J. periodont. Res. 3 : 96-110.

1968

Cell contacts in oral epithelia

HOLGER T H I L A N D E R AND GUNNAR D . B L O O M

Departments of Oral Surgery and Histology, University of Umea, Sweden.

Biopsies from normal, non-inflamed buccal gingiva, palatal gingiva, retromolar and upper vestibular
mucosa were obtained from 12 adult patients for an electron microscopical investigation of cell contacts
in the epithelial layers. The junctional complexes found were desmosomes, tight junctions and inter-
mediate junctions. In parakcratotic or poorly keratinized epithelium (e.g. retromolar and vestibular
mucosa, gingiva) the desmosomes maintained their typical appearance in all layers. In highly keratinized
epithelium (e.g. palatal gingiva) the structure of the desmosomes changed in the stratum corneum to a
three-layered structure between the thickened cell membranes. The tight junctions were classified as
maculae occludentes in the basal layer and the stratum spinosum and as zonulae occludentes in the stra-
tum granulosum and the stratum corneum. The intermediate junctions were rather numerous and of
different length. The principal mode of attachment of clear cells to adjoining epithelial cells seemed to be
of this type. At the tissue surface the orifices of the intercellular spaces were closed by tight junctions
or modified desmosomes. No such junctional complexes were found at the orifices of the intercellular
spaces to the basement membrane. In the light of the findings some permeability conditions of oral
epithelia are discussed.

Introduction and the guinea pig) Farquhar & Palade

In 1864 Schultze and Bizzozero, independent
of each other, described the spinous processes
of epidermal cells and assumed that they af-
forded means of mutual adherence between
the cells. Later Bizzozero (1870) assumed the
processes of adjacent cells to be in end-to-end
contact, thus forming non-continuous inter-
cellular bridges. This opinion was, however,
contested by Ranvier (1879, 1882) who stated
that intracellular fibrils passed from one cell to
another through "true" intercellular bridges.
Thereafter a copious and controversial litera-
ture on this subject has been published.
The first ultrastructural description of
epithelial cell contacts was given by Porter
(1954) on larvae of Amblystoma punctatum
and in man by Selby (1955, 1956). A detailed
analysis of the fine structure of desmosomes in
human epidermis was then published by Od-
land in 1958. In an investigation of different The material consists of biopsies from the buc-
epithelia (glands and cavity organs of the rat cal gingiva, the palatal gingiva and the oral
(1963) found, besides the desmosomes, two
junctional complexes: The zonula occludens
(tight junction) and the zonula adherens (inter-
mediate junction) .
In human gingiva desmosomes and hemi-
desmosomes have been described by e.g. List-
garten (1964), Stern (1965) and Schroeder and
Theilade (1966). In this latter investigation
maculae or zonulae occludentes were also de-
scribed.
As some controversial concepts exist on the
fine structure of junctional complexes this in-
vestigation was carried out on the different
epithelial cell contacts found in oral tissues
such as the buccal gingiva, the palatal gingiva
and the lining mucosa of the retromolar re-
gion and upper vestibular fornix.
Material and Methods
 CELL CONTACTS IN O R A L EPITHELIA 97

