The purpose of this study was to evaluate the oral health-related quality of
life of patients treated with implant-supported mandibular overdentures and to compare
the attachment systems used.
Material and Methods
The presence of myofibroblasts as well as transforming growth
factor-beta1 was examined in twenty cases of fibrous epulis and 22
ossifying fibrous epulis, using immunohistochemistry.
Results
Myofibroblasts positive for alpha smooth muscle actin and vimentin but
negative to desmin were found in 20% and 45% in fibrous epulis and
ossifying fibrous epulis, respectively. Myofibroblasts were distributed
in areas with and without inflammatory infiltration and their presence
in inflammatory areas was not related with the degree of inflammatory
infiltration. A percentage of 21 - 60% of fibroblasts and chronic
inflammatory cells expressed transforming growth factor-beta1 in all
cases.
Conclusions
These data suggest that transforming growth factor-beta1 and
myofibroblasts contribute to the formation of collagenous connective
tissue in fibrous epulis and ossifying fibrous epulis. Myofibroblasts
are mainly presented in ossifying fibrous epulis than in fibrous epulis.
It seems to be no relationship between the presence of myofibroblasts
and the degree of inflammatory infiltration of the lesions.
Myofibroblasts (MFs) are specialized fibroblasts characterized by the
presence of contractile apparatus [1] and are responsible for
synthesizing enzymes involved in extracellular matrix degeneration,
tissue remodelling and wound healing [2]. Furthermore, these cells are
involved in several fibrotic diseases, such as pulmonary fibrosis,
interstitial lung fibrosis, liver cirrhosis, renal fibrosis and
scleroderma, thus being responsible for overproduction of extracellular
matrix molecules, such as collagen type I [3,4]. Although fibroblasts
are considered to be the main progenitor cells of MFs, pericytes and
vascular smooth muscle cells may be transformed into MFs [5].
Transforming growth factor-beta (TGF-β) is a large family of
structurally related growth and differentiation factors including
activins and bone morphogenic proteins. There are three TGF-β isoforms,
TGF-β1, TGF-β2 and TGF-β3 with distinct and overlapping activities, such
as control of mesenchymal cell proliferation and differentiation, wound
healing and extracellular matrix production [6,7]. TGF-β1 is present in
epithelia, connective tissue and mononuclear inflammatory cells [7]. It
is well documented that TGF-β1 plays the principal role in the
trans-differentiation of fibroblasts into MFs [4], as well as
fibroblasts' proliferation and collagen secretion in pathologic
conditions [8,9].
Fibrous epulis (FE) or peripheral fibroma and ossifying fibrous epulis (OFE)
or peripheral ossifying fibroma consists of common reactive fibrous
overgrowths of the gingiva caused by chronic irritation. FE consists of
interlacing collagen bundles but areas with chronic inflammatory
infiltration may also be presented [10,11]. On the other hand, OFE is
composed of high fibrocellular tissue containing little or plenty of
bone, cementum-like material, and dystrophic calcification and
peripherally, there is a less fibrocellular tissue that is infiltrated
by chronic inflammatory cells, mostly lymphocytes and plasma cells. The
stratified epithelium of the lesion may be ulcerated [11,12].
The expression of TGF-β1 in both FE and OFE has not been investigated in
detail, so far. Few studies have focused on the presence of MFs. Their
controversial results were based on a small number of cases [13-15].
Furthermore, the relation of MFs and the inflammatory infiltration was
described only in one study on FE [16].
The aim of the present study was to detect immunohistochemically the
presence of myofibroblasts and transforming growth factor-beta1 in
fibrous and ossifying-fibrous epulis in an attempt to note their
possible contribution in the formation of collagenous connective tissue
of the lesions. Also, a possible relationship between the presence of
myofibroblasts and degree of inflammatory infiltration was examined.
