Mouse Bone Research Articles

Single-cell transcriptomics reveals novel chondrocyte and osteoblast subtypes and their role in knee osteoarthritis pathogenesis

Research on treating knee osteoarthritis (KOA) is becoming more challenging due to a growing number of younger patients being affected. The pathogenesis of KOA is complex for being a multifactorial disease affecting the entire joint, with remodeling of subchondral bone playing a key role in the degeneration of the overlying cartilage. Therefore, this study constructed a bipedal postmenopausal KOA mouse model to better understand how the interplay between subchondral bone remodeling and cartilage degeneration contributes to KOA development. A single-cell atlas of the osteochondral composite tissue was established. Furthermore, three novel subtypes of chondrocytes, including Smoc2+ angiogenic chondrocytes, Angptl7+ angiogenic chondrocytes, and Col1a1+ osteogenic chondrocytes, were identified in femoral condyles of KOA mice. In addition, the Angptl7+ chondrocytes promoted angiogenesis in the subchondral bone of KOA mice by interacting with endothelial cells via the FGF2-FGFR2 signaling pathway. The number of H-type vessels was increased in the subchondral bone, recruiting osteoprogenitor cells and facilitating osteogenesis in KOA mice. Sparc+ osteoblasts have negatively regulated bone mineralization and osteoblastic differentiation, aggravated the pathological remodeling of subchondral bone, and promoted the progression of KOA. The above findings have offered new targets and opened up an avenue for the therapeutic intervention of KOA.

ATAD2 Drives Prostate Cancer Progression to Metastasis.

Metastasis accounts for the overwhelming majority of cancer deaths. In prostate cancer and many other solid tumors, progression to metastasis is associated with drastically reduced survival outcomes, yet the mechanisms behind this progression remain largely unknown. ATAD2 (ATPase family AAA domain containing 2) is an epigenetic reader of acetylated histones that is overexpressed in multiple cancer types and usually associated with poor patient outcomes. However, the functional role of ATAD2 in cancer progression and metastasis has been relatively understudied. Here we employ genetically engineered mouse models of prostate cancer bone metastasis, as well as multiple independent human cohorts, to show that ATAD2 is highly enriched in bone metastasis compared to primary tumors and significantly associated with the development of metastasis. We show that ATAD2 expression is associated with MYC pathway activation in patient datasets and that, at least in a subset of tumors, MYC and ATAD2 can regulate each other's expression. Using functional studies on mouse bone metastatic cell lines and innovative organ-on-a-chip bone invasion assays, we establish a functional role for ATAD2 inhibition in diminishing prostate cancer metastasis and growth in bone. Implications: Our study highlights ATAD2 as a driver of prostate cancer progression and metastasis and suggests it may constitute a promising novel therapeutic target.

Leveraging Metabolomics and Ionomics to Illuminate Aluminum-Induced Toxicity in Mouse Organs

Aluminum, a widely prevalent environmental pollutant, has been established to exert toxic effects on multiple organs in the human body. To gain a comprehensive understanding of these toxic effects and the mechanisms involved, this study aimed to assess potential correlations between metabolites and ion data through metabolomic and ionomic analyses. We sought to explore the intricate impact of aluminum on various organs in mice. Gas chromatography-mass spectrometry (GC-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) were employed to conduct metabolomic and ionomic analyses on the hippocampus, cortex, heart, liver, spleen, lung, kidney, bone, intestine, stomach, and serum of both control and aluminum-exposed mice. Besides, histological examinations and behavioral experiments were conducted. Multivariate analysis revealed 91 differential metabolites across various organs, primarily encompassing amino acids, fatty acids, and carbohydrates. The implicated abnormal metabolic pathways included amino acid metabolism, arachidonic acid metabolism, and glutathione metabolism. Additionally, alterations in the homeostasis of ions such as manganese, zinc, selenium, iron, copper, phosphorus, magnesium, and calcium were observed in various organs, potentially influencing the activity of critical enzymes. The changes in these potential biomarkers and ions suggest toxic mechanisms of aluminum exposure involving oxidative stress, inflammatory responses, cellular signal dysregulation, disruption of key enzyme activities, and impaired energy metabolism. This study provides a novel perspective on understanding the toxic mechanisms of aluminum exposure, potentially contributing to the prevention and treatment of aluminum toxicity.

Aerosol inhalation of rhIL-10 improves acute lung injury in mice by affecting pulmonary neutrophil phenotypes through neutrophil-platelet aggregates.

