doi:10.1111/iej.
12906
REVIEW
Disclosing the physiology of pulp tissue for vital
pulp therapy
W. L. O. da Rosa, E. Piva & A. F. da Silva
Department of Restorative Dentistry, School of Dentistry, Federal University of Pelotas, Pelotas, Brazil
Abstract
da Rosa WLO, Piva E, da Silva AF. Disclosing the
physiology of pulp tissue for vital pulp therapy. International
Endodontic Journal, 51, 829–846, 2018.
The discovery that dentine is a reservoir of bioactive
molecules that can be recruited on demand has
attracted efforts to develop new protocols and materi-
als for vital pulp therapy (VPT). The noncollagenous
proteins (NCPs) present in the dentine extracellular
matrix (ECM) include growth factors (TGF-b1, BMP-7,
FGF-2, IGF-1 and IGF-2, NGF and GDNF), extracellu-
lar matrix molecules (DSP, DPP, BSP, DMP-1 and
DSPP) and both anti-inflammatory and pro-inflamma-
tory chemokines and cytokines (TNF-a, IL-1, IL-6 and
IL-10). Molecules such as DSP and DPP are mainly
expressed by odontoblasts, and they are cleaved prod-
ucts from dentine sialophosphoprotein (DSPP). Some
molecules, such as TGF-b1, specifically interact with
decorin/biglycan in dentine. Although TGF-b1
increases the expression and secretion of NGF in
Introduction
Pulp tissue can be affected as the result of deep caries
lesions and accidental trauma or by preparation tech-
niques used during the restoration of teeth affected by
carious lesions (Mattos et al. 2014). Thus, protection
of the pulp by applying capping agents directly or
Correspondence: Adriana Fernandes da Silva, Federal
University of Pelotas, Faculty of Dentistry, Department of
Restorative Dentistry, Goncßalves Chaves St., 457/503, Zip
Code: 96015-560, Pelotas, RS, Brazil (e-mail: adriana@
ufpel.edu.br).
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
human pulp cells, NGF induces mineralization and
increases the expression of DSPP and DMP-1. Further-
more, GDNF may act as a cell survival factor and
mitogen during tooth injury and repair. Pulp capping
materials, such as MTA and calcium hydroxide, can
solubilize bioactive dentine molecules (TGF-b1, NGF
and GDNF) that stimulate tertiary dentinogenesis. The
binding of these signalling molecules leads to activa-
tion of several signalling transduction pathways
involved in dentinogenesis, odontoblast differentiation
and inflammatory responses, such as the p38 MAPK,
NF-kb and Wnt/b-catenin signalling pathways.
Understanding the cascade of cellular and molecular
events underlying the repair and regeneration pro-
cesses provides a reasonable new approach to VPT
through a targeted interaction between tooth tissue
and bioactive molecules.
Keywords: dentinogenesis, molecular biology,
protein expression, pulp biology, signal transduction.
Received 21 May 2017; accepted 30 January 2018
indirectly on pulp tissue is called vital pulp therapies
(VPTs) (Da Rosa et al. 2017).
In recent years, with the progress of regenerative
and molecular approaches, it is known that the effi-
cacy of direct and indirect pulp capping might be
affected by the biomaterials used and their biological
properties. Some studies have suggested that the effect
of using calcium hydroxide or glass–ionomer cement
on dentine caries is not superior to the use of an inert
material (such as wax; Corralo & Maltz 2013), in
indirect pulp capping of primary (Marchi et al. 2006,
Casagrande et al. 2010, Schwendicke et al. 2015) or
permanent teeth (Baratieri et al. 2002, Whitworth
et al. 2005, Wegehaupt et al. 2009, Pereira et al.
International Endodontic Journal, 51, 829–846, 2018 829
 Proteins for vital pulp therapy da Rosa et al.
2017). On the other hand, for direct pulp capping an
overall success rate of 80.5% for MTA has been
reported when compared with calcium hydroxide
(59% success rate) up to 123 months (Mente et al.
2014). Hence, there is an ongoing quest to find pulp
capping agents that have improved physico-mechani-
cal properties, and potentiate the best biological
response (Da Rosa et al. 2017). For this reason, there
are considerable efforts in the field of regenerative
and molecular dentistry to use more biological
approaches, as the use of biomaterials is not exten-
sively developed considering our biological knowledge
(Sadaghiani et al. 2016).
The definition of regenerative endodontic proce-
dures such as ‘as biologically based procedures
designed to replace damaged structures’ (Murray et al.
2007) does not distinguish if the procedures involve
regeneration or repair (Smith et al. 2016). Regenera-
tion is the formation of a physiological-like dentine
tissue, while repair is the formation of a new tissue
resembling the native pulp–dentine complex at the
histologic level with the expected physiological func-
tions (Smith et al. 2016). Although the ultimate goal
of VPT is the complete regeneration of tissues lost to
caries or traumatic injuries, it is unlikely that the cur-
rent therapies are capable of stimulating the forma-
tion of native dentine (Smith et al. 2016). In this
context, reactionary and reparative dentinogenesis
are two types of tertiary dentine formation that may
occur and involve primary odontoblasts and dental
Figure 1 Two types of tertiary dentine formation: (a) reactionary dentinogenesis that occurs following moderate dentine injury
if primary odontoblasts are present to secrete dentine; (b) reparative dentinogenesis that occurs if odontoblasts have been dam-
aged or destroyed and if the formation of tertiary dentine is performed after stem/progenitor cell recruitment by odontoblast-
like cells.
830 International Endodontic Journal, 51, 829–846, 2018
pulp stem cells, respectively, as shown in Fig. 1. Reac-
tionary dentinogenesis occurs after moderate dentine
injury if primary odontoblasts are present to secrete
dentine. However, localized dental tissue damage may
result in the death of primary odontoblasts, and
reparative dentinogenesis occurs if tertiary dentine is
formed after stem/progenitor cell recruitment by
odontoblast-like cells (Smith et al. 2012). Further-
more, the extracellular dentine matrix contains bioac-
tive molecules that are sequestered within dentine
during dentinogenesis, including growth factors,
cytokines and other matrix molecules, which are cap-
able of stimulating dentine secretory activity via
odontoblasts or odontoblast-like cells (Smith et al.
2012).
Thus, the main goal of current research in the field
is to develop more biocompatible dental materials that
can fulfil the physico-mechanical requirements and
can perform the desired biological functions to gener-
ate the most appropriate tissue response, without elic-
iting undesirable local or systemic effects. Advances
in understanding the applicability of bioactive mole-
cules in VPT targeted to stimulate molecular path-
ways that lead to tissue repair or regeneration could
promote the best biological response with biomaterials
based on the stimulation and upregulation of tooth
substrates (Da Rosa et al. 2016). Thus, this review
intends to bring into context the current knowledge
on tertiary dentine formation (reparative and reac-
tionary dentinogenesis) and the current challenges
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd

regarding the use of bioactive molecules to improve
VPT.
Review
Dentinogenesis under physiological and
pathological conditions
Dentine is predominantly a mineralized connective tis-
sue with a collagenous matrix and hydroxyapatite
crystals, which was traditionally considered inert, with
limited biological responsiveness (Piva et al. 2014).
Fortunately, current knowledge has indicated that
extracellular dentine matrix contains bioactive mole-
cules, which allows the exploration of other molecular
mechanisms involved in regenerative and reparative
events of the dentine–pulp complex (Smith et al.
2012). The main terminally differentiated cells capable
of generating new tubular dentine are the odontoblasts,
which secrete primary and secondary dentine before
and after tooth eruption. The process of dentine secre-
tion, known as dentinogenesis, continues throughout
the life of a tooth, whereas tertiary dentine is formed in
response to injuries to protect the pulp tissue from dam-
age during VPT (De Lima et al. 2015, Da Rosa et al.
2016). In addition, differently from the process that
occurs during physiological odontogenesis, tertiary
dentinogenesis occurs in a pathologic microenviron-
ment with less regulation (Smith et al. 2016). For
instance, when tooth tissues are injured by caries or
trauma, the dissolution and release of bioactive dentine
molecules may occur. The complex molecules involved
in dentine repair and the dynamics of the disease pro-
cess are not yet fully understood, and they will be dis-
cussed in the following sections.
Which molecules are involved in odontoblast secretory
activity during tertiary dentinogenesis?
Tertiary dentinogenesis comprises a cascade of molec-
ular events intended to induce odontoblast secretory
activity. The binding of signalling molecules may lead
to receptor phosphorylation, which activates several
signalling transduction pathways, as represented in
Fig. 2 and described in Table 1. Amongst the path-
ways involved in dentinogenesis, the induction of
MAPK and PI3K/AKT/mTOR, which are essential ser-
ine–threonine kinases, is involved in cell proliferation,
apoptosis, adhesion and migration. The MAPK family
is comprised of three main subfamilies: p38 MAPK
(Fig. 2A), ERK (Fig. 2F) and JNK (Fig. 2G). Amongst
these, p38 MAPK is activated after phosphorylation
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
da Rosa et al. Proteins for vital pulp therapy
and translocates into the nucleus to activate specific
genes. It appears to be central to the transcriptional
control of odontoblasts, playing an important role in
the regulation of odontoblast secretory activity (Simon
et al. 2010, 2011). Moreover, the c-Jun N-terminal
kinases are members of the MAPK family activated by
growth factors, environmental stress and cytokines.
JNK is activated after phosphorylation and is translo-
cated to the nucleus to modulate odontoblast activity
(Fig. 2G).
Furthermore, bioactive molecules, such as TGF-b1
and BMP-2, may affect the proliferation, collagen
turnover and differentiation of dental pulp cells by
activating the TGF-b/Smad signalling pathway. SMAD
proteins are expressed in dental pulp cells and are
active in response to members of TGF-b family (He
et al. 2004). Smads 1, 5 and 8 serve principally as
substrates for BMPs, whereas Smads 2, 3 and 4 are
activated after TGF-b1 signalling, which is repre-
sented in Fig. 2B. Identifying the molecular events
involved in the odontoblast secretory activity makes it
possible to find new ways to modulate this activity
with biomaterials that can potentiate the dentine–
pulp biological response.
Molecular events involved in inflammatory responses
Dentine repair is a balance between inflammatory
response and reparative events that prevent irre-
versible pulp inflammation (Simon et al. 2010). Den-
tine is permeable due its tubular structure, and after
tooth injuries, the diffusion of a variety of molecules,
such as bioactive proteins, bacteria and toxins, can
occur and initiate inflammatory events. Although a
low level of inflammation stimulates dentine repair
events, exacerbated inflammation impairs dentinogen-
esis by many mechanisms, including cell death
(Cooper et al. 2010). The inflammatory response
requires activation of the intercellular signalling path-
ways, such as the NF-jb and MAPK pathways (ERK,
JNK and, in particular, the p38 MAPK pathway;
Botero et al. 2010, Cvikl et al. 2016). The NF-jb
pathway is activated by cytokine receptors, such as
TNF-a and interleukins. Phosphorylation of Ijb leads
to its ubiquitination and proteasomal degradation,
which releases NF-jb. The NF-jB complexes are fur-
ther activated by post-translational modifications
(phosphorylation, acetylation, glycosylation), translo-
cate to the nucleus and then induce the target gene
expression (Fig. 2E). In addition, MAPK/ERK pathway
is activated by growth factor and cytokine receptors.
The components of the pathway vary in response to
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 Proteins for vital pulp therapy da Rosa et al.

Figure 2 Representation of the cascade of molecular events that occurs after stimulation by signalling molecules can induce
odontoblast secretory activity, inflammatory responses of dental pulp cells (odontoblasts, fibroblasts, dental pulp stem cells),
odontoblast proliferation and differentiation to reach dentine formation: (A) p38 MAPK Pathway; (B) TGFb-Smad signalling
pathway; (C) PI3K/AKT/mTOR pathway; (D) Wnt/b-catenin signalling pathway; (E) NF-kb pathway; (F) MAPK/ERK pathway;
(G) JNK pathway. The main effects of the pathways are shown in Table 1.

