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Original article
Folia Med. Fac. Med. Univ. Saraeviensis 2019; 53(1):
3-9foliamedica.mf.unsa.ba
Immunohistochemical expression of matrix metalloproteinase MMP-2
and MMP-9 in healthy and inflamed human dental pulp
Irmina Tahmiščija1*, Svjetlana Radović2, Silvana Jukić-Krmek3,
Alma Konjhodžić1, Lajla Hasić-Branković1, Aida Džanković1, Samra
Korać11 Department of Restorative dentistry and Endodontics,
Faculty of Dentistry, University of Sarajevo, Sarajevo, Bosnia and
Herzegovina2 Department of Pathology, Faculty of Medicine,
University of Sarajevo, Sarajevo, Bosnia and Herzegovina3
Department of Endodontics and Restorative Dentistry, School of
Dental Medicine, University of Zagreb, Croatia
*Corresponding authorIrmina TahmiščijaDepartment of Restorative
dentistry and EndodonticsFaculty of DentistryUniversity of
SarajevoBolnička 4a, 71000 SarajevoBosnia and Herzegovinae-mail:
[email protected]
AbstractObjective: The aim of this study was to investigate the
impact of inflammation on expression of MMP-2 and MMP-9, as well as
to identify the cellular sources of these enzymes in human dental
pulps using immunohistochemistry.Methods: Fifty-four irreversibly
inflamed samples of dental pulp were used as the experimental
group. Fifty-one healthy pulps, obtained from teeth extracted for
orthodontic reasons, were used as the control group. The tissue
samples were forma-lin-fixed, paraffin-embedded, and cut into
sections at 3- 4 μm. An immunohistochemical study was performed
using mono-clonal antibodies against MMP-2 and MMP-9. Evaluation of
the immunohistochemical expression was determined by the
semi-quantitative method and scored as follows: no staining (score
0), less than 10% of stained cells (score 1), less than 30% of
weakly stained or strongly but incompletely stained cells (score
2), and more than 30% of strongly and completely stained cells
(score 3).Results: Immunohistochemical analysis revealed a
significantly greater expression of MMP-9 in inflamed than in
healthy den-tal pulps (Mann–Whitney U, p=0.0001). In contrast,
there was no difference in the expression of MMP-2 between these
two groups (Mann–Whitney U, p=0.907). MMP-2 and MMP-9
immunoreactivity was detected the most frequently in endothe-lial
cells.Conclusions: MMP-9 is highly overexpressed in inflamed
den-tal pulps. There are no differences in the expression of MMP-2
between healthy and inflammed dental pulps. Endothelial cells
represent the major cellular source of MMP-9, as well as MMP-2, in
healthy and inflamed dental pulps.Keywords: dental pulp,
inflammation, MMP-2, MMP-9, im-munohistochemistry
© 2019 Folia Medica Facultatis Medicinae Universitatis
Saraeviensis. All rights reserved.
IntroductionMatrix metalloproteinases (MMPs), collectively known
as matrixins, are a family of structurally relat-ed enzymes
dependent on zinc, which are capable of disintegrating different
components of the extracellu-lar matrix (ECM). These enzymes are
involved in both normal and pathological tissue remodeling [1].The
fundamental role of MMPs during the develop-ment and remodeling of
oral tissues has been demon-strated in several studies. These
enzymes are involved in the development of enamel and enamel
fluorosis [2,3]. In addition to that, MMPs also participate in the
remodeling of the organic dentin matrix. It has been proven that
activation of MMP-2 and MMP-9 plays a key role in the degradation
of dentin collagen during caries progression [4]. The expression of
MMPs strong-ly correlates to periodontal diseases, since they are a
major factor in the breakdown of collagen during peri-odontal
tissue destruction [5,6]. Controlled remodel-ing of the ECM, which
is essential for the growth and invasion of oral tissue tumors, is
mediated through the activity of MMPs [7]. Evidence of
collagenolytic and gelatinolytic activities in partially
demineralized den-tin, treated with either etch-and-rinse or
self-etch ad-hesives, confirms the participation of these
endoprote-ases in the disruption of incompletely resin-infiltrated
collagen fibrils within the hybrid layer [8,9].The inflammatory
process in the dental pulp leads to degradation of the ECM
proteins. Different endopep-tidases, which act on the various
structural proteins, are included in this matrix turnover. Thus,
the gelatinases (MMP-2 and MMP-9) are involved in the degradation
of denatured gelatins: laminin, elastin, fibronectin and basement
membrane zone- associated collagen. Previ-ous studies have
suggested that MMPs may play an im-
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TAHMIŠČIJA ET AL: IMMUNOHISTOCHEMICAL EXPRESSION OF MMP-2 AND
MMP-9 IN DENTAL PULP
Folia Med. Fac. Med. Univ. Saraeviensis 2019; 54(1): 24-30
foliamedica.mf.unsa.ba
portant role in pulpitis development and progression [10-14].The
aim of this study was to investigate the impact of inflammation on
the expression of MMP-2 and MMP-9, as well as to identify the
cellular sources of these enzymes in human dental pulps, using
monoclonal mouse anti-human antibodies.
