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Glycosphingolipids (GSLs) are key constituents of membrane bilayers playing a role in structural integrity, cell signalling in microdomains, endosomes and lysosomes, and cell death pathways. Conversion of ceramide into GSLs is controlled by GCS (glucosylceramide synthase) and inhibitors of this enzyme for the treatment of lipid storage disorders and specific cancers. With a diverse range of functions attributed to GSLs, the ability of the GSC inhibitor, eliglustat, to reduce myeloma bone disease was investigated. In pre-clinical models of multiple myeloma, osteoclast-driven bone loss was reduced by eliglustat in a mechanistically separate manner to zoledronic acid, a bisphosphonate that prevents osteoclast-mediated bone destruction. Autophagic degradation of TNF receptor-associated factor 3 (TRAF3), a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. By altering GSL composition, eliglustat prevented lysosomal degradation whilst exogenous addition of missing GSLs rescued TRAF3 degradation to restore osteoclast formation in bone marrow cells from myeloma patients. This work highlights the clinical potential of eliglustat as a therapy for myeloma bone disease. Furthermore, using eliglustat as a lysosomal inhibitor in osteoclasts may widen its therapeutic uses to other bone disorders such as bone metastasis, osteoporosis and inflammatory bone loss.

BACKGROUND: Recent studies have shown that the occurrence and prevention of osteoporosis often focus on the cellular molecular level, and the mechanism of related signaling pathways is an important way to further understand osteoporosis. At present, traditional Chinese medicine has been proved to play a significant role in alleviating osteoporosis. Kaempferol as an emerging Chinese herbal extract has become the focus of clinical and basic research due to its antiosteoporosis effectiveness and mechanism of action. OBJECTIVE: To further understand the mechanism underlying the anti-osteoporosis effect of kaempferol active monomer through regulation of related signaling pathways by analyzing and collating domestic and foreign literature. METHODS: “Kaempferol, osteoporosis, osteoblasts, osteoclasts, bone marrow mesenchymal stem cells, signaling pathways” were used as Chinese and English search terms to search CNKI, WanFang, VIP, PubMed, Web of Science and Embase databases for relevant literature published from database inception to February 2023. RESULTS AND CONCLUSION: Kaempferol affects the occurrence and progression of osteoporosis to varying degrees by participating in the regulation of differentiation, proliferation and apoptosis of bone marrow mesenchymal stem cells, osteoblasts and osteoclasts. Kaempferol can prevent and treat osteoporosis by regulating various signaling pathways. Kaempferol can promote the proliferation and differentiation of osteoblasts and inhibit the formation of osteoclasts by interfering with the Wnt/β-catenin signaling pathway to regulate β-catenin protein counting and the formation of β-catenin-TCf/LEF complex. Kaempferol interferes with the RANK/RANKL pathway to maintain the dynamic balance of osteoclasts and bone homeostasis. Kaempferol can promote bone formation by intervening with the PI3K/Akt signaling pathway to upregulate the levels of related osteogenic factors Runx2 and Osterix and promote bone cell calcification. Kaempferol interferes with osteoclast differentiation and inhibits reactive oxygen species activity by regulating the ER/ERK pathway. Kaempferol inhibits the expression of ERK, JNK, p38/MAPK and decreases reactive oxygen species production by interfering with the MAPK pathway, thus protecting osteogenesis. Kaempferol enhances the expression of osteogenic factors, bone morphogenetic protein-2, p-Smad1/5/8, β-catenin and Runx2, inhibits the expression of Peroxisome proliferation-activated receptor, and promotes the differentiation and proliferation of osteoblasts through the BMP/Smad pathway. [ABSTRACT FROM AUTHOR]

Oxytocin (OT) is a posterior pituitary hormone that, in addition to its role in regulating childbirth and lactation, also exerts direct regulatory effects on the skeleton through peripheral OT and oxytocin receptor (OTR). Bone marrow mesenchymal stem cells (BMSCs), osteoblasts (OB), osteoclasts (OC), chondrocytes, and adipocytes all express OT and OTR. OT upregulates RUNX2, BMP2, ALP, and OCN, thereby enhancing the activity of BMSCs and promoting their differentiation towards OB rather than adipocytes. OT also directly regulates OPG/RANKL to inhibit adipocyte generation, increase the expression of SOX9 and COMP, and enhance chondrocyte differentiation. OB can secrete OT, exerting influence on the surrounding environment through autocrine and paracrine mechanisms. OT directly increases OC formation through the NκB/MAP kinase signaling pathway, inhibits osteoclast proliferation by triggering cytoplasmic Ca2+ release and nitric oxide synthesis, and has a dual regulatory effect on OCs. Under the stimulation of estrogen, OB synthesizes OT, amplifying the biological effects of estrogen and OT. Mediated by estrogen, the OT/OTR forms a feedforward loop with OB. Apart from estrogen, OT also interacts with arginine vasopressin (AVP), prostaglandins (PGE2), leptin, and adiponectin to regulate bone metabolism. This review summarizes recent research on the regulation of bone metabolism by OT and OTR, aiming to provide insights into their clinical applications and further research. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusion and Implications Most inhibitors of histone deacetylases (HDACs) are not selective and are cytotoxic. Some have anti‐inflammatory activity in disease models, but cytotoxicity prevents long‐term uses in non‐fatal diseases. Inhibitors selective for class IIa HDACs are much less cytotoxic and may have applications in management of chronic inflammatory diseases.LL87 is a novel HDAC inhibitor examined here for HDAC enzyme selectivity. It was also investigated in macrophages for cytotoxicity and for inhibition of lipopolysaccharide (LPS)‐stimulated cytokine secretion. In a rat model of collagen‐induced arthritis, LL87 was investigated for effects on joint inflammation in Dark Agouti rats. Histological, immunohistochemical, micro‐computed tomography and molecular analyses characterise developing arthritis and anti‐inflammatory efficacy.LL87 was significantly more inhibitory against class IIa than class I or IIb HDAC enzymes. In macrophages, LL87 was not cytotoxic and reduced both LPS‐induced secretion of pro‐inflammatory cytokines, and IL6‐induced class IIa HDAC activity. In rats, LL87 attenuated paw swelling and clinical signs of arthritis, reducing collagen loss and histological damage in ankle joints. LL87 decreased immune cell infiltration, especially pro‐inflammatory macrophages and osteoclasts, into synovial joints and significantly reduced expression of pro‐inflammatory cytokines and tissue‐degrading proteases.A novel inhibitor of class IIa HDACs has been shown to have an anti‐inflammatory and anti‐arthritic profile distinct from current therapies. It is efficacious in reducing macrophage infiltration and joint inflammation in a chronic model of rat arthritis. [ABSTRACT FROM AUTHOR]

Diet‐induced obesity is associated with enhanced systemic inflammation that limits bone regeneration. HDAC inhibitors are currently being explored as anti‐inflammatory agents. Prior reports show that myeloid progenitor‐directed Hdac3 ablation enhances intramembranous bone healing in female mice. In this study, we determined if Hdac3 ablation increased intramembranous bone regeneration in mice fed a high‐fat/high‐sugar (HFD) diet. Micro‐CT analyses demonstrated that HFD‐feeding enhanced the formation of periosteal reaction tissue of control littermates, reflective of suboptimal bone healing. We confirmed enhanced bone volume within the defect of Hdac3‐ablated females and showed that Hdac3 ablation reduced the amount of periosteal reaction tissue following HFD feeding. Osteoblasts cultured in a conditioned medium derived from Hdac3‐ablated cells exhibited a four‐fold increase in mineralization and enhanced osteogenic gene expression. We found that Hdac3 ablation elevated the secretion of several chemokines, including CCL2. We then confirmed that Hdac3 deficiency increased the expression of Ccl2. Lastly, we show that the proportion of CCL2‐positve cells within bone defects was significantly higher in Hdac3‐deficient mice and was further enhanced by HFD. Overall, our studies demonstrate that Hdac3 deletion enhances intramembranous bone healing in a setting of diet‐induced obesity, possibly through increased production of CCL2 by macrophages within the defect. [ABSTRACT FROM AUTHOR]

BACKGROUND: Interleukin-8 is an important cytokine that has been found to play an important role in bone regeneration through multiple pathways. OBJECTIVE: To comprehensively review the action mechanism of interleukin-8 effects on bone regeneration to provide ideas for the following studies on interleukin-8. METHODS: By searching the China National Knowledge Infrastructure database for articles published from January 1999 to February 2023 and PubMed database for articles published from January 1985 to February 2023 reporting the role of interleukin-8 in bone-associated cells and vascularisation. Chinese and English search terms were "interleukin-8, bone repair, bone metabolism, mesenchymal stem cells, osteoblasts, osteoclasts, vascularization". The initial review yielded 508 articles in English and Chinese, and a total of 51 articles were included for review and analysis according to the inclusion and exclusion criteria. RESULTS AND CONCLUSION: According to the existing research, interleukin-8 can promote bone cell regeneration and assist bone healing through multiple pathways, which is mainly divided into three aspects: (1) Promote the proliferation and differentiation of bone cells such as mesenchymal stem cells and osteoblasts, and promote the development of cells in the direction of promoting bone healing; (2) interleukin-8 can promote angiogenesis and provide sufficient nutrition and oxygen for bone tissue, thus further improving the quality and stability of bone healing. (3) The appearance of interleukin-8 facilitates the expression of hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinase, which can create a microenvironment conducive to bone regeneration, thus promoting the regeneration and repair of bone tissue. In summary, interleukin-8 plays an important role in bone healing by promoting osteoblast proliferation and differentiation, facilitating angiogenesis and improving the mechanical properties of bone regeneration, as well as influencing bone metabolism through osteoclasts, mesenchymal stem cells, and other action sites. [ABSTRACT FROM AUTHOR]

