing dental structures in rats aged 13 to 27 days. A substantially greater part of the nerve fibres was immunoreactive to CGRP and SP than to NPY. In the bell stage, nerve fibres immunoreactive to PGP 9.5, CGRP and SP were found in the dental follicle but not in the dental papilla and stellate reticulum. In the advanced bell stage, after initiation of dentine and enamel formation, PGP 9.5, CGRP- and SP-immunoreactive fibres were found in the dental papilla, while the first NPY-immunoreactive fibres were observed in the papilla when root formation started. Concomitant with the beginning of root development, a subodontoblastic nerve plexus was gradually formed and PGP 9.5-, CGRP- and SP-immunoreactive fibres were found within the dentinal tubules. From the start of root formation, CGRP-, SP- and NPY-immunoreactive nerves were shown in the developing periodontal ligament, although a mature distribution pattern was not observed until root formation was nearly completed. Ameloblasts, odontoblasts and cell-like structures in the outer enamel epithelium and within the dental lamina were PGP 9.5-immunoreactive at the bell stage. As the tooth matured, the immunolabelling gradually decreased, but was still present in some odontoblasts after tooth eruption. NSE-immunoreactive, cell-like structures were found in the periphery of the dental follicle, and persisted close to alveolar bone in the periodontal ligament when the tooth reached occlusion. Hence, it may be concluded that sensory nerves containing SP and CGRP are present in the pulp in advance of sympathetic nerves immunoreactive to NPY.

Microcirculation of human fetal tooth buds: a SEM study of corrosion casts.

The microcirculation of tooth buds at the bell stage obtained from 5-month-old human fetuses was studied using corrosion casting and scanning electron microscopy. Each tooth bud has two independent vascular networks: one of the enamel organ and one of the dental papilla. Both systems are supplied by vertical branches of the inferior alveolar artery. The vascular bed of the enamel organ consists of capillaries relatively uniform in shape, forming a moderately dense network with irregular meshes. In contrast, the vasculature of dental papilla is extremely dense and its vessels show a sinusoidal character and signs of a vivid angiogenesis. The cast surfaces of capillaries in both vascular systems show the presence of tiny blebs probably representing extravasations of the casting medium through endothelial fenestrations.

Microvascular architecture of the enamel organ in the rat-incisor maturation zone. Scanning and transmission electron microscopic studies.

In order to clarify the microvascular architecture and ultrastructural features of the capillary vessels related to transendothelial transport of metabolites, scanning electron microscopy of tissues digested by HCl-collagenase and of vascular corrosion casts as well as thin-section, tracer, and freeze-fracture replications were employed to study the maturation zone of rat-incisor enamel organ. The enamel-organ maturation zone was shown to have a well-developed, dense capillary plexus. The capillary vessels were distributed along furrows formed by the enamel-organ papillary ridges. In central regions they formed a regular, blindlike network; in the peripheral regions, however, they formed an irregular, circular network. Everywhere except in the nuclear and perinuclear regions, the very thin capillary-vessel endothelial walls were pierced with numerous fenestrations. Such fenestrations were evident in endothelial walls facing the ameloblast-layer site. In tracer experiments, intravenously injected horseradish peroxidase passed through the fenestrations in the endothelial walls to diffuse throughout the enamel-organ extracellular spaces. It did not, however, pass through intercellular spaces or transendothelial channels. The dense, regular distribution of highly fenestrated capillaries in the enamel organ is thought to make possible the rapid transcapillary exchange of various metabolites between the vascular system and the ameloblast and papillary layers that is necessary for enamel maturation.

Inhibition of morphogenesis and stimulation of vascular proliferation in embryonic tooth cultures by a sarcoma growth factor preparation.

Sarcoma growth factor (SGF) induces proliferation and anchorage-independent growth of nonmalignant cells. It competes with epidermal growth factor (EGF) for the EGF-receptors at the cell surface. SGF-like factors have recently been isolated from embryos, suggesting that SGFs may represent embryonic forms of EGF. Therefore, we have tested whether SGF preparations affect organogenesis and differentiation of cultured embryonic tissues. The embryonic tooth rudiments were cultivated in the presence of SGF and EGF. Stimulation of vascularization was seen in both of these organ cultures. Therefore, we propose that endothelial cells may be target cells for SGF, and SGF may be involved in the control of vascularization during embryogenesis. SGF and also, to a certain extent, EGF profoundly inhibited morphogenesis and differentiation of the tooth germ, with concomitant stimulation of vascularization. Analysis of cell proliferation revealed that some cell types of the tooth germ did not respond to SGF by proliferation, while a stimulation by EGF was observed. Nevertheless, tooth morphogenesis was also slightly inhibited by EGF, suggesting that growth factors which enhance proliferation do not necessarily stimulate morphogenesis and differentiation. Since the SGF preparations contain several factors, the effects observed could be due to the action of one or more factors.

