Heparanase, heparan sulfate and perlecan distribution along with the vascular penetration during stellate reticulum retraction in the mouse enamel organ.

OBJECTIVE: Immunohistochemical and gene expression profiles of heparanase were determined in murine molar tooth germs from their embryonic to postnatal stages, paying special attention to neovascularization within the enamel organ, which is poorly vascularized before birth. DESIGN: Protein and gene expression profiles of heparanase, heparan sulfate (HS), vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), and perlecan were comparatively examined by immunohistochemistry and in-situ hybridization, respectively, in mouse mandibular molar tooth germs from embryonic day 11.5 to postnatal day 6. At the same time, their mRNA expression levels were also confirmed by reverse transcriptase-polymerase chain reaction using laser-captured microdissection of enamel organ tissues. RESULTS: Stellate reticulum cells highly expressed perlecan but only slightly expressed heparanase and HS in their embryonic days. On and after postnatal day 1, the expressions of heparanase became dramatically higher in the stellate reticulum, while HS disappeared leaving the immunopositivity for perlecan core protein. Immunohistochemically, HS was enhanced around blood vessels which were newly formed after birth within the enamel organs, whose volume was also regressive. Similar expression patterns were obtained for VEGF and TGF-beta1. CONCLUSIONS: Such synchronized expression modes among the HS metabolism-related molecules suggested that heparanase plays an important role in degradation of HS chains, which is closely related to vascular penetration into the stellate reticulum, which may be one of the driving forces for the postnatal regression of the enamel organ.

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.

Distribution of the epithelial rests of Malassez and their relationship to blood vessels of the periodontal ligament during rat tooth development.

BACKGROUND: There is some evidence that the epithelial cell rests of Malassez partition the root surface from the periodontal ligament blood vessels, and may protect the root from resorption. OBJECTIVE: The aim of the present study was to determine the distributions of the epithelial rests of Malassez (ERM) and blood vessels in the periodontal ligament (PDL) of the developing rat first molar before, during and after emergence. METHODS: Four Sprague-Dawley rats were sacrificed at two days, one week, two weeks, three weeks, four weeks and six weeks of age. After processing, the maxillae were embedded in paraffin, and sectioned longitudinally and transversely. The sections were stained with a double immuno-histochemical technique which utilised a keratin antibody AE1-AE3 (1:2,000) and an endothelial antibody Factor VIII (1:10,000) to enable simultaneous labelling of ERM and blood vessels. ERM and blood vessel counts were obtained from the mesio-buccal roots of three week, four week and six week-old rats, whilst qualitative observations were made for the earlier developmental stages. RESULTS: ERM cells and cell clusters were found in the tooth third of the PDL width at the three, four and six week stages. Cells and cell clusters increased in number with age, especially in the upper third of the mesio-buccal root. The largest numbers of cells and clusters were found on the distal surfaces of the roots in all age groups. Cells and clusters in all root surfaces increased from three to four weeks, but decreased from four to six weeks. The greatest number of blood vessels was found in the bone-side third of the PDL. The distal surface had the highest proportion of blood vessels, and the palatal surface the least proportion. The number of blood vessels in all surface quadrants did not vary much from three to four weeks of age, but increased from four to six weeks of age, possibly as a reaction to tooth emergence and occlusal function. Physiological root resorption was only observed after tooth emergence, and appeared to be related to loss of continuity of the ERM network and the incursion of blood vessels. CONCLUSIONS: Orthodontic root resorption can be regarded as an exaggerated response to loss of PDL homeostatic control, possibly mediated by the epithelial rests of Malassez.

Localization of specific binding sites for 125I-TGF-beta1 to fenestrated endothelium in bone and anastomosing capillary networks in enamel organ suggests a role for TGF-beta1 in angiogenesis.

Previous studies have shown endothelial cells to be a major target for endocrine TGF-beta in several soft tissues in the normal growing rat. The potent effect of TGF-beta1 on bone formation prompted us to analyze in detail the localization of specific binding sites for endocrine TGF-beta in hard tissues. At 2.5 minutes after injection of 125I-TGF-beta1, specific binding, as demonstrated by quantitative radioautography, was localized to fenestrated endothelium participating in angiogenesis in the vascular invasion region of the growth plate in bone as well as to anatomizing capillary networks in the maturation zone of the enamel organ. At 15 minutes after injection, the bound ligand was internalized into endocytic vesicles of endothelial cells. In bone, quantitation revealed significant differences in receptor density between endothelia undergoing proliferation vs those in a state of elongation and anastomosis with neighboring endothelial cells. In the rat incisor, specific binding of 125I-TGF-beta1 to endothelium correlated with increased formation of anastomotic capillary networks. These studies identify differential specific binding sites of 125I-TGF-beta1 in angiogenically active endothelium, providing an important link between TGF-beta1, the endothelium, and hard tissue development.