different stimuli, but usually include a set of adaptors
linking the receptor to a guanine nucleotide exchange
factor, which transmits the signal to small GTP-bind-
ing proteins (RAS) and in turn activates the core unit
of the cascade composed of a MAPKKK (RAF), a
MAPKK (MEK1/2) and finally a MAPK (ERK1/2;
Fig. 2F).
Odontoblasts located at the periphery of the pulp
are the first cells to encounter products of the infec-
tious process, such as bacteria and released dentine
matrix constituents. Components from cariogenic bac-
teria (e.g. lipopolysaccharides, lipoteichoic acids, flag-
ellin, peptidoglycans and lipoproteins) are detected by
toll-like receptors (TLRs), mainly TLR-1 to TLR-6 and
TLR-9, which are expressed in odontoblasts and pul-
pal fibroblasts (Chang et al. 2005). Their binding acti-
vates the NF-jb pathway, which is responsible for
regulating the molecular inflammatory response
(Chang et al. 2005, Cooper et al. 2010). Other recep-
tors from dental pulp cells include the cytoplasmic
NOD-like receptors (NLRs) and retinoic acid-inducible
gene RIG-like receptors (RLRs) (Creagh & O’Neill
2006). Knowledge of the immunomodulatory path-
ways in tertiary dentinogenesis can be determinant of
whether tooth will remain vital or not, and new bio-
logical therapies must be focused on identifying mole-
cules involved in inflammatory events (De Lima et al.
2015).
Amongst signalling molecules, a complex variety of
pro-inflammatory and anti-inflammatory molecules
within the dentine matrix with important roles in
pulp response to infections, that is IL-1a, IL-1b, IL-4,
IL-6, IL-8, IL-10, IL-12 and TNF-a, have been
reported (Cooper et al. 2010). Cytokines and chemoki-
nes are produced in response to NF-jb signalling and
are able to regulate the immune and inflammatory
responses by binding to their specific receptors
(Cooper et al. 2010). The activation of the JNK, PI3K/
AKT/mTOR, p38 MAPK, MAPK/ERK and NF-jb
pathways in the presence of TNF-a has also been

