J Periodont Res 2003; 38; 237–241 Copyright
Blackwell Munksgaard Ltd

Printed in the UK. All rights reserved

JOURNAL OF PERIODONTAL RESEARCH
ISSN 0022-3484

C. S. A. Araujo1,2, E. Graner1,
Histomorphometric O. P. Almeida1, J. J. Sauk3,4,
R. D. Coletta1

characteristics and
1
Department of Oral Pathology, Dental School,
University of Campinas, Piracicaba, S¼oPaulo
Brazil, 2Department of Pathology, Dental School,

expression of epidermal
Paranaense University, Umuarama, Paran ,
Brazil, 3Department of Diagnostic Sciences and
Pathology, Dental School, University of Maryland
at Baltimore, Baltimore, MD, USA and

growth factor and its receptor 4

Greenebaum Cancer Center, University of
Maryland at Baltimore, Baltimore, MD, USA

by epithelial cells of normal

gingiva and hereditary
gingival fibromatosis
Araujo CSA, Graner E, Almeida OP, Sauk JJ, Coletta RD. Histomorphometric
characteristics and expression of epidermal growth factor and its receptor on
epithelial cells of normal gingiva and hereditary gingival fibromatosis. J Periodont
Res 2003; 38; 237–241.
Blackwell Munksgaard, 2003

