of Dental Medicine Elizabeth

and Surgery. University of Melbourne, Street. Melbourne. Victoria 3000. Australia

Summary-The establishment of contacts between reaggregating, dissociated, bovine, odontogenie epithelial cells was studied in vitro using time-lapse cinephotomicrography and transmission and scanning electron microscopy. Brief contacts between fine cell processes were noted by cinephotomicrography when the cells were distributed sparsely in the early cultures .but, as shown by electron microscopy, other morphological forms of contact developed as the cell density increased. Desmosomes were only observed at confluence and after the cells had been in culture for 3 weeks. It was concluded that intercellular contacts increased in complexity and surface area as the age of the cultures and cell density increased.


tion, were placed in a sterile 1Oml plastic centrifuge tube containing 5 ml Dulbecco phosphate buffered saline (PBS) [Commonwealth Serum Laboratories (CSL), Melbourne] supplemented with 20 per cent (v/v) fetal calf serum (FCS) at 4’C and transported to the laboratory in a flask of crushed ice.

and morAlthough the details of the molecular phological events in cellular recognition are little understood. the morphological aspects of cell contact events may represent, in a relatively gross fashion, phenomena associated with cell recognition (Loor, 1977); e.g. microvilli may be so concerned (Pugh-Humphreys and Sinclair, 1970; Ben-Shaul and Moscona, 1975). The desmosomes between epithelial cells are implicated in cellular adhesive interactions (Overton, 1975). Previously (Orams. Sim and Reade. 1974), it was found. by time-lapse cinephotomicrography, that dissociated cells of mesenchymal and epithelial odontogenie origin were mobile in cell culture and that movement of either eritire cells or their extensions resulted in the re-establishment of intercellular contacts in association with and the separation of cells into aggregates of one or the other cell type. Our aim was to examine the contacts between odontogenic epithelial cells in rirro.

Separation 0s rpitheliw front footh grrfns Upon arrival at the laboratory, the tooth germs were rinsed 3 times with PBS and then placed in 0.25 per cent (w/v) trypsin solution (CSL, Melbourne) at ambient temperature for 1 h. During this time, the dental follicle cells became detached from the underlying dental epithelium without mechanical intervention. This allowed the freed cells to be discarded during subsequent rinsing. The absence of fibroblast-like cells, as determined by light and electron microscopy, in the cultures prepared from the remaining tissue confirmed that such cells had been removed by this procedure. After three further rinses in PBS, each developing tooth was dissected in 5 ml PBS in sterile culture dishes (Falcon, 1006) using a dissecting microscope at x25 magnification. The buccal cusps were isolated and epithelium removed with a cataract knife from areas external to the mineralizing matrix. All tissue manipulations were done in a laminar flow cabinet and the material resulting from these manipulations was pooled.

MATERIALS AND METHODS .4nirna/s 12 bovine fetuses of approximately 16 weeks gestation, as assessed by crown-rump measurement (Roberts, 1971). were obtained from Protean Ltd, Richmond, Victoria, Australia.

Preparation of cell suspensions

Surgical rernooal 0s fnohr tooth germ After killing a pregnant cow, the horn of the uterus containing the fetus was removed and its external surface swabbed with 70 per cent (v/v) ethanol. A 10-l 5 cm incision was made through the uterine wail, the fetal membranes were incised and the fetus removed. The umbilical cord was severed .and the fetus decapitated. Antero-posterior incisions were made in the palatal eminences to expose the developing third and fourth maxillary deciduous molars which were gently removed. Forty-eight developing teeth, after collec293

Pieces of epithelium were incubated in 5 ml of 0.25 per cent (w/v) trypsin solution for 15 min at 37°C and the suspension centrifuged at 240 g for 10 min at 2°C. After removal of the supematant, the cells were resuspended in 2 ml of RPM1 1640 medium (CSL, Melbourne) plus 20 per cent v/v FCS.. Single cells were obtained by filtering the suspension through double thickness sterile lens tissue, tested for viability and diluted to 2.5 x lo6 cells/ml. Cell suspensions were then either plated as multiple separated drops to Rose chambers for cinephotomicrography or placed in plastic culture dishes and incubated for 3@-60min at 37°C in a humidified 5 per cent CO,/air atmosphere.

