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The mechanistic regulation of bone mass in aged animals is poorly understood. In this study, we examined the role of SIRT6, a longevity-associated factor, in osteocytes, using mice lacking Sirt6 in Dmp-1-expressing cells (cKO mice) and the MLO-Y4 osteocyte-like cell line. cKO mice exhibited increased osteocytic expression of Sost, Fgf23 and senescence inducing gene Pai-1 and the senescence markers p16 and Il-6, decreased serum phosphate levels, and low-turnover osteopenia. The cKO phenotype was reversed in mice that were a cross of PAI-1-null mice with cKO mice. Furthermore, senescence induction in MLO-Y4 cells increased the Fgf23 and Sost mRNA expression. Sirt6 knockout and senescence induction increased HIF-1α binding to the Fgf23 enhancer sequence. Bone mass and serum phosphate levels were higher in PAI-1-null aged mice than in wild-type mice. Therefore, SIRT6 agonists or PAI-1 inhibitors may be promising therapeutic options for aging-related bone metabolism disruptions.

To maintain normal function of cartilage tissue normally, the presence of a sufficient amount of type II collagen and aggrecan is essential, and their synthesis is tightly regulated. Therefore, understanding the mechanisms that control the expression of type II collagen and aggrecan would be useful for understanding gene expression changes in diseases such as osteoarthritis. Recently, we have identified two pairs of enhancer elements, termed E1 and E2 in the type II collagen gene and Ea and Eb in the aggrecan gene. However, their different mechanisms of action remained unclear. Thus, the central aim of this study was to clarify the different transcriptional regulation mediated through each enhancer element. To this end, we established different stable reporter cell lines that express a reporter gene under the control of different enhancer elements using a silent reporter system we previously constructed. Using these cell lines, we found that dexamethasone, forskolin, and trichostatin A affect the gene expression of type II collagen and aggrecan via different enhancer elements. Moreover, we clarified that E1 and E2 enhancer activities are regulated through distinct epigenetic modifications by histone deacetylase 10 and sirtuin 6.

Sarcopenia is among the most common medical problems of the aging population worldwide and a major social concern. Here, we explored the therapeutic potential of TM5484, a novel orally available PAI-1 inhibitor, to prevent sarcopenia. The sarcopenic phenotypes of the calf muscle of 12- and 6-month-old middle-aged mice were compared. Although significant decline of isometric gastrocnemius muscle force was detected in the older untreated mice, those administered TM5484 had significantly greater calf muscle force, as determined using isometric measurements by electrical stimulation. Histological analysis indicated that cross-sectional gastrocnemius muscle fibers in untreated older mice were thinner than those in younger mice; however, TM5484-treated group showed thicker fibers than younger mice. Treatment with TM5484 for 6 months enhanced Igf1, Atrogin-1, Mt-Co1, and Chrna1 mRNA expression in the mice gastrocnemius muscle, with increased serum IGF-1 concentration. TM5484 induced dose-dependent Igf1, Atrogin-1, and Chrna1 expression in C2C12 myoblastic cells, confirming cell autonomous effect. Further, the presence of plasmin for 72 h caused significantly increased Igf1 expression in C2C12 cells. These findings suggest that oral PAI-1 inhibitors represent a promising therapeutic candidate for preventing sarcopenia progression in humans.

The aim of this study was to examine the effect of long-term locking plate fixation on the cortical bone of the canine radius. Locking compression plates were fixed to the left and right radius in dogs (n = 3). The left radius was fixed with a locking head screw (Locking Plate group, LP). The locking compression plate was compressed periosteally in the right radius using a cortex screw (Compression Plate group, CP). Radial bones from dogs that were euthanized for other purposes were collected as an untreated control group (Control group). After euthanasia at 36 weeks following plate fixation, radial bones were evaluated for bone mineral density and underwent histological analysis. Bone metabolic markers were analyzed by quantitative polymerase chain reaction (qPCR). Statistical analyses were performed for comparisons between groups. The LP group showed no significant difference in bone mineral density after plate fixation, whereas the CP group showed significantly lower bone mineral density. Histological analysis indicated that the number of osteoclasts and rate of empty lacunae increased significantly in the CP group relative to the Control and LP groups. qPCR analysis indicated increased expression of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-6, and tumor necrosis factor ligand superfamily member 11 in the CP group, whereas Runt-related transcription factor 2, an osteoblast marker, was similar in all groups. The expression of hypoxia-inducible factor-1α in the CP group was also increased relative to that in the Control and LP groups. Thus, locking plate fixation is a biologically superior fixation method that does not cause implant-induced osteoporosis in the bone in the long term.

The purpose of the present study was to confirm if proanthocyanidin-rich grape seed extract (GSE) had the ability to improve bone health such as bone loss, bone healing, and implant osseointegration (defined as the direct connection between bone tissue and an implant) in ovariectomized (OVX) animals. We demonstrated that daily oral administration of GSE prevented bone loss in the lumbar vertebrae and femur in OVX mice. In addition, osteoclastogenesis in the lumbar spine bone of OVX mice, as assessed by histological and histomorphometric analyses, was accelerated but GSE prevented this dynamization, suggesting that GSE could counteract OVX-induced accelerated osteoclastogenic activity. In rats, OVX clearly impaired the healing of defects created on the calvaria, and GSE overcame this OVX-impaired healing. In the same way, osseointegration of a tibial implant in rats was retarded by OVX, and GSE counteracted the OVX-induced poor osseointegration, likely promoting bone healing by preventing imbalanced bone turnover. These results suggest that orally administered GSE improved implant osseointegration by mitigating the impaired bone health induced by OVX as a model of estrogen deficiency.

