Clinical and Laboratory Research
144 Congenital Anomalies 2016; 56, 144–153
INVITED REVIEW ARTICLE
Odontoblasts: Specialized hard-tissue-forming cells in the dentin-pulp
complex
Nobuyuki Kawashima, and Takashi Okiji
1
Department of Pulp Biology and Endodontics, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo
Medical and Dental University (TMDU), Tokyo, Japan
ABSTRACT Odontoblasts are specialized cells that
produce dentin and exhibit unique morphological characteristics;
i.e., they extend cytoplasmic processes into dentinal tubules.
While osteoblasts, which are typical hard-tissue-forming cells,
are generated from mesenchymal stem cells during normal and
pathological bone metabolism, the induction of odontoblasts only
occurs once during tooth development, and odontoblasts survive
throughout the lives of healthy teeth. During the differentiation
of odontoblasts, signaling molecules from the inner enamel
epithelium are considered necessary for the differentiation of
odontoblast precursors, i.e., peripheral dental papilla cells. If
odontoblasts are destroyed by severe external stimuli, such as
deep caries, the differentiation of dental pulp stem cells into
odontoblast-like cells is induced. Various bioactive molecules,
such as non-collagenous proteins, might be involved in this
process, although the precise mechanisms responsible for
odontoblast differentiation have not been fully elucidated.
Recently, our knowledge about the other functional activities of
odontoblasts (apart from dentin formation) has increased. For
example, it has been suggested that odontoblasts might act as
nociceptive receptors, and surveillance cells that detect the
invasion of exogenous pathogens. The regeneration of the
dentin-pulp complex has recently gained much attention as a
promising future treatment modality that could increase the
longevity of pulpless teeth. Finally, congenital dentin anomalies,
which are concerned with the disturbance of odontoblast
functions, are summarized.
Key Words: dental pulp stem cells, dentin pulp complex, dentinogenesis
imperfecta, odontoblasts
INTRODUCTION
Teeth are made up of three unique hard tissues, enamel, dentin, and
cementum. Enamel and cementum cover the surface of the tooth
crown and root, respectively, and dentin comprises the main portion
of the tooth. The dental pulp is the only non-mineralized connective
Correspondence: Nobuyuki Kawashima, DDS, PhD, Department of Pulp
Biology and Endodontics, Division of Oral Health Sciences, Graduate School
of Medical and Dental Sciences, Tokyo Medical and Dental University
(TMDU), Tokyo, Japan, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549,
Japan. Email: kawashima.n.endo@tmd.ac.jp
Received January 23, 2016; revised and accepted April 22, 2016.
© 2016 Japanese Teratology Society
doi: 10.1111/cga.12169
tooth tissue that is surrounded by dentin and rich in blood vessels
and nerve fibers. Odontoblasts are present in the periphery of the
dental pulp and exhibit unique morphological characteristics; i.e.,
they extend cytoplasmic processes termed odontoblast processes into
the dentinal tubules within dentin. The close anatomical and
functional relationships between dentin and dental pulp tissue have
led to the development of the concept of the "dentin/pulp complex".
Although the principal role of odontoblasts is the formation of dentin,
there is increasing evidence that they also act as nociceptors and
defensive cells in the dental pulp. The purpose of this article is to
review current knowledge about the differentiation mechanisms,
functional properties, and regeneration of odontoblasts.
TOOTH DEVELOPMENT
Tooth development is initiated from a localized thickening of dental
epithelium, which then proliferates into the oral mesenchyme and forms
a band of epithelial tissue termed the dental lamina (E6-7w in human,
E12d in mouse). At the bud stage (E9-10w in human, E13d in mouse),
spherical to ovoid epithelial condensation is formed at the distal end of
the dental lamina, which is termed the enamel organ. The exact numbers
of enamel organs are grown at the programmed or scheduled areas of
dental lamina in each animal. Mesenchymal condensation around the
enamel organ is observed simultaneously. The epithelial components
are separated from the adjacent mesenchyme by a basement membrane,
and the signals from epithelium to mesenchyme are delivered via the
basement membrane, and vice versa. Such signal delivery is essential
for tooth development, which is evidenced by the fact that the oral
mesenchyme separated from the oral epithelium fails to form teeth
(Mina and Kollar 1987).
At the cap stage (E11w in human, E15d in mouse), the end of the
enamel organ is thickened, and invagination is induced. The complex
of the oral epithelium and mesenchyme is termed the tooth germ. The
outer and inner layers of the enamel organ become outer and inner
enamel epithelium, respectively, and the space formed between them
is termed the stellate reticulum. The condensed mesenchyme
surrounded by the inner enamel epithelium is termed the dental
papillae, the origin of the dental pulp tissue and odontoblasts. The
dental follicle is the mesenchyme surrounding the tooth germ, and
is the origin of the cementum, periodontal ligament, and alveolar
bone. Moreover, there are areas of cell condensation in the central
area of the inner enamel epithelium; this is termed the enamel knot,
in which Sonic hedgehog (Shh), bone morphogenetic proteins
(BMPs), and fibroblast growth factors (FGFs) are expressed (Thesleff
2003). Therefore, the enamel knot is considered as a signaling center
of tooth development, and is essential for the morphogenesis of tooth
crowns. The enamel knots formed in the cap and bell stages are
 Odontoblasts and the dentin-pulp complex 145

termed the primary and secondary enamel knot, respectively, and they
are involved in cusp formation of tooth crowns.
