Toxicity of dioxin to developing teeth and salivary glands

Transcription

1 Department of Pediatric and Preventive Dentistry and Department of Oral Pathology Institute of Dentistry University of Helsinki Finland Toxicity of dioxin to developing teeth and salivary glands An experimental study Anu Kiukkonen Helsinki University Biomedical Dissertations No. 77 ACADEMIC DISSERTATION To be presented with the permission of the Faculty of Medicine of the University of Helsinki, for public examination in the main auditorium of the Institute of Dentistry on June 16 th, 2006, at 12 noon.

2 Supervised by: Docent Pirjo-Liisa Lukinmaa, DDS, PhD Department of Oral Pathology Institute of Dentistry University of Helsinki, Finland Professor Satu Alaluusua, DDS, PhD Department of Pediatric and Preventive Dentistry Institute of Dentistry University of Helsinki, Finland Reviewed by: Professor Jorma Toppari, MD, PhD Department of Physiology Department of Pediatrics University of Turku, Finland Professor Seppo Vainio, MSc, PhD Department of Biochemistry Biocenter Oulu University of Oulu, Finland ISBN (paperback) ISBN (PDF) ISSN Yliopistopaino Helsinki 2006

3 CONTENTS LIST OF ORIGINAL PUBLICATIONS... 7 ABBREVIATIONS... 8 ABSTRACT INTRODUCTION REVIEW OF THE LITERATURE General features of organogenesis Outlines of tooth development and structure Distinctive features of tooth formation Tooth types Initiation and morphogenesis Morphological features Cellular characteristics Molecular mechanisms Dental cell differentiation Dental hard tissues Cellular origin Formation and structure of the enamel Formation and structure of the dentin Abnormal tooth development and structure Genetic aberrations Environmental defects Defects caused by certain chemicals and radiotherapy Outlines of salivary gland structure and development Anatomical and functional characteristics Branching morphogenesis Morphological features Cellular and molecular mechanisms Dioxins General characteristics of dioxins and related compounds Human exposure Effects on humans General effects Effects on developing teeth Effects on animals The concepts of short-term effects and long-term effects Acute lethality

4 Range of responses to TCDD toxicity Developmentally toxic effects Molecular mechanisms of TCDD toxicity AhR-ARNT pathways AhR-dependent, ARNT-independent mechanisms AhR-independent mechanisms AIMS OF THE STUDY MATERIALS AND METHODS Approval of the study protocols (I-V) Chemicals (I-V) In vivo studies (II, IV) Animals and animal care Experimental design Macroscopic and stereomicroscopic examination of rat incisors Histological and immunohistochemical studies In vitro studies (I, III, V) Organ culture Stereomicroscopic examination Processing of the specimens for histological and immunohistochemical studies Histological and immunohistochemical stainings Detection of apoptotic cells by the TUNEL method Detection of proliferating cells by BrdU labelling and immunohistochemical staining In situ hybrization Statistics (II, V) RESULTS Effect of TCDD on mouse molar tooth morphogenesis and early stages of dental hard tissue formation in vitro (I, III) Morphological changes Stereomicroscopic findings Histological findings Cellular characteristics (I) Apoptosis Cell proliferation Molecular alterations (III) Expression of Dspp Expression of Bono Expression of MMP Effect of TCDD on rat molar hard tissue formation in vivo (II) Mineralization of the enamel Mineralization of the dentin AhR and CYP1A1 expression in dental cells Effect of TCDD on rat incisor formation in vivo and its relation to the sensitivity of a rat strain to TCDD acute lethality (IV)

5 Morphological changes Stereomicroscopic findings Histological findings Effect of TCDD on mouse submandibular gland branching morphogenesis in vitro (V) Morphological changes Stereomicroscopic findings Histological findings Cellular findings Apoptosis and cell proliferation Molecular characteristics CYP1A1 induction Effect of exogenous EGF Expression of FN and effect of exogenous FN DISCUSSION Research frame and methodological aspects (I-V) Morphological characteristics of toxicity of TCDD to developing teeth and salivary glands Dependence of TCDD effects on the stage of tooth development and tooth type (I, II, IV) Interference by TCDD with salivary gland branching morphogenesis (V) Cellular and molecular mechanisms of toxicity of TCDD to developing teeth and salivary gland Effects of TCDD on apoptosis and cell proliferation (I, V) AhR as the mediator of toxicity of TCDD to developing teeth and salivary gland (II, IV, V) The role of EGFR in the mediation of developmental toxicity of TCDD to teeth and salivary gland (V) An alternative mechanism of TCDD action based on modification of the effects of other agents (V) The role of DSPP in retarded dentin mineralization of embryonic mouse molar teeth (III) CONCLUSIONS REFERENCES ACKNOWLEDGEMENTS

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7 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original articles, which are referred to in the text by their Roman numerals. I Partanen A-M, Kiukkonen A, Sahlberg C, Alaluusua S, Thesleff I, Pohjanvirta R, Lukinmaa P-L. Developmental toxicity of dioxin to mouse embryonic teeth in vitro: arrest of tooth morphogenesis involves stimulation of apoptotic program in the dental epithelium. Toxicol Appl Pharmacol 2004; 194: Erratum in: Toxicol Appl Pharmacol 2005; 202: Copyright (2004, 2005) with permission from Elsevier. II Gao Y, Sahlberg C, Kiukkonen A, Alaluusua S, Pohjanvirta R, Tuomisto J, Lukinmaa P-L. Lactational exposure of Han/Wistar rats to 2,3,7,8- tetrachlorodibenzo-p-dioxin interferes with enamel maturation and retards dentin mineralization. J Dent Res 2004; 83: Copyright (2004) with permission from the International and American Associations for Dental Research. III Kiukkonen A, Sahlberg C, Lukinmaa P-L, Alaluusua S, Peltonen E, Partanen A-M. 2,3,7,8-Tetrachlorodibenzo-p-dioxin specifically reduces mrna for the mineralization-related dentin sialophosphoprotein in cultured mouse embryonic molar teeth. (submitted for publication). IV Kiukkonen A, Viluksela M, Sahlberg C, Alaluusua S, Tuomisto JT, Tuomisto J, Lukinmaa P-L. Response of the incisor tooth to 2,3,7,8-tetrachlorodibenzo-pdioxin in a dioxin-resistant and a dioxin-sensitive rat strain. Toxicol Sci 2002; 69: Copyright (2002) with permission from the Society of Toxicology. V Kiukkonen A, Sahlberg C, Partanen A-M, Alaluusua S, Pohjanvirta R, Tuomisto J, Lukinmaa P-L. Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling. Toxicol Appl Pharmacol 2006; 212: Copyright (2006) with permission from Elsevier. I thank the publishers for their permissions to reprint the original papers. 7

