/. Periodontal Res.

Id: 1-11

The role of gingival connective tissue in

determining epithelial differentiation
T. KAEKINO, N . P . LANG AND HARALD LOE
Department of Periodontology, Royal Dental College, Aarhus, Denmark

Free grafts of connective tissue, without epithelium, were transplanted from either the kera-
tinized gingiva or the non-keratinized alveolar mucosa (controls) into areas of the alveolar
mucosa in seven monkeys. The grafts were placed in pouches created in the connective
tissue as close as possible to the overlying epithelium. After 3 ^ weeks, the transplants
were exposed by removal of the overlying tissue in order to allow epithelialization from
the surrounding non-keratinized alveolar mucosa. The transplants were ex.£imined clinically
and histologicaily at time periods between 1 and 12 months.
The gingiva! connective tissue grafts became covered with keratiuized epithelium dis-
playing the same characteristics as those of normal gingivai epithelium. The alveolar mu-
cosa transplants were covered with non-keratinized epithelium. This indicates that gin-
gival connective tissue is capable of inducing the formation of a keratinized gingival epi-
thelium.

(Received for publication Oct. 3, 1974; accepted Dec. 6, 1974)

previous study (Karring, 0stergaard & Loe

Introdudion
The mechanisms responsible for the main-
tenance of specific features of the epithelium
covering the various surface regions of the
adult mammalian organisms are not well
understood. Changes in tissue morphology
can occur as a result of environmental stim-
uli. For example, the epidermis may dis-
play an increased thicktiess of the keratin
layer in response to abrasion (MacKenzie
1973). On the other hand, if split thickness
grafts from skin or mucous membranes are
transplanted to foreign sites on the surface
of the body, the epithelium retains its orig-
inal specificity (Krohti 1955, Billingliam &
Silvers 1963, 1968, Smith 1970). These stu-
dies indicate that the characteristic features
of the epithelium in various regions of the
adult organism are genetically determined
rather than the result of functional adapta-
tion. We have confirmed these findings in a
1971) in which tissue grafts of kera-
tinized gingiva and non-keratinized alveolar
mucosa were transposed. Both these tissues
retained their clinical and histologie charac-
teristics in the new environment. However,
despite the fact that in this experiment the
non-keratinized alveoiar mucosa transplants
were placed io close contact with the teeth,
a narrow border of keratinized gingiva de-
veloped immediately adjacent to the tooth
surface. Apparently, the connective tissue of
this new gingiva originated from the peri-
odontal membrane, whereas the epithelium
most likely proliferated from the adjacent
graft of the non-keratinized alveolar mu-
cosa. On the basis of these findings, it was
suggested that the periodontal connective
tissue possesses the capability oí inducing
the differentiation of epithelial cells into
keratinized gingivai epithelium.
Most studies favor the hypothesis that epi-
 KARRING,, LANG A N D L Ö E
thelial specificity is maintained by morpho-
genetic stimuli from the underlying connec-
tive tissue or is in some way controlled by
an interaction between the two tissues (Bil-
Iingham & .Silvers 1965, Gillette 1971, Kri-
kos 1971). However, some experiments have
indicated that certain epithelia innately pos-
sess the mechanism which determine their
morphology (Beer & Billingham 1970, Bil-
lingham & Silvers 1971).
The ptirpose of the present investigation
was to study the role of gingival connective
tissue in determining the differentiation of
the overlying epithelium.
Material and Aflethods
Seven adult green monkeys were used in the
study. Under intraperitoneal sodium pento-
barbital anesthesia, free connective tissue
transplants (approx. 10x5X2 mm.) of the
maxillary gingiva were prepared as follows.
The crevicular epithelium was eliminated
using a reverse bevel incision. The epithe-
lium covering the oral aspect of the gingiva
was removed as a single piece by free hand
dissection, using a sharp scalpel, and dis-
carded. The gingival connective tissue with-
out epithelium was subsequently removed
from the bone using a pedosteal elevator.
In the buccal alveolar mucosa adjacent to
the mandibular premolars and molars, a
total of 28 pouches were created by blunt
dissection as close as possible to the over-
lying epithelium (Fig. lA). The gingival
connective tissue transplants were placed
into 14 of these pouches. Serving as coti-
trols, the other 14 pouches received similar
transplants obtained froni alveolar mucosal
connective tissue. The connective tissue once
inserted into the pouch assumed a corru-
gated form. There was no attempt to orient
any particular surface toward the vestibule.
After placement of the transplants, the en-
trance to the pouches was closed by sutures.
The sutures were removed after one week.
Three to four weeks later the connective
tissue grafts were exposed to the oral cavity
by removing the overlying alveolar mucosa
(Fig. IB), allowing the wottnded areas to he
re-epithelialized from the surrounding non-
keratinized epithelium. The exposed areas
were examined clinically and histologically
after 1, 2, 3, 4, 6, 8, 11 and 12 months.
Upon sacrifice of the animals the mandibles
were dissected free and fixed in 10 per cent
neutral buffered formalin. After 1 week of
fixation, those portions of the jaws which
contained the transplanted connective tissue
were removed from the rest of the mandible
and decalcified in EDTA. Following décal-
cification and dehydration, the specimens
were embedded in paraffin. The entire graft
site was serially sectioned in a bucco-lingual
orientation at 8 microns and stained with
hematoxylin and eosin, Rhodamin B, (Clau-
sen & Dabelsteen 1969), PAS-reagent or
Weigert's stain for elastic fibers.
Identification of the Grafts
The connective tissue and the covering epi-
thelium of the normal gingiva and alveolar
mucosa demonstrate specific histological dif-
ferences (Lozdan & Squier 1969, Karring,
0stergaard & Loe 1971). The following
characteristics were used to facilitate the
identification of the grafted connective tis-
sue and to determine the specificity of the
covering epithelium.
1. While the gingival connective tissue con-
tains few elastic fibers in association with
blood vessels, they are numerous and
evenly distributed throughout the connec-
tive tissue of the alveolar mucosa.
2. The configuration of the basement mem-
brane of the gingiva is characterized by
long connective tissue papillae and slender
epithelial projections. In the alveolar
mucosa the connective tissue papillae are
shorter and the epithelial projections are
wider,
3. The gingival epithelium displays a distinct
 C O N N E C T I V E T I S S U E A N D E P I T H E L I A L D I F F E R E N T I A T I O N

