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Endodontic Topics 2012, 20, 5284 2012 John Wiley & Sons A/S
All rights reserved ENDODONTIC TOPICS 2012
1601-1538

The dentinpulp border:

a dynamic interface between hard
and soft tissues
LEO TJDERHANE & MARKUS HAAPASALO

The dentinpulp border is a dynamic interface where odontoblasts form the organic matrix and mineralize it to
form dentin. Even though dentin and odontoblasts are often compared to other mineralized tissues (bone and
osteoblasts), dentin is in many ways a unique tissue, and odontoblasts are unique cells with distinct morphological
and functional differences from osteoblasts. Unlike osteoblasts, odontoblasts may remain vital for the persons
lifetime. Dentin formation and mineralization is also in some ways different from the bone formation. Dentin
contains dentinal tubules that may be patent all the way from the dentinenamel junction to the dentinpulp
border, making dentin (and pulp tissue) theoretically accessible to oral microbes and other noxious stimuli during
or immediately after the destruction of enamel or cementum covering dentin. Odontoblasts are the first cells
facing the external milieu (oral cavity), and together with the pulp tissue they must be able and ready to react to
external microbial or other irritations. Recent discoveries actually indicate that odontoblasts are better equipped
to respond to external stimuli than previously believed. In addition to forming dentin, odontoblasts may have
sensory functions, and they may also sense and respond to microbial antigens in much the same manner as
immunological cells. Both of these functions indicate that odontoblasts are more active in the regulation of the
defensive reactions of the dentinpulp complex, including fine-tuning pulp inflammatory reactions. The aim of
this review is to provide an update on the current knowledge of the different aspects of dentinogenesis, as well as
the potential roles of odontoblasts on other functions in the dentinpulp complex.

Received 25 September 2010; accepted 17 July 2011.

Differentiation of odontoblasts and
initial dentin formation
Odontoblasts are derived from embryonic connective
ectomesenchymal cells from the cranial neural crest.
The primitive oral epithelium thickens and prolifer-
ates into the underlying ectomesenchyme, resulting in
the formation of the dental lamina, the enamel organ,
and the dental papilla. A number of epithelial
ectomesenchymal interactions participate in the initia-
tion of tooth formation (1,2). In the early phases of
odontogenesis, the proliferation of epithelial cells
occurs at the bud, cap, and bell stages. The differen-
tiation of odontoblasts begins at the tip of the future
cusps at the early bell stage, after the shape of the
crown has been established. Signaling molecules from
the primary and secondary enamel knots regulate the
differentiation of the post-mitotic outer cells of the
dental papilla into odontoblasts (3,4) (Fig. 1). A
number of growth factors have been indicated to par-
ticipate in the process (5), but it is also possible that
some other proteins such as amelogenin or even pep-
tides may participate in this inductive phenomenon
(6). The differentiating odontoblasts start the secre-
tion of predentinal proteins before the initiation of
enamel matrix secretion by ameloblasts.
During the early stages of odontogenesis, from the
initiation to the final bell stage where cells undergo
final differentiation, the basement membrane (BM) is
present in epitheliomesenchymal interactions. The

52

Fig. 1. Schematic representation of the association of
enamel knotdental papilla interplay during tooth mor-
phogenesis and odontoblast differentiation. (a) During
the cap stage, the primary enamel knot expresses signal-
ing molecules, which regulate the formation of the
dental papilla (arrows). (b) During the bell stage, signals
from the secondary enamel knots induce the initiation of
odontoblast differentiation (arrows). (c) During the late
bell stage, the odontoblast (OB) differentiation pro-
ceeds. After initiation of odontoblast differentiation, the
differentiation signals may come from the enamel knot
(black double-ended arrows), from the further differen-
tiated odontoblasts (white arrow), or from both. The
basement membrane (BM) is degraded and gradually
disappears. The odontoblasts secrete mantle dentin
(MD) and induce the terminal differentiation of amelo-
blasts (AB) (black arrows). Modified from Thesleff
et al., 2001 (4).
Dentinpulp border
basement membrane is essential for odontoblast dif-
ferentiation, indicating cellmatrix interactions in the
signaling between the two tissues (3) (Figs. 1 and 2).
Type III collagen disappears from the BM during
terminal odontoblast differentiation (Fig. 1), while
fibronectin surrounding pre-odontoblasts accumulates
with decorin at the apical pole of polarizing cells. Other
proteins related to the BM and possibly involved in
odontoblast differentiation include at least tenascin
and amelogenin (3). Signals from the odontoblasts
then pass to the overlying epithelium, triggering the
terminal differentiation of ameloblasts. Ameloblast dif-
ferentiation has been shown to require the presence of
functional odontoblasts or predentin matrix (7,8)
(Fig. 1). At the onset of predentin formation, the BM
disappears, presumably degraded by matrix metallo-
proteinases (MMPs) (9), most likely controlled by the
tissue inhibitors of matrix metalloproteinases (TIMPs)
(10). The distal part of pre-secretory ameloblasts pro-
trudes into the non-mineralized predentin, and the
initial inner aprismatic layer of enamel starts to form
along the mantle dentin. For a detailed description of
the molecular mechanisms controlling the cytodiffer-
entiation of tooth-forming cells, the reader is referred
to a recent review by Tompkins (5), and the genes
considered essential in each phase of the development
are reviewed by Tummers & Thesleff (11). It is inter-
esting to note, however, that out of 65 gene defects
known to affect tooth morphogenesis and formation in
transgenic mice, only 11 are related exclusively to
dentin or root (12).
After differentiation, odontoblasts begin the forma-
tion of the organic matrix. The first collagen fibrils are
large (0.10.2 mm) in diameter and arranged at a right
angle to the BM between the epithelium and dental
papilla. These collagen fibrils, together with the dental
papilla ground substance, constitute the organic
matrix of the mantle dentin. Odontoblasts increase in
size, contact each other to form a uniform cell layer,
and begin to move toward the pulp, leaving behind
the odontoblast process. At the same time, the forma-
tion of apatite crystals begins, first in the matrix vesi-
cles (MVs) produced by odontoblasts (13,14). MVs
are 50150 nm spherical, membrane-limited bodies
only visible with a transmission electron microscope
(TEM). They are believed to originate by budding
from the plasma membrane, even though the mem-
brane composition is somewhat different from the
parent membrane. MVs are enriched with tissue-
53
Tjderhane & Haapasalo
Fig. 2. Principles of odontoblast life cycle. Pre-
odontoblasts are short (approximately 15 mm) cylindral
cells in which the polarization of cell organelles is mainly
limited to the nucleus. The cell is still closely associated
with the inner dental epithelium (Ep) and basement
membrane (BM). Secretory odontoblasts increase in
size with numerous and highly polarized intracellular
organelles. Transitional odontoblasts, with reduced
secretory activity, become narrower and have a lower
level of polarization of organelles. The autophagic vacu-
oles, most likely responsible for the degradation of the
intracellular components, appear. Finally, aged odonto-
blasts have a small number of organelles, a nucleus
located more centrally in relation to other organelles,
and large vacuoles. They decrease in size and polariza-
tion, together with a small size and number of rough
endoplastic reticulum, Golgi apparatus, and mitochon-
dria. Primary cilia are present in all stages. Ep: inner
dental epithelium; BM: basement membrane; OP: odon-
toblast process; PD: predentin; JC: junctional complex;
SG: secretory granule; G: Golgi apparatus; Ce: centriole;
cl: primary cilia; m: mitochondria; Col: collagen; rER:
rough endoplasmic reticulum; N: nucleus; nu: nucleolus;
Va: vacuole. Figure reproduced with permission from
Couve, 1986 (252).
nonspecific alkaline phosphatase (TNAP), nucleotide
pyrophosphatase phosphodiesterase (NPP1/PC-1),
annexins (ANX), phosphatidyl serine (PS), and matrix
metalloproteinases (MMPs) (15). TNAP is the most-
54
studied of the MV proteins and has long been impli-
cated as a key factor in mineralization. Still, even
though the role of MVs in the initial mineralization
of dentin and other hard tissues is generally accepted,
the exact nature and extent of that role is controver-
sial (15), and the mechanisms through which MVs
initiate matrix mineralization in these tissues remain
unclear. It is known, however, that the first dentin
mineral crystals appear within MVs, in a manner
similar to that of immature bone and calcifying car-
tilage (16). After the crystallites rupture the vesicles,
their growth continues until they fuse with adjacent
crystallite clusters (17,18). MVs have also been impli-
cated to be involved in the mineralization of repara-
tive dentin under injury (19,20), but not in
physiological mineralization of circumpulpal dentin
(i.e. physiological primary and secondary dentin
formed after mantle dentin formation) (21).
The mechanism by which mineralization spreads
from the MVs to the surrounding organic matrix is not
completely clear since collagen alone does not induce
matrix-specific mineral formation from calcium phos-
phate solutions (22). Dechichi and co-workers (23)
investigated the relationship between proteoglycans
and collagen fibrils during the early mantle dentin
mineralization process. Using subsequent EDTA and
chondroitinase treatments, the authors proposed a
model of interaction between proteoglycans and col-
lagen fibrils in the initiation of mineral crystal nuclea-
tion in mantle dentin (Fig. 3a). In this model,
proteoglycan would concentrate in the collagen gap
zone, with the protein core surrounding the collagen
fibril, and glycosaminoglycan (GAG) chains would
penetrate inside the fibrils. Inside the fibril, the
calcium ions related to the GAG chains would start the
crystal nucleation (23). Further work is needed to
confirm the model, as well as to examine whether or
not this would also be the collagen mineralization
sequence in circumpulpal dentin.
As well, it has been suggested that the proteinaceous
continuum between enamel and dentin and the orien-
tation of enamel proteins by dentin crystals control the
initial mineralization in the DEJ (24). The proteins
responsible could be, for example, amelogenins
secreted by ameloblasts (25) and tuftelins secreted by
odontoblasts during the initial phases of DEJ develop-
ment (26).
Mineralization of the outer dentin layer could
induce an immobilization of these proteins (24).
 Dentinpulp border

Fig. 3. Schematic representation of proposed mechanisms of collagen mineralization in dentin. (a) Suggested specific
stereochemical arrangement of proteoglycan molecules and collagen fibrils in the gap region, initiating the intrafibrillar
hydroxyapatite crystal formation in mantle dentin. GAG: glycosaminoglycan with Ca2+ ions. Modified from Dechichi
et al., 2007 (23). (b) Extrafibrillar mineral plates are present between individual collagen fibrils. The intrafibrillar
minerals are located in the gap region between collagen molecules. The size and orientation of the individual collagen
molecules create 40 nm gap zones with 27 nm overlap zones, which give the collagen the typical periodicity. Modified
from Bertassoni et al., 2009 (116). (c) Control of intrafibrillar mineralization prior to extrafibrillar mineralization via
a mechanism called mineralization by inhibitor exclusion (121123,150). In the extrafibrillar space, inactive crystal
nucleator(s) are activated by tissue-nonspecific alkaline phosphatase (and possibly other enzymes). Activated nucleators
initiate the crystal formation. Fetuin interacts with small initial crystals, inhibiting crystal growth. Small crystals
(< 6 kDa protein size) can enter the intrafibrillar space, but fetuin can not due to its large size. In the intrafibrillar space,
crystals can grow since the inhibitor is excluded. Modified from Price et al., 2009 (123).

However, the origin of these proteins is still unknown,
as differentiating odontoblasts seem to transiently
produce enamel proteins, such as amelogenin, and
differentiating ameloblasts may produce some dentin
proteins (27). Perhaps the only protein that can safely
be excluded from this group is ameloblastin. Amelo-
blastin is the second most abundant extracellular
matrix protein produced by the ameloblasts (28), and
transiently expressed by differentiating odontoblasts
(2931), during tooth root formation (32) and
craniofacial bone development (33). However, appar-
ently normal dentin can be seen in ameloblastin
knockout mice despite a practically complete loss of
enamel formation (34). Regardless of the mechanism,
the mineralization of organic matrix completes the
formation of the first layer of dentin in dental papilla,
called mantle dentin.
Odontoblasts
Dentinogenically active odontoblasts are tall cells, and
their morphological polarization is unique in the
group of collagen-synthesizing cells. Odontoblasts
form a single layer of cells between dentin and pulp,

55
Tjderhane & Haapasalo

Fig. 4. (a) Schematic representation of the dentinpulp border. Odontoblast processes (OBP) penetrate through
predentin (PD) into mineralized intertubular dentin (ITD) in dentinal tubules. The cell processes are devoid of
cytoplasmic organelles and do not make contact with the tubular walls. Peritubular dentin (PTD) formation does not
start immediately at the mineralization front, but some distance from it, still without direct contact with the
odontoblast process. Modified from Mjr 1985 (285). (bc) Odontoblast cell layer (arrowhead) firmly attached to
mineralized dentin (MD) covering the pulp chamber wall in a fractured human tooth. The terminal ends of the
odontoblast in the pulp chamber away from the fracture line are partially covered with the cell-poor layer of Weil
(arrow). (b) Higher magnification of the odontoblastpredentinmineralized dentin interface. Reproduced with
permission from Tjderhane et al., 1998 (96).

numbering up to 45,000 cells per mm2 (35). The cell
body is located on a pulpal wall of dentin, and cyto-
plastic processes are inserted into tubules of mineral-
ized dentin. The cell bodies are from 20 to 40 mm tall,
depending on dentinogenic activity, and 3 to 5 mm
wide (Fig. 2; Figs. 4a and b). A large nucleus is located
in the basal portion of the cell, close to the pulp,
while the well-defined Golgi apparatus, rough endo-
plastic reticulum (RER), and several mitochondria are
located in a dentinal direction from the nucleus.
Numerous membrane-bound granules, filaments, and
microtubules are also present in the cytoplasm (17,
18) (Fig. 2; Fig. 4a).
The polarity of odontoblasts has been based on their
columnar morphology at the secretory phase and their
highly polarized spread of cytoplasmic organelles (35).
However, we have recently demonstrated that the
odontoblast plasma membrane is also polar, confirm-
ing the truly polarized nature of the cells (36,37).
These new findings indicate that the odontoblast
process plasma membrane is basolateral, and the cell
body facing the pulp tissue is apical. This type of
distribution of apical and basolateral membrane
surfaces is similar to that found in epithelial cells (38),
but opposite to that of osteoblasts (39). While the
exact role of odontoblast membrane polarity is not

56

known, it may be formed during odontoblast differ-
entiation, relate to the polarized secretory activity of
the cell (i.e. dentinogenesis), reflect the role of nutri-
ent uptake through the apical membrane (on the
pulpal side) (37), or be related to the possible sensory
function of the odontoblasts (40) (for details, see
below).
Adjacent odontoblasts in a secretory phase are
attached together with extensive junctional complexes
and with gap junctions. The junctional complex con-
sists of a tight junction (known as the zonula occlu-
dens), a zonula adherens, and a desmosome-like
junction (35). Tight junctions are thought to partici-
pate in the final polarization of odontoblasts, leading
to the redistribution of organelles and polarized exo-
cytosis (41). These junctions form a stable barrier
between cell bodies and predentin (4244), where
their role may be to restrain the transportation of
mineral ions between the cells (4446). In developing
rat teeth, tight junctions between differentiating
odontoblasts contain abundant amounts of zona
occludens-1 (ZO-1, also called tight junction
protein-1, TJP-1), a cytoplasmic junctional protein
closely related to intracellular actin filaments. Occludin
and claudins, two classes of transmembrane proteins
functionally related to the sealing of tight junctions
(4749), are present in fewer quantities. ZO-1 and
claudin-1, -10, and -11 are also present in mature
human odontoblasts (37). However, occludin is not
present in secretory rat (49) or human (37) odonto-
blasts. The role of occludin in tight junction formation
and function has recently been questioned, while ZO
seems to be essential (reviewed in 50). Therefore, the
absence of occludin in post-differentiation odonto-
blasts does not necessarily indicate impaired tight junc-
tion function.
Unlike other polarized cells (e.g. epithelial cells),
odontoblast tight junctions may be more related to
crescent cell polarization and differentiation than to
cell layer permeability (51). Opinions about the
tightness of the junctions between odontoblasts vary
(52), and it has been shown that the stability of the
barrier is disturbed at least as a response to trauma
(45,46,53,54). Additionally, interodontoblastic colla-
gen fibers, so-called von Korffs fibers, may pass
through the odontoblastpredentin layer from pulp
into dentin (44,55,56), and lanthanium was
observed to penetrate predentin in small amounts at
the locations of these fibers (44).
Dentinpulp border
Odontoblast processes
The odontoblast cell process is a gradually narrowing
cytoplasmic process that penetrates into mineralized
dentin, filling the lumen of dentinal tubules. The
process is composed of the main trunk, with a diameter
of 0.5 to 1 mm, and of thinner (0.10.2 mm) lateral
branches (Fig. 5). The odontoblast process lacks cell
organelles but contains secretory vacuoles, numerous
longitudinally arranged actin filaments, and microtu-
bules (18,35).
One of the longest-lasting and most controversial
questions in research on the dentinpulp complex has
been the extent of odontoblast processes into dentinal
tubules. The controversies are often related to the
techniques used to study the processes, as discussed
extensively by Torneck (35). For example, scanning
electron microscopy (SEM) images have been used
both to demonstrate that odontoblast processes pen-
etrate throughout the dentin thickness, and to show
that the process length is only about 0.7 mm from the
dentinpulp border. Since SEM only shows the surface
structure, not the content of the process, the studies
claiming that the process reaches the DEJ have been
criticized as representing the tubular lining mem-
brane called the lamina limitans, not the process itself.
Transmission electron microscopy (TEM) with con-
ventional fixation has failed to demonstrate the odon-
toblast processes in outer dentin, also indicating that
the penetration depth would be limited. These studies
have been criticized for not being able to adequately
fix the processes deep in dentin, or for the artifactual
loss of processes in the outer dentin due to retraction
of processes by intracellular contractile elements in the
cell processes. Indeed, using cryofixation (which pre-
sumably prevents the contraction), odontoblast proc-
esses have been shown to reach the DEJ. However, it
has been claimed that cryofixation causes an outward
pressure from the pulp chamber toward the DEJ,
pushing the odontoblasts and their processes into
tubules. This would result in the presence of odonto-
blast processes in areas where they do not normally
exist. Immunohistochemical studies with antibodies
against cytoskeletal proteins (presumably strictly intra-
cellular) have shown that the majority of dentinal
tubules exhibit these proteins through the entire thick-
ness of dentin. Finally, cryofixation TEM studies have
questioned the actual presence of lamina limitans,
which actually may be only an artifactual finding rep-
57
Tjderhane & Haapasalo
resenting the condensate of intratubular proteins
caused by the fixation processes (35).
Byers & Sugaya (57) attempted to solve the contro-
versy by using carbocyanine dye Di-I and a gelatin
embedding technique to observe the cell membrane
lipids of odontoblast processes. They found that, while
the tubules stained all the way to the DEJ in developing
teeth, in the teeth of older animals (rat and monkey),
a Di-I technique and F-actin labeling, demonstrated that
b length of the processescan be as little as 0.1 to
c Higher magnification of the area marked with a square
58
the processes did not penetrate further than 0.3 mm
into coronal dentin, but to the outer third or even
occasionally dentincement junction in root dentin.
The findings were supported by Yoshiba et al. (58) in
human teeth, using the same method combined with
immunohistochemical staining of F-actin (a cytoskel-
etal protein) and TEM: even though the authors could
see faint labeling all the way to the DEJ with the Di-I
technique, they concluded that the staining was due to
a large amount of lipids present in mantle dentin (58).
To add to the confusion, Tsuchiya et al. (59), using the
the odontoblast processes penetrated all the way to the
enamel-free surface of cuspal dentin in rats regardless of
age, and to the DEJ of the cusp region in adult mon-
keys, while in root dentin the processes were withdrawn
with age (59). It would thus seem that, even though the
majority of studies indicate that the odontoblast cell
processes would not extend far from the dentinpulp
border, the issue still remains controversial.
The differences in observations in odontoblast
process penetration depth may alsoat least partially
be due to the different species used for the studies.
The thickness of mature dentinand the potential
0.2 mm in small animals such as mice, but up to and
even more than 5 mm in primates, including humans.
From a cell biology point of view, it may be difficult to
believe that the odontoblast processes would penetrate
through the full thickness of mature human tooth
dentin. Since dentin does not have a vascular supply,
Fig. 5. Cross-section of a mouse molar embedded in
acrylic resin and examined with back-scattered electron
microscopy. (a) The artefactual 10 mm fracture, caused
by the dehydration of the sample, exposed several odon-
toblast processes. Magnification 500 ; bar 10 mm. (b)
in Figure 5a. The width of the processes (1 to 2 mm)
indicates that they are main processes, instead of lateral
branches, which would be about 0.1 to 0.2 mm. Other
processes (arrows) run along the wall of the fractures at
sites where the dentinal tubules were cut along the
tubular direction. Magnification 5,000 ; bar 1 mm. (c)
SEM of dentin fracture parallel to the dentinal tubules.
Odontoblast processes are retained in some tubules, one
of them with apparent ramification (arrow), and one
partially loose process that has turned across the direc-
tion of the tubules. In one tubule where the process has
been lost, a narrow structure, possibly a nerve fiber, can
be detected (arrowheads).