This study investigates the therapeutic effects of recombinant human IL-10 (rhIL-10) administered via aerosol inhalation in acute lung injury (ALI), with a particular focus on neutrophils. It explores how rhIL-10, in the presence of platelets, modulates neutrophil polarization to ameliorate acute lung injury. Initially, the ALI model established in mice demonstrated that aerosol inhalation of rhIL-10 significantly mitigated the cytokine storm in the lungs, reduced pulmonary edema, and alleviated histopathological damage to lung tissue. Additionally, rhIL-10 administration was found to decrease neutrophil infiltration and platelet activation in the lungs of mice, inhibiting the formation of platelet-neutrophil aggregates (PNAs) and promoting the differentiation of neutrophils toward an anti-inflammatory phenotype in the presence of platelets. Subsequently, primary neutrophils and platelets were isolated from mouse bone marrow and blood to explore the underlying mechanisms. The results indicated that rhIL-10 promotes the expression of the signal transducer and activator of transcription 3 (STAT3) and the suppressor of cytokine signaling 3 (SOCS3) in neutrophils while inhibiting the activation of the nuclear factor kappa B (NF-κB) and the NF-κB inhibitor (IκB), which in turn enhances CD40 expression. This interaction facilitates the formation of PNAs and influences neutrophil phenotype differentiation. Furthermore, the application of the STAT3 phosphorylation inhibitor Stattic and CD40 antibody in vivo provided further validation of this potential mechanism. In conclusion, these results indicate that aerosol inhalation of rhIL-10 effectively ameliorates ALI. The underlying mechanism may involve the modulation of the neutrophil STAT/SOCS-IκB/NF-κB-CD40 signaling pathway, promoting interactions between neutrophils and platelets that facilitate the differentiation of neutrophils toward an anti-inflammatory phenotype.

IFN alpha signaling drives hematopoietic stem cells malfunction under acute inflammation.

Inflammation stimulation regulates the activity of hematopoietic stem cells (HSCs) through direct-sensing and cytokine-mediation. It is known that HSCs directly sense lipopolysaccharide (LPS), a classical infection-related inflammatory signal, via toll like receptor 4 (TLR4) and subsequently become active. However, the mechanism underlying the activity change of HSCs induced by LPS remains incompletely disclosed. Here we explored that under LPS stimulation, the activation of interferon alpha (IFNα) signal pathway resulted in the activation and exhaustion of HSCs in vitro, indicating HSCs directly responded to LPS through the downstream IFNα signal pathway. We also discovered the increased production of IFNα in mice bone marrow and expression of interferon-α/β receptor (IFNAR) on mice HSCs after LPS stimulation. Creatine, an IFNα inhibitor, could reverse the activation and prevent the exhaustion of HSCs caused by LPS by suppressing the expressions of genes associated with the IFNα signal pathway both in vitro and in vivo. Furthermore, we found that the IFNAR deficiency in mice effectively protected HSCs from activation, elevated apoptosis and impaired reconstitution ability under LPS stimulation in vivo. This finding further supports the notion that LPS activates and injures HSCs indirectly via promoting IFNα secretion in the bone marrow environment. Overall, our findings reveal that LPS causes the injury to HSCs either through direct or cytokine-mediated indirect activation of the IFNα signal pathway.

The Effect of Anti-Activin Receptor Type IIA and Type IIB Antibody on Muscle, Bone and Blood in Healthy and Osteosarcopenic Mice.

