Kazuto Hoshi

The Primary Calcification in Bones Follows Removal of Decorin and Fusion of Collagen Fibrils

To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19‐day postcoitum embryonic rat calvariae. Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts. In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 nm in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils. In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side. Where calcification was progressed, this fusion was even more so. Some very large fibrils exhibited complicated contours, 400 nm or more in diameter. Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification. The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.

Immunolocalization of tissue non-specific alkaline phosphatase in mice

Abstractu2002Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-μm tissue slices, and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP. Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes.

Targeted disruption of cadherin-11 leads to a reduction in bone density in calvaria and long bone metaphyses

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin‐11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin‐11 in osteoblastogenesis, cadherin‐11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round‐shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild‐type littermates. These results show that absence of cadherin‐11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin‐11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.

Morphological characterization of skeletal cells in Cbfa1-deficient mice

To clarify the mechanisms by which core-binding factor-alpha1 (Cbfa1), an essential transcription factor in osteogenesis, functions in osteoblast matrix formation, as well as in chondrocyte differentiation and osteoclastic bone resorption, Cbfa1-deficient embryonic mice were investigated ultrastructurally and histocytochemically at 18.5 days postcoitum. In homozygotic mice, both endochondral and intramembranous ossification were arrested, although bone tissue had already formed at this stage in the wild type. The tibiae of homozygotic mice were characterized by calcified cartilage and alkaline phosphatase (ALP)-positive perichondrium, whereas membranous structures indicating the presence of ALP activity in the lateral portion were observed in the calvariae, rather than the bone tissue. Most of the ALP-positive perichondrial cells in homozygotic tibiae possessed a spindle-shaped cell contour and small cytoplasm, the extracellular matrix of which contained neither type I collagen nor calcifying matrix vesicles. In contrast, some perichondrial cells at the very middle part of tibiae became flattened. In the vicinity of these cells, a thin layer of type I collagen-based calcified matrix, containing osteopontin, bone sialoprotein, or osteocalcin, was observed. In the cartilage of mutant mice, we observed a hypoplasic zone of proliferative chondrocytes, the flattening of hypertrophic chondrocyte-like cells, and calcified chondrocytes which, while not degraded, did display a high level of cell function. Mononuclear osteoclastic cells were found in the perichondrium, near calcified chondrocytes, in mutant mice. Multinuclear osteoclasts possessing H+-ATPase and ruffled borders were also present, although only in limited numbers. Neither the development of ruffled borders nor intracellular polarization was complete. Because the majority of osteogenic cells in Cbfa1-deficient mice can neither form nor calcify the bone matrix, Cbfa1 principally plays essential roles in osteoblastic differentiation and bone matrix formation. Cbfa1 also affects both the proliferation and the differentiation of chondrocytes, whereas its absence prevents normal osteoclast formation and related functions.

Localizational Alterations of Calcium, Phosphorus, and Calcification-Related Organics Such as Proteoglycans and Alkaline Phosphatase During Bone Calcification

To further approach the mechanisms of bone calcification, embryonic rat calvariae were observed at electron microscopic level by the means of fine structures and various cytochemical localizations, including nonspecific proteoglycan (PG) stained by cuprolinic blue (CB), decorin, chondroitin sulfate, hyaluronan, and alkaline phosphatase (ALP), as well as the elemental mapping of calcium (Ca) and phosphorus (P) by energy‐filtering transmission electron microscopy (EFTEM). In the calvariae, calcification advanced as the distance from osteoblasts increased. Closer to the osteoblasts, the osteoid was marked by an abundance of CB‐positive PGs around collagen fibrils. After crystallization within matrix vesicles, calcified nodules formed and expanded, creating a coherent calcified matrix. The sizes of CB‐positive PG‐like structures diminished as calcification proceeded. Although small CB‐positive structures were accumulated in early stage‐calcified nodules, they were localized along the periphery of larger calcified nodules. Cytochemical tests for decorin, chondroitin sulfate, and hyaluronan determined their presence in the areas around collagen fibrils of the osteoid, as well as in and around calcified nodules, whereas ALP was found in the matrix vesicles, as well as in and around the calcified nodules. Ca tended to localize at the PG sites, while P often mapped to the collagen fibril structures, in the uncalcified matrix. In contrast, Ca/P colocalization was visible in and around the calcified nodules, where ALP and smaller CB‐positive structures were observed. The difference in the localization patterns of Ca and P in uncalcified areas may limit the local [Ca2+][PO43−] product, leading to the general inhibition of hydroxyapatite crystallization. The downsizing of CB‐positive structures suggested enzymatic fragmentation of PGs. Such structural alterations would contribute to the preservation and transport of calcium. ALP possesses the ability to boost local phosphate anion concentration. Therefore, structurally altered PGs and ALP may cooperate in Ca/P colocalization, thus promoting bone calcification.

