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From the Laboratoire Interface Matrice Extracellulaire
Biomatériaux,*
Equipe d’Acceuil 2198,
Faculté d’Odontologie, Université de la
Méditerranée, Marseille, France; the Centre d’Etude des
Biomateriaux et Interfaces,
Equipe d’Acceuil
2068, Institut Fédératif de Recherche 53, Faculté
d’Odontologie, Reims, France; and the Department of Cell and Molecular
Biology,
Medical Nobel Institute, Karolinska
Institutet, Stockholm, Sweden
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-internexin, which are expressed in neuronal tissues. Nuclear lamins
belong to the type V IFs, whereas type VI is represented by nestin.
However, nestin can be also included in type IV, based on similarities
in exon/intron structure. Little is yet known about the specific functions of individual intermediate filament genes, but there is an emerging picture that IFs are important for the organization and function of cells and tissues. For example, keratin gene mutations are the cause of genetic skin diseases such as epidermolysis bullosa simplex and epidermolytic hyperkeratosis.3 Similarly, cardiac, skeletal, and smooth muscle phenotypes have been demonstrated in mice with targeted deletion of the desmin gene.4,5 Furthermore, mutations in the human desmin gene can cause myopathy.6,7 Functional inactivation of vimentin in mice results in dilated blood vessels, whereas in double-knockout mice for vimentin and GFAP, scar formation after central nervous system injury is compromised.8
Nestin is mainly expressed at early stages of central nervous system and muscle development and is replaced by neurofilaments and GFAP in nervous tissue9 and by desmin in muscle10,11 at more advanced developmental stages. Nestin is also expressed in other tissues, such as in heart,12 neural crest,9 and testis.13 In most of these tissues, nestin is generally expressed early during development and is down-regulated in mature tissues.9,10 Nestin can, however, become re-expressed in pathological conditions, for example as a result of injury, trauma, or tumors. Nestin is thus up-regulated in reactive astrocytes after brain injury14-16 and in tumors of the central and peripheral nervous systems.17-19
Our previous work revealed a strict temporospatial pattern of nestin expression during rodent tooth development.20 The developing tooth is an excellent model for studying the phenomena of spatial organization, symmetry, morphogenesis, and organ-specific cytodifferentiation. Teeth develop as a result of reciprocal inductive interactions between the oral ectoderm and cranial neural crest-derived mesenchymal cells. During advanced stages of tooth development, some of the ectomesenchymal cells differentiate into odontoblasts, synthesizing the dentin matrix. Odontoblasts form a layer with an epithelial appearance that serves as a protective barrier for the tooth pulp. Nestin is expressed in both epithelial and mesenchymal components of the developing rodent teeth and its expression progressively becomes restricted to differentiating odontoblasts.20 By contrast to other organs, nestin is not down-regulated in mature functional odontoblasts. These data suggest that nestin may be involved in both odontoblast differentiation and function. It remains uncertain, however, whether nestin is important for tooth homeostasis and regeneration. Here, extending our previous studies to human teeth, we examined the pattern of nestin expression in embryonic and permanent intact teeth, as well as in injured and carious teeth. Furthermore, we studied the regulation of nestin expression in a well-established culture system of human pulp cells in vitro.21 Our study shows that the distribution of nestin is dependent on the differentiation and functional status of odontoblasts and that its expression is regulated by bone morphogenic protein-4 (BMP4).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation and characterization of the rabbit anti-nestin antiserum, which is raised against the c-terminal region of rat nestin, has been already described.22 This antiserum specifically identifies nestin in immunohistochemistry and in Western blots10,17 and does not cross-react with other known IFs.22,23 Furthermore, this antiserum generates the same pattern in immunohistochemistry and Western blots as the monoclonal antibody against human nestin (no. 4350; Lendahl U, unpublished observations). Vector Vectastain ABC kit was purchased from Biosys (Compiègne, France). For the preparation of the culture media, all materials were purchased from Gibco BRL (Life Technologies Inc., Grand Island, NY). Other chemicals were obtained from Sigma (St. Louis, MO).
For cultures, minimum essential medium (MEM) was supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 UI/ml penicillin, 100 µg/ml streptomycin (Biowhittaker, Gagny, France), and 0.25 µg/ml amphotericin B (Fungizone, Biowhittaker).