In the bell stage (E14w in human, E17 in mouse), connection of the
enamel organ to the oral epithelium disappears, and the tooth germ
becomes an independent tissue. The dental papillae cells attached to
the inner enamel epithelial cells via a basement membrane start to
differentiate into odontoblasts, which is initiated at the future cusp area.
The inner enamel epithelium attaching to odontoblasts differentiate into
ameloblasts. As will be described later, signaling molecules from the
inner enamel epithelium are considered necessary for the differentiation
of the peripheral dental papilla cells into odontoblasts. In the process of
odontoblast differentiation, odontoblasts move away from the basement
membrane to the dental papillae, keeping an attachment to the basement
membrane, which results in the formation of the odontoblastic process.
The basement membrane becomes discontinuous and disappears at the
initial stage of dentin mineralization (Frank and Nalbandian 1989). In
the late bell stage (E18w in human, E19 in mouse, Fig. 1A), mineral
deposition is initiated, and odontoblasts and ameloblasts form dentin
and enamel, respectively. The dentin formation slightly precedes the
enamel formation. Differentiated odontoblasts secret their characteristic
organic matrix called dentin matrix (see 3.1. Functions of odontoblasts
under physiological conditions and Table 1). Mineralization of dentin
starts from the deposition of minerals in the dentin matrix. The joining
area of inner and outer enamel epithelium, called the cervical loop, forms
the epithelial root sheath enclosing the dental papillae, and the root
formation is induced.
MORPHOLOGY AND DEVELOPMENT OF
ODONTOBLASTS
Odontoblasts are long-living post-mitotic cells that align along the
dentin-pulp interface, where they maintain pre-dentin and dentin
apposition throughout the whole life of a tooth (Sasaki and Garant
1996). Odontoblasts are similar to neurons and cardiomyocytes in that
they are basically stable and are not replaced. They extend
cytoplasmic processes into the dentinal tubules in dentin and form a
single layer of tall columnar and highly polarized cell bodies. Each
odontoblast has a single eccentrically located large oval nucleus in
its basal region (Fig. 1B). Mature odontoblasts contain rough and
smooth endoplasmic reticulum, Golgi apparatus, and mitochondria
and synthesize various proteins related to dentinogenesis (Sasaki
and Garant 1996). Gap junctions between odontoblasts, which mainly
consist of connexin 43, might be involved in the transduction of
signals from stimulated odontoblasts to the surrounding odontoblasts
(Fried et al. 1996).
As stated above, odontoblasts differentiate from the cells of the dental
papilla, which are derived from cranial neural crest cells (Ruch et al.
1995; Ruch 1998). During the course of tooth development, including
odontoblast differentiation, epithelial-mesenchymal interactions are
essential; signals (signaling molecules) from the inner enamel epithelium
are necessary for the differentiation of odontoblasts from the peripheral
dental papilla cells that are in contact with the basal laminae, which
are located between the inner enamel epithelium and the dental papilla
(Thesleff et al. 2001; Thesleff and Mikkola 2002; Thesleff 2003).
Although the precise mechanisms responsible for odontoblast
differentiation remain unclear, various factors have been reported to
induce it. The members of the BMP family are the most widely studied
of the growth factors involved in the differentiation of odontoblasts and
osteoblasts. BMP2 (Nakashima et al. 1994; Iohara et al. 2004; Saito
et al. 2004; Yang et al. 2007; Yang et al. 2008), BMP4 (Nakashima
et al. 1994), and BMP7 (Lin et al. 2007; Wang et al. 2010b) have been
reported to induce odontoblast differentiation. Although it is clear that
these BMPs are strong inducers of mineralization, they cannot determine
the direction of odontoblast differentiation by themselves. In other words,
BMP are able to induce osteoblast marker expression in various cells, but
can only do so in odontoblast-committed cells. Transforming growth
factor-β (TGF-β) (Liang et al. 1990; Liang et al. 1992; Nakashima
et al. 1994; Sloan and Smith 1999; Dobie et al. 2002; Li et al. 2011) is
another growth factor that is known to be involved in odontoblast
differentiation. However, its effects on pulp cell differentiation are
complex, and it has also been reported to have negative effects on this
process (Shirakawa et al. 1994; Tai et al. 2008). The other growth factors
involved in odontoblast differentiation include epidermal growth factor
(Liang et al. 1990; Liang et al. 1992), FGF2 (Nakao et al. 2004;
Shimabukuro et al. 2005; Morito et al. 2009; Shimabukuro et al. 2009),

Fig. 1 HE staining of the dental pulp of rat upper incisors (A) The initiation of odontoblast differentiation is observed at the late bell stage, and the dental papillae
cells that are in contact with the inner enamel epithelium differentiate into odontoblasts. AB, alveolar bone; D, dentin; DP, dental papillae; OB, odontoblasts;
SR, stellate reticulum; right figure, high power view of the boxed area, bar: 100 μm. (B) Odontoblasts localize at the periphery of the dental pulp and extend
their cytoplasmic processes into the dentin. Mature odontoblasts possess columnar cell bodies, which are connected by gap junctions. Signal transduction is
induced via these junctions. BV, blood vessel; D, dentin; OB, odontoblasts; PD, predentin; bar, 100 μm. HE staining of the mouse upper incisor tooth germ at
the late bell stage. Reprinted from Kriangkrai et al. (2006) with permission (A).