surface keratinization whereas in the alve-
olar mucosa, cell nticlei are regularly pre-
sent ill the superficial cell layer,
4. The alveolar mucosa shows PAS-positive
material in the upper spinous cell layers
whereas the gingival epithelium does not.
pearance and size of these tissue areas did
not change.
The 4 gingival connective tissue trans-
plants which failed to show surface eleva-
tion and the 14 control areas grafted with

Results

CHtiical Observations
The surface epithelium of the alveolar tnu-
cosa covering the cotmective tissue trans-
plants in the pouches did not show any signs
of changes prior to the surgical exposure.
However, the position of the transplants
could be easily identified since the grafted
areas were slightly elevated and the grafts
were firm and movable in relation to the
underlying bone.
One month following exposure, most recip-
ient sites of the gingival connective tissue
transplants appeared slightly reddish and in-
flamed but were distinctly less reddish than
those of the alveolar mucosal connective tis-
sue grafts. After 2 months, 10 of the 14
surgically exposed gingival connective tissue
transplants demonstrated surface elevations
of varying sizes together with a surface ap-
pearance resembling that of normal keratin-
ized gingiva (Fig. lC). A minor reduction in
size seemed to take place between 2 and 3
months following the exposure, but during
the subsequent observation periods, the ap-

Fig. 1. Transplantation of gingival connective tissue

into areas of the non-keraiinized alveolar mucosa. A.
Instrument inserted into ,s pouch created in the alveo-
lar mucosa as close as possible to the overlying epi-
thelium. B. Surgical exposure of gingival (G) and al-
veolar mucosal (AM) connective tissue grafts to aE-
low for re-epithelial izatioo from the surrounding non-
keratinized alveolar mucosa. C. Connective tissue
grafts twelve months after the surgical exposure. An
area [G) with a surface appearance similar to that of
normal keratinized gingiva (NG) has developed at the
recipient site for the gingival connective tissue graft.
The area grafted with alveoSar mucosal connective tis-
sue (AM) cannot be distinguished from that of the
surrounding tissue.
 KARRING, LANG AND LÖE

alveolar mucosal connective tissue were

slightly redder than the adjacent alveolar
mucosa 2 months following wounding.
Three to 12 months following exposure
these areas appeared normal and could not
be distinguished from the surrounding mu-
cosa.

