Molecular Aspects of Tocrth
Pathpgenesis and Repair:
in vivo and in vitro Models
Imad About, Thimios A. Mitsiadis*
Faculte d'Odontologie, Universite de la Mediterranee, 27 Boulevard Jean
Moulin, 13385 Marseille Cedex 5, France; "corresponding author,
mitsiadis.e@odontologie.univ-mrs.fr
Adv Dent Res 15:59-62, August, 2001
Abstract Several growth factors and extracellular matrix
molecules, which are expressed during embryonic tooth
development, are re-expressed in dental tissues under
pathological conditions. Pathological conditions such as
caries lesions and dental injuries are often lethal to the
odontoblasts, which are then replaced by other pulp cells.
These cells are able to differentiate into odontoblast-like
cells and produce a reparative dentin. Here we demonstrate
the in vivo distribution of several molecules in human
permanent teeth under normal and pathological conditions.
The intermediate filament protein nestin, which is a marker
of young odontoblasts, is absent from old permanent teeth.
Similarly, the Notch protein, which is involved in cell fate
specification and is localized in the sub-odontoblastic cell
layer during odontogenesis, is not detected in adult dental
tissues. In carious and injured teeth, nestin is expressed in a
selective manner in odontoblasts surrounding the injury
site, while Notch is expressed in the sub-odontoblastic layer
of cells. We reproduced this physiological event in an in
vitro culture system. Pulp cells cultured in the presence of
(3-glycerophosphate formed mineralization nodules. As
odontoblasts, pulp cells contributing to the nodule
formation express type I collagen, osteonectin, dentin
sialophosphoprotein, and nestin. In this in vitro assay
system, nestin is up-regulated after local application of
Bone Morphogenetic Protein 2 and 4. Fourier transform
infrared microspectroscopy showed that both the organic
and the mineral compositions of the nodules have the
characteristics of human dentin and differ from those of
enamel and bone. These findings show that both the
molecular and the mineral characteristics of the human
dentin matrix are respected in the in vitro culture
conditions.
Introduction
D
uring tooth development, inductive epithelial-
jmesenchymal interactions lead to the differentiation of
'ectomesenchymal pulp cells into odontoblasts. These
cells express specific gene products that will form the
highly mineralized extracellular matrix of dentin.
Hydroxyapatite forms the main inorganic part of dentin, while
the organic components consist mostly of type I collagen (Butler
and Ritchie, 1995). Fewer amounts of the non-collagenous
proteins decorin, biglycan, osteonectin, osteocalcin, osteopontin,
bone sialoprotein, and dentin matrix protein 1, which are
detected in the bone matrix, are also found in the dentin.
However, two extracellular matrix proteins have been shown to
be specific for the dentin matrix: the dentin sialoprotein (DSP)
and the dentin sialophosphoprotein (DSPP) (Butler and Ritchie,
1995). Furthermore, dentin is a reservoir of growth factors such
as transforming growth factor beta (TGF3), bone morphogenetic
proteins (BMPs), and fibroblast growth factors (FGFs), since these
molecules are captured in the dentin matrix (Ruch et al., 1995).
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In pathological conditions involving mild dentin lesions
(i.e., caries lesions), the activity of odontoblasts is stimulated to
elaborate reactionary dentin. This stimulation may be the effect
of several signaling molecules (i.e., TGF(31, BMP-2) liberated
from the dentin during the demineralization process (Tziafas et
al., 2000). In contrast, pathological conditions involving a
violent stress (i.e., deep cavity preparation) lead to odontoblast
disintegration. In this case, newly formed odontoblast-like
cells, possibly originating from dental pulp fibroblasts,
elaborate a reparative dentin (Tziafas et al., 2000). The
deposition of reparative dentin may increase in vivo after local
application of a pulp-capping medication that contains
signaling molecules such as BMPs (Nakashima, 1994). Thus, it
seems that specific signaling molecules are necessary to
stimulate both proliferation and differentiation of pulp cells.
Several in vitro models have been proposed for the study of the
mechanisms underlying reactionary dentin production. In
addition, many attempts have been carried out to reproduce in
vitro the conditions necessary for dental pulp cell
differentiation into odontoblasts, thus simulating the events of
in vivo reparative dentin production.
The comparative study of molecules involved in
dentinogenesis during normal and pathological conditions may
indicate several common molecular mechanisms for dental tissue
development and repair. For this purpose, normal (embryonic
and adult), carious, and injured human teeth were collected and
used for the detection of several molecules. We mostly studied
the expression of Notch, which is involved in the processes of cell
fate specification and cell proliferation, and nestin, which is an
excellent marker of differentiated odontoblasts, to understand
the mechanisms leading to the generation of odontoblast-like
cells under pathological conditions.
