Biomedical subjects

H Magloire

Publications and source records attributed to H Magloire.

At least 37 records · Page 2Linked to original sources

PubMed1996

An in vitro model of human dental pulp repair.

Pulp tissue responds to dentin injury by laying down reactionary dentin secreted by existing odontoblasts or reparative dentin elaborated by odontoblast-like cells that differentiated from precursor cells in the absence of inner dental epithelium and basement membrane. Furthermore, growth factors or active dentin matrix components are fundamental signals involved in odontoblast differentiation. In vitro, dental pulp cells cultured under various conditions are able to express typical markers of differentiation, but no culture system can re-create pulp response to dentin drilling. This paper reports the behavior of thick slices from human teeth drilled immediately after extraction and cultured from 3 days to 1 month. Results show that the damaged pulp beneath the cavity is able to develop, in vitro, some typical aspects correlated to tissue healing, evidenced by cell proliferation (BrdU-positive cells), neovascularization (positive with antitype-IV collagen antibodies), and the presence of functional (3H proline-positive) cuboidal cells close to the injured area. After 30 days of culture, elongated spindle-shaped cells can be seen aligned along the edges of the relevant dentin walls, whereas sound functional odontoblasts are well-preserved beneath healthy areas. This tissue recovery leads us to believe that such a culture model will be a useful system for testing factors regulating pulp repair.

Adolescent↗

PubMed1995

Immunocytochemical localization of fibronectin and a 165-kDa membrane protein in the odontoblast layer under initial carious lesions in man.

The possible role of fibronectin in dental tissue repair was investigated by comparing its distribution and that of the 165-kDa fibronectin-binding membrane protein (165 kDa-FnBP) in odontoblasts underlying carious and sound dentine. By immunoperoxidase and light microscopy, fibronectin was localized in the dentine underlying the carious lesion, mainly on the surface of the tubule walls, whereas it could not be detected in neighbouring sound zones. The antibody to the 165 kDa-FnBP strongly reacted with the membrane of odontoblasts underlying the lesion, although those facing sound dentine did not express this antigen. Ultrastructurally the 165 kDa-FnBP was localized in the cell membrane at the apical portion of odontoblasts, including the process membrane, beneath the initial lesion; fibronectin was detected in the dentinal area close to the process, and also in contact with its external surface. By a high-resolution immunogold procedure, the proteins were colocalized at the external surface of odontoblast processes. These data suggest that fibronectin present in human carious dentine could modulate the behaviour of underlying odontoblasts by means of newly expressed 165 kDa-FnBP.

Adhesins, Bacterial↗

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

Immunohistochemical localization of L-type calcium channels in the developing first molar of the rat during odontoblast differentiation.

To study the presence of L-type calcium channels during the different steps of odontoblast differentiation, a specific monoclonal antibody against 1,4-dihydropyridine receptors was used in combination with avidin-biotin-peroxidase complex labelling. Staining was seen on the cell bodies of pre-odontoblasts, concentrated at the apical pole (distal portion) of functional odontoblasts and localized on cell bodies and processes of mature odontoblasts. Thus calcium channels were expressed at the onset of differentiation and maintained in the differentiated cells but with some changes of localization. It is suggested that these channels may facilitate the entry of calcium to act as a second messenger for cellular polarization or be involved in the transcellular transport of calcium to the mineralizing front.

Animals↗

PubMed1993

Selective inhibition of collagen synthesis by fluoride in human pulp fibroblasts in vitro.

Human dental pulp cells were cultured in fluoridated mediums (0, 1, 10, 25 ppm) in order to study the biological effect of the ion regarding the cellular metabolism: cell growth, alkaline phosphatase (ALP) activity, and protein synthesis. The results indicated a decrease of the cell growth at 25 ppm and a dose-dependent decrease of the ALP activity. Type I collagen immunoperoxidase staining, radioimmunoassay quantitation, and analysis of type I and III collagens mRNA levels showed an inhibition of collagen production and gene expression. In contrast, fibronectin production and gene expression were not affected by fluoride. The treatment did not influence the qualitative pattern of the different mRNA species. Of the three collagen chains, the alpha 1(I) was the most affected. These data suggest that fluoride does not exert a general depletive effect on human dental pulp cells but rather a selective inhibition on collagen production.

