[Structure of teeth in mature ovarian teratoma].

MATERIAL AND METHODS: The authors investigated three mature ovarian dermoid cysts that were found to contain atypically developed teeth (one tooth in one cyst; 2 teeth in another cyst, and a set of 8 teeth with jawbone fragments in the third one). Their structure was examined using the epoxy resin plastination technique to obtain plastinated sections. RESULTS: Some of these teratomous teeth have obvious signs of alteration in both dentin and enamel, which may be referred to as fluorosis or macular enamel hypoplasia in one case whereas other sections indicate a caries lesion. CONCLUSION: These facts are contradictory to the concept of the exogenous nature of dental caries, which is accepted in dentistry.

[Histological analysis of dental follicles after unerupted mandibular third molar extraction]. / Analyse histologique des follicules dentaires après germectomies des troisièmes molaires mandibulaires.

INTRODUCTION: The extraction of third mandibular tooth germ (M3) is often prophylactic to avoid orthodontic treatment relapse and to prevent infectious or tumoral diseases developing from the dental sac. The purpose of this study was to screen for early histopathological modification of dental follicles (inflammatory, infiltration, or epithelial metaplasia) after extraction of third mandibular tooth germ (M3) on asymptomatic patients. The secondary objective was to study the proliferative activity of the epithelium by dosing the anti Ki-67 antibody. PATIENTS AND METHOD: Twenty dental follicles extracted from 12 boys and eight girls between 14 and 18 years of age were examined under phototonic microscopy. The proliferative activity of the epithelium was assessed by immuno-histochemistry. RESULTS: Three dental follicles presented with focal epidermoid metaplasia of the epithelium, without odontogenic tumoral proliferation. In all other cases, the cylindrical epithelial cell structure was normal. A mild chronic inflammatory infiltrate was present in 30% of the cases. Immuno-histochemical analysis revealed labeling of very rare epithelial lining cells, slightly more in cases presenting with metaplasia. DISCUSSION: The prevalence of early morphological changes of dental sac is low. This histo-morphological study does not support the systematic extraction of asymptomatic mandibular tooth germs (M3).

Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells.

In tooth development matrix metalloproteinases (MMPs) are under the control of several regulatory mechanisms including the upregulation of expression by inducers and downregulation by inhibitors. The aim of the present study was to monitor the occurrence and distribution pattern of the extracellular matrix metalloproteinase inducer (EMMPRIN), the metalloproteinases MMP-2 and MT1-MMP and caveolin-1 during the cap and bell stage of rat molar tooth germs by means of immunocytochemistry. Strong EMMPRIN immunoreactivity was detected on the cell membranes of ameloblasts and cells of the stratum intermedium in the bell stage of the enamel organ. Differentiating odontoblasts exhibited intense EMMPRIN immunoreactivity, especially at their distal ends. Caveolin-1 immunoreactivity was evident in cells of the internal enamel epithelium and in ameloblasts. Double immunofluorescence studies revealed a focal co-localization between caveolin-1 and EMMPRIN in ameloblastic cells. Finally, western blotting experiments demonstrated the expression of EMMPRIN and caveolin-1 in dental epithelial cells (HAT-7 cells). A substantial part of EMMPRIN was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. The differentiation-dependent co-expression of MMPs with EMMPRIN in the enamel organ and in odontoblasts indicates that EMMPRIN takes part in the induction of proteolytic enzymes in the rat tooth germ. The localization of EMMPRIN in membrane rafts provides a basis for further investigations on the role of caveolin-1 in EMMPRIN-mediated signal transduction cascades in ameloblasts.

Diverse effects of c-src deficiency on molar tooth development and eruption in mice.

