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PubMed1999

Immunohistochemical analysis of bcl-2 family proteins in benign and malignant ameloblastomas.

To study the possible roles of bcl-2 family proteins in oncogenesis and cytodifferentiation of odontogenic epithelium, the expression of bcl-2, bcl-x and bax proteins was examined immunohistochemically in tooth germs and various types of ameloblastoma. In ameloblastomas, bcl-2 and bcl-x proteins were expressed intensely in peripheral columnar cells and weakly in central polyhedral cells, while bax protein reactivity was low. In variants of ameloblastoma, no expression of these proteins was found in the keratinizing areas or granular cell clusters. Basal cell ameloblastomas showed intense reactivity for bcl-2 and bcl-x proteins, but slight or no reactivity for bax protein. These results indicate that in ameloblastomas the bcl-2 family proteins might function primarily as anti-apoptotic factors, reflecting proliferative activity. Malignant ameloblastomas showed decreased reactivity for bcl-2 protein and increased levels of bax protein as compared with benign ones.

Ameloblastoma↗
PubMed1980

Ultrastructure of the human enamel organ. II. Internal enamel epithelium, preameloblasts, and secretory ameloblasts.

The fine structure of internal enamel epithelium, preameloblasts and secretory ameloblasts in primary tooth germs (bell stage) from four human foetuses was investigated. The characteristics of the differentiation of internal enamel epithelium via preameloblasts to secretory ameloblasts are described. The internal enamel epithelium consists of a row of low differentiated prismatic cells separated from the dental papilla by a distinct even basal lamina. In the preameloblasts the rough endoplasmic reticulum cisterns and mitochondria increase in number, the Golgi complexes become extensive and take up a distal position, and secretory granules are formed. Furthermore, the basal lamina is removed by coated vesicles, and proximally and distally in the cells a complex of zonulae adhaerentes, terminal webs and gap junctions is formed. The secretory ameloblasts make up a layer of highly differentiated cells demonstrating typical merocrine secretion.

Ameloblasts↗
PubMed1975

[Transfer of 45Ca during amelogenesis studied by autoradiography and electron microscopy].

45Ca transfer through the stratum intermedium and the secreting ameloblasts towards enamel has been studied by quantitative electron microscopical autoradiography in tooth-germs of newborn cats following intravenous injection of the isotope. Two transfer pathways were demonstrated. The relatively more important direct path passed through the stratum intermedium and ameloblast intercellular spaces and reached the enamel directly. The second pathway consisted in an intracellular transfer through the ameloblasts. 45Ca penetrated the cell through its basal pole. The mitochondria were the most highly labeled organelles at the different experimental time intervals studied. A maximum of radioactivity was respectively noticed at 30 min and 1 h in the endoplasmic reticulum and the Golgi apparatus. A total absence of silver grains was noted over the secretory ameloblastic bodies. At 6 h, the highest labeling was observed over enamel and especailly over the inner enamel along the enameldentin junction.

Ameloblasts↗
PubMed2006

Inhibition of apoptosis in early tooth development alters tooth shape and size.

Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Dlx and other homeobox genes in the morphological development of the dentition.

The dentition is a segmental system whose evolution and morphology bears analogy to the evolution of segmentation in the vertebral column and limb. Combinatorial expression of members of the large "Hox" class of homeobox regulatory genes has been shown to play an important role in positional specification in these skeletal systems. This raises the possibility that homeobox genes are also used for positional specification in the dentition, and several homeobox genes are known to be expressed in developing teeth. To identify additional dentally expressed homeobox genes, cDNA from from murine tooth germs at 9.5, 14.5, and 17.5 days gestational age was amplified by PCR using sets of degenerate primers to the homeodomains of 18 different classes of homeobox genes. Amplification products were cloned and sequenced and compared to known gene sequences. To date this approach has confirmed the presence of Msx1, Msx2, Dlx1, and Dlx2, and identified several other homeobox genes not previously known to be expressed in teeth: Dbx, MHox, and Mox2A, plus an a additional Dlx gene, Dlx7. The Msx and Dlx genes are the best current candidates for a combinatorial mechanism that controls the differentiation of structures within and between teeth, and perhaps also the evolution of those structures.

