doi:10.1111/iej.

12627

REVIEW
Angiogenic mechanisms of human dental pulp and
their relationship with substance P expression in
response to occlusal trauma

~ o-Gomez1,
J. Caviedes-Bucheli1, J. F. Gomez-Sosa2, M. M. Azuero-Holguin1, M. Ormen
1 3
V. Pinto-Pascual & H. R. Munoz
1
Centro de Investigaciones Odontologicas, Pontificia Universidad Javeriana, Bogota, Colombia; 2Department of Endodontics,
Universidad Central de Venezuela, Caracas, Venezuela; and 3Postgraduate Department of Endodontics, Universidad de San
Carlos de Guatemala, Guatemala

Abstract trauma. Articles included in this review were

Caviedes-Bucheli J, Gomez-Sosa JF, Azuero-Holguin
MM, Ormen ~ o-Gomez M, Pinto-Pascual V, Munoz HR.
Angiogenic mechanisms of human dental pulp and their
relationship with substance P expression in response to occlusal
trauma. International Endodontic Journal, 50, 339–351, 2017.
Angiogenesis is the formation of new blood vessels
based on a pre-existing vasculature. It comprises two
processes, sprouting of endothelial cells and the
division of vessels due to abnormal growth of the
microvasculature. It has been demonstrated that
substance P (SP) can induce angiogenesis either by
modulating endothelial cell growth (direct mecha-
nism) or by attracting cells with angiogenic potential
to the injury site (indirect mechanism). Therefore, the
purpose of this article is to review the angiogenic
mechanisms that regulate mineralized tissue forma-
tion in human dental pulp tissue and their relation-
ship with SP expression as a defence response to
stimuli such as the masticatory function and occlusal
searched in PubMed, Scopus and ISI Web of Science
databases, combining the following keywords: human
dentine pulp, angiogenesis, angiogenic growth factors,
neuropeptides, substance P, neurogenic inflammation,
dentine matrix, dentinogenesis, occlusal trauma and
dental occlusion. It is concluded that human dental
pulp tissue responds to occlusal trauma and mastica-
tory function with a neurogenic inflammatory phe-
nomenon in which SP plays an important role in the
direct and indirect mechanisms of angiogenesis by the
action evoked via NK1 receptors at different cells,
such as fibroblasts, endothelial and inflammatory
cells, leading to new blood vessel formation which are
needed to stimulate mineralized tissue formation as a
defence mechanism.
Keywords: angiogenesis, angiogenic growth fac-
tors, human dental pulp, masticatory function, neuro-
genic inflammation, occlusal trauma, substance P.
Received 13 October 2015; accepted 3 March 2016

extracellular matrix, blood vessels and nerve endings.

Introduction
The human dental pulp is a loose connective tissue
containing different types of cells, collagen fibres,
Correspondence: Javier Caviedes Bucheli, Centro de Investi-
gaciones Odontologicas, School of Dentistry, Pontificia
Universidad Javeriana, Carrera 7 No. 40-62, Bogota, Colom-
bia (Tel.: +5716105694; e-mail: javiercaviedes@gmail.com)
During mastication, it undergoes mechanical stress,
and whenever the masticatory function exceeds the
tolerance and adaptive capacity of both the pulp and
the periodontal ligament, occlusal trauma takes place,
stimulating the pulp nerve fibres to release neuropep-
tides, which are substances that take part in the
process of transmission and modulation of pain and
inflammatory process as a defence mechanism
(Mattuella et al. 2007a, Caviedes-Bucheli et al. 2008,

