Osteoclast Differentiation Research Articles

Gingival mesenchymal stem cells derived from patients with rheumatoid arthritis treat experimental arthritis

AbstractA therapeutic strategy using mesenchymal stem cells (MSCs) has been accepted as a novel therapy for treating rheumatoid arthritis (RA). Human gingiva‐derived MSCs (GMSCs) are superior in regulating immune responses. Autologous MSCs are the optimal candidate to avoid the potential risks of allogenic MSCs. However, whether autologous GMSCs from RA patients are therapeutic remains unknown. In this study, we compared the therapeutic efficacy of GMSCs derived from patients with RA (RA‐GMSCs) and that from health donors (H‐GMSCs) in vivo and in vitro. Then, we utilized RNA‐sequencing, the molecular and cellular assays to determine the immunomodulatory molecules that contribute to the therapeutic effect of RA‐GMSCs on both collagen‐induced arthritis (CIA) and humanized synovitis models. We demonstrated that GMSCs derived from patients with RA (RA‐GMSCs) and health donors (H‐GMSCs) shared a similar expression of immunomodulatory molecules. Moreover, RA‐GMSCs were as effective as H‐GMSCs in suppressing T‐cell proliferation, proinflammatory cytokines secretion, as well as osteoclast differentiation in vitro. In addition, RA‐GMSCs had a robust therapeutic effect on the CIA model. Importantly, RA GMSCs can survive for at least 24 days in a CIA mouse model and can be distributed in the spleen, lymph nodes, and joints. Specifically, RA‐GMSCs decreased the frequency of Th1 and Th17 cells whereas enhanced Treg cells, reducing the joint histopathological scores of lymphocytes, osteoclasts, and cartilages. Moreover, RA‐GMSCs were also effective in suppressing inflamed synoviocyte proliferation, migration, and invasion in vitro, and cartilage invasion in a humanized synovitis model in vivo. Our study implies that manipulation of RA‐GMSCs is therapeutic in CIA mice and humanized synovitis models and may have therapeutic potential in RA patients using autologous GMSCs in the future.

Paired single-cell RNA sequencing and T cell receptor sequencing reveals both pro- and anti-inflammatory roles of T cells in patients with calcific aortic valve disease

Abstract Background Calcified aortic valve disease (CAVD) is a chronic unresolvable inflammatory disorder that affects 25% of adults over 65 years old, leading to aortic stenosis, heart failure, and death [1]. Since the molecular mechanisms of its pathogenesis are not well understood, therapies to treat root causes of the disease remain to be developed, with valve replacement as the major intervention strategy currently. Increasing evidence indicates that substantial T cells infiltrate in the aortic valve during calcification, and clonal expanded CD8+ T cells have been identified in the calcificed aortic valve by bulk T cell receptor (TCR) repertoire sequencing [2]. Purpose To decipher T cell heterogenity and understand the precise mechanisms of T cell subsets involving CAVD progression as a basis for novel therapeutic targets. Methods In Cohort 1, we collected aortic valves from CAVD (n=3) and healthy (n=2) patients for single-cell RNA sequencing (scRNA-seq). In Cohort 2, we performed paired scRNA-seq and TCR sequencing (scTCR-seq) on aortic valves obtained from CAVD (n=3) and healthy (n=3) patients to evaluate both gene expression and clonality. Pathway enrichment analysis and cell-cell interaction analysis elucidated the function of T cells in the calcificied valves. Immunohistochemistry and fluorescence-activated cell sorting were recruited to verify the key findings. Results scRNA-seq demonstrated a conversion from anti-inflammatory regulatory T (Treg) cells towards pro-inflammatory T helper 17 (Th17) cells in the calcificied aortic valve. Paired scRNA-seq and scTCR-seq revealed CAVD-related signaling pathways (osteoclast differentiation, NOD-like receptor signaling pathways, IL-17 signaling pathways) significantly upregulated in highly expanded CD8+ T cells in the calcified valve, with upregulation of pro-inflammatory mediators such as IFNG, CCL4 and CCL5, suggesting pro-inflammation functions in clonally expanded T cells. Moreover, we characterized tissue-resident memory T cells, which resembled similar transcriptome and TCR clonality as exhausted T cells in calcified valves. These cells up-regulated anti-inflammation-related transcriptomes but their cellular fraction decreased in calcified valves, implying anti-inflammatory functions of these tissue-resident memory and exhausted T cells. Conclusion This study elucidates transcriptomic and immune profiling of T cells in the calcified aortic valve, therefore shedding light on the pathophysiological mechanisms underlying aortic valve calcification from the novel perspective of tissue-specific T lymphocytes, providing promising targets for immune checkpoint-based therapeutic interventions.