Amino Acid Sequence↗
PubMed1994

Cholesterol in the distal portions of differentiating and fully differentiated rat odontoblasts observed by freeze-fracture.

Freeze-fracture replicas of rat molar tooth germs in conjunction with the 3-beta-hydroxysterol marker filipin were used to study the distribution of cholesterol in the distal portions of odontoblast plasma membrane. Filipin-sterol deformations (interactions) appeared as clusters interspersed with deformation-free regions on the distal plasma membrane of early differentiating odontoblasts. In fully differentiated odontoblasts, the whole distal membrane, including the process, was occupied by packed deformations with no free regions. It seems that early developing odontoblasts are poorer in cholesterol than the fully differentiated cells. As the content and/or distribution of 3-beta-hydroxysterols (cholesterol) is known to influence membrane fluidity, the low cholesterol content of early differentiating odontoblasts might be related to the fluidity required for the budding off of matrix vesicles.

Animals↗
PubMed1997

Immunohistochemical demonstration of bcl-2 protein in ameloblastoma.

The bcl-2 gene is known through its product, bcl-2 protein, which prevents apoptosis, and also by its expression in development, especially in sites characterized by epithelial-mesenchymal interactions. In the developing tooth germ, bcl-2-protein is expressed in the epithelial component. In this study, we examined the expression of bcl-2 protein immunohistochemically in 25 ameloblastomas using a monoclonal antibody against anti-human bcl-2 oncoprotein. In all 25 cases, bcl-2 protein was found mainly in the outer layer of tumor cells, whereas the inner cells (stellate reticulum-like cells and squamoid cells) were negative. The bcl-2 protein is therefore thought to play a role in maintaining the stem-cell population in the peripheral layers of the tumor nests from which proliferating cells can be recruited.

Adolescent↗
PubMed2002

Amelogenin gene expression in porcine odontoblasts.

Amelogenin is the major organic component in the enamel matrix of developing teeth and plays an important role in enamel biomineralization. Amelogenin has been reported to be a specific secretory product of ameloblasts. In this study, we examined amelogenin gene expression in various cell layers prepared from a porcine permanent tooth germ using reverse transcription-polymerase chain-reaction (RT-PCR). Amelogenin amplification products were detected only in the secretory ameloblast layer after 20 cycles of PCR. After 30 cycles of PCR, amelogenin amplification products were detected in secretory and maturation-stage ameloblasts and in odontoblasts. The relative levels of amelogenin gene expression in secretory and maturation-stage ameloblasts and odontoblasts were determined. Secretory ameloblasts expressed over 1000 times the level of amelogenin mRNA found in odontoblasts. Amelogenin gene expression in odontoblasts was confirmed in an erupted porcine permanent first molar, which has no ameloblasts. Amelogenin PCR amplification products were identified from 4 different alternatively spliced transcripts in the ameloblast samples, and the same spliced forms were detected in the odontoblast samples.

Ameloblasts↗
PubMed2003

Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130.

The functions of p107 and p130, members of the retinoblastoma family, include the control of cell cycle progression and differentiation in several tissues. Our previous studies suggested a role for p107 and p130 in keratinocyte differentiation in vitro. We now extend these data using knockout animal models. We found impaired terminal differentiation in the interfollicular keratinocytes of p107/p130-double-null mice epidermis. In addition, we observed a decreased number of hair follicles and a clear developmental delay in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice showed altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Besides normal hair formation, follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, were also observed in the p107/p130 skin transplants. Finally, the hair abnormalities in p107/p130-null skin were associated with altered Bmp4-dependent signaling including decreased DeltaNp63 expression. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions.

Animals↗
PubMed1986

Cell-matrix interactions: influence of noncollagenous proteins from dentin on cultured dental cells.

