Key words:
Endodontic treatment - endotoxins - meta-analysis - systematic review
INTRODUCTION
A great diversity of Gram-negative bacteria has been identified in root canal infection,[1]
[2]
[3]
[4]
[5] especially species belonging to Prevotella, Porphyromonas and Fusobacterium spp.[6]
[7] The cell wall of Gram-negative bacteria contains lipopolysaccharide (LPS), a complex
molecule, which is generally referred to as endotoxin.[8]
Clinical studies have detected endotoxins in infected root canals,[1]
[2]
[3]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] and most of them have suggested a correlation between endotoxin levels and development
of clinical symptoms whereas others have not demonstrated this association.[4]
[9]
[12]
[13]
[15]
[19]
[20]
[21]
[22] Endotoxins are potent inflammatory agents, which activate classical and alternative
pathways of complement system.[23]
[24] Complement activation releases biologically active peptides, which mediate a number
of aspects of the inflammatory process.[23] LPS may evoke pain through activation of the Hageman factor or through neurotoxic
properties when acting on presynaptic nerve terminals,[24] direct sensitization of nociceptors,[25] sensitization and up-regulation of the transient receptor potential cation channel,
sub-family V, member 1 (TRPV1).[26]
[27]
Endotoxins have been found to stimulate stimulation of bone resorption in tissue culture.[28]
[29]
[30]
[31] Some clinical studies have demonstrated a positive correlation between higher levels
of endotoxin in infected root canals and larger area of periapical bone destruction,
whereas others have failed.[4]
[18]
[20]
[32] A more complex network between different inflammatory mediators seems to be implicated
in the development of clinical symptoms and radiographic features.[32] The LPS released from infected root canal triggers the synthesis of IL-1 and TNF-alpha,[30]
[33] which in turn up-regulates the production of MMP (matrix Metalloproteinase) by macrophages
to promote periapical bone resorption.[30] Furthermore, LPS stimulates bone resorption by enhancing RANKL (receptor activator
of NF-κB ligand) through activation of toll-like receptor-2 in osteoblasts[31] as well as inhibitory effect on osteoblast differentiation.[29]
It is important to highlight that an overall analysis of data from studies correlating
presence of endotoxins with clinical and radiographic features has not been performed
yet. One of the main advantages of a systematic review with a statistical approach,
such as meta-analysis, is to address sources of bias in order to produce the most
valid and precise estimate of effect as possible.[34] Therefore, the main purpose of this study was to conduct an extensive systematic
review of the literature on endotoxin in endodontic infections in order to elucidate
the relationship between endotoxin levels and presence of clinical signs/symptoms
and radiographic features in patients with endodontic infection.
MATERIALS AND METHODS
Review question
Is there a relationship between endotoxin levels and presence of clinical/radiographic
symptoms in patients with periapical periodontitis?
Inclusion and exclusion criteria
Studies were included based on the following inclusion criteria:
-
Clinical studies in humans
-
Endodontic infection must be present
-
Quantification of endotoxin and description of the test
-
Evaluation of clinical/radiographic symptoms
-
Publication in English.
Studies were eliminated if the inclusion criteria were not met or if they presented
any of the following exclusion criteria:
-
In vitro or animal study, case report, review article, or opinion article
-
Lack of mean level of endotoxin and standard deviation, or data that allow their calculation
-
Lack of evaluation of the endotoxin levels
-
Lack of description of the type of endodontic infection
-
The publication was based on a population from another study
-
Study evaluating endotoxin in primary teeth
-
The same levels of endotoxins described by an author in more than one study
-
Lack of clear definition of the method used for endotoxin detection.
Search strategy
The literature was searched to identify published articles analysing the presence
of endotoxin in endodontic infection. Electronic searches were performed on Medline/PubMed,
Embase, Cochrane Library, Scielo, Science Direct, Web of Knowledge and Scopus databases
and also in grey literature on Google Scholar from where we have analysed the first
100 articles for identification of relevant studies published up to December 2016.
The search was conducted by using Medical Subject Heading (MeSH) terms and other free
terms as follows: “Periapical periodontitis, OR periapical diseases, OR dental pulp
diseases, OR apical periodontitis, OR endodontic infection AND endotoxin, OR LPS.”