The Biphasic Activity of Auricularia Auricula-Judae Extract on Bone Homeostasis Through Inhibition of Osteoclastogenesis and Modulation of Osteogenic Activity.

Osteoporosis arises from the disturbance of bone homeostasis, a process regulated by osteoblasts and osteoclasts. The treatment and prevention of bone metabolic disorders resulting from an imbalance in bone homeostasis require the use of agents that effectively promote both bone formation and anti-resorptive effects. Therefore, an investigation was carried out to determine the potential of the edible mushroom Auricularia auricula-judae in modulating bone remodeling by inhibiting RANKL-induced osteoclastogenesis and enhancing BMP-2-stimulated osteoblast differentiation. Moreover, this study assessed the mode of action of the Auricularia auricula-judae extracts. The staining of tartrate-resistant acid phosphatase (TRAP), a marker for osteoclast activity, demonstrated that Auricularia auricula-judae water extract (AAJWE) inhibited the formation of multinucleated osteoclasts while exhibiting no cytotoxic effects. The study demonstrated that AAJWE reduced RANKL-induced osteoclast differentiation by inhibiting c-Fos/NFATc1 through the inhibition of ERK and JNK phosphorylation during the RANKL-induced osteoclast differentiation. Moreover, AAJWE exhibited a dose-dependent induction of ALP expression in the presence of BMP-2 during osteoblast differentiation. The AAJWE strengthened BMP-2-induced osteogenesis through the activation of Runx2 and Smad phosphorylation. Therefore, AAJWE emerges as a promising candidate for both prevention and therapy owing to its biphasic effect, which aids in the preservation of bone homeostasis.

Dennd2c Negatively Controls Multinucleation and Differentiation in Osteoclasts by Regulating Actin Polymerization and Protrusion Formation

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages.

Autophagy Regulator Rufy 4 Promotes Osteoclastic Bone Resorption by Orchestrating Cytoskeletal Organization via Its RUN Domain

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4’s potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption.

Antimicrobial peptide GL13K-Modified titanium in the epigenetic regulation of osteoclast differentiation via H3K27me3

Antimicrobial peptide GL13K-Modified titanium in the epigenetic regulation of osteoclast differentiation via H3K27me3

Propionibacterium freudenreichii MJ2-derived extracellular vesicles inhibit RANKL-induced osteoclastogenesis and improve collagen-induced rheumatoid arthritis

Rheumatoid arthritis causes excessive bone loss by stimulating osteoclast differentiation. Extracellular vesicles are valuable disease markers, conveyors of distant cell-to-cell communication, and carriers for drug delivery. The aim of this study was to investigate the anti-osteoclastogenic effects of extracellular vesicles derived from dairy Propionibacterium freudenreichii MJ2 (PFEVs) and the improvement effect of PFEVs on collagen-induced arthritis (CIA) animal model. PFEVs were observed by scanning electron microscopy, transmission electron microscopy, nanoparticle tracking analysis, and LC–MS/MS. The inhibitory activity of PFEVs against receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation was investigated in RAW 264.7 cells. PFEVs significantly decreased the expression levels of genes and proteins related to osteoclast differentiation. PFEVs decreased RANK–RANKL binding. In a CIA mouse model, PFEVs treatment significantly reduced arthritis scores and collagen-specific immunoglobulins. PFEVs treatment also reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines. The anti-inflammatory effects were confirmed by H&E staining, and PFEVs treatment inhibited osteoclastogenesis in the CIA mouse model. In conclusion, PFEVs inhibited osteoclast differentiation by inhibiting RANK–RANKL signaling, thereby decreasing the expression of osteoclast differentiation-related genes. PFEVs also improved collagen-induced arthritis by inhibiting inflammation and osteoclastogenesis.