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 339–351, 2017 339
 Angiogenesis and substance P Caviedes-Bucheli et al.
2011b). This response is necessary for pulp defence
and repair to take place in cases such as pulpitis,
degenerative pulp calcification and necrosis.
When the pulp is submitted to mechanical stress, it
undergoes an ageing process where perfusion
decreases, affecting the tissue nutrition and oxygena-
tion. Simultaneously, in an attempt to adapt to its
new condition, the pulp creates new blood vessels.
This process is known as angiogenesis and it can
occur by direct and indirect mechanisms. This phe-
nomenon is also regulated by numerous growth fac-
tors as well as by endogenous substances such as
substance P (SP), which is necessary to fulfil the
needs for nutrients and oxygen of the areas that
require them (Ikeda et al. 1998, Caviedes-Bucheli
et al. 2008, Killough et al. 2009, Saghiri et al. 2015).
Although it has been demonstrated that SP is
released when thermal, chemical or mechanical stim-
uli such as occlusal trauma, masticatory function and
when orthodontics movements are applied to the
tooth (Caviedes-Bucheli et al. 2005, 2010, 2011a,b,
Javed et al. 2015), little is known about its relation-
ship with angiogenesis as a response of pulp tissue to
occlusal trauma.
Occlusal trauma has been associated with pulp cal-
cification phenomena. The mechanism by which the
pulp produces tertiary dentine is preceded by the for-
mation of new blood vessels. As perfusion of the pulp
decreases in the presence of harmful stimuli, the tis-
sue should be resupplied with essential fluids in order
that the repair processes take place optimally. Vascu-
lar neoformation will carry out the function of provid-
ing oxygen and nutrients to the damaged tissue to
make the dental pulp viable in spite of the dentine
apposition that characterizes the calcification phe-
nomena (Ikeda et al. 1998, Tran-Hung et al. 2008,
Marchionni et al. 2009, Zhang et al. 2011).
The purpose of this review was to analyse the role
of SP in the angiogenic mechanisms that regulate
mineralized tissue formation in the human dental
pulp as a defence response to stimuli such as the mas-
ticatory function and occlusal trauma.
Materials and methods
The methodology followed in this article includes the
search and review of articles published about the role
of SP in the induction of the angiogenic mechanisms
of the human pulp tissue response to occlusal trauma
and the masticatory function. The articles were
searched in PubMed, Scopus and ISI Web of Science
340 International Endodontic Journal, 50, 339–351, 2017
databases using keywords listed in MeSH Terms,
Index key Words and Key Word Plus of each data-
bases.
Search strategy
In the three databases used, the keywords chosen
were human dental pulp in combination with angio-
genesis, angiogenic growth factors, neuropeptides,
substance P, neurogenic inflammation, dentine
matrix, dentinogenesis, occlusal trauma and dental
occlusion. Two reviewers carried out the evaluation
of the eligibility and search for relevant data indepen-
dently; a third reviewer was selected for the resolution
of disagreement during the evaluation process.
Inclusion and exclusion criteria
The inclusion criteria considered were:
• Review articles and research studies about the
human dental pulp, either in vivo or in vitro;
• Articles that meet the quality standards required
by AMSTAR (Shea et al. 2007) and PRISMA
(Urrutia & Bonfill 2010) guides, published in Eng-
lish journals with impact factors Q1 and Q2 from
January 1990 to August 2015 and
• Articles that evaluate the angiogenic mechanisms
of the human dental pulp and the role of sub-
stance P prior to the formation of mineralized tis-
sue as a response to the masticatory function and
occlusal trauma.
Articles about animal models or cell lines studies
on other tissues that do not refer to human dental
pulp as well as studies where medicaments are used
as endodontic therapy to produce angiogenesis were
excluded.
Literature review
Dental pulp microcirculation and innervation
The dental pulp is a loose connective tissue of mes-
enchymal origin that lies inside the rigid walls of the
dentine, which together form the so-called pulp–den-
tine complex. This complex is considered a functional
and structural unit that acts as a sensorial system
which can respond to different stimuli (Mattuella
et al. 2007a). The dental pulp has several functions,
including the formation of hard tissues such as den-
tine, the production of tertiary dentine to compensate
the loss of hard tissues caused by mechanical and
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd

chemical trauma and providing cell nutrition and tis-
sue vitality (Arana-Chavez & Massa 2004, Cooper
et al. 2010).
In the presence of occlusal trauma several cells
induce defensive and repairing processes within the
pulp–dentine complex. Amongst these cells, the odon-
toblasts are responsible for producing the reactive ter-
tiary dentine depending on the intensity and duration
of the stimulus; secondly, the dental pulp stem cells
(DPSCs) take part in the process of tertiary dentine
production; and finally, the human dental pulp fibrob-
lasts (HDPF) that can be found in a greater propor-
tion are responsible for the collagen matrix that
becomes mineralized just before reparative tertiary
dentine formation (Arana-Chavez & Massa 2004, Kil-
lough et al. 2009, Rosa et al. 2011, Saghiri et al.
2015).
The cells that take part in the pulp-repairing pro-
cess respond to signals from molecules in the blood-
stream and the dentine matrix, which reach the pulp
due to a microcirculatory system characterized by the
presence of arterioles, capillaries and venules whose
main functions are the regulation of dental pulp local
interstitial medium and the elimination of waste prod-
ucts (Li et al. 2011, Zhang et al. 2011). A singular
characteristic of pulp microvasculature is the presence
of arteriovenous anastomoses (AVA), which partici-
pate in the regional control of the blood flow due to
its dilation or contraction in the presence of thermal,
chemical or mechanical irritation (Matthews &
Andrew 1995, Heyeraas & Berggreen 1999, Rodd &
Boissonade 2002), such as occlusal trauma, where an
alteration in the vasculature takes place, thereby
increasing vascular permeability and vascular stasis
and decreasing the resistance of the vessels and caus-
ing oedema. All these changes are caused not only by
the local control mechanisms of the blood vessels
(presence of pre- and post-capillary sphincters) but
also by the main control performed by the nervous
system in the regulation of pulp blood flow due to the
release of neurotransmitters by the sympathetic,
parasympathetic and somatosensory fibres such as SP
(Kim 1990, Olgart 1996, Rodd & Boissonade 2003,
Caviedes-Bucheli et al. 2008, Lee et al. 2013).
The pulp tissue is innervated by fibres from the
sympathetic, the parasympathetic and the somatosen-
sory systems. The sympathetic nervous system
reduces pulp blood flow (vasoconstriction) due to the
release of neurotransmitters as neuropeptide Y (NPY).
The parasympathetic fibres stimulate vasodilation due
to the release vasoactive intestinal peptide (VIP).
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
Caviedes-Bucheli et al. Angiogenesis and substance P
Somatosensory fibres are responsible for the produc-
tion of several neuropeptides that regulate the pulpal
blood flow such as SP, calcitonin gene-related peptide
(CGRP) and neurokinin A (NKA) which inhibit the
sympathetic activity through vasodilation (Wakisaka
1990, Olgart 1996, Rodd & Boissonade 2003,
Caviedes-Bucheli et al. 2008).
Substance P is a neuropeptide found in large quan-
tities in immunoreactive nerve fibres and has been
also found in both the human dental pulp and den-
tine in immunohistochemical studies (Wakisaka
1990). In the pulp centre, reactive SP nerve fibres are
associated with large blood vessels, whereas in the
periphery they are associated with smaller ones. On
the other hand, nerve fibres not related to the blood
vessels, pass into the subodontoblastic layer and
branch out in the pre-dentine, some of them crossing
into the dentine (Rodd & Boissonade 2003). SP is
released into the dentine–pulp complex by neurons as
a response to thermal, chemical or mechanical nox-
ious stimuli, including orthodontic movement, masti-
catory function and occlusal trauma (Caviedes-
Bucheli et al. 2010, 2011a,b).
Moreover, SP is not able to cross cell membranes;
therefore, target cells must express the correspondent
receptor on their plasma membrane, which must
have a binding site on the extracellular surface of the
cell. These receptors bind the natural agonist when
present in the extracellular environment and generate
intracellular signals to regulate other functions within
the cell (Caviedes-Bucheli et al. 2007). Substance P
receptor (NK1) is expressed in most inflammatory
cells such as mast cells and macrophages as well as
in other connective tissue cells (Caviedes-Bucheli et al.
2008). It has been demonstrated that SP receptor
expression in human pulps is significantly greater
during clinical inflammatory phenomena (Table 1)
(Awawdeh et al. 2002, Bowles et al. 2003, Caviedes-
Bucheli et al. 2006, 2007).
Occlusal trauma and neurogenic inflammation
The pulp suffers physiological modifications through-
out its life due to phenomena such as mastication. It
also reacts in the presence of pathological situations
such as caries, cavity preparation, dental materials
and occlusal trauma (Tziafas 1994, von Bohl et al.
2012, Lee et al. 2013), which stimulate the nerve
fibres of the periodontal ligament and the pulp releas-
ing neuropeptides to regulate all the vascular and cel-
lular processes that occur in the premature pulp
International Endodontic Journal, 50, 339–351, 2017 341
 Angiogenesis and substance P Caviedes-Bucheli et al.