F. R. P. Sim. H. J. Orams.


Additional RPM1 medium plus 20 per cent (v/v) FCS was added and incubation continued with change of medium every 3 days. The cultures were examined either continuously by cinephotomicrography or terminated at 6, 14 and 21 days for electron microscopic examination. Tirnr-lapse


Using a Bolex HI6 reflex tine-camera attached to a Zeiss Plankton microscope and activated by a Paillard-Wild Variotimer, the cultures in Rose chambers were photographed at three frames/min on Recordak fine grain film 7460 [Kodak (Australia) Pty. Ltd., Melbourne]. The processed films were later studied using an L-W Photo-Optical Data Analyser (L-W Photo Inc.. Van Nuys, California). Cell movement and mitosis in particular were analysed using as follows: a Data Reduction Screen, No. 1012 (L-W Photo Inc.. 1972) was calibrated by projecting an image of a Clay Adams Neubauer Bright Line haemocytometer square of 50jtm so that it exactly coincided with one large square on the screen. Thus each large square on the screen represented 50itm2 and each small square 10 jtm2. Because the original filming rate was three framesimin, the temporal parameter in minutes was obtained by counting the number of elapsed frames during a particular movement or episode and dividing by 3. Prcparution microscopy

of cd/ (TEM)





Cell cultures were fixed in situ in the plastic culture dishes by removing the culture medium and washing the cells 3 times with 0.1 M sodium cacodylate buffer. pH 7.4. for 1 h at 4’C. They were then post-fixed in osmium tetroxide and washed 3 times in de-ionized water keeping the temperature at 4°C. The fixed cultures were dehydrated in graded ethanol at 4°C and. after a final wash in alcohol at room temperature. were infiltrated with Araldite. Capsules filled with Araldite were then inverted over selected areas which were previously shown by phase contrast microscopy to contain cells in contact. The capsules plus attached cell monolayers were fractured from the plastic culture dishes and thin sections (5@60 nm) were cut with an LKB Ultrotome III. The sections were picked up on 200 mesh grids and stained with saturated uranyl acetate in 70 per cent (v/v) ethyl alcohol and water for 5 min followed by lead citrate (Reynolds. 1963) for 5 min. Sections were examined using a Phillips EM 300 electron microscope at 60 kV. Staining



with wthrniwl


The procedure was the same as above. except that the glutaraldehyde mixture contained 0.1 mM ruthenium red (Chroma-Gesellschaft. Schmidt. Stuttgart) and the osmium tetroxide mixtures contained 1 mg/ml ruthenium red. Preparation of cdl cultures for scanning rlectron scopy (SEM)


Cultures grown on glass coverslips were terminated by removing the medium, washing the cells 3 times with 0.1 M sodium cacodylate buffer, pH 7.4, and fixing the cells with a solution of 5 per cent (v/v) glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, for 1 h

S. S. Prime

and P. C. Reade

at 4°C. After washing 3 times with 0.1 M sodium cacodylate, pH 7,4, the cultures were dehydrated in graded ethanol, washed 3 times in acetone, then partially plastic-embedded by a technique adapted from that used by Cleveland and Schneider (1969) for ocular tissues. The cultures were rolled for 12 h in a mixture of equal parts of acetone and Araldite at room temperature. Then they were transferred to 100 per cent Araldite (plus accelerator) for 3 h at room temperature followed by 3&40 min at 60°C. This last step was a critical one and was watched carefully so that as soon as the Araldite mixture became viscous the cultures were removed from the oven and immediately rinsed with warm acetone. The surfaces of the cover slips and attached monolayers were further washed with a jet of warm acetone to remove all traces of Araldite from the surface. Finally, polymerization of the Araldite within the cells was completed at 60°C for 48 h. The embedded cultures were attached to metal stubs. gold-coated under vacuum in a Dynavac High Vacuum Coating Unit and examined with a Cambridge Stereo-Scan scanning electron microscope S4-10, operating at 20 kV. RESULTS