large teeth should either need special mechanisms to
support very long cell processes in dentin or the proc-
esses should pull away after dentin exceeds a certain
thickness in order to stay near the vascular pulp (57,
60). According to Pashley (60), the ability of the
odontoblast cell body to support cytoplasmic proc-
esses of that length is questionable. In human teeth,
the volume of odontoblast processes in full-thickness
dentin would be approximately 2,300 to 2,400 mm3.
The volume of the columnar odontoblast cell body
would be approximately 600 mm3. Even more impor-
tantly, the diffusion distance from the nearest capillary
(providing oxygen and nutrients) would be 3 mm, a
distance not known to exist in any other cell in human
tissues. These calculations led Pashley (60) to con-
clude that it is unlikely that the odontoblasts could
maintain such a long process. However, it may also be
questioned whether oxygen or nutrient support
would be needed in close proximity of odontoblast
processes, since all of the cytoplasmic organelles
(nucleus, Golgi complex, etc.) are located in the main
body of the cell.
Dentinogenesis
In different biomineralization processes, it is always
the cell responsible for the synthesis of mineralized
tissue that directs the mineral formation process. The
biomineralization of collagenous matrix requires
several controlled events to occur simultaneously: (a)
cellular synthesis of extracellular matrix molecules,
including type I collagen and non-collagenous pro-
teins; (b) cell-derived accumulation of mineral ions;
(c) cell-directed modification of the matrix to allow
precise and controlled mineralization; (d) deposition
of initial mineral crystals; and (e) regulated growth and
accumulation of hydroxyapatite, perhaps accompanied
by further remodeling of the matrix (61,62). The rate
of dentin formation is dependent on the age and func-
tion of the tooth and the site in which the cell is
situated (63). Young odontoblasts are active in secre-
tion, while older cells produce predentin at a much
slower rate, although the formation never actually
ceases as long as the cell is alive.
A layer of unmineralized predentin separates odon-
toblasts from mineralized dentin. It consists mainly of
collagen, glycoproteins, and proteoglycans (35). In
this 10 to 30-mm-wide layer, the organic matrix organ-
izes before mineralization and, on the dentinal side of
Dentinpulp border
predentin, there is a sharp mineralization front in
which the organic matrix mineralizes (52).
Formation of extracellular matrix
Synthesis of matrix components during primary den-
tinogenesis occurs in the cell bodies, but the exo- and
endocytotic activities occur either directly from the
main cell body into the predentin, or in odontoblast
processes. The ECM for intertubular dentin is formed
in the predentin zone in which the collagen molecules
synthesized in odontoblast aggregate to form collagen
fibrils as well as the collagen mesh of dentin. During
the time between the initial collagen molecule aggre-
gation and mineralization of the ECM, the composi-
tion of the matrix is modified: components are added
and subtracted before reaching the mineralization
front. The density of this collagenous network
increases toward the mineralization front (Fig. 6a).
Secretion of collagen
The exocytosis of collagen occurs near the odontoblast
cell bodies, and before mineralization, the intra- and
intermolecular cross-linking takes place (Figs. 6 and
7). Mineralization occurs via heterogeneous nuclea-
tion instead of matrix vesicle formation; actually, cir-
cumpulpal dentin, formed after mantle dentin
formation, appears to be the only mesodermal hard
tissue in which matrix vesicles are not involved
in appositional mineralization (21). Odontoblasts
control the mineralization process by regulating the
composition of predentin and adding mineral ions and
other mineralization-related components into the
mineralization front.
The predentin maturates from the poorly organized
proximal zone (close to the odontoblast cell body)
into well-organized collagen matrix with precisely
localized non-collagenous components in the distal
zone (close to the mineralization front). During this
maturation process, the thickness and banding of col-
lagen fibrils change (64), indicating the dynamic
maturation of predentin collagen prior to mineraliza-
tion (Figs. 6be; Fig. 7). A similar maturation process
has been observed to occur during the conversion of
unmineralized osteoid into mineralized bone, includ-
ing thickening of collagen fibrils and a loss of non-
collagenous mineralization-related proteins (65). The
mechanically tight, interwoven structure and high
59
Tjderhane & Haapasalo

a b d

c e

Fig. 6. (a) TEM image at the odontoblastpredentinmineralized dentin interface. The mineralized dentin (MD)
exhibits high electron opacity, while in the underlying unmineralized predentin (Pd) with collagenous and non-
collagenous ECM components, the opacity gradually increases from the odontoblasts toward the mineralization front.
Since predentin is not mineralized, the increased opacity demonstrates the increased level of organization of the ECM,
with less free space between collagen fibers. An odontoblasts process (P) arising from the odontoblast cell body (O)
crosses the entire predentin and penetrates into a tubule of mineralized dentin. Some branches (double arrowheads)
of the odontoblast process are clearly observed in other odontoblast process. Magnification 5,400 . Reproduced with
permission from Arana-Chavez et al., 2004 (18). (bd) Organization of collagen in three zones of predentin. (b)
Proximal zone, with thin fibrils of approximately 30 nm diameter. The fibrils only fill about 30% of the available space.
No fibril bundles are observed. (c) Central zone, where the fibrils are 50 to 70 nm thick. The bundle formation can
already be seen, and the fibrils fill about 50 to 70% of the available space. (d) Distal zone: the collagen fibrils are closely
packed and bundle formation is obvious, with very little free space available. The increase in fibril diameter compared
to central and proximal zones is obvious, and is approximately 150 nm. Thus, during ECM maturation, the collagen
fibrils thicken and pack closer to each other, to ultimately form a dense network with extremely narrow spaces between
the fibrils. (e) Averaged banding patterns of collagen fibrils in dentin, bone, and soft tissue ECM: (1) proximal
predentin; (2) central predentin; (3) distal predentin zone. Clear change is banding, most likely reflecting the increased
order of the structure, but possibly also the differences in the non-collagenous proteins in the three predentin zones.
The banding of dentin collagen is virtually identical to that found in bone (4), and strikingly different from
non-mineralizable soft tissue collagen in rat tail tendons (5). Figures be reproduced with permission from Beniash
et al., 2000 (64).

level of intermolecular cross-linking are believed to
cause the high degree of resistance of dentin collagen
to thermal and proteolytic degradation (6669). The
same seems to apply to at least some non-collagenous
proteins (70).
The relatively constant thickness of predentin
throughout dentinogenesis has been proposed to indi-
cate that specific controlling events are involved in
ECM maturation (71). In many biological systems,
the control of connective tissue ECM is delivered via
regulated proteolysis and activation/inhibition cas-
cades by proteolysis involving enzymes such as matrix
metalloproteinases (MMPs) or cysteine cathepsins.
According to this widely accepted concept, it has
been suggested that the maturation and subsequent
mineralization of predentin involves (among other
factors) a series of proteinase and inhibitor events
(7274).
In order to explain the mineralization of the ECM,
the characteristics of collagen matrix have been
studied. As bone and dentin collagens differ from most
soft tissue collagens by the absence of type III collagen

60
 Dentinpulp border

Fig. 7. Factors involved in dentin matrix formation, maturation, and mineralization. (a) Ca2+ ion transport in
odontoblasts. Ca2+ ions are transferred from serum via Ca2+ channels and Na+/Ca2+-exchangers. To maintain physi-
ological cytosolic Ca2+ concentration, Ca2+-activated ATPase (Ca-ATPase) is needed for the extrusion of excess calcium.
Cell organelles participate in cytosolic Ca2+ activity buffering: mitochondria have an electrophoretic uniporter for the
intake and Na+/Ca2+-exchangers for the extrusion of Ca2+ ions. The Golgi complex and endoplastic reticulum are also
involved in Ca2+ regulation. Intravesicular Ca-ATPase-dependent accumulation of Ca2+ is needed for the controlled
transport of Ca2+ in the mineralization front. Even though odontoblasts are responsible for the majority of Ca2+
transport into the mineralization front, some calcium may also enter via an intercellular route despite the presence of
tight junctionsat least in cases of odontoblast cell layer integrity disruption (arrow with question marks). (bc) The
vast majority (in intact teeth, possibly all) of dentin organic matrix components are processed by odontoblasts and
secreted into predentin from the proximal part of the cell body (e.g. collagen), along the maturing predentin, or
directly at or close to the mineralization front (especially acidic dentin proteins responsible for mineralization, such
as DPP). The increase of DMP1 toward the mineralization front, and the differential presence of PGs (Dec: decorin)
or their GAG side-chains (KS: keratin sulphate; CS: chondroitin sulphate) in different parts of predentin indicate
either increased transportation along maturing predentin, or (most likely) enzymatic modifications of the proteins in
the predentin. At least MMP-2, -3, and -20, PHEX, and BMP1/Tolloid-like proteinases have been indicated to
participate in protein processing during predentin maturation. The concentration of MMP-3 has been shown to
increase toward the mineralization front, while MMP-2 levels seem fairly constant in predentin, but diminish at some
distance from the mineralization front. Due to protein processing, and since predentin contains proteins that are not
seen in mineralized dentin (e.g. type III collagen), odontoblasts also need to have a transport system that excludes the
unwanted proteins and degradation products from predentin. (d) Odontoblasts are responsible for the synthesis,
transport, and extrusion of dentinal fluid, proteins, and mineral ions needed for peritubular dentin formation and
tubular occlusion with age, even though at least part of the tubular occlusion (e.g. under caries or cavity preparations)
may be due to serum-derived proteins after disruption of the odontoblast cell layer integrity. The figure is modified
and data have been obtained from references 72, 142, 157, 231, and 286.

(75) and intermolecular cross-linking geometry (76, Non-collagenous proteins

77), it has been suggested that these factors are impor-
tant for mineralization (75). However, a more popular
idea seems to be that the collagen network primarily
functions as a support for non-collagenous dentin
proteins, which in turn initiate and control crystal
formation and growth (52,62,72,7882).
Various non-collagenous proteins are essential for
predentin organization, maturation, and mineraliza-
tion (Fig. 7). These proteins participate in collagen
fibrillogenesis and collagenous matrix organization,
prevent premature mineralization, and initiate miner-

61
Tjderhane & Haapasalo
alization when the matrix is ready to be mineralized.
Enzymes participate in the organization by modifying
the non-collagenous proteins in predentin, but pos-
sibly also by degrading and eliminating unwanted
collagenous proteins prior to mineralization. The
various roles of proteoglycans (PGs) and their
glycosaminoglycan (GAG) side-chains in predentin
metabolism are discussed in detail below. As the role
of glycoproteins in dentin formation is mainly con-
sidered to be the regulation of hydroxyapatite crystal
formation, their presence and role in dentino-
genesis is discussed later in relation to mineralization
mechanisms.
Overall, the research has highlighted the diverse
potential roles for the various proteoglycan (PG)
species and their degradation products in regulating
matrix formation, modulating cellular activity, and
directing hydroxyapatite crystal growth and morphol-
ogy. PGs are widely accepted as modulators of bio-
mineralization, including dentin mineralization (72).
PGs can be defined as one or more glycosaminogly-
cans (GAGs) covalently attached to specific core pro-
teins. Different PG profiles are present in the different
soft and mineralized tissues, and the PG profile of
dentin differs from that observed in predentin. Moreo-
ver, the concentration of PGs is different in different
parts of predentin (Figs. 7b and c), implicating their
significance in matrix maturation and the control of
mineralization (72,83) (Fig. 7). GAGs appear to be
important in dictating the functional characteristics of
the PG. The GAGs identified in predentin include
hyaluronan (HA), dermatan sulphate (DS), chondroi-
tin 4-sulphate (C4S), chondroitin 6-sulphate (C6S),
keratan sulphate (KS), heparan sulphate (HS), and
heparin. The initial pool of PGs may participate in
matrix collagen fibrillogenesis and organization, but
PGs act as inhibitors of mineralization. For example,
versican may be important in the initial formation of
the extracellular matrix by acting as a model or a
scaffold (83).
Hyaluronan (HA)-containing versican is practically
the only large aggregating proteoglycan observed in
the predentindentin interface (83). However, several
Small Leucine-Rich Proteoglycans/proteins (SLRPs)
are abundantly present in predentin, and widely con-
sidered essential in collagen fibril formation and
arrangement. SLRPs related to dentin formation
include biglycan, decorin, fibromodulin, osteoad-
herin, lumican, mimecan (osteoglycin), and asporin
62
(72,84,85). The function of the SLRPs depends on
both their core protein and the GAG side-chains
attached to the core protein. The core proteins allow
the SLRPs to interact with fibrillar collagen, regulat-
ing fibril diameter during formation and possibly
fibrilfibril interactions in the extracellular matrix.
They may also limit access of the collagen-degrading
enzymes to their cleavage sites, protecting the fibrils
from proteolytic damage (86). It has been proposed
that several predentinal SLRPs (at least decorin, bi-
glycan, osteoadherin, and fibromodulin) may par-
ticipate in collagen fibrillogenesis and/or matrix
organization and maturation prior to mineralization,
as well as in the regulation of the mineralization
process itself (72,83,8789). Recent studies have
indicated that biglycan, decorin, and fibromodulin
each have distinct and partially overlapping roles: they
all promote dentin mineralization, and biglycan and
fibromodulin also regulate collagen fibrillogenesis in
dentinogenesis (89,70). Biglycan and fibromodulin
increase the diameter of the collagen fibrils through-
out predentin, indicating the control of the lateral
fusion of collagen fibrils by inhibiting collagen
fibrillogenesis in predentin. The interaction of SLRPs
is not confined to the fibrillar collagens, as they may
also interact with many other macromolecules includ-
ing growth factors such as EGF, TGF-b, and TNF-a
(86).
While the PGs may inhibit mineralization during
dentin organic matrix formation and maturation, they
are also believed to promote dentin mineralization.
For example, the different terminal parts of the same
non-collagenous protein can act both as an inhibitor
and an inducer of the mineralization (72). The non-
uniform presence of PGs or GAGs in different parts of
predentin (Figs. 7b and c) most likely reflects the
changes in the predentinal matrix, and the different
roles of different PG components in matrix maturation
and mineralization. In addition, it has been suggested
that different pools of the same PGs as are present
(and modified by degradation) in predentin may be
separately secreted into the mineralization front, with
potentially different role(s) than in more proximal pre-
dentin (72,83). Conflicting data has been presented
regarding the role of SLRPs in the initial mineral
formation. PGs have been considered both as miner-
alization inhibitors and nucleators (91), depending on
the research models used (92). Most likely, the role of
PGs in mineralization varies in different parts of the