C-src deficiency is characterized by osteopetrosis due to impaired bone resorption by hypofunctional osteoclasts and the resultant failure of tooth eruption. In preliminary observations, we frequently encountered erupted molars in c-src deficient mice unlike in other osteopetrotic animals. Here we examine the effects of c-src deficiency on the development of molar teeth with an emphasis on the spatial relation of growing teeth with the surrounding bones. In c-src deficient mice, the magnitude of tooth impaction differed considerably among the types of molars; all maxillary 1st molars were totally impacted deep in the alveolar sockets, whereas most mandibular 1st molars fully erupted into oral cavity. Distribution of osteoclasts in the alveolar bone was identical among all types of molars, and electron microscopy revealed signs of bone resorbing activity in these osteoclasts despite the absence of a ruffled border. From early development, the alveolar space was much narrower in the upper molar tooth germs than in the lower ones in both wild type and homozygous animals, and particularly so in the upper 1st molars. Current observations thus indicate a significant contribution of "hypofunctional osteoclasts" in c-src deficient mice in molar tooth development except for the upper 1st molars, which appear to require highly functional osteoclasts to gain sufficient space for them to grow normally. Taken together, these findings on the seemingly tooth-type specific effects of c-src deficiency on the development and eruption of molar teeth in c-src deficient mice can be attributed to the given differential spatial relation of the respective tooth germs with the surrounding bones in the presence of hypofunctional osteoclasts.

Collagen analysis in human tooth germ papillae

The extracellular matrix (ECM) performs a very important role in growth regulation and tissue differentiation and organization. In view of this, the purpose of this study was to analyze the collagen, the major organic component of dental pulp ECM, in papillae of human tooth germs in different developmental phases. The maxillas and mandibles of 9 human fetuses ranging from 10 to 22 weeks of intrauterine life were removed and 16 tooth germs (1 in the cap stage, 8 in the early bell stage and 7 in the late bell stage) were obtained. The pieces were processed for histological analysis and stained with hematoxylin-eosin, Masson's Trichrome and picrosirius staining technique. Both types of collagen in the dental papilla were only detected by the picrosirius staining technique under polarized light microscopy. Type III collagen was detected in all specimens. Type I collagen was present in focal areas of the dental papilla only in some specimens. In conclusion, the findings of this study showed that type III collagen is a regular component of the papillae of human tooth germs whereas type I collagen is present in a significantly lesser amount.

A matriz extracelular (MEC) tem um papel importante na regulação do crescimento e na diferenciação e organização dos tecidos. Com base nestes aspectos o objetivo do deste estudo foi analisar o colágeno, maior componente orgânico da MEC da polpa dentária, na papila de germes dentários humanos, em diferentes fases do desenvolvimento. Foram obtidos fragmentos de maxilas e mandíbulas de 9 fetos humanos com 10 a 22 semanas de vida intra-uterina, dos quais foram analisados 16 germes dentários (1 em estágio de capuz, 8 em estágio de campânula precoce e 7 em estágio de campânula tardia). Secções histológicas seriadas foram coradas com hematoxilina e eosina, tricrômico de Masson e técnica de coloração do picrosirius. Ambos os tipos de colágeno na papila dentária foram somente detectados pela técnica de coloração do picrosirius usando microscopia de luz polarizada. Colágeno tipo III foi detectado em todas as amostras. Colágeno tipo I estava presente em áreas focais da papila dental em algumas amostras. Concluiu-se que o colágeno tipo III mostrou-se um componente regular da papila de germes dentários humanos, enquanto o colágeno tipo I esteve presente em quantidade significativamente menor.

Tooth development in vitro in two teleost fish, the cichlid Hemichromis bimaculatus and the cyprinid Danio rerio.

A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.

Rat enamel contains RP59: a new context for a protein from osteogenic and haematopoietic precursor cells.

We have recently identified a protein, RP59, in bone marrow cells and young osteoblasts, in cells involved in bone repair and in young erythroblasts and megakaryocytes. Here, we report immunohistochemical data at the light- and electron-microscope level indicating that RP59 is also present in newly secreted tooth enamel of the rat and in ameloblasts, the formative cells. In enamel matrix, RP59 was located proximal to secretory ameloblasts only, i.e. in newly secreted material. Distal enamel and enamel in association with maturation stage ameloblasts were unlabelled. Secretory ameloblasts contained RP59 in the matrix-proximal region including Tomes' processes, post-secretory ameloblasts in the cell-matrix interface. Western blotting of proteins from tooth germs identified RP59 as a band at 90 kD, co-migrating with RP59 from bone marrow and spleen. Antisera versus a chemically synthesised RP59 peptide and versus a bacteria-synthesised protein fragment reacted in the same manner. In situ hybridisation of tooth tissue revealed RP59 RNA specifically in ameloblasts. The reverse transcription/polymerase chain reaction method identified tooth RNA coding for RP59. Sequence analysis indicated that RP59 RNA from tooth and marrow had the same sequence. An internal sequence motif was found in rat RP59 resembling a signal implicated in secretion of the chicken "engrailed" gene product. The findings indicate that RP59 is a genuine product of ameloblasts and that it is secreted in the course of enamel formation together with other matrix components.

