Advances in Dental Research

Advanceshttp://adr.sagepub.com/ in Dental Research

Molecular Aspects of Tooth Pathogenesis and Repair: in vivo and in vitro Models Imad About and Thimios A. Mitsiadis ADR 2001 15: 59 DOI: 10.1177/08959374010150011501 The online version of this article can be found at: http://adr.sagepub.com/content/15/1/59

Published by: http://www.sagepublications.com

On behalf of: International and American Associations for Dental Research

Additional services and information for Advances in Dental Research can be found at: Email Alerts: http://adr.sagepub.com/cgi/alerts Subscriptions: http://adr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav

Downloaded from adr.sagepub.com at Universitaet Zuerich.Psychologisches Institut on March 17, 2011 For personal use only. No other uses without permission.

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, [email protected] 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 epithelialjmesenchymal 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).

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

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

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 tissues—for example, the nervous system and muscle (Lendahl et al., 1990).

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).


60

About & Mitsiadis

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 inflammatory 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 subodontoblastic 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, differentiation of odontoblast-like cells and hard tissue formation have been shown after pulp amputation in vivo 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.

Fig. 2 — Formation of mineralization nodules in human dental pulp explants in vitro after p-glycerophosphate treatment. (A) Phase-contrast microscopy showing the formation of nodules after 3 wks of treatment. Immunohistochemical localization of osteonectin (B), collagen I (C), and nestin (D) expression in the mineralization nodules.

In vitro Conditions For cell cultures, normal extracted immature third molars were used. Each dental pulp was divided into two groups, cultured either without or with B-glycerophosphate. In the first group, the explants were cultured in MEM medium. In the second group, the explants were cultured in the same medium supplemented with 2 mM B-glycerophosphate. Three weeks after culture in the presence of Bglycerophosphate, regular and fiber-like structures started to appear from the explant border and extending toward the periphery (About et al., 2000a). This was followed by the deposition of mineral crystals along and within the fibrous structures, and this mineralization front continued to expand during the eight-week culture procedure (Fig. 2A). The cells in direct contact with the nodules exhibited a polarized morphology similar to that observed in vivo. However, formation of dentinal tubuli was not observed. No mineralization was observed in control cultures (without B-glycerophosphate). Type I collagen immunoreactivity was strong and uniform in all p-glycerophosphate-treated cells. The expression of collagen I was also evident in the mineralization nodules (Fig. 2C). Osteonectin (Fig. 2B) and dentin sialophosphoprotein (data not shown) were expressed in these cells as well as in the nodules. Nestin is also up-regulated in dental pulp cells which have differentiated to odontoblast-like cells and secrete the matrix of the mineralization nodules in the in vitro assay system (Fig. 2D). This confirms the re-expression of nestin in odontoblast-like cells and the potential of pulp cells to differentiate into cells secreting dentin matrix.

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 obtained from the mineralization nodules in vitro were compared with those obtained in vivo, they revealed an analogy with those of the dentin, with peaks and bands of the same aspects at the same locations. Moreover, when the mineralization spectra obtained in vitro were compared with other tooth hard tissues, major differences were observed when they were compared with the spectra obtained from the enamel and the alveolar bone. For explant culture, human dental pulps were minced with scalpels and then rinsed with PBS. After being minced, the explants were cultured in MEM medium supplemented with 2 mM B-glycerophosphate. BMPs and FGFs were used to pre-load agarose beads to concentrations from 100 to 200 (jLg/mL. Beads were transferred to the tops of human dental pulp explants, and after 24 hrs of culture, the explants were fixed in 4% PFA and processed for whole-mount immunohistochemistry as described previously. Although the molecular interactions underlying reparative processes are not well-understood, signaling molecules of the TGFB superfamily seem to be important to hard tissue formation after pulp injury. It has been shown that TGFB1 and BMPs may induce odontoblast differentiation (Ruch et al., 1995) and upregulate Deltal expression in dental mesenchyme in vitro (Mitsiadis et al., 1998). Taken together, these results suggest that members of the TGFB superfamily may also be involved in regulating nestin and Deltal after injury. We followed the expression of nestin by whole-mount immunohistochemistry. Analysis of the explants shows nestin immunoreactivity in pulp cells surrounding beads containing BMP4. BMP4 up-regulated nestin expression in a wide area of cells surrounding the bead. This is the first example of a signaling molecule directly influencing nestin expression.


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 nestinpositive 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

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.

References About I, Bottero MJ, DeDenato P, Camps J, Franquin JC, Mitsiadis TA (2000a). Human dentin production in vitro. ExpCell Res 258:33-41. About I, Maquin D, Lendahl U, Mitsiadis TA (2000b). Nestin expression in embryonic and adult human teeth under normal and pathological conditions. Am J Pathol 157:287-295. Butler WT, Ritchie H (1995). The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39:169-179. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H (2000). Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 66:129-138. Lendahl U, Zimmerman LB, McKay RDG (1990). CNS stem cells express a new class of intermediate filament protein. Cell 60:585-595. Mitsiadis TA, Hirsinger E, Lendahl U, Goridis C (1998). DeltaNotch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation. Dev Biol 204:420-431. Mitsiadis T, Fried K, Goridis C (1999). Reactivation of DeltaNotch signaling after injury: complementary expression patterns of ligand and receptor in dental pulp. Exp Cell Res 246:312-318. Nakashima M (1994). Induction of dentin formation on canine amputated pulp by recombinant human bone morphogenetic proteins (BMP)-2 and -4. / Dent Res 73:1515-1522. Ruch JV, Lesot H, Begue-Kirn C (1995). Odontoblast differentiation. Int J Dev Biol 39:51-68. Terling C, Rass A, Mitsiadis TA, Fried K, Lendahl U, Wroblewski J (1995). Expression of the intermediate filament nestin during rodent tooth development. Int J Dev Biol 39:947-956. Tziafas D, Smith AJ, Lesot H (2000). Designing new treatment strategies in vital pulp therapy. / Dent 28:77-92. Van Mullem PJ (1991). Healing of the guinea pig incisor after partial pulp removal. Endod Dent Traumatol 7:164-176.


62

About & Mitsiadis

Adv Dent Res 15:59-62, August, 2001