Osteoclast Expression Research Articles

Signal crosstalk between cytokine RANKL and AhR signaling in osteoclasts controls bone homeostasis

Abstract The nuclear receptor aryl hydrocarbon receptor (AhR) plays a crucial role in receptor activator of nuclear factor (NF)-kB ligand (RANKL)-mediated osteoclastogenesis. A considerable amount of evidence reveals that AhR is also a key modulator of skeletal remodeling. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than the expression of signature osteoclast genes such as NFATc1, c-Src, integrin avb3, and cathepsin K. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and mitogen-activated protein kinase as well as NF-kB, while their response to the macrophage colony-stimulating factor remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in a RANKL/c-Fos-dependent manner. Further, ligand activation of AhR by the smoke toxin benzo [a] pyrene (BaP) accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest the new role that the RANKL-AhR-c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone.

Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

The axonal guidance proteins semaphorin (Sema)4D and Sema3A play important roles in communication between osteoclasts and osteoblasts. As stimulation of adenosine A2A receptors (A2AR) regulates both osteoclast and osteoblast function, we asked whether A2AR regulates both osteoclast and osteoblast expression of Semas. In vivo bone formation and Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1 protein expression were studied in a murine model of wear particle-induced osteolysis. Osteoclast/osteoblast differentiation were studied in vitro as the number of tartrate-resistant acid phosphatase+/Alizarin Red+ cells after challenge with CGS21680 (A2AR agonist, 1 µM) or ZM241385 (A2AR antagonist, 1 µM), with or without Sema4D or Sema3A (10 ng/ml). Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1, and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) expression was studied by RT-PCR and Western blot. β-Catenin activation and cytoskeleton changes were studied by fluorescence microscopy and Western blot. In mice with wear particles implanted over the calvaria, CGS21680 treatment increased bone formation in vivo, reduced Sema4D, and increased Sema3A expression compared with mice with wear particle-induced osteolysis treated with vehicle alone. During osteoclast differentiation, CGS21680 abrogated RANKL-induced Sema4D mRNA expression (1.3 ± 0.3- vs. 2.5 ± 0.1-fold change, P < 0.001, n = 4). PlexinA1, but not Neuropilin-1, mRNA was enhanced by CGS21680 treatment. CGS21680 enhanced Sema3A mRNA expression during osteoblast differentiation (8.7 ± 0.2-fold increase, P < 0.001, n = 4); PlexinB1 mRNA was increased 2-fold during osteoblast differentiation and was not altered by CGS21680. Similar changes were observed at the protein level. CGS21680 decreased RANKL, increased OPG, and increased total/nuclear β-catenin expression in osteoblasts. Sema4D increased Ras homolog gene family, member A phosphorylation and focal adhesion kinase activation in osteoclast precursors, and CGS21680 abrogated these effects. In summary, A2AR activation diminishes secretion of Sema4D by osteoclasts, inhibits Sema4D-mediated osteoclast activation, and enhances secretion of Sema3A by osteoblasts, increasing osteoblast differentiation and diminishing inflammatory osteolysis.-Mediero, A., Wilder, T., Shah, L., Cronstein, B. N. Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

Local administration of nuclear factor of activated T cells (NFAT) c1 inhibitor to suppress early resorption and inflammation induced by bone morphogenetic protein-2.

Nuclear factor of activated T cells (NFAT)-c1 is known as a key regulator in osteoclast differentiation and immune response. This study is a follow-up to our previous study showing the antiresorptive activity of VIVIT, a peptide type NFATc1 inhibitor, using absorbable collagen sponge (ACS). This study aimed to investigate the effective concentration range of local VIVIT that suppresses early excessive osteoclast activation and inflammation induced by high-dose recombinant human bone morphogenetic protein (rhBMP)-2 and concomitantly enhances bone healing in a rat critical-sized calvaria defect model. High-dose rhBMP-2 (40 μg/defect) alone significantly increased in vivo osteoclast activation and expression of the inflammatory cytokines interleukin-1β and transforming necrosis factor-α on the scaffold at 7 days after surgery. However, rhBMP-2 had no direct effect on osteoclast activation in vitro. Osteoclast activation by rhBMP-2 was significantly suppressed by combined treatment with VIVIT at concentrations of 75 and 150 μM, but not at 15 μM, whereas suppression of inflammation occurred at all doses of VIVIT. Microcomputed tomography at 4 and 8 weeks after implantation revealed that the combination of rhBMP-2 and VIVIT at 75 μM VIVIT led to a greater bone fraction at the initial defect area, compared with rhBMP-2 alone. These findings revealed that local administration of VIVIT at certain concentrations has multiple positive effects that weaken early excessive osteoimmunological responses and enhance bone healing after rhBMP-2 administration. VIVIT has the potential to expand the therapeutic area of high-dose rhBMP-2 therapy to inflammatory bone loss. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1299-1310, 2018.

Umbilical cord extracts improve osteoporotic abnormalities of bone marrow-derived mesenchymal stem cells and promote their therapeutic effects on ovariectomised rats

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the most valuable source of autologous cells for transplantation and tissue regeneration to treat osteoporosis. Although BM-MSCs are the primary cells responsible for maintaining bone metabolism and homeostasis, their regenerative ability may be attenuated in postmenopausal osteoporosis patients. Therefore, we first examined potential abnormalities of BM-MSCs in an oestrogen-deficient rat model constructed by ovariectomy (OVX-MSCs). Cell proliferation, mobilisation, and regulation of osteoclasts were downregulated in OVX-MSCs. Moreover, therapeutic effects of OVX-MSCs were decreased in OVX rats. Accordingly, we developed a new activator for BM-MSCs using human umbilical cord extracts, Wharton’s jelly extract supernatant (WJS), which improved cell proliferation, mobilisation and suppressive effects on activated osteoclasts in OVX-MSCs. Bone volume, RANK and TRACP expression of osteoclasts, as well as proinflammatory cytokine expression in bone tissues, were ameliorated by OVX-MSCs activated with WJS (OVX-MSCs-WJ) in OVX rats. Fusion and bone resorption activity of osteoclasts were suppressed in macrophage-induced and primary mouse bone marrow cell-induced osteoclasts via suppression of osteoclast-specific genes, such as Nfatc1, Clcn7, Atp6i and Dc-stamp, by co-culture with OVX-MSCs-WJ in vitro. In this study, we developed a new activator, WJS, which improved the functional abnormalities and therapeutic effects of BM-MSCs on postmenopausal osteoporosis.

Expression of Osteoprotegrin and Osteoclast Level in Chronic Apical Periodontitis Induced with East Java Propolis Extract.

Introduction:The objective of this animal study was to promote East Java propolis as a potential natural intracanal medicament for periapical chronic apical periodontitis bone resorption through evaluating the expression of osteoprotegrin (OPG) and osteoclast level.Methods and Materials:Propolis extract was produced using a maceration procedure. Thirty Wistar rats were divided into three groups. In group I, the control group, the first upper right molar constituted a healthy tooth. In group II, containing rodents with experimentally chronicapical periodontitis, infection with Enterococcus faecalis ATCC29212 106 CFU was performed. In group III, the treatment group, after being injected with E faecalis, 10 μL propolis was applied. It required 21 days to induce post-pulp chronic apical periodontitis infection. The rats were euthanised for immunohistochemical examination in order to measure the expression of OPG and to count histologically the number of osteoclast. Result:The expression of OPG and osteoclast constituted 17.5±1.58 and 6.4±0.96 in group I, 10±2 and 16.2±1.31 in group II and 17±1.69 and 7.5±1.08 in group III. Group I presented the highest level of OPG expression but the lowest level of osteoclast expression. There were significant differences between groups II and III and group I regarding OPG and osteoclast expression (P<0.05). Conclusion:East Java Propolis was a potential intracanal medicament promoting an increase in osteoprotegerin expression and a decrease in the number of osteoclasts thereby inhibiting osteoclastogenesis.

Resolvin E1 Inhibits Osteoclastogenesis and Bone Resorption by Suppressing IL-17-induced RANKL Expression in Osteoblasts and RANKL-induced Osteoclast Differentiation

Resolvin E1 (RvE1) derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid is known to be a potent pro-resolving lipid mediator that prevents chronic inflammation and osteoclastogenesis. We investigated the inhibitory effects of RvE1 on osteoclastogenesis and bone resorption to clarify its therapeutic potential for rheumatoid arthritis (RA). Receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was assessed with tartrate-resistant acid phosphatase staining. RANKL-induced bone resorption was assessed by the measurement of pit formation using calcium phosphate-labeled fluorescent polyanionic molecules in RAW264.7 cells as osteoclast precursors. The effects of RvE1 on the RANKL-induced mRNA expression of osteoclast-specific genes and transcriptional factors such as c-fos and nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells were measured by quantitative real-time PCR. The distribution of NFATc1 induced by RANKL was evaluated by immunofluorescence staining in RAW264.7 cells. To analyze the mechanism of the inhibitory effect of RvE1 on osteoclastogenesis, we measured IL-17-induced RANKL mRNA expression in MC3T3-E1 osteoblast cells treated with RvE1 using quantitative real-time PCR and determined the level of prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay. RvE1 significantly suppressed RANKL-induced osteoclast differentiation and bone resorption. RvE1 inhibited the RANKL-induced mRNA expression of osteoclast-specific genes along with the transcription factors NFATc1 and c-fos. Moreover, NFATc1 translocation from the cytoplasm to the nucleus of RAW264.7 cells was suppressed following RvE1 treatment. RvE1 also inhibited IL-17-induced RANKL mRNA expression and PGE2 production in MC3T3-E1 cells. RvE1 inhibited osteoclastogenesis and bone resorption by suppressing RANKL-induced NFATc1 and c-fos expression in osteoclasts and IL-17-induced RANKL expression through the autocrine action of PGE2 in osteoblasts. Our data suggest RvE1 as a new therapeutic target of RA.

8-Nitro-cGMP is a promoter of osteoclast differentiation induced by RANKL

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.

Icariin ameliorates dexamethasone‑induced bone deterioration in an experimental mouse model via activation of microRNA‑186 inhibition of cathepsin K.

The present study aimed to investigate bone deterioration in glucocorticoid-induced osteoporosis (GIOP) mice, and the anti-osteoporosis effect and underlying molecular mechanism of icariin. Dexamethasone (DSM) treatment was demonstrated to facilitate the induction of hypercalciuria in GIOP mice. Icariin treatment reversed the dexamethasone (DXM)-induced disequilibrium of calcium homeostasis and bone resorption, and increased serum alkaline phosphatase, tartrate resistant acid phosphatase, osteocalcin and deoxypyridinoline. Haematoxylin and eosin staining revealed an increase in disconnections and separation in the trabecular bone network of the tibial proximal metaphysis, in the GIOP group. Icariin treatment reversed the DXM-induced trabecular deleterious effects, and stimulated bone remodeling in GIOP mice. Furthermore, the results demonstrated that the mRNA and protein expression of cathepsin K were significantly increased in GIOP mice, compared with the control group. Icariin treatment may suppress the expression of cathepsin K in the tibia of GIOP mice. The levels of microRNA (miR)-186 were markedly reduced in the tibia of GIOP mice compared with control group; however, this was inhibited by icariin treatment. Bioinformatics analysis demonstrated that miR-186 regulates cathepsin K via binding to the upstream 3′-untranslated region. Furthermore, transfection with miR-186 mimics resulted in inhibition of cathepsin K expression, whereas miR-186 inhibitors facilitated cathepsin K expression in osteoclasts. In conclusion, the present study demonstrated the protective effects of icariin against bone deteriorations in the experimental GIOP mice, and the underlying mechanism was mediated, at least partially, via activation of miR-186-mediated suppression of cathepsin K. These results provide evidence to support the use of icariin as a therapeutic approach in the management of glucocorticoid-induced bone loss, and the disequilibrium of calcium homeostasis.

Inhibitory effect of quercetin on titanium particle-induced endoplasmic reticulum stress (ERS)-related apoptosis and in vivoosteolysis.

Wear particle induced periprosthetic osteolysis is the main cause of aseptic loosening of orthopedic implants. The aim of the present study is to determine the protective effect of quercetin (QUE) against titanium (Ti) particle induced endoplasmic reticulum stress (ERS) related apoptosis and osteolysis. In the present study, RAW264.7 cells were pretreated with different concentrations (40, 80, and 160 μmol/l) of QUE for 30 min and then treated with Ti particle (5 mg/ml) for 24 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC Apoptosis Detection Kit, respectively. Protein and mRNA expressions of ERS-related genes were examined by Western blot and real-time PCR, respectively. The release of inflammatory cytokines was detected by ELISA. Then, a mouse calvarial osteolysis model was established. Histological sections of calvaria were stained with Hematoxylin-Eosin (H&E) or tartrate-resistant acid phosphatase (TRAP). The results showed that Ti particle reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of Ti particle were dramatically inhibited by QUE pretreatment. Interestingly, we found that QUE also significantly reduced Ti particle induced up-regulation of the expression levels of protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1), glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and caspase-3 and enhanced the down-regulation of Bcl-2. In addition, QUE decreased Ti particle-induced inflammatory cytokines release from RAW264.7 cells. Moreover, treatment with QUE markedly decreased osteoclast number. In a mouse calvarial osteolysis model, QUE inhibited Ti particle induced osteolysis in vivo by inhibiting osteoclast formation and expressions of ERS-related genes. In conclusion, QUE can protect RAW264.7 cells from Ti particle induced ERS-related apoptosis and suppress calvarial osteolysis in vivo.

The synergistic effects of Sr and Si bioactive ions on osteogenesis, osteoclastogenesis and angiogenesis for osteoporotic bone regeneration.

Bioactive ions released from bioceramics play important roles for bone regeneration; however, it is unclear how each of ionic compositions in complex bioceramics exerts its specific effect on bone regeneration. The aim of present study is to elucidate the functional effects of Sr and Si ions in complex bioceramics on the regeneration of osteoporotic bone. A model bioceramic with Sr and Si-containing components (SMS) was successfully fabricated and the effects of ionic products from SMS bioceramics on the osteogenic, osteoclastic and angiogenic differentiation of rBMSCs-OVX and RANKL-induced osteoclasts were investigated. The results showed that SMS bioceramics could enhance ALP activity and expression of Col 1, OCN, Runx2 and angiogenic factors including VEGF and Ang-1. SMS bioceramics not only rebalanced the ratio of OPG/RANKL of OVX-BMSCs at early stage, but also repressed RANKL-induced osteoclast formation and expression of TRAP, DC-STAMP, V-ATPase a3, and NFATc1. The synergistic effects of Sr and Si ions were further investigated as compared with the similar concentration of Sr and Si ions alone. It was found that Sr and Si ions possessed synergistic effects on osteogenesis, osteoclastogenesis and angiogenesis, attributed to the dominant effects of Sr ions on enhancing angiogenesis and repressing osteoclastogenesis, and the dominant effects of Si ions on stimulating osteogenesis. The in vivo study using critical-size mandibular defects of OVX rat models showed that SMS bioceramics could significantly enhance bone formation and mineralization as compared with β-TCP bioceramics. It is suggested that SMS bioceramics may be a promising biomaterial for osteoporotic bone regeneration. To our knowledge, this is the first time to elucidate the specific effect of each ion from bioceramics on osteogenesis, osteoclastogenesis and angiogenesis for osteoporotic bone regeneration, paving the way to design functional biomaterials with complex compositions for tissue engineering and regenerative medicine.

Coil poly(ethylene glycol)-brush-like poly(L-lysine) block copolymers as efficient nanocarriers for insulin delivery

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.

Abstract 5803: Role of Neuropilin 2 in osteoclasts promoting prostate cancer bone metastasis