Histological Observations
Alveolar Mucosa Transplants (Controls) —
At one month following surgical exposure,
the graft and surrounding alveolar mucosal \
connective tissue were infiltrated by inflam-
matory cells. The connective tissue beneath
the epithelium demonstrated very delicate
elastic fibers. The epithelium covering the
wounded area was thinner than that of the
adjacent tissue and had an almost flat epi-
thelium-connective tissue interface.
The epithelium in 2 to 12 month control
specimens was thicker than that of one-
month specimens and exhibited "rete pegs"
similar to those of the normal alveolar mu-
cosa. A PAS-positive reaction was consistent-
ly seen in the upper spinous cell layers and
cell nuclei were always visible in the sur-
face cells. Fig. 2. Photomicrograph of section thr( u^h £.3 7 o-'h
In many 2 to 12 month control specimens, oid recipient site for an aiveoiar mucosai connective
tissue transpiant. The transpiant has partiy degsner-
the transplanted mucosal connective tissue ated. Some coarse eiastic fibers JEF) are seen in the
was difficult to identify although disrupted, residual tissue. The regenerated connectjve tissue
coarse and heavily stained elastic fibers contains deiicate eiastic fibers (arrows) and is cover-
ed with a non-iieratinized epitheiium. Weigerts stain
facilitated the identification of the wounded for elastic fibers: orig. magn.: x 130.
area (Fig. 2). The elastic fibers in the newly
formed connective tissue in the wounded
area tended to be thicker with time (Fig. 2). mal thickness was regularly present between
However, even in the 12 month specimens, the graft and the underlying periostetim.
the fibers had not reached the same thick- The graft and the surrounding tissue both
ness as those seen in the surrounding non- were heavily infiltrated with inflammatory
wounded alveolar mucosa. cells. The epithelium which directlj' covered
the gingival connective tissue transplant was
Gingival Connective Tissue Transplants thin. However, in localized areas the super-
The gingival connective tissue in sections ficial cell layers showed signs of keratiniza-
through the grafted areas one month follow- tion as indicated by the absence of cell nu-
ing exposure could easily he identified due clei and a positive reaction with the Rhod-
to the absence of elastic fibers. A zone of amie B stain. The epithelium-connective tis-
connective tissue with elastic fibers of nor- sue interface exhibited a few "rete pegs"
 CONNECTIVE TISSUE AN D E P I T H E L IA L D I F F E R E N T I A T I O N 5

