Review Article
Pulp Inflammation Diagnosis from Clinical to
Inflammatory Mediators: A Systematic Review
Marjorie Zanini, DDS, MSc,* Elisabeth Meyer, DDS,* and St
ephane Simon, DDS, PhD, HDR*
Abstract
Introduction: Similar to other tissues, the dental pulp
mounts an inflammatory reaction as a way to eliminate
pathogens and stimulate repair. Pulp inflammation is
prerequisite for dentin pulp complex repair and regener-
ation; otherwise, chronic disease or pulp necrosis oc-
curs. Evaluation of pulp inflammation severity is
necessary to predict the clinical success of maintaining
pulp vitality. Clinical limitations to evaluating in situ in-
flammatory status are well-described. A molecular
approach that aids clinical distinction between revers-
ible and irreversible pulpitis could improve the success
rate of vital pulp therapy. The aim of this article is to re-
view inflammatory mediator expression in the context of
clinical diagnosis. Methods: We searched PubMed and
Cochrane databases for articles published between
1970 and December 2016. Only published studies of in-
flammatory mediator expression related to clinical diag-
nosis were eligible for inclusion and analysis. Results:
Thirty-two articles were analyzed. Two molecular ap-
proaches were described by study methods, protein
expression analysis and gene expression analysis. Our
review indicates that interleukin-8, matrix metallopro-
teinase 9, tumor necrosis factor-a, and receptor for
advanced glycation end products expression increase
at both the gene and protein levels during inflammation.
Conclusions: Clinical irreversible pulpitis is related to
specific levels of inflammatory mediator expression.
The difference in expression between reversible and irre-
versible disease is both quantitative and qualitative. On
the basis of our analysis, in situ quantification of in-
flammatory mediators may aid in the clinical distinction
between reversible and irreversible pulpitis. (J Endod
2017;43:10331051)
Key Words
Dental pulp, diagnosis, gene expression, inflammation,
molecular pattern
From the *UFR dodontologie, Universit
e Paris Diderot, Paris, France; Groupe Hospitalier Piti
e Salp^etriere-Charles Foix, Paris, France; and UMRS INSERM 1138
Team 5, Centre de recherche des Cordeliers, Paris, France.
Address requests for reprints to Dr St
ephane Simon, Oral Biology and Endodontixs, University of Paris Diderot (Paris 7), 75006 Paris, France. E-mail address:
simendo@gmail.com
0099-2399/$ - see front matter
Copyright 2017 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2017.02.009
JOE Volume 43, Number 7, July 2017
T he goal of any restor-
ative or endodontic
procedure should be to
Signicance
Because of the limitations of accuracy of clinical
diagnosis tools, outcomes after vital pulp therapy
maintain dental pulp vitality
are unpredictable. Molecular-based diagnostic
and functionality without
strategies are promising and may be relevant to
patient discomfort. Pulp tis-
determine proper clinical indications for vital pulp
sue is necessary for tooth
therapies and optimize prognosis.
nutrition, innervation, and
immunocompetency.
Maintaining dental pulp vitality increases tooth mechanical resistance and long-term sur-
vival (1, 2). Therefore, pulp vital therapies such as indirect pulp capping, stepwise caries
excavation, direct pulp capping, and pulp chamber pulpotomy may be preferable to root
canal treatment in some situations. Unfortunately, the success of vital pulp therapy varies
greatly, especially for direct pulp capping after carious excavation (31.8%91.3%) (3, 4).
Clinical early failures (within days or weeks) are multifactorial but certainly may be related
to improper diagnosis of pulp disease. Indeed, poor pulp status evaluations may result in
underestimation of pulp inflammation severity. This oversight can lead to irreversible pulp
inflammation and pulp tissue necrosis, which results in spontaneous and persistent pain
after therapy.
Proper evaluation of pulp inflammation and choice of appropriate materials
before therapy are the keys to improving pulp vital therapy success. Tricalcium sili-
catebased cements have excellent sealing properties and bioactive properties because
they induce good-quality hard tissue barriers (57). Accurate diagnosis requires
knowledge of pulp inflammation severity and the likelihood of response to
endodontic procedures. Intense or long-term pulpal inflammation that is due to unre-
solved infection or inflammation precludes regeneration (8). Because inflammation
aids in pathogen elimination and repair stimulation, control of pulp inflammation
severity is necessary for vital pulp therapy success (9, 10).
Currently, pain quality and history and responses to pulp sensitivity tests are the
only clinical tools available to evaluate pulp inflammation severity (11, 12). Pulpal
diseases are often clinically classified by using the criteria of the American
Association of Endodontists (AAE) (13). The terms reversible or irreversible pulpitis
simply reflect the intention of the practitioner to keep or remove vital pulp; therapy
could be attempted on a tooth with reversible pulpitis, as defined by the absence of
spontaneous pain or absence of pain after stimulation. Root canal treatment remains
the therapy of choice for irreversible pulpitis because the pulp is too deeply inflamed
to expect recovery.
Unfortunately, the AAE clinical categorization has limitations. Histologic observa-
tions show no correlation between clinical diagnosis and in situ pulp status (14, 15).
Histologic evaluation of tissues clinically diagnosed as irreversible symptomatic pulpitis
Dental Pulp Inflammation Diagnosis 1033
Review Article
do not show deep inflammation (16), whereas pulp necrosis may occur
in asymptomatic or low-grade symptomatic patients whose disease
could be wrongly diagnosed as reversibly inflamed. Therefore, pain
characterization that is based on clinical tests seems inadequate to reli-
ably differentiate between reversible and irreversible pulp inflamma-
tion. Because clinical diagnosis is unreliable, the outcome of vital
pulp therapy is unpredictable, and practitioners may completely remove
the pulp more often than is necessary to limit postoperative pain and
infection.
Pulp inflammation involves several biological processes evaluable
at the macroscopic, microscopic, and molecular levels. Macroscopic
changes are mainly noticeable at the vascular level (eg, vasodilatation)
(17, 18). Increased immune cell numbers are remarkable on
microscopic examination (19). Release of multiple inflammatory bio-
molecules is also observed. The molecular immune response may pre-
cede the cellular immune response, suggesting that cytokines and other
signaling molecules are synthesized and secreted by dentin pulp com-
plex host cells before immune cell recruitment and activation (20).
The quality and quantity of these mediators are key to inflammatory
evolution, especially with respect to the type of tissue immune response
generated. Some mediators guide and amplify the inflammatory process.
Type 1 cytokines (eg, interferon-g, interleukin [IL]-2, IL-12, tumor ne-
crosis factor [TNF]-a) orchestrate strong cellular immune responses,
Identification
particularly intense phagocytic activity. Other mediators are responsible
for tissue repair. Type 2 cytokines (eg, IL-10, IL-4) suppress macrophage
activation and phagocytosis and stimulate B-cell proliferation and differ-
entiation into plasma cells after resolution of cell-mediated inflammation
(21, 22). Furthermore, the carious lesion model describes deeper
carious lesions as having greater quantities of inflammatory mediators
and more pulp inflammation (20, 23).
Inflammatory mediators orchestrate the inflammatory process in-
side the pulp tissue. Consequently, the molecular phase precedes the
macroscopic and microscopic inflammatory changes; thus, it would
be instructive to study their expression inside pulpal tissue both quali-
tatively and quantitatively relative to the severity of the pulpal disease.
The aim of this study was to review articles concerning inflammatory
molecule expression related to clinical diagnosis to identify key inflam-
matory mediators and describe their expression profile in inflammatory
pulp disease.
Methods
We performed a literature review of expression of any inflamma-
tory mediators related to clinical diagnosis to create a list of potential
expression patterns. This systematic review was conducted by using
Preferred Reporting Items for Systematic reviews and Meta Analyses