Grape‐seed extract (GSE) is rich in proanthocyanidins (polymers of flavan‐3‐ols). GSE is well known to have various beneficial effects to health. The objective of this study was to examine the effect of dietary GSE on the intestinal microbiota in ovariectomized (OVX) mice as a model of menopause. Phylum‐level analyses using 16S rRNA‐targeted group‐specific polymerase‐chain reaction primers in fecal samples collected 8 weeks postoperatively from OVX mice revealed that the proportion of Firmicutes and Bacteroidetes populations became imbalanced as compared with that in sham‐operated control mice. That is, the ratio of Firmicutes:Bacteroidetes populations in the OVX group were increased significantly. When OVX animals were given dietary GSE, the imbalanced proportion of Firmicutes and Bacteroidetes populations was normalized to that seen in control mice. In addition, the body weight of OVX animals measured at 6 weeks postoperatively was significantly higher than that in sham‐operated control animals. Dietary GSE also prevented OVX animals from increasing body weight. Thus, we postulated that GSE can improve imbalanced populations of intestinal microbiota, leading to prevention of obesity under conditions of not only menopause but morbidity. The GSE has a great potential to be a functional food to improve dysbiosis in post‐menopausal women.

Osteoporosis is a progressive bone disease caused by an imbalance between bone resorption and formation. Recently, plasminogen activator inhibitor-1 (PAI-1) was shown to play an important role in bone metabolism using PAI-1-deficient mice. In this study, we evaluated the therapeutic benefits of novel, orally available small-molecule PAI-1 inhibitor (iPAI-1) in an estrogen deficiency-induced osteoporosis model. Eight-week-old C57BL/6J female mice were divided into three groups: a sham + vehicle (Sham), ovariectomy + vehicle (OVX + v), and OVX + iPAI-1 (OVX + i) group. iPAI-1 was administered orally each day for 6 weeks starting the day after the operation. Six weeks of iPAI-1 treatment prevented OVX-induced trabecular bone loss in both the femoral bone and lumbar spine. Bone formation activity was significantly higher in the OVX + i group than in the OVX + v and Sham groups. Unexpectedly, OVX-induced osteoclastogenesis was partially, but significantly reduced. Fluorescence-activated cell sorting analyses indicated that the number of bone marrow stromal cells was higher in the OVX + i group than that in the OVX + v group. A colony-forming unit-osteoblast assay indicated enhanced mineralized nodule formation activity in bone marrow cells isolated from iPAI-1-treated animals. Bone marrow ablation analysis indicated that the remodeled trabecular bone volume was significantly higher in the iPAI-1-treated group than that in the control group. In conclusion, our results suggest PAI-1 blockade via a small-molecule inhibitor is a new therapeutic approach for the anabolic treatment of postmenopausal osteoporosis.

Osteoarthritis (OA) is a chronic degenerative joint disorder commonly associated with metabolic syndrome. As ageing and obesity has a great impact on the initiation/severity of OA, herein we sought to investigate the involvement of Sirt6 in the crosstalk between ageing and metabolic syndrome/OA. Sirt6 haploinsufficiency in mice promoted the expression of inflammatory cytokines in the IPFP. Enhanced inflammation of the IPFP in the aged Sirt6 ± HFD group was paralleled with accelerated OA change, including osteophyte growth and chondrocyte hypertrophy. Conversely, mesenchyme-specific Sirt6-deficient mice revealed both attenuated chondrocyte hypertrophy and proteoglycan synthesis, although chondrocyte senescence was enhanced as shown in the aged WT mice. Thus Sirt6 has key roles in the relationship among ageing, metabolic syndrome, and OA.

Grape-seed extract (GSE) is rich in proanthocyanidins (polymers of flavan-3-ols). GSE is well known to have various beneficial effects to health. The objective of this study was to examine the effect of dietary GSE on the intestinal microbiota in ovariectomized (OVX) mice as a model of menopause. Phylum-level analyses using 16S rRNA-targeted group-specific polymerase-chain reaction primers in fecal samples collected 8 weeks postoperatively from OVX mice revealed that the proportion of Firmicutes and Bacteroidetes populations became imbalanced as compared with that in sham-operated control mice. That is, the ratio of Firmicutes:Bacteroidetes populations in the OVX group were increased significantly. When OVX animals were given dietary GSE, the imbalanced proportion of Firmicutes and Bacteroidetes populations was normalized to that seen in control mice. In addition, the body weight of OVX animals measured at 6 weeks postoperatively was significantly higher than that in sham-operated control animals. Dietary GSE also prevented OVX animals from increasing body weight. Thus, we postulated that GSE can improve imbalanced populations of intestinal microbiota, leading to prevention of obesity under conditions of not only menopause but morbidity.The GSE has a great potential to be a functional food to improve dysbiosis in post-menopausal women.

Purpose: To evaluate intraoperatively the effects of femoral offset and head size on stability of the hip joints in total hip arthroplasty (THA) via posterior approach. Methods: Thirty cementless THAs were included in this study. After acetabular shell and femoral broach fixation, trial reduction was repeated using a femoral neck (5- to 8-mm higher or standard offset) and a head (26 mm or 32 mm). To evaluate joint stability, range of internal rotation (IR) in hip flexion prior to posterior subluxation and range of external rotation (ER) in hip extension were measured. Results: The high-offset neck provided significantly (approximately 10°) greater range of IR to subluxation than the standard-offset neck. No hips ended in anterior subluxation by ER. The head size did not have significant effects on the stability. Conclusion: Results suggest that the 5- to 8-mm greater femoral offset might be effective in preventing instability in primary THA.

