Introduction
Asymptomatic apical periodontitis (AAP) corresponds to the inflammation and destruction of periradicular tissues caused by bacterial infection of dental pulp. It is the most common consequence of untreated dental caries and leads frequently to tooth loss. The hallmark of AAP is the presence of an apical lesion (AL) that results from destruction of hard and soft apical tissues [
1,
2].
The generation of reactive oxygen species (ROS), namely superoxide, hydroxyl and nitric oxide radicals, hydrogen peroxide and hypochlorous acid, represents an important pathogenic mechanism for diseases associated with phagocytic infiltration and bone resorption [
3,
4] as a host defense mechanisms against the invading pathogen [
5]. Accordingly, neutrophils obtained from peripheral blood of AAP subjects have shown increased production of hydrogen peroxide and superoxide anion, which tend to normalize after surgical treatment [
6,
7]. Nevertheless, oxidant status in apical tissues remains unknown. As oxidant effects are additive, measuring the total oxidant (TOS) and antioxidant status (TAS) of a sample can provide a new and practical approach [
8,
9].
Matrix metalloproteinases (MMPs) are zinc and calcium-dependent endopeptidases that function at neutral pH. Fibrillar collagens are the major components of periodontal extracellular matrix. During periodontal homeostasis and pathologic conditions, they are cleaved into smaller fragments by collagenases (MMPs -1, -8, and -13) and further degraded by active gelatinases (MMPs -2 and -9) and other non specific tissue proteinases [
10]. MMP-9 and MMP-2 have been identified through immunohistochemistry in experimentally-induced apical periodontitis in animal models where they were proposed to play a role in both, the initiation and progression of apical periodontitis [
11,
12]. Previous works have demonstrated increased mRNA expression levels of MMP-9 in apical granulomas in comparison to cysts [
13,
14], as well as MMP-9 activity levels in apical exudates from acute versus apical abscesses [
15]. Furthermore, recent preliminary studies reported for the first time that gingival crevicular fluid (GCF) composition changes in AAP-affected teeth, showing increments in MMP-9 activity, frequency of detection of MMP-2 and total protein content in comparison to healthy controls [
16,
17]. Despite gelatinase activity has been reported in oral fluids during AAP, no previous determination of MMP-9 and -2 activities has been performed in apical lesions to support that these findings actually reflect apical status. Study of GCF particularly represents a novel approach in the search of biomarkers for apical periodontitis. In order to further understand whether changes in GCF reflect apical status, these changes should be demonstrated in both, apical lesions and GCF.
Additionally, new evidence of an oxidative regulation of MMP expression and activity in chronic periodontitis is emerging [
18], but this association has never been reported in AAP, despite both diseases share common pathogenic mechanisms. Consequently, it is proposed that apical lesions present higher MMP-2 and MMP-9 activity compared to healthy PDL in association with oxidant status and that these changes can be reflected in GCF.
The aim of this study was first, to characterize and associate oxidant balance and the activity levels of MMP -2 and -9 in apical lesions and healthy periodontal ligament; and second, to determine whether potential changes in oxidant balance were reflected in GCF from AAP-affected teeth.
Discussion
Periapical lesions result from the dynamic encounter between microbial factors and host immune response in subjects with clinical diagnosis of asymptomatic apical periodontitis. Both, granulomas and radicular cysts, are considered to represent two different stages of development of the same inflammatory process and are characterized by leukocyte infiltration. These cells represent important regulators of extracellular matrix turnover and destruction, representing the major source of bone-resorbing mediators, including reactive oxygen species and MMPs [
5,
6,
16].
The results of the current study support that an oxidative imbalance along with increments in MMP-9 and MMP-2 levels and activity might play a role in the development and/or progression of apical lesions. Furthermore, oxidative imbalance at the expense of reduced antioxidant levels was also reflected in GCF of AAP teeth, in comparison to healthy controls and endodontically treated teeth.
Leukocytes and resident fibroblasts from periodontal tissues are known to secrete MMPs, including MMPs -1, -2, -3, -8, -9 and -13 [
21‐
24]. During the last few years, MMPs -2 and -9 have shown to be over expressed at the mRNA level in apical granulomas [
14,
15] and higher activity levels have been reported in apical exudates from acute apical abscesses, as well as in GCF from teeth affected with asymptomatic apical periodontitis versus healthy teeth [
16]. The results of the current study complement the previous findings reported in GCF, showing significantly higher activity levels of both, proenzymes and active forms of MMP-9 and MMP-2 in apical lesions when compared to healthy periodontal ligaments. Additionally, tissue localization of MMP-9 and MMP-2 was confirmed in apical granulomas by imunohistochemistry [
13]. The study of GCF as a source of potential biomarkers for AAP represents a novel approach that needs further validation. Overall, these data support that changes in MMP activity in GCF are similar as those for apical lesions.
