Possible mechanisms underlying the regulation of postmenopausal osteoporosis by follicle-stimulating hormone.

Objective: To explore the possible mechanisms by which follicle-stimulating hormone (FSH) regulates postmenopausal osteoporosis through the FSH/FSH receptor (FSHr)/G protein/C/EBPß/heat shock protein 90 alpha (HSP90α) signalling pathways. Methods: We measured serum FSH, luteinising hormone (LH), and HSP90α levels in the serum and adipose tissue of women of childbearing age and menopausal status. In the in vivo studies, 12 B57CL female mice were divided equally into Sham, OVX, and OVX + FSHr Blocker groups. Serum levels of alkaline phosphatase, FSH, and HSP90α, along with StRACP vitality, were determined, and femur micro-computed tomography was performed. Additionally, FSH, FSHr, G protein, C/EBPß, and HSP90α levels were assessed using quantitative polymerase chain reaction. Finally, we divided the human multiple myeloma cell line U266 into three groups. The activity of tartrate-resistant acid phosphatase (TRAP) in the supernatant at different stages was detected, and myeloma cells were stained with TRAP. Results: HSP90α levels in adipose tissue supernatant and serum were lower in women of childbearing age than in menopausal women (P < 0.05). Serum FSH and HSP90α levels demonstrated a strong correlation. Treatment with FSHr blockers resulted in decreased FSH, FSHr, G protein, C/EBPß, and HSP90α levels in mice. TRAP staining of osteoclast-like cells exhibited a significantly higher intensity in the M-CSF + RANKL + recombinant HSP90α group than in the M-CSF + RANKL and blank control groups (P < 0.05). Conclusions: Our results indicate that FSH promotes HSP90α secretion by adipocytes via the FSHr/G protein/C/EBPß pathway. This mechanism affects osteoclast activity and exacerbates osteoporosis.

Suppressing ERp57 diminishes osteoclast activity and ameliorates ovariectomy-induced bone loss via the intervention in calcium oscillation and the calmodulin/calcineurin/Nfatc1 pathway.

Background: Increased osteoclast activity constitutes the primary etiology of excessive bone erosion in postmenopausal osteoporosis. ERp57, otherwise referred to as protein disulfide isomerase A3 (PDIA3), plays a crucial role in the regulation of intracellular calcium signaling. This is documented to exert a profound impact on osteoclast differentiation and functionality. Methods: To ascertain the potential role of ERp57 in disease progression, prevention, and treatment, network pharmacology and bioinformatics analyses were conducted in relation to postmenopausal osteoporosis and ERp57 inhibitor (Loc14). Then, subsequent experimental verifications were employed in vitro on osteoclast and osteoblast, and in vivo on ovariectomy (OVX) mice models. Results: Multiple enrichment analyses suggested that the "calcium signaling pathway" may constitute a potential avenue for therapeutic intervention by Loc14 in the treatment of postmenopausal osteoporosis. In vitro experiments demonstrated inhibition of ERp57 could block osteoclast differentiation and function by interfering with the expression of osteoclast marker genes (Traf6, Nfatc1, and Ctsk). Further mechanisms studies based on calcium imaging, qPCR, and WB established that ERp57 inhibitor (Loc14) could obstruct calcium oscillation in osteoclast precursor cells (OPCs) by limiting the entry sources of cytosolic Ca2+ and interfering with calmodulin/calcineurin/Nfatc1 pathway. Evidence from Micro-CT scanning and double calcein labeling confirmed that the application of Loc14 in vivo could alleviate bone loss and partially reversed the osteogenic impairment caused by OVX in mice. Conclusions: Our findings proved the suppressive effects of Loc14 on osteoclastogenesis via attenuating calcium oscillation and associated singling pathways, providing ERp57 as a potential therapeutic target for postmenopausal osteoporosis.

Lysosomal biogenesis and function in osteoclasts: a comprehensive review.