8 ABBREVIATIONS AEC 3-amino-9-ethylcarbazole AhR Aryl hydrocarbon receptor AI Amelogenesis imperfecta ARNT Aryl hydrocarbon receptor nuclear translocator (protein) Barx BarH1 homologue in vertebrates (transcription factor) BCIP 5-bromo-4-chloro-3-inodyl phosphate BMP Bone morphogenetic protein BrdU 5 -bromo-2 -deoxyuridine BSP Bone sialoprotein CYP1A1 Cytochrome P450 1A1 DD Dentin dysplasia DI Dentinogenesis imperfecta Dlx Distal-less homologue in vertebrates (transcription factor) DMBA 7,12-dimethylbenz[a]anthracene DMEM Dulbecco s modified Eagle s medium DMP Dentin matrix protein DMSO Dimethyl sulfoxide DPP Dentin phosphoprotein DSP Dentin sialoprotein DSPP Dentin sialophosphoprotein E Embryonic day EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EGFR Epidermal growth factor receptor EROD Ethoxyresorufin-O-deetylase FCS Foetal calf serum FGF Fibroblast growth factor FN Fibronectin Gli Glioma-associated oncogene homologue (transcription factor) HE Haematoxylin and eosin H/W Han/Wistar (rats) Hh Hedgehog HIF Hypoxia-inducible factor IACUC Institutional Animal Care and Use Committee IGF Insulin-like growth factor IGFBP-rP10 Insulin-like growth factor binding protein-related protein 10 L-E Long-Evans (rats) Lef Lymphoid enhancer-binding factor 1 (transcription factor) Lhx Lim-homeobox domain gene (transcription factor) MAPK Mitogen-activated protein kinase MEPE/OF45 Matrix extracellular phosphoglycoprotein MIH Molar-incisor-hypomineralization MMP Matrix metalloproteinase Msx Msh-like genes in vertebrates (transcription factor) NBT Nitro blue tetrazolium chloride NF-κB Nuclear factor-κb No. Number 8

9 OPN PAH Pax PCB PCDD PCDF PFA Pn Shh TCDD TdT TGF TNF TUNEL USEPA Wnt Osteopontin Non-halogenated polycyclic aromatic hydrocarbon Paired box homeotic gene (transcription factor) Polychlorinated biphenyl Polychlorinated dibenzo-p-dioxin Polychlorinated dibenzofuran Paraformaldehyde Postnatal Sonic hedgehog 2,3,7,8-tetrachlorodibenzo-p-dioxin Terminal deoxynucleotidyl transferase Transforming growth factor Tumour necrosis factor Terminal deoxynucleotidyl transferase (TdT) -mediated nick end labelling U.S. Environmental Protection Agency Wingless homologue in vertebrates (transcription factor) 9

10 ABSTRACT Dioxins are ubiquitous environmental pollutants having unequivocal health effects on various species, including humans. In experimental animals e.g. carcinogenic, reproductive, endocrine, immunologic and developmental effects have been described. While most vertebrate species are responsive to the developmental toxicity of dioxins, their sensitivities to various toxic end points differ. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), is a potent modulator of epithelial cell growth and differentiation. The majority of its effects are thought to be mediated by the aryl hydrocarbon receptor (AhR). Previous studies show that developing human teeth may be sensitive to dioxins and that TCDD is developmentally toxic to rodent teeth. The aim of the present thesis work was to clarify morphological outcomes and cellular and molecular mechanisms of developmental toxicity of TCDD to rat and mouse teeth from the initiation to the formation and mineralization of the dental matrices and mouse salivary gland branching morphogenesis. For the in vitro studies, mouse embryonic teeth and salivary gland explants were cultured in a Trowell type organ culture with TCDD at various concentrations and examined stereomicroscopically. Apoptosis and cell proliferation were localized in tissue sections. The expression of dentin sialophosphoprotein (Dspp), Bono1 and matrix metalloproteinase-20 (MMP-20) was studied by in situ hybridization. The roles of epidermal growth factor (EGF) receptor signalling and fibronectin (FN) in the mediation of TCDD effects on submandibular gland were studied by culturing explants with EGF or FN alone and in combination with TCDD. To see in vivo if the effect of TCDD on the continuously erupting rat incisor is associated with the sensitivity of a rat strain to TCDD acute lethality, dioxinsensitive Long-Evans (L-E) rats and dioxin-resistant Han/Wistar (H/W) rats with a mutated AhR allele determining the exceptional resistance of the rat strain to TCDD acute lethality were exposed to TCDD. The treatment began when the rats were 10 weeks old and continued for 20 weeks. The incisors were examined with a stereomicroscope and processed for histological examination. To test if TCDD interferes with mineralization of rat molars, H/W rat dams were administered a single dose of 50 or 1000 µg/kg TCDD one day after delivery. Tissue sections of the pup heads were analysed at post-natal days 9 and 22. The expression of AhR and cytochrome P450 (CYP) 1A1 in the dental cells was localized by immunohistochemical staining. The results showed that TCDD dose-dependently interfered with the formation of the molar and incisor teeth. Molar tooth development was arrested in vitro when the exposure began at the initiation stage. Exposure at later stages resulted in reduced tooth size and low cusps. The effects were associated with accelerated apoptosis in those dental epithelial cells that are predestined to undergo apoptosis during normal morphogenesis. TCDD also impaired the function of secretory ameloblasts and odontoblasts in vivo, which resulted in delayed or defective mineralization of molar teeth. The impaired mineralization after TCDD exposure was accompanied by decreased expression of AhR and CYP1A1, suggesting mediation of the TCDD effect by the AhR pathway. Specific reduction of Dspp expression could explain retarded mineralization of dentin. Dentin and enamel formation was impaired in the rat incisors after TCDD exposure and the impairment was independent of the resistance of the rat strain to the acute lethality of TCDD. TCDD decreased epithelial branching morphogenesis of cultured mouse submandibular glands. The impairment was associated with induction of CYP1A1 and involved reduced EGF receptor signalling. In conclusion, TCDD exposure in target organs is likely to have activated the AhR pathway with the consequent activation of 10