predentin mineralization frontdentin interface, and is
related to protein modifications. Dentinal PGs show
an increased binding affinity for hydroxyapatite during
the transition from predentin to dentin, with an
approximately 2.5-fold increase in affinity between
predentin and mineralization front PGs, and an
approximately 7.5-fold affinity increase between min-
eralization front and dentin PGs (93). The differences
may at least partially be related to the differences in
GAGs in different fractions (93).
In the predentindentin interface, SLRPs predomi-
nate, especially the chondroitin sulphate (CS) SLRPs
decorin and biglycan. Other SLRPs present include at
least the keratan sulphate (KS)-rich PGs fibromodulin
and lumican, and osteoadherin (72,83,87,88). The
apparent reduction in CS toward the mineralization
front (Figs. 7b and c) is conceivably a feature of MMPs
(Figs. 7c and d) cleaving the decorin protein core and
thus liberating a proportion of the GAG (72). Modifi-
cation of the PGs before or during calcification may
actually be an important or even the major function of
the neutral proteinases in predentin, as has been sug-
gested by the growth plate of the cartilage (94). The
elimination of GAG side-chains from the PG core
protein by MMPs (and possibly by other proteinases) is
believed to be the reason for the small amount of GAGs
in dentin (72). The core protein is believed to be
sequestered in the collagen gap zone, where it would
promote mineralization. Alternatively, GAG degrada-
tion may lead to the release of calcium (72,95), which
may contribute to the amount of calcium needed for
mineralization. The main candidates of PG modifica-
tions are the various MMPs (at least MMP-2, -3, -8, and
-9) synthesized by odontoblasts (9699).
In general, an absence of a single PG seems to have
an effect on mineralization. Loss of biglycan, fibro-
modulin, or decorin have all been shown to impair
dentin mineralization (89,90), albeit with different
severity, as the absence of decorin seems to have a
more pronounced effect than biglycan (89,90). As the
loss of different PGs causes different outcomes in con-
tinuously growing rodent incisors and in molars (89,
90), the possibility of the presence of compensatory
mechanisms to minimize the damage exists. Despite
the structural similarity, the compensation mechanisms
of dentin formation and mineralization do not seem to
involve changes in the expression of other SLRPs, but
rather the alteration of the expression and/or process-
ing of SIBLINGs (89).
Dentinpulp border
Phospholipids
There is some evidence that lipids, more specifically
phospholipids, may play a role in dentin formation and
mineralization (reviewed in 100). Phospholipids are
present in predentin, organized as a network of lipo-
somes or vesicles, and located between collagen fibrils.
Even though their role is not exactly known, they may
participate in dentinogenesis in several ways. They may
participate in the transportation of collagen fibrils
from proximal predentin to the distal part. In vitro,
lipoproteins have been shown to participate with PGs
in collagen fibrillation (101). This may also be the case
in predentin, as phospholipids share the same locations
as PGs in both predentin and dentin. Alternatively, the
interaction of phospholipids and PGs may be involved
in the mineralization process. PGs interacting with
lipids contribute to the formation of mineralized
atherosclerotic plaque (102104), and the same may
apply to dentin (100). Liposomes may contribute to
calcium transport toward the mineralization front. It
has been proposed that, since matrix vesicles are not
present in circumpulpal dentinogenesis, these lipo-
somes might take on the role of matrix vesicles, since
both matrix vesicles and liposomes are formed by lipids
(100). Phospholipids have been shown to be present
in peritubular dentin together with PGs (105), which
may support their collective contribution to the dentin
mineralization process. This finding is important since
the authors used the technique that allows for the
examination of untreated dentin (TOF-SIMS) (105,
106), which avoids the possibility of artifactual asso-
ciation caused by dentin treatments (100).
The origin of phospholipids in predentin and dentin
is also controversial. Odontoblasts are widely accepted
as the cells responsible for the constituents of dentin,
but this may at least partially not be the case with
phospholipids. Goldberg and co-workers (107) dem-
onstrated the incorporation of [3H]choline-labeled
lipid precursor in dentin prior to the uptake into odon-
toblast and predentin. The authors speculated that the
albuminfatty acid complex passes between odonto-
blasts, crossing the distal junctional complexes
between the odontoblasts, and diffuses into dentin
prior to any cellular uptake and phospholipid synthesis
by the odontoblasts (100,107). However, the work
was performed with continuously growing rodent
incisors, which may differ in many respects from teeth
with limited growth (52), such as human teeth.
63
Tjderhane & Haapasalo
Whether the same results would apply to human teeth
remains to be demonstrated.
Mineralization of
extracellular matrix
In dentinogenesis, calcium and other mineral elements
must be transported from the pulpal vascular network
across the odontoblastic cell layer and predentin to the
mineralization front of the predentindentin interface.
Extracellular enrichment of calcium in predentin indi-
cates calcium sequestration by predentin matrix com-
ponents (108110).
In mammalian mineralized tissues, the mineral forms
in two distinct type I collagen fibril places: (a) intrafi-
brillar (within the collagen fibrils between collagen
molecules) and (b) extrafibrillar (on the surface of the
collagen fibrils) (111) (Fig. 3). In both cases, the
mineral crystals are bound either directly to collagen or
through a link facilitated by non-collagenous proteins.
Most likely the apatite crystals first nucleate in the gap
zone, followed by secondary mineralization of the
interstitial positions between the fibrils (112) (Fig. 3b).
Mechanical properties of collagen fibrils in the direc-
tion of the long axis of the fibrils increase dramatically
with increased mineral content (112). The intrafibrillar
mineral is mostly responsible for the strength of min-
eralized dentin (113115). These findings may have
clinical significance: attempts to remineralize partially
or totally demineralized collagen should aim for crys-
tallization in the gap zones (115,116).
The mineral in bones and teeth is a poorly crystal-
line, carbonate-rich analog of the naturally occurring
mineral hydroxyapatite. The mineral contains consid-
erable amounts of carbonate (CO3) and variable
amounts of other ions, for example Cl-, F-, Mg2+, Sr2+,
Zn2+, and Pb2+. More than 45 elements are found in
trace amounts in dentin (117). Therefore apatite in
bone, dentin, and enamel has also been called bio-
logical apatite and carbonatoapatite. Ion substitu-
tion may occur within the crystal or in the water layer
absorbed on the apatite surface (117). Alterations in
the ion constituents of dentin mineral affect its crys-
tallinity, stability, and solubility.
Despite the apparent similarities, the mineral prop-
erties of bones and teeth are different. Dentin mineral
crystals are larger and more perfect than bone mineral
crystals, with different stoichiometries and composi-
tions (118). Boskey (62) suggests several factors that
64
may cause these differences in mineral properties.
Odontoblasts are more polarized than osteoblasts, the
rate of matrix formation is different, and the collagen
fibrils in predentin and newly formed dentin are
thicker and more oriented than in non-mineralized
osteoid and newly formed bone. As well, the extent to
which the tissue is remodeled after minerals are first
deposited is different. Osteocalcin, present in lower
concentrations in dentin where little or no remodeling
occurs, is important for recruiting osteoclasts to resorb
bone. Finally, the nature and relative amounts of the
mineral-associated proteins are different between bone
and dentin (62).
The induction of crystal nucleation calls for specific
nucleation sites with electric or other properties that
initiate the formation of solid calcium phosphates from
a solution (119). In addition, the regulation of growth
is believed to result from the interactions between
crystal and protein structures (62). Most of the pro-
teins associated with biomineralization attain their
anionic characters by post-translational modifications,
modulated by enzymes. The expression and activity of
these enzymes, as well as the nature of the modifica-
tions, may explain the tissue specificity that determines
where and when crystal growth will appear (62).
Circumpulpal dentin mineralization also seems to be
exceptional among hard tissues with respect to the
need for matrix vesicles in the mineralization process.
A certain population of matrix vesicles (6070/
100 mm2) is involved in the appositional mineraliza-
tion process in most mesodermal hard tissues, in
addition to their role in initial mineral induction in
these tissues. However, this is not the case in cir-
cumpulpal dentin formation, where matrix vesicles are
not required for the appositional mineralization
process (21). Instead, exclusive localization of dentin
phosphoproteins in circumpulpal dentin layers must
occur in order to facilitate appositional mineralization
at the calcification front (21).
Mineralization front
Even though standard histochemical staining may
indicate a very sharp mineralization front, the process
is most likely more gradual. Physical fixation (high
pressure freezing and freeze substitution) reveals a
0.5 to 2-mm-thick intermediary zone where only the
collagen fibers are mineralized. In fully mineralized
dentin, the intercollagenous spaces are also mineral-
 Dentinpulp border

ized (120). This transitional zone between predentin

and dentin, called metadentin, correlates well with
a
the proposed biomineralization scheme in which
intrafibrillar mineralization precedes interfibrillar
crystal growth (Fig. 3c) (121123) (for details, see
below).
The shape of the predentinmineralized dentin
interface is usually fairly linear, but mineralized globu-
lar protrusions also occur frequently (Figs. 8 and 9).
These protrusions, called calcospherites, are thought
to represent heterogeneous deposition of crystals,
which form the globules with continuous crystal
growth (124). Globular mineralization is most visible
in mantle dentin, and it is also believed to occur in
circumpulpal dentin when the dentin deposition b
occurs rapidly (124). However, when Tsurumachi and
co-workers (125) analyzed the mineralization front of
teeth from young (10 to 19 years) and older (46 to 62
years) patients from the pulpal side with SEM, marked
differences were observed between the groups. In
young subjects, the mineralization front in the pulp
chamber, cervical, and mid-root areas appeared as a
more or less level layer of fused calcospherites: only in
the apical root did approximately 30% of the teeth
present with a partially network- or ridge-like miner-
alization front. In the older group, fused calcosphe-
rites were still predominant in the coronal, cervical, Fig. 8. Structure of the mineralization front. (a) Histo-
and mid-root dentin (94%, 94%, and 75%, respec- logical appearance of the dentinpulp border. Predentin
tively) while in the apical part, approximately 80% of (PD) is clearly delineated by mineralized dentin (D) on
the teeth contained mostly other forms of the miner- the other side, and by the odontoblast layer (OB) on the
pulpal side. A distinct border between the odontoblasts
alization front. Also in the older group, spherical and
and non-mineralized predentin is readily seen (arrow-
structureless mineralizations, not seen in younger head). Also the mineralization front appears as a sharp,
teeth, were observed in the apical root (125). The but not straight, line. Instead, globular structures pro-
morphological appearance also indicates that the min- truding toward odontoblasts represent mineralization
through calcospherites (arrows). The apparent separa-
eralization in circumpulpal dentin is globular (Fig. 9).
tion of some calcospherites from the mineralization
However, with a slower dentin formation rate, the front is most likely due to the location of sectioning, and
mineralization front appears more linear in histological the calcospherite would actually be in contact with the
sections. The mineralization pattern may change with mineralization front in the adjacent area. CLW: cell-free
age; in apical dentin, irregularities in mineralization layer of Weil. (b) SEM image of the mineralization front
after removing all of the pulpal organic components
dramatically increase with age. mechanically and with sodium hypochlorite (NaOCl),
and fracturing the dentin. An uneven, undulating min-
eralization front due to the calcospherites is apparent.
Transport of mineral ions
In most cells, the intracellular Ca2+ concentration is an intracellular second messenger; several systems for
much lower than that in extracellular fluids (126), and slow or rapid Ca2+ translocation across cell membranes
most of the Ca2+ ions are complexed and stored in have been identified (126). The principles of odonto-
intracellular organic structures. The main goal of Ca2+ blast Ca2+ transportation are presented in Figure 7a.
transporting systems is to maintain a low and steady When calcified tissue is formed, mineralization of the
Ca2+ activity due to the importance of the Ca2+ ion as collagenous ECM requires a large influx of calcium and

65
Tjderhane & Haapasalo
a after removal of the root pulp tissue, odontoblasts,
b seen in the mantle dentin area) is not a common finding
c dependent intravesicular accumulation of Ca2+ ions
phosphate ions to form hydroxyapatite. Odontoblasts
actively transport calcium from pulp into predentin and
the mineralization front, as predentin has three times
more Ca2+ ion activity than pulp does (127,128). Sep-
arate mechanisms for the inflow and extrusion of Ca2+
function on cell surfaces (129). Voltage-gated Ca2+
channels (130,131) (L-type Ca2+ channels) play a cru-
cial role in calcium intake because their inhibition
significantly impairs radio-labeled calcium intake into
dentin (127). The channels seem to be highly selective
66
Fig. 9. Calcospherites in the mineralization front
and predentin with full-strength NaOCl. (a) A low-
magnification SEM image demonstrates the calcosphe-
rites uniformly cover the whole mineralized dentin
surface. (b) Higher magnification shows the calcosphe-
rites with different sizes and different phases of fusion
with adjoining calcospherites and mineralized dentin
below. The smallest calcospherites (approximately 8 mm:
white arrows) may contain only one or two dentinal
tubules, while the largest ones (from 20 mm to 40 mm:
white arrowheads) contain several tubules. (c) With high
magnification, the smooth continuity of the single cal-
cospherite with the underlying dentin, as well as the
continuity of the dentinal tubules (white arrows), is
apparent, explaining why globular dentin (frequently
in normal circumpulpal dentin in histological sections.
as the odontoblast layer in a healthy tooth is imperme-
able to lanthanum ion (La3+) (43,44), an ion
mimicking Ca2+. Ca2+ extrusion occurs through an
ATP-dependent process and Na+/Ca2+ antiport (132).
Ca2+ activated ATPase is present in dentinogenically
active odontoblasts (133135), and located in vesicle
membranes in the distal part of the cell body and in
the cell plasma membrane itself (132,136). ATP-
demonstrates the involvement of transport Ca-ATPase
(129,137,138). In osteoblasts, Ca2+ extrusion at the
matrix-facing pole of the cells (the mineralization site)
most likely occurs through Na+/Ca2+ exchangers, based
on the highly asymmetrical localization to the
substrate-adherent side of the cells (139). Na+/Ca2+
exchangers are expressed and functionally active in
native odontoblasts (132) and cultured odontoblast-
like cells (MRPC-1 cells) that form mineralized nodules
in culture (140,141), with a strikingly similar expres-
sion pattern to that of osteoblasts (140). Considering
the identical phenotypes of the odontoblast and oste-
oblast with regards to Na+/Ca2+ exchanger expression,
it has been hypothesized that these exchangers are
responsible for translocating Ca2+ ions into predentin
close to the mineralization front (140). While the
phosphate transport system in native odontoblasts is
not well known, the presence of functionally active
Na+-Pi co-transporters in MRPC-1 cells indicates that it
would be the mechanism for the odontoblasts to
provide phosphate for dentin mineralization (141).
Inside odontoblasts, Ca2+ fluxes are regulated by
transporters in different cellular membranes (Fig. 7a).

