Abstract 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.

Melatonin has diverse effects, and has been reported to promote bone formation in addition to regulating the sleep–wake cycle. In the present study, we investigated the effects of melatonin on bone metabolism using ovariectomized (OVX) rats; a model of postmenopausal osteoporosis. Here, we focused on the differences in bone formation when melatonin was subcutaneous injected at day or early night. The OVX rats were injected with melatonin once daily (0.8 or 8 mg/head) between 11:00 to 14:00 or 18:00 to 19:30 for the day or early night, respectively, for six weeks. After completion of the injection, the femur and tibia in the OVX rats were dissected under general anesthesia and examined by quantitative computed tomography (pQCT) and histological analysis, respectively. Interestingly, the trabecular bone mineral density in the femur metaphysis of the OVX rats receiving 8 mg/head melatonin at early night was higher than those receiving melatonin during the day and they recovered to a similar level as the rats with sham treatment. In the diaphysis, the pQCT analysis results indicated that there was no significant difference in bone mineral density between the day and early night melatonin-injected OVX rats. Histological analysis of the secondary trabecular bone in the tibia of the OVX rats, revealed that the bone matrix area of the group receiving 8 mg/head melatonin at early night was higher compared with that of the day group and had a significant difference compared with OVX treatment rats. Taken together, the subcutaneous melatonin injection in OVX rats at early night was found to promote trabecular bone formation better than melatonin injection during the day. The timing of melatonin injection is a crucial factor when examining the influence of bone metabolism.

We previously reported that the oral administration of melatonin from 4 to 20 months to male mice improved femoral bone strength and bone density during the aging. Additionally, melatonin receptor, MT2, was immunologically detected in both osteoblasts and osteoclasts of the mouse femoral bone. Thus, melatonin can act on both osteoblasts and osteoclasts to maintain bone strength during the aging process. Here, we analyzed plasma calcium (Ca2+), magnesium (Mg2+), and inorganic phosphorus ([PO4]3-) in 20-month-old male mice with or without administration melatonin (15-20 mg/kg/day) in drinking water. We found that plasma Ca2+ and Mg2+ levels in melatonin-treated mice increased significantly as compared with control mice. In [PO4]3-, melatonin administration tended to increase its plasma level, but did not reach statistical significance. The potential association between these divalent ions and metabolism markers of femoral bone was also examined. In the femoral diaphysis, the plasma Ca2+ and Mg2+ concentrations were positively correlated with periosteal and endosteal circumference which were significantly associated with the Strength Strain Index. Therefore, melatonin treatment enlarged femoral diaphysis and enhanced bone strength by increasing mineral depositions. In addition, the plasma melatonin levels were significantly positive correlation with total bone density and critical thickness in the femoral diaphysis. Since we had not observed the primary trabecular bone and osteoclasts in 20-month-old mice previously, it is suggested that plasma Ca2+ and Mg2+ are not elevated due to bone resorption. The increased plasma Ca2+ and Mg2+ by melatonin may originate from the intestinal absorption of these ions since melatonin binds to the vitamin D3 receptor, its activation is known to promote the intestinal absorption of Ca2+.

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.

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.

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.

Objective: It is essential to have sufficient numbers of cells for bone regeneration. We previously reported that LIPUS exposure to rodent fractured femurs or sockets of extracted teeth recruited cells from sites remote to the lesion through peripheral blood vessels (Kumagai, Takeuchi et al, J Orthop Res 2012 and Hidaka et al, Int J Anal Bio-Sci 2015). In the present study, we tried to show LIPUS exerted its anabolic action systemically on tricalcium phosphate (β-TCP) discs implanted in the back of rats, which were not directly treated with LIPUS. Materials and Methods: Four pairs of male Sprague Dawley rats were operated on as previously described. A week later, 3 β-TCP discs were implanted in the dorsal aspect of sham operated control rats and the experimental rats. On and following the day of implantation, the 2 discs closer to the rat head only in one of the pair were exposed to LIPUS every 24 hours for 2 weeks as described. Results and Discussion: Except in one rat, which showed enlarged spleen and adrenal body, no abnormality was found. Body weight, histology, and transfer of injected fluorescence-conjugated protein from one to the other rat suggested the operation was successful. In the β-TCP discs of LIPUS-untreated side rats, we observed invaded cells and blood vessels present in numbers between those of the LIPUS-exposed and the control rats. Real-time PCR and immunohistochemistry analysis also showed consistent results. Given these findings, the systemic effect of LIPUS may be more influential than we used to assume.

Pigs are occasionally used in toxicity and pharmacology studies in European countries but rarely in Japan. On the other hand, in pharmacology studies, pigs have been used in skin irritation and absorption tests as one of the alternate animals. However, in recent years, the production of min-pigs maintained under the quality management has been established in Japan and these animals are used actively in each field. The accumulation of the basic data for mini-pigs increases steadily and pigs are used while confirming whether the projects can be correctly conducted only in pigs each time.

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

Porous materials show low Young's moduli and excellent bonding to living bone. However, the strength of such materials is often insufficient in the initial stage of implantation. Thus, the objective of this study was to increase strength and Young's modulus of porous titanium by filling the pores with a biodegradable mixture of polyglycolide (PGA) and calcium carbonate (CaCO3). The mixture was introduced into porous Ti (porosity: 60%) using a heat injection technique. When the pores were filled with PGA-CaCO3, compressive strength and Young's moduli were drastically increased and influenced by the content of CaCO3. In an animal test, unfavourable autopsy findings could not be confirmed in rats and no bone was observed in the pores of the Ti-PGA composite. Decomposition of PGA lowered the surrounding pH, it was found to inhibit bone formation in the pores. Addition of CaCO3 to PGA could suppress the decrease in pH.