Fig.1. Survey micrograph of the basal cell layer. Palatal gingiva. Intercellular spaces are rather wide and interdigitating mi-
crovilli numerous. The border between epithelium and connective tissue appears irregular. The basement membrane can
clearly be seen. x8000.
98 THILANDER AND BLOOM
mucosa (retromolar regions and upper vesti-
bular fornix) of 12 adult patients (from 17 to
40 years old). The biopsies were obtained from
normal, clinically noninflamed tissues by exci-
sion with scalpel. The specimens were imme-
diately immersed in ice-cold 1 per cent os-
mium tetroxide buffered at pH 7.6 with phos-
phate buffer (Millonig, 1961) and cut into
smaller pieces. The fixation time was 2-3 hours
in the osmium fixative. The specimens were
then dehydrated in graded ethanols and fi-
nally embedded in Epon. Sections for orien-
tation were obtained by cutting 1-2 micron
thick sections in an ultramicrotome. These
sections were placed on ordinary glass slides
and were stained in toluidin blue. After final
trimming the specimens were sectioned in an
LKB ultrotome using glass knives. The thin
sections were stained with uranyl acetate or
double stained with uranyl acetate and lead
citrate. The sections were examined in a Sie-
mens Elmiskop IA or a Philips electron mi-
croscope EM 200.
Results
Stratum basale
The intercellular space is rather wide and is
therefore clearly seen even at low magnifica-
tion. The cell peripheries are very uneven and
cytoplasmic processes of different size and
shape frequently interdigitate (Fig. I). Desmo-
somes are rather evenly distributed around the
cells. The structure of desmosomes is by now
rather wellknown (Fig. 2). They consist of
areas of strict parallelism of adjacent cell-
membranes. The attachment plaques back the
inner leaflet of the cell membrane and cyto-
plasmic filaments (tonofilaments) converge on
the inner aspect of the attachment plaques.
Between the attachment plaques there is a
typical lamellated structure consisting of four
layers of rather low electron density, separa-
ted by three more osmiophilic layers. The
latter present themselves as two lateral dense
lines and a central one, the intercellular con-
tact layer. Of these three dense layers, the two
lateral dense lines seem to represent the outer
leafiet of the cell membrane of the adjacent
cells can therefore not be considered as inter-
cellular structures. The distance between the
inner surfaces of the attachment plaques is
about 300-350 A and the distance between the
dense layers about 75 A.
Tight junctions (zonula occludens) (Fig. 2)
can be observed rather frequently. This is a
junctional complex in which the outer mem-
brane leaflets of adjoining cells have come
together and fused. These fused outer leaflets
are seen in the electron microscope as a cen-
tral dense layer. The tight junctions in stratum
basale are rather short and follow a straight
or slightly wavy course. The distance between
the inner membrane leaflets of the two cells
is between 100-150 A.
The cell membranes of adjacent cells often
run parallel for 0.2-0.8 n and with a distance
between the outer membrane leaflets of about
200-300 A. The intercellular space is here oc-
cupied by a homogeneous, amorphous ma-
terial. In our opinion these regions could be
characterized as intermediate junctions (zo-
nulae adherens) (Fig. 2).
Between the epithelial cells of this layer so-
called clear cells are frequently interspersed
(Fig. 3o). The cell membrane of this cell type
runs a more even course than that of the epithe-
lial cell and the intercellular space is in most
places rather narrow. No desmosomes or
tight junctions have been observed in connec-
tion with these ceils. The cell membrane of the
clear cells runs for rather long distances (up to
2 n) parallel with that of the adjoining epithe-
lial cell and the width of the intercellular space
is about 200-300 A (Fig. 36). The intercellular
substance is homogenous and amorphous and
shows a rather low electron density. These re-
gions thus show the characteristics of inter-
mediate junctions.
No characteristic pattern could be detected
in this epithelial zone as to the distribution of
and relation between desmosomes, tight junc-
tions and intermediate junctions nor was any
 C E L L C O N T A C T S IN O R A L EPITHELIA 99

Fig. 2. Junctional complexes in the basal cell layer at higher magnification. (A). Desmosomes
and intermediate junctions. x160000. (B). Desmosome and tight junction. x135000. (C).
Desmosomes, tight junctions and intermediate junction. x115000. (D). Desmosome and
intermediate junctions, x 160000. The lateral dense line is continuous with the outer leaflet
of the cell membrane (A and B).
100 THILANDER AND BLOOM