Intervertebral disc degeneration (IVDD) greatly affects the quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP because the cells disappear with age and are replaced by fibrochondrocyte-like cells. Because IVDD develops as early as within the first year of life, we used canines as a model to investigate the in vitro mechanisms underlying IVDD. The mechanism underlying age-related IVDD, however, is poorly understood. Several research groups have suggested that Wnt/β-catenin signaling plays an important role in IVDD. However, the role of Wnt/β-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/β-catenin signaling could enhance Runx2 expression in IVDD and lead to IVD calcification. Nucleus pulposus (NP) tissue was obtained from Beagle dogs after evaluation of the degeneration based on magnetic resonance imaging (MRI). Histological analysis showed that lack of Safranin-O staining, calcified area, and matrix metalloproteinase (MMP) 13-positive cells increased with progression of the degeneration. Furthermore, the levels of β-catenin- and Runx2-positive cells also increased. Real-time reverse-transcription polymerase chain reaction analysis showed that the MRI signal intensity and mRNA expression levels of β-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced β-catenin accumulation and Runx2 expression. In contrast, FH535 inhibited LiCl-induced upregulation. These results suggest that Runx2 transcript and protein expression, potentially in combination with β-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs.

Oxidative stress contributes to the pathogenesis of age-related diseases as well as bone fragility. Our previous study demonstrated that copper/zinc superoxide dismutase (Sod1)-deficient mice exhibit the induction of intracellular reactive oxygen species (ROS) and bone fragility resulting from low-turnover bone loss and impaired collagen cross-linking (Nojiri et al. J Bone Miner Res. 2011;26:2682–94). Mechanical stress also plays an important role in the maintenance of homeostasis in bone tissue. However, the molecular links between oxidative and mechanical stresses in bone tissue have not been fully elucidated. We herein report that mechanical unloading significantly increased intracellular ROS production and the specific upregulation of Sod1 in bone tissue in a tail-suspension experiment. We also reveal that Sod1 loss exacerbated bone loss via reduced osteoblastic abilities during mechanical unloading. Interestingly, we found that the administration of an antioxidant, vitamin C, significantly attenuated bone loss during unloading. These results indicate that mechanical unloading, in part, regulates bone mass via intracellular ROS generation and the Sod1 expression, suggesting that activating Sod1 may be a preventive strategy for ameliorating mechanical unloading–induced bone loss. © 2013 American Society for Bone and Mineral Research.

Objective and Background Periodontitis is an inflammatory disorder of the supporting tissue of teeth, which is composed of gingival soft tissue, cementum covering the tooth root, alveolar bone and periodontal ligament. The receptor activator of nuclear factor kappa B ligand (RANKL) is known to be an essential factor for osteoclastogenesis. Recent clinical studies indicate that levels of RANKL in the gingival crevicular fluid are increased while levels of its decoy receptor, osteoprotegerin (OPG), are decreased in patients with periodontitis. Although the gingival sulcus is composed of gingival tissue, RANKL and OPG expression in gingival epithelial cells is not fully understood. The aim of this study is to investigate the expression of RANKL and OPG in gingival tissue and which factors regulate RANKL expression in gingival epithelial cells. Material and Methods Reverse transcriptase polymerase chain reaction analysis, western blotting and immunohistochemistry were performed to confirm RANKL and OPG expression in gingival epithelial cells (GECs) and in gingival tissue. Immunostaining was also examined to confirm tumor necrosis factor (TNF)-α and TNF receptor type 1 (TNFR1) expression in gingival tissue. Ca9-22 cells, a human gingival epithelial cell line and human primary GECs were treated with TNF-α. Ca9-22 cells were treated by antibodies against TNF receptors, an inhibitor and an activator of protein kinase A (PKA) signaling and inhibitors of p38, Erk and NF-κB signaling to examine TNF-α-RANKL signaling pathways. Results RANKL mRNA and protein were expressed in GECs. Immunohistochemistry also showed RANKL expression in gingival tissue. On the other hand, the reverse transcriptase polymerase chain reaction and immunohistochemistry assay showed that GECs did not express OPG. In addition, TNF-α and TNFR1 proteins were expressed in junctional epithelium. TNF-α increased RANKL expression in GECs. TNF-α-induced RANKL expression was inhibited by an antibody against TNFR1 and an inhibitor of PKA signaling. Surprisingly, forskolin, a PKA activator, increased TNF-α-induced RANKL expression. Conclusion RANKL, TNF and TNFR1 were coexpressed in junctional epithelium of gingival tissue. TNF-α induced RANKL expression via TNFR1 and PKA signaling in GECs of junctional epithelium.

Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a(-/-) mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a(-/-) mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1(-/-) and Sema3aosx(-/-) mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin(-/-) and Sema3anestin(-/-) mice) had low bone mass, similar to Sema3a(-/-) mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin(-/-) mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin(-/-) mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a(-/-) mice, such as olfactory development, were identified in Sema3asynasin(-/-) mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a(-/-) mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

A metabolic syndrome (MetS) is accompanied by hyperuricemia, during which xanthine oxidoreductase (XOR) catalyzes the production of uric acid. In the cohort study, a correlation between uric acid concentration in the synovial fluid and osteoarthritis (OA) incidence is observed. The purpose of our study was to elucidate XOR function in terms of correlation between MetS and OA. Seven week-old male C57BL6J mice were fed normal diet (ND) or high fat diet (HFD) with or without febuxostat (FEB), a XOR inhibitor. HFD stimulated xanthine oxidase activity in the IPFP and the visceral fat. OA changes at the site of the knee joints had progressed due to HFD, but these changes were reduced upon FEB administration. IL-1β expression in the HFD group was increased in accordance with the enhancement of NLRP3 or iNOS expression in the IPFP, whereas it was inhibited by FEB administration. In the organ culture system, when the IPFP was stimulated with insulin, IL-1β expression was increased in accordance with the increase of NLRP3 expression; however, they were reduced by FEB administration. Based on the above results, we showed that inflammasome activation accompanied by an increase in XOR activity contributed to IPFP inflammation followed by OA progression.

Obesity is a risk factor for osteoarthritis (OA). To investigate the roles of increased mechanical loading in the onset of obesity-induced OA, knee joints were histologically analyzed after applying a tail suspension (TS) model to a high-fat diet (HFD)-induced OA model. Mice were divided into four groups: normal diet (ND) with normal loading (NL) group; HFD with NL group; ND with TS group; and HFD with TS group. Whole knee joints were evaluated by immunohistological analysis. The infrapatellar fat pad (IPFP) was excised and mRNA expression profiles were compared by qPCR analysis. After twelve weeks of the diet, body weight was increased by HFD in both the NL group and TS group. Upon histological analysis, the irregularity of the surface layer of articular cartilage was observed only in the NL+HFD group. Osteophyte area increased as a result of HFD in both the NL and TS groups, although osteophyte area in the TS+HFD group was smaller than that of the NL+HFD group. In the evaluation of the IPFP by qPCR, adipokines and inflammatory cytokines also increased as a result of HFD. While TGF-β increased as a result of HFD, the trend was slightly lower in the TS group, in parallel with osteophyte area. To detect apoptosis of articular chondrocytes, TUNEL staining was employed. TUNEL-positive cells were abundantly observed in the articular cartilage in the HFD mice regardless of mechanical loading. IPFP inflammation, enhanced chondrocyte apoptosis, and osteophyte formation were seen even in the TS group as a result of a HFD. In all, these data demonstrate that HFD contributed to osteophyte formation through mechanical loading dependent and independent mechanisms.

To evaluate the effects of distal design of a proximally coated femoral component on periprosthetic bone remodeling, we prospectively performed 21 one-stage bilateral total hip arthroplasties using a distally tapered and a distally cylindrical stem with the same proximal design, randomized to side. All hips showed good outcomes clinically and radiographically at the final follow-up, average of 7 years postoperatively. Cancellous condensation was always found in Gruen's zones 2 and 6 around the cylindrical stem and in regions between zones 2 and 3 and between zones 6 and 5 around the tapered stem. Bone mineral density of Gruen's zones 2 and 6 was significantly lower around the tapered stem. These results suggested more distal loading in hips with the tapered stem than in those with the cylindrical stem.

Intervertebral disc degeneration induced by mechanical compression is an important issue in spinal disorder research. In this study, the biomechanical aspect of the rat tail model was investigated. An external loading device equipped with super-elastic TiNi springs was developed to apply a precise load to the rat tail. By using this device, rat tail discs were subjected to compressive stress of 0.5 or 1.0 MPa for 2 weeks. Discs in the sham group received an attachment of the device but no loading. After the experimental period, first the intact tail with peripheral tissues (PT) such as tendon and skin and then the retrieved disc without PT were subjected to a uniaxial tension–compression test; biomechanical characteristics such as range of motion (ROM), neutral zone (NZ), and hysteresis loss (HL) were evaluated. Furthermore, the load-bearing contribution of PT in the intact tail was estimated by comparing the load–displacement curves obtained by the mechanical tests performed with and without PT. The experimental findings revealed that the continuous compressive stress induced reduction in disc thickness. The intact tail demonstrated decreases in ROM and NZ as well as increases in HL. On the other hand, the retrieved disc demonstrated increases in ROM, NZ, and HL. Further, a significant increase in the load-bearing contribution of PT was indicated. These findings suggest that the load-bearing capacity of the disc was seriously deteriorated by the application of compressive stress of 0.5 or 1.0 MPa for 2 weeks.

The causes of early degeneration and calcification of the nucleus pulposus in the chondrodystrophoid dog are poorly understood, and the underlying molecular mechanism of this process has not yet been clearly defined. Type X collagen is one of the key molecules in endochondral bone growth and development, especially matrix calcification. The relationship between type X collagen and disc degeneration and calcification in chondrodystrophoid dogs has not yet been studied. We analyzed the expression of type X collagen in degeneration and calcification of the intervertebral disc in chondrodystrophoid dogs, using type X collagen immunohistochemistry. Control intervertebral discs were collected from five dogs (4 female, 1 male, average age 1.3 years, beagle breed). Degenerated intervertebral discs were surgically removed from 11 canine patients with intervertebral disc extrusion (1 female, 10 male, average age 5.1 years, dachshund breed) in Nippon Veterinary and Animal Science University. All extruded disc samples showed hypertrophic changes and clustering of cells, typical features observed in the degenerated nucleus pulposus. The relative expression of type X collagen in the degenerated nucleus pulposus (84.3 +/- 11.0%) was significantly increased compared to the control nucleus pulposus (5.4 +/- 5.4%). Our findings suggest that type X collagen might contribute to the development of degeneration or calcification in the nucleus pulposus of the chondrodystrophoid dog.

