Excellent clinical and radiological outcomes after arthroscopic reduction and double row-suture bridge for large-sized greater tuberosity fractures of the humerus.

PURPOSE: Currently, there is limited information on the clinical outcomes of arthroscopic reduction and double-row suture bridge fixation for large greater tuberosity fractures of the proximal humerus. This study aimed to evaluate the radiological and clinical outcomes of arthroscopic reduction and double-row suture bridge fixation for these fractures, hypothesizing that arthroscopic reduction and double-row suture bridge fixation is a safe, effective and minimally invasive treatment for large greater tuberosity fractures. METHODS: This retrospective study analysed patients with large greater tuberosity fractures (fracture fragment ≥30 mm in diameter) who underwent arthroscopic reduction and double-row suture bridge fixation and had a follow-up period exceeding 2 years. The anatomic reduction was confirmed by assessing the step-off on radiographs immediately after surgery, and the radiologic union time was recorded. At the final follow-up, range of motion and functional outcome scores were evaluated. Additionally, any surgery-related complications were evaluated. RESULTS: Fifteen patients with a mean follow-up of 57.7 ± 23.1 months were included in the study. The mean fracture fragment size was 32.5 ± 2.4 mm, with a mean displacement of 5.1 ± 1.6 mm. Immediately postsurgery, 13 of 15 patients (86.7%) had a fracture step-off of <3 mm, with an average union time of 3 months. At the final follow-up, patients demonstrated excellent outcomes, with an average forward flexion of 167 ± 9.7° and external rotation of 70 ± 16.3. Functional outcome scores showed significant improvement compared with preoperative scores (p < 0.001). No major surgery-related complications were reported. CONCLUSIONS: Arthroscopic reduction and double-row suture bridge fixation for large-sized greater tuberosity fractures is safe and shows good fracture reduction and excellent clinical outcomes. Therefore, this surgical method can be considered an alternative to open reduction for large greater tuberosity fractures. LEVEL OF EVIDENCE: Level IV.

Comparison of blood loss between intra-articular microporous polysaccharide hemospheres powder and tranexamic acid following primary total knee arthroplasty.

Total knee arthroplasty (TKA) is associated with substantial blood loss and tranexamic acid (TXA) effectively reduces postoperative bleeding. Although it is known that there is no difference between intravenous or intra-articular (IA) injection, the general interest is directed towards topical hemostatic agents regarding thromboembolic events in high-risk patients. This study aimed to compare the blood conservation effects of IA MPH powder and TXA in patients undergoing primary TKA. We retrospectively analyzed 103 patients who underwent primary TKA between June 2020 and December 2021. MPH powder was applied to the IA space before capsule closure (MPH group, n = 51). TXA (3 g) was injected via the drain after wound closure (TXA group, n = 52). All patients underwent drain clamping for three postoperative hours. The primary outcome was the drain output, and the secondary outcomes were the postoperative hemoglobin (Hb) levels during the hospitalization period and the perioperative blood transfusion rates. An independent Student's t-test was used to determine differences between the two groups. The drain output in the first 24 h after surgery was significantly higher in the MPH group than in the TXA group. The postoperative Hb levels were significantly lower in the MPH group than in the TXA group. In patients with simultaneous bilateral TKA, there was a significant difference in the blood transfusion volumes and the rates between groups. It is considered that IA MPH powder cannot replace IA TXA because of an inferior efficacy in reducing blood loss and maintaining postoperative Hb levels in the early postoperative period after primary TKA. Moreover, in the case of simultaneous bilateral TKA, we do not recommend the use of IA MPH powder because it was notably less effective in the field of transfusion volume and rate.

Blood Compatibility of Drug-Inorganic Hybrid in Human Blood: Red Blood Cell Hitchhiking and Soft Protein Corona.

A drug-delivery system consisting of an inorganic host-layered double hydroxide (LDH)-and an anticancer drug-methotrexate (MTX)-was prepared via the intercalation route (MTX-LDH), and its hematocompatibility was investigated. Hemolysis, a red blood cell counting assay, and optical microscopy revealed that the MTX-LDH had no harmful toxic effect on blood cells. Both scanning electron microscopy and atomic force microscopy exhibited that the MTX-LDH particles softly landed on the concave part inred blood cells without serious morphological changes of the cells. The time-dependent change in the surface charge and hydrodynamic radius of MTX-LDH in the plasma condition demonstrated that the proteins can be gently adsorbed on the MTX-LDH particles, possibly through protein corona, giving rise to good colloidal stability. The fluorescence quenching assay was carried out to monitor the interaction between MTX-LDH and plasma protein, and the result showed that the MTX-LDH had less dynamic interaction with protein compared with MTX alone, due to the capsule moiety of the LDH host. It was verified by a quartz crystal microbalance assay that the surface interaction between MTX-LDH and protein was reversible and reproducible, and the type of protein corona was a soft one, having flexibility toward the biological environment.

Physicochemical Properties and Hematocompatibility of Layered Double Hydroxide-Based Anticancer Drug Methotrexate Delivery System.

A layered double hydroxide (LDH)-based anticancer delivery system was investigated in terms of crystalline phase, particle size, hydrodynamic radius, zeta potential, etc. through in vitro and in vivo study. Size controlled LDH with anticancer drug methotrexate (MTX) incorporation was successfully prepared through step-by-step hydrothermal reaction and ion-exchange reaction. The MTX-LDH was determined to have a neutral surface charge and strong agglomeration in the neutral aqueous condition due to the surface adsorbed MTX; however, the existence of proteins in the media dramatically reduced agglomeration, resulting in the hydrodynamic radius of MTX-LDH being similar to the primary particle size. The protein fluorescence quenching assay exhibited that MTX readily reduced the fluorescence of proteins, suggesting that the interaction between MTX and proteins was strong. On the other hand, MTX-LDH showed much less binding constant to proteins compared with MTX, implying that the protein interaction of MTX was effectively blocked by the LDH carrier. The in vivo hemolysis assay after intravenous injection of MTX-LDH showed neither significant reduction in red blood cell number nor membrane damage. Furthermore, the morphology of red blood cells in a mouse model did not change upon MTX-LDH injection. Scanning electron microscopy showed that the MTX-LDH particles were attached on the blood cells without serious denaturation of cellular morphology, taking advantage of the cell hitchhiking property.

Polyethylenimine-functionalized cationic barley ß-glucan derivatives for macrophage RAW264.7 cell-targeted gene delivery systems.

Cationic barley ß-glucan derivatives (bGPEIs) with various polyethylenimine 2k (PEI2k) graft degrees were synthesized by periodate oxidation of backbone vicinal diols and reductive amination of PEI2k for gene delivery systems. bGPEIs could form positively charged (∼40 mV zeta-potential) and nano-sized (∼150 nm) spherical polyplexes. Cytotoxicity of bGPEIs was concentration and PEI graft degree-dependent. bGPEIs showed higher transfection efficiency and intracellular localization ability than PEI25k in RAW264.7 cell, especially in serum condition. High cellular uptake of bGPEI polyplexes at 4 °C in RAW264.7 cells suggested that bGPEIs would possess specific interaction ability with membrane receptors of RAW264.7 cells. In addition, bGPEIs could activate RAW264.7 cells, inducing the secretion of cytokine, tumor necrosis factor-α (TNF-α). Therefore, bGPEIs showed a potential for macrophage RAW264.7 cell-targeted gene delivery systems.

Immunotoxicity of titanium dioxide nanoparticles via simultaneous induction of apoptosis and multiple toll-like receptors signaling through ROS-dependent SAPK/JNK and p38 MAPK activation.

BACKGROUND: Titanium dioxide nanoparticles (TiO2 NPs) represent a scientific breakthrough in the areas of biological and medicinal applications. Interaction of TiO2 NPs with components of innate immune system remains elusive. AIM: This study explored in vitro immunotoxicity of murine macrophage RAW 264.7 to TiO2 NPs (20 nm, negative charge) and its underlying molecular mechanism by way of immunoredox profiling. MATERIALS AND METHODS: In this study, chemically synthesized BSA-functionalized TiO2 NPs (20 nm, negative charge) were characterized and immunotoxicity was investigated on RAW 264.7 cells. RESULTS: We found that reactive oxygen species levels significantly increased with increasing nitric oxide production, whereas depleting endogenous antioxidant super oxide dismutase as well as nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels. Furthermore, NPs exposure increased the expression of apoptotic factors such as BAX, BIM, and PUMA with disruption of mitochondrial membrane potential (Δψm) that lead to decrease in immunocytes. Molecular immune profiling revealed the activation of multiple toll-like receptors (TLRs) 4/9/12/13 simultaneously with the phosphorylation of p-p38MAPK and p-SAPK/c-Jun N-terminal kinase (JNK) compared to untreated control. CONCLUSION: Collectively, this study shows that the molecular nature of TiO2 SA20(-) NP-induced immunotoxicity in RAW 264.7 macrophage is simultaneous induction of immunocyte apoptosis and multiple TLRs signaling through oxidative stress-dependent SAPK/JNK and p38 mitogen-associated protein kinase activation. This is the first study to address newer molecular mechanism of TiO2 SA20(-) NP-induced immunotoxicity.

Layered Double Hydroxide Nanomaterials Encapsulating Angelica gigas Nakai Extract for Potential Anticancer Nanomedicine.

We prepared hybrids consisting of Angelica gigas Nakai (AGN) root or flower extract and layered double hydroxide (LDH) for potential anticancer nanomedicine, as decursin species (DS) in AGN are known to have anticancer activity. Dimethylsulfoxide solvent was determined hybridization reaction media, as it has affinity to both AGN and LDH moiety. In order to develop inter-particle spaces in LDH, a reversible dehydration-rehydration, so-called reconstruction route, was applied in AGN-LDH hybridization. Quantitative analyses on AGN-LDH hybrids indicated that the content of DS was two times more concentrated in the hybrids than in extract itself. Using X-ray diffraction, FT-IR spectroscopy, scanning electron microscopy, and zeta-potential measurement, we found that AGN extract moiety was incorporated into inter-particle spaces of LDH nanoparticles during the reconstruction reaction. Time-dependent DS release from hybrids at pH 7.4 (physiological condition) and pH 4.5 (lysosomal condition) exhibited a pH-dependent release of extract-incorporated LDH hybrids. An anticancer activity test using HeLa, A549, and HEK293T cells showed that the AGN-LDH hybrid, regardless of extract type, showed enhanced anticancer activity compared with extract alone at an equivalent amount of DS, suggesting a nanomedicine effect of AGN-LDH hybrids.

Nanolayered hybrid mediates synergistic co-delivery of ligand and ligation activator for inducing stem cell differentiation and tissue healing.

Cellular behaviors, such as differentiation, are regulated by complex ligation processes involving cell surface receptors, which can be activated by various divalent metal cations. The design of nanoparticle for co-delivery of ligand and ligation activator can offer a novel strategy to synergistically stimulate ligation processes in vivo. Here, we present a novel layered double hydroxide (LDH)-based nanohybrid (MgFe-Ado-LDH), composed of layered MgFe hydroxide nanocarriers sandwiching the adenosine cargo molecule, maintained through an electrostatic balance, to co-deliver the adenosine (Ado) ligand from the interlayer spacing and the Mg2+ ion (ligation activator) through the dissolution of the MgFe nanocarrier itself. Our findings demonstrate that the MgFe-Ado-LDH nanohybrid promoted osteogenic differentiation of stem cells through the synergistic activation of adenosine A2b receptor (A2bR) by the dual delivery of adenosine and Mg2+ ions, outperforming direct supplementation of adenosine alone. Furthermore, the injection of the MgFe-Ado-LDH nanohybrid and stem cells embedded within hydrogels promoted the healing of rat tibial bone defects through the rapid formation of fully integrated neo-bone tissue through the activation of A2bR. The newly formed bone tissue displayed the key features of native bone, including calcification, mature tissue morphology, and vascularization. This study demonstrates a novel and effective strategy of bifunctional nanocarrier-mediated delivery of ligand (cargo molecule) and activation of its ligation to receptor by the nanocarrier itself for synergistically inducing stem cell differentiation and tissue healing in vivo, thus offering novel design of biomaterials for regenerative medicine.

A novel synthesis of an Fe3+/Fe2+ layered double hydroxide ('green rust') via controlled electron transfer with a conducting polymer.

This report examines the influence of a conducting polymer on the crystal growth of labile green rust (GR) through hybridization with polypyrrole. All hybrids used in this study were prepared via one-pot co-precipitation at neutral pH, with specific stoichiometric ratios among all chemical species. The role of the conducting polymer and the effective stoichiometric ratio were demonstrated to facilitate the crystal growth of GR.

Cytotoxicity, Intestinal Transport, and Bioavailability of Dispersible Iron and Zinc Supplements.

Iron or zinc deficiency is one of the most important nutritional disorders which causes health problem. However, food fortification with minerals often induces unacceptable organoleptic changes during preparation process and storage, has low bioavailability and solubility, and is expensive. Nanotechnology surface modification to obtain novel characteristics can be a useful tool to overcome these problems. In this study, the efficacy and potential toxicity of dispersible Fe or Zn supplement coated in dextrin and glycerides (SunActive FeTM and SunActive ZnTM) were evaluated in terms of cytotoxicity, intestinal transport, and bioavailability, as compared with each counterpart without coating, ferric pyrophosphate (FePP) and zinc oxide (ZnO) nanoparticles (NPs), respectively. The results demonstrate that the cytotoxicity of FePP was not significantly affected by surface modification (SunActive FeTM), while SunActive ZnTM was more cytotoxic than ZnO-NPs. Cellular uptake and intestinal transport efficiency of SunActive FeTM were significantly higher than those of its counterpart material, which was in good agreement with enhanced oral absorption efficacy after a single-dose oral administration to rats. These results seem to be related to dissolution, particle dispersibility, and coating stability of materials depending on suspending media. Both SunActiveTM products and their counterpart materials were determined to be primarily transported by microfold (M) cells through the intestinal epithelium. It was, therefore, concluded that surface modification of food fortification will be a useful strategy to enhance oral absorption efficiency at safe levels.

Nicotinamide phosphoribosyltransferase inhibits receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation in vitro.

The adipokine nicotinamide phosphoribosyltransferase (Nampt), also known as pre-B-cell colony-enhancing factor or the insulin-mimetic hormone visfatin, has a crucial role in the conversion of nicotinamide to nicotinamide mononucleotide during biosynthesis of the coenzyme nicotinamide adenine dinucleotide. Previous reports have demonstrated the inhibitory effects of Nampt on osteoclast formation from human peripheral blood mononuclear cells and CD14+ monocytes. However, the effects of Nampt on bone marrow macrophage (BMM)­derived osteoclastogenesis and its precise role in the process remain unclear. The present in vitro study used recombinant Nampt and BMMs as osteoclast precursors demonstrated that Nampt suppresses receptor activator of nuclear factor­κB ligand (RANKL)­induced osteoclastogenesis by decreasing the phosphorylation of various early signal transducers, including c­Jun N­terminal kinase, Akt, glycogen synthase kinase­3 ß, Bruton's tyrosine kinase and phospholipase C γ­2. In addition, western blotting and reverse transcription­quantitative polymerase chain reaction analysis indicated that Nampt downregulates the mRNA and protein expression levels of c­Fos and nuclear factor of activated T cells, cytoplasmic 1, leading to a decrease in the expression of osteoclast­specific genes including tartrate­resistant acid phosphatase, osteoclast­associated receptor and cathepsin K. However, the bone­resorbing activity of mature osteoclasts treated with Nampt was similar to untreated control osteoclasts. This finding indicates that Nampt exerts its anti­osteoclastogenic activity by targeting osteoclast precursor cells rather than mature osteoclasts. Consequently, the present study demonstrated that Nampt acts as a negative regulator of RANKL­mediated differentiation of BMMs into osteoclasts, suggesting the potential therapeutic targets to treat bone-related disorders such as osteoporosis.

Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo.

Dendrobium moniliforme (DM) is a well-known plant-derived extract that is widely used in Oriental medicine. DM and its chemical constituents have been reported to have a variety of pharmacological effects, including anti-oxidative, anti-inflammatory, and anti-tumor activities; however, no reports discuss the beneficial effects of DM on bone diseases such as osteoporosis. Thus, we investigated the relationship between DM and osteoclasts, cells that function in bone resorption. We found that DM significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation; DM directly induced the down-regulation of c-Fos and nuclear factor of activated T cells c1 (NFATc1) without affecting other RANKL-dependent transduction pathways. In the later stages of osteoclast maturation, DM negatively regulated the organization of filamentous actin (F-actin), resulting in impaired bone-resorbing activity by the mature osteoclasts. In addition, micro-computed tomography (µ-CT) analysis of the murine model revealed that DM had a beneficial effect on lipopolysaccharide (LPS)-mediated bone erosion. Histological analysis showed that DM attenuated the degradation of trabecular bone matrix and formation of TRAP-positive osteoclasts in bone tissues. These results suggest that DM is a potential candidate for the treatment of metabolic bone disorders such as osteoporosis.

Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo.

Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer. However, no reports are available for verifying the pharmacological effects of ebselen on major metabolic bone diseases such as osteoporosis. In this study, we observed that ebselen suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its µ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation and osteoclast formation in the bone tissues. Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate (NMDA) receptor. These results indicate that ebselen is a potentially safe drug for treating metabolic bone diseases such as osteoporosis.

Protocatechuic Acid Attenuates Osteoclastogenesis by Downregulating JNK/c-Fos/NFATc1 Signaling and Prevents Inflammatory Bone Loss in Mice.

Protocatechuic acid (PCA) plays a critical role in nutritional metabolism; it is a major metabolite of anthocyanins, which are flavonoids with a range of health benefits. PCA has a variety of biological activities including anti-oxidant, antiinflammatory, anti-apoptosis, and anti-microbial activities. However, the pharmacological effect of PCA, especially on osteoclastogenesis, remains unknown. We examined the effect of PCA on receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption. PCA dose-dependently inhibited RANKL-induced osteoclast differentiation in mouse bone marrow macrophages (BMMs) and suppressed the bone-resorbing activity of mature osteoclasts. At the molecular level, PCA suppressed RANKL-induced phosphorylation of JNK among MAPKs only, without significantly affecting the early signaling pathway. PCA also suppressed RANKL-stimulated expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1) at the mRNA and protein levels, without altering c-Fos mRNA expression. Additionally, PCA down-regulated the expression of downstream osteoclastogenesis-related genes including ß3-integrin, DC-STAMP, OC-STAMP, Atp6v0d2, CTR, and CtsK. Mice treated with PCA efficiently recovered from lipopolysaccharide-induced bone loss in vivo. Thus, PCA inhibits RANKL-induced osteoclast differentiation and function by suppressing JNK signaling, c-Fos stability, and expression of osteoclastic marker genes. These results suggest that PCA could be useful in treatment of inflammatory bone disorders.

Niclosamide suppresses RANKL-induced osteoclastogenesis and prevents LPS-induced bone loss.

Niclosamide (5-chloro-salicyl-(2-chloro-4-nitro) anilide) is an oral anthelmintic drug used for treating intestinal infection of most tapeworms. Recently, niclosamide was shown to have considerable efficacy against some tumor cell lines, including colorectal, prostate, and breast cancers, and acute myelogenous leukemia. Specifically, the drug was identified as a potent inhibitor of signal transducer and activator of transcription 3 (STAT3), which is associated with osteoclast differentiation and function. In this study, we assessed the effect of niclosamide on osteoclastogenesis in vitro and in vivo. Our in vitro study showed that receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast differentiation was inhibited by niclosamide, due to inhibition of serine-threonine protein kinase (Akt) phosphorylation, inhibitor of nuclear factor-kappaB (IκB), and STAT3 serine(727). Niclosamide decreased the expression of the major transcription factors c-Fos and NFATc1, and thereafter abrogated the mRNA expression of osteoclast-specific genes, including TRAP, OSCAR, αv/ß3 integrin (integrin αv, integrin ß3), and cathepsin K (CtsK). In an in vivo model, niclosamide prevented lipopolysaccharide-induced bone loss by diminishing osteoclast activity. Taken together, our results show that niclosamide is effective in suppressing osteoclastogenesis and may be considered as a new and safe therapeutic candidate for the clinical treatment of osteoclast-related diseases such as osteoporosis.

WHI-131 Promotes Osteoblast Differentiation and Prevents Osteoclast Formation and Resorption in Mice.

The small molecule WHI-131 is a potent therapeutic agent with anti-inflammatory, antiallergic, and antileukemic potential. However, the regulatory effects of WHI-131 on osteoblast and osteoclast activity are unclear. We examined the effects of WHI-131 on osteoblast and osteoclast differentiation with respect to bone remodeling. The production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts in response to interleukin (IL)-1 or IL-6 stimulation decreased by 56.8% or 50.58%, respectively, in the presence of WHI-131. WHI-131 also abrogated the formation of mature osteoclasts induced by IL-1 or IL-6 stimulation. Moreover, WHI-131 treatment decreased RANKL-induced osteoclast differentiation of bone marrow-derived macrophages, and reduced the resorbing activity of mature osteoclasts. WHI-131 further decreased the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) by almost twofold, and significantly downregulated the mRNA expression of the following genes: tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), DC-STAMP, OC-STAMP, ATP6v0d2, and cathepsin K (CtsK) compared with the control group. WHI-131 further suppressed the phosphorylation of protein kinase B (Akt) and degradation of inhibitor of kappa B (IκB); Ca(2+) oscillation was also affected, and phosphorylation of the C-terminal Src kinase (c-Src)-Bruton agammaglobulinemia tyrosine kinase (Btk)-phospholipase C gamma 2 (PLCγ2) (c-Src-Btk-PLCg2 calcium signaling pathway) was inhibited following WHI-131 treatment. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway was activated by WHI-131, accompanied by phosphorylation of STAT3 Ser727 and dephosphorylation of STAT6. In osteoblasts, WHI-131 caused an approximately fourfold increase in alkaline phosphatase activity and Alizarin Red staining intensity. Treatment with WHI-131 increased the mRNA expression levels of genes related to osteoblast differentiation, and induced the phosphorylation of Akt, p38, and Smad1/5/8. Furthermore, 5-week-old ICR mice treated with WHI-131 exhibited antiresorbing effects in a lipopolysaccharide-induced calvaria bone loss model in vivo and increased bone-forming activity in a calvarial bone formation model. Therefore, the results of this study show that WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Thus, WHI-131 could be a useful pharmacological agent to treat osteoporosis by promoting bone growth and inhibiting resorption.

Stauntonia hexaphylla (Lardizabalaceae) leaf methanol extract inhibits osteoclastogenesis and bone resorption activity via proteasome-mediated degradation of c-Fos protein and suppression of NFATc1 expression.

BACKGROUND: Natural plants, including common vegetables and fruits, have been recognized as essential sources for drug discovery and the development of new, safe, and economical medicaments. Stauntonia hexaphylla (Lardizabalaceae) is widely distributed in Korea, Japan, and China, and is a popular herbal supplement in Korean and Chinese folk medicine owing to its analgesic, sedative, and diuretic properties. However, the exact pharmacological effects of S. hexaphylla extract, particularly its effect on osteoclastogenesis, are not known. METHODS: Osteoclast differentiation and function were identified with tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay, and the underling mechanisms were determined by real-time RT-PCR and western blot analysis. RESULTS: S. hexaphylla was found to inhibit early-stage receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without cytotoxicity and bone-resorbing activity in mature osteoclasts in a dose-dependent manner. This S. hexaphylla-mediated blockade of osteoclastogenesis involved abrogation of the NF-κB, ERK, and c-Src-Btk-PLCγ2 calcium signal pathways. Interestingly, we found that S. hexaphylla down-regulated RANKL-associated c-Fos protein induction by suppressing its translation. Furthermore, ectopic overexpression of c-Fos and NFATc1 rescued the inhibition of osteoclast differentiation by S. hexaphylla. Furthermore, S. hexaphylla inhibited the c-Fos- and NFATc1-regulated expression of genes required for osteoclastogenesis, such as TRAP, OSCAR, ß3-integrin, ATP6v0d2, and CtsK. CONCLUSIONS: These findings suggest that S. hexaphylla might be useful for the development of new anti-osteoporosis agents.

Harpagoside Inhibits RANKL-Induced Osteoclastogenesis via Syk-Btk-PLCγ2-Ca(2+) Signaling Pathway and Prevents Inflammation-Mediated Bone Loss.

Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devil's claw) that is reported to have anti-inflammatory effects; however, these effects have not been investigated in the context of bone development. The current study describes for the first time that HAR inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro and suppresses inflammation-induced bone loss in a mouse model. HAR also inhibited the formation of osteoclasts from mouse bone marrow macrophages (BMMs) in a dose-dependent manner as well as the activity of mature osteoclasts, including filamentous actin (F-actin) ring formation and bone matrix breakdown. This involved a HAR-induced decrease in extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation, leading to the inhibition of Syk-Btk-PLCγ2-Ca(2+) in RANKL-dependent early signaling, as well as the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which resulted in the down-regulation of various target genes. Consistent with these in vitro results, HAR blocked lipopolysaccharide (LPS)-induced bone loss in an inflammatory osteoporosis model. However, HAR did not prevent ovariectomy-mediated bone erosion in a postmenopausal osteoporosis model. These results suggest that HAR is a valuable agent against inflammation-related bone disorders but not osteoporosis induced by hormonal abnormalities.

Overexpression of prohibitin-1 inhibits RANKL-induced activation of p38-Elk-1-SRE signaling axis blocking MKK6 activity.

Prohibitin-1 (PHB) regulates diverse cellular processes by controlling several signaling pathways. In this study, we investigated the functional involvement of PHB in osteoclast differentiation. PHB expression was time-dependently increased by RANKL in BMMs. However, the retroviral over-expression of PHB strongly inhibited the expression of c-Fos and NFATc1, and activation of p38-Elk-1-SRE signaling pathway. Anti-osteoclastogenic action of PHB was significantly inhibited by constitutively active forms of MKK6, but not Elk-1. Collectively, PHB negatively regulates the formation of mature osteoclasts via inhibition of MKK6 activity that affects the activation of the p38-Elk-1 signaling axis required for the expression of c-Fos and NFATc1.

CTRP3 acts as a negative regulator of osteoclastogenesis through AMPK-c-Fos-NFATc1 signaling in vitro and RANKL-induced calvarial bone destruction in vivo.

Adipokines derived from adipocytes are important factors that act as circulating regulators of bone metabolism. C1q/tumor necrosis factor (TNF)-related Protein-3 (CTRP3) is a novel adipokine with multiple effects such as lowering glucose levels, inhibiting gluconeogenesis in the liver, and increasing angiogenesis and anti-inflammation. However, the effects and the mechanisms of CTRP3 on bone metabolism, which is regulated by osteoblasts and osteoclasts, have not been investigated. Here, we found that CTRP3 inhibited osteoclast differentiation induced by osteoclastogenic factors in bone marrow cell-osteoblast co-cultures, but did not affect the ratio of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to osteoprotegerin (OPG) induced by osteoclastogenic factors in osteoblasts. We also found that CTRP3 inhibited osteoclast differentiation from mouse bone marrow macrophages (BMMs) induced by RANKL in a dose-dependent manner without cytotoxicity. Functionally, CTRP3 inhibited the F-actin formation and bone resorbing activity of mature osteoclasts. Pretreatment with CTRP3 significantly inhibited RANKL-induced expression of c-Fos and nuclear factor of activated T-cells (NFATc1), essential transcription factors for osteoclast development. Surprisingly, the activation of AMP-activated protein kinase (AMPK) was considerably increased by pretreatment with CTRP3 for 1h. The CTRP3-stimulated AMPK activation was also maintained during RANKL-induced osteoclastogenesis. CTRP3 did not affect RANKL-induced p38, ERK, JNK, Akt, IκB, CREB, and calcium signaling (Btk and PLCγ2). These results suggest that CTRP3 plays an important role as a negative regulator of RANKL-mediated osteoclast differentiation by acting as an inhibitor of NFATc1 activation through the AMPK signaling pathway. Furthermore, CTRP3 treatment reduced RANKL-induced osteoclast formation and bone destruction in mouse calvarial bone in vivo based on micro-CT and histologic analysis. In conclusion, these findings strongly suggest that CTRP3 deserves new evaluation as a potential treatment target in various bone diseases associated with excessive osteoclast differentiation and bone destruction.