Inhibits Osteoclast Differentiation Research Articles

Reduced form of Galectin-1 Suppresses Osteoclastic Differentiation of Human Peripheral Blood Mononuclear Cells and Murine RAW264 Cells In Vitro.

Galectin-1 (Gal-1) is an evolutionarily conserved sugar-binding protein found in intra- and extracellular spaces. Extracellularly, it binds to glycoconjugates with β-galactoside(s) and functions in various biological phenomena, including immunity, cancer, and differentiation. Under extracellular oxidative conditions, Gal-1 undergoes oxidative inactivation, losing its sugar-binding ability, although it exhibits sugar-independent functions. An age-related decrease in serum Gal-1 levels correlates with decreasing bone mass, and Gal-1 knockout promotes osteoclastic bone resorption and suppresses bone formation. However, the effect of extracellular Gal-1 on osteoclast differentiation remains unclear. Herein, we investigated the effects of extracellular Gal-1 on osteoclastogenesis in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage RAW264 cells. Recombinant Gal-1 suppressed the macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand-dependent osteoclast formation, actin ring formation, and bone-resorption activity of human PBMCs. Similar results were obtained for RAW264 cells. Gal-1 knockdown increased osteoclast-like cell formation, suggesting that it affected differentiation in an autocrine-like manner. Oxidized Gal-1 slightly affected differentiation, and in the presence of lactose, the differentiation inhibitory effect of galectin-1 was not observed. These findings suggest that extracellular Gal-1 inhibits osteoclast differentiation in a β-galactoside-dependent manner, and an age-related decrease in serum Gal-1 levels may contribute to reduced osteoclast activity and decreasing bone mass.

Periodontal ligament cells-derived exosomes promote osteoclast differentiation via modulating macrophage polarization

Several studies have demonstrated that exosomes (Exos) are involved in the regulation of macrophage polarization and osteoclast differentiation. However, the characteristics as well as roles of exosomes from human periodontal ligament cells (hPDLCs-Exos) in M1/M2 macrophage polarization and osteoclast differentiation remain unclear. Here, periodontal ligament cells were successfully extracted by method of improved Type-I collagen enzyme digestion. hPDLCs-Exos were extracted by ultracentrifugation. hPDLCs-Exos were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting (WB). Osteoclast differentiation was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR), WB and tartrate-resistant acid phosphatase (TRAP) staining. M1/M2 macrophage polarization were evaluated by RT-qPCR and WB. The results showed hPDLCs-Exos promoted osteoclast differentiation and M2 macrophage polarization, but inhibited M1 macrophage polarization. Moreover, M1 macrophages inhibited osteoclast differentiation, whereas M2 macrophages promoted osteoclast differentiation. It has shown that hPDLCs-Exos promoted osteoclast differentiation by inhibiting M1 and promoting M2 macrophage polarization.

Latest Progress of LIPUS in Fracture Healing: A Mini-Review.

The use of low-intensity pulsed ultrasound (LIPUS) for promoting fracture healing has been Food and Drug Administration (FDA)-approved since 1994 due to largely its non-thermal effects of sound flow sound radiation force and so on. Numerous clinical and animal studies have shown that LIPUS can accelerate the healing of fresh fractures, nonunions, and delayed unions in pulse mode regardless of LIPUS devices or circumstantial factors. Rare clinical studies show limitations of LIPUS for treating fractures with intramedullary nail fixation or low patient compliance. The biological effect is achieved by regulating various cellular behaviors involving mesenchymal stem/stromal cells (MSCs), osteoblasts, chondrocytes, and osteoclasts and with dose dependency on LIPUS intensity and time. Specifically, LIPUS promotes the osteogenic differentiation of MSCs through the ROCK-Cot/Tpl2-MEK-ERK signaling. Osteoblasts, in turn, respond to the mechanical signal of LIPUS through integrin, angiotensin type 1 (AT1), and PIEZO1 mechano-receptors, leading to the production of inflammatory factors such as COX-2, MCP-1, and MIP-1β fracture repair. LIPUS also induces CCN2 expression in chondrocytes thereby coordinating bone regeneration. Finally, LIPUS suppresses osteoclast differentiation and gene expression by interfering with the ERK/c-Fos/NFATc1 cascade. This mini-review revisits the known effects and mechanisms of LIPUS on bone fracture healing and strengthens the need for further investigation into the underlying mechanisms.

MCP-5 suppresses osteoclast differentiation through Ccr5 upregulation.

Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand(RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.

Strontium-Doped Mesoporous Bioactive Glass-Loading Bisphosphonates Inhibit Osteoclast Differentiation and Prevent Osteoporosis in Ovariectomized Mice

Postmenopausal osteoporosis, a metabolic bone disease associated with aging that affects bones throughout the body, is emerging as an urgent public health concern and imposes a substantial healthcare burden on society. The clinical application of bisphosphonate, the primary treatment for osteoporosis, is limited owing to the drug’s severe complications. Herein, we investigate the synthesis and utilization of strontium-doped mesoporous bioactive glass loaded with alendronate (ALN@Sr-MBG) as a novel therapeutic agent for osteoporosis, to explore its potential as an alternative to alendronate (ALN). Strontium-doped mesoporous bioactive glass (Sr-MBG) was synthesized using the sol–gel method, while ALN@Sr-MBG was obtained via incorporating Sr-MBG into an alendronate saturated solution. The bioactivities of ALN@Sr-MBG, including biotoxicity, inflammation inhibition, and anti-osteoclast differentiation, were investigated in vitro using CCK-8, flow cytometry, tartrate-resistant acid phosphatase (TRAP) staining, and RT-PCR assays. Animal models were established by surgically removing the ovaries from non-pregnant female C57/BL mice, followed by weekly intraperitoneal injections of ALN@Sr-MBG, ALN, or excipients. After 8 weeks, the mice femurs were extracted and analyzed through micro-CT scanning and hematoxylin-eosi, osteoblastic, and osteogenic staining. These in vitro findings demonstrate that ALN@Sr-MBG displays enhanced biological efficacy compared to ALN in terms of inflammation inhibition, osteogenesis promotion, and osteoclastogenesis inhibition. Furthermore, micro-CT analysis revealed that ALN@Sr-MBG significantly augments bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), while reducing trabecular separation (Tb.Sp) and the structural model index (SMI) in mice with ovariectomy-induced osteoporosis. The osteoblast and osteogenic staining results indicate the enhanced bioactivities of ALN@Sr-MBG in promoting bone formation and inhibiting bone resorption compared to ALN. In vitro and in vivo assessments further confirmed that ALN@Sr-MBG exhibits superior anti-osteoporotic bioactivity compared to ALN. This study’s findings confirm the potential of ALN@Sr-MBG as a novel alternative therapy to ALN and a drug candidate for the treatment of osteoporosis.

Dietary Puerarin Translocates to Femur and Suppresses Osteoclast Differentiation in Ovariectomized Mice.

Osteoporosis is characterized by bone loss and deterioration in bone microstructure, leading to bone fragility. It is strongly correlated with menopause in women. Previously, we reported that diets supplemented with a kudzu (Pueraria lobata) vine extract suppressed bone resorption in ovariectomized (OVX) mice, a postmenopausal model. The main isoflavone in kudzu is puerarin (daidzein-8-C-glycoside). Puerarin (daidzein-8-C-glycoside), which is main isoflavone of kudzu, probably contributes to the beneficial effect. However, the underlying mechanism is unclear. Therefore, the nutrikinetics of puerarin and the comparison with the suppressive effects of kudzu isoflavones on osteoclast differentiation was examined in this study. We demonstrated that orally administered puerarin was absorbed from the gut and entered the circulation in an intact form. In addition, puerarin accumulated in RAW264.7 pre-osteoclast cells in a time-dependent manner. Tartrate-resistant acid phosphatase activity was decreased by puerarin treatment in a concentration-dependent manner in RAW264.7 cells stimulated with the receptor activator of nuclear factor kappa-B ligand. Ovariectomy-induced elevated bone resorption was suppressed, and the fragile bone strength was improved by puerarin ingestion in the diet. These findings suggested that orally administered puerarin was localized in bone tissue and suppressed bone resorption and osteoclastogenesis in ovariectomized mice.

Cinobufagin Suppresses Lipid Peroxidation and Inflammation in Osteoporotic Mice by Promoting the Delivery of miR-3102-5p by Macrophage-Derived Exosomes.

Cinobufagin, the primary active compound in toad venom, is commonly used for anti-tumor, anti-inflammatory, and analgesic purposes. However, its specific bone-protective effects remain uncertain. This research aims to ascertain the bone-protective properties of cinobufagin and investigate underlying mechanisms. Mice were ovariectomized to establish an osteoporosis model, followed by intraperitoneal injections of cinobufagin and cinobufagin-treated RAW.264.7-derived exosomes for therapy. MicroCT, HE staining, and TRAP staining were employed to evaluate bone mass and therapeutic outcomes, while mRNA sequencing and immunoblotting were utilized to assess markers of bone metabolism, inflammation, and lipid peroxidation. Osteoblast and osteoclast precursor cells were differentiated to observe the impact of cinobufagin-treated exosomes derived from RAW264.7 cells on bone metabolism. Exosomes characteristics were studied using transmission electron microscopy and particle size analysis, and miRNA binding targets in exosomes were determined by luciferase reporting. In ovariectomized mice, cinobufagin and cinobufagin-treated exosomes from RAW264.7 cells increased trabecular bone density and mass in the femur, while also decreasing inflammation and lipid peroxidation. The effect was reversed by an exosomes inhibitor. In vitro experiments revealed that cinobufagin-treated exosomes from RAW264.7 cells enhanced osteogenic and suppressed osteoclast differentiation, possibly linked to Upregulated miR-3102-5p in RAW-derived exosomes. MiR-3102-5p targets the 3'UTR region of alox15, thereby suppressing its expression and reducing the lipid peroxidation process in osteoblasts. Overall, this study clarified cinobufagin's bone-protective effects and revealed that cinobufagin can enhance the delivery of miR-3102-5p targeting alox15 through macrophage-derived exosomes, demonstrating anti-lipid peroxidation and anti-inflammatory effects.

Farrerol suppresses osteoclast differentiation and postmenopausal osteoporosis by inhibiting the nuclear factor kappa B signaling pathway

Excessive bone resorption caused by upregulated osteoclast activity is a key factor in osteoporosis pathogenesis. Farrerol is a typical natural flavanone and exhibits various pharmacological actions. However, the role and mechanism of action of farrerol in osteoclast differentiation regulation remain unclear. This study aimed to evaluate the effects and mechanism of farrerol on the inhibition of osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin staining, and the pit formation assay were performed to examine the differentiation and functions of osteoclasts in vitro. The expression of proteins associated with the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways was analyzed by western blotting. Dual X-ray absorptiometry, microcomputed tomography, and histopathological and immunohistochemical analyses were performed to determine the therapeutic effect of farrerol in vivo bone loss prevention. The effects of farrerol on osteoblastic bone formation were assessed using alkaline phosphatase, alizarin red S staining, and calcein-alizarin red S double labeling. Farrerol inhibited osteoclastogenesis and bone resorption in osteoclasts by suppressing nuclear factor kappa B signaling rather than mitogen-activated protein kinase signaling in vitro. Farrerol protected mice against ovariectomy-induced bone loss by inhibiting osteoclast-mediated bone resorption, instead of promoting osteoblast-mediated bone formation in vivo. The findings of the current study revealed that farrerol is a potential therapeutic agent for osteoporosis.

Engineering tunable dual peptide hybrid coatings promote osseointegration of implants

Utilizing complementary bioactive peptides is a promising surface engineering strategy for bone regeneration on osteogenesis. In this study, we designed block peptides, (Lysine)6-capped RGD (K6-(linker-RGD)3) and OGP (K6-linker-(YGFGG)2), which were mildly grafted onto PC/Fe-MPNs through supramolecular interactions between K6 and PC residues on the MPNs surface to form a dual peptide coating, named PC/Fe@K6-RGD/OGP. The properties of the block peptides coating, including mechanics, hydrophilicity, chemical composition, etc., were detailly characterized by various techniques (ellipsometry, quartz crystal microbalance, X-ray photoelectron spectroscopy, water contact angle, scanning electronic microscopy and atomic force microscopy). Importantly, the RGD/OGP ratio can be well adjusted, which allowed optimizing the RGD/OGP ratio to endow significantly enhanced osteogenic activity of MC3T3-E1 cells through the Wnt/β-catenin pathway, while also promoting cell adhesion, immune regulation, inhibiting osteoclast differentiation and oxidative stress reduction. In vivo, the optimized RGD/OGP coatings promoted bone regeneration and osseointegration around implants in rats with bone defects. In conclusion, rationally designed PC/Fe@K6-RGD/OGP coating integrated RGD and OGP bioactivities, providing a convenient approach to enhance bioinert implant surfaces for bone regeneration.

Isolation of Pro-Osteogenic Compounds from Euptelea polyandra That Reciprocally Regulate Osteoblast and Osteoclast Differentiation.

Plants contain a large number of small-molecule compounds that are useful for targeting human health and in drug discovery. Healthy bone metabolism depends on the balance between bone-forming osteoblast activity and bone-resorbing osteoclast activity. In an ongoing study searching for 22 plant extracts effective against osteoporosis, we found that the crude extract of Euptelea polyandra Sieb. et Zucc (E. polyandra) had osteogenic bioactivity. In this study, we isolated two compounds, isoquercitrin (1) and astragalin (2), responsible for osteogenic bioactivity in osteoblastic MC3T3-E1 cells from the leaf of E. polyandra using column chromatography and the spectroscopic technique. This is the first report to isolate astragalin from E. polyandra. Compounds (1) and (2) promoted osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and alizarin red S stain-positive calcium deposition, while simultaneously suppressing tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation in RAW264.7 cells at non-cytotoxic concentrations. Isoquercitrin (1) and astragalin (2) increased the expression of osteoblastic differentiation genes, Osterix, ALP, and Osteoprotegerin in the MC3T3-E1 cells, while suppressing osteoclast differentiation genes, TRAP, Cathepsin K, and MMP 9 in the RAW264.7 cells. These compounds may be ideal targets for the treatment of osteoporosis due to their dual function of promoting bone formation and inhibiting bone resorption.

Regulating Chondro-Bone Metabolism for Treatment of Osteoarthritis via High-Permeability Micro/Nano Hydrogel Microspheres.

Destruction of cartilage due to the abnormal remodeling of subchondral bone (SB) leads to osteoarthritis (OA), and restoring chondro-bone metabolic homeostasis is the key to the treatment of OA. However, traditional intra-articular injections for the treatment of OA cannot directly break through the cartilage barrier to reach SB. In this study, the hydrothermal method is used to synthesize ultra-small size (≈5nm) selenium-doped carbon quantum dots (Se-CQDs, SC), which conjugated with triphenylphosphine (TPP) to create TPP-Se-CQDs (SCT). Further, SCT is dynamically complexed with hyaluronic acid modified with aldehyde and methacrylic anhydride (AHAMA) to construct highly permeable micro/nano hydrogel microspheres (SCT@AHAMA) for restoring chondro-bone metabolic homeostasis. In vitro experiments confirmed that the selenium atoms scavenged reactive oxygen species (ROS) from the mitochondria of mononuclear macrophages, inhibited osteoclast differentiation and function, and suppressed early chondrocyte apoptosis to maintain a balance between cartilage matrix synthesis and catabolism. In vivo experiments further demonstrated that the delivery system inhibited osteoclastogenesis and H-vessel invasion, thereby regulating the initiation and process of abnormal bone remodeling and inhibiting cartilage degeneration in SB. In conclusion, the micro/nano hydrogel microspheres based on ultra-small quantum dots facilitate the efficient penetration of articular SB and regulate chondro-bone metabolism for OA treatment.

Nrf2 Mitigates RANKL and M-CSF Induced Osteoclast Differentiation via ROS-Dependent Mechanisms.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to be a negative regulator of osteoclast differentiation, but the precise mechanisms have not yet been established. We examined the precise roles of Nrf2 in regulating antioxidants and reactive oxygen species (ROS) levels, especially the cytoplasmic and mitochondrial ROS during osteoclastogenesis in vitro. In the current study, we found that the absence of Nrf2 promotes osteoclast differentiation in bone-marrow-derived macrophages (BMMs) and RAW 264.7 cells. The receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) significantly lowered the levels of Nrf2 and its downstream antioxidant enzymes at mRNA and/or protein levels during osteoclast differentiation in the BMMs of mice and RAW 264.7 mouse leukemic monocytes. Compared to the wild-type cells, Nrf2-deficient cells exhibited heightened sensitivity to both transient RANKL-induced cytoplasmic ROS and prolonged RANKL and M-CSF-induced cytoplasmic and mitochondrial ROS accumulation. Furthermore, exogenous antioxidant agents, including N-acetyl-cysteine (NAC), diphenyleneiodonium chloride (DPI), and mitoquinone mesylate (MitoQ), exhibited substantial capability to suppress the elevation of ROS levels during osteoclast differentiation induced by Nrf2 deficiency, and they consequently inhibited osteoclast differentiation augmented by the lack of Nrf2. The activation of phosphorylated c-FOS resulting from elevated ROS promoted osteoclast differentiation. The inhibition of c-FOS blocked osteoclast differentiation, which was elevated by Nrf2-deficiency. Taken together, these data reveal that Nrf2 effectively decreased the accumulation of intracellular ROS and the phosphorylation of c-FOS during osteoclastic differentiation by regulating antioxidant enzymes and subsequently inhibited RANKL-induced osteoclast differentiation.

MicroRNA-155 targets SOCS1 to inhibit osteoclast differentiation during orthodontic tooth movement

BackgroundMicroRNA-155 (miR-155) is a multifunctional miRNA whose expression is known to be involved in a range of physiological and pathological processes. Its association with several oral diseases has been established. However, the specific role of miR-155 in orthodontic tooth movement remains unclear. In this study, we investigated the impact of miR-155 on osteoclast differentiation and orthodontic tooth movement models, aiming to explore the underlying mechanisms.MethodsIn this experiment, we utilized various agents including miR-155 mimic, miR-155 inhibitor, as well as non-specific sequences (NC mimic & NC inhibitor) to treat murine BMMNCs. Subsequently, osteoclast induction (OC) was carried out to examine the changes in the differentiation ability of monocytes under different conditions. To assess these changes, we employed RT-PCR, Western blotting, and TRAP staining techniques. For the orthodontic tooth movement model in mice, the subjects were divided into two groups: the NaCl group (injected with saline solution) and the miR-155 inhibitor group (injected with AntagomiR-155). We observed the impact of orthodontic tooth movement using stereoscopic microscopy, micro-CT, and HE staining. Furthermore, we performed RT-PCR and Western blotting analyses on the tissues surrounding the moving teeth. Additionally, we employed TargetScan to predict potential target genes of miR-155.ResultsDuring osteoclast induction of BMMNCs, the expression of miR-155 exhibited an inverse correlation with osteoclast-related markers. Overexpression of miR-155 led to a decrease in osteoclast-related indexes, whereas underexpression of miR-155 increased those indexes. In the mouse orthodontic tooth movement model, the rate of tooth movement was enhanced following injection of the miR-155 inhibitor, leading to heightened osteoclast activity. TargetScan analysis identified SOCS1 as a target gene of miR-155.ConclusionsOur results suggest that miR-155 functions as an inhibitor of osteoclast differentiation, and it appears to regulate osteoclasts during orthodontic tooth movement. The regulatory mechanism of miR-155 in this process involves the targeting of SOCS1.

Quercetin suppresses ovariectomy-induced osteoporosis in rat mandibles by regulating autophagy and the NLRP3 pathway.

With the aging population and the popularity of implant prostheses, an increasing number of postmenopausal osteoporosis (PMOP) patients require implant restorations; however, poor bone condition affects the long-term stability of implant prostheses. This study aimed to investigate the therapeutic effect of quercetin (QR) compared with alendronate (ALN), the primary treatment for PMOP, on mandibular osteoporosis (OP) induced by ovariectomy (OVX) in female rats. Adult female rats were treated with QR (50 mg/kg/day), ALN (6.25 mg/kg/week) by gavage for 8 weeks, chloroquine (CQ, 10 mg/kg/twice a week), and cytokine release inhibitory drug 3 (MCC950, 10 mg/kg/three times a week) by intraperitoneal injection for 8 weeks after bilateral OVX. Blood samples were collected prior to euthanasia; the mandibles were harvested and subjected to micro-computed tomography (micro-CT) and pathological analysis. QR administration controlled weight gain and significantly improved the bone microstructure in OVX rats, increasing bone mass, and bone mineral density (BMD), reducing bone trabecular spacing, and decreasing osteoclast numbers. Western blotting, real-time quantitative PCR (RT-qPCR), and serum markers confirmed that QR inhibited interleukin- 1β (IL-1β) and interleukin-18 (IL-18) on the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) protein 3 (NLRP3) pathway thereby inhibiting osteoclast differentiation, immunofluorescence and western blotting also confirmed that QR inhibited autophagy in OVX rats and suppressed the number of tartrate-resistant acid phosphatase (TRAP)-stained positive osteoclasts. The findings suggest that QR may protect the bone structure and prevent bone loss in osteoporotic rats by inhibiting the NLRP3 pathway and autophagy in osteoclasts with comparable effects to ALN, thus QR may have the potential to be a promising alternative supplement for the preventive and therapeutic treatment of PMOP.

Epigenetic landscape analysis reveals the significance of early reduced chromatin accessibility in osteoclastogenesis.

Recently improved techniques could provide snapshots of chromatin structure generated based on chromatin accessibility. Since chromatin accessibility determines transcriptional potential, it has been attempted in a variety of cell systems. However, there has been no genome-wide analysis of chromatin accessibility for the entire murine osteoclast (OC) differentiation process. We performed an Assay for Transposase-Accessible Chromatin (ATAC)-sequencing (seq) during RANKL-induced OC differentiation and found that global chromatin accessibility decreased, especially early in OC differentiation. The global histone H3K27Ac level, an active histone modification mark, was diminished during OC differentiation by western blot and histone extract experiments. Its genomic enrichment was also reduced based on publicly available H3K27Ac chromatin immunoprecipitation (ChIP)-seq data. ATAC-seq and H3K27Ac ChIP-seq data demonstrated that RANKL induced a less accessible chromatin state during OC differentiation. Restoration of reduced H3K27Ac, presumably representing accessible states upon acetate treatment, suppresses OC differentiation by provoking immune-related gene expression. Subsequential integrative analysis of ATAC-seq, RNA-seq after acetate treatment, and H3K27Ac ChIP-seq reveals that Irf8 and its downstream targets are the most vulnerable to chromatin accessibility changes and acetate supplementation. Taken together, our study generated chromatin accessibility maps during the whole OC differentiation and suggested perturbation of chromatin accessibility might be a potential therapeutic strategy for excessive OC diseases.

Mesenchymal stem cell-derived apoptotic bodies alleviate alveolar bone destruction by regulating osteoclast differentiation and function

Periodontitis is caused by overactive osteoclast activity that results in the loss of periodontal supporting tissue and mesenchymal stem cells (MSCs) are essential for periodontal regeneration. However, the hypoxic periodontal microenvironment during periodontitis induces the apoptosis of MSCs. Apoptotic bodies (ABs) are the major product of apoptotic cells and have been attracting increased attention as potential mediators for periodontitis treatment, thus we investigated the effects of ABs derived from MSCs on periodontitis. MSCs were derived from bone marrows of mice and were cultured under hypoxic conditions for 72 h, after which ABs were isolated from the culture supernatant using a multi-filtration system. The results demonstrate that ABs derived from MSCs inhibited osteoclast differentiation and alveolar bone resorption. miRNA array analysis showed that miR-223-3p is highly enriched in those ABs and is critical for their therapeutic effects. Targetscan and luciferase activity results confirmed that Itgb1 is targeted by miR-223-3p, which interferes with the function of osteoclasts. Additionally, DC-STAMP is a key regulator that mediates membrane infusion. ABs and pre-osteoclasts expressed high levels of DC-STAMP on their membranes, which mediates the engulfment of ABs by pre-osteoclasts. ABs with knock-down of DC-STAMP failed to be engulfed by pre-osteoclasts. Collectively, MSC-derived ABs are targeted to be engulfed by pre-osteoclasts via DC-STAMP, which rescued alveolar bone loss by transferring miR-223-3p to osteoclasts, which in turn led to the attenuation of their differentiation and bone resorption. These results suggest that MSC-derived ABs are promising therapeutic agents for the treatment of periodontitis.

LncRNA-Gm5532 regulates osteoclast differentiation through the miR-125a-3p/TRAF6 axis.

Long noncoding RNAs (lncRNAs) are important regulators of bone metabolism. In this study, lncRNA microarray analysis was used to identify differentially expressed lncRNAs in differentiated osteoclasts. lncRNA-Gm5532 is highly expressed during osteoclast differentiation. lncRNA-Gm5532 knockdown impairs osteoclast formation and bone resorption. Mechanistic experiments show that lncRNA-Gm5532 functions as a competing endogenous RNA (ceRNA) and acts as a sponge for miR-125a-3p, which promotes TNF receptor-associated factor 6 (TRAF6) expression. miR-125a-3p mimics suppress osteoclast differentiation and TAK1/NF-κB/MAPK signaling. The miR-125a-3p inhibitor reverses the negative effects of siGm5532 on osteoclast differentiation. In summary, our study reveals that lncRNA-Gm5532 functions as an activator in osteoclast differentiation by targeting the miR-125a-3p/TRAF6 axis, making it a novel biomarker and potential therapeutic target for osteoporosis.

Water Extract of Desalted Salicornia europaea Inhibits RANKL-Induced Osteoclast Differentiation and Prevents Bone Loss in Ovariectomized Mice

Osteoporosis, which is often associated with increased osteoclast activity due to menopause or aging, was the main focus of this study. We investigated the inhibitory effects of water extract of desalted Salicornia europaea L. (WSE) on osteoclast differentiation and bone loss in ovariectomized mice. Our findings revealed that WSE effectively inhibited RANKL-induced osteoclast differentiation, as demonstrated by TRAP staining, and also suppressed bone resorption and F-actin ring formation in a dose-dependent manner. The expression levels of genes related to osteoclast differentiation, including NFATc1, ACP5, Ctsk, and DCSTAMP, were downregulated by WSE. Oral administration of WSE improved bone density and structural parameters in ovariectomized mice. Dicaffeoylquinic acids (DCQAs) and saponins were detected in WSE, with 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA being isolated and identified. All tested DCQAs, including the aforementioned types, inhibited osteoclast differentiation, bone resorption, and the expression of osteoclast-related genes. Furthermore, WSE and DCQAs reduced ROS production mediated by RANKL. These results indicate the potential of WSE and its components, DCQAs, as preventive or therapeutic agents against osteoporosis and related conditions.

Extracellular Vesicles from Human Adipose Tissue-Derived Mesenchymal Stem Cells Suppress RANKL-Induced Osteoclast Differentiation via miR122-5p.

Researchers are increasingly interested in cell therapy using mesenchymal stem cells (MSCs) as an alternative remedy for osteoporosis, with fewer side effects. Thus, we isolated and characterized extracellular vesicles (EVs) from human adipose tissue-derived MSCs (hMSCs) and investigated their inhibitory effects on RANKL-induced osteoclast differentiation. Purified EVs were collected from the supernatant of hMSCs by tangential flow filtration. Characterization of EVs included typical evaluation of the size and concentration of EVs by nanoparticle tracking analysis and morphology analysis using transmission electron microscopy. hMSC-EVs inhibited RANKL-induced differentiation of bone marrow-derived macrophages (BMDMs) into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by EV treatment of osteoclasts. In addition, EVs decreased RANKL-induced phosphorylation of p38 and JNK and expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. To elucidate which part of the hMSC-EVs plays a role in the inhibition of osteoclast differentiation, we analyzed miRNA profiles in hMSC-EVs. The results showed that has-miR122-5p was present at significantly high read counts. Overexpression of miR122-5p in BMDMs significantly inhibited RANKL-induced osteoclast differentiation and induced defects in F-actin ring formation and bone resorption. Our results also revealed that RANKL-induced phosphorylation of p38 and JNK and osteoclast-specific gene expression was decreased by miR122-5p transfection, which was consistent with the results of hMSC-EVs. These findings suggest that hMSC-EVs containing miR122-5p inhibit RANKL-induced osteoclast differentiation via the downregulation of molecular mechanisms and could be a preventive candidate for destructive bone diseases.

Inter-Donor Variability in Osteoclastogenesis and Responses to Osteoprotegerin (OPG), Von Willebrand Factor (vWF), and Factor VIII (FVIII)

Background: Patients with hemophilia A (HA) commonly exhibit reduced bone mineral density. We hypothesize that osteoclast differentiation may be influenced by the absence of factor VIII (FVIII), as well as other complement-associated proteins such as von Willebrand factor (vWF). Combinatory effects of vWF and recombinant FVIII (rFVIII) on osteoclast differentiation have been described previously, however the effects of rFVIII or vWF on osteoclast precursors across individual donors in the presence of osteoprotegerin (OPG), an important inhibitor of osteoclast differentiation, have not been systematically addressed. Aim: The main aim of this study is to investigate the effect of vWF, FVIII, or a combination of both factors, on osteoclastogenesis (induced by receptor activator of NF-κB ligand, RANKL) in differentiating osteoclasts from individual human donors, in the presence or absence of OPG. Methods: CD14+ monocytes (osteoclast progenitors) were isolated from male healthy donors and cultured for 11 days in medium with 10% fetal calf serum (FCS), according to established protocols. The response of these osteoclast precursors to RANKL and OPG was measured by culturing them under the following conditions: macrophage colony-stimulating factor (M-CSF) (25 ng/ml), M-CSF + RANKL (60 ng/ml) and M-CSF + RANKL + OPG (concentration range 30-60 ng/mL) with cells from each donor. Cells were stained for presence of tartrate-resistant acid phosphatase (TRAP), actin ring, and number of nuclei, and the osteoclast-occupied surface was measured using a semi-automated imaging and analysis protocol. Subsequently, cells were cultured either in 10% human serum (HS) or 10% FCS and exposed to vWF (5 or 10 IU/mL), rFVIII (10 IU/mL), or both vWF + rFVIII for 7, 11 or 14 days in the presence of M-CSF, RANKL and OPG. Morphology was assessed by the aforementioned stainings, TRAP enzymatic activity was determined in the cell culture supernatant, and cathepsin K (CATK) expression was measured using qPCR. The potential of vWF and/or rFVIII to decrease osteoclastogenesis in the presence of OPG was measured through osteoclast-occupied surfaces or osteoclast numbers, with or without TRAP enzymatic activity and/or CATK expression. Donors were categorized as OPG-responding or OPG-non-responding based on the relative reduction of osteoclast formation in the presence of OPG. Results: The initial response to RANKL and OPG was determined in 7 donors. Two OPG-non-responding (donors #1 and #2) and 5 OPG-responding donors (donors #3-7) were identified (Figure 1A). Most donors showed a reduced osteoclastogenesis in presence of OPG and vWF. In donor #2, in whom osteoclastogenesis in the presence of OPG was already low, a further reduction by vWF could not be achieved. Of note, a clear inhibition was observed in this donor without OPG (Figure 1B). The effects of rFVIII were more donor-dependent than those with vWF or vWF + rFVIII, with 4 of 7 donors showing reduced osteoclastogenesis. The results were influenced by the type of serum (HS or FCS) used. In all donors, and irrespective of the medium type, the combination of vWF + rFVIII reduced osteoclast formation, indicating a potentially beneficial effect of vWF + rFVIII compared with vWF or rFVIII alone (Figure 1B). Conclusion: Under inhibitory conditions (i.e. in the presence of OPG), rFVIII and vWF (alone or combined) decreased osteoclastogenesis in vitro. This may indicate that vWF and rFVIII, either alone or in combination, can influence the potency of OPG to inhibit osteoclastogenesis. However, this effect was not consistent in all donors. Currently, we are looking into the mechanisms that may explain the differential behavior of osteoclasts derived from OPG-responding and OPG-non-responding donors, as well as studying these effects in cells from patients with HA. We speculate that the absence of FVIII may lead to less efficient inhibition of OPG and increased osteoclastogenesis, which could explain lower bone mineral density observed in HA.