MATERIAL AND METHODS
Twenty cases of FE and 22 cases of OFE were retrieved from the archives
of the Department of Oral Medicine and Oral Pathology, Dental School of
Aristotle University of Thessaloniki, Greece. Cases with and without
inflammatory infiltrations in both FE and OFE were included. Serial, 4
μm sections from paraffin-embedded tissues stained with haematoxylin and
eosin (for the confirmation of diagnosis) and for immunohistochemistry,
as well. Patients had given informed consent and the whole study was
performed according to the Declaration of Helsinski II.
The inflammatory infiltration was graded as absent, mild, moderate and
severe. In cases with different degrees of inflammatory infiltration,
the most frequent degree was recorded in association with the presence
of MFs.
Monoclonal antibodies against alpha smooth muscle actin (alpha-SMA),
vimentin, and desmin and polyclonal antibody against TGF-β1 were used.
Endogenous peroxidase activity was quenched with 3% H2O2
for 10 min at room temperature. Then the sections were pre-treated for
antigen retrieval (Table 1). Sections that were intended for the
detection of alpha-SMA, vimentin and desmin were incubated with normal
rabbit serum, and those for the detection of TGF-β1 with normal mouse
serum at a dilution of 1:20 for 30 min at room temperature. Sections
were incubated with monoclonal and polyclonal antibodies (Table 1) and
sections incubated with normal mouse serum were used as negative
control. Envision/horse radish peroxidase (HRP) ChemMate/TechMate
detection system (Dako, Glostrup, Denmark) was performed for the
detection of all antigens using the autostainer Ventana (Ventana Med
Systems Inc Tuscon, AZ, USA), and the reaction was developed using
diaminobenzidine. Haematoxylin was used as counterstain. Sections from a
leiomyoma and placenta were used as positive control for desmin and
TGF-β1, respectively.
Source, clone and pretreatment for antigen retrieval
Antibody
Source
Clone
Pretreatment
Dilution
Incubation
(min)
Alpha smooth muscle actin
Dako, Glostrup, Denmark
1A4
No treatment
1:100
30
Vimentin
Dako, Glostrup, Denmark
V9
Microwave
Citrate buffer 0.01M
pH 7.2 ,95°C, 15 min
1:100
30
Desmin
BioCare, Carmino Diablo,
CA, USA
D33
Microwave
Citrate buffer 0.01M
pH 7.2, 95°C, 15 min
1:100
30
Transforming
growth factor-beta1
Spinge Biosense, Pleasaton,
CA, USA
Polyclonal
Microwave
Citrate buffer 0.01M
pH 6, 95°C, 10 min
1:25
30
alpha-SMA and desmin were used in conjunction with morphology to
identify MFs, which are usually appeared as spindle shaped and sometimes
stellate. MFs' presence was counted as a percentage of all spindle
shaped and stellate cells, as reported previously [14]: (0) no staining,
(+) weak staining of 1% - 20% of all spindle shaped and stellate cells,
(++) intense staining of 21% - 60% spindle shaped and stellate cells and
(+++) intense staining of more than 60% of spindle shaped and stellate
cells. Fibroblasts and inflammatory cells expressing TGF-β1 were
evaluated together using the method mentioned above. Five hundred cells
from 5 fields of each section were enumerated as the percentage of
positive MFs and cells expressing TGF-β1. Sections were examined by two
of the authors (AE, DA) independently of each other. Sections were
re-examined when there were differences, and discussion was occasionally
necessary to establish uniformity.
Statistical analysis
Statistical analysis was performed using the chi-squared test (x2)
and statistical significance level was defined at P = 0.05.
RESULTS
MFs positive for alpha-SMA and vimentin but negative for desmin were
found in 4 of 20 cases (20%) of FE. Half of the cases did not contain
inflammatory cells and 2 of these cases showed a percentage of intense
presence of MFs (Figure 1A). In the rest 10 cases of FE, where areas
with and without inflammatory cells were concurrently presented in the
same case, two of the cases with weak and intense presence of MFs were
observed, respectively (Table 2). The inflammatory infiltration in these
cases consisted of plasma cells lymphocytes and occasionally macrophages
(Figure 1B).
