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D Septier

Publications and source records attributed to D Septier.

At least 55 records · Page 3Linked to original sources

Dental mineralization.

Extracellular matrix components and cell-derived microstructures are implicated in mineralization processes which occur in dental tissues. The respective role(s) of collagenic and non-collagenic matrix components are reviewed: phosphorylated and non-phosphorylated proteins, proteoglycans and phosphpholipids. Space-filling amphiphilic molecules seem to play an important role in the preorganization and oriented deposition of calcium phosphate on structures serving more or less as passive support in dentine as well as in enamel.

Amelogenesis↗

Iodoplatinate visualization of phospholipids in rat incisor predentine and dentine, compared with malachite green aldehyde.

The iodoplatinate (IP) reaction, a selective method for visualization of phospholipids, was applied to the predentine and dentine of rat incisors and compared with malachite green aldehyde (MG) fixation/staining. Spot tests indicated (1) that IP specifically stains phospholipids, but not amino acids, displaying as do phospholipids, quaternary ammonium groups; and (2) phosphatidylserine and sphingomyelin were also stained by MGA. Although this reagent is known to interact with phosphorus, phosphoproteins remained unstained. In the rat incisor, an IP-positive network including granules and thin filaments was seen in predentine in the inter-collagen spaces, in many cases closely associated with collagen fibres and their periodic striations. In dentine, positively stained needle-like structures were located along individual collagen fibres, or at the surface of groups of collagen fibres. This staining pattern was unchanged on sections of material pretreated with acetone, whereas the staining was abolished or markedly reduced when the samples were treated either with chloroform/methanol or phospholipase C prior to the IP reaction. Pretreatment of the samples with hyaluronidase promoted subsequent diffusion of the staining. A very similar staining pattern was observed with MGA, in accordance with earlier reports. The present findings validate the histochemical results reported previously on the distribution and potential role(s) of phospholipids in dentine biomineralization.

Animals↗

Cuprolinic blue visualization of cytosolic and membrane-associated glycosaminoglycans in the rat junctional epithelium and gingival epithelia.

The gingiva of rat molars was studied at the light microscope level using glutaraldehyde as fixative, Cuprolinic Blue for visualizing polyanionic glycosaminoglycans and the autometallographic technique for enhancing the copper signal of the cationic dye. The polyanions were located inside the epithelial cells in the junctional epithelium, whereas a network located along either the plasma membrane or the intercellular spaces, or both, of the gingival oral epithelium and sulcular oral epithelium was evident with autometallography. In these cases, positive staining was limited to the basal and spinous layers, the granular and keratinized layers being unstained. With the transmission electron microscope, electron-dense aggregates were seen in the gingival lamina propria, in the basement membrane and along the plasma membrane of the keratinocytes of the basal and spinous layers of the gingival and sulcular oral epithelia. In the junctional epithelium, Cuprolinic Blue-positive granules, 25 nm in diameter, were seen in the cytoplasm. Together with some vesicles containing electron-dense material, they may account for the staining process noted after autometallography. When the ultra-thin sections were digested with bovine testicular hyaluronidase, the staining was abolished. This indicates that glycosaminoglycans were primarily responsible for the staining pattern visualized with these methods. In the junctional epithelium, the cytosolic location of the 25 nm granules reflects either transcellular transfer between the plasma membrane and the nucleus or accumulation of glycosaminoglycans in this group of keratinocytes. The glycoconjugates located inside vesicles or vacuoles are related to endocytosis and lysosomal degradation. Interstitial glycosaminoglycans seen in the two types of oral epithelium may play a role in the diffusion of water and nutriments.

Animals↗

Suramin-induced mucopolysaccharidosis in rat incisor.

