A PAI-1 antagonist ameliorates hypophosphatemia in the Hyp vitamin D-resistant rickets model mouse.

Congenital fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets/osteomalacia is a rare bone metabolism disorder characterized by hypophosphatemia and caused by genetic abnormalities that result in excessive secretion of FGF23. Hyp mice are a model of X-linked hypophosphatemia (XLH) caused by deletion of the PHEX gene and excessive production of FGF23. The purpose of this study was to investigate the potential of TM5614 as a therapeutic agent for the treatment of congenital FGF23-related hypophosphatemic rickets and osteomalacia in humans by administering TM5614 to Hyp mice and examining its curative effect on hypophosphatemia. After a single oral administration of TM5614 10 mg·kg-1 to female Hyp mice starting at 17 weeks of age, the serum phosphate concentration increased with a peak at 6 h after administration. ELISA confirmed that TM5614 administration decreased the intact FGF23 concentration in the blood. Expression of 25-hydroxyvitamin D-1α-hydroxylase protein encoded by Cyp27b1 mRNA in the kidney was suppressed in Hyp mice, and treatment with 10 mg·kg-1 of TM5614 normalized the expression of 25-hydroxyvitamin D-1α-hydroxylase protein and Cyp27b1 mRNA in the kidneys of these mice. Our data indicate that oral administration of TM5614 ameliorates hypophosphatemia in Hyp mice, suggesting that TM5614 may be an effective treatment for congenital FGF23-related hypophosphatemic rickets and osteomalacia.

Osteolytic Bone Loss and Skeletal Deformities in a Mouse Model for Early-Onset Paget's Disease of Bone with PFN1 Mutation Are Treatable by Alendronate.

A novel osteolytic disorder due to PFN1 mutation was discovered recently as early-onset Paget's disease of bone (PDB). Bone loss and pain in adult PDB patients have been treated using bisphosphonates. However, therapeutic strategies for this specific disorder have not been established. Here, we evaluated the efficiency of alendronate (ALN) on a mutant mouse line, recapitulating this disorder. Five-week-old conditional osteoclast-specific Pfn1-deficient mice (Pfn1-cKOOCL) and control littermates (33 females and 22 males) were injected with ALN (0.1 mg/kg) or vehicle twice weekly until 8 weeks of age. After euthanizing, bone histomorphometric parameters and skeletal deformities were analyzed using 3D µCT images and histological sections. Three weeks of ALN administration significantly improved bone mass at the distal femur, L3 vertebra, and nose in Pfn1-cKOOCL mice. Histologically increased osteoclasts with expanded distribution in the distal femur were normalized in these mice. Geometric bone shape analysis revealed a partial recovery from the distal femur deformity. A therapeutic dose of ALN from 5 to 8 weeks of age significantly improved systemic bone loss in Pfn1-cKOOCL mice and femoral bone deformity. Our study suggests that preventive treatment of bony deformity in early-onset PDB is feasible.

SIRT6-PAI-1 axis is a promising therapeutic target in aging-related bone metabolic disruption.

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.

Effect of CD44 signal axis in the gain of mesenchymal stem cell surface antigens from synovial fibroblasts in vitro.

Tissue-residing mesenchymal stromal/stem cells (MSCs) have multipotent characteristics that are important for adult tissue homeostasis and tissue regeneration after injury. We previously reported that fibroblastic cells isolated from the synovial membrane in the knee joint give rise to cells with MSC characteristics in a two-dimensional culture. To explore the molecular mechanisms underlying these hyperplastic properties, we performed time-course surface antigen expression analyses during in vitro culture. Cells freshly isolated from the synovial membrane rarely contained cells that met the criteria (CD45-CD73+CD90+CD105+). However, the number of cells expressing MSC antigens increased on day 7. Flow cytometric analysis indicated that cells positive for either CD73 or CD90 were specifically derived from cells positive for CD44. CD44 expression was upregulated during culture, and CD105+ cells were specifically derived from the CD44 highly expressing cells. In addition, depletion of hyaluronic acid (HA), a major ligand of CD44, decreased the number of CD105+ cells, whereas supplementation with HA increased their number. These data suggest that intracellular signals activated by CD44 play an important role in the formation and/or maintenance of MSCs.

