J.periodom.Res. 2: 46-52.

1967

Electron microscopic features of the

newly formed epithelial attachment
after gingival surgery
A preliminary report

MAX A,.LISTGARTEN
Department of Periodontology Faculty of Dentistry University of Toronto, Canada

Deciduous first molars with the buccal gingiva in siiii were obtained from three year old cynomologcs
monkeys 2 months and 4 months after gingivectomy. Of the four specimens examined to date, all
showed formation of a new epithehal attachment consisting of hemidesmosomes and a basement lamina.
In some areas this attachment apparatus connected the cells direetiy to the tooth surface. In other
areas, "cuticular" structures were interposed between the attachment apparatus and the tooth surface.
Although the layers intervening between the attachment apparatus and the tooth surface may repre.sent
acquired pellicles, they may also be due to denaturation of superficial layers of cementum. Any factor
atfecling the integrity of the epithelial tissue, the attachment apparatus or these "cuticles" could result
in breakdown of the epithelial attachment.

A recent study of tiie gingivo-dental junction
of man (Listgarten, 1966 a) has detnonstrated
the existence in clinically normal tissues of an
attachment apparatus between the cells of the
epithelial cufF and the tooth. The attachment
apparatus cotisisted of hemidesmosomes and
a cementing layer resembling the basement
lamina observed at the interface of a variety
of epithelium - connective tissue jutictions.
In addition, it vi'as noted that at least two types
of "cuticular" structures were interposed be-
tween the epithelium and the tooth. When this
occurred the epithelial cells were attached to
these "cuticles" rather than to the tooth itself.
One of these "cuticular" structures referred to
as a type A "cuticle" was found near the
cemento-enamel junction, over enamel sur-
faces which had been exposed to contact with
the surrounding connective tissue, as a result
of a focal degeneration or shrinkage of the
reduced enamel epithelium prior to eruption
(Listgarten 1966b). The other, or type B
cuticle appeared to be dependent for its. forma-
tion on the presence of the epitheiial attach-
ment.
A study of unerupted human teeth after
cotnpletion of amelogenesis (Listgarten 1966 b)
demonstrated the presence of a similar attach-
ment apparatus between the cells of the re-
duced enamel epithelium and the adjacetit
enamel surface. It should be pointed out, how-
ever, that a type B cuticle was never found in
the regioo of the attachment, in unerupted
teeth.
Some investigators (Becks 1929, Cohen
1962) have published data which, they sug-
gested, indicate that only reduced ameloblasts
are capable of providing a real attachment
to the tooth. Furthermore, we know from the
results of other studies (Engler, Ramf jord and

This investigation was supported by Grant DA-136 from the National Research Council of Canada,
Associate Committee on Dental Research,
 NEWLY FORMED EPITHELIAL ATTACHMENT 47

Fig.1. Cell of epitfielialattachmBm(E) is connected to the surface of root CBmentuni (C) by an