© 2016 Japanese Teratology Society

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Table 1 Components of the dentin extracellular matrix
Collagen
Type I, Type III, Type V
Non-collagenous proteins
Proteoglycans
chondroitin sulfate (biglycan, decorin),
heparan sulfate (entactin, perlecan)
keratan sulfate
dermatan sulfate
Phosphorylated matrix proteins (SIBLINGs)
DSPP (DSP, DPP)*, DMP-1, OPN, MEPE, IBSP
Non-phosphorylated matrix proteins
matrix Gla1 protein, OC (BGLAP2), osteonectin (SPARC3)
Growth factors
TGF-β (TGF-β1, TGF-β2, TGF-β3)
BMP (BMP-2, BMP-4, BMP-7)
FGF (FGF-2)
IGF (IGF-1, IGF-2)
PDGF, VEGF4, NGF5
Others
MMP6s (MMP-1, MMP-2, MMP-3, MMP-9, MMP-20)
TIMP7s (TIMP-1, TIMP-2, TIMP-3)
Annotation
1: gamma-carboxyglutamate
2: bone gamma-carboxyglutamate protein
3: secreted protein acidic and rich in cysteine
4: vascular endothelial growth factor
5: nerve growth factor
6: matrix metalloproteinase
7: tissue inhibitors of metalloproteinases
For other abbreviations, please refer to the original text.
* DPP and DSP are the cleavage products of DSPP.
growth differentiation factor 11 (Nakashima et al. 2002; Nakashima et al.
2004), platelet-derived growth factor (PDGF) (Yokose et al. 2004),
hepatocyte growth factor (Li et al. 2009), and insulin-like growth factors
(IGF) (Onishi et al. 1999). Odontoblast differentiation is modulated by
various molecules, including twist (Galler et al. 2007), Nuclear factor
I-C (Lee et al. 2009), Notch (Zhang et al. 2008; Sun et al. 2010), Wnt
(Scheller et al. 2008; Koizumi et al. 2013), Runx2, Sp7 (Chen et al.
2009), Runx3 (Zheng et al. 2007), and DLX3 (Choi et al. 2010).
However, the specific factor(s) or gene(s), including master genes,
required for commitment to odontoblast differentiation remain unclear.
PHYSIOLOGICAL AND PATHOLOGICAL
FUNCTIONS OF ODONTOBLASTS
Functions of odontoblasts under physiological conditions
Odontoblasts, which are also called “dentinoblasts”, are responsible
for the formation of dentin and pre-dentin, which is an immature
mineralized tissue. The major organic component of dentin is
collagen type I (about 90%), and collagen type III and type V have
also been detected in dentin (Goldberg et al. 2011). Non-collagenous
proteins comprise a relatively small percentage of the organic matrix
of dentin, but are considered to play important roles. Proteoglycans,
including chondroitin sulfate (biglycan and decorin), heparan sulfate
(entactin and perlecan), keratan sulfate, and dermatan sulfate, are
© 2016 Japanese Teratology Society
N. Kawashima and T. Okiji
present in dentin. The members of the SIBLING (small integrin-binding
ligand, N-linked glycoprotein) family (Huang et al. 2008; Zhang et al.
2010), which possess the arginine-glycine-aspartate (RGD) integrin
binding site and multiple phosphorylation sites in their amino acid
sequences, are well known dentin and bone non-collagenous proteins.
The members of this family include dentin sialophosphoprotein (DSPP),
dentin matrix protein 1 (DMP-1), osteopontin (OPN), matrix extracellular
phosphoglycoprotein (MEPE), and integrin-binding sialoprotein (IBSP).
The typical components of the dentin extracellular matrix are summarized
in Table 1.
Laminins, a major component of the basement membrane, and
fibronectin, an extracellular matrix glycoprotein and adhesion molecule,
have historically been reported to be present at the epithelial-mesenchymal
junction (Lesot et al. 1981). However, they are not classified as
dentin extracellular matrix proteins. Laminins promote odontoblast
differentiation by inducing dentin sialoprotein synthesis (Yuasa
et al. 2004), and fibronectin is involved in the polarization of
odontoblasts (Lesot et al. 2001).