Embryonic Tissues
Human fetal tissues were obtained from legal abortions. The material comprised teeth from 5 fetuses aged 6–18 gestational weeks (g.w.). The gestation age was estimated from the fetal foot length and from the date of last menstruation of the mother. Embryos were noninfected, and all tissues were both macroscopically and microscopically normal. The fetuses were fixed immediately by the obstetrician in 10% buffered formalin for 48 hours to 5 days according to the fetus size. Maxillary and mandibular jaws from 7- to 15-week-old embryos and fetuses were embedded in Paraplast at 56°C; the samples from fetuses aged 16 to 18 g.w. were decalcified for 3 weeks in formic acid/10% formalin before embedding in Paraplast. Sections 4 to 6 µm thick were used for immunohistochemistry.
This study was carried out in compliance with the French legislation, after approval of the Regional Ethics Committee of Development and Reproduction of the Unité de Formation et de Recherche of Medicine of Rheims-France (Department of Developmental Biology, INSERM 314).
Permanent Teeth
The teeth used in this study were of three types: unerupted third molars extracted during normal treatment of 17-year-old patients, premolars of 11- to 15-year-old adolescents, and mature intact and carious teeth of 40-year-old patients. The teeth were freshly extracted and used in this study with the patient’s informed consent. The extracted teeth were fixed in 10% neutral buffered formalin for 24 hours, demineralized in sodium formiate for 21 days, and then embedded in paraffin wax. They were serially sectioned (6-µm-thick sections) and then processed for immunohistochemistry.
Cavities Preparation and Tooth Processing
Cavities were prepared in intact first premolars scheduled for extraction at the Dental Care Center of Marseille. Cavities were prepared first by means of an intermittent application of an Airotor with water coolant to remove the enamel. Cavities 2 to 3 mm wide and 1 to 1.3 mm deep were then cut into the tooth dentin with a bur using the least possible pressure. The pulp chambers were not exposed during the preparation of the cavities. The walls of the cavities were immediately conditioned with a 3% hydrogen peroxide solution and dried with an extremely light stream. The cavities were then restored with the calcium hydroxide product Dycal (Dentsply, York, PA) covered by IRM (De Trey Dentsply, Zurich, Switzerland), a temporary filling material.
After a postoperative interval of 9 weeks, the teeth were extracted using a local anesthetic with the patient’s informed consent.
Teeth with cavities or carious lesions were fixed in 10% neutral buffered formalin for 24 hours, demineralized in sodium formiate for 21 days, and then routinely processed and embedded in paraffin wax.
Explant Culture of Human Pulp
Immediately after extraction of the third molars of the 17-year-old patients, the teeth were swabbed with 70% (v/v) alcohol and the soft tissue was removed with curettes. The teeth were then washed with sterile phosphate-buffered saline (PBS) and transferred into a laminar flow tissue culture hood to perform the rest of the procedures under sterile conditions. The apical part of the teeth was removed with scalpels, and the dental pulps were gently removed with forceps. Each dental pulp was minced separately with scalpels and then rinsed with PBS. After mincing, each tooth pulp explant was cultured in 100-mm diameter culture dishes (Becton Dickinson Labware, Lincoln Park, NJ) in MEM supplemented with 2 mmol/L ß-glycerophosphate (Sigma Chemical Co.). The explants were cultured at 37°C in a humidified atmosphere of 5% CO2, 95% air and the culture medium was changed every other day. Confluent cultures were collected by trypsinization (0.2% trypsin and 0.02% EDTA). The cells were plated at 3 x 103/cm2 on tissue culture-treated 8-chambered glass slides (Becton Dickinson Labware). After 3 weeks of culture, the cells were fixed with 70% ethanol for 1 hour at 4°C and processed for immunohistochemistry.
Recombinant Protein and Treatment of the Beads
BMP4 (a gift of the Genetics Institute, Cambridge, MA) was used to preload agarose beads (1 µl of a solution of 100–250 µg/ml per 10 beads). As a control, we used beads preloaded with 0.2% bovine serum albumin (BSA) in PBS. Beads were transferred on top of human dental pulp explants, and after 24 hours of culture in absence of ß-glycerophosphate the explants were fixed in 4% paraformaldehyde and processed for whole mount immunohistochemistry as described previously.24,25
Immunohistochemistry on Sections and on Cell Cultures
Immunoperoxidase staining on sections was done as previously described.24,26 Briefly, the sections were deparaffinized, exposed to a 0.3% solution of hydrogen peroxide in methanol, and then incubated overnight at 4°C with the anti-nestin antibody diluted 1:1500 in PBS containing 0.2% BSA. Peroxidase was revealed by incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction solution. After staining the sections were mounted with Eukitt (Labonord, Villeneuve d’Ascq, France). In control sections the primary antibodies were omitted.