Lysosomes serve as catabolic centers and signaling hubs in cells, regulating a multitude of cellular processes such as intracellular environment homeostasis, macromolecule degradation, intracellular vesicle trafficking and autophagy. Alterations in lysosomal level and function are crucial for cellular adaptation to external stimuli, with lysosome dysfunction being implicated in the pathogenesis of numerous diseases. Osteoclasts (OCs), as multinucleated cells responsible for bone resorption and maintaining bone homeostasis, have a complex relationship with lysosomes that is not fully understood. Dysregulated function of OCs can disrupt bone homeostasis leading to the development of various bone disorders. The regulation of OC differentiation and bone resorption for the treatment of bone disease have received considerable attention in recent years, yet the role and regulation of lysosomes in OCs, as well as the potential therapeutic implications of intervening in lysosomal biologic behavior for the treatment of bone diseases, remain relatively understudied. This review aims to elucidate the mechanisms involved in lysosomal biogenesis and to discuss the functions of lysosomes in OCs, specifically in relation to differentiation, bone resorption, and autophagy. Finally, we explore the potential therapeutic implication of targeting lysosomes in the treatment of bone metabolic disorders.

Exploring the associations between elevated plasma SP-D levels and OSCAR gene expression as potential biomarkers in patients with COPD: a cross-sectional study.

Background: Chronic obstructive pulmonary disease (COPD) imposes a substantial burden on patients and healthcare systems. Spirometry is the most widely used test to diagnose the disease; however, a surrogate marker is required to predict the disease pattern and progression. Objectives: The aim of the current study was to explore the association of elevated levels of plasma surfactant protein D (SP-D) with gene expression of osteoclast-associated receptor (OSCAR) and lung functions as potential diagnostic biomarkers of COPD. Methods: This cross-sectional study employed convenience sampling. As men compose the majority of patients in the outpatient department and with smoking being common among Pakistani men, choosing men offered a representative sample. Using a post-bronchodilator forced expiratory volume in the first second (FEV1) to a forced vital capacity (FVC) of less than 0.70 (FEV1/FVC <0.7), COPD patients were diagnosed on spirometry (n = 41). Controls were healthy individuals with FEV1/FVC >0.7 (n = 41). Plasma SP-D levels were measured using an enzyme-linked immunosorbent assay (ELISA). The gene expression of OSCAR was determined by real-time polymerase chain reaction (qPCR) and subsequently analyzed by the threshold cycle (Ct) method. Statistical Package for Social Sciences (SPSS) version 20 was used for statistical analysis. Results: The mean BMI of controls (25.66 ± 4.17 kg/m2) was higher than that of cases (23.49 ± 2.94 kg/m2 (p = .008)). The median age of controls was 49 years (interquartile range (IQR) 42.0-65.0 years) and that of cases was 65 years (IQR = 57.50-68.50). SP-D concentration was not significantly higher in COPD patients [4.96 ng/mL (IQR 3.26-7.96)] as compared to controls [3.64 ng/mL (IQR 2.60-8.59)] (p = .209). The forced expiratory ratio (FEV1/FVC) and FEV1 were related to gene expression of OSCAR (p = <.001). The gene expression of OSCAR was significantly related to SP-D (p = .034). A multiple regression model found FEV1 and FVC to have a significant effect on the gene expression of OSCAR (p-values <0.001 and 0.001, respectively). Conclusion: Gene expression of OSCAR was increased in COPD patients and related directly to SP-D levels and inversely to lung functions in cohort of this study, suggesting that OSCAR along with SP-D may serve as a diagnostic biomarker of COPD.

Increased nerve growth factor expression and osteoclast density are associated with subchondral bone marrow lesions in osteoarthritic knees.