Introduction: Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Methods and Results: Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Steroid-induced osteonecrosis of the femoral head is a refractory disease in the field of orthopedics. There is no definitive idea to fully explain its pathogenesis. With the increased research on the active ingredients of Panax notoginseng interfering with the signaling pathways related to various diseases, the active ingredients of Panax notoginseng that treat steroid-induced necrosis of the femoral head via the regulation of relevant signaling pathways have gradually become a hot research topic. OBJECTIVE: To systematically summarize the literature on the pathological mechanism of steroid-induced osteonecrosis of the femoral head and the regulation of signaling pathways by the active ingredients of Panax notoginseng in recent years, thereby providing a reference for the follow-up study on the active ingredients of Panax notoginseng in the treatment of this disease. METHODS: CNKI, WanFang, and PubMed were searched for relevant literature with the key words of “glucocorticoid, steroid-induced osteonecrosis of the femoral head, pathological mechanism, signaling pathway, Panax notoginseng, active ingredient” in Chinese and English. Documents related to the pathological mechanism of steroid-induced osteonecrosis of the femoral head as well as related to the intervention of active ingredients of Panax notoginseng on the signaling pathway of steroid-induced osteonecrosis of the femoral head were retrieved. A total of 63 documents were finally included according to the inclusion and exclusion criteria. RESULTS AND CONCLUSION: The main ingredients of Panax notoginseng include Panax notoginseng saponins, ginsenoside, Panax notoginseng saponins, quercetin, kaempferol, etc. Panax notoginseng saponins, ginsenoside Rb1 and quercetin can promote bone repair and angiogenesis by acting on the transforming growth factor-β/bone morphogenetic protein pathway. Panax notoginseng saponins, ginsenoside CK and kaempferol can promote osteogenic differentiation and lipid metabolism by acting on the Wnt/β-catenin pathway. Panax notoginseng saponins and Panax notoginseng saponins R1/R2 act on the MAPK pathway to inhibit osteoclastogenesis and promote bone repair. Panax notoginseng saponins, ginsenoside Rb2 and quercetin can inhibit osteoclast proliferation and promote osteoblastic differentiation by acting on the RANKL/RANK/OPG pathway. Panax notoginseng saponins, quercetin and kaempferol can repair vascular injury and promote osteogenesis by acting on the hypoxia-inducible factor-1α pathway. Panax notoginseng saponins R1, quercetin combined with hydroxyapatite nanoparticles, Panax notoginseng saponins combined with polyethylene-L-lactic acid and other biomaterials have good research prospects in the treatment of steroid-induced osteonecrosis of the femoral head. The active ingredients of Panax notoginseng can regulate the signaling pathways related to steroid-induced osteonecrosis of the femoral head through various mechanisms, and play an active intervention role in the disease. However, the depth and breadth of relevant research are insufficient at present, and the future research should be based on the existing mechanism to explore the specific mechanism of Panax notoginseng regulating different pathways and the interaction between pathways, which will be beneficial to the multi-development of the active ingredients of Panax notoginseng in the treatment of steroid-induced osteonecrosis of the femoral head. [ABSTRACT FROM AUTHOR]

BACKGROUND: The development of steroid-induced osteonecrosis of the femoral head is a complex process involving multiple mechanisms. There is still no standard therapeutic drug for early intervention of this disease. Current studies have shown that baicalein has various pharmacological activities such as regulating lipid metabolism, bone metabolism, apoptosis and anti-oxidative stress, which provides an idea for the prevention and treatment of steroid-induced osteonecrosis of the femoral head. OBJECTIVE: To observe the preventive effect of baicalein against steroid-induced osteonecrosis of the femoral head and to investigate its possible mechanism. METHODS: Thirty-six 10-week-old male Sprague-Dawley rats were randomly divided into three groups (n=12 per group): blank control group, model group, and baicalein intervention group. In the model group and baicalein intervention group, intraperitoneal lipopolysaccharide and intramuscular injection of methylprednisolone sodium succinate were performed for modeling, while normal saline was used as a substitute for the modeling drug in the blank control group. Baicalein 300 mg/kg was administered by gavage (once a day for 6 weeks) at the time of initial intramuscular glucocorticoid injection in the baicalein intervention group, and baicalein was replaced by normal saline in the other two groups. The serum level of malondialdehyde in rats was detected at 2 weeks of the experiment. Blood lipid indicators and bone formation metabolic markers were detected at 6 weeks of the experiment, the histomorphometric changes of the femoral head were analyzed by hematoxylin-eosin staining, anti-tartaric acid phosphatase staining and TUNEL staining, and the femoral head was subjected to Micro-CT scanning and three-dimensional reconstruction of the bone in order to analyze the alterations of bone tissue structure and parameters. RESULTS AND CONCLUSION: The serum levels of malondialdehyde, triglyceride, β-collagen type I carboxy-terminal peptide were increased and the serum levels of bone specific alkaline phosphatase and pre-collagen type I amino-terminal peptide were decreased in the model group compared with the blank control group (P < 0.05). The serum level of malondialdehyde decreased in the baicalein intervention group compared with the model group (P < 0.05), but there was no significant difference between the baicalein intervention group and blank control group (P > 0.05). The serum level of triglyceride was higher in the baicalein intervention group than the blank control group (P < 0.05), but had no significant difference between the baicalein intervention group and model group (P > 0.05). There were also no significant differences in the levels of bone specific alkaline phosphatase and β-collagen type I carboxy-terminal peptide between the baicalein intervention group and the other two groups (P > 0.05). The serum level of the baicalein intervention group was lower in the baicalein intervention group than the blank control group (P < 0.05) but had no significant difference between the baicalein intervention group and model group (P > 0.05). Histomorphological analysis of the femoral head showed that the rate of bone empty lacuna, osteoclast counting and cell apoptosis rate in the femoral head of model group rats were significantly higher than those of the other two groups (P < 0.05). There was a significant increase in the number of adipocytes in the bone marrow cavity of the femoral head, bone trabeculae were thinned and sparsely arranged with more disruptions in the continuity. The incidence of osteonecrosis was higher in the model group (75%) than in the baicalein intervention group (25%; bilateral and unilateral exact significance results were both 0.05). There was also an increase in the number of adipocytes in the bone marrow cavity of the femoral head in the baicalein intervention group, and the trabecular changes were roughly similar to those in the model group. Micro-CT results showed that bone volume fraction, trabecular thickness, trabecular number, and bone mineral density decreased and trabecular separation increased in the model group compared with the blank control group (P < 0.05). Overall significant bone mass loss was observed in the model group. Bone tissue parameters in the baicalein intervention group were significantly improved than those in the model group, which were reflected in bone volume fraction, trabecular thickness and trabecular separation (P < 0.05), and trabecular number and bone mineral density had no significant difference between the baicalein intervention group and blank control group (P > 0.05). Although baicalein failed to significantly ameliorate dyslipidemia and promote bone formation in rats with steroid-induced osteonecrosis of the femoral head, it could reduce the incidence of steroid-induced osteonecrosis of the femoral head by reducing oxidative stress damage, decreasing cell apoptosis, inhibiting osteoclasts, suggesting its effectiveness in the early prevention of steroid-induced osteonecrosis of the femoral head. [ABSTRACT FROM AUTHOR]

BACKGROUND: Some studies have found that local application of alendronate can promote osteogenesis, but less is reported on the process of distraction osteogenesis. OBJECTIVE: To observe the promoting effect of alendronate on rapid mandibular distraction in a rabbit model and explore its possible mechanism. METHODS: Thirty-six male New Zealand white rabbits were randomly divided into groups A, B and C (n=12 per group) after operation and rapid distraction (3- day delay period followed by 3-day distraction at 1.5 mm/12 hours). At the 1st, 3rd and 7th days of the consolidation period, animal were injected with 200 μg/kg alendronate in group A and 100 μg/kg alendronate in group B, while those in group C were treated as controls. CT scanning and dual energy X-ray bone mineral density measurement were performed at 4 and 8 weeks of the consolidation period. After the radionuclide scanning was completed at the 4th week, several animals were sacrificed and the samples were collected for western blot assay and tartrate resistant acid phosphatase staining. A three-point bending test was performed after the animals were sacrificed at the 8th week. RESULTS AND CONCLUSION: CT results showed that bone formation in the distraction space of group B was significantly better than that in groups A and C. At the 4th week, the bone mineral density in group B was (0.092±0.010) g/cm², which was 1.26 times higher than that in group A (P < 0.001) and 1.28 times higher than that in group C (P < 0.001). At the 8th week, the bone mineral density in group B was (0.175±0.029) g/cm², which was 1.38 times higher than that in group A (P < 0.001) and 1.45 times higher than that in group C (P < 0.001). Tartrate resistant acid phosphatase staining showed that the number of osteoclast-like cells in group C were 2.83 times more than that in group A (P < 0.001) and 2.21 times more than that in group B (P < 0.001). The radionuclide intensity was higher in group C than in groups A and B. Western blot assay results showed that the expression of Runx2 was significantly stronger in group B than in groups A and C. The maximum biomechanical load in group B was (158.48 ± 23.21) N, which was 1.26 times higher than that in group A (P=0.007) and 1.31 times higher than that in group C (P=0.003). To conclude, the low concentration of alendronate may promote rapid distraction osteogenesis of the rabbit mandible by inhibiting osteoclast signals. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a chronic disease that causes pain, morbidity, and disability. The main strategy for OA treatment focuses on inflammation suppression, inhibition of osteoclastogenesis, and protection of articular cartilage. These functions cannot be performed effectively by monotherapy. Therefore, an effective drug delivery system is required, capable of containing and controlling the efflux of various drugs to alleviate osteoclastogenesis, protect cartilage and subchondral bone, and suppress inflammation. In this work, an encapsulation system is constructed using a self-healing chitosan hydrogel and allocated compound drugs. The self-healing gel is composed of branched-functionalized chitosan, created by simultaneously using polycaprolactone polyethylene glycol azide as a block polymer and the host-guest assembly of β-cyclodextrin and adamantane. Inhibitors of the NFkB pathway are loaded into the cavities of β-cyclodextrin and the spring-like structure of the block polymer, which can be rapidly released upon joint friction (due to the reassembly of β-cyclodextrin and adamantane by shear stress and the stretch of the block polymer). In vitro experiments using BMMs and the ATDC5 cell line confirm that the developed hydrogel can simultaneously suppress osteoclastogenesis and induce chondrogenesis. Additionally, a model of knee arthritis in C57 mice was used to confirm that this double-crosslinked encapsulation system can lubricate the knee joint surface and provide adequate protection on demand through shear-responsive drug release. [ABSTRACT FROM AUTHOR]