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

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.

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.

Event Abstract Back to Event Histological characteristics of formed bone around porous titanium-polyglycolide composites Masato Ueda1*, Azusa Seki2 and Masahiko Ikeda1 1 Kansai University, Faculty of Chemistry, Materials and Bioengineering, Japan 2 Hamri Co., Ltd., Japan Introduction: Mechanical properties of metallic materials can be controlled by not only alloy design [1] but also constructing appropriate structure [2]. A porous material with adequate pore structure showing appropriate mechanical properties has long been sought as the ideal bone substitute [3], because it exhibits low Young’s modulus and bone ingrowth. However, bone ingrowth into the pores makes the Young’s modulus and the strength increase after implantation. Therefore, it is necessary to implant porous devices showing lower strength than that of living bone. The purpose of this study was to improve the strength of porous implants by filling biodegradable plastic into the pores. In addition, histological observation was carried out for newly formed bone around the implants. Experimental: The Polyglycolide (PGA) powder and porous Ti (porosity: 60 %) were put in a die and pressed up to 2 MPa at 210-240 ºC by using a high frequency induction heating equipment. Seven-week-old male Sprague Dawley rats (Charles River Japan Inc., Tokyo, Japan) and porous Ti (φ2 × 5 mm) were used in the present study. The specimens were implanted in the tibial metaphysis of the rats. After period of 4 or 12 weeks, histological observation of surrounding tissue was performed. Results and Discussion: Penetration of PGA to the centre could be confirmed (Fig.1). The filling fraction was measured to be 70 %. The porous Ti shows a proof stress of about 40 MPa. The compressive strength drastically increased by filling the pores with PGA, it reached more than 100 MPa. This strength is comparable to that of cortical bone. Figure 1 – Appearance (a) and cross-section (b) of porous Ti-PGA composite. Unfavourable autopsy findings such as suppuration, bleeding, hyperplasia of the connective tissue could not be confirmed. Villanueva Goldner staining allows a clear observation of newly formed bone in relevant parts; it stains the osteoid red and the mineralised bone green (Fig. 2). In the porous Ti, mineralised bone was almost completely conducted to the pores from the cortical bone. Osteoid was also formed in contact portion of cancellous bone or bone marrow. In contrast, nothing was observed in the pores of the Ti-PGA composite. This tendency was also observed in the samples for 4 weeks. PGA makes pH of surrounding region low by decomposition. It is important to control decomposition behaviour of PGA for controlling bone formation in the Ti-PGA composites. Figure 2 – Light microscopy histological sections in (a) porous Ti and (b) porous Ti-PGA composite after 12 weeks. Conclusions: 1. Compressive strength of porous Ti was drastically improved by filling the pores with PGA. It was comparable to that of cortical bone. 2. In the porous Ti, mineralised bone was almost completely conducted to the pores from the cortical bone after 12 weeks. Complexed PGA inhibited the bone formation in the pores of porous Ti.It is important to control decomposition behaviour of PGA. The authors would like to thank the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Grants-in-Aid for Scientific Research (Grant no: 26630355) for providing financial support to this project.References:[1] M. Niinomi; M. Nakai; J. Hieda Acta Biomaterialia 2012, 8, 3888.[2] N. Nomura; T. Kohama; I. H. Oh; S. Hanada; A. Chiba; M. Kanehira; K. Sasaki Mater. Sci. Eng. C 2005, 25, 330.[3] M. Takemoto; S. Fujibayashi; M. Neo; J. Suzuki; T. Kokubo; T. Nakamura Biomaterials 2005, 30, 6014. Keywords: in vivo, Implant, mechanical property, Biodegradable material Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Composites: polymeric, ceramic and metallic Citation: Ueda M, Seki A and Ikeda M (2016). Histological characteristics of formed bone around porous titanium-polyglycolide composites. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01383 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Masato Ueda, Kansai University, Faculty of Chemistry, Materials and Bioengineering, Suita, Japan, Email1 Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Masato Ueda Azusa Seki Masahiko Ikeda Google Masato Ueda Azusa Seki Masahiko Ikeda Google Scholar Masato Ueda Azusa Seki Masahiko Ikeda PubMed Masato Ueda Azusa Seki Masahiko Ikeda Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Porous materials show low Young's moduli and excellent bonding to living bone. However, the strength of such materials is often insufficient in the initial stage of implantation. Thus, the objective of this study was to increase the strength of porous titanium by filling the pores with polyglycolide (PGA), a biodegradable plastic. PGA powder was prepared via the thermal decomposition of sodium chloroacetate at 433 K. The PGA was then introduced into the pores of porous Ti (porosity: 60%) using two methods: (i) centrifugal packing and heating and (ii) heat injection. In the latter method, almost all pores were filled by PGA; the filling fraction was measured to be 65-85% regardless of the injection temperature. When the pores in the porous Ti were filled with PGA, the compressive strength increased drastically from 40 to 100 MPa. The increased strength is comparable to that of cortical bone. In addition, the strength increased with increasing injection temperature. In an animal test, unfavourable autopsy findings, such as suppuration, bleeding, and hyperplasia of the connective tissue, could not be confirmed in rats and no bone was observed in the pores of the Ti-PGA composite. Decomposition of PGA lowered the surrounding pH, it was found to inhibit bone formation in the pores of the porous Ti. It is important to control the decomposition rate of PGA.

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.

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 18months. 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 R2=0.748, p<0.001) and breaking energy (model R2=0.702). Stiffness was affected by Tb.Th, enzymatic immature cross-link levels and their ratio (model R2=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.