Objective: The objective of this study was to examine the histomorphometric

features and evaluate the expression of epidermal growth factor (EGF) and
transmembranic receptor (EGFr) and the proliferative potential of epithelial cells
from normal and hereditary gingival fibromatosis (HGF) gingival tissues.
Background: EGF is a multifunctional cytokine with a variety of biological effects
including stimulation of cell proliferation by binding to its specific EGFr.
Methods: Immunohistochemistry was performed to measure EGF and EGFr
expression and the epithelial cell proliferation was determined by measuring
proliferating cell nuclear antigen (PCNA).
Results: Histomorphometric evaluation indicated that in HGF the mean height of
the epithelial papillae was higher compared to the normal gingiva (NG), whereas
mean epithelial area and number of epithelial papillae were quite similar in both
groups. The EGF and EGFr positive cells were observed in the basal, spinous and
granular cell layers of both normal and HGF tissues, with a gradual reduction
from the basal layer. Although the expressions of EGF and EGFr in the control
group were significantly higher than those from HGF, in HGF the epithelial Dr Ricardo D. Coletta, Discipline of Oral
papilla tips showed increased number of proliferating cells and elevated expression Pathology, University of Campinas Dental
School, Caixa Postal 52, 13414-018
of EGF and EGFr. There was a correlation between the proliferative potential of
Piracicaba-SP, Brazil
epithelial cells and the expression of EGF or EGFr only in the epithelial papilla Tel: + 55 19 34125315
tips of HGF gingiva. Fax: + 55 19 34125218
e-mail: coletta@fop.unicamp.br
Conclusion: Our data suggest that EGF and EGFr in the oral epithelium of HGF
Key words: hereditary gingival fibromatosis;
gingiva may stimulate epithelial cell proliferation, with the resultant apical histomorphometry; epidermal growth factor;
migration of the oral epithelium and formation of the slender deep epithelial epidermal growth factor receptor; proliferation
papillae; however, without hyperplastic alterations. Accepted for publication March 14, 2002
238 Araujo et al.
Hereditary gingival fibromatosis
(HGF) is an uncommon oral condition
characterized by a slow, progressive
enlargement of the gingiva, involving
both the maxilla and mandible (1).
HGF may manifest as an isolated
finding or in association with other
features, as part of a syndrome. As
such, HGF has been recorded in
association with hypertrichosis, mental
retardation, epilepsy, progressive hear-
ing loss and abnormalities of the
extremities, particularly fingers and
toes (2–7). In most of the cases, an
autosomal dominant mode of inherit-
ance with variable penetrance and
expressivity is involved; however, the
disease may be found as an autosomal
recessive disorder (8, 9). The histolog-
ical appearance of HGF is character-
ized by a mucosa in which the
hyperplastic epithelium contains long
slender papillae that extend into a
dense connective tissue that is rich in
collagen fibers (10, 11). In addition
to collagen, HGF and other gingi-
val overgrowths contain increased
amounts of other extracellular matrix
molecules, including proteoglycans,
glycosaminoglycans and fibronectin
(12, 13).
Epidermal growth factor (EGF) was
first isolated from mice submandibular
salivary glands and characterized as a
small single polypeptide of 6 kDa (14).
EGF exerts its variety of effects
through binding to its transmembranic
receptor, which shows an intrinsic
protein tyrosine kinase activity (15).
The biological response of EGF
receptor (EGFr) triggered after EGF
binding varies from mitogenesis to
apoptosis, stimulation to inhibition of
protein synthesis, migration to differ-
entiation or dedifferentiation even in
the same cell depending on the context,
which includes cell density, type of
matrix, other cytokines, and position
within a cell colony (16). However, the
molecular bases of these responses are
only now being defined (17). EGF and
its receptor EGFr have been demon-
strated by immunohistochemistry in
the gingival tissues of periodontally
healthy individuals and patients with
periodontitis (18–21). These in vivo and
in vitro studies have shown high levels
of expression of EGF and EGFr in the
basal cells of the gingival epithelium.
Moreover, an up-regulation of these
peptides occurs during the inflamma-
tory response of the underlying con-
nective tissue. Therefore, EGF and its
receptor might be important mediators
of epithelial modifications found in
HGF gingiva. Our purpose was to
evaluate the morphological pattern of
the gingival epithelium of HGF tissues
in comparison with the epithelium of
the normal gingiva (NG). Addition-
ally, we analyzed the expression of
EGF and EGFr in NG and HGF
gingival epithelial cells and correlated
its with the proliferative potential, as
revealed by the immunohistochemical
expression of proliferating cellular
nuclear antigen (PCNA).
Materials and methods
Gingival samples
Gingival tissue samples were obtained
by gingivectomy/gingivoplasty from
10 patients affected by HGF and from
10 patients under periodontal treatment
at the Orocentro – Center for the Study
of Oral Diseases at the University of
Campinas Dental School, Piracicaba-
SP, Brazil. All HGF patients were
members of the same family, and only
fibrous and non-inflamed gingival
overgrowth tissues were selected. The
mean age of HGF group was
23.2 ± 8.3 years and included five
males and five females. Control sam-
ples were derived from non-inflamed or
hyperplastic gingiva of five males and
five females, with a mean age of
26.2 ± 4.9 years. All gingival samples
were fixed in formalin and embedded
in paraffin for hematoxylin and eosin
staining (H&E) or immunohistoche-
mistry. The study protocol was
approved by the Ethical Committee in
Research at the University of
Campinas Dental School. All patients
were informed about the study’s
purpose before they consented to
participate.
Histomorphometry
Histomorphometric analyzes were
performed with the aid of an image
computer analyzer (Kontron 400,
Zeiss, Germany) in 10 sections from
each sample. Six-micrometer semiserial
sections (one in each 30 lm) were
made on the mesial-distal orientation
and stained with H&E. Oral epithelial
area, height and number of epithelial
papillae were measured in 0.5 linear cm
of gingiva. From these data the mean
was calculated for each sample,
expressed in lm or lm2/linear cm of
gingiva, and used for statistical analy-
sis.
Immunohistochemistry
Immunohistochemical analyzes were
performed using the streptavidin–
biotin peroxidase complex method.
Briefly, paraffin sections (3-lm thick)
were cut and mounted on silane-coated
glass slides. Following deparaffiniza-
tion and hydration in graded alcohol
solutions, the sections were treated
with 3% H2O2 followed by antigen
retrieval with 10 mM citric acid,
pH 6.0, in a microwave for 24 min,
divided in 2 cycles. After washing with
phosphate-buffered saline (PBS), the
sections were treated with 1% bovine
serum albumin (BSA, Sigma Chemical
Co., St. Louis, MO, USA) in PBS for
1 h, and then incubated with primary
antibodies for 16 h at 4C. The pri-
mary antibodies used were as follows:
polyclonal rabbit antihuman EGF
diluted 1 : 50 (Oncogene Research
Products, Boston, MA, USA), mono-
clonal mouse antihuman EGFr diluted
1 : 50 (Dako Corp., Carpenteria, CA,
USA) and monoclonal mouse antihu-
man PCNA diluted 1 : 3000 (Dako).
Subsequent incubations were made
with biotinylated IgG followed by
streptavidin–biotin peroxidase complex
(StrepABC Complex/HRP Duet kit,
Dako). Reactions were developed by
incubating the sections with 0.6 mg/ml
3,3¢-diaminobenzidine tetrahydrochlo-
ride (DAB, Sigma) containing 0.01%
H2O2 and counterstained with Mayer’s
hematoxylin. Positive and negative
controls were included in all reactions.
For immunohistochemical assess-
ment, a reproductible semiquantitative
analysis was performed as previously
described (22). After scanning and
examination of the entire section, five
representative areas were evaluated

