SummaryMatrix vesicle-mediated mineralization is an orchestrated sequence of ultrastructural and biochemical events that lead to crystal nucleation and growth. The influx of phosphate ions into the matrix vesicle is mediated by several proteins such as TNAP, ENPP1, Pit1, annexin and so forth. The catalytic activity of ENPP1 generates pyrophosphate (PPi) using extracellular ATPs as a substrate, and the resultant PPi prevents crystal overgrowth. However, TNAP hydrolyzes PPi into phosphate ion monomers, which are then transported into the matrix vesicle through Pit1. Accumulation of Ca2+ and PO43− inside matrix vesicles then induces crystalline nucleation, with calcium phosphate crystals budding off radially, puncturing the matrix vesicle’s membrane and finally growing out of it to form mineralized nodules.

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To elucidate which of elevated serum concentration of inorganic phosphate (Pi) or disrupted signaling linked to αklotho/fibroblast growth factor 23 (FGF23) is a predominant regulator for senescence-related degeneration seen in αKlotho-deficient mice, we have examined histological alteration of the periodontal tissues in the mandibular interalveolar septum of αKlotho-deficient mice fed with Pi-insufficient diet. We prepared six groups of mice: wild-type, kl/kl, and αKlotho mice with normal diet or low-Pi diet. As a consequence, kl/kl and αKlotho mice showed the same abnormalities in periodontal tissues: intensely stained areas with hematoxylin in the interalveolar septum, dispersed localization of alkaline phosphatase-positive osteoblasts and tartrate-resistant acid phosphatase-reactive osteoclasts, and accumulation of dentin matrix protein 1 in the osteocytic lacunae. Although kl/kl mice improved these histological abnormalities, αKlotho mice failed to normalize those. Gene expression of αKlotho was shown to be increased in kl/kl specimens. It seems likely that histological abnormalities of kl/kl mice have been improved by the rescued expression of αKlotho, rather than low concentration of serum Pi. Thus, the histological malformation in periodontal tissues in αKlotho-deficient mice appears to be due to not only increased concentration of Pi but also disrupted αklotho/FGF23 signaling.

Objectives: The aim of this study is a biological application of focused ion beam-scanning electron microscopy (FIB-SEM) to demonstrate serial sectional images of skeletal tissues, here presenting the ultrastructure of 1) cartilaginous extracellular fibrils and 2) osteoblastic cytoplasmic processes.Methods: Seven weeks-old female wild-type mice were fixed with half-Karnovsky solution and subsequent OsO4, and the tibiae were extracted for block staining prior to observation under transmission electron microscope (TEM) and FIB-SEM.Results: TEM showed the fine fibrillar, but somewhat amorphous ultrastructure of the intercolumnar septa in the growth plate cartilage. Alternatively, FIB-SEM revealed bundles of stout fibrils at regular intervals paralleling the septa's longitudinal axis, as well as vesicular structures embedded in the cartilaginous matrix of the proliferative zone. In the primary trabeculae, both TEM and FIB-SEM showed several osteoblastic cytoplasmic processes on the osteoid, with numbers higher than those seen in the bone matrix. FIB-SEM revealed the agglomeration of cytoplasmic processes beneath the osteoblasts, which formed a tubular continuum extending from those cells. Based on these findings, we postulated that osteoblasts not only extend their cytoplasmic processes through to the bone matrix, but also stack these cell processes on the osteoid of the primary trabeculae. Conclusion:Taken together, it is likely that FIB-SEM imaging strategy on serial sections may successfully deliver new insights on the ultrastructure of cartilage and bone tissues. words