832 International Endodontic Journal, 51, 829–846, 2018 © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd

Table 1 Description and main effects of key pathways involved in cell differentiation and proliferation, odontoblast secretory
activity and inflammatory response
Pathway
p38 MAPK Pathway (p38 Mitogen-Activated Protein Kinases)
TGFb-Smad Signalling Pathway (Transforming Growth
Factor/Small Mother Against Decapentaplegic Pathway)
PI3K/AKT/mTOR Pathway (Phosphatidylinositol 3-Kinase/
Protein Kinase B/Mechanistic Target of Rapamycin
Pathway)
Wnt/b-catenin Signalling Pathway
NF-kb Pathway (key Nuclear Factor kappa b Pathway)
MAPK/ERK Pathway (Mitogen-Activated Protein Kinase/
Extracellular signal-Regulated Kinase Pathway)
JNK Pathway (c-Jun N-terminal Kinase Pathway)
reported (Goda et al. 2015, Shin et al. 2015). The
protein products of the JNK pathway are involved in
apoptosis, neurodegeneration, cell differentiation and
proliferation, inflammation and cytokine production
mediated by the activation of transcription factors
(Goda et al. 2015). In addition, other molecules
involved in tertiary dentinogenesis have well-known
anti-inflammatory properties and modulate defence
and repair processes, such as TGF-b1 and ADM
(Cooper et al. 2010; Da Silva et al. 2018).
Environmental stimulus involved in dentinogenesis
Different stimuli (i.e. thermal, mechanical and chemi-
cal) can activate dentinogenesis. To respond to these
stimuli, odontoblasts express members of the transient
receptor potential (TRP) family. There are several
TRP receptors in odontoblasts, including the transient
receptor potential cation channel subfamily V chan-
nels (TRPV) (Burns et al. 2016). TRPVs are able to
respond to a variety of stimuli. For instance, studies
have suggested that the expression of TRP melastatin
subfamily member 8 (TRPM8) and TRP ankyrin sub-
family member 1 (TRPA1) in odontoblasts is linked to
the detection of low-temperature stimulation of the
dentine surface (Tsumura et al. 2013). Recent studies
reported the expression of TRPA1 channels in odonto-
blasts that are activated by alkaline stimuli, which
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
da Rosa et al. Proteins for vital pulp therapy
Main effects
Implicated in cell differentiation and proliferation, odontoblast
secretory activity and inflammatory response (Simon et al. 2010,
2011, Cooper et al. 2014, Shin et al. 2015, Cvikl et al. 2016).
Implicated in cell differentiation and proliferation (Chang et al.
2015) and odontoblast secretory activity (Lucchini et al. 2002,
He et al. 2004).
Implicated in cell differentiation (Kim et al. 2011), odontoblast
secretory activity (Zhang et al. 2015) and inflammatory
response (Shin et al. 2015).
Implicated in odontoblast differentiation and proliferation (Wang
et al. 2012a, Yoshida et al. 2016).
Implicated in inflammatory responses, innate and adaptive
immunity and stress responses (Chang et al. 2005, Cooper et al.
2010, 2014, Shin et al. 2015, Cvikl et al. 2016).
Implicated in cell differentiation and proliferation (Bento et al.
2013, Wang et al. 2016b), odontoblast secretory activity (Zhang
et al. 2015) and inflammatory responses (Botero et al. 2010, Shin
et al. 2015, Cvikl et al. 2016).
Implicated in cell differentiation, odontoblast secretory activity and
inflammatory responses (Goda et al. 2015, Shin et al. 2015,
Zhang et al. 2015).
could occur after application of some alkaline pulp
capping agents for VPT, such as calcium hydroxide or
mineral trioxide aggregate (MTA) (Kimura et al.
2016). Additionally, to mediate reactionary dentino-
genesis, there could be some mechanism involved in
the activation of TRP channels and TRPV1, TRPV2
and TRPV4 (Tsumura et al. 2013).
The role of dental pulp stem cells in tertiary
dentinogenesis
Reparative dentinogenesis is a more complex process
requiring dental pulp stem cell (DPSC) recruitment
followed by the signalling of odontoblast-like cell dif-
ferentiation and dentine secretion (Smith et al. 2012).
DPSCs can differentiate into odontoblasts and other
cell lineages, such as osteoblasts, chondrocytes and
neuronal progenitor cells (Piva et al. 2014). They are
present within niches, which provide a microenviron-
ment responsible for maintaining DPSCs in their
undifferentiated state (Smith et al. 2016). These stem
cells can be stimulated by many bioactive proteins,
and the odontoblast-like cells responsible for repara-
tive dentinogenesis are probably the newly differenti-
ated DPSCs that receive adequate stimulus in their
niches. There are different niches of stem cells in the
dental pulp tissue, but little is known about the stem-
ness of each niche. Differences in the mineral
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 Proteins for vital pulp therapy da Rosa et al.
composition of dentine formed by cells differentiated
from DPSCs and SCAPs have also been reported (Vol-
poni et al. 2015). During physiological odontogenesis,
the inner enamel epithelium-derived growth factors
that are immobilized on the dental basement mem-
brane induce the differentiation of peripheral cells of
the dental papilla into odontoblasts. In addition,
bioactive molecules, including FGF-1, TGF-b1 and
TGF-b3, play an essential role in these processes
(Smith et al. 2012).
Studies have suggested that the PI3K/AKT/mTOR
pathway is activated during DPSC differentiation (Kim
et al. 2011) and that the MAPK/ERK pathway
(Fig. 2F) is a regulator of DPSC endothelial differentia-
tion (Botero et al. 2010, Bento et al. 2013). The cas-
cade is activated by growth factors, environmental
stress and cytokines. Activated PI3K (phosphoinosi-
tide 3-kinase) phosphorylates and activates AKT (ser-
ine/threonine kinase), subsequently activating mTOR,
which can affect the transcription of proteins (see
Fig. 2C). The MAPK/ERK signalling pathway was
recently shown to be involved in adenosine 50 -tripho-
sphate (ATP)-mediated odontoblastic differentiation
and could participate in pulp tissue repair, inducing
pulp cells into odontogenic differentiation by binding
and activating the multiple purinergic P2 receptors
(Wang et al. 2016b).
In addition, the Wnt/b-catenin signalling pathway
seems to play an essential role in tertiary dentinogene-
sis (Yoshioka et al. 2013, Han et al. 2014) by promot-
ing the proliferation and odontoblastic differentiation
of DPSCs (Wang et al. 2012a, Yoshida et al. 2016)
and stem cells from the apical papilla (SCAP) (Wang
et al. 2012b). This pathway is also known as the
canonical pathway. The signalling of Wnt after bind-
ing to the Frizzled receptor and low-density lipopro-
tein-related receptor protein (LRP) causes the
inactivation of the Axin⁄APC⁄GSK-3b complex and sta-
bilization of cytoplasmic b-catenin. This results in an
accumulation of cytoplasmic b-catenin, which can
translocate to the nucleus, where it forms a complex
with members of T-cell factor/lymphoid enhancer fac-
tor (TCF/LEF) (Fig. 2D). The expression of b-catenin
has been reported in odontoblast-like cells lining the
inner surface of reparative dentine (Yoshioka et al.
2013, Han et al. 2014). Furthermore, b-catenin could
enhance the odontoblastic differentiation of dental
pulp cells through activation of the transcription fac-
tor Runx2, which might be the mechanism involved
in odontoblastic differentiation (Han et al. 2014).
834 International Endodontic Journal, 51, 829–846, 2018
The role of bioactive molecules in physiological
and pathological dentinogenesis
Advances in the field of oral biology research have
allowed the identification and better understanding of
a diverse group of bioactive signalling molecules
involved in dentine reparative or regenerative events.
This large group of noncollagenous proteins includes
cytokines, growth factors (Table 2), extracellular
matrix molecules (SIBLINGs, osteocalcin, SLRPs;
Table 3), neuropeptides and neurotrophic factors
(Table 4; Smith et al. 2012, 2016, Piva et al. 2014).
Although some bioactive molecules are associated
with dentine through specific binding, as in the case
of TGF-b1 with decorin/biglycan of dentine (Baker
et al. 2009), many molecules are probably seques-
tered within the dentine through a nonspecific associ-
ation with the dentine mineral phase (Smith et al.
2016).
The effects of bioactive molecules on tertiary den-
tine formation have long been recognized with differ-
ent molecules of natural or synthetic origin applied in
both direct pulp capping and indirect pulp capping
and in pulpotomies performed in animal studies (Da
Rosa et al. 2016). Molecules from the TGF-b super-
family, including TGF-b1, BMPs, FGF-2 and DMP-1,
were able to induce tertiary dentinogenesis with a
lower initial inflammatory response (Da Rosa et al.
2016). Although many studies have focused on the
effects of individual molecules, their effects are syner-
gistic and may differ significantly from those when
they are present individually (Piva et al. 2014). The
correct integration of these molecules is likely to be a
key factor in these events. A pool of soluble dentine
matrix proteins extracted from animal teeth was able
to mimic the effects of recombinant proteins when
applied in direct (Smith et al. 1990) or indirect pulp
capping procedures (Smith et al. 2001, Duque et al.
2006), which was probably better able to represent
the complex cocktail of molecules involved in tertiary
dentinogenesis. Further studies are necessary to iden-
tify which bioactive dentine components are crucial
for signalling odontoblasts and odontoblast-like cell
differentiation.
TGF-b1 remains sequestrated within the dentine
matrix and plays an important role in odontogenesis,
especially in odontoblast differentiation. This bioactive
molecule has been extensively studied and may be
released during carious disease or by biomaterials that
solubilize dentine mineralized components, such as
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
 da Rosa et al. Proteins for vital pulp therapy

Table 2 Main functions of cytokines and growth factors in dentine–pulp complex repair and regenerative events

