Miki Abe

Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization

Summary Matrix 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.

Localization of Minodronate in Mouse Femora Through Isotope Microscopy

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 transmission electron microscopy (TEM). Eight-week-old male ICR mice received [15N]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [15N]-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.

Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM)

The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes’ cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.

Histological Effects of the Combined Administration of Eldecalcitol and a Parathyroid Hormone in the Metaphyseal Trabeculae of Ovariectomized Rats

Summary Intermittent administration of human parathyroid hormone (1-34) (hPTH(1-34)) promotes anabolic action in bone by stimulating bone remodeling, while eldecalcitol, an analog of active vitamin D3, suppresses osteoclastic bone resorption, and forms new bone by minimodeling. We have examined the biological effects of combined administration of eldecalcitol and hPTH(1-34) on 9-week-old Wistar rats that underwent an ovariectomy (OVX) or Sham operation. They were divided into a Sham group, OVX with vehicle (OVX group), OVX with 10 µg/kg/day of hPTH(1-34) (PTH group), OVX with 20 ng/kg/day of eldecalcitol (eldecalcitol group) or OVX with 10 μg/kg/day of hPTH(1-34), and 20 ng/kg/day of eldecalcitol (combined group) for 4 or 8 weeks. As a consequence, the combined group showed a marked increase in bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) than OVX and had the highest bone mineral density (BMD) compared with other groups. OVX and PTH groups exhibited a high osteoblastic surface/bone surface (Ob.S/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS) indices and many TRAP-reactive osteoclasts. Contrastingly, eldecalcitol and combined groups tended to attenuate the indices of osteoclastic surface/bone surface (Oc.S/BS) and Ob.S/BS than that the other groups. The combined group revealed histological profiles of minimodeling- and remodeling-based bone formation. Thus, the combined administration of eldecalcitol and hPTH(1-34) augments their anabolic effects by means of minimodeling and remodeling.

Chronological immunolocalization of sclerostin and FGF23 in the mouse metaphyseal trabecular and cortical bone

To assess the chronological participation of sclerostin and FGF23 in bone metabolism, this study tracked the immunolocalization of sclerostin and FGF23 in the metaphyses of murine long bones from embryonic day 18 (E18) through 1 day after birth, 1 week, 2 weeks, 4 weeks, 8 weeks, and 20 weeks of age. We have selected two regions in the metaphyseal trabeculae for assessing sclerostin and FGF23 localization: close to the chondro-osseous junction, i.e., bone modeling site even in the adult animals, and the trabecular region distant from the growth plate, where bone remodeling takes place. As a consequence, sclerostin-immunopositive osteocytes could not be observed in both close and distant trabecular regions early at the embryonic and young adult stages. However, osteocytes gradually started to express sclerostin in the distant region earlier than in the close region of the trabeculae. Immunoreactivity for FGF23 was observed mainly in osteoblasts in the early stages, but detectable in osteocytes in the later stages of growth in trabecular and cortical bones. Fgf23 was weakly expressed in the embryonic and neonatal stages, while the receptors, Fgfr1c and αKlotho were strongly expressed in femora. At the adult stages, Fgf23 expression became more intense while Fgfr1c and aKlotho were weakly expressed. These findings suggest that sclerostin is secreted by osteocytes in mature bone undergoing remodeling while FGF23 is synthesized by osteoblasts and osteocytes depending on the developmental/growth stage. In addition, it appears that FGF23 acts in an autocrine and paracrine fashion in fetal and neonatal bones.

Histochemical assessment for osteoblastic activity coupled with dysfunctional osteoclasts in c-src deficient mice

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-/- or c-src-/- mice. Osteopetrotic c-fos deficient (c-fos-/-) mice have no osteoclasts, while c-src deficient (c-src-/-) mice, another osteopetrotic model, develop dysfunctional osteoclasts due to a lack of ruffled borders. c-fos-/- mice possessed no tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts, and showed very weak tissue nonspecific alkaline phosphatase (TNALPase)-reactive mature osteoblasts. In contrast, c-src-/- mice had many TNALPase-positive osteoblasts and TRAPase-reactive osteoclasts. Interestingly, the parallel layers of TRAPase-reactive/osteopontin-positive cement lines were observed in the superficial region of c-src-/- bone matrix. This indicates the possibility that in c-src-/- mice, osteoblasts were activated to deposit new bone matrices on the surfaces that osteoclasts previously passed along, even without bone resorption. Transmission electron microscopy demonstrated cell-to-cell contacts between mature osteoblasts and neighboring ruffled border-less osteoclasts, and osteoid including many mineralized nodules in c-src-/- mice. Thus, it seems likely that osteoblastic activities would be maintained in the presence of osteoclasts, even if they are dysfunctional.

Immunolocalization of osteocyte-derived molecules during bone fracture healing of mouse ribs

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.