Records identified
through data bases
searching
(n = 431)

Additional records titles Records screened Records excluded

Screening

through backward search (n = 75) (n = 371)

(n = 79)

Records abstract Records excluded

screened (n =53)
Records abstract (n = 22)
excluded
(n = 50)
Eligibility

29 Full-text articles assessed Full-text articles excluded,

for eligibility with reasons
(n =51) (n = 19)
Included

Studies included in
qualitative synthesis
(n = 32)

Figure 1. Flow diagram of records.

1034 Zanini et al. JOE Volume 43, Number 7, July 2017

JOE Volume 43, Number 7, July 2017
TABLE 1. Selected Studies Analysing Protein Expression Of Inflammatory Mediators
Clinical diagnosis, sample
Authors, date (references) size
Cohen et al, 1985 (44) Normal pulps, n = 20
Reversible pulpitis, n = 30
Irreversible symptomatic
pulpitis, n = 13
Proctor et al, 1991 (45) Normal pulp
Irreversible symptomatic
pulpitis or necrotic pulp
Hahn and Falkler, 1992 (46) Normal pulp, n = 15
Irreversible symptomatic
pulpitis, n = 38
Dental Pulp Inflammation Diagnosis
1035
Sample collection
Extraction of teeth and
extirpation of pulp tissue.
Pulp tissue extirpation with
spoon excavator or sterile
barbed broach.
Extirpated pulp dissected
into pieces and tissue
culture.
Objective of the study
To measure and compare
levels of PGE2 and PGF2a in
painful and asymptomatic
human dental pulps.
To determine whether CRP
levels in pulp could be
correlated with histologic
disease status of pulp and
with systemic blood levels
of the protein.
To investigate the humoral
immune responses of
clinically symptomatic
pulpitis and normal pulps
by allowing antibodies in
SF to react with
predominant bacteria
isolated from deep carious
lesions.
ELISA with pooled SFs
Methods Results
Radioimmunoassay Mean value of PGE2 in
reversible pulpitis group is
significantly higher than
that for normal pulp group
(P < .05).
Mean value of PGE2 in
irreversible symptomatic
pulpitis group is
significantly higher than
that for normal pulp group
and reversible pulpitis
group (P < .01).
Mean PGF2a in irreversible
symptomatic pulpitis is
significantly higher than
the 2 other groups
(P < .01).
PGF2a values for normal
pulp group and reversible
pulpitis group were not
significantly different
(P > .05).
ELISA assay Only normal versus inflamed
pulp contrast was
significant (P < .05).
Correlation between
serum CRP and pulp CRP
was not significant
(P = .160).
Histologic analysis Histologic classification
Normal pulp, n = 5
Inflamed pulp, n = 11
Necrotic pulp, n = 6
Double diffusion Irreversible symptomatic
in agar assay pulpitis group: 73.68%
reacted with goat anti-
human Y chain serum, and
only 1 specimen had
detectable IgA as well as
IgG in SF.
No IgM was detected.
Normal pulp group: IgG
Review Article
was detected in 73.33%.
Neither IgA nor IgM was
detected in SF.
No difference of mean
antibody levels between
irreversible and normal
groups of reactivity of SF to
all microorganisms except
(continued )
 1036
TABLE 1. (continued )
Clinical diagnosis, sample
Authors, date (references)
Zanini et al.
Rauschenberger et al, 1994
(30)
JOE Volume 43, Number 7, July 2017
Rauschenberger et al, 1997
(47)
size Sample collection
Normal pulp, n = 9 Normal pulp group:
Irreversible symptomatic extraction of teeth and
pulpitis, n = 12 extirpation of pulp tissue.
Irreversible symptomatic
pulpitis group: pulp
extirpation with spoon
excavator
One half in buffered
formalin for histologic
examination and one half
stored at 70 .
Extraction of pulp and To determine whether IL-2
extraction of pulp tissue.
Objective of the study
To determine whether a
relationship exists
between levels of PMN
proteases (PMN-E, PMN-
CG) and the common
inhibitor (A2-M) in healthy
and inflamed dental pulps.
can be detected in normal
Review Article
Methods Results
Lactobacillus casei subsp.
casei and L. acidophilus.
Irreversible pulpitis group
showed significantly
higher levels of antibody
to L. acidophilus when
compared with normal
group. Levels of antibody
reacting with L. casei
subsp. casei were
approaching significantly
higher level in normal
group when compared
with irreversible group.
Regardless of strain of
microorganisms tested,
large range of variation of
antibody levels was noted
in both irreversible and
normal groups.
Histologic analysis 9 are classified as healthy
5 are classified as mildly
inflamed
7 are classified as
moderately inflamed to
severely inflamed.
ELISA Moderate to severely
inflamed pulpal tissues
had significantly greater
concentrations of PMN-E
than healthy or mildly
inflamed tissues (P < .05).
Mildly inflamed tissues had
significantly less PMN-CG
concentrations than
normal or moderately to
severely inflamed pulpal
tissues (P < .05).
No significant correlation
in healthy or mildly
inflamed pulpal tissues
concerning PMN-E, PMN-
CG, A2-M (P = .005).
Significant correlation
between A2-M and PMN-
CG in moderately to
severely inflamed pulpal
sample group (P = .05).
Histologic analysis 14 are classified as healthy
8 are classified as mildly
JOE Volume 43, Number 7, July 2017

