Nestin Expression in Embryonic and Adult Human Teeth under Normal and Pathological Conditions
2000; Elsevier BV; Volume: 157; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64539-7
ISSN1525-2191
AutoresImad About, D. Laurent‐Maquin, Urban Lendahl, Thimios A. Mitsiadis,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoNestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivodistribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitro assay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation. Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivodistribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitro assay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation. The cytoskeleton is formed by three types of filamentous structures: microtubuli, microfilaments, and intermediate filaments (IFs). The IF family consists of more than 50 distinct proteins capable of forming morphologically similar filaments in different cell types.1Fuchs E Weber K Intermediate filaments: structure, dynamics, function, and disease.Annu Rev Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1272) Google Scholar, 2Parry DAD Steinert PM Intermediate filament structure. R. G. Landes and Co., Austin TX1995Google Scholar The IFs can be divided into six types, based on sequence homology and exon/intron organization in the gene. Keratins belong to the type I and II IFs, expressed mainly in epithelia. Desmin, vimentin, glial fibrillary acidic protein (GFAP), and peripherin form the type III IFs. Type IV comprises the neurofilaments and α-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.3Fuchs E Coulombe PA Of mice and men: genetic skin diseases of keratin.Cell. 1992; 69: 899-902Abstract Full Text PDF PubMed Scopus (122) Google Scholar Similarly, cardiac, skeletal, and smooth muscle phenotypes have been demonstrated in mice with targeted deletion of the desmin gene.4Capetanaki Y Milner DJ Weitzer G Desmin in muscle formation and maintenance: knockouts and consequences.Cell Struct Funct. 1997; 22: 103-116Crossref PubMed Scopus (172) Google Scholar, 5Li Z Colucci-Guyon E Pincon-Raymond M Mericskay M Pournin S Paulin D Babinet C Cardiovascular lesions and skeletal myopathy in mice lacking desmin.Dev Biol. 1996; 175: 362-366Crossref PubMed Scopus (287) Google Scholar Furthermore, mutations in the human desmin gene can cause myopathy.6Munoz-Marmol AM Strasser G Isamat M Coulombe PA Yang Y Roca X Vela E Mate JL Coll J Fernandez-Figueras MT Navas-Palacios JJ Ariza A Fuchs E A dysfunctional desmin mutation in a patient with severe generalized myopathy.Proc Nat Acad Sci USA. 1998; 95: 11312-11317Crossref PubMed Scopus (228) Google Scholar, 7Goldfarb LG Park KY Cervenakova L Gorokhova S Lee HS Vasconcelos O Nagle JW Semino-Mora C Sivakumar K Dalakas MC Missense mutations in desmin associated with familial cardiac and skeletal myopathy.Nat Genet. 1998; 19: 402-403Crossref PubMed Scopus (440) Google Scholar 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.8Pekny M Johansson CB Eliasson C Stakeberg J Wallen A Perlmann T Lendahl U Betsholtz C Berthold CH Frisen J Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin.J Cell Biol. 1999; 145: 503-514Crossref PubMed Scopus (336) Google ScholarNestin is mainly expressed at early stages of central nervous system and muscle development and is replaced by neurofilaments and GFAP in nervous tissue9Lendahl U Zimmerman LB McKay RDG CNS stem cells express a new class of intermediate filament protein.Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2757) Google Scholar and by desmin in muscle10Sejersen T Lendahl U Transient expression of the intermediate filament nestin during skeletal muscle development.J Cell Sci. 1993; 106: 1291-1300PubMed Google Scholar, 11Kachinsky AM Dominov JA Miller JB Myogenesis and the intermediate filament protein, nestin.Dev Biol. 1994; 165: 216-228Crossref PubMed Scopus (110) Google Scholarat more advanced developmental stages. Nestin is also expressed in other tissues, such as in heart,12Kachinsky AM Dominov JA Miller JB Intermediate filaments in cardiac myogenesis: nestin in the developing mouse heart.J Histochem Cytochem. 1995; 43: 843-847Crossref PubMed Scopus (124) Google Scholar neural crest,9Lendahl U Zimmerman LB McKay RDG CNS stem cells express a new class of intermediate filament protein.Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2757) Google Scholar and testis.13Frojdman K Pelliniemi LJ Lendahl U Virtanen I Eriksson JE The intermediate filament protein nestin occurs transiently in differentiating testis of rat and mouse.Differentiation. 1997; 61: 243-249Crossref PubMed Google Scholar In most of these tissues, nestin is generally expressed early during development and is down-regulated in mature tissues.9Lendahl U Zimmerman LB McKay RDG CNS stem cells express a new class of intermediate filament protein.Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2757) Google Scholar, 10Sejersen T Lendahl U Transient expression of the intermediate filament nestin during skeletal muscle development.J Cell Sci. 1993; 106: 1291-1300PubMed Google Scholar 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 injury14Frisen J Johansson CB Torok C Risling M Lendahl U Rapid, widespread, and longlasting induction of nestin contributes to the generation of glial scar tissue after CNS injury.J Cell Biol. 1995; 131: 453-464Crossref PubMed Scopus (433) Google Scholar, 15Lin RCS Matesic DF Marvin M McKay RD Brustle O Re-expression of the intermediate filament nestin in reactive astrocytes.Neurobiol Dis. 1995; 2: 79-85Crossref PubMed Scopus (201) Google Scholar, 16Holmin S Almqvist P Lendahl U Mathiesen T Adult nestin-expressing subependymal cells differentiate to astrocytes in response to brain injury.Eur J Neurosci. 1997; 9: 65-75Crossref PubMed Scopus (153) Google Scholar and in tumors of the central and peripheral nervous systems.17Dahlstrand J Collins PV Lendahl U Expression of the class VI intermediate filament nestin in human central nervous system tumors.Cancer Res. 1992; 52: 5334-5341PubMed Google Scholar, 18Tohyama T Lee VMY Rorke LB Marvin M McKay RDG Trojanowski J Nestin expression in embryonic human neuroepithelium and in human neuroepithelial tumor cells.Lab Invest. 1992; 66: 303-313PubMed Google Scholar, 19Florenes VA Holm R Myklebost O Lendahl U Fodstad O Expression of the neuroectodermal intermediate filament nestin in human melanomas.Cancer Res. 1994; 54: 354-356PubMed Google ScholarOur previous work revealed a strict temporospatial pattern of nestin expression during rodent tooth development.20Terling C Rass A Mitsiadis TA Fried K Lendahl U Wroblewski J Expression of the intermediate filament nestin during rodent tooth development.Int J Dev Biol. 1995; 39: 947-956PubMed Google Scholar 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.20Terling C Rass A Mitsiadis TA Fried K Lendahl U Wroblewski J Expression of the intermediate filament nestin during rodent tooth development.Int J Dev Biol. 1995; 39: 947-956PubMed Google Scholar 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.21Hao JJ Shi JN Niu ZY Xun WX Yue L Xiao MZ Mineralized nodule formation by human dental papilla cells in culture.Eur J Oral Sci. 1997; 105: 318-324Crossref PubMed Scopus (20) Google Scholar 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).Materials and MethodsMaterialsPreparation and characterization of the rabbit anti-nestin antiserum, which is raised against the c-terminal region of rat nestin, has been already described.22Sjöberg G Edström L Lendahl U Sejersen T Myofibers from Duchenne/Becker muscular dystrophy and myositis express the intermediate filament nestin.J Neuropathol Exp Neurol. 1994; 53: 416-423Crossref PubMed Scopus (36) Google Scholar This antiserum specifically identifies nestin in immunohistochemistry and in Western blots10Sejersen T Lendahl U Transient expression of the intermediate filament nestin during skeletal muscle development.J Cell Sci. 1993; 106: 1291-1300PubMed Google Scholar, 17Dahlstrand J Collins PV Lendahl U Expression of the class VI intermediate filament nestin in human central nervous system tumors.Cancer Res. 1992; 52: 5334-5341PubMed Google Scholar and does not cross-react with other known IFs.22Sjöberg G Edström L Lendahl U Sejersen T Myofibers from Duchenne/Becker muscular dystrophy and myositis express the intermediate filament nestin.J Neuropathol Exp Neurol. 1994; 53: 416-423Crossref PubMed Scopus (36) Google Scholar, 23Sjöberg G Jiang WO Ringertz NR Lendahl U Sejersen T Colocalization of nestin and vimentin/desmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy.Exp Cell Res. 1994; 214: 447-458Crossref PubMed Scopus (104) Google Scholar 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 TissuesHuman 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 TeethThe 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 ProcessingCavities 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 PulpImmediately 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 β-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 × 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 BeadsBMP4 (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 β-glycerophosphate the explants were fixed in 4% paraformaldehyde and processed for whole mount immunohistochemistry as described previously.24Mitsiadis TA Muramatsu T Muramatsu H Thesleff I Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis.J Cell Biol. 1995; 129: 267-281Crossref PubMed Scopus (126) Google Scholar, 25Mitsiadis TA Hirsinger E Lendahl U Goridis C Delta-notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation.Dev Biol. 1998; 204: 420-431Crossref PubMed Scopus (88) Google ScholarImmunohistochemistry on Sections and on Cell CulturesImmunoperoxidase staining on sections was done as previously described.24Mitsiadis TA Muramatsu T Muramatsu H Thesleff I Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis.J Cell Biol. 1995; 129: 267-281Crossref PubMed Scopus (126) Google Scholar, 26Mitsiadis TA Dicou E Joffre A Magloire H Immunohistochemical localization of nerve growth factor (NGF) and NGF receptor (NGF-R) in the developing first molar tooth of the rat.Differentiation. 