ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 17
OBJECTIVE: To investigate the histopathological altera-
tions in gingival tissue of streptozotocin (STZ) diabetic
rats and the expressions of ADAMTS-5 and TNF-α to
better understand the effects of diabetes on oral mucosa.
STUDY DESIGN: Twenty Wistar rats, divided into
control and diabetic groups, received a single dose of
STZ (55 mg/kg) ip. The rats were sacrificed, and gin-
gival tissues were dissected and fixed in formalin for
routine paraffin protocol. Hematoxylin-eosin and im­
munohistochemical staining (ADAMTS-5 and TNF-α)
were applied and sections were examined under a mi-
croscope.
RESULTS: The control group revealed regular place-
ment of epithelial cells. In the diabetic group signifi-
cant thinning of the epithelial cell layer, degeneration
of the cells in the papillary region, pyknosis in nuclei,
and inflammatory cell infiltration were seen. Negative
ADAMTS-5 expression was observed in parakerati­
nized epithelium of the control group. The diabetic
group showed positive ADAMTS-5 expression in the
epithelial cells. In the control group, TNF-α positive
expression was observed in some of the gingival epi-
thelial and vascular endothelial cells. In the diabetic
group, TNF-α expression was observed in basal lamina,
endothelial cells, and fibroblast cells and dilated blood
vessels.
CONCLUSION: In diabetics, hypertrophic changes of
the gingival epithelium with the epithelial permeability
alteration and the presence of intraepithelial inflamma-
tory cells eventually gave rise to degenerative lesions
and epithelial ulceration. (Anal Quant Cytopathol
Histpathol 2020;42:17–22)
Keywords: ADAMTS-5, dental caries, dental health
surveys, diabetes, diabetes complications, diabe-
tes mellitus, gingiva, histopathology, immunohisto­
chemistry, mouth mucosa, oral health, periodontal
diseases, TNF-α.
Diabetes mellitus is a metabolic disease character­
ized by chronic hyperglycemia. Diabetes mellitus
is responsible for tooth deprivation both by decay
and periodontal disease.1 Patients with diabetes
are susceptible to infection, and they have a high
prevalence of periodontal disease.2,3 Periodontal
disease is a well-known complication of diabetes.4
The epidemiologic data and animal model studies
have shown evidence for the pathophysiology of
periodontal disease as a complication of diabetes
mellitus.5 Periodontal disease was expressed as
the sixth complication of diabetes mellitus and as
a possible risk factor for poor metabolic control in
diabetic patients.6 The development of periodon-
tal disease is related to the response to microbial
attack. In diabetic patients, the response of im­
mune and inflammatory cells to microbial attack
results in gingivitis and periodontitis.7 Gingiva has
intact vascular tissue to resume oxygen and nu-
trients through the tissues and remove waste pro-
ducts to maintain proper tissue function. Diabetes
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4201-0017/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Effects on Gingiva of Diabetes Mellitus
Induced by Streptozotocin in Rats
Fikret I
·
pek, Ph.D.
From the Department of Periodontology, Faculty of Dentistry, University of Dicle, Diyarbakır, Turkey.
Dr. I
·
pek is Associate Professor and Dentist.
Address correspondence to: Fikret I
·
pek, Ph.D., Department of Periodontology, Faculty of Dentistry, University of Dicle, University
Street, Diyarbakır 21280, Turkey (melihsemih21@hotmail.com).
Financial Disclosure: The author has no connection to any companies or products mentioned in this article.

2. is associated with periodontal vascular changes
because of the increased thickness of basement
membrane in small blood vessels. In case of dam-
age in the endothelial cells of vessels, vascular
complications arise.8 All these factors together lead
to a deterioration in oxygen diffusion, metabolic
waste elimination, leukocyte migration, and dif-
fusion of immune factors. Poor metabolic control
and longer duration of the disease worsen vascular
changes in the gingival vascular bed, resulting in
vascular abnormalities in the gingival tissue of
diabetics.9 Kido et al3 showed that wound healing
in the gingiva of diabetic rats is delayed and that
poor healing is associated with impaired fibroblast
function via oxidative stress.