Material and MethodsSample selectionThe study was approved by a
local ethics committee (School of Dentistry, Sarajevo, Bosnia and
Herzegovi-na, number of approval letter: 09-622-2/11) and
per-formed in accordance with the guidelines of the Decla-ration of
Helsinki for Human Research.All samples were collected
prospectively during regu-lar therapy of patients who visited the
School of Den-tal Medicine in Sarajevo for a period of one calender
year. The samples of inflamed pulp were collected during endodontic
treatment. The samples of healthy pulp were obtained from
caries-free premolars after ex-traction. Histological and
immunohistochemical anal-ysis were performed at Department of
Pathology at the Faculty of Medicine in Sarajevo.Fifty-four (54)
samples of dental pulps from premo-lars, clinically diagnosed as
irreversibly inflamed, were used as the experimental group. The
diagnosis criteria included: history of spontaneous and/or
lingering pain in response to cold and/or heat stimulus, clinically
and radiographically evident carious exposure of the pulp chamber,
and radiographically normal periapical ap-pearance. Fifty-one (51)
samples of healthy pulp ex-tirpated from clinically healthy
premolars with fully developed roots, which were extracted for
orthodontic reasons, were used as the control group.Immediately
after the extirpation, the tissue was placed in 10% neutral
buffered formalin, embedded in par-affin and cut with a microtome
to a thickness of 3-4 micrometers. For each tissue sample, three
different depths of cut were made, which were first stained with
hematoxylin-eosin (HE). Observation of HE stained sections involved
determination of the form and inten-sity of inflammation.Samples
with insufficient tissue, as well as samples on which tissue
necrosis was observed, were excluded from the study (nine
samples).Immunohistochemical stainingThree to four micrometer thick
sections of the dental pulp were mounted on
3-aminopropyltriethoxysilane (APES) - coated slides, deparaffinised
in xylene and rehydrated via graded ethanol solutions. Then
sections were rinsed with distilled water and washed three
times
with PBS (pH 7.4). Heat-induced pretreatment for an-tigen
retrieval (sections were immersed in a 10 mmol/L citrated buffer,
pH 6.0, at 60°C for 5 min) was carried out prior to incubation with
the primary antibody. The endogenous peroxidase activity was
inhibited by incu-bation of the samples with 0.3% hydrogen
peroxidase in methanol for 30 minutes at the temperature of 4°C.
After blocking the non-specific reactions with 10% normal rabbit
serum, the sections were incubated with the primary antibody
against MMP-2 (Clone 17B11, Leica Biosystems, Novocastra,
Newcastle, UK) and MMP-9 (Clone 15W2, Leica Biosystems,
Novocas-tra, Newcastle, UK) for two hours. The sections were
subsequently incubated with anti-mouse secondary an-tibody
conjugated with avidin-biotin-peroxidase com-plex. The color
reaction was developed using 3–3’di-amino-benzidine (DAB). Finally,
the sections were counterstained with Mayer’s hematoxylin, mounted
and cover slipped.Evaluation of immunohistochemical stainingThe
slides were examined by light microscopy using an Olympus BX40
microscope (Artisan Scientific, Champaign, Illinois, USA) at
magnification of 400×. The product of the immunohistochemical
reaction was detected in the cytoplasm of the endothelial cells,
fi-broblasts and inflammatory cells. MMP-2 and MMP-9 positive cells
were identified by their brown color, ranging from light brown to
dark brown. The cytoplas-mic staining intensity was scored
semi-quantitative-ly, according to a previously described method
[15], as follows: no staining (score 0), less than 10% of stained
cells (score 1), less than 30% of weakly stained or strongly but
incompletely stained cells (score 2) and more than 30% of strongly
and completely stained cells (score 3). Statistical
analysisComparison of MMP-2 and MMP-9 expression be-tween healthy
and inflamed dental pulp was evaluated by the Mann-Whitney U test.
