Shohei Higashi

Uptake of horseradish peroxidase by bone cells during endochondral bone development.

SummaryTo investigate the mechanisms whereby bone cells absorb organic bone-matrix components during endochondral bone development, rat humeri were examined, employing horseradish peroxidase as a soluble protein tracer.Intravenously-injected peroxidase filled the osteoid layer and penetrated into the osteocyte lacunae and canaliculi, but did not enter the mineralized bone matrix. Whereas osteocytes rarely took up exogenous peroxidase, osteoblasts and osteoclasts actively endocytosed peroxidase in pinocytotic coated vesicles, tubular structures, and vacuoles. They also formed endocytotic vacuoles containing peroxidase in the Golgi area. The Golgi apparatus and dense bodies of these bone cells were, however, free of reaction products. Osteoclast ruffled borders were responsible for peroxidase absorption. In the osteoblast, osteocyte and osteoclast, endogenous peroxidatic reaction was detected only in mitochondria and not in other membrane-bounded vesicles and bodies. These results strongly suggest that both osteoblasts and osteoclasts participate in the resorption of bone-matrix organic components during bone remodelling.

Morphology and Permeability of Junctional Complexes in Maturing Ameloblasts of Rat Incisors

Thin sections of newborn rat incisors were examined by tracer experiments and freeze-fracture replication in order to clarify the morphology and permeability of ameloblast junctional complexes in the maturation stage. Ameloblast junctional complexes consisted of gaps and tight junctions at the proximal and distal ends. Whereas the proximal junctional complexes sealed extracellular spaces incompletely, the distal ones formed complete, belt-like barriers around the cell. Tight junctions of these junctional complexes, however, were composed of both continuous and discontinuous rows of particles with various spaces among them. Intravenously injected horseradish peroxidase (HRP) reached the enamel surface through the extracellular spaces among ameloblasts and was absorbed by ameloblasts of the ruffled borders. Pinocytosis and transcellular migration of HRP could not be demonstrated in maturing ameloblasts except in the ruffled border zone.

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.

Thin-Section, Tracer, and Freeze-Fracture Study of the Smooth-Ended Maturation Ameloblasts in Rat Incisors

The morphology and functional roles of the smooth-ended maturation ameloblasts (SAs) of rat incisors were examined by means of routine thin sections, tracer experiments, and freeze-fracture replication. SAs possessed two sets of junctional complexes consisting of tight junctions (fasiae occludentes) and gap junctions at the proximal and distal ends. Neither the proximal nor the distal junctional complex formed a complete barrier around the cell; intravenously injected horseradish peroxidase (HRP) reached the developing enamel surface through SAs extracellular spaces. SA supranuclear cytoplasm included such various cytoplasmic vesicles as the multivesicular body (MVB), large and vacuoles, dense body, and coated vesicles. The HRP that reached the enamel surface was incorporated into some coated vesicles and small vacuoles via coated pits and membrane invaginations of the distal cell surface of SA. Then, in the process of time, it migrated into the MVB and large endocytic vacuoles. These results indicate that the SA layer forms an extracellular transfer route for metabolites between papillary layer and the enamel surface and that SAs absorb exogenous protein.

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

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

Correlated Observations and Analysis of Maturation-ameloblast Morphology and Enamel Mineralization

A combined HCI-collagenase digestion technique and scanning electron microscopy were used to isolate the enamel organ and to confirm the presence of maturation ameloblasts of both ruffle-ended (RA) and smooth-ended (SA) types on maturing enamel in kitten permanent tooth germs. EDTA perfusion of animals fixed with aldehyde produced two or three belt-like shallow grooves (from 30 to 100 μm wide) running horizontally through the maturing enamel surface, coinciding closely with the SA distribution pattern. In animals that had been perfusion-fixed with unbuffered osmium tetroxide containing 2.5% potassium pyroantimonate, SEM-EDX analysis detected K in a superficial enamel layer overlaid by the SA layer. Potassium concentration decreased gradually toward the deeper layers. Very little K penetrated the enamel under the RA layer. Energy-dispersive x-ray analysis of Ca and P concentrations in the enamel revealed an even distribution of these elements throughout the superficial layer of maturing enamel. These results suggest that the SA layer forms an access route for K and EDTA and that, in spite of the obvious morphological and functional differences between RA and SA, the maturing enamel surfaces overlaid by these two cell types show similar degrees of mineralization.

Energy-dispersive X-ray microanalysis and scanning electron microscopy of developing and mature cat enamel.

Calcium and phosphorus distribution in forming, maturing and mature enamel of cat teeth and the microstructures manifest in all these were examined in fractured enamel from the dentine-enamel junction toward the enamel surface. concentrations of both Ca and P increased gradually from the forming enamel, through the maturing enamel and into the mature enamel. The innermost layer, adjacent to the dentine-enamel junction showed the greatest and the superficial layer the lowest concentration of Ca. Still the mature enamel of the erupted tooth was not yet completely mineralized and Ca and P concentrations only slightly higher than those in maturing enamel. Molar Ca:P ratio of each enamel stage was lower than that of pure crystalline hydroxyapatite. Simultaneously-performed SEM observations revealed microstructural changes in the enamel: in the forming-front layer of the forming enamel, the enamel was a rough, immature structure but began to show more compact, tighter structures as concentrations of Ca and P altered. The results suggest that the enamel organ exercises intense cellular control over increases of Ca and P concentration during the formation and maturation stages of amelogenesis.

Three-Dimensional Observations of Accessory Canals in Mature and Developing Rat Molar Teeth

The structure, distribution and formation of accessory canals in the developing and mature molar teeth of rat mandibular jaws were investigated with scanning electron microscopy and with three-dimensional image analysis using serial light-microscopic sections. In the initial stage of the accessory canal formation, most of the canals appeared in the gaps of the epithelial root sheaths formed by their approaching each other in the initial stage of the root formation. However, some of the canals appeared in the slits which may be formed by the destruction of the epithelial root sheath in the root apex regions. When the gaps and slits were invaded by blood vessels, the regions surrounding the vessels did not mineralize but became accessory canals. Usually, an accessory canal with one blood vessel connected the periodontal ligament to the dental pulp; however, in some cases, the canals were broken off midway following the destruction of the vessels.

Formation of Tight and Gap Junctions in the Inner Enamel Epithelium and Preameloblasts in Human Fetal Tooth Germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.