Kanako Tsuboi

Frequency of Teriparatide Administration Affects the Histological Pattern of Bone Formation in Young Adult Male Mice

Evidence supports that daily and once-weekly administration of teriparatide, human (h)PTH(1-34), enhance bone mass in osteoporotic patients. However, it is uncertain whether different frequencies of hPTH(1-34) administration would induce bone formation similarly in terms of quantity and quality. To investigate that issue, mice were subjected to different frequencies of PTH administration, and their bones were histologically examined. Frequencies of administration were 1 time/2 days, 1 time a day, and 2 and 4 times a day. Mice were allocated to either to control or to 3 different dosing regimens: 80 μg/kg of hPTH(1-34) per injection (80 μg/kg per dose), 80 μg/kg of hPTH(1-34) per day (80 μg/kg · d), or 20 μg/kg of hPTH(1-34) per day (20 μg/kg · d). With the regimens of 80 μg/kg per dose and 80 μg/kg · d, high-frequency hPTH(1-34) administration increased metaphyseal trabecular number. However, 4 doses per day induced the formation of thin trabeculae, whereas the daily PTH regimen resulted in thicker trabeculae. A similar pattern was observed with the lower daily hPTH(1-34) dose (20 μg/kg · d): more frequent PTH administration led to the formation of thin trabeculae, showing a thick preosteoblastic cell layer, several osteoclasts, and scalloped cement lines that indicated accelerated bone remodeling. On the other hand, low-frequency PTH administration induced new bone with mature osteoblasts lying on mildly convex surfaces representative of arrest lines, which suggests minimodeling-based bone formation. Thus, high-frequency PTH administration seems to increase bone mass rapidly by forming thin trabeculae through accelerated bone remodeling. Alternatively, low-frequency PTH administration leads to the formation of thicker trabeculae through bone remodeling and minimodeling.

Healing of osteonecrosis of the jaw (ONJ) after discontinuation of denosumab in a patient with bone metastases of colorectal cancer: a case report and hypothesis.

Osteonecrosis of the jaw (ONJ) is associated with the use of bisphosphonates (BPs), denosumab, and antiangiogenic drugs; however, the pathophysiology of medication-related ONJ (MRONJ) remains unknown. Recent advances in therapies for diseases that affect bone remodeling have led to the development of agents that inhibit the receptor activator of nuclear factor-kappa B ligand (RANKL). One such inhibitor is denosumab, a highly specific, fully human immunoglobulin G2 monoclonal antibody against RANKL. We report a case of ONJ that developed following dental extraction in a patient treated for metastatic colorectal cancer with denosumab. At the first medical examination at our hospital, her clinical presentation was indistinguishable from stage 2 MRONJ, classified according to the 2014 American Academy of Oral Medicine position paper. Discontinuation of denosumab was directed by her oncologist, and we prescribed oral antibiotics and irrigated the exposed area of bone. Seven months after denosumab cessation, the sequestrum of the anterior part of the mandible was naturally shed and the site was healed. Denosumab and BPs have significantly different mechanisms of action. The effects of denosumab on bone turnover are more rapid and reversible than those of BPs. Discontinuation of denosumab may be effective in the management of denosumab-related ONJ, depending on the primary tumor control.

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.

Histological assessment for femora of ovariectomized obesity (db/db) mice carrying mutated leptin receptor.

In order to provide a clue to understand the interplay between leptin and estrogen, we have examined femoral metaphyses of ovariectomized db/db mice carrying a mutated leptin receptor. We performed ovariectomy (OVX) or sham-operation (sham) on 12-week old female wild-type and db/db mice, and then, after 8 weeks, divided the animals into four groups: wild-type sham, wild-type OVX, db/db sham and db/db OVX. Samples from all groups were prepared for histochemical and ultrastructural examinations. As a result, db/db sham mice showed a reduced number and thickness of metaphyseal trabeculae and excessive adipose tissue when compared to wild-type sham mice. The wild-type OVX group exhibited markedly diminished trabecular number, as well as lower populations of osteoblasts and osteoclasts in comparison to wild-type sham group. On the other hand, trabecular numbers were similar for the two db/db groups, suggesting that the effect of the ovariectomy, i.e., estrogen deficiency may be lessened in this animal model. Leptin receptor was mainly found in osteoblasts and in bone marrow stromal cells including adipocytes. In addition, the expression of estrogen receptor did not seem to change after OVX in wild-type mice and in db/db mice. Both db/db sham and OVX mice featured many adipocytes close to the metaphyseal chondro-osseous junction, while osteoblasts accumulated glycogen granules and lipid droplets. Therefore, it seems likely that the disruption of leptin signaling in db/db mice shifts the cell differentiation cascade towards the adipocyte lineage, resulting in an osteoporotic bone independently of estrogen deficiency.

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.