Table 1 Summary of vascular and inflammatory effects of substance P

Dental pulp target cell

(expressing NK1 receptor) Vascular effects Inflammatory effects

Substance P • Mast cell • Blood flow regulation • Immune system stimulation

• Macrophage
• Mesenchymal cell
• Endothelial cell
• FibroblastTakahashi
et al. (1992)
• Vasodilation
• Plasma extravasation Kim (1990),
Olgart (1996), Caviedes-Bucheli
et al. (2008)
• Chemotaxis
• Increased macrophages action
• Increased collagen formation
Trantor et al. (1995),
Caviedes-Bucheli et al. (2008)

ageing forming tertiary reparative dentine as a
response to harmful stimuli (Rodd & Boissonade
2003, Caviedes-Bucheli et al. 2011a, Lee et al. 2013).
Occlusal trauma is the condition in which the mas-
ticatory function exceeds the tolerance of the liga-
ment and the dental pulp, causing damages on both
tissues. Studies have shown that the application of
excessive occlusal loads disturbs pulp metabolism gen-
erating changes in the pulp and periodontal fibres, as
well as other inflammatory reactions and vascular
alterations. This response depends on the intensity
and magnitude of the force and the pulp tissue condi-
tion. After this form of stimuli takes place, the defence
mechanisms of the pulp include the production of ter-
tiary dentine and inflammation (Ikeda et al. 1998,
Caviedes-Bucheli et al. 2011a, Lee et al. 2013).
Occlusal trauma can be classified as primary or sec-
ondary. The former takes place in the pulp and in the
periodontal ligament due to the application of a
higher-than-normal force on the dental structures
with a periodontal support system in optimal condi-
tions. This case includes orthodontic movements,
‘high’ or overextended restorations and parafunc-
tional habits such as bruxism. Secondary occlusal
trauma takes place when normal and higher-than-
normal forces are applied on teeth with previous
supporting tissue alterations caused by periodontal
disease or previous trauma (Jin & Cao 1992, Poiate
et al. 2009).
There is evidence that human fibroblasts can pro-
duce SP and that neuropeptides regulate the expres-
sion of angiogenic growth factors in fibroblasts,
suggesting that such cells also play a role in neuro-
genic inflammation (Tran-Hung et al. 2006, El Karim
et al. 2009). There is a close relationship between the
periodontal ligament and the pulp which has been
considered as synergic; therefore, once the periodontal
tissues become inflamed due to mechanical loads, the
inflammation is quickly transferred to the dental pulp.
Likewise, any inflammation that initially affects the
pulp will be transferred to the periodontal tissues.
This type of inflammation is neurogenic in nature
because the nervous system controls both the vascu-
lar and the immune system responses. This process
describes the stimulation of peripheral neurons which
results in neuropeptides release, altering the vascular
processes causing vasodilatation, increasing the per-
meability of the vessels and facilitating the accumula-
tion of leucocytes (Kim 1990, Ikeda et al. 1998,
Caviedes-Bucheli et al. 2008, 2011a).
Substance P release takes place when mechanical
stimuli are applied to the pulp–dentine complex. It
plays an important role in inflammation due to its
interaction with the endothelial cells that cause the
opening of the intercellular pores allowing fluid
release. Additionally, it causes the production of his-
tamine by the granulocytes favouring plasma extrava-
sation. It also activates the immune system and
promotes chemotaxis by recruiting and regulating
other inflammatory cells, an effect that occurs after
the activation of cells through the receptors present
on their surface. These receptors are called neu-
rokinin 1, 2 and 3 (NK1, NK2 and NK3). When the
SP is found in high concentrations, it can stimulate
receptors NK2 and NK3 in some peripheral tissues,
but it binds with more affinity to NK1 (Awawdeh
et al. 2002, Caviedes-Bucheli et al. 2007, Nakashima
et al. 2009).
However, on the other hand, SP plays an important
role in pulp repair after injury. Tissue repair induced
by SP occurs due to the chemotactic properties of the
neuropeptide, which favours the recruitment of sev-
eral cellular lineages such as myeloid, lymphoid, den-
dritic cells, macrophages, epithelial progenitor cells
and DPSCs from the blood. Finally, SP promotes the
differentiation and proliferation of endothelial cells,
which are needed for tissue repairing process to take
place (El Karim et al. 2009, Kohara et al. 2010). Sev-
eral studies indicate that tissue repair involves the
formation of new vessels. During occlusal trauma,