Adolescent↗

PubMed1993

Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar.

Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of non-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75NGFR and p140prototrk) on responsive cells. The continuously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF, proNGF and p75NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75NGFR was correlated with differentiation event only in dental mesenchyme: polarizing odontoblasts expressed p75NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.

Animals↗

PubMed1993

[Response of odontoblastic and pulpal cells to carious lesions].

The odontoblast responds to caries by the formation of sclerotic as well as reparative dentin. Sclerotic dentin is deposited during the early stages of the dentinal injury. It is characterized by the amplification of the collagen synthesis and the increase in alkaline phosphatase activity in the odontoblastic cell layer. Reparative dentin will be deposited under the sclerotic zone after the destruction of odontoblasts. At this stage, specific components from damaged dentinal tissues and/or odontoblastic necrotic debris will trigger pulpal cells to elaborate a cartilage-like matrix layer (fibrodentin). The latter may induce pulpal odontoblast-like cells to give rise to the tubular reparative dentin. Thus, pulpal cell response seems to be similar to bone-cell response to injury. Molecular signals responsible for this tissular healing remain largely unknown, but dentin is a potential source of matrical or soluble organic molecules that may be released after demineralization. Some of these factors have been identified in the sound tissue (glycoproteins, proteoglycans, growth factors, ...), but their role in the stimulation of the elaboration of the cicatricial tissue remains to be elucidated.

Cell Differentiation↗

PubMed1992

Immunohistochemical localization of nerve growth factor (NGF) and NGF receptor (NGF-R) in the developing first molar tooth of the rat.

Nerve growth factor (NGF) is a well established target-derived trophic factor supporting sympathetic and sensory innervation in the peripheral tissues as well as cholinergic innervation in the brain. Despite its name, NGF may have broader biological functions early in development in a wide range of non-neuronal differentiating cells. The many effects of NGF are directly dependent on initial binding of NGF to specific plasma membrane receptors on target cells. Here we use immunohistochemical methods to show that NGF and its receptor (NGF-R) are localized in a variety of embryonic epithelial and mesenchymal cells in the rat developing molar tooth. Dental cells known to play important roles in morphogenesis and inductive tissue interactions show NGF-like reactivity. Thus, labelling is seen in epithelial preameloblasts and mesenchymal odontoblasts. We also show a transient expression of NGF-R in restricted parts of the dental epithelium (inner dental epithelium) and dental mesenchyme differentiating cells (post-mitotic, polarizing odontoblasts). The expression patterns of NGF are different to those of NGF-R during embryogenesis and this is illustrated in detail in the developing tooth. The histochemical findings reported here support the notion that NGF may have multiple roles during morphogenetic and cytodifferentiation events in the tooth.

Animals↗

PubMed1992

Odontoblast response under carious lesions.

The local regulation of odontoblast response to caries is viewed through initiation and elaboration of sclerotic as well as reparative dentin. Dentin tissue represents a multiple source of potent environment factors when teeth are affected by the demineralization phases of carious process. Some of them have already been identified in sound tissue (matrix glycoproteins, proteoglycans, growth factors, Bone Morphogenetic Protein) and may act on the cell through membrane receptors. Thus, the amplification in collagen synthesis and alkaline phosphatase activity previously observed during sclerotic dentin deposition can be related to the interaction between matrix signals and cell receptors such as the 165 kDa protein shown only by odontoblasts under the affected zone. Similarly, under established lesions generating cell death, the specific matrix made of odontoblasts debris and damage tissues, probably rich in active molecules, may trigger pulp cells to elaborate a cartilage-like layer (identified by type II and XI collagen) followed by odontoblast-like cells to give rise to abnormal tubular dentin. Here, odontoblast response is identical to bone-cells response to injury. What remains to be elucidated concern: The nature of signals found in carious dentin (matrix components, growth factors, bacterial products). The nature and regulation of expression of cell membrane receptors during tooth repair. How the odontoblast produces specific responses to each of these signaling molecules will be the focus of important new investigations.

Cell Differentiation↗

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

Immunoblotting and cytochemical characterization of human enamel proteins.

