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This study evaluated the long-term effects of the cathepsin K inhibitor ONO-5334 on bone mass and strength in ovariectomised (OVX) cynomolgus monkeys. Animals were assigned to one of the following six groups: Sham (non-OVX), OVX control treated with vehicle, ONO-5334 1.2, 6 or 30 mg/kg/day, p.o., or alendronate (ALN) 0.05 mg/kg/2 weeks, i.v. for 16 months. Peripheral quantitative computed tomography (pQCT) analysis revealed that ONO-5334 increased not only trabecular bone mineral density (BMD) but also cortical BMD in the distal radius and the lumbar vertebra. ONO-5334 and ALN suppressed the deterioration of trabecular architecture by micro-CT analysis in the distal radius. Assessments of bone strength showed that ONO-5334 increased maximum load at the distal and midshaft radius. The linear regression lines between bone mass and strength in the lumbar vertebra were tended to be shifted towards increasing bone strength in the ONO-5334 6 and 30 mg/kg groups compared with the ALN groups. This indicated that bone strength was higher in the ONO-5334 groups than the ALN group, even though bone mineral content (BMC) and BMD were comparable. Subpopulation analysis revealed that, at similar integral BMC or BMD level, cortical bone mass for ONO-5334 was higher than for ALN; the opposite effects were observed for trabecular bone. In conclusion, ONO-5334 preferentially increased cortical bone, which may provide a greater contribution to bone strength. Since these results support a different mode of action for ONO-5334 compared with that of ALN, ONO-5334 may offer new therapeutic options to patients with osteoporosis.

A link between vascular calcification and bone anomalies has been suggested in chronic kidney disease (CKD) patients with low bone turnover disease. We investigated the vascular expression of osteocyte markers in relation to bone microarchitecture and mineralization defects in a model of low bone turnover CKD rats with vascular calcification. CKD with vascular calcification was induced by 5/6 nephrectomy followed by high calcium and phosphate diet, and vitamin D supplementation (Ca/P/VitD). CKD + Ca/P/VitD group (n = 12) was compared to CKD + normal diet (n = 12), control + normal diet (n = 8) and control + Ca/P/VitD supplementation (n = 8). At week 6, tibia, femurs and the thoracic aorta were analysed by Micro-Ct, histomorphometry and for expression of osteocyte markers. High Ca/P/VitD treatment induced vascular calcification only in CKD rats, suppressed serum parathyroid hormone levels and led to higher sclerostin, DKK1 and FGF23 serum levels. Expression of sclerostin, DKK1 and DMP1 but not FGF23 were increased in calcified vessels from CKD + Ca/P/VitD rats. Despite low parathyroid hormone levels, tibia bone cortical thickness was significantly lower in CKD + Ca/P/VitD rats as compared to control rats fed a normal diet, which is likely the result of radial growth impairment. Finally, Ca/P/VitD treatment in CKD rats induced a bone mineralization defect, which is likely explained by the high calcitriol dose. In conclusion, Ca/P/VitD supplementation in CKD rats induces expression of osteocyte markers in vessels and bone mineralisation anomalies. Further studies should evaluate the mechanisms of high dose calcitriol-induced bone mineralisation defects in CKD.

Minodronic acid is an aminobisphosphonate that is an antagonist of purinergic P2X2/3 receptors involved in pain. The aim of this study was to investigate the action and distribution of minodronic acid and the potential for P2X2/3 receptor antagonism based on the estimated concentration of minodronic acid. Microlocalization of radiolabeled minodronic acid was examined in the femur of neonatal rats. The bone-binding characteristics of minodronic acid and morphological changes in osteoclasts were analyzed in vitro. The minodronic acid concentration around bone resorption lacunae was predicted based on bone binding and the shape of lacunae. In microautoradiography, radioactive silver grains were abundant in bone-attached osteoclasts and were detected in calcified and ossification zones and in the cytoplasm of osteoclasts but not in the hypertrophic cartilage zone. In an osteoclast culture with 1 µM minodronic acid, 65% of minodronic acid was bound to bone, and C-terminal cross-linking telopeptide release was inhibited by 96%. Cultured osteoclasts without minodronic acid treatment formed ruffled borders and bone resorption lacunae and had rich cytoplasm, whereas those treated with 1 µM minodronic acid were not multinucleated, stained densely with toluidine blue, and were detached from the bone surface. In the 1 µM culture, the estimated minodronic acid concentration in resorption lacunae was 880 µM, which is higher than the IC 50 for minodronic acid antagonism of P2X2/3 receptors. Thus, inhibition of P2X2/3 receptors around osteoclasts may contribute to the analgesic effect of minodronic acid.

Mature osteoblasts have three fates: as osteocytes, quiescent lining cells, or osteoblasts that undergo apoptosis. However, whether intermittent parathyroid hormone (PTH) can modulate the fate of mature osteoblasts in vivo is uncertain. We performed a lineage-tracing study using an inducible gene system. Dmp1-CreERt2 mice were crossed with Rosa26R reporter mice to obtain targeted mature osteoblasts and their descendants, lining cells or osteocytes, which were detected using X-gal staining. Rosa26R:Dmp1-CreERt2(+) mice were injected with 0.25 mg 4-OH-tamoxifen (4-OHTam) on postnatal days 5, 7, 9, 16, and 23. In a previous study, at 22 days after the last 4-OHTam, most LacZ+ cells on the periosteal surface were inactive lining cells. On day 25 (D25), the mice were challenged with an injection of human PTH (1-34, 80 μg/kg) or vehicle daily for 10 (D36) or 20 days (D46). We evaluated the number and thickness of LacZ+ osteoblast descendants in the calvaria and tibia. In the vehicle group, the number and thickness of LacZ+ osteoblast descendants at both D36 and D46 significantly decreased compared to D25, which was attenuated in the PTH group. In line with these results, PTH inhibited the decrease in the number of LacZ+/osteocalcin-positive cells compared to vehicle at both D36 and D46. As well, the serum levels of sclerostin decreased, as did the protein expression of sclerostin in the cortical bone. These results suggest that intermittent PTH treatment can increase the number of periosteal osteoblasts by preventing mature osteoblasts from transforming into lining cells in vivo.

Quantitative analysis of the orientational distribution of biological apatite (BAp) crystals is proposed as a new index of bone quality. This study aimed to analyze BAp c-axis orientation in ovariectomized (OVX) monkeys treated with amino-bisphosphonates minodronic acid and alendronate as reference. Sixty female monkeys aged 9-17 years were divided into five groups: one sham group and four OVX groups. The sham group and one OVX group were treated daily with vehicle for 17 months. The other three groups were treated daily with minodronic acid at doses of 0.015 and 0.15 mg/kg, and alendronate at 0.5 mg/kg orally, respectively. The seventh lumbar vertebrae were subjected to analysis of the preferential BAp c-axis orientation in the ventral cortical bone. The BAp c-axis orientation along the craniocaudal axis was significantly increased in the OVX monkeys. The high dose of minodronic acid suppressed the OVX-induced increase in the BAp c-axis orientation, whereas alendronate showed a non-significant tendency to suppress the increase in the orientation. In analysis with other parameters, the BAp c-axis orientation was positively correlated with bone formation indices in biochemical markers and bone histomorphometry and negatively correlated with the increase in lumbar bone mineral density. On the other hand, the BAp c-axis orientation was not correlated with bone resorption indices, except for the eroded surface. These results indicate that the increase in BAp c-axis orientation was ameliorated by minodronic acid treatment in OVX monkeys, mainly by suppression of bone formation increase.