Normal pulp, n = 16 One half in buffered and inflamed pulpal inflamed

Irreversible symptomatic formalin for histologic
pulpitis, n = 12 examination and one half
stored at 70 .
Barkhordar et al, 1999 (48) Normal pulp, n = 8 Normal pulp group: To study the level of IL-6 in
Irreversible symptomatic extraction of teeth and
pulpitis, n = 6 extraction of pulp tissue.
Irreversible symptomatic
pulpitis group: non
precise.
Huang et al, 1999 (49) Normal pulp, n = 15 Normal pulp group: To determine levels of IL-8
Irreversible symptomatic extraction of teeth and
pulpitis, n = 14 extraction of pulp tissue.
Irreversible symptomatic
pulpitis: pulp extirpation
with endodontic spoon
excavator (coronal pulp)
and file (radicular pulp).
Dental Pulp Inflammation Diagnosis
tissues classified by clinical 3 are classified as
symptoms and histologic moderately inflamed
assessment. 3 are classified as severely
inflamed.
ELISA IL-2 detected in all vital pulp.
Significant differences in
IL-2 concentrations in
clinically normal and
irreversibly inflamed
pulpal tissues (P < .05).
ELISA Healthy pulp tissues showed
inflamed human pulps by marginal amount of the
using ELISA. cytokine.
Significant amounts of IL-6
were detected in all
inflamed specimens,
compared with normal
pulp group (P non-precise).
ELISA Normal pulp group: 53% of
expression in normal samples show detectable
human dental pulps as well but low level of IL-8.
as in pulps that show signs Irreversible symptomatic
of irreversible pulpitis group: 71%
symptomatic pulpitis. demonstrated detectable
and elevated levels of IL-8
Significant difference
P < .05.
Immunohistochemistry Intensity of IL-8 staining in
normal samples varied
from negative to weakly
stained.
Intensity of IL-8 staining in
inflamed samples varied
from weak to marked.
Strong IL-8 staining was
seen in macrophages and
lymphocytes, frequently in
endothelial cells, and
occasionally in pulpal
fibroblasts.

Rodd and Boissonade, 2000 Normal pulp, n = 19 Extraction of pulp and To investigate expression of Immunocytochemistry Increase of SP expression in
(50) Moderately carious teeth, extraction of pulp tissue. SP within human teeth in pulp horn as follows: in
n = 22 both health and disease grossly carious
Grossly carious teeth, and to seek a correlation teeth > moderately carious
n = 21 between reported pain teeth > intact teeth
Of the grossly carious history and SP expression. (P < .001).

Review Article
specimens: Increase of SP expression in
Irreversible asymptomatic subodontoblastic nerve
pulpitis, n = 11 plexus as follows: in grossly
Irreversible symptomatic carious teeth > moderately
pulpitis, n = 10 carious teeth > intact teeth
(P < .05).
(continued )
1037
 1038
TABLE 1. (continued )
Clinical diagnosis, sample
Authors, date (references)
Zanini et al.
Awawdeh et al, 2002 (51)
Irreversible symptomatic
Anderson et al, 2002 (52)
Irreversible symptomatic
Gusman et al, 2002 (53)
Irreversible symptomatic
JOE Volume 43, Number 7, July 2017
size Sample collection
Normal pulp, n = 20 Extraction of teeth and pulp
extirpation with barbed
pulpitis, n = 46 broach or Hedstro m file.
Normal pulp, n = 24 Extraction of teeth and
extraction of pulp with
pulpitis, n = 32 forceps.
Necrotic pulp, n = 23
Normal pulp, n = 18 Normal pulp group:
extraction of teeth and
pulpitis, n = 17 extraction of pulp.
Irreversible symptomatic
pulpitis group: pulp
extirpation with Hedstro m
files and spoon excavator.
Objective of the study
To compare levels of SP, NKA,
and CGRP in human dental
pulp tissue from painful
and non-painful teeth.
To confirm whether there are
significant differences in
IL-2 levels within human
pulpal tissues diagnosed as
normal, irreversibly
inflamed, or necrotic by
commonly accepted
clinical diagnostic testing
procedures.
To study levels of certain
selected MMPs, their
interdependence, and the
overall gelatinolytic
activity in both clinically
healthy and inflamed
human dental pulps.
Review Article
Methods Results
Increase of SP expression in
mid-coronal pulp as
follows: in grossly carious
teeth > moderately carious
teeth > intact teeth
(P < .05).
Increase of SP expression in
3 regions in grossly carious
teeth.
Significant expression of
SP in painful teeth
compared with
asymptomatic teeth
(P < .05).
Radioimmunoassay Mean concentrations of SP,
NKA, and CGRP were
significantly higher in pulp
tissue from painful teeth
compared with non-
painful teeth (SP, P < .05;
CGRP, P < .05; NKA,
P < .0001).
Significant association
between severity of pain
(visual analogue scale) and
levels of SP (P < .05), NKA
(P < .001), CGRP (P < .05).
ELISA No statistical difference
between experimental
groups.
ELISA MMP-1 level is below
detection limit for both
clinically healthy and
irreversible symptomatic
pulpitis group.
Levels of MMP-2 and MMP-
3 were significantly lower
in symptomatic versus
clinically healthy pulp
(P < .001).
MMP-9 value was
significantly higher in
inflamed pulp than in
normal pulp (P < .001).
JOE Volume 43, Number 7, July 2017
Shin et al, 2002 (54) Normal pulps, n = 10
Irreversible symptomatic
pulpitis, n = 12
Irreversible asymptomatic
pulpitis, n = 12
Pezelj-Ribaric et al, 2002 (55) Normal pulp, n = 20
Reversible pulpitis, n = 20
Irreversible symptomatic
Dental Pulp Inflammation Diagnosis
pulpitis, n = 20
Artese et al, 2002 (56) Normal pulp, n = 25
Irreversible symptomatic
pulpitis, n = 25
1039
Extirpation of pulp tissue To evaluate tissue levels of
(during root canal MMP-1, MMP-2, and MMP-
treatment). 3, and their distributions in
inflamed human dental
pulps and periapical
lesions.
Extraction of teeth and To determine whether TNF-a
extraction of pulp tissue. could be detected in
normal, symptomatic, and
asymptomatic pulpal
tissues classified according
to clinical symptoms.
Non-precise To evaluate immunostaining
for VEGF and factor VIII in
normal healthy and
inflamed dental pulps.
Gelatinolytic activity Degradation bands were
significantly more
abundant and intense in
irreversible symptomatic
pulpitis groups compared
with healthy group
(P < .001).
Immunohistochemistry Irreversible symptomatic
pulpitis: MMP-1 and MMP-
3 were localized
predominantly in
infiltrating neutrophils
and macrophages.
Immunoreactivity of MMP-
1 and MMP-3 was stronger
and more frequent than
that of MMP-2.
Irreversible asymptomatic
pulpitis:
MMP-1, MMP-2, and MMP-
3 were expressed in
chronic inflammatory
cellsplasma cells,
lymphocytes, and
macrophages.
ELISA Concentration of MMP-1 in
inflamed group than in
normal pulp (P < .05).
Level of MMP-2 was
significantly higher in
irreversible symptomatic
pulpitis than in control
(P < .05).
Levels of MMP-3 were
significantly higher in
irreversible symptomatic
pulpitis than in control
group (P < .05).
ELISA Expression of TNF-a in all vital
pulp samples.
Difference in
concentrations was
statistically significant
between irreversible
symptomatic and
reversible pulpitis samples
(P = .000).
Review Article
Immunohistochemistry Expression of VEGF was
strongly positive in
inflammatory infiltrate of
irreversible pulpitis.
VEGF expression in stromal
cells in healthy pulps
ranged from 20% to 100%
(continued )
 1040

