Yasunori Sakakura

Ultrastructural evidence for a possible secretory function of Merkel cells in the barbels of a teleost fish, Cyprinus carpio

SummaryExamination of barbels of the carp (Cyprinus carpio) revealed cells showing the characteristics of Merkel cells. Some ultrastructural features of these cells suggest a secretory function.

Migration of Merkel cells in the labial mucous epithelium of adult rabbits following mental nerve resection

SummaryMerkel cells in the lower labial mucosa of adult rabbits were studied electron microscopically, 9, 21, 28, and 50 days after resection of the mental nerves. By day 9, nerve fibers were completely retracted from the epithelial layer of the mucosa. On and after day 21, Merkel cells were located not only in the basal layer but also in the prickle or more superficial cell layers. The ultrastructure of the migrating Merkel cells was unchanged, both as to the amount and location of the specific cored granules in the cytoplasm, until the cells reached the granular cell layer. The position of the migrating Merkel cells differed from cell to cell, and migration continued for at least 50 days. A remarkably large number of immature Merkel cells was observed in the basal and suprabasal cell layers of the denervated epithelium even by day 50. Therefore, the possibility of the reproduction of Merkel cells exists. The migrating Merkel cells, as well as the keratinocytes in the same cell layer, had degenerated drastically in the parakeratinized cell layer. This seems to indicate that the Merkel cells belong to the line of keratinocytes.

The development of mandibular molar tooth germs isografted in the mouse spleen.

Tooth germs taken from 13-day-old embryonic mice and isografted in the spleen of adult mice were examined by light and electron microscopy. Well-organized tooth structures from the early cap stage to fully developed and mineralized mature teeth were obtained up to day 60 after transplantation. Germs on day 2 were similar to those prior to the onset of grafting but reached the late cap stage of development on day 4. On day 6, enamel and dentine formation were initiated and inner enamel epithelium and dental papilla cells were polarized. On days 10-15, enamel-matrix secretion was completed and almost all ameloblasts had become resorptive enamel epithelium. India ink injected from the recipient caudal vein accumulated to the capillaries within the pulp throughout the newly-formed vessels. On day 20, defined root formation had begun but occasionally irregular and cellular osteodentine was formed in root areas. On day 30, transplants were covered with reduced enamel epithelium and acellular cementum was formed at the root areas together with rudimentary periodontal ligament fibres. Cellular cementum became thicker up to day 40. There was little evidence of cellular infiltration from recipient tissue up to day 60. The spleen seems to be a suitable site for transplantation of tooth germs.

Hemopoietic sites and development of eosinophil granulocytes in the loach, Misgurnus anguillicaudatus

SummaryUnique eosinophils, each of which contained only one eosinophilic granule, have been found in the peripheral blood of the loach (itMisgurnus anguillicaudatus). Several loach organs have been studied by light and electron microscopy to determine the hemopoietic site of this cell type. Eosinophils are produced mainly in the spleen and to a small extent in the kidney, but not in other organs.Presumed myeloblasts are identified as large lymphoid cells containing a number of small-dense granules (diameter, 0.12–0.16 μm) in the cytoplasm. These granules have been observed throughout eosinophilopoiesis but they are most abundant in the promyelocyte stage. The largest cells have been identified as myelocytes which contain a number of large granules (diameter, 0.7–1.4 μm) with electron-dense crystalline cores. These large granules are present from the myelocyte to metamyelocyte stage. Metamyelocytes differ from myelocytes in having more large granules. Mature eosinophils are morphologically similar to metamyelocytes but are characterized by the presence of only one very large electron-dense granule (diameter, 2.5–2.8 μm) with a crystalline core.The nature of these granules has been studied by enzyme digestion using pepsin and trypsin. The results indicate that the crystalline cores are almost pure protein.

