Toxicity of dioxin to developing teeth and salivary glands : An experimental study

Dioxins are ubiquitous environmental poisons having unequivocal adverse health effects on various species. The majority of their effects are thought to be mediated by the aryl hydrocarbon receptor (AhR). Developing human teeth may be sensitive to dioxins and the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to rodent teeth. Mechanisms of TCDD toxicity can be studied only experimentally. The aim of the present thesis work was to delineate morphological end points of developmental toxicity of TCDD in rat and mouse teeth and salivary glands in vivo and in vitro and to characterize their cellular and molecular background. Mouse embryonic teeth and submandibular gland explants were grown in organ culture without/with TCDD at various concentrations, examined stereomicroscopically and processed for histological examination. The effects of TCDD on cellular mechanisms essential for organogenesis were investigated. The expression of various genes eliciting the response to TCDD exposure or involved in tooth and salivary gland development was studied at the mRNA and/or protein levels by in situ hybridization and immunohistochemistry. Association of the dental effects of TCDD with the resistance of a rat strain to TCDD acute lethality was analyzed in two lactationally exposed rat strains. The effect of TCDD on rat molar tooth mineralization was studied in tissue sections. TCDD dose- and developmental stage-dependently interfered with tooth formation. TCDD prevented early mouse molar tooth morphogenesis and altered cuspal morphology by enhancing programmend cell death, or apoptosis, in dental epithelial cells programmed to undergo apotosis. Cell proliferation was not affected. TCDD impaired mineralization of rat molar dental matrices, possibly by specifically reducing the expression of the mineralization-related dentin sialophosphoprotein gene shown in cultured mouse teeth. The impaired mineralization of rat teeth was accompanied by decreased expression of AhR and the TCDD-inducible xenobiotic-metabolozing enzyme P4501 A1 (CYP1A1), suggesting mediation of the TCDD effect by the AhR pathway. The severe interference by TCDD with rat incisor formation was independent of the genotypic variation of AhR determining the resistance of a rat strain to TCDD acute lethality. The impairment by TCDD of mouse submandibular gland branching morphogenesis was associated with CYP1A1 induction and involved blockage of EGF receptor signalling. In conclusion, TCDD exposure is likely to have activated the AhR pathway in target organs with the consequent activation of other signalling pathways involving developmentally regulated genes. The resultant phenotype is organ specific and modified by epithelial-mesenchymal interactions and dependent on dose as well as the stage of organogenesis at the time of TCDD exposure. Teeth appear to be responsive to TCDD exposure throughout their development.

Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

Cattle Teeth in Graves : Interpretations of animal bones found in Finnish inhumation graves (ca AD 550-1700)

The nature of a burial is always ritualistic. This is often forgotten when dealing with Finnish inhumation burials containing animal bones. Only the animal bones found close to the deceased have traditionally been thought to have a ritualistic purpose. The animal bones found in the filling of the grave, which is still part of the burial, has on the other hand, often been neglected in the previous research. In this Master s thesis I will discuss the function and interpretation of animal bones in graves. The base of this study is six sites, all of different nature, from Finland. Luistari in Eura is from the western coast and is dated to Late Iron Age (and possibly Medieval period), the Medieval hamlet of Finno is situated in Espoo which is situated on the southern coast. Two town burials, Turku and Porvoo, are also included in this study. The graves from Turku are dated to Late Medieval period and Early Renaissance, whereas the cemetery in Porvoo is from the 18th century. Visulahti in Mikkeli is from the Late Iron Age and represents Eastern Finnish burial tradition, the same as Suotniemi from Käkisalmi parish, which is nowadays part of Russia. While parts of the animal bones had already been analysed before, the author also analysed animal bones for the purpose of the present Master´s thesis. The bones were compared to the burial contexts, when possible. Based on the comparisons I have made interpretations which might explain the existence of animal bones in the graves. The interpretations are among others sacrifice, commemoration meals and animal burials. The site could also have been a settlement site prior to the graves, thus the bones in the graves would belong to the settlement phase. When comparing the date of the studied sites, the town burials are later and the animal bones are probably related to previous or contemporary use of the sites as graveyards. On top of this there does not seem to be much difference in burial tradition between Eastern and Western Finland, although at least from the hamlet burials of Finno there are aspects that could be linked to Eastern burials. In making the interpretations I have taken into consideration the aspects of belief during different time periods when they could be accounted as relevant. Also the problems with bone preservation were relevant and challenging for the study. Often only the hardest substance of the skeleton, namely teeth, has been preserved. For this reason the quality of the archaeological documentation was a key issue in this study. In producing quality interpretations of the animal bones in graves, the bones, contexts and their relationship to the surrounding site should be documented with care.

