Cellular function of osteocytes in normal and αklotho-deficient mice

During the last decade, osteocyte-derived factors i.e., sclerostin, dentin matrix protein-1, fibroblast growth factor 23 (FGF23) that reduces serum phosphate concentration by mediating FGF receptor 1c/αklotho in the kidney, have been highlighted for osteocytes’ fine-turned regulation on bone remodeling and phosphate homeostasis. Osteocytes are interconnected through gap junctions between their cytoplasmic processes, and thereby, build upon the functional syncytia, referred to as the osteocytic lacunar-canalicular system (OLCS). Osteocytes appear to communicate surrounding osteocytes and osteoblasts by means of two possible pathways of molecular transport throughout the OLCS : One is a passageway of their cytoplasmic processes, and the other is a pericellular space in the osteocytic canaliculi. The regularly-oriented OLCS in mature compact bone appears to efficiently serve for molecular transport, mechanosensing and targeted bone remodeling that would erase microdamages in bone. In a disrupted signaling state of FGF23/αklotho, serum concentration of phosphate would be markedly-elevated. Despite highly-elevated serum phosphate, αklotho -deficient mice revealed defective mineralization in bone matrix. OLCS in αklotho -deficient mice were irregularly-distributed and the connectivity of cytoplasmic processes of osteocytes was very poor, so that osteocytes did not seem to form functional syncytia. Therefore, osteocytes’function cooperated with other bone cells, rather than serum concentration of calcium/phosphate, and this seems to play a central role in maintaining bone mineralization. In this review, the biological function of the regularly-arranged OLCS in a normal state will be introduced, as well as dysfunctional osteocytes in αklotho-deficient state, using animal models

マウス歯胚の象牙芽細胞層におけるpodoplanin, CD44およびendomucinの局在 [全文の要約]

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Localization of minodronate in bone

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 (15N-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and TEM. Eight-weeks-old male ICR mice received 15N-minodronate (1mg/kg) intravenously and were sacrificed after three hours, 24 hrs, one week and one month. Isotope microscopy showed that 15N-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hrs 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 hrs after minodronate administration. The numbers of TRAP-positive osteoclasts and ALP-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

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

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

The diversity of preosteoblastic morphology : Preosteoblastic response to parathyroid hormone

The current concept of a preosteoblast is a precursor of an osteoblast, which is regarded as a transient cell type during osteoblastic differentiation. We have previously demonstrated different phenotypes of preosteoblasts expressing Runx2, ALPase, and BrdU incorporation. Transmission electron microscopy revealed following four distinct preosteoblastic cell types : 1) cells rich in rough endoplasmic reticulum (rER) but with a few vesicles and vacuoles (ERrich/vesicle-poor preosteoblasts), 2) cells extending their cytoplasmic processes connecting distant cells, with a small amount of scattered cisterns of rER and many vesicles and vacuoles (ER-poor/vesicle-rich preosteoblasts), 3) translucent cells showing few dispersed cell organelles and irregular cell shape with a translucent cytoplasm (translucent cells), and 4) small cells without developed cell organelles (small undifferentiated cells). ER-rich/vesicle-poor preosteoblasts were often closely adjacent to mature osteoblasts and therefore appeared to be ready for differentiation into osteoblasts. In contrast, after the administration of parathyroid hormone (PTH), ER-poor/vesicle-rich preosteoblasts rather than ER-rich/vesicle-poor cells significantly increased in number, forming a huge meshwork overlying mature osteoblasts. Thus, ERpoor/vesicle-rich preosteoblasts appeared to respond well to PTH. We also attempted to unveil the cellular behavior of these preosteoblasts against PTH and to dissect the role of osteoclasts on the mediation of PTH anabolic actions. PTH stimulated the proliferation of ER-poor/vesicle-rich preosteoblasts and bone formation in mature osteoblasts. However, an increased population of ER-poor/vesicle-rich preosteoblasts appears to require cell coupling from osteoclasts to differentiate into ER-rich/vesicle-poor preosteoblasts and mature osteoblasts. Without osteoclasts, PTH could induce neither preosteoblastic differentiation into mature osteoblasts nor subsequent bone formation. In this mini-review, we will introduce preosteoblasts in vivo consisting of several cell types with different ultrastructural properties and PTH action on preosteoblasts