The statistical signifi-cance of differences between MMP-2 and
MMP-9 ex-pression in different cell populations was estimated by
the chi-square test. All data analyses were carried out using the
Statistical Package for Social Sciences, version 20 (SPSS Inc.,
Chicago, IL, USA).
ResultsImmunohistochemical analysis revealed a significant-ly
greater expression of MMP-9 in inflamed than in healthy dental
pulps (Mann–Whitney U, p=0.0001). In contrast, there was no
difference in expression of MMP-2 between these two groups
(Mann–Whitney U, p=907). MMP-9 immunoreactivity was detected the
most frequently in endothelial cells (n=33) (Figure
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TAHMIŠČIJA ET AL: IMMUNOHISTOCHEMICAL EXPRESSION OF MMP-2 AND
MMP-9 IN DENTAL PULP
Folia Med. Fac. Med. Univ. Saraeviensis 2019; 54(1):
24-30foliamedica.mf.unsa.ba
imental and control groups (chi-square, p=0.88) were observed,
Table 1. Comparison of MMP-2 expression in fibroblasts would have
made little sense due to the negligible number of positive
samples.
1) then in inflammatory cells (n=16) and fibroblasts (n=13)
(Figure 2).Simultaneous expression of MMP-9 in endothelial cells,
inflammatory cells and fibroblasts was observed in nine cases; in
endothelial cells and fibroblasts in three cases; and in
endothelial and inflammatory cells in six cases (Figure 3). MMP-2
immunoreactivity in inflamed pulp was de-tected in endothelial
cells in 11 cases and only twice in inflammatory cells (Figure 4).
As shown in Figure 5., no MMP-2 immunoreactivity was detected in
the 41 tissue samples in the experimen-tal group.There was a
statistically significant difference between the experimental and
control group in the expression of MMP-9 in endothelial cells
(chi-square, p=0.0001), Table 1, as well as in fibroblasts
(chi-square, p= 0.0001), Table 2. No statistically significant
differences in the expres-sion of MMP-2 in the endothelial cells of
the exper-
Figure 1. MMP-9 positive endothelial cells (arrows) in inflamed
pulp (IH, X400).
Figure 2. MMP-9 positive inflammatory cells and fibro-blasts in
inflamed pulp (IH, X400).
Figure 3. MMP-9 immunostaining localizes to edothe-lial and
inflammatory cells in inflamed dental pulp (IH, X400).
Figure 4. Very few MMP-2– mild positive inflammatory cells (IH,
X200).
Figure 5. Inflamed dental pulp with MMP-2 negative
immunostaining (IH, X400).
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TAHMIŠČIJA ET AL: IMMUNOHISTOCHEMICAL EXPRESSION OF MMP-2 AND
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Folia Med. Fac. Med. Univ. Saraeviensis 2019; 54(1): 24-30
foliamedica.mf.unsa.ba
As shown in Table 3., there was a statistically significant
difference between the experimental and control group in
cytoplasmic staining intensity of MMP-9 (Mann–Whitney U, p=0.0001),
while was not statistically sig-nificant differences in cytoplasmic
staining intensity of MMP-2 (Mann–Whitney U, p=0.907).