Anisotropic, gradient and metal-like mechanical behaviors of teeth and their implications on tooth functions

The anisotropy and gradient of the elastic modulus and the hardness of teeth were investigated by means of instrumented indentation method. Such properties are attributed to the unique microstructures of teeth based on scanning electron microscopic analysis. By comparing the relationship between the ratio of hardness to the reduced elastic modulus and the ratio of elastic unloading work to the total work of teeth in course of indentation to those of other materials, we found that the material behaviors of teeth display metal-like characteristics rather than ceramics as considered traditionally. These material behaviors and relevant functions are discussed briefly.

Analysis of growth layers in the teeth of Tursiops truncatus using light microscopy, microradiography, and SEM

Preliminary results show microradiography and scanning electron microscopy (SEM) to be more accurate methods of accessing growth layer groups (GLGs) in the teeth of Tursiops truncatus than transmitted light microscopy. Microradiography shows the rhythmic deposition of mineral as alternating radiopaque and radiolucent layers. It improves the resolution of GLGs near the pulp cavity in older individuals, better than either SEM or light microscopy. SEM of etched sections show GLGs as ridges and grooves which are easily counted from the micrograph. SEM also shows GLGs to be composed of fine incremental layers of uniform size and number which may allow for more precise age determination. Accessory layers are usually hypomineralized layers within the hypermineralized layer of the GLG and are more readily distinguishable as such in SEM of etched sections and microradiographs than in thin sections viewed under transmitted light. The neonatal line is hypomineralized, appearing translucent under transmitted light, radiolucent in a microradiograph, and as a ridge in SEM. (PDF contains 6 pages.)

Age determination and age related factors in the teeth of Western North Atlantic bottlenose dolphins.

Teeth were taken from 120 bottlenose dolphins, Tursiops truncatus, which had stranded on the mid-Atlantic coast of the United States. The number of annual growth layer groups (GLGs) for each animal was used to construct a growth curve. The growth rate of coastal North Atlantic Ocean Tursiops is similar to other cetaceans in having a high initial rate of growth, with no differences in growth between females and males. In females, the first dentinal GLG is thickest and is followed by GLGs which become progressively narrower. In males, the second GLG is thicker than the first; GLGs beyond number two become progressively smaller but at a slower rate than in females. In males and females, the translucent layer makes up proportionally larger parts of the GLG as the animal ages, but in males the percent translucent layer remains constant at about 50% while in females it continues to increase up to about 70% of the GLG. These two factors, GLGs width and translucent layer width, indicate that the sex and age of the animal influence the deposition of GLGs. Incremental layers are also present, averaging 12 per GLG, and seem similar to incremental layers described in other marine mammals. A plot of the relationship of percent growth of the last GLG to time of death suggests that the deposition of GLGs is relatively constant, at least during the first half of the year, and that North Atlantic Ocean Tursiops give birth in the fall as well as in the spring. (PDF contains 31 pages.)

Estimating age of spotted and spinner dolphins (Stenella attenuata and Stenella longirostris) from teeth

This paper is an account of preparation and examination techniques and criteria used to estimate age in decalcified and stained tooth thin sections from spinner and spotted dolphins. A dentinal growth layer group (GLG), composed of two thin light and two thicker dark-stained layers, is deposited annually. The GLG component layers are variably visible, but the "ideal" pattern and successive thinning of dentinal GLGs are used as a guide to determine GLG limits. Age-specific thicknesses of dentinal GLGs found in Hawaiian spinner dolphin teeth seem to be applicable to teeth of spotted dolphins and can be used as an aid in locating GLG boundaries. Cementa1 GLGs are composed of a dark-stained and alightly stained layer and usually are deposited at a rate of one per year, but may be deposited every other year or two or three times per year. Two slightly different methods of counting dentinal GLGs are presented, along with guidelines for determining whether dentinal or cementa1 GLG counts provide the best estimate of age for a specimen. (PDF contains 23 pages.)

Marginal adaptation and performance of bioactive dental restorative materials in deciduous and young permanent teeth

Objective: The aim of this study was to investigate the adaptation of different types of restorations towards deciduous and young permanent teeth. Materials and Methods: Class V cavities were prepared in deciduous and young permanent teeth and filled with different materials (a conventional glass-ionomer, a resin-modified glass-ionomer, a poly-acid-modified composite resin and a conventional composite resin). Specimens were aged in artificial saliva for 1, 6, 12 and 18 months, then examined by SEM. Results: The composite resin and the polyacid-modified composite had better marginal adaptation than the glass-ionomers,though microcracks developed in the enamel of the tooth. The glass-ionomers showed inferior marginal quality and durability, but no microcracking of the enamel. The margins of the resin-modified glass-ionomer were slightly superior to the conventional glass-ionomer. Conditioning improved the adaptation of the composite resin, but the type of tooth made little or no difference to the performance of the restorative material. All materials were associated with the formation of crystals in the gaps between the filling and the tooth; the quantity and shape of these crystals varied with the material. Conclusions: Resin-based materials are generally better at forming sound, durable margins in deciduous and young permanent teeth than cements, but are associated with microcracks in the enamel. All fluoride-releasing materials give rise to crystalline deposits.