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Biomedical subjects

M L Couble

Publications and source records attributed to M L Couble.

At least 19 recordsLinked to original sources
PubMed1995

Ultrastructural characterization of mesenchymal and epithelial cells co-cultured from human dental root apical explants.

Previous studies have shown the role of cell-cell and cell-matrix interactions in the differentiation of the specific secretory cells of the tooth. In order to elucidate the mechanisms implicated in root dentin formation, we developed a co-culture system of human pulpal mesenchymal and epithelial root sheath cells. Root tips of premolars were cultured in Eagle's basal Medium supplemented with fetal calf serum, ascorbic acid, antibiotics and, for some of them, with sodium beta-glycerophosphate. After 60 days of culture, cells were prepared for light and electron microscopy. Three main cell types were observed: (1) polygonal mesenchymal cells showing a functional polarity and producing a dense network of tactoid collagenous fibers. The latter had a specific circular organization that delimited small lacunae around the cells and mineralized in the presence of beta-glycerophosphate; (2) spindle-shaped mesenchymal cells mainly localized inside epithelial-mesenchymal knots and synthesizing an abundant collagenous matrix; and (3) epithelial cells lying on the plastic culture dish, on the dense collagenous matrix, or on spindle-shaped cells. Epithelial cells deposited a structured basement membrane when they were lying on the plastic culture dish or on spindle-shaped cells. On the contrary, no basement membrane was found when epithelial cells were overlying the dense collagenous network. Immunoelectron microscopic analysis of type IV collagen and laminin indicated that these two specific basement membrane components were produced by all cell types. These results show that the co-culture system should be valuable for (1) studying the in vitro formation of human dental root hard tissues, (2) characterizing cell-cell and cell-matrix interactions implicated in dental basement membrane production, and (3) isolating populations of cells implicated in dental root formation.

Adolescent↗
PubMed1994

Qualitative study of collagenous and noncollagenous glycoproteins of the human healthy keratinized mucosa surrounding implants.

The purpose of this study was to analyse the distribution of interstitial collagenous and noncollagenous glycoproteins of keratinized mucosa surrounding successful endosseous implants. Biopsies were incubated with highly purified antibodies against types I, III, IV collagen, laminin and fibronectin and routinely observed by immunofluorescence staining. Whereas no significative difference in the distribution of collagenous components was observed in comparison with healthy human gingiva, the collagen fibers of the connective tissue attachment ran parallel to the long axis of the implant. In 50% of the biopsies the gingival connective tissue underlying the junctional epithelium was rich in inflammatory cells and poor in collagenous components. However, the increased staining of type III collagen and the intense presence of fibronectin in this area reflect the very important remodeling ability of the local keratinized mucosa.

Collagen↗
PubMed1991

Morphological and immunocytochemical characterization of cultured rat incisor cervical epithelial cells.

Epithelial cells from the cervical loop of the rat incisor were isolated by co-culture of apical explants with growth-arrested 3T3 fibroblasts. The epithelial phenotype of the expanding outgrowths was confirmed 10 days after the seeding of the explants by phase-contrast microscopy and immunocytochemical identification of cytokeratins. After 3 weeks in culture, the epithelial cells covered the entire surface of the coverslips and were then passaged. Subcultures gave rise to a confluent sheet within 10-12 days. Light and electron microscopy showed that confluent cervical epithelial cells generally reconstituted a bi-layered structure similar to Hertwig's epithelial sheath. Epithelial cells from the rat palate, cultured and subcultured according to the same procedure, organized themselves in 5-6 cell layers, the upper cells having generally a squamous morphology. Synthesis of extracellular matrix molecules by rat incisor cervical epithelial cells was studied with specific antibodies. These cells failed to produce type I collagen, but synthesized all the major basement membrane components (type IV collagen, laminin, heparan sulphate proteoglycan and fibronectin). These observations suggest that the culture conditions allowed the reconstitution of a typical Hertwig's epithelial sheath by rat incisor cervical epithelial cells.

Animals↗
PubMed1991

Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium hydroxide-containing cement.

