アブストラクト(15巻1号:The Bulletin of Kanagawa Dental College)

The Bulletin of Kanagawa Dental College

English

Title : A Morphological Study on Bone Remodeling Sites of Mouse Calvaria Using a Modified Tannin-Ferrocyanide・OsO4 Method
Subtitle : ORIGINAL ARTICLES
Authors : Seigyo So
Authors(kana) :
Organization : Department of Oral Anatomy, Kanagawa Dental College
Journal : The Bulletin of Kanagawa Dental College
Volume : 15
Number : 1
Page : 43-63
Year/Month : 1987 / 3
Article : Original article
Publisher : Kanagawa Odontological Society
Abstract : [Abstract] The present study is intended to supply, with respect to the study of bone remodeling, a new approach enabling us to distinguish the variety of cells within the differentiating bone cell population and at the same time facilitating us to investigate the cellular kinetics on or along the bone surface during bone remodeling. A staining method which is modified from the Ferrocyanide-reduced・OsO4 method and Tannin-Ferrocyanide・OsO4 method is introduced in this study. The Ferrocyanide-reduced OsO4 method, applied by Karnovsky, has been considered capable of distinguishing an osteoblast from a preosteoblast owing to the presence of glycogen deposits within the preosteoblast contributed by the addition of potassium ferrocyanide into postfixative. But to make a distinction between the osteogenic cells and osteoclastic cells is still impossible. The Tannin-Ferrocyanide・OsO4 method, proposed by Gen Takahashi, in which the specimens are prefixed with glutaraldehyde and tannic acid, followed by postfixing with a mixture of potassium ferrocyanide and osmic acid has been suggested to produce a much more enhanced contrast of the cell surface and intracellular components, such as the mitochondria and filaments. A modified ruthenium red staining method, described by Takahashi et al., seems to be able to delineate the external cell surface and identify intracellular components. Neither of the methods mentioned above is able to clearly distinguish the cell types at the bone remodeling sites of a mouse calvaria unless the histochemical approaches are applied. While a modified staining method propounded in the present study is used, a distinguished staining characteristic enabling to distinguish the osteogenic cells from the osteoclastic cells is formed, leading to the possibility of investigating the cellular kinetics within the bone remodeling sites of the mouse calvaria. The procedures are as follows: 1) The specimens are prefixed with 2.5% glutaraldehyde in a 0.1 M cacodylate buffer at pH 7.3 for overnight, followed by rinsing with a 0.08 M cacodylate buffer containing 0.18 M sucrose several times. 2) Incubated with a mixture of 1% tannic acid and 1.5% potassium ferrocyanide for 90 min at room temperature in the dark. 3) Postfixed with a 0.1 M cacodylate-buffered 1% osmic acid for 90 min in the dark. Taking advantage of this staining characteristic, an intensive observation on time-lapse changes of the bone remodeling sites of parietal bone of the mouse calvariae from 16 days of gestation to 3 days after birth has been carried out in order to elucidate the respective role of bone cells during bone remodeling. The cells with known morphological characteristic of osteoclast precursors, namely, a low nucleoplasmic ratio, conspicuous numbers of mitochondria and cytoplasmic granules appear dense-staining, while the cells for bone formation, namely, preosteoblasts and osteoblasts reveal faint-staining. Hence a clear investigation on the cellular events of bone remodeling not only in a light microscopic level but also in an electron microscopic level is under control. A striking observation on the differentiation of a preosteoclast is that once the preosteoclast attaches to the bone surface and demonstrates coated pits on the interface regions of plasm membrane, the cytoplasm of the portion underneath the interface region changes from dense-staining to faint-staining, indicating the process of undergoing a transition from the preosteoclast to the osteoclast. Only by 16 days of gestation can the first typical preosteoclast be found around the perivascular area in the endocranial periosteum of parietal bone of the mouse calvaria. After this occurs, they progressively approach toward and reach the bone surface by sending long cytoplasmic pseudopods between the lining osteogenic cells. At this time the osteoblasts lining the bone surface stop synthesizing the matrix and become flatten. When advancing their way to the bone surface, they conserve the typical morphological characteristics and show dense staining until they first reach the bone surface and show coated pits on the interface regions of plasma membrane against the bone surface. At this stage the number of preosteoclasts in the endocranial periosteum compared to that in the ectocranial periosteum shows an overwhelming majority. The number of preosteoclasts progressively increases and reaches a peak in 18 days of gestation, then succeeds by a gradual increase in the multinucleated osteoclasts. After this stage the number of osteoclasts increases progressively and the blood vessels around them show increased fenestrations on the endothelial cells. The intensive observation on the correlation of the number of fenestrations to that of the osteoclastic cells indicate that the more markedly the osteoclastic cells appear, the more the number of fenestrations increases in the endothelial cells.
Practice : Dentistry
Keywords : Osteoclast Precursor, Preosteoclast, Osteoclast, Osteoblast, Preosteoblast, Modified Tannin-Ferrocyanide・OsO4 Method, Cellular Kinetics