Molecules (Symbol) Main functions in dentine–pulp complex events

Cytokines
Interleukins (IL) Pro-and anti-inflammatory molecules within the dentine matrix, including IL-1a, IL-1b, IL-4
(Hahn et al. 2000), IL-6 (Barkhordar et al. 1999), IL-8, IL-10 (Hahn et al. 2000) and IL-12
(Cooper et al. 2010). IL-1a and -1b act on the pulp response to infections (Cooper et al.
2010), whereas IL-8 is basally expressed by odontoblasts (Levin et al. 1999). During
pathologic events, previous studies have shown increased levels of IL-4, IL-6 and IL-10
(Barkhordar et al. 1999, Hahn et al. 2000)
Tumour necrosis factor-a (TNF-a) Lipopolysaccharide (LPS) leads to the upregulation of vascular endothelial growth factor
(VEGF) and silent information regulator protein 1 (SIRT1) via the PI3K/AKT/mTOR, p38
MAPK, MAPK/ERK, JNK and NF-jb pathways (Shin et al. 2015)
Growth factors
Adrenomedullin (ADM) Involved in p38 activation via stimulation of odontoblastic differentiation (Simon et al. 2010)
and enhanced proliferation of DPSCs through activation of the JNK pathway
(Zhu et al. 2016)
Bone morphogenetic Induced tertiary dentinogenesis in vivo (Nakashima 1994a,b), odontoblastic differentiation
protein-2 (BMP-2) and the induction of dentine sialophosphoprotein (DSPP) (Iohara et al. 2004,
Chen et al. 2008)
Bone morphogenetic protein-4 Induced tertiary dentinogenesis in vivo (Nakashima 1994a,b) and enhanced odontoblastic
(BMP-4) differentiation (About et al. 2000)
Bone morphogenetic protein-7 Induced tertiary dentinogenesis in vivo (Rutherford et al. 1993, Jepsen et al. 1997, Andelin
(BMP-7/OP-1) et al. 2003, Bezerra Da Silva et al. 2008), and promoted DPSC differentiation
(Suzuki et al. 2011)
Epidermal growth factor (EGF) Involved in enhancing neuronal differentiation of DPSCs (Arthur et al. 2008) and stem cells
of the apical papilla (SCAP) (Vanacker et al. 2014)
Fibroblast growth factor-2 Induced tertiary dentinogenesis in vivo (Kikuchi et al. 2007, Ishimatsu et al. 2009); affects
(FGF-2) osteogenic differentiation of human DPSCs (Qian et al. 2015) and also upregulates
chemokines in vitro (Kim et al. 2010b)
Insulin-like growth factor 1 and IGF-1 induced tertiary dentinogenesis in vivo (Lovschall et al. 2001) and promoted
2 (IGF-1 and -2) proliferation and differentiation of DPSCs and SCAP into mineralizing phenotypes
(Wang et al. 2012b, Feng et al. 2014)
Placental growth factor (PGF) Involved in angiogenesis (Kinnaird et al. 2004) and osteogenic differentiation of
mesenchymal stem cells (MSC) (McCoy et al. 2013)
Platelet-derived growth factor Involved in angiogenesis (Morelli et al. 2011) and chemotaxis of MSCs (Fiedler et al. 2002)
(PDGF) and in the process of odontoblastic differentiation (Yokose et al. 2004) with other growth
factors (Kim et al. 2010a)
Transforming growth factor-b1 Induced tertiary dentinogenesis in vivo (Begue-Kirn et al. 1992, Kalyva et al. 2010,
(TGF-b1) Li et al. 2014), activated Smad2, Smad3 and Smad4 (Unterbrink et al. 2002, He et al. 2004)
and induced upregulation of dentine matrix by odontoblasts (Dobie et al. 2002)
Transforming growth factor-b2 Gene profiles identified during the process of DPSC mineralization (Liu et al. 2007)
(TGF-b2)
Transforming growth factor-b3 Involved in the process of odontoblastic differentiation (Sloan & Smith 1999,
(TGF-b3) Huojia et al. 2005)
Vascular endothelial growth Induced endothelial cell survival and differentiation of new blood vessels (Ferrara & Henzel
factor (VEGF) 1989, Ferrara 2000). Also induced the vasculogenic differentiation of DPSC and stem cells
from human exfoliated deciduous teeth (SHED) via activation of the Wnt/b-catenin
signalling pathway (Zhang et al. 2016) and induced ERK and AKT phosphorylation
(Sakai et al. 2010)

EDTA (Sadaghiani et al. 2016), calcium hydroxide
(Graham et al. 2006) and MTA (Tomson et al. 2007).
TGF-b1 can regulate bioactive molecules probably
through p38 phosphorylation (Cooper et al. 2010).
Previous studies have shown that other molecules,
such as TNF-a and ADM, have similar patterns of
p38 MAPK activation (Simon et al. 2010, Zhu et al.
2016).
In addition, bone morphogenetic proteins (BMP)
are a family of signalling molecules involved in the
formation of many tissues, such as bones and teeth
(Smith et al. 2012). Whilst dentine contains BMP

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 Proteins for vital pulp therapy da Rosa et al.

Table 3 Main functions of extracellular matrix molecules in dentine–pulp complex repair and regenerative events.

Molecules (Symbol) Main functions in dentine–pulp complex events

Extracellular matrix molecules (SIBLINGs, osteocalcin, SLRPs)

Bone sialoprotein (BSP)
Dentine matrix protein-1 (DMP-1) Expressed by odontoblasts (Toyosawa et al. 2004) plays an important role in dentine
Dentine sialophosphoprotein
(DSPP)
Dentine sialoprotein (DSP)
Dentine phosphoprotein (DPP)
Matrix extracellular
phosphoglycoprotein (MEPE)
Osteocalcin (OC)
Osteopontin (OPN)
Small leucine-rich
proteoglycans (SLRPs)
Induced tertiary dentinogenesis in vivo (Decup et al. 2000, Six et al. 2002) and is also
present in tertiary dentine (Moses et al. 2006). It is involved in the initial formation of
hydroxyapatite crystals, acting as an inhibitor of the growth of the crystals thereafter
(Qin et al. 2004, Boskey et al. 2008)
mineralization and maturation of predentin to dentine during dentinogenesis and is involved
in the inflammatory process, activating the synthesis of interleukin-6 and interleukin-8
(IL-6 and IL-8) from pulp fibroblasts (Abd-Elmeguid et al. 2012)
Expressed by odontoblasts is the most abundant noncollagenous protein in the dentine
(Yamamoto et al. 2015) and plays an essential role in the biomineralization process of
predentin (Sreenath et al. 2003). DSPP is processed by proteases into three major domains:
dentine sialoprotein (DSP), dentine glycoprotein (DGP) and dentine phosphoprotein (DPP)
(MacDougall et al. 1997, Yamamoto et al. 2015). Leptin stimulated DSPP protein expression
in human dental pulp (Mart
ın-Gonza
lez et al. 2015)
DSP promotes growth, migration and odontoblastic differentiation of dental pulp cells
in vitro (Lee et al. 2012) and is also able to promote the migration and pro-inflammatory
activation of immune system cells (Tani-Ishii et al. 1995, Silva et al. 2004, 2005)
Involved in the process of dentine mineralization, binding to collagen and initiating formation
of hydroxyapatite crystals in dentine (Butler & Ritchie 1995)
Expressed in the odontoblasts during tooth formation (MacDougall et al. 2002) with an
inhibitory role in mineralization (Gowen et al. 2003). Member of the bone matrix protein
family (Smith et al. 2012)
Most abundant in the bone extracellular matrix and is also detected in the odontoblast
cell body (Papagerakis et al. 2002)
Expressed in the dentine and has an inhibitory effect on hydroxyapatite crystal formation
and growth (Boskey et al. 1993, 2002). Recent studies showed that rosiglitazone (RSG)
upregulated osteopontin expression (De Lima et al. 2015)
Present in the predentine, such as decorin and biglycan, with a role in mineralization events
via their ability to bind to calcium (Embery et al. 2001, Goldberg et al. 2003). Another
SLRP is the PLAP-1, an extracellular matrix protein mainly expressed in the periodontal
ligament, which regulates BMP-2, FGF-2 and TGF-b activity (Yamada et al. 2007,
Tomoeda et al. 2008, Awata et al. 2015)