In addition to the plasma membrane, at least the endo-
plasmic reticulum and the mitochondrial inner mem-
brane participate through an active transmembraneous
transport system (142). When entering odontoblasts,
Ca2+ may be taken up by buffering organelles such as
mitochondria and the endoplasmic reticulum, or be
translocated directly to the plasma membrane at the
distal end of the cells, to be extruded into the extracel-
lular region at the mineralization front. Ca2+-binding
proteins may also regulate calcium fluxes between intra-
cellular compartments and in the interactions with cy-
toskeletal proteins and membrane components. In den-
tinogenically active odontoblasts, several Ca2+-binding
proteins (such as calmodulin, 28-kDa calbindin, parval-
bumin, and annexin VI) are present (143147).
It is interesting to note that with odontoblast-like
cells in vitro, dramatic changes in cellular Pi and Ca2+
uptake occur concomitantly with mineral formation,
but several days after the cell had established a mature
odontoblast-like phenotype, defined by the synthesis
of dentin-specific matrix proteins (141). This indicates
that initiation of mineralization is not merely depend-
ent on the synthesis and secretion of a matrix (which
is, of course, necessary to induce and regulate mineral
crystal formation), but also that functional and effi-
cient cellular Pi and Ca2+ ion membrane transport
systems are required for odontoblast-mediated miner-
alization (141).
Mechanisms of dentin matrix
mineralization
Type I collagen fibrils play several critical roles in
bone mineralization. Collagen fibrils provide the
aqueous compartment in which mineral crystals grow,
and water within the fibrils is then replaced with min-
erals. The physical structure of the collagen fibril acts
as a gatekeeper that allows molecules smaller than a
6 kDa protein to freely access the water within the
fibril, but prevents molecules larger than a 40 kDa
protein from entering the fibril (Fig. 3c) (121). Mol-
ecules smaller than a 6 kDa protein can directly inter-
act with apatite crystals growing within the fibril. One
of these small proteins related to biomineralization
could be osteocalcin. However, proteins that are too
large to penetrate the collagen fibril may still have
important roles in mineralization control. Some large
proteins present in dentin, such as osteopontin (148)
and fetuin (149), may potently inhibit apatite forma-
Dentinpulp border
tion or growth in vitro. These large protein inhibitors
of calcification may actually promote collagen fibril
mineralization by selectively inhibiting apatite growth
outside the fibril. This role has recently been proven
at least for fetuin (122,123,150). Another protein
that may be involved is bone sialoprotein, which ini-
tially acts as a nucleator of the apatite crystals; when
the mineral grows on the collagen matrix, it acts as an
inhibitor in directing the growth of the crystals
(151). Toroian and co-workers (121) suggest that
bone sialoprotein (BSP) (among others) may gener-
ate apatite crystal nuclei outside of the collagen fibril,
and that these small crystals (up to about 12 hydroxy-
apatite unit cells) can then diffuse into the interior of
the fibril and grow (121). Tissue-nonspecific alkaline
phosphatase (TNAP) is also essential in the process,
at least in vitro (Fig. 3c) (123).
Dentin acidic proteins
Dentin acidic non-collagenous proteins are believed to
control biomineralization by reducing the activation
energy required for nucleation and formation of
apatite. One of these proteins, widely considered a key
factor in biomineralization, is dentin matrix protein 1
(DMP1) (Fig. 7b). DMP1 belongs to the family of
SIBLING (small integrin-binding ligand, N-linked
glycoproteins) proteins (for review, see the article on
dentin non-collagenous proteins in the next issue).
DMP1 has a potent calcium binding capacity and
binds with high affinity to the N-telopeptide of fibrillar
collagen, located at the edge of the gap region of the
collagen fibril (152). It has been shown to participate
in the initiation and modulation of collagen minerali-
zation both in vivo and in vitro (152154). The ear-
liest localization of DMP1 in dentin is observed in the
Golgi region and nucleus of differentiating odonto-
blasts (155). Later, when mineralization spreads from
the matrix vesicles to the surrounding matrix, DMP1
is present extracellularly around the mineralizing glob-
ules. In fully mineralized mantle dentin, DMP1 is
present mainly at the peritubular dentin and at the
mineralization front between dentin and predentin
(155157), presumably the metadentin zone. As the
early unmineralized mantle dentin matrix does not
contain DMP1, it is likely that the onset of mineral
deposition into the matrix vesicles is independent of
DMP1, but the interactions between DMP1 and col-
lagen fibrils may be essential for matrix-mediated min-
67
Tjderhane & Haapasalo
eralization (152154). Indeed, Gajjeraman and
co-workers (158) demonstrated the in vitro minerali-
zation of type I collagen with DMP1, with great
resemblance to dentin in both ultrastructural (with
calcospherite-type mineralization) and mechanical
properties (elastic modulus). The fundamental role of
DMP1 in collagen matrix mineralization is further
indicated by the finding that the N-terminal domain of
DMP1 stabilizes amorphous calcium phosphate
during the growth phase and inhibits hydroxyapatite
formation, while the C-terminal domain is required
for crystal formation. This is supported by the recent
finding that the proteoglycan form of the DMP1
N-terminal fragment, called DMP1-PG (159), is
abundant in predentin (156). It is important to under-
stand that both type I collagen and DMP1 are
required for the synthesis of needle-shaped hydroxy-
apatite crystals with high crystallinity (158), as
observed in dentin and bone. These findings lend
strong support to previous suggestions that, in miner-
alized tissues, type I collagen defines the space neces-
sary for crystal growth, size, and morphology, while
acidic proteins such as DMP1 are important in the
nucleation, transformation, and growth of hydroxy-
apatite crystals.
Another non-collagenous protein important in
dentin mineralization is dentin sialophosphoprotein
(DSPP), or rather its proteolytically processed prod-
ucts, N-terminal dentin sialoprotein (DSP) and
C-terminal dentin phosphoprotein (DPP, also called
phosphophoryn, PP) (160). Dentin sialoprotein also
occurs in a proteoglycan form (161163), called
DSP-PG (163). Human and animal genetic studies
have shown that mutations in the DSPP gene result in
mineralization defects in dentin and bone (reviewed in
164). The chemical features of DPP and DSP are
remarkably different from each other, and considering
that they are encoded by a single mRNA, the amount
is also dramatically different (approximately 10 times
more DPP is produced). Originally, DSPP/DSP/DPP
were believed to be tooth-specific, but DSPP or its
product expression was later shown in bone, cemen-
tum, and some non-mineralized tissues, although in
much lower levels than in dentin (164).
As with the chemical structure, the localization and
roles in biomineralization of DSP and DPP also differ
significantly. DSP is more abundant in predentin than
in mineralized dentin (163,165,166), and in mineral-
ized dentin, it is localized with the odontoblast proc-
68
esses along the dentinal tubules (166,167), but not in
mineralized compartments (163). The functions of
DSP in dentin formation are not clear. At best, it may
act as a weak mineral nucleator (62,168), possibly
involved in the initiation of dentin mineralization, but
not in the maturation of this tissue (169). The pres-
ence of the DSP-PG in predentin, but not in mineral-
ized dentin, indicates that a major portion of the
proteoglycan form of DSP is removed prior to the
mineralization of collagen (163). Because of the com-
parative proteolytic processing and distribution of
DMP1 and DSPP N- and C-terminal products (160,
167), it would be tempting to speculate that DSP
(with a function similar to the N-terminal domain of
DMP1) would prevent premature mineralization,
while DPP (with a function similar to the C-terminal
domain of DMP1) would induce and control the
growth of mineral crystals.
DPP has a high affinity for type I collagen (170) and
readily binds large amounts of calcium (171,172),
forming insoluble aggregates in the presence of Mg
and Ca ions (173,174). Therefore, DPP is believed to
be directly involved in controlling the nucleation and
growth of hydroxyapatite crystals during dentin min-
eralization. In the predentindentin interface, DPP is
transported directly to the mineralization front (175,
176) (Fig. 7b), where it binds to collagen fibrils, pro-
moting the formation of initial apatite crystals. During
the mineralization process, DPP is believed to be one
of the proteins that bind to the growing hydroxyapa-
tite crystal faces and inhibit or slow crystal growth,
controlling the size and shape of the apatite crystals
(164).
With regard to other SIBLINGs in dentin forma-
tion, it has been suggested that BSP has a dual role in
nucleation induction and control of crystal growth
similar to that of DPP (177). Meanwhile, osteopontin
(OPN) has been indicated to act as an effective inhibi-
tor of apatite formation and growth (148,151,178
180).
Proteolytic enzymes in the dentin
mineralization process
Recent studies strongly indicate that different kinds of
enzymes act in concert in proteolysis during dentin
mineralization. DSPP first seems to be cleaved by bone
morphogenetic protein-1/Tolloid-like proteinase
(BMP1/Tolloid-like proteinases) into fully active DSP

and DPP. DSP is then further degraded (and possibly
inactivated) by MMP-2, MMP-20, and possibly other
proteinases (164).
A PHEX (a phosphate-regulating gene with
endopeptidase activity on the X chromosome) muta-
tion causes human X-linked hypophosphatemic rickets
(XLH), which is characterized by bone and dentin
hypomineralization and widening of the osteoid and
predentin, growth retardation, and impaired renal
phosphate reabsorption. A body of data has suggested
that the PHEX protein is the major proteinase respon-
sible for processing DMP1, functioning synergistically
with BMP1/Tolloid-like proteinases in the proteolytic
processing of DMP1 precursors (reviewed in 177).
However, DMP1 and DSPP fragments are present in
the predentin and dentin of Hyp mice with the PHEX
mutation, indicating that the PHEX protein is not the
enzyme solely responsible for the proteolytic process-
ing of DMP1 and DSPP (181).
Tertiary dentin formation
It has been suggested that tertiary dentin is formed by
odontoblasts and/or odontoblast-like cells that have
been differentiated after the beginning of irritation.
Lesot et al. (182) were the first to suggest that dentin
formed by the primary odontoblasts should be called
reactionary dentin, whereas newly differentiated odon-
toblasts or odontoblast-like cells are involved in
reparative dentin formation. This classification, which
can often be easily distinguished in histological sec-
tions, has been widely accepted.
When there is intensive irritation or severe damage,
the dentinpulp complex may respond with reparative
dentin formation formed by a new generation of
replacement odontoblasts or odontoblast-like cells
(182) (Figs. 10a and b). Even though the fate of the
primary odontoblasts is not fully understood, it is
widely believed that damaged odontoblasts degenerate
(183187) or go through apoptosis (Fig. 10a) and are
eliminated by macrophages (187,188). However, the
majority of the experiments on the responses of dental
pulp have been carried out on sound teeth with cavity
preparation, presenting a situation different from the
one seen clinically (189). Whether the reaction is
similar in clinical conditions, in which the damage
even in the worst case is significantly slower, is still
unknown because designing well-controlled experi-
ments is obviously quite difficult. At least some
Dentinpulp border
affected odontoblasts could survive and participate on
the deposition of reparative dentin after cavity prepa-
ration (53,190).
The appropriate term for the response to early
and/or slowly progressing external irritation would be
reactionary dentin, as it is formed by the primary
odontoblasts with an increased secretion rate com-
pared to secondary dentinogenesis (182). If the irrita-
tion is reasonably low grade (for example,
physiological attrition) and reactionary dentin is
formed slowly, it is indistinguishable from secondary
dentin and can only be seen as an increased amount of
mineralized dentin (Figs. 10c and d). The dentin more
or less retains is tubularity. Reactionary dentin forma-
tion has been a target of numerous studies; however,
the mechanisms regulating the onset and intensity of
reactionary dentin under injury still remain at least
partially unclear. Even in well-controlled experiments
with reasonably low variability in the intensity of den-
tinal damage, variable rates of reactionary dentin for-
mation have been observed (191,192). These studies
indicate that the secretion of reactionary dentin is a
more complex process than an automatic response in
odontoblasts to cavity restoration. It has been sug-
gested that the gene expression profile in mature
odontoblasts differs from that found in newly formed,
dentinogenically active odontoblasts (193). Even
though the reversal of gene expression has not been
demonstrated, it is possible that when reactionary den-
tinogenesis occurs, the cells may resume their earlier
expression pattern. The factors affecting the reaction-
ary dentin formation rate include at least the age of the
patient, residual dentin thickness and cavity dimen-
sions (number of cut dentinal tubules), restorative
material, extension of damage to the odontoblast cell
process or intratubular elements, nerve damage, and
the presence and solubilization of growth factors from
the dentin matrix (191,192). After severe injury,
primary odontoblasts are replaced by other cells that
are able to differentiate into odontoblast-like cells and
produce true reparative dentin. This process mimics
the early phases of tooth development and morpho-
genesis (194); may include cell proliferation, migra-
tion, and organization; and results in the initiation of
matrix synthesis with subsequent calcification (2).
Understanding the cellular and molecular changes
which occur in odontoblasts at different stages of their
life cycle, or the factors regulating the differentiation
and recruitment of replacement odontoblasts, could
69
Tjderhane & Haapasalo

Fig. 10. A model proposed for the fate of odontoblasts in pathological and healthy teeth. (a) In cases of intensive
external irritation (deep and/or active caries, extensive wear, or trauma), bacteria and/or dentinal growth factors and
other bioactive factors may induce local odontoblast (OB) destruction and apoptosis. Simultaneously, the factors
released from dentin, by dying odontoblasts or other cells, may attract the pulp stem cells to migrate and differentiate
into replacement odontoblasts. The process results in local synthesis of reparative dentin (RD), usually distinctly
different from primary dentin (lack of dentinal tubules, lamellar osteodentin-type calcification, etc.). (b) Back-
scattered electron microscope image of a rat molar fissure, dentin, and pulp tissue in a tooth with an experimentally
induced dentinal caries (DC) lesion. Reparative dentin (RD) formation as a response to the caries is clearly different
from the primary dentin (D). Also ectopic calcification (EC) in the pulp tissue can be seen. E: enamel. (c) In intact
teeth or teeth with physiological wear or other mild irritation, slow but continuous dentin formation (either
physiological or reactionary dentin formation by primary odontoblasts) leads to a decrease in the pulpal space. This
causes crowding among the original odontoblasts, calling for apoptosis of selected cells (black cells). Normal tubular
dentin formation continues with the remaining primary odontoblasts, with no need for replacement odontoblasts. (d)
In a rat molar with caries limited to the enamel and with only slight signs of demineralization under the dentinenamel
junction, reactionary dentin formation is indicated by the moderate increase in dentin formation on the pulpal side
of the bottom of the fissure (between arrows). Ectopic calcification in the pulp tissue is not present. Figures a and c
are modified with permission from Mitsiadis et al., 2008 (287); Figures b and d are modified with permission from
Hietala et al., 1993 (288).

facilitate the design of pharmacological tools to replacement odontoblasts is beyond the focus of this
manage and control this healing process. While the review, the reader is referred to recent articles and
molecular mechanisms involved in the reactivation of reviews for a detailed treatise of this interesting area (2,
odontoblast activity and initiation and the control of 194197).

70

Dentinal fluid
The space between the odontoblast process and the
tubule wall (the periodontoblastic space) is believed to
be filled with dentinal fluid, but absolute certainty of
the origin (or even existence) of dentinal fluid has not
been achieved (35). It has been suggested that the
odontoblast cell layer forms a functional barrier that
mostly restrains the passage of fluids, ions, and other
molecules along the extracellular pathway (4446).
Furthermore, it has also been suggested that, at least
in the situations not involving external stimulus
causing tissue damage (e.g. caries, cavity preparation,
abrasion), dentinal fluid content is strictly under odon-
toblast control (44,46) (Fig. 7d). Most studies meas-
uring dentinal fluid flow and consistency are based on
cavity preparation during which the tight junctions
between odontoblasts may be altered (45,46,53) and
fluid from the pulpeither from the pulp tissue extra-
cellular space, from the blood vessels, or bothmay
diffuse into the dentin. Therefore the fluid would be a
physiological response to the trauma (46,53,198,
199), and it is commonly suggested that dentinal fluid
originates from pulpal tissues (200). However, even in
intact teeth, studies with radioisotopes (201,202) and
fluorescence dye (203,204) have shown that a trans-
port mechanism exists between blood circulation and
dentin. Whether these components are transported by
the odontoblasts or via an intercellular pathway is not
known (199). Studies on ouabain and colchicine
(which disturb microtubule-guided transcellular trans-
port) and with calcium antagonists indicate that active
transport is involved (128,205), and it has been sug-
gested that odontoblasts regulate the transport of sub-
stances into predentin and dentin (4446,128).
Dentin also contains several serum proteins (at least
albumin, IgG, transferrin, fetuin-A, and superoxide
dismutase 3 [SOD3]; for details, see the review by
Mazzoni et al. in the next issue) and at least some of
them may be mainly present in dentinal tubules. The
genome-wide database of mature human odontoblast
gene expression profiles (Gene Expression Omnibus
[GEO] database # GSM215142) (206) indicates that
only transferrin is expressed by odontoblasts. There-
fore, serum proteins most likely have a passage to
dentinal fluid even in intact teeth. However, the pres-
ence of SOD3 (84) and the significantly higher con-
centration of fetuin-A in dentin compared to serum
(up to 100 to 200-fold) (207) at least strongly indi-
Dentinpulp border
cates that active transport systems by the odontoblasts
are involved for the serum proteins. In this article, the
expression dentinal fluid is used regardless of the
tissue from which the fluid may originate.
Despite the controversy surrounding the origin of
dentinal fluid, it is widely accepted that dentin sensi-
tivity to extrinsic irritants is mediated via alterations in
hydraulic conductance (reviewed in 200 and 208).
Hydrodynamic theory implicates fluid movement
through dentinal tubules as a transducting mechanism
in the production of dentin sensitivity (209), human
tooth being more sensitive to outward than inward
flow (210). Dentin may be considered to be a physi-
ological barrier that works in harmony with neurovas-
cular elements in the pulp to maintain the health of the
tissues (200,211). When dentin is exposed to external
stimuli for the first time, fluid shifts across the dentin
and activates a pulpal neurovascular response which,
along with the sensation of pain, likely greatly
increases the extracellular fluid volume to eliminate the
deleterious stimulus from the dentin tubules (200).
The excitability of pulpal nerves may be affected by
odontoblasts (208). The magnitude of this reaction is
dependent on the magnitude of the stimulation (200)
and the permeability of dentin (200,208). The factors
affecting the permeability of dentin include at least
dentinal tubule size and patency, reparative dentin for-
mation, the integrity of the odontoblast layer and the
presence of odontoblast processes in tubules, and the
flow rate and consistency of dentinal fluid (200,208,
212).
Dentinal fluid also affects the success of adhesive
restorative procedures. Increased dentinal wetness,
due to the increased size of dentinal tubules and fluid
flow, makes successful bonding in deep cavities (close
to pulp) more difficult than in superficial dentin
(reviewed in 213). Dentinal fluid may cause degrada-
tion of hydrophilic adhesives, but may also increase the
collagen degradation rate in the hybrid layer, both
leading to a decrease in bond strength durability
(213).
In carious teeth, dentinal fluid is considered to be a
protective factor through the occlusion of dentinal
tubules (especially in slowly progressing, chronic
lesions) (214) and part of the innate response of the
dentinpulp complex to the deposition of intratubular
immunoglobulins (215). Both the quality and the
quantity of immunoglobulins seem to vary according
to caries depth and intensity even in uninfected
71
Tjderhane & Haapasalo
tubules (215). It has also been suggested that dentinal
tubules contain superoxide dismutase 3 (SOD3) (84),
an antioxidant that in other tissues protects them from
oxidative stress (216). Since SOD3 is generally con-
sidered to be extracellular SOD, present e.g. in plasma
and lymph, it is possibly a component of dentinal fluid.
Some evidence exists that physiological dentinal fluid
flow, at least in rat molars with active dentinogenesis,
may be controlled by the endocrine system. It has been
suggested that a factor called parotid hormone affects
the dentinal fluid flow rate (199,204,217224). This
hormone has been isolated from porcine parotid
glands (225) and can be revealed by radioimmu-
noassay (RIA) in pig plasma (226); similarly active
factors have been isolated from bovine and rat pa-
rotid tissue (227,228). The involvement of the
hypothalamusparotid gland endocrine axis has also
been demonstrated (217), and a parotid hormone-
releasing factor has been partially purified from
porcine thalamushypothalamus tissue (221). A syn-
thetic 30-amino acid parotid hormone has been syn-
thesized, with biological activity equal to the
respective parotid gland-purified hormone in enhanc-
ing intradentinal fluid movement, as measured by
intradentinal dye penetration in rat molars (229).
Dentinal fluid movement is also affected by other
extrinsic factors. A high sucrose diet proved to be a
powerful depressant of dentinal fluid flow (199,218).
Various chemical substances known to have a car-
iostatic effect have been shown to increase dentinal
fluid flow (204,219,220), whereas a factor transvers-
ing the direction of dentinal fluid flow toward the pulp
significantly increased caries (203). As well, parasym-
pathetic stimulants (203) reactivated dentinal fluid
flow that had been depressed by a high sucrose diet or
parotidectomy/total sialoadenoctomy (203). Devitali-
zation of pulp reduces caries in rats receiving a car-
iogenic diet, and desalivation causes a marked increase
in caries scores in intact but not in devitalized teeth
(220,230). Numerous studies have also demonstrated
that high sucrose diets reduce dentin formation during
active dentinogenesis (reviewed in 231). Collectively,
these findings have provided evidence for the assump-
tion that centrifugal dentinal fluid flow plays a systemic
protective role against bacterial acidogenic challenge
(reviewed in 232). Interestingly, removal of parotid
but not submandibular/sublingual salivary glands also
significantly decreases dentin formation in rats with
active (primary) dentinogenesis (233,234).
72
Odontoblasts as sensory cells
In addition to (or instead of) the classic hydrodynamic
theory as to the cause of pain, odontoblasts themselves
may at least partially be responsible for the pain sen-
sation. Two types of mechanosensitive K+ channels and
a Na+ channel are expressed in human odontoblast-like
cells in vitro (235237). The TREK-1 K+ channel has
also been demonstrated in humans in vivo, located
specifically at the terminal web where the odontoblasts
are connected to each other (237). At least in vitro,
human odontoblast-like cells are excitable, generating
action potentials when electrically stimulated (238).
Together these findings already strongly indicate that
the odontoblasts themselves might sense mechanical
stress through these channels and produce the action
potentials that are transmitted to adjacent nerve
endings. Supporting this suggestion, several members
of the transient receptor potential (TRP) family, espe-
cially those belonging to the TRPV (vanilloid)
group, have been detected in odontoblast-like cells in
culture (40,239,240). At least TRPV1 is also present
in rat odontoblasts in vivo (241). These receptors
are directly related to the cellular mechanism of
temperature-sensing and nociception. TRPV1 was the
first and most extensively studied member in this
receptor family. It was first identified in ganglion
neurons but is also highly expressed in spinal and
peripheral nerve terminals, as well as in multiple non-
neuronal cell types. TRPV1 is broadly involved in
nociception and contributes to the detection and inte-
gration of painful chemical and thermal stimuli. It is
activated by vanilloid compounds (including capsai-
cin), but also by multiple other stimuli including mod-
erate heat and low ( 5.9) pH (242). The activation
of TRPV1 occurs upon depolarization (242), and both
in stomach epithelium (243) and in odontoblasts
(241), it has been suggested that the function is
related to the delivery of sensation and protection
against external noxious stimuli. Since epithelial cells
(as well as neuronal cells) have membrane polarization
similar to odontoblasts (36,37), and the expression of
mechanosensitive K+ channel TREK-1 is highly polar-
ized to the odontoblast cell bodies toward the pulp
(237), odontoblast membrane polarity may be related
to the sensory function of these cells. Interestingly, a
recent study by Yeon and co-workers (244) did not
observe TRPV expression or function in rat odonto-
blast, apparently in contrast to previous findings