Review Article
TABLE 1. (continued )
Clinical diagnosis, sample
Authors, date (references) size Sample collection Objective of the study Methods Results
Zanini et al.

and in irreversible pulpitis

Bowles et al, 2003 (57) Normal pulp, n = 8
Irreversible pulpitis, n = 16
Piattelli et al, 2004 (58) Normal pulp, n = 23
Irreversible symptomatic
pulpitis, n = 20
JOE Volume 43, Number 7, July 2017
Pulp exposure and insertion
of microdialysis probe into
pulp tissue.
Extraction of teeth and
extraction of pulp tissue
with excavator.
To determine in vivo pulpal
levels of immunoreactive
SP in human teeth with
diagnosis of normal pulp
or irreversible
symptomatic pulpitis.
To evaluate and compare
TGF-b1 expression in
healthy pulps and in those
with irreversible pulpitis.
ranged from 0% to 100%;
the difference was
significant (P = .05).
Decrease of microvessel
density in irreversible
symptomatic pulpitis
(P = .001).
Microdialysis Levels of induced SP collected
by microdialysis were
much higher in irreversibly
inflamed pulp versus
normal pulp (P = .001).
Immunohistochemistry Healthy pulps:
In 5 cases, more than
50 cells were positive to
TGF-b1 in odontoblastic-
subodontoblastic layer.
In 6 cases, positive cells
were between 10 and
50 cells, whereas 12 cases
were negative.
Irreversible symptomatic
pulpitis:
In 15 cases, more than
50 cells were positive to
TGF-b1 in odontoblastic-
subodontoblastic layer.
In 2 cases, positive cells
were between 10 and
50 cells, whereas 3 cases
were negative.
Higher expression of TGF-
b1 was found in
odontoblastic-
subodontblastic layer of
irreversible pulpitis, and
this difference is

