Mouse Tooth Germs Research Articles

A Chondroitin Sulfate Epitope in Mammalian Dental Pulp and its Developmental Expression in Mouse Dental Papilla

The molecular specificity of the dental papilla of a bell-stage tooth was studied by production of dental-papilla-reactive monoclonal antibodies (Mabs). One of the Mabs, designated 7C5, recognized an epitope present in glycosaminoglycan. Several lines of evidence suggested that the 7C5-epitope consists of chondroitin 6-sulfate. The Mab did not react with mouse dental epithelium, but reacted uniformly with mesenchymal tissue in the mandibular process and accumulated in the dental sac and in the papilla of bell-stage tooth germs. The 7C5-staining was lost from the differentiating odontoblasts, while the staining in the molar tooth papilla was accumulated in the subodontoblastic layer. In the developing mouse incisor, the 7C5-epitope was restricted to the lingual-posterior area. The 7C5-epitope was also present in pulpal tissue and predentin of different types of teeth of various mammalian species, including man, sheep, swine, and rat. Collagenase pre-treatment of tissue sections abolished the bulk of the 7C5-reactivity in peridental mesenchyme during embryonic stages while leaving the staining of the dental papilla intact. In newborn and adult teeth, collagenase also impaired the reactivity in the pulp except for the subodontoblastic layer. This suggests the existence of different subpopulations of the 7C5-epitope containing proteoglycans in dental papilla and pulp. A high-molecular-weight proteoglycan, sensitive to chondroitinase ABC but not to heparinase or heparitinase, was immunoprecipitated by 7C5 from extracts of bell-stage mouse tooth germs. We suggest that the evolutionary conservation of chondroitin 6-sulfate in the dental pulp reflects its properties as non-terminally differentiated tissue and perhaps the retention of a potential to differentiate to odontoblasts.

Effects of Low Power Laser Irradiation on the Mineralization of Tooth Germ in Mice

The effect of low power laser (diode laser) irradiation on the mineralizationand alkaline phosphatase activity in the tooth germ was examined.Mandibular first molarsfrom 17-day-old mouse embryos were cultured with or without low power laser irradiation (the irradiation time were 0, 1, 3 or 5 minutes per day). The calcium content and alkalinephosphatase activity in the tooth germ increased gradually from 0 to 7 days in culture, theincrease of calcium content being preceded by that of alkaline phosphatase activity. The irradiation by low power laser (5 minutes) increased the alkaline phosphatase activity in thetooth germ cultured for 3 days, without affecting the calcium content. There was no increaseof alkaline phosphatase activity in the tooth germ cultured and irradiated with low powerlaser for 7 days. However the irradiation by low power laser (5 minutes) increased calciumcontent in the tooth germ.These results suggest that low power laser irradiation may accelerate the mineralizationstage of the tooth germ. For evaluating the action on mineralization in the tooth germ, irradiation of 5 minutes per day was optimal. (J. Jpn. Soc. Laser Dent. 4: 27-31, 1993 Reprint requests to Dr. Arakawa)

Effects of Low Power Laser Irradiation on the Mineralization of Tooth Germ in Mice

Recently, much attention has been given to various biological effects to the reaction from low power laser irradiation. Among these effects, it is widely admitted thatwound healing is advanced by low power laser irradiation. In the present study, the effectof low power diode laser irradiation on the growth process of teeth as examined using theorgan culture of tooth germs dissected from 17-day-old mouse embryos. The irradiation of30mW was applied to tooth germs for 1 to 7 days for 5 minutes per day and the effect on thetooth germ was evaluated by biochemical analysis and histological observation.Irradiation for 1, 3, 5 or 7 days did not increase the alkaline phosphatase activity in the tooth germ. Irradiation for 5 or 7 days increased the calcium content in the tooth germ.Histologically, irradiation for 5 days increased the quantity of enamel, irradiation for 7days increased the quantity of enamel and dentine. These results suggest that irradiation oflow power diode laser accelerate the mineralization of the tooth germ. For evaluating theaction on mineralization in the tooth germ, irradiation for 7 days was optimal. (J. Jpn. Soc. Laser Dent. 4: 39-43, 1993 Reprint requests to Dr. Isobe)

P-455 - Effects of calcitonin (CT), 1,25-dihydroxyvitamln D3 (1,25-(OH)2D3) and parathyroid hormone (PTH) on mineralization of tooth germ in mice.