342 International Endodontic Journal, 50, 339–351, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Caviedes-Bucheli et al. Angiogenesis and substance P

there is a decrease in the nutritional and oxygen con-
tribution in the pulp followed by the apposition of ter-
tiary dentine as a defence and repairing mechanism.
It is possible to infer from the literature that such a
process is preceded by a vascular neoformation, a pro-
cess known as angiogenesis (Derringer et al. 1996,
Roberts-Clark & Smith 2000, Tran-Hung et al. 2008,
Kohara et al. 2010, Zhang et al. 2011).
Angiogenesis
Angiogenesis is the formation of new vessels based on
a pre-existing vasculature. It comprises two processes,
the sprout of endothelial cells and the division of
vessels by the abnormal growth of the microvascula-
ture. The organs derived from the ectoderm and
the mesoderm are vascularized by angiogenesis, a
process which occurs physiologically during embryo-
genesis and remains as a normal condition during
inflammation and tissue repair (Derringer et al. 1996,
Tran-Hung et al. 2006, Marchionni et al. 2009,
Saghiri et al. 2015).
The stages of angiogenesis (Fig. 1) involve the
breaking of the vascular membrane which consists of
the fragmentation of the basal layer and the extracel-
lular matrix of the blood vessels due to the proteolytic
action of some enzymes such as metalloproteinases
(MMPs) and plasminogen activators, which are segre-
gated by the endothelial cells and the macrophages
after the stimulation. After this vasculature breaking,
cell migration starts due to chemotactic signals and
multiplication (by mitosis) of the endothelial cells at
the perivascular space. Then, there is enlargement of
the sprout of the future blood vessel that will be
located near the pre-existing blood vessels. Blood cap-
illaries start to sprout and fold themselves to form the
lumen of the vessel and then join each other at their
tips until forming handle-shape ramifications. Blood
flow starts to circulate after the formation of such
ramifications which will also produce new sprouts
until the formation of a new capillary plexus. Finally,
the apposition of the pericytes and the smooth muscle
cells takes place to give support to the newly synthe-

INJURED TISSUE
MMPs and plasminogen activators produced
Proteolytic action
by macrophages and endothelial cells

Fragmentation of the basal membrane and

extracellular matrix of the blood vessels
Promoting

Chemotaxis, proliferation, migration and adhesion of endothelial cells

Collagen type 1 and
angiogenic growth factors

Formation of capillary sprouts branching

Apposition

Pericytes and smooth muscle cells in newly formed blood vessels

scaffold

Extracellular matrix surrounding the new blood vessels

generating

Blood vessel at the injury site:

ANGIOGENESIS
Figure 1 Phases of angiogenesis: the breaking of the vascular membrane through the proteolytic action of enzymes such as
metalloproteinase and plasminogen activators secreted by the endothelial cells and macrophages after stimulation. The macro-
phages, granulocytes and fibroblasts produce growth factors with angiogenic potential, such as VEGF, PDGF and bFGF. Conse-
quently, migration and multiplication of the endothelial cells begin at the perivascular space, and enlargement of the sprout of
the future blood vessel occurs. Finally, the apposition of pericytes and smooth muscle cells takes place which will function as a
support to the recently synthesized vessels.