This study was aimed to verify the cellular interplay between vascular endothelial cells and surrounding cells in the chondro-osseous junction of murine tibiae. Many CD31-positive endothelial cells accompanied with Dolichos Biflorus Agglutinin lectin-positive septoclasts invaded into the hypertrophic zone of the tibial epiphyseal cartilage. MMP9 immunoreactive cytoplasmic processes of vascular endothelial cells extended into the transverse partitions of cartilage columns. In contrast, septoclasts included several large lysosomes which indicate the incorporation of extracellular matrices despite no immunopositivity for F4/80 －a hallmark of macrophage/monocyte lineage. In addition, septoclasts were observed in c-fos-/- mice but not in Rankl-/- mice. Unlike c-fos-/- mice, Rankl-/- mice showed markedly-expanded hypertrophic zone and the irregular shape of the chondro-osseous junction. Immunoreactivity of PDGF-bb, which involved in angiogenic roles in the bone, was detected in not only osteoclasts but also septoclasts at the chondro-osseous junction. Therefore, septoclasts appear to assist the synchronous vascular invasion of endothelial cells at the chondro-osseous junction. Vascular endothelial cells adjacent to the chondro-osseous junction possesses endomucin but not EphB4, whereas those slightly distant from the chondro-osseous junction were intensely positive for both endomucin and EphB4, while being accompanied with ephrinB2-positive osteoblasts. Taken together, it is likely that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2.

Minodronate is highlighted for its marked and sustained effects on osteoporotic bones. To determine the duration of minodronate's effects, we have assessed the localization of the drug in mouse bones through isotope microscopy, after labeling it with a stable nitrogen isotope ([(15)N]-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and transmission electron microscopy (TEM). Eight-week-old male ICR mice received [(15)N]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [(15)N]-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hr after minodronate administration, histochemistry and TEM showed osteoclasts with well-developed ruffled borders. However, osteoclasts were roughly attached to the bone surfaces and did not feature ruffled borders at 24 hr after minodronate administration. The numbers of tartrate-resistant acid phosphatase-positive osteoclasts and alkaline phosphatase-reactive osteoblastic area were not reduced suddenly, and apoptotic osteoclasts appeared in 1 week and 1 month after the injections. Von Kossa staining demonstrated that osteoclasts treated with minodronate did not incorporate mineralized bone matrix. Taken together, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect.

Since osteoblastic activities are believed to be coupled with osteoclasts, we have attempted to histologically verify which of the distinct cellular circumstances, the presence of osteoclasts themselves or bone resorption by osteoclasts, is essential for coupled osteoblastic activity, by examining c-fos

We employed a well-standardized murine rib fracture model to assess the distribution, in the cortical bone, of three important osteocyte-derived molecules-dentine matrix protein 1 (DMP1), sclerostin and fibroblast growth factor 23 (FGF 23). Two days after the fracture, the periosteum thickened, and up to the seventh day post-fracture, the cortical surfaces were promoting neoformation of two tissue types depending on the distance from the fracture site: chondrogenesis was taking place near the fracture, and osteogenesis distant from it. The cortical bones supporting chondrogenesis featured several empty lacunae, while in the ones underlying newly-formed woven bone, empty lacunae were hardly seen. DMP1-immunopositive osteocytic lacunae and canaliculi were seen both close and away from the fracture. In contrast, the region close to the fracture had only few sclerostin-and FGF23-immunoreactive osteocytes, whereas the distant region revealed many osteocytes immunopositive for these markers. Mature cortical bone encompassing the native cortical bone was observed at two-, three-and four-weeks post-fracture, and the distribution of DMP1, sclerostin and FGF23 appeared to have returned to normal. In summary, early stages of fracture healing seem to be important for triggering chondrogenesis and osteogenesis that may be regulated by osteocytes via their secretory molecules.Fracture healing is a sequence of biological processes that include new formation of cartilage and bone. The tissue composed of newly-formed cartilage and bone, referred to as "callus", is the product of a coordinated physiological cascade that involves proliferation and differentiation of various lineages of inflammatory cells, angioblasts, fibroblasts, chondroblasts, and osteoblasts (24, 32). The initial cellular event is supposed to be cell replication of periosteal mesenchymal cells in the tissues surrounding the fracture site. After that, chondroblastic and osteoblastic differentiation ensues to promote the growth of a scaffold of cartilaginous and bony tissue, which is needed for initial bridging of the fracture gap. Through endochondral ossification, bone tissue gradually replaces the cartilaginous callus that was essential for primary stabilization of the fractured bone. There are reports showing that periosteal mesenchymal cells respond to locally increased levels of growth factors and cytokines, and may differentiate into chondrocytes or osteoblasts (41), so that, the periosteal mesenchymal environment appears to influence such cell fate (4). In spite of the local ef-