Caviedes-Bucheli et al, 2004 Normal pulp, n = 5 Normal pulp: extraction of
(59) Irreversible symptomatic teeth and extraction of
pulpitis, n = 5 pulp.
Induced pulp Irreversible symptomatic
inflammation, n = 5 pulpitis and induced pulp
inflammation groups: pulp
extirpation with sterile
barbed broach.
To determine tissue levels of
CGRP in human pulpal
samples collected from
teeth with clinical
diagnosis of acute
irreversible pulpitis, in
teeth with induced
inflammation, and in teeth
statistically significant
(P = .0002).
Flow cytometry Significant statistical
differences were found
between percentages of
CGRP expression in healthy
pulps and pulps with acute
irreversible pulpitis
(P < .005).
No difference between
induced inflammation
JOE Volume 43, Number 7, July 2017
Nakanishi et al, 2005 (60)
Tsai et al, 2005 (73)
Dental Pulp Inflammation Diagnosis
Caviedes-Bucheli et al, 2006
(61)
Caviedes-Bucheli et al, 2007
(62)
1041
Normal pulp, n = 5 Extraction of teeth and
Irreversible symptomatic extraction of pulp with
pulpitis, n = 8 explorer and/or spoon
excavator.
Normal pulp, n = 14 Extraction of teeth and
Irreversible symptomatic extraction of pulp tissue
pulpitis, n = 14 with spoon excavator.
Normal pulp, n = 6 Normal pulp: extraction of
Irreversible symptomatic teeth and extraction of
pulpitis, n = 6 pulp.
Induced pulp Irreversible symptomatic
inflammation, n = 6 pulpitis and induced pulp
inflammation groups: pulp
extirpation with sterile
barbed broach.
Normal pulp, n = 5 Normal pulp: extraction of
Irreversible symptomatic teeth and extraction of
with clinically normal
pulps.
To study MIP-3a and CCR6 Immunohistochemistry
expressing cells in normal
and inflamed dental pulp
tissue and to elucidate
whether MIP-3a
contributes to
pathogenesis of pulpitis.
To study levels of MMP-9 in Immunohistochemistry
both clinically healthy and
inflamed human dental
pulps.
To study these neuropeptides Radioimmunoanalysis
and understand their role
in pulp neurophysiology.
To compare SP receptor
expression in human pulp
group and irreversible
symptomatic group
(P = .08).
Normal pulp group:
MIP-3a was detectable,
whereas CCR6 was not
detectable.
Irreversible symptomatic
pulpitis group:
All inflamed pulp
contained MIP-3a
producing and CCR6-
positive cells. MIP-3a was
detected in CD68-positive
monocytes/macrophages
adjacent to carious lesions.
Few microvascular
endothelial cells located
near the bacterial invasion
displayed MIP-3a
immunoreactivity. CCR6
expression was observed in
infiltrating lymphocytes.
MMP-9 staining was detected
in clinically healthy pulps.
MMP-9 staining was
stronger in inflamed pulps
than in clinically healthy
pulps.
MMP-9 was observed
mainly in cytoplasm of
odontoblasts, fibroblasts,
inflammatory cells, and
endothelial cells in
inflamed pulps.
Significantly greater MMP-
9 expression was noted in
inflamed human dental
pulps than those of
clinically healthy pulps
(P = .02).
5 neuropeptides (SP, CGRP,
NKA, NPY, VIP) were
identified in all samples.
Significantly higher
expression of
neuropeptides in pulps
Review Article
with irreversible
symptomatic pulpitis
(P < .0001), except for VIP,
which is unaltered in the 3
groups (P > .05).
Radioreceptor assay SP receptor expression was
found in all samples.
(continued )
 1042
TABLE 1. (continued )
Clinical diagnosis, sample
Authors, date (references) size
Zanini et al.
pulpitis, n = 5
Induced pulp
inflammation, n = 5
Sattari et al, 2009 (63) Normal pulp, n = 16
Irreversible asymptomatic
pulpitis (pulp polyp), n = 16
Silva et al, 2009 (64) Normal pulp, n = 10
Reversible pulpitis, n = 10
JOE Volume 43, Number 7, July 2017
Review Article
Sample collection Objective of the study Methods Results
pulp. tissue samples collected Statistically significant
Irreversible symptomatic from teeth having clinical difference was found in all
pulpitis and induced pulp diagnosis of acute 3 groups (P < .01).
inflammation groups: pulp irreversible pulpitis, Statistical difference was
extirpation with sterile healthy pulps, and teeth found between normal
barbed broach. with induced pulp group and
inflammation. irreversible symptomatic
pulpitis group (P < .005).
Statistical difference was
found between induced
inflammation group and
irreversible symptomatic
pulpitis group (P < .05).
Normal pulp group: To evaluate possible ELISA IgE was found in 100% of
extraction of teeth and relationship between pulp pulp polyps and 50% of
extraction of pulp. polyp formation and normal pulps.
Irreversible asymptomatic allergy. Histamine was found in
pulpitis group: extirpation 100% of pulp polyps and
of pulp with sterile 12.5% of normal pulps; the
excavator. difference is significant
(P < .001).
Significant positive
correlation was found
between concentration of
histamine and pulp polyp
formation (P < .001).
IL-4 was found in 62.5% of
pulp polyps and 18.75% of
normal pulps. The
difference is significant
(P < .01).
IL-12 was found in 25% of
pulp polyps and 12.5% of
normal pulps (the
difference is not
statistically significant).
Normal pulp: extraction of To analyze presence, Immunohistochemistry Normal pulp group:
teeth and extraction of location, distribution, and Presented negative
pulp. concentration of these immunolabeling for IL-1b
Reversible pulpitis group: cytokines in healthy and and IL-8 antibodies.
during pulpectomy inflamed dental pulps by Reversible pulpitis group:
procedure. immunohistochemistry Presented intense staining
and ELISA. specific for both
antibodies and also
specific and restricted to
inflammatory cells.
ELISA Statistical difference was
found between control
and inflamed pulp both for
IL-1b and for IL-8 (P < .001).
JOE Volume 43, Number 7, July 2017
Kangarlou Haghighi et al,
2010 (66)
Zehnder et al, 2011 (65)
Abd-Elmeguid et al, 2012 (67)
Accorsi-Mendonca et al, 2013
(68)
Dental Pulp Inflammation Diagnosis
1043
Normal pulp, n = 20 Extraction of teeth and pulp
Reversible pulpitis, n = 20 extraction.
Normal pulp, n = 12 Dentinal fluid collection
Irreversible symptomatic
pulpitis, n = 19
Normal pulp, n = 5 Extraction of teeth and pulp
Reversible and irreversible extraction.
pulpitis, n = 40
Normal pulp, n = 10 Normal pulp group:
Irreversible symptomatic extraction of teeth and
pulpitis, n = 10 extraction of pulp.
Irreversible symptomatic
pulpitis group: extirpation
of pulp during root canal
treatment.
To determine relation
between pulpal
neuropeptides (SP and
CGRP) and caries.
To determine whether MMP-
9 levels in dentinal fluid
were detectable and
differed between pulps
from symptomatic teeth
diagnosed with
irreversible pulpitis and
healthy counterparts.
To examine presence of DMP- Immunohistochemistry
1 in inflamed pulps and
understand its role in
dental pulp inflammation.
To investigate gelatinolytic
activity of MMP-2 and
MMP-9, TIMP-2, MPO
expression in healthy and
inflamed dental pulps.
ELISA CGRP was found in 5% and
20% of intact and carious
samples, respectively.
SP was present in 40% and
60% of intact and carious
samples, respectively.
SP level is significantly
increased in reversible
pulpitis group compared
with normal pulp group
(P < .05).
MMP-9 assay Normal pulp group:
No detectable level of
MMP-9 was found.
Irreversible symptomatic
group: detectable levels of
MMP-9 were found in 7
samples.
Level of MMP-9 was
significantly higher in
irreversible symptomatic
pulpitis group (P < .05).
Normal pulp group:
DMP-1 was not expressed
in mature teeth.
Inflamed pulp group:
DMP-1 was present in all
samples and localized in
both subodontoblastic cell
layer in the periphery and
the center of the pulp
tissue. Intense DMP-1
staining in areas of fibrosis
and calcification was
found.
ELISA Statistically significant
difference in levels of
TIMP-2 was found
between healthy and
inflamed groups (P < .005).
Zymography Higher diversity of band
degradation was observed
in inflamed pulp when
compared with healthy
group.
Degradation bands of
total MMP-2 were
Review Article
significantly more intense
in inflamed group
(P = .038)
MPO assay Higher amounts of MPO
concentrations were
found in inflamed group
(P = .038).
(continued )
 1044
TABLE 1. (continued )
Clinical diagnosis, sample
Authors, date (references) size
Zanini et al.
Abd-Elmeguid et al, 2013 (69) Normal pulp, n = 30
Reversible pulpitis, n = 23
Irreversible symptomatic
pulpitis, n = 12
Tancharoen et al, 2014 (74) Normal pulp, n = 15
Irreversible symptomatic
pulpitis, n = 15
JOE Volume 43, Number 7, July 2017
Sample collection Objective of the study
Extraction and pulp To localize OCN in inflamed
extirpation. pulps, to distinguish
different levels of OCN in 2
stages of pulp
inflammation.
Extraction of teeth pulp To investigate expression of
tissue was removed by RAGE in human dental
using sterile dental probe pulpitis.
and forceps.
Review Article
Methods Results
Multiplex assay OCN expression in reversible
pulpitis is significantly
higher than in irreversible
pulpitis, and both were
significantly higher than in
controls.
OCN expression in
reversible pulpitis was
positively correlated to
expression of VEGF, FGF,
MIP-1b, monocyte-derived
chemokine, monocyte
chemoattractant protein-
1, IL-17, and soluble IL-2
receptor a and negatively
correlated with that of IL-
1a, IL-1b, IL-8, GM-CSF, and
MIP-1 a.
Immunohistochemistry Normal teeth:
No evidence of OCN
expression was found.
Reversible and irreversible
pulpitis tissues had higher
OCN levels compared with
control tissues.
OCN levels were higher in
reversible pulpitis
compared with irreversible
pulpitis. OCN in inflamed
tissues was localized in
cells and matrix around
calcification areas and in
cells around blood vessels.
Immunohistochemistry In healthy tissues: RAGE
signal was low in
odontoblastic and
subodontoblastic layers,
stromal pulp fibroblast-
like cells, and endothelial-
like cells lining.
In inflamed tissues:
Abundant RAGE labeling
in both odontoblastic and
subodontoblastic cell
layers, in odontoblast
processes in tubules of the
predentin, stromal pulp
fibroblast-like cells, and
endothelial-like cells lining
in pulpitis tissues.
JOE Volume 43, Number 7, July 2017

Mente et al, 2016 (70) Normal pulp, n = 8
Reversible pulpitis, n = 11
Irreversible symptomatic
pulpitis and anti-
inflammatory drugs
(NSAID), n = 20
Irreversible symptomatic
pulpitis and absence of
anti-inflammatory drugs
(NSAID), n = 6
RAGE expression in
infiltrating inflammatory
cells in interstitial
connective tissues was
intensely stained.
Western blot RAGE protein quantity,
normalized to b-actin,
showed significant
differences in RAGE
expression in pulpitis
group compared with
normal pulp group
(P < .001).
Blood sample from exposed To determine level of MMP-9 and TIMP-1 MMP-9 levels in normal pulp
pulp by using inflammation markers of assay group were significantly
microcapillary tube. proteolytic enzymes. different from those in
teeth with reversible
pulpitis or irreversible
pulpitis (P = .006, P < .001,
P < .001, respectively).
Statistically significant
difference was observed
between MMP-9 levels in
reversible pulpitis group
and irreversible
symptomatic pulpitis
group (without NSAID)
(P = .005).
MMP-9 and TIMP-1 levels
showed very highly
significant correlation
regardless of group
assignment with positive
correlation coefficient
(P < 001).