All references were tabulated by using the software EndNote X6. Duplicate references
were excluded. Titles, abstracts and key-words were screened by two reviewers (DDR
and LLF) based on inclusion and exclusion criteria. The cases of disagreement between
authors were solved after discussion. After initial screening of titles and abstract,
the full articles were evaluated by the same two reviewers. In addition to the electronic
search, a hand search of the reference lists of the selected articles was performed.
Pre-defined data collection worksheets were used for assessment of each selected publication.
Data extraction
Relevant data were extracted from the selected articles based on study description
(i.e., setting, sample), endotoxin detection method, type of endodontic infection
(i.e., acute or chronic) and presence of clinical or radiographic features. Furthermore,
in order to conduct the meta-analysis, the mean level and standard deviation of endotoxins
were gathered.
The authors were contacted when further clarifications regarding the study methodology
or results were required. Data were extracted by two reviewers (DDR and LLF) independently
by using pre-piloted data extraction forms. In case of disagreement, discussions were
held to resolve and reach consensus. All the stages of this systematic review were
supervised by a third reviewer (GGN) who has expertise in systematic review methodology.
Quality assessment
The articles were systematically evaluated and the quality of the methodology was
assessed. For each study, the following parameters recorded: authors’ names, date
of publication, study design, sample size and included subjects. The evidence level
was determined according to guidelines provided by The Centre for Evidence-Based Medicine
at Oxford.
Statistical analysis
Five different meta-analyses were conducted in order to evaluate the levels of endotoxin
in the presence of each clinical symptom as follows: pain on palpation (POP), tenderness
to percussion (TTP), previous episode of pain (PEP), exudation (EX) and size of radiographic
lesion greater than 2 mm (SRL). For each model, the standardized mean difference was
obtained with fixed- and random-effect models. If heterogeneity was present (P < 0.05; I2 ≥ 50%), the random-effect model was employed.[35] Median and range values were converted into mean and standard deviation according
to Hozo et al.[36] The analyses were conducted by using the software RevMan 5.3 (The Nordic Cochrane
Center, Copenhagen, Denmark, 2014).
Methods used to quantify endotoxins
With regard to the methods used to quantify endotoxins, two studies used Gel Clot
LAL assay,[19]
[20] two used Endpoint Chromogenic LAL assay,[9]
[13] four used Kinetic Turbidimetric LAL assay.[1]
[2]
[11]
[12] All these LAL tests use the principle of a serine protease catalytic coagulation
cascade activated by Factor C, the first component in the cascade which is a protease
zymogen activated by endotoxin binding.[37]
This pathway activates downstream a proclotting enzyme into a clotting enzyme (i.e.,
coagulogen into coagulin).[37] The Gel Clot LAL assay and Kinetic Turbidimetric LAL assay use coagulogen by monitoring
its conversion into coagulin, which begins to form a gel clot, thus increasing the
turbidity. The Endpoint Chromogenic LAL assays uses synthetic peptide-pNA substrate,
which is cleaved by the clotting enzyme, imparting a yellow color to the solution.
The strength of the yellow color (determined at an optical density [OD] = 405 nm)
resulting from chromogenic LAL substrate and turbidity (determined at an OD = 340
nm) due to the conversion of coagulogen is correlated with the endotoxin concentration
from the standard curve.
It is important to point out that the sensitivity of these tests depends on the time
point (single or multiple time point) of the progress of the LAL reaction leading
to the coagulogen conversion recorded. In the Endpoint Chromogenic LAL assay, OD is
recorded at single time (at ≈ 16 minutes) (0.1-1 EU/mL). In Kinetic Turbidimetric
LAL assay, OD is read at multiple time points because the reaction proceeds with no
termination step (z 60 minutes), which allows the concentration of endotoxin to be
quantified over a wider range sensitivity (0.01-100 EU/mL in the turbidimetric methods).