DiscussionDuring the progression of inflammation, proteolysis of
the ECM is one of the key events. In this process, the most
important role is played by MMPs.The expression of MMP-9 in
inflamed dental pulp has been encompassed by several studies that
used different tests. Thus, the levels of MMP-1, MMP-2, MMP-3 and
MMP-9 in clinically healthy and inflamed dental
pulps were determined using enzyme-linked immu-nosorbent assay
(ELISA) [16]. As the overexpression of MMP-9 in inflamed dental
pulp was positively correlated with gelatinolytic activity, it was
concluded that MMP-9 may be a potential marker protein for
assessment of the dental pulp status. Using real-time polimerase
chain reaction (RT-PCR) and immunohis-tochemistry, expression of
MMP-9 was shown to be significantly increased in inflamed in
comparison to healthy pulps [17]. Although immunohistochemical
expression of MMP-9 was observed in odontoblasts, fibroblasts,
inflammatory and endothelial cells, the number of the pulp samples
in which the mentioned cells were positive for MMP-9 was not
specified. A pi-lot study was conducted on the basis of the
aforemen-tioned research findings. In this study, a fluorescent
as-
Table 1. Immunohistochemical expression of MMP-2 and MMP-9 in
endothelial cells
GroupTotal
Statistical analysisexperimental control χ2 test
p-value
MMP-2
negativeCount 43 40 83
0.023 0.880
% within MMP-2 in endothelial cells 51.8% 48.2% 100.0%% within
group 79.6% 78.4% 79.0%
positiveCount 11 11 22
% within MMP-2 in endothelial cells 50.0% 50.0% 100.0%% within
group 20.4% 21.6% 21.0%
TotalCount 54 51 105
% within MMP-2 in endothelial cells 51.4% 48.6% 100.0%% within
group 100.0% 100.0% 100.0%
MMP-9
negativeCount 21 44 65
24.973 0.0001
% within MMP-2 in endothelial cells 32.3% 67.7% 100.0%% within
group 38.9% 86.3% 61.9%
positiveCount 33 7 40
% within MMP-2 in endothelial cells 82.5% 17.5% 100.0%% within
group 61.1% 13.7% 38.1%
TotalCount 54 51 105
% within MMP-2 in endothelial cells 51.4% 48.6% 100.0%% within
group 100.0% 100.0% 100.0%
GroupTotal
Statistical analysisexperimental control χ2 test
p-value
MMP-9
negativeCount 41 51 92
14.013 0,0001
% within MMP-2 in endothelial cells 44.6% 55.4% 100.0%% within
group 75.9% 100.0% 87.6%
positiveCount 13 0 13
% within MMP-2 in endothelial cells 100.0% 0.0% 100.0%% within
group 24.1% 0.0% 12.4%
TotalCount 54 51 105
% within MMP-2 in endothelial cells 51.4% 48.6% 100.0%% within
group 100.0% 100.0% 100.0%
Table 2. Immunohistochemical expression of MMP-9 in
fibroblasts
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24-30foliamedica.mf.unsa.ba
say was used to test the levels of MMP-9 in the dentin-al fluid
collected during the preparation of teeth with a clinical diagnosis
of irreversible pulpitis or required replacement fillings [18].
Despite the many limitations of the study, the fact that MMP-9 was
detected only in the dentinal fluid samples of the teeth with
clinical diagnosis of irreversible pulpitis indicates the possible
significance of MMP-9 as a potential molecular pre-dictor in the
assessment of the dental pulp condition. Comparison of
gelatinolytic activity in ten healthy and ten inflamed pulp samples
once again confirmed an in-creased level of MMP-9 in inflamed
dental pulps [19]. In our research, the immunohistochemical
expression of MMP-9 was significantly higher in the irreversibly
inflamed than in the healthy pulp with a very high level of
significance (p = 0.0001), which supports the previous findings. In
inflamed pulps, MMP-9 was the most frequently detected in
endothelial cells, followed by the inflammatory cells and
fibroblasts. In healthy pulps, MMP-9 was detected exclusively in
endothelial cells in seven cases. It is known that most of MMPs are
synthesized as inactive zymogens that require acti-vation. Several
cytokines (IL-1, TNF-α) and bacterial toxins induce relief of MMPs
from pulpal cells, which has been proven in cell cultures [20-23].
These data in-dicates the possibility that MMP-9 is deposited in
the pulp cells in the form of precursors, which are released during
inflammation influenced by bacterial products and inflammatory
cytokines. The results of our study
support the assumption that MMP-9 plays a key me-diating role in
the degradation of the inflamed pulp tissue.While there are quite
consistent data regarding the ex-pression of MMP-9 in inflamed
dental pulp, the re-spective available data for MMP-2 are rather
conflict-ing. A study in which the quantification of matrixins was
performed by ELISA concluded that the level of MMP-2 was
significantly higher in inflamed than in healthy pulps [24]. The
same study, which used im-munohistochemistry, established that the
MMP-2 was weakly expressed in inflammatory cells and fibroblasts
tested groups. This is in contrast to the results of our research.