A new culture method assuring the three-dimensional development of the mouse embryonic molar toothIn vitro

SummaryMandibular first molars from 17-day-old mouse embryos were cultivatedin vitro for 10 days using a new organ culture method. This method consisted of using a small glass dish and an agar chamber to slowly float the molars to the gas-medium interface where they were maintained by the surface tension of the medium. The molars developed three-dimensionally through the use of this method. Five cusps with refractile dental matrix were recognized from the occlusal side. It was easy to determine antero-posterior and bucco-lingual orientation of the molars. Thick sections of the bucco-lingual plane showed the normal cytodifferentiation of ameloblasts and odontoblasts. The thick enamel layer was formed to the extent of the fissure region between cusps. Furthermore, a great quantity of stippled material accumulated between the enamel and the ameloblasts. The stippled material displayed a meshlike fibrilar structure, and at the mineralization front, it seemed that the fibrils of stippled material adjacent to the enamel had been transformed into needlelike enamel crystals. In addition. bundles of the fibrils similar to enamel crystals were found in the stippled material. As the floatation method resulted in the three-dimensional development of the molars and the development of the enamel without the presence of histological disturbances, it was considered superior to other culture methods for the facilitation of orientation and the development of cusps.

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 explants 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 explants.

In vitro Effects of Calcitonin and/or Parathyroid Hormone on Odontogenesis of Mouse Embryonic Molars

Mandibular first molars of mouse embryos were cultivated for examination of the effects of calcitonin (CT) and/or parathyroid hormone (PTH) on the odontogenesis of the molars, and for determination of whether and how CT, which is a PTH antagonist, has an influence on the effect of PTH on odontogenesis. On the second day, the inner enamel epithelium in the control group had already differentiated into pre-ameloblasts. Typical odontoblasts had secreted a layer of pre-dentin. On the fourth day of culture, the pre-ameloblasts achieved terminal differentiation into secretory ameloblasts, and enamel and dentin had already been deposited. PTH (1 unit/mL) inhibited the odontogenesis of the cultured molars during the designated culture periods (two and four days), while CT (0.5 unit/mL) stimulated odontogenesis. On the second day, the development of the molars in the CT + PTH group showed an intermediate stage between the control and PTH-treated explants, but on day 4 it corresponded to that of the controls. Moreover, when the molars exposed to PTH for two days were untreated and treated with CT for an additional two days, the former produced a small yuantity of enamel matrix, while the latter formed a large amount of the matrix. These histological findings were also supported by a morphometric analysis of the enamel matrix in the cultured molars. The present results suggest that CT stimulates, but PTH suppresses, the odontogenesis of the mouse embryonic molars, and that CT is an antagonist to the inhibitory effect of PTH on odontogenesis.

Effects of parathyroid hormone on odontogenesis of the mouse embryonic molar toothIn vitro

SummaryMandibular first molars of 17-day-old mouse embryos were culturedin vitro to examine the histological effects of various concentrations of parathyroid hormone (PTH) on odontogenesis of the molars. PTH did not affect the cytodifferentiation of mesenchymal cells into preodontoblasts but inhibited that of preodontoblasts into odontoblasts. Consequently, the odontoblasts failed to undergo dentinogenesis. On the other hand, inner enamel epithelium achieved terminal cytodifferentiation into secretory ameloblasts and these cells partially formed enamel in spite of the absence of dentin. All treated molars showed the same histological disturbances and these effects were independent of PTH dose. The present study indicated that PTH had an influence on mesenchyme-derived cells, inhibiting both the differentiation of odontoblasts and the formation of predentin and dentin.

A simple, disposable, and improved organ culture system for maintaining three-dimensional development of mouse embryonic molars

SummaryMandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and ligual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.

Influence of 1,25-dihydroxyvitamin D3 on cell proliferation during odontogenesis of the mouse embryonic molarsIn vitro

SummaryMandibular first molars from 17-day-old mouse embryos were cultured for 2 and 4 days in alpha-MEM supplemented with 10% newborn bovine serum containing 0.02% ethanol or 0.1, 1.0 or 10 ng/ml 1,25-(OH)2D3. After embedding, every 6th section was stained and mitotic features of inner dental epithelium (IDE) and dental papilla cells (DP) were counted under a light microscope. On the 2nd day, no significant differences were observed in the IDE and DP mitotic indexes among the control, ethanol, and the three vitamin D groups. On the 4th day, the indexes for all groups decreased. But the IDE indexes for all vitamin D groups were significantly different from those for the control (P<0.01) and ethanol (P<0.05) groups, whereas the DP index was significant (P<0.05) only in the 10 ng/ml vitamin D group. The present results suggest that vitamin D3 may have an influence on cell proliferation of IDE and DP in mouse embryonic molarsinvitro.