Immunolocalization of matrix metalloproteinases-2 and -9 during apical periodontitis development

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Immunolocalization of matrix metalloproteinases-2and -9 during apical periodontitis development

Mauro V. Corotti a,h, Willian F. Zambuzzi b,h, Katiucia B.S. Paiva c,Renato Menezes d, Lidiane C. Pinto e, Vanessa S. Lara e, Jose M. Granjeiro f,g,*a Laboratory of Biochemistry, Department of Biological Sciences, University of Sao Paulo, Bauru, SP, Brazilb Laboratory of Bioassays and Signal Transduction, Department of Biochemistry, Institute of Biology, University of Campinas,

Campinas, SP, Brazilc Laboratory of Molecular Pathology, Department of Oral Pathology, Dental School, University of Sao Paulo, Sao Paulo, SP, BrazildDepartment of Oral Biology and Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh,

Pittsburgh, PA, USAe Laboratory of Pathology, Department of Stomatology, University of Sao Paulo, Bauru, SP, BrazilfDepartment of Cell and Molecular Biology, Institute of Biology, Fluminense Federal University, Niteroi, RJ, BrazilgCell and Molecular Biology Center (Nucell), University of Sao Paulo, Sao Paulo, SP, Brazil

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 7 6 4 – 7 7 1

a r t i c l e i n f o

Article history:

Accepted 17 April 2009

Keywords:

Apical periodontitis

Metalloproteinases

Extracellular matrix

Bone remodelling

a b s t r a c t

Objective: The objective of this study was to determine the expression of matrix metallo-

proteinase-2 (MMP-2) and -9 (MMP-9) during apical periodontitis development.

Methods: Using an experimental design of induced periapical lesions in rats and immuno-

histochemistry assay as investigative tool, the MMP-2 and MMP-9 expression and distribu-

tion were evaluated at 3, 7, 14, 21, 30, 60 and 90 days after coronary access and pulp exposure

of the first left mandibular molar to the oral environment. Two blind observers scored the

immunoreactivity. A semi-quantitative analysis was performed.

Results: Except at day 3, MMP-2 and MMP-9 immunostaining was observed in all experi-

mental periods. The MMP-2 ( p = 0.004) and MMP-9 (p = 0.005) immunostaining was higher in

the period between 7 and 21 days. They were mainly observed in cells surrounding the apical

foramen and adjacent periapical areas. Cells into the hypercementosis areas were strongly

stained while both osteoblasts and osteoclasts presented discrete staining along of this

study. No staining was observed on epithelial walls. At 30, 60 and 90 days, the subjacent

connective tissue presented intense MMP-2 and MMP-9 immunostaining in mononuclear

cells (suggestive of fibroblasts, macrophages, infiltrating neutrophils and lymphocytes).

Conclusion: The results observed in this study suggest that MMP-2 and MMP-9 play a critical

role in the development of inflammatory periapical lesions, probably involved in the

extracellular matrix (ECM) degradation during the initial phase of the lesion development.

# 2009 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Cell and Molecular Biology, Institute of Biology, Fluminense Federal University, Outeiro de SaoJoao Baptista, s/n, Campus do Valonguinho, Zip Code: 24.020-150, Niteroi, RJ, Brazil. Tel.: +55 21 2629 2324; fax: +55 21 3701 1617.

E-mail address: [email protected] (J.M. Granjeiro).h These authors contributed equally to this study.

avai lable at www.sc iencedi rec t .com

journal homepage: www.intl.elsevierhealth.com/journals/arob

0003–9969/$ – see front matter # 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2009.04.013