Microvascular system of the rat incisor enamel organ. A scanning electron microscopic study of vascular corrosion casts.

The rat incisor is a commonly used model in studies of tooth eruption, amelogenesis and effects of mechanical loading on the dental and periodontal tissues. The purpose of this study was to assess the three-dimensional architecture of the microvascular bed of the rat incisor enamel organ, to describe the direction of blood flow, and to provide a histometric assessment of the vascular categories that can be statistically analyzed. Vascular corrosion casts were prepared and examined by scanning electron microscopy. The microvasculature of the labial periodontal space was arranged in three distinct layers. The inner layer in direct relation to the enamel organ consisted of a capillary network which was drained by short venules at the cemento-enamel junction. The intermediate layer consisted of arterioles oriented parallel to the long axis of the incisor mainly mid-labially, branching off smaller arterioles to the capillary network. The outer layer was formed by flattened sinusoid vessels of larger caliber. Blood supply was from the anterior superior alveolar artery branches through the arterioles into the capillary network. Drainage was postero-laterally along the cemento-enamel junction via short venules which emptied into the sinusoid vessels, finally to flow through Volkmann's canals into the alveolar bone via small venules. The findings demonstrate that the microvasculature of the rat incisor enamel organ has an exceptionally high level of physiologically-adapted structural organization.

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 of the upper major incisor in the rabbit.

The ultrastructure of the ameloblasts in the rabbit major incisor was investigated previously by Okada (1983) and the amelogenetic process was classified into six zones/stages. The present paper deals with changes in the microvascular architecture and ultrastructure of the blood capillaries in proportion to the amelogenetic process in the upper major incisor of the rabbit utilizing the acryl plastic injection method. Three different vascular layers were observed in the periodontal spaces of the major incisor of the rabbit. The inner vascular network consisted of a capillary network supplying the enamel organ and its meshes have vigorously changed during the amelogenesis. The capillary network was observed to be in the shape of a ladder with a continuous wall in the proliferation zone, to appear as round meshes with a fenestrated wall in the differentiation zone, as polygonal meshes with abundant fenestrations in the secretion zone, as ovoid meshes with fenestrations in the early maturation zone, and finally as coarse and avoid meshes with a continuous wall again in the late maturation and regression zones. In the intermediate layer, arterioles and venules were located close to the capillary network, and the arterioles were derived from the short and long branches of the anterior superior alveolar artery. In the outer layer, a sinusoid network was observed to be in contact with the alveolar wall and received blood from the capillary network as well as venous vessels in the alveolar bone. The ladder-shaped capillary network mentioned above was thought to represent an intermediate form towards the succeeding zone, in which the round meshes may be suitable for supplying the nutrient elements that are needed in the differentiation of the inner enamel epithelial cells. The polygonal and ovoid meshes may be favorable for the transport of various necessary metabolic materials that are involved in the enamel ground substance formation and calcium deposition within a very short period.

Vascularization of the enamel organ in developing molar teeth of rats--scanning electron microscope study of corrosion casts.

Vascularization of the enamel organ was demonstrated in the developing upper first molar teeth of rats from the 19-day embryo to 5 days after birth employing the vascular casting/scanning electron microscope method. Capillaries were first observed in the enamel organ at the 21-day embryo. By that time, with the beginning of differentiation of the inner enamel epithelium into ameloblasts, mesenchymal cells situated in close proximity to the inner enamel epithelium had begun to differentiate into odontoblasts, but deposition of organic substances had not commenced. The occurrence of blood capillaries before the nutritional supply through the dental papilla was interrupted by the deposition of dentin and enamel, may possibly be due to the high nutritional requirements of the ameloblasts following differentiation from the inner enamel epithelium. With the advance of dentin and enamel formation, many capillaries entered the enamel organ and finally formed a flattened vascular network next to the stratum intermedium. These findings suggest that the capillaries in the enamel organ should be regarded as a change which affords a rapid and sufficient supply of metabolic substances necessary for amelogenesis. The newly developed capillaries in the enamel organ grew first by sprouting and later by loop formation.