attachment apparatus consistiog of hemidesmosomes (small arrows) aod a space containing
granular basemarit lamina material (BL). Note that cemental collagen fibrils, extend right to. the
cementum surface. ^ 30.000'.
 LiSTGARTEN
Hitiiker 1965, McHugh and Zander 1965)
that the epithelial attachment, depending on
its stage of development, may consist primarily
of reduced enamel epithelium, a combination
of reduced enamel epithelium and cells derived
from oral epithelium, or primarily of cells
derived from the oral epithelium. Thus the
possibility that reduced amelobiasts mediate
the actual attachment even at a late stage of
eruption cannot be entirely dismissed.
The present experiment was, therefore,
designed to learn whether or not an attach-
ment consisting of hemidesmosomes and a
basement lamina could be produced de novo
from oral epithelium, in the absence of reduced
enamel epithelium.
Materials and Methods
Under nembutal arjaesthesia gingivectomies
were performed in one half of the mouth on
the buccal and oral aspects of maxillary and
mandibular quadrants of two cynomologus
monkeys approximately 3 years of age. Par-
ticular attention was paid to the thorough
removal of any soft tissue still adhering to the
tooth surfaces including the ioterproximal
areas. The tooth surfaces were carefully
planed with curettes to insure the complete
removal of any portion of the existing epithe-
lial attachment. The resulting soft tisstie mar-
gin was located approximately 2 mm. apical
to the cemento-enamel junction. The animals
received a single intra-muscular injection of
500,000 units of peoicillin G postoperatively.
The operated areas healed uneventfully, with-
out the application of a periodontai dressing.
Two and four months postoperatively, the
first deciduous molars were extracted with the
overlying buccal gingiva in situ.
The teeth were immediatelyfixedin ice cold
5 %, cocodylate buffered glutaraldehyde fixa-
tive for one hour. The specimens were then
trimmed with an "air-rotor", demineralized in
0.25 M Versene for two weeks, post-fixed in
2 % veronai-acetate buffered osmic acid and
embedded in epon in the routine manner.
Details of the technique have been published
elsewhere (Listgarten 1966a).
Thin sections cut at 0.1 microns were col-
lected on naked grids and stained with a
saturated alcoholic solution of uranyl acetate.
Some grids were double stained with lead
citrate according to the method of Venable
and Coggeshal! (1965).
Results
Although at the time the gingivectomies were
performed, the gingiva appeared clinically
healthy, slight to moderate marginal gingivitis
was evident at the time the specimens were
collected.
Despite the inflammation, however, it was
observed that in all cases the newly formed
epithelial attachment was morphologically
similar to that observed in the normal state
(Listgarten r966a). Since no significant differ-
ences existed between the samples collected
two months post-operatively and those col-
lected after a four month interval, the restilts
to follow apply equally to all samples.
The cell membrane of the epithelial cells
facing the tooth surface had differentiated in
localized areas into typical hemidesmosomes
(Stem 1965). The cell membrane was located
approximately 1000 A from the surface to
which the cell was attached. The space be-
tween tbe cell was filled with a granular sub-
stance probably representing basement lamina
material. This basement lamina material could
not be readily resolved into a lamina densa and
a lamina lucida (Stern 1965). This had also
been observed to be the case in the normal
epithelial attachment (Listgarten 1966a).
Although epithelial ceils could be directly
attached to the root surface by the attachment
apparatus described above (Fig. 1), "cuticular"
structures were occasionally interposed be-
tween the cells and the tooth surface. Fig. 2
shows a granular layer interposed between the
basement lamina material and the root cemen-
 iNEWLY FORMED EPiTHELIAL ATTACHMENT

Fig.2. Epithelial' attachmentlo the tooth surface is mediated bv a granular layer (X) which occupies
the region between the basement lamina (BL) and the cementum surface. Although X may repre-
sent an acquired "cuticular" structure, it is also possible that it represents a denatured region of
superficial cementum in which the collagen fibrils have lost their periodicity C, cementum: E,
epithelial cell; D, desmosomes: arrows, hemidesmosomes. " 30,000.
50 LISTGARTEN
turn. Normal root cementum is readily rec-
ognized because of its clear content of fibrillar
collagen. Fig. 3 shows a denser "cuticle", of
irregular width, in a corresponding location to
that shown in Fig. 2. This dense cuticle does
not have the same granular appearance as the
granular layer shown in the previous illustra-
tion. There appears to be a suggestion of ap-
positional lines in its structure.
Discussion
The importance of the above findings relate
first to the ability of oral epithelium to recon-
stitute an attachment apparatus similar to that
observed in the normal state, consisting essen-
tially of hemidesmosomes and a granular,
more or less homogeneous basement lamina.
It indicates, furthermore, that to achieve this
the presence of reduced enamel epithelium is
not needed, at least as far as an epithelial
attachment to the root surface is con-
cerned.
The results also suggest that "cuticles" may
act as mediators in the attachment of epithelial
cells to the tooth surface. The "cuticular"
structure shown in Fig. 2 may result from ad-
sorption to a hydroxyapatite surface, of break-
down products from surrounding connective
tissue. Such breakdown products could be
produced daring the repair phase, following a
gingivectomy. Seivig (1966) has recently sug-
gested that in periodontal disease, breakdown
products of the connective tissue may con-
tribtite to the formation of acquired pellicles.
On the other hand, an appearance similar to
that of an acquired pellicle could be rendered
if a superficial layer of cementum underwent
some pathologic alteration in the course of
periodontal disease or following surgery. Such
a process could result in loss of the typical col-
lagen striations, possibly as a result of tissue
or bacterial coUagenase or as a result of leuko-
cytic hydroiases acting on previously dena-
tured collagen.
The granular layer shown in Fig. 2 could,
therefore, represent a superficial layer of dena-
tured cementum, rather than an acquired pel-
licle. Whatever the origin of this layer it is to
be noted that a new attachment has been
formed to its surface by the epithelial cells of
the cuff.
Fig. 3 resembles the type B cuticle which is
present in a corresponding location in the
normal epithelial attachment (Listgarten
1966a). However, certain subtle differences
such as the lack of mottling and the suggestion
of appositional lines raise the possibility that
these may be different structures. Formation
of the type B cuticle appears to require con-
tinuous contact with the cells of the epithelial
attachment over a prolonged period of time,
the cuticle being wider in the more corona!
portion of the attachment than in the more
apical part which may be completely devoid
of it, or only show a tenuous layer (unpub-
lished observations). This relationship did not
appear to exist with respect to the dense layer
depicted in Fig. 3. It is interesting to speculate
whether the two structures are similar or
whether the layer shown in Fig. 3 represents a
pillicle acquired after surgery, possibly of the
same nature as the artifically produced pellicle
of Hodson (1966), which is primarily com-
posed of "conglutinated" erythrocytes. The
type B cuticle, on the other hand, may depend
on the epithelial cells for its formation and
could result from the gradual, local accumula-
tion of inspissated basement lamina material.
The presence of ctiticular structures of
varied origins as mediators of the epithelial
attachment suggests that there may be different
potential targets to a pathogenic stimulus caus-
ing epithelial detachment. Disruption of the
attachment could be produced intra-epithelial-
ly by factors affecting cellular cohesion,, that
is factors acting on the desmosomes. Factors
acting on the hemidesmosomes and basement
lamina could equally result in such a detach-
ment. It has been shown that these are not
necessarily identical (Coman 1961, Berwick
 NEWLY FORMED EPITHELIAL ATTACHMENT 51