ED-71, a novel analog of 1alpha,25-(OH)2 D3, increases bone mass to a greater extent than alfacalcidol, an 1alpha,25-(OH)2 D3 prodrug. In this study, we used a murine bone marrow ablation model to compare the effect of ED-71 on bone formation and resorption in vivo with that of 1alpha,25-(OH)2 D3. We discovered that bone matrix remodeling occurring within the first week after bone marrow ablation was enhanced by a single injection of ED-71, but not by 1alpha,25-(OH)2 D3. This enhancement was associated with an increase in bone surface. Trabecular bone resorption occurring from 1 to 2 weeks after the procedure was suppressed by a single injection of ED-71, but not 1alpha,25-(OH)2 D3, with treated mice exhibiting a reduction in osteoclast numbers, despite increases in osteoblast surface. As seen with the single injection, daily administration of ED-71 also enhanced bone modeling. Bone marrow osteoblast differentiation was also augmented by ED-71 pretreatment. Furthermore, ED-71 treatment immediately after bone marrow ablation enhanced angiogenesis within the bone marrow cavity via enhancement of VEGF(120) expression. In this paper, we clearly demonstrate that ED-71 is an orally administered small molecular weight compound with an anabolic effect on bone metabolism.

The purpose of this retrospective study is to report the incidence of osteolysis and evaluate the significance of screening computed tomography (CT) compared to plain radiography in detecting osteolysis after total hip arthroplasty with metal-on-highly cross-linked polyethylene bearings.We retrospectively reviewed 264 primary cementless total hip arthroplasties of 211 patients, 24 males, 187 females, who received postoperative screening CT scan in addition to radiography at postoperative 7-10 years (average 8.2 years). First-generation highly cross-linked polyethylene was used in all cases.On the plain radiographs, no acetabular osteolysis (0%) and two cases of femoral osteolysis (0.8%) were found in the follow-up period. No osteolysis was newly found by screening CT scan.Very low incidence of osteolysis after total hip arthroplasty with highly cross-linked polyethylene at postoperative 7-10 years was confirmed, and routine screening CT scan for detecting osteolysis in this setting was not supported from this study.

Osteoblastic differentiation is regulated by various factors, including hormones and transcription factors. Runt-related transcription factor 2 (Runx2) is an essential player in osteoblastogenesis and transactivates its molecular target by creating a protein complex with its hetero-dimeric partner core binding factor beta (Cbfb). However, the molecular regulation of Cbfb expression remains unknown. Here, we identified miR-145 as a crucial regulator of Cbfb expression. The expression of miR-145 increased during osteoblastogenesis, indicating that miR-145 works as an inhibitor of osteoblastogenesis. Stable expression of miR-145 decreased endogenous Cbfb expression and inhibited osteoblastogenesis, in cooperation with miR-34c. Furthermore, miR-145 decreased bone regeneration in vivo. Our results indicate that miR-145 physiologically regulates osteoblast differentiation and bone formation via Cbfb expression by forming a regulatory microRNA network.

Osteocytes are major bone cells that play a crucial role in maintaining the quality of and healing damage to bone tissue. The number of living osteocytes and canalicular networks declines in an age-dependent manner. However, the pathological effects of mitochondrial redox imbalances on osteocytes and bone metabolism have not been fully elucidated. We generated mice lacking mitochondrial superoxide dismutase 2 (Sod2) in osteocytes. Like an aged bone, Sod2 depletion in the osteocytes positively enhanced the production of cellular superoxide in vivo. A bone morphological analysis demonstrated that the Sod2-deficient femurs showed remarkable bone loss in an age-dependent manner. Interestingly, Sod2 loss induced markedly disorganized osteocytic canalicular networks and decreased the number of live osteocytes. Furthermore, Sod2 deficiency significantly suppressed bone formation and increased bone resorption concomitant with the upregulation of sclerostin and receptor activator of NF-κB ligand (RANKL). In vitro experiments also revealed that treatment with paraquat, a superoxide inducer in mitochondria, promoted the RANKL expression via, in part, ERK phosphorylation. These findings demonstrate that the mitochondrial superoxide induced in osteocytes by Sod2 ablation causes age-related bone loss due to the impairment of canalicular networks and bone metabolism via the deregulation of the sclerostin and RANKL expression.

Hydroxyapatite/hyaluronic acid (HAp/HyA) and hydroxyapatite/chondroitin sulfate (HAp/ChS) microparticles, which show the high adsorption ability of proteins, high biocompatibility and osteoconductivity, are potential scaffolds for a time-controlled BMP release. The present study evaluated the biocompatibility and osteoconductivity of the composites after injection into bone defect. Drilled bone holes were made at tibia and femur of Japanese white rabbits, and HAp/HyA or HAp/ChS was implanted into each bone hole using an injection syringe. After 2 and 4 weeks of implantation, rabbits were sacrificed and histological observations were conducted with HE, TRAP, and ALP staining. Histological observations revealed that HAp/ChS has superior biocompatibility compared with HAp/HyA, and 20% HAp/ChS promotes bone formation as well as osteoblast activities compared with lower ratios of HAp/ChS.

Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages, Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix-loop-helix (bHLH) type transcription factor. is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG-box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways, To address this point, we investigated scleraxis and Sox9 rnRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons, In the early stage of mesenchymal tissue development at 10.5 d.p.c., scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c.. scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage, scleraxis expression was closely associated with, but distinct from, formation of skeletal primordia, At 13.5 d.p.c., scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then. at 15.5 d.p.c., scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of scleraxis expression during embryonic development of tendon precursor cells in close association with that of So,0 expression in chondrogenic cells in skeletal tissues. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Intervertebral disc degeneration (IVDD) greatly affects the quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP because the cells disappear with age and are replaced by fibrochondrocyte-like cells. Because IVDD develops as early as within the first year of life, we used canines as a model to investigate the in vitro mechanisms underlying IVDD. The mechanism underlying age-related IVDD, however, is poorly understood. Several research groups have suggested that Wnt/β-catenin signaling plays an important role in IVDD. However, the role of Wnt/β-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/β-catenin signaling could enhance Runx2 expression in IVDD and lead to IVD calcification. Nucleus pulposus (NP) tissue was obtained from Beagle dogs after evaluation of the degeneration based on magnetic resonance imaging (MRI). Histological analysis showed that lack of Safranin-O staining, calcified area, and matrix metalloproteinase (MMP) 13-positive cells increased with progression of the degeneration. Furthermore, the levels of β-catenin- and Runx2-positive cells also increased. Real-time reverse-transcription polymerase chain reaction analysis showed that the MRI signal intensity and mRNA expression levels of β-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced β-catenin accumulation and Runx2 expression. In contrast, FH535 inhibited LiCl-induced upregulation. These results suggest that Runx2 transcript and protein expression, potentially in combination with β-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs.

Osteoclastic bone resorption requires a number of complex steps that are under the control of local regulatory molecules. Osteopontin is expressed in osteoclasts and is also present in bone matrix; however, its biological function has not been fully understood. To elucidate the role of osteopontin in the process of osteoclastic bone resorption, we conducted ectopic bone implantation experiments using wild-type and osteopontin knockout mouse. In the wild-type group, bone discs from calvariae implanted ectopically in muscle were resorbed, and their mass was reduced by 25% within 4 weeks. In contrast, the mass of the bone discs from calvariae of osteopontin knockout mice was reduced by only 5% when implanted in osteopontin knockout mice. Histological analyses indicated that the number of osteoclasts associated with the implanted bones was reduced in the osteopontin knockout mice. As osteopontin deficiency does not suppress osteoclastogenesis per se, we further examined vascularization immunohistologically and found that the number of vessels containing CD31-positive endothelial cells around the bone discs implanted in muscle was reduced in the osteopontin knockout mice. Furthermore, sc implantation assays indicated that the length and branching points of the newly formed vasculatures associated with the bone discs were also reduced in the absence of osteopontin. In this assay, tartrate-resistant acid phosphatase-positive area of the bone discs was also reduced in the osteopontin knockout mice, indicating further the link between the osteopontin-dependent vascularization and osteoclast accumulation. The bone resorption defect could be rescued by topical administration of recombinant osteopontin to the bones implanted in muscle. These observations indicate that osteopontin is required for efficient vascularization by the hemangiogenic endothelial cells and subsequent osteoclastic resorption of bones.

Sirtuin 6 (Sirt6) is a mammalian homologue of NAD+-dependent histone deacetylase Sir2. Although Sirt6−/− mice exhibit growth retardation, the role of Sirt6 in cartilage metabolism is unclear. The aim of this study was to investigate the Sirt6 signaling pathway in cartilage metabolism. Immunohistological evaluation of the tibial growth plate in Sirt6−/− mice exhibited impaired proliferation and differentiation of chondrocytes, reduced expression of Indian hedgehog (Ihh) and a senescent phenotype. When Sirt6 was knocked down in chondrocytes in vitro, expression of Ihh and its downstream genes were reduced. Impaired differentiation by Sirt6 silencing was completely rescued by administration of a Hh signal agonist. When sirtuins were activated, chondrocyte differentiation was enhanced together with activation of Ihh signal and these effects were abrogated by Sirt6 silencing. ChIP assay revealed the affinity of ATF4 to the Ihh promoter was markedly decreased by Sirt6 knockdown. These data indicate Sirt6 directly controls proliferation and differentiation of chondrocytes.

Intervertebral disc (IVD) degeneration greatly affects quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP, because the cells disappear with age and are replaced by fibrochondrocyte-like cells. However, because IVD develops as early as within the first year of life, we used canines as a model to investigate in vitro the mechanisms underlying IVD degeneration. Specifically, we evaluated the potential of a three-dimensional (3D) culture of healthy NP as an in vitro model system to investigate the mechanisms of IVD degeneration. Agarose hydrogels were populated with healthy NP cells from beagles after performing magnetic resonance imaging, and mRNA expression profiles and pericellular extracellular matrix (ECM) protein distribution were determined. After 25 days of 3D culture, there was a tendency for redifferentiation into the native NP phenotype, and mRNA levels of Col2A1, COMP, and CK18 were not significantly different from those of freshly isolated cells. Our findings suggest that long-term 3D culture promoted chondrodystrophic NP redifferentiation through reconstruction of the pericellular microenvironment. Further, lipopolysaccharide (LPS) induced expression of TNF-α, MMP3, MMP13, VEGF, and PGES mRNA in the 3D cultures, creating a molecular milieu that mimics that of degenerated NP. These results suggest that this in vitro model represents a reliable and cost-effective tool for evaluating new therapies for disc degeneration.

Oxidative stress contributes to the pathogenesis of age-related diseases as well as bone fragility. Our previous study demonstrated that copper/zinc superoxide dismutase (Sod1)-deficient mice exhibit the induction of intracellular reactive oxygen species (ROS) and bone fragility resulting from low-turnover bone loss and impaired collagen cross-linking (Nojiri et al. J Bone Miner Res. 2011;26:2682-94). Mechanical stress also plays an important role in the maintenance of homeostasis in bone tissue. However, the molecular links between oxidative and mechanical stresses in bone tissue have not been fully elucidated. We herein report that mechanical unloading significantly increased intracellular ROS production and the specific upregulation of Sod1 in bone tissue in a tail-suspension experiment. We also reveal that Sod1 loss exacerbated bone loss via reduced osteoblastic abilities during mechanical unloading. Interestingly, we found that the administration of an antioxidant, vitamin C, significantly attenuated bone loss during unloading. These results indicate that mechanical unloading, in part, regulates bone mass via intracellular ROS generation and the Sod1 expression, suggesting that activating Sod1 may be a preventive strategy for ameliorating mechanical unloading-induced bone loss.