Furthermore, another group of authors, who also used ELISA, points
out that the level of MMP-2 was significantly lower in the group
with symptomat-ic pulpitis than in healthy pulps [16]. In our
research, there was no significant difference in the quantity and
intensity of immunohistochemical expression of MMP-2 between
healthy and inflamed pulps. What is interesting is that the
expression of MMP-2 was al-most completely restricted to the
endothelium of the vasculature in both the experimental and the
control group. Immunohistochemistry was used to analyze the
expression of MMP-2 and MMP-9 in the pulp of teeth that are subject
to orthodontic traction [25]. The anal-ysis revealed that in a very
small sample of the control group (four healthy pulp samples) the
expression of the aforementioned matrixins was mainly limited to
the
Cytoplasmic staining intensity of MMP-2
group score N Mann-Whitney U Z p
experimental
0 41
1363,5 -0,117 0,907
1 122 13 0
Total 54
control
0 391 122 03 0
Total 51
Cytoplasmic staining intensity of MMP-9
experimental
0 19
616 -5,537 0,0001
1 122 113 12
Total 54
control
0 441 52 23 0
Total 51
Table 3. Cytoplasmic staining intensity of MMP-2 and MMP-9
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TAHMIŠČIJA ET AL: IMMUNOHISTOCHEMICAL EXPRESSION OF MMP-2 AND
MMP-9 IN DENTAL PULP
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foliamedica.mf.unsa.ba
endothelial cells, which is consistent with the results of our
research. Given that our research showed that the expression of
MMP-2 was almost identical in healthy and inflamed pulp, there
remains an open question as to what its role in the dental pulp. It
is known, howev-er, that matrix metalloproteinases have numerous
roles in development of both physiological and pathological
conditions. The real role of MMP-2 in the pulp tissue and the
reasons why its expression is restricted mainly to the endothelium
of the vasculature could be an in-teresting subject for further
studies.
ConclusionThe conclusions of this study are the following:
1. MMP-9 is highly overexpressed in inflamed dental pulps.
2. There are no differences in the immunohistochem-ical
expression of MMP-2 between healthy and in-flammed dental
pulps.
3. Dental pulp inflammation contributes to the ex-pression of
MMP-9, while it has no influence on the expression of MMP-2.
4. Endothelial cells represent a major cellular source of MMP-9,
as well as MMP-2, in healthy and in-flamed human dental pulps.
Declaration of interest The authors declare no conflicts of
interest.
References[1] Hannas AR, Pereira JC, Granjeiro JM, Tjäderhane L.
The
role of matrix metalloproteinases in the oral environment. Acta
Odontol Scand. 2007;65(1):1-13.
[2] Bartlett JD, Beniash E, Lee DH, Smith
CE. Decreased min-eral content in MMP-20 null
mouse enamel is prominent during the maturation stage. J
Dent Res. 2004;83(12):909-13.
[3] Zhang Y, Yan Q, Li W, DenBesten PK. Fluoride down-reg-ulates
the expression of matrix metalloproteinase-20 in hu-man fetal tooth
ameloblast-lineage cells in vitro. Eur J Oral Sci. 2006;114(suppl
1):105–10.
[4] Chaussain-Miller C, Fioretti F, Goldberg M, Menashi S. The
role of matrix metalloproteinases (MMPs) in human caries. J Dent
Res. 2006;85(1):22-32.
[5] Birkedal-Hansen H. Role of matrix
metalloproteinases in human periodontal diseases. J
Periodontol. 1993;64(5 Sup-pl):474-84.
[6] Lee W, Aitken S, Sodek J, McCulloch CA. Evidence of a
direct relationship between neutrophil collagenase activity
and per-iodontal tissue destruction in vivo: role of active
enzyme in human periodontitis.
J Periodontal Res. 1995;30(1):23-33.
[7] Vilen ST, Salo T, Sorsa T, Nyberg P. Fluctuating roles of
ma-trix metalloproteinase-9 in oral squamous cell carcinoma.
ScientificWorldJournal. 2013;(3):920595.
[8] Mazzoni A, Carrilho M, Papa V, Tjäderhane L, Gobbi P, Di
Lenarda R, et al. MMP-2 assay within the hybrid layer cre-ated by a
two-step etch-and-rinse adhesive: biochemical and
immunohistochemical analysis. J Dent. 2011;39 (7):470-7.
[9] Nishitani Y, Yoshiyama M, Wadgaonkar B, Elrod D, Breschi L,
Mannello F, et al. Activation of gelatinolytic/collagenolyt-ic
activity in dentin by self-etching adhesives. Eur J Oral Sci.