Anti-Activin Receptor Type IIA and Type IIB antibody (αActRIIA/IIB ab) is a recently developed drug class that targets the activin receptor signalling pathway. Inhibition of receptor ligands (activins, myostatin, growth differentiation factor 11, etc.) can lead to skeletal muscle hypertrophy, bone formation, and increased haematopoiesis. Despite the αActRIIA/IIB ab, bimagrumab, having progressed to clinical trials, two crucial questions about αActRIIA/IIB ab therapy remain: Does αActRIIA/IIB ab influence bone metabolism and bone strength similarly to its generic classmates (activin receptor-based ligand traps)? Does αActRIIA/IIB ab affect red blood cell parameters, thereby increasing the risk of thromboembolism, similar to its generic classmates? Therefore, the aim of the present study was to investigate the therapeutic potential of αActRIIA/IIB ab in a mouse model of concurrent sarcopenia and osteopenia and to investigate the effect on bone and haematopoiesis in more detail. In C57BL/6JRj mice, combined sarcopenia and osteopenia were induced locally by injecting botulinum toxin A into the right hindlimb, resulting in acute muscle paresis. Immediately after immobilization, mice received twice-weekly intraperitoneal injections with αActRIIA/IIB ab (10 mg/kg) for 21 days, after which they were sacrificed. Muscle mass, skeletal muscle fibre size and Smad2 expression were analysed in the rectus femoris and gastrocnemius muscles. Bone mass and bone microstructure were analysed in the trabecular bone at the distal femoral metaphysis, while the cortical bone was analysed at the femoral mid-diaphysis. In a substudy, the effect on haematopoiesis was explored 2 and 7 days after a single αActRIIA/IIB ab (30 mg/kg) injection in C57BL/6JRj mice. αActRIIA/IIB ab caused a large increase in muscle mass in both healthy (+21%) and immobilized (sarcopenic and osteopenic) (+12%) mice. Furthermore, αActRIIA/IIB ab increased trabecular bone (bone volume fraction) for both healthy (+65%) and immobilized (+44%) mice. For cortical bone, αActRIIA/IIB ab caused a small, but significant, increase in bone area (+6%) for immobilized mice, but not for healthy mice. Treatment with αActRIIA/IIB ab did not change red blood cell count, haemoglobin concentration or mean cell volume after either 2 or 7 days. Treatment with αActRIIA/IIB ab caused a significant increase in both skeletal muscle mass and bone parameters in both healthy and immobilized mice, suggesting a potential in the treatment of concurrent osteopenia and sarcopenia. Interestingly, the bone anabolic effect of the treatment was much more pronounced on trabecular bone than on cortical bone. There was no pronounced effect of short-term treatment on haematopoiesis.

Study of Podoplanin-Deficient Mouse Bone with Mechanical Stress

Objective: We investigated morphological differences in osteocyte processes between aged mice and our original podoplanin-conditional knockout (cKO) mice in which the floxed exon 3 of podoplanin was deleted by Dmp-1-driven Cre (Dmp1-Cre;PdpnΔ/Δ). Methods: SEM observation on osteocyte cell process, histochemistry for bone remodeling with mechanostress, and RT-PCR for RANKL and M-CSF in podoplanin cKO mouse bone with mechanostress was investigated. Results: SEM observations showed fewer and thinner osteocyte processes in femurs from 23-week-old Dmp1-Cre;PdpnΔ/Δ mice than from 23-week-old wild-type mice, while the numbers of osteocyte processes in femurs and calvarias were similar in 23-week-old Dmp1-Cre;PdpnΔ/Δ mice and 48-week-old wild-type mice. Furthermore, cell process numbers in femurs and calvarias were significantly smaller in 23-week-old Dmp1-Cre;PdpnΔ/Δ mice than in 48-week-old wild-type mice. In the test for differences in alveolar bone resorption under mechanical stress between Dmp1-Cre;PdpnΔ/Δ and wild-type mice, the area of TRAP-positive resorption pits was larger in wild-type mice than in Dmp1-Cre;PdpnΔ/Δ mice. In a quantitative tissue PCR analysis, the mRNA expression levels of RANKL and M-CSF in alveolar bone under mechanical stress were significantly lower in Dmp1-Cre;PdpnΔ/Δ mice than in wild-type mice. These results suggest that a reduction in cell process formation in osteocytes with podoplanin cKO affected the absorption of alveolar bone under mechanical stress in Dmp1-Cre;PdpnΔ/Δ mice. Conclusions: In podoplanin-deficient bone, the deformation of osteocyte processes by mechanical stimuli is not recognized as a stress due to the lower number of cell processes with podoplanin deficiency; therefore, the production of osteoclast migration/differentiation factors by activated osteocytes is not fully induced and macrophage migration to alveolar bone with mechanical stress appeared to be suppressed. These results indicate that podoplanin-dependent osteocyte process formation indirectly plays a key role in sensing mechanical stress in bone.

GDF11 restores the impaired function of EPCs-MA by promoting autophagy: GDF11 ameliorates endothelial progenitor cell aging by promoting autophagy