P-455 - Effects of calcitonin (CT), 1,25-dihydroxyvitamln D3 (1,25-(OH)2D3) and parathyroid hormone (PTH) on mineralization of tooth germ in mice.

P-343 - The in vitro effect of caicitonin on the mineralization of tooth germ in mice. -Comparison with that of parathyroid hormone and 1,25-dihydroxyvitamin D3

P-343 - The in vitro effect of caicitonin on the mineralization of tooth germ in mice. -Comparison with that of parathyroid hormone and 1,25-dihydroxyvitamin D3

Effects of ascorbate-deficiency on collagen secretion and resorption in cultured mouse incisor germs.

The effects of ascorbic acid deficiency on mouse incisors, grown in vitro, has been investigated at the histological and cytological levels. In this model, continuously growing mouse incisors are characterized by the existence of different type of predentin-dentin matrix on its lingual (root-analogue) and labial (crown-analogue) surface and the absence of enamel on the lingual surface. Our observations indicated that ascorbate-deficiency affected the behavior of mouse tooth germs in vitro: odontoblast differentiation was disturbed and morphological evidence for odontoblast-mediated collagen resorption were observed. An abnormal amorphous predentin-dentin matrix existed and the basement membrane was prematurely disrupted. The dentin mineralization, as well as functional differentiation of ameloblasts were strongly hampered. Chronic deficiency led to disorganization of the dental tissues.

Chondroitin sulfates in developing mouse tooth germs

Abstract The role of glycosaminoglycans and proteoglyans during ontogenesis is not known. The developing tooth offers a potentially important model for studies of structure-function relationships. In this study, we have analysed the temporal and spatial expression of chondroitins of differing sulfation patterns in embryonic molars and incisors. For this purpose, we have used monoclonal antibodies (Mabs) specific for unsulfated, 4-sulfated, and 6-sulfated forms of chondroitin in conjunction with indirect immuno-fluorescence or immunoperoxidase labeling. Unsulfated chondroitin was not detected in embryonic teeth. Chondroitin 4- and chondroitin 6-sulfates were present in the stellate reticulum but otherwise they were confined to the dental mesenchyme. The 3B3 and MC21C-epitope, which are markers of 6-sulfated chondroitin, were uniformly distributed in the dental mesenchyme during the bud stage; they disappeared from the dental papilla of the cusps and of the anterior region of the incisor as development proceeded. These epitopes were absent from the basement membrane and from the predentin. In the odontoblastic cell lineage, the 3B3 and MC21C-epitopes were detected only between preodontoblasts at an early stage of differentiation. The monoclonal antibody 2B6 served as a probe to localize chondroitin 4-sulfate. This glycosaminoglycan was detected as early as the dental lamina stage but its expression was restricted to the basement membrane of the teeth until the late bell stage. After the onset of cusp formation, strong staining was also observed over the occlusal region of the dental papilla while the cervical region of the dental papilla remained 256-negative. Incisors at the bell stage exhibited a decreasing gradient of immunostaining by 2B6 from their anterior region to their posterior end. The extracellular matrix surrounding preodontoblasts reacted with 2B6 and the predentin, produced by the odontoblasts, was also intensely labeled with this antibody. Comparison between immunostaining with 3B3 and 2B6, on consecutive sections revealed a mutually exclusive pattern of distribution of the corresponding epitopes during odontogenesis. Furthermore, in the continuously growing incisor, a striking positive correlation was found between the immunostaining patterns produced by 3B3 and MC21C and the mitotic indices along the anterior-posterior axis of the tooth. Hence, sulfation of chondroitin seems developmentally regulated. We postulate that changes in the sulfation pattern of chondroitin might play a role in ontogenesis by locally altering the functional properties of the extracellular matrix.