Two weeks after a single injection of suramin, the secretory and post-secretory ameloblasts of the rat incisor were filled with large lysosome-like vacuoles. At the light-microscope level, these vacuoles were positively stained with Alcian blue when MgCl2 was used at a critical electrolyte concentration varying between 0.1 and 0.3 M, whereas no staining appeared when MgCl2 varied between 0.7 and 0.9 M. Hyaluronidase digestion markedly reduced but did not totally abolish the staining, indicating that glycosaminoglycans were accumulated inside these vacuoles. Examination of these cells with the electron microscope revealed a polymorphic population of large vesicles, filled to various degrees with cetylpyridinium chloride (CPC)-positive and malachite green aldehyde (MGA)-positive material. The same pattern was observed in secretory odontoblasts but to a lesser extent. In the extracellular matrix, suramin-induced alterations appeared as large defects occurring during enamel formation. In predentin and dentin, the number and/or size of electron-dense aggregates resulting from CPC and MGA fixation, were enhanced in the suramin-injected rats. These aggregates were largely reduced or suppressed respectively by hyaluronidase digestion and chloroform/methanol treatment of the sections. The accumulation of glycosaminoglycans and phospholipids reported here inside ameloblasts and odontoblasts and in predentin and dentin supports the occurrence of suramin-induced mucopolysaccharidosis and lipidosis in this experimental animal model.

Ameloblasts↗

[Proteoglycans-phospholipids interactions: roles in dentine mineralization].

Electron-histochemical visualization of proteoglycans was carried out in the predentine and dentine of rat incisors. Using various techniques proteoglycans were seen to be located between the collagen fibres in predentine, whereas they were observed at the surface of groups of collagen fibres in dentine. The same distribution was found when electron histochemical techniques aiming to visualize phospholipids were used. This co-distribution may play role in the mineralization processes.

Animals↗

[Lysosomal storage diseases, genetic or drug-induced? effect of glycosaminoglycan and sphingolipid disorders on dental tissues].

In vivo studies were carried out on dental tissues of rat incisor after a single injection of suramin, a drug which induces mucopolysaccharidosis-like disease. Accumulation of lysosome-like structures was seen in secretory ameloblasts and odontoblasts. In vitro studies on embryonic tooth germ buds showed similar changes when they were cultured in presence of suramin. Anti-phospholipid immunolabelling revealed a developmentally regulated temporo-spatial pattern. Radiolabeling with 3H-suramin indicated cytosolic and nuclear incorporation. The drug acting as polyanion interacted directly with predentine. 35S sulphate incorporation was impaired by the drug. Another lysosomal storage disease, the sphingolipidosis, Krabbe's disease was also investigated in human. Changes were observed in pulp cells and as a consequence in dentin. Enamel also displayed many changes. Pharmacological or genetically acquired diseases constitute models providing insights on the role played by glycosaminoglycans and phospholipids in biomineralization.

Ameloblasts↗

Localization of malachite green positive lipids in the matrix of bone nodule formed in vitro.

An electron histochemical study was carried out on bone nodules formed in vitro in collagenase-released calvarial cells in order to visualize the lipid components of the extracellular matrix (EM). The malachite green aldehyde fixative technique, which allows both preservation and staining of some phospholipids of the extracellular matrix, was used. Controls were performed on sections demineralized, and then submitted to lipid extraction with a chloroformmethanol mixture (2/1 v/v) and to glycosaminoglycans digestion with 0.5% bovine testicular hyaluronidase to verify specificity for lipid staining. This allowed us to visualize the lipids (1) in the osteoid as granules associated to ribbon-like structures connected to the collagen fibers, (2) as electrondense deposits seen as dots on the outer surface membrane of the matrix vesicles, and (3) in the mineralized matrix as roundish patches formed of needle-shaped materials and at the mineralization front as individual ones. This study demonstrated that at the EM level, the lipids are present in the osteoid at locations very similar to what have been observed for the glycosaminoglycans, and in the mineralized matrix as components of the crystal ghosts.

Animals↗

Structural variations of different oral basement membranes revealed by cationic dyes and detergent added to aldehyde fixative solution.

The ultrastructural appearance of different types of basement membrane was studied using histochemical methods for visualizing glycosaminoglycans. Samples of rat gingiva and mouse molar germ tissue were fixed either with glutaraldehyde, glutaraldehyde-ruthenium hexammine trichloride (RHT), glutaraldehyde-Cuprolinic Blue (CB) or cetylpyridinium chloride-glutaraldehyde (CPC). Ultrathin sections were stained with uranyl acetate and lead citrate. The results showed that the conventional trilaminar structure of the basement membrane was observed after glutaraldehyde and CB fixation. In contrast, after CPC or RHT fixation, the appearance of the basement membrane was homogeneous without any evidence of a lamina lucida. Furthermore, after single fixation with CPC, the ultrastructure of different basement membranes from oral tissues showed some differences in appearance which were related to their localizations, functions, or both.

