Tokio Nawa

Histochemical and immunohistochemical analysis of the mechanism of calcification of Meckel's cartilage during mandible development in rodents

It is widely accepted that Meckels cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckels cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckels cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckels cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckels cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral‐type mechanism of calcification.

Mouse Meckel's cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture

We reported that when Meckels cartilage was transplanted ectopically, chondrocytes transformed into osteocyte‐like cells accompanying the extracellular calcified matrix. However, we could not determine whether the osteocyte‐like cells were derived from host tissues or from Meckels cartilage itself. Therefore, we examined whether the Meckels cartilage chondrocytes, which have a retrogressive ultimate fate, are capable of inducing the observed calcification and further transform into osteocyte‐like cells in culture.

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.

Ultrastructural localization of calcium in mechanoreceptors of the oral mucosa

SummaryCytochemical localization of Ca2+ in Meissner corpuscles and Merkel cell-neurite complexes in the palatine mucosa of the Mongolian gerbil was studied by a combined oxalate antimonate-microwave irradiation procedure. The reaction products obtained were identified as calcium antimonate by EGTA solubility and X-ray microanalysis. Meissner corpuscles in the normal palatine rugae could be roughly classified into three types by amount and localization of Ca2+. Type I corpuscles were characterized by a high Ca2+ content in both the terminal axoplasm and caveolae of the lamellar plates, type II, by a low Ca2+ content in the terminal axoplasm and a high Ca2+ content in the lamellar cytoplasm. Type III corpuscles showed intermediate characteristics. Palatine rugae stimulated mechanically during fixation contained an increased number of type I corpuscles. On the other hand, two patterns were distinguished in the distribution of Ca2+ in Merkel cells in palatine rugae fixed under normal conditions. One showed abundant Ca2+ dispersed throughout the cell, while in the other, Ca2+ was specifically localized in the Golgi apparatus and mitochondria. Similar distribution patterns also were observed in palatine rugae that had received mechanical stimulus during fixation. Axon terminals of most Merkel cell-neurite complexes in normal palatine rugae were poor in axoplasmic Ca2+, whereas those in most Merkel cell-neurite complexes in mechanically stimulated palatine rugae contained abundant Ca2+ in their axoplasm.

Formation of the sphenomandibular ligament by Meckel's cartilage in the mouse: possible involvement of epidermal growth factor as revealed by studies in vivo and in vitro.

Abstract. In mammals, the midportion of the soft tissue of Meckels cartilage at the degenerating stage forms a ligament known as the sphenomandibular ligament. To clarify the mechanism of formation of this ligament by Meckels cartilage in mouse, we examined the effects of epidermal growth factor (EGF) on the chondrocytes in terms of the proliferation and differentiation of cells and calcification of the matrix in vivo and in vitro. The effects of EGF were examined by immunohistochemical staining, with EGF-soaked beads, by electron microscopy, and by general histochemical analysis of proteoglycans and calcification. Analysis of labeling with bromodeoxyuridine (BrdU) and the rate of cell growth revealed that EGF enhanced DNA synthesis and the proliferation of Meckels chondrocytes. Histological findings in organ culture and in cell culture, with and without the application of EGF-soaked beads, revealed that EGF inhibited the differentiation of cells to chondrocytes and induced phenotypic changes in fibroblastic cells. The inhibition of alkaline phosphatase activity that resulted from exposure to EGF was accompanied by prolonged calcification of the matrix. Whole-mount staining revealed that subcutaneous injection of EGF enhanced the disappearance of Meckels cartilage. Our results suggest a possible mechanism whereby the midportion of Meckels cartilage remains uncalcified and is rapidly transformed into the sphenomandibular ligament.

Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression.