Immunohistochemical study of the relationship between extracellular matrix and root bifurcation in the mouse molar

In order to investigate epithelial-mesenchymal interaction during root bifurcation, the distribution of type III and I collagen, fibronectin and laminin in the epithelial-mesenchymal junction between the dental epithelia (epithelial diaphragm and interradicular process) and the cells of the dental papilla (or pre-odontoblasts) was examined, using maxillary first molar tooth germs of CF1 mice from day 1–16 after birth. Three-dimensional reconstructions of the immunofluorescent patterns were made from serial sections of tooth germs from day 3–9, stained with the antibodies against the collagens. The findings were as follows. (1) Type III collagen was first seen in the epithelial-mesenchymal junction at the tip of the interradicular process, where it sprouted from the epithelial diaphragm, and spread along the interradicular process toward its base, accompanied its extension, and then disappeared on completion of root bifurcation. No staining was seen in the epithelial-mesenchymal junction at the epithelial diaphragm during and after root bifurcation. (2) Type I collagen appeared in the epithelial-mesenchymal junction at the base of the interradicular process, where it sprouted from the epithelial diaphragm and spread toward the tip of the interradicular process, following its extension, and increased on completion of the root bifurcation. No staining was seen in the epithelial-mesenchymal junction at the epithelial diaphragm during or after root bifurcation. (3) Fibronectin and laminin remained constant in the epithelial-mesenchymal junction, both at the interradicular process and the epithelial diaphragm, during and after root bifurcation. These findings suggest that type III collagen may play a significant role in the early stage of root bifurcation in the molar.

Immunohistochemical localization of type IV collagen in mouse tooth germ: an ultrastructural study.

Localization of type IV collagen was analyzed at the ultrastructural level in mouse embryonic molars by using a preembedding technique. Cryostat sections were incubated with type IV collagen antibody and then treated with the peroxidase-antiperoxidase complex. This antibody was visualized at the epithelio-mesenchymal interface. Labeling was intense and uniformly distributed throughout the basement membrane. However, it was mainly restricted to the lamina densa. No immunostaining was detectable in the lamina lucida but it was crossed by fine filaments that appeared as projections from the lamina densa to the epithelial cell plasma membrane. At the mesenchymal aspect of the basement membrane, projections of labeled material extended from the lamina densa in the underlying dental mesenchyme. At the presecretory stage of odontoblasts, these projections were in close connection with mesenchymal cell processes.

Guanine salvage by organ cultures of mouse tooth germs.

The effect of mycophenolic acid (MPA) which inhibits the biosynthesis of guanosine monophosphate (GMP) in organ cultures of mouse tooth germs can be partially counteracted by adding guanine to the MPA cultures. This may be due to salvaging guanine by the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), or to competition for a common membrane carrier involved in mediated transport of both guanine and hypoxanthine in normal biosynthesis and also of MPA. Experiments were carried out to compare the effect of either hypoxanthine or guanine on the MPA-caused inhibition. While addition of guanine to the MPA cultures (MPAG) supports growth equal to controls and development of dental-enamel junction (DEJ) to a level intermediate between control and MPA the addition of hypoxanthine (MPAHX) supports growth and DEJ development not better than MPA. This indicates that guanine is salvaged by HGPRT to GMP while hypoxanthine, salvaged to inosinic acid (inosinic monophosphate, IMP) is ineffective because the MPA inhibition is on the pathway from IMP to GMP.

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.

Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth

Although local epithelial-mesenchymal tissue interactions which are presumably mediated by extracellular matrix molecules are important regulators of tooth morphogenesis and differentiation, our studies have indicated that these developmental processes also depend on circulating molecules. The iron-carrying serum protein transferrin is necessary for the early morphogenesis of mouse tooth in organ culture ( A-M. Partanen, I. Thesleff, and P. Ekblom, 1984 Differentiation 27, 59–66 ). In the present study we have examined the effects of other growth factors on mouse tooth germs grown in a chemically defined medium containing transferrin. Fibroblast growth factor and platelet derived growth factor had no detectable effects but epidermal growth factor (EGF) inhibited dramatically the morphogenesis of teeth, and prevented odontoblast and ameloblast cell differentiation. EGF stimulated cell proliferation in the explants measured as [ 3H]thymidine incorporation in DNA. However, when the distribution of dividing cells was visualized in autoradiographs, it was observed that cell proliferation was stimulated in the dental epithelium but was inhibited in the dental mesenchyme. The inhibition of cell proliferation in the dental mesenchyme apparently caused the inhibition of morphogenesis. We do not know whether the dental epithelium or mesenchyme was the primary target for the action of EGF in the inhibition of morphogenesis. It is, however, apparent that the response of the dental mesenchymal cells to EGF (inhibition of proliferation) is regulated by their local environment, since EGF enhanced proliferation when these cells were disaggregated and cultured as monolayers. This indicates that the organ culture system where the various embryonic cell lineages are maintained in their original environment corresponds better to the in vivosituation when the roles of exogenous growth factors during development are examined.