Fig.3. Dense ciiiticular struciure (Y) between basement lamina (BL) and cementum (C). This
material differs morphologically from: the granular layer observed iin Fig.2. It may repiresent an
acquired cuticle possibly associated witti postoperative haemorrhage. ^ 35,000.
52 LISTGARTEN
and Coman 1962). Finally, factors capable of
breaking up the acquired pellicles or dena-
tured layers on the tooth surface could also
cause a detachment of the epithelial cuff.
References
Becks, H. 1929. Normal and pathological pocket
formation. J. Amer. dent. Ass. 16: 2167-2188.
Berwick, L. and D. R. Coman. 1962. Some
chemical factors in cellular adhesion and stick-
iness. Cancer Res. 22: 982-986.
Cohen, B. 1962. A study of the periodontal
epithelium. Brit. dent. J. 112: 55-64.
Coman, D. R. 1962. Adhesiveness and stickiness:
Two independent properties of the cell surface.
Cancer Res. 21: 1436-1438.
Engler, W. O., S. P. Ramfjord and J. J. Hiniker.
1965. Development of epithelial attachment
and gingival sulcus in rhesus monkeys. J.
Periodont. 36: 44-57.
Hodson, J. J. 1966. A critical review of the dental
cuticle with special reference to recent invest-
igations. Int. dent. J. 16: 350-384.
Listgarten, M. A. 1966 a. Electron microscopic
study of the gingivodentai junction of man.
Amer. J. Anat. 119: 147-178.
Listgarten, M. A. 1966 b. Phase-contrast and
electron microscopic study of the junction
between reduced enamel epithelium and
enamel in unerupted human teeth. Arch, oral
Biol. I I : 999-1016.
McHugh, W. D. and H. A. Zander. 1965. Cell
division in the periodontium of developing
and erupted teeth. Dent. Pract. dent. Rec. 15:
451-457.
Selvig, K. A. 1966. Ultrastructural changes in
cementum and adjacent connective tissue in
periodontal disease. Acta odont. scand. 24:
459-500.
Stern, I. B. 1965. Electron microscopic observa-
tions of oral epithelium. I. Basal ceils and the
basement membrane. Periodontics 3: 224-238.
Venable, J. H. and R. Coggeshall. 1965. A sim,
plified lead citrate stain for use in electron
electron microscopy. J. Cell Biol. 25: 407-408.
Address:
Dr. Max A.Listgarten
Departmefil of Periadatitoiogy
University of Toronto
124. Edward Street
Toronto 2
Ontario