1. Introduction

Apical periodontitis is an inflammatory disorder of periradi-

cular tissues caused by persistent microbial infection within

the root canal system of the affected tooth.1 It is viewed as a

dynamic encounter between microbial factors and host

defenses at the interface between infected radicular pulp

and periodontal ligament that results in local inflammation,

resorption of hard tissues, destruction of other periapical

tissues, and eventual formation of various histopathological

categories of apical periodontitis, commonly referred to as

periapical lesions.2 As a result of bone and apical periodontal

extracellular matrix degradation, an apical granuloma or cyst

could be formed.2,3

Matrix metalloproteinases (MMPs), collectively called

matrixins, form a multi-gene family within the metallopro-

teinase class of endopeptidases that mediate the degradation

of practically all extracellular matrix (ECM) molecules,

including native and denatured collagen.4–7 Over the past

years, 24 different MMPs have been cloned, of which 23 are

found in humans.6–9 Matrix metalloproteinase (MMP)-2 and

MMP-9 are known to cleave specifically type 4 collagen, which

is a major structural component of basement membrane. This

is an early step of inflammation.

It has been proposed that MMPs play an important role in

the pulp and periapical tissue destruction during inflamma-

tion process.10–12 With the purpose of evaluating the tissue

levels of matrix metalloproteinase-2 and -9 and their

distribution in periapical lesions, a disease model in Wistar

rat molar was employed to better understand the molecular

and cellular mechanisms governing the onset and progression

of periapical lesions.

2. Material and methods

2.1. Animals and experimental group characterization

After proper IRB approval, obtained from Animal Research

Ethics Committee, Universityof Sao Paulo, BauruDentalSchool,

21 male Wistar rats (8-week old) were used in the study.

Periapical lesions were induced as previously described.13

All animals were maintained with water and food ad libitum,

throughout the entire experimental period.

2.2. Experimental induction of periapical lesions

At day 0, the animals were anesthetized (2% Xylazin-

Anasedan1 Vetbrands, Jacareı, SP, Brazil and 10% Ketamine-

Agener Uniao1, Uniao Quımica Farmaceutica Nacional S.A.,

Embu-Guacu, SP, Brazil) and mounted on a jaw retraction

board. Pulp exposure was made at the mesial fossa of left

mandibular first molars using # 1/4 round bur to the depth of

bur diameter. The exposed teeth were left open to the oral

environment (without any protection/cover) during the entire

experimental period, with the objective of contaminating the

root canal system and consequently inducing the periapical

lesion formation.13

The animals were randomly distributed in seven groups

(n = 3) and killed at 3, 7, 14, 21, 30, 60 and 90 days after pulp

exposure. Hereafter, the hemi-maxillae were collected and

fixed in phosphate-buffered formalin (10%) for 24 h, deminer-

alized in 0.05 M EDTA (pH 7.2), and dehydrated in an ethanol-

graded series, clarified in xylol. Paraffin wax-embedded

sections were processed for histological and immunohisto-

chemical analysis. Serial sections (5 mm of thickness) were cut

and mounted on Dako slides (Dako–cat. # S3003, Carpinteria,

CA, USA).

2.3. Immunohistochemical assay

Sections were submitted to the indirect immunoperoxidase

method.14–16 Briefly, the sections were deparaffinized through

immersion in xylene and ethanol followed by incubation with

3% hydrogen peroxide diluted in phosphate-buffered saline

(PBS; pH 7.2) for 40 min. Thereafter, the sections were

incubated with goat anti-MMP-2 (Santa Cruz Biotechnology,

sc-6838, Santa Cruz, CA, USA) and goat anti-MMP-9 (Santa

Cruz Biotechnology, sc-6840, Santa Cruz, CA, USA), diluted in

1% PBS-bovine serum albumin (BSA), at 4 8C overnight.

Following the incubation period, the sections were washed

in PBS with Triton X-100 p.a. (Merck, Darmstadt, Germany) and

incubated with biotinylated rabbit anti-goat IgG (Pierce,

Rockford, IL, USA) antibodies in 1% PBS-BSA for 60 min at

room temperature. Sections were washed in PBS with Triton

X-100 p.a. and incubated with avidin–biotin complex (Dako,

K0492, DAKO, Glostrup, Denmark) for 45 min at room

temperature. Further sections were incubated in a solution

with 3,3-diaminobenzidine plus H2O2 (DAKO, Glostrup, Den-

mark) for 5 min at room temperature. After washing with

distilled water, the slides were counterstained with Mayers’

haematoxylin. Breast cancer sections were used as positive

control for MMP-217 and MMP-9.18 Negative controls consisted

of sections in which primary antibodies were omitted and

replaced with non-immune murine serum (DAKO, Glostrup,

Denmark) or 1% PBS-BSA.

Immunoreactivity was evaluated by two blind observers,

who independently assessed the immunostaining by means of

light microscopy at 40� magnification high power field.

2.4. Semi-quantitative image analysis

Two calibrated investigators analyzed the intensity of the

immunostaining for MMP-9 and MMP-2 under 40�magnifica-

tion according to the following criteria: (0) no staining, (1) light

staining, (2) moderate staining and (3) intense staining. The

mean and standard deviation of the samples were analyzed by

using unpaired t-test with Welch correction (Instat, GraphPad,

Software, Oberlin, San Diego, CA, USA).

3. Results

In order to validate our results, positive and negative controls

were carried out during all experiments (Fig. 1).

Immunostaining for both MMP-2 and MMP-9 was observed

in almost all experimental periods, except on day 3 when the

lesion had not yet been established due to partial pulp vitality.

The MMP-2 and MMP-9 immunoexpression did not differ

significantly between 7–21 days (p > 0.05, unpaired t-test with

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 7 6 4 – 7 7 1 765


Welch correction) and 30–90 days (p > 0.05). However, the

MMP-2 and MMP-9 immunostaining at 7, 14 and 21 days

was higher than at 30, 60 and 90 days (p < 0.05, unpaired t-test

with Welch correction) (Fig. 5).

In the earliest periods, an acute inflammatory infiltrate

was predominant with a suggestive intense presence

of leukocytes, notably infiltrating neutrophils. The

immunostaining was intense for both MMP-2 and MMP-9

(Fig. 2A–D).

In the subsequent periods, the immunostaining was not

intense as previously described; it was dispersed and mainly

observed in isolated mononuclear cells.

After day 30, cells in the interface of the granulomatous

and exudative zones19 showed immunostaining for both

MMPs (Fig. 3A and B). A few cells presented immunostain-

ing in the fibrous zone, except for mononuclear cells

suggestive of fibroblasts, lymphocytes and macrophages

(Fig. 4A and B).

Fig. 1 – Serial sections of human breast cancer (hBC) biopsies were used as positive control for both MMPs. Letters A and C

represent MMP-2 and MMP-9 staining respectively (positive control). Negative controls consisted of sections of hBC in

which primary antibodies were omitted and replaced with non-immune murine serum (B) and (D). Letters (E) and (F)

summarize the negative control by using sections of 14th day of periapical lesion development. Magnification: 40T.



The periodontal ligament in the distant areas from the

lesion presented rare and smooth staining that became

intensified in the proximity of the exudative zone.19 Cemen-

toblast-like cells were noted strongly stained in hypercemen-

tous areas after 21 days (Fig. 4C).

Endothelial cells also demonstrated immunostaining for

both MMP-2 and MMP-9 in all periods (Fig. 4D). Sixty percent of

the cases assessed presented an epithelium after 30 days of

lesion development, however, no staining was found in any

period for both MMPs.

At the interface of the periapical lesion with the bone

tissue, rare cells suggestive of osteoblasts demonstrated

cytoplasmatic immunostaining (Fig. 4E). In addition, the most

of osteoclast-like cells presented weak cytoplasmatic staining

in all the experimental periods (Fig. 4F). On the other hand,

osteocyte did not present positive for both MMP-2 and MMP-9

staining in any of the assessed periods (Fig. 4F).