We examined whether Meckels cartilage of embryonic mice, 17 days in utero, undergo the cellular transformation into the osteocyte-like phenotype under organ culture conditions. Explants were grown by our original pithole method modified Trowell-type cultures for up to 4 weeks at 37° C under 5% CO2 in air. Specimens were examined using histological procedures including immunostaining and electron microscopy. In addition, the effects of β-glycerophosphate on matrix calcification were also examined in cultures with or without β-glycerophosphate. Addition of β-glycerophosphate induced calcification at a higher level, but calcium mineral deposition occurred regardless of the addition of β-glycerophosphate to the culture medium. Light and electron microscopic analyses showed that freshly isolated chondrocytes prior to cell culture had typical hypertrophic morphology, but shortly after commencement of culture, they showed morphological modifications. The cells showing chondrocytic phenotypes became basophilic elliptical cells, and eventually transformed into flattened osteocyte-like cells. Bone-like features for cellular elements were characterized by spindle-shaped cells with elongated processes accompanying bone-specific thickbanded collagen fibrils. Immunostaining showed that at 2 weeks in culture, type I and type II collagens coexisted in the matrix, but subsequently type II collagen synthesis ceased and was replaced by type I collagen synthesis. Immunofluorescent labeling for osteocalcin was noted first in the peripheral cells by 1 week, but at 3 weeks this reaction spread to the central zone in explants. Alkaline phosphatase activity (ALPase) was expressed on the cells in the central zone prior to calcium mineral deposition as shown by von Kossas reaction at 3 weeks in culture. These results showed that Meckels cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression.

Immunocytochemical expression of type I and type II collagens by rat Meckel’s chondrocytes in culture during phenotypic transformation

In culture, chondrocytes of Meckels cartilage can differentiate further to become bone-type collagen-synthesizing cells. Here, the replacement of type II collagen by type I collagen, accompanying expression of the osteocytic phenotype, was analysed by double immunofluorescence staining, histochemistry and electron microscopy. After 1 week in culture, formation of a toluidine blue-positive matrix, demonstrating the synthesis of cartilaginous proteoglycans, and the expression of type II collagen were detected. After 2 weeks, immunoreactivity specific for type II collagen was detected along the cartilaginous areas of the nodules, and type I collagen appeared in association with the immunopositive extracellular matrix around spindle-shaped cells. Electron microscopy revealed that the extracellular matrix at this stage was composed of homogeneous fine fibrils of type II collagen and thick cross-banded bundles of type I collagen: there was also continuity between the type I and II collagens. Double immunofluorescence staining of 3 week-old cultures revealed that type II collagen had been replaced by type I which was synthesized by small round cells that appeared at the top of the nodules. With further passage of time in culture, the distribution of type I collagen expanded further towards the peripheral areas from the central areas of the nodules. The present combination of ultrastructural analysis and double immunofluorescence staining shows that the transition from synthesis of cartilage-specific type II collagen to expression of type I collagen occurred sequentially in spindle-shaped cells located at the top of nodules in conjunction with the further differentiation of Meckels cartilage cells.

Expression of osteopontin in Meckel's cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis

Abstract The localization of osteopontin (OP) was examined in Meckel’s cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix.

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.

Postnatal differentiation of Merkel cells in the rat palatine mucosa, with special reference to the timing of peripheral nerve development and the potency of cell mitosis.

The origin and mechanism of the differentiation and proliferation of Merkel cells are enigmatic. A preliminary study in our laboratory showed that Merkel cells in the rat palatine mucosa emerge after birth. This is in contrast to the case of similar cells in the skin that differentiate during the embryonic period prior to the establishment of peripheral nerve innervation. We studied immunohistochemically the developmental timings of Merkel cells and peripheral nerves in the rat palatine mucosa using antibodies to cytokeratins 18 and 20, PGP 9.5, and CGRP using developing palates of prenatal and postnatal rats. We also studied the potency of mitosis in Merkel cells by immunohistochemistry using antibodies for a cell proliferation marker Ki67 and cyclin D-kinase inhibitors p16, p21 and p27. It was shown that Merkel cells in the rat palatine mucosa differentiate postnatally, after the development of peripheral nerve fiber terminals was almost established. The emergence and increase in number of Merkel cells progressed in an anterior-to-posterior wave. Newly appearing Merkel cells were usually negative for anti-cytokeratin 20 antibody but gained affinity for the antibody with progress of maturation. All Merkel cells in the palatine mucosa were negative for anti-Ki67 antibody but positive for anti-p27 antibody. These results indicate that Merkel cells in the rat palatine mucosa are not responsible for the development of peripheral nerve fiber terminals and that these cells differentiate in situ from intraepithelial stem cells.