The ADAMTS (a disintegrin and metalloprotein­
ase with thrombospondin motifs) family consists
of 19 associated proteases that act in the forma-
tion of the extracellular matrix.10 Their functions
include collagen processing, cleavage of the ma-
trix proteoglycans, inhibition of angiogenesis, and
blood coagulation homeostasis. They are also in­
volved in organogenesis, inflammation, and fer-
tility. ADAMTS-5 is also known as aggrecan-2, a
major component of the extracellular matrix and
ADAMTS-5 gene known to be expressed in the
bladder, cervix, esophagus, etc.11
Tumor necrosis factor alpha (TNF-α) is a 17-kDa
protein and a proinflammatory pleiotropic cyto­
kine. TNF-α is activated by macrophages and has
a crucial role in the modulation of cellular re-
sponses (inflammation and fibrosis) in injured tis-
sue.12 The aim of this study was to investigate the
histopathological alterations in gingival tissue of
streptozotocin-induced diabetic rats and to dem­
onstrate the expressions of ADAMTS-5 and TNF-α
to better understand the effects of diabetes on oral
mucosa.
Materials and Methods
Experimental Procedure
The present work was performed in accordance
with the guidelines for the Care and Use of Labo-
ratory Animals from Dicle University Experimental
Animal Research Center, Diyarbakır, Turkey. The
diabetes induction and histopathological and im­
munohistochemical analysis methods were mod­
ified from Uysal et al.13 Adult male Wistar rats
(n=20) were divided into 2 groups. The control
group rats (n=10) were fed with standard rat
chow and drinking water ad libitum for 8 weeks.
The diabetic group rats (n=10) received a single
dose of streptozotocin (55 mg/kg) (Sigma, USA),
dissolved in sodium citrate buffer (0.1 M, pH 4. 5)
and administered by intraperitoneal injection. Be­
fore the experiment the subjects were fasted for
12 hours, then fed with standard rat chow and
drinking water after 4 hours. After fasting of 2
days and 12 hours, blood glucose levels were
measured from the tail end with a capillary blood
glucose meter, and rats with a value of ≥250 mg/
dL were considered as diabetic and included in the
experiment. At the end of the experimental period
the animals were sacrificed by decapitation. Gin­
gival tissues were dissected immediately. Samples
were fixed with neutral buffered formalin solu­
tion, dehydrated in a graded series of ethanol, and
embedded in paraffin wax. Next, 4–6 µm sections
were cut with a microtome (Rotary Microtome,
RM2265, Leica Biosystems, Germany) and mounted
on coated slides. The sections were stained with
hematoxylin and eosin and observed under light
microscope (Nikon Eclipse 80i).
Immunohistochemical Staining
An antigen retrieval process was performed in
citrate buffer solution (pH 6.0) 2 times, first for
7 minutes and then for 5 minutes, in a microwave
oven at 700 W. They were allowed to cool to room
temperature for 30 minutes and were washed in
distilled water for 5 minutes twice. Endogenous
peroxidase activity was blocked in 0.1% hydrogen
peroxide for 15 minutes. Ultra V block (Histostain-
Plus Kit, Invitrogen, Carlsbad, California, USA)
was applied for 10 minutes prior to the applica-
tion of the primary antibodies ADAMTS-5 (cata-
log no. ab41037, Abcam, Cambridge, Massachu­
setts, USA) and TNF-α (catalog no. sc52746, 1/200,
Santa Cruz Biotechnology, USA) overnight. The
secondary antibody (Histostain-Plus Kit, Invitro­
gen) was applied for 20 minutes. Then the slides
were exposed to streptavidin-peroxidase for 20
minutes. Diaminobenzidine (DAB, Invitrogen) was
used as a chromogen. Negative control slides were
prepared as mentioned above but omitting the
primary antibodies. After counterstaining with
Mayer’s Hematoxylin stain, washing in tap water
for 5 minutes and in distilled water for 2×5 min,
the slides were mounted.