Typical odontoblasts with highly polarized cell bodies (Fig. 1)
become flattened as the tooth ages (Couve et al. 2013). While most
osteoblasts become embedded in bone tissue after their differentiation
(Dallas and Bonewald 2010), odontoblasts never become embedded in
dentin. Odontoblasts typically synthesize DSPP (Begue-Kirn et al.
1998; Narayanan et al. 2006) and DMP-1 (Narayanan et al. 2006),
and these proteins are therefore considered to be specific markers of
odontoblasts, although they are also synthesized by osteoblasts in
small amounts (Qin et al. 2003; Staines et al. 2012). DSPP mutations
cause inherit anomalies, including the dentinogenesis imperfecta,
which will be mentioned in the latter part.
The dentin that forms before tooth eruption, which is designated
primary dentin, forms at a rate of 4–8 μm/day, while the dentin that
forms after tooth eruption (secondary dentin) develops at a rate of
0.5 μm/day (Bleicher 2014).
Functions of odontoblasts under pathological conditions
Reparative dentin formation
Dentin acts as a physiological barrier that protects the dental pulp from
exogenous noxious stimuli, and the thickness of the dentin barrier can
be increased via the formation of reactionary and/or reparative dentin
in response to external stimuli, including caries lesions, as a self-
defense mechanism. Such additional dentin is classified into
reactionary dentin, which is formed from pre-existing odontoblasts,
and reparative dentin, which is elaborated by odontoblast-like cells that
form after the death of the original odontoblasts (Aguiar and Arana-
Chavez 2007). Mild noxious stimuli induce upregulated dentin
synthesis activity in odontoblasts (Smith et al. 1995), and the
synthesized reactionary dentin exhibits anatomical, biochemical, and
functional similarities to primary and secondary dentin (Bleicher
2014). Several cytokines and growth factors have been suggested to
be involved in the activation of odontoblasts and the induction of
reactionary dentin synthesis, but the precise mechanisms responsible
for this process remain unclear (Smith et al. 2001). Markedly noxious
stimuli, including severe caries lesions, can destroy odontoblasts. This
leads to the differentiation of odontoblast-like cells from dental pulp
stem cells (DPSC), as will be described later in this article. The
reparative dentin in rodent incisors is reported to be strongly
immunoreactive for OPN, a non-collagenous protein abundant in bone
matrix but not usually immunodetected in primary and secondary
dentin (Aguiar and Arana-Chavez 2007). The properties of reparative
dentin are dependent on the differentiation features of the odontoblast-
like cells, and those exhibiting osteoblastic features may form bone-
like hard tissues rather than dentin (Goldberg et al. 2011).
 Odontoblasts and the dentin-pulp complex
Recognition of noxious stimuli
External stimuli, such as those from caries lesions, induce pain, which
acts as an alarm system for warning of the invasion of exogenous
pathogenic factors into the body. There has been a long discussion
as to whether odontoblastic processes are involved in pain perception.
The hydrodynamic theory (Brännström 1986) is still the most widely
accepted theory for explaining dentin sensation. It states that the flow
of fluid in dentinal tubules triggers the activation of nerve endings
located near the pulpal ends of the dentinal tubules. However, there
is increasing evidence that odontoblasts function as sensory cells for
detecting nociceptive signals, and odontoblastic processes act as
sensors that are capable of detecting various signals. Odontoblasts
possess several ion channels that participate in nociception and signal
propagation, such as voltage-gated sodium channels (Allard et al.
2006; Byers and Westenbroek 2011). Recently, the expression of
several members of the transient receptor potential superfamily of
ion channels has been detected in odontoblasts, indicating that
odontoblasts might be able to detect and/or transduce sensory
physiological stimuli, such as thermal, mechanical, and chemical
stimuli (de Souza et al. 2009; El Karim et al. 2011; Kim et al. 2012;
Sato et al. 2013). Odontoblasts also express acid-sensing ion
channels, which act as pH sensors for detecting pH fluctuations
(Sole-Magdalena et al. 2011), and TREK-1, a mammalian
mechanosensitive K+ channel involved in polymodal pain perception
(Alloui et al. 2006).
HOST DEFENSE
Bacteria or bacterial products that invade through dentinal tubules that
have been exposed to the oral cavity by caries lesions or dental trauma
might first be detected by odontoblasts (odontoblastic processes). This
notion is supported by the fact that odontoblasts express various
specialized pattern recognition receptors against pathogen-associated
molecules, including Toll-like receptors (TLR), and nucleotide-
binding oligomerization domain (NOD)-like receptors (Mogensen
2009). TLR2 recognizes lipoteichoic acid (LTA), which is one of the
components of the cell walls of Gram-positive bacteria (Takeda et al.