4. Discussion

The biological model used in this study is considered

satisfactory for observing the periapical lesions development

in all of its phases.13,20 Based on histological outcomes and

current literature, two distinct phases were observed: (1)

growth/acute phase (between 7 and 21 days), generally caused

by micro-organisms residing in or invading from the apical

root canal into the periapical tissues,2 and (2) a chronic phase

(period longer than 21 days), caused by a prolonged presence

of microbial irritants leading to a shift in the infiltrating

Fig. 2 – Inflammatory periapical lesions development. Both MMPs-2 and -9 were expressed by infiltrating mononuclear cells

surrounding the apical foramen and adjacent periapical areas. The panel was arranged as follows—7th day: (A) MMP-2 and

(B) MMP-9; 14th day: (C) MMP-2 and (D) MMP-9; 21st day: (E) MMP-2 and (F) MMP-9. Magnification: 40T.



neutrophils-dominated lesion to a macrophage, lymphocyte,

and plasma-cell-rich one, encapsulated in collagenous con-

nective tissue.2,20

The observed temporal-spatial distribution of both MMP-2

and MMP-9 in this study suggests that these enzymes are

strongly expressed during the acute phase of the disease (7–21

days, Figs. 2 and 4) by cells resident around the lesion. On the

other hand, we also observed a decrease in the expression of

MMP-2 and MMP-9 during the chronic phase of the lesion (30–

90 days, Figs. 3 and 4). Of note, altogether, MMPs are part of a

class of zinc dependent endopeptidases that are capable of

degrading almost all the ECM components.7,9 Among them,

collagenase activities are critical for fibrillar collagen break-

down, which represents the main constituents of period-

ontium and alveolar bone. As collagens are cleaved into

smaller fragments, they denature at body temperature and are

further degraded by gelatinases (MMP-2 and MMP-9) and other

non-specific tissue proteinases.7,21,22 These MMPs have been

associated with periodontal tissue destruction in other

conditions such as chronic periodontitis and peri-implantitis

disease.23,24

Normal tissues do not store MMPs, and constitutive

expression is minimal. MMPs are transcriptionally regulated

by growth factors, cytokines, and extracellular matrix (ECM)

components.25 The role of these mediators over dissemination

of infection and pulpal necrosis is not completely understood.

Nevertheless, as extracellular matrix degradation by MMPs

can be induced by proinflammatory cytokines, it is possible

that these enzymes could have an active role in the

pathogenesis of pulpal and periapical damage.

It has been proposed that apical and chronic periodontitis

may share some pathogenic mechanisms that include

inflammatory mediators like IL-1, IL-6, TNF-a, and MMPs.

The production of MMPs during the acute phase of the lesion is

induced by interleukin-1 (IL-1) and IL-6 produced by neutro-

phils present in the exudative zone of the granulomas.19 MMP-

9 in particular, is involved in the recruitment of leukocytes to

an inflammatory site, by affecting development of allergen-

specific T cell responses or perhaps by influencing expression

of inflammatory mediators such as cytokines and chemo-

kines.26

In this study, we observed a discrete expression of MMP-2

and MMP-9 in the chronic phase of the lesion (Fig. 5), which

could suggest that the reduction in the number of PMNs in

the chronic phase could interfere with the expression of IL-

1a and IL-6, thus diminishing the activation of MMP-2 and

MMP-9. Our results are in agreement with a previous study

where MMP-2 was virtually absent in chronic periodontitis

(Fig. 5).27

MMPs could also act in a protective manner inhibiting the

development of bacterium-induced periapical tissue destruc-

tion (through the processing anti-inflammatory cytokines and

chemokines) as recently reported in periodontitis.28 MMP

inhibition was demonstrated to affect pulpal and periapical

inflammation, increasing the rate of periapical bone lesion

formation.29

Fig. 3 – Inflammatory periapical lesions development. MMP-2 and -9 immunostaining was less intense than that observed

in the period of 7–21 days. The panel was arranged as follows—30th day: (A) MMP-2 and B MMP-9; 90th day: (C) MMP-2 and

(D) MMP-9. Magnification: 40T.



