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Boron neutron capture therapy (BNCT) with p-boronophenylalanine (BPA) is expected to have less effect on the decrease in normal bone strength than X-ray therapy. However, the compound biological effectiveness (CBE) value necessary to convert the boron neutron capture reaction (BNCR) dose into a bioequivalent X-ray dose has not been determined yet. The purpose of this study was to evaluate the influence of BNCT on normal bone in mice and to elucidate the CBE factor. We first searched the distribution of BPA in the normal bone of C3H/He mice and then measured the changes in bone strength after irradiation. The CBE value was determined when the decrease in bone strength was set as an index of the BNCT effect. The 10B concentrations in the tibia after subcutaneous injection of 125, 250 and 500 mg/kg BPA were measured by prompt gamma-ray spectroscopy and inductively coupled plasma (ICP)-atomic emission spectrometry. The 10B mapping in the tibia was examined by alpha-track autoradiography and laser ablation-ICP-mass spectrometry. The 10B concentration increased dose-dependently; moreover, the concentrations were maintained until 120 min after BPA administration. The administered 10B in the tibia was abundantly accumulated in the growth cartilage, trabecular bone and bone marrow. The bone strength was analyzed by a three-point bending test 12 weeks after irradiation. The bending strength of the tibia decreased dose-dependently after the irradiation of X-ray, neutron and BNCR. The CBE factor was obtained as 2.27 by comparing these dose-effect curves; the value determined in this study will enable an accurate dosimetry of normal bone.

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Osteocytes, osteoblasts (bone-forming cells), and osteoclasts (bone-resorbing cells) are the primary types of cells that regulate bone metabolism in mammals. Sclerostin produced in bone cells activates osteoclasts, inhibiting bone formation; excess production of sclerostin, therefore, leads to the loss of bone mass. Fish scales have been reported to have morphological and functional similarities to mammalian bones, making them a useful experimental system for analyzing vertebrate bone metabolism in vitro. However, whether fish scales contain cells producing sclerostin and/or osteocytes has not been determined. The current study demonstrated, for the first time, that sclerostin-containing cells exist in goldfish scales. Analysis of the distribution and shape of sclerostin-expressing cells provided evidence that osteoblasts produce sclerostin in goldfish scales. Furthermore, our results found that osteocyte-like cells exist in goldfish scales, which also produce sclerostin. Finally, we demonstrated that microgravity in outer space increased the level of sclerostin in the scales of goldfish, a finding suggesting that the induction of sclerostin is the mechanism underlying the activation of osteoclasts under microgravity.

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Melatonin has recently been found to be a possible new regulator of bone metabolism. However, the influence of melatonin in natural age-related osteoporosis has not been fully elucidated yet, although there have been some reports regarding postmenopausal osteoporosis with melatonin treatments. The present study investigated the effects of long-term melatonin administration during the aging process on bone metabolism. Using quantitative computed tomography methods, we found that the total bone density of both the femur metaphysis and diaphysis decreased significantly in 20-month-old male mice. In the metaphysis, both trabecular bone mass and Polar-Strength Strain Index (SSI), which is an index of bone strength, decreased significantly. Judging from bone histomorphometry analysis, trabecular bone in 20-month-old male mice decreases significantly with age and is small and sparse, as compared to that of 4-month-old male mice. Loss of trabecular bone is one possible cause of loss of bone strength in the femoral bone. In the metaphysis, the melatonin administration group had significantly higher trabecular bone density than the non-administration group. The Polar-SSI, cortical area, and periosteal circumference in the diaphysis was also significantly higher with melatonin treatments. Since the melatonin receptor, MT2, was detected in both osteoblasts and osteoclasts of the femoral bone of male mice, we expect that melatonin acts on osteoblasts and osteoclasts to maintain the bone strength of the diaphysis and metaphysis. Thus, melatonin is a potential drug for natural age-related osteoporosis.

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The following articles have been retracted by the Editorial Board of Experimental Animals, because some parts of their contents were published elsewhere.

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Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

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The following articles have been retracted by the Editorial Board of Experimental Animals, because some parts of their contents were published elsewhere.

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The following articles have been retracted by the Editorial Board of Experimental Animals, because some parts of their contents were published elsewhere.

