Abstract is tertiary dentinogenesis that occurs secondaryAbstract is tertiary dentinogenesis that occurs secondary


An acute inflammatory response is crucial for
initiating healing and repair and the interplay between acute
inflammation and regeneration is well illustrated in the pulp-dentine complex.
An example of this is tertiary dentinogenesis that occurs secondary to caries
induced pulp inflammation. Tertiary dentinogenesis is also induced by dental
materials like calcium hydroxide (Ca(OH2)), or mineral trioxide aggregate (MTA).
Odontoblasts play a pivotal role in regeneration as their functions are
upregulated to form dentine. In severe inflammation and following the death of
odontoblasts, the dental pulp stem cells (DPSCs) are activated and recruited to
the site of injury to differentiate into odontoblast-like cells that produce new
dentine. DPSCs and odontoblasts have important immunological functions and
express components of the innate immune defense system. Inflammasomes
constitute an essential component of the innate immune response and we
hypothesize that the innate immune response through inflammasomes can be
harnessed for tooth repair and dentine regeneration via the recruitment and
differentiation of DPSCs. The dental pulp is an accessible organ and the
availability of DPSCs provides an ideal human injury model that can be used to
study inflammasome activation and subsequent signalling events leading to
repair and regeneration.

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literature review will be divided into three main sections. The first section
will discuss the dentine-pulp complex and its role in inflammation and repair,
the second section will cover the role of DPSCs in the interplay between
inflammation and regeneration, and the last section will explore the AIM2 and the
NLRP3 inflammasomes and their role as mediators of the innate immune response
in the dentine-pulp complex.


The tooth is
composed of three layers of hard tissue namely, enamel dentine and cementum. Encased
within this hard tissue is loose connective tissue known as the dental pulp. The
dental pulp is composed of fibroblasts, immune cells, dental pulp cells and
odontoblasts. The primary dentinogenesis starts at the late bell stage, when
the mesenchymal cells of the dental papilla are induced by mature internal enamel
epithelium (IEE) to differentiate into odontoblasts. Odontoblasts will secrete primary
dentine which will induce the IEE to differentiate into ameloblasts.
Ameloblasts will subsequently secrete enamel but it cannot be regenerated following
tissue injury as ameloblasts will be lost. However, dentine can be regenerated
following injury as odontoblasts activity can be upregulated or odontoblast-like
cells can be generated from stem/progenitor cells in the pulp during tertiary
dentinogenesis1–4 The primary function of odontoblasts
is dentine formation during primary dentinogenesis and throughout a tooth’s
life as secondary/physiological dentinogenesis. Tertiary dentinogenesis illustrates
the tooth’s ability to repair itself, it happens as a response to external
stimuli; caries/traumatic/chemical injury3,5,6, ( Figure 1). If the stimulus
is of low intensity this will lead to mild inflammation and the odontoblasts
secretory activity will be upregulated leading to reactionary tertiary dentine3,5–11 which is similar to primary
and secondary/physiological dentine 12. On the other hand, if the
stimulus is more intense, this will cause odontoblast death. , as a result,
stem/progenitor cells in the pulp will be recruited to differentiate into
odontoblast-like cells that will form reparative tertiary dentine3,5–11. At the end, the two types of
tertiary dentine; reactionary or reparative, will create a new layer of dentine
that will protect the pulp. This layer is clinically known as  a dental bridge6.

1: A- Illustrates the two types of
tertiary dentinogenesis:  1) The reactionary
dentinogenesis occurs as a result of mild stimuli and leads to the
upregulation of odontoblasts secretory activity. 2) The reparative
dentinogenesis occurs as a result of odontoblast death which leads to stem/progenitor
cell recruitment and their differentiation into odontoblast-like cells that
will form reparative dentine. (P. Cooper, Yusuke Takahashi a,b, Lee
W. Grahama, Stephane Simona,c, Satoshi Imazato b, Anthony J. Smith . 2010)
B- A longitudinal section of a tooth; enamel, dentine and pulp are seen
with a dentinal bridge of reparative dentin formed in response to caries.
C- A histological section of the
tooth shows the odontoblastic layer composed of odontoblasts and
odontoblast-like cells that formed tertiary dentine.