Objective—To investigate the relationship between runt-related transcription factor 2 (RUNX2) expression in canine nucleus pulposus (NP) cells and intervertebral disk aging in chondrodystrophoid dogs. Animals—7 healthy Beagles (mean age, 35.6 months) and 11 Dachshunds with herniated disks (mean age, 61 months). Procedures—All dogs underwent MRI examination of the thoracic and lumbar vertebral column immediately before sample collection under general anesthesia. The disk center–to–CSF T2-weighted signal intensity ratio was determined for healthy Beagles. Samples of NP were obtained from nonherniated disks in healthy Beagles and from herniated disks during surgical treatment of hospitalized Dachshunds. Samples were evaluated for RUNX2 and matrix metalloproteinase 13 transcript expression via reverse transcriptase PCR assay; RUNX2 protein expression was evaluated via immunohistochemical analysis, and correlation between these variables and age of dogs was evaluated. A 3′ and 5′ rapid amplification of cDNA ends method was used to identify the RUNX2 coding region. Results—RUNX2 cDNA had > 97% conservation with the human cDNA sequence and approximately 95% conservation with the mouse cDNA sequence; RUNX2 and matrix metalloproteinase 13 mRNA expression and RUNX2 protein expression in NP cells were positively correlated with age. The disk center–to–CSF T2-weighted signal intensity ratio was negatively correlated with RUNX2 protein expression in the NP of healthy dogs. Conclusions and Clinical Relevance—Results indicated that RUNX2 mRNA and protein expression in the NP are enhanced in aging intervertebral disks in dogs.

Ossification of the Posterior Longitudinal Ligament (OPLL) is a disease that is characterized by the ectopic calcification of the ligament; however, the pathogenesis of OPLL remains to be investigated. We attempted to identify the in vivo role of Runx2, a master regulator of osteoblast differentiation and skeletal mineralization, in the pathogenesis of OPLL. The expression of Runx2 in the ligament was examined using in situ hybridization and immunohistochemistry and by monitoring the activity of a LacZ gene that was inserted into the Runx2 gene locus. To investigate the functional role of Runx2, we studied ENPP1(ttw/ttw) mice, a mouse model of OPLL, that were crossed with heterozygous Runx2 mice to decrease the expression of Runx2, and we performed histological and quantitative radiological analyses using 3D-micro CT. Runx2 was expressed in the ligament of wild-type mice. The induction of Runx2 expression preceded the development of ectopic calcification in the OPLL-like region of the ENPP1(ttw/ttw) mice. Runx2 haploinsufficiency ameliorated the development of ectopic calcification in the ENPP1(ttw/ttw) mice. Collectively, this study demonstrated that Runx2 is expressed in an OPLL-like region, and its elevation is a prerequisite for developing the complete OPLL-like phenotype in a mouse model of OPLL.

Osteoarthritis (OA) is a common disease in the elderly due to an imbalance in cartilage degradation and synthesis. Heterotopic ossification (HO) occurs when ectopic masses of endochondral bone form within the soft tissues around the joints and is triggered by inflammation of the soft tissues. Procyanidin B3 (B3) is a procyanidin dimer that is widely studied due to its high abundance in the human diet and antioxidant activity. Here, we evaluated the role of B3 isolated from grape seeds in the maintenance of chondrocytes in vitro and in vivo. We observed that B3 inhibited H(2)O(2)-induced apoptosis in primary chondrocytes, suppressed H(2)O(2)- or IL-1ß-induced nitric oxide synthase (iNOS) production, and prevented IL-1ß-induced suppression of chondrocyte differentiation marker gene expression in primary chondrocytes. Moreover, B3 treatment enhanced the early differentiation of ATDC5 cells. To examine whether B3 prevents cartilage destruction in vivo, OA was surgically induced in C57BL/6J mice followed by oral administration of B3 or vehicle control. Daily oral B3 administration protected articular cartilage from OA and prevented chondrocyte apoptosis in surgically-induced OA joints. Furthermore, B3 administration prevented heterotopic cartilage formation near the surgical region. iNOS protein expression was enhanced in the synovial tissues and the pseudocapsule around the surgical region in OA mice fed a control diet, but was reduced in mice that received B3. Together, these data indicated that in the OA model, B3 prevented OA progression and heterotopic cartilage formation, at least in a part through the suppression of iNOS. These results support the potential therapeutic benefits of B3 for treatment of human OA and heterotopic ossification.

Objective—To evaluate effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs. Animals—12 healthy female Beagles. Procedures—A mid-diaphyseal transverse osteotomy (stabilized with an intramedullary pin) of the right tibia was performed in each dog. The carprofen group (n = 6 dogs) received carprofen (2.2 mg/kg, PO, q 12 h) for 120 days; the control group (6) received no treatment. Bone healing and change in callus area were assessed radiographically over time. Dogs were euthanized 120 days after surgery, and tibiae were evaluated biomechanically and histologically. Results—The osteotomy line was not evident in the control group on radiographs obtained 120 days after surgery. In contrast, the osteotomy line was still evident in the carprofen group. Callus area was significantly less in the carprofen group, compared with the area in the control group, at 20, 30, and 60 days after surgery. At 120 days after surgery, stiffness, elastic modulus, and flexural rigidity in the carprofen group were significantly lower than corresponding values in the control group. Furthermore, histologic evaluation revealed that the cartilage area within the callus in the carprofen group was significantly greater than that in the control group. Conclusions and Clinical Relevance—Long-term administration of carprofen appeared to inhibit bone healing in dogs that underwent tibial osteotomy. We recommend caution for carprofen administration when treating fractures that have delays in healing associated with a reduction in osteogenesis as well as fractures associated with diseases that predispose animals to delays of osseous repair.