1992; 49: 47-61Crossref PubMed Scopus (58) Google Scholar 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).ResultsNestin Expression in the Developing Deciduous Human Tooth GermsIn 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).Figure 2Immunohistochemical localization of nestin in sections of developing permanent human teeth. A: Crown area. Nestin immunoreactivity is observed in the cell bodies and processes of the odontoblasts. Note that the other pulp cells are negative for nestin. B: In the cusp of the crown region nestin staining is seen only in the odontoblastic processes. C: Nestin immunoreactivity is observed in some odontoblast processes near the dentin-enamel junction. D–F: From the middle of the crown (D) until the apical part of the root (F), nestin staining decreases in the odontoblastic processes and is limited in the odontoblastic cell bodies at the apical root area (F). Note the faint immunoreactivity in cells of the subodontoblastic layer at the root area (E). G: Section passing through the dotted line of A. Note the dentinal tubuli and the positive staining representing the odontoblastic processes. H: Immunoreactivity is observed in some pulp cells at the proximity of blood vessels (asterisks). d, dentin; o, odontoblasts; p, pulp; soc, subodontoblastic cells; t, dentinal tubuli. Scale bars, 50 μm (A, B, D–F,H) and 35 μm (C and G).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Continued growth of the tooth germ leads to the bell stage of tooth development (Figure 1C). 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 TeethIn 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 TeethNestin 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).Figure 3Immunohistochemical localization of nestin in sections of normal and carious mature permanent human teeth. A: Nestin staining is absent from the normal mature teeth. B: In carious teeth, bacterial infiltration of dentin is shown (asterisks) after a gram staining for tissues. C: In carious teeth, nestin reactivity is found in some odontoblastic processes approaching the dentin-enamel junction and near of the carious front (asterisks). D: In the carious front (asterisks), nestin immunostaining is absent, while the staining is strong in the odontoblastic processes near to the carious front. E: Nestin immunoreactivity is absent in the odontoblast bodies (asterisks) of the inflamed pulp. F: Nestin is observed in odontoblast bodies facing the carious irritation and cells in the proximity of the dilated blood vessels (asterisks). d, dentin; o, odontoblasts; p, pulp. Scale bar, 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nestin Expression in the Mature Permanent Human Teeth after InjuryNine 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).Figure 4Immunohistochemical localization of nestin in sections of mature permanent human teeth after cavity preparation. A: Hematoxylin-eosin staining. Nine weeks after the cavity preparation, reactionary dentin (asterisk) is seen near to the site of the injury. Frames represent the sites shown in B and C. B: Nestin is absent from in the newly formed odontoblasts. C and D: Nestin reactivity is observed in the odontoblast processes at a site distal of the injury site. Note the weak staining in the odontoblastic bodies. D: Section passing through the dotted line of C. c, cavity; d, dentin; o, odontoblasts; p, pulp; pd, predentin. Scale bars, 80 μm (A) and 50 μm (B–D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nestin Expression in Human Tooth Pulp Cells in VitroAfter 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 β-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 β-glycerophosphate (Figure 5F). Nestin immunoreactivity was observed in pulp cells treated with β-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 β-glycerophosphate were nestin-negative (Figure 5G).Figure 5Nestin immunoreactivity in human dental pulp cells in vitro after β-glycerophosphate treatment. A: Phase contrast microscopy showing multilayered cells and the mineralization nodule formation after three weeks of culture in presence of β-glycerophosphate. B: Confluent pulp cells treated with β-glyceropho
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