(241). However, Yeon and others performed a single-
cell analysis of isolated adult rat odontoblasts, the
method of which required separating the cell from the
dentin, and the cells had to be kept cold (35C) to
maintain cell viability. It is therefore possible that cell
membrane polarity and the expression of proteins
related to the polarity had been lost.
Another possible mechanism for sensory function of
the odontoblasts is the expression of primary cilia.
Primary cilia are single, non-motile flagellar organelles
present on nearly all vertebrate cells, where they serve
a diverse set of signaling functions (245). Primary cilia
also function as flow sensors in kidney tubule epithelial
cells (246) and in osteoblasts and osteocytes (247,
248). In bone cells, cilia are responsible for changes in
cellular activity after mechanical stimulation (248),
suggesting that the bone cell primary cilia flow-sensing
function participates in bone remodeling (249). There
has also been indications that they are chondrocyte
mechanosensory organelles (250,251).
Odontoblasts express primary cilia (252,253)
(Fig. 2) and a recent study indicates their role in termi-
nal differentiation of odontoblasts and in signals that
influence cell movement toward the pulp (254). The
cilia may respond to mechanical stimulus from dentinal
fluid movements (255) or other signals either from the
dentin or pulp extracellular milieu, or both. The
primary cilium is a polarized structure and an intimate
link between the primary cilium and the establishment
and maintenance of cell polarity has been suggested in
several studies (245). The establishment of cell polarity
in epithelial cells is a prerequisite for ciliogenesis;
however, the presence of the cilium itself might exert an
effect in maintaining cell polarity (245). Primary cilia
disassemble when cells enter the cell cycle, and retrac-
tion of the primary cilium and re-entry into mitosis are
tightly co-ordinated, even though ciliary disassembly is
not a prerequisite for cell cycle entry. In odontoblasts,
primary cilia are observed in the apical pole of cells in
vivo (252,254) (Fig. 2), as is also the case with bone
cells (248) and epithelial cells (256). In in vitro
odontoblast-like cells, primary cilia have been found
only after morphological and functional differentiation
(253). Mice deficient in the oral facial digital type I
protein (OFD1), a protein closely related to ciliogen-
esis (257), demonstrate a lack of odontoblast differen-
tiation, disorganized cusp formation, and marked
overall dentin defects (254). Overall, these findings
indicate that odontoblast primary cilia may be closely
Dentinpulp border
related to cell differentiation (252) and membrane
polarization (36,37). It is important to note, however,
that mesenchymal cells in general are ciliated even
though they lack apicobasal polarity (245).
Odontoblasts as
immunological cells
Odontoblasts are the first dental cells to encounter the
bacteria entering dentin from the oral cavity, repre-
senting the first line of cellular defense for the host. In
that sense, the role of odontoblasts is similar to that of
epithelial cells e.g. in gut lumen, and the similar
plasma membrane polarity between these highly dif-
ferentiated post-mitotic cells is interesting (36,37).
Recent findings convincingly suggest that the role of
odontoblasts in the initiation and development of
dentinpulp complex inflammatory and/or immune
responses may be stronger than previously believed
(258262), and the odontoblast response to oral
pathogens differs from that seen in pulp fibroblasts
(261,263).
Toll-like receptors (TLRs) are a group of transmem-
brane glycoproteins that recognize microbial and viral
particles, e.g. bacterial cell wall components (including
lipopolysaccharide [LPS] and lipoteichoic acid
[LTA]), fungal proteins, and viral and bacterial RNAs
and DNAs (264). TLRs have an important role in the
early activation of innate immune responses such as the
secretion of pro-inflammatory cytokines and chemo-
kines to recruit inflammatory cells, production of anti-
microbial peptides, and maturation of dendritic cells
(262). Out of the 10 TLRs identified in humans,
TLR16 and TLR9 genes are expressed in human
cultured odontoblast-like cells in vitro (259,262). In
principle, odontoblast-expressed TLRs would be
capable of recognizing practically all essential bacterial
components (262).
The best-known TLRs in odontoblasts are TLR2
and TLR4. They have been recognized in situ in tissue
cultures of human mature (fully differentiated) odon-
toblasts (260) using a model where the odontoblasts
are cultured while still attached to dentin (96). TLR2
participates in the recognition of LTA and other
Gram-positive bacterial components. TLR2 has been
observed in mouse (265) and human (262) molar
odontoblast layers under carious lesions as a re-
sponse to Gram-positive bacteria, which are the main
micro-organisms in small caries lesions (266). Even
73
Tjderhane & Haapasalo
though the TLR2 response of newly differentiated
odontoblast-like cells and mature fully differentiated
odontoblasts to Gram-positive bacterial components is
different (259,260), it is likely that odontoblasts are
equipped to react to the microbial flora prevalent in
small or moderate-sized caries lesions (262). TLR4,
meanwhile, recognizes the LPS from Gram-negative
bacteria, which are dominant in deep caries lesions
approaching pulp (267). TLR4 protein expression
in the odontoblasts of healthy teeth has been well
demonstrated (260,268), and activation of TLR4-
expressing mature human odontoblasts by LPS
induces the production of several pro-inflammatory
cytokines (260).
Defensins are a group of small (35 kDa) peptides
with broad-spectrum antimicrobial activity. Two sub-
families, the a- and the b-defensins, are found in
humans (269); of the four human b-defensins (hBDs),
at least three (hBD13) are also present in saliva (270
272). They originate from oral mucosal keratinocytes
and salivary glands, and together possess strong anti-
microbial effects against Gram-negative bacteria, yeast
and viruses (270272), and Str. mutans (273). hBD-1
and -2 are also expressed by odontoblasts both in vivo
and in vitro (274,275), where it has been suggested
that they play a role in the innate host defense of
human dental pulp (274). In odontoblast-like cells,
both hBD-2 (275) and heat-killed Str. mutans (276)
increase IL-6 and -8 gene expression. Interestingly,
hBD-2 is a host-derived ligand for TLR4 (277), and
the inhibition of central TLR signaling molecule phos-
phoinositide 3-kinase (PI3K) (278) blocks the Str.
mutans-induced increase in IL-6 and -8 gene expres-
sion (276). Overall, the data indicates that, in addition
to antimicrobial functions, odontoblast-derived hBDs
may also participate in the auto- or intracrine regula-
tion of odontoblast immunodefense.
Class II major histocompatibility complex (MHC)-
positive cells, especially those with dendritic profiles,
participate in the recognition and the processing of
antigenic substances in the dental pulp and serve as
antigen-presenting cells (210,279). In humans, the
dendritic cells in the odontoblastic layer are arranged
in and just beneath the odontoblastic layer in a manner
that indicates that each cell controls its own territory
(279). Some of these cells extend their cytoplasmic
processes into dentinal tubules (279281). Odonto-
blast layer dendritic cells also situate close to nerve
fibers, suggesting potential interactions between den-
74
dritic cells and nerves (279). The finding that dendritic
cell processes in healthy tooth predentin may contact
several odontoblast processes in a manner similar to
nerve fibers indicates that they may also have some
regulatory function on the odontoblasts under physi-
ological conditions (280). However, hBD-2 is a host-
derived ligand for TLR4 that induces dendritic cell
activation (277). Since in vitro studies indicate that
odontoblasts and dendritic cells respond differentially
to microbial components (282), it is possible that a
complex interplay occurs between odontoblasts and
dendritic cells in the odontoblastpredentin layer.
Even though odontoblasts appear to have a distinct
role in the regulation of the initiation and progress of
the dentinpulp complex inflammatory response,
much more research is needed before any clinical treat-
ment approaches are justified. First of all, the opinions
regarding potential treatment strategies vary. At least a
local inflammatory response to caries and other exter-
nal irritation is inevitable and some authors have
stressed the prerequisite nature of inflammation for
pulp repair (196,261); others call for novel therapeutic
strategies to prevent odontoblast-initiated inflamma-
tory responses in the dentinpulp border and pulp
tissue (262). Secondly, the role of other contributing
factors (especially dentin-bound growth factors) needs
to be taken into account for clinical therapeutic
models since TGF-b has been shown to affect inflam-
matory interleukin production in mature human
odontoblasts (283), inhibit TLR2 and TLR4 expres-
sion, and attenuate odontoblast-like cell responses to
caries pathogens in vitro (284). Much of the current
information has been gained via in vitro studies using
cultured odontoblast-like cells that, while providing
important basic information, differ significantly from
the clinical reality. The complexity of the dentinpulp
complex calls for detailed in vivo or in situ studies,
with approaches as close as possible to clinical reality.
Conclusion
The dentinpulp complex interface appears to be
much more than just a layer of odontoblasts with
mineralizing predentin. It is dynamic in nature and
able to respond to various external signals. Most likely
this response is much more complicated than previ-
ously thought, with potentially poorly understood sys-
tematic regulation and the ability to participate in both
sensory and immunological responses. A fundamental

understanding of these aspects may facilitate future
methods of treating inflamed pulp by attempting to
limit the destructive processes in pulp tissue and to
augment the healing and protective processes against
chemical or microbial damage.
References
1. Cobourne MT, Sharpe PT. Tooth and jaw: molecular
mechanisms of patterning in the first branchial arch.
Arch Oral Biol 2003: 48: 114.
2. Mitsiadis TA, Graf D. Cell fate determination during
tooth development and regeneration. Birth Defects Res
C Embryo Today 2009: 87: 199211.
3. Ruch JV, Lesot H, Bgue-Kirn C. Odontoblast differ-
entiation. Int J Dev Biol 1995: 39: 5168.
4. Thesleff I, Kernen S, Jernvall J. Enamel knots as
signaling centers linking tooth morphogenesis and
odontoblast differentiation. Adv Dent Res 2001: 15:
1418.
5. Tompkins K. Molecular mechanisms of cytodifferen-
tiation in mammalian tooth development. Connect
Tissue Res 2006: 47: 111118.
6. Veis A. Amelogenin splice gene products: potential
signaling molecules. Cell Mol Life Sci 2003: 60:
3055.
7. Karcher-Djuricic V, Staubli A, Meyer JM, Ruch JV.
Acellular dental matrices promote functional differen-
tiation of ameloblasts. Differentiation 1985: 29: 169
175.
8. Zeichner-David M, Diekwisch T, Fincham A, Lau E,
MacDougall M, Moradian-Oldak J, Simmer J, Snead
M, Slavkin HC. Control of ameloblast differentiation.
Int J Dev Biol 1995; 39: 6992.
9. Heikinheimo K, Salo T. Expression of basement mem-
brane type IV collagen and type IV collagenases
(MMP-2 and MMP-9) in human fetal teeth. J Dent
Res 1995: 74: 12261234.
10. Sahlberg C, Reponen P, Tryggvason K, Thesleff I.
Timp-1, -2 and -3 show coexpression with gelatinases
A and B during mouse tooth morphogenesis. Eur J
Oral Sci 1999: 107: 121130.
11. Tummers M, Thesleff I. The importance of signal
pathway modulation in all aspects of tooth develop-
ment. J Exp Zool B Mol Dev Evol 2009: 312B: 309
319.
12. Bei M. Molecular genetics of tooth development. Curr
Opin Genet Dev 2009: 19: 504510.
13. Katchburian E. Membrane-bound bodies as initiators
of mineralization of dentine. J Anat 1973: 116: 285
302.
14. Katchburian E. Initiation of mineral deposition in
dentine. Calcif Tissue Int 1977: 22: 179184.
15. Golub EE. Role of matrix vesicles in biomineralization.
Biochim Biophys Acta 2009: 1790: 15921598.
16. Bonucci E. Crystal ghosts and biological mineraliza-
tion: fancy spectres in an old castle, or neglected
Dentinpulp border
structures worthy of belief? J Bone Miner Metab 2002:
20: 249265.
17. Ten Cate AR. Dentinogenesis. In: Ten Cate AR, ed.
Oral Histology: Development, Structure and Function,
4th edn. Mosby Inc., 1994: 147168.
18. Arana-Chavez VE, Massa LF. Odontoblasts: the cells
forming and maintaining dentine. Int J Biochem Cell
Biol 2004: 36: 13671373.
19. Sela J, Tamari I, Hirschfeld Z, Bab I. Transmission
electron microscopy of reparative dentin in rat molar
pulps. Primary mineralization via extracellular matrix
vesicles. Acta Anat 1981: 109: 247251.
20. Hirschfeld Z, Bab I, Tamari I, Sela J. Primary miner-
alization of dentin in rats after pulp capping with
calcium-hydroxide. J Oral Pathol 1982: 11: 426433.
21. Takano Y, Sakai H, Baba O, Terashima T. Differential
involvement of matrix vesicles during the initial and
appositional mineralization processes in bone, dentin,
and cementum. Bone 2000: 26: 333339.
22. Saito T, Arsenault AL, Yamauchi M, Kuboki Y, Cren-
shaw MA. Mineral induction by immobilized phos-
phoproteins. Bone 1997: 21: 305311.
23. Dechichi P, Biffi JC, Moura CC, de Ameida AW. A
model of the early mineralization process of mantle
dentin. Micron 2007: 38: 486491.
24. Bodier-Houll P, Steuer P, Meyer JM, Bigeard L,
Cuisinier FJ. High-resolution electron-microscopic
study of the relationship between human enamel and
dentin crystals at the dentinoenamel junction. Cell
Tissue Res 2000: 301: 389395.
25. Nanci A, Kawaguchi H, Kogaya Y. Ultrastructural
studies and immunolocalization of enamel proteins in
rodent secretory stage ameloblasts processed by
various cryofixation methods. Anat Rec 1994: 238:
425436.
26. Diekwisch TG, Ware J, Fincham AG, Zeichner-David
M. Immunohistochemical similarities and differences
between amelogenin and tuftelin gene products during
tooth development. J Histochem Cytochem 1997: 45:
859866.
27. Nanci A. Enamel: composition, formation, and struc-
ture. In: Nanci A, ed. Ten Cates Oral Histology. 7th
edn. Mosby Elsevier, 2008: 141190.
28. Hu JC, Yamakoshi Y, Yamakoshi F, Krebsbach PH,
Simmer JP. Proteomics and genetics of dental enamel.
Cells Tissues Organs 2005: 181: 219231.
29. Bgue-Kirn C, Krebsbach PH, Bartlett JD, Butler WT.
Dentin sialoprotein, dentin phosphoprotein, enam-
elysin and ameloblastin: tooth-specific molecules that
are distinctively expressed during murine dental differ-
entiation. Eur J Oral Sci 1998: 106: 963970.
30. Hao J, He G, Narayanan K, Zou B, Lin L, Muni T,
Ramachandran A, George A. Identification of differ-
entially expressed cDNA transcripts from a rat odon-
toblast cell line. Bone 2005: 37: 578588.
31. Simmons D, Gu TT, Krebsbach PH, Yamada Y,
MacDougall M. Identification and characterization of
a cDNA for mouse ameloblastin. Connect Tissue Res
1998: 39: 312.
75
Tjderhane & Haapasalo
32. Fong CD, Slaby I, Hammarstrm L. Amelin: an
enamel-related protein, transcribed in the cells of epi-
thelial root sheath. J Bone Miner Res 1996: 11: 892
898.
33. Spahr A, Lyngstadaas SP, Slaby I, Pezeshki G.
Ameloblastin expression during craniofacial bone
formation in rats. Eur J Oral Sci 2006: 114: 504511.
34. Wazen RM, Moffatt P, Zalzal SF, Yamada Y, Nanci A.
A mouse model expressing a truncated form of ame-
loblastin exhibits dental and junctional epithelium
defects. Matrix Biol 2009: 28: 292303.
35. Torneck CD. Dentinpulp complex. In: Ten Cate AR,
ed. Oral Histology: Development, Structure and Func-
tion, 5th edn. Mosby Inc., 1998: 150196.
36. Tjderhane L, Mesterton S, Ilvesaro J, Pkknen V,
Metsikk K, Salo T, Tuukkanen J. Cell membrane
polarity of mature human odontoblasts. Int Endod J
2009: 42: 1136.
37. Tjderhane L, Mesterton S, Ilvesaro J, Pkknen V,
Metsikk K, Soini S, Salo T, Tuukkanen J. Cell mem-
brane polarity of mature human odontoblasts. J Dent
Res (submitted for publication).
38. Matter K, Mellman I. Mechanisms of cell polarity:
sorting and transport in epithelial cells. Curr Opin Cell
Biol 1994: 6: 545554.
39. Ilvesaro J, Metsikk K, Vnnen K, Tuukkanen J. 55.
Polarity of osteoblasts and osteoblast-like UMR-108
cells. J Bone Miner Res 1999: 14: 13381344.
40. Magloire H, Maurin JC, Couble ML, Shibukawa Y,
Tsumura M, Thivichon-Prince B, Bleicher F. Topical
review. Dental pain and odontoblasts: facts and
hypotheses. J Orofac Pain 2010: 24: 335349.
41. Arana-Chavez VE, Katchburian E. Freeze-fracture
studies of the distal plasma membrane of rat odonto-
blasts during their differentiation and polarisation. Eur
J Oral Sci 1998: 106(Suppl 1): 132136.
42. Boyde A, Reith EJ, Jones J. Intercellular attachments
between calcified collagenous tissue forming cells in
the rat. Cell Tissue Res 1978: 191: 507512.
43. Bishop MA. Evidence for tight junctions between
odontoblasts in the rat incisor. Cell Tissue Res 1985:
239: 137140.
44. Bishop MA, Yoshida S. A permeability barrier to lan-
thanum and the presence of collagen between odon-
toblasts in pig molars. J Anat 1992: 181: 2938.
45. Turner DF, Marfurt CF, Sattelberg C. Demonstration
of physiological barrier between pulpal odontoblasts
and its perturbation following routine restorative pro-
cedures: a horseradish peroxidase tracing study in the
rat. J Dent Res 1989: 68: 12621268.
46. Turner DF. Immediate physiological response of
odontoblasts. Proc Finn Dent Soc 1992: 88(Suppl I):
5563.
47. Joo SM, Arana-Chavez VE. Expression of connexin
43 and ZO-1 in differentiating ameloblasts and odon-
toblasts from rat molar tooth germs. Histochem Cell
Biol 2003: 119: 2126.
48. Joo SM, Arana-Chavez VE. Tight junctions in differ-
entiating ameloblasts and odontoblasts differentially
76
express ZO-1, occludin, and claudin-1 in early odon-
togenesis of rat molars. Anat Rec A Discov Mol Cell
Evol Biol 2004: 277: 338343.
49. Hoshino M, Hashimoto S, Muramatsu T, Matsuki M,
Ogiuchi H, Shimono M. Claudin rather than occludin
is essential for differentiation in rat incisor odonto-
blasts. Oral Dis 2008: 14: 606612.
50. Tsukita S, Yamazaki Y, Katsuno T, Tamura A, Tsukita
S. Tight junction-based epithelial microenvironment
and cell proliferation. Oncogene 2008: 27: 6930
6938.
51. Arana-Chavez VE, Katchburian E. Development of
tight junctions between odontoblasts in early dentino-
genesis as revealed by freeze-fracture. Anat Rec 1997:
248: 332338.
52. Linde A, Goldberg M. Dentinogenesis. Crit Rev Oral
Biol Med 1993: 4: 679728.
53. Chiego DJ Jr. An ultrastructural and autoradiographic
analysis of primary and replacement odontoblasts fol-
lowing cavity preparation and wound healing in the rat
molar. Proc Finn Dent Soc 1992: 88(Suppl 1): 243
256.
54. Byers MR, Lin KJ. Patterns of fluoro-gold entry into
rat molar enamel, dentin, and pulp. J Dent Res 2003:
82: 312317.
Bishop MA, Malhotra M, Yoshida S. Interodontoblas-
tic collagen (von Korff fibers) and circumpulpal dentin
formation: an ultrathin serial section study in the cat.
Am J Anat 1991: 191: 6773.
56. Stratmann U, Schaarschmidt K, Wiesmann HP, Plate
U, Hhling HJ, Szuwart T. The mineralization of
mantle dentine and of circumpulpal dentine in the rat:
an ultrastructural and element-analytical study. Anat
Embryol 1997: 195: 289297.
57. Byers MR, Sugaya A. Odontoblast processes in dentin
revealed by fluorescent Di-I. J Histochem Cytochem
1995: 43: 159168.
58. Yoshiba K, Yoshiba N, Ejiri S, Iwaku M, Ozawa H.
Odontoblast processes in human dentin revealed
by fluorescence labeling and transmission electron
microscopy. Histochem Cell Biol 2002: 118: 205
212.
59. Tsuchiya M, Sasano Y, Kagayama M, Watanabe M.
The extent of odontoblast processes in the dentin is
distinct between cusp and cervical regions during
development and aging. Arch Histol Cytol 2002: 65:
179188.
60. Pashley DH. Dynamics of the pulpodentin complex.
Crit Rev Oral Biol Med 1996: 7: 104133.
61. Boskey A. Noncollagenous matrix proteins and their
role in mineralisation. Bone Miner 1989: 6: 111123.
62. Boskey AL. Biomineralization: an overview. Connec
Tissue Res 2003: 44(Suppl 1): 59.
63. Kawasaki K, Tanaka S, Ishikawa T. On the incremental
lines in human dentine as revealed by tetracycline
labeling. J Anat 1977: 123: 427436.
64. Beniash E, Traub W, Veis A, Weiner S. A transmission
electron microscope study using vitrified ice sections of
predentin: structural changes in the dentin collagenous