The onset of osteonecrosis of the femoral head (ONFH) is intimately associated with the extensive administration of glucocorticoids (GCs). Long‐term stimulation of GCs can induce oxidative stress in both osteoclasts (OCs) and osteoblasts (OBs), resulting in the disturbance of bone remodelling. An alkaloid named crebanine (CN) demonstrates pharmacological properties including anti‐inflammation and reactive oxygen species (ROS) modulation. Our objective is to assess the therapeutic potential of CN in treating ONFH and elucidate the associated underlying mechanisms. The network pharmacology analysis uncovered that CN played a role in regulating ROS metabolism. In vitro, CN demonstrated its ability to reduce the dexamethasone (DEX)‐stimulated generation of OCs and suppress their resorptive function by downregulating the level of osteoclast marker genes. Concurrently, CN also mitigated DEX‐induced damage to OBs, facilitating the restoration of osteoblast marker gene expression, cellular differentiation and function. These effects were achieved by CN augmenting the antioxidant system to reduce intracellular ROS levels. Furthermore, in vitro results were corroborated by micro‐CT and histological data, which also showed that CN attenuated MPS‐induced ONFH in mice. This study highlights the therapeutic potential of CN in counteracting GCs‐induced ONFH. [ABSTRACT FROM AUTHOR]

Osteoclasts are the cells primarily responsible for inflammation‐induced bone loss, as is particularly seen in rheumatoid arthritis. Increasing evidence suggests that osteoclasts formed under homeostatic versus inflammatory conditions may differ in phenotype. While microRNA‐29‐3p family members (miR‐29a‐3p, miR‐29b‐3p, miR‐29c‐3p) promote the function of RANKL‐induced osteoclasts, the role of miR‐29‐3p during inflammatory TNF‐α‐induced osteoclastogenesis is unknown. We used bulk RNA‐seq, histology, qRT‐PCR, reporter assays, and western blot analysis to examine bone marrow monocytic cell cultures and tissue from male mice in which the function of miR‐29‐3p family members was decreased by expression of a miR‐29‐3p tough decoy (TuD) competitive inhibitor in the myeloid lineage (LysM‐cre). We found that RANKL‐treated monocytic cells expressing the miR‐29‐3p TuD developed a hypercytokinemia/proinflammatory gene expression profile in vitro, which is associated with macrophages. These data support the concept that miR‐29‐3p suppresses macrophage lineage commitment and may have anti‐inflammatory effects. In correlation, when miR‐29‐3p activity was decreased, TNF‐α‐induced osteoclast formation was accentuated in an in vivo model of localized osteolysis and in a cell‐autonomous manner in vitro. Further, miR‐29‐3p targets mouse TNF receptor 1 (TNFR1/Tnfrsf1a), an evolutionarily conserved regulatory mechanism, which likely contributes to the increased TNF‐α signaling sensitivity observed in the miR‐29‐3p decoy cells. Whereas our previous studies demonstrated that the miR‐29‐3p family promotes RANKL‐induced bone resorption, the present work shows that miR‐29‐3p dampens TNF‐α‐induced osteoclastogenesis, indicating that miR‐29‐3p has pleiotropic effects in bone homeostasis and inflammatory osteolysis. Our data supports the concept that the knockdown of miR‐29‐3p activity could prime myeloid cells to respond to an inflammatory challenge and potentially shift lineage commitment toward macrophage, making the miR‐29‐3p family a potential therapeutic target for modulating inflammatory response. [ABSTRACT FROM AUTHOR]

This study provides a detailed understanding of the preclinical pharmacokinetics and metabolism of ELP‐004, an osteoclast inhibitor in development for the treatment of bone erosion. Current treatments for arthritis, including biological disease‐modifying antirheumatic drugs, are not well‐tolerated in a substantial subset of arthritis patients and are expensive; therefore, new treatments are needed. Pharmacokinetic parameters of ELP‐004 were tested with intravenous, oral, and subcutaneous administration and found to be rapidly absorbed and distributed. We found that ELP‐004 was non‐mutagenic, did not induce chromosome aberrations, non‐cardiotoxic, and had minimal off‐target effects. Using in vitro hepatic systems, we found that ELP‐004 is primarily metabolized by CYP1A2 and CYP2B6 and predicted metabolic pathways were identified. Finally, we show that ELP‐004 inhibits osteoclast differentiation without suppressing overall T‐cell function. These preclinical data will inform future development of an oral compound as well as in vivo efficacy studies in mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bone is one of the most prevalent sites of metastases in various epithelial malignancies, including breast cancer and this metastasis to bone often leads to severe skeletal complications in women due to its osteolytic nature. To address this, we devised a novel drug delivery approach using an Alendronate (ALN) functionalized self-assembled porous crystalsomes for concurrent targeting of Oleanolic acid (OA) and ALN (ALN + OA@NCs) to bone metastasis. Initially, the conjugation of both PEG-OA and OA-PEG-ALN with ALN and OA was achieved, and this conjugation was then self-assembled into porous crystalsomes (ALN + OA@NCs) by nanoemulsion crystallization. The reconstruction of a 3D single particle using transmission electron microscopy ensured the crystalline porous structure of ALN + OA@NCs, was well aligned with characteristic nanoparticle attributes including size distribution, polydispersity, and zeta potential. Further, ALN + OA@NCs showed enhanced efficacy in comparison to OA@NCs suggesting the cytotoxic roles of ALN towards cancer cells, followed by augmentation ROS generation (40.81%), mitochondrial membrane depolarization (57.20%), and induction of apoptosis (40.43%). We found that ALN + OA@NCs facilitated inhibiting osteoclastogenesis and bone resorption followed by inhibited osteolysis. In vivo activity of ALN + OA@NCs in the 4 T1 cell-induced tibia model rendered a reduced bone loss in the treated mice followed by restoring bone morphometric markers which were further corroborated bone–targeting effects of ALN + OA@NCs to reduce RANKL-stimulated osteoclastogenesis. Further, In vivo intravenous pharmacokinetics showed the improved therapeutic profile of the ALN + OA@NCs in comparison to the free drug, prolonging the levels of the drug in the systemic compartment by reducing the clearance culminating the higher accumulation at the tumor site. Our finding proposed that ALN + OA@NCs can effectively target and treat breast cancer metastasis to bone and its associated complications. [Display omitted] [ABSTRACT FROM AUTHOR]

(1) Background: Methylphenidate (MP) and amphetamine (AMP) are psychostimulants that are widely prescribed to treat Attention Deficit Hyperactivity Disorder (ADHD) and narcolepsy. In recent years, 6.1 million children received an ADHD diagnosis, and nearly 2/3 of these children were prescribed psychostimulants for treatment. The purpose of this review is to summarize the current literature on psychostimulant use and the resulting effects on bone homeostasis, biomechanical properties, and functional integrity. (2) Methods: Literature searches were conducted from Medline/PubMed electronic databases utilizing the search terms "methylphenidate" OR "amphetamine" OR "methylphenidate" AND "bone health" AND "bone remodeling" AND "osteoclast" AND "osteoblast" AND "dopamine" from 01/1985 to 04/2023. (3) Results: Of the 550 publications found, 44 met the inclusion criteria. Data from identified studies demonstrate that the use of MP and AMP results in decreases in specific bone properties and biomechanical integrity via downstream effects on osteoblasts and osteoclast-related genes. (4) Conclusions: The chronic use of psychostimulants negatively affects bone integrity and strength as a result of increased osteoclast activity. These data support the need to take this into consideration when planning the treatment type and duration for bone fractures. [ABSTRACT FROM AUTHOR]

Surface pre-reacted glass-ionomer (S-PRG) is a new bioactive filler utilized for the restoration of decayed teeth by its ability to release six bioactive ions that prevent the adhesion of dental plaque to the tooth surface. Since ionic liquids are reported to facilitate transepithelial penetration, we reasoned that S-PRG applied to root caries could impact the osteoclasts (OCs) in the proximal alveolar bone. Therefore, this study aimed to investigate the effect of S-PRG eluate solution on RANKL-induced OC-genesis and mineral dissolution in vitro. Using RAW264.7 cells as OC precursor cells (OPCs), TRAP staining and pit formation assays were conducted to monitor OC-genesis and mineral dissolution, respectively, while OC-genesis-associated gene expression was measured using quantitative real-time PCR (qPCR). Expression of NFATc1, a master regulator of OC differentiation, and the phosphorylation of MAPK signaling molecules were measured using Western blotting. S-PRG eluate dilutions at 1/200 and 1/400 showed no cytotoxicity to RAW264.7 cells but did significantly suppress both OC-genesis and mineral dissolution. The same concentrations of S-PRG eluate downregulated the RANKL-mediated induction of OCSTAMP and CATK mRNAs, as well as the expression of NFATc1 protein and the phosphorylation of ERK, JNK, and p38. These results demonstrate that S-PRG eluate can downregulate RANKL-induced OC-genesis and mineral dissolution, suggesting that its application to root caries might prevent alveolar bone resorption. [ABSTRACT FROM AUTHOR]