• • •"•••,' \

• • • . •«•

CT

L'
Â

V
-••- '. .-• •• ". *• '.••••".'•."•

' I >

Fig. 3. Photomicrographs of consecutive sections through a 12 months old recipient site for a gingival connec-
tive tissue graft. A. The transpianted connective tissue (CT) can easily be identified due to the absence of
elastic fibers- The transition (arrows) between eiastic aiveoiar mucosal connective tissue and the niOn-eSastic
gingivai conneotive tissue is sudden and distinct. B. Adjacent secticn showing that the gingivai connective
tissue transplant (CT) is covered with a keratinized epitheJium identical to that of the normal gingiva. C. High
magnification of the transition between aiveoiar mucosaj' and Iransplanted gingival connective tissue in Fig.
3A. D,. High magnification of Fig. 3B showing that the transition farrows) between keratinized and non-kera-
tinized epithelium corresponds exactiy to the junction between eiastic and non-elastic connective tissue {C}.. A..
and C : Weigerts stain for elastic iibers.. B. and D.: Hematoxyián and eosin. Oräg. magn.: A. and B.: x 20. C.
and D.: x 60.
 K A R R I N G ,
and was markedly different from that of
adjacent alveolar mucosa. The gingival con-
nective tissue transplants could easily be
identified in the 2 to 12 month specimens
(Fig. 3A) except in the four which failed to
demonstrate clinical signs of keratinization.
In the latter specimens, the grafted areas
were occupied by connective tissue contain-
ing delicate elastic fibers.
The junction between the non-elastic con-
nective tissue of the gingival grafts and the
elastic connective tissue of the surrounding
alveolar mucosa was usually distinct (Fig.
3A and C). However, a few specimens show-
ed a transitional zone of connective tissue
containing delicate elastic fibers with a
course different from those of the surround-
ing alveolar mucosa. The connective tissue
immediately beneath the epithelium was al-
ways free of elastic fibers. Epithelial cords
or nets were not observed in the connective
tissue of any gingival or mucosal graft site.
The epithelium covering the transplanted
gingival connective tissue exhibited a distinct
keratin layer at the surface (Fig. 3B and
3D). As is characteristic for norm..al gin-
gival epithelium, there was no PA.S-positive
reaction in the upper spinous cell layers. In
most specimens the configuration of the
epithelium-connective tissue, interface fea-
tured long and slender epithelial projections
and resembled that seen in normal gingiva
(Fig. 3B). In some specimens, however,
a reduction in the length of the "rete pegs"
in the entire or in localized areas of the
graft was noticed. The transition between
the keratinized epithelium of the gingival
transplants and the non-keratinized epithe-
lium of the surrounding alveolar mucosa
was generally sudden and distinct (Fig. 3D)
and corresponded exactly with the junction
between the non-elastic and elastic connec-
tive tissue (Fig. 3C). In specimens display-
ing a transitional zone of connective tissue
with thin elastic fibers the corresponding
junction between keratinized and non-kera-
L A N G A N D L Ö E
tinized epithelium tended to be less distinct.
The findings in 2 to 12 month specimens
were identical and no changes were ob-
served which could be related to the time
elapsed since the exposure of the grafts.
Discussion
Conservation of Epithelial Specificity
This study has shown that gingival connec-
tive tissue transplanted to the non-kera-
tinized alveolar mucosa and subsequently
wounded becomes covered with a keratin-
ized epithelium indistinguishable from that
of the normal gingiva. The characteristics of
this epithelium were maintained at least one
year after exposure of the tissue grafts.
In the histologie specimens of the gin-
gival connective tissue grafts which failed to
demonstrate clinical surface characteristics
similar to those of keratinized gingiva, the
transplanted connective tissue could not be
identified. This indicates that the grafts have
failed to survive transplantation or possibly
might have been removed during their surgi-
cal exposure.
The areas where the grafts had disap-
peared were occupied by new connective
tissue containing thin elastic fibers. These
areas were always covered by epithelium
displaying the same features as those of the
alveolar mucosa. Thus, it is apparent that
the mere development of granulation or scar
tissue does not lead tO' the formation of a
keratinized epithelium. The same conclusion
can be drawn on the basis of the results
from the control areas which were always
covered with non-keratinized epithelium
similar to that of the adjacent alveolar mu-
cosa.
Usually the junction between the trans-
planted non-elastic gingival connective tis-
sue and the elastic connective tissue of the
alveolar mucosa was extremely distinct. In
these specimens this junction coincided com-
pletely with the transition between keratin-
 CONNECT,IVE TISSUE A N D E P I T H E L I: A L D I F F E R E N T I A T I O N 7

