Effects of beta-D-xyloside on morphogenesis and cytodifferentiation in cultured embryonic mouse molars.

Embryonic mouse molars were grown on a semi-solid medium supplemented with 2 mM beta-D-xylopyranoside (beta-xyloside), a specific inhibitor of proteoglycan synthesis. The induced glycosaminoglycan depletion in the extracellular matrix was monitored by immunohistochemistry employing monoclonal antibodies to chondroitin 4- and chondroitin 6-sulfates. beta-Xyloside inhibited formation of the dental bell and delayed the appearance of the first odontoblasts. Odontoblast functional differentiation proceeded in the absence of chondroitin sulfate in the basement membrane. Predentin secreted in the presence of beta-xyloside triggered the polarization of ameloblasts, but did not allow the maintenance of polarized odontoblasts. These results support the hypothesis that, in the tooth germ, chondroitin sulfate proteoglycans participate in the regulation of cell kinetic-dependent morphogenesis (Mark et al., 1990. Differentiation 43, 37-50). On the other hand, the possibility that chondroitin sulfate might play a role in odontoblast terminal differentiation is definitively ruled out.

Membrane-cytoskeleton interactions: inhibition of odontoblast differentiation by a monoclonal antibody directed against a membrane protein.

It is known that high-molecular-weight (HMW) membrane proteins mediate interactions with constituents of the extracellular matrix and/or with cytoskeletal elements. To study participation of HMW membrane proteins in odontoblast or ameloblast differentiation, an immunological approach has been adopted. Antibodies directed against membrane proteins (Mr, 110-190) from mouse embryos have been produced by the hybridoma technique. Supernatants of hybridoma cultures were screened for their ability to stain dental tissues and also tested for their biological activities on dental cells in primary culture or on developing tooth germs in organ culture. An IgM monoclonal antibody, MC16A16, directed against a 165-kDa antigen present in plasma membrane preparations, reacted strongly with the dental epithelium and weakly with the mesenchyme. MC16A16 also reacted with the cell surface of nonpermeabilized cultured dental cells and could detach epithelial cells cultured on glass, but not mesenchymal cells which maintained vinculin-containing focal contacts. This antibody, which affected the organization of dental-cell microfilaments in primary culture, also inhibited the polarization of odontoblasts, but not that of ameloblasts.

Monoclonal antibodies against mouse molar papilla: preliminary indirect immunofluorescence.

A variety of monoclonal antibodies have been produced against native dental papilla and used to stain frozen sections. Five different staining patterns corresponding to unidentified antigens were documented. The suitability of monoclonal antibodies for probing dental cell lineages is discussed.

The lingual (root analogue) and the labial (crown analogue) mouse incisor dentin promotes ameloblast differentiation.

Previous studies have suggested that mouse molar ameloblast differentiation was triggered by the predentin-dentin. Knowing that enamel is absent on the lingual surface of the mouse incisor, the aim of this study was to compare in heterotopic tissue recombinations the behavior of mouse molar inner dental epithelium associated with lingual or labial mouse incisor dentin. It was shown that root-analogue and crown-analogue incisor dentin promotes ameloblast differentiation of competent molar inner dental epithelium.

Dental cell interaction with extracellular-matrix constituents: type-I collagen and fibronectin.

It has been suggested that, during odontoblast differentiation, the extracellular matrix present at the epitheliomesenchymal junction modulates the activity of the cytoskeleton by means of membrane constituents (proteins, proteoglycans or gangliosides). To investigate this, we studied the interaction of iodinated fibronectin and type-I collagen with dissociated dental tissues and with membrane proteins prepared from these tissues. Isolated dental papillae and enamel organs were cultured for increasing periods of time in the presence of iodinated proteins. Fibronectin and type-I collagen were preferentially bound to dental papillae; however, after 6 h of incubation, fibronectin no longer interacted with the dental papillae, and the bound radioactivity was released. In the meantime, de novo synthesized fibronectin was deposited in the extracellular matrix of the dental papillae. Membrane proteins were prepared from isolated enamel organs and dental papillae. After sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, these proteins were transferred to nitrocellulose by electroblotting and then incubated in the presence of either 125I-labelled fibronectin or 125I-labelled type-I collagen. Autoradiography confirmed the preferential interaction of fibronectin with the dental papilla. Fibronectin interacted with three high-molecular-weight proteins (Mr, 145,000, 154,000 and 185,000), which were not detected when membranes were prepared from enamel organs. Under the same conditions, type-I collagen did not interact with membrane proteins. The known interaction of type-I collagen with the plasma membrane of dental-papilla cells might be mediated either by another constituent of the extracellular matrix or by cell-surface-associated proteoglycans.