Animals↗

Ultrastructure of inter-odontoblastic fibres in the rat molar.

Radially directed inter-odontoblastic collagen fibres were observed in ultra-thin sections of the radicular pulp/predentine complex of rat molars. Bundles of fibres crossed the distal junctional complexes of the odontoblasts, went through the whole thickness of the predentine and were incorporated into the mineralized dentine. Scanning electron microscopy showed that bundles of radial fibres are not found between the coronal odontoblasts of the rat molar but only in the root. The radial fibres were inserted into the predentine along vertical long-axial crests. The intercrest interval was not tightly fixed and the pattern of fibre-bundle insertion did not show a regular periodicity.

Animals↗

Autometallographic visualization of glycosaminoglycans in the tongue mucosa of rats using cuprolinic blue and enzymatic digestions.

In order to visualize by light microscopy the glycosaminoglycans (GAGs) in the rat tongue mucosa, the tissue was fixed with cuprolinic-blue (CB)-aldehyde and the staining enhanced by autometallographic (AM) procedure. As other polyanions were also detected, enzymatic digestions with hyaluronidase, chondroitinase ABC and pronase were performed on these tissues in order to test the specificity of the staining. Chondroitinase ABC caused a dramatic decrease of silver grains in the lamina propria whereas hyaluronidase and pronase induced only discrete or no modification. This supported the concept that the GAGs visualized by CB and autometallography in this area as dermatan sulphate. The other polyanions (mostly DNA and RNA) seen in the epithelial layers were unaffected by these enzyme treatments.

Animals↗

Immunolocalization of a 110 kD molecule and a 150 kD molecule in rat incisor and mandibular bone.

In the present study, antibodies against rat dental proteoglycans were used to characterize and localize the proteoglycans in rat incisor and mandibular tissues. Polyclonal rabbit antibodies were raised against a CPC-precipitated fraction of a sulfated dental extract. In unpurified dental extract these antibodies recognized two molecules of 110 kD and 150 kD. The 150 kD molecule was susceptible to chondroitinase ABC digestion but the 110 kD molecule resisted this enzymatic degradation. Immunocytochemically these two molecules were seen to be located in the pulp, the enamel organ and the mandibular bone. In each tissue only the periphery of the cells was stained and not the intracellular compartment. In the mineralized area of bone, dentin and forming enamel no staining was seen. These results indicate common epitopes in the proteoglycans from pulp, predentin, enamel organ and bone. Some differences were found in the nature of tooth and bone proteoglycans.

Animals↗

Zinc deficiency-induced changes in the lipid composition and ultrastructure of rat incisor teeth.

The lipids and ultrastructure of the forming, maturing and erupted parts of incisors were compared in rats fed a zinc-deficient diet for 28 days, pair-fed rats and control rats. The lipid levels in the forming portions of zinc-deficient incisors were 30-50% below control levels and were associated with longer Tomes' processes in the secretory ameloblasts, porosities in the forming enamel and fewer malachite green-aldehyde-phospholipid aggregates in the predentine. No marked structural changes were seen in the erupted portions of the teeth, although variations in lipid composition were detected both in the maturing and erupted parts. No differences were found between teeth from the 3 groups of animals for Na, Mg, Cl, Ca and P signals with the electron microprobe.

Animals↗

Visualization of glycosaminoglycans in rat incisor predentin and dentin with cetylpyridinium chloride-glutaraldehyde as fixative.

Using cetylpyridinium chloride (CPC) in glutaraldehyde as fixative, we observed sinuous fiber-like structures 300-500 nm long and 7-14 nm thick in the spaces between the collagen fibers of rat incisor predentin. Small granules and fibrils were also detected. Electron-dense vesicles were seen inside the odontoblast processes. The plasma membrane was irregularly stained with material that adhered to its surface. In demineralized dentin, needle-like structures were seen at the periphery of globular structures which were not stained. Staining the sections with Alcian blue did not greatly improve the visualization of CPC-precipitated glycosaminoglycans. The specificity of staining was assessed on serial sections by selective dissociation of glycosaminoglycan aggregates with 2 M calcium chloride and their digestion by bovine testicular hyaluronidase. The glycosaminoglycans were probably combined with lipids, because treatment of the sections with a chloroform/methanol mixture removed the CPC-induced precipitates from both predentin and dentin.

