Tadashi Ninomiya

MyD88 But Not TRIF Is Essential for Osteoclastogenesis Induced by Lipopolysaccharide, Diacyl Lipopeptide, and IL-1α

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll–IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88−/−) mice and TRIF-deficient (TRIF−/−) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1α stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF−/− mice, but not MyD88−/− mice. These factors stimulated receptor activator of nuclear factor-κB ligand mRNA expression in TRIF−/− osteoblasts, but not MyD88−/− osteoblasts. LPS stimulated IL-6 production in TRIF−/− osteoblasts, but not TRIF−/− macrophages. LPS and IL-1α enhanced the survival of TRIF−/− osteoclasts, but not MyD88−/− osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88−/− mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.

Evaluation of Pharmaceuticals With a Novel 50-Hour Animal Model of Bone Loss

Osteoporosis remains a major public health problem through its associated fragility fractures. Several animal models for the study of osteoporotic bone loss, such as ovariectomy (OVX) and denervation, require surgical skills and several weeks to establish. Osteoclast differentiation and activation is mediated by RANKL. Here we report the establishment of a novel and rapid bone loss model by the administration of soluble RANKL (sRANKL) to mice. Mice were injected intraperitoneally with sRANKL and used to evaluate existing anti‐osteoporosis drugs. sRANKL decreased BMD within 50 h in a dose‐dependent manner. The marked decrease in femoral trabecular BMD shown by pQCT and the 3D images obtained by μCT were indistinguishable from those observed in the OVX model. Histomorphometry showed that osteoclastic activity was significantly increased in the sRANKL‐injected mice. In addition, serum biochemical markers of bone turnover such as Ca, C‐telopeptide of type 1 collagen (CTX), and TRACP5b were also significantly increased in the sRANKL‐injected mice in a dose‐dependent manner. Bisphosphonates (BPs), selective estrogen receptor modulators (SERMs), and PTH are commonly used for the treatment of osteoporosis. We successfully evaluated the effects of anti–bone‐resorbing agents such as BPs, a SERM, and anti–RANKL‐neutralizing antibody on bone resorption in a couple of weeks. We also evaluated the effects of PTH on bone formation in 2 wk. A combination of sRANKL injections and OVX made it possible to evaluate a SERM. The sRANKL model is the simplest, fastest, and easiest of all osteoporosis models and could be useful in the evaluation of drug candidates for osteoporosis.

Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L‐glutamate. Osteoclasts secrete L‐glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+‐dependent manner. KCl‐ and ATP‐dependent secretion of L‐glutamate was absent in osteoclasts prepared from VGLUT1−/− knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L‐glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1−/− mice develop osteoporosis. Thus, in bone‐resorbing osteoclasts, L‐glutamate and bone degradation products are secreted through transcytosis and the released L‐glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

Experimental Spinal Fusion With Recombinant Human Bone Morphogenetic Protein-2 Delivered by a Synthetic Polymer and β-Tricalcium Phosphate in a Rabbit Model

Study Design. An experimental animal study to achieve posterolateral intertransverse process spine fusion with recombinant bone morphogenetic protein in combination with a new delivery system. Objective. To evaluate the efficacy of a new synthetic biodegradable bone-inducing material containing recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone-graft substitute for posterolateral intertransverse process fusion in a rabbit model. Summary of Background Data. rhBMP-2, a powerful bone-inducing cytokine, has been used as a bone graft substitute in combination with animal-derived collagen to achieve spinal fusion in animal models. However, the minimum dose of rhBMP-2 required to obtain solid posterolateral intertransverse process fusion was high on the basis of previous reports (>100 &mgr;g in rabbit models). To improve the efficacy, performance of rhBMP-2, and the safety of the delivery system for this protein, a more sophisticated system is required. Methods. To fabricate one implant for one-side L4–L5 intertransverse process fusion, &bgr;-tricalcium phosphate (&bgr;-TCP) powder (300 &mgr;g), a polymer gel (PLA-DX-PEG block copolymer; 300 &mgr;g) and rhBMP-2 (7.5, 15, or 30 &mgr;g) were mixed and manually shaped to resemble a rod. Through a posterolateral approach, two implants were placed on both sides (1 per side) by surgery so as to bridge the transverse processes of adult New Zealand white rabbits (n = 27). In control animals, implants without rhBMP or autogenous cortico-cancellous bone chips from the iliaccrest were placed in a similar location. The lumbar vertebrae were recovered 6 weeks after surgery. The posterolateral fusion was examined by manual palpation, radiography, biomechanical testing, and histology. Results. Rabbits that received 15 or 30 &mgr;g of rhBMP-2 showed consistent fusion. However, solid fusion was seen in 2 of 5 rabbits with autografting and rabbits that received 7.5 &mgr;g of rhBMP-2. Fusion was not observed in the rabbits that did not receive rhBMP-2. Conclusions. Consistent spinal fusion was obtained by implanting a biodegradable bone-inducing implant composed of &bgr;-TCP, PLA-DX-PEG, and rhBMP-2 within a period of 6 weeks. The rhBMP-2 doses required for the spinal fusion were significantly lower than those reported previously.