Results
Histopathological Examinations
Examination of the control group revealed that
the epithelial cells in the dentogingival junction
18 Analytical and Quantitative Cytopathology and Histopathology®
I
·
pek et al

3. showed regular placement along the basal lamina
towards the apical, while desquamation was ob­
served in some cells in the apical. Although the
microscopic papillae in this region were not deep,
it was observed that the parakeratinized feature of
the epithelium towards the tooth surface formed
epithelial clusters in the connective tissue area
where the papillary structures deepened. Fibril-
lary structures of the connective tissue were irre-
gularly tight, and solitary connective tissue cells
were distributed (Figure 1A). In the diabetic group,
significant thinning of the epithelial cell layer in
the dentogingival sulcus area, degeneration of the
cells in the papillary region from the basal lamina
to the connective tissue, pyknosis in nuclei, inflam-
matory cell infiltration around blood vessels, and
dilation of blood vessels were seen. Extensions of
the extracellular matrix between collagen fibrils in
the connective tissue area and apoptotic changes
in the cells were observed (Figure 1B).
Immunohistochemistry Analysis
ADAMTS-5 Immunoexpression
In the control group, ADAMTS-5 expression was
positive in the epithelial cells of the dentogin-
gival sulcus, while ADAMTS-5 expression was
negative in the parakeratinized epithelium in the
neck region. Expression of ADAMTS-5 was posi-
tive in fibroblast cells in the cluster in the connec-
tive tissue area (Figure 2A). In the diabetes group,
ADAMTS-5 positive expression was seen in the
desquamated sections of the epithelial cells in the
gingival junction and in the cells around the sec­
ondary papillae. In the connective tissue below the
neck region, an increase in ADAMTS-5 positive
expression was observed in inflammatory cells and
fibroblasts and collagen fibers (Figure 2B).
TNF-α Immunoexpression
In the control group, TNF-α positive expression
was observed in some of the gingival epithelial
cells and endothelial cells in the blood vessels
(Figure 3A). In the diabetic group, increased TNF-α
expression in basal lamina and connective tissue
of epithelial cells with dentogingival junction, pos­
itive TNF-α expression in endothelial cells, and
fibroblast cells in dilated blood vessels were ob-
served (Figure 3B).
Discussion
Hyperglycemia, as a common feature of diabetes
mellitus, is a cause of different pathogenic mech­
anisms influencing endothelial function. Diabetes
mellitus results in the development of large and
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Effects on Gingiva of Diabetes Mellitus
Figure 1 (A, B) Control group: Regular placement along the basal lamina towards the apical in the epithelial cells in the dentogingival
junction, desquamation in some cells in the apical. Parakeratinized feature of the epithelium towards the tooth surface formed epithelial
clusters in the connective tissue area where the papillary structures deepened. Irregularly tight fibrillary structures of the connective
tissue, solitary connective tissue cells. H-E staining, bar=20 µm. Diabetic group: significant thinning of the epithelial cell layer in the
dentogingival sulcus area, degeneration of the cells in the papillary region from the basal lamina to the connective tissue, pyknosis in
nuclei, inflammatory cell infiltration around blood vessels and dilation of blood vessels. Extensions of the extracellular matrix between
collagen fibrils in the connective tissue area and apoptotic changes in the cells. H-E staining, bar=50 µm.

4. micro-vessel damage.14 Changes in inflammatory
cell infiltration that occurs after one week of step­
tozotocin treatment in the gingivomucosal tissue
was reported by Fehér et al.7
In one study, a gingival wound was created
with the underlying periosteum. Because of re-
section from the periosteum, wounds were al-
lowed to heal only from the gingival wound mar-
gin. In control rats, wound healing proceeded with
blood clot accumulation, inflammation, prolifera­
tion, and remodeling in approximately 7 days, but
wound healing was significantly delayed in dia-
betic rats because of the high glucose level. Here
histological analysis revealed extension of imma-
ture epithelium and completion of wound closure
on days 3 and 7 in control rats, but granulation
was observed on days 3, 7, and 21 in diabetic
rats.3 According to Kido et al,3 delayed gingival
wound healing in diabetic rats was caused by im-
paired proliferation and migration of fibroblasts.
Dysfunction of the fibroblasts may be caused by
high glucose-induced insulin resistance via oxi­
dative stress. There are several studies on the
relationship between periodontitis and diabetes
such that healing and tissue regeneration after
nonsurgical periodontal treatment15-17 and surgical
procedures18 are impaired in the dental practice
of diabetic patients.