Cells of the Pulp-Dentine complex and their role in inflammation and

Immune cells:

The pulp-dentine
complex is important in the interplay between inflammation and regeneration. The
pulp is loose connective tissue derived from the dental papilla. It is composed
of odontoblasts lined peripherally, fibroblasts and DPSCs needed for repair and
regeneration 813 14. The pulp is highly innervated
with sensory nerves and is rich in vascularity and capillary networks14. The pulp also contains immunocompetent
cells; like dendritic cells (DC), macrophages, mast cells 14,15 and natural killer (NK) cells6,16–18.  DCs are the major antigen presenting cells in
the pulp, they are present in an immature state and are attracted by chemokines
secreted by odontoblasts to accumulate in the odontoblastic layer close to
carious dentine 12,17–23.  DCs can influence the innate and adaptive
immunity by the cytokines they secrete as they present their antigens to naïve
T helper cells (Th0) activating them to 
differentiate into Th1,  Th2, Th17
or induced T regulatory cells (iTreg). This differentiation is determined by
the cytokines present in thesurroundings 6. Some types of DCs like tolerogenic
DCs regulate inflammation by  T cell
depletion, by inducing Treg differentiation or by secreting many immune modulating
mediators 6,24. T lymphocytes are important
residents of the dental pulp; they are divided into T helper cells (CD4+)
and cytotoxic T cells (CD8+). CD4+ T helper cells as previously mentioned are
also classified into Th1, Th2  and Th17 cells.
 Th1 cells produce  IL-2 and interferon-gamma (IFN-?) which activates
macrophages whereas Th2 produces cytokines such as IL-4,5,6, and IL-10 which
induce the proliferation and differentiation of B-lymphocytes  6,14. B-lymphocytes  are present in the pulp but in small numbers18 they can differentiate into
either plasma cells secreting antibodies or memory B-cells25. All of these immune cells
secrete pro-inflammatory cytokines to remove the noxious stimuli but they can also
secrete anti-inflammatory cytokines to regulate inflammation, as an example of
anti-inflammatory cytokines is IL-10 which is mainly secreted by macrophages 17. It plays a central role in
limiting host immune response to pathogens by 
promoting the development of Treg cells thus preventing unnecessary
inflammation, it  has also been found to
be upregulated in inflamed pulps challenged with bacteria6,23,26–28 Other anti-inflammatory
mediators  include soluble Tumor Necrosis
Factor-receptor (TNF-R), Treg cells, heme-oxygenase-1, nitric oxide (NO) and  IL-1 receptor antagonist.14,23,27,29


formation is also important in the interplay between inflammation and
regeneration. The extracellular matrix protein family named small integrin
binding ligand N linked glycoproteins (SIBLINGS) plays an essential role in
dentine regeneration and regulates hydroxyl apatite crystal formation. This
family includes; dentine sialophosphoprotien (DSPP), dentine matrix protein-1 (DMP-1),
osteopontin(OPN), matrix extracellular phosphoglycoprotien(MEPE) and integrin
binding sialoprotein (IBSP) 30–32. In addition, dentine sialoprotein
(DSP)  is  able to stimulate the recruitment of
pro-inflammatory immune cells33–35. Many growth factors are present
in dentine such as transforming growth factor beta (TGF-b), Insulin growth
factor 1 and 2 (IGF-1,2), fibroblast growth factor 2 (FGF2) and various
angiogenic growth factors36–39. TGF-?1, TGF-?3 or bone morphogenetic
protein (BMP-7) can upregulate odontoblasts secretory activity therefore
inducing reactionary dentinogenesis 40,41 TGF-?1, FGF2, BMP-2, BMP-4 can
induce reparative dentinogenesis by recruiting cells from dental pulp inducing
their proliferation and differentiation into odontoblast-like cells 41–44. Matrix metalloproteinases (MMPs)
and tissue inhibitors of MMPs are secreted by odontoblasts, they are biological
activators of SIBLINGs, and growth factors45,46. All of these molecules
become fossilized in dentine during dentinogenesis and are later released
during the demineralization of dentine in injury or during the carious process.
These molecules are important because they can organize regenerative and
defensive responses in the tooth3,6.  Another example of the link between
inflammation and repair can be illustrated by MTA and Ca(OH)2 which are dental
materials known to stimulate tertiary dentinogenesis. It has been shown that after
their application inflammation is usually histologically observed prior to
healing. Their regenerative effects may be due to their antibacterial
properties, calcium release and their ability to release entrapped growth
factors from dentine.5 47–51