The cement produced microcrystals of calcite by reaction with culture medium supplemented with calf serum. Human dental pulp cells seeded on such a substrate preferentially adhered and aggregated around the microcrystals. Immunofluorescence and immunogold labelling revealed a high affinity of serum fibronectin molecules for the calcite crystals. At 4 weeks in culture, the cells had various features of differentiated odontoblasts, notably nuclear polarization, typical appearance of the Golgi apparatus, synthesis of type I collagen and absence of type III, and apical accumulation of actin and vimentin. These cells also elaborated a collagenous extracellular matrix which did not mineralize.

Adolescent↗
PubMed1991

Differential expression of type I and type III collagen genes during tooth development.

Collagen gene expression during mouse molar tooth development was studied by quantitative in situ hybridization techniques. Different expression patterns of type I and type III collagen mRNAs were observed in the various mesenchymal tissues that constitute the tooth germ. High concentration for pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were found within the osteoblasts. We found that the cellular content of type I collagen mRNAs in the odontoblasts varies throughout the tooth formation: whereas mRNA concentration for pro-alpha 1(I) collagen decreases and that of pro-alpha 2(I) increases, during postnatal development. Moreover, different amounts of pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were observed in crown and root odontoblasts, respectively. Type III collagen mRNAs were detected in most of the mesenchymal cells, codistributed with type I collagen mRNAs, except in odontoblasts and osteoblasts. Finally, this study reports differential accumulation of collagen mRNAs during mouse tooth development and points out that type I collagen gene expression is regulated by distinct mechanisms during odontoblast differentiation process. These results support the independent expression of the collagen genes under developmental tissue-specific control.

Animals↗
PubMed1991

Isolation and characterization of rat alveolar bone cells.

Samples of rat alveolar bone were first treated by collagenase digestion and then used as explants for cell culture. The cells obtained were subcultured and characterized by morphological and functional criteria. Their alkaline phosphatase activity was increased after incubation in 1,25-(OH)2 vitD3 10(-8) M whereas with gingival cells it did not change. The bone derived-cells organized nodular structures, synthesized type I collagen, Gla-protein, few type III collagen, and fibronectin. In the defined culture conditions no mineralization was observed. However, the method used allows to obtain cells from rat alveolar bone displaying some features of the osteoblastic phenotype.

Alveolar Process↗
PubMed1990

Expression of fibronectin and type I collagen by human dental pulp cells and gingiva fibroblasts grown on fibronectin substrate.

Specific antibodies and indirect immunoperoxidase labelling were used to study the intracellular production of collagen and fibronectin by cells grown on fibronectin-coated glass; the same cell populations seeded on uncoated glass were used as controls. Strong intracellular staining for type I collagen was seen in all cases, but immunostaining for fibronectin was very faint or negative in both gingival and pulp cells grown on the fibronectin substrate, in contrast to control cells. Thus, fibronectin substrate inhibited fibronectin synthesis by the cultured cells, but did not seem to influence type I collagen synthesis.

Cells, Cultured↗
PubMed1990

Ultrastructural and immunocytochemical study of bone-derived cells cultured in three-dimensional matrices: influence of chondroitin-4 sulfate on mineralization.

Bone-derived cells were cultured in three-dimensional reconstituted matrices made of type I collagen or type I collagen chondroitin-4-sulfate. As observed by microscope, their characteristics were as follows: The cells deposited a faint extracellular matrix mainly composed of type I collagen. In the collagen-chondroitin-sulfate sponge fibers, a calcification process, which involved the deposition of hydroxyapatite crystals, was demonstrated. Mineralization occurred only in collagen chondroitin sulfate sponge fibers when seeded with bone-derived cells and was not seen with nonosteogenic cells, such as gingival fibroblasts. Gla protein was intracellularly visualized in both types of sponges seeded with bone-derived cells while an extracellular secretion was seen only in the collagen chondroitin sulfate sponge fibers where calcification occurred. These results suggest that collagen chondroitin sulfate promotes in vitro mineralization of three-dimensional collagen matrices when seeded with bone-derived cells.

Animals↗
PubMed1989

Immunohistochemical study of types I, III and IV collagen in diseased human gingiva of patients with rapidly progressive periodontitis: a light and electron microscopic study.