Morphological examination of bone synthesis via direct administration of basic fibroblast growth factor into rat bone marrow

Woven bone induced by direct injection of basic fibroblast growth factor (bFGF) into rat bone marrow was examined. On the first day after injection, fibrous tissues formed in the treated region of the bone marrow. Tissue‐nonspecific alkaline phosphatase (TNAPase)‐immunopositive osteoblastic cells and osteopontin immunopositive‐extracellular matrices were observed in the fibrous tissues, indicating bone induction. On the fifth day, the bFGF‐induced bone was found broadly in the bone marrow. In the originally existing bone, osteopontin‐immunoreactivity was observed at cement lines, but not in the fully calcified matrix, whereas the woven bone displayed immunoreactivity throughout the matrix. Numerous TRAPase‐positive osteoclasts were present on the surfaces of the woven bone, but no obvious cement line was observed. Therefore, both bone formation and resorption appeared highly active, without normal cellular coupling equilibrated between bone formation and resorption performed by osteoblasts and osteoclasts. On the tenth day, the bFGF‐induced bone was almost replaced by bone marrow. Thus, bone formation actively occurred in the first half of the experimental period, whereas bone resorption came to be predominant thereafter. This study demonstrated that bFGF stimulates bone formation, which, however, is subject to subsequent resorption, probably due in part to the absence of coordinated cellular coupling between osteoclasts and osteoblasts. Microsc. Res. Tech. 41:313–322, 1998.

An immunohistochemical study on hard tissue formation in a subcutaneously transplanted rat molar

While dental pulp undergoes calcification following tooth replantation or transplantation, we actually know little about these mechanisms. We therefore conducted histological and immunohistochemical evaluations of mineralized tissue that formed in the pulp of rat maxillary molar transplanted into abdominal subcutaneous tissue. One, 2, 3, and 4xa0weeks post-transplantation, the teeth were investigated immunohistochemically using antibodies to osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin sialoprotein (DSP), and tissue non-specific alkaline phosphatase (TNAP). In the 1stxa0week after transplantation, cell-rich hard tissue was formed at the root apex. At 2xa0weeks, formations of hard tissue, with few cells in the root canals and bone-like tissue in the coronal pulp chamber, were noted. After 3 and 4xa0weeks, the amounts of these hard tissues were increased. The immunolocalization of OCN, OPN, and BSP was seen strongly in coronal and apical hard tissues, but weakly in the root hard tissue. Conversely, DSP localized in the root hard tissue, but not in other newly formed hard tissues. At 1xa0week, TNAP localized along the periphery of the apical hard tissue and the lower surfaces of root predentin. These results demonstrate that the newly formed hard tissues in the pulp cavity of subcutaneously transplanted molars could be classified into three types, suggesting that these might be formed by type-specific cells.

Observation of human dentine by focused ion beam and energy-filtering transmission electron microscopy.

Molar dentine was sliced into 100u2003nm ultrathin sections, by means of a focused ion beam, for observation by energy‐filtering transmission electron microscopy (EFTEM). Within the matrix, crystals approximately 10u2003nm wide and 50–100u2003nm long were clearly observed. When carbon and calcium were mapped in electron spectroscopic images by EFTEM, carbon failed to localize in crystals. However, it was found in other regions, especially those adjacent to crystals. Because carbon localizations were thought to reflect the presence of organic components, carbon concentration in regions near crystals suggested the interaction of crystals and organics, leading to organic control of apatite formation and growth. Ca was present in almost all regions. The majority of Ca localizing in regions other than crystals may be bound to organic substances present in dentine matrix. These substances are thought to both accumulate Ca and act as reservoirs for crystallization of apatite in dentine.

Matrix Vesicle Calcification in Bones of Adult Rats

Abstract. To clarify the calcification mechanism that functions in bone formation in adult rats, the ultrastructure of tibial trabeculae and calvarial endostea obtained from 8- to 18-month-old rats was investigated morphologically, and compared with that of 19.5-day post-coitum fetal rats. In both samples, osteoid was observed between the activated osteoblasts and the calcified matrix, which contained matrix vesicles enclosed by a biological membrane. Some of these vesicles contained needle-like crystals thought to be hydroxyapatite, suggesting probable matrix vesicle calcification. These results indicate that matrix vesicle function not only in the initial calcification that occurs during embryonic ossification but also contribute to bone formation in adults.