2006;114(2):160-6.
[10] Panagakos FS, O’Boskey JF Jr, Rodriguez E. Regulation of
pulp cell matrix metalloproteinase production by cytokines and
lipopolysaccharides. J Endod. 1996;22(7):358-61.
[11] O’Boskey FJ Jr, Panagakos FS. Cytokines stimulate matrix
metalloproteinase production by human pulp cells during long-term
culture. J Endod. 1998;24(1):7-10.
[12] Chang YC, Yang SF, Hsieh YS. Regulation of matrix
met-alloproteinase-2 production by cytokines and pharma-cological
agents in human pulp cell cultures. J Endod.
2001;27(11):679-682.
[13] Chang YC, Lai CC, Yang SF, Chan Y, Hsieh YS. Stimula-tion
of matrix metalloproteinases by black-pigmented Bacte-roides in
human pulp and periodontal ligament cell cultures. J Endod.
2002;28(2):90-93.
[14] Huang FM, Yang SF, Hsieh YS, Liu CM, Yang LC, Chang
YC. Examination of the signal transduction
pathways in-volved in matrix metalloproteinases-2 in human pulp
cells. Oral Surg Oral Med Oral Pathol Oral Radiol
En-dod. 2004;97(3):398-403.
[15] Bagnoli F, de Oliveira VM, da Silva MA, Taromaru GC,
Ri-naldi JF, Aoki T. The interaction between aromatase,
met-alloproteinase 2, 9 and CD44 in breast cancer. Rev. Assoc. Med.
Bras. 2010;56(4):472-7.
[16] Gusman H, Santana RB, Zehnder M. Matrix metallo-proteinase
levels and gelatinolytic activity in clinically healthy and
inflamed human dental pulps. Eur J Oral Sci. 2002;110(5):353-7.
-
30
TAHMIŠČIJA ET AL: IMMUNOHISTOCHEMICAL EXPRESSION OF MMP-2 AND
MMP-9 IN DENTAL PULP
Folia Med. Fac. Med. Univ. Saraeviensis 2019; 54(1):
24-30foliamedica.mf.unsa.ba
[17] Tsai CH, Chen YJ, Huang FM, Su YF, Chang YC. The
up-regulation of matrix metalloproteinase-9 in inflamed human
dental pulps. J Endod. 2005;31(12):860-2.
[18] Zehnder M, Wegehaupt FJ, Attin T. A first study on the
use-fulness of matrix metalloproteinase 9 from dentinal fluid to
indicate pulp inflammation. J Endod. 2011;37(1):17-20.
[19] Accorsi-Mendonça T , Silva EJ, Marcaccini AM, Gerlach RF,
Duarte KM, Pardo AP, et al. Evaluation of gelatinases, tissue
inhibitor of matrix metalloproteinase-2, and myeloperox-idase
protein in healthy and inflamed human dental pulp tissue. J Endod.
2013;39(7):879-82.
[20] Panagakos FS, O’Boskey JF Jr, Rodriguez E. Regulation of
pulp cell matrix metalloproteinase production by cytokines and
lipopolysaccharides. J Endod. 1996;22(7):358-61.
[21] Nakata K, Yamasaki M, Iwata T, Suzuki K, Nakane A,
Na-kamura H. Anaerobic bacterial extracts influence production
of matrix metalloproteinases and their inhibitors by human
dental pulp cells. J Endod. 2000;26(7):410-3.
[22] O’Boskey FJ Jr, Panagakos FS. Cytokines stimulate matrix
metalloproteinase production by human pulp cells during long-term
culture. J Endod. 1998;24(1):7-10.
[23] Chang YC, Yang SF, Hsieh YS. Regulation of matrix
met-alloproteinase-2 production by cytokines and pharma-cological
agents in human pulp cell cultures. J Endod.
2001;27(11):679-82.
[24] Shin SJ, Lee JI, Baek SH, Lim SS. Tissue levels of matrix
metalloproteinases in pulps and periapical lesions. J Endod.
2002;28(4):313-5.
[25] Leone A, Mauro A, Spatola GF, Provenzano S, Caradonna C,
Gerbino A, et al. MMP-2, MMP-9, and iNOS expression in human dental
pulp subjected to orthodontic traction. An-gle Orthod.
2009;79(6):1119-25.