Normal Conditions
For the study of normal dentinogenesis, we used human dental
fetal tissues (gestation week 18, g.w. 18), which were obtained
after legal abortions. This study was carried out in compliance
with French legislation, after approval of the Regional Ethics
Committee of the Marseille Hospital Center (CCPPRB
Marseille I). The tissues were fixed immediately in 10%
buffered formalin for 5 days. The samples were then
decalcified for 3 wks in formic acid/10% formalin prior to
being embedded in Paraplast. Dentinogenesis is initiated at the
tip of the cusp during the bell stage of tooth development (g.w.
18). The pulp cells adjoining the dental epithelium differentiate
into odontoblasts, and start to secrete the organic matrix of
dentin. We used immunohistochemistry for the detection of
several molecules such as nestin and Notch.
Nestin is an intermediate filament, which is expressed
predominantly in the developing nervous system and muscles
(Lendahl et ah, 1990). During dentinogenesis, nestin
immunoreactivity was observed in the odontoblasts and pulp
fibroblasts of the cusp area (About et al., 2000b). Nestin expression
was also evident in the processes of the odontoblasts up to the
Key Words
Nestin, human, tooth, differentiation, dentin, odontoblast, culture.
Presented at the International Meeting on Signaling Mechanisms in
Dentin Development, Regeneration, and Repair: from Bench to
Clinic, held at Thessaloniki, Greece, November 10-11, 2000
59

Fig. 1 Immunohistochemical localization of nestin in sections of normal and
pathological human teeth. (A) Nestin immunoreactivity is observed in the cell
bodies and processes of the odontoblasts of the developing adult teeth. (B) In
carious teeth, nestin is observed in odontoblast bodies facing the carious
irritation and cells near the dilated blood vessels. (C) Hematoxylin-eosin staining.
Nine weeks after the cavity preparation, reparative dentin is seen near the site of
the injury. (D) Nestin is observed in odontoblasts at a site distal to the injury site.
Abbreviations: c, cavity; d, dentin; o, odontoblasts; p, pulp; rd, reparative dentin.
dentin-enamel junction. A staining gradient was observed in the
odontoblasts from the cervical loop to the cusp region: The
cervical loop was negative, while the immunostaining increased
toward the cusp region. In the 17-year-old developing third
molars, nestin expression was restricted to odontoblasts.
Distribution was seen in both the cell bodies and the processes of
the odontoblasts (Fig. 1A). The distribution of nestin in the
odontoblasts exhibited a gradient following their maturation
state. In mature odontoblasts, nestin immunoreactivity was
observed only in their processes, while in young odontoblasts,
nestin immunostaining was restricted to the cell bodies.
Immunoreactivity for nestin was also observed in some pulp
fibroblasts near blood vessels. In adult teeth (40 yrs old), we could
not detect immunoreactivity for nestin. The nestin expression
pattern in human teeth differs from that reported in the teeth of
rodents (Terling et ah, 1995). Nestin is down-regulated from
odontoblasts in mature permanent human teeth, whereas nestin
expression is maintained in odontoblasts of aged rats. The reason
for this species difference in expression is not known, but it may
be the difference in life span between rodents and humans. The
expression pattern of nestin in embryonic human teeth, i.e., a
transient expression during development of an organ, is quite
similar to nestin expression seen in other tissuesfor example,
the nervous system and muscle (Lendahl et al., 1990).
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60 About & Mitsiadis
The Notch signaling pathway controls cell fate commitment
during development of a wide range of tissues. Previously, we
have studied the expression of the Notch receptors and its
ligand Deltal during tooth development (Mitsiadis et al., 1998).
During dentinogenesis, the Deltal and Notch genes showed
complementary expression patterns: Deltal is expressed in
differentiating odontoblasts, whereas Notch expression is
confined to sub-odontoblastic cells, suggesting a role for Notch
signaling in the control of odontoblast differentiation. Both
Notch and Delta were absent from adult dental tissues.
Pathological-Dentin Repair Conditions
Carious and injured teeth were collected for the study of dental
tissue reactions under pathological conditions. Teeth were
fixed in 10% neutral-buffered formalin for 24 hrs,
demineralized in sodium formiate for 21 days, and then
embedded in paraffin wax.
Micro-organisms are involved in both decalcification and
proteolysis of the dentin during the process of dental caries.
During dentin decalcification, the activity of the odontoblasts
is stimulated, leading to the production of reactionary dentin.
If the irritation increases, the odontoblasts may be
disintegrated. Pulp cells then replace the missing odontoblasts,
which differentiate into odontoblast-like cells and start the
secretion and deposition of reparative dentin. The pulp
volume is reduced with the elaboration of reactionary and
reparative dentin. In carious teeth, nestin immunoreactivity
was seen in cells surrounding the caries lesion. Nestin is
distributed in the processes of mature odontoblasts situated
near the carious front, but was absent in the carious front level.