Mature enamel proteins (tuft proteins) and fetal enamel proteins were extracted by an homogenizing buffer method, subjected to SDS-PAGE and immunoblotted with a polyclonal antibody raised against the mature enamel proteins. Both fetal and tuft proteins were recognized by this immunoblotting. With the same antibody, immunolocalization of the developing enamel proteins was done on semi-thin-sections of human fetal tissue at the secretory stage, using an immunoperoxidase technique. Specific labelling of the enamel protein matrix was observed. It is concluded that a polyclonal antibody against mature enamel proteins (anti-tuft) can recognize the developing protein matrix at the secretory stage. This suggests that a common antigenic determinant is maintained throughout the course of amelogenesis in human enamel.

Adult↗

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↗

PubMed1991

Extracellular matrix and intermediate filaments in the first stages and repair of experimental gingivitis in man.

By the indirect immunoperoxidase labelling procedure the expressions of type I and III collagens, laminin and fibronectin and of KL1 cytokeratin and vimentin were examined in the first stages and repair of experimental gingivitis in young subjects. A two month longitudinal study was performed using the tissue from buccal marginal gingival biopsies of four subjects taken sequentially at five specific times: before and during plaque accumulation, and during plaque elimination. The sites examined microscopically were the coronal half of the junctional epithelium and the underlining infiltrated connective tissue fraction. No clinical change could be observed during the study period. Histological examination showed reversible cellular changes during the accumulation of plaque. There were increases in vascularization and cellularity and loss of collagen. They recovered 56 days after plaque elimination their baseline level. Electron microscopic examination showed myofibroblastic aspects in some fibroblasts. The changes in the expression of laminin, fibronectin and KL1 in the J.E. might be due to a proliferation rate enhancement, and suggest an adaptation to the alterations brought about by the inflammatory process. They also reinforce the hypothesis that this epithelium resembles a developmental tissue. Type I collagen demonstrated the "collagen loss-repair" cycle shown in connective tissue by the histological study. The rise in type III collagen and the vimentin fall, both at the initial stage, suggest that these protein profiles may yield information for clinical research purposes during the very early inflammatory process. The variations in fibronectin indicate its key role in the early inflammatory and repair processes. Finally, the variations in matrix and cytoskeletal proteins variations, as well as the morphological modifications observed, were nearly all reversible.

Adolescent↗

PubMed1991

[A model of an in vitro biological assay controlled by immunofluorescence and scanning electron microscopy].

This study was designed to complete the cell culture test for the cytotoxicity assessment of dental material presented by Christen et al. (1989) and Regad et al. (1989). Two liner materials were selected for this experiment which had already been subjected to both in vivo and in vitro tests. After 2, 7 and 14 days of contact with the test materials, the cell responses were evaluated under the light microscope by their growth pattern and morphological aspects. Cell synthesis of fibronectin and type I and III collagen was studied by immunofluorescence. Surface appearance of the cells in contact with the materials as well as their structures were observed by scanning electron microscopy. This study confirmed the viability of the in vitro cytotoxicity assessment methods with cell cultures. The results also demonstrated the potentials of this method in the early screening of dental materials for their biological compatibility.

Acrylic Resins↗

PubMed1990

In vitro mineralization of a three-dimensional collagen matrix by human dental pulp cells in the presence of chondroitin sulphate.

These matrices were used as cell culture substrates to investigate the influence of extracellular molecules on mineralization. Pulp cells seeded in type I collagen or type I collagen-chondroitin-4-sulphate sponges were able to grow and were morphologically similar to cells responsible for reparative dentine formation in vivo. In sponges consisting of collagen only, the cells elaborated an abundant new matrix which became organized with time and consisted of collagen fibres surrounded by fibrillar material, but no mineralization was observed. In collagen-chondroitin sulphate sponges, cells deposited less and poorly organized matrix; in these, calcification occurred, increasing with time, and at the ultrastructural level, small needle-like crystals containing calcium and phosphorus were scattered throughout the sponge fibres. These observations suggest that chondroitin sulphate might influence in vitro calcification induced by pulp cells.

Cells, Cultured↗

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↗