The distribution of type I, III and IV collagens and their ultrastructural organization have been studied in diseased gingival connective tissue of patients with rapidly progressive periodontitis. This disease is characterized by acute destruction of the gingival collagenous components. The use of an immunofluorescent procedure has shown that the diseased connective tissue was made up of both type I and III collagens but that type III collagen was less resistant to acute inflammation. Ultrastructural immunolabelling, using the peroxidase procedure has shown that the large, dense bundles of type I collagen of PI, the main pattern of organization of the gingival connective tissue offered a better resistance to acute destruction than PII, a loose pattern of organization mainly composed of type III collagen. Type IV collagen was exclusively located in degraded lamina densa of basement membrane.

Adult↗
PubMed1988

Distribution and organization of the elastic system fibres in healthy human gingiva. Ultrastructural and immunohistochemical study.

The ultrastructural distribution and organization of the elastic system fibres, i.e. oxytalan, elaunin and elastic fibres, were studied by transmission electron microscopy and by an immunohistochemical method for the detection of elastin in healthy human gingiva. The morphological distribution of these fibres was characterized by the presence of oxytalan, elaunin and elastic fibres, respectively, in the upper, medium, and deep layers of gingival connective tissue. Anti-elastin antibody reacted with microfibrils and amorphous material of the elastic system fibres throughout the gingival connective tissue. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin at their surface.

Adult↗
PubMed1987

Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silkgland.

Four mRNA of 10.5, 9.0, 4.0, and 2.8 kb are made from the sericin Ser1 gene by alternative maturation of a unique mRNA precursor. By means of RNA blots and in situ hybridization, we investigated variations in the distribution of these mRNA during the last larval instar in different territories of the middle silkgland. Taken together, the results from these two techniques show that 150 out of the 266 cells of this region of the organ express the Ser1 gene, but accumulate distinct mature mRNA species. Of these 150 cells 42 are specialized in a processing pathway resulting in the production of the 2.8-kb Ser1 mRNA throughout the larval instar. The 108 others perform successively three distinct splicing pathways leading to a development-dependent accumulation of, respectively, the 4.0-, the 10.5-, and the 9.0-kb mRNA. This suggests the occurrence of two switches in the splicing capacities of these cells during the fifth instar. The middle silkgland cells also express another sericin gene (Ser2) which encodes two mRNA of 5.4 and 3.1 kb, also arising by differential splicing. At the beginning of development, all the middle silkgland cells express this gene but, as development proceeds, expression becomes restricted to only the anterior cells. The biological consequence of this topological and temporal regulation of the mode of expression of these two genes is the sequential secretion and layering of the different sericins around the silk thread.

Animals↗
PubMed1987

Evaluation of the biocompatibility of a Ni-Cr-Mo dental alloy with human gingival explant culture in vitro: morphological study, immunodetection of fibronectin, and collagen production.

This study was undertaken to evaluate the biocompatibility of a Ni-Cr-Mo dental casting alloy by an in vitro explant culture method. Through ultrastructural features, the immunolabelling of fibronectin, and the immunodetection of type I and III collagen production, we investigated the behaviour of gingival cells (both fibroblasts and epithelial cells) in contact with the alloy in comparison with control cultures. Our results indicate that cultured gingival cells present a well preserved ultrastructure and synthesized fibronectin (the main glycoprotein involved in adhesion to substrates). The alteration of collagen production concerned only type III collagen which decreased significantly in the cultures on the dental alloy.

Cells, Cultured↗
PubMed1986

Ultrastructure of multilayered cultures of human gingival epithelial cells: attachment to enamel surfaces in vitro.

Human gingival cells were collected using an enzymatic procedure and seeded on a feeder layer of irradiated mouse 3T3 fibroblasts. Epithelial cells generate stratified colonies ultimately forming an epithelium which was studied using electron microscopy. When this epithelium-like structure was dispased and transferred to enamel surfaces, the relationship between basal cells and enamel corresponded to half-desmosomes and a discontinuous extracellular matrix.

Adult↗