Acellular dental matrices promote functional differentiation of ameloblasts.

EDTA treatment of post-natal mouse molars made possible the isolation of cell-free dental matrices composed of basal lamina, predentin, dentin and enamel. Trypsin-isolated dental papillae and enamel organs from embryonic-mouse mandibular molars were combined with isolated matrices and cultured in vitro. In such recombinations, functional odontoblasts were never observed. On the other hand, competent preameloblasts in contact with the epithelial side of occlusal predentin overtly differentiated. Matrices treated with guanidine-EDTA or acetic acid were unable to promote the functional differentiation of ameloblasts. These data are discussed in terms of the epitheliomesenchymal interactions involved in odontogenesis.

Behaviour of odontogenic epithelial cells in primary culture.

Odontogenic epithelial cells in primary culture easily adhere to the substratum through close and focal contacts. The adhesion processes of well-spread cells are found to be heterogeneous with respect to sensitivity to divalent cations/chelating agents. When cells are detached by mechanical forces, material remains attached to the substratum, which shows an enrichment in actin, intermediate filament components, and a protein with a MW of 130,000 daltons. The cytoskeleton is visualized after Triton-X 100 extraction. Microfilaments are abundant at the ventral side of cells in contact with the substratum. Intermediate filaments and microfilament bundles are observed close to the nuclear surface. Stress fibers end and ravel out at the ventral side of the cell in sites corresponding to focal contacts. In some regions cells in contact with the substratum deposit extracellular material with the ultrastructural aspect of a basal lamina. This material is associated to hemidesmosomes in early stages.

[Basement membranes: general aspects, role in odontogenesis]. / Membranes basales: aspects généreaux, rôle dans l'odontogenèse.

Basement membranes are interposed between epithelial and mesenchymal cells and surround muscles. This particular extracellular matrix was first considered as a static structure, assuming cohesiveness of adjacent tissues. However during the last ten years, basement membranes were shown to be continuously renewed and to present compositional and structural modifications. During development changing basement membranes play important roles during histomorphogenesis and cytodifferentiation and are involved in normal and pathological behavior of adult tissues.

Neutral morphogenetic activity of epithelium in heterologous tissue recombinations.

To assess the existence of specific and nonspecific epithelial instructions for mesenchymal cell differentiation we compared homospecific and heterospecific mouse and quail tissue recombinations. In heterospecific recombinants between trypsin-dissociated mouse molar mesenchyme and quail epithelia neither odontoblasts nor chondrocytes differentiated. Cartilage appeared if the quail epithelium was contaminated with homologous limb mesenchyme and odontoblasts differentiated if the mouse dental epithelium was contaminated with dental papilla cells.

Cytological response of the basal epithelial cell surface to the dental basement membrane.

Reassociations of trypsin-isolated dental or foot epithelia and trypsin or EDTA-isolated dental papillae were cultured for short and long time periods on a semi solid medium. After trypsin treatment the basement membrane (BM) is removed. After EDTA treatment an intact BM remains associated with the dental papilla. Cytochalasin B was added to a series of cultures. These experiments demonstrate the behaviour of the basal epithelial cell surface in the presence or absence of an intact dental basement membrane. In the presence of the dental basement membrane the epithelia restore a flat surface within one hour.

Facts and hypotheses concerning the control of odontoblast differentiation.

Numerous studies using amphibians have demonstrated that preodontoblasts emerging from the dental papilla are derived from cranial neural crest cells [4, 12, 46, 64]. However this has not been established for mammals. The history of odonotogenesis begins during the early stages of cranial-facial development when the maxillary and mandibular processes processes develop. Continuous epithelio-mesenchymal interactions condition the histogenesis and morphogenesis of the teeth [24-26, 43, 44, 49, 51, 58] as well as the terminal differentiation of odontoblasts and ameloblasts [23, 47, 52, 54, 59, 61, 67]. During recent years a considerable amount of experimental data relating to differentiation of odontoblasts has been published. We summarize these data and attempt to integrate them in deductive hypothesis concerning the control of odontoblast differentiation.

Synthesis of collagen type I, type I trimer and type III by embryonic mouse dental epithelial and mesenchymal cells in vitro.