Assessment of clinical and radiographic features
Percussion is a diagnostic procedure used to assess the condition of a body part by
means of tapping, with a painful response indicating periradicular inflammation.[38]
Palpation is a diagnostic procedure used to assess the condition of periapical inflammation
by performing a firm pressure with the pad of the finger or with a cotton swab buccal
or lingual gingiva apical to a suspected tooth. A painful response may indicate periapical
inflammation.[38]
According to the AAE glossary,[38] percussion is a diagnostic procedure used to assess the condition of a body part
by means of tapping, with a painful response indicating periradicular inflammation.
The tooth, which is sensitive to percussion, has a periapical diagnosis of symptomatic
apical periodontitis.[39]
[40]
In the diagnostic of the periapical periodontitis, marked visual signals may leave
the clinician uncertain as to whether the structures have in fact normal morphology
or pathological alterations.[40] Strategies for the radiographic diagnosis of periapical pathosis take this doubt
into account through calibration of the observers and plans for handling of borderline
and disagreement cases.[40] Therefore, for this study we have agreed that a 2 mm radiolucency in periapical
radiograph would be a parameter to determine the presence of a major radiographic
lesion.
RESULTS
Electronic search revealed 385 articles. After removing 141 duplicate articles, 244
articles were included for title and abstract screening. Twenty-nine articles were
included for full-text appraisal, including one article identified in the reference
list. Twenty-one articles were excluded after full-text assessment. Subsequently,
eight studies met the inclusion criteria in this systematic review. [Figure 1] displays the flowchart of the study selection. The samples in the included studies
totaled 231 individuals. Six studies were conducted in Brazil,[1]
[2]
[9]
[11]
[12]
[13] one in Japan[20] and one in the United States[19] [Table 1]. With regard to the methods used to quantify endotoxins, two studies used Gel Clot
LAL assay,[19]
[20] two used Endpoint Chromogenic LAL assay,[9]
[13] four used Kinetic Turbidimetric LAL assay[1]
[2]
[11]
[12] [Table 1]. Meta-analysis revealed that individuals with tenderness to percussion (TTP) (P = 0.04; I2 57%) and previous episode of pain (PEP) (P = 0.001; I2 81%) presented higher levels of endotoxin than their counterparts [Figure 2]. No association between endotoxin concentration and pain on palpation (POP) was
noted (P = 0.87; I2 45%). A higher level of endotoxins was associated with the presence of
exudation in infected root canals (EX) (P = 0.0007; I2 0%) and larger size of radiographic lesion (SRL >2 mm) (P = 0.02; I2 68%). Meta-analysis and graph for symptoms and radiographic features are
shown in [Figure 2].
Figure 1: Flowchart of the articles screened in the review process
Figure 2: Forest plot with the evaluation of exudation, previous episode of pain, pain on palpation,
size of radiographic lesion, and tenderness to percussion. Endotoxin concentration
(mean and standard deviation values in EU/mL)
Table 1:
Studies included in the systematic review. Study design, subjects and sample size,
diagnosis of symptoms, method of endotoxin detection and evidence level
Authors
|
Year
|
Country
|
Study design
|
Subjects
|
Diagnosis of symptoms
|
Method of Detection
|
Evidence level according to Oxford Centre for Evidence Based on Medicine
|
TTP: Tenderness to percussion, POP: Pain on palpation, SRL: Size of radiographic lesion,
EX: Exudation, PEP: Previous episode of pain
|
Schein & Schilder
|
1975
|
USA
|
Cross-sectional
|
20 Patients with Primary Infection
|
SRL, PEP
|
Gel Clot LAL Assay
|
D
|
Horriba et al.
|
1991
|
Japan
|
Cross-sectional
|
30 Patients with Primary Infection
|
EX, SRL, PEP
|
Gel Clot LAL Assay
|
D
|
Jacinto et al.
|
2005
|
Brazil
|
Cross-sectional
|
50 Patients with Primary Infection
|
TTP, POP, PEP
|
Endpoint Chromogenic LAL Assay
|
D
|
Martinho & Gomes
|
2008
|
Brazil
|
Cross-sectional
|
24 Patients with Primary Infection
|
TTP, POP, PEP
|
Endpoint Chromogenic LAL Assay
|
D
|
Martinho et al.
|
2010a
|
Brazil
|
Cross-sectional
|
21 Patients with Primary Infection
|
TTP, POP, EX, SLR
|
Kinetic Turbidimetric LAL Assay
|
C
|
Martinho et al.