Abstract Objective Our study aimed to assess the effects of Growth and differentiation factor 11 (GDF11) on the function of endothelial progenitor cells in middle-age individuals (EPCs-MA) isolated from mouse bone marrow and to explore the mechanistic relationship between GDF11 and age-related ALP impairment. Methods Bone marrow-derived EPCs were isolated, culture and GDF11 treatment. In vivo, the mice model of myocardial ischemia (MI) was induced by permanent ligation of the left anterior descending coronary artery (LAD) and mice were randomly divided into MI group and EPCs transplantation group (EPCs-Y, EPCs-MA, EPCs-MA/GDF11). The positive effect of GDF11 treatment of EPCs-MA on MI was verified by echocardiography and the average ratio of fibrotic area to left ventricular (LV) area. In vitro, the effect of GDF11 on ameliorating EPCs aging by promoting autophagy was confirmed by transwell assay, immunofluorescence staining, characterization of EPCs ultrastructure through transmission electron microscope (TEM), lysosome imaging and Western blot. Result Our findings demonstrate that GDF11 enhances the migration capacity of EPCs-MA and improves recovery of impaired cardiac function after myocardial infarction (MI) in mice, with EPCs isolated from young mice (EPCs-Y) as controls. Moreover, GDF11 restored functional phenotypes of EPCs-MA to levels akin to EPCs-Y, promoting the expression of CD31, endogenous NO synthase, and the restoration of von Willebrand factor (vWF) and CDH5 expression patterns, as well as the formation of Weibel-Palade bodies—key organelles for storage and secretion in endothelial cells and EPCs. Furthermore, GDF11 significantly enhanced the autophagic clearance capability of EPCs-MA by promoting ALP. Conclusions Our results suggest that GDF11 ameliorates cardiac function impairment by restoring the activities of EPCs from aging mice through enhanced ALP. These findings suggest that GDF11 may hold therapeutic potential for improving aging-related conditions associated with declined autophagy.

Artificial light at night effects glucose metabolism in the developing jawbone by inhibiting melatonin secretion

Objective: To investigate the effects of artificial light at night on the growth of mandibles in mice and its regulatory mechanisms. Methods: A mouse model of artificial light at night (night light pollution group) and normal lighting (normal light group) was established by controlling light exposure time, with 4 mice in each group. Micro-CT was employed to analyze the differences in bone quantities of the mandibles between the two groups. Real-time fluorescence quantitative PCR (RT-qPCR) was used to examine the expression levels of osteogenic differentiation and metabolism-related genes in the cortical bone and condylar ossification center of the mandibles. Enzyme-linked immunosorbent assay was utilized to assess the diurnal variation of serum melatonin concentrations between the two groups. The artificial light at night experimental group received daily timed injections of a defined dose of melatonin to restore the diurnal variation of serum melatonin concentration in the mice, while the normal light group and the artificial light at night control group received the same volume of saline. Bone quantities, mandibular tissue morphologies, ossification differentiation in the condylar region and cortical bone, as well as glucose metabolism expression differences were assessed across the three groups. Results: The cortical bone thickness of the mandibles in the artificial light at night group [(0.196±0.017) mm] was significantly less than that in the control group [ (0.228±0.007) mm] (P=0.029). The bone volume fraction of the condylar ossification center in the artificial light at night group [(36.90±1.09) %] was significantly lower than that in the normal light group [(54.24±1.49) %] (P<0.001). The length of the mandible in the artificial light at night group [(10.86±0.17) mm] was significantly shorter than that in the normal light group [(11.41±0.32) mm] (P=0.032). RT-qPCR results indicated that the expressions of osteogenic-related genes alkaline phosphatase, liver/bone/kidney (Alpl), osteocalcin (Ocn), Runt-related transcription factor 2 (Runx2), and osterix (Osx) in the cortical bone and condylar ossification center of mice in the artificial light at night group were significantly lower than those in the normal light group (all P<0.05). The expression of metabolism-related genes protein kinase, AMP activated alpha 1 (Prkaa1), V-type proton ATPase subunit d 1 (Atp6v0d1), and cytochrome C oxidase subunit Ⅳ isoform 1 (Cox4i1) in the cortical bone and condylar ossification center of mice in the artificial light at night group were also significantly reduced compared to normal mice (all P<0.01). The serum melatonin concentration peaked 8 hours after lights off in the normal light group, whereas the artificial light at night group exhibited a significantly reduced nocturnal serum melatonin concentration with no apparent peak compared to the normal light group. Micro-CT results demonstrated that after artificial light at night group mice received timed melatonin supplementation daily, the thickness of cortical bone, the bone volume fraction of the condylar ossification center, and the length of the mandible were all significantly higher than those in the artificial light at night group (all P<0.05). Histological staining results indicated that the cortical bone structure of the mandibles in the melatonin supplementation group was more organized than that of the artificial light at night group, with higher brain and muscle ARNT-like 1 (BMAL1) expression (P=0.003). RT-qPCR results further showed that the expression levels of Prkaa1, Atp6v0d1, and Cox4i1 in the cortical bone and condylar ossification center significantly increased in the melatonin supplementation group compared to the artificial light at night group (all P<0.05), but still significantly lower than those in the normal light group (all P<0.05). Additionally, the RT-qPCR results further revealed that the expression levels of osteogenic differentiation-related genes Alpl, Ocn, and Runx2 in the