Objectives: Subchondral bone marrow lesions (BMLs) detected on magnetic resonance imaging in knee osteoarthritis (OA) are associated with knee pain, though the mechanisms remain unknown. Increased nerve growth factor (NGF) expression and osteoclast density in subchondral bone appear to be the key features associated with bone pain in knee OA. Therefore, we aimed to identify associations among NGF, osteoclasts, and BMLs in knee OA. Methods: Twenty tibial plateaus were obtained from patients undergoing total knee arthroplasty for medial knee OA with BMLs at the medial tibial plateau (MTP). Osteochondral tissue samples from the weight-bearing part of the MTP, with and without BML, and from the weight-bearing part of the lateral tibial plateau (LTP), without BML, were collected. NGF expression and density of osteoclasts were compared among the three osteochondral tissue types. Results: MTP bone with BMLs exhibited significantly higher NGF expression in bone marrow space and osteochondral channel, and higher osteoclast density than MTP bone without BML and LTP bone. The mean differences in NGF-positive area in the bone marrow space and the percentage of NGF-positive channels between MTP bones with and without BML were 9.0% (95% confidence interval [CI]: 5.9-12.1%) and 23.1% (95% CI: 11.3-35.0%), respectively. The difference in osteoclast density between MTP bones with and without BML was 0.6 osteoclasts per mm (95% CI: 0.3-0.9 osteoclasts per mm). Conclusions: Increased NGF expression and osteoclast density are associated with subchondral BMLs in knee OA, contribute to understanding the mechanisms underlying BML-related bone pain in knee OA.

Incidence and prevention of skeletal-related events in multiple myeloma patients: A population-based real-world experience.

Novel treatments in multiple myeloma (MM) could influence the incidence of skeletal-related events (SREs). We aimed to examine the incidence of SRE and the preventive use of osteoclast inhibitors (OIs) in a cohort of MM patients in the era of modern treatment. In this real-world retrospective study, we included 199 patients with a diagnosis of MM between January 1, 2010, and December 31, 2019, with follow-up at St. Olavs University Hospital. Data was extracted from The Myeloma Registry of Central Norway. SREs occurred in 46% of patients at baseline and 55.8% during follow-up. Excluding baseline SREs, the incidence rate was 29 (95% confidence interval: 26-33) per 100 person years. 48% experienced > 1 SRE. The incidence of SREs was highest at baseline followed by a gradual increase in each subsequent line of treatment. The first two years after diagnosis 80% received bisphosphonates (BPs). The proportion of recommended dosage was 46%. Only two cases (1.2%) of symptomatic hypocalcemia and one case (0.6%) of osteonecrosis of the jaw were identified. SREs are still a common problem in an era of novel treatment. Cumulative dosage of BPs was lower than recommended, and treatment with BPs was safe in this population.

Zoledronic acid relieves steroid-induced avascular necrosis of femoral head via inhibiting FOXD3 mediated ANXA2 transcriptional activation.

BACKGROUND: Zoledronic acid (ZOL) is a type of bisphosphonate with good therapeutic effects on orthopaedic diseases. However, the pharmacological functions of ZOL on steroid-induced avascular necrosis of femoral head (SANFH) and the underlying mechanism remain unclear, which deserve further research. METHODS: SANFH models both in vivo and in vitro were established by dexamethasone (Dex) stimulation. Osteoclastogenesis was examined by TRAP staining. Immunofluorescence was employed to examine autophagy marker (LC3) level. Cell apoptosis was analyzed by TUNEL staining. The interaction between Foxhead box D3 protein (FOXD3) and Annexin A2 (ANXA2) promoter was analyzed using ChIP and dual luciferase reporter gene assays. RESULTS: Dex aggravated osteoclastogenesis and induced osteoclast differentiation and autophagy in vitro, which was abrogated by ZOL treatment. PI3K inhibitor LY294002 abolished the inhibitory effect of ZOL on Dex-induced osteoclast differentiation and autophagy. FOXD3 overexpression neutralized the downregulation effects of ZOL on Dex-induced osteoclasts by transcriptionally activating ANXA2. ANXA2 knockdown reversed the effect of FOXD3 overexpression on ZOL-mediated biological effects in Dex-treated osteoclasts. In addition, ZOL improved SANFH symptoms in rats. CONCLUSION: ZOL alleviated SANFH through regulating FOXD3 mediated ANXA2 transcriptional activity and then promoting PI3K/AKT/mTOR pathway, revealing that FOXD3 might be a target for ZOL in SANFH treatment.

Nanographene Oxide Promotes Angiogenesis by Regulating Osteoclast Differentiation and Platelet-Derived Growth Factor Secretion.