A local bone anabolic effect of rhFGF2-impregnated gelatin hydrogel by promoting cell proliferation and coordinating osteoblastic differentiation.

UNLABELLED The bone anabolic effect of rhFGF2 is attributed to activation of proliferation and differentiation of osteoblasts. Concomitant up-regulation of Runx2 and Bmp2 implies a coordinative function of FGF/FGFR signaling on osteoblast differentiation. INTRODUCTION Duration and tissue concentration of growth factor exposure are important in tissue regeneration. This study analyzed the availability of rhFGF2 using a sustained release gelatin hydrogel system. To examine biological aspects of the bone anabolic effect, we carried out morphological and cell proliferation assays together with gene expression analyses of osteoblast related genes induced by rhFGF2 using localizing and quantifying procedures in vivo. MATERIALS AND METHODS Bone formation induced by implantation of gelatin hydrogel impregnated with 20 microg rhFGF2 (rhFGF2(+)) onto mice maxillae was analyzed by micro computed tomography, proliferating cell nuclear antigen (PCNA) immunohistochemistry, in situ hybridization and quantitative real time polymerase chain reaction combined with laser microdissection (LMD-QPCR). RESULTS The bony maxilla was augmented to 1.58 times its original volume (p=0.002) by the implantation of rhFGF2(+) gelatin hydrogel. An increased number of PCNA-positive nuclei were observed among differentiated osteoblasts as well as undifferentiated mesenchymal cells. Fgfr1, Fgfr2 and Runx2 were shown to be co-expressed mainly in differentiated osteoblasts but also in osteoblast marker-negative spindle-shaped cells that were scattered within the outer layer of hyperplastic periosteum. LMD-QPCR revealed up-regulation of Bmp2 expression accompanied by increased transcription of Fgfr1, Fgfr2 and Runx2 by rhFGF2 controlled release. CONCLUSIONS rhFGF2 sustained release results in bone formation on the maxilla by positively regulating the expansion and differentiation of osteoblastic cells. It is suggested that FGF/FGFR signaling coordinates a bone anabolic effect by simultaneously activating RUNX2 and BMP2 pathways. The gelatin hydrogel system, which enables a sustained slow rate of release of rhFGF2 in tissue has advantages of optimizing bone regeneration.

Micro-computed tomography newly developed for in vivo small animal imaging

ObjectivesThe aim of this paper is to report a newly developed micro-computed tomography system for in vivo use.MethodsThe system was composed of a micro-focus X-ray tube and an image intensifier (I.I.), both of which rotated around the object stage. A guinea pig and a rat were examined. The anesthetized animal was set on the secure object stage. Images of the head of the guinea pig and the tibia/knee joint of the rat were taken. In addition, an image of the rat’s tail was taken. The reconstruction and the image viewing were carried out using I-View software.ResultsThe voxel matrix was 512 × 512 × 384. The voxel sizes ranged from 10 × 10 × 10 µm to 100 × 100 × 100 µm. The exposure time was 17 s, and the reconstruction time was 150 s. The head of the guinea pig and the tibia/knee joint of the rat were observed clearly under 100-µm and 30-µm voxels, respectively. The trabecular bone of the tail was also observed clearly under a 10-µm voxel.ConclusionsThe newly developed micro-computed tomography system makes it possible to obtain images of anesthetized animals set on a secure object stage. Clear bone images of the small animals could be obtained within a short time.

Intermittent PTH Administration Stimulates Pre‐Osteoblastic Proliferation Without Leading to Enhanced Bone Formation in Osteoclast‐Less c‐fos−/− Mice

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH‐driven bone anabolism. After administering PTH intermittently to wildtype and c‐fos−/− mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH‐treated wildtype mice, whereas in the osteoclast‐deficient c‐fos−/− mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double‐positive for alkaline phosphatase and BrdU, suggesting active pre‐osteoblastic proliferation. Ultrastructural examinations showed two major pre‐osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c‐fos−/− mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH‐administered wildtype mice. We concluded that the absence of osteoclasts in c‐fos−/− mice might hinder PTH‐driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.