activity, pulp fibroblasts express mRNA transcripts for
the BMP receptors BMPR-IA, BMPR-IB, ActR-1 and
BMPR-II (Gu et al. 1996). In vivo studies have also
shown that BMP-2, BMP-4 and BMP-7 induced ter-
tiary dentine formation after their application in
direct pulp capping (Da Rosa et al. 2016).
It has also been demonstrated that PLAP-1, an
extracellular matrix protein that is mainly expressed
in the periodontal ligament, regulates BMP-2 and
FGF-2 activity through direct interaction, and TGF-b
through indirect interaction (Awata et al. 2015). One
of the main functions of these proteins is to bind
bioactive molecules and regulate their activation, dis-
tribution and presentation to cells. The functions of
the bioactive molecule FGF-2, for instance, are regu-
lated by PLAP-1 through the formation of the FGF-2–
FGFR1 complex via direct binding (Awata et al.
2015). The effect of FGF-2 on odontoblast secretion is
probably due to the binding of this molecule to FGF
receptor (FGFR), which leads to the receptor phospho-
rylation of intrinsic tyrosine residues, activating many
signalling transduction pathways including MAPK
and PI3K/AKT/mTOR (Liang et al. 2012). Other
growth factors that were also able to induce tertiary
dentinogenesis in vivo were EGF, IGF-1 and IGF-2,
and PDGF (Da Rosa et al. 2016).
Regarding extracellular matrix molecules, including
the SIBLINGs, osteocalcin and SLRPs, recent findings
have shown that rosiglitazone (RSG), a synthetic full
agonist of the transcription factor peroxisome prolifer-
ator-activated receptor gamma, has an anti-inflamma-
tory effect on lipopolysaccharide-induced pulp
inflammation; decreases human dental pulp cell pro-
liferation; and upregulates osteopontin (OPN) expres-
sion (De Lima et al. 2015). Another important
extracellular matrix molecule for reparative/

836 International Endodontic Journal, 51, 829–846, 2018 © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
 da Rosa et al. Proteins for vital pulp therapy

Table 4 Main functions of neuropeptides, neurotrophic factors and other molecules in dentine–pulp complex repair and regen-
erative events

Molecules (Symbol) Main functions in dentine–pulp complex events

Neuropeptides
Calcitonin (CT)
Calcitonin gene-related
peptide (CGRP)
Neuropeptide Y (NPY)
Substance P (SP)
Vasoactive intestinal
polypeptide (VIP)
Neurotrophic factors
Brain-derived neurotrophic
factor (BDNF)
Glial cell line-derived growth
factor (GDNF)
Growth/differentiation
factor 15 (GDF-15)
Nerve growth factor (NGF)
Other molecules
Semaphorin 3A (Sema3A)
Copine 7 (CPN7)
Involved in inflammatory responses and decrease in the degree of inflammation in vivo
(Cullum & Kline 1985).
Pro-inflammatory vasodilator that is released after activation of transient receptor potential
cation channel subfamily V member 1 (TRPV1) in neurons (Brain et al. 1985, Burns et al. 2016).
Calcitonin receptor-like receptor (CRLR) is expressed in pulp during a clinical inflammatory
process (Caviedes-Bucheli et al. 2005, 2008).
Expressed in human dental pulp from sympathetic nerves originating in the superior cervical
ganglion (Luthman et al. 1992, Uddman et al. 1998, Rodd & Boissonade 2002), in the
subodontoblastic plexus and also as free nerves in the odontoblastic layer projecting towards
the dentine, particularly in the pulp horn areas (El Karim et al. 2006).
Expressed in nerve fibres within the tooth pulp and dentine, mainly in the subodontoblastic layer
where they branch towards predentine, with some SP fibres penetrating into dentine
(Wakisaka et al. 1984, Caviedes-Bucheli et al. 2008). The SP receptor expression in pulp
increases during an inflammatory processes (Caviedes-Bucheli et al. 2007).
Expressed in dental pulp neurons; the VIP fibres are present at the subodontoblastic layer
(Caviedes-Bucheli et al. 2008). Its expression remains unchanged during inflammatory
processes (Toyosawa et al. 2004).
Promotes neuronal growth and axonal targeting and may bind to glycosaminoglycans, which
is a possible mechanism for interaction and storage in the extracellular dentine matrix
(De Almeida et al. 2014).
Expressed in mesenchymal pulp cells and (pre)odontoblasts (Nosrat et al. 1998, 2002).
Promoted nerve regeneration in vivo (Fiore et al. 2009) and pulp cell survival/proliferation
(Woodnutt et al. 2000) and is involved in odontoblast differentiation (De Vicente et al. 2002).
Involved in the promotion of axonal regeneration and function after injury with an important
role in neuronal maintenance (Wang et al. 2015).
Induced odontoblast differentiation and is implicated in dentinogenesis (Mitsiadis & Luukko
1995, Amano et al. 1999, Arany et al. 2009).
Induced tertiary dentinogenesis in vivo, and also migration, chemotaxis, proliferation and
odontoblastic differentiation of DPSCs in vitro; expressed in neural crest cells
(Yoshida et al. 2016).
Induced tertiary dentinogenesis in vivo and odontoblast differentiation in vitro
(Choung et al. 2016).