11 other signalling pathways involving developmentally regulated genes. The resultant phenotype is organ specific and modified by epithelial-mesenchymal interactions and apart from the dose, dependent on the stage of organogenesis at the time of TCDD exposure. 11

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13 1. INTRODUCTION Dioxins and related compounds are ubiquitous environmental pollutants that microorganisms and chemical agents are incapable of degrading. These halogenated aromatic hydrocarbons arise from combustion and as by-products of various industrial processes. They are fat-soluble and enriched in the food chain. The major source of dioxins for humans is food. A variety of dioxin effects, including developmental effects, have been established in experimental animals but apart from chloracne, resulting from heavy and/or prolonged exposure, effects on humans are more or less controversial. Clinical findings suggest that depending on the level, children s developing teeth can be sensitive to environmental dioxins via mother s milk (Alaluusua et al., 1996, 1999; Alaluusua and Lukinmaa, 2006). It has also been found that the prevalence of developmental enamel defects and congenitally missing teeth in subjects accidentally exposed to high amounts of dioxin is increased (Alaluusua et al., 2004). Experimental studies show that the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), is toxic to developing rodent teeth and that the lowest doses arresting rat tooth development in vivo (Kattainen et al., 2001) are of the same order of magnitude as those that have been associated with developmental dental defects in children. TCDD is a potent modulator of epithelial cell growth and differentiation. Therefore, organs forming as a result of interactions between the epithelium and the mesenchyme, for example teeth, salivary glands, mammary glands and prostate, are putative targets of TCDD action. Early stages of tooth and salivary gland development share many features in common. However, whereas the functional portion of the fully developed salivary gland comprises the epithelial component solely, teeth are formed of both epithelial and mesenchymal matrices which eventually undergo mineralization. Teeth are sensitive to dioxins even at later stages of development which are no longer under the control of epithelial-mesenchymal interactions. TCDD has been found to interfere with rat and mouse tooth development in vivo and in vitro. The effects depend on dose and developmental stage of the tooth as well as tooth type. Lactational exposure of rats to TCDD arrests third molar development within a limited time span of exposure (Kattainen et al., 2001, Lukinmaa et al., 2001; Miettinen et al., 2002). While TCDD does not appear to interfere with the differentiation of tooth-forming cells of the continuously erupting rat incisor (Lukinmaa et al., 2001), it disturbs deposition and mineralization of the enamel and dentin matrices, leading to colour defects and pulpal perforation (Alaluusua et al., 1993; Lukinmaa et al., 2001). Exposure of cultured mouse embryonic molars can arrest tooth morphogenesis and perturb cell differentiation and the formation and mineralization of the dental matrices. These effects are dependent on the expression of the epidermal growth factor receptor (EGFR) (Partanen et al., 1998). TCDD exposure arrests molar root development (Lukinmaa et al., 2001). To my knowledge, effects of TCDD on developing salivary glands have not been reported. The vast majority of TCDD effects, including developmental effects, are thought to be mediated by the aryl hydrocarbon receptor (AhR) (Pohjanvirta and Tuomisto, 1994). AhR is an intracellular transcription factor that upon activation by ligand binding dimerizes with the AhR nuclear translocator (ARNT) protein and thereby initiates a cascade of events leading to the transcription of e.g. xenobiotic-metabolizing enzymes such as cytochrome P4501A1 (CYP1A1) (Schmidt and Bradfield, 1996). Physiological high-affinity ligands of AhR are currently unknown but the receptor is essential for normal mammalian development 13

14 (Fernandez-Salguero et al., 1995, 1997; Schmidt et al., 1996; Lund et al., 2003) and it takes part in the regulation of the cell cycle and apoptosis (Nebert et al., 2000). AhR and ARNT are expressed in embryonic mouse teeth already in the epithelial bud and the expression level increases in secretory ameloblasts and odontoblasts (Sahlberg et al., 2002). Their co-expression during TCDD-sensitive stages of tooth development suggests that the toxic effects of TCDD to developing teeth are mediated via the AhR-ARNT pathway. Neither protein is known to play a role in tooth development. Resistance to the acute lethality of TCDD, which is primarily determined by a mutated AhR allele (Pohjanvirta et al., 1998, Tuomisto et al., 1999), varies markedly not only among animal species but also among strains (Pohjanvirta and Tuomisto, 1994). The resistance of a rat strain to the acute lethality of TCDD does not necessarily predict responsiveness of the strain to other toxic and developmental effects of TCDD (Tuomisto et al., 1999; Simanainen et al., 2002). Besides conferring resistance to the acute lethality of TCDD, the mutation, which involves the transactivation domain of AhR, also accounts for the exceptional resistance of Han/Wistar (H/W) rats to hepatotoxicity (Tuomisto et al., 1999), liver tumour promotion activity (Viluksela et al., 2000), and bone effects (Jämsä et al., 2001) of TCDD, but does not account for e.g. the CYP1A1 induction or thymic atrophy. Developmental dental effects of TCDD appear to be closer to the latter category, even though minor differences in response have been reported between strains with different resistance to TCDD acute lethality (Kattainen et al., 2001). These studies were undertaken to characterize developmental toxicity of TCDD at morphological, cellular and molecular levels as seen in rat and mouse teeth and mouse salivary glands in vivo and in vitro. 14