|
2011a
|
Brazil
|
Cross-sectional
|
21 Patients with Primary Infection
|
TTP, POP, EX
|
Kinetic Turbidimetric LAL Assay
|
C
|
Endo et al.
|
2012
|
Brazil
|
Cross-sectional
|
15 Patients with Primary Infection
|
TTP, POP, SRL
|
Kinetic Turbidimetric LAL Assay
|
C
|
Gomes et al.
|
2012
|
Brazil
|
Cross-sectional
|
30 Patients with Primary and Secondary Infection
|
TTP, POP, SRL
|
Kinetic Turbidimetric LAL Assay
|
C
|
DISCUSSION
To the best of our knowledge, this is the first systematic review and meta-analysis
evaluating the relationship between endotoxin levels and presence of clinical signs/symptoms
and radiographic features in patients with endodontic infection. The data obtained
in this study revealed that individuals having teeth with tenderness to percussion
as well as previous episode of pain showed higher levels of endotoxin than their counterparts.
Additionally, larger size of radiographic lesion and presence of root canal exudation
were associated with higher levels of endotoxin.
The data obtained in this study revealed that individuals with tenderness to percussion
showed higher contents of endotoxins than their counterparts. Also, a correlation
was found between higher levels of endotoxins and patients with previous episode of
pain. These correlations are consistent with the hypothesis that LPS in clinical infections
is related to the production of pain and mechanical allodynia. This hypothesis is
strengthened by preclinical studies demonstrating that injection of LPS into rats
produces nocifensive behavior and mechanical allodynia.[41]
There are different mechanisms that can evoke pain due to bacterial LPS.[25]
[26]
[27] It is well accepted that the innate immune response against bacterial contents leads
to the release of different inflammatory mediators, including interleukin 1- beta
(IL-1β), prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF-α), which can
sensitize nociceptors. Higher levels of PGE2 have been related to teeth with clinical
symptomatology.[3]
[42]
[43] Higher levels of PGE2 were found in macrophage supernatants stimulated with bacterial
contents from teeth showing tenderness to percussion.[3] Thus, higher contents of IL-1β have also been detected in teeth with clinical symptomatology.[44],[45] Martinho et al.[1] not only indicated higher levels of endotoxins in teeth with tenderness to percussion,
but also found higher levels of IL-1β in macrophage supernatants stimulated with bacterial
contents from teeth with this clinical symptomatology. Thereby, IL-1 is a potent stimulus
for PGE-2 release.[46]
Besides, increased sensitization and up-regulation of TRPV1 constitutes a potential
mechanism by which TNF-alpha mediates inflammatory hyperalgesia and pain. It has been
demonstrated that trigeminal neurons express both LPS receptors (TLR4 and CD14) leading
to the hypothesis that bacterial byproducts might directly activate or sensitize trigeminal
nociceptors. The sensitization and up-regulation of transient receptor potential cation
channel, sub-family V, member 1 (TRPV1), has been addressed.[26]
[27] Ferraz et al.,[25] by using cultures of rat trigeminal neurons, demonstrated that pre-treatment with
LPS produced a significant increase in the capsaicin-evoked release of calcitonin
gene-related peptide (CGRP) compared to vehicle pre-treatment, thus showing sensitization
of the capsaicin receptor TRPV1 by LPS. Additionally, the authors showed co-localization
of the LPS receptor (toll-like receptor 4, TLR4) with CGRP-containing nerve fibers.
Diogenes et al.[26] found that (i) LPS binds to receptors in trigeminal neurons by means of competitive
binding, (ii) LPS evokes a concentration-dependent increase in the intracellular calcium
accumulation (Ca(2+))(i) and inward currents, and (iii) LPS significantly sensitizes
TRPV1 to capsaicin measured by (Ca(2+))(i) release of calcitonin gene-related peptide
and inward currents. All these together imply that LPS is capable of directly activating
trigeminal neurons and sensitizing TRPV1 via a TLR4-mediated mechanism.