Expression and role of Lhx8 in murine tooth development.

We examined the expression and possible functions of Lhx8, a member of the LIM-homeobox gene family, during tooth morphogenesis of the mouse. Lhx8 was expressed in the dental mesenchyme between the bud and early bell stage of the molar tooth germ. Tooth germ explants from embryonic day 12.5 mice treated for 5 to 7 days with antisense-oligodeoxynucleotides (AS-ODN) against Lhx8 showed a marked decrease in the number of mesenchymal cells. The explants treated with AS-ODN for 11 to 14 days were filled with a large number of undifferentiated epithelial cells and a limited number of undifferentiated mesenchymal cells, but did not contain a tooth germ. Treatment of explants with AS-ODN for 7 days suppressed the proliferation of dental mesenchymal cells and induced apoptosis; the latter was confirmed by histochemical and ultrastructural examinations. Moreover, the expression of Lhx6, Msx1, Msx2, Bmp4 and Gsc, which are also known to be involved in tooth morphogenesis, were suppressed after the application of AS-ODN against Lhx8 for 7 days. The present results suggest that Lhx8 plays an important role in the survival of mesenchymal cells of the tooth germ during development.

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs.

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.

Lectin binding patterns of odontogenic epithelium in the rat during various phases of molar tooth development.

Light and electron microscopic investigations of lectin binding patterns in rat tooth germs were undertaken in order to elucidate glycoconjugate localization in cells of the reduced enamel epithelia and their derivatives. It was found that Ulex europaeus agglutinin (UEA-1), peanut agglutinin (PNA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) exhibited variable reactivity patterns with different epithelia. UEA-1 was reactive with cells of the stratum intermedium and stellate reticulum in the tooth germ but unreactive with ameloblasts, outer enamel epithelial cells, and junctional epithelium at later stages. Reaction patterns of PNA in these cells differed from those of UEA-1. Results indicated that inner and outer cells of the reduced enamel epithelium are heterogeneous with regard to lectin binding patterns.

Apoptosis is involved in the disappearance of the diastemal dental primordia in mouse embryo.

Three transient dental primordia (D1, D2 and D3) exist in the upper diastema in mouse embryos and their regression is associated with the presence of cell death. In order to specify the type of cell death and its temporo-spatial distribution, staining with hematoxylin, supravital staining with Nile Blue, TUNEL method, electron microscopic analysis and computer assisted 3-D reconstructions were performed. These data demonstrated that apoptosis is involved in the disappearance of the diastemal dental rudiments. Apoptosis occurred first with prevalence in the buccal part of the epithelium of the diastemal dental primordia and extended later to the whole epithelium of the dental rudiments and the dental lamina interconnecting them with the incisor and molar epithelia. Cell death occurred only sporadically in the adjacent mesenchyme. The prospective upper diastema in mouse embryos may provide a model for studies of developmental determination of toothless areas in the jaw as well as a tool for analyses of regulatory mechanisms of programmed cell death in morphogenesis.

Alternative splicing of amelogenins.

Amelogenins comprise as much as 90% of the protein in the developing enamel matrix. Separating amelogenins by gel electrophoresis reveals a complex of polypeptides with apparent mobilities ranging from low molecular weight species on up to 28,000 Daltons. A major objective of our research is determine the extent to which alternative RNA splicing contributes to this heterogeneity. We have cloned seven alternatively spliced mouse amelogenin mRNAs. The predicted translation products of these messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. The 194 residue amelogenin is the only mouse amelogenin to include a polypeptide segment encoded by exon 4, which has a deduced amino acid sequence of KSHSQAINTDRTAL. Antibodies were raised against synthetic exon 4 encoded polypeptides and used to immunostain histologic tooth sections. These data indicate that alternatively spliced amelogenin mRNAs are translated into protein and secreted into the enamel matrix.

Experimental odontomas in osteopetrotic op/op rats.