Another study conducted on patients with peri-
odontal disease and type 1 diabetes mellitus
showed that the histological analysis with HE
staining of gingival mucosa revealed epithelial
projections of the stratified pavimentous epithe-
lium to the underlying connective tissue and as-
pects of exocytosis. The penetration of the im-
mune system cells into the layers of the covering
gingival epithelium leads to degenerative lesions
of the epitheliocytes with decrease of cell adhe­
sion and appearance of ulcerations noticed in the
studied patients.19 It was observed in the intra­
epithelial level that polymorphonuclear leukocytes
and lymphocytes in relation with epitheliocyte
absorption and intercellular bridge disintegra-
tion was apparent. The presence of the inflam­
matory cells at the intraepithelial level suggested
an increased vascular and gingival epithelium per-
meability, with degenerative places over the epi­
theliocytes that finally evolved into ulcerations.
As the diabetes progresses at the epithelial level,
the atrophy continues with the reduction in num-
ber of the squamous cell layers, the deletion of the
interpapillary ridges, and the tendency for recti­
linear epithelium-chorion limit.20
A study by Balakhonov et al21 demonstrated that
patients with a history of diabetes mellitus of 5–15
years and/or predominance of atrophic changes
20 Analytical and Quantitative Cytopathology and Histopathology®
I
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pek et al
Figure 2 (A, B) Control group: positive ADAMTS-5 expression in the epithelial cells of the dentogingival sulcus, negative ADAMTS-5
expression in parakeratinized epithelium in the neck region. Positive ADAMTS-5 in fibroblast cells in the cluster in the connective
tissue area. ADAMTS-5 immunostaining, bar=50 µm. Diabetic group: positive ADAMTS-5 expression in the desquamated sections of the
epithelial cells in the gingival junction and in the cells around the secondary papillae. In the connective tissue below the neck region,
increase in ADAMTS-5 positive expression in inflammatory cells and fibroblasts and collagen fibers. ADAMTS-5 immunostaining,
bar=20 µm.

5. in gingival biopsy preparations show a mucosal
thinning at the alveolar processes and atrophy of
the stratified squamous epithelium, with the ap-
pearance of keratinization centers. As a result of
diabetes, the epithelial cell layer in the dentogin-
gival sulcus area is considerably thinner, and the
cells in the papillary region degenerate from the
basal lamina to the connective tissue, along with
inflammatory cell infiltration around the blood ves-
sels. Extension of the extracellular matrix between
the collagen fibrils in the connective tissue area
and apoptotic changes in the cells were observed
(Figure 1B).
Numerous studies showed that there is a link
between periodontal disease and diabetes because
diabetic patients develop more severe periodon-
tal disease. It was also reported that diabetes in-
fluences the evolution of periodontal disease by
immune/inflammatory response. In the diabe-
tic group, ADAMTS-5 positive expression in the
segregated portions of epithelial cells in the gum
junction and around the secondary papilla also
showed an increase in ADAMTS-5 positive ex-
pression in connective tissue, inflammatory cells,
fibroblasts, and collagen fibers under the neck
region (Figure 2B). A high level of blood glucose
seems to accelerate the secretion of inflammatory
mediators and, consequently, an excessive peri-
odontal response.22 It was verified that diabetes
patients have higher IL-6, IL-1β, and TNF-α levels
as a response to the general irritation caused by
bacterial plaque.23,24 In the diabetic group in our
study, increased TNF-α expression in basal lamina
and expression of positive TNF-α expression in
endothelial cells and connective tissue fibroblast
cells of epithelial cells by dentogingival fusion
were observed (Figure 3B). Diabetes has been
shown to increase inflammation, angiogenetic ef­
fect, and collagen synthesis, and cytokine activity
may be significantly affected.
In conclusion, we can suggest that in diabetics,
hypertrophic changes of the gingival epithelium
with the epithelial permeability alteration and the
presence of the intraepithelial inflammatory cells
eventually give rise to degenerative lesions and
epithelial ulceration.
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