regenerative events in the dentine–pulp complex is
DMP-1. DMP-1 is a noncollagenous calcium-binding
protein that plays an essential role in dentine miner-
alization (Padovano et al. 2015) and is involved in
the inflammatory process by activating the synthesis
of IL-6 and IL-8 from pulp fibroblasts (Abd-Elmeguid
et al. 2012). A study that evaluated synthetic peptides
derived from DMP-1 showed that they can bind to
type I collagen and promote nucleation of hydroxyap-
atite when exposed to calcium and phosphate ions
in vitro (Padovano et al. 2015).
Additionally, the most abundant noncollagenous
protein in the dentine matrix is DSPP, which is
expressed by odontoblasts and cleaved into DSP, DGP
and DPP (Piva et al. 2014). DSPP gene expression is
downregulated by lipoteichoic acid (Durand et al.
2006). Furthermore, DSPP has been used as an indi-
cator of odontoblastic differentiation as it is expressed
by odontoblast-like cells underlying the reparative
dentine (Mart
ın-Gonz
alez et al. 2015). TNF-a has
been reported to increase the expression of DSPP in
dental pulp cells in vitro (Paula-Silva et al. 2009).
Other molecules linked to dentine–pulp reparative/
regenerative events are neuropeptides and neu-
rotrophic factors, which are expressed in odontoblasts
that originate from cranial neural crest cells. Neu-
ropeptides are peptide neurotransmitters or neuro-
modulators, such as CT, CGRP, NY, SP and VIP
(Smith et al. 2012). They are synthesized and released
from neurons that are part of the neuronal network

© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 51, 829–846, 2018 837
 Proteins for vital pulp therapy da Rosa et al.
(plexus of Raschkow) within the dentine–pulp com-
plex and trigger biological effects by activating recep-
tors located on the plasma membranes of target cells.
As the immune system cells present neuropeptide
receptors on their cell surfaces, neuropeptides stimu-
late the target cell activating receptors after release
(Caviedes-Bucheli et al. 2008). These molecules help
to control the neuro-inflammatory response to the
pulp tissue defence during disease, and afferent nerves
respond to bacterial antigens by neuropeptide release
(Smith et al. 2012).
Furthermore, neurotrophic factors have also been
involved in the development and control of nerve cells
and non-neuronal tissues. Some of these molecules
are expressed by dental pulp cells and odontoblasts,
including NGF and BDNF (Smith et al. 2012). Knowl-
edge about the mechanism of this interaction and
storage of these proteins in the dentine matrix has
been limited. Studies have suggested that BDNF can
bind to glycosaminoglycans (Kanato et al. 2009) and
that GDNF can bind the extracellular proteoglycan
heparin sulphate (Rider 2006). Furthermore, it has
been demonstrated that in carious disease, pulp
fibroblasts express C5aR, which plays a critical role in
the inflammatory process, is a major component of
innate immunity and has also been implicated in the
control of BDNF secretion (Chmilewsky et al. 2016).
Recently, other bioactive molecules have been
demonstrated to act in dentine–pulp biological events,
such as Semaphorin 3A (Sema3A) (Yoshida et al.
2016) and Copine 7 (CPNE7) (Choung et al. 2016,
Table 4). Sema3A and its receptor neuropilin 1
(Nrp1) are expressed in neural crest cells and play an
important role in embryogenesis (development of
blood vessels, peripheral nerves, skeletal tissues) and
in dentine regeneration via the canonical Wnt/b-cate-
nin signalling pathway (Schwarz et al. 2009). In
addition, a recent in vivo study reported that these
proteins can stimulate tertiary dentine formation in
direct pulp capping (Yoshida et al. 2016). Moreover,
CPNE7 was another protein that could induce odon-
toblast differentiation in vitro and stimulate tertiary
dentinogenesis in vivo (Choung et al. 2016). CPNE7, a
soluble dental epithelium-derived factor, is a member
of the calcium-dependent phospholipid-binding pro-
tein family. Furthermore, this molecule was able to
promote the expression of odontoblastic markers, such
as DSPP mRNA and DSP protein, in vitro (Oh et al.
2015). Further research is needed to evaluate the effi-
cacy of using these different bioactive molecules alone
and mainly together to better understand this
838 International Endodontic Journal, 51, 829–846, 2018
complex mechanism for optimizing new treatments
for VPT.
Current challenges and future directions
New strategies for VPT are currently being evaluated
via two main routes. The first of these involves the
exploitation of bioactive molecules naturally seques-
tered within the dentine by biomaterials that can
potentiate their release. Findings have demonstrated
the solubilization of these molecules by pulp capping
agents such as calcium hydroxide cements, MTA
(Graham et al. 2006, Tomson et al. 2007, 2016).
Other potential ways to release bioactive molecules
from the dentine matrix in a clinical situation may be
using EDTA, phosphoric acid and citric acid (Sadaghi-
ani et al. 2016). MTA can also induce odontoblastic
differentiation of human DPSCs via the MAPK (Rathi-
nam et al. 2015), BMP/Smad (Jung et al. 2015),
Wnt/b-catenin (Chen et al. 2016) and NF-jb sig-
nalling pathways (Rathinam et al. 2015) and was
able to activate the p38 MAPK pathway (Huang et al.
2015, Rathinam et al. 2015).
MTA-derived materials have been suggested in
recent years as pulp capping agents, such as calcium
silicate, calcium phosphate and calcium aluminate-
based cements. They have improved properties com-
pared to MTA, such as good sealing correlated to
expansion, being able to set in the presence of fluids,
releasing ions such as calcium and having good bio-
logical properties (Da Rosa et al. 2017). Calcium sili-
cate-based cements (e.g. iRoot BP Plus; Innovative
Bioceramix, Vancouver, Canada, and Biodentine; Sep-
todont, Saint Maur des Foss
es, France) have been
reported to induce odontoblastic differentiation by
activating the MAPK/ERK (Jung et al. 2015), NF-jb,
p38 MAPK and JNK pathways (Rathinam et al.
2015). These agents also promote dental pulp cell
migration and pulp repair involving the FGFR-
mediated ERK 1/2, JNK and PI3K/AKT/mTOR path-
ways (Zhang et al. 2015). Similar outcomes regarding
tertiary dentine formation and inflammatory response
between calcium silicate-based cement (Biodentine)
and MTA (ProRoot MTA; Dentsply Sirona, Ballaigues,
Switzerland) have been demonstrated (Nowicka et al.
2013, De Rossi et al. 2014, Cuadros-Fernandez et al.
2015). Moreover, when used for indirect pulp cap-
ping, the calcium silicate-based cement had similar
clinical results to those of a glass–ionomer cement
(Fuji IX, GC Corporation, USA) after 12 months of fol-
low-up (Hashem et al. 2015). Novel treatment options
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd

for VPT can potentiate the biological effect of the
material through a targeted interaction with dental
tissues to release the bioactive binding molecules for
tertiary dentine formation. Regardless of the material
used, Mente et al. (2014) concluded that a delay of
≥2 days in the placement of a permanent restoration
after direct pulp capping resulted in an increased risk
of failure. Thus, an efficient and immediate sealing of
the cavity using restorative materials is imperative to
increase the success rate of VPT (Mente et al. 2014).
The second approach involves the use of bioactive
molecules incorporated into new materials. However,
the introduction of bioactive materials into routine
clinical use faces significant challenges. The preserva-
tion of protein activity, storage and adequate tem-
porospatial release without being detrimental to the
pulp presents significant challenges that need to be
overcome (Piva et al. 2014, Smith et al. 2016). The
application of a wide variety of bioactive molecules in
VPT has previously been proposed, with the use of
different molecules of natural or synthetic origin
being applied in both direct pulp capping (Andelin
et al. 2003, Ishimatsu et al. 2009) and indirect pulp
capping (Duque et al. 2006, Kalyva et al. 2010) and
in pulpotomies performed in animal studies (Bezerra
Da Silva et al. 2008, Ko et al. 2010). Additionally,
reports have indicated that systemic alterations, such
as diabetes, could compromise the healing capacity of
dental tissues (M€ uller et al. 2015). Moreover, the
complex in vivo scenario may differ from that demon-
strated by controlled in vitro and in vivo studies, and
may involve other biomolecules that could reduce or
prevent the bioavailability or activity of dentinogene-
sis biomolecules. In a clinical scenario, bioactive
materials for pulp capping should balance the activity
of molecules that enhance dentinogenesis and target
the antidentinogenesis molecules. There is still a very
limited understanding of the interplay amongst many
bioactive molecules in tertiary dentinogenesis, and
more in-depth knowledge of this aspect is crucial to
future optimization of new biological approaches for
VPT.
Strategies involving delivery systems have been
proposed to control the release kinetics of bioactive
molecules and potentiate odontoblast activity, as in
the case of biomaterials with multiple layers and in
the encapsulation of bioactive molecules inside poly-
lactic-co-glycolic acid (PLGA) microspheres (Da Rosa
et al. 2016). Some carriers that have previously been
proposed were collagen (Hu et al. 1998, Kikuchi et al.
2007, Bezerra Da Silva et al. 2008), gelatin (Goldberg
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
da Rosa et al. Proteins for vital pulp therapy
et al. 2001; Ishimatsu et al. 2009), agarose (Chaus-
sain et al. 2009), sodium alginate (Oliva-Rodriguez
et al. 2011), chitosan (Li et al. 2014). Current in vitro
evidence suggests other potential drug delivery sys-
tems for bioactive molecules, such as biodegradable
polymer matrix of lactide and glycolide (Mathieu et al.
2013), porous silk fibroin scaffolds (Yang et al. 2015),
poly-2-hydroxyethyl methacrylate (polyHEMA)-based
hydrogel (Takeda et al. 2015), tricalcium phosphate
microsphere–hydrogel composite (Lee et al. 2014). In
addition, biomaterials that can be used as scaffolds for
VPT have recently been suggested and could enhance
proliferation and differentiation of human dental pulp
cells, such as polycaprolactone/submicron bioactive
glass hybrid composites (Wang et al. 2016a), a chi-
tosan-based scaffold (Bellamy et al. 2016) and keratin
hydrogel (Sharma et al. 2016).
Delivery systems with multiple layers (Li et al.
2014) and encapsulation of bioactive molecules inside
polylactic-co-glycolic acid (PLGA) microspheres
(Zhang et al. 2008) have previously been suggested.
Aubeux et al. (2016) reported that silated-hydroxy-
propyl methylcellulose used as a hydrogel scaffold
was also able to extract dentine matrix proteins from
dentine powder. Additionally, other molecules have
been suggested as potential stimulators for odonto-
genic differentiation from human dental pulp cells,
such as a natural collagen cross-linking agent geni-
pin, that could also improve physical properties of col-
lagen scaffolds (Kwon et al. 2015), an actin-severing
and actin-capping protein called adseverin (Li et al.
2015), and Nell-like molecule-1 (Nell-1) (Liu et al.
2016). Strategies to clinically translate our biological
understanding of pulp regeneration into improved
patient management approaches could overcome the
shortcomings of biomaterials available on the market
at present (Simon & Smith 2014).
Conclusions
The reparative and regenerative events of the den-
tine–pulp complex involve cell migration, proliferation
and differentiation of dental pulp cells. Knowledge
about these multistep processes of bioactive molecules
in the signalling of transduction pathways involved in
dentinogenesis, odontoblast differentiation and inflam-
matory response has allowed considerable advances
in this field of research in recent years. Several
in vitro and in vivo studies have demonstrated that
bioactive molecules act upon numerous cell types,
specifically odontoblasts and odontoblast-like cells, but
International Endodontic Journal, 51, 829–846, 2018 839
 Proteins for vital pulp therapy da Rosa et al.
their adequate release in an active form is still not
fully understood. Additionally, an adequate and early
sealing of the cavity is imperative for the success of
VPT regardless of the approach used. Moreover, the
high cost of biomaterials containing these molecules
may hinder their clinical application. Finally, even
with substantial challenges to understand signals yet
unknown as well as control them by means of inno-
vative biomaterials, the new treatments for VPT based
on these bioactive molecules offer exciting opportuni-
ties to promote the most appropriate tissue response
for tertiary dentinogenesis.
Acknowledgements
The present research was financially supported by the
National Council of Technological and Scientific
Development (CNPq/Universal # 456972/2014-5).
The authors acknowledge that some references
directly associated with this review have been omitted
due to limitations imposed by journal guidelines. The
authors declare no potential conflict of interests with
respect to the authorship and/or publication of this
article.
Conflict of interest
The authors have stated explicitly that there are no
conflict of interests in connection with this article.
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