of cdl



(a) Sub-conj7urnt cells. In sub-confluent cultures. leading lamellae were generated by cells at any point on their periphery: this usually indicated a preferred direction of future bodily movement. If such movement occurred. cells proceeded across the glass substratum at an average velocity of 6Obtm/h. Occasionally, cells did not translocate but remained stationary and formed multiple. extended cell processes with ruffled membranes at their tips. These processes moved by a gliding action with a variation in velocity of 10&300 /(m/h. They appeared to be acting as sensors of the substratum and the surfaces of adjacent cells and exhibited sporadic protrusion and withdrawal movements. (b) Confltrent cells. At confluence. which usually occurred by the end of the first week. cells became grouped together as pavemented. monolayered sheets of polygonal ceils which intermittently made contact with each other temporarily with contacts often lasting for less than 1 min. These characteristics were regarded as typical of epithelial cells in culture. The most notable features of confluent cultures were the tentative nature of intercellular contacts. the greater number of microvilli present compared to sub-confluent cultures and the increased number of microvillar protrusions and withdrawals over any given time period. when compared with sub-confluent cultures. Cells in contact-inhibited monolayers entered mitosis, and as a prelude to this, contracted circumferentially, thus increasing the intercellular space. The intercellular bridges of cells entering mitosis were therefore elongated, and were ultimately broken as various distances from the cell body. As the cell assumed a more spherical form, all contact with neighbouring cells was lost and substratum-attached material was observed at the site previously occupied by its periphery. Surrounding cells often retained a connection to the substratum-attached material by


Epithelial cell contacts way of intercellular bridges. Mitosis took 20 25min from the beginning of prophase to the completion of telophasc. After telophase. daughter cells prepared to spread on the glass coverslip to occupy the space within the substratum-attached material which appeared in the form of arcs of two different sizes. The daughter cell within the larger arc was the first to begin spreading. Neither daughter cell spread beyond the line demarcated by the substratumattached material but produced I /cm cell extensions which made contact with neighbouring cells. Once a cell had spread to occupy the arc. it maintained intermittent contacts with neighbouring cells by these cell extensions. These cells thus behaved in a similar manner to the cells already established in the monolayer. The difference in size of the daughter cells was apparently related to the morphology of the cellular material which remained attached to the glass substratum. Topoyruphy

of cdl


(a) Scanning ektron micro.scopJ*. In the &day cultures. cells in less densely-packed areas were observed in contact with each other by fine cell extensions, some of which ramified over the surfaces of the opposing cells. This type of contact was described as a ramifying contact (Fig. 1). Cells in more denselypacked areas were in contact by fine cell extensions resembling the intercellular bridges common to epitheliat cells (Fig. 2). In areas of more intimate contact, intertwining microvilli which produced a chain-mail appearance were observed (Fig. 3). Stereo-pair scanning electron micrographs showed the intricate 3-dimensional intertwining of these microvilli. When the cells became more densely packed. there was an increase in the complexity of intercellular contact by microvilli, with involvement of a greater proportion of the cell surfaces and close contact between one cell and several others. (b) Transruission rlrcrron ruicroscopJ*. In the 6-day cultures. the following morphological forms of contact were observed: contact by the surface coat of one cell extension with the surface coat of an extension from another cell as demonstrated by the positive ruthenium red staining (Fig 4); abutting contact by a cell extension against the lateral border of a neighbouring cell (Fig. 5); contact between neighbouring cells via complexes formed by cell extensions (Fig. 6): a closer type of contact in which cells were in apposition at areas along their borders (Fig. 7). The ultrastructure of these four types of contacts was as follows: in the first two types (Figs. 4 and 5). there appeared to have been no membrane specialization; contact involved only the surface-coat material of the participating cells. However, where there was abutting contact (Fig. 5), the surface coats of the two cells appeared to be fused and condensed. In both the first two types of contacts, heavy concentrations of aligned microfilaments were a prominent feature. They were arranged as a central core within cell extensions and microvilli. In the third type (Fig. 6). intercellular contact appeared to be via a complex of intertwining cell extensions. The microfilament bundles streaming from the contact area appeared to end in a large microfilament network aligned parallel to the cell surface. In the fourth type of contact listed,