2003). TLR2 is expressed in odontoblasts (Staquet et al. 2008), and
the synthesis of several chemokines including chemokine ligand
(CCL)2, C-X-C motif ligand (CXCL)1, CXCL2, CXCL8, and
CXCL10 is induced by TLR2 signaling (Fargues et al. 2010). These
chemokines might induce the recruitment of immature dendritic cells
(Durand et al. 2006), which could be involved in the paraodontoblastic
accumulation of dendritic-like cells under dentinal caries lesions (Okiji
et al. 1997; Sakurai et al. 1999). TLR4 recognizes lipopolysaccharide
(LPS), which is the major component of the outer membrane of Gram-
negative bacteria (Takeda et al. 2003). Odontoblasts express TLR4 and
produce several proinflammatory cytokines including interleukin
(IL)-1 and tumor necrosis factor and chemokines including CCL20
and IL-8 in response to TLR4 signaling (Veerayutthwilai et al. 2007).
TLR2 and TLR4 signaling induced by LTA and LPS, respectively,
have been suggested to have negative effects on the activity of
alkaline phosphatase, the expression of DSPP and osteocalcin (OC),
and nodule formation (Nomiyama et al. 2007; Yamagishi et al.
2011). NOD2, which is also known as caspase recruitment domain-
containing protein 15, is involved in inflammasome assembly and
the activation of caspase-1 (Stutz et al. 2009). NOD2 is expressed in
odontoblasts, and the synthesis of IL-1 and IL-18 is induced via
NOD2 signaling (Keller et al. 2011) .
Beta-defensins are cationic, broad-spectrum antimicrobial peptides
that form channel-like micropores that disrupt membrane integrity
(Sahl et al. 2005). They are mainly produced by epithelial and
147
immune cells and are involved in the resistance of epithelial and
mucosal surfaces to microbial invasion (Farges et al. 2015). They
have been detected in odontoblasts, suggesting that these molecules
contribute to pulpal host defense (Dommisch et al. 2007;
Veerayutthwilai et al. 2007; Paris et al. 2009; Farges et al. 2015).
Beta-defensins induce IL-6 and IL-8 expression in odontoblast-like
cells in vitro (Dommisch et al. 2007).
ODONTOBLAST PRECURSORS, DPSC, AND
PULP REGENERATION
Odontoblast-like cells forming reparative dentin
In the presence of severe exogenous stimuli, such as the expansion of
caries lesions and pulpal exposure to the oral cavity by trauma or
cavity preparation, odontoblasts are destroyed, and DPSC might
differentiate into odontoblast-like cells to form reparative dentin. It
is not known how odontoblast precursors are recruited. In earlier
studies, pulpal fibroblasts were regarded to be candidates for the
source of regenerated odontoblasts (Yamamura 1985). Pericytes,
which are observed around the endothelial cells of capillaries, have
also been considered as a potential source of newly generated
odontoblasts (Feng et al. 2011). Recently, a population of
mesenchymal stem cells (MSC) has been isolated from dental pulp
tissue, and these cells are generally referred to as DPSC (Gronthos
et al. 2000).
Several non-collagenous proteins, such as those belonging to the
SIBLING family, induce the differentiation and mineralization of
odontoblasts or pulp cells, although some have negative effects on
these processes. For example, MEPE (Wang et al. 2010a; Wang
et al. 2011) and periostin (Zhou et al. 2015) negatively regulate the
expression of odontoblastic markers. In addition, Notch is a receptor
that is involved in the development of various tissues/cells and has
negative effects on both osteoblast differentiation (Shindo et al.
2003) and odontoblast differentiation (Sun et al. 2010). Controlling
odontoblast differentiation is key to the induction of pulp tissue
regeneration.
DPSC
Dental pulp stem cells display multipotency and have the ability to
differentiate into osteoblasts, adipocytes, and neuronal cells. They
were first isolated from the pulp tissue of permanent human teeth
and were designated postnatal DPSC (Gronthos et al. 2000). These
postnatal DPSC were often selected based on their high growth rate.
However, the DPSC collected using this method comprised a mixed
population and are not “pure” stem cells. By using CD105 (Iohara
et al. 2011) or STRO-1 (Yang et al. 2007; Yang et al. 2009) as typical
MSC markers, DPSC can be isolated using a fluorescence-activated
cell sorting system, but the total volume of collected cells is low. Filter
isolation methods involving chemotactic factors such as granulocyte
colony stimulating factor have since been developed, which makes
it possible to isolate pure DPSC easily and in relatively large volumes
(Murakami et al. 2013; Horibe et al. 2014). Three-dimensional
spheroid cultures, which resemble the natural and physiological
conditions found in tissues, induce odontoblastic and osteoblastic
marker expression (Fig. 2) and nodule formation in dental pulp cells
(Yamamoto et al. 2014).