matrix prior to mineralization. J Struct Biol 2000: 132:
212225.
65. Hoshi K, Kemmotsu S, Takeuchi Y, Amizuka N,
Ozawa H. The primary calcification in bones follows
removal of decorin and fusion of collagen fibrils. J Bone
Miner Res 1999: 14: 273280.
66. Veis A, Schlueter RJ. The macromolecular organiza-
tion of dentine matrix collagen. I. Characterization of
dentine collagen. Biochemistry 1964: 3: 16501657.
67. Schlueter RJ, Veis A. The macromolecular organiza-
tion of dentin matrix collagen. II. Periodate degrada-
tion and carbohydrate cross-linking. Biochemistry
1964: 3: 16571665.
68. Armstrong SR, Jessop JL, Winn E, Tay FR, Pashley
DH. Denaturation temperatures of dentin matrices. I.
Effect of demineralization and dehydration. J Endod
2006: 32: 638651.
69. Armstrong SR, Jessop JL, Winn E, Tay FR, Pashley
DH. Effects of polar solvents and adhesive resin on the
denaturation temperatures of demineralised dentin
matrices. J Dent 2008: 36: 814.
70. Sulkala M, Tervahartiala T, Sorsa T, Larmas M, Salo T,
Tjderhane L. Matrix metalloproteinase-8 (MMP-8) is
the major collagenase in human dentin. Arch Oral Biol
2007: 52: 121127.
71. Butler WT, Brunn JC, Qin C. Dentin extracellular
matrix (ECM) proteins: comparison to bone ECM and
contribution to dynamics of dentinogenesis. Connect
Tissue Res 2003: 44(Suppl 1): 171178.
72. Embery G, Hall R, Waddington R, Septier D,
Goldberg M. Proteoglycans in dentinogenesis. Crit
Rev Oral Biol Med 2001: 12: 331349.
73. Tjderhane L, Palosaari H, Wahlgren J, Larmas M,
Sorsa T, Salo T. Human odontoblast culture method:
the expression of collagen and matrix metalloprotein-
ases (MMPs). Adv Dent Res 2001: 15: 5558.
74. Butler WT, Brunn JC, Qin C, McKee MD. Extracel-
lular matrix proteins and the dynamics of dentin for-
mation. Connect Tissue Res 2002: 43: 301307.
75. Butler WT. Dentin collagen: chemical structure and
role in mineralization. In: Linde A, ed. Dentin and
Dentinogenesis, Volume II. CRC Press, 1984: 37
53.
76. Kuboki Y, Mechanic GL. Comparative molecular dis-
tribution of cross-link in bone and dentin collagen.
Structurefunction relationships. Calcif Tissue Int
1982: 34: 306308.
77. Mechanic GL, Katz EP, Henmi M, Noyes C,
Yamauchi M. Locus of a histidine-based, stable trifunc-
tional, helix to helix collagen cross-link: stereospecific
collagen structure of type I skin fibrils. Biochemistry
1987: 26: 35003509.
78. Lussi A, Crenshaw MA, Linde A. Induction and inhi-
bition of hydroxyapatite formation by rat dentine
phosphoprotein in vitro. Arch Oral Biol 1988: 33:
685691.
79. Glimcher MJ. Mechanism of calcification: role of col-
lagen fibrils and collagen-phosphoprotein complexes
in vitro and in vivo. Anat Rec 1989: 224: 139153.
Dentinpulp border
80. Linde A, Lussi A, Crenshaw MA. Mineral induction by
immobilized polyanionic proteins. Calcif Tissue Int
1989: 44: 286295.
81. Boskey AL, Maresca M, Doty S, Sabsay B, Veis
A. Concentration-dependent effects of dentin phos-
phophoryn in the regulation of in vitro hydroxyapatite
formation and growth. Bone Miner 1990: 11: 5565.
82. Lussi A, Linde A. Mineral induction in vivo by dentin
proteins. Caries Res 1993: 27: 241248.
83. Waddington RJ, Hall RC, Embery G, Lloyd DM.
Changing profiles of proteoglycans in the transition of
predentine to dentine. Matrix Biol 2003: 22: 153
161.
84. Park ES, Cho HS, Kwon TG, Jang SN, Lee SH, An
CH, Shin HI, Kim JY, Cho JY. Proteomics analysis of
human dentin reveals distinct protein expression pro-
files. J Proteome Res 2009: 8: 13381346.
85. Kalamajski S, Oldberg A. The role of small leucine-rich
proteoglycans in collagen fibrillogenesis. Matrix Biol
2010: 29: 248253.
86. Roughley PJ. The structure and function of cartilage
proteoglycans. Eur Cell Mater 2006: 12: 92101.
87. Couble ML, Bleicher F, Farges JC, Peyrol S, Lucchini
M, Magloire H, Staquet MJ. Immunodetection of
osteoadherin in murine tooth extracellular matrices.
Histochem Cell Biol 2004: 121: 4753.
88. Lucchini M, Couble ML, Romeas A, Staquet MJ,
Bleicher F, Magloire H, Farges JC. Alpha v beta 3
integrin expression in human odontoblasts and
co-localization with osteoadherin. J Dent Res 2004:
83: 552556.
89. Goldberg M, Septier D, Oldberg A, Young MF,
Ameye LG. Fibromodulin-deficient mice display
impaired collagen fibrillogenesis in predentin as well as
altered dentin mineralization and enamel formation.
J Histochem Cytochem 2006: 54: 525537.
90. Goldberg M, Septier D, Rapoport O, Iozzo RV,
Young MF, Ameye LG. Targeted disruption of two
small leucine-rich proteoglycans, biglycan and decorin,
excerpts divergent effects on enamel and dentin for-
mation. Calcif Tissue Int 2005: 77: 297310.
91. Sugars RV, Milan AM, Brown J O, Waddington R J,
Hall RC, Embery G. Molecular interaction of
recombinant decorin and biglycan with type I collagen
influences crystal growth. Connect Tissue Res 2003:
44(Suppl. 1): 189195.
92. Goldberg M, Rapoport O, Septier D, Palmier K, Hall
R, Embery G, Young M, Ameye L. Proteoglycans in
predentin: the last 15 micrometers before mineraliza-
tion. Connect Tissue Res 2003: 44: 184188.
93. Milan AM, Sugars RV, Embery G, Waddington RJ.
Dentinal proteoglycans demonstrate an increasing
order of affinity for hydroxyapatite crystals during the
transition of predentine to dentine. Calcif Tissue Int
2004: 75: 197204.
94. Dean DD, Schwartz Z, Muniz OE, Gomez R, Swain
LD, Howell DS, Boyan BD. Matrix vesicles are
enriched in metalloproteinases that degrade proteogly-
cans. Calcif Tissue Int 1992: 50: 342349.
77
Tjderhane & Haapasalo
95. Embery G, Rees S, Hall R, Rose K, Waddington R,
Shellis P. Calcium- and hydroxyapatite-binding prop-
erties of glucuronic acid-rich and iduronic acid-rich
glycosaminoglycans and proteoglycans. Eur J Oral Sci
1998: 106(Suppl 1): 267273.
96. Tjderhane L, Salo T, Larjava H, Larmas M, Overall
CM. A novel organ culture method to study the func-
tion of human odontoblasts in vitro: gelatinase expres-
sion by odontoblasts is differentially regulated by
TGF-b1. J Dent Res 1998: 77: 14861496.
97. Hall R, Septier D, Embery G, Goldberg M.
Stromelysin-1 (MMP-3) in forming enamel and
predentine in rat incisor-coordinated distribution
with proteoglycans suggests a functional role. Histo-
chem J 1999: 31: 761770.
98. Palosaari H, Wahlgren J, Larmas M, Rnk H,
Sorsa T, Salo T, Tjderhane L. The expression of
MMP-8 in human odontoblasts and dental pulp cells is
down-regulated by TGF-b1. J Dent Res 2000: 79:
7784.
99. Palosaari H, Pennington CJ, Larmas M, Edwards DR,
Tjderhane L, Salo T. Expression profile of matrix
metalloproteinases (MMPs) and tissue inhibitors of
MMPs in mature human odontoblasts and pulp tissue.
Eur J Oral Sci 2003: 111: 117127.
100. Goldberg M, Septier D. Phospholipids in amelogenesis
and dentinogenesis. Crit Rev Oral Biol Med 2002: 13:
276290.
101. Pentikinen MO, rni K, Lassila R, Kovanen PT.
The proteoglycan decorin links low density lipopro-
teins with collagen type I. J Biol Chem 1997: 272:
76337638.
102. Jarrold BR, Bacon WL, Velleman SG. Expression and
localization of the proteoglycan decorin during the
progression of cholesterol induced atherosclerosis in
Japanese quail: implications for interactions with col-
lagen type I and lipoproteins. Atherosclerosis 1999:
146: 299308.
103. Kovanen P, Pentikinen MO. Decorin links low-
density lipoproteins (LDL) to collagen: a novel
mechanism for retention of LDL in the athero-
sclerotic plaque. Trends Cardiovasc Med 1999: 9:
8691.
104. Olin KL, Potter-Perigo S, Barrett PHR, Wight TN,
Chait A. Biglycan, a vascular proteoglycan, binds
differently to HDL2 and HDL3: role of apoE.
Arterioscler Thromb Vasc Biol 2001: 21: 129135.
105. Gotliv BA, Veis A. Peritubular dentin, a vertebrate
apatitic mineralized tissue without collagen: role of a
phospholipid-proteolipid complex. Calcif Tissue Int
2007: 81: 191205.
106. Gotliv BA, Veis A. The composition of bovine peritu-
bular dentin: matching TOF-SIMS, scanning electron
microscopy and biochemical component distributions.
New light on peritubular dentin function. Cells Tissues
Organs 2009: 189: 1219.
107. Goldberg M, Lcolle S, Vermelin L, Benghezal A,
Septier D, Godeau G. [3H]choline uptake and
turnover into membrane and extracellular matrix
78
phospholipids, visualized by radioautography in the rat
incisor dentin and enamel. Calcif Tissue Int 1999: 65:
6672.
108. Nicholson WA, Ashton BA, Hhling HJ, Quint P,
Schreiber J, Ashton IK, Boyde A. Electron microprobe
investigations into the process of hard tissue forma-
tion. Cell Tissue Res 1977: 177: 331345.
109. Wiesmann HP, Plate U, Hhling HJ, Barckhaus RH,
Zierold K. Analysis of early hard tissue formation in
dentine by energy dispersive X-ray microanalysis and
energy-filtering transmission electron microscopy.
Scanning Microsc 1993: 7: 711718.
110. Wiesmann HP, Hhling HJ, Zierold K, Barckhaus
RH. Elemental distributions in predentin associated
with dentin mineralization in rat incisor. Connect
Tissue Res 1995: 33: 179184.
111. Landis WJ. Mineral characterization in calcifying
tissues: atomic, molecular and macromolecular
perspectives. Connect Tissue Res 1996: 34: 239246.
112. Landis WJ, Librizzi JJ, Dunn MG, Silver FH. A study
of the relationship between mineral content and
mechanical properties of turkey gastrocnemius tendon.
J Bone Miner Res 1995: 10: 859867.
113. Kinney JH, Pople JA, Driessen CH, Breunig TM,
Marshall GW, Marshall SJ. Intrafibrillar mineral may
be absent in dentinogenesis imperfecta type II (DI-II).
J Dent Res 2001: 80: 15551559.
114. Kinney JH, Habelitz S, Marshall SJ, Marshall GW. The
importance of intrafibrillar mineralization of collagen
on the mechanical properties of dentin. J Dent Res
2003: 82: 957961.
115. Balooch M, Habelitz S, Kinney JH, Marshall SJ, Mar-
shall GW. Mechanical properties of mineralized colla-
gen fibrils as influenced by demineralization. J Struct
Biol 2008: 162: 404410.
116. Bertassoni LE, Habelitz S, Kinney JH, Marshall SJ,
Marshall GW Jr. Biomechanical perspective on the
remineralization of dentin. Caries Res 2009: 43:
7077.
117. Posner AS, Tannenbaum PJ. The mineral phase of
dentin. In: Linde A, ed. Dentin and Dentinogenesis,
Volume II. CRC Press, 1984: 1736.
118. Arnold S, Plate U, Wiesmann HP, Stratmann U, Kohl
H, Hohling HJ. Quantitative analysis of the biomin-
eralization of different hard tissues. J Microsc 2001:
202: 488494.
119. Landis WJ, Paine MC, Glimcher MJ. Electron micro-
scopic observations of bone tissue prepared anhy-
drously in organic solvents. J Ultrastruct Res 1977:
59: 133.
120. Goldberg M, Septier D. A comparative study of the
transition between predentin and dentin, using various
preparative procedures in the rat. Eur J Oral Sci 1996:
104: 269277.
121. Toroian D, Lim JE, Price PA. The size exclusion
characteristics of type I collagen: implications for
the role of noncollagenous bone constituents in
mineralization. J Biol Chem 2007: 282: 22437
22447.