15 2. REVIEW OF THE LITERATURE 2.1. General features of organogenesis Concomitant cell proliferation and differentiation are strictly regulated in time and place and as a final outcome specific cells form tissues and organs. The number of cells in each organ is regulated by apoptosis and cell proliferation (see below) and it determines the size and form of an organ. In the course of gastrulation, ectoderm, mesoderm and endoderm form and they gradually differentiate during organogenesis to separate types of tissues and organs. The epithelial cells of the stomatodeum are ectodermal in origin and the mesenchymal cells, which also participate in the development of teeth, salivary glands and facial bones, migrate from the neural crest. Migration is directed by interactions between cells and/or cells and extracellular matrix (Fig.1). In many organs inductive interactions prevail between the epithelium and the mesenchyme throughout development. It is typical for epithelial-mesenchymal interactions that both tissues can be active messengers depending on the developmental stage and the organ. Soluble messenger molecules function reciprocally and the signalling continues throughout organogenesis. Hox-proteins regulate the patterning of the embryo in the cranialcaudal direction. Patterning genetically determines the places of future organs and begins before the initiation of morphogenesis. epithelial-mesenchymal interactions lungs mammary gland hair tooth salivary gland kidney Figure 1. Many organs share the same phenomena of epithelial-mesenchymal interactions during early development. 15

16 2.2. Outlines of tooth development and structure Distinctive features of tooth formation Tooth development is genetically regulated, but it is susceptible to environmental effects. Initiation of tooth development, tooth morphogenesis and differentiation of the dental cells are directed by a series of interactions between the epithelium and mesenchyme (Kollar and Baird, 1970a, b; Lumsden, 1988; Thesleff et al., 1995). As the development proceeds, the source of instructions for tooth formation shifts from the epithelium to the mesenchyme (Mina and Kollar, 1987). Once cytodifferentiation has been completed, the enamel-forming ameloblasts, dentin-forming odontoblasts and cementum-forming cementoblasts start the formation and mineralization of the specific dental matrices (Kollar and Baird, 1970a, b). Since tooth matrices are not remodelled as occurs in bone, developmental defects caused by severe disturbances (genetic or non-genetic) are permanent. This makes tooth a good model organ for studies on not only normal but also aberrant development Tooth types Vertebrate species that have teeth can be classified as homodont or heterodont ones. The tooth shapes of heterodont animals can be divided to four families: incisors, canines, premolars and molars. Incisors have one cusp and develop in the anterior part of the mouth whereas molars have more cusps and are situated more posteriorly. Different animal species have teeth from different tooth families. s and mice, for example, have only incisors and molars while rabbits have incisors, premolars and molars. Human teeth and rat and mouse molars have limited growth (brachyodont teeth), which makes comparisons between species possible. Unlike human teeth, rat and mouse molars do not form enamel at the cusp tips and rat molars have a thick layer of cementum over the roots (Shellis and Berkovitz, 1981). In contrast to the molars, rat and mouse incisors erupt continuously (Fig. 2; hypsodont teeth). In response to the physiological attrition at the incisal tooth end, new tooth substance is constantly generated at the basal, germinative end. The incisor tooth starts to develop when the mouse embryo is 11 days old and the rat embryo is 14 days old. Mineralization of the incisor begins in the mouse and rat just before birth (embryonic day E20 and E21, respectively). Both species are born without incisors, which erupt after birth after 10 days. The rat incisor reaches the attrition level within 16 days of birth and the dental material is replaced about every 62 days (Shellis and Berkovitz, 1981; Madhukar et al., 1984). Thus, incisors provide a good tool to study tooth development simultaneously from stem cell proliferation and differentiation to the completion of matrix formation. 16

17 e d c M1 b p o M2 M3 cl a Figure 2. Illustration of the continuously erupting rat mandibular incisor tooth as viewed sagittally. e, enamel; d, dentin; a, ameloblasts; o, odontoblasts; p, pulp; c, cementum; b, bone; cl, cervical loop; M1, first molar; M2, second molar; M3, third molar. Heterodont vertebrates with teeth of limited growth (Fig. 3) may have two sets of teeth (diphyodonty), unlike homodont vertebrates which have continuously erupting and constantly wearing teeth (polyphyodonty) (Butler, 1981). Of the two sets of teeth of humans and some animals the first are deciduous teeth and the second permanent teeth. The permanent tooth develops from the same dental lamina as the primary tooth and is seen as a bud lingually to the primary tooth germ. The development of permanent teeth continues in different ways depending on tooth shape (Ten Cate et al., 2003). 17

18 a e o o d c p Figure 3. Illustration of a rat mandibular molar tooth of limited growth as viewed sagittally. e, enamel; d, dentin; a, ameloblasts; o, odontoblasts; p, pulp; c, cementoblasts Initiation and morphogenesis Morphological features Tooth development (Fig. 4) starts when the oral epithelium locally thickens and proceeds via successive morphogenetic (bud, cap, bell) stages to cell differentiation and the subsequent formation and mineralization of the dental matrices (Thesleff and Nieminen, 1998). The thickened epithelium forms the dental lamina, which indicates the future dental arch (Jernvall and Thesleff, 2000). The mesenchyme underneath the epithelium condenses at the site of the first molar tooth of E11 mice. The dental lamina grows down to the mesenchyme and forms a bud (the bud stage). In a 14-day-old mouse embryo the epithelium folds and further invaginates to the mesenchyme. The epithelium traps the mesenchyme and forms a cap-like structure (the cap stage). At the cap stage the dental epithelium comprises the outer and inner epithelia separated by the lace-like stellate reticulum. Together these elements constitute the enamel organ. Mesenchymal parts of the tooth germ comprise the dental papilla and the dental follicle. During the early bell stage, the shape of the cusps of the E16 mouse begins to emerge and at a later bell stage (E18) ameloblasts and the opposing odontoblasts differentiate and start to polarize. When the cuspal morphogenesis is completed the tooth shifts to the secretory stage and the dental matrices are excreted and mineralized. At this point root development is still incomplete and it continues after the tooth has erupted into the oral cavity. In E13-14 mice the second molar starts to develop posterior to the first molar in the same way as the first molar and after 2-3 days the third molar development starts. Tooth development begins in the anterior parts of the oral cavity and proceeds posteriorly. Development of the mandibular 18