When caries progresses rapidly, the blood vessels of the pulp
dilate, and scattered inflammatory cells become evident in the
pulp. Nestin is expressed in dying odontoblasts facing the
irritation front and forming the dead tracts, as well as in
inflammatory cells close to the dilated blood vessels (Fig. IB).
Notch2 immunoreactivity is also observed in blood vessels and
inflammatory cells. This indicates a correlation between
nestin/Notch up-regulation and inflammatory events.
Since the dentin matrix can be seen as a reservoir of
signaling molecules such as BMPs and FGFs, these molecules
can be released from the matrix and can diffuse to reach
adjacent cells during dentin decalcification. Dental pulp cells
in the vicinity of the lesion, under the influence of the
diffused BMP4, could then differentiate into odontoblast-like
cells and start the secretion and deposition of the reparative
dentin matrix.
Injured teeth were used after cavity preparation in intact
first premolars of 15-year-old adolescents. Cavities were
prepared in dentin with a bur under the least possible
pressure. The cavities were restored with a calcium hydroxide
product. After a post-operative interval of 9 wks, the teeth
were extracted with the patients' informed consent. Nine
weeks after cavity preparation, reparative dentin deposition
was seen near the injury site (Fig. 1C). This reparative dentin
matrix was synthesized by the newly formed odontoblasts,
replacing the dying odontoblasts after the injury.
Nestin immunoreactivity was not detected at the site of
the reparative dentin production 9 wks after the lesion (About
et al., 2000b). The absence of the staining at the site of dentin
production may be due to the delay between cavity
preparation and tooth extraction. However, nestin staining
was evident at a distance from the cavity preparation (Fig.
ID). The exact function of nestin has not been established, but
its expression in the cell bodies suggests a role in dentin
matrix synthesis. Odontoblasts are accompanied by nerve
fibers, but direct contacts between these two structures have
not been reported. It has been shown recently that the
distribution and expression of nestin in myofibers are
regulated by innervation, suggesting a similar effect in
odontoblasts (About et al, 2000b).
Adv Dent Res 1 5:59-62, August, 2001
 In teeth with a pulp lesion,
Notch2 immunoreactivity was strong
in the pulp mesenchyme located
close to the site of the injury
(Mitsiadis et al., 1999). A surprising
result was that Notch2 expression is
activated not only in pulp cells close
to the injury, but also at the apex of
the roots, suggesting that these sites
represent important cell pools from
which different pulp cell types will
derive after injury. Although blood
vessels irrigating the crown pulp
were negative for Notch2, a strong
staining was found in vascular
structures traversing the roots of the
injured teeth. Deltal expression was
activated in vascular structures of
the root, reflecting either ingrowth of
new blood vessels or an inflam-
matory reaction (Mitsiadis et al.,
1999). The expression pattern of
Deltal in the roots of injured teeth is
complementary to that of Notch2,
which is expressed in the sub-
odontoblastic layer of cells.
The processes of pulp healing
and regeneration can be stimulated
by the local application of signaling
molecules such as BMP-2 and BMP-4
(Nakashima, 1994). However, differ- Fig. 2 Formation of mineralization nodules in human dental pulp explants in vitro after p-glycerophosphate
entiation of odontoblast-like cells treatment. (A) Phase-contrast microscopy showing the formation of nodules after 3 wks of treatment.
and hard tissue formation have been Immunohistochemical localization of osteonectin (B), collagen I (C), and nestin (D) expression in the
shown after pulp amputation in vivo mineralization nodules.
without the addition of any of these
signaling molecules (Van Mullem,
1991). This may be due to the
expression of TGFB1, BMP-2, BMP-4,
and BMP-7 by human pulp cells. In an attempt to show that these mineralization nodules have
the characteristics of dentin, we used Fourier transform infrared
microspectroscopy (FTIR-MS). When the FTIR-MS spectra
In vitro Conditions obtained from the mineralization nodules in vitro were compared
For cell cultures, normal extracted immature third molars were with those obtained in vivo, they revealed an analogy with those
used. Each dental pulp was divided into two groups, cultured of the dentin, with peaks and bands of the same aspects at the
either without or with B-glycerophosphate. In the first group, same locations. Moreover, when the mineralization spectra
the explants were cultured in MEM medium. In the second obtained in vitro were compared with other tooth hard tissues,
group, the explants were cultured in the same medium major differences were observed when they were compared with
supplemented with 2 mM B-glycerophosphate. the spectra obtained from the enamel and the alveolar bone.