ized and non-keratinized epitheiium sug- Billingham (1970) who reported that follow-
gesting that epithelial differentiation is de- ing the transplantation of tongue mucosa or
termined by the underlying connective tis- skin grafts to the uteri of estrogen treated
sue. This result supports our proposal that rats, the lingual epithelium and epidermis
gingival connective tissue is capable of in- migrated onto the endometrical surface, pro-
ducing the formation of a keratinized gin- gressively replacing the native epithelium.
gival epithelium (Karring, 0stergaard& Loe On this alien connective tissue substrate
1971). both these epithelia maintained their orig-
inal specificity.
Mechanisrrts Responsible for Determination From the foregoing, it would appear that
of Epithelial Differentiation some epithelia, under certain circumstances,
Whereas it is generally agreed that various retain their ability to express their original
characteristics of the epidermis in different morphology when exposed to an alien con-
sites of the adult organism results from in- nective tissue. This problem obviously war-
ductive mechanisms exerted by the under- rants further investigation. The "recombi-
lying mesoderm during the embryonic de- nant" technique as used in the experiments
velopment of skin (Cairns & Saunders 1954, by Billingham & Silvers (1967) is extremely
McLoughlin 1961, Rawles 1963, Biliingham difficult. The possibility of some retained
& Silvers 1965, McLoughlin 1968, Siavkin connective tissue or epithelium on the re-
et al. 1972), different results have been ob- spective grafts cannot currently be discount-
tained relative to the role of connective tis- ed.
sue in determining the differentiation of ma- We believe the model system described in
ture epithelium. this report is well suited for the study of the
Various recombination experiments in influence of connective tissue on epithelial
adult rodents using dermis and epidermis differentiation for the following reasons:
from the trunk, ear and sole of the foot 1. Both the connective tissue and epithelium
showed that the dermal tissue consistently of the mucous membrane in primates dis-
determined the type of epidermis produced, play verj' distinguishable morphologic
suggesting that epithelial basal cells are features in various regions of the oral
capable of producing several different types cavity.
of stratified squamous epithelium (Billing- 2. This system does tiot require the trauma
ham & Silvers 1967). This observation cor- of etizymatic separation and re-combina-
roborates the finding that epidermis or oral tion of the epithelium and connective tis-
epithelium devoid of native connective tis- sue.
sue either did not sui-vive when transplanted 3. There is usually adequate tissue and ade-
to a new environment or developed morph- quate space for transplantation.
ologic characteristics resembling those of The presence of numerous elastic fibers
epithelium normally resident in the recipient in the alveolar mucosa facilitated the identi-
site (Van Scott & Reinertson 1961). fication of the transplanted connective tissue
On the other hand, some studies have in- in the present study. This made possible the
dicated that tongue, esophagus and hamster precise determination of the relationship be-
cheek pouch epithelium are resistant to the tween the two types of connective tissue and
influence of some alien types of connective the overlying epithelium. Thus, although the
tissue and retain their original specificity technique used for the preparation of the
(Billingham ,& Silvers 1967). This view has connective tissue grafts does not completely
been supported in a recent study of Beer & rule out that some epithelial cells might
 KARRING,
have been left in a few areas of some grafts,
this factor obviously is of less importance.
Since the transition between keratinized and
non-keratiniz.ed epithelium invariably and
exactly corresponded to the junction be-
tween the non-elastic and elastic connective
tissue, it is tmlikeiy that the epithelium
covering the exposed connective tissue grafts
has derived from such epithelial remnants.
This could take place if the connective tis-
sue transplants had become epithelialized by
proliferation from these remnants while
positioned in the surgically created pouch.
Another possibility is that a mechanism was
operating which would prevent the epithe-
lium from migrating beyond the hmits of its
own native connective tissues, allowing the
cells from these residual epithelial cells as
well as the alveolar mucosa to migrate ex-
actly to the junction between the two types
of connective tissue. If the first possibility
had occurred, epithelial remnants would
have been present in the connective tissue
of the graft site after the gingival exposure,
since no attempts were made to orientate
the connective tissue grafts in the pouches.
Epithelial remnants were not seen in any
graft site. The second possibility may be
excluded on the basis of recent studies de-
monstrating that the migration of epithelial
cells onto alien connective tissue does occur
(MacCallum, Lillie & Karring 1975).
It must, therefore, be concluded that the
epitheiium of the graft has developed