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 339–351, 2017 343
 Angiogenesis and substance P Caviedes-Bucheli et al.

sized vessels (Tran-Hung et al. 2006, Zhang et al.
2011, Bronckaers et al. 2013, Saghiri et al. 2015).
The extracellular matrix surrounding the vessels
constitutes a scaffold for the new vascular endothe-
lium, as it favours proliferation, migration and adhe-
sion of the endothelial cells in three dimensions. In
addition, as a consequence of the generation of con-
tractile mechanical forces in the extracellular matrix,
the endothelial cells establish some form of orienta-
tion based on tension. This process allows the forma-
tion of networks of interconnected blood vessels just
where the injury is located, where collagen type 1
intervenes on the endothelial cells shape change. The
main angiogenic factor is the vascular endothelial
growth factor (VEGF), which induces angiogenesis
and also favours the expression of integrins on the
microvascular endothelial cells (Derringer et al. 1996,
Artese et al. 2002, Tran-Hung et al. 2008, Krishnan
& Davidovitch 2009, Nakashima et al. 2009).
The lack of collateral perfusion in the pulp makes it
one of the most sensitive tissues in humans. There is
evidence indicating that in the presence of occlusal
trauma, the oxygen levels in the pulp decrease so that
cell damage, vascular disorders and inflammation
may occur. However, hypoxia-inducing factor-1a
(HIF-1a) and VEGF expression in DPSC and HDPF are
then enhanced, and therefore, angiogenesis takes
place. This explains why vascular neoformation plays
a fundamental role in repairing the pulp tissue as
these new vessels will be in control of fulfilling the
metabolic and oxygenation needs of the pulp by the
mediation of the pulp tissue nervous system through
neuropeptides such as SP (Derringer et al. 1996, Der-
ringer & Linden 1998, Aranha et al. 2010, Kim et al.
2014, Zhang et al. 2014).
Therefore, SP can induce angiogenesis either by
direct mechanisms such as the growth modulation of
the endothelial cells or by indirect mechanisms such
as the attraction of the cells with angiogenic potential
to the location of the injury (Fig. 2) (Kohara et al.
2010).
Indirect mechanism
Several studies confirm that SP performs chemotactic
activities to carry granulocytes from the blood flow to
the location of the inflammatory injury (Kohara et al.
2010, Murakami et al. 2013). SP binds to granulo-

ANGIOGENIC MECHANISMS

Indirect Mechanism Direct Mechanism

SP SP

Binds to Binds to
NK1-R Granulocytes NK1-R Macrophages NK1-R Fibroblasts NK1-R Endothelial cells

To release To release

VEGF ANG2 TGF- GM-CSF VEGF FGF-2 VEGF FGF

bFGF EGF bFGF IGF bFGF PIGF EGF integrin
TGF- HB-EGF PDGF VEGF EGF HGF
PDGF Leptin
Endothelial cells
proliferation and
migration

New blood vessel

Figure 2 Angiogenic mechanism induced by SP. Left side: Indirect mechanism. SP binds to NK1 receptors in granulocytes and
macrophages to release growth factors with angiogenic potential, therefore promoting new blood vessel formation. Right side:
Direct mechanism. SP binds to NK1 receptors in fibroblasts and endothelial cells inducing the release of growth factors with
angiogenic potential, promoting proliferation and migration of endothelial cells to form new blood vessels.

344 International Endodontic Journal, 50, 339–351, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Caviedes-Bucheli et al. Angiogenesis and substance P

cytic cells through NK1 and makes them release
proinflammatory cytokines that have angiogenic and
angiostatic functions. Table 2 summarizes the action
of some of these cytokines, including the vascular
endothelial growth factor (VEGF), the basic fibroblas-
tic growth factor (bFGF), the transforming growth fac-
tor-beta (TGF-b), angiogenin, leptin, angiopoietin 2
(ANG-2), the epidermal growth factor (EGF), the hep-
arin-binding epidermal growth factor (HB-EGF) and
the platelet-derived growth factor (PDGF) (Derringer &
Linden 2007, Mattuella et al. 2007a,b, El Karim et al.
2009, Kohara et al. 2010, Virtej et al. 2013).
Vascular endothelial growth factor is a heparin-
binding protein with specific affinity to endothelial
cells and plays a key role in angiogenesis. This glyco-
protein family includes six members, VEGF-A, B, C, D
and E, and PDGF, which have autocrine and para-
crine effects in the dental pulp. Factors VEGF-A and
VEGF-B are closely related to the angiogenic phe-
nomenon as they stimulate endothelial cellular prolif-
eration and increase vascular permeability (Mattuella
et al. 2007a). VEGF also seems to induce the differen-
tiation of human dental pulp cells into endothelial
cells, and their ligands are expressed in DPSCs and
HDPFs in hypoxic conditions. It is also known that
blood vessels and immune cells are equipped with
VEGFR-2 and VEGFR-3 (Mattuella et al. 2007a,b,
Marchionni et al. 2009, Dissanayaka et al. 2012,
Janebodin et al. 2013, Virtej et al. 2013).
Angiogenin and ANG-2 have functions related to
the proliferation of endothelial cells. In the presence
of VEGF, ANG-2 causes proliferation and migration of
endothelial cells and stimulates the formation of new
vessels, whereas angiogenin causes the neovascular
formation from bFGF and VEGF. Factor HB-EGF and
leptin also have angiogenic potential inducing the for-
mation of structures similar to blood vessels (El Karim
et al. 2009).
Moreover, the activation of macrophages by SP
adds to its indirect angiogenic function. Macrophages
are key stimulators of angiogenesis as they have sev-
eral functions during the process of vascular neofor-
mation. One of these functions is the production of
metalloproteinases which degrade the basal mem-
brane and the extracellular matrix of the vessel walls
to allow migration of endothelial cells in the early
stages of angiogenesis. They also promote the release
of a large number of growth factors which can induce
the proliferation and migration of cells containing
that capillary endothelium as well as the release of
more angiogenic factors such as the tumoral growth
factor alpha (TGF-a), the granulocyte–monocyte col-