122. Price PA, Toroian D, Lim JE. Mineralization by inhibi-
tor exclusion: the calcification of collagen with fetuin.
J Biol Chem 2009: 284: 1709217101.
123. Price PA, Toroian D, Chan WS. Tissue-nonspecific
alkaline phosphatase is required for the calcification
of collagen in serum: a possible mechanism for
biomineralization. J Biol Chem 2009: 284: 4594
4604.
124. Nanci A. Dentinpulp complex. In: Nanci A, ed. Ten
Cates Oral Histology. 7th ed. Mosby Elsevier, 2008:
191238.
125. Tsurumachi T, Huang TJ, Zhan W, Hayashi M, Ogiso
B. Scanning electron microscopic study of dentinal
pulpal walls in relation to age and tooth area. J Oral Sci
2008: 50: 199203.
126. Carafoli E. Intracellular calcium homeostasis. Annu
Rev Biochem 1987: 56: 395433.
127. Lundgren T, Nannmark U, Linde A. Calcium ion
activity and pH in the odontoblastpredentin region:
ion-selective microelectrode measurements. Calcif
Tissue Int 1992: 50: 134136.
128. Lundgren T, Linde A. Calcium ion transport kinetics
during dentinogenesis: effects of disrupting odonto-
blast cellular transport systems. Bone Miner 1992: 19:
3144.
129. Granstrm G, Linde A. ATP-dependent uptake of
Ca2+ by a microsomal fraction from rat incisor
odontoblasts. Calcif Tissue Int 1981: 33: 125
128.
130. Lundgren T, Linde A. Voltage-gated and nonvoltage-
gated calcium uptake pathways in the rat incisor odon-
toblast plasma membrane. Calcif Tissue Int 1997: 60:
7985.
131. Lundgren T, Nilsson M, Ritchie HH, Linde A. Junc-
tional proteins and Ca2+ transport in the rat
odontoblast-like cell line MRPC-1. Calcif Tissue Int
2001: 68: 192201.
132. Lundgren T, Linde A. Na+/Ca2+ antiports in mem-
branes of rat incisor odontoblasts. J Oral Pathol 1988:
17: 560563.
133. Linde A, Magnusson BC. Inhibition studies of alkaline
phosphatases in hard tissue-forming cells. J Histochem
Cytochem 1975: 23: 342347.
134. Granstrm G, Linde A. A comparison of ATP-
degrading enzyme activities in rat incisor odontoblasts.
J Histochem Cytochem 1976: 24: 10261032.
135. Granstrm G, Jontell M, Linde A. Separation of odon-
toblast Ca2+-ATPase and alkaline phosphatase. Calcif
Tissue Int 1979: 27: 211217.
136. Granstrm G, Linde A, Nygren H. Ultrastructural
localization of alkaline phosphatase in rat incisor
odontoblasts. J Histochem Cytochem 1978: 26: 359
368.
137. Granstrm G. Further evidence of an intravesicular
Ca2+-pump in odontoblasts from rat incisors. Arch
Oral Biol 1984: 29: 599606.
138. Lundgren T, Linde A. Regulation of free Ca2+ by
subcellular fractions of rat incisor odontoblasts. Arch
Oral Biol 1987: 32: 463468.
Dentinpulp border
139. Stains JP, Gay CV. Asymmetric distribution of func-
tional sodium-calcium exchanger in primary osteo-
blasts. J Bone Miner Res 1998: 13: 18621869.
140. Lundquist P, Lundgren T, Gritli-Linde A, Linde A.
Na+/Ca2+ exchanger isoforms of rat odontoblasts and
osteoblasts. Calcif Tissue Int 2000: 67: 6067.
141. Lundquist P, Ritchie HH, Moore K, Lundgren T,
Linde A. Phosphate and calcium uptake by rat
odontoblast-like MRPC-1 cells concomitant with min-
eralization. J Bone Miner Res 2002: 17: 18011813.
142. Linde A, Lundgren T. From serum to the mineral
phase. The role of the odontoblast in calcium transport
and mineral formation. Int J Dev Biol 1995: 39: 213
222.
143. Hubbard MJ, Bradley MP, Kardos TB, Forrester IT.
Calmodulin-like activity in a mineralizing tissue: the
rat molar tooth germ. Calcif Tissue Int 1981: 33:
545548.
144. Celio MR, Norman AW, Heizmann CW. Vitamin
D-dependent calcium-binding protein and parvalbu-
min occur in bones and teeth. Calcif Tissue Int 1984:
36: 129130.
145. Goldberg M, Escaig F, Feinberg J, Weinman S.
Ultrastructural localization of calmodulin in rat incisor
ameloblasts and odontoblasts during the early stages of
development. Adv Dent Res 1987: 1: 227235.
146. Magloire H, Joffre A, Azerad J, Lawson DEM. Locali-
zation of 28 kDa calbindin in human odontoblasts.
Cell Tissue Res 1988: 254: 341346.
147. Goldberg M, Feinberg J, Lecolle S, Kaetzel MA,
Rainteau D, Lessard JL, Dedman JR, Weinman S.
Co-distribution of annexin VI and actin in secretory
ameloblasts and odontoblasts of rat incisor. Cell Tissue
Res 1991: 263: 8189.
148. Boskey AL, Maresca M, Ullrich W, Doty SB, Butler
WT, Prince CW. Osteopontin-hydroxyapatite interac-
tions in vitro: inhibition of hydroxyapatite formation
and growth in a gelatin-gel. Bone Miner 1993: 22:
147159.
149. Schinke T, Amendt C, Trindl A, Pschke O,
Mller-Esterl W, Jahnen-Dechent W. The serum
protein a2-HS glycoprotein/fetuin inhibits apatite
formation in vitro and in mineralizing calvaria cells: a
possible role in mineralization and calcium homeosta-
sis. J Biol Chem 1996: 271: 2078920796.
150. Toroian D, Price PA. The essential role of fetuin in the
serum-induced calcification of collagen. Calcif Tissue
Int 2008: 82: 116126.
151. Hunter GK, Kyle CL, Goldberg HA. Modulation of
crystal formation by bone phosphoproteins: structural
specificity of the osteopontin-mediated inhibition of
hydroxyapatite formation. Biochem J 1994: 300: 723
728.
152. He G, George A. Dentin matrix protein 1 immobilized
on type I collagen fibrils facilitates apatite deposition in
vitro. J Biol Chem 2004: 279: 1164911656.
153. He G, Dahl T, Veis A, George A. Dentin matrix
protein 1 initiates hydroxyapatite formation in vitro.
Connect Tissue Res 2003: 44: 240245.
79
Tjderhane & Haapasalo
154. He G, Dahl T, Veis A, George A. Nucleation of apatite
crystals in vitro by self-assembled dentin matrix
protein 1. Nat Mater 2003: 21: 552558.
155. Massa LF, Ramachandran A, George A, Arana-Chavez
VE. Developmental appearance of dentin matrix
protein 1 during the early dentinogenesis in rat molars
as identified by high-resolution immunocytochemis-
try. Histochem Cell Biol 2005: 124: 197205.
156. Maciejewska I, Qin D, Huang B, Sun Y, Mues G,
Svoboda K, Bonewald L, Butler WT, Feng JQ, Qin C.
Distinct compartmentalization of dentin matrix
protein 1 fragments in mineralized tissues and cells.
Cells Tissues Organs 2009: 189: 186191.
157. Orsini G, Ruggeri A, Mazzoni A, Nato F, Falconi M,
Putignano A, Di Lenarda R, Nanci A, Breschi L.
Immunohistochemical localization of dentin matrix
protein 1 in human dentin. Eur J Histochem 2008: 52:
215220.
158. Gajjeraman S, Narayanan K, Hao J, Qin C, George A.
Matrix macromolecules in hard tissues control the
nucleation and hierarchical assembly of hydroxyapa-
tite. J Biol Chem 2007: 282: 11931204.
159. Qin C, Huang B, Wygant JN, McIntyre BW, McDon-
ald CH, Cook RG, Butler WT. A chondroitin sulfate
chain attached to the bone dentin matrix protein 1
NH2-terminal fragment. J Biol Chem 2006: 281:
80348040.
160. Butler WT. Macromolecules of extracellular matrix:
determination of selective structures and their func-
tional significance. Connect Tissue Res 2008: 49: 383
390.
161. Yamakoshi Y, Hu JC, Fukae M, Iwata T, Kim JW,
Zhang H, Simmer JP. Porcine dentin sialoprotein is a
proteoglycan with glycosaminoglycan chains contain-
ing chondroitin 6-sulfate. J Biol Chem 2005: 280:
15521560.
162. Qin C, Brunn JC, Baba O, Wygant JN, McIntyre BW,
Butler WT. Dentin sialoprotein isoforms: detection
and characterization of a high molecular weight
dentin sialoprotein. Eur J Oral Sci 2003: 111: 235
242.
163. Huang B, Sun Y, Maciejewska I, Qin D, Peng T,
McIntyre B, Wygant J, Butler WT, Qin C. Distribu-
tion of SIBLING proteins in the organic and inorganic
phases of rat dentin and bone. Eur J Oral Sci 2008:
116: 104112.
164. Prasad M, Butler WT, Qin C. Dentin sialophospho-
protein in biomineralization. Connect Tissue Res 2010:
51: 404417.
165. Bronckers AL, DSouza RN, Butler WT, Lyaruu DM,
van Dijk S, Gay S, Wltgens JH. Dentin sialoprotein:
biosynthesis and developmental appearance in rat
tooth germs in comparison with amelogenins, osteo-
calcin and collagen type-I. Cell Tissue Res 1993: 272:
237247.
166. Baba O, Qin C, Brunn JC, Wygant JN, McIntyre
BW, Butler WT. Colocalization of dentin matrix
protein 1 and dentin sialoprotein at late stages of rat
molar development. Matrix Biol 2004: 23: 371379.
80
167. Qin C, Baba O, Brunn JC, McKee MD, Bonewald L,
Butler WT. Dentin matrix protein 1 (DMP1) and
dentin sialophosphoprotein (DSPP) share unique
properties including tissue localization, proteolytic
processing, and high molecular weight forms. In:
Sodek J, Landis W, eds. Proceedings of the 8th ICCBMT.
Toronto: University of Toronto Press, 2005: 174
177.
168. Boskey A, Spevak L, Tan M, Doty SB, Butler WT.
Dentin sialoprotein (DSP) has limited effects on in
vitro apatite formation and growth. Calcif Tissue Int
2000: 67: 472478.
169. Suzuki S, Sreenath T, Haruyama N, Honeycutt C,
Terse A, Cho A, Kohler T, Mller R, Goldberg M,
Kulkarni AB. Dentin sialoprotein and dentin phospho-
protein have distinct roles in dentin mineralization.
Matrix Biol 2009: 28: 221229.
170. Stetler-Stevenson WG., Veis A. Type I collagen shows
a specific binding affinity for bovine dentin phospho-
phoryn. Calcif Tissue Int 1986: 38: 135141.
171. Zanetti M, de Bernard B, Jontell M, Linde A. Ca2+-
binding studies of the phosphoprotein from rat-incisor
dentine. Eur J Biochem 1981: 113: 541545.
172. Marsh ME. Binding of calcium and phosphate ions to
dentin phosphophoryn. Biochemistry 1989: 28: 346
352.
173. Kuboki Y, Fujisawa R, Aoyama K, Sasaki S. Calcium-
specific precipitation of dentin phosphoprotein: a new
method of purification and the significance for the
mechanism of calcification. J Dent Res 1979: 58:
19261932.
174. Marsh ME. Self-association of calcium and magnesium
complexes of dentin phosphophoryn. Biochemistry
1989: 28: 339345.
175. Weinstock M, Leblond, CP. Radioautographic visuali-
zation of the deposition of a phosphoprotein at the
mineralization front in the dentin of the rat incisor.
J Cell Biol 1973: 56: 838845.
176. Rabie AM, Veis A. An immunocytochemical study of
the routes of secretion of collagen and phosphophoryn
from odontoblasts into dentin. Connec. Tissue Res
1995: 31: 197209.
177. Qin C, Baba O, Butler WT. Post-translational modifi-
cations of sibling proteins and their roles in osteogen-
esis and dentinogenesis. Crit Rev Oral Biol Med 2004:
15: 126136.
178. Goldberg HA, Hunter GK. The inhibitory activity of
osteopontin on hydroxyapatite formation in vitro.
Ann N Y Acad Sci 1995: 760: 305308.
179. Wada T, McKee MD, Steitz S, Giachelli CM.
Calcification of vascular smooth muscle cell cultures:
inhibition by osteopontin. Circ Res 1999: 84: 166
178.
180. Boskey AL, Spevak L, Paschalis E, Doty SB, McKee
MD. Osteopontin deficiency increases mineral content
and mineral crystallinity in mouse bone. Calcif Tissue
Int 2002: 71: 145154.
181. Zhang B, Sun Y, Chen L, Guan C, Guo L, Qin C.
Expression and distribution of SIBLING proteins in