Objective To investigate the molecular mechanisms underlying particle-induced osteolysis, we focused on osteopontin (OPN), a cytokine and cell-attachment protein that is associated with macrophage chemoattractant and osteoclast activation. Methods We compared OPN protein levels in human periprosthetic osteolysis tissues with those in osteoarthritis (OA) synovial tissues. To investigate the functions of OPN during particle-induced osteolysis in vivo, titanium particles were implanted onto the calvaria of OPN-deficient mice and their wild-type (WT) littermates. Mice were killed on day 10 and evaluated immunohistologically. The effects of OPN deficiency on the secretion of inflammatory cytokines were examined using cultured bone marrow–derived macrophages (BMMs). BMMs from OPN-deficient and WT mice were cultured with titanium particles for 12 hours, and the concentrations of inflammatory cytokines in the conditioned media were measured by enzyme-linked immunosorbent assay. Results Expression of OPN protein was enhanced in human periprosthetic osteolysis tissues as compared with OA synovial tissues. In the particle-induced model of osteolysis of the calvaria, bone resorption was significantly suppressed by OPN deficiency via inhibition of osteoclastogenesis, whereas an inflammatory reaction was observed regardless of the genotype. Results of immunostaining indicated that OPN protein was highly expressed in the membrane and bone surface at the area of bone resorption in WT mice. When BMMs were exposed to titanium particles, the concentration of proinflammatory cytokines, such as tumor necrosis factor α, interleukin-1α (IL-1α), IL-1β, and IL-6, as well as chemotactic factors, such as monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α, in the conditioned medium were significantly reduced by OPN deficiency. Whereas phagocytic activity of BMMs was not attenuated by OPN deficiency, phagocytosis-mediated NF-κB activation was impaired in OPN-deficient BMMs. These data indicated that OPN was implicated in the development of particle-induced osteolysis via the orchestration of pro-/antiinflammatory cytokines secreted from macrophages. Conclusion OPN plays critical roles in wear debris–induced osteolysis, suggesting that OPN is a candidate therapeutic target for periprosthetic osteolysis.

A porous hydroxyapatite/collagen composite (HAp/Col) was developed that consists of hydroxyapatite nanocrystals and atelocollagen. In this study, cylindrical (diameter: 5 mm, height: 3 mm) porous HAp/Col implants with different pore sizes (diameter: 160 or 290 microm) were prepared, and the influences of pore size and implanted volume were evaluated using a rabbit bone defect model. In the implant groups, one or three (diameter: 5 mm, total height: 9 mm) implants were transplanted into bone holes created in the anteromedial site of the proximal tibiae, while a group without implantation was used as a control. Histological observation revealed that at two weeks after implantation, bone formation was initiated not only from the periosteum but in regions where the implants bordered on bone marrow. At four weeks, bone formation expanded from the marrow cavity side into the center of the implants, particularly in those implants with large pores. At twelve weeks, four implant groups showed repair of cortical defects and implant absorption, which was thought to be the result of natural bone remodeling mechanisms. The control group showed bone formation developed from the periosteum without bone induction in the marrow cavity, and at four weeks, the bone hole was almost healed. pQCT analysis revealed that the expansion rates of bone tissue were higher in the large-pore implant groups than in the small-pore groups. These data demonstrate the osteoconductivity of porous HAp/Col and the importance of its porous structure.

Objective Runx2 is a transcription factor that regulates chondrocyte differentiation. This study was undertaken to address the role of the different Runx proteins (Runx1, Runx2, or Runx3) in chondrocyte differentiation using chondrocyte-specific Runx–transgenic mice, and to study the importance of the QA domain of Runx2, which is involved in its transcriptional activation. Methods Runx expression was analyzed in the mouse embryo by in situ hybridization. Overexpression of Runx1, Runx2 (lacking the QA domain [ΔQA]), or Runx3 was induced in chondrocytes in vivo, to produce α(1)II-Runx1, α(1)II-Runx2ΔQA, and α(1)II-Runx3 mice, respectively, for histologic and molecular analyses. Runx expression was also examined in an experimental mouse model of mechanical stress–induced intervertebral disc (IVD) degeneration and in human patients with IVD degeneration. Results Runx1 expression was transiently observed in condensations of mesenchymal cells, whereas Runx2 and Runx3 were robustly expressed in prehypertrophic chondrocytes. Similar to α(1)II-Runx2 mice, α(1)II-Runx2ΔQA and α(1)II-Runx3 mice developed ectopic mineralization of cartilage, but this was less severe in the α(1)II-Runx2ΔQA mice. In contrast, α(1)II-Runx1 mice displayed no signs of ectopic mineralization. Surprisingly, α(1)II-Runx1 and α(1)II-Runx2 mice developed scoliosis due to IVD degeneration, characterized by an accumulation of extracellular matrix and ectopic chondrocyte hypertrophy. During mouse embryogenesis, Runx2, but not Runx1 or Runx3, was expressed in the IVDs. Moreover, both in the mouse model of IVD degeneration and in human patients with IVD degeneration, there was significant up-regulation of Runx2 expression. Conclusion Each Runx protein has a distinct, yet overlapping, role during chondrocyte differentiation. Runx2 contributes to the pathogenesis of IVD degeneration.