Table 2 Angiogenic growth factors and proteins released from granulocytes and macrophages attracted by SP-induced chemo-
taxis

Dental pulp Growth factors and

cells attracted proteins with
by chemotaxis angiogenic functions Biological effect

Substance P Granulocytes Vascular endothelial Stimulate the proliferation and migration of endothelial cells and
growth factor (VEGF) increase vascular permeability and maturation of sprouted capillary
A, B, C, D, E. vessels Mattuella et al. (2007a,b), Virtej et al. (2013)
Angiopoietin 2 (ANG2) Proliferation and migration of endothelial cells and stimulates the
formation of new blood vessels Kohara et al. (2010)
Angiogenin Proliferation of endothelial cells and activation of bFGF
Saghiri et al. (2015)
bFGF Stimulation of neurovascular formation El Karim et al. (2009)
Leptin Formation of structures similar to blood vessels Derringer &
HB-EGF Linden (2007)
TGF-b
EGF
PDGF
Macrophages Metalloproteinases Degradation of the basal membrane during initial stages of
angiogenesis (Saghiri et al. 2015)
TGF-a Proliferation and migration of endothelial cells and increase
GM-CSF release of more angiogenic factors Murakami et al. (2013)
bFGF
IGF
PDGF
VEGF

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 339–351, 2017 345
 Angiogenesis and substance P Caviedes-Bucheli et al.

ony-stimulating factor (GM-CSF), the basic fibroblast SP-induced angiogenic changes in human dental
growth factor (bFGF), the insulin like growth factor pulp and periodontal ligament in response to
(IGF), PDGF and VEGF (Table 2) (Murakami et al. masticatory function and occlusal trauma
2013, Saghiri et al. 2015).
It has been reported that changes in the pulp and
Some of these growth factors, that is the hepatocyte
periodontal tissues vary according to the intensity
growth factor (HGF), the placental growth factor
and force of the masticatory stimuli. The application
(PIGF) and bFGF, are stimulated by other neuropep-
of force generates compression and tension zones on
tides such as the CGRP. This peptide has some func-
the periodontal ligament. The compression zones are
tions during the early stages of angiogenesis,
associated with an increase in cellular activity, disor-
suggesting that the mechanism for the formation of
ganization of the fibres of the ligament and bone
new vessels is complex and involves more than one
resorption. In the tension zones, there is enlargement
neuropeptide. It is also important to mention that SP
of such fibres as well as formation of bone and apical
rarely acts alone. Quite the opposite, it coexists along
cement. Additionally, in the compression zones, dam-
with other nerve fibres that produce CGRP and NKA.
age to the periodontal ligament, venous thrombosis,
It has also been claimed that NKA and SP have simi-
haemorrhage, necrosis and hyalinization of the peri-
lar synergic effects because they share the same
odontal ligament occur (Jin & Cao 1992, Poiate et al.
receptors (Caviedes-Bucheli et al. 2006, El Karim et al.
2009, Caviedes-Bucheli et al. 2011a).
2009).
The presence of SP in the periodontal ligament after
excessive occlusal load has been demonstrated. It can
Direct mechanism therefore be inferred that due to the presence of the
neuropeptide, the cells that express NK1 receptors on
It has been reported that the biological mechanism
their surface release numerous angiogenic factors in
for the direct induction of angiogenesis by SP is
order to reverse the hypoxia caused by the decrease
caused by its interaction with the endothelial cells
of blood flow in occlusal trauma (Fig. 3). Therefore,
and the fibroblasts that take part in the repairing pro-
angiogenic changes take place when clastic cells in
cess of the connective tissue (El Karim et al. 2009,
the compression zone respond to SP with root resorp-
Kohara et al. 2010). SP increases the differentiation
tion, whilst blastic cells in the tension zone respond to
and proliferation of endothelial cells because they
SP with tissue apposition, as a compensatory defence
express NK1 receptors on their surface. It also induces
mechanism of PDL that depends on duration and
the formation of structures similar to capillaries thus
direction of the force created by occlusal trauma and
increasing vascular density (Fig. 2). Angiogenesis is
masticatory function (Motohira et al. 2007, Poiate
affected by the concentration of neuropeptides as the
et al. 2009, Caviedes-Bucheli et al. 2011a).
angiogenic phenomenon produced by SP is dose
The changes that take place in the dental pulp and
dependent. In other words, the more intense and pro-
PDL potentiate each other as both are mesenchymal in
longed the occlusal trauma, the higher the SP release,
origin and are related via the apical foramen and the
and therefore a greater differentiation and prolifera-
lateral canals (Ikeda et al. 1998, Caviedes-Bucheli et al.
tion of the vascular endothelium will occur (Derringer
2011a). However, very little is known about the
& Linden 2007, Caviedes-Bucheli et al. 2008, 2011a,
response of the pulp to excessive occlusal loads. In spite
Kohara et al. 2010).
of the lack of studies on the subject, it is well known
It has also been observed that in the presence of
that occlusal trauma causes neurogenic inflammation
interleukin-1 (IL-1), SP acts in a synergic way and
in the pulp characterized by changes similar to pulp
can cause vascular neoformations. The presence of
ageing (Caviedes-Bucheli et al. 2008, Lee et al. 2013).
IL-1 in pulp inflammation has been widely described
In the presence of occlusal loads, the pulp becomes
(Killough et al. 2009, Kohara et al. 2010). Some stud-
inflamed in a localized manner. By the chemoattrac-
ies confirm also the presence of NK1 receptors on the
tant properties of the SP, it reacts by adding cellular
surface of the fibroblasts. These cells can produce
components to the cell-free zone in the tissue adjacent
growth factors with angiogenic potential such as
to the injury. Most of these cells have morphologic
VEGF, bFGF, fibroblastic growth factor-2 (FGF-2), pla-
characteristics of fibroblasts and undifferentiated cells,
cental-like growth factor (PIGF), epidermal growth
and only a few of them are inflammatory. The forma-
factor (EGF) and hepatocyte growth factor (HGF)
tion of new capillaries induced by SP is evident by the
(Tran-Hung et al. 2006, El Karim et al. 2009).