A2-M, a2 macroglobulin; CCR6, chemokine receptor 6; DMP, dentin matrix protein; FGF, fibroblast growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; Ig, immunoglobulin; MIP, macrophage inflammatory protein; MPO, myeloperoxidase; NSAID, nonsteroidal
anti-inflammatory drug; OCN, osteocalcin; PMN-CG, PMN cathepsin G; PMN-E, PMN elastase; SF, supernatant fluid; VIP, vasoactive intestinal peptide.
Dental Pulp Inflammation Diagnosis

Review Article
1045
Review Article
(PRISMA) guideline principles (24). The flow diagram of included re-
cords is shown in Figure 1. The focused question was proposed by using
principle of Population, the Intervention (or exposure), the appropriate
Control or comparator, and the Outcomes of interest (PICO): Which
mediators are related to inflammatory pulpal disease?
Selection Criteria
Articles that met the following criteria were included in the final
selection: articles that concern human permanent mature teeth, articles
with a clear clinical diagnosis of inflammatory pulp disease (in accor-
dance with the AAE), studies that included a search for inflammatory
mediators, and articles that were published in English.
Literature Search
The electronic databases PubMed and Cochrane were searched by
using a key search terms combination relevant to the research topic
(Fig. 1): [PULP TISSUE] AND [GENE OR PROTEIN] EXPRESSION]
AND [DIAGNOSIS]. Articles published between 1970 and December
2016 were included in this screening process. We also considered
grey literature according to recommendations from Yaylali and Alacam
(25). In addition, to increase the exhaustivity of the research, the refer-
ence lists of selected articles were screened to find studies that might
qualify for this review (backward research). This search was made
by 2 independent investigators (M.Z., S.S.).
Data Extraction and Analysis
A first group of 22 publications was selected, and backward
research produced 79 additional articles, 29 of which were eligible.
Full texts of qualified articles were then read and selected. Among the
51 full-text articles assessed for eligibility, exclusion criteria were
applied to disqualify the following articles: an article reporting a tech-
nical development (26), studies in which the clinical diagnosis was not
described (2737), in vivo study (38), cell culture studies (39, 40), a
study with immature teeth (41), studies in which the comparison with a
disease state was not made (29, 39, 42), and a study in which only
macroscopic and microscopic changes had been investigated (43).
Results
Selected Studies
This review included 32 articles (Tables 1 and 2). The analysis
showed that authors used methods of messenger RNA or protein
quantification of inflammatory mediators during inflammation; 28
addressed protein expression profiles (30, 4470), 2 addressed
gene expression profiles (71, 72), and 2 addressed both molecular
and gene expression (73, 74).
Sampling Methods
Sampling involved tooth extraction in 24 articles (30, 44, 4853,
55, 5864, 6669, 7174) and canal treatment in 15 articles (45, 46,
53, 54, 57, 59, 6165, 68, 7072).
Method Variability
Various methods were used to evaluate protein and gene expres-
sion: enzyme-linked immunosorbent assay (ELISA) (30, 4549,
5255, 6366, 68, 70), multiplex assay (69), radioimmunoassay
(44, 51, 61, 62), immunohistochemistry (49, 54, 56, 58, 60, 64, 67,
69, 73, 74), microdialysis (57), zymography (68), immunocytochem-
istry (50, 59), Western blot (74), and reverse transcriptase polymerase
chain reaction (RT-PCR) (7174). The ELISA test was the most used
technique in the selected articles.
1046 Zanini et al.
Variability of the Molecular Types
Table 3 lists all the types of molecules investigated in the included
studies of this review.
Prostaglandins (PGE2 and PGF2a), C-reactive protein (CRP), IL-6,
substance P (SP), calcitonin-related gene peptide (CGRP), neurokinin
A (NKA), neuropeptide Y (NPY), transforming growth factor (TGF)-b1,
and vascular endothelial growth factor (VEGF) were studied at the
protein level to compare teeth clinically diagnosed with irreversible
symptomatic pulpitis with teeth with normal pulp.
Increased matrix metalloproteinase (MMP)-9, TNF-a, IL-8, and
receptor for advanced glycation end products (RAGE) expression
was evident at both gene and protein levels for teeth clinically diagnosed
with irreversible pulpitis compared with teeth with normal pulp.
Because RAGE has been investigated only once in the context of the
research question, further studies should be conducted to determine
the reliability of this inflammatory mediator. Contradictory results for
MMP-2 and MMP-3 prevent us from drawing conclusions regarding
these mediators (53, 54); further studies are required.
Expression patterns of MMP-9, TNF-a, RAGE, and Il-8 are synthe-
sized in Figure 2.
Discussion
The main limitation of this study is the lack of quality studies.
Furthermore, the variability in study methods and sample sizes are
secondary limitations that preclude meta-analysis.
The results of this systematic review suggest that the gene and pro-
tein expression of IL-8, TNF-a, MMP-9, and RAGE increase inside pulp
samples of teeth clinically diagnosed with irreversible symptomatic
pulpitis.
IL-8 is produced by several pulpal cells including macrophages,
lymphocytes, fibroblasts, and endothelial cells (75). In in vitro condi-
tions, odontoblast cells exhibit low level IL-8 expression that signifi-
cantly increases with pathogen-associated molecular pattern
stimulation, especially lipopolysaccharide. This increased IL-8 expres-
sion is correlated with increased polymorphonuclear neutrophils
(PMNs) within the pulp (49) because IL-8 induces neutrophil chemo-
taxis and release of degradation enzymes during degranulation. Thus,
IL-8 is often described as the primary regulatory molecule in the acute
inflammatory response, and elevated levels may perpetuate and exacer-
bate the acute inflammatory response. This activity may explain the re-
view findings of increased IL-8 expression in pulp tissue with
irreversible pulpitis. Furthermore, human fibroblast nemosis is the pro-
cess of cell activation and death that generates inflammatory mediators
such as PGs and growth factors (76). Reported increased secretory IL-8
expression in human dental pulpal fibroblast (HDPF) spheroids sug-
gests that the HDPF nemosis response promotes the secretion of chemo-
kines such as IL-8 in vitro (77). Fibroblast nemosis has gained
attention because of its potent role in pulp repair. HDPF spheroids
have been shown to promote human dental pulp stem cell migration
(77); however, further studies are required to elucidate the role of
IL-8 in nemosis and pulp regeneration.
TNF-a is a pleiotropic molecule that increases leukocyte toxicity,
stimulates acute phase inflammatory proteins, and induces inflamma-
tory cytokine production. TNF-a is an anti-inflammatory mediator
essential to the dental pulp response to infection (55, 64). Its
synthesis by macrophages is often stimulated by toll-like receptor ligand
binding in the presence of bacteria. Because inflammation is a prereq-
uisite to regeneration, certain inflammatory mediators have a role in
both inflammation and regeneration. Both roles are concentration-
dependent and time- or context-dependent. Interestingly, TNF-a synthe-
sis may be induced directly in response to extraction of matrix proteins
JOE Volume 43, Number 7, July 2017
JOE Volume 43, Number 7, July 2017
TABLE 2. Selected Studies Analyzing Gene Expression of Inflammatory Mediators
Authors, date (references)
Zehnder et al, 2003 (71)
Tsai et al, 2005 (73)
Kokkas et al, 2007 (72)
Dental Pulp Inflammation Diagnosis
Tancharoen et al, 2014 (74)
1047
Clinical diagnosis, sample size
Normal pulp, n = 13
Irreversible symptomatic
pulpitis, n = 11
Normal pulp, n = 14
Irreversible symptomatic
pulpitis, n = 14
Normal pulp, n = 6
Reversible pulpitis, n = 6
Irreversible symptomatic
pulpitis, n = 6
Normal pulp, n = 15