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Calcium phosphate cements (CPCs), consisting of a mixture of calcium phosphate powders and setting liquid, have been widely used in orthopedic applications. One of the drawbacks of CPCs is their poor resorbability in the living body, which hinders substitution with natural bones. One of the strategies to facilitate the resorption of CPCs is the incorporation of bioresorbable or water-soluble pore-generating particles (porogens), such as gelatin, in the CPC matrices. In spite of numerous reports, however, little is known about the effect of the dissolution/resorption rate of the porogens on concomitant bone regeneration. In the present study, we prepared preset CPCs dispersed with 10 mass% of low-endotoxin gelatin particles 200-500 µm in diameter having different heat-treatment histories, therefore exhibiting different dissolution rate, and then the obtained CPC/gelatin composites were evaluated for in vivo resorption and concomitant in vivo bone formation behaviors. As the results, the dispersion of gelatin particles markedly promoted in vivo resorption of CPC, and enhanced concomitant bone formation, connective tissue formation, osteoblast proliferation, and vascularization. The dissolution/resorption rate was able to be controlled by changing the up-front heat-treatment temperature. In particular, when CPC/gelatin composites were implanted in distal metaphysis of rabbits, the optimum dissolution/resorption was attained by heat-treating gelatin particles at 383 K for 24 h before dispersing in CPC. Quick resorption of calcium phosphate cement and concomitant bone formation by dispersing properly heat-treated with gelatin particles.

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The effects of low-intensity pulsed ultrasound (LIPUS) on osteoclastogenesis were examined using fish scales that had both osteoclasts and osteoblasts. The binding of the receptor activator of NF-κB ligand (RANKL) in osteoblasts to the receptor activator of NF-κB (RANK) in osteoclasts induced osteoclastogenesis. Therefore, we focused on RANK/RANKL signaling. After 6 h of incubation following LIPUS treatment, mRNA expression of RANKL increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 h of incubation, and then the mRNA expression of functional genes was significantly up-regulated after 12 h of incubation. However, the mRNA expression of osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 h of incubation and tended to further increase after 12 h of incubation. At 24 h of incubation, osteoclastic functional genes' mRNA expression decreased to the level of the control. Furthermore, we performed an in vivo experiment with goldfish. Two weeks after daily LIPUS exposure, osteoclastic marker enzymes tended to decrease while osteoblastic marker enzymes were activated. The regeneration rate of the LIPUS-treated scales was significantly higher than that of the control scales. Thus, LIPUS moderately activates osteoclasts and induces bone formation.

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Teriparatide (TPTD) is known to increase the cortical thickness and porosity. The purpose of the present study was to determine whether switching from TPTD to ibandronate (IBN) would be useful for improving cortical bone parameters as assessed using high-resolution quantitative computed tomography (HR-QCT) analyses in mature rabbits. Forty-two female New Zealand white rabbits (18-22 weeks old) were randomized into six groups of 7 animals each as follows: 4-week vehicle administration group, 4-week TPTD administration group (20 µg/kg, subcutaneously [s.c.], daily), 12-week vehicle administration group, 4-week TPTD administration + 8-week vehicle administration group, 4-week TPTD administration + 8-week lower-dose IBN administration group (20 µg/kg, s.c., every 4 weeks), and 4-week TPTD administration + 8-week higher-dose IBN administration group (100 µg/kg, s.c., every 4 weeks). After the 4- or 12-week experimental period, the cortical bone of the distal femoral diaphysis was processed for HR-QCT analysis. The 4-week TPTD administration increased the pore ratio, number, and density as well as the cortical area, thickness, and bone mineral content (BMC), without significant influencing the volumetric bone mineral density (BMD). The 4-week TPTD administration + 8-week vehicle administration decreased the pore ratio, number, and density as well as the cortical area and thickness, compared with the 4-week TPTD administration, but the pore ratio, cortical area, and thickness were still higher compared with the 12-week vehicle administration. The 4-week TPTD administration + 8-week higher-dose IBN administration, but not the 4-week TPTD administration + 8-week lower-dose IBN administration, increased the cortical area, thickness, BMC, and volumetric BMD and decreased the pore ratio, but not the pore number or density, compared with the 4-week TPTD administration + 8-week vehicle administration. These results suggest that higher-dose IBN after TPTD therapy has a beneficial effect on the BMC, volumetric BMD, cortical area, thickness, and porosity in mature rabbits.