19 molars starts before that of the maxillary molars and in both jaws it proceeds in the mesial to distal direction and, as regards an individual tooth, also in the coronal to apical direction. Early stages of tooth development (initiation, morphogenesis and cell differentiation) are governed by inductive interactions between the epithelium and the subjacent mesenchyme. Up to the bud stage the epithelium guides tooth development (Mina and Kollar, 1987) and at this stage the oral epithelium can induce tooth development even with a non-dental neural crest-derived mesenchyme (Lumsden, 1988). When development proceeds the command comes from the mesenchyme at the late bud stage and accordingly, the dental mesenchyme can induce tooth development with a non-dental epithelium (Kollar and Baird, 1970b; Mina and Kollar, 1987). Thus the epithelium decides whether the teeth are formed or not, and the mesenchyme determines the tooth type. Not only developing teeth but also several other organs (limb and notochord) have a centre which guides morphogenesis. In teeth the signalling centres, called enamel knots, are transient epithelial structures which are found at different stages of tooth development (Fig. 4). In mice a signalling centre is first detected in the beginning of the early bud stage (E11.5-E12) (Keränen et al., 1998; Jernvall and Thesleff, 2000). The enamel knot is fully developed at the cap (E14) stage and it can be detected in histological sections as a compact cell entity. The cells of the enamel knot itself are nonproliferative and they disappear by apoptosis (Vaahtokari et al., 1996a; Jernvall et al., 1998). At the early bell stage (E16) the molar morphology starts to form and the enamel knots appear at the developing cusp tips. These signalling centres direct the height and places of tooth cusps (Jernvall and Thesleff, 2000). Signalling molecules from four families, i.e. transforming growth factor β/bone morphogenetic protein (TGFβ/BMP), fibroblast growth factor (FGF), hedgehog (Hh) and Wnt, have thus far been localized in enamel knots (Jernvall et al., 1994; Vaahtokari et al., 1996b; Jernvall and Thesleff, 2000). Initiation (E10) Initiation (E11) Bud stage (E12-13) Cap stage (E14-15) Bell stage (E16-E18) Secretory stage (Pn2-) dental lamina placode outer epithelium stellate reticulum enamel ectomesenchyme inner epithelium dental papilla dental follicle dentin pulp Initiation Morphogenesis Cell differentiation Matrix formation Figure 4. Illustration of the morphological stages of mouse molar tooth development. E, embryonic day; Pn, postnatal day. 19

20 Cellular characteristics A basic requirement for organogenesis is not only cell proliferation but also programmed cell death, or apoptosis. Cell proliferation is detected through the active morphogenesis of the tooth both in the epithelium and mesenchyme (Fig. 5). At the early bud stage proliferation is evident in the dental epithelial cells and mesenchymal cells adjacent to the basement membrane. At the late bud stage proliferation is more prominent in the mesenchyme. At the cap stage the epithelial enamel knot shows no proliferation. The rest of the dental epithelium including the epithelial cervical loop, which guides root formation, and mesenchyme show cell proliferation (Vaahtokari et al., 1991; Vainio et al., 1991). During the bell stage cell proliferation is detected in cervical mesenchymal cells and preameloblasts in the intercuspal areas (Vainio et al., 1991). Initiation (E10-11) Bud stage (E12-13) Cap stage (E14-15) Late bell stage (Pn0) epithelial cervical loop Initiation Cell differentation Figure 5. Illustration of cell proliferation (dark gray) during morphogenesis of mouse molar tooth (modified after Vaahtokari et al., 1991; Vainio et al., 1991). E, embryonic day; Pn, postnatal day. By contributing to the control of the number of cells and thereby tissue modelling, apoptosis plays a major role in not only normal development (Vaux and Korsmeyer, 1999) but also during various pathological processes (Rich et al., 2000). Apoptosis in cells proceeds via two major pathways, which both involve activation of caspase proteinases but operate mostly independently. In the death-receptor pathway apoptosis is triggered by binding of extracellular ligand molecules to death receptors belonging to the tumour necrosis factor (TNF) family. The mitochondrial pathway is controlled by members of the Bcl2 protein family including both pro- and anti-apoptotic factors that regulate each other (Hengartner, 2000). Expression of the Bcl2-family proteins is temporospatially regulated during tooth development (Krajewski et al., 1998; Kondo et al., 2001). 20

21 At the initiation stage of tooth development no apoptosis is detected. At the early bud stage apoptosis has been localized in cells of the dental epithelium adjacent to the oral epithelium. At a later bud stage (E13) apoptosis extends from the oral epithelium to the tip of the bud. At the cap stage apoptosis is located in the primary enamel knot and the dental lamina. At the early bell stage (E16) apoptosis is seen in the outer dental epithelium, secondary enamel knots, stratum intermedium cells and it is restricted to the dental lamina. When a molar tooth has reached the late bell stage, apoptosis is detected in ameloblasts and is still present in stratum intermedium and extends to stellate reticulum cells (Vaahtokari et al., 1996a; Fig. 6). About 50% of ameloblasts may die during enamel maturation. Twenty-five percent of ameloblasts may undergo apoptosis (Smith and Warshawsky, 1977; Joseph et al., 1999). Early bud stage (E12) Bud stage (E13) Cap stage (E14-15) dental lamina Late bell stage (Pn0) stellate reticulum a Initiation primary enamel knot outer dental secondary epithelium enamel knots Cell differentation Figure 6. Illustration of the distribution pattern of apoptosis (dark gray) during mouse molar tooth morphogenesis (modified after Vaahtokari et al., 1996a). E, embryonic day; Pn, postnatal day; a, ameloblasts Molecular mechanisms The transcription of genes is regulated by signalling networks involving intracellular molecules. Developing teeth share common signalling mechanisms with other organs but their function differs in various organs. Signalling molecules, also called growth factors, are small peptides that mediate cell and tissue interactions. They bind to specific receptors at the surface of the target cell and through complex intracellular signalling networks activate transcription factors in the nucleus. Transcription factors can cause multiple effects in cells so as to regulate cell cycle, cell adhesion and production of new receptors on cell surfaces (Thesleff 21