with high power. Scoring of the
immunohistochemical results were
performed by two authors, who were
blinded with regard to the clinical data.
At each selected field, epithelial cells
were classified with respect to staining
intensity ranging from 0 to 4, and the
percentage of positive cells estimated.
A numerical value for each field was
then calculated by multiplying the
proportion of positive cells (decimal
equivalent of percentage of cells) by the
numerical value of that intensity. For
statistical analysis the mean of each
sample was used. Expression levels of
each marker in NG and HGF samples
were based on the total score as fol-
lows: negative – score 0 to 0.5; low –
score 0.6 to 1; high – score 1.1 to 4.
Statistical analysis
Non-parametric one-way analysis of
variance (ANOVA) was used to test
group effects based on Wilcoxon test.
To compare the histomorphomet-
ric measurements, Student’s t-test
(bi-caudal) was used. Correlations of
immunohistochemical scores between
groups were assessed by Spearman’s
rank correlation test (rs). In our com-
parisons, P < 0.05 was considered to
indicate statistical significance.
Results
Histological examination of gingival
overgrowth tissue specimens stained
with H&E revealed a well-structured
epithelium with elongated and thin
papillae inserted in deep fibrous con-
nective tissue. Short areas of epithelial
atrophy were eventually found between
these papillae. All HGF samples
showed the epithelial papillae sig-
nificantly higher than the control
group (P < 0.005; Table 1). The
height in HGF group ranged between
398 and 526 lm/papilla, with a median
Table 1. Mean values of height and number of papillae and of epithelial area in the NG and
HGF groups. The data correspond to the mean ± SD of 10 samples for each group
Height of papillas (lm) Number of papillas
NG 359.1 ± 64.4 8.1 ± 4.2
HGF 467.6 ± 41.8* 10.5 ± 6.8
*P < 0.005.
EGF and EGFr in NG and HGF gingiva
of 473.2 lm (mean 467.6 ± 41.8 lm/
papilla), whereas for NG group height
ranged between 263 and 439 lm/
papilla, with a median of 384.34 lm
(mean 359.1 ± 64.4 lm/papilla). There
were no statistical differences in epi-
thelial area and number of epithe-
lial papillae between both groups
(Table 1). The mean area of HGF
gingival epithelium was 144.7 ±
29.5 · 103 lm2 compared to 135.8 ±
22.8 · 103 lm2 in the control group.
In both NG and HGF gingiva,
positive staining for EGF was detected
throughout the cytoplasm and mem-
brane of epithelial cells, whereas EGFr
was only visualized in the membrane
(Fig. 1). Nuclear immunoreactivity for
PCNA was clearly and easily identified
in both groups. Nuclei with a clear
brown color, regardless of the intensity
of staining, were interpreted as posit-
ive. In all specimens, the immuno-
staining for EGF, EGFr and PCNA
was more often positives in the inner
compartment (basal cells) than in the
outer compartment (spinous and
granular cells). Compared with HGF,
the control group showed a signifi-
cantly higher expression of EGF
(P < 0.05) and EGFr (P < 0.01;
Table 2). However, when considering
only epithelial papilla tips of HGF
group, higher EGF, EGFr and PCNA
expression were observed in compar-
ison with those from the control group
(P < 0.01 for EGF and EGFr, and
P < 0.05 for PCNA). A high correla-
tion was observed between EGF
and EGFr expression (rs ¼ 0.66,
P < 0.05). There was also a highly
significant correlation between staining
intensity and the percentage of positive
cells for EGF as well as EGFr
(rs ¼ 0.80, P < 0.001). The expression
pattern of PCNA in HGF samples was
comparable to that of the control
(Table 2). Correlation between EGF
or its receptor and PCNA was only
Area of epithelium (· 103 lm2)
135.8 ± 22.8
144.7 ± 29.5
239
observed in HGF epithelial papilla
tips (rs ¼ 0.66, P < 0.05 for EGF;
rs ¼ 0.56, P < 0.05 for EGFr).
Discussion
It is accepted that HGF is a disease of
genetic origin, but the mechanism that
leads to the accumulation of excessive
amounts of gingival tissue is unknown.
The abnormal accumulation of con-
nective tissue has traditionally been
regarded to result from local increases
in collagen synthesis, which is consis-
tent with the histological evidences.
Although the exact genetic alteration
remains unclear, our understanding of
the biochemical mechanisms involved
in this condition are evolving. For
example, our group has recently dem-
onstrated that both impairment of
connective tissue and excessive pro-
duction of extracellular matrix are
equally important in the pathogenesis
of HGF (23).
Until recently most attention in
the investigation of HGF has been
focused on the connective tissue
alterations. Herein, we have deter-
mined that HGF gingiva has higher