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Bazedoxifene (BZA) is used for the treatment of post-menopausal osteoporosis. To elucidate changes in collagen, mineralization, and structural properties and their relationship to bone strength after treatment with BZA in ovariectomized (OVX) monkeys, the levels of collagen and enzymatic immature, mature, and non-enzymatic cross-links were simultaneously examined, as well as trabecular architecture and mineralization of vertebrae. Adult female cynomolgus monkeys were divided into 4 groups (n=18 each) as follows: Sham group, OVX group, and OVX monkeys given either 0.2 or 0.5mg/kg BZA for 18 months. Collagen concentration, enzymatic and non-enzymatic pentosidine cross-links, whole fluorescent advanced glycation end products (AGEs), trabecular architecture, mineralization, and cancellous bone strength of vertebrae were analyzed. The levels of enzymatic immature and mature cross-links, bone volume (BV/TV), and trabecular thickness (Tb.Th) in BZA-treated groups were significantly higher than those in the OVX control group. In contrast, the trabecular bone pattern factor (TBPf), the structure model index (SMI), the enzymatic cross-link ratio, and the levels of pentosidine and whole AGEs in BZA-treated groups were significantly lower than those in the OVX control group. Stepwise logistic regression analysis revealed that BV/TV, Tb.Th, TbPf, and pentosidine or whole AGEs independently affected ultimate load (model R(2)=0.748, p<0.001) and breaking energy (model R(2)=0.702). Stiffness was affected by Tb.Th, enzymatic immature cross-link levels and their ratio (model R(2)=0.400). Treatment with BZA prevented OVX-induced deterioration in the total levels of immature enzymatic cross-links and AGEs accumulation and structural properties such as BV/TV, Tb.Th, and TbPf, which contribute significantly to vertebral cancellous bone strength.

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This study investigated the effects of combined teriparatide (an anabolic agent) and monthly risedronate (an anti-resorptive agent) therapy on cancellous bone mass in orchidectomized (ORX) rats. Fifty 14-week-old male Sprague-Dawley rats were randomized into five groups of ten animals each: sham-operation + vehicle; ORX + vehicle; ORX + risedronate (90 µg/kg subcutaneous, every 4 weeks); ORX + teriparatide (30 µg/kg subcutaneous, three times per week); and ORX + risedronate + teriparatide. After the 12-week experimental period, cancellous bone in the tibial proximal metaphysis was examined by static and dynamic histomorphometric analyses. ORX decreased bone volume per total volume (BV/TV) and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Risedronate increased BV/TV and Tb.N above the sham control values, while teriparatide prevented the ORX-induced decrease in BV/TV and increased trabecular width (Tb.Wi) above sham control levels. Risedronate decreased Tb.Sp below control values, while teriparatide prevented the ORX-induced increase in Tb.Sp. The combination of teriparatide and risedronate further increased BV/TV and Tb.N and decreased Tb.Sp as a result of suppression of bone remodeling, compared with teriparatide alone. These results suggest that teriparatide and monthly risedronate exert different effects on cancellous bone structure and thus have additive effects on cancellous bone mass in ORX rats.

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Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

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High-dose glucocorticoids reduce cortical bone gain in rats. The aim of the present study was to examine the effect of the intermittent administration of human parathyroid hormone (1-34) (hPTH[1-34]) on cortical bone in rats treated with high-dose prednisolone (PSL). Twenty-five female Sprague-Dawley rats (6 weeks old) were randomized into the following three groups: a vehicle administration (control) group, a PSL (10 mg/kg s.c., 5 times a week) administration group, and a PSL + hPTH(1-34) (30 µg/kg s.c., 3 times a week) administration group. After 8 weeks of treatment, the bone mineral density (BMD) of the femoral diaphysis was determined using peripheral quantitative computed tomography, and a static bone histomorphometric analysis was performed on the tibial diaphysis. PSL administration induced a decrease in the BMD of the femoral diaphysis, compared with the control group, as well as decreases in the total tissue area, cortical area, percent cortical area, and periosteal perimeter and increases in the marrow area, percent marrow area, and endocortical perimeter of the tibial diaphysis, compared with the control group. The intermittent administration of hPTH(1-34) to PSL-treated rats attenuated PSL-related changes in the BMD of the femoral diaphysis and the percent cortical area, marrow area, percent marrow area, and endocortical perimeter of the tibial diaphysis. The findings of the present study suggest that the intermittent administration of hPTH(1-34) improves cortical BMD, acts on the endocortical bone surface, and improves cortical bone geometry, in rats treated with highdose PSL.