Matrix-mediated epitheliomesenchymal interactions control dental cytodifferentiations. Experiments were performed in order to study the effects of noncollagenous proteins extracted from dentin on cultured enamel organs and dental papillae. Seven noncollagenous protein fractions were prepared from rabbit incisor dentin and used as substrates to coat Millipore filters. Embryonic mouse tooth germs were dissociated and the isolated tissues were cultured for 4 days on these different substrates as well as on noncoated Millipore filters. When compared to control cultures, only two protein fractions affected the behaviour of epithelial cells. A slight elongation of the cell body and a preferential localization of the nuclei at the basal pole of the cells in contact with the filter was observed with protein fractions 5 and 6. When dental papillae were cultured on Millipore filters coated either with protein fraction 2 or fraction 6, the mesenchymal cells in contact with the filter elongated, polarized and demonstrated a high metabolic activity. Such modifications in the cell organization, implying changes in the cytoskeleton organization and, or, activity, never occurred spontaneously or in the presence of isolated collagens (I-V), laminin or fibronectin.

Amino Acids↗
PubMed1995

Molecular analysis of rat dentin sialoprotein.

Dentin sialoprotein (DSP) is a noncollagenous protein originally isolated from rat dentin. Because it is made by odontoblasts that are actively synthesizing dentin. DSP may play an important role in dentinogenesis. We have isolated a full length DSP cDNA from a rat odontoblast/dental pulp cDNA library (Ritchie et al. [1994] J. Biol. Chem. 269:3698-3702) which codes for a 17 residue signal peptide and a 366 residue, 53 kDa mature protein. In situ hybridization revealed DSP mRNA expression by odontoblasts, but no other cells, in jaws from newborn rat. Northern analysis of various rat tissues demonstrated the presence of DSP transcripts in newborn tooth germs and 21 day old rat incisors. Moreover, multiple transcripts of 4.6 kb and 1.5 kb were found in these two tissues. To better understand the origin of these DSP mRNA multiple transcripts, we have isolated two rat genomic clones. Digestion of each clone with EcoRI followed by Southern analysis revealed that DSP cDNA hybridized to a 4 kb fragment in a lambda dash clone and to a 6 kb fragment in a cosmid clone. Since DSP cDNA hybridized to a 6 kb EcoRI fragment and a 4 kb EcoRI fragment obtained from a rat liver genomic cDNA digested with EcoRI, the multiple DSP mRNA transcripts are most likely derived from two related DSP genes which coexist in the rat genome.

Animals↗
PubMed2000

Ultrastructural preservation of rat embryonic dental tissues after rapid fixation and dehydration under microwave irradiation.

Adequate preservation of the cells and matrix of mineralising tissues remains difficult, as organic components and initial mineral deposits may be lost during conventional processing for electron microscopy. In this study, we have reduced significantly the processing time using microwave irradiation. Rat molar tooth germs were fixed in 4% glutaraldehyde + 4% formaldehyde with 0.1 M sodium cacodylate in a laboratory microwave oven for two periods of 20 s with a maximal temperature of 37 degrees C. After conventional washing and post-fixation, specimens were dehydrated in graded ethanols under microwave irradiation for a total of 7 min 20 s. For comparison, some specimens were processed by conventional methods. After embedding, ultrathin sections were examined by electron microscopy. In differentiating ameloblasts and odontoblasts, plasma membranes, mitochondria, rough endoplasmic reticulum, the Golgi complex, together with all other cytoplasmic organelles exhibited excellent preservation. Microtubules, microfilaments and coated vesicles were particularly evident. Crystal-like mineral deposits were conspicuously present in relation to dentine matrix vesicles and collagen fibrils as well as in enamel matrix. The matrix of forming enamel had a globular electron-lucent appearance. It is concluded that this is a rapid method which provides a preserved or even improved morphology.

Ameloblasts↗
PubMed2001

The distribution of retinaldehyde dehydrogenase-2 in rat and human orodental tissues.