Localization of DPSCs
Localization of DPSCs in the dental pulp tissues is not clear, primarily
because there is no specific marker of DPSCs. A historical study have
shown that 3H-thymidine labeled odontoblast-like cells are present at
the pulp exposure site of calcium hydroxide-capped monkey teeth,
© 2016 Japanese Teratology Society
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Fig. 2 Immunohistological detection of DMP-1 expression in dental pulp cells. The differentiation of dental papillae cells into odontoblasts is initiated by the inner
enamel epithelium, but the odontoblastic differentiation of dental pulp cells or dental pulp stem cells is also induced in adult mature pulp tissues in the
absence of the enamel epithelium. DMP-1 (an odontoblast marker) expression was induced in 3D-spheroid cultures of dental pulp cells, especially at the
periphery of the spheroid bodies (A: cultured for 1 day, B: cultured for 14 days). On the contrary, no DMP-1 expression was seen in 2D-cultured dental pulp
cells (C). Bars: 50 μm. Reprinted from Yamamoto et al. (2014) with permission.
suggesting that the pulp deep from the exposure site contains DPSCs
that differentiate into odontoblast-like cells following multiple DNA
replication and migration (Fitzgerald et al. 1990). Most of the MSCs
are reported to be present in the periphery of blood vessels, which is
thought to be a stem cell niche (Kolf et al. 2007). The side population
(SP) cells are regarded as stem cells, and are relatively large and dark
cells when labeled with the Hoechst dye (Goodell 2005). Relatively
elevated activity of ABC transporters may be involved in the removal
of Hoechst dye (Zhou et al. 2001), and thus high expression of ABC
transporters may be useful in the identification of SP cells. SP cells are
identified in the dental pulp tissues (Iohara et al. 2006; Iohara et al.
2008), and high ABC transporter expression is detected at the
periphery of blood vessels (Iohara et al. 2006). These results suggest
that the periphery of the blood vessels is one of the niches of DPSCs
in the dental pulp tissues.
STRO-1 is thought to be a typical mesenchymal stem cell marker
(Kolf et al. 2007), and STRO-1+ DPSCs isolated from rat dental pulp
tissues are high-quality stem cells (Yu et al. 2010). STRO-1+ cells
locate within the perivascular cell population and express the pericyte
marker 3G5 (Shi and Gronthos 2003).
Notch signaling is evolutionarily conserved and operated in many
cell types and at various stages during development (Bray 2006;
Andersson et al. 2011; Guruharsha et al. 2012). Subodontoblastic
cells under the newly formed odontoblast-like cells highly express
Notch2, suggesting the involvement of Notch signaling in the
differentiation of DPSCs into odontoblast-like cells (Mitsiadis et al.
1999; Mitsiadis et al. 2011). In a rodent incisor model, Notch and
FGF10 are involved in the maintenance of stem cell niches located
at the end of the tooth (Harada et al. 1999; Harada et al. 2002), and
Shh released from nerve bundle is associated with the maintenance
of Gli1+ stem cells in the apex of incisors (Zhao et al. 2014). Rodent
incisors continue to grow throughout life, and their roots develop
continuously. Therefore, stem cell niches are present in their apex.
Dental pulp regeneration
The progression of caries lesions induces pulpal inflammation, and
total pulp removal (pulpectomy) is used to treat irreversible pulpitis
and prevent the establishment of pulp necrosis and apical
periodontitis. In the resultant “pulpless” or “non-vital” teeth, marked
progression of caries lesions can easily occur because of the loss of
the alarm system (pain), immune defense system, and physical barrier
formation (reparative dentin). Tooth root fractures can also occur in
non-vital teeth. Therefore, the conservation of pulp tissue is essential
for increasing tooth longevity. The pulp revascularization procedure,
which involves the regeneration of pulp tissue by host blood, bone
marrow, or periodontal ligament-derived stem cells in immature
© 2016 Japanese Teratology Society
N. Kawashima and T. Okiji
non-vital teeth, is one of the current hot topics in clinical endodontics
(Wigler et al. 2013). In this procedure, hard-tissue formation is
observed in successful cases, but the resultant hard tissue is not always
dentin-like and can exhibit a bone/cementum-like structure instead
(Wang et al. 2010c). Thus, regeneration of the dentin/pulp complex
via the transplantation of DPSC into the root canals of non-vital teeth
has recently gained much attention as a future therapeutic approach
(Nakashima and Iohara 2014). During pulp regeneration therapy, it
is very important to induce both hard and soft tissue regeneration.
The in vivo transplantation of DPSC using scaffold materials, such
as hydroxyapatite and poly(lactic-co-glycolic acid), results in hard
tissue formation by the transplanted DPSC (Gronthos et al. 2000;
La Noce et al. 2014). However, it remains unclear whether the hard-
tissue-forming cells differentiate from the transplanted DPSC or from
host tissue-derived stem cells whose differentiation is induced by the
transplanted DPSC. However, it is known that the hard-tissue-
forming cells express odontoblastic marker genes, such as the DSPP
gene. The hard tissues that form after the transplantation of DPSC
exhibit bone-like features instead of dentin-like features (Gronthos
et al. 2000; Huang et al. 2010). Such formation of bone-like tissue
(which is known as osteodentin) is also observed after direct pulp
capping, a clinical method in which a protective material such as
calcium hydroxide or mineral trioxide aggregate is applied to exposed
vital dental pulp tissues to induce the closure of the exposed site with
new hard tissue (Citron 1977; Nowicka et al. 2013).
