217269a0Nature2175125196801202692700028-0836196810.1038/217269a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v217/n5125issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue217269a0Odontogenesis: Cell-Cell Interactions in vitro
AU  - SLAVKIN, H. C.
AU  - BEIERLE, J.
AU  - BAVETTA, L. A.Department of Biochemistry, School of Dentistry, University of Southern California, Los Angeles, California.EMBRYONIC interactions of epithelial and mesenchymal tissue before organogenesis have been observed in several organ systems1-4. These phenomena have been tested by isolating embryonic tissues from their in vivo environment and transplanting them in an artificial environment from which no morphogenetic signals can be received. The chorio-allantoic membrane (CAM) of chick provides a versatile site for the maintenance and further differentiation of embryonic organs5,6. One such organ, the tooth primordium, arises as a result of interactions between epithelial and mesenchymal tissues and offers a model system for developmental studies.Recently, studies of the trans-filter interaction of embryonic rodent incisor tissues have shown that overt differentiation occurs in both separated epithelial and mesenchymal tissues7. These results and those of numerous other investigators8?12 suggest that the interaction of epithelial and mesenchymal tissues is essential for tooth formation.
To examine the morphogenetic potential of cells from these tissues, an experiment was designed to evaluate cell-cell interactions before histogenesis. Maxillary incisors were removed from the embryos of New Zealand white rabbits at the twentieth day of gestation. At this stage of tooth development both enamel and dentine formation have begun in the coronal portion of the organ, and most tissues which have been formed show various degrees of differentiation (Fig. 1).
Fig. 1. Maxillary incisor in the embryo of New Zealand white rabbit at day 20 of gestation before excision. The whole tooth rudiment is encapsulated in alveolar bone. The rectangle contains the cervical loop region from which dissociated inner enamel epithelium and mesenchymal papillae cells were obtained. E, Epithelial cells; M, mesenchymal cells (x 50).
Apical or cervical regions of these incisor tooth germs were excised. The isolated cervical regions were tryp-sinized to dissociate the inner enamel epithelium from the mesodermal papilla by the methods of Moscona13. The isolated epithelial and mesenchymal tissues were further treated with 0.4 per cent trypsin to obtain single cell suspensions. The isolated cells, in a calcium-magnesium free phosphate buffered saline at pH 7.2, were pipetted onto the CAM of 8-day embryonated hens' eggs and incubated at 37.5 C for periods of up to 10 days. Three sets of experiments (twenty grafted eggs in a group) were used to test epithelial cells only, mesenchymal cells only and the interaction when both cell types were mixed together in culture. Tissue and cell dissociations were monitored by phase microscopy in order to ensure that there were relatively clean cell suspensions of both inner enamel epithelium and mesodermal papillae. At various periods during the experiment, xenogeneic cell preparations were dissected with the adjacent CAM and were immediately placed in Hollande-Bourn's solution. The tissues were sectioned at 5m and stained with periodic acid-Sehiff's (PAS) reagent. There is a variety of cell types within the original separated tissue preparations; it might therefore be anticipated that these originally dissimilar cells would display different patterns of mobility, differentiation and survival. The test for viability of the xenogeneic cells in these culture conditions was based upon the frequency of mitotic figures. Additional quantitation of the surviving epithelial and mesenchymal cell suspensions before and after the experiment has not yet been performed. The viability of the host chick embryo was determined by daily candling.
A positive interaction is considered to be represented by one or both cell types undergoing morphological differentiation.
When either one of the cell suspensions was cultivated independently on the CAM, no indication of tooth formation was observed (Fig. 2). Both homogeneous xenograft groups exhibited a continued proliferation of cells, reflected by numerous mitotic figures. The isolates appeared as randomly dispersed cells with neither apparent orientation nor continued differentiations; no re-aggregation of the isolates was observed.