Previous works support that MMP -2, MMP-9 and MMP-13 play important roles in both, the initiation and progression of inflammatory bone resorption and soft periodontal tissue breakdown during pathological processes, including chronic periodontitis [
21,
25‐
27]. Under inflammatory conditions, bone resorptive mediators, like interleukin-1 and prostaglandin E2, induce a marked expression of RANKL and MMPs, such as MMP-13, -3 and -2 by osteoblasts and MMP-9 by activated osteoclasts and leukocytes [
1,
28]. Additionally, MMP-9 activity is thought to act over preosteoclast recruitment and migration [
29].
High levels of oxidants in tissues perturb the normal redox balance and shift cells into a state of oxidative stress [
30‐
32]. In the current study, we evidenced an oxidant imbalance in favor to ROS in both, apical lesions and GCF from AAP teeth versus healthy controls and endodontically-treated teeth. Evidence has led ROS to become increasingly implicated in the damage of extracellular matrix components from connective tissue incurred during inflammatory diseases [
33]. At the cellular level, ROS activate redox-sensitive transcription factors, including NF-κB and AP-1, thereby causing indirect tissue damage and exacerbation of inflammation [
9]. A positive correlation between TOS and the bone resorptive area and a negative correlation between TOS and TAS was found in apical lesions, but not in healthy PDL. An imbalance in favor to ROS might stimulate apical bone loss. Recent evidence has shown that ROS might play an osteolytic role by suppressing bone formation through inhibition of osteoblastic differentiation, and by stimulating osteoclast differentiation and bone resorption [
34] through induction of receptor activator of NFκB ligand (RANKL). Additionally, higher MMP-2 expression and activation in response to ROS has been reported [
3,
35,
36], evidencing a key link between ROS production, MMP-mediated proteolysis and bone resorption, which could play a central role in the progression of apical lesions. In line with these findings, an association between TOS and active MMP-2 was found in the current study, suggesting that pro-oxidant status and MMP-mediated proteolysis might be cooperative in nature during AAP progression. In support of this, an association between active MMP-8 and myeloperoxidase has recently been reported during chronic periodontitis progression [
18].
Additionally, based on the fact that MMP-9 can induce proMMP-2 activation
in vitro[
37,
38], the finding of a positive correlation between active MMP-9 and active MMP-2 suggests that this activation mechanism might also occur in vivo. A limitation of this study was the age difference found between AAP groups and their respective controls. Healthy erupted teeth are obtained for orthodontic purposes mostly in young patients. Nevertheless, a lack of association between age, TOS and MMPs supports that the differences found resulted from shifting of apical status. A deeper knowledge of the mechanisms involved in destruction of apical periodontium will contribute to the development of improved methods of diagnosis, treatment and follow-up for side-diagnostic tools.
GCF represents a simple, non-invasive and useful tool in monitoring inflammation and treatment response in marginal periodontal diseases [
39,
40], but it has rarely been studied in apical periodontitis. MMP activity or active forms in GCF, particularly of MMP--13, MMP-9 and MMP-8, have previously been associated with progression and/or severity of marginal chronic periodontitis [
26,
41]. Recently, changes in GCF composition, involving higher gelatinase activity [
16] and protein concentration, were reported in AAP in comparison to healthy controls [
17]. These studies provide preliminary evidence supporting that GCF might reflect the health status of the apical tissues, as well as apical disease progression. In line with these reports, the present study shows the occurrence of an oxidant imbalance in apical lesions and also in GCF from AAP teeth, whereas endodontic treatment appears to restore antioxidant status to its normal levels. Considering that treatment outcome is difficult to predict based solely upon clinical and radiographic criteria and classical samples for endodontic purposes are invasive, GCF measurement of gelatinase activities along with the oxidant balance might represent a useful side- diagnostic tool, but further studies are needed to confirm whether GCF can reflect apical inflammation, its resolution and apical healing.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DA, GM, OAV, GJ: Contributed to study design and funding acquisition, enrolment of study subjects, performance of clinical evaluation and treatments, data interpretation and manuscript preparation; VMA, PR, G-S J, MS, MV: Contribution of study design, sample preparation, biochemical and immunobiochemical analyses, data interpretation and manuscript preparation; HM: Study conception and design, funding acquirement, supervision of laboratory procedures, data analysis and interpretation, manuscript writing and preparation. All authors read and approved the final manuscript.