Fig.3. (A). Clear cell in the basal cell layer. Palatal

gingiva. No desmosomes can be detected in con-
nection with this cell, x 19000. (B). Higher mag-
nification of an intermediate junction between the
clear cell in (A) (framed area) and an epithelial cell.
X 95000.
 CELL C O N T A C T S IN O R A L EPITHELIA 101

significant difference observed between speci- 5 and 6). The desmosomes are numerous. At
mens from different oral regions. the border zone to fully keratinized cells the
desmosomes show a composite structure (Fig.
Stratum spinosum 8). The inner leaflet of the cell membrane of
In this layer the intercellular space is as stratum corneum cell shows a continuous
wide as or even wider than that observed in the thickening. The attachment plaques of these
stratum basale. Cytoplasmic projections inter- cells blend with this thickened membrane. On
digitate freely betvi'een cells (Fig. 4). The des- the stratum granulosum side there is no
mosomes seem to be more numerous than in thickened inner leaflet outside the desmosome.
the basal layer. No differences in the fine struc- The substance between the attachemnt plaques
ture of desmosomes could be detected be- seems to be more opaque, but its lamellated
tween these two layers. Tight junctions and character is still clearly seen.
intermediate junctions are also of rather fre- Tight junctions are very common and can
quent occurrence. in this layer be very long, especially in the
border zone adjacent to the stratum corneum.
Stratum granulosum Intermediate junctions are also very common
The intercellular space is diminished in this (Fig. 7). Especially in specimens taken from the
layer compared to the stratum spinosum (Fig. palatal mucosa, which is highly keratinized

Fig.4. Survey micrographs of the stratum spinosum in the buccal gingiva. Desmosomes are numerous. x 4 500.
102 THILANDER AND BLOOM

Fig. 5. Lower part of the stratum granulosum in vestibular mucosa. The intercellular space shows an irregular course. Des-
mosomes are numerous, x 26000.

the intercellular connections of the upper To the first group belong the specimens
stratum granulosum, except for focally ex- from the lining mucosa (retromolar and vesti-
panded parts, consist only of desmosomes, bular areas) and some of the gingival speci-
tight junctions or intermediate junctions mens. Here the stratum corneum is thin and
(Fig. 6). the intercellular spaces are rather wide and
show an irregular course, due to rather broad
Stratum corneum cytoplasmic projections which extend from
The appearance of cellular contacts in this the cells (¥\g.9a). The desmosomes are nu-
layer seems to depend on the degree keratini- merous. They are mostly rather short and
zation of the specimen. This description will have the typical lamellated appearance pre-
therefore be divided in two parts, one for viously described (Fig.96 and c). The attach-
specimens with a parakeratotic or poorly kera- ment plaques are well defined as there is usu-
tinized epithelium and one for highly kera- ally no thickening of the cell membrane. Tight
tinized epithelium. junctions are found mostly in the outermost
 CELL C O N T A C T S
layer, where the intercellular space has nar-
rowed down. At the orifices of the intercellu-
lar spaces on the tissue surface tight junctions
are frequently observed.
In highly keratinized epithelium (palatal
gingiva, some regions of gingival mucosa) the
picture is different. The intercellular space is
here very narrow and only focally widened.
Furthermore it shows a straighter course than
deeper in the epithelium (Fig. lOo). The junc-
tional elements can be described as modified
desmosomes and tight junctions. The modi-
fied desmosomes are very common and occur
at short intervals. In the desmosome area the
cell membranes of apposing cells run strictly
Fig. 6. Upper part of the stratum granulosum in palatal gingiva. The intercellular space is narrow and rather straight, x 36 000
IN O R A L EPITHELIA 103
parallel. The membranes are thickened and no
attachment plaques are visible. Sometimes
there is a small concentration of a dense amor-
phous material in the subjacent cytoplasm.
Between the thickened cell membranes there
is a three-layered structure, a central, thick,
osmiophilic band bounded by two narrow, less
electron dense, lines (Fig. 106). Sometimes a
denser intermediate line in the central osmio-
philic band can be detected. At the orifices of
the intercellular spaces at the tissue surface
there is a modified tight junction, e.g. a struc-
tural complex similar to the modified desmo-
somes described above. However, the di-
stance between the cell membranes is gene-
104 THILANDER AND BLOOM

Fig.7. Higher magnification of cell contacts in the stratum granulosum. (A). Intermediate
junction. Vestibular mucosa. x 160000. (B). Tight junction. Vestibular mucosa. x 160000.
(C). Desmosomes and intermediate junction. Palatal gingiva. x 11 5000.