Other than
regulating dentine synthesis, odontoblasts have immune functions. Due to their
location peripherally lining the pulp, the extension of their odontoblastic
processes through the dentinal tubules and because they express pattern
recognition receptors (PRRs) 6,17,19,22,52–54, odontoblasts are the first  cells to encounter any external stimuli. The
PRRs are receptors which can sense a broad range of stimuli like pathogen
associated molecular patterns, (PAMPS), which are conserved molecular
structures produced by microbes 12,22,55,56,  or danger associated molecular patterns
(DAMPs)which are host biomolecules secreted by cells under high stress 57,58. The PRRs are a family of
five main members; Toll-Like Receptors (TLRs), Nod-Like Receptors(NLRs),
RIG-like Receptors(RIRs), AIM2-like receptors (ALRs), and C-type lectin
receptors53,59–62. PRRs can be further
characterized depending on their location; transmembrane receptors like TLRs , some
TLRs are endosomal bound in the cell, or intracellular receptors (cytoplasmic)
like NLRs, ALRs, RLRs. 53,59,61,62. Some of these PRRs can form
multiprotein complexes known as inflammasomes, as first termed by Tshopp63. Examples of inflammasomes
are  NLRP1,NLRP3, NLRC4 and AIM2 (HIN-200
family member), and Pyrin59,63,64.

have been shown to express all TLR1-6 and TLR9s genes 19,22,52,53,65. TLR2 can sense the cell wall
component of Gram positive bacteria; Lipoteochic acid (LTA) leading to the
secretion of certain pro-inflammatory cytokines such as IL-8 by odontoblasts
and fibroblasts. LTA also induces DC recruitment and cytokine production19,20,26. Odontoblasts have been shown
to downregulate their primary function of dentine secretion and upregulate
their innate immune functions when challenged with LTA19. The TLR4 can sense Gram
negative bacterial components; like Lipopolysaccharide (LPS), inducing  the secretion of TNF-?, IL-1B, IL-8 and others12,52,66. IL-8 is secreted in low
amounts by odontoblasts, it  attracts
neutrophils 6,67,68. TLR signaling also leads to the
production of antimicrobial peptides by odontoblasts like beta defensin 69–72. Odontoblasts can also regulate
inflammation by limiting  its intensity, as
odontoblast-like cells with TLR2 engagement, have been shown to upregulate IL-10
in vitro26. The dental pulp cells like odontoblasts,
fibroblasts and  immune cells express
other PRRs like the NLRs 20,21,53,73–76. NLRP3 , a member of the NLRs,
has been detected in the dental pulpal fibroblasts, odontoblasts, and vascular
endothelial cells of the pulp74,76. This shows how the pulp-dentine
complex is well equipped to elicit an innate immune response when detecting
noxious stimuli.  

Dental pulp stem cells (DPSCs):