i.e. close cell-border to cell-border contact (Fig. 7). the cell-surface coat had apparently been condensed between the cells at the contact sites but specialized structures were not observed. In the t4-day confluent, but not densely-packed, cultures. the types of contacts were mainly simple cellextension to cell-extension contacts and close cellborder to cell-border contacts similar to those of the 6-day cultures. In the 2l-day cultures. the cells were densely packed and polygonal. The contacts between these cells were numerous and were either simple cell-extension to cell-extension contacts by microvilli, or complex junctions not seen in the earlier cultures where areas of membrane specialization and associated concentrations of filaments were typical of desmosomes (Fig. 8).


Our findings show that the complexity of intercellular contacts and .proportion of cell-surface area involved increase as cultures of odontogenic epithelium increase in age and cell density. The active participation of surface carbohydrates in recognition phenomena was shown by Ashwell and Morel1 (1974); in our study, the ruthenium red-positive cellsurface coat was involved in all cell contacts examined. This cationic dye stains carbohydrate moieties at the cell surface (Luft. 1971) and the positive staining is evidence that carbohydrate at the cell surface participates in intercellular contact. We observed that a condensation of coat material occurred as the cell membranes became more closely associated, e.g. in the abutting contacts, the cell-border to cell-border contacts and in the desmosomes. Extensive microvillus formation was observed, especially during the establishment of initial contacts. Various workers have suggested that microvilli are involved in cell-recognition events at the level of making initial contacts between cells and producing closer associations of cell surfaces (reviewed by Loor, 1977). We showed that microfilaments occurred as central cores in the microvilli; Ishikawa. BischolT and Hertzer (1969) showed that the villi of gastric epithelium contain microfilament bundles. In accord with present understanding of the mechanisms of cell mobility (reviewed by Pollard and Weihing, 1974). we suggest that it is the activity of the microlilamcnts which pro duces the mobility and changes of form which were constant features of the processes of the odontogenic epithetial cells observed in cirro. Some contacts, especially the simple cell-extension to cell-extension type, persisted for periods of a few seconds before breaking because of movement of the processes. On the other hand, Lazarides (1976), from a study of human lung cells in rirro. suggested that in contacts which persist for long periods, the microfilaments could act to stabilize the cell surface by a rigidity conferred upon them by the preferential binding of tropomyosin to the actin of the microfilaments. If this is correct, the filaments in contacts which persist for longer periods could be involved in stabilizing rather, than in moving the contacting part of a cell. Overton (1975) has described the stabilizing effect of desmo-


F. R. P. Sim. H. J. Orams.

S. S. Prime and P. C. Readc

somcs where the associated filaments are approximately twice the diameter of microfilaments and are considered to bc composed mainly of myosin. Although the morphological details of cell contacts described here came mainly from fixed material, it appeared that as reaggregating odontogenic epithelial cells became more closely packed the complexity and stability of some cell contacts increased. It is not clear whether the morphological forms of contacts we describe are associated with stages of desmosome formation. Desmosome formation was a late event which occurred after, rather than during, the establishment of close associations between cells. As Overton (1975) has shown. this could be due to the stage of development of the tissue from which the cell suspensions were originally prepared or it could be related to some limiting conditions in the culture system used. Ackno~rlc,t/y~,/ilmrs-We are grateful to Misses S. Watkins and D. Seward and to Mr. D. Rowler for their excellent technical assistance. to Mrs. E. Dinnage for typing the manuscript and to the National Health and Medical Research Council of Australia for financial support.