[Structural organization of the microcirculatory bed of the primordia of human deciduous teeth]. / Strukturnaia organizatsiia mikrotsirkuliatornogo rusla zachatkov molochnykh zubov cheloveka.

By means of injection and noninjection techniques structural organization of microcirculatory bed in the deciduous (milk) teeth germs have been studied in human fetuses, newborns and children. A definite dependence of the angioarchitectonics on the developmental stage of every tooth has been stated. Morphological differentiation of separate parts of the microcirculatory ways in the dental papilla is connected with dentinogenesis, and in the dental saccule--with amelogenesis, periodontum and alveolar wall formation. Those dental germ zones which have a peculiar functional importance (peripheral parts of the dental papilla, dental saccule in the area of the epithelial dental organ) are characterized by the presence of certain structural-functional apparatuses which provide increasing contact areas between blood and the tissues. Intraorganic circulatory bed of the dental papilla consists of identically constructed vascular complexes. The conformity coefficient between the arteriolar inlet and the venular outlet of the dental papilla microcirculatory ways during dentinogenesis is between 0.16--0.3. It has been stated that devices regulating the blood stream in the deciduous tooth germs (anastomoses, sphincters, arcades, etc.) are forming during the antenatal and early postnatal periods.

Adaptation of arterioles to moving capillaries.

The capillary network of the enamel organ of the continuously growing mandibular incisor of the rat is supplied by a series of arteries which, after penetrating the bone, are disposed in a row in the periodontium along the tooth and linked together by anastomoses. The branches from these arteries are subjected to adaptive changes consistent with a forward movement of the capillaries in relation to the arteries. The mechanism which renders this movement possible appears to be the following: Each artery in the row supplies a section of the passing plexus by coupling and uncoupling short-lived arterioles, which go through a cycle of proliferation, elongation and degeneration. Proliferation takes place at the posterior end of the section, where new arterioles replace discarded arterioles from the preceding artery. By growing in length, the arterioles keep pace with the migrating capillary network. When the capillaries are within reach of the next artery, the arterioles disconnect. Thereafter they become obliterated and die in the intermediate zone of the periodontium, to which they have been gradually displaced by proliferation of the inner-tooth-related layer of the periodontal connective tissue.

The vascular pattern in the papillary region of rat incisor and molar tooth enamel organ.

Scanning electron microscopy of the enamel organ of rat incisor and molar teeth in the maturation stage of amelogenesis revealed two vascularization patterns of the papillary layer. In one pattern, the anastomosing capillaries formed loops of varying sizes around spherical or somehwat oblong papillae. In the second pattern, the capillaries were parallel to each other embedded in furrows between long ridges of papillary cells. It is postulated that each of these two patterns may be associated with a specific stage in the process of enamel maturation.

Blood supply of the rat periodontal space during amelogenesis as studied by the injection replica SEM method.

Distribution of blood vessels at the labial periodontal space of the rat lower incisor teeth was studied, using Mercox-resin vascular casts, which were coated with gold-palladium and observed in a scanning electron microscope (SEM). Three different layers were identified in the vascular bed of the periodontium. In the inner layer the enamel organ was supplied by a blood capillary network that changed from a circular mesh to a ladder-like pattern during amelogenesis. The middle layer was supplied by small arteries and arterioles. Small arteries originated from the inferior alveolar artery; arterioles, arising from them, became blood capillaries. In the outer layer, the sinusoid veins continued with the blood capillaries which ran into the proximal and the distal sites of the inner layer. This venous layer is located near the alveolar bones. As capillary networks change in pattern during amelogenesis, the circular mesh is considered convenient to provide the required materials for the proliferation and differentiation of inner enamel epithelial cells as well as for early enamel matrix formation, whereas the ladder pattern seems suitable to supply numerous organic or inorganic materials for the advanced enamel matrix formation and calcification.