The tooth
harbors many  mesenchymal stem cells, the
DPSCs were the first type to be isolated from the human dental pulp and were
named postnatal DPSCs by Gronthos et al in 2000 13. Other dental stem cells
include stem cells from exfoliated deciduous teeth (SHED)77,  periodontal ligament stem cells (PDLSC)78,  the dental follicle precursor cells (DFPC)79 and stem cells from the
apical papilla, (SCAP)80 81. The DPSCs have been shown to
express mesenchymal stem cell markers such as CD105, CD13, and CD73, this means
that they are of mesenchymal phenotype 82.
CD105 is a component of the TGF-? receptor complex; therefore it is
highly inclined to bind to TGF-?1 and TGF-?382. The dental stem cells have
been shown to be multipotent meaning that they can give rise to different cell
lineages like  osteogenic 83,84, odontogenic13, chondrogenic, adipogenic and
neurogenic 85,86.  Although the dental stem cells seem to be guided
more towards odontogenic development87 88. Many receptors for growth
factors and mediators have been identified in dental stem cells;  growth factors like TGF-?81,87,89 ,  BMPs 89–92and others which can be  secreted by immune cells and/or released
during the demineralization of dentine. These growth factors can bind to receptors
expressed by dental stem cells. TGF-?1 can stimulate DPSC recruitment and
differentiation 93–95, also stromal cell derived
factor 1 (SDF-1) can induce DPSC recruitment 90 96. Therefore, these factors play
important roles in mediating wound healing and repair by recruiting stem cells,
inducing their proliferation and/or differentiation. The pulp also incorporates
progenitor cells or undifferentiated cells that can differentiate into
odontoblast-like cells. Progenitor cells expressing the STRO-1 stem cell marker
also express C5a receptor, this receptor binds to C5a which is an anaphylatoxin
and a member of the complement system. It has been shown that when a C5a
gradient is induced, progenitor cells are led by this gradient to the site of
injury/inflammation to  differentiate
into odontoblast-like cells that form reparative dentine97. Also progenitor cells
expressing CXCR4 are responsive to SDF-1 which is upregulated during inflammation,
therefore progenitor cells can be attracted to the site of tissue
injury/inflammation 98. It has also been shown that
SDF-1 and CXCR4 mRNAs are increased in expression in inflamed dental pulps 90. This adds to the many
examples of interplay between inflammation and repair in the pulp-dentine


The NLRP3 and AIM2 inflammasomes:

The NLRP3 is a cytoplasmic
PRR that senses a broad range of PAMPs from bacterial, viral, protozoal pathogens99 and DAMPS like DNA, ATP 100uric acid, reactive oxygen
species (ROS)101 as well as environmental
irritants; asbestos, silica or alum99,102. When these patterns are
detected, the NLRP3 activates  into an
inflammasome59,64. The NLRP3 receptor is composed
of three domains: carboxy-terminal Leucine-rich-repeat (LRR) domain, a central
nucleotide-binding and oligomerizatio domain (NACHT)and at the amino terminal a
pyrin domain (PYD)59,60,64. The LRR domain is thought to
function as a ligand sensor but direct binding to it is inconclusive103. The PYD domain works as an
intermediate for homotypic protein-protein interactions with the  (ASC) adapter apoptosis associated speck-like
protein containing a caspase activating recruitment domain(CARD)73. ASC is important for
downstream signaling during oligomerization and is composed of two domains
PYD-CARD 104,105. NLRP3 oligomerizes into an
inflammasome after receiving two signals, the first signal primes the cell by
activating NF?? pathway via TLRs to produce pro-forms of IL-1? and IL-18. The
second signal is provided by an NLRP3 activating agent. It has been shown that all
second signals cause K+ efflux leading to NLLRP3-inflammasome assembly64,106. This assembly happens by
recruiting inactive caspase-1 through the ASC protein to the NLRP3 scaffold,
thus activating caspase-176,104,107,108, (Figure 2).  In turn activated caspase-1 cleaves IL-1? and
IL-18 into their active forms to be secreted, thus triggering innate immune
defenses 104,109,110. Recent studies have shown
that NLRP3 inflammasome is expressed in the dental pulp. NLRP3 and
caspase-1  have been shown in the
odontoblastic layer of healthy pulp and expressed in vascular endothelial cells
74. Teeth with irreversible
pulpitis have also been shown to have more functional NLRP3 and caspase-1 than
in healthy and reversibly inflamed pulps. NLRP3 and caspase-1 have been shown
to be expressed by the pulp fibroblasts with NLRP3-inflammasome activated after
stimulation by LPS as a first signal and ATP as a second signal76. This provides evidence of
the importance of NLRP3 inflammasome in the pulpal defense against external


Figure 2: NLRP3 inflammasome structure and the two signals
for activation: (Kigerl, Kristina A. de Rivero Vaccari, Juan Pablo Dietrich,
W. Dalton Popovich, Phillip G.Keane, Robert W.2014)
NLRP3 inflammsome is composed of NLRP3 scaffold,
ASC protein (PYD-CARD) and caspase-1.
 Signal 1
happens when a TLR ligand binds to the TLR causing movement of NFkb into
the nucleus and the transcription of pro-IL-18 and pro-IL-1B.
Signal 2 induces NLRP3 inflammasome activation and
cleavage of pro-IL-18 and pro-IL-1B by caspase-1 to their active forms to
be secreted.