the predentin/dentin and mandible of Hyp mice. Oral
Dis 2010: 16: 453464.
182. Lesot H, Bgue-Kirn C, Kubler MD, Meyer JM,
Smith AJ, Cassidy N, Ruch JV. Experimental induc-
tion of odontoblast differentiation and stimulation
during reparative process. Cells Materials 1993: 3:
201207.
183. Ohshima H. Ultrastructural changes in odontoblasts
and pulp capillaries following cavity preparation in rat
molars. Arch Histol Cytol 1990: 53: 423438.
184. DSouza RN, Bachman T, Baumgardner KR, Butler
WT, Litz M. Characterization of cellular responses
involved in reparative dentinogenesis in rat molars.
J Dent Res 1995: 74: 702709.
185. Bronckers AL, Lyaruu DM, Goei W, Litz M, Luo G,
Karsenty G, Wltgens JH, DSouza RN. Nuclear DNA
fragmentation during postnatal tooth development of
mouse and hamster and during dentin repair in the rat.
Eur J Oral Sci 1996: 104(2(Pt 1)):102111.
186. Kitamura C, Kimura K, Nakayama T, Toyoshima K,
Terashita M. Primary and secondary induction of
apoptosis in odontoblasts after cavity preparation of rat
molars. J Dent Res 2001: 80: 15301534.
187. Ohshima H, Nakakura-Ohshima K, Takeuchi K,
Hoshino M, Takano Y, Maeda T. Pulpal regeneration
after cavity preparation, with special reference to close
spatio-relationships between odontoblasts and immu-
nocompetent cells. Microsc Res Tech 2003: 60: 483
490.
188. Ohshima H, Kawahara I, Maeda T, Takano Y. The
relationship between odontoblasts and immunocom-
petent cells during dentinogenesis in rat incisors: an
immunohistochemical study using OX6-monoclonal
antibody. Arch Histol Cytol 1994: 57:435447.
189. Paterson RC. Pulp response in sound and carious
teeth: a pilot study. Oral Surg Oral Med Oral Pathol
1981: 51: 209212.
190. Ohshima H, Nakakura-Ohshima K, Yamamoto H,
Maeda T. Responses of odontoblasts to cavity prepa-
ration in rat molars as demonstrated by immunocyto-
chemistry for heat shock protein (Hsp) 25. Arch Histol
Cytol 2001: 64: 493501.
191. Murray PE, About I, Lumley PJ, Franquin JC,
Remusat M, Smith AJ. Human odontoblast cell
numbers after dental injury. J Dent 2000: 28: 277
285.
192. Murray PE, About I, Lumley PJ, Smith G, Franquin
JC, Smith AJ. Postoperative pulpal and repair
responses. J Am Dent Assoc 2000: 131: 321329.
193. Simon S, Smith AJ, Lumley PJ, Berdal A, Smith G,
Finney S, Cooper PR. Molecular characterization of
young and mature odontoblasts. Bone 2009: 45: 693
703.
194. Mitsiadis TA, Rahiotis C. Parallels between tooth
development and repair: conserved molecular mech-
anisms following carious and dental injury. J Dent Res
2004: 83: 896902.
195. Goldberg M, Smith AJ. Cells and extracellular matrices
of dentin and pulp: a biological basis for repair and
Dentinpulp border
tissue engineering. Crit Rev Oral Biol Med 2004: 15:
1327.
196. Cooper PR, Takahashi Y, Graham LW, Simon S,
Imazato S, Smith AJ. Inflammationregeneration
interplay in the dentinepulp complex. J Dent 2010:
38: 687697.
197. Tziafas D. Dentinogenic potential of the dental pulp:
facts and hypotheses. Endod Topics 2010: 17: 42
64.
198. Vongsavan N, Matthews B. Fluid flow through cat
dentine in vivo. Arch Oral Biol 1992: 37: 175185.
199. Leonora J, Tieche JM, Steinman RR. The effect of
dietary factors on intradentinal dye penetration in the
rat. Arch Oral Biol 1992: 37: 733741.
200. Pashley DH. Dentine permeability and its role in the
pathobiology of dentine sensitivity. Arch Oral Biol
1994: 39(Suppl): 73S80S.
201. Sognnaes RF, Shaw JH, Bogoroch R. Radiotracer
studies on bone, cementum, dentin and enamel of
rhesus monkeys. Am J Physiol 1955: 180: 408420.
202. Potts TV, Cunningham T, Finkelstein MJ, Silverberg-
Strumfeld L. The movement of radioactive molecules
across dentine in vivo in the dog. Arch Oral Biol 1985:
30: 353357.
203. Steinman RR. Pharmacologic control of dentinal fluid
movement and dental caries in rats. J Dent Res 1968:
47: 720724.
204. Steinman RR, Leonora J. Effect of infusing selected
chemical compounds on dentinal fluid movement in
the rat. J Dent Res 1975: 54: 567569.
205. Litovsky B, Arancibia S. [Changes induced by ouabain
and colchicine in the transdentinal flow estimated in
vivo by perfusion: push-pull of the incisors of rats].
J Biol Buccale 1988: 16: 209214.
206. Pkknen V, Vuoristo JT, Salo T, Tjderhane L.
Comparative gene expression profile analysis between
native human odontoblasts and pulp tissue. Int Endod
J 2008: 41: 117127.
207. Thomas M, Leaver AG. Identification and estimation
of plasma proteins in human dentine. Arch Oral Biol
1975: 20: 217218.
208. Holland GR. Morphological features of dentine and
pulp related to dentine sensitivity. Arch Oral Biol
1994: 39(Suppl): 3S11S.
209. Brnnstrm M. A hydrodynamic mechanism in the
transmission of pain-producing stimuli through the
dentine. In: Anderson DJ, ed. Sensory Mechanisms in
Dentine. Pergamon, 1963: 7379.
210. Charoenlarp P, Wanachantararak S, Vongsavan N,
Matthews B. Pain and the rate of dentinal fluid flow
produced by hydrostatic pressure stimulation of
exposed dentine in man. Arch Oral Biol 2007: 52:
625631.
211. Matthews B, Vongsavan N. Interactions between
neural and hydrodynamic mechanisms in dentine and
pulp. Arch Oral Biol 1994: 39(Suppl): 87S-95S.
212. Thomas HF. The dentinpredentin complex and its
permeability: anatomical overview. J Dent Res 1985:
64(Spec Iss): 607612.
81
Tjderhane & Haapasalo
213. Pashley DH, Tay FR, Breschi L, Tjderhane L,
Carvalho RM, Carrilho M, Tezvergil-Mutluay A. State
of the art etch-and-rinse adhesives. Dent Mater 2011:
27: 116.
214. Mjr IA. Dentin permeability: the basis for under-
standing pulp reactions and adhesive technology. Braz
Dent J 2009: 20: 316.
215. Hahn CL, Liewehr FR. Innate immune responses of
the dental pulp to caries. J Endod 2007: 33: 643
651.
216. Levin ED. Extracellular superoxide dismutase (EC-
SOD) quenches free radicals and attenuates age-
related cognitive decline: opportunities for novel drug
development in aging. Curr Alzheimer Res 2005: 2:
191196.
217. Leonora J, Steinman RR. Evidence suggesting the
existence of a hypothalamic-parotid gland endocrine
axis. Endocrinology 1968: 83: 807815.
218. Steinman RR, Leonora J. Relationship of fluid trans-
port through the dentin to the incidence of dental
caries. J Dent Res 1971: 50: 15361543.
219. Steinman RR, Leonora J. Effect of selected dietary
additives on the incidence of dental caries in the rat.
J Dent Res 1975: 54: 570577.
220. Steinman RR, Leonora J, Singh RJ. The effect of
desalivation upon pulpal function and dental caries in
rats. J Dent Res 1980: 59: 176185.
221. Tieche JM, Leonora J. Biological and chemical evi-
dence for the existence of a porcine hypothalamic
parotid hormone-releasing factor. Biochem Biophys Res
Commun 1989: 159: 899906.
222. Leonora J, Tieche JM, Steinman RR. Further evidence
for a hypothalamus-parotid gland endocrine axis in the
rat. Arch Oral Biol 1993: 38: 911916.
223. Leonora J, Tieche JM, Steinman RR. Stimulation of
intradentinal dye penetration by feeding in the rat.
Arch Oral Biol 1993: 38: 763767.
224. Tieche JM, Leonora J, Steinman RR. High-sucrose
diet inhibits basal secretion of intradentinal dye
penetration-stimulating parotid hormone in pig.
J Appl Physiol 1994: 76: 218222.
225. Tieche JM, Leonora J, Steinman RR. Isolation and
partial characterization of a porcine parotid hormone
that stimulates dentinal fluid transport. Endocrinology
1980: 106: 19942005.
226. Leonora J, Tieche JM, Celestin J. Physiological factors
affecting secretion of parotid hormone. Am J Physiol
1987: 252: E477E484.
227. Yamamoto T, Kobayashi M, Kobayashi M. Purification
and properties of dentinal fluid transport stimulating
substance from bovine parotid gland. Chem Pharm
Bull 1986: 34: 12031211.
228. Yamamoto T, Kobayashi M, Kobayashi M, Yamamoto
M, Nomura M, Aonuma S. Isolation and amino acid
sequence of dentinal fluid transport-stimulating
peptide from rat parotid glands. Chem Pharm Bull
1986: 34: 38033811.
229. Zhang Q, Szalay AA, Tieche JM, Kyeyune-Nyombi E,
Sands JF, Oberg KC, Leonora J. Cloning and func-
82
tional study of porcine parotid hormone, a novel
proline-rich protein. J Biol Chem 2005: 280: 22233
22244.
230. Steinman RR. Physiologic activity of the pulpdentin
complex. Quintessence Int 1985: 16: 723726.
231. Tjderhane L, Hietala E-L, Huumonen S, Larmas M.
The effect of high sucrose diet on mineralized tissues.
In: Pandalai SD, ed. Recent Research Development in
Nutrition. Research Signpost, 2001: 126.
232. Roggenkamp C. Dentinal Fluid Transport. Loma
Linda: Loma Linda University Press, 2004.
233. Leonora J, Tjderhane L, Tieche JM. Effect of dietary
carbamyl phosphate on dentine apposition in rat
molars. Arch Oral Biol 2002: 47: 147153.
234. Leonora J, Tjderhane L, Tieche JM. Parotid gland
function and dentin apposition in rat molars. J Dent
Res 2002: 81: 259264.
235. Allard B, Couble ML, Magloire H, Bleicher F. Char-
acterization and gene expression of high conductance
calcium-activated potasium channels displaying mech-
anosensitivity in human odontoblasts. J Biol Chem
2000: 275: 2555625561.
236. Allard B, Magloire H, Couble ML, Maurin JC,
Bleicher F. Voltage-gated sodium channels confer
excitability to human odontoblasts: possible role in
tooth pain transmission. J Biol Chem 2006: 281:
2900229010.
237. Magloire H, Lesage F, Couble ML, Lazdunski M,
Bleicher F. Expression and localization of TREK-1 K+
channels in human odontoblasts. J Dent Res 2003: 82:
542545.
238. Allardt B, Couble ML, Magloire H, Bleicher F.
Characterization and gene expression of high-
conductance calcium-activated potassium channels
displaying mechanosensitivity in human odontoblasts.
J Biol Chem 2000: 275: 2555625561.
239. Kim YS, Kim YJ, Paik SK, Cho YS, Kwon TG,
Ahn DK, Kim SK, Yoshida A, Bae YC. Expres-
sion of metabotropic glutamate receptor mGluR5
in human dental pulp. J Endod 2009: 35: 690
694.
240. Son AR, Yang YM, Hong JH, Lee SI, Shibukawa Y,
Shin DM. Odontoblast TRP channels and thermo/
mechanical transmission. J Dent Res 2009: 88: 1014
1019.
241. Okumura R, Shima K, Muramatsu T, Nakagawa K,
Shimono M, Suzuki T, Magloire H, Shibukawa Y. The
odontoblast as a sensory receptor cell? The expression
of TRPV1 (VR-1) channels. Arch Histol Cytol 2005:
68: 251257.
242. Pedersen SF, Owsianik G, Nilius B. TRP channels: an
overview. Cell Calcium 2005: 38: 233252.
243. Nozawa Y, Nishihara K, Yamamoto A, Nakano M,
Ajioka H, Matsuura N. Distribution and characteriza-
tion of vanilloid receptors in the rat stomach. Neurosci
Lett 2001: 7: 31373151.
244. Yeon KY, Chung G, Shin MS, Jung SJ, Kim JS, Oh SB.
Adult rat odontoblasts lack noxious thermal sensitivity.
J Dent Res 2009: 88: 328332.

245. Gerdes JM, Davis EE, Katsanis N. The vertebrate
primary cilium in development, homeostasis, and
disease. Cell 2009: 137: 3245.
246. Praetorius HA, Spring KR. The renal cell primary
cilium functions as a flow sensor. Curr Opin Nephrol
Hypertens 2003: 12: 517520.
247. Whitfield JF. Primary ciliumis it an osteocytes
strain-sensing flowmeter? J Cell Biochem 2003: 89:
233237.
248. Malone AM, Anderson CT, Tummala P, Kwon RY,
Johnston TR, Stearns T, Jacobs CR. Primary cilia
mediate mechanosensing in bone cells by a calcium-
independent mechanism. Proc Natl Acad Sci USA
2007: 104: 1332513330.
249. Xiao ZS, Quarles LD. Role of the polycytin-primary
cilia complex in bone development and mechanosens-
ing. Ann N Y Acad Sci 2010: 1192: 410421.
250. McGlashan SR, Jensen CG, Poole CA. Localization of
extracellular matrix receptors on the chondrocyte
primary cilium. J Histochem Cytochem 2006: 54:
10051014.
251. McGlashan SR, Knight MM, Chowdhury TT, Joshi P,
Jensen CG, Kennedy S, Poole CA. Mechanical loading
modulates chondrocyte primary cilia incidence and
length. Cell Biol Int 2010: 34: 441446.
252. Couve E. Ultrastructural changes during the life cycle
of human odontoblasts. Arch Oral Biol 1986: 31:
643651.
253. Magloire H, Couble ML, Romeas A, Bleicher F.
Odontoblast primary cilia: facts and hypotheses. Cell
Biol Int 2004: 28: 9399.
254. Thivichon-Prince B, Couble ML, Giamarchi A,
Delmas P, Franco B, Romio L, Struys T, Lambrichts I,
Ressnikoff D, Magloire H, Bleicher F. Primary cilia of
odontoblasts: possible role in molar morphogenesis.
J Dent Res 2009: 88: 910915.
255. Magloire H, Couble ML, Thivichon-Prince B, Maurin
JC, Bleicher F. Odontoblast: a mechano-sensory
cell. J Exp Zool B Mol Dev Evol 2009: 312B: 416
424.
256. Rodat-Despoix L, Delmas P. Ciliar functions in the
nephron. Pflugers Arch 2009: 458: 179187.
257. Ferrante MI, Zullo A, Barra A, Bimonte S, Messaddeq
N, Studer M, Doll P, Franco B. Oral-facial-digital
type I protein is required for primary cilia formation
and left-right axis specification. Nat Genet 2006: 38:
112117.
258. Levin LG, Rudd A, Bletsa A, Reisner H. Expression of
IL-8 by cells of the odontoblast layer in vitro. Eur J
Oral Sci 1999: 107: 131137.
259. Durand SH, Flacher V, Romeas A, Carrouel F,
Colomb E, Vincent C, Magloire H, Couble M-L,
Bleicher F, Staquet MJ, Lebecque S, Farges JC.
Lipoteichoic acid increases TLR and functional che-
mokine expression while reducing dentin formation in
in vitro differentiated human odontoblasts. J Immunol
2006: 176: 28802887.
260. Veerayutthwilai O, Byers MR, Pham TT, Darveau RP,
Dale BA. Differential regulation of immune responses
Dentinpulp border
by odontoblasts. Oral Microbiol Immunol 2007: 22:
513.
261. Goldberg M, Farges JC, Lacerda-Pinheiro S, Six N,
Jegat N, Decup F, Septier D, Carrouel F, Durand S,
Chaussain-Miller C, Denbesten P, Veis A, Poliard A.
Inflammatory and immunological aspects of dental
pulp repair. Pharmacol Res 2008: 58: 137147.
262. Farges JC, Keller JF, Carrouel F, Durand SH, Romeas
A, Bleicher F, Lebecque S, Staquet MJ. Odontoblasts
in the dental pulp immune response. J Exp Zool B Mol
Dev Evol 2009: 5: 425436.
263. Staquet MJ, Durand SH, Colomb E, Romas A,
Vincent C, Bleicher F, Lebecque S, Farges JC. Differ-
ent roles of odontoblasts and fibroblasts in immunity.
J Dent Res 2008: 87: 256261.
264. Takeuchi O, Akira S. Pattern recognition receptors and
inflammation. Cell 2010: 140: 805820.
265. Mutoh N, Tani-Ishii N, Tsukinoki K, Chieda K,
Watanabe K. Expression of toll-like receptor 2 and 4 in
dental pulp. J Endod 2007: 33: 11831186.
266. Love RM, Jenkinson HF. Invasion of dentinal tubules
by oral bacteria. Crit Rev Oral Biol Med 2002: 13:
171183.
267. Martin FE. Carious pulpitis: microbiological and his-
topathological considerations. Aust Endod J 2003: 29:
134137.
268. Jiang HW, Zhang W, Ren BP, Zeng JF, Ling JQ.
Expression of toll like receptor 4 in normal human
odontoblasts and dental pulp tissue. J Endod 2006: 32:
747751.
269. Schrder JM. Epithelial peptide antibiotics. Biochem
Pharmacol 1999: 57: 121134.
270. Abiko Y, Nishimura M, Kaku T. Defensins in saliva and
the salivary glands. Med Electron Microsc 2003: 36:
247252.
271. Tao R, Jurevic RJ, Coulton KK, Tsutsui MT, Roberts
MC, Kimball JR, Wells N, Berndt J, Dale BA. Salivary
antimicrobial peptide expression and dental caries
experience in children. Antimicrob Agents Chemother
2005: 49: 38833888.
272. Dale BA, Tao R, Kimball JR, Jurevic RJ. Oral antimi-
crobial peptides and biological control of caries. BMC
Oral Health 2006: 6(Suppl 1): S13.
273. Nishimura E, Eto A, Kato M, Hashizume S, Imai S,
Nisizawa T, Hanada N. Oral streptococci exhibit
diverse susceptibility to human beta-defensin-2: anti-
microbial effects of hBD-2 on oral streptococci. Curr
Microbiol 2004: 48: 8587.
274. Dommisch H, Winter J, Ail Y, Dunsche A, Tiemann
M, Jepsen S. Human beta-defensin (hBD-1, -2)
expression in dental pulp. Oral Microbiol Immunol
2005: 20: 163166.
275. Dommisch H, Winter J, Willebrand C, Eberhard J,
Jepsen S. Immune regulatory functions of human
beta-defensin-2 in odontoblast-like cells. Int Endod J
2007: 40: 300307.
276. Dommisch H, Steglich M, Eberhard J, Winter J,
Jepsen S. Phosphatidylinositol-3-kinase inhibitor
LY 294002 blocks Streptococcus mutans-induced inter-
83
Tjderhane & Haapasalo
leukin (IL)-6 and IL-8 gene expression in
odontoblast-like cells. Int Endod J 2008: 41: 763
771.
277. Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E,
Shakhov A, Chertov O, Shirakawa AK, Farber JM,
Segal DM, Oppenheim JJ, Kwak LW. Toll-like recep-
tor 4-dependent activation of dendritic cells by beta-
defensin 2. Science 2002: 298: 10251029.
278. Hazeki K, Nigorikawa K, Hazeki O. Role of phospho-
inositide 3-kinase in innate immunity. Biol Pharm Bull
2007: 30: 16171623.
279. Jontell M, Okiji T, Dahlgren U, Bergenholtz G.
Immune defense mechanisms of the dental pulp. Crit
Rev Oral Biol Med 1998: 9: 179200.
280. Ohshima H, Maeda T, Takano Y. The distribution and
ultrastructure of class II MHC-positive cells in human
dental pulp. Cell Tissue Res 1999: 295: 151158.
281. Ohshima H, Nakakura-Ohshima K, Takeuchi K,
Hoshino M, Takano Y, Maeda T. Pulpal regeneration
after cavity preparation, with special reference to close
spatio-relationships between odontoblasts and immu-
nocompetent cells. Microsc Res Tech 2003: 60: 483
490.
282. Keller JF, Carrouel F, Colomb E, Durand SH,
Baudouin C, Msika P, Bleicher F, Vincent C, Staquet
MJ, Farges JC. Toll-like receptor 2 activation by
84
lipoteichoic acid induces differential production of
pro-inflammatory cytokines in human odontoblasts,
dental pulp fibroblasts and immature dendritic cells.
Immunobiology 2010: 215: 5359.
283. Pkknen V, Vuoristo J, Salo T, Tjderhane L. Effects
of TGF-b1 on interleukin profile of human dental pulp
and odontoblasts. Cytokine 2007: 4: 4451.
284. Horst OV, Tompkins KA, Coats SR, Braham PH,
Darveau RP, Dale BA. TGF-b1 inhibits TLR-
mediated odontoblast responses to oral bacteria.
J Dent Res 2009: 88: 333338.
285. Mjr IA. Dentin-predentin complex and its permeabil-
ity: pathology and treatment overview. J Dent Res
1985: 64(Spec No): 621627.
286. Boushell LW, Kaku M, Mochida Y, Bagnell R,
Yamauchi M. Immunohistochemical localization of
matrixmetalloproteinase-2 in human coronal dentin.
Arch Oral Biol 2008: 53: 109116.
287. Mitsiadis TA, De Bari C, About I. Apoptosis in devel-
opmental and repair-related human tooth remodeling:
a view from the inside. Exp Cell Res 2008: 314; 869
877.
288. Hietala EL, Tjderhane L, Larmas M. Dentin caries
recording with Schiffs reagent, fluorescence, and
back-scattered electron image. J Dent Res 1993: 72:
15881592.