Irreversible symptomatic
pulpitis, n = 15
Sample collection
Normal pulp group: extraction
of teeth and collection of pulp
with excavator.
Irreversible symptomatic
pulpitis: extirpation of pulp
with excavator and Hedstro m
file.
Extraction of teeth and
extraction of pulp tissue with
spoon excavator.
Normal pulp group: extraction
of teeth and extirpation of
pulp with spoon excavator OR
extraction of pulp with
barbed broach.
Reversible and irreversible
symptomatic pulpitis groups:
extirpation of pulp with
barbed broach.
Extraction of teeth and pulp
tissue was removed by using
sterile dental probe and
forceps.
Objective of the study
To investigate differences in
cytokines gene expression
between pulps from healthy
teeth and from symptomatic
vital teeth with severe caries
lesions.
To study levels of MMP-9 in both
clinically healthy and
inflamed human dental pulps.
To evaluate whether correlation
between TNF-a gene
expression in human dental
pulp and clinically defined
reversibility of pulp
inflammation could be
detected.
To investigate expression of
RAGE in human dental
pulpitis.
Methods Results
RT-PCR Significantly higher mRNA levels
for IL-6 (P < .05) and especially
IL-8 and IL-18 in symptomatic
versus healthy pulps (P < .005).
Contents of IL-1a and IL-1b
mRNA between 2 groups
scantily failed to reach
statistical significance (P = .06
and .26, respectively).
Significant correlations were
observed between IL-1a and
IL-1b, between IL-6 and IL-18,
and between Il-8 and IL-18
(P < .05).
RT-PCR Significantly greater MMP-9
expression was noted in
inflamed human dental pulps
(2.1) than those of clinically
healthy pulps (P < .05).
RT-PCR TNF-a expression detected in all
3 groups.
Statistical difference in
irreversible symptomatic
group compared with either
normal pulp group (P = .002)
or reversible pulpitis group
(P = .015).
No statistical difference
between normal pulp group
and reversible pulpitis group
(P = .7).
RT-PCR Overexpression of RAGE mRNA
in pulpitis samples (P < .001).
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Review Article
released by bacterial demineralization (78). TNF-a can activate the p38 in short-term cultures but inhibits mineralization in long-term cultures
mitogen-activated protein kinase pathway and induce odontoblast-like (82). Interestingly, short-term exposure of cells to TNF-a (6 and
cell differentiation into dental pulp stem cells by increasing dentin sia- 12 hours) induces apoptosis with VEGF upregulation and nuclear factor
loprotein and dentin phosphoprotein expression and forming tertiary kappa B signaling, whereas long-term (chronic) exposure (14 days)
dentin (79). High TNF-a concentrations downregulate alkaline phos- increases proliferation and shortens the telomere (83). TNF-a triggers
phatase, osteopontin, osteocalcin, osterix, and Runx2 expression in neutrophils to release large amounts of reactive oxygen intermediates
dental pulp stem cells, whereas low TNF-a concentrations produce and promotes neutrophil degranulation (8486). TNF-a also plays a
the opposite biological effects (80). Thus, high TNF-a concentrations crucial role in neutrophil survival, particularly through induction of
inhibit pulpal cell mineralization through the Wnt/b-catenin pathway. apoptosis; however, the mechanisms underlying this activity have not
These findings have been confirmed in mesenchymal stem cells (81) been defined (87).
and human apical papilla cells (82). On the basis of our review, pulp MMPs are a family of 24 zinc-dependent endopeptidases that are
tissues clinically diagnosed with irreversible pulpitis show increased responsible for structural extracellular matrix (ECM) protein degradation
TNF-a expression, which may inhibit pulp repair and induce apoptosis. (88). MMPs are essential to tissue homeostasis and play a role in patho-
A precise TNF-a cutoff point may be necessary to determine the pulp logic conditions, especially pulpal inflammation. MMPs alter cellular ad-
status at which regeneration is impossible. TNF-a temporal and contex- hesive properties to aid in cellular migration through the ECM. Their role
tual profiles may also influence the pulp repair process. TNF-a may in tissue destruction is evident but not well-understood. Pulp tissue
benefit odontoblast survival in chronic disease but limit odontoblast- destruction is partially regulated by MMPs and tissue inhibitors of
like cell differentiation from stem or progenitor cell populations until MMPs (TIMPs) (88). MMP family proteins have dual roles in inflamma-
infection resolution. TNF-a promotes apical papilla cell mineralization tion pathogenesis, stimulation of protective innate and adaptive immune
functions and tissue destruction (89). Some MMPs are involved in pulpal
repair. MMP-3 enhances proliferation, migration, and survival of human
TABLE 3. Molecule Types in the Included Studies umbilical vein endothelial cells in vitro (90). Furthermore, topical appli-
cation of MMP to injured pulp tissues of rat incisors accelerates tissue
Molecule types References
regeneration, angiogenesis, and reparative dentin formation (90).
Prostaglandins (Pg) (44) MMP-3 produces anti-inflammatory effects by decreasing the number
C-reactive protein (CRP) (45)
Immunoglobulins (Ig) (46, 63)
of macrophages and antigen presenting cells and significantly inhibiting
Interleukins IL-6 expression (91). MMP-9 or gelatinase (a collagenase family mem-
IL-1a and IL-1ra (69) ber) is secreted primarily by PMNs, which are abundant in inflamed tis-
IL-1b (64, 69, 71) sues compared with healthy tissues. Because their main role is to degrade
IL-1 (71)
IL-2 (47, 52)
collagen extracellular ground substance, MMP-9s are usually considered
IL-4 (69) as markers of pulp tissue breakdown (92). The role of MMPs in pulpal
Il-6 (48, 69, 71) disease is supported by periodontal studies of MMP levels (especially
IL-7 (69) MMP-9) in gingival crevicular fluid (9396). MMP-9 levels increase
Il-8 (49, 64, 69) with severity of the periodontal destruction such that MMP-9 could serve
Il-18 (71)
IL-13 (69) as a marker of connective tissue breakdown (96).
IL-15 (69) There is evidence for IL-8, TNF-a, and MMP-9 linkage, which is
Neurogenic mediators based on findings that TNF-a and IL-8 can induce rapid MMP-9
Substance P (SP) (50, 51, 57, 61) zymogen release in whole human blood (97). Furthermore, these 3 bio-
Calcitonin gene-related peptide (CGRP) (51, 59, 61, 66) molecules are associated with neutrophils; IL-8 induces neutrophil
Neurokinin A (NKA) (51, 61)
Neuropeptide Y (NPY) (61) chemotaxis, TNF-a is a powerful priming neutrophil agonist, and
Vasoactive intestinal peptide (VIP) (61) MMP-9 is secreted primarily by PMNs. Pulpitis is clearly a PMN-
Matrix metalloproteinase (MMP) driven inflammation; it usually starts with neutrophil invasion into the
MMP-2 (53, 54, 68) pulp adjacent to infected dentin. Neutrophils actively defend against
MMP-3 (53, 54, 68)
MMP-9 (53, 65, 68, 70, 73) pulp infection; however, they can also induce irreversible tissue dam-
Cytokines age. Neutrophils release MMP to recruit immune cells to the pulp
Tumor necrosis factor a (TNF-a) (55, 69, 72) and release reactive oxygen intermediates, including superoxide an-
TNF-b (69) ions, hydrogen peroxide, hydroxyl radicals, and other potent
Macrophage inflammatory protein 1 (69)
receptor a (MIP-1ra)
MIP-1b (69)
MIP-3a (60) Healthy pulp Irreversible Symptomatic Pulpitis
Interferon-g (69)
Growth factors
Vascular endothelial growth factor (56, 69) MMP-9 (55, 67, 72, 75, 80)
(VEGF)
Fibroblast growth factor (FGF) (69)
Platelet-derived growth factor (PDGF) (69) TNF (57, 74)
Transforming growth factor b (TGF-b) (58)
Receptor for advanced glycation end (74)
RAGE (76)
products (RAGE)
Cathepsin G (30)
a2 macroglobulin (30) Il-8 (51, 66, 83)
Matrix dentin proteins
Osteocalcin (OCN) (69) Figure 2. Expression patterns of MMP-9, TNF-a, RAGE, and Il-8 according to
Dentin matrix protein 1 (DMP-1) (67)
the clinical diagnosis.