REFERENCES Ashwell G. and Morel1 A. G. 1974. The role of surpace carbohydrates in the hepatic recognition and transport of circulating glyco-proteins. Aclr. En-_,rrt~o/. 41. 99- 128. Ben-Shaul Y. and Moscona A. A. 1975. Scanning electron

Plate Fig. 1. SEM ramification.

microscopy of embryonic neural retina cell surfaces. Expl. C-r//. Rrs. 95. I9 I 204. Cleveland P. H. and Schneider C. W. 1969. A simple method of preserving ocular tissue for scanning microscopy. Vision Rcs. 9. 1401 1402. lshikawa H.. Bischoff R. and Holtzer H. 1969. Formation of arrow head complexes with heavy-meromyosin in ;I variety of cell types: .I. Cc// Biol. 43.. 312-328. Lazarides E. 1976. Two aeneral classes of cvtoolasmic actin filaments in tissue cultured cells: The rile bf tropomyosin. .I. .supro~~~o/cc.Srrucr. 5, 53 I-563. Loor F. 1977. Structure and dynamics of the lymphocyte surface. In: B und T Cells /n~n~unr Rrccynition (Edited by Loor F. and Roelants G. E.) pp. 153-189. John Wiley. New York. Luft J. H. 1971. Ruthenium red and violet I. Chemistry. purification. methods of use for electron microscopy and mechanisms of action. A~lur. RN. 171. 347-368. Orams H. J.. Sim F. R. P. and-Rcade P. C. 1974. An in rirro and in riro study of dissociated tooth germs of rats. Archs owl Biol. 19. 285-291. Overton J. 1975. Experiments with junctions of the adhaerens type. Clrrr. Top. deal. Biol. IO. l-34. Pollard P. D. and Weihing R. R. 1974. Actin and myosin and cell movement. Criricul Rvriws in Biochwrisrry. Vol. I I. pp. I 65. Chemical Rubber Co.. Cleveland. Ohio. Pugh-Humphrcys R. G. P. and Sinclair W. J. 1970. Ultrastructural studies relating to the surface morphology of cultured cells. J. Cell Sci. 6. 477 484. Reynolds E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17. 208-212. Roberts S. J. 1971. Vurcrinury Oh.sfmic.\ und Gvnirul LX\wscs. 2nd Edn. p. 19. Ithaca. New York.


of a 6-day culture of odontogenic epithelial cells showing intercellular Fine ramifying micro-extensions from cell A overlap the surface many points of mutual contact. x 3CKIO

contact by extension of cell B. providing

Fig. 2. SEM of a 6-day culture of odontogenic epithelial ceils showing contact between cells via cell extensions from the cell bodies (cb) of two adjacent cells resulting in structures resembling the intercellular bridges (ib) of epithelial cells. x 7500 Fig. 3. SEM of a 6-day culture of odontogenic epithelial cells showing a complex intertwining arrangement of microvilli from the cell bodies (cb) of two adjacent cells. presenting a chain mail appearance. x7500 Fig. 4. TEM of a 6-day culture of odontogenic epithelial cells showing a cell-extension IO cell-extension type contact. Cell extensions A and B are in contact with each other via ruthenium red-positive surface coat material. Note microfilaments in cell extension A. x 88,tXO Fig. 5. TEM of a 6-day culture of odontogenic epithelial cells showing a cell extension in contact with_cell (B) in cell-extension to cell-body type contact. Ruthenium red-positive material lies within the area of contact. Microfilaments (mfh arranged in meshwork formation. are present in cell B. x 88.000 Fig. 6. TEM of a 6-day culture of odontogenic epithelial cells showing two cells in contact via a complicated intertwining of cell extensions. Microfilaments (mf) are aligned at right angles to the cell surface, and deeper within the cell, microfilament bundles are aligned parallel to the surface and as meshworks. x 16,000 Fig. 7. TEM

of a 6-day


of odontogenic epithelial cells showing (arrows). x 70,000

close lateral-border


Fig. 8. TEM of a 2l-day culture of odontogenic epithelial cells showing desmosomes (arrows). with associated microfilament conuzntrations and the desmosomal plaque stained intensely with ruthenium red x 70,000

Epithelial cell contacts

Plate 1.


Morphology of contacts between bovine odontogenic epithelial cells in vitro.

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