Epithelial and mesenchymal dental cells were grown in primary monolayer culture and the ability of both cell types to synthesize interstitial collagens was investigated. Pepsin-solubilized collagens were analyzed by CM-cellulose chromatography and both cell types were found to synthesize collagen type I, type III and type I trimer. The collagen phenotype of mesenchymal cells (type I: 82.4%, type III: 8.5%, type I trimer: 9.1%) was different from that of epithelial cells (type I: 71.8%, type III: 9.5%, type I trimer: 18.7%). The radioactivity incorporated into collagen molecules by mesenchymal cells was 34-times greater than the radioactivity incorporated by epithelial cells. This result agreed with previous observations obtained from tissue culture experiments (Lesot, H. and Ruch, J.V. (1979) Biol. Cell. 34, 23--37) which indicated a low synthesis of interstitial collagens by isolated dental epithelia when compared to isolated dental mesenchymes.

[In vitro differentiation of primitive molar tissue from mouse embryo]. / Différenciation in vitro du matériel molaire présomptif de l'embryon de souris.

The developing molar tissues of the lower jaw of 13, 14 and 15 day Mouse embryos were removed and cultured in vitro. Complete molar dentition differentiated.

[Role of dental pulp in the histogenesis of the enamel organ]. / Rôle de la pulpe dentaire dans l'histogenèse de l'organe adamantin.

The histological study of in vitro cultured associations between dental pulps and outer dental epithelium showed that the predontoblasts initiated the histogenesis of the enamel organ and particularly the differentiation of the inner dental epithelium.

Action of 5-bromodeoxyuridine on tooth germs "in vitro." I. - Effects on cytodifferentiation.

Sixteen or eighteen day old mouse embryonic first lower molars were treated with 5-bromodeoxyuridine (BrdU) in vitro. --In 16-day old tooth germs cytodifferentiation of both odontoblasts and ameloblasts was inhibited. This inhibition was reversible. --In 18-day old tooth germs, the odontoblasts localized at the top of the principal cusps (post-mitotic cells) differentiated normally and secreted predentin. In these conditions ameloblasts localized in front of functional odontoblasts differentiated, although they were able to incorporate BrdU. --In others experiments, control and BrdU-treated tooth germs were dissociated into enamel organs and pulps. Cultivated associations between either control or BrdU treated pulps with either control or BrdU treated enamel organs were analyzed. It appeared that the primary effect of BrdU might result in its incorporation in the preameloblasts which were no longer able to interact normally with the preodontoblasts.

Basement membrane reconstitution and cytodifferentiation of odontoblasts in isochronal and heterochronal reassociations of enamel organs and pulps.

The restoration of the basement membrane and the terminal differentiation of odontoblasts were studied in iso- and heterochronal reassociations between dental mesenchyme and enamel organs. It was suggested that the cytodifferentiation of odontoblasts was triggered by the basement membrane secreted by the enamel organ of a specific stage.

Action of 5-bromodeoxyuridine on tooth germs "in vitro". II. - Effects on collagen synthesis.

The first lower molar tooth germs removed from 16-day-old mouse embryos were cultured for 2 days on a standard medium and then for 24 hours on the same medium containing BrdU (treated tooth germs) or not (controls). We attempted to study the effects of 5-Bromodeoxyuridine on the type I and type III collagen synthesis 24 (stage 16 + 4) and 72 (stage 16 + 6) hours after the incorporation of this thymidine analogue. At stage 16 + 4, type I and type III collagen were synthesized both in control and BrdU-treated tooth germs. However BrdU induced quantitative modifications in the type I collagen synthesis which might be explained by modifications in the turnover of this type of collagen. At stage 16 + 6, the BrdU treatment resulted in the inhibition of the terminal differentiation of odontoblasts. Consequently, the normal amplification of collagen type I synthesis could not occur. However, both control and BrdU-treated tooth germs synthesized type I and type III collagen. Quantitatively, the synthesis of type III collagen was slightly affected by BrdU treatment.

[Ultrastructural aspects of reconstitution of the basal membrane in associations of dental components cultured in vitro]. / Aspects ultrastructuraux de la reconstitution de la membrane basale dans des associations de constituants dentaires cultivées in vitro.

Separation of the enamel organ and pulp of mice tooth germs by trypsin removed the basal lamina. Within 18 hours in cultivated reassociations of enamel organ and pulp, a new lamina was deposited. When the epithelial component was cultivated alone, no basal lamina formed.