Plasma Membranes Of Odontoblasts Research Articles

Polarity of Mature Human Odontoblasts

Odontoblast polarization is based on histological appearance as columnar cells with asymmetric disposition of organelles and plasma membrane domains. However, little is known about the odontoblast plasma membrane organization. We investigated odontoblast membrane polarity using influenza virus hemagglutinin and vesicular stomatitis virus glycoprotein as model proteins in mature human odontoblast organ culture. We also examined the distribution patterns of aquaporin 4 and 5, which are basolateral and apical proteins in epithelial cells, respectively. Confocal microscopy immunofluorescence and electron microscopy demonstrated that the apical markers located at the surface toward pulp and basolateral markers located at the plasma membrane of odontoblast processes. Therefore, odontoblast plasma membrane polarity was different from that in epithelial cells. Also, certain lectins stained odontoblast processes while others stained the soma, reflecting the different natures of their membrane domains. Strong ZO-1 and weaker claudin expression suggest weak tight junctions in the odontoblasts. TGF-β1 showed a tendency to reinstate the expression of selected TJ genes, indicating that TGF-β1 may control odontoblast cell layer integrity by controlling tight junction protein expression.

Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarisation.

We have examined freeze-fracture replicas of maxillary first molar tooth germs of newborn rats at early stages of dentinogenesis to study the development of tight junctions in the distal plasma membrane of differentiating odontoblasts. In addition, freeze-fracture was combined with filipin to observe the distribution of cholesterol on the distal plasma membrane of odontoblasts during differentiation. Only gap junctions were present in early differentiating odontoblasts. The distal plasma membrane exhibited low cholesterol content, which might indicate high fluidity. With the beginning of mineral deposition in matrix-vesicles, the first signs of tight junction formation were observed. Further development revealed increasingly complex focal tight junctions. In later stages, when mineralisation is observed progressing to the fibrillar and non-fibrillar constituents of the matrix, well developed focal tight junctions were detected. Concomitantly, cholesterol in distal portions of the odontoblast plasma membrane increased, indicating, probably, a higher rigidity. Thus, a distal plasma membrane domain is established, odontoblasts become fully differentiated, and partial compartmentalisation of matrix occurs. At this stage, odontoblasts may be able to secrete specific matrix molecules to ensure the progression of mineralisation.

Development of tight junctions between odontoblasts in early dentinogenesis as revealed by freeze-fracture.

Mature odontoblasts possess junctional structures constituted by adherens, gap, and tight junctions. Although adherens and gap junctions appear early between odontoblasts, there is no information on the appearance and development of tight junctions between odontoblasts. In this study, we have examined freeze-fracture replicas of early dentinogenesis to study the development of tight junctions between odontoblasts and to determine whether these junctions are of zonular or macular type. Upper first molar tooth germs of Wistar rats between 1 and 3 days old were fixed in buffered 4% glutaraldehyde/4% formaldehyde and subsequently cryoprotected with cacodylate-buffered glycerol. Freeze-fracture replicas were obtained in a Balzers 301 apparatus, and early stages of dentinogenesis were examined in a Jeol 100 CX II electron microscope. In the stage of early dentine matrix prior to mineralization, odontoblasts exhibit only gap junctions. With the progression of development, the distal plasma membranes of odontoblasts show numerous short tight junctions formed by fused particles and grooves. In the stage of advanced mineralization, branched and continuous rows of fused particles or grooves constitute tight junctions of the focal or macular type. The present study shows that tight junctions of focal or macular type appear on distal plasma membrane of early odontoblasts during differentiation. Formation of tight junctions indicates the establishment of a distal membrane domain and maturation of odontoblasts. These events occur as mantle dentine formation ceases and circumpulpar dentine formation begins.

Voltage-gated calcium channels and nonvoltage-gated calcium uptake pathways in the rat incisor odontoblast plasma membrane.