Geranylgeranyl diphosphate synthase inhibition impairs osteoclast differentiation, morphology and resorptive activity

Abstract Nitrogen bisphosphonates (NBPs), such as zoledronic acid, target the enzyme farnesyl diphosphate synthase (FDPS) in the isoprenoid biosynthetic pathway (IBP), and are the frontline treatment for osteolytic bone diseases. The strong affinity of these agents for bone limits their distribution out of the skeleton. Geranylgeranyl diphosphate synthase (GGDPS) is directly downstream to FDPS in the IBP and novel GGDPS inhibitors such as RAM2061 have been shown to have key drug-like features including prolonged half-life, metabolic stability, and systemic distribution. Furthermore, RAM2061 exerts anti-neoplastic benefits in mouse models of multiple myeloma and Ewing sarcoma. Therefore, we are interested in determining the potential impact of RAM2061 on osteoclast biology and bone remodeling. Studies utilizing undifferentiated RAW264.7 cells demonstrated that treatment with RAM2061 depletes cells of GGPP, impairs protein geranylgeranylation and induces markers of the unfolded protein response pathway and apoptosis. Differentiation of RAW264.7 cells to mature osteoclasts is disrupted by RAM2061, resulting in decreased numbers of mature osteoclasts, altered morphology and decreased tartrate resistant acid phosphatase activity. Treatment of fully differentiated RAW264.7 cells with RAM2061 led to decreased resorptive activity. Confocal microscopy studies revealed that RAM2061 disrupts Cdc42 localization, inhibiting proper actin ring formation in osteoclasts. No significant impact on bone turnover markers or bone histomorphology was observed following a 3-week treatment of CD-1 mice with RAM2061, although decreased numbers of osteoclasts were observed. Liquid chromatography–tandem mass spectrometry studies confirmed accumulation of RAM2061 in bone from the in vivo studies as well as hydroxyapatite binding in vitro. In conclusion, these studies are the first to demonstrate the anti-osteoclastic activity of GGDPS inhibitor treatment and support future studies exploring the therapeutic benefit of this novel therapy in the setting of pathological bone remodeling.

Effects of coagulation factors on bone cells and consequences of their absence in haemophilia a patients

Haemophilia is associated with reduced bone mass and mineral density. Due to the rarity of the disease and the heterogeneity among the studies, the pathogenesis of bone loss is still under investigation. We studied the effects of coagulation factors on bone cells and characterized in a pilot study the osteoclastogenic potential of patients’ osteoclast precursors. To evaluate the effect of coagulation factors on osteoclasts, we treated Healthy Donor-Peripheral Blood Mononuclear Cells (HD-PBMC) with Factor VIII (FVIII), von Willebrand Factor (VWF), FVIII/VWF complex, activated Factor IX (FIXa), activated Factor X (FXa) and Thrombin (THB). FVIII, VWF, FVIII/VWF, FXa and THB treatments reduced osteoclast differentiation of HD-PBMC and VWF affected also bone resorption. Interestingly, PBMC isolated from patients with moderate/severe haemophilia showed an increased osteoclastogenic potential due to the alteration of osteoclast precursors. Moreover, increased expression of genes involved in osteoclast differentiation/activity was revealed in osteoclasts of an adult patient with moderate haemophilia. Control osteoblasts treated with the coagulation factors showed that FVIII and VWF reduced ALP positivity; the opposite effect was observed following THB treatment. Moreover, FVIII, VWF and FVIII/VWF reduced mineralization ability. These results could be important to understand how coagulation factors deficiency influences bone remodeling activity in haemophilia.

Viscoelastic Hydrogel Modulates Phenotype of Macrophage-Derived Multinucleated Cells and Macrophage Differentiation in Foreign Body Reactions.

Biomaterial-induced macrophage-derived multinucleated cells (MNCs) are often observed at or near material implantation sites, yet their subtypes and roles in tissue repair and wound healing remain unclear. This study compares material-induced MNCs to cytokine-induced MNCs using both in vitro and in vivo models. 3D-embedded Raw264.7 cells and rat bone marrow-derived monocytes (BMDMs), with or without cytokines such as IL-4 and RANKL, were characterized for their MNC morphologies and subtypes via in situ immunocytochemistry and flow cytometry. Macrophage polarization and osteoclastic differentiation were assessed through NO production, arginase activity, and tartrate-resistant acid phosphatase levels. 3D matrix-induced MNCs expressed the same phenotypic heterogeneity as the IL-4 and RANK-treated ones. 3D matrix-induced MNCs displayed the same phenotypic heterogeneity as those treated with IL-4 and RANKL. A high viscoelastic matrix (1006.48 ± 92.29 Pa) induced larger populations of proinflammatory and osteoclast-like MNCs, whereas a low viscoelastic matrix (38.61 ± 7.56 Pa) supported active differentiation and gene expression across pro-, anti-inflammatory, and osteoclast-like macrophages. Matrix viscoelasticity also influenced the effects of IL-4 and RANKL on macrophage-derived MNC polarization. In an in vivo subcutaneous implantation model, medium to high viscoelastic matrices exhibited higher populations of CD86+ and RANK+ MNCs, while low viscoelastic matrices showed higher populations of CD206+ MNCs. These findings suggest that matrix viscoelasticity modulates macrophage differentiation and MNC phenotype, with low viscoelastic matrices potentially favoring anti-inflammatory MNCs and macrophage differentiation suitable for subcutaneous implantation.

Matrix-bound nanovesicles alleviate particulate-induced periprosthetic osteolysis.

Aseptic loosening of orthopedic implants is an inflammatory disease characterized by immune cell activation, chronic inflammation, and destruction of periprosthetic bone, and is one of the leading reasons for prosthetic failure, affecting 12% of total joint arthroplasty patients. Matrix-bound nanovesicles (MBVs) are a subclass of extracellular vesicle recently shown to mitigate inflammation in preclinical models of rheumatoid arthritis and influenza-mediated "cytokine storm." The molecular mechanism of these anti-inflammatory properties is only partially understood. The objective of the present study was to investigate the effects of MBV on RANKL-induced osteoclast formation in vitro and particulate-induced osteolysis in vivo. Results showed that MBV attenuated osteoclast differentiation and activity by suppressing the NF-κB signaling pathway and downstream NFATc1, DC-STAMP, c-Src, and cathepsin K expression. In vivo, local administration of MBV attenuated ultrahigh molecular weight polyethylene particle-induced osteolysis, bone reconstruction, and periosteal inflammation. The results suggest that MBV may be a therapeutic option for preventing periprosthetic loosening.

An Integrated Microcurrent Delivery System Facilitates Human Parathyroid Hormone Delivery for Enhancing Osteoanabolic Effect.

Human parathyroid hormone (1-34) (PTH) exhibits osteoanabolic and osteocatabolic effects, with shorter plasma exposure times favoring bone formation. Subcutaneous injection (SCI) is the conventional delivery route for PTH but faces low delivery efficiency due to limited passive diffusion and the obstruction of the vascular endothelial barrier, leading to prolonged drug exposure times and reduced osteoanabolic effects. In this work, a microcurrent delivery system (MDS) based on multimicrochannel microneedle arrays (MMAs) is proposed, achieving high efficiency and safety for PTH transdermal delivery. The internal microchannels of the MMAs are fabricated using high-precision 3D printing technology, providing a concentrated and safe electric field that not only accelerates the movement of PTH but also reversibly increases vascular endothelial permeability by regulating the actin cytoskeleton and interendothelial junctions through Ca2+-dependent cAMP signaling, ultimately promoting PTH absorption and shortening exposure times. The MDS enhances the osteoanabolic effect of PTH in an osteoporosis model by inhibiting osteoclast differentiation on the bone surface compared to SCI. Moreover, histopathological analysis of the skin and organs demonstrated the good safety of PTH delivered by MDS in vivo. In addition to PTH, the MDS shows broad prospects for the high-efficiency transdermal delivery of macromolecular drugs.

Employing single-cell RNA sequencing coupled with an array of bioinformatics approaches to ascertain the shared genetic characteristics between osteoporosis and obesity.

This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously. Using weighted gene co-expression network analysis (WGCNA), we analyzed datasets from the Gene Expression Omnibus (GEO) database to identify co-expressed gene modules in OB and OP. These modules underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and protein-protein interaction analysis to discover Hub genes. Machine learning refined the gene selection, with further validation using additional datasets. Single-cell analysis emphasized specific cell subpopulations, and enzyme-linked immunosorbent assay (ELISA), protein blotting, and cellular staining were used to investigate key genes. WGCNA revealed critical gene modules for OB and OP, identifying the Toll-like receptor (TLR) signalling pathway as a common factor. TLR2 was the most significant gene, with a pronounced expression in macrophages. Elevated TLR2 expression correlated with increased adipose accumulation, inflammation, and osteoclast differentiation, linking it to OP development. Our study underscores the pivotal role of TLR2 in connecting OP and OB. It highlights the influence of TLR2 in macrophages, driving both diseases through a pro-inflammatory mechanism. These insights propose TLR2 as a potential dual therapeutic target for treating OP and OB.

Circulating Baseline CXCR3+Th2 and Th17 Cell Proportions Correlate With Trabecular Bone Loss After 48 Weeks of Biological Treatment in Early Rheumatoid Arthritis.

The high prevalence of osteoporosis in rheumatoid arthritis (RA) is due to inflammation that stimulates differentiation of osteoclasts, a process involving circulating monocytes and T cell-derived factors. The aim of this study was to evaluate relations between circulating monocytes, T cell subsets, and changes in bone characteristics before and after treatment with biological disease-modifying antirheumatic drugs (bDMARDs) in RA. Thirty patients with untreated early RA who met the American College of Rheumatology/EULAR 2010 criteria were included. Data were collected before and 48 weeks after treatment with methotrexate (MTX) together with one of three bDMARDs (abatacept, tocilizumab, or certolizumab pegol). Disease activity was measured using the Clinical Disease Activity Index, swollen or tender joint counts, C-reactive protein levels, and erythrocyte sedimentation rates. Proportions of monocyte and CD4+ T cell subsets in blood samples were analyzed by flow cytometry. Bone densitometry was performed using high-resolution peripheral quantitative computed tomography (HR-pQCT). HR-pQCT revealed an overall decrease in cortical (P = 0.009) and trabecular (P = 0.034) bone mineral density, although a subset of patients showed no bone loss after 48 weeks of treatment. The overall bone loss was not associated with age, body mass index, sex, intraarticular glucocorticoid injections, or baseline disease activity. Loss of trabecular bone volume fraction correlated with high proportions of circulating CXCR3+Th2 cells (r = -0.38, P = 0.04) and CXCR3+Th17 cells (r = -0.36, P = 0.05) at baseline. Similarly, no loss of trabecular bone volume fraction correlated with high proportions of regulatory T cells (r = 0.4, P = 0.03) at baseline. However, the associations were not significant when corrected for confounders and multiple testing. MTX together with bDMARDs efficiently reduce disease activity but only prevent bone loss in a subset of patients with RA after 48 weeks of treatment. The correlations of circulating baseline T helper cell and regulatory T cell populations with trabecular bone changes suggest a potential novel role for these cells in systemic bone homeostasis during early RA.

Amentoflavone maintaining extracellular matrix homeostasis and inhibiting subchondral bone loss in osteoarthritis by inhibiting ERK, JNK and NF-κB signaling pathways

Amentoflavone (AF), a plant biflavone isolated from Selaginella sinensis ethanol extract, is characterized by anti-inflammatory and anti-oxidant properties. According to previous studies, inflammation and oxidative stress are closely related to the pathophysiology of osteoarthritis (OA). However, the effects and mechanisms of AF on OA have not been elucidated.To investigate the inhibitory effects and its molecular mechanism of AF on extracellular matrix (ECM) degradation stimulated by IL-1β as well as subchondral bone loss induced by RANKL in mice chondrocytes. Quantitative PCR was used to detect the mRNA expression of genes related to inflammation, ECM, and osteoclast differentiation. Protein expression level of iNOS, COX-2, MMP13, ADAMTS5, COL2A1, SOX9, NFATc1, c-fos, JNK, ERK, P65, IκBα was measured by western blotting. The levels of TNF-α and IL-6 in the supernatants were measured by ELISA. The amount of ECM in chondrocytes was measured using toluidine blue staining. The levels of Aggrecan and Col2a1 in chondrocytes were measured using immunofluorescence. Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and immunofluorescence were used to detect the effect of AF on osteoclast differentiation and bone resorption. The effect of AF on destabilization of the medial meniscus (DMM)-induced OA mice can be detected in hematoxylin–eosin (H&E) staining, Safranin O green staining and immunohistochemistry.AF might drastically attenuated IL-1β-stimulated inflammation and reduction of ECM formation by blocking ERK and NF-κB signaling pathways in chondrocytes. Meanwhile, AF suppressed the formation of osteoclasts and the resorption of bone function induced by RANKL. In vivo, AF played a protective role by stabilizing cartilage ECM and inhibiting subchondral bone loss in destabilization of the medial meniscus (DMM)-induced OA mice, further proving its protective effect in the development of OA. Our study show that AF alleviated OA by suppressing ERK, JNK and NF-κB signaling pathways in OA models in vitro and DMM-induced OA mice, suggesting that AF might be a potential therapeutic agent in the treatment of OA.

Orthosilicic acid inhibits human osteoclast differentiation and bone resorption.

Silicon (Si), which is present in the diet in the bioavailable form of orthosilicic acid (OSA) and is detected as a dissolution product of certain bone-substitute materials, is suggested to promote bone health, and enhance bone healing, respectively. Silicon has been shown to stimulate osteoblastic cell differentiation and function, although the effect of Si on human osteoclasts is unclear. The present study investigated the direct effects of Si on human osteoclast differentiation, gene expression, and bone resorption. Human CD14+ monocytes were isolated from buffy coats and cultured with M-CSF and RANKL in medium without or with Si (50 μg/ml; constituting 75% OSA). The effects of Si on osteoclast differentiation were evaluated by TRAP-staining and the expression levels of CtsK, CalcR, TRAP, and DC-STAMP measured by RT-qPCR. The effect of Si on the expression level of AQP9, which encodes a potential Si transporter, was also analyzed. Bone resorption was determined based on the number of resorption pits formed when the RANKL-stimulated monocytes were cultured on bone slices, and by the levels of type I collagen fragments released into the cell culture medium. Silicon significantly inhibited the number of TRAP+ multinucleated cells and the expression of osteoclast related genes but increased the late expression of AQP9. Furthermore, Si significantly inhibited the number of resorption pits and the amount of collagen fragments in the medium when cells were cultured on bone slices. Our results demonstrate that OSA inhibits RANKL-stimulated human osteoclast differentiation, gene expression of osteoclast phenotypic markers, and bone resorption.

Measuring Calcium Levels in Bone-Resorbing Osteoclasts and Bone-Forming Osteoblasts.

Bone remodeling is a crucial, dynamic process that renews bone and maintains mineral homeostasis. It consists of several steps, including osteoclastic bone resorption and osteoblastic bone formation and mineralization. Intracellular calcium signaling is essential for osteoclast and osteoblast differentiation and activity. Here, we describe the differentiation of human osteoclasts and osteoblasts in vitro and provide common methods to determine cell differentiation and activity. We then describe protocols for measuringintracellular calcium in these cells using Fura2-AM.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq revealed key genes for bone metastasis and chemoresistance in prostate cancer.

Prostate cancer (PCa) is a serious malignancy. The main causes of PCa aggravation and death are unexplained resistance to chemotherapy and bone metastases. This study aimed to investigate the molecular mechanisms associated with the dynamic processes of progression, bone metastasis, and chemoresistance in PCa. Through comprehensive analysis of single-cell RNA sequencing (scRNA-seq) data, Gene Expression Omnibus (GEO) tumor progression and metastasis-related genes were identified. These genes were subjected to lasso regression modeling using the Cancer Genome Atlas (TCGA) database. Tartrate-resistant acid phosphatase (TRAP) staining and real-time quantitative PCR (RT-qPCR) were used to evaluate osteoclast differentiation. CellMiner was used to confirm the effect of LDHA on chemoresistance. Finally, the relationship between LDHA and chemoresistance was verified using doxorubicin-resistant PCa cell lines. 7928 genes were identified as genes related to tumor progression and metastasis. Of these, 7 genes were found to be associated with PCa prognosis. The scRNA-seq and TCGA data showed that the expression of LDHA was higher in tumors and associated with poor prognosis of PCa. In addition, upregulation of LDHA in PCa cells induces osteoclast differentiation. Additionally, high LDHA expression was associated with resistance to Epirubicin, Elliptinium acetate, and doxorubicin. Cellular experiments demonstrated that LDHA knockdown inhibited doxorubicin resistance in PCa cells. LDHA may play a potential contributory role in PCa initiation and development, bone metastasis, and chemoresistance. LDHA is a key target for the treatment of PCa.

Maltobionic acid protects against ovariectomy‐induced osteoporosis by suppressing bone resorption

AbstractBackgroundOsteoporosis is a debilitating disease characterized by decreased bone density. In this study, we evaluated the anti‐osteoporotic effect of maltobionic acid (MB), one of the components of honey, and calcium maltobionate (MBCa) on bone density and metabolism using a mouse model of osteoporosis. The underlying mechanisms of MB and MBCa action were also investigated.ResultsOvariectomized (OVX) mice were fed diet containing MB or MBCa for 80 days, and femoral bone mineral content and mineral density (BMD) were evaluated. As expected, OVX reduced BMD; however, the administration of MB and MBCa significantly prevented this decrease. Furthermore, MB and MBCa treatment significantly reduced the serum levels of tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a bone resorption marker, and significantly increased the levels of serum calcitonin, compared to those in the OVX control group. In vitro, the relatively high‐dose levels of MB and MBCa inhibited osteoclast differentiation by decreasing the protein expression of nuclear factor of activated T‐cell cytoplasmic 1 (NFATc1), the master transcription factor of osteoclastogenesis.ConclusionOur results showed that osteoporotic mice treated with MB and MBCa had improved bone density and bone metabolism compared to the OVX controls. Moreover, it was hypothesized herein that the suppressive effect on osteoclast differentiation slightly contributed to these results concomitantly. These findings suggest that the intake of MB or MBCa may contribute to the maintenance of bone health, including the prevention of primary osteoporosis.

Therapeutic effect of bloodletting on bone deterioration induced by hypobaric hypoxia in young rats

ObjectivesHigh-altitude regions, comprising hypoxic conditions, are associated with different altitude-induced pathologies, including a reduction in bone density. Elucidating the mechanisms underlying bone degradation in such environments and developing targeted interventions and therapeutics is important. Bloodletting therapy has promising clinical applications, but its effects on the skeletal system and bone homeostasis are not well understood. The aim of this study was to investigate the effects of a hypobaric hypoxia environment on specific femoral morphological and structural properties, including bone volume, cortical thickness, and trabecular microarchitecture, in juvenile Sprague–Dawley (SD) rats, and to explore the potential modulating effects of a bloodletting intervention on these parameters. MethodsMale SD rats, 6 weeks of age, were subjected to a simulated hypobaric hypoxia environment, replicating a 5000-m altitude, for 12 weeks. For the bloodletting intervention group, rats were subjected to a weekly 500 μL tail vein blood withdrawal. Micro-CT technology, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining were employed to comprehensively assess the femoral microstructure, tissue architecture, and cellular morphology. Additionally, immunofluorescence analysis was conducted to quantify the expression of key proteins, and transcriptome analysis was performed to identify differentially expressed genes. ResultsExposure of rats to hypobaric hypoxia led to a significant reduction in bone mineral content, trabecular bone number, and cortical bone thickness, suggesting a deterioration of bone microstructure. Additionally, the hypoxic environment upregulated the expression of RANKL and HIF-1α, while downregulating RUNX2. Notably, although bloodletting intervention did not significantly reverse these bone structural changes, transcriptome analysis revealed its regulatory influence on the expression of key genes, particularly Mmp2, Fosl2, and URS0000B2A65A, which are implicated in pathways governing the hypoxic response, osteoclast differentiation, and PI3K–Akt signaling. ConclusionThis study highlights the detrimental effect of hypobaric hypoxia on the bone microstructure of juvenile rats and underscores the therapeutic potential of bloodletting to ameliorate this condition. Additionally, our study on the regulatory mechanisms mediating bloodletting's effects on gene expression offers fresh perspectives on bone alterations. It suggests promising avenues for the development of novel preventative measures and targeted therapies to address the challenges posed by related bone disorders.