Fibrous epulis. A = myofibroblasts positive for alpha smooth
muscle actin in area without inflammatory infiltration. B = in
area with severe inflammatory infiltrate (hematoxylin and eosin
stain, original magnification x100).
Presence of myofibroblasts (MFs) in different areas of 10 cases
of fibrous epulis
Number of cases
with MFs
Number of cases
Degree of inflammatory
infiltration
Areas with inflammatory infiltration
1
5
Mild
1
5
Moderate
Areas without inflammatory infiltration
2
10
Absent
Inflammatory infiltration and MFs were not found in 5 of 22 cases of OFE.
In the other 17 cases of OFE, in the periphery of the central, highly
fibrocellular and mizeralized part of the lesion, chronic inflammatory
infiltration from plasma cells, lymphocytes and occasional macrophages
could be observed. The presence or absence of MFs in the central part
and areas with different degrees of inflammatory infiltration around the
central part, as well as the percentages of MFs in 17 cases of OFE are
presented in Table 3 and appeared in
Figure 2A, B. Although, the
frequency of MFs between FE and OFE was statistically significant (P <
0.001), however, the presence of MFs in areas with inflammatory cells
was not related to the degree of inflammatory infiltration in both FE
and OFE (Tables 2 and
3).
Myofibroblasts (MFs) in different areas of 17 cases of ossifying
fibrous epulis, degrees of inflammatory infiltration and
percentage of myofibroblasts
N
MFs in
the central part
MFs in areas
without inflammatory infiltration
around the central part
MFs in areas
with inflammatory infiltration
around the central part
Degree of inflammatory
infiltration
MFs
(%)
3
-
-
-
mild
0
4
-
-
-
severe
0
1
-
+
+
mild
1 - 20
2
+
-
+
mild
1 - 20
2
+
-
+
moderate
1 - 20
3
+
+
+
severe
21 - 60
2
+
+
-
moderate
21 - 60
N = number of cases.
Ossifying fibrous epulis. A = myofibroblasts positive for alpha
smooth muscle actin in central area
(hematoxylin and
eosin stain, original magnification x200).
B = Beyond the vessels, no positive for alpha smooth muscle
actin myofibroblasts are presented in area with severe
inflammatory infiltrate around the central area of the lesion
(hematoxylin and eosin stain, original magnification x100).
TGF-β1 expression was intensely seen in fibroblasts, plasma cells,
lymphocytes and macrophages in both FE and OFE (Figure 3A, B). Also, a
percentage of 21 - 60% of cells expressing TGF-β1 was found in all
examined cases.
Fibroblasts and inflammatory cells express transforming growth
factor-beta 1 in areas with chronic inflammatory infiltrate. A =
fibrous epulis. B = ossifying fibrous epulis
(hematoxylin and
eosin stain, original magnification x100).
DISCUSSION
The results of the current study showed that MFs and TGF-β1 are likely
involved in the collagen formation in FE and OFE, but in contrast no
relationship was found between the presence of MFs and the degree of
inflammatory infiltration in both FE and OFE. Immunohistochemically, MFs
may have a variable phenotype including those that express only vimentin
(V type); vimentin and alpha-SMA (VA type); vimentin, alpha-SMA and
desmin (VAD type) and vimentin, alpha-SMA, smooth muscle myosin heavy
chains and/no desmin (VAM or VAMD type). The expression of alpha-SMA is
considered to be the main biochemical marker of myofibroblastic
differentiation [17]. In
the current study the detection of MFs was based on the combined
immunohistochemical profile (vimentin and alpha-SMA) as well as their morphology indicating that MFs of FE and OFE belong to the VA type.
Interestingly, the presence of MFs in FE has not been reported in
previous studies [13,15]. This finding could be explained by the small
number of cases in previous studies and differences in the immunohistochemical procedure. The findings of the present study
regarding OFE are in accordance to a previous study by Garcia de Marcos
et al. [14] that reported the presence of MFs positive for vimentin and
alpha-SMA, whereas, Damasceno et al. [15] did not reveal the presence of MFs. The results of our study suggest that MFs may contribute in the
formation of collagenous connective tissue more frequently in OFE than
in FE.