Osteopetrosis is an autosomal recessive disease in several mammalian species. Osteopetrotic op/op rats suffer from complete failure of tooth eruption related to reduced bone resorption. In our earlier studies, op/op rats grafted with bone marrow cells 3 days after birth were cured of the disease and their molar eruption was restored. However, the incisors failed to erupt and their proliferating ends were distorted, forming odontomas. The purpose of the present investigation was to study the odontogenic tissues in the odontomas, using the correlated techniques of radiography and microradiography of undecalcified material, together with histology of decalcified material and scanning electron microscopy.

Localization of ornithine decarboxylase in mouse teeth. An in vitro and in vivo study.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, and thus in tissue growth and development, has been localized in mouse dental tissues, in vivo as well as in vitro by light and electron microscopic autoradiography with radiolabeled alpha-difluoromethylornithine ([3H]DFMO). Mandibular first molar germs from day-18 fetuses were incubated in vitro in the presence of [3H]DFMO and processed for autoradiography. For ODC localization in vivo, 3-day old puppies received [3H]DFMO by injection. As controls, puppies were injected either with unlabeled DFMO, or with cycloheximide before administration of isotope. Kidneys and mandibles were excised and processed for autoradiography. In vitro, labeling was found in all cell types of the tooth germ, but with a more intense labeling in ameloblasts and odontoblasts. In both these, radioactivity decreased from the tip of the cusps to the cervical loop. In vivo the binding of [3H]DFMO in cells of the ameloblast and odontoblast lineages, respectively, showed a gradual increase form the posterior end of the incisor to its anterior end. The distribution of radioactivity in the kidney was in accordance with findings by others. Both the kidney and tooth cell labeling decreased strongly after cycloheximide treatment. The results show that ODC is expressed in tooth-forming cells, and that ODC is not only present in differentiating cells but occurs at higher amounts in mature, secreting cells. The findings suggest that polyamines have a central role in tooth development.

SEM contribution to the morphological study of dental structures in ovarian cystic teratoma.

This study in scanning electron microscopy (SEM) was carried out to observe the possible pathologic aspect of five teeth grossly arranged in normal sequence and of their surrounding tissues, found inside an ovarian cystic teratoma. Though the general morphology of the teeth was nearly normal, several anomalies affected the different mineralized dental tissues such as enamel hypoplasia, irregular growth of cementum, altered predentin layer, and immature osteofibrous bony outgrowths.

Immunolocalization of transforming growth factor beta 1 and epidermal growth factor receptor epitopes in mouse incisors and molars with a demonstration of in vitro production of transforming activity.

Day-14 lower incisors and day-18 first lower molars of mouse embryos produced in vitro transforming activities for non-confluent NRK cells co-cultured in agar, and mitogenic activities for exponentially growing NRK and BHK cells. The patterns of distribution of TGF beta 1 and EGF receptor, both known to regulate cell proliferation, differentiation and transformation in vitro and suspected to play important roles in developmental processes, were studied during mouse odontogenesis by means of indirect immunofluorescence on fixed or frozen fixed sections. TGF beta 1 epitopes were detected in the stellate reticulum of day-13 to day-16 incisors and of molars from day-17 onwards. Dental mesenchyme of day-14 incisors and postnatal molars, and peridental mesenchyme of bud and cap stage molars and incisors were also stained by TGF beta 1 antibodies. EGF receptor was localized in the enamel organs of incisors and molars; the inner dental epithelium and later the outer dental epithelium rapidly became negative while the stellate reticulum remained stained. Incisor apical mesenchyme showed an intense reaction with EGF receptor antibodies after birth.

Regional odontodysplasia: new histopathological data.

Light microscopy, microradiography, SEM and TEM of 4 tooth follicles in a 12-year-old caucasian girl presenting regional odontodysplasia showed widespread globular dentin, calcifications located in the enlarged pulp chambers, hypoplastic and hypomineralized enamel. Hypomineralized strands were sandwiched between two normal enamel layers, which indicates that amelogenesis, interrupted for a while, has once more become established. The enamel surface was covered with calcoglobules. Numerous rounded calcifications were scattered within the dental follicles. Some of these occurred in microfibrils (possibly oxytalan fibers), distinct from collagen fibers. Calcification of the sheath surrounding epithelial rests was a conspicuous feature. The fibroblasts in contact with calcifications developed numerous cytoplasmic extensions, which suggests that they may have assumed a phagocytosis behaviour.

Growth factors and tooth development.