346 International Endodontic Journal, 50, 339–351, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd

SP
SP
SP
SP SP
SP SP
R E
Intensity
and Force
VEGF
bFGF
Masticatory
Stimuli TGF-
PIGF
REVERT HYPOXIA
Figure 3 Response of the periodontal ligament (PDL) to occlusal trauma and masticatory function. Angiogenic changes take
place when clastic cells in the compression zone respond to SP with root resorption, whilst blastic cells in the tension zone
respond to SP with tissue apposition, as a compensatory defence mechanism of PDL that depends on duration and direction of
the force created by occlusal trauma and masticatory function.
presence of erythrocytes produced by small blood
extravasations (Table 3) (El Karim et al. 2009,
Kohara et al. 2010, Caviedes-Bucheli et al. 2011a).
In addition, vascular alterations mediated by SP
also take place, such as arteriosclerotic changes of the
blood vessels in which the tunica intima thickens
whilst the adventitia is calcified lowering the blood
flow towards the pulp tissue. Nerve fibres also
undergo a progressive mineralization of all their mem-
branes, and fat deposits are also observed around the
odontoblastic layer, the cell-rich zone and the walls of
the capillaries. The accumulation of interstitial liquid
provokes the presence of vacuoles of odontoblasts
coming from the separation of these cells from the
dentine wall. There is a net of atrophic reticular fibres
related to the high content of interstitial liquid and to
the decrease in the number of pulp cells. As ageing
progresses, the pulp continues to lose cellular content,
whereas the number of collagen fibres continues to
rise. The remaining odontoblasts have a smaller size
and are flat in shape. The number of capillaries and
nerve fibres has a tendency to decrease, and simulta-
neously, collagen is produced to form a completely
fibrous pulp (Morse 1991, Goga et al. 2008, Cooper
et al. 2010, Lee et al. 2013).
Latent odontoblasts may become activated by SP
signalling, and therefore, the production of tertiary
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
Caviedes-Bucheli et al. Angiogenesis and substance P
Granulocytes
SP Fibroblasts
SP
Endothelial cells
NK1 Receptor
Clastic Cell
Blast Cell
L E A S E
ANG-2 Tension
Zone
Compression
EGF Zone
Apposition
HB-EGF mineralized
Resorption tissue
Leptin mineralized
tissue
dentine starts with a mixture of dentine deposits and
direct deposits of mineral crystals inside the dentine
tubules which reduces their permeability (Fig. 4). This
process is therefore called tubular or sclerotic dentine
and is the result of increasing dentine deposits and
the occlusion of the tubules due to the precipitation of
crystals in a short period of time. In order to produce
reactive tertiary dentine, it is necessary that the effect
of the harmful stimulus be much longer than that
needed for sclerotic dentine formation (Roberts-Clark
& Smith 2000, Arana-Chavez & Massa 2004, Zhang
et al. 2011).
After a longer period of time or after increasing the
occlusal load, the reaction will be characterized by a
noticeable cellular infiltration observed on the injury
site and the formation of microabscesses due to the
compartmentalization capacity of the pulp. Polymor-
phonuclear and mononuclear leucocytes are also
observed in the affected area. The nuclei of the odon-
toblasts can be seen in the tubules due to the increase
in fluid pressure and numerous vascular neoforma-
tions surrounding the intense cellular infiltration
(Mattuella et al. 2007a, Li et al. 2011, Lee et al.
2013). There is also evidence of FGF-2, PDGF and
VEGF in the extracellular matrix. These are released
in a neurogenic inflammatory process by a SP stimu-
lus in order to contribute to the repairing response of
International Endodontic Journal, 50, 339–351, 2017 347
 Angiogenesis and substance P Caviedes-Bucheli et al.