rally shorter than usual (~ 250 A). Sometimes
the orifice of the intercellular space may con-
sist of a desmosome (Fig. 11 b).
The connection epithelium - connective tissue
The border between epithelium and under-
lying connective tissue is irregular with a wavy
course (Fig. 1). The two tissues are separa-
ted by a 400-700 A thick osmiophilic zone,
the basement membrane or the lamina densa.
The basement lamina densa consists of a finely
granular or sometimes filamentous substance
Between the cell membranes of the basal cells
and the basement membrane (lamina densa)
 C E L L C O N T A C T S IN O R A L
there is a less electron dense, homogeneous
zone of about 300-500 A in width. This clear
zone has been named lamina lucida. At irregu-
lar intervals hemidesmosomes are present
(Fig. 11). This structure consists of an attach-
ment plaque, about 200 A thick, in contact
with the inner leaflet of the cell membrane and
a peripheral density in the lamina lucida locat-
EPITHELIA 105
ed at a distance of 200-250 A from the attach-
ment plaque. The substance between these two
structures is more osmiophilic than the rest of
lamina lucida and furthermore shows a lamel-
lar appearance. Mid-way between the peri-
pheral density and the attachment plaque a
dense line corresponding to the lateral dense
line of desmosomes, can be discerned. This re-
Fig.8. (A). Border zone stratum granulosum-stratum cor-
neum in vestibular mucosa. The cell membrane is thickened
at the stratum corneum side, x 33000. (B). A composite
desmosome at higher magnification, x 90000.
106 THILANDER AND BLOOM

presents the outer leaflet of the cell membrane. membrane and even from areas of the cell
Often filamentous structures seem to extend membrane between the hemidesmosomes to
from the peripheral density to the basement the basement membrane. (Fig. 11 b).

Fig.9. (A). Stratum corneum. Retromolar

mucosa. Intercellular spaces are rather wide
and show an irregular course. Desmosomes
are numerous. x5500. (B). Desmosomes at
higher magnification. They are short and show
a typical lamellated appearance, x 90000.
 CELL CONTACTS IN O R A L EPITHELIA 107

Ik

Fig.10. (A). Stratum corneum in palatal gingiva. Intercellular spaces narrow and rather straight. The cell junctions show a
modified appearance. x43000. (B) A modified junctional complex at higher magnification. x145000. (C). The orifice of
an intercellular space at the tissue surface. The orifice consists of a modified cell junction. Detritus on the tissue surface.
X 88000.
108 TH I LAN DER AND BLOOM

At the orifices of the intercellular spaces to
the lamina lucida no desmosomes or tight
junctions have been observed.
Discussion
The presence of so-called tight junctions in
gingival epithelium has recently been re-
ported by Schroeder and Theilade (1966).
They could not, however, decide whether the
tight junctions observed in the stratum basale,
spinosum and granulosum were maculae or
zonulae occludentes, according to the defini-
tion given by Farquhar and Palade (1963,
1965). According to these authors zonulae oc-
cludentes are belts of membrane fusion and
in maculae occludentes the fusion of adjoining
cell membranes is limited to very small areas
or spots. In stratum basale and stratum
spinosum, independent of sectioning angle,
the tight junctions found in the present ma-
terial always seemed to be rather short,
shorter than the desmosomes in the same stra-
ta. They therefore seem to represent maculae