Retinoic acid is an important signalling molecule in embryological development and continues to be important in the adult animal because it modulates growth and differentiation in many epithelial tissues. The distribution of the enzyme retinaldehyde dehydrogenase-2 (RALDH 2), which is involved in the synthesis of retinoic acid, was studied using immunocytochemical techniques in: (1) the developing orodental region of rats aged between 15 days in utero and 6 months; and (2) in archival human autopsy material consisting of abdominal skin and mucosa from various regions of the mouth. In developing tooth germs, RALDH 2 was absent in the enamel organ and dental papilla, its presence only being noted at the periphery of the dental follicle adjacent to parts of the developing alveolar crypt. In adult teeth, the presence of RALDH 2 was limited to blood vessels in the periodontal ligament. In embryos, the connective tissue beneath the nasal epithelium and the meninges stained strongly positively for RALDH 2, as did the connective tissue beneath nasal epithelium in an adult rat. Both keratinized and non-keratinized human oral epithelia and abdominal skin stained positively for RALDH 2. Staining was present throughout the stratified epithelium, except in the keratinized layer and in the basal layer associated with the dorsal surface of the tongue. In addition, the adnexia as well as the ductal lining of mucous glands stained positively for RALDH 2.

Adult↗
PubMed2006

Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats.

Newborn rats were treated with sodium alendronate to study how enamel is formed and the effect of alendronate during early odontogenesis. Ultrastructural analysis combined with high-resolution immunocytochemistry for amelogenin was carried out. Twelve rats were subjected to daily SC injections of sodium alendronate (2.5 mg/kg/day) for 3 days on their dorsal region, whereas three rats were daily injected with saline solution as a control. Molar tooth germs from 3-day-old rats were fixed under microwave irradiation in 0.1% glutaraldehyde + 4% formaldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. The specimens were left undecalcified, postfixed with osmium tetroxide, dehydrated, and embedded in LR White resin. Ultrathin sections were incubated with a chicken anti-24-kDa rat amelogenin antibody, a secondary antibody, and finally with a protein A-gold complex. Large patches of amelogenin were present over the unmineralized mantle dentin and at early secretory ameloblasts. At more advanced stages, they were also detected at the enamel matrix, as well as in the mineralized dentin, at the periodontoblastic space of the dentinal tubules, and at the predentin. It is likely that the main effect of alendronate at early stages of odontogenesis is the increase of synthesis/secretion of amelogenin, promoting its deposition within the forming dentin and enamel.

Alendronate↗
PubMed1999

Ultrastructural verification of anchoring role of lamina fibroreticularis of dental basement membrane in odontogenesis.

In a previous study of the developing tooth a characteristic fibrillar layer associated with the basement membrane of the inner enamel epithelium was found to be a highly specialized lamina fibroreticularis of the basement membrane which is unusually rich in basotubules, 10 nm wide microfibril-like structures. In this study this layer was further examined in detail in the hope of ultrastructurally elucidating its role in odontogenesis. Tooth germs of the monkey (Macaca fuscata) were processed for thin section observations. Dental papilla cell processes were inserted into the lamina fibroreticularis and their surface was closely associated with numerous parallel basotubules. With high-resolution observations the space between the surface and nearest basotubules as well as the spaces between neighbouring basotubules were bridged by 1.5-3 nm wide filaments running perpendicular to the axis of basotubules. These results indicate that the processes of dental papilla cells are linked to groups of basotubules by means of 1.5-3 nm wide filaments. Immunoperoxidase staining showed the presence of fibronectin along basotubules as well as within the space between the process and basotubule. This result, together with the comparison of these filaments with microfibril-associated 1.2-3 nm wide fibronectin filaments and the reported abundance of fibronectin in the basement membrane area during odontogenesis, indicates that these 1.5-3 nm wide filaments are composed of fibronectin. After immunostaining for amyloid P component, done with the rat tissue because of the nature of an available antiserum, basotubules in the lamina fibroreticularis were positively stained, as has been shown in basotubules/microfibrils in other locations. Microfibrils function as anchoring rods by interlinking connective tissue components to one another and to the cells. Basotubules, thought to be basement membrane-incorporated microfibrils, in the lamina fibroreticularis in this study are also likely to function as an anchoring device to immobilize dental papilla cells along the basement membrane. Such an arrangement of mesenchymal cells is known to be crucial for the successful differentiation of odontoblasts in the developing tooth.

Animals↗
PubMed1980

Ultrastructure of the ameloblast-stratum intermedium border during ameloblast differentiation.

The molar tooth germs of newborn mice were isolated and the zone of ameloblast differentiation studied with the electron microscope. Special attention was paid to the changes that occur in the contacts such as desmosomes, tight junctions and close attachments associated with cell interdigitations between the inner enamel epithelium (IEE) and the stratum intermedium (SI) cells from the beginning of differentiation to the onset of enamel secretion. These changes are accompanied by variations in the width and configuration of the intercellular space separating the two cell layers. The terminal web persists from the beginning of cell elongation to the appearance of Tomes's process. Through its attachment to the zonulae adherentes, a terminal web-lateral junction system is created which stabilizes the differentiating ameloblasts. Also the SI cell layer is interpreted as a stabilizer for the IEE layer, particularly during the differentiation phase. This interpretation does not conflict with information that the SI cells transport materials required for enamel secretion.

Ameloblasts↗
PubMed1989

Morphological evidence of the formation of intracellular collagen fibrils in the embryonic mouse molar odontoblasts induced by colchicine administration.

The effects of colchicine on collagen formation were examined ultrastructurally using secretory odontoblasts in mouse molar tooth germs isografted to the spleen for 1 week. Colchicine in concentrations of 0.025 or 0.05 mg/0.1 ml was injected intravenously 12-24 h prior to harvesting. Colchicine induced the disruption of the Golgi apparatus and caused the accumulation of various types of Golgi-associated vacuoles containing collagenous fibrillar structures. Many vacuoles containing fine particles, nonstriated parallel filaments, banding patterns with a periodicity of approximately 63-nm intervals, and occasionally segment-long-spacing-like assemblies were aggregated in the cytoplasm during the experimental period. These morphological changes in vacuole contents may reflect the initial steps for polymerization of the intracellular collagen fibrils. The majority of the aggregated vacuoles were degraded by fusion with lysosomes but banded filamentous material in some vacuoles appeared to polymerize into the collagen fibrils with native structures. These results suggested that in unsecreted vacuoles accumulated in the odontoblasts as a result of colchicine administration the polymerization of collagen fibrils with native structures can occur.

Animals↗
PubMed1986

Correlated observations and analysis of maturation-ameloblast morphology and enamel mineralization.

A combined HCl-collagenase digestion technique and scanning electron microscopy were used to isolate the enamel organ and to confirm the presence of maturation ameloblasts of both ruffle-ended (RA) and smooth-ended (SA) types on maturing enamel in kitten permanent tooth germs. EDTA perfusion of animals fixed with aldehyde produced two or three belt-like shallow grooves (from 30 to 100 micron wide) running horizontally through the maturing enamel surface, coinciding closely with the SA distribution pattern. In animals that had been perfusion-fixed with unbuffered osmium tetroxide containing 2.5% potassium pyroantimonate, SEM-EDX analysis detected K in a superficial enamel layer overlaid by the SA layer. Potassium concentration decreased gradually toward the deeper layers. Very little K penetrated the enamel under the RA layer. Energy-dispersive x-ray analysis of Ca and P concentrations in the enamel revealed an even distribution of these elements throughout the superficial layer of maturing enamel. These results suggest that the SA layer forms an access route for K and EDTA and that, in spite of the obvious morphological and functional differences between RA and SA, the maturing enamel surfaces overlaid by these two cell types show similar degrees of mineralization.

Ameloblasts↗