Function of OPG as a traffic regulator for RANKL is crucial for controlled osteoclastogenesis

The amount of the receptor activator of NF‐κB ligand (RANKL) on the osteoblastic cell surface is considered to determine the magnitude of the signal input to osteoclast precursors and the degree of osteoclastogenesis. Previously, we have shown that RANKL is localized predominantly in lysosomal organelles, but little is found on the osteoblastic cell surface, and consequently, the regulated subcellular trafficking of RANKL in osteoblastic cells is important for controlled osteoclastogenesis. Here we have examined the involvement of osteoprotegerin (OPG), which is currently recognized as a decoy receptor for RANKL, in the regulation of RANKL behavior. It was suggested that OPG already makes a complex with RANKL in the Golgi apparatus and that the complex formation is necessary for RANKL sorting to the secretory lysosomes. It was also shown that each structural domain of OPG is indispensable for exerting OPG function as a traffic regulator. In particular, the latter domains of OPG, whose physiologic functions have been unclear, were indicated to sort RANKL molecules to lysosomes from the Golgi apparatus. In addition, the overexpression of RANK‐OPG chimeric protein, which retained OPG function as a decoy receptor but lost the function as a traffic regulator, inhibited endogenous OPG function as a traffic regulator selectively in osteoblastic cells and resulted in the upregulation of osteoclastogenic ability despite the increased number of decoy receptor molecules. Conclusively, OPG function as a traffic regulator for RANKL is crucial for regulating osteoclastogenesis at least as well as that as a decoy receptor.

An In Vivo 3D Micro-CT Evaluation of Tooth Movement After the Application of Different Force Magnitudes in Rat Molar

OBJECTIVE To investigate the precise longitudinal change in the periodontal ligament (PDL) space width and three-dimensional tooth movement with continuous-force magnitudes in living rats. MATERIALS AND METHODS Using nickel-titanium closed-coil springs for 28 days, 10-, 25-, 50-, and 100-g mesial force was applied to the maxillary left first molars. Micro-CT was taken in the same rat at 0, 1, 2, 3, 10, 14, and 28 days. The width of the PDL was measured in the pressure and tension sides from 0 to 3 days. Angular and linear measurements were used to evaluate molar position at day 0, 10, 14, and 28. The finite element model (FEM) was constructed to evaluate the initial stress distribution, molar displacement, and center of rotation of the molar. RESULTS The initial evaluation of PDL width showed no statistical differences among different force magnitudes. Tooth movement was registered 1 hour after force application and gradually increased with time. From day 10, greater tooth movement was observed when 10 g of force was applied. The FEM showed that the center of rotation in the molar is located in the center of five roots at the apical third of the molar roots. CONCLUSION The rats molar movement mainly consists of mesial tipping, extrusion of distal roots, intrusion of mesial root, palatal inclination, and mesial rotation. Although the initial tooth movement after the application of different force magnitudes until day 3 was not remarkably different, 10 g of force produced more tooth movement compared with heavier forces at day 28.

Prostaglandin E2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors.

Prostaglandin E2 (PGE2) has been proposed to be a potent stimulator of bone resorption. However, PGE2 itself has been shown to directly inhibit bone-resorbing activity of osteoclasts. We examined the role of PGE2 in the function of mouse osteoclasts formed in vitro. Bone marrow macrophage osteoclast precursors expressed PGE2 receptors EP1, EP2, EP3β, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by receptor activator of NF-κB ligand and macrophage colony-stimulating factor. In contrast, functional EP1 was continuously expressed in mature osteoclasts. PGE2 as well as calcitonin caused intracellular Ca2+ influx in osteoclasts. However, PGE2 and 17-phenyltrinol-PGE2 (an EP1 agonist) failed to inhibit actin-ring formation and pit formation by osteoclasts cultured on dentine slices. When EP4 was expressed in osteoclasts using an adenovirus carrying EP4 cDNA, both actin-ring and pit-forming activities of osteoclasts were inhibited in an infectious unit-dependent manner. Treatment of EP4-expressing osteoclasts with PGE2 further inhibited their actin-ring and pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are similar to those that are calcitonin receptor-mediated. Thus, osteoclast precursors down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts to escape inhibitory effects of PGE2 on bone resorption.