Our results revealed that higher levels of endotoxins were associated with the presence
of exudation in infected root canals. Exudate is defined as fluid, cells and plasma
proteins which escaped from the vascular system and accumulates in a tissue or tissues,
usually being the result of inflammation.[38] The presence of exudation in root canal reflects an acute inflammation in periapical
lesion.[42]
Gram-negative bacterial LPSs are one of the mainly potent stimuli for macrophage cells
in the release of PGE2.[47]
[48] A positive correlation between number of Gram-negative bacterial species and levels
of PGE2 macrophage secretion was revealed.[3] PGE2 is both directly and indirectly implicated in most of the inflammatory and
destructive changes occurring in apical lesions (e.g., vasodilatation), thus increasing
vascular permeability and collagen degradation.[49] Takayama et al.[42] stated that the PGE2 concentration in periapical exudate could reflect the state
of the disease activity in periapical periodontitis. Additionally, IL-1β and TNF-α
are also implicated in exudation.[50]
[51]
[52]
[53] Therefore, a more complex network among these and other different cytokines seems
to play a role in exudation.[32]
Higher levels of endotoxin have been found in root canals with larger size of radiolucent
(>2 mm). Previous studies demonstrated that the endotoxin content of teeth with radiolucent
areas is five times as great as that of teeth without them.[54]
[55] Such a correlation is not inconsistent with the hypothesis that LPS in clinical
infections is related to bone destruction.
This hypothesis is strengthened by pre-clinical studies showing that injection of
LPS into animal tissues induces periapical bone destruction.[30]
[56]
[57] The LPS released from the infected root canal triggers the synthesis of IL-1 and
TNF-α from macrophages, which in turn up-regulates the production of MMP-1[30] and serves primarily for degrading non-mineralized extracellular matrix.[58]
[59] Stimulation of osteoclastogenesis by MMP-1 and generating collagen degradation fragments
on bone surfaces has also been proposed.[60]
Studies have also demonstrated that MMP-1-expressing macrophages increase consistently
as the lesion expands, which implies the involvement of MMP-1 in the production of
macrophages in periapical destruction.[61]
[62] These authors have also shown that LPS induces the expression of inducible nitric
oxide synthesis (iNOS) and transforming growth factor-β1 (TGF-β1) genes.[63]
[64] Furthermore, LPS stimulates bone resorption by enhancing the receptor activator
of NF-κB ligand (RANKL) through activation of toll-like receptor 2 in osteoblasts[31] as well as by inhibiting osteoblast differentiation.[29] Porphyromonas endodontalis LPS inducing RANKL by osteoblast[65] and Porphyromonas gingivalis exacerbating ligature-induced RANKL-dependent alveolar bone resorption via differential
regulation of toll-like receptor 2 (TLR2) and TLR4[66] are reported. It was demonstrated that the complex interplay between different cytokines
plays a role in periapical bone destruction, including significant (-) correlations
between IL-6 and IL-1β as well as PGE2 and (+) correlation between TNF-α and PGE2.[32]
It is important to highlight that not only the levels of endotoxin are implicated
with the presence/development of symptoms and severity of bone destruction, but also
the bacterial community involved in the infection, its interplay (synergism/antagonism),[2]
[6]
[67] and consequently, the type of bacterial LPS and its lipid A structure.[68]
[69] Among the Gram-negative bacteria, black-pigmented anaerobes, including Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens have been implicated as pathogens associated with the presence/development of symptoms
and severity of bone destruction.[2]
[6]
[7]
[67] Importantly, the LPS structure shows considerable heterogeneity among bacterial
species,[69]
[70]
[71] thus evoking different patterns of inflammatory response.[33]
[50]
[69] It is worth to point out that the lipid A structure of the LPS molecule (i.e., hydrophobic
component located in the outer leaflet of the outer membrane, responsible for the
toxic effects of infections with Gram-negative bacteria)[72] can suffer modulation from the environment, particularly, regarding hemin concentration[70] and temperature.[73] Hence, LPS molecule can also vary among different strains of single species, and
consequently exhibit different inflammatory potentials.[70]
[71]
[73]
Overall, this meta-analysis provides strong evidence that endotoxin are related with
the presence of clinical signs/symptoms and radiographic features in patients with
endodontic infection.
Financial support and sponsorship
Nil.