An imbalanced system of angiogenesis-osteoblasts-osteoclasts is regarded as the main factor in bone remodeling dysfunction diseases or osseointegration loss. Osteoclast precursors are the key cells that accelerate bone-specific angiogenesis and maintain normal osteoblast and osteoclast function. Graphene oxide is an effective scaffold surface modification agent with broad application prospects in bone tissue engineering. However, the effect of graphene oxide on the interaction between osteoclasts and angiogenesis has not yet been elucidated. In this study, a rat calvarial defect model was established and treated with an electrochemically derived nanographene oxide (ENGO) hydrogel. Higher angiogenesis and platelet-derived growth factor (PDGF) B in preosteoclasts were observed in the ENGO group compared with that in the control group. Moreover, in vitro experiments demonstrate the efficacy of ENGO in substantially reducing the expression of the receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast-associated markers and inhibiting bone resorption activity. Additionally, ENGO enhances the secretion of the osteoclast-derived coupling factor PDGF-BB and promotes angiogenesis. Our investigation revealed the crucial role of isocitrate dehydrogenase 1 (IDH1) in the ENGO-mediated regulation of osteoclast differentiation and PDGF-BB secretion. The decreased expression of IDH1 reduces the level of histone lysine demethylase 7A (KDM7A) and subsequently increases the H3K9me2 level in the cathepsin K promoter region. In summary, we found that ENGO promotes angiogenesis by inhibiting the maturity of RANKL-induced osteoclasts and enhancing PDGF-BB secretion. These results indicate that ENGO holds promise for the application in fostering osteoclast-endothelial cell crosstalk, providing an effective strategy for treating bone resorption and osteoclast-related bone loss diseases.

Trifolirhizin protects ovariectomy-induced bone loss in mice by inhibiting osteoclast differentiation and bone resorption.

Background: Osteoporosis is a debilitating condition characterized by reduced bone density and microstructure, leading to increased susceptibility to fractures and increased mortality, particularly among older individuals. Despite the availability of drugs for osteoporosis treatment, the need for targeted and innovative agents with fewer adverse effects persists. Trifolirhizin, a natural pterostalin derived from the root of Sophora flavescens, has been previously studied for its effects on certain anticancer and antiinflammatory. The impact of trifolirhizin on the formation and function of osteoclasts remain unclear. Purpose: Herein, the possible roles of trifolirhizin the formation and function of osteoclasts and the underlying mechanism were explored. Methods: Bone marrow-derived macrophages (BMMs) were employed to evaluate the roles of trifolirhizin on steoclastogenesis, bone absorption and the underlying mechanism in vitro. Bone loss model was established by ovariectomy(OVX) in mice in vivo. Results: Trifolirhizin repressed osteoclastogenesis, bone resorption induced by receptor activator of nuclear factor kappa B ligand (RANKL) in vitro. Mechanistically, trifolirhizin inhibits RANKL-induced MAPK signal transduction and NFATc1 expression. Moreover, trifolirhizin inhibited osteoclast marker gene expression, including NFATc1, CTSK, MMP9, DC-STAMP, ACP5, and V-ATPase-D2. Additionally, trifolirhizin was found to protect against ovariectomy(OVX)-induced bone loss in mice. Conclusion: Trifolirhizin can effectively inhibit osteoclast production and bone resorption activity. The results of our study provide evidence for trifolirhizin as a potential drug for the prevention and treatment of osteoporosis and other osteolytic diseases.

Mn2+ vs Co2+ substitution into ß-TCP: Structural details and bone cells response.

This work investigated the range of substitution of two biologically relevant ions, namely Mn2+ and Co2+, into the structure of ß-tricalcium phosphate, as well as their influence on bone cells response. To this aim, ß-TCP was synthesized by solid state reaction in the presence of increasing amount of the substituent ions. The results of the X-ray diffraction analysis reveal that just limited amounts of these ions can enter into the ß-TCP structure: 15 at% and 20 at% for cobalt and manganese, respectively. Substitution provokes aggregation of the micrometric particles and reduction of the lattice constants. In particular, the dimension of the c-parameter exhibits a discontinuity at about 10 at% for both cations, although with different trend. Moreover, Rietveld refinement demonstrates a clear preference of both manganese and cobalt for the octahedral site (V). The influence of these ions on cell response was tested on osteoblast, osteoclast and endothelial cells. The results indicate that the presence of manganese promotes a good osteoblast viability, significantly enhances the expression of osteoblast key genes and the angiogenic process of endothelial cells, while inhibiting osteoclast resorption. At variance, osteoblast viability appears reduced in the presence of Co samples, on which osteoblast genes reach higher expression than on ß-TCP just in a few cases. On the other hand, the results clearly show that cobalt significantly stimulates the angiogenic process and inhibits osteoclast resorption.

The "Forgotten" Subtypes of Breast Carcinoma: A Systematic Review of Selected Histological Variants Not Included or Not Recognized as Distinct Entities in the Current World Health Organization Classification of Breast Tumors.

Breast carcinoma is the most common cancer in women. Nineteen different subtypes of breast carcinomas are recognized in the current WHO classification of breast tumors. Except for these subtypes, there are a number of carcinomas with special morphologic and immunohistochemical features that are not included in the 5th WHO classification, while others are considered special morphologic patterns of invasive breast carcinoma of no special type. In this manuscript, we systematically review the literature on four different subtypes of invasive breast carcinoma, namely lymphoepithelioma-like breast carcinoma, breast carcinoma with osteoclast-like giant cells, signet-ring breast carcinoma, and metaplastic breast carcinoma with melanocytic differentiation. We describe their clinicopathological characteristics, focusing on the differential diagnosis, treatment, and prognosis.

In vitro osteoclast differentiation enhanced by hepatocyte supernatants from high-fat diet mice.

Non-alcoholic fatty liver disease (NAFLD) is associated with abnormal bone metabolism, potentially mediated by elevated levels of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-ɑ) and interleukin 6 (IL-6). This study aims to investigate the direct regulatory effects of liver tissues on osteoblast and osteoclast functions in vitro, focusing on the liver-bone axis in NAFLD. Twelve-week-old C57BL/6 mice were fed either a control diet or a high-fat diet (HFD) for 12 weeks. Bone structural parameters were assessed using microCT. Primary hepatocyte cultures were established from control and HFD-fed C57BL/6 mice, as well as IL-6-/- and TNF-α-/- mice. The supernatants from these hepatocyte cultures were used to induce differentiation in bone marrow cell-derived osteoblasts and osteoclasts in vitro. Results showed that mice on a HFD exhibited increased lipid infiltration in liver and bone marrow tissues, alongside reduced bone mass. Moreover, the supernatants from hepatocyte cultures from mice on a HFD displayed elevated TNF-α and IL-6 levels. These supernatants, particularly those derived from HFD-fed and IL-6-/- mice, significantly enhanced osteoclast differentiation in vitro. In contrast, supernatants from TNF-α-/- mice did not significantly affect osteoblast or osteoclast differentiation in vitro. In conclusions, this current study suggested that fatty liver tissues may negatively impact bone metabolism. Additionally, knockout of TNF-α and IL-6 genes revealed distinct influence on osteoblast and osteoclast functions, highlighting the complex interplay between live pathology and bone health.

SNX10 regulates osteoclastogenic cell fusion and osteoclast size in mice.

Bone-resorbing osteoclasts (OCLs) are formed by differentiation and fusion of monocyte precursor cells, generating large multi-nucleated cells. Tightly-regulated cell fusion during osteoclastogenesis leads to formation of resorption-competent OCLs, whose sizes fall within a predictable physiological range. The molecular mechanisms that regulate the onset of OCL fusion and its subsequent arrest are, however, largely unknown. We have previously shown that OCLs cultured from mice homozygous for the R51Q mutation in the vesicle trafficking-associated protein sorting nexin 10, a mutation that induces autosomal recessive osteopetrosis in humans and in mice, display deregulated and continuous fusion that generates gigantic, inactive OCLs. Fusion of mature OCLs is therefore arrested by an active, genetically-encoded, cell-autonomous, and SNX10-dependent mechanism. In order to directly examine whether SNX10 performs a similar role in vivo, we generated SNX10-deficient (SKO) mice and demonstrated that they display massive osteopetrosis and that their OCLs fuse uncontrollably in culture, as do homozygous R51Q SNX10 (RQ/RQ) mice. OCLs that lack SNX10 exhibit persistent presence of DC-STAMP protein at their periphery, which may contribute to their uncontrolled fusion. In order to visualize endogenous SNX10-mutant OCLs in their native bone environment we genetically labelled the OCLs of wild-type, SKO and RQ/RQ mice with EGFP, and then visualized the three-dimensional organization of resident OCLs and the pericellular bone matrix by two-photon, confocal, and second harmonics generation microscopy. We show that the volumes, surface areas and, in particular, the numbers of nuclei in the OCLs of both mutant strains were on average 2-6 fold larger than those of OCLs from wild-type mice, indicating that deregulated, excessive fusion occurs in the mutant mice. We conclude that the fusion of OCLs, and consequently their size, are regulated in vivo by SNX10-dependent arrest of fusion of mature OCLs.

Osteoclasts (OCLs) are cells that degrade bone. These cells are generated by fusion of monocyte precursor cells, but the mechanisms that regulate this process and eventually arrest it are unknown. We had previously shown that OCLs cultured from mice carrying the R51Q mutation in the protein sorting nexin 10 (SNX10) lose their resorptive capacity and become gigantic due to uncontrolled fusion. To examine whether SNX10 is required for OCL fusion arrest also in vivo, we inactivated the Snx10 gene in mice and fluorescently labelled their OCLs and OCLs of R51Q SNX10 mice, isolated their femurs, and used advanced 3D microscopy methods to visualize OCLs within the bone matrix. As expected, mice lacking SNX10 exhibited excessive bone mass, indicating that their OCLs are inactive. OCLs within bones of both mutant mouse strains were on average 2-6-fold larger than in control mice, and contained proportionally more nuclei. We conclude that OCL fusion is arrested in control, but not SNX10 mutant, mice, indicating that the sizes of mature OCLs are limited in vivo by an active, SNX10-dependent mechanism that suppresses cell fusion.

Trifolirhizin reduces osteoclast formation and prevents inflammatory osteolysis by inhibiting RANKL-induced activation of NF-κB and MAPK signaling pathways and ROS.

Inflammatory osteolysis is often caused by the excessive activation of osteoclasts stimulated by bacterial products such as lipopolysaccharide. The natural flavonoid trifolirhizin (TRI) has anti-inflammatory properties; however, its function in inflammatory bone lysis remains unclear. This study aimed to elucidate the potential regulatory mechanisms of TRI in osteoclasts.Tartrate-resistant acid phosphatase (TRAP) staining, acid secretion assays, podosomal actin belt fluorescence staining, and bone resorption assays were used to investigate the effects of TRI on osteoclast differentiation and bone resorption. A reactive oxygen species (ROS) measurement kit was used to detect the effect of TRI on ROS levels in osteoclasts. The effects of TRI on genes and signaling pathways related to osteoclast differentiation were determined by quantitative polymerase chain reaction (qPCR) and western blotting. A mouse model of lipopolysaccharide-mediated inflammatory osteolysis was established, and the effects of TRI treatment on bone mass were observed using micro-CT and histological examination. Mechanistically, TRI reduced ROS production by inhibiting receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, and by upregulating the expression levels of the anti-ROS enzymes heme oxygenase-1 (HO-1) and catalase (CAT), which contributed to the degradation of ROS, ultimately leading to a decrease in osteoclastogenesis. TRI inhibited osteoclast formation and ameliorated lipopolysaccharide (LPS)-mediated inflammatory osteolysis. Thus, TRI may be a candidate agent for anti-inflammatory osteolysis.

Differential effects of clopidogrel and/or aspirin on the healing of tooth extraction wound bone tissue.

Introduction: A multitude of variables influence the healing of tooth extraction wounds, and delayed or non-healing extraction wounds might complicate later prosthodontic therapy. In this research, we analyzed the effects of systemic clopidogrel and aspirin alone or in combination on the healing of tooth extraction wounds in mice in order to provide experimental evidence for the healing of extraction wounds in patients who are clinically treated with the two medicines. Methods: 7-week-old ICR mice were randomly divided into four groups: control group (CON), clopidogrel group (CLOP), aspirin group (ASP), and clopidogrel combined with aspirin group (CLOP + ASP); left upper first molar was extracted, after which mice in 1 week of adaptive feeding, CLOP/ASP/CLOP + ASP groups were respectively administered with clopidogrel (10 mg/kg/d), aspirin (15 mg/kg/d), clopidogrel (10 mg/kg/d)+aspirin (15 mg/kg/d), and the control group was given an equal amount of 0.9% saline by gavage. Mice in each group were euthanized at 14 and 28 days postoperatively, and the maxilla was extracted. The tissues in the extraction sockets were examined using MicroCT and sectioned for HE staining, Masson staining, and TRAP staining, and immunohistochemistry staining (for TRAP, RANKL and osteoprotegerin). Results: MicroCT analysis showed that at day 14, BS/BV was significantly lower in CLOP and CLOP + ASP groups compared to control and ASP groups, while BV/TV, Tb.Th was significantly higher. At day 28, BV/TV was significantly higher in the CLOP + ASP group compared to the CLOP group, with p < 0.05 for all results. HE staining and Masson trichrome staining findings revealed that at day 28, the mesenchyme in the bone was further decreased compared to that at day 14, accompanied with tightly arranged and interconnected bone trabeculae. In the quantitative analysis of Masson, the fraction of newly formed collagen was significantly higher in the CLOP group in comparison with that in the CON group (p < 0.05). At day 14, the ASP group had substantially more TRAP-positive cells than the CLOP and CLOP + ASP groups (p < 0.05). In immunohistochemical staining, RANKL expression was found to be significantly higher in the ASP group than those in the other three groups at day 28 (p < 0.05); OPG expression was significantly higher in the CLOP group and the CLOP + ASP group compared with that at day 14, and was higher than that in the ASP group at day 14 and day 28. OPG/RANKL was significantly higher in the CLOP and the CLOP + ASP groups than in the ASP group (p < 0.05). Conclusion: Clopidogrel alone promotes osteogenesis in the extraction wound, whereas aspirin alone inhibits alveolar bone healing. When the two drugs were combined, the healing effect of the extraction wound was more similar to that of the clopidogrel alone group. These results indicated that clopidogrel could promote the healing of the tooth extraction wound, and neutralize the adverse effect of ASP on osteogenesis when the two drugs were used in combination.

Resveratrol reversed rosiglitazone administration induced bone loss in rats with type 2 diabetes mellitus.

Rosiglitazone (RSG), as an insulin-sensitizing drug to treat type 2 diabetes mellitus (T2DM) is reported to decrease bone quality and increase bone fracture risk. The multiple off-target effects of Resveratrol (RSV), a natural specific agonist of Sirtuin1 (Sirt1) with pro-osteoblastogenesis and anti-adipogenesis effects, on bone loss in T2DM are still under discussion. In this study, successfully ovariectomized rats were fed with high-fat diet and STZ (HFD/STZ) to induced T2DM mice. RSV alone, RSG alone or co-administration of RSV and RSG were given orally to T2DM rats for 8 weeks to determine whether RSV administration had any prevention effect on T2DM osteoporosis. Bone mesenchymal stem cells (BMSCs) and bone marrow­derived macrophages (BMMs) were cultured under high glucose condition and were induced to osteoblasts or adipocytes and osteoclasts, respectively. µCT and HE staining showed that in T2DM osteoporotic rats, RSV co-administration prevents RSG induced-bone loss. ELISA results confirmed that RSV suppressed osteoclast activity and promoted osteoblast activity in diabetic osteoporosis rats and RSG-administrated diabetic osteoporosis rats. In vitro study showed that RSV significantly reversed RSG induced inhibition on osteogenesis and promotion on adiopogenesis of BMSC under high glucose (HG). Moreover, RSV significantly reverse RSG induced osteoclast formation and mature under HG. Taken together, these findings uncover a previously unappreciated anti-osteoporosis effect of concomitant treatment with RSV in RSG-administrated diabetic rats, suggesting the clinical use of RSV as an adjuvant in the treatment of T2DM for preventing or reversing RSG administration-associated bone loss.

Upscaling Osteoclast Generation by Enhancing Macrophage Aggregation Using Hollow Microgels.

Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated.

pH-Responsive Mesoporous Silica Nanoparticles Loaded with Naringin for Targeted Osteoclast Inhibition and Bone Regeneration.

Background: It is well-established that osteoclast activity is significantly influenced by fluctuations in intracellular pH. Consequently, a pH-sensitive gated nano-drug delivery system represents a promising therapeutic approach to mitigate osteoclast overactivity. Our prior research indicated that naringin, a natural flavonoid, effectively mitigates osteoclast activity. However, naringin showed low oral availability and short half-life, which hinders its clinical application. We developed a drug delivery system wherein chitosan, as gatekeepers, coats mesoporous silica nanoparticles loaded with naringin (CS@MSNs-Naringin). However, the inhibitory effects of CS@MSNs-Naringin on osteoclasts and the underlying mechanisms remain unclear, warranting further research. Methods: First, we synthesized CS@MSNs-Naringin and conducted a comprehensive characterization. We also measured drug release rates in a pH gradient solution and verified its biosafety. Subsequently, we investigated the impact of CS@MSNs-Naringin on osteoclasts induced by bone marrow-derived macrophages, focusing on differentiation and bone resorption activity while exploring potential mechanisms. Finally, we established a rat model of bilateral critical-sized calvarial bone defects, in which CS@MSNs-Naringin was dispersed in GelMA hydrogel to achieve in situ drug delivery. We observed the ability of CS@MSNs-Naringin to promote bone regeneration and inhibit osteoclast activity in vivo. Results: CS@MSNs-Naringin exhibited high uniformity and dispersity, low cytotoxicity (concentration≤120 µg/mL), and significant pH sensitivity. In vitro, compared to Naringin and MSNs-Naringin, CS@MSNs-Naringin more effectively inhibited the formation and bone resorption activity of osteoclasts. This effect was accompanied by decreased phosphorylation of key factors in the NF-κB and MAPK signaling pathways, increased apoptosis levels, and a subsequent reduction in the production of osteoclast-specific genes and proteins. In vivo, CS@MSNs-Naringin outperformed Naringin and MSNs-Naringin, promoting new bone formation while inhibiting osteoclast activity to a greater extent. Conclusion: Our research suggested that CS@MSNs-Naringin exhibited the strikingly ability to anti-osteoclasts in vitro and in vivo, moreover promoted bone regeneration in the calvarial bone defect.

XAF1 promotes osteoclast apoptosis by antagonizing the XIAP-caspase axis.

Background: Over-activated osteoclast (OC) is a major cause of diseases related to bone loss and bone metabolism. Both bone resorption inhibition and apoptosis induction of osteoclast are crucial in treating these diseases. X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is an important interferon-stimulated and apoptotic gene. However, how XAF1 regulates bone formation and remodeling is unknown. Methods: We generate global and chimeric Xaf1 knockout mouse models and utilize these models to explore the function and mechanism of XAF1 in regulating bone formation and remodeling in vivo and in vitro. Results: We show that XAF1 depletion enhances osteoclast generation in vitro. XAF1 knockout increases osteoclast number and bone resorption, thereby exacerbating bone loss in both OVX and osteolysis models. Activation of XAF1 with BV6 (a potent XIAP inhibitor) suppresses osteoclast formation. Mechanistically, XAF1 deletion decreases osteoclast apoptosis by facilitating the interaction between XIAP and caspase-3/7. Conclusions: Our data illustrates an essential role of XAF1 in controlling osteoclastogenesis in both osteoporosis and osteolysis mouse models and highlights its underlying mechanism, indicating a potential role in clinical treatment.The translational potential of this article: The translation potential of this article is that we first indicated that osteoclast apoptosis induced by XAF1 contribute to the progression of osteoporosis and osteolysis, which provides a novel strategy in the prevention of osteoporosis and osteolysis.