Aldehydes↗

A radioautographic comparison of in vivo 3H-proline and 3H-serine incorporation in the pulpal dentine of rat molars: variations according to the different zones.

3H proline and 3H serine were injected intraperitoneally to rats which were killed 4 and 24 hours later. The incorporation of the labelled precursors was studied in the odontoblasts, predentine and dentine of the first lower molars. After radioautography, statistically significant differences in grain density appeared between the furcation, occlusal and lateral areas of the pulp chamber. The incorporation of predentine components into dentine was faster in the lateral than in the occlusal area, the slowest being detected in the furcation area. These differences may be related to the distribution of occlusal forces in the rat molar.

Animals↗

Effect of tunicamycin on glycogen accumulation in the stratum intermedium and odontoblasts of rat incisor.

Repeated injection of rats with tunicamycin over two days induced a 1- to 5-fold increase in glycogen. This accumulation occurred in the stratum intermedium of the enamel organ and in young secretory odontoblasts. In rats injected over 3 days, the number of glycogen particles was at least 10 times larger than in control rats, and large glycogen accumulations were observed in the cytosol of these two groups of cells. These results were obtained by staining with periodic acid-thiocarbohydrazide and silver proteinate, a specific method for the detection of glycoconjugates containing vic-glycol groups. The existence of a relationship between these local cytosolic accumulations of glycogen and the developmental stage of certain groups of cells was shown by the changes that occurred in glycogen distribution. The present results suggest that the stratum intermedium supplies energy for precursor transport.

Animals↗

A comparative ultrahistochemical study of glycosaminoglycans with cuprolinic blue in bone formed in vivo and in vitro.

Histochemical and morphological studies have shown that proteoglygans (PG) are involved in mineralization process in vivo but such studies have not yet been conducted in vitro. A comparative histochemical study in electronic microscopy of the localization, organization, and morphology of the PG was performed with bones of calvaria rat formed in vivo and bone nodules formed in vitro from osteoblastic cells in culture. For this investigation, we used a cationic phthalocyanin dye, cuprolinic blue, in a critical electrolyte concentration which simultaneously stained the glycosaminoglycans and demineralized the bone. This histochemical technique demonstrated (1) osteoblast cells in vitro synthesized PG which were included in the matrix formed. (2) These PG were found in the calcified and uncalcified matrix both in vivo and in vitro. In the uncalcified matrix, PG were either free with a granular or rodlike structure or tightly connected to the periphery of the collagen fiber. Contrarily, in the calcified matrix, PG formed dense filamentous reticular patches between the collagen fibers. (3) Similarities in localization, organization, and morphology were noted in PG of bone formed de novo in vitro and in vivo with the exception of the mineralization front, where the staining in vivo compared with in vitro was faint or absent.

Animals↗

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue.

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.

Animals↗

Visualization of predentine matrix components and endocytic structures in rat incisor odontoblasts with tannic acid.

Rat incisor odontoblasts and predentine fixed with tannic acid-glutaraldehyde-osmium tetroxide (Tago) were compared with those obtained by prior incubation in tannic acid-Ringer before conventional fixation with glutaraldehyde-osmium-tetroxide (Tari) The Tago method allowed visualization of complex glycoconjugates along the plasma membrane, in the pericellular spaces and in the intercellular predentine matrix. The non-collagenous proteins, proteoglycans and lipids were seen as granules and thin filaments located between the collagen fibers and at their surface. The collagen fibers themselves were also stained. The Tari method which was used to visualize exocytosis, mainly revealed endocytosis in the form of large intracellular vacuoles containing tannic acid and stained proteoglycans. It is suggested that tannic acid-Ringer incubation prior to fixation increases the endocytosis of the matrix components, which acculumates in these large vesicles.

Animals↗