On cell cultures, the cells were permeabilized for 15 minutes with 0.5% Triton X-100 in PBS before immunohistochemistry. Peroxidase was revealed by incubation with 3-amino-9-ethylcarbazole (AEC) reaction solution and then the slides were mounted with Aquamount (BDH Laboratory Supplies, Gurr, UK).
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Results |
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In 5-week-old human embryos, the oral epithelium proliferates into
the subjacent mesenchyme and forms a series of epithelial ingrowths
into the neural crest-derived mesenchyme at sites corresponding to the
position of the future deciduous teeth. From this point tooth
development proceeds in three descriptive stages: the bud, cap, and
bell stage. At 6 g.w. (Figure 1A)
,
the epithelial ingrowth gives rise to the epithelial dental bud. At
this stage, immunoreactivity for nestin was not observed in either the
epithelial or mesenchyme components (Figure 1D)
. During the cap stage
of development (8–15 g.w.), the dental epithelium forms the enamel
organ and the mesenchyme condenses to form the dental papilla (Figure 1B)
. At this stage, nestin was absent from cells of the enamel
epithelium and the dental papilla (Figure 2E)
.
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. Dentinogenesis is initiated at the tip of the
cusp and the tooth shape (crown morphology) is apparent. The pulp cells
adjoining the dental epithelium differentiate into odontoblasts, and
start to secrete the organic matrix of dentin. After predentin
mineralization the inner enamel epithelial cells differentiate into
ameloblasts and start to secrete enamel matrix proteins. During the
bell stage (18 g.w.), nestin immunoreactivity was observed in the
odontoblasts and pulp fibroblasts of the cusp area (Figure 1, F and G)
.
Nestin was also expressed in the odontoblastic processes up to the
dentin-enamel junction (Figure 1H)
and in pulp cells (Figure 1, G and I)
. A staining gradient was observed in the odontoblasts from the
cervical loop to the cusp region: the cervical loop was negative
(Figure 1J)
, whereas the immunostaining increased toward the cusp
region (Figure 1, F, G, and I)
. Labeling was absent from all dental
epithelial cells. Nestin Expression in Developing and Mature Permanent Teeth
In the 17-year-old developing third molars, nestin expression was
restricted to odontoblasts. Distribution was seen in both the
odontoblast cell bodies (Figure 2, A and D
–F) and the odontoblast
processes (Figure 2, B, C, and G)
. The distribution of nestin in the
odontoblasts exhibited a gradient after the maturation state. In more
mature odontoblasts, in the cusp region of the crown, nestin
immunoreactivity was observed in their processes, but not in their cell
bodies (Figure 2B)
. Some of the odontoblast processes reach the
dentin-enamel junction and were nestin-positive (Figure 2C)
. In the
younger odontoblasts, ie, in the apical root region, nestin
immunostaining was restricted to the cell bodies (Figure 2F)
. In
odontoblasts situated at the intermediate area, between the cusps and
the apical root regions, nestin expression decreased in the odontoblast
processes and increased in the cell bodies, following the crown-root
direction (Figure 2, A, D, and E)
. Immunoreactivity for nestin was also
observed in some pulpal fibroblasts in the proximity of blood vessels
(Figure 2H)
.
Nestin Expression in Carious Human Teeth
Nestin immunoreactivity was completely absent in intact teeth from
a 40-year-old patient (Figure 3A)
, but
nestin staining is observed in mature carious teeth. Expression was
seen in cells surrounding the carious lesion (Figure 3B)
. Nestin is
distributed in the processes of mature odontoblasts situated in the
proximity of the carious front (Figure 3, C and D)
, but nestin
immunoreactivity was absent in bacteria located at the carious front
level (Figure 3D)
. In advanced carious lesion, an inflammation can be
observed in the pulp of the teeth. Disintegrated odontoblastic cell
bodies and pulp fibroblasts facing the lesion were negative for nestin
in such conditions (Figure 3E)
. In some cases, the carious lesions are
responsible for the hyperemia of the pulp. During hyperemic conditions,
nestin staining is observed in the cell bodies of the odontoblasts
facing the lesion and, in pulp fibroblasts, near the dilated blood
vessels (Figure 3F)
.