The Absent in Melanoma
2 (AIM2) is a member of the HIN200 family, it’s a cytosolic double
standard  DNA (dsDNA) receptor that senses
 dsDNA from host, virus or bacteria 111,112 AIM2 is composed of the C-
terminal HIN domain which binds to the dsDNA and an N-Terminal pyrin domain. It
oligomerizes into an inflammasome by recruiting caspase-1 through ASC protein
leading to caspase-1 activation. In turn caspase-1  induces IL-1? 
and IL-18 maturation and secretion 111,113–116(Figure 3). AIM2 inflammasome
is expressed in the odontoblast layer of healthy dental pulp with a stronger
expression in fibroblasts and inflammatory cells of inflamed pulp, this suggests
that AIM2 plays an important role in the immune defense in the dentine-pulp
complex117. A recent study showed that the
activation of the AIM2 inflammasome is regulated by the expression level of
AIM2-receptor.Moreover, any block in the AIM2 inflammasome pathway will inhibit
IL-1? secretion.118

 As mentioned, NLRP3 and AIM2 inflammasomes
lead to IL-1? and IL-18 secretion, which are important cytokines that stimulate
inflammation and integrate innate and adaptive immune responses. IL-1? has  different functions extending from organizing
normal body functions like sleep, body temperature to regulating immune
responses in inflammation and injury 119 120 121. It is a powerful
pro-inflammatory cytokine that is important in the defense against infections.
Its pro-inflammatory functions encourage the infiltration of immunocompetent
cells, increasing the expression of adhesion molecules, and the induction of
cytokines and chemokines, thus encouraging the healing process 121. The main sources of IL-1?
are hematopoietic cells like macrophages, monocytes, DCs, NK cells and B cells 121. As previously illustrated, activation
of IL-1? happens in a caspase-1 dependent way via inflammasomes, but exceptions
do exist121. IL-18 was first obsereved as
an IFN-? inducing cytokine in endotoxemic mice122, , it’s activation is also
caspase-1 dependent via inflammasomes but exceptions exist. It has a role in
Th1 and Th2 responses acting with IL-12 or without it respectively123, it  also promotes IL-17 expression by Th17 cells121. Therefore, the activation of
the innate immune system by AIM2 and NLRP3 inflammasomes indicates their
importance in pulp defense against various foreign stimuli. Moreover, NLRP3 and
AIM2 inflammasomes induce a pro-inflammatory type of cell death called
pyroptosis. Pyroptosis is characterized by the entrance of water, swelling of
the cell , plasma membrane tearing and liberation of cytoplasmic content124. It differs from apoptosis
but the DNA fragmentation  is similar to
that of apoptosis125.             

Figure 3: AIM2 receptor and inflammasome.
A-       AIM2 receptor
can detect dsDNA from virus, bacteria or host. It then oligomerizes into an
AIM2 inflammasome.. (Petr Broz and Vishva M.Dixit 2016)
AIM2 inflammasome 
is composed of AIM2 receptor, ASC protein and caspase-1.
(Lamnkafi  and Vishva M.Dix.2014)











In conclusion:

Our preliminary
data with fluorescent immunohistochemistry showed expression of AIM2 and NLRP3
inflammasomes in healthy pulp tissue, but more intense staining in inflamed
pulps (Figure 4). DPSCs are usually quiescent
but they have a quick response to injury 82 so by using novel human dental injury models and DPSCs
we will investigate the effects of inflammasome signalling on DPSCs recruitment
and differentiation.