Fig. 11. Border zone epithelium - connective tissue. (A). Palatal gingiva Hemidesmoso-
mes and basement membrane, x 100000. (B). Vestibular mucosa. Filamentous structures
extending from the cell membrane and the peripheral density of the hemidesmosomes to the
basement membrane, x 140000.
 CELL CONTACTS
occludentes. In the stratum corneum, in re-
gions with a high degree of keratinization, and
in the stratum granulosum most of the tight
junctions were rather long and seem to re-
present zonulae occludentes. According to the
theories of Farquhar and Palade (1963, 1965)
zonulae occludentes act as barriers which im-
pede the flow of water, ions and small water-
soluble molecules along the intercellular
spaces of the epithelium. The presence of tight
junctions in the upper strata of oral epithelium
could, in the light of these theories, explain
the findings that trypan blue (Thilander 1961)
or flourescin (Brill and Bjorn 1959) could not
pass through the oral mucous membrane ex-
cept for areas within the gingival pocket. The
upper strata is by these junctions divided into
numerous sub-compartments and the per-
meability conditions may be dependent on the
number and position of zonulae occludentes.
Highly keratinized epithelia such as the palatal
epithelium and parts of the gingival epithe-
lium may therefore act as an effective barrier
against penetration of different substances
from the tissue surface. Mucosa with poorly
keratinized epithelium such as in specimens
from e.g. the retromolar and vestibular areas
would not seem to offer the same protection
due to the limitation of tight junctions only
to the outermost cell-layer and the presence
of rather wide intercellular spaces immediate-
ly below this layer.
Besides their influence on diffusion condi-
tions of the intercellular spaces, zonulae occlu-
dentes have been ascribed, as have the macu-
lae occludentes, the facilitation of ion ex-
change between adjoining cells (Farquhar and
Palade 1965), and to represent intercellular
attachment devices. The desmosomes are the
most important attachment devices and they
can be characterized as areas of more perma-
nent attachment. They seem to be rather re-
sistent structures. By treatment of human oral
epithelium with different enzymes, a general
widening of the intercellular spaces was ob-
tained, but this did not seem to affect the des-
IN O R A L EPITHELIA 109
mosomes (Thilander 1963). There has been
some disagreement concerning the structure of
the desmosomes. In Listgarten's opinion the
desmosomes in oral epithelia show a loss of
the intercellular contact layer in the superfi-
cial portion of stratum spinosum and further
up alterations in the cell membranes adjacent
to the desmosomes. Similar alterations have
been found by Horstmann and Knopp (1958)
in rat foot epithelium. According to Schroeder
and Theilade (1966) the desmosomes in hu-
man gingival epithelium maintain their dense
triple layered structure in all epithelial layers
even to the most superficial cells. In our ma-
terial we have found that the structure of
desmosomes in higher strata seems to be de-
pendent on the degree of epithelial keratiniza-
tion. The differences of opinion may there-
fore be due to regional differences among the
various authors' specimens. The reported
early loss of the intercellular contact layer in
the desmosomes may also be due to sectioning
directions; we have found that this layer may
disappear upon a slightly oblique sectioning.
Another point of discussion in the literature
is whether the lateral dense lines represent the
outer leaflet of the cell membrane or not. It
has been suggested that the intercellular con-
tact layer represents the fusion of this outer
leaflet (Fasske and Themann 1958) and also
that the lateral dense line is intercellularly
located (Roth and Clark 1964). In our ma-
terial we have found the lateral dense lines to
represent the outer leaflet of the cell membrane
and the intercellular contact layer to lie
"truly" intercellularly. This also seems to be
the case with respect to the corresponding
dense lines described by us in hemidesmo-
somes.
The existence of zonulae adherence (inter-
mediate junction) has not previously been
reported in human oral epithelium. According
to the definition given by Farquhar and Palade
(1963) the zonula adherence is characterized
by the presence of an intercellular space
(~ 200 A) occupied by a homgeoneous, appa-
110 THILANDER AND BLOOM
rently amorphous material of low density, by
strict parallelism of the adjoining cell mem-
branes over distances of 0,2 n and by con-
spicuous bands of dense material located in
the subjacent cytoplasmic matrix. The inter-
mediate junctions we have observed in oral
epithelia fulfill this definition except for the
bands of dense material in the subjacent cyto-
plasmic matrix. They may therefore represent
modified intermediate junctions. Their func-
tion is yet not clear but it is possible that they
are more or less temporary intercellular attach-
ment zones. The principal mode of attachment
of clear cells to adjoining epithelial cells seems
to be of this type.
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Department of Oral Surgery
University of Umed
Umea 6, Sweden