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The purpose of the present study was to determine whether teriparatide and monthly minodronic acid would have an additive effect on cancellous bone mass in ovariectomized rats. Seven-week-old female Sprague-Dawley rats were randomized into five groups of 10 animals each, including a sham-operation+vehicle group, an ovariectomy (OVX)+vehicle group, an OVX+minodronic acid (6 µg/kgs.c., every 4 weeks) group, an OVX+teriparatide (20 µg/kgs.c., daily) group, and an OVX+minodronic acid+teriparatide group. After the 12-week experimental period, static and dynamic histomorphometric analyses were performed on the cancellous bone of the tibial proximal metaphysis. OVX decreased the bone volume per total volume (BV/TV) and the trabecular number (Tb.N) and increased the trabecular separation (Tb.Sp) as a result of increased bone remodeling. Minodronic acid prevented the OVX-induced decreases in BV/TV, while teriparatide increased the BV/TV and trabecular width (Tb.Wi) beyond the values of the sham controls. Minodronic acid prevented, but teriparatide only mitigated, the OVX-induced decrease in Tb.N, although both drugs similarly prevented the OVX-induced increase in Tb.Sp. A combination of teriparatide and minodronic acid further increased the BV/TV and Tb.N and decreased the Tb.Sp as a result of the suppression of bone remodeling, compared with teriparatide alone. These results suggest the differential effect of teriparatide and monthly minodronic acid on cancellous bone structure and the additive effect of the two drugs on cancellous bone mass in OVX rats.

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Various techniques for forming TiO(2) coatings on Ti have been investigated for the improvement of the osteoconductivity of Ti implants. However, it is not clear how the oxidizing process affects this osteoconductivity. In this study, TiO(2) coatings were prepared using the following three processes: anodizing in 0.1 M H(3)PO(4) or 0.1 M NaOH aqueous solution; thermal oxidation at 673 K for 2 h in air; and a two-step process of anodizing followed by thermal oxidation. The oxide coatings were evaluated using SEM, XRD, and XPS. The water contact angle on the TiO(2) coatings was measured as a surface property. The osteoconductivity of these samples was evaluated by measuring the contact ratio of formed hard tissue on the implanted samples (defined as the R(B-I) value) after 14 d implantation in rats' tibias. Anatase was formed by anodizing and rutile by thermal oxidation, but the difference in the TiO(2) crystal structure did not influence the osteoconductivity. Anodized TiO(2) coatings were hydrophilic, but thermally oxidized TiO(2) coatings were less hydrophilic than anodized TiO(2) coatings because they lacked in surface OH groups. The TiO(2) coating process using anodizing without thermal oxidation gave effective improvement of the osteoconductivity of Ti samples.

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Renal insufficiency induces cortical bone loss in rats. The present study examined the influence of vitamin K(2) on renal function, cortical bone mass, and bone strength in rats with renal insufficiency. Thirty male Sprague-Dawley rats (8 weeks old) were randomized by the stratified weight method to the following three groups of 10 animals each: sham operation (control), 5/6 nephrectomy, and 5/6 nephrectomy + oral vitamin K(2) (menaquinone-4, menatetrenone, 30 mg/kg, 5 days/week). Treatment was initiated 10 days after surgery. After 6 weeks of treatment, samples of serum, urine, and bone (femur and tibia) were obtained. Renal function was evaluated, bone histomorphometric analysis was performed on the tibial diaphysis, and the bone mineral density (BMD) and mechanical strength of the femoral diaphysis were determined by peripheral quantitative computed tomography and a three-point bending test, respectively. Nephrectomy induced renal dysfunction, as indicated by increased levels of serum creatinine and urea nitrogen along with a decrease of creatinine clearance; and it also decreased BMD without significantly affecting bone strength at the femoral diaphysis. Vitamin K(2) improved renal function parameters but did not significantly influence BMD at the femoral diaphysis. However, vitamin K(2) decreased the bone marrow area of the tibial diaphysis and increased the stiffness of the femoral diaphysis. These findings suggest that administration of vitamin K(2) improves renal function and increases cortical bone strength without altering BMD in rats with renal insufficiency.