Three weeks after culture in the presence of B- For explant culture, human dental pulps were minced
glycerophosphate, regular and fiber-like structures started with scalpels and then rinsed with PBS. After being minced,
to appear from the explant border and extending toward the the explants were cultured in MEM medium supplemented
periphery (About et al., 2000a). This was followed by the with 2 mM B-glycerophosphate. BMPs and FGFs were used to
deposition of mineral crystals along and within the fibrous pre-load agarose beads to concentrations from 100 to 200
structures, and this mineralization front continued to (jLg/mL. Beads were transferred to the tops of human dental
expand during the eight-week culture procedure (Fig. 2A). pulp explants, and after 24 hrs of culture, the explants were
The cells in direct contact with the nodules exhibited a fixed in 4% PFA and processed for whole-mount
polarized morphology similar to that observed in vivo. immunohistochemistry as described previously.
However, formation of dentinal tubuli was not observed. Although the molecular interactions underlying reparative
No mineralization was observed in control cultures (without processes are not well-understood, signaling molecules of the
B-glycerophosphate). TGFB superfamily seem to be important to hard tissue formation
Type I collagen immunoreactivity was strong and uniform after pulp injury. It has been shown that TGFB1 and BMPs may
in all p-glycerophosphate-treated cells. The expression of induce odontoblast differentiation (Ruch et al., 1995) and up-
collagen I was also evident in the mineralization nodules (Fig. regulate Deltal expression in dental mesenchyme in vitro (Mitsiadis
2C). Osteonectin (Fig. 2B) and dentin sialophosphoprotein et al., 1998). Taken together, these results suggest that members of
(data not shown) were expressed in these cells as well as in the the TGFB superfamily may also be involved in regulating nestin
nodules. Nestin is also up-regulated in dental pulp cells which and Deltal after injury. We followed the expression of nestin by
have differentiated to odontoblast-like cells and secrete the whole-mount immunohistochemistry. Analysis of the explants
matrix of the mineralization nodules in the in vitro assay shows nestin immunoreactivity in pulp cells surrounding beads
system (Fig. 2D). This confirms the re-expression of nestin in containing BMP4. BMP4 up-regulated nestin expression in a wide
odontoblast-like cells and the potential of pulp cells to area of cells surrounding the bead. This is the first example of a
differentiate into cells secreting dentin matrix. signaling molecule directly influencing nestin expression.
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Adv Dent Res 1 5:59-62, August, 2001 Molecular Aspects of Tooth Pathogenesis and Repair 61
 Conclusion
Nestin and Notch are involved in the dynamic processes
triggered by pulp injury. Notch up-regulation in the injured
pulp may represent an early molecular event in dental tissue
repair processes, since expression is observed early after injury
(Mitsiadis et ah, 1999). Notch2 seems to be the most important
receptor in injured molars, since its expression in pulp cells
was predominant. During dentinogenesis, Notch is expressed
in cells of the sub-odontoblastic layer, which are probably
committed to an odontoblastic fate (Mitsiadis et ah, 1998). By
contrast, nestin is detected in odontoblasts (About et ah, 2000b).
During regeneration of injured molars, Notch2 expression may
be activated in undifferentiated sub-odontoblastic cells which
are engaged in a differentiation pathway leading to nestin-
positive odontoblasts and/or pulp fibroblasts. In this way,
activation of both nestin and Notch after injury or during
regeneration may ensure a continuous balance between
differentiated odontoblasts and progenitors committed to
becoming odontoblasts. These results highlight the similarities
between developmental and regenerative processes and add
further weight to the notion that activation of Notch and nestin
is instrumental in tooth homeostasis.
Cultured human pulp cells are able to differentiate into
odontoblast-like cells and to secrete dentin matrix in vitro. The
differentiation of dental pulp cells into odontoblast-like cells is
shown by morphological and, most notably, by molecular
criteria. The cells are polarized in contact with the
mineralization nodules, and synthesize collagen I, DSPP, and
osteonectin (Couble et ah, 2000). This extracellular mineralized
matrix shows the main characteristics of the dentin produced
in vivo. The synthesized extracellular matrix molecules formed
organic fiber-like structures, and these fibrils were the
initiation sites of mineral crystal formation. In spite of the
absence of the tubuli in the matrix produced in vitro, both the
organic and mineral composition of the nodules is similar to
the composition of dentin. This is confirmed by the expression
of nestin, which characterizes the odontoblasts, and the
analogy between the FTIR-MS spectra obtained in vitro and
those of the dentin in vivo.
Taken together, these results show that both the molecular
and the mineral characteristics of the human dentin matrix are
respected in the culture conditions. This system represents a
useful model for the study of many pathological conditions
affecting the human teeth leading to reparative dentin
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62
production. Moreover, this culture system may be useful for
the study of new biomedical products used in restorative
dentistry, and most particularly after direct pulp-capping.
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About & Mitsiadis Adv Dent Res 15:59-62, August, 2001