1048 Zanini et al. JOE Volume 43, Number 7, July 2017


enzymes. Gradual tissue necrosis is thus initiated by microabscess for-
mation, which then leads to pulp necrosis (98, 99). By extrapolation,
high IL-8 and MMP-9 levels indicate increased neutrophil levels and
could be considered markers of tissue breakdown.
Molecular diagnosis may be defined as techniques analyzing both
the genome and proteome by applying molecular biology to medical
testing. These strategies have revolutionized clinical medicine, particu-
larly in the fields of infectious diseases, oncology, pharmacogenomics,
and prenatal genetic testing. Testing is based on expressions of bio-
markers, which have been used objectively as indicators of physiologic
health, pathogenic processes, and pharmacologic responses to therapy.
The first step toward molecular diagnosis of dental pulp disease is
identification of key inflammatory mediators and increased understand-
ing of their role in inflammation and regeneration. Because of the
complicated nature of inflammatory pulp disease, it is unlikely that a sin-
gle mediator will provide sufficient information to diagnose irreversible
inflammatory disease. The second step is to determine cutoff points for
tests using these inflammatory mediators. Cutoff points will be based on
levels of pulp tissue inflammatory mediators judged to reflect the limits of
reversible pulp tissue disease. The third step is to develop clinical tools
and methods to detect these biomolecules with qualitative and quantita-
tive accuracy. The methods should be feasible in clinical practice and
executable chairside to allow for their adoption into therapy. On the basis
of findings from this systematic review, evaluation of protein expression
appears to be the most appropriate method for clinical evaluation of
dental pulp inflammation. Compared with other immunoassay methods,
ELISA appears to be the most accurate, sensitive, and specific test for mo-
lecular evaluation of dental pulp inflammation. According to the review, 2
types of sampling are suggested, dentinal fluid sampling and pulp blood
analysis. The dentinal fluid may serve as a liquid biopsy to assess the state
of the underlying pulp by determining component concentrations of the
pulpal tissue fluid (100). Dentinal fluid is easily collected and considered
a serum-derived tissue fluid containing serum proteins and immuno-
globulins (the protein concentration is about one fifth that of plasma).
Thus, dentinal fluid may serve as a patient specific diagnostic test for
pulp disease (100, 101). This method is theoretically promising
because it is noninvasive. However, the difficulty of collecting sufficient
dentinal fluid makes its use clinically impractical (101, 102). Blood
evaluation at the site of pulp exposure has been tested (57, 70, 71),
but its main disadvantage is its invasiveness. However, pulpal blood
collection using heparinized microcapillary tubes or microdialysis has
proven practical and reliable (70).
Conclusion and Perspectives
Because dental pain descriptions are subjective, clinical diagnosis
alone is unreliable to accurately determine pulp inflammation severity
and pulp tissue ability to respond to endodontic procedures. As a result,
pulp vital therapy is usually performed following empirical decision-
making criteria. The need for new pulp diagnostic tools is increasingly
evident. Molecular-based strategies are promising and may be relevant
to determine appropriate clinical indications for vital pulp therapy and
to optimize prognosis after pulp vitality therapy.
By providing more precise diagnoses, molecular-based strategies
will change the decision-making of dental practitioners and enhance
patient assessment and treatment toward customized therapies.
Decreasing root canal overtreatment provides both biological and so-
cioeconomic advantages for patients.
Acknowledgments
The authors deny any conflicts of interest related to this study.
JOE Volume 43, Number 7, July 2017
Review Article
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