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The present study examined changes of cancellous and cortical bone in rats with combined methionine-choline deficiency (MCD). In addition, the effects of vitamin K2 on cortical and cancellous bone mass and hepatic lipids were investigated in rats with MCD. Six-week-old male Sprague-Dawley rats were randomized into three groups of ten, including an age-matched control (standard diet) group, an MCD diet group, and an MCD diet+vitamin K2 (menatetrenone at 30mg/kg/d orally, 5 times a week) group. After the one-month experimental period, histomorphometric analysis was performed on cortical and cancellous bone from the tibial diaphysis and proximal metaphysis, respectively, while histological examination of the liver was performed after staining with hematoxylin and eosin and Oil Red O. MCD rats displayed weight loss, diffuse and centrilobular fatty changes of the liver, and a decrease of the cancellous bone volume per tissue volume (BV/TV) and percent cortical area (Ct Ar) as a result of decreased trabecular, periosteal, and endocortical bone formation along with increased trabecular and endocortical bone resorption. Administration of vitamin K2 to rats with MCD attenuated weight loss, accelerated the decrease of cancellous BV/TV due to an increase of bone remodeling, and ameliorated the decrease of percent Ct Ar by increasing periosteal and endocortical bone formation. Vitamin K2 administration also prevented MCD-induced diffuse fatty change of the liver. These findings suggest a beneficial effect of vitamin K2 on cortical bone mass and hepatic lipid metabolism in rats with MCD. The loss of cancellous bone mass could possibly have been due to re-distribution of minerals to cortical bone.

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The purpose of the present study was to examine the effect of vitamin K2 on cancellous and cortical bone mass in rats with streptozotocin (STZ)-induced type 1 diabetes. Twenty-seven male Sprague-Dawley rats aged 12 weeks were randomized by the weight-stratified method into the following three groups: age-matched control group, STZ + vehicle group, and STZ + vitamin K2 group. STZ (40 + 50 mg/kg) was administered intravenously twice during the initial 1-week period. Vitamin K2 (menatetrenone, 30 mg/kg) was administered orally 5 days a week. After 12 weeks of treatment, the serum glucose concentration and femoral length and weight were measured and histomorphometric analysis was performed on the cancellous and cortical bone of the distal femoral metaphysis and femoral diaphysis, respectively. STZ administration induced hyperglycemia and a decrease in femoral weight. The STZ + vehicle group also showed cancellous osteopenia due to a decrease in the number of osteoblasts/bone surface (N.Ob/BS) and the osteoblast surface (ObS)/BS without any significant changes in bone-resorption parameters, but it did not have a significant decrease in cortical bone mass. Administration of vitamin K2 to STZ-treated rats prevented the development of hyperglycemia and a decrease in femoral weight. Vitamin K2 also prevented cancellous osteopenia by inhibiting the decrease in N.Ob/BS and ObS/BS without significantly affecting bone-resorption parameters, but it did not significantly increase cortical bone mass. These results suggest that vitamin K2 has beneficial effects on glucose concentration and cancellous bone mass in rats with STZ-induced type 1 diabetes.

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Red ginger (Zingiber officinale var. Rubra) has been prescribed as an analgesic for arthritis pain in Indonesian traditional medicine. The surface color of the rhizome is purple because of the anthocyanidins in its peel. We prepared 40% ethanolic extract from dried red ginger (red ginger extract [RGE]) and evaluated its anti-inflammatory activity using acute and chronic inflammation models. In an acetic acid-induced mouse writhing model, RGE (10-100 mg/kg) suppressed both the frequency of writhing and the increase in permeability of abdominal capillaries. On the other hand, continuous treatment with RGE (10 mg/kg) significantly (P < .05) suppressed footpad edema in a rat adjuvant arthritis model. To clarify the anti-inflammatory mechanism of RGE, we examined the effect on prostaglandin (PG) and nitric oxide (NO) production from mouse leukemic monocytes (RAW264 cells) stimulated by lipopolysaccharide. RGE (3 and 10 microg/mL) significantly (P < .05) suppressed PGE(2) production, while it also suppressed NO production at 100 microg/mL. After bioassay-guided separation of RGE, we found that [6]-shogaol and gingerdiols suppressed NO production. Red dye fractions presumed to be proanthocyanidins also suppressed NO production at 100 microg/mL. Consequently, we found a potent suppressive effect of RGE on acute and chronic inflammation, and inhibition of macrophage activation seems to be involved in this anti-inflammatory effect. [6]-Shogaol, gingerdiols, and proanthocyanidins were identified as constituents that inhibited NO production.

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