through the migration of non-keratinizing
epithelial cells of the surrounding alveolar
mucosa and that upon passing the junction
between the two types of connective tissue,
the gingival connective tissue has induced
their differentiation into cells with the same
characteristics as those of keratinized gin-
gival epithelium.
Thus, the present study has demonstrated
that the gingival connective tissue controls
the differentiation of the covering epithe-
lium and that the differentiation of the epi-
LANG A N D L Ö E
thelial cells of the non-keratinized alveolar
mucosa is not innately determined. These
conclusions are consistent with the finding
of Stambaugh & Gordon (1973) who ob-
served an inductive influence of palatal con-
nective tissue on epithelial differentiation,
but disagree with results presented by Berni-
moulin & Lange (1973).
In the present study the quality of the epi-
thelium of the alveolar mucosa covering the
connective tissue transplants did not change
prior to the surgical exposure. This same
observation was made following transplan-
tation of palatal connective tissue beneath
the epithelium of the alveolar mucosa (Bo-
tero & Stallard 1974). Thus, in order for the
connective tissue to exert its inductive in-
fluence it must be in direct contact with the
epithelium.
Clinical Significance
It is generally believed that the characteris-
tic features of the gingival tissue portrays
the functional demands placed upon the
gingiva by the abrasive action of foods (Or-
ban 1957, Pfeifer 1963). Conversely, how-
ever, the present study has shown that the
characteristics of the gingival tissue are con-
trolled by intrinsic mechanisms inherent to
the gingival connective tissue. This new con-
cept is essential to the understanding of the
healing subsequent to various surgical proce-
dures commonly used in periodontal therapy.
It is generally accepted that the presence
of a certain width of keratinized gingiva is
important for gingival health (Gottsegen
1954, Nabers 1954, Friedman 1957, Ochsen-
bein 1960, Friedman & Levine 1960, Car-
ranza & Carraro 1970). To this end, various
surgical procedures have been designed
(Gottsegen 1954, Ariaudo & Tyrell 1957,
Friedman 1962, Bohannan 1962a, b, Robin-
son & Agnew 1963, Carranza & Carraro
1970). Following exposure of the periosteum
(split flap) or alveolar bone denudation an
 C O N N E C T I V E TISSUE AND iEPITHELIAL
increased width of keratinized gingiva is
expected to occur due to changes in func-
tion and environment by the displacement
of muscle attachments and the extension of
vestibular depth. Supposedly the regenerat-
ing tissue is then subjected to mechanical
stimuli which make it adapt to functional
requirements similar lo those of the kera-
tinized gingiva (Ivancie 1957, Bradley, Grant
& Ivancie 1959, Pfeifer 1963). Other muco-
gingival procedures such as the "apically
repositioned flap" (Friedman 1962) and the
"periosteai fenestration" technique (Robin-
son & Agnew 1963) are essentially based on
a similar hypothesis and may be considered
to be modifications of the periosteai reten-
tion or denudation procedures.
Although an increased width of gingiva is
frequently achieved following such surgical
techniques, the post-operative results are
less predictable (Ochsenbein I960, Arnold
& Hatchett 1962, Bohannan 1962b, Don-
nenfeld, Marks & Giickman 1964, Carraro
et ai 1964, Pennel et al. 1965, Costich &
Ramfjord 1968, Ramfjord & Costich 1968,
Carranza & Carraro 1970). The finding of
the present study that the gingiva! connec-
tive tissue determines the differentiation of
the overlying epithelium suggests that the
success or failure in extending the width of
the gingiva by surgical means rests with the
origin of the regenerating tissue. Granula-
tion tissue proliferating from the alveolar
mucosa will produce a non-keratinized epi-
thelium, whereas granulation tissue origina-
ting from the supra-alveolar connective tis-
sue or possibly the superficial part of the
periodontal membrane will produce a kera-
tinized gingival epithelium. Since during the
healing of mucogingival wounds, granula-
tion tissue is likely to originate mainly from
these areas (Wilderman, Wentz & urban
1961, Arnold & Hatchett 1962, Pfeifer 1963,
Wilderman 1963, Karring et al. 1975) the
post-surgical level of the mucogingival line
will depend on the relative rate of prolifer-
D I F F E R E N T I A T I O N
ation of the granulation tissue from these
sources.
Split thickness grafts of palatal or gingival
tissue retain their original specificity when
transplanted to the alveolar mucosa (Kar-
ring, 0stergaard & Loe 1971). This may
suggest that the use of transplants is a more
efficacious method for predetermining the
post-surgical width of the gingiva than the
commonly used mucogingival procedures.
Although the present study was designed
to examine tnechanisms responsible for the
differentiation of gingival epithelium, it
also emphasizes the importance of basic
biological knowledge in the development
of clinical procedures.
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E P I T H E L i A L D I F F E R E N T I A T I O N II
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