Ultrastructure of the effects of calcitonin on the development of mouse tooth germs in vitro

Mandibular first molars, from 17-day-old embryos, were cultivated in control medium or medium containing 0.1, 0.01 or 0.001 unit/ml of calcitonin (CT) for periods up to 10 days. In untreated tooth germs, cells of the dental papilla differentiated into pre-odontoblasts up to 4 days and predentine was seen on day 6. Cells treated with 0.1 unit/ml of CT differentiated into pre-odontoblasts up to 4 days, but no predentine was formed even after 10 days in culture. With 0.01 unit/ml, cells differentiated into odontoblasts, and had already secreted predentine a few days earlier than the untreated group. With 0.001 unit/ml, the developing germs were similar to the control expiants during the entire 10-day cultivation period. The proportional area of rough endoplasmic reticulum to cytoplasm of the odontoblasts was low at 0.1 unit/ml of CT and high at 0.01 unit/ml compared to the untreated expiants.

Glycosaminoglycans in Embryonic Mouse Teeth and the Dissociated Dental Constituents

The nature, amounts, and distribution of glycos-aminoglycans (GAG) before and during odontoblast terminal differentiation were studied. GAG have been isolated from intact mouse tooth germs and from dissociated dental epithelia and dental papillae after labeling with [ 3H] glucos-amine or 35SO 4 2− as precursor. The kinds and relative amounts of 3H-labeled GAG were analyzed by chromatography on a DEAE-cellulose column and cellulose thin-layer sheets. The amounts of individual GAG relative to total GAG were determined from the elution profiles, whereas their nature was identified by the selective removal of chromatographic peaks after enzymatic or chemical degradation. We found hyaluronate and probably a minute quantity of heparan sulfate in the dental epithelium, while hyaluronate, heparan sulfate, and chondroitin sulfate were the main types of GAG in the dental papilla. The chondroitin sulfate recovered was further fractionated by cellulose thin-layer chromatography into two isomers, namely chondroitin-4-sulfate (the major component) and chondroitin-6-sulfate. Changes in the elution profile from DEAE-cellulose chromatography of tooth GAG extracted from different developmental stages suggest that modifications of GAG occur during odontogenesis. Alcian blue staining localized large amounts of hyaluronate and sulfated GAG along the epithelio-mesenchymal junction. Tissue specificity and changing patterns of GAG were demonstrated during odontogenesis.

Autoradiographic visualization of glycoproteins and glycosaminoglycans in the epithelio-mesenchymal interface of developing mouse tooth germ.

The turnover of basement membrane macromolecules during tooth morphogenesis and odontoblast differentiation was examined by light microscopic autoradiography using 3H-fucose, 35S-sulfate and 3H-glucosamine. Marked incorporation into the basement membrane was found throughout the progressive development. Pulse-chase experiments and prelabeling of tissue components indicated that glycoproteins and glycosaminoglycans in the dental basement membrane are mainly derived from the enamel epithelium. During odontoblast differentiation, incorporation was increased at the epitheliomesenchymal interface at the site of differentiating mesenchymal cells. From these sites the label also disappeared rapidly. This suggests that the active remodeling of extracellular matrix is related to odontoblast differentiation.

Diazo-oxo-norleucine (DON)-induced alterations in the extracellular matrix of the mouse tooth germ

In a previous study we have shown that the glutamine analogue diazo-oxo-norleucine (DON) inhibits the differentiation of mesenchymal cells into odontoblasts in the developing tooth. In the present study we have studied the effect of DON on the formation of the extracellular matrix by using light microscopic autoradiography and transmission electron microscopy. The inhibition of odontoblast differentiation was accompanied by alterations in the extracellular matrix at the epithelio-mesenchymal interface. The interface was reduced in space and filled with filamentous material, and the organization of collagen fibers in more advanced tooth germs was disturbed. DON also reduced the incorporation of [ 35S]sulphate into the basement membrane region, whereas no marked change was observed in the incorporation of [ 3H]fucose. These results suggest that DON affected the cell—matrix interaction which is believed to control the differentiation of odontoblast.