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Nine weeks after the cavity preparation, reactionary dentin
deposition was seen near to the injury site (Figure 4A)
. This reactionary dentin matrix is
synthesized by newly formed odontoblasts (odontoblast-like cells)
replacing dying odontoblasts after the injury. Nestin immunoreactivity
was not detected at the site of the reactionary dentin production 9
weeks after the lesion (Figure 4B)
, but was evident at a distance from
the cavity preparation. Nestin was distributed mainly in the
odontoblast processes (Figure 4, C and D)
, whereas a weak staining was
found in the odontoblast cell bodies (Figure 4C)
.
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After 3 weeks of culture of human pulp explants, fiber-like
structures started to appear from the explant border and extended
toward the peripheral parts. This was followed by the deposition of
mineral crystals along and within the fibrous structures in pulp
explants cultured in presence of ß-glycerophosphate. This
mineralization front continued to expand during the 8-week culture
procedure; furthermore, new nodules were formed during this culture
period. The cells in direct contact with the nodules exhibited a
polarized morphology similar to that observed in vivo
(Figure 5A)
. Mineral nodules were not
observed in pulp explants cultured in absence of ß-glycerophosphate
(Figure 5F)
. Nestin immunoreactivity was observed in pulp cells treated
with ß-glycerophosphate (Figure 5, B
-E). The staining was very strong
in polarized cells contacting the mineralization nodules (Figure 5, C and D)
, whereas a faint staining was observed in cells at a distance
from the nodules. Pulp cells cultured in absence of
ß-glycerophosphate were nestin-negative (Figure 5G)
.
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It has been shown that BMPs are involved in odontoblast
differentiation,27
suggesting that these signaling
molecules may play a role in the regulation of nestin expression in the
dental pulp mesenchyme. To test this, we placed beads releasing BMP4 on
top of dental pulp explants from a 17-year-old patient and followed the
expression of nestin by whole mount immunohistochemistry. Analysis of
the explants showed nestin immunoreactivity in pulp cells surrounding
beads containing BMP4 (Figure 6, A
-C).
BMP4 up-regulated nestin expression in a wide area of cells surrounding
the bead. As a control, we used beads soaked in BSA, and in no case was
nestin expression induced (Figure 6, D and E)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we show that the IF protein nestin is expressed during human tooth development, is down-regulated in adult teeth, and finally is up-regulated under pathological conditions such as carious lesions and dental wound healing. The nestin expression pattern in human teeth differs from that previously reported in the teeth of rodents.20 Nestin is first expressed at the bell stage of the developing human teeth, whereas in the rat molars nestin was first detected at the bud stage. Nestin expression is down-regulated in odontoblasts of mature permanent human teeth, whereas nestin expression is maintained in odontoblasts of adult rats. Finally, nestin expression in the human teeth is restricted to the odontoblasts and pulp fibroblasts, whereas in the rat molars nestin is also expressed in cells of the dental epithelium. The reason for this species difference in expression is not known, but may be the difference in life span between rodents and human, the single continuously erupting dentition of rodents, or the different use of teeth. The expression pattern of nestin in embryonic human teeth, ie, a transient expression during development of the organ, is quite similar to nestin expression seen in other tissues, eg, the nervous system and muscle.9,10
In the developing deciduous tooth germs, nestin expression in the odontoblasts increases from the apical to the cusp area following a cytodifferentiation gradient. In the developing permanent teeth, the inverse gradient is observed: nestin expression is maintained only in the processes, but not in the cell bodies, of the odontoblasts of the cusp area, where they have completed their maturation. Nestin distribution decreases in the odontoblast processes and increases in the cell bodies toward the apical area. The exact function of nestin has not been established, but its expression in the cell bodies suggests a role in the dentin matrix synthesis, while its presence in the odontoblast processes is not clear. Odontoblast processes are accompanied by small nerve fibers in the dentinal tubuli, 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,28 suggesting a similar effect in odontoblasts.