Short cytoplasmic isoform of IL1R1/CD121a mediates IL1ß induced proliferation of synovium-derived mesenchymal stem/stromal cells through ERK1/2 pathway.

Objectives: IL1ß enhances proliferation of synovial mesenchymal stem/stromal cells (synMSCs) although they don't express its receptor, IL1R1/CD121a, on the cell surface. This study was aimed to elucidate the underlying mechanisms of IL1ß-mediated growth promotion. Methods: Human synMSCs were isolated from the suprapatellar synovial membrane. Cell proliferation was measured by MTT. Flowcytometric analyses were performed for surface antigen expression. Intracellular signaling pathway was analyzed by western blotting, immunocytochemistry and Q-PCR. Results: IL1ß enhanced proliferation through IL1R1/CD121a because IL1 receptor antagonist (IL1Ra) completely inhibited it. Expression analyses indicated that a short isoform of IL1R1/CD121a is expressed in synMSCs. Immunocytochemistry indicated that IL1R1/CD121a was majorly localized to the cytoplasm. Western blotting indicated that IL1ß induced delayed timing of the ERK1/2 phosphorylation and IκBα degradation in synMSCs. Q-PCR analyses for IL1ß-target genes indicated that cyclin D was specifically downregulated by a MAPK/ERK inhibitor, U0126, but not by a NFκB inhibitor, TPCA-1. In contrast, the expression of inflammatory cytokines such as IL1α and IL6 are significantly decreased by TPCA-1 but less effectively decreased by U0126. Conclusion: Our data indicated that the cytoplasmic IL1R1/CD121a transduced IL1ß signal in synMSCs. And the growth-promoting effect of IL1ß can be separated from its inflammatory cytokine-inducing function in synMSCs.

Effects of long-term plate fixation with different fixation modes on the radial cortical bone in dogs.

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.

A novel PAI-1 inhibitor prevents ageing-related muscle fiber atrophy.

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.

Proanthocyanidin-rich grape seed extract improves bone loss, bone healing, and implant osseointegration in ovariectomized animals.

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.

Expression of type II collagen and aggrecan genes is regulated through distinct epigenetic modifications of their multiple enhancer elements.

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.

A small-molecule PAI-1 inhibitor prevents bone loss by stimulating bone formation in a murine estrogen deficiency-induced osteoporosis model.

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.

Proanthocyanidin-Rich Grape Seed Extract Modulates Intestinal Microbiota in Ovariectomized Mice.

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. PRACTICAL APPLICATION: The GSE has a great potential to be a functional food to improve dysbiosis in post-menopausal women.

Intraoperative evaluation of the effects of femoral component offset and head size on joint stability in total hip arthroplasty.

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.

Pleiotropic Functions of High Fat Diet in the Etiology of Osteoarthritis.

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-ß 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.

Xanthine oxidoreductase activation is implicated in the onset of metabolic arthritis.

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-1ß 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-1ß 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.

Limited significance of screening computed tomography after cementless total hip arthroplasty with highly cross-linked polyethylene at 7-10 years of follow-up.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

MicroRNA-145 regulates osteoblastic differentiation by targeting the transcription factor Cbfb.

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.

Pivotal role of Sirt6 in the crosstalk among ageing, metabolic syndrome and osteoarthritis.

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.

Mitochondrial superoxide in osteocytes perturbs canalicular networks in the setting of age-related osteoporosis.

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.

Enhancement of Runx2 expression is potentially linked to ß-catenin accumulation in canine intervertebral disc degeneration.

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/ß-catenin signaling plays an important role in IVDD. However, the role of Wnt/ß-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/ß-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 ß-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 ß-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced ß-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 ß-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs.