Inhibits Osteoclast Maturation Research Articles

Kurarinone, a flavonoid from Radix Sophorae Flavescentis, inhibits RANKL-induced osteoclastogenesis in mouse bone marrow-derived monocyte/macrophages.

Inflammation-induced osteoclast proliferation is a crucial contributor to impaired bone metabolism. Kurarinone (KR), a flavonoid extracted from the Radix Sophorae Flavescentis, exhibits notable anti-inflammatory properties. Nevertheless, the precise influence of KR on osteoclast formation remains unclear. This study's objective was to assess the impact of KR on osteoclast activity in vitro and unravel its underlying mechanism. Initially, a target network for KR-osteoclastogenesis-osteoporosis was constructed using network pharmacology. Subsequently, the intersecting targets were identified through the Venny platform and a PPI network was created using Cytoscape 3.9.1. Key targets within the network were identified employing topological algorithms. GO enrichment and KEGG pathway analysis were then performed on these targets to explore their specific functions and pathways. Additionally, molecular docking of potential core targets of KR was conducted, and the results were validated through cell experiments. A total of 83 target genes overlapped between KR and osteoclastogenesis-osteoporosis targets. Enrichment analysis revealed their role in inflammatory response, protein tyrosine kinase activity, osteoclast differentiation, and MAPK and NF-κB signaling pathways. PPI analysis and molecular docking demonstrate that key targets MAPK14 and MAPK8 exhibit more stable binding with KR compared to other proteins. In vitro experiments demonstrate that KR effectively inhibits osteoclast differentiation and bone resorption without cellular toxicity. It suppresses key osteoclast genes (NFATc1, c-Fos, TRAP, MMP9, Ctsk, Atp6v2), hinders IκB-α degradation, and inhibits ERK and JNK phosphorylation, while not affecting p38 phosphorylation. The results indicate that KR may inhibit osteoclast maturation and bone resorption by blocking NF-κB and MAPK signaling pathways, suggesting its potential as a natural therapeutic agent for osteoporosis.

Anti-Siglec-15 Antibody Prevents Marked Bone Loss after Acute Spinal Cord Injury-Induced Immobilization in Rats.

Rapid and extensive sublesional bone loss after spinal cord injury (SCI) is a difficult medical problem that has been refractory to available interventions except the antiresorptive agent denosumab (DMAB). While DMAB has shown some efficacy in inhibiting bone loss, its concurrent inhibition of bone formation limits its use. Sialic acid-binding immunoglobulin-like lectin (Siglec)-15 is expressed on the cell surface of mature osteoclasts. Anti-Siglec-15 antibody (Ab) has been shown to inhibit osteoclast maturation and bone resorption while maintaining osteoblast activity, which is distinct from current antiresorptive agents that inhibit the activity of both osteoclasts and osteoblasts. The goal of the present study is to test a Siglec-15 Ab (NP159) as a new treatment option to prevent bone loss in an acute SCI model. To this end, 4-month-old male Wistar rats underwent complete spinal cord transection and were treated with either vehicle or NP159 at 20 mg/kg once every 2 weeks for 8 weeks. SCI results in significant decreases in bone mineral density (BMD, -18.7%), trabecular bone volume (-43.1%), trabecular connectivity (-59.7%), and bone stiffness (-76.3%) at the distal femur. Treatment with NP159 almost completely prevents the aforementioned deterioration of bone after SCI. Blood and histomorphometric analyses revealed that NP159 is able to greatly inhibit bone resorption while maintaining bone formation after acute SCI. In ex vivo cultures of bone marrow cells, NP159 reduces osteoclastogenesis while increasing osteoblastogenesis. In summary, treatment with NP159 almost fully prevents sublesional loss of BMD and metaphysis trabecular bone volume and preserves bone strength in a rat model of acute SCI. Because of its unique ability to reduce osteoclastogenesis and bone resorption while promoting osteoblastogenesis to maintain bone formation, Siglec-15 Ab may hold greater promise as a therapeutic agent, compared with the exclusively antiresorptive or anabolic agents that are currently used, in mitigating the striking bone loss that occurs after SCI or other conditions associated with severe immobilization. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Aucubin Impeded Preosteoclast Fusion and Enhanced CD31hi EMCNhi Vessel Angiogenesis in Ovariectomized Mice.

Osteogenesis is tightly correlated with angiogenesis during the process of bone development, regeneration, and remodeling. In addition to providing nutrients and oxygen for bone tissue, blood vessels around bone tissue also secrete some factors to regulate bone formation. Type H vessels which were regulated by platelet-derived growth factor-BB (PDGF-BB) were confirmed to couple angiogenesis and osteogenesis. Recently, preosteoclasts have been identified as the most important source of PDGF-BB. Therefore, inhibiting osteoclast maturation, improving PDGF-BB secretion, stimulating type H angiogenesis, and subsequently accelerating bone regeneration may be potent treatments for bone loss disease. In the present study, aucubin, an iridoid glycoside extracted from Aucuba japonica and Eucommia ulmoides, was found to inhibit bone loss in ovariectomized mice. We further confirmed that aucubin could inhibit the fusion of tartrate-resistant acid phosphatase (TRAP)+ preosteoclasts into mature osteoclasts and indirectly increasing angiogenesis of type H vessel. The underlying mechanism is the aucubin-induced inhibition of MAPK/NF-κB signaling, which increases the preosteoclast number and subsequently promotes angiogenesis via PDGF-BB. These results prompted that aucubin could be an antiosteoporosis drug candidate, which needs further research.

Exercised accelerated the production of muscle-derived kynurenic acid in skeletal muscle and alleviated the postmenopausal osteoporosis through the Gpr35/NFκB p65 pathway.

BackgroundReduced serum estrogen levels in postmenopausal patients not only aggravate bone loss but also impact myokine secretion. Emerging evidence has revealed the importance of myokines in bone metabolism, and exercise can interfere with the secretion of myokines. However, few studies have explored the impact of exercise on myokine secretion in the postmenopausal osteoporosis (PMOP) process.MethodsTen-weeks-old C57B/L6 female mice were used for constructing the postmenopausal osteoporosis model. The expression levels of kynurenine aminotransferases (Kats) were detected by RT-PCR and Western Blot. The concentration of serum kynurenic acid (Kyna) was detected by HPLC-MS. Micro-CT analysis was used for determine the changes of bone mineral density and the microstructure. The primary osteoblast and osteoclast were isolated from mice to determine the effect and mechanism of Kyna on the bone formation and resorption.ResultsIn our research, we found a lower serum level of muscle-derived kynurenic acid (Kyna) in PMOP model mice, accompanied by a decreased level of kynurenine aminotransferases (Kats) in the gastrocnemius muscle. Moreover, treadmill-running exercise upregulated the muscle levels of KATs and increased the serum concentration of Kyna, which was positively correlated with the alleviation of bone loss. Furthermore, we found that exogenous Kyna treatment alleviated bone mineral loss and microstructure destruction in PMOP mice by inhibiting osteoclast maturation and increasing osteoblast viability. Mechanistically, we observed that Kyna reduced the NFκB p65 phosphorylation level by activating the Gpr35 receptor, which inhibited NFATc1 expression in osteoclasts and upregulated Runx2 expression in osteoblasts.ConclusionOur results revealed that the muscle levels of Kats and serum level of Kyna were negatively correlated with the severity of PMOP. Exercise intervention and exogenous Kyna treatment alleviated the impairment of bone microstructure through the Gpr35 receptor, paving the way for a novel therapeutic intervention in PMOP.The Translational potential of this articleThis study provides evidences that Kyna could increase the osteoblastgenesis and inhibit the osteoclastgenesis, which could be a novel therapeutic approach for osteoporosis treatment.

Oroxin B Attenuates Ovariectomy-Induced Bone Loss by Suppressing Osteoclast Formation and Activity.

BackgroundOsteoclasts are the major players in bone resorption and have always been studied in the prevention and treatment of osteoporosis. Previous studies have confirmed that a variety of flavonoids inhibit osteoporosis and improve bone health mainly through inhibiting osteoclastogenesis. Oroxin B (OB) is a flavonoid compound extracted from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent, exerts potent antitumor and anti-inflammation effect, but its effect on osteoclastogensis remains unknown.MethodsWe comprehensively evaluated the effect of OB on the formation and function of osteoclasts and the underling mechanism by bone marrow-derived macrophage in vitro. In vivo, we used mice ovariectomized model to verify the protective effect of OB.ResultsOB was found to inhibit osteoclast formation and bone resorption function in vitro, in a dose-dependent manner and the increased osteoclastic-related genes induced by RANKL (NFATc1, c-fos, cathepsin K, RANK, MMP9 and TRAP) were also attenuated following OB treatment. Mechanistical investigation showed OB abrogated the increased phosphorylation level of MAPK and NF-κB pathway, and diminished the expression of the vital transcription factors for osteoclastogenesis. OB also prevented ovariectomy (OVX)-induced bone loss by inhibiting osteoclast formation and activity in mice.ConclusionOur study demonstrated that OB may act as an anti-osteoporosis agent by inhibiting osteoclast maturation and attenuating bone resorption.

Osteosarcoma-Derived Small Extracellular Vesicles Enhance Tumor Metastasis and Suppress Osteoclastogenesis by miR-146a-5p.

Osteosarcoma is the most frequent type of primary bone tumor in children and adolescents, thus care for patients with malignant osteosarcoma is strongly required. The roles of small extracellular vesicles (SEVs) in enhancing metastases have been demonstrated in multiple tumors, but they are still poorly understood in osteosarcoma. Hence, this study investigated the effects of SEVs on progression and the tumor microenvironment in mice and patients. In an orthotopic implantation study, we found that osteosarcoma-derived SEVs had the potential to enhance metastases and angiogenesis. In addition, osteosarcoma-derived SEVs decreased the number of mature osteoclasts in vivo. In vitro osteoclastogenesis studies revealed that the inhibition of osteoclast maturation by osteosarcoma-derived SEVs was mediated by suppressing the NF-κB signal pathway. MicroRNA analysis of SEVs from different malignant human osteosarcomas revealed that miR-146a-5p was involved in the inhibition of osteoclastogenesis. In osteosarcoma patients, lower numbers of osteoclasts in biopsy specimens at the first visits were correlated with higher malignancy. These findings indicated that osteosarcoma-derived SEVs enhance distant metastasis of osteosarcomas by inhibiting osteoclast maturation, which may be a useful prognostic marker. This diagnostic method may enable to predict malignancy at early stage, and help to provide optimal care to patients with risk of high malignancy.

Anti-perimenopausal osteoporosis effects of Erzhi formula via regulation of bone resorption through osteoclast differentiation: A network pharmacology-integrated experimental study

Ethnopharmacological relevanceErzhi formula (EZF) consists of Ecliptae herba (EH) and Fructus Ligustri Lucidi (FLL) at a ratio 1:1, and constitutes a well-known formula in China that is commonly used for treating menopausal diseases. Aim of the studyIn this study, we explored the pharmacologic actions and potential molecular mechanisms underlying EZF's action in preventing and treating osteoporosis. Materials and methodsThe active components and related targets of EZF's anti-osteoporotic effects were predicted by network pharmacology, and functional enrichment analysis was also performed. We then used an osteoporosis model of ovariectomized (OVX) mice to detect the effects of EZF on osteoporosis. ResultsThe results from network pharmacology identified a total of 10 active ingredients from EH and 13 active ingredients from FLL that might affect 65 potential therapeutic targets. GO enrichment analysis revealed that EZF affected bone tissue primarily via hormone (particularly estradiol)-related pathways and bone resorption by osteoclast differentiation. KEGG analysis demonstrated that bone-related factors such as Runt-related transcription factor 2 (Runx2), Ca2, estrogen receptor1 (ESR1), androgen receptors (AR), and TNFα served as the primary targets during osteoclastic differentiation. In vivo experiments showed that the formula significantly improved the diminution in estrogen and the subsequent uterine atrophy induced by ovariectomy (P < 0.01 or 0.05), implying that the EZF exerted its actions via regulation of estradiol and the nourishing effects of the uterus in OVX mice. Dual-energy X-ray absorptiometry and micro-CT showed that EZF significantly inhibited bone loss and improved bone micro-architecture by statistically increasing the number of bone trabeculae and decreasing the separation of bone trabeculae in OVX mice (P < 0.01 or 0.05); EZF also inhibited bone loss and enhanced bone-fracture load. Furthermore, we confirmed that EZF reduced the calcium concentrations, augmented protein and mRNA levels for Runx2 in the bone marrow, and reduced PPARγ levels. RANKL—a key downstream regulatory protein of many targets that was referred to in our results of network pharmacology as being involved in the regulation of osteoclastogenesis—was significantly diminished by EZF; it also elevated OPG content. In addition, we used monocytes of bone-marrow origin to detect the effects of the potential components of EZF on osteoclast differentiation and found that wedelolactone, oleanolic acid, echinocystic acid, luteolin, and luteolin-7-o-glucoside significantly inhibited osteoclast differentiation from monocytes induced by 25 ng/mL MCSF and 50 ng/mL RANKL (P < 0.01 or 0.05). ConclusionsOur present study indicated that EZF significantly inhibited the bone loss induced by OVX in mice by its regulation of estradiol combined with the nourishing effect of the uterus, and that it also attenuated bone resorption by decreasing the RANKL/OPG ratio so as to inhibit osteoclast maturation.

P0885THE RELATIONSHIP BETWEEN BONE REGULATORY MARKERS AND BONE TURNOVER IN RENAL OSTEODYSTROPHY

Abstract Background and Aims Renal osteodystrophy is common in advanced chronic kidney disease (CKD) patients and is characterized by abnormal bone turnover and mineralization. Parathyroid hormone (PTH) increases bone turnover through osteoblast and osteoclast activation. Osteoprotegerin (OPG) is a decoy receptor of receptor activator of nuclear factor kappa-β ligand and thus, inhibits osteoclast maturation. Meanwhile, sclerostin is an inhibitor of the Wnt signalling pathway and thus, inhibits osteoblast maturation. We aimed to assess the relationship between these bone regulatory markers and bone turnover as assessed by bone histomorphometry and bone turnover markers (BTMs). Method We recruited 43 CKD patients with eGFR&amp;lt;30ml/min/1.73m2 or on dialysis. Fasting serum samples were analysed using Immunodiagnostic Systems automated assays (Boldon, UK) for intact PTH (iPTH) and BTMs such as bone alkaline phosphatase (bALP) and intact procollagen type 1 N-terminal propeptide (intact PINP) which are bone formation markers, and tartrate-resistant acid phosphatase 5b (TRAP5b) which is a bone resorption marker. OPG and sclerostin were analysed using manual ELISA by Biomedica (Vienna, Austria). Trans-iliac bone biopsy was performed after tetracycline labelling. Bone samples were analysed using quantitative histomorphometry. Normal bone turnover was defined as bone formation rate/bone surface (BFR/BS) of 18 - 38µm3/µm2/year. Spearman rank correlation was used to test the relationship between the variables. Results Median BFR/BS was 32.12 (IQR 17.76 – 48.25) um3/um2/year. 26% of patients had low and 40% had high bone turnover. iPTH and OPG were positively correlated with BFR/BS (rho = 0.42, p&amp;lt;0.01 and rho = 0.36, p&amp;lt;0.05 respectively). Sclerostin was not correlated with BFR/BS. Furthermore, sclerostin did not correlate with bALP and intact PINP whereas OPG correlated with TRAP5b (rho = 0.43, p&amp;lt;0.01). iPTH correlated with bALP (rho = 0.62, p&amp;lt;0.001), intact PINP (rho = 0.62, p&amp;lt;0.001) and TRAP5b (rho = 0.50, p = 0.001). Conclusion Circulating levels of iPTH and OPG were modestly associated with bone turnover but sclerostin was not. There are likely to be bone regulators other than iPTH, OPG and sclerostin which regulate bone turnover in renal osteodystrophy.

Individualized treatment with denosumab in children with osteogenesis imperfecta \u2013 follow up of a trial cohort

BackgroundOsteogenesis imperfecta (OI) is a rare disease leading to hereditary bone fragility. Nearly 90% of cases are caused by mutations in the collagen genes COL1A1/A2 (classical OI) leading to multiple fractures, scoliosis, short stature and nonskeletal findings as blue sclera, hypermobility of joints, bone pain and delayed motor function development. Bisphosphonates are used in most moderate and severely affected patients assuming that an increase of bone mineral density might reduce fractures and bone pain in patients with OI. Denosumab as a RANK ligand antibody inhibiting osteoclast maturation has been approved for osteoporosis treatment in adults. First data from small clinical trials promised a high efficacy of Denosumab in children with OI. Aim of this analysis was a retrospective evaluation of an individualized biomarker-associated treatment regime with Denosumab in 10 children with classical OI which were followed for 1 year after their participation in a pilot trial with Denosumab. Therefore urinary deoxypyridinoline levels were evaluated frequently as an osteoclastic activity marker and depending on that levels Denosumab injections were scheduled individually.MethodsTen patients (age range: 6.16–12.13 years; all participated in the former OI-AK phase 2 trial (NCT01799798)) were included in the follow-up period. Denosumab was administered subcutaneously depending on the individual urinary excretion course of deoxypyridinoline (DPD/Crea) as osteoclastic activity marker with 1 mg/kg body weight. DPD/Crea levels were evaluated before denosumab administration and afterwards. If patients present after an initial decrease after injection with a re-increase up to the DPD/crea level before Denosumab injection next dosage was planned. Changes of areal bone mineral density (aBMD) using dual energy x-ray absorptiometry of the lumbar spine after 12 month was evaluated. Safety was assessed by bone metabolism markers and side effect reporting.ResultsDuring follow-up mean relative change of lumbar aBMD was − 6.4%. Lumbar spine aBMD z-Scores decreased from − 1.01 ± 2.61 (mean ± SD) to − 1.91 ± 2.12 (p = 0.015). Mobility changed not significantly (GMFM-88 -6.49 ± 8.85% (p = 0.08). No severe side effects occurred. Dose intervals could be extended in the mean from 12 weeks previously to 20.3 weeks.ConclusionsOn average, it was possible to prolong the intervals between drug administrations and to reduce the total dose about by 25% without a decrease of mobility or change of vertebral shape despite a reduction of lumbar aBMD during 1 year of biomarker-directed Denosumab treatment. Further trials are necessary to balance side effects and highest efficacy in children.

Glycyrrhizic acid suppresses osteoclast differentiation and postmenopausal osteoporosis by modulating the NF-κB, ERK, and JNK signaling pathways

Postmenopausal osteoporosis, mainly caused by osteoclast-induced bone resorption, has become a global public health burden. Natural compounds are emerging as potential therapeutics for postmenopausal osteoporosis. In vitro osteoclastogenesis assay was conducted to investigate the effect of Glycyrrhizic acid (Gly) on osteoclast differentiation without cytotoxity. We applied bone resorption pit assessment and F-actin immunofluoresence to explore the effect of Gly on osteoclasts function in vitro. RT-qPCR was used to evaluate the expression level of RANKL-induced osteoclast-specific gene. Western blotting was conducted for analyzing potential mechanisms of inhibitory influence of Gly on the formation and function of osteoclasts in vitro. Ovariectomized (OVX) mice model, micro-CT and histomorphometry was used to survey the potential effect of Gly on the resorption of bone in vivo. Gly inhibited osteoclast differentiation and function without significant cytotoxicity at a dose of no more than 8 mM in vitro. Gly attenuated mRNA expression of osteoclast-specific genes including NFATc1, c-fos, TRAP, CTR, cathepsin K, and V-ATPase d2 in vitro. Gly inhibited degradation of IκBα, phosphorylation of ERK and JNK without disturbing phosphorylation of p38 after treating osteoclasts in vitro. In OVX mice, Gly attenuates osteoclast formation and preserves bone mass and trabecular structure. Gly can effectively inhibit osteoclast maturation and bone resorption by suppressing NF-κB, ERK, and JNK pathway in vitro and exhibits an osteoprotective effect in OVX mice.

Effects of intermedin on proliferation, apoptosis and the expression of OPG/RANKL/M-CSF in the MC3T3-E1 osteoblast cell line.

Bone remodeling is a vital physiological process of healthy bone tissue in humans. It is characterized by the formation of bone by osteoblasts and its resorption by osteoclasts, and the bone resorbed by osteoclasts is replaced through the differentiation and activity of osteoblasts. Imbalances in this vital process lead to pathological conditions, including osteoporosis. Intermedin (IMD) as a newly discovered peptide in the calcitonin (CT) family of peptides, which shares similar functions with CT, calcitonin gene-related peptide and amylin in bone resorption. However, the mechanism underlying its effect remains to be elucidated. This was investigated in the present study using the osteoblastic MC3T3-E1 cell line, which was treated with different doses of IMD (0, 1, 10 and 100 nM). Cell proliferation, apoptosis and the expression of receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG) and macrophage colony-stimulating factor (M-CSF) were measured following treatment using multiple detection techniques, including an MTT assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction and western blot analysis. The resulting data demonstrated that IMD significantly inhibited the apoptosis of MC3T3-E1 cells induced by serum-free culture and dexamethasone, however, no significant effects on MC3T3-E1 cell proliferation were observed. IMD had additional functions on the MC3T3-E1 cells, including inhibition of the expression of RANKL and M-CSF, and promotion of the expression of OPG. Previous studies have also demonstrated that RANKL and M-CSF are two vital factor produced by osteoblasts to promote the maturation and differentiation of osteoclasts, and it has been reported that IMD can inhibit the osteoclast formation stimulated by RANKL and M-CSF. Together with these findings, the present study concluded that IMD reduces bone resorption by inhibiting osteoblast apoptosis, decreasing the RANKL/OPG ratio and the expression of M-CSF, and inhibiting osteoclast maturation and differentiation.

DHEA promotes osteoblast differentiation by regulating the expression of osteoblast-related genes and Foxp3(+) regulatory T cells.

Several studies have reported that dehydroepiandrosterone (DHEA) promotes osteoblast proliferation and inhibits osteoblast apoptosis and that DHEA inhibits osteoclast maturation. However, whether DHEA regulates osteoblast differentiation remains unclear. The present study first examined the effect of DHEA on bone morphology in vivo. DHEA was found to increase bone volume (BV), bone mineral density (BMD), and the number of trabeculae in bone (Th.N) and it was found to decrease trabecular spacing in bone (Th.sp) in ovariectomized (OVX) mice. Next, the effect of DHEA on osteoblast differentiation was examined in vitro and osteoblastogenesis-related marker genes, such as Runx2, Osterix, Collagen1, and Osteocalcin, were also detected. DHEA increased osteoblast production in mesenchymal stem cells (MSCs) cultured in osteoblastogenic medium, and DHEA increased the expression of Runx2 and osterix, thereby increasing the expression of osteocalcin and collagen1. Immune cells and bone interact, so changes in immune cells were detected in vivo. DHEA increased the number of Foxp3(+) regulatory T cells (Tregs) in the spleen but it did not affect CTLA-4 or IL-10. When MSCs were treated with DHEA in the presence of Tregs, alkaline phosphatase (ALP) activity increased. Osteoblasts and adipocytes are both generated by MSCs. If osteoblast differentiation increases, adipocyte differentiation will decrease, and the reverse also holds true. DHEA was found to increase the number of adipocytes in osteoblastogenic medium but it had no effect on the number of adipocytes and expression of PPARγ mRNA in adipogenic medium. This finding suggests that osteoblasts may be involved in adipocyte production. In conclusion, the current results suggest that DHEA can improve postmenopausal osteoporosis (PMO) by up-regulating osteoblast differentiation via the up-regulation of the expression of osteoblastogenesis-related genes and via an increase in Foxp3(+) Tregs.

Propolis decreases lipopolysaccharide‐induced inflammatory mediators in pulp cells and osteoclasts

Intracanal medicaments are used to disinfect the root canal system, reduce interappointment pain and inflammation, and prevent resorption. Bacterial components such as lipopolysaccharide (LPS) are implicated in the development of pulpal and periapical inflammation and inducing osteoclastogenesis. Propolis is a natural, non-toxic substance collected from bee's wax that has been used for many years in folk medicine. Propolis has been demonstrated to have antibacterial and anti-inflammatory properties. Our previous studies have shown that propolis inhibits osteoclast maturation. However, the effect of propolis on the inflammatory response of pulp cells and osteoclasts has not been explored. The purpose of this study was to evaluate whether propolis alters the inflammatory response of three endodontically relevant cell lines: mouse odontoblast-like cells (MDPC-23), macrophages (RAW264.7), and osteoclasts. Cells were exposed to 0-20 ug ml(-1) LPS to induce an inflammatory response, in the presence of propolis or vehicle control. Culture supernatants were collected after 6 and 24 h, and expression of multiple soluble mediators was determined using Luminex(®) multiplex technology. Propolis was effective in reducing secretion of the LPS-induced inflammatory cyto/chemokines: IL-1α, IL-6, IL-12(p70), IL-15, G-CSF, TNF-α, MIP-1α, MCP-1, and IP-10. Our results demonstrate that propolis suppresses the LPS-induced inflammatory response of key cells within the root canal system.

Effect of Hydrostatic Pressure on Bone Regeneration Using Human Mesenchymal Stem Cells

Mechanics is increasingly being recognized as the fourth essential factor in bone tissue engineering next to cell, scaffold, and growth factors. The development of bioprocessors has made it possible to simulate the in vivo mechanics that are needed to generate three-dimensional (3D) bone constructs. However, although hydrostatic pressure (HP) is a dominant and constant mechanical strain on bone cells in vivo, little is known about the effect of HP applied via perfusion bioprocessors on in vitro human bone marrow-derived mesenchymal stem cell (hMSC) behavior. hMSCs underwent primary culture for three passages before being seeded into hydroxyapatite (HA) scaffolds. The scaffolds were incubated for 3 weeks in an automated bioprocessor under cyclic HP. Scaffolds exposed to atmospheric pressure (AP) served as the comparator. Osteogenic differentiation medium was employed for both the HP and AP groups. Immediately before and 1, 2, and 3 weeks after incubation, the scaffolds were harvested for histological, immunohistochemical, and gene expression analyses. Cells were only found in the AP scaffold surfaces, whereas in the HP group, they were distributed evenly throughout the scaffolds. Immunohistochemical analysis revealed that the HP group expressed higher levels of osteocalcin (OC), osteopontin (OP), osteonectin (ON), and collagen type 1 (Col1) than the AP group during the 3-week process. Gene expression analysis revealed that the HP group expressed higher levels of ON, Col1, alkaline phosphatase, and integrin β5 than the AP group at the 1-, 2-, and 3-week timepoints. The HP group also expressed higher levels of core-binding factor α-1 (Cbfa1) at the 2- and 3-week timepoints and higher levels of OP and OC at the 1-week timepoint. Their proliferating cell nuclear antigen levels were lower at the 1- and 2-week timepoints. HP enhances cellular viability and improves osteogenic differentiation and maturation, although somewhat at the expense of proliferation and self-renewal of MSCs. Possible negative effects of the bioprocessor-induced HP on bone regeneration were not observed. Further, the mechanotransductive molecule integrin β5 was expressed at high levels after HP stimulation and may enhance migration, promote differentiation, and inhibit osteoclast maturation during HP-driven osteogenesis in vitro.

The role of RANK-ligand inhibition in cancer: the story of denosumab.

The diagnosis of bone metastases is an event with certain consequences for the patient. They often mean pain and can also mean pathological fractures, hypercalcemia, and spinal cord compression, all synonymous with a diminished quality of life and often also hospitalization. Since the advent of the intravenous bisphosphonates, things began to look a bit brighter for patients with bone metastases-bone destruction was kept at bay a little longer. The next generation of bone metastasis treatments is well on its way in clinical development, and among them, the most advanced drug is denosumab. Denosumab is a fully human monoclonal antibody that inhibits osteoclast maturation, activation, and function by binding to receptor activator of nuclear factor kappa B ligand, with the final result being a reduced rate of bone resorption. In this review, we give an overview of relevant preclinical and clinical data regarding the use of denosumab in patients with solid tumors in general and prostate cancer in particular.

Effect of alendronate on orthodontic tooth movement in rats

Osteoclastic activity is required for orthodontic force to move teeth through alveolar bone. Bisphosphonates are drugs that inhibit osteoclast maturation, function, and survival. The aim of this study was to assess orthodontic tooth movement in rats receiving bisphosphonate treatment. Two groups of Sprague-Dawley rats were used. The rats in the treatment group received 7 mg per kilogram of body weight per week of alendronate sodium, and those in the control group received no drugs. A coil spring exerting a constant 50-g force was activated across the span from the central incisors to the first molar. As the first molar tipped mesially, a diastema between the first and second molars was created. Vinyl polysiloxane impressions were poured in die stone, and the diastema was measured indirectly with a charged-couple device microscope camera and Optimas software (Media Cybernetics, Newburyport, Mass). Statistical analysis with repeated-measures analysis of variance showed less orthodontic tooth movement in the alendronate group compared with control group (0.06 vs 0.24 mm at 2 weeks, and 0.45 vs 1.06 mm at 4 weeks; P = 0.0004 for the alendronate vs control main effect). This study demonstrated an inhibitory effect of alendronate administration on orthodontic tooth movement in a rat model.

V-ATPase V0 subunit d2–deficient mice exhibit impaired osteoclast fusion and increased bone formation

Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H(+)) ATPase (v-ATPase) V(0) domain (Atp6v0d2). Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.

Inhibition of Prostate Tumor Growth and Bone Remodeling by the Vascular Targeting Agent VEGF121/rGel

The pathophysiology of tumor growth following skeletal metastases and the poor response of this type of lesion to therapeutic intervention remains incompletely understood. Vascular endothelial growth factor (VEGF)-A and its receptors play a role in both osteoclastogenesis and tumor growth. Systemic (i.v.) treatment of nude mice bearing intrafemoral prostate (PC-3) tumors with the vascular ablative agent VEGF(121)/recombinant gelonin (rGel) strongly inhibited tumor growth. Fifty percent of treated animals had complete regression of bone tumors with no development of lytic bone lesions. Immunohistochemical analysis showed that VEGF(121)/rGel treatment suppressed tumor-mediated osteoclastogenesis in vivo. In vitro treatment of murine osteoclast precursors, both cell line (RAW264.7) and bone marrow-derived monocytes (BMM), revealed that VEGF(121)/rGel was selectively cytotoxic to osteoclast precursor cells rather than mature osteoclasts. VEGF(121)/rGel cytotoxicity was mediated by Flt-1, which was down-regulated during osteoclast differentiation. Analysis by flow cytometry and reverse transcription-PCR showed that both BMM and RAW264.7 cells display high levels of Flt-1 but low levels of Flk-1. Internalization of VEGF(121)/rGel into osteoclast precursor cells was suppressed by pretreatment with an Flt-1 neutralizing antibody or by placenta growth factor but not with an Flk-1 neutralizing antibody. Thus, VEGF(121)/rGel inhibits osteoclast maturation in vivo and it seems that this process is important in the resulting suppression of skeletal osteolytic lesions. This is a novel and unique mechanism of action for this class of agents and suggests a potentially new approach for treatment or prevention of tumor growth in bone.

The nitrogen-containing bisphosphonate, zoledronic acid, influences RANKL expression in human osteoblast-like cells by activating TNF-alpha converting enzyme (TACE).

Bisphosphonates are used to prevent osteoclast-mediated bone loss. Zoledronic acid inhibits osteoclast maturation indirectly by increasing OPG protein secretion and decreasing transmembrane RANKL expression in human osteoblasts. The decreased transmembrane RANKL expression seems to be related to the upregulation of the RANKL sheddase, TACE. Bisphosphonates (BPs) exhibit high affinity for hydroxyapatite mineral in bone and are used extensively to treat malignancy-associated bone disease and postmenopausal bone loss by inhibiting osteoclast (OC)-mediated bone resorption. We examined the effect of the most potent nitrogen-containing BP available, zoledronic acid (ZOL), on the expression of RANKL and osteoprotegerin (OPG), critical factors in the regulation of OC formation and activation, in primary osteoblast (OB)-like cells derived from human bone, using flow cytometry, ELISA, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ immunofluorescence staining, and Western blotting. Our studies show that ZOL, while not significantly affecting RANKL or OPG gene expression, markedly increased OPG protein secretion and reduced transmembrane RANKL protein expression in OB-like cells. The reduction in transmembrane RANKL expression was preceded by a marked increase in the expression of the metalloprotease-disintegrin, TNF-alpha converting enzyme (TACE). In addition, the decreased transmembrane expression of RANKL could be partially reversed by a TACE inhibitor, TAPI-2. Our studies indicate that ZOL, in addition to its direct effects on mature OCs, may inhibit the recruitment and differentiation of OCs by cleavage of transmembrane RANKL in OB-like cells by upregulating the sheddase, TACE.

Circulating osteoprotegerin in women during GnRH-agonist treatment and their relationships with mineral components and biomarkers of bone turnover

A novel cytokine termed osteoprotegerin (OPG) that is also called osteoclastogenesis-inhibitory factor, which inhibits osteoclast maturation and activity, was recently isolated. In order to determine the influence of estrogen deficiency on the levels of circulating OPG in women, we studied the changes in the levels of circulating OPG in 10 Japanese women ages 25–49 (mean ± SD, 34.0 ± 6.9) years with endometriosis receiving gonadotropin-releasing hormone agonists (GnRH-a) therapy. We further analyzed whether the levels of circulating OPG have relations with the levels of the biomarkers of bone turnover or those of circulating mineral components in these patients during GnRH-a treatment. The patients were treated with a monthly injection of 3.75 mg leuprolide acetate depot for 6 months. In all patients, the concentrations of serum estradiol decreased after 6 months of GnRH-a treatment. The bone mineral density of the lumber spines in these patients significantly ( P < 0.01) decreased (percentage change: mean ± SD, −5.4 ± 2.1%), while circulating OPG levels significantly ( P < 0.01) increased after 6 months of treatment. The values of circulating OPG had significant correlations with those of urinary pyridinoline ( r = 0.59, P < 0.01), urinary deoxypylridinoline (Dpd) ( r = 0.46, P < 0.05), and serum alkaline phosphatase ( r = 0.66, P < 0.01) but not with those of serum carboxy-terminal propeptide of type I procollagen during GnRH-a treatment. The values of circulating OPG also correlated significantly with those of serum calcium (Ca) and phosphorus (P) ( r = 0.65 and 0.72, P < 0.01). Further analyses revealed that the percentage change in the value of circulating OPG had a significant correlation with that of urinary Dpd ( r = 0.84, P < 0.01). These results suggest that circulating OPG levels rise against the increase in osteoblastic bone resorption and circulating Ca levels in the case of estrogen deficiency, possibly as a compensatory mechanism serving to limit circulating Ca levels and bone density.