Although nestin can form homodimers,29 it requires the presence of other IFs for proper filament formation.2,29,30 Nestin transfected into IF-negative cells fails to generate an IF network,30 and, in mice targeted for both vimentin and GFAP, the nestin protein is diffusely spread in reactive astrocytes.8 Nestin is not the only IF protein expressed in odontoblasts and pulp cells of the teeth. Desmin31 and vimentin32 are expressed in dental pulp cells, but only vimentin is detected in odontoblasts.31-33 Taken together, these data suggest that nestin may copolymerize with vimentin to form IFs in the cell body and process of the odontoblastic cell.
Nestin in Irritated Adult Human Teeth
Microorganisms are involved in both decalcification and proteolysis of the dentin during the process of dental caries. The pulp defends itself against the lesion of caries; in response to the oncoming process of decay, the tubuli of the dentin gradually become calcified, provided that the odontoblasts remain vital. In the event that the irritation increases, the odontoblasts degenerate and form dead tracts. In response to further irritation, the secretory activity of the remaining vital odontoblasts is stimulated to elaborate reactionary dentin. In effect, the pulp volume is reduced with the elaboration of reactionary dentin, and the aging process of the pulp is accelerated. Nestin is re-expressed in the processes of odontoblasts surrounding the carious lesion, suggesting a role for nestin in the elaboration of the reactionary dentin (hypercalcification). The pulp does not become inflamed until wide areas of dentinal tubuli are decalcified. When the caries progress more rapidly than the elaboration of reactionary dentin, 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. This indicates a correlation between nestin up-regulation and inflammatory events.
Odontoblasts are directly damaged by cavity preparations involving the dentin. The degenerated odontoblasts may affect the neighboring odontoblasts, which, in turn, die. Disturbance of the odontoblastic layer affects the elaboration of dentin: the quantity and the quality of the deposited dentin matrix change. In this case, newly formed odontoblast-like cells, originating from dental pulp cells,34,35 replace the injured odontoblasts and elaborate the reparative dentin.36-38 Despite the absence of nestin at the site of the injury, which may be due to the delay between cavity preparation and tooth extraction, nestin is localized to the processes of odontoblasts located at a distance from the injury site. These results suggest that nestin may be involved in reparative processes, most notably in the secretory activity of the odontoblastic cell after dental injury. It is worth noting that nestin is often re-expressed under pathological conditions. Nestin is, for example, up-regulated in reactive astrocytes after central nervous system injury14,15 and in different types of tumors in the nervous system.17-19 Similarly, nestin is up-regulated in pathological conditions such as Duchenne’s and Becker’s muscular dystrophy22 and in tumors of muscle origin.39
Regulation of Nestin Expression in Dental Pulp
BMPs are good candidates for involvement in signaling within the dental mesenchyme. During the late bell stage of mouse tooth development, BMP2 and BMP4 are expressed in differentiating odontoblasts.40,41 Furthermore, BMPs have been shown to induce odontoblast differentiation in vitro.27 Here, we show that BMP4 up-regulates nestin expression in the mesenchyme when applied to dental pulp explants. This is the first example of a signaling molecule directly influencing nestin expression, and may explain some aspects of nestin expression during embryonic development, for example, in migrating neural crest cells.42,43
Nestin is also up-regulated in dental pulp cells which have differentiated to odontoblast-like cells and secreted the matrix of the mineralization nodules in an in vitro assay system.21 This confirms the re-expression of nestin in the secretory odontoblast-like cells and the potential of pulpal cells to differentiate into dentin secretory cells. Dentin matrix can be seen as a reservoir for signaling molecules such as BMPs and fibroblast growth factors. During dentin decalcification, after a carious irritation, these signaling molecules are released from the matrix and can diffuse to reach adjecent cells. 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.
In conclusion, the present results demonstrate that nestin is expressed not only during tooth development but also under pathological conditions such as carious and operative lesions of the tooth. The limited nestin distribution remaining in differentiated odontoblasts may be associated with some IF functions during dentin matrix deposition. Finally, nestin expression in dental pulp cells and odontoblasts is up-regulated by BMP4.
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Acknowledgements |
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Footnotes |
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Supported by institutional grants from the Université de la Méditerranée and by specific grants of the Ligue Nationale Contre le Cancer and the Association pour la Recherche sur le Cancer (ARC).
Accepted for publication April 3, 2000.
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