Experimental induction of cementogenesis on the enamel of transplanted mouse tooth germs

First and second maxillary molar tooth germs with their surrounding bone were removed from 9-day-old mice, freed of the reduced enamel epithelium, re-inserted crown downwards in their bony crypts and then transplanted in the subcutaneous tissue of hosts of the same age and litter. Grafts were removed 14 days later and prepared for light and electron microscopy. In the areas where the reduced enamel epithelium was missing, a layer of cemen-tum-like tissue was present on the enamel surface, always associated with cells showing the typical features of cementoblasts. A thin electron-lucent layer of fine fibrillar material separated the enamel surface from the new hard tissue which was composed of densely-packed collagen mixed with a ground substance. Where the cementum-like tissue was thick, cells were trapped in the collagenous matrix. This cementogenesis on enamel was strictly dependent on the absence of the reduced enamel epithelium. Thus, when exposed to follicular tissue, the surface of immature enamel appears to exert an influence on follicular cells and stimulate cementogenesis. This hypothesis could explain the presence of overgrowths of cementum in the cervical region of tooth crowns where the reduced enamel epithelium may be particularly vulnerable.

Collagen type I trimer synthesis by cultured embryonic mouse molars.

Embryonic mouse tooth germs were cultured in vitro and the collagens type I, type III and type I trimer were purified and biochemically characterized. Collagen type I trimer has been identified by means of CM-cellulose chromatography, CNBr peptide analysis, pepsin resistance and molecular sieve chromatography. Already before the odontoblasts were functional, this molecule was found to be a constituent of the dental extracellular matrix. However, the synthesis of collagen type I trimer was considerably increased when odontoblasts polarized and became functional. the incorporation of 5-bromodeoxyuridine into the dental cells inhibited the polarization of odontoblasts as well as the amplification of collagen type I and type I trimer synthesis.

Developmental arrest of mouse tooth germs in vitro by 6-diazo-5-oxonorleucine (DON).

Molars and incisors were excised from the mandibles of 15-day-old mouse embryos and explanted to agar-solidified Eagle's basal medium with 10% fetal calf serum and to medium containing 6-diazo-5-oxonorleucine (DON), a glutamine analog which inhibits glycoprotein and glycosaminoglycan synthesis. The concentration of DON ranged from 5-100 microgram/ml; the lowest dose at which consistent results were obtained was 75 microgram/ml. At the end of a 4-day culture period, controls displayed characteristic incisiform or molariform morphologies and possessed odontoblasts and preameloblasts. The growth and development of DON-treated germs, however, was arrested and such germs remained morphologically and histologically similar to the developmental state attained at the time of explantation. DON-treated germs also contained less abundant Alcian blue staining material than controls. Development arrest by DON was prevented by addition of glutamine but not by glucosamine or the purine analog amino imidazol carboximine. Suppressed germs resumed normal development when removed from medium containing 75 microgram/ml of DON to control medium. These results suggest that glycoprotein or glycosaminoglycans play a role in epithelial-mesenchymal inductive interactions.

Ultrastructural studies of effects of proline analogue L-azetidine-2-carboxylic acid on in vitro development of mouse tooth germ

The effects of L-azetidine (an analogue of proline) on the development of mouse tooth germs cultivated in vitro were studied. Tooth germs removed on the 18 th day were cultivated in the presence of 50, 100 or 150μg/0.5ml L-azetidine. Light microscopically, after 4 days of culture in the presence of L-azetidine, the growth of tooth germs was clearly inhibited, while predentine was almost non-existent. The suppressed tooth germs which were removed from the medium containing L-azetidine and placed in a control medium for 2 days resumed development. Electron microscopically, at 50μg/0.5ml L-azetidine, odontoblasts showed the reduction and vesiculation of r-ER and many lysosomal granules containing abnormal fibers which were about a quarter of SLS in length appeared in the cytoplasms of odontoblasts. At 100 or 150μg/0.5ml Lazetidine, following the prominent increase of cytosegresomes and a moderate or severe loss of cytoplasmic organelles in the cytoplasms, the odontoblasts became gradually small in size. Also, the inner enamel epithelial cells, which had been in the progressive stage of the differentiation to ameloblasts, displayed regression and disorganization. After being removed from the medium containing L-azetidine, the inhibited odontoblasts in a control medium for 2 days showed varying degrees of differentiation, and the inner enamel epithelium once again began to be polarized to come ameloblasts. These results suggest that L-azetidine has reversible inhibitory effects on the cytodifferentiation and function of odontoblasts. The relationship between cytodifferentiation and the function of odontoblasts, and the development of tooth germs are discussed in some detail.