Abstract Background: Bone metastasis is one of the major clinical concerns that causes skeletal related malignancies and increased mortality. Bone is one of the preferred sites for metastatic prostate cancer. The tumor cells interact with bone cells (osteoblasts and osteoclasts) resulting in an imbalance in the bone homeostasis causing metastatic bone disease. Unlike other cancers, the bone maturation promoted by metastatic prostate cancer (PCa) leads to increased osteoblastic activity resulting in osteoblastic bone lesions. However, bone resorption promoted by PCa cells precedes bone production leading to release of growth factors that aid PCa cells to develop into an overt metastasis. Our preliminary data indicated a non-tyrosine kinase receptor Neuropilin 2 (NRP2) is expressed in osteoclasts (OC) induced by PCa cells. The objective of our current study is to study the role of NRP2 in PCa-induced OCs. We hypothesize that PCa-induced NRP2 expression in OC is necessary for low osteolytic activity and that it negatively regulates the OCs leading to PCa bone metastasis. Methods: Mouse OC precursors were isolated from bone marrow of C57BL/6 mice and differentiated into OCs under conditions of RANKL and M-CSF and in conditioned medium (CM) collected from PCa cell line LNCaP C4-2B (promotes high osteoblastic and low osteoclastic activity) and PC3 (predominantly osteoclastic activity) to mimic the conditions in normal bone and PCa bone metastasis. NRP2 expression at protein and mRNA was evaluated. TRAP staining and activity were conducted to confirm the differentiation of OCs. Results &amp; Discussion: We observed that NRP2 was expressed in OCs induced under standard conditions and in PC3 and C4-2B CM. TRAP staining and activity confirmed the differentiation of OCs under these conditions. Interestingly, depletion of NRP2 and treatment either in standard conditions or C4-2B CM exhibited a drastic increase in osteoclastogenesis. However, NRP2-depleted OC precursors when treated with PC3 CM showed no change in osteoclastogenesis. These findings advocate a role of NRP2 in inhibiting osteoclastic activity in PCa bone metastasis and osteolytic PCa evades NRP2 inhibition. Protein analysis of the time course of NRP2 in OC in PC3 CM and C4-2B CM showed a differential NRP2 expression. This difference in the NRP2 levels can be impacted at either transcription or post-translational level. No change was observed in the NRP2 expression at transcriptional level in both the conditions suggesting that this difference can be due to either translation or rapid degradation of NRP2. Using in-vitro and transgenic mice, we will examine how PC3 CM-induced OCs escapes the inhibition of NRP2. We will also determine whether NRP2 expression regulates the transcription of genes involved in OC differentiation and activation. Together, this approach will elucidate the role of NRP2 axis on OCs in promoting PCa-induced bone metastasis and will aid in determining whether NRP2 axis can be a therapeutic target. Note: This abstract was not presented at the meeting. Citation Format: Navatha shree Polavaram, Arup Bag, Sohini Roy, Samikshan Dutta, Kaustubh Datta. Role of Neuropilin 2 in osteoclasts promoting prostate cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5803. doi:10.1158/1538-7445.AM2017-5803

Osteoclast profile of medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy: a comparison with osteoradionecrosis and osteomyelitis

BackgroundThe medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy [MRONJ (BP)] is characterized by non-healing exposed bone in the maxillofacial region. The pathogenesis of MRONJ (BP) is not fully understood. Giant, hypernucleated, inactive osteoclasts were found in MRONJ (BP) tissues, which indicated that accelerated cell–cell fusion might play a role. Dendritic cell-specific transmembrane protein (DC-STAMP) is associated with the cell–cell fusion of osteoclasts and precursor cells. Tartrate-resistant acid phosphatase (TRAP) is essential for osteoclastic bone resorption. The cell–cell fusion, as part of the osteoclastogenesis, and the resorptive activity can determine the morphology of osteoclasts. This study analyzed jaw bone from patients with MRONJ (BP), osteomyelitis (OM) and osteoradionecrosis (ORN) because a comparison with the osteoclast profiles of OM and ORN is essential for characterizing the osteoclast profile of MRONJ (BP).MethodsFormalin-fixed routine jaw bone specimens from 70 patients [MRONJ (BP) n = 30; OM: n = 15, ORN: n = 15, control: n = 10] were analyzed retrospectively for osteoclast quantity, morphology and the expression of TRAP and DC-STAMP. The specimens were processed for hematoxylin and eosin staining (H&E), histochemistry (TRAP) and immunohistochemistry (anti-DC-STAMP) and were analyzed via virtual microscopy.ResultsThe quantity, diameter and nuclearity of osteoclasts were significantly higher in MRONJ (BP) specimens than in OM, ORN and control specimens. Giant, hypernucleated osteoclasts were detected in MRONJ (BP) specimens only. Osteoclastic TRAP expression was lower in MRONJ (BP) and ORN specimens than in OM and control specimens. The DC-STAMP expression of osteoclasts and mononuclear cells was significantly higher in MRONJ (BP) and ORN specimens than in OM and control specimens.ConclusionsThis study indicates that the osteoclast profile of MRONJ (BP) is characterized by osteoclast inactivation and a high cell–cell fusion rate; however, the presence of giant, hypernucleated osteoclasts cannot be attributed to increased DC-STAMP-triggered cell–cell fusion alone. The incidental characterization of the osteoclast profiles of OM and ORN revealed differences that might facilitate the histopathological differentiation of these diseases from MRONJ (BP), which is essential because their therapies are somewhat different.