Prior to the in vivo transplantation of DPSC, root canal dentin can
be treated with sodium hypochlorite solution to remove organic
substances (remaining pulp cells, bacteria, etc.), which is a routine
endodontic procedure. However, it should be noted that such
chemical modification of the dentin surface might negatively
influence the differentiation of odontoblasts that are in contact with
dentin (Ring et al. 2008; Trevino et al. 2011). Thus, the optimal root
canal irrigant(s) for this purpose requires further research before the
establishment of pulp regeneration therapy.
CONGENITAL ANOMALIES IN DENTIN
(TABLE 2)
Dentinogenesis Imperfecta Type I (DGI-I)
DGI-I is the oral manifestation of deficient collagen formation that is
mainly associated with osteogenesis imperfecta (OI; See the “6.6
Osteogenesis Imperfecta” section below). DGI-I demonstrates dentin
alterations similar to DGI-II and DGI-III, such as defective dentin
mineralization (Majorana et al. 2010; see below)
 Odontoblasts and the dentin-pulp complex 149

Table 2 Congenital anomalies in dentin

Syndrome Type OMIN* Inheritance** Gene Prevalence

Dentinogenesis Imperfecta (DGI) I see osteogenesis imperfecta (OI) type I-A
II 125490 AD DSPP (4q22.1) 1 in 6000–8000
III 125500 AD DSPP (4q21.3)
Dentin Dysplasia (DD) I 125400 AD SMOC2 (6q27) 1 in 10000
II 125420 AD DSPP (4q21.3)
Osteogenesis Imperfecta (OI) 1 in 10 000–20000
I-A 166200 AD COL1A1 (17q21.31-
q22.05)/COL1A2 (17q22.1)
III*** 259420 AD/AR
IV-B 166220 AD
X 613848 AR SERPINH1 (11q13)
Ehlers-Danlos syndrome I 130000 AD COL1A1 (17q21.33)/COL5A1
(9q34.3)/COL5A2 (2q32.2)
VII-C 225410 AR ADAMTS2 (5q35.3)
Spondylometaphyseal dysplasia 184260 AR COL1A1, COL2A1
associated with joint laxity and DGI
Elashy-Waters Brachio-skeleto-genital 211380 AR ?
syndrome
Immuno-osseous dysplasia, 242900 AR SMARCAL1 (2q35)
Schimke type

* Online Mendelian Inheritance in Man® (http://www.omim.org/)

** AD: Autosomal dominant, AR: Autosomal recessive
*** with DGI (50%)

Dentinogenesis Imperfecta Type II (DGI-II)
In the DGI-II, anomalies are typically observed in dentin. The color of
dentin is bluish-brown, amber and opalescent, and dentin is easily
worn away by occlusion (Barron et al. 2008). The shape of crown is
bulbous and cervical constriction at the root junction is observed in
X-ray photographs. The pulp chamber is narrow or totally obliterated.
Roots are short. Both deciduous and permanent teeth are affected. The
enamel tends to chip from the underlying softer dentin and variable
degrees of attrition can be observed, especially in the permanent
dentition. The number of crystallites decreases and thus a significant
decrease in the total mineral content is observed in dentin. Estimated
incidence in the United States is between 1:6,000 and 1:8,000
(Witkop 1957).
Mutations in the DSPP gene have been identified in several
congenital dentin anomalies, including DGI-II and III. The absence
of DSPP could affect the functions of odontoblasts, such as secretion
of other extracellular matrix proteins and mineral ion transport, leading
to compromised dentin formation. In fact, DSPP KO mice show
widened predentin area and defective dentin mineralization, which is
a similar phenotype to DGI-II and III patients (Sreenath et al. 2003).
Full-length DSPP is cleaved to yield dentin sialoprotein (DSP) and
dentin phosphoprotein (DPP), two principal noncollagenous proteins
of mature dentin matrices (MacDougall et al. 1997), and DPP and
DSP are present in approximately 10:1 (Prasad et al. 2010). DPP
may be involved in the nucleation and modulation of apatite crystal
formation (Prasad et al. 2010). DPP is also reported to work as a
coactivator in bone morphogenetic protein-2 (BMP2) (Saito et al.
2004), and activate the Smad signaling (Jadlowiec et al. 2006). In
DPP KO mice, disturbance of dentin mineralization similar to
DSPP KO mice is observed, but dentin volume of the DPP KO
mice is thicker than that of DSPP KO mice (Suzuki et al. 2009).
These findings suggest that both DSP and DPP contribute to the
initiation and maturation of mineralization, but DPP is mainly
involved in the dentin mineralization. In DGI-I and II, odontoblast
differentiation is commonly disturbed in its early stage (Takagi and
Sasaki 1988), but differentiation of odontoblasts and dentin
formation are induced in DGI.
In teeth suffering DGI-II, exposed dentin is susceptible to caries
and control of oral hygiene including application of fluoride by
professional oral hygienists is essential. In order to prevent the
attrition, restoration using metal crown and/or composite resin may
be recommended (American Academy on Pediatric Dentistry Council
on Clinical A 2008).
Dentinogenesis Imperfecta Type III (DGI-III)
DGI-III was found in a population of Brandywine (an inbred tri-
racial population) isolated from Maryland and Washington DC.
Phenotype of DGI-III is more severe than that of DGI-II (Dong
et al. 2005).
Dentin Dysplasia Type I (DD-I)
Typical phenotype of DD is short rooted teeth (rootless teeth) with
sharp conical apical constrictions and irregular hyper-growth of
dentin in the pulp chamber, which induces total pulpal obliterations
(Wesley et al. 1976). The enamel and coronal dentin are well formed,
but the radicular dentin lacks histological organization (MacDougall
et al. 2006). Both dentitions are affected. The candidate gene of
DD-I is reported to be SMOC2 (Bloch-Zupan et al. 2011).
Dentin Dysplasia Type II (DD-II)
Clinically the primary dentition in DD-II appears opalescent, and
the pulp chambers are obliterated, which resembles DGI. However,

© 2016 Japanese Teratology Society

150
unlike DGI, the permanent teeth in DD-II are normal in color and
on radiographs have a thistle-tube pulp chamber configuration with
pulp stones (Dean et al. 1997). An asp6-to-tyr missense mutation in
the bicistronic DSPP gene is identified in a family with DD-II
(Rajpar et al. 2002).
Osteogenesis Imperfecta (OI)
Osteogenesis imperfecta (OI) comprises a heterogeneous group
of diseases characterized by the susceptibility to bone fractures
with variable severity and, in most cases, with presumed or
proven defects in collagen type I biosynthesis (van Dijk et al.
2011). In approximately 90% of individuals with osteogenesis
imperfecta, mutations in either of the genes encoding the pro-
α1 or pro-α2 chains of type I collagen (COL1A1 or COL1A2)
can be identified (Basel and Steiner 2009). Other clinical
manifestations include short stature, blue sclerae, dentinogenesis
imperfecta, and hearing loss (van Dijk et al. 2011). OI is
classified into Type I to XVII, but types I and IV account for
considerably more than half of all OI cases (Steiner et al.
1993). Subtypes of OI accompanied by DGI are Type IA
(Paterson et al. 1983), III (Basel and Steiner 2009), IVB (Levin
et al. 1978), and X (Christiansen et al. 2010).
DGI-associated syndromes
Abnormal dentin formation with variable features is also observed in the
following syndromes; Ehlers-Danlos syndrome type I (De Coster et al.
2007) and type VIIc (De Coster et al. 2007), spondylometaphyseal
dysplasia associated with joint laxity and dentinogenesis imperfecta
(Goldblatt et al. 1991; Bonaventure et al. 1992; Unger et al. 2008),
Elashy-Waters Brachio-skeleto-genital syndrome (Castori et al. 2010),
immuno-osseous dysplasia, Schimke type (da Fonseca 2000), and
odontodysplasia (Regional odontodysplasia, not listed in Table 2,
Kantaputra et al. 2002).
CONCLUDING REMARKS
Odontoblasts are long-living post-mitotic cells that are specialized
to form dentin. However, there is increasing evidence to support the
notion that odontoblasts also function as nociceptors and defensive
cells in the dental pulp. The regeneration of odontoblasts from
DPSC takes place when odontoblasts are damaged by severe
noxious stimuli. Various factors are involved in the differentiation
of the progenitors of odontoblasts, but the precise mechanisms
responsible for odontoblast differentiation remain unclear.
Recently, total pulp regeneration via the transplantation of DPSC
into the pulp chamber has been demonstrated in animal models
(Iohara et al. 2011; Nakashima and Iohara 2014). Elucidating the
mechanisms responsible for odontoblast differentiation would
allow the induction of effective odontoblast differentiation from
DPSC or other stem cells, which would enable the development
of clinical whole pulp regeneration methods involving complete
odontoblast differentiation.
ACKNOWLEDGEMENTS
The authors thank Drs Mioko Yamamoto, Sonoko Noda, Kentaro
Hashimoto, Michiko Tsuji, Shoichi Suzuki, Toshiko Furutera, and
Sachiko Iseki (TMDU) for their contribution to this review. This
research was partially supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology, Japan (MEXT grants #25293386 and #25253101
to NK).
© 2016 Japanese Teratology Society
N. Kawashima and T. Okiji
DISCLOSURE
The authors declare that they have no conflicts of interest.
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