22 and Mikkola, 2002; Gilbert, 2003). The regulation and function of these signalling networks is complicated and as yet are not fully understood. Many families of signalling factors are known, for example, Wnt, Hh, TGFβ, FGF and EGF families (Gilbert, 2003). The signalling molecules have been localized at the sites of epithelial-mesenchymal interaction suggesting that they mediate inductive signalling. In the beginning of tooth development some of these signalling factors exist in the epithelium where they possibly induce the mesenchyme to condense and form the dental papilla (Neubüser et al., 1997; Mandler and Neubüser, 2001; Thesleff and Mikkola, 2002). Signalling molecules of the EGF family, including EGF and transforming growth factor α (TGFα), bind to EGFR, a transmembrane protein tyrosine kinase playing an important role in the control of cell growth and differentiation, especially during development (Carpenter and Wahl, 1991). EGF has several functions, but the most basic effect is the promotion of cell proliferation (Cohen 1962; Carpenter and Wahl, 1991). Activation of EGFR by EGF (or TGFα) leads to the activation of an intracellular signalling cascade (Ullrich and Schlessinger, 1990). EGFR null mutant mice show an almost normal tooth morphology up to the stage of early mineralization (Partanen et al., 1998) but die during the first week of their lives (Miettinen et al., 1995) so that the effect of EGFR-deficiency on later formation and mineralization of the dental matrices is not known. Previous organ culture studies have shown that the biological effects of exogenous EGF on development essentially depend on its concentration and that the range of concentrations in terms of a given effect is rather narrow (Vaahtokari et al., 1996a). In mouse teeth, EGFR expression has been localized in the dental epithelium throughout development. At the bud stage (E13) the dental epithelium, excluding the future inner dental epithelium, and dental mesenchyme express EGFR. In E14 early capstaged teeth EGFR is expressed in the outer dental epithelium and in the follicle while the expression in the mesencyme is slightly decreased compared to the bud-staged tooth. At the bell stage (E17) and secretory stage (Pn1) EGFR expression is seen in cells of the outer dental epithelium, stellate reticulum and stratum intermedium of the enamel organ, and also in the dental follicle. At the bell stage differentiating odontoblasts express EGFR but by the secretory stage the expression has disappeared (Partanen et al., 1995; Fig. 7). 22

23 Initiation (E10-11) Bud stage (E13) Cap stage (E14) outer dental epithelium Bell stage (E17) stellate reticulum Secretory stage (Pn1) dental follicle odontoblasts outer dental epithelium Initiation Cell differentation Matrix formation Figure 7. Illustration of EGFR expression during mouse molar tooth formation (modified after Partanen et al., 1995). The expression is stronger in areas marked with dark gray than in those marked with light gray. E, embryonic day; Pn, postnatal day. Homeobox genes encode transcription factors which direct the patterning of teeth. The distalless homeobox proteins (Dlx) guide tooth patterning in the maxilla and mandible. Mice with double null mutation in Dlx1/Dlx2 do not develop maxillary molars and Dlx5/Dlx6 mutant mandibles have a maxillary identity (Thomas et al., 1997; Depew et al., 2002). When the homeobox gene Barx1 is expressed ectopically in the presumptive incisor mesenchyme the incisor tooth transforms to a molar type tooth (Tucker et al., 1998). Homeobox genes Msx, Dlx, Pax and Lhx, coding for transcription factors, have been linked to tooth development (Vainio et al., 1993; Neubüser et al., 1997; Thomas et al., 1997; Grigoriou et al., 1998). Tooth development is arrested at the bud stage in transgenic mice having a mutation in Lef1 (van Genderen et al., 1994; Kratochwil et al., 1996), Msx1 (Satokata and Maas, 1994; Chen et al., 1996), Pax9 (Peters et al., 1998), and a mutation in both Gli2 and Gli3 (Hardcastle et al., 1998), which substantiates the criticalness of the bud stage in terms of continuation of tooth development. Dental mesenchyme expresses Lef1, Msx1 and Pax9, which are regulated by BMPs (included in the TGFβ signalling factor family) and FGFs (Vainio et al., 1993; Neubüser et al., 1997; Sasaki et al, 2005). BMP4 has been shown to partly rescue tooth development in Msx1 null mutant mice. Therefore, it has been suggested that Msx1 regulates mesenchymal Bmp4 expression (Chen et al., 1996). FGF4, on the other hand, has been shown to rescue tooth development in Lef1 null mutant mice (Kratochwil et al., 2002). As for defective human tooth development, several families with oligodontia (congenital lack of six or more teeth) have been shown to carry mutations in Msx1 or Pax9 (Lammi et al., 2003; Nieminen et al., 2003). Mutations in both Msx1 and Msx2 genes in mice arrests tooth development at the initiation stage, indicating redundancy in the functions of Msx1 and Msx2 in the dental mesenchyme during the initiation stage (Bei and Maas, 1998). Gli2 and Gli3 23

24 mediate sonic hedgehog (Shh) signalling, which occurs in dental epithelium during tooth development (Iseki et al., 1996). Wnt is one of the major signalling factor families participating in the maintenance of cell homeostasis. Lef1 is a protein mediating Wnt signalling and is expressed at different stages of tooth development but has been shown to be necessary only during epithelial signalling at the bud stage (Kratochwil et al., 1996, 2002). Recently it has been reported that a mutation in the AXIN2 gene, encoding a protein also participating in Wnt signalling, causes hereditary tooth agenesis and predisposes to colorectal cancer (Lammi et al., 2004). Thus, Wnt signalling is necessary for normal tooth development as well in humans Dental cell differentiation Of the three principal cusps of mouse and rat molars, the first (mesial) starts to form at the cap stage of tooth development. During the bell stage the enamel organ folds and forms all cusps of the molar tooth. Cells of the inner epithelium differentiate into ameloblasts and the mesenchymal cells of dental papilla, facing ameloblasts, differentiate into odontoblasts. Up to the start of matrix secretion, these two cell layers are separated by a basement membrane at their distal ends. Like tooth morphogenesis, differentiation of the dental cells is regulated by epithelial-mesenchymal interactions. Roots start to develop only when morphogenesis of the crown has been completed (Ten Cate et al., 2003). The polarized cells of the inner dental epithelium (future ameloblasts) induce the differentiation of the peripheral dental papilla cells to odontoblasts and during this process the basement membrane is of vital importance (Thesleff et al., 1977; Lesot et al., 1986). Cellmatrix interactions possibly contribute to the polarization of odontoblasts. For several decades it has been known that dentin matrix is needed for the differentiation of ameloblasts (Huggins et al., 1934), and after that a number of studies aimed at clarifying the nature of the epithelial-mesenchymal interactions have been undertaken. Cell-cell contacts, cell-matrix contacts or signalling molecules, or all of these together, contribute to ameloblast differentiation (Karcher-Djuricic et al., 1985), but even so, the mechanism is not known in detail. When odontoblasts start to form predentin the basement membrane undergoes degradation allowing cell-cell contacts between odontoblasts and ameloblasts. It has not, however, been possible to establish any key role for this mechanism. While acellular dentin matrix can induce ameloblast differentiation (Karcher-Djuricic et al., 1985), purified extracellular matrix molecules have not been shown to possess such inductive capability. Several signalling molecules, such as BMPs, TGFβs, FGFs and insulin-like growth factors (IGFs), have been linked to odontoblast differentiation in vitro (Finkelman et al., 1990; Cam et al., 1992; Bègue-Kirn et al., 1994) and their localization in developing teeth suggests that they play a role during development (Ruch et al., 1995; Åberg et al., 1997; Ruch, 1998; Unda et al., 2000). It has been hypothesized that the number of cell cycles is involved in terminal differentiation of odontoblasts (Lisi et al., 2003). BMP2, BMP4 and WNT10b are expressed in preameloblasts (Nadiri et al., 2004). Of BMPs, BMP2 and BMP4 induce the differentiation of ameloblasts in vitro (Bègue-Kirn et al., 1994; Coin et al., 1999). 24

25 Dental hard tissues Cellular origin The dental enamel, dentin and cementum are tooth-specific mineralized tissues formed by ameloblasts, odontoblasts and cementoblasts, respectively. After having differentiated as a result of epithelial-mesenchymal interactions, these cells become post-mitotic and secretory. Cells of the oral epithelium, which are ectodermal in origin, form the enamel organ at the cap stage of tooth development and at the bell stage cells of the inner dental epithelium differentiate into ameloblasts. Ameloblasts induce differentiation of the opposing dental papilla cells into odontoblasts but they do not become secretory until a small amount of predentin has been laid down by odontoblasts. The dental papilla, which is formed from neural crest-derived ectomesenchymal cells, also gives rise to pulpal cells. During root development the epithelial cervical loop, comprising the inner and outer dental epithelia, forms the Hertwig s epithelial root sheath. After having induced the differentiation of root odontoblasts, followed by the start of dentinogenesis, cells of the root sheath disintegrate and undergo resorption. The small amount of enamel proteins secreted by the root sheath cells (Slavkin et al., 1988, 1989) before its degradation then induces the differentiation of mesenchymal cells of the dental follicle into cementoblasts (MacNeil and Thomas, 1993). Thus the first-formed (intermediate) cementum is, in fact, epithelial in origin (Lindskog, 1982a, b; Lindskog and Hammarström, 1982). Of the dental hard tissues the cementum most closely resembles bone. Morphologically it can be divided to acellular, cervically located, and cellular, more apically located cementum, which is deposited slowly but continually. External circumstances markedly influence the accumulation rate and, consequently, the thickness of the cellular cementum Formation and structure of the enamel Enamel is the hardest and the only mineralized tissue known to be epithelial in origin. Differentiated ameloblasts are tall columnar and polarized cells. Deposition of enamel matrix starts from cusp tips and proceeds cervically. Amelogenins and non-amelogenins (mainly enamelins) are major enamel proteins secreted by ameloblasts. Mineralization of enamel starts concomitantly with the commencement of matrix deposition and up to one third of the final mineral content is achieved during primary mineralization (Nanci, 2003a). Matrix metalloproteinase-20 (MMP-20) and kallikrein-4 are major proteinases that degrade enamel matrix proteins to be transported back to ameloblasts during secondary mineralization, or maturation (Hu et al., 2002). Maturation is thought to start when the final thickness of enamel at a given site is achieved and it is phasic. A prerequisite for normal maturation of the enamel is an adequate degradation of the organic matrix. The basic structural unit of enamel is the enamel rod, each rod being a product of four ameloblasts. Correspondingly, one ameloblast participates in the formation of four rods (Nanci, 2003a). When the tooth erupts the enamel loses its contact with living cells. The main component of mature enamel is hydroxyapatite. Only about 4% of mature enamel is organic material and water and the remaining 96% is mineral (Sarkar et al., 1997). 25