Tsubota et al. reported that MMP-8 expression in

osteoblasts, osteocytes, periodontal ligament cells, cemen-

toblasts, and cementocytes may be involved in remodelling

the periodontium during tooth eruption.30 A similar process

of periodontium remodelling was observed during periapi-

cal pathology installation and development. Here, period-

ontal ligament cells presented intense immunostaining for

both MMP-2 and MMP-9 when they are close to the lesion. In

addition, osteoblast- and osteoclast-like cells showed dis-

crete staining in all periods evaluated, while osteocytes

were not positive for MMP-2 and MMP-9 staining. These

events suggest that during the initial development of the

Fig. 4 – Some biological events found during inflammatory periapical lesions development. (A and B) Mononuclear cells

(fibroblast- and macrophage-like cells) at 21st day presenting MMP-2 immunostaining mainly localized in the connective

tissue (CT). The photomicrography (C) brings out the hypercementous areas (H) presenting cementoblast-like cells

immunopositive to MMP-9 (31st day). In all periods in this study, the presence of endothelial cells immunostained for both

enzymes was observed. Thus, (D) shows endothelial cells immunostained for MMP-9 (21st day). The (E) and (F) detail the

bone tissue and bring out some bone cells immunostained. In (E) osteoblast-like cells were immunopositive to MMP-9 (large

arrow, 21st day) and (F) immunostaining for both MMPs was not observed in osteocyte-like cell (arrows). Also in (F), note

that osteoclast-like cells were slightly immunostained (*, 21st day). Magnification: 40T.



apical periodontitis, MMP-2 and MMP-9 could be acting

in the ECM degradation favouring the lesion expansion

and together with other cytokines, e.g. RANKL/RANK/

OPG,31,32 can also contribute to bone destruction.

However, future studies are necessary to elucidate this

mechanism.

A sequel to granuloma formation is the proliferation of the

epithelial cell rests of Malassez associated with the inflam-

mation, which may lead to the development of an inflamma-

tory radicular cyst.33,34 In our experimental model, the longest

experimental periods (30–90 days), 60% of the cases presented

epithelial proliferation. However, with the limitation of the

lesion size and the limitations of the investigative tool used,

we are not able to affirm that these lesions were radicular cysts

or infer about any correlation between the expression of MMP-

2 and MMP-9 and the periapical granuloma-radicular cyst

transition.

MMP-13 (collagenase-3), a natural substrate for MMP-2,

has been immunodetected in periapical epithelized lesions in

humans.12 In fact, MMP-13 seems to contribute to the

migration of epithelial cells and granulomatous tissue

invasion during the conversion of a periapical granuloma

with epithelium into a radicular cyst.12 Moreover, it is well

known that MMP-13 activity in gingival tissue extracts and

gingival crevicular fluid (GCF) from periodontitis sites are

pathologically elevated when compared with the periodontal

healthy gingival tissue and GCF.35,36 However, in the present

animal model study we did not observe epithelial cells

staining for both MMP-2 and MMP-9, suggesting that MMP-13

would not be active.

Indeed, MMPs perform an important function during bone

turnover16,37 and the lesion expansion probably involves

interactions of some MMPs with their endogenous inhibitors

(TIMPs and RECK).38 The results observed in this study suggest

that MMP-2 and MMP-9 play a role in the development of

inflammatory periapical lesions, probably involved in the

extracellular matrix turnover during the initial phase of the

lesion development.

Acknowledgments

The authors are grateful to Post-Doc scholarship from

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo

(FAPESP) for WFZ (proc. no. 08/53003-9) and Thematic Project

(proc. no. 01/10707). CNPq (grant no. 475721/2003-9).

Funding: None.

Competing interests: None declared.

Ethical approval: Not required.

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Immunolocalization of matrix metalloproteinases-2 and -9 during apical periodontitis development

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