TLR2 Signaling Pathway in Alveolar Bone Osteogenesis Induced by Aloe vera and Xenograft (XCB).

The aim of this study was to find the role of TLR2 signaling pathway in reducing osteoclast activity and promoting osteoblast growth by inducing a combination of Aloe vera and cancellous bovine xenograft (XCB) into dental extraction socket. Forty-eight Cavia cobayas were used. They were divided into eight groups (n=6). For control group, their mandibular incisors were extracted and filled with PEG. For treatment groups, they were extracted and filled with XCB, Aloe vera and the combination of Aloe vera and XCB. The first four groups were sacrificed after 7 days and the other groups after 30 days. Immunohistochemistry and histopathology examination were conducted to examine TLR2, TNFa, OPG, collagen-1, and the osteoblast and osteoclast expressions. The expressions of TLR2, OPG and Collagen-1, as well as the number of osteoblast were increased. Meanwhile, the expressions of TNFa and osteoclast were decreased. The study finding was that TLR2 signaling pathway influenced alveolar bone osteogenesis process by reducing osteoclast activity and stimulating osteoblast growth induced by the combination of Aloe vera and XCB.

Ketogenic Diet Compromises Both Cancellous and Cortical Bone Mass in Mice.

To clarify osteoporotic effects of ketogenic diet (KD) on cancellous and cortical bone compared with ovariectomy (OVX) in mice. Forty female C57BL/6J 8-week-old mice were randomly divided into SD+Sham, SD+OVX, KD+Sham, and KD+OVX groups, and fed for 12weeks. The distal femur of trabecular bone and the middle femur of cortical bone were evaluated with Micro-CT scanning. The maximum bending force and stiffness of the tibia were calculated using a three-point bending test. Osteoblast and osteoclast expression of femur were identified using tartrate-resistant acid phosphatase (TRAP), collagen type I (CoLI), and osteocalcin (OCN) staining. A 2-factor analysis of variance was used to evaluate effects of KD and OVX on radiological, biomechanical, and histological parameters. KD resulted in not only remarkable cancellous bone decline comparable to OVX, but also unique cortical bone reduction. The maximum bending force and stiffness decreased in the KD+Sham and KD+OVX groups but did not change in the SD+OVX group. The KD+OVX led to significantly higher expression in TRAP and noticeably lower expression in CoLI when compared with other groups. Both KD+Sham and SD+OVX prominently increased expression in TRAP, but decreased expression in CoLI. There was no significant difference in OCN among the four groups. The present results suggest that KD compromises both the cancellous and cortical bone architecture of long bones while OVX only in cancellous bone architecture. A combination of KD and OVX may lead to more bone loss.

Gallium enhances reconstructive properties of a calcium phosphate bone biomaterial.

Calcium phosphate (CaP)-based biomaterials are commonly used in bone reconstructive surgery to replace the damaged tissue, and can also serve as vectors for local drug delivery. Due to its inhibitory action on osteoclasts, the semi-metallic element gallium (Ga) is used for the systemic treatment of disorders associated with accelerated bone resorption. As it was demonstrated that Ga could be incorporated in the structure of CaP biomaterials, we investigated the biological properties of Ga-loaded CaP biomaterials. Culturing bone cells on Ga-CaP, we observed a decrease in osteoclast number and a downregulation of late osteoclastic markers expression, while Ga-CaP upregulated the expression of osteoblastic marker genes involved in the maturation of bone matrix. We next investigated in vivo bone reconstructive properties of different Ga-loaded biomaterials using a murine bone defect healing model. All implanted biomaterials showed a good osseointegration into the surrounding host tissue, accompanied by a successful bone ingrowth and bone marrow reconstruction, as evidenced by histological analysis. Moreover, quantitative micro-computed tomography analysis of implants revealed that Ga enhanced total defect filling. Lastly, we took advantage for the first time of a particular mode of non-linear microscopy (second harmonic generation) to quantify in vivo bone tissue reconstruction within a CaP bone substitute. By doing so, we showed that Ga exerted a positive impact on mature organized collagen synthesis. As a whole, our data support the hypothesis that Ga represents an attractive additive to CaP biomaterials for bone reconstructive surgery. Copyright © 2017 John Wiley & Sons, Ltd.