Odontoblasts participate actively in the transport and accumulation of Ca2+ ions to the mineralization front during dentinogenesis. These cells are known to carry membrane-bound ATP-driven pumps and Na+/Ca2+ antiports for Ca2+ extrusion, but little is known about Ca2+ influx mechanisms into these cells. It has been shown that the administration of Ca2+ channel blockers in vivo strongly impairs Ca2+ uptake in the mineral phase during dentinogenesis in the rat; the present in vitro study is aimed at further elucidating odontoblast Ca2+ uptake mechanisms. Dissected rat incisor odontoblasts exhibited a pronounced fluorescence when incubated with a fluorescently-labeled (STBodipy) dihydropyridine, which is specific for voltage-gated Ca2+ channels of the L-type, and this binding was competitively abolished by nifedipine. As assayed by fluorescence spectrometry, odontoblast Ca2+ uptake was enhanced by the agonistic dihydropyridine BAYK-8644 (5 micro;M) as well as by plasma membrane depolarization in a high K+ (120 mM) medium. The Ca2+ uptake after depolarization was impaired by nifedipine (5 micro;M). When treated with the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA; 10 micro;M), a nonvoltage-gated uptake of 45Ca2+ was identified. This uptake was not influenced by nifedipine (20 micro;M) but was impaired by lanthanum ions (200 micro;M). A nonvoltage-gated uptake of Mn2+ into CPA-treated cells could be traced using the fura-2 quenching technique. This CPA-induced Ca2+ flux was not caused by an alteration of the plasma membrane potential, as assayed with di-8-ANEPPS. The results demonstrate that Ca2+ flux into dentinogenically active odontoblasts occurs through voltage-gated Ca2+ channels of the L-type and by nonvoltage-gated, agonist-sensitive Ca2+ uptake pathways.

Structure and organization of odontoblasts.

Differentiation of odontoblasts involves cell-to-cell recognition, contact stabilization involving the formation of attachment specializations, cytoplasmic polarization, development of the protein synthetic and secretory apparatus, and the active transport of mineral ions. The secretory odontoblast is characterized by an extensive rough-surfaced endoplasmic reticulum, a highly developed Golgi complex, and the presence of specific secretion granules. Type I collagen, a major constituent of dentin matrix, appears to be secreted by the odontoblast into predentin at the proximal portion of the odontoblast process, the major cytoplasmic process extending from the odontoblast cell body into the dentin. The odontoblast process contains a rich network of microtubules and microfilaments. The proximal portion of the process is also a site of fluid-phase endocytosis. Adjacent odontoblasts are held together by numerous macula adherens junctions and a well-developed distal junctional complex adjacent to be predentin. Junctional strands of the occludens type have been observed to be a component of this junctional complex. Tracer studies employing horseradish peroxidase indicate that this junctional complex does not form a tight barrier to the diffusion of tissue fluid from the interodontoblast spaces into the predentin. Many well-developed gap junctions are formed between adjacent odontoblasts and between odontoblasts and the fibroblasts that make up the subodontoblastic layer. Ca-ATPase activity is demonstrated in the Golgi complex and mitochondrial cristae and along the distal plasma membranes of odontoblasts. ALPase activity is also intense along the entire odontoblast cell surface. The osmium tetroxide-pyroantimonate technique for calcium localization demonstrates prominent reaction precipitates in mitochondria of odontoblasts. Energy-dispersive x-ray microanalysis of anhydrously fixed and processed odontoblasts detected Ca and P peaks throughout the cytoplasm. A sulfur peak is noted in the distal cytoplasm of odontoblasts and in matrix vesicles. Together, these results demonstrate the complexity and variety of cell functions involved in dentinogenesis.

Cholesterol in the distal portions of differentiating and fully differentiated rat odontoblasts observed by freeze-fracture

Freeze-fracture replicas of rat molar tooth germs in conjunction with the 3-β-hydroxysterol marker filipin were used to study the distribution of cholesterol in the distal portions of odontoblast plasma membrane. Filipin-sterol deformations (interactions) appeared as clusters interspersed with deformation-free regions on the distal plasma membrane of early differentiating odontoblasts. In fully differentiated odontoblasts, the whole distal membrane, including the process, was occupied by packed deformations with no free regions. It seems that early developing odontoblasts are poorer in cholesterol than the fully differentiated cells. As the content and/or distribution of 3-β-hydroxysterols (cholesterol) is known to influence membrane fluidity, the low cholesterol content of early differentiating odontoblasts might be related to the fluidity required for the budding off of matrix vesicles.

A histochemical study by light and electron microscopy of the distribution of dipeptidyl peptidase-IV activity in the human dental pulp

This activity was demonstrated in blood vessels, fibroblast-like cells, odontoblasts and Schwann cells surrounding non-myelinated axons. It was present on both the luminal and abluminal plasma membrane of endothelial cells. The plasma membrane of fibroblast-like cells and their processes had patches of fine electron-dense end-product corresponding to DPP-IV activity, while the plasma membrane of odontoblasts was loaded with a consistent reaction product. DPP-IV activity in the non-myelin-forming Schwann cells was in the plasma membrane of the free surface in contact with the extracellular matrix as well as in the plasma membrane in close contact with the axolemma. The plasma membrane of Schwann cells producing myelin sheath had no positive reaction for DPP-IV. The DPP-IV activity in the plasma membrane of fibroblast-like cells and odontoblasts may be associated with fibronectin-mediated adhesion to collagen, whereas the membrane-bound DPP-IV activity in endothelium and non-myelin-forming Schwann cells may be involved in cleavage of neuropeptides of other biologically active peptides.

Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs

In order to examine the synthesis and secretion of enamel protein by ameloblasts in their early stages of development, immunohistochemical localization was carried out at light and electron microscopic levels using a monoclonal antibody produced in a preliminary experiment. Materials used were tooth germs of mandibular first molars of rats at 0-5 days after birth. Immunoblot analysis after two-dimensional electrophoresis revealed that antigen molecules recognized by the monoclonal antibody were amelogenins of 26-28 kDa (pI, 6.6-7.0). An immunohistochemical examination using this monoclonal antibody demonstrated that the presecretory ameloblasts in their early stages of differentiation both synthesized amelogenin and secreted through a classical merocrine secretory pathway. In some presecretory ameloblasts as well as ameloblasts we observed the distended cisternae of rough endoplasmic reticulum (rER) which demonstrated heterogenous immunolabelling. The immunolabellings were also detected in the predentin as well as the intercellular spaces of odontoblasts and dental pulp cells which indicated penetration of amelogenin from the presecretory ameloblast layer to the dental pulp. The presence of coated pits at the plasma membrane of odontoblasts in close proximity to enamel protein along with the immunolabelling of lysosomes of the odontoblasts suggests the phagocytosis of the enamel protein into the odontoblasts. These observations suggest the possibility that the penetration of enamel protein toward the dental pulp and odontoblasts plays a role in the interaction between ameloblasts and odontoblasts.

Localization of calcium and phosphorus in early predentin-matrix components by electron spectroscopic imaging (ESI)-analysis in rat molars

The subcellular distribution of the inorganic elements calcium (Ca) and phosphorus (P) was studied in the first-formed dentin matrix during initial mineralization in neonatal rat molars. This most peripheral matrix region is comprised of a proteoglycan-rich ground substance, interwoven by a collagenous network, matrix vesicles, aperiodic fibrils derived from the dental basal lamina, and apical odontoblastic cell processes. All matrix components may possibly serve as templets for mineral deposition during initial calcification of first-formed mantle dentin and predentin. By means of the very sensitive ESI-analysis we studied the subcellular localization of Ca and P and their possible association with distinct organic extracellular matrix components and odontoblasts. Ca-signals were found in the ground substance, at striated collagen fibrils and plasma membranes of odontoblasts in the cuspal early matrix region, but occurred only sparsely in the ground substance of the more distal matrix region where odontoblast processes attach to aperiodic fibrils of the dental basal lamina. Ca was generally absent in matrix vesicles. In contrast, P-signals were found in matrix vesicles, at aperiodic fibrils and at the plasma membranes of odontoblasts. Ca and P co-localized at striated collagen fibrils (type I or II). These results suggest that striated collagen fibrils might serve as primary deposition sites for calcium phosphate during early biological calcification of organic extracellular macromolecules.

Na+/Ca2+ antiports in membranes of rat incisor odontoblasts

When incubated in the presence of Na+ ions, dissected rat incisor odontoblasts extruded Ca2+ ions, as detected by tetracycline fluorimetry. Also odontoblast mitochondria, obtained by subcellular fractionation and monitored by ion-specific mini-electrode technique, were found to extrude Ca2+ upon addition of Na+ ions. These findings demonstrate the presence of Na+/Ca2+ antiports in the odontoblast plasma membrane as well as in the odontoblast mitochondrial inner membrane. In addition, evidence was found for an ATP-dependent Ca2+ ion extrusion across the plasma membrane in these cells.

Ultrastructural localization and gradient of activity of alkaline phosphatase activity during rodent odontogenesis.

The ultrastructural localization and gradient of activity of alkaline phosphatase were studied with respect to cell differentiation, matrix synthesis, and matrix mineralization in the incisor and molar teeth of 4-day-old Sprague-Dawley rats. The animals were perfused intracardially at room temperature with 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4) with 3-4% sucrose. The jaws were dissected, immersion-fixed for 24 h, and the incisor and molar tooth germs removed. These were determined in 10% EDTA in NaOH (pH 7.4) with 7% sucrose. After reactivation of the enzyme with 0.1M MgCl in Tris-maleate buffer (pH 7.4) at 4 degrees C, the medium consisting of 6 ml 3% sodium beta-glycerophosphate, 4 ml 0.2M Tris-HCl buffer (pH 9.2), 3 ml 1.6% MgSO4, 12 ml 0.5% lead citrate (pH congruent to 12), and 2.1 g sucrose. The pH was adjusted to 9.2 with 0.2M HCl, the volume made up to 30 ml, and the solution centrifuged for 10 min at 5000 rpm. Control teeth were incubated in medium minus the substrate. Finally, the specimens were routinely post-fixed and embedded for sectioning and examination with a Philips 300 electron microscopy. A gradient of alkaline phosphatase activity was mapped along the developing teeth in the cells of the stratum intermedium, the proximal borders of the ameloblasts, the early dentine matrix, the predentine-dentine border, matrix vesicles, and the plasma membranes of odontoblasts and subodontoblast cells. The gradient of alkaline phosphatase activity was evident in the forming tooth from the cervical loop to the crown apex and was related to the cellular events, matrix synthesis, and matrix mineralization occurring during odontogenesis.

Ultrastructural and cytochemical studies on the matrix vesicle calcification in the teeth of the killifish, Oryzias latipes.

Tooth germs of killifish were examined by both ultrastructural and cytochemical methods. The ultrastructure of the dentin in the early calcification stage of its development resembled that of mammals. Numerous extracellular membrane-bounded vesicles, matrix vesicles, were seen in the predentinal space, especially abundantly in the basal portion of the tooth germ. They were more numerous and more uniform in appearance than in any other higher vertebrates. Where calcification was going to start, slightly identifiable needles of apatite crystals were found in the vesicular structures. Crystals increase in number filling up the vesicles and then ther surroundings, finally to form many calcified spherules. Where calcification was more extensive, the matrix vesicles were no longer visible having been buried calcified among spherules. Contrary, to popular belief, there was no relationship seen between collagen fibrils and the initial deposits of minerals, that is, collagen fibrils did not seem to work as the initial site of calcification. Instead, matrix vesicles and plasma membranes of odontoblasts likely were involved in calcification as they both showed alkaline phosphatase activity. The ultrastructural and cytochemical findings from this study thus indicated that calcification of the killifish tooth germs was initiated not in the enamel, but in their dentin by the matrix vesicles which were liberated into the dentin matrix from odontoblasts mainly by way of fragmentation of cell processes.