Osteoporosis, a metabolic bone disorder characterized by decreased bone mass per unit volume, poses a significant global health burden due to its association with heightened fracture risk and adverse impacts on patients' quality of life. This review synthesizes the current understanding of the pathophysiological mechanisms underlying osteoporosis, with a focus on key regulatory pathways governing osteoblast and osteoclast activities. These pathways include RANK/RANKL/OPG, Wingless-int (Wnt)/β-catenin, and Jagged1/Notch1 signaling, alongside the involvement of parathyroid hormone (PTH) signaling, cytokine networks, and kynurenine in bone remodeling. Pharmacotherapeutic interventions targeting these pathways play a pivotal role in osteoporosis management. Anti-resorptive agents, such as bisphosphonates, estrogen replacement therapy/hormone replacement therapy (ERT/HRT), selective estrogen receptor modulators (SERMs), calcitonin, anti-RANKL antibodies, and cathepsin K inhibitors, aim to mitigate bone resorption. Conversely, anabolic agents, including PTH and anti-sclerostin drugs, stimulate bone formation. In addition to pharmacotherapy, nutritional supplementation with calcium, vitamin D, and vitamin K2 holds promise for osteoporosis prevention. However, despite the availability of therapeutic options, a substantial proportion of osteoporotic patients remain untreated, highlighting the need for improved clinical management strategies. This comprehensive review aims to provide clinicians and researchers with a mechanistic understanding of osteoporosis pathogenesis and the therapeutic mechanisms of existing medications. By elucidating these insights, this review seeks to inform evidence-based decision-making and optimize therapeutic outcomes for patients with osteoporosis. [ABSTRACT FROM AUTHOR]

This article explores the potential side effect of osteonecrosis of the jaw (ONJ) associated with the use of bisphosphonates, medications used to treat bone diseases. The exact mechanism of how bisphosphonates contribute to ONJ is not fully understood, but it is believed to involve the inhibition of osteoclast differentiation and angiogenesis. The article suggests that local therapies targeting osteoclast differentiation in the jaw, such as inhibiting Glycogen synthase kinase-3 beta (GSK3b), administering geranyl-geraniol (GG), or using magnesium (Mg), may be potential treatments for ONJ. However, further studies are needed to explore these potential treatments. The article also mentions the potential use of teriparatide, a recombinant form of parathyroid hormone, in stimulating bone formation. Further research is needed to investigate these potential treatments for osteonecrosis of the jaw. [Extracted from the article]

Background: Osteoclast hyperactivation due to the pathological overproduction of reactive oxygen species (ROS) stimulated by glucocorticoids (GCs) is one of the key drivers behind glucocorticoid-induced osteonecrosis of the femoral head (GIONFH). The insulin degrading enzyme (IDE), a conserved Zn2+ metallo-endopeptidase, facilitates the DNA binding of glucocorticoid receptor and plays a substantial role in steroid hormone-related signaling pathways. However, the potential role of IDE in the pathogenesis of GIONFH is yet undefined. Methods: In this study, we employed network pharmacology and bioinformatics analysis to explore the impact of IDE inhibition on GIONFH with 6bK as an inhibitory agent. Further evidence was collected through in vitro osteoclastogenesis experiments and in vivo evaluations involving methylprednisolone (MPS)-induced GIONFH mouse model. Results: Enrichment analysis indicated a potential role of 6bK in redox regulation amid GIONFH development. In vitro findings revealed that 6bK could attenuate GCs-stimulated overactivation of osteoclast differentiation by interfering with the transcription and expression of key osteoclastic genes (Traf6, Nfatc1, and Ctsk). The use of an H2DCFDA probe and subsequent WB assays introduced the inhibitory effects of 6bK on osteoclastogenesis, linked with the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2)-mediated antioxidant system. Furthermore, Micro-CT scans validated that 6bK could alleviate GIONFH in MPS-induced mouse models. Conclusions: Our findings suggest that 6bK suppresses osteoclast hyperactivity in GCs-rich environment. This is achieved by reducing the accumulation of intracellular ROS via promoting the Nrf2-mediated antioxidant system, thus implying that IDE could be a promising therapeutic target for GIONFH. [ABSTRACT FROM AUTHOR]

Introduction: Amultitude 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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Tumor necrosis factor-α is a broadly acting inflammatory cytokine that plays an important role in the immune inflammatory response of the body. The current study concluded that tumor necrosis factor-α has significant biological effects on a variety of bone tissue cells. OBJECTIVE: To summarize the expression and action pathways of tumor necrosis factor-α in osteoblastic and osteoclastic cells to further elucidate the regulatory role of tumor necrosis factor-α on bone tissue cells. METHODS: PubMed and CNKI were searched until March 2023, and the Chinese search terms included “tumor necrosis factor α, osteoblast, osteoclast, osteoclast, osteoprogenitor”; the English search terms included “TNF-α, osteoblast, osteoclast, osteocyte, osteoprogenitor cell”. The corresponding criteria were established according to the research needs, and the final literature was screened. Finally, 77 articles were included for review. RESULTS AND CONCLUSION: (1) Tumor necrosis factor-α is participating in regulating the recruitment, appreciation, and differentiation of osteoprogenitor cells, but leads to osteoprogenitor cell stripping and death under specific environments. It also participates in bone resorption directly or indirectly through secreted enzymes. (2) Tumor necrosis factor-α can increase the level of inflammatory factors in the environment by activating relevant signaling pathways in osteoclast lineage cells or directly induce the generation of osteoclasts in specific environments. (3) Tumor necrosis factor-α can inhibit osteogenic differentiation by activating nuclear factor-κB signaling pathway, inhibiting the expression of transcription factors such as RUNX2 and Osterix, and inducing apoptosis and necrotizing apoptosis in osteoblasts. (4) Tumor necrosis factor-α inhibits osteogenesis and promotes osteoclastogenesis by activating the nuclear factor-κB signaling pathway in osteocytes and inducing cytokines such as RANKL, SOST, and DKK1, while enhancing apoptosis of the osteocytes, as well as bone resorption around the apoptotic bone tissue. (5) Taken together, the effect of tumor necrosis factor-α in bone tissue is mainly to inhibit osteogenesis and promote osteoclastosis. The biological effect of tumor necrosis factor-α in bone tissue cells is usually dependent on the activation of tumor necrosis factor receptor and nuclear factor-κB signaling pathways. (6) The interaction of tumor necrosis factor-α with other tissue cell types surrounding bone tissue and its role in bone immune regulation still deserve attention in future studies. [ABSTRACT FROM AUTHOR]

BACKGROUND: Abnormal activation of osteoclasts plays an important role in the bone destruction due to spinal tuberculosis. During the pathogenesis of osteoporosis, miR-155 knockdown activates adenosine phosphate-dependent protein kinase (AMPK) by increasing the expression of leptin receptors, thereby inhibiting osteoclast differentiation and bone resorption. However, the role of miR-155/leptin receptor(LEPR)/AMPK axis in the bone destruction due to spinal tuberculosis remains unclear. OBJECTIVE: To investigate the role and mechanism of miR-155/LEPR/AMPK axis in tuberculin-induced osteoclast formation. METHODS: RAW264.7 cells were cultured and treated with different concentrations of purified protein derivative (PPD) (1.0, 2.5, 5.0, 10.0 IU/mL) and transfected with negative control (NC) sequence or miR-155 inhibitor, NC siRNA sequence or LEPR siRNA sequence. Tartrate resistant acid phosphatase staining was used to detect the number of osteoclasts. Fluorescence quantitative PCR was used to detect the expression of miR-155. Western blot was used to detect the expression of LEPR and p-AMPK. Double luciferase reporter gene was used to verify miR-155 targeting LEPR. RESULTS AND CONCLUSION: Compared with the control group, the number of osteoclasts and the expression level of miR-155 significantly increased, while the expression level of LEPR and p-AMPK significantly decreased in 2.5, 5.0, and 10.0 IU/mL PPD groups (P < 0.05). Compared with NC+5.0 IU/mL PPD group, the number of osteoclasts and the expression level of miR-155 significantly decreased, while the expression level of LEPR and p-AMPK significantly increased in the miR-155 inhibitor+5.0 IU/mL PPD group (P < 0.05). Compared with the NC group, the fluorescence activity of LEPR wild-type double luciferase reporter gene was increased in the miR-155 inhibitor group, and decreased in the miR-155 mimic group (P < 0.05). Compared with si-NC+miR-155 inhibitor+5.0 IU/ mL PPD group, the expression level of miR-155 had no significant change, the number of osteoclasts significantly increased, and the expression levels of LEPR and p-AMPL significantly decreased in si-LEPR+miR-155 inhibitor+5.0 IU/mL PPD group (P < 0.05). To conclude, tuberculin can induce osteoclast formation by increasing miR-155 expression and inhibiting downstream LEPR expression and AMPK activation. [ABSTRACT FROM AUTHOR]

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre‐expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl(Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL), the number of TRAP‐positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild‐type (WT) G9a‐expressing adenovirus in G9a KO cells restored the number of TRAP‐positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock‐out (cKO) mice by crossing G9afl/fl mice with RankCre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C‐terminal telopeptide of type I collagen (CTX), a bone‐resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone‐resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function. [ABSTRACT FROM AUTHOR]

Bone functions not only as a critical element of the musculoskeletal system but also serves as the primary lymphoid organ harboring hematopoietic stem cells (HSCs) and immune progenitor cells. The interdisciplinary field of osteoimmunology has illuminated the dynamic interactions between the skeletal and immune systems, vital for the maintenance of skeletal tissue homeostasis and the pathogenesis of immune and skeletal diseases. Aberrant immune activation stimulates bone cells such as osteoclasts and osteoblasts, disturbing the bone remodeling and leading to skeletal disorders as seen in autoimmune diseases like rheumatoid arthritis. On the other hand, intricate multicellular network within the bone marrow creates a specialized microenvironment essential for the maintenance and differentiation of HSCs and the progeny. Dysregulation of immune–bone crosstalk in the bone marrow environment can trigger tumorigenesis and exacerbated inflammation. A comprehensive deciphering of the complex "immune–bone crosstalk" leads to a deeper understanding of the pathogenesis of immune diseases as well as skeletal diseases, and might provide insight into potential therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background: Angelicin, which is found in Psoralea, can help prevent osteoporosis by stopping osteoclast formation, although the precise mechanism remains unclear. Methods: We evaluated the effect of angelicin on the oxidative stress level of osteoclasts using ovariectomized osteoporosis model rats and RAW264.7 cells. Changes in the bone mass of the femur were investigated using H&E staining and micro-CT. ROS content was investigated by DHE fluorescence labelling. Osteoclast-related genes and proteins were examined for expression using Western blotting, immunohistochemistry, tartrate-resistant acid phosphatase staining, and real-time quantitative PCR. The influence of angelicin on osteoclast development was also evaluated using the MTT assay, double luciferin assay, chromatin immunoprecipitation, immunoprecipitation and KAT6A siRNA transfection. Results: Rats treated with angelicin had considerably higher bone mineral density and fewer osteoclasts. Angelicin prevented RAW264.7 cells from differentiating into osteoclasts in vitro when stimulated by RANKL. Experiments revealed reduced ROS levels and significantly upregulated intracellular KAT6A, HO-1, and Nrf2 following angelicin treatment. The expression of genes unique to osteoclasts, such as MMP9 and NFATc1, was also downregulated. Finally, KAT6A siRNA transfection increased intracellular ROS levels while decreasing KAT6A, Nrf2, and HO-1 protein expression in osteoclasts. However, in the absence of KAT6A siRNA transfection, angelicin greatly counteracted this effect in osteoclasts. Conclusions: Angelicin increased the expression of KAT6A. This enhanced KAT6A expression helps to activate the Nrf2/HO-1 antioxidant stress system and decrease ROS levels in osteoclasts, thus inhibiting oxidative stress levels and osteoclast formation. [ABSTRACT FROM AUTHOR]

Aim: The goal of this study was to investigate the potential effects of an immunotherapeutic drug targeting STING to suppress the overreactive innate immune response and relieve the bone defect in apical periodontitis. Methodology: We established an apical periodontitis mouse model in Sting−/− and WT mice in vivo. The progression of apical periodontitis was analysed by micro‐CT analysis and H&E staining. The expression level and localization of STING in F4/80+ cells were identified by IHC and immunofluorescence staining. RANKL in periapical tissues was tested by IHC staining. TRAP staining was used to detect osteoclasts. To clarify the effect of STING inhibitor C‐176 as an immunotherapeutic drug, mice with apical periodontitis were treated with C‐176 and the bone loss was identified by H&E, TRAP, RANKL staining and micro‐CT. Bone marrow‐derived macrophages (BMMs) were isolated from Sting−/− and WT mice and induced to osteoclasts in a lipopolysaccharide (LPS)‐induced inflammatory environment in vitro. Moreover, WT BMMs were treated with C‐176 to determine the effect on osteoclast differentiation by TRAP staining. The expression levels of osteoclast‐related genes were tested using qRT‐PCR. Results: Compared to WT mice, the bone resorption and inflammatory cell infiltration were reduced in exposed Sting−/− mice. In the exposed WT group, STING was activated mainly in F4/80+ macrophages. Histological staining revealed the less osteoclasts and lower expression of osteoclast‐related factor RANKL in Sting−/− mice. The treatment of the STING inhibitor C‐176 in an apical periodontitis mice model alleviated inflammation progression and bone loss, similar to the effect observed in Sting−/− mice. Expression of RANKL and osteoclast number in periapical tissues were also decreased after C‐176 administration. In vitro, TRAP staining showed fewer positive cells and qRT‐PCR reflected decreased expression of osteoclastic marker, Src and Acp5 were detected during osteoclastic differentiation in Sting−/− and C‐176 treated BMMs. Conclusions: STING was activated and was proven to be a positive factor in bone loss and osteoclastogenesis in apical periodontitis. The STING inhibitor C‐176 administration could alleviate the bone loss via modulating local immune response, which provided immunotherapy to the treatment of apical periodontitis. [ABSTRACT FROM AUTHOR]

AIM: The study aimed to explore the effect of curcumin (Cur) on lipopolysaccharide (LPS)-induced RAW264. 7 cell-derived osteoclasts, together with its underlying mechanism. METHODS: An osteoclast model was established by treating RAW264. 7 cells with LPS. The viability of the cells was assessed by CCK-8 assays and osteoclast formation was evaluated by tartrate-resistant acid phosphatase (TRAP) activity. The levels of reactive oxygen species (ROS), Fe2+, glutathione (GSH), and malondialdehyde (MDA) were examined by biochemical assays. Mitochondrial morphology was assessed by transmission electron microscopy. The mRNA and protein levels of p53, glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) were determined by RT-qPCR and Western blot, respectively. RESULTS: Treatment with LPS successfully induced osteoclasts formation in RAW264. 7 cells. The TRAP results showed that compared with the LPS-treated group, the number of osteoclasts and TRAP activity in the curcumin-treated group decreased dose-dependently (P<0. 01). Compared with the control group, the LPS+Erastin group showed significantly increased TRAP activity (P<0. 01), while after curcumin treatment, the TRAP activity declined in a dose-dependent manner (P<0. 01). The results of the biochemical tests showed that compared with the control group, the LPS+Erastin group had significantly elevated levels of ROS, Fe2+, and MDA, while the GSH level was significantly reduced (P< 0. 01, and compared with the LPS+Erastin group, the ROS, Fe2+, and MDA levels in the curcumin group decreased (P< 0. 01) and GSH levels increased (P<0. 01). These effects were all dose-dependent. Transmission electron microscopy showed that compared with the LPS group, the LPS+Erastin group had reduced mitochondrial cristae and increased membrane density, while after treatment with curcumin, both these effects were reversed. The RT-qPCR and Western blot results showed that compared with the control group, the mRNA and protein levels of p53 in the LPS+Erastin group were significantly increased, while those of of SLC7A11 and GPX4 were significantly reduced (P<0. 01). After curcumin treatment, the p53 mRNA and protein levels were reduced while the levels of SLC7A11 and GPX4 were increased (P<0. 01). CONCLUSION: Curcumin can inhibit lipopolysaccharide-induced differentiation of RAW264. 7 cells into osteoclasts, and its mechanism may be related to the inhibition of the p53/SLC7A1 1/GPX4 signaling pathway. [ABSTRACT FROM AUTHOR]

Increasing epidemiological evidence has shown that PM 2.5 exposure is significantly associated with the occurrence of osteoporosis. It has been well demonstrated that PM 2.5 exposure enhanced the differentiation and function of osteoclasts by indirectly causing chronic inflammation, while the mechanism in osteoblasts remains unclear. In our study, toxic effects were evaluated by direct exposure of 20–80 μg/ml PM 2.5 to MC3T3-E1 cells and BMSCs. The results showed that PM 2.5 exposure did not affect cell viability via proliferation and apoptosis, but significantly inhibited osteoblast differentiation in a dose-dependent manner. Osteogenic transcription factors Runx2 and Sp7 and other biomarkers Alp and Ocn decreased after PM 2.5 exposure. RNA-seq revealed TGF-β signaling was involved in PM 2.5 exposure inhibited osteoblast differentiation, which led to P-Smad1/5 and P-Smad2 reduction in the nucleus by increasing the ubiquitination and degradation of Smad4. At last, the inflammation response increased in MC3T3-E1 cells with PM 2.5 exposure. Moreover, the mRNA levels of Mmp9 increased in bone marrow-derived macrophage cells treated with the conditional medium collected from MC3T3-E1 cells exposed to PM 2.5. Overall, these results indicated that PM 2.5 exposure inhibits osteoblast differentiation and concurrently increases the maturation of osteoclasts. Our study provides in-depth mechanistic insights into the direct impact of PM 2.5 exposure on osteoblast, which would indicate the unrecognized role of PM 2.5 on osteoporosis. • PM 2.5 exposure significantly inhibits osteoblast differentiation. • PM 2.5 inhibits TGFβ-Smad signaling via ubiquitin-dependent degradation of Smad4. • Smad4 replenish recovers the impaired differentiation with PM 2.5 treatment. • PM 2.5 elicits osteoblastic inflammatory response and promotes osteoclast function. [ABSTRACT FROM AUTHOR]

Background: Understanding human stem cell differentiation into osteoblasts and osteoclasts is crucial for bone regeneration and disease modeling. Numerous morphological techniques have been employed to assess this differentiation, but a comprehensive review of their application and effectiveness is lacking. Methods: Guided by the PRISMA framework, we conducted a rigorous search through the PubMed, Web of Science and Scopus databases, analyzing 254 articles. Each article was scrutinized against pre-defined inclusion criteria, yielding a refined selection of 14 studies worthy of in-depth analysis. Results: The trends in using morphological approaches were identified for analyzing osteoblast and osteoclast differentiation. The three most used techniques for osteoblasts were Alizarin Red S (mineralization; six articles), von Kossa (mineralization; three articles) and alkaline phosphatase (ALP; two articles) followed by one article on Giemsa staining (cell morphology) and finally immunochemistry (three articles involved Vinculin, F-actin and Col1 biomarkers). For osteoclasts, tartrate-resistant acid phosphatase (TRAP staining) has the highest number of articles (six articles), followed by two articles on DAPI staining (cell morphology), and immunochemistry (two articles with VNR, Cathepsin K and TROP2. The study involved four stem cell types: peripheral blood monocyte, mesenchymal, dental pulp, and periodontal ligament. Conclusion: This review offers a valuable resource for researchers, with Alizarin Red S and TRAP staining being the most utilized morphological procedures for osteoblasts and osteoclasts, respectively. This understanding provides a foundation for future research in this rapidly changing field. [ABSTRACT FROM AUTHOR]

Evidences shows that T helper 17 (Th17) and regulatory T (Treg) cells imbalance plays a critical role in bone lesions of MM patients. Therefore, regulating the Th17/Treg imbalance may be beneficial for bone lesions in MM. Ten MM mice complicated with bone lesions were established and divided into the halofuginone (HF) group and the PBS group. After treatment, tibia and fibula from both groups were scanned by micro-CT. Osteoclasts and osteoblasts were validated by histochemical staining and ELISA. Th17 and Treg cells were tested by flow cytometry. The correlations between Th17/Treg cell ratio and osteoclasts, osteoblasts and bone remodeling were analyzed using the Spearman relative analysis. After treatment, mice in the HF group had an increase in trabecular bone volume fraction and thickened cortex, but a decrease in trabecular separation compared to mice in the PBS group.Tartrate-resistant acid phosphase (TRAP) + osteoclasts and its biomarker TRACP5b in serum were reduced, while alkaline phosphatase (ALP) + osteoblasts and its biomarker N-terminal propeptide of type 1precollagen (P1NP) in serum were accreted in the HF group. Th17/Treg cell ratio in halofuginone-treated mice was 0.85 ± 0.05, and was significantly lower than that in PBS-treated mice, which was 1.51 ± 0.03. In addition, it showed that the Th17/Treg cell ratio was significantly and positively associated with osteoclasts, but was significantly and negatively associated with osteoblasts and bone remodeling. Halofuginone plays a critical role in the amelioration bone lesions in MM, as it can inhibit osteoclastogenesis and enhance osteoblastogenesis by regulating the Th17/Treg cell balance. [ABSTRACT FROM AUTHOR]

Aging‐related bone loss is driven by various biological factors, such as imbalanced bone metabolism from decreased osteoblast and increased osteoclast activities. Various transcriptional and post‐transcriptional factors increase osteoclast activity with aging; however, studies regarding the post‐translational regulators of osteoclast activity are still limited. The ubiquitin E3 ligase Pellino‐1 is a well‐known post‐translational regulator of inflammation. However, how Pellino‐1 expression regulation affects osteoclast differentiation remains unclear. This study determined that Pellino‐1 levels are reduced in bone marrow monocytes (BMMs) from 40‐week‐old mice compared to 4‐week‐old mice. Interestingly, conditional Knockout (cKO) of Pellino‐1 in 6‐week‐old mice resulted in decreased bone mass, reduced body size, and lower weight than in Pellino‐1 floxed mice; however, these differences are not observed in 20‐week‐old mice. The increased number of tartrate‐resistant acid phosphatase (TRAP)‐positive cells and serum levels of C‐terminal telopeptides of type I collagen, a marker of bone resorption, in 6‐week‐old Pellino‐1 cKO mice implied a connection between Pellino‐1 and the osteoclast population. Enhanced TRAP activity and upregulation of osteoclast genes in BMMs from the cKO mice indicate that Pellino‐1 deletion affects osteoclast differentiation, leading to decreased bone mass and heightened osteoclast activity. Thus, targeting Pellino‐1 could be a potential gene therapy for managing and preventing osteoporosis. [ABSTRACT FROM AUTHOR]

Our research explores the interplay between Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) and the host's inflammatory response in molar/incisor pattern periodontitis (MIPP). Cdt disrupts phosphatidylinositol-3,4,5-triphosphate (PIP3) signaling, influencing cytokine expression through canonical and non-canonical inflammasome activation as well as nuclear factor-κB (NF-κB) activation, leading to inflammation in MIPP. THP-1 differentiated macrophages (TDMs) exposed to Cdt exhibited an upregulation of pro-inflammatory genes and subsequent cytokine release. We analyzed the ability of a small molecule therapeutic, LGM2605, known for its anti-inflammatory properties, to reduce pro-inflammatory gene expression and cytokine release in Cdt-exposed and Aa-inoculated TDMs. LGM2605's mechanism of action involves inhibiting NF-κB while activating the Nrf2–transcription factor and antioxidants. Herein, we show that this small molecule therapeutic mitigates Cdt-induced pro-inflammatory cytokine expression and secretion. Our study also further defines Cdt's impact on osteoclast differentiation and maturation in MIPP. Cdt promotes increased TRAP+ cells, indicating heightened osteoclast differentiation, specific to Cdt's phosphatase activity. Cathepsin K levels rise during this process, reflecting changes in TRAP distribution between control and Cdt-treated cells. Exploring LGM2605's effect on Cdt-induced osteoclast differentiation and maturation, we found TRAP+ cells significantly reduced with LGM2605 treatment compared to Cdt alone. Upon LGM2605 treatment, immunocytochemistry revealed a decreased TRAP intensity and number of multinucleated cells. Moreover, immunoblotting showed reduced TRAP and cathepsin K levels, suggesting LGM2605's potential to curb osteoclast differentiation and maturation by modulating inflammatory cytokines, possibly involving Nrf2 activation. In summary, our research reveals the intricate connections between Cdt, pro-inflammatory cytokines, and osteoclast differentiation, offering novel therapeutic possibilities for managing these conditions. [ABSTRACT FROM AUTHOR]

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis. [ABSTRACT FROM AUTHOR]

In recent times, there has been growing attention towards exploring the nutritional and functional aspects of potato protein, along with its diverse applications. In the present study, we examined the anti‐osteoclast properties of potato protein hydrolysate (PP902) in vitro. Murine macrophages (RAW264.7) were differentiated into osteoclasts by receptor activator of nuclear factor‐κB ligand (RANKL), and PP902 was examined for its inhibitory effect. Initially, treatment with PP902 was found to significantly prevent RANKL‐induced morphological changes in macrophage cells, as determined by tartrate‐resistant acid phosphatase (TRAP) staining analysis. This notion was further supported by F‐actin analysis using a confocal microscope. Furthermore, PP902 treatment effectively and dose‐dependently down‐regulated the expression of RANKL‐induced osteoclastogenic marker genes, including TRAP, CTR, RANK, NFATc1, OC‐STAMP, and c‐Fos. These inhibitory effects were associated with suppressing NF‐κB transcriptional activation and subsequent reduced nuclear translocation. The decrease in NF‐κB activity resulted from reduced activation of its upstream kinases, including I‐κBα and IKKα. Moreover, PP902 significantly inhibited RANKL‐induced p38MAPK and ERK1/2 activities. Nevertheless, PP902 treatment prevents RANKL‐induced intracellular reactive oxygen species generation via increased HO‐1 activity. The combined antioxidant and anti‐inflammatory effects of PP902 resulted in significant suppression of osteoclastogenesis, suggesting its potential as an adjuvant therapy for osteoclast‐related diseases. [ABSTRACT FROM AUTHOR]

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1β with the NCoR/HDAC3 complex, resulting in the activation of PGC1β and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.

Abstract Osteoblasts and osteoclasts play an important role in maintaining the structural integrity of bone tissue, in which osteoclasts degrade bone structure and osteoblasts restore bone tissue. The imbalance of osteoblast and osteoclast function can lead to many bone-related diseases, such as osteoporosis and inflammatory osteolysis. The drug that can both promote bone formation and inhibit bone loss will be able to treat those diseases. In this study, it was found that LMK-235, an selective HDAC4/5 inhibitor, inhibited the differentiation and maturation of osteoclasts by regulating NF-κB and p-Smad2/3 signaling pathways via inhibition of HDAC4. At the same time, we found that LMK-235 promoted osteoblast mineralization by upregulating Runx2 expression via inhibition of HDAC4. In vivo, LMK-235 was able to alleviate lipopolysaccharide (LPS)-induced calvarial osteolysis and promote the repair of bone defects. Taken together, LMK-235 suppresses osteoclast differentiation and promotes osteoblast formation by inhibiting HDAC4. This may provide a valuable treatment for bone diseases caused by abnormal osteoclast bone resorption and osteoblast bone regeneration.

Martina Trentini,1,&ast; Ugo D’Amora,2,&ast; Alfredo Ronca,2 Luca Lovatti,2 José Luis Calvo-Guirado,3 Danilo Licastro,4 Simeone Dal Monego,4 Lucia Gemma Delogu,5 Mariusz R Wieckowski,6 Shlomo Barak,7 Oleg Dolkart,7 Barbara Zavan1 1Translational Medicine Department, University of Ferrara, Ferrara, 44121, Italy; 2Institute of Polymers, Composites and Biomaterials - National Research Council (IPCB-CNR), Naples, 80125, Italy; 3Faculty of Health Sciences, Universidad Autonoma de Chile, Santiago de Chile, 7500912, Chile; 4AREA Science Park, Trieste, 34149, Italy; 5Biomedical Science Department University of Padova, Padova, Italy; 6Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; 7Magdent B.S.R. 3, Bnei Brak, Tel Aviv, Israel&ast;These authors contributed equally to this workCorrespondence: Alfredo Ronca; Barbara Zavan, Email alfredo.ronca@cnr.it; barbara.zavan@unife.itIntroduction: In the process of bone regeneration, a prominent role is played by macrophages involved in both the initial inflammation and the regeneration/vascularization phases, due to their M2 anti-inflammatory phenotype. Together with osteoclasts, they participate in the degradation of the bone matrix if the inflammatory process does not end. In this complex scenario, recently, much attention has been paid to extracellular communication mediated by nanometer-sized vesicles, with high information content, called exosomes (EVs). Considering these considerations, the purpose of the present work is to demonstrate how the presence of a pulsed electromagnetic field (PEMF) can positively affect communication through EVs.Methods: To this aim, macrophages and osteoclasts were treated in vitro with PEMF and analyzed through molecular biology analysis and by electron microscopy. Moreover, EVs produced by macrophages were characterized and used to verify their activity onto osteoclasts.Results: The results confirmed that PEMF not only reduces the inflammatory activity of macrophages and the degradative activity of osteoclasts but that the EVS produced by macrophages, obtained from PEMF treatment, positively affect osteoclasts by reducing their activity.Discussion: The co-treatment of PEMF with M2 macrophage-derived EVs (M2-EVs) decreased osteoclastogenesis to a greater degree than separate treatments. Keywords: PEMF, exosomes, macrophages, bone, osteoclast

Abstract Background Osteoclast hyperactivation due to the pathological overproduction of reactive oxygen species (ROS) stimulated by glucocorticoids (GCs) is one of the key drivers behind glucocorticoid-induced osteonecrosis of the femoral head (GIONFH). The insulin degrading enzyme (IDE), a conserved Zn2+ metallo-endopeptidase, facilitates the DNA binding of glucocorticoid receptor and plays a substantial role in steroid hormone-related signaling pathways. However, the potential role of IDE in the pathogenesis of GIONFH is yet undefined. Methods In this study, we employed network pharmacology and bioinformatics analysis to explore the impact of IDE inhibition on GIONFH with 6bK as an inhibitory agent. Further evidence was collected through in vitro osteoclastogenesis experiments and in vivo evaluations involving methylprednisolone (MPS)-induced GIONFH mouse model. Results Enrichment analysis indicated a potential role of 6bK in redox regulation amid GIONFH development. In vitro findings revealed that 6bK could attenuate GCs-stimulated overactivation of osteoclast differentiation by interfering with the transcription and expression of key osteoclastic genes (Traf6, Nfatc1, and Ctsk). The use of an H2DCFDA probe and subsequent WB assays introduced the inhibitory effects of 6bK on osteoclastogenesis, linked with the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2)-mediated antioxidant system. Furthermore, Micro-CT scans validated that 6bK could alleviate GIONFH in MPS-induced mouse models. Conclusions Our findings suggest that 6bK suppresses osteoclast hyperactivity in GCs-rich environment. This is achieved by reducing the accumulation of intracellular ROS via promoting the Nrf2-mediated antioxidant system, thus implying that IDE could be a promising therapeutic target for GIONFH.

Abstract Background Angelicin, which is found in Psoralea, can help prevent osteoporosis by stopping osteoclast formation, although the precise mechanism remains unclear. Methods We evaluated the effect of angelicin on the oxidative stress level of osteoclasts using ovariectomized osteoporosis model rats and RAW264.7 cells. Changes in the bone mass of the femur were investigated using H&E staining and micro-CT. ROS content was investigated by DHE fluorescence labelling. Osteoclast-related genes and proteins were examined for expression using Western blotting, immunohistochemistry, tartrate-resistant acid phosphatase staining, and real-time quantitative PCR. The influence of angelicin on osteoclast development was also evaluated using the MTT assay, double luciferin assay, chromatin immunoprecipitation, immunoprecipitation and KAT6A siRNA transfection. Results Rats treated with angelicin had considerably higher bone mineral density and fewer osteoclasts. Angelicin prevented RAW264.7 cells from differentiating into osteoclasts in vitro when stimulated by RANKL. Experiments revealed reduced ROS levels and significantly upregulated intracellular KAT6A, HO-1, and Nrf2 following angelicin treatment. The expression of genes unique to osteoclasts, such as MMP9 and NFATc1, was also downregulated. Finally, KAT6A siRNA transfection increased intracellular ROS levels while decreasing KAT6A, Nrf2, and HO-1 protein expression in osteoclasts. However, in the absence of KAT6A siRNA transfection, angelicin greatly counteracted this effect in osteoclasts. Conclusions Angelicin increased the expression of KAT6A. This enhanced KAT6A expression helps to activate the Nrf2/HO-1 antioxidant stress system and decrease ROS levels in osteoclasts, thus inhibiting oxidative stress levels and osteoclast formation.

Background: Osteomyelitis (OM) is an inflammatory condition of bone characterized by cortical bone devascularization and necrosis. Dysregulation of bone remodelling is triggered by OM. Bone remodelling is precisely coordinated by bone resorption and formation via a reversal phase. However, the cellular and molecular mechanisms underlying bone remodelling failure after osteomyelitis remain elusive. Methods: To elucidate the cellular and molecular mechanism underlying bone healing after osteomyelitis, we employed single-cell RNA sequencing (scRNA-seq) to depict the atlas of human cortical bone in normal, infected and reconstructed states. Dimensionality reduction by t-stochastic neighbourhood embedding (t-SNE) and graph-based clustering were applied to analyse the detailed clusters of osteoclast lineages. After trajectory analysis of osteoclast lineages over pseudotime, real-time PCR and immunofluorescence (IF) staining were applied to identify marker gene expression of various osteoclast lineages in the osteoclast induction model and human bone sections, respectively. The potential function and communication of osteoclasts were analysed via gene set enrichment analysis (GSEA) and CellChat. The chemotactic ability of mesenchymal stem cells (MSCs) and osteoclast lineage cells in various differentiation states was determined by transwell assays and coculture assays. The effects of various osteoclast lineages on the osteogenic differentiation potential of MSCs were also determined by using this coculture system. A normal mouse tibia fracture model and an osteomyelitis-related tibia fracture model were generated via injection of luciferase-labelled Staphylococcus aureus to verify the relationships between a novel osteoclast lineage and MSCs. Then, the infection was detected by a bioluminescence imaging system. Finally, immunofluorescence staining was used to detect the expression of markers of MSCs and novel osteoclast lineages in different remodelling phases in normal and infected bone remodelling models. Results: In this study, we constructed a cell atlas encompassing normal, infected, and reconstructed cortical bone. Then, we identified a novel subset at the earlier stage of the osteoclast lineage that exhibited increased expression of IDO1, CCL3, and CCL4. These IDO1highCCL3highCCL4high cells, termed osteostaticytes (OSCs), were further regarded as the reservoir of osteoclasts in the reversal phase. Notably, OSCs exhibited the highest chemotactic activity, surpassing other lineage subsets. We also discovered that cells at the earlier stage of the osteoclast lineage play a significant role in recruiting mesenchymal stem cells (MSCs). Finally, the data revealed that OSCs might be positively related to the occurrence of bone MSCs and the contribution of bone remodelling. Conclusion: Collectively, our findings revealed a novel stage (OSC) within the osteoclast lineage, potentially representing elusive bone reversal cells due to its increased chemotactic ability towards MSCs and potential contribution to bone remodelling. This study provides valuable insights into the intricate mechanisms of the reversal phase during bone remodelling and unveils potential therapeutic strategies for diseases associated with bone uncoupling. Translational potential of this article: This study identified a new subset, referred to as IDO1(plus symbol) CCL3(plus symbol) CCL4(plus symbol) osteostaticytes which displayed the highest chemotactic activity among all osteoclast lineages and may serve as reversal cells in bone remodelling. These findings offer new insights and insights for understanding bone reversal-related diseases and may serve as novel therapeutic targets for conditions such as osteomyelitis and delayed bone healing.

Copper is an essential trace element for the human body. Abnormalities in copper metabolism can lead to bone defects, mainly by directly affecting the viability of osteoblasts and osteoclasts and their bone remodeling function, or indirectly regulating bone metabolism by influencing enzyme activities as cofactors. Copper ions released from biological materials can affect osteoblasts and osteoclasts, either directly or indirectly by modulating the inflammatory response, oxidative stress, and rapamycin signaling. This review presents an overview of recent progress in the impact of copper on bone metabolism.Translational potential of this article: The impact of copper on bone metabolism can provide insights into clinical application of copper-containing supplements and biomaterials.

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.

Osteoarthritis (OA) is the most prevalent degenerative joint disease worldwide. Effective management for early-stage OA is crucial. Denosumab (DS) has been widely used to treat osteoporosis (OP) and rheumatoid arthritis, but its potential for managing OA remains clear. We assessed the effects of DS on osteoclast activity and chondrocyte apoptosis using tartrate-resistant acid phosphatase (TRAP) assay, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, and TUNEL staining. To assess the impact of DS on the NF-κB pathway, we performed Western blot and immunofluorescence staining. Additionally, we used an OA model to explore the influence of DS on subchondral bone remodeling and cartilage degeneration in vivo. We found that DS hindered receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis by inhibiting the activity of the NF-κB pathway. Besides, DS alleviated reactive oxygen species (ROS)-induced apoptosis in chondrocytes by regulating the expression of genes related to apoptosis. Moreover, we observed an attenuation of OA-related subchondral bone remodeling and cartilage degeneration in vivo. Our findings indicate that DS could effectively suppress osteoclast activity and chondrocyte apoptosis, thereby mitigating OA-related subchondral bone remodeling and cartilage degeneration. These results provide a mechanistic basis for using DS to treat OA.

BACKGROUND: Previous studies have shown that puerarin can inhibit the differentiation of osteoclasts, and the expression of Notch signaling pathway-related proteins such as Notch1, HES1, and Jagged1 is decreased. However, the specific mechanism of the Notch1 signaling pathway for the inhibition of osteoclast differentiation by puerarin is not clear. OBJECTIVE: To explore the effect of Notch signaling pathway on puerarin inhibiting the differentiation of mouse macrophage Raw264.7 into osteoclasts. METHODS: Raw264.7 cells were divided into seven groups for intervention culture. Blank control group was cultured in high-sugar DMEM medium; the osteoclast induction group was cultured in osteoclast induction medium; the puerarin intervention group was cultured with 50 μmol/L puerarin at the same time of osteoclast induction; Notch1 siRNA control group, Notch1 siRNA group, Notch1 overexpression control group and Notch1 overexpression group were transfected with Notch1 siRNA control sequence, Notch1 siRNA, Notch1 overexpression control plasmid and Notch1 overexpression plasmid, respectively, and then cultured with osteoclast induction medium and puerarin. The number and size of osteoclasts were observed by tartrate-resistant acid phosphatase staining, the skeleton formation of osteoclasts was observed by F-actin staining, and the gene expression level of osteoclast formation markers was detected by RT-PCR. RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining results showed that puerarin intervention could inhibit the generation of osteoclasts, Notch1 silencing could further reduce the number of osteoclasts, while the number of osteoclasts in the osteoclast-induced group increased significantly after Notch1 overexpression. The results of F-actin showed that Raw264.7 cells could form a well-defined F-actin ring after osteoclast induction. Puerarin intervention would inhibit the formation of cytoskeleton, and Notch1 silencing could aggravate the inhibitory effect of cytoskeleton formation, while Notch1 overexpression could alleviate this inhibitory effect of puerarin. RT-PCR results showed that puerarin could inhibit the mRNA expression levels of tartrateresistant acid phosphatase, Cathepsin K and c-Fos, the expression of the above-mentioned three factors decreased significantly after Notch1 gene silencing, and Notch1 overexpression could upregulate the expression of these factors. These finding indicate that puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Sepsis-induced systemic inflammation leads to rapid bone mass loss; however, there is a lack of effective treatments. Ulinastatin is an antiinflammatory drug, but its protective effect and mechanism on bone under sepsis-induced systemic inflammation are still unclear. OBJECTIVE: To explore whether ulinastatin can relieve acute bone loss caused by lipopolysaccharide. METHODS: (1) Animal experiment. Thirty male C57BL/6 mice were randomly divided into three groups (n=10 per group): control group, model group and experimental group. The control group was injected intraperitoneally with normal saline, the model group was injected intraperitoneally with lipopolysaccharide, and the experimental group was injected intraperitoneally with lipopolysaccharide and ulinastatin. In the experimental group, ulinastatin was injected continuously for 3 days. After intraperitoneal injection of ulinastatin for 14 days, femoral tissues were taken for CT scanning and pathological observation. (2) Cell experiment. C57BL/6 mouse primary osteoblasts were isolated and divided into three groups: the control group was routinely cultured, lipopolysaccharide was added to the model group, and lipopolysaccharide with ulinastatin was added to the experimental group. Cell proliferation and osteogenic differentiation were detected. C57BL/6 mouse bone marrow mononuclear cells were isolated and divided into three groups: the control group was routinely cultured, lipopolysaccharide was added to the model group, and lipopolysaccharide and ulinastatin were added to the experimental group. Osteoclast differentiation was detected. RESULTS AND CONCLUSION: (1) Animal experiment. CT scanning and hematoxylin-eosin staining showed that bone mass in lipopolysaccharide-treated mice was reduced but increased after treatment with ulinastatin. Tartrate resistant acid phosphatase staining showed that the number of osteoclasts in bone tissue increased in the model group, but significantly decreased in the experimental group compared with the model group. (2) Cell experiment. Cell counting kit-8 assay showed that lipopolysaccharide treatment inhibited the proliferation of osteoblasts, and ulinastatin elevated the proliferation of osteoblasts after lipopolysaccharide treatment. Alkaline phosphatase staining, alizarin red staining and osteogenesis-related gene (alkaline phosphatase, Runx2, osteocalcin, osteoblastin, nuclear factor κB receptor-activating factor ligand, osteoprotegerin) detection showed that lipopolysaccharide treatment inhibited osteogenic differentiation of osteoblasts and elevated the nuclear factor κB receptor-activating factor ligand/osteoprotegerin ratio; ulinastatin did not have any significant effect on the reduction of osteoblast function induced by lipopolysaccharide but decreased the nuclear factor κB receptor-activating factor ligand/osteoprotegerin ratio. Tartrate resistant acid phosphatase staining and osteoclast-related gene (tartrate resistant acid phosphatase and matrix metalloproteinase 9) detection showed that lipopolysaccharide treatment could promote osteoclast differentiation of bone marrow monocytes, while ulinastatin could inhibit lipopolysaccharide-induced osteoclast differentiation of bone marrow monocytes. (3) Overall, ulinastatin can significantly inhibit lipopolysaccharide-induced bone loss, mainly through promoting osteoblast proliferation and directly or indirectly inhibiting osteoclast differentiation to alleviate bone loss and achieve osteoprotective effects. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recent studies have shown that research on naringin anti-osteoporosis mostly stays in in vitro and in vivo experiments. Understanding the mechanism of related signaling pathways and the expression of related proteins and some specific genes is an important way to deeply understand naringin anti-osteoporosis. At present, traditional Chinese medicine has been confirmed to have a significant role in anti-osteoporosis. Naringin is one of the main active ingredients in Rhizoma Drynariae. Its effectiveness and mechanism of action against osteoporosis have been gradually recognized by scholars, and its clinical and basic research has been gradually emphasized. OBJECTIVE: To analyze and summarize the research progress of naringin in anti-osteoporosis in vitro and in vivo, thereby providing some ideas for the next step to study its related mechanism of action. METHODS: The relevant literatures included in CNKI and PubMed database were searched with the Chinese search terms of “naringin, osteoporosis, traditional Chinese medicine compound, pathogenesis, signaling pathway, bone marrow mesenchymal stem cells, osteoblasts, osteoclasts” in Chinese and English, respectively. The corresponding criteria were established according to the research needs, and finally 69 articles were included for review. RESULTS AND CONCLUSION: Naringin blocks the increase in the number of osteoclasts and adipocytes, the decrease in the number of osteocytes and osteocalcin (+) cells induced by fructose-rich diet, and promotes the secretion of Sema3A from osteoblasts and osteocytes, thereby enhancing local bone formation and inhibiting osteoclast production by activating the Wnt/β-catenin pathway. Naringin is an important way to induce autophagy of osteoblasts, but autophagy-related proteins participate in osteoblast differentiation and bone formation. Lack of autophagy in osteoblasts reduces mineralization and leads to an imbalance in the number of osteoblasts and osteoclasts, which results in bone loss and decreased bone density. The composite scaffold loaded with naringin can be used as a necessary carrier for bone defect repair and has excellent bone repair properties. Naringin can also accelerate the growth of new bone tissue by increasing the local contents of bone morphogenetic protein 2 and vascular endothelial growth factor. Naringin can regulate bone metabolism and inhibit oxidative stress via ERK, PI3K/Akt and Wnt signaling pathways to improve osteoporosis, which can play a good role in preventing and controlling the disease. However, the depth and breadth of the relevant research is insufficient. Based on the mechanism of the current study, we should investigate the specific mechanisms by which naringin regulates different pathways and inter-pathway interactions in the future, which will be beneficial to the multifaceted development of naringin used in the treatment of osteoporosis.. [ABSTRACT FROM AUTHOR]

BACKGROUND: Careful regulation of bone immune response during repair of bone scaffold is important for bone regeneration. OBJECTIVE: To review the influence of bone immune response on bone repair and the design of bone tissue engineering scaffold with regulating bone immune function and its application in bone repair. METHODS: Relevant articles published from 1973 to 2023 were retrieved from Science Direct, PubMed, Web of Science, and CNKI databases. English search terms were “osteoimmunology, macrophages, bone repair materials, bone scaffold, bone defects, bone regeneration”. Chinese search terms were “bone immunity, macrophages, bone repair material, bone stent, bone defect, bone regeneration”. Totally 80 articles of the latest research progress in this field were summarized and analyzed. RESULTS AND CONCLUSION: (1) A detailed review was conducted on the important time points in the origin and development process of bone immunity, and it was explained that macrophages, as important members of the bone immune regulatory system, can be divided into two phenotypes: M1 (pro-inflammatory) and M2 (anti-inflammatory), and play a key role in different stages of bone regeneration. During the inflammatory phase, M1 type macrophages can activate osteoclasts, initiate tissue repair processes, and participate in the reconstruction of bone microvascular networks. On the other hand, during the bone tissue regeneration process in the later stages of inflammation, sustained high expression of M1 type macrophages can hinder the formation of new bones. During the repair phase, M2 macrophages can secrete osteogenic cytokines, stimulate osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells, and promote bone formation. On the other hand, long-term activation of M2 macrophages can increase the secretion of fibrogenic molecules, leading to excessive formation of scar tissue and delaying the healing process. Therefore, regulating macrophages to undergo phenotype transformation at appropriate stages and constructing an immune microenvironment beneficial for osteogenesis has great significance for bone regeneration. (2) In the process of designing bone scaffolds with bone immune regulation characteristics, the physical and chemical properties such as scaffold roughness, pore structure, stiffness, hydrophilicity, surface charge, and surface functional groups can be changed to affect non-specific protein and cell adhesion, thereby affecting the interaction between bone scaffolds and the immune system. By designing surface functional coatings of bioactive substances such as hydroxyapatite, bioactive glass, metal ions, extracellular matrix, drugs, cytokines, and exosomes, the immune microenvironment can be actively regulated by releasing bioactive substances after implantation into the body, affecting macrophage polarization and crosstalk between macrophages and bone cells, and promoting more M2 polarization of macrophages, so as to build a bone immune microenvironment that is conducive to bone regeneration. (3) Based on the research and development of bone tissue engineering scaffolds, in addition to focusing on the direct regulatory factors of stem cell osteogenic differentiation, this article also proposes that attention should be paid to the management of the immune microenvironment of stem cell differentiation. By regulating the appropriate bone immune microenvironment, more stem cell osteogenic differentiation can be induced; the osteogenic efficiency of the scaffold can be enhanced, and the concept of “bone immune regulatory characteristics” can be condensed; deeply elucidated the multi-directional regulatory role of the bone immune microenvironment and introduced the existing strategies for changing the physicochemical properties and surface functional coating of scaffolds to endow them with bone immune regulatory potential, providing new ideas for guiding the development of a new generation of bone tissue engineering scaffolds with bone immune regulatory characteristics. However, the bone immune microenvironment is a dynamic equilibrium state, and most of the existing regulatory strategies do not consider the dynamic matching of regulation. Therefore, the research and development of intelligent bone immune regulatory scaffolds with efficient and targeted regulation of the immune microenvironment will be a key focus of attention for scholars in future. [ABSTRACT FROM AUTHOR]