Table 3 Summary of the processes mediated by SP that occurs in the dental pulp during occlusal trauma

Pulp cell Function

Human dental • Increase collagen fibres Caviedes-Bucheli et al. (2008)

pulp fibroblast • Synthesize growth factors with angiogenic potential El Karim et al. (2009),
(HDPF) Killough et al. (2009)
• Stimulate matrix mineralization Trantor et al. (1995)
Dental pulp • Differentiate into endothelial cells for vascular neoformation Aranha et al. (2010)
stem cell • Differentiate into fibroblasts for the synthesis of collagen fibres, tertiary dentine and new
(DPSC) blood vessels Rosa et al. (2011)
• Differentiate into odontoblast-like cells to produce tertiary dentine Arana-Chavez & Massa
(2004), Rosa et al. (2011)
Inflammatory cell • Promote chemotaxis of macrophages, and polymorphonuclear and mononuclear leucocytes
Caviedes-Bucheli et al. (2008)
Endothelial cell • Increase differentiation and proliferation for the formation of new blood vessels
Kohara et al. (2010)
Granulocyte • Release cytokines with angiogenic functions Saghiri et al. (2015)
• Synthesize growth factors with angiogenic potential Mattuella et al. (2007a,b),
Virtej et al. (2013)

Granulocytes
SP Fibroblasts
SP
Macrophages
SP SP SP
Endothelial cells
Odontoblast-like cells

SP
Substance P
Collagen
Release growth factors NK1 receptor
SP

Angiogenesis
New capillary plexus
Pulp
SP
Calcification
SP
Intensity and Force

Collagen Formation
Masticatory Stimuli

Tertiary dentine formation Pulp Calcification

as defense
mechanism

Figure 4 Response of human dental pulp to occlusal trauma and masticatory function. Angiogenic changes are mediated by
SP through direct and indirect mechanisms where cells such as macrophages, granulocytes, endothelial cells and fibroblasts
release angiogenic factors that induce the formation of new blood vessels. Those vessels are necessary for the formation of col-
lagen and tertiary dentine by redifferentiated fibroblasts, similar to the odontoblasts and odontoblast-like cells as a defence
mechanism.

the pulp–dentine complex (Roberts-Clark & Smith
2000, Tran-Hung et al. 2008, Zhang et al. 2011,
2014).
If the damage persists, odontoblasts will not be able
to defend the pulp as they are post-mitotic cells. It is
at this stage, and due to the action of different types
of cells, that the repairing dentine starts forming
under the damaged tubular region by increasing the
normal collagen portion of the pulp and decreasing
the cellular content. The harmful stimulus can
destroy the subjacent odontoblasts, and the pulp
might need a repopulation of the destroyed odonto-
blast layer based on the differentiation of cells which
can be undifferentiated perivascular cells, pulp fibrob-

348 International Endodontic Journal, 50, 339–351, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd

lasts or cells formed by the odontoblastic lineage but
had not been exposed to final epithelial influence dur-
ing dentinogenesis (Tziafas 1994, Cooper et al. 2010,
Smith et al. 2012).
The changes that take place in the connective tis-
sue as a response to both occlusal trauma and masti-
catory function are mediated mainly by the
fibroblasts. These cells respond to mechanical stimuli
due to a cascade of signals which come from the
extracellular matrix and are received by the receptors
located on their surface such as the NK1 specific for
the SP. Such signals also affect the morphology as
well as the proliferation and differentiation of fibrob-
lasts, which contain proteins called kinases that are
sensitive to the stress caused by the extracellular
matrix. These proteins activate phosphorylation sites
recognized by several molecular signals that modify
the operation of the fibroblasts (El Karim et al. 2009,
Krishnan & Davidovitch 2009, Kohara et al. 2010).
Once stimulated by SP, the fibroblasts express angio-
genic factors that contribute to the formation of new
blood vessels by either direct or indirect mechanisms
which are active participants in tissue remodelling
associated with occlusal trauma. Under normal and
pathological conditions, the new vessels supply the
periodontal ligament, the pulp and their surrounding
tissues with oxygenation and nutrients necessary for
their metabolic activities. They also provide tissues
with hormones and a wide variety of endogenous
substances (Tran-Hung et al. 2008, Killough et al.
2009, Krishnan & Davidovitch 2009).
If the stimulus persists after the inflammation pro-
cess is diagnosed, the vascular neoformation cannot
supply oxygen and nutrients to the tissues, the vascu-
lar return system also collapses, and the result will be
the accumulation of mediators, liquid and plasma, as
this system was in charge of removing the inflamma-
tory mediators. This would provoke a vicious cycle in
which the pulp located inside a cavity with limited
expansion capacity will not be able to defend itself,
and therefore, pulp necrosis will occur by coagulation.
This type of necrosis is characterized by the coagula-
tion of intracellular proteins due to a low level of oxy-
gen. It is important to highlight that only 7% of root
canals that become calcified will develop pulp necrosis
(Holcomb & Gregory 1967). This low percentage of
necrotic pulps is probably due to the continuous for-
mation of vessels induced by the constant release of
SP which provides the tissue with the capacity to
remain viable (Killough et al. 2009, Levin et al.
2009).
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
Caviedes-Bucheli et al. Angiogenesis and substance P
Conclusion
Human dental pulps respond to occlusal trauma and
to masticatory function via a neurogenic inflamma-
tory process in which SP plays an important role in
the direct and indirect mechanisms of angiogenesis by
triggering the NK1 receptors of cell populations such
as fibroblasts, endothelial and inflammatory cells,
leading to the formation of new blood vessels which
are needed to form mineralized tissue as a defence
mechanism.
Acknowledgements
Authors would like to express gratitude to Mr. Wilson
Guacaname Rodriguez, from the Biblioteca General
Pontificia Universidad Javeriana, for his assistance
and collaboration in the use of library databases.
Conflict of interest
The authors have stated explicitly that there are no
conflict of interests in connection with this article.
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