When both cell preparations were mixed and suspended on the CAM, an induction was observed which resulted in the initiation of tooth formation (Fig. 3). A well defined PAS-positive interface, analogous to that reported to occur in vivo14?15, separated the interacting cells. In several xenografts within this group, the cell-cell interaction was preceded by the bud stage of tooth development. The similarity between in vivo and in vitro development is shown by condensation of the mesenchymal cells, a specific PAS-positive future dentino-enamel junction, and regularity of the tall columnar inner enamel epithelial cells. The temporal differentiation of these epithelial cells is shown in vivo and in vitro by differentiation of squamous cells into cuboidal then columnar inner enamel epithelial cells with a subsequent alteration in nuclear polarity. The differentiation of the mesodermally derived odontoblasts is undoubtedly related to interaction with the inner enamel epithelium. Earlier workers had postulated this fundamental relationship, primarily because of the observed differentiation of inner enamel epithelium into ameloblasts before differentiation of odontoblasts16?18. The maximum duration of these experiments (10 days) might not allow sufficient time for the mesenchymal cells to differentiate into odontoblasts in vitro.
The analysis of these events during tooth development in vitro shows that the morphogenetic potential is closely related to the properties of both interacting cell types, not exclusive to either one. Thereafter, tooth formation must be regarded as a complex temporal differentiation involving both types of interacting cells. After re-aggregation, the events in vitro show a striking developmental similarity to those occurring in vivo.
Fig. 2. Mesenchymal cell suspension explanted onto the CAM for 10 days of cultivation. The isolated cell suspensions of either mesenchymal or epithelial tissue fail to initiate tooth formation and do not re-aggregate or orientate when cultivated on the CAM (x c. 370).
Fig. 3. When both cell preparations were mixed and cultivated for'10 days on the CAM, tooth formation was initiated. E, Inner enamel epithelium; M, mesenchymal papilla. Arrow points to PAS-positive future dentino-enamel junction (x c. 370).
This investigation was supported in part by a US Public Health Service grant, a training grant and a research career award to L. A, Bavetta, from the National Institute for Dental Research. We thank Drs S. Allerton and M. Nimni for help and encouragement, and Mrs Zoya Trirogoff for technical assistance.Saunders, , J. W., Cairns, , J. M., and Gasseling, , M. T., J. Morphol., 101, 57 (1957).ArticleRawles, , M. E., J. Embryol. Exp. Morphol, 11, 765 (1963).PubMedISIChemPortAuerbach, , R., Develop. Biol., 2, 271 (1960).ArticlePubMedISIChemPortGrobstein, , C., J. Exp. Zool., 124, 383 (1953).ArticleISIGlasstone, , S., J. Anat., 88, 392 (1954).PubMedISIChemPortSlavkin, , H. C., and Bavetta, , L. A., Experientia (in the press).Koch, , W., J. Exp. Zool., 165, 155 (1967).ArticlePubMedISIChemPortHay, , M. F., Arch. Oral Biol., 3, 86 (1961).PubMedISIChemPortLefkowitz, , W., Bodecker, , C. F., and Mardfin, , D. F., J. Dent. Rest., 32, 749 (1953).ChemPortPaynter, , K. L., and Hunt, , A. M., Arch. Oral Biol., 9, 611 (1964).ISIPourtois, , M., J. Embryol. Exp. Morphol., 12, 391 (1964).PubMedChemPortSlavkin, , H. C., and Bavetta, , L. A., Arch. Oral Biol. (in the press).Moscona, , A. A., Exp. Cell Res., 22, 455 (1961).ArticlePubMedISIChemPortHunt, , A. M., and Paynter, , K. J., Arch. Oral Biol., 8, 65 (1963).PubMedISIChemPortZussman, , W. V., Oral Surg., Oral Med. and Oral Path., 21, 217 (1966).ChemPortGlasstone, , S., Proc. Roy. Soc., B, 126, 315 (1938).Huggins, , C. B., McCarroll, , H. R., and Dahlberg, , A. A., J. Exp. Med., 60, 199 (1934).ArticleFleming, , H. S., J. Dent. Res., 31, 166 (1952).PubMedISIChemPort