and deeper epithelial papillae, and
that EGF and EGFr expression pat-
terns are positively correlated with the
proliferative potential of the cells of
papilla tip area, as measured by
PCNA positivity. Interestingly, EGF
and EGFr expression in NG samples
are higher compared with HGF sam-
ples, when all epithelial layers are
considered. However, expression of
EGF and EGFr in NG group is not
associated with proliferative potential
of epithelial cells.
The few previous studies comparing
the histological features of NG and
HGF gingiva agree that the gingival
enlargement is primarily the result of a
marked increase and thickening of the
collagenous bundles in the connective
tissue stroma (3, 24–28). However, they
are divergent regarding the epithelial
features. Farrer-Brown et al. (26)
reported epithelial hyperplasia from
acanthosis in gingival specimens from
four patients affected with HGF, but
only in those areas associated with
chronic inflammation. Fletcher (28)
and others (3, 24, 25) also described
240 Araujo et al.
Fig. 1. Immunohistochemical detection of EGF (A) and EGFr (B) in normal gingiva. EGFr
was expressed as a specific cell-surface labeling, whereas EGF immunoreactivity was
localized in cytoplasm and membrane of the cells. For both EGF and EGFr antibodies, the
most intense labeling is seen on the basal cells; in contrast, the superficial cell layers are free
from label (immunostaining, · 200).
epithelial hyperplasia in HGF speci-
mens with intense inflammatory cell
infiltration. In the present study, the
epithelial area of HGF and NG group
was quite similar; however, our sam-
ples were free of inflammatory infil-
A spinous and granular cells. These
B mechanisms responsible for such an
tration. Raeste et al. (27) found
epithelial hyperplasia and prominent
papillae in areas with intense chronic
inflammation, whereas areas without
inflammation showed only long and
deep epithelial papillae. We have also
found epithelial hyperplasia in
inflamed specimens from patients with
HGF (data not shown), which were
not included in this study. Inflamma-
tion may render the cells more per-
missive to small peptides as growth
factors, originating from nearby cells
such as macrophages and resident
fibroblasts, stimulating cell prolifer-
ation and hiperplastic changes. It is
interesting that in the drug-induced
gingival overgrowth, epithelial hyper-
plasia is probably a response to fibrosis
or inflammation instead of effects of
drug.
The studies presented here revealed
that immunohistochemical reactivity
for EGF and EGFr in both NG and
HGF specimens were more pro-
nounced in the basal layer, progres-
sively diminishing in intensity in
results indicate that their synthesis
occurs in the basal cells and is inter-
rupted when the cells stratify and dif-
ferentiate. Upper-layer keratinizing
cells were negatives. Some interspeci-
men variation in EGF and EGFr
expression was observed in both nor-
mal and HGF patients. Occasional
faint positivity was found in restricted
areas of both groups, which may be
associated with different levels of cel-
lular differentiation. Although all
patients showed healthy gingiva, some
small foci of inflammatory cells (not
included in histomorphometric analy-
sis) were observed in both groups.
Irwin et al. (18) related an increase in
the expression of EGFr in inflamed
tissue, suggesting that besides the
cellular differentiation status, the
response to EGF may be modulated by
alterations in receptor number. The
up-regulation of EGFr expression are
not yet understood, and a number of
factors released from activated macr-
ophages and lymphocytes may play a
role in this context.
In summary, we have determined
that epithelial cells from the papilla tips
have increased expression of EGF and
EGFr as well as elevated proliferative
potential. Furthermore, high levels of
EGF and EGFr in the papilla tips were
significantly associated with increased
expression of PCNA. A high density of

Table 2. Expression of EGF, EGFr and PCNA in NG and HGF groups
EGF score EGFr score
£ 1.0 >1.0 P £ 1.0 >1.0
NG 3 7 1 9
HGF 6 4 0.05 4 6
EGF receptor and PCNA positive cells
in the basal layer may indicate a role for
7.
EGF in promoting proliferation and
apical migration of the epithelial
papillae in HGF gingiva. This complex
pattern of response indicates that fur- 8.
ther studies are required to determine
the precise interactions that occur
between EGF and EGFr in the regula-
tion of proliferation of epithelial cells
9.
from HGF gingival.
Acknowledgments
10.
Support for this research was provided
from grant 1999/08191-0 from Fun-
dação de Amparo à Pequisa do Estado
11.
de São Paulo-FAPESP, Brazil (to
RDC).
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