Evidence that activation of ASIC1a by acidosis increases osteoclast migration and adhesion by modulating integrin/Pyk2/Src signaling pathway.

Osteoclast adhesion and migration are responsible for osteoporotic bone loss. Acidic conditions promote osteoclastogenesis. ASIC1a in osteoclasts is associated with acid-induced osteoclastogenesis through modulating transcription factor NFATc1 activation. However, the influence and the detailed mechanism of ASIC1a in regulating osteoclast adhesion and migration, in response to extracellular acid, are not well characterized. In this study, knockdown of ASIC1a was achieved in bone marrow macrophage cells using small interfering RNA (siRNA). The adhesion and migration abilities of osteoclast precursors and osteoclasts were determined by adhesion and migration assays, in vitro. Bone resorption was performed to measure osteoclast function. Cytoskeletal changes were assessed by F-actin ring formation. αvβ3 integrin expression in osteoclasts was measured by flow cytometry. Western blotting and co-immunoprecipitation were performed to measure alterations in integrin/Pyk2/Src signaling pathway. Our results showed that blockade of ASIC1a using ASIC1a-siRNA inhibited acid-induced osteoclast precursor migration and adhesion, as well as osteoclast adhesion and bone resorption; we also demonstrated that inhibition of ASIC1a decreased the cell surface αvβ3 integrin and β3 protein expression. Moreover, blocking of ASIC1a inhibited acidosis-induced actin ring formation and reduced Pyk2 and Src phosphorylation in osteoclasts and also inhibited the acid-induced association of the αvβ3 integrin/Src/Pyk2. Together, these results highlight a key functional role of ASIC1a/αvβ3 integrin/Pyk2/Src signaling pathway in migration and adhesion of osteoclasts.

Phenotypic Spectrum in Osteogenesis Imperfecta Due to Mutations in TMEM38B: Unraveling a Complex Cellular Defect.

Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Clinical and bone material phenotype description and osteoblast differentiation studies. Natural history study in pediatric research centers. Eight patients with type XIV OI. Clinical examinations included bone mineral density, radiographs, echocardiography, and muscle biopsy. Bone biopsy samples (n = 3) were analyzed using histomorphometry, quantitative backscattered electron microscopy, and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Type XIV OI clinical phenotype ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara, and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband lumbar spine bone density z score was reduced [median -3.3 (range -4.77 to +0.1; n = 7)] and increased by +1.7 (1.17 to 3.0; n = 3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast, and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased expression of late and mineralization-related markers. Predominance of trimeric intracellular cation channel type B over type A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. OI type XIV has a bone histology, matrix mineralization, and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities.

Cyclin-Dependent Kinase Inhibitor-1-Deficient Mice are Susceptible to Osteoarthritis Associated with Enhanced Inflammation.

Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle-related proteins play a role in OA pathology. Cyclin-dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3-signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21-knockout (p21-/- ) mice were used to develop an in vivo OA model, and C57BL/6 (p21+/+ ) mice with the same background as the p21-/- mice were used as controls. Morphogenic changes were measured using micro-CT, IL-1β serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21-deficient DMM-model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild-type mice. Immunohistochemistry results revealed p21-/- mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP-3 and MMP-13 expression through IL-1β-induced NF-κB signaling. p21-/- mice also showed subchondral bone destruction according to micro-CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21-/- mice displayed increased serum IL-1β levels, and isolated chondrocytes from p21-/- mice indicated elevated MMP-3 and MMP-13 expression with phosphorylation of IκB kinase complex in response to IL-1β stimulation, whereas treatment with a specific p-IκB kinase inhibitor attenuated MMP-3 and MMP-13 expression. Our results indicated that p21-deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL-1β-induced NF-κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment. © 2017 American Society for Bone and Mineral Research.

The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis.

The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR-/- mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos-dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR-/- mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone.