Ginkgetin attenuates bone loss in OVX mice by inhibiting the NF-κB/IκBα signaling pathway.

Background: Osteoporosis is a disease associated with bone resorption, characterized primarily by the excessive activation of osteoclasts. Ginkgetin is a compound purified from natural ginkgo leaves which has various biological properties, including anti-inflammation, antioxidant, and anti-tumor effects. This study investigated the bone-protective effects of ginkgetin in ovariectomized (OVX) mice and explored their potential signaling pathway in inhibiting osteoclastogenesis in a mouse model of osteoporosis. Methods: Biochemical assays were performed to assess the levels of Ca, ALP, and P in the blood. Micro CT scanning was used to evaluate the impact of ginkgetin on bone loss in mice. RT-PCR was employed to detect the expression of osteoclast-related genes (ctsk, c-fos, trap) in their femoral tissue. Hematoxylin and eosin (H&E) staining was utilized to assess the histopathological changes in femoral tissue due to ginkgetin. The TRAP staining was used to evaluate the impact of ginkgetin osteoclast generation in vivo. Western blot analysis was conducted to investigate the effect of ginkgetin on the expression of p-NF-κB p65 and IκBα proteins in mice. Results: Our findings indicate that ginkgetin may increase the serum levels of ALP and P, while decreasing the serum level of Ca in OVX mice. H&E staining and micro CT scanning results suggest that ginkgetin can inhibit bone loss in OVX mice. The TRAP staining results showed ginkgetin suppresses the generation of osteoclasts in OVX mice. RT-PCR results demonstrate that ginkgetin downregulate the expression of osteoclast-related genes (ctsk, c-fos, trap) in the femoral tissue of mice, and this effect is dose-dependent. Western blot analysis results reveal that ginkgetin can inhibit the expression of p-NF-κB p65 and IκBα proteins in mice. Conclusion: Ginkgetin can impact osteoclast formation and activation in OVX mice by inhibiting the NF-κB/IκBα signaling pathway, thereby attenuating bone loss in mice.

Caspase-8 promotes scramblase-mediated phosphatidylserine exposure and fusion of osteoclast precursors.

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

Impdh2 deficiency suppresses osteoclastogenesis through mitochondrial oxidative phosphorylation and alleviates ovariectomy-induced osteoporosis.

Abnormalities in osteoclastic generation or activity disrupt bone homeostasis and are highly involved in many pathologic bone-related diseases, including rheumatoid arthritis, osteopetrosis, and osteoporosis. Control of osteoclast-mediated bone resorption is crucial for treating these bone diseases. However, the mechanisms of control of osteoclastogenesis are incompletely understood. In this study, we identified that inosine 5'-monophosphate dehydrogenase type II (Impdh2) positively regulates bone resorption. By histomorphometric analysis, Impdh2 deletion in mouse myeloid lineage cells (Impdh2LysM-/- mice) showed a high bone mass due to the reduced osteoclast number. qPCR and western blotting results demonstrated that the expression of osteoclast marker genes, including Nfatc1, Ctsk, Calcr, Acp5, Dcstamp, and Atp6v0d2, was significantly decreased in the Impdh2LysM-/- mice. Furthermore, the Impdh inhibitor MPA treatment inhibited osteoclast differentiation and induced Impdh2-cytoophidia formation. The ability of osteoclast differentiation was recovered after MPA deprivation. Interestingly, genome-wide analysis revealed that the osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation, were impaired in the Impdh2LysM-/- mice. Moreover, the deletion of Impdh2 alleviated ovariectomy-induced bone loss. In conclusion, our findings revealed a previously unrecognized function of Impdh2, suggesting that Impdh2-mediated mechanisms represent therapeutic targets for osteolytic diseases.

New mechanistic understanding of osteoclast differentiation and bone resorption mediated by P2X7 receptors and PI3K-Akt-GSK3ß signaling.

OBJECTIVE: Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3ß signaling pathway. METHODS: An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3ß signaling pathway. RESULTS: Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3ß signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD. CONCLUSION: P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3ß signaling pathway.

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Mitochondria from osteolineage cells regulate myeloid cell-mediated bone resorption.

Interactions between osteolineage cells and myeloid cells play important roles in maintaining skeletal homeostasis. Herein, we find that osteolineage cells transfer mitochondria to myeloid cells. Impairment of the transfer of mitochondria by deleting MIRO1 in osteolineage cells leads to increased myeloid cell commitment toward osteoclastic lineage cells and promotes bone resorption. In detail, impaired mitochondrial transfer from osteolineage cells alters glutathione metabolism and protects osteoclastic lineage cells from ferroptosis, thus promoting osteoclast activities. Furthermore, mitochondrial transfer from osteolineage cells to myeloid cells is involved in the regulation of glucocorticoid-induced osteoporosis, and glutathione depletion alleviates the progression of glucocorticoid-induced osteoporosis. These findings reveal an unappreciated mechanism underlying the interaction between osteolineage cells and myeloid cells to regulate skeletal metabolic homeostasis and provide insights into glucocorticoid-induced osteoporosis progression.

Bone microstructural characteristics or positional changes of condyle head affect short-term condyle head resorption after orthognathic surgery.

Condylar resorption occurs in some cases after orthognathic surgery, and the risk factors associated with postoperative condylar head resorption have been extensively described. Nevertheless, even in cases with a combination of risk factors, postoperative condylar resorption may not appear. This study analyzed the microstructure and three-dimensional positional change of the condylar bone via imaging in patients who have undergone bimaxillary orthognathic surgery to determine whether the microstructure or condylar position differs between patients with and without postoperative condylar resorption. Among asymptomatic patients who underwent bimaxillary surgery between April 2021 and March 2022 at our department, 17 patients were analyzed, limited to "female," "skeletal Class II," and "high-angle cases," which are known risk factors for mandibular head resorption. Multidetector computed tomography was performed on these patients before and 6 months after surgery, and the bone microstructure of the condylar head and the three-dimensional positional changes of the condylar bone and the proximal bony fragments were compared with the presence of postoperative condyle resorption using the bone morphology software TRI/3D-BON. Patients with condylar bone abnormalities before surgery and those with high trabecular bone density can develop postoperative resorption if the condyle is misaligned by surgery.

Aging decreases osteocyte peri-lacunar-canalicular system turnover in female C57BL/6JN mice.

Osteocytes engage in bone resorption and mineralization surrounding their expansive lacunar-canalicular system (LCS) through peri-LCS turnover. However, fundamental questions persist about where, when, and how often osteocytes engage in peri-LCS turnover and how these processes change with aging. Furthermore, whether peri-LCS turnover is associated with natural variation in cortical tissue strain remains unexplored. To address these questions, we utilized confocal scanning microscopy, immunohistochemistry, and scanning electron microscopy to characterize osteocyte peri-LCS turnover in the cortical (mid-diaphysis) and cancellous (metaphysis) regions of femurs from young adult (5 mo) and early-old-age (22 mo) female C57BL/6JN mice. LCS bone mineralization was measured by the presence of perilacunar fluorochrome labels. LCS bone resorption was measured by immunohistochemical marker of bone resorption. The dynamics of peri-LCS turnover were estimated from serial fluorochrome labeling, where each mouse was administered two labels between 2 and 16 days before euthanasia. Osteocyte participation in mineralizing their surroundings is highly abundant in both cortical and cancellous bone of young adult mice but significantly decreases with aging. LCS bone resorption also decreases with aging. Aging has a greater impact on peri-LCS turnover dynamics in cancellous bone than in cortical bone. Lacunae with recent peri-LCS turnover are larger in both age groups. While peri-LCS turnover is associated with variation in tissue strain between cortical quadrants and intracortical location for 22 mo mice, these associations were not seen for 5 mo mice. The impact of aging on decreasing peri-LCS turnover may have significant implications for bone quality and mechanosensation.

Exploring the impact of osteoprotegerin on osteoclast and precursor fusion: Mechanisms and modulation by ATP.

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.

Age-Related Decrease in Pellino-1 Expression Contributes to Osteoclast-Mediated Bone Loss.

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.

Mechanical protein polycystin-1 directly regulates osteoclastogenesis and bone resorption.

Mechanical loading is required for bone homeostasis, but the underlying mechanism is still unclear. Our previous studies revealed that the mechanical protein polycystin-1 (PC1, encoded by Pkd1) is critical for bone formation. However, the role of PC1 in bone resorption is unknown. Here, we found that PC1 directly regulates osteoclastogenesis and bone resorption. The conditional deletion of Pkd1 in the osteoclast lineage resulted in a reduced number of osteoclasts, decreased bone resorption, and increased bone mass. A cohort study of 32,500 patients further revealed that autosomal dominant polycystic kidney disease, which is mainly caused by loss-of-function mutation of the PKD1 gene, is associated with a lower risk of hip fracture than those with other chronic kidney diseases. Moreover, mice with osteoclast-specific knockout of Pkd1 showed complete resistance to unloading-induced bone loss. A mechanistic study revealed that PC1 facilitated TAZ nuclear translocation via the C-terminal tail-TAZ complex and that conditional deletion of Taz in the osteoclast lineage resulted in reduced osteoclastogenesis and increased bone mass. Pharmacological regulation of the PC1-TAZ axis alleviated unloading- and estrogen deficiency- induced bone loss. Thus, the PC1-TAZ axis may be a potential therapeutic target for osteoclast-related osteoporosis.

Therapeutic avenues in bone repair: Harnessing an anabolic osteopeptide, PEPITEM, to boost bone growth and prevent bone loss.

The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Targeting SAT1 prevents osteoporosis through promoting osteoclast apoptosis.

Osteoporosis is a systemic bone disease characterized by decreased bone mass that is tightly regulated by the coordinated actions of osteoclasts and osteoblasts. Apoptosis as a precise programmed cell death involves a cascade of gene expression events which are mechanistically linked to the regulation of bone metabolism. Nevertheless, the critical biomolecules involved in regulating cell apoptosis in osteoporosis remain unknown. To gain a deeper insight into the relationship between apoptosis and osteoporosis, this study integrated the sequencing results of human samples and using a machine learning workflow to overcome the limitations of a single study. Among all immune cell populations, we assessed the apoptotic level and portrayed the distinct subtypes and lineage differentiation of monocytic cells in osteoporotic tissues. Osteoclasts expressed a higher level of Spermidine/spermine-N1-Acetyltransferase1 (SAT1) during osteoclastogenesis which prevented osteoclasts apoptosis and facilitate osteoporosis progression. In addition, Berenil, one potent SAT1 inhibitor, increased osteoclast apoptosis and reversed the bone loss in the femurs of a murine ovariectomy model. In summary, Berenil promotes osteoclast apoptosis, inhibits the bone resorption and improves the abnormal bone structure in vitro and in vivo models by targeting SAT1, demonstrating its potential as a precise therapeutic strategy for clinical osteoporosis treatment.

Characterization and quantification of in-vitro equine bone resorption in 3D using µCT and deep learning-aided feature segmentation.

High cyclic strains induce formation of microcracks in bone, triggering targeted bone remodeling, which entails osteoclastic resorption. Racehorse bone is an ideal model for studying the effects of high-intensity loading, as it is subject to focal formation of microcracks and subsequent bone resorption. The volume of resorption in vitro is considered a direct indicator of osteoclast activity but indirect 2D measurements are used more often. Our objective was to develop an accurate, high-throughput method to quantify equine osteoclast resorption volume in µCT 3D images. Here, equine osteoclasts were cultured on equine bone slices and imaged with µCT pre- and postculture. Individual resorption events were then isolated and analyzed in 3D. Modal volume, maximum depth, and aspect ratio of resorption events were calculated. A convolutional neural network (CNN U-Net-like) was subsequently trained to identify resorption events on post-culture µCT images alone, without the need for pre-culture imaging, using archival bone slices with known resorption areas and paired CTX-I biomarker levels in culture media. 3D resorption volume measurements strongly correlated with both the CTX-I levels (p < 0.001) and area measurements (p < 0.001). Our 3D analysis shows that the shapes of resorption events form a continuous spectrum, rather than previously reported pit and trench categories. With more extensive resorption, shapes of increasing complexity appear, although simpler resorption cavity morphologies (small, rounded) remain most common, in acord with the left-hand limit paradigm. Finally, we show that 2D measurements of in vitro osteoclastic resorption are a robust and reliable proxy.

Nodal negatively regulates osteoclast differentiation by inducing STAT1 phosphorylation.

Several members of the transforming growth factor beta (TGF-ß) superfamily regulate the proliferation, differentiation, and function of bone-forming osteoblasts and bone-resorbing osteoclasts. However, it is still unknown whether Nodal, a member of the TGF-ß superfamily, serves a function in bone cells. In this study, we found that Nodal did not have any function in osteoblasts but instead negatively regulated osteoclast differentiation. Nodal inhibited RANKL-induced osteoclast differentiation by downregulating the expression of pro-osteoclastogenic genes, including c-fos, Nfatc1, and Blimp1, and upregulating the expression of antiosteoclastogenic genes, including Bcl6 and Irf8. Nodal activated STAT1 in osteoclast precursor cells, and STAT1 downregulation significantly reduced the inhibitory effect of Nodal on osteoclast differentiation. These findings indicate that Nodal activates STAT1 to downregulate or upregulate the expression of pro-osteoclastogenic or antiosteoclastogenic genes, respectively, leading to the inhibition of osteoclast differentiation. Moreover, the inhibitory effect of Nodal on osteoclast differentiation contributed to the reduction of RANKL-induced bone loss in vivo.

Gallium and silver-doped titanium surfaces provide enhanced osteogenesis, reduce bone resorption and prevent bacterial infection in co-culture.

Bacterial infection remains a significant problem associated with orthopaedic surgeries leading to surgical site infection (SSI). This unmet medical need can become an even greater complication when surgery is due to malignant bone tumor. In the present study, we evaluated in vitro titanium (Ti) implants subjected to gallium (Ga) and silver (Ag)-doped thermochemical treatment as strategy to prevent SSI and improve osteointegration in bone defects caused by diseases such as osteoporosis, bone tumor, or bone metastasis. Firstly, as Ga has been reported to be an osteoinductive and anti-resorptive agent, its performance in the mixture was proved by studying human mesenchymal stem cells (hMSC) and pre-osteoclasts (RAW264.7) behaviour. Then, the antibacterial potential provided by Ag was assessed by resembling "The Race for the Surface" between hMSC and Pseudomonas aeruginosa in two co-culture methods. Moreover, the presence of quorum sensing molecules in the co-culture was evaluated. The results highlighted the suitability of the mixture to induce osteodifferentiation and reduce osteoclastogenesis in vitro. Furthermore, the GaAg surface promoted strong survival rate and retained osteoinduction potential of hMSCs even after bacterial inoculation. Therefore, GaAg-modified titanium may be an ideal candidate to repair bone defects caused by excessive bone resorption, in addition to preventing SSI. STATEMENT OF SIGNIFICANCE: This article provides important insights into titanium for fractures caused by osteoporosis or bone metastases with high incidence in surgical site infection (SSI) because in this situation bacterial infection can become a major disaster. In order to solve this unmet medical need, we propose a titanium implant modified with gallium and silver to improve osteointegration, reduce bone resorption and avoid bacterial infection. For that aim, we study osteoblast and osteoclast behavior with the main novelty focused on the antibacterial evaluation. In this work, we recreate "the race for the surface" in long-term experiments and study bacterial virulence factors (quorum sensing). Therefore, we believe that our article could be of great interest, providing a great impact on future orthopedic applications.

Vasorin as an actor of bone turnover?

Bone diseases are increasing with aging populations and it is important to identify clues to develop innovative treatments. Vasn, which encodes vasorin (Vasn), a transmembrane protein involved in the pathophysiology of several organs, is expressed during the development in intramembranous and endochondral ossification zones. Here, we studied the impact of Vasn deletion on the osteoblast and osteoclast dialog through a cell Coculture model. In addition, we explored the bone phenotype of Vasn KO mice, either constitutive or tamoxifen-inducible, or with an osteoclast-specific deletion. First, we show that both osteoblasts and osteoclasts express Vasn. Second, we report that, in both KO mouse models but not in osteoclast-targeted KO mice, Vasn deficiency was associated with an osteopenic bone phenotype, due to an imbalance in favor of osteoclastic resorption. Finally, through the Coculture experiments, we identify a dysregulation of the Wnt/ß-catenin pathway together with an increase in RANKL release by osteoblasts, which led to an enhanced osteoclast activity. This study unravels a direct role of Vasn in bone turnover, introducing a new biomarker or potential therapeutic target for bone pathologies.

Temporal patterns of osteoclast formation and activity following withdrawal of RANKL inhibition.

Rebound bone loss following denosumab discontinuation is an important clinical challenge. Current treatment strategies to prevent this fail to suppress the rise and overshoot in osteoclast-mediated bone resorption. In this study, we use a murine model of denosumab treatment and discontinuation to show the temporal changes in osteoclast formation and activity during RANKL inhibition and withdrawal. We show that the cellular processes that drive the formation of osteoclasts and subsequent bone resorption following withdrawal of RANKL inhibition precede the rebound bone loss. Furthermore, a rise in serum TRAP and RANKL levels is detected before markers of bone turnover used in current clinical practice. These mechanistic advances may provide insight into a more defined window of opportunity to intervene with sequential therapy following denosumab discontinuation.

Stopping denosumab, a medication commonly used to improve bone mass by blocking formation of bone resorbing osteoclasts, leads to a rebound loss in the bone which was gained during treatment. Current strategies to prevent this bone loss fail in most cases as they are unable to prevent the rise and overshoot in bone resorption by osteoclasts. Thie stems from an incomplete understanding of how osteoclasts behave during denosumab treatment and after treatment is discontinued. We use a mouse model of this phenomenon to show how osteoclast formation and activity changes throughout this process. We show that increases in the processes that drive the formation of osteoclasts can be detected in the circulation before bone loss occurs. These findings could therefore provide insight into a targeted 'window of opportunity' to intervene and prevent the rebound bone loss following stopping denosumab in patients.

The histone methyltransferase ASH1L protects against bone loss by inhibiting osteoclastogenesis.

Absent, small, or homeotic1-like (ASH1L) is a histone lysine methyltransferase that generally functions as a transcriptional activator in controlling cell fate. So far, its physiological relevance in bone homeostasis and osteoclast differentiation remains elusive. Here, by conditional deleting Ash1l in osteoclast progenitors of mice, we found ASH1L deficiency resulted in osteoporosis and potentiation of osteoclastogenesis in vivo and in vitro. Mechanistically, ASH1L binds the promoter of the Src homology 3 and cysteine-rich domain 2 (Stac2) and increases the gene's transcription via histone 3 lysine 4 (H3K4) trimethylation modification, thus augmenting the STAC2's protection against receptor activator of nuclear factor kB ligand (RANKL)-initiated inflammation during osteoclast formation. Collectively, we demonstrate the first piece of evidence to prove ASH1L as a critical checkpoint during osteoclastogenesis. The work sheds new light on our understanding about the biological function of ASH1L in bone homeostasis, therefore providing a valuable therapeutic target for the treatment of osteoporosis or inflammatory bone diseases.