26 Formation and structure of the dentin Despite marked differences in their structural characteristics, dentin and pulp form an intimate anatomical and functional complex. In contrast to ameloblasts, the dentin-forming odontoblasts remain as functional cells through the whole life of the tooth. They are columnar in shape and their nuclei are located on the pulpal side of the cell body. Odontoblasts first secrete a layer of predentin to be located next to the enamel in a fully developed tooth and with the progressing dentinogenesis they draw away from ameloblasts. Unlike enamel starting to mineralize concomitantly with matrix deposition, dentin starts to mineralize after a short delay, a permanent sign of which is the unmineralized predentin pulpally to dentin. Characteristically, predentin and dentin are traversed by dentinal tubules each containing a cytoplasmic extension of an odontoblast. The tubules reach the dentin-enamel junction where they branch but how far the cell processes extend is contradictory (Nanci, 2003b). Dentin is composed of organic macromolecules and hydroxyapatite mineral. The mineral content of mature dentin is about 70% by weight, 20% is organic material and 10% is water. Once mineralization has started the mineral content of dentin (and bone) rapidly approaches its final level, in contrast to the enamel. The organic dentin matrix is mainly composed of type I collagen. The structures of predentin and dentin differ by the organization of collagen and also by the composition of structural proteins other than collagen (Septier et al., 1998; Beniash et al., 2000). Dentin phosphoprotein (DPP) and dentin sialoprotein (DSP) are major non-collagenous proteins of dentin encoded by the dentin sialophosphoprotein gene (Dspp). Dspp codes for one big protein which is post-translationally cleaved to DPP and DSP. MMP- 20 has been suggested to participate in the cleavage of DSPP (Bègue-Kirn et al., 1998). Recently, mrna for DSPP was also found in bone (Qin et al., 2002). DPP has been suggested to be involved in the initiation of early apatite crystal formation at the predentindentin border (Butler and Ritchie, 1995; Rabie and Veis, 1995; Butler, 1998) and it may also contribute to the regulation of crystal growth as mineralization of dentin proceeds (Butler and Ritchie, 1995; Butler, 1998). It has been shown in vitro that DSP has only limited effects on apatite formation and growth, but as yet its definite function is unknown (Boskey et al., 2000; Butler et al., 2003; Qin et al., 2004). Bono1 (also named insulin-like growth factor binding protein-related protein 10; IGFBP-rP10) is one of the macromolecules comprising dentin, and has been localized not only in dentin but also in bone and soft tissues (James et al., 2004; Shibata et al., 2004). Mutations in genes coding for the major dentin proteins play key roles in the aetiology of inherited developmental defects manifested primarily in dentin Abnormal tooth development and structure Dental defects can be classified by their heredity or the possible association with a generalized disease or disease complex versus restriction to teeth. A common way to classify dental defects is to divide them into genetic and non-genetic (environmental) defects Genetic aberrations Genetic diseases causing structural changes in teeth can be restricted to dental tissues or they can be part of a wider disease complex or syndrome. An ultimate form of a defect affecting all dental tissues is tooth agenesis. Nonsyndromic tooth agenesis has been attributed to e.g. dominant mutations in transcription factors MSX1, PAX9 and AXIN2 (Vastardis et al., 1996; Stockton et al., 2000; Nieminen et al., 2001; Lammi et al., 2003, 2004). 26

27 Genetic aberrations restricted to the enamel are termed amelogenesis imperfecta (AI). If the teeth clinically resemble those seen in AI patients but the defect is associated with a disease complex or syndrome, it is not classified as AI. AI is characterized by clinical and genetic heterogeneity. AI can be inherited as autosomal dominant or recessive or, less frequently, X- linked traits (Bäckman and Holmgren, 1988; Stephanopoulos et al., 2005). To date, mutations have been identified in genes coding for enamel matrix proteins (Kim et al., 2004, 2005), enzymes involved in degradation of matrix proteins associated with enamel maturation (Ozdenir et al., 2005) and in the homeobox gene DLX3 (Dong et al., 2005). At least 14 classical forms based on clinical features and the mode of inheritance have been described (Witkop, 1988). Heritable human dentin defects is the designation for a group of autosomal dominant conditions in which the dentin matrix is structurally abnormal and defectively mineralized. Teeth are discoloured and susceptible to attrition and/or fracture of the enamel. The diseases resemble each other clinically and morphologically with colour change, aberrant shape (usually obliteration) of the pulp and abnormal tubular pattern as main characteristics. There are two main types, dentinogenesis imperfecta (DI) and dentin dysplasia (DD). DI is divided into three (currently two) and DD into two subgroups (Shields et al., 1973). Type I DI, originally included in the classification, is the dental manifestation of the generalized connective tissue disorder osteogenesis imperfecta. Types II and III DI as well as type II DD have been attributed to different mutations in DSPP. In humans DSPP is located on chromosome 4q21 within a gene cluster where genes for osteopontin (OPN), bone sialoprotein 1 (BSP1), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE/OF45) are also located. All of these genes code for proteins participating in tooth and/or bone development (MacDougall et al., 1997, 2002; Fisher et al., 2001). At least seven different mutations have been described in DSPP in the region coding for DSP (Kim et al., 2005) that cause DI type II. In one family a deletion and an insertion was found in DPP causing type III DI (Dong et al., 2005) and one mutation in DSP has been found in a family with type II DD (Rajpar et al., 2002). A clear indication of Dspp having a crucial role in the mineralization of dentin matrix is that Dspp null mutant mice have similar dental defects to patients with type III DI (Sreenath et al., 2003) Environmental defects Dental defects caused by the environment, also called acquired defects, can be divided into local and systemic defects. Localized defects are seen in one tooth or asymmetrically in several teeth. The most common of this type of defect is seen in traumatised teeth. Over a hundred systemic factors or incidents have been linked to disturbed tooth development, among them, nutritional deficiencies, severe diseases, some drugs and chemicals including environmental pollutants (Pindborg, 1982; Alaluusua et al., 1996, 1999). Since the schedule of tooth development is well-known, the time of origin of an environmental disturbance can be traced back Defects caused by certain chemicals and radiotherapy Fluorides at appropriate concentrations prevent dental caries but when the concentration is too high they cause fluorosis in developing teeth. The resultant defects are dependent on the exposure level. Mild fluorosis appears as opacities of enamel and a severe form is manifested as hypomineralized enamel, which is discoloured brown and fractures easily. Fluorosis can be 27