The effects of various growth factors on tooth development were studied in organ cultures of mouse embryonic tooth germs. Transferrin was shown to be a necessary growth factor for early tooth morphogenesis. Transferrin was required for the development of bud- and early cap-staged teeth, and it was shown to be the only serum protein that was needed by early cap-staged teeth in organ culture. Promotion of tooth morphogenesis and dental cell differentiation was shown to be based on the stimulation of cell proliferation. The roles of polypeptide growth factors in tooth development were studied by adding these factors to the transferrin-containing chemically-defined culture medium which supports early tooth morphogenesis and cell differentiation. Fibroblast growth factor or platelet-derived growth factor did not affect cell proliferation or morphogenesis of tooth germs in culture. On the contrary, epidermal growth factor (EGF) stimulated cell proliferation in tooth explants, but at the same time inhibited tooth morphogenesis and dental cell differentiation. Autoradiographic localization of proliferating cells revealed that dental tissues responded to EGF with different proliferation rates. The responsiveness to EGF was stage-dependent, early cap-staged teeth were sensitive to EGF but late cap-staged and bell-staged teeth developed normally in the presence of EGF in the culture medium. The presence and distribution of receptors for both transferrin and EGF were studied in mouse embryonic teeth at various developmental stages by incubating freshly-separated tooth germs with 125Iodine-labeled transferrin or EGF, and then processing the tissues for autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in the extracellular matrix of the dental follicle during tooth eruption.

Eruption of the third and fourth mandibular premolars in the dog begins at 15 weeks of age, is dependent upon the dental follicle, and is complete by 23 weeks. Our study covered the period from 12 to 20 weeks, and revealed several changes in extracellular matrix structure and organization of the follicle which correlate with specific physiological events in eruption. First, the average DNA content per follicle reached a maximum at 14 weeks. Two weeks later, follicle size had increased 1.3- to 2.4- times. Second, the collagen content of follicles increased 2.5-fold over the study period, with two-thirds of this increase over the last four weeks. Type I collagen was the major collagen at all stages of follicular development. The amount of proteoglycan rose 45% from 16 to 20 weeks of age. Third, the ultrastructure of the dental follicle prior to eruption (12 weeks) indicated a disorganized interstitial connective tissue matrix; during eruption, two size classes of fibrils were observed which clustered together in linearly aligned bundles. Fourth, gel electrophoretic analyses resolved more than twenty follicle proteins with the major species a Mr = 95k glycoprotein. Immunoblotting demonstrated only one minor component was derived from serum. Comparison of noncollagenous proteins from different aged follicles indicated that three small polypeptides (Mr = 20-25 k) were present primarily at 16 weeks, the same time at which root elongation begins. A different sequence of changes was exhibited by two other proteins of Mr = 13 and 15 k. These findings may serve as biochemical markers of stages of dental follicle development and facilitate a search for local control mechanisms.

Nerve growth to tooth buds after homotopic or heterotopic autotransplantation.

Feline permanent incisor tooth buds (bell stage) were autotransplanted to mandibular alveolar sockets (homotopic site) or to the submandibular subcutis or the leg (heterotopic sites). This was done in 34 kittens aged 1-2 months. After survival times of 3-8 months the animals were fixed by glutaraldehyde perfusion. A total of 56 mineralized teeth, which had developed at the recipient sites, were removed, demineralized and processed for light microscopic (LM) general evaluation. Fourty-four teeth, which were judged to be grossly normal in the LM, were selected for electron microscopic (EM) analysis with respect to the occurrence of pulpal nerve fibres. The highest proportion of normal teeth (16 of 16) was obtained from the alveolar site, followed by the submandibular (11 of 14) and hindlimb (17 of 26) sites. Most of the grossly normal grafts possessed pulpal axons (37 of 44). The alveolar grafts were all innervated and exhibited a largely normal appearance qualitatively and in terms of percentage of myelinated fibres. The proportion of innervated pulps was lower among the heterotopic mandibular (10 of 11) and hindlimb (11 of 17) grafts. In addition, signs of nerve fibre degeneration appeared more frequently at the heterotopic sites. On the basis of these findings, and in view of the results of other workers, we conclude that tooth germs are attractive targets for all divisions of the trigeminal nerve and for cutaneous nerves outside the trigeminal system. However, the morphological picture tends to become increasingly abnormal with increasing distance from the normal locus.