FE is considered to originate from mesenchymal cells of gingival and
periosteum [11], and OFE is considered to originate from cells of
periodontal ligament and periosteum [12]. Although immunohistochemical
studies did not reveal the presence of MFs [13,18], cell cultures of
fibroblasts from healthy gingiva and periodontal ligament demonstrated
that a few cells express alpha-SMA [19,23]. It remains to be determined
whether the alpha-SMA positive cells in gingiva and periodontal ligament
represent a permanent or modulated fibroblastic population.
Differentiation from fibroblast into MF phenotype has been proposed to
be dependent on local environmental cues, including accumulation of
biologically active TGF-β1 [20], extracellular matrix-integrin
interactions [21] and mechanical stress [1]. The reverse
MF-to-fibroblast differentiation is also possible under insulin-like
growth factor-1 [22], and basic fibroblast growth factor influence [23].
Possibly the absence of MFs in many of our cases may be due to
alterations or lack of local environmental cues, or the presence of
insulin like growth factor-1 and/or basic fibroblast growth factor.
TGF-β1 is believed to participate in fibroblastic differentiation and
alpha α-SMA expression in fibroblasts in vitro and in vivo
[1]. Connective tissue cells, macrophages, neutrophils, lymphocytes and
plasma cells express TGF-β1 [7,24] that has paracrine and autocrine
effect [5]. Our results showed that MFs were not always present in areas
containing different degree of inflammatory infiltration. Similar
findings were reported in another study of FE, as well [16]. These
results suggest that TGF-β1 of chronic inflammatory cells possibly is
not always involved in the recruitment of mesenchymal cells and their
trans-differentiation into MFs. Noteworthy, MFs were constantly
presented in cases with different degree of chronic inflammatory
infiltrate in obstructive pancreatitis [25], whereas in oral squamous
cell carcinoma, MFs tended to be inversely related to the infiltration
with mononuclear inflammatory cells [26]. Extracellular matrix molecules
and cytokines may affect the bioactivity of TGF-β1. Fibronectin domain
ED-A is crucial for myofibroblastic phenotype induction [27], whereas decorin, tumour necrosis factor-α, interferon-γ, and interleukin-1
inhibit the trans-differentiation of MFs [28-31].
In the current study 21 - 60% of fibroblasts and chronic inflammatory
cells found to express TGF-β1 in all cases of FE and OFE. This result
may indicate that there was not relationship between the presence of MFs
and percentage of cells expressing TGF-β1. Although the expression of
TGF-β1 was similar in all examined cases of FE and OFE, it can be
hypothesized that the absence of MFs in many cases may be due to the
lack of other than TGF-β1 local environmental cues, or presence of
extracellular matrix molecules/cytokines affecting TGF-β1 bioactivity.
Further investigation is needed to examine the local environmental cues
and growth factors/cytokines implication in the differentiation of MFs
in FE and OFE. TGF-β1 has a marked effect on exracellular matrix
composition by attracting fibroblasts and leading to the synthesis and
secretion of extracellular matrix molecules, such as collagen type I,
fibronectin and tenascin [32,33]. It is possible that autocrine and/or
paracrine effect of TGF-β1 of fibroblasts cause collagen overproduction,
as well.
CONCLUSIONS
The results of the present study suggest that transforming growth
factor-beta1 likely contributes in the formation of collagenous
connective tissue in fibrous epulis and ossifying fibrous epulis,
whereas myofibroblasts more often in ossifying fibrous epulis than in
fibrous epulis. Also, there is no obvious relationship between the
presence of myofibroblasts and the degree of inflammatory infiltration
in both fibrous epulis and ossifying fibrous epulis.
ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS
The authors declare that they have no conflict of interests.
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