An ultrastructural study of the papillary layer and its vascular bed in the kitten enamel organ.

Ultrastructure and three-dimensional architecture of the papillary layer and associated capillaries in the enamel organ of 2-3-month-old kittens were examined by means of routine thin sections, freeze-fracture, and scanning electron microscopy of the tissues digested by HCl-collagenase and of vascular corrosion casts. Outwardly, the papillary layer formed gently sloping upheavals, but did not show prominent papillary ridges. The papillary cells were characterized by a high concentration of intramembranous particles on the plasma membrane P-face, by numerous hemi-annular gap junctions between the cell process of one papillary cell and the cell body of another host cell, and by annular gap junctional vesicles in the subsurface cytoplasm. Some annular gap junctions appeared partially degenerated. These findings led us to speculate that these annular gap junctions are produced by the endocytosis of gap junctional membranes from the cell surface into the subsurface cytoplasm. Capillaries were distributed on the enamel organ within shallow furrows between the papillary upheavals. A part of these capillaries penetrated deeply into the enamel organ but did not contact the ameloblasts. The endothelial walls of the capillaries were pierced with many endothelial fenestrations, especially when facing the papillary layer. The endothelial cell also contained numerous micropinocytotic vesicles throughout its entire cytoplasm. These findings suggest that the papillary cells and associated capillaries are highly specialized for transport of solutes and molecules between the vascular region and the enamel organ during the phase of enamel maturation.

In vivo demonstration by radioautography of binding sites for insulin in liver, kidney, and calcified tissues of the rat.

An in vivo binding assay using radioautography was employed to visualize insulin receptors in rat tissues. Two and one-half minutes after the intravenous injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with lactated Ringer's solution followed by perfusion with glutaraldehyde. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. Nonspecific binding of labeled insulin was noted in the proximal convoluted tubules of the kidney cortex, prebone and adjacent bone, predentin and adjacent dentin, and enamel. Specific binding sites were observed at the periphery of hepatocytes, over osteoblasts, and in relation to the endothelial cells of fenestrated capillaries within the papillary layer of the maturation zone of the incisors.

[Cellular changes at the level of the outer enamel epithelium during vascularization of the enamel organ in the rat]. / Modifications cellulaires au niveau de l'épithélium adamantin externe au cours de la vascularisation de l'organe de l'émail chez le rat.

In the albinos rat, at day 20 in utero, the outer epithelial layer of the first molar is depressed by numerous capillaries. The mesenchymal cells of the follicle present cytoplasmic extensions which are in close relationship with the basement lamina of the external epithelial layer with fibrillar material adherent to the basement lamina. The endothelial cells of the capillaries also present cytoplasmic extensions in contact with the basement lamina. At day 21 in utero, the capillaries penetrate deeply into the stellate reticulum. The fact that the epithelium which surrounds the capillaries is separated from the latter by a basement membrane indicates that the vascular penetration induces a very deep invagination of the epithelium and the basement membrane. This vascular penetration could play a role in the transport of precursors towards the secretory ameloblasts. The mesenchymal and endothelial tissues of the capillaries could have an inductive role on the external epithelial layer through the basement membrane, leading to a disorganization of the enamel organ.

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.

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.

Immunohistochemical evidence for the intracellular formation of basement membrane collagen (type IV) in developing tissues.

Antibodies to type IV collagen obtained from the basement membrane of the mouse EHS tumor were incubated with sections of rat incisor teeth and other tissues for immunostaining by direct or indirect methods. In all locations, the immunostaining was pronounced in basement membranes in which it was restricted to the "basal lamina" layer, from which "bridges" often extended to nearby basal laminae. Usually no immunostaining was detectable in the cells associated with the basement membranes. However, examination of the capillaries at the posterior extremity of the rat incisor tooth, where tissues are at an early stage of development, showed immunostaining not only of the basement membrane, but also of the endothelial cells. The staining was localized in rough endoplasmic reticulum cisternae, some Golgi saccules and their peripheral distensions, and structures believed to be secretory granules. These findings suggest that the synthesis of type IV collagen proceeds along the classical secretory pathways through rough endoplasmic reticulum and Golgi apparatus. At the same time, immunostaining was usually lacking in the cells of the capillaries that had migrated about 2 mm away from the posterior end of the incisor tooth and also in the cells of most other tissues examined, even though the associated basal laminae were reactive. It is, therefore, presumed that the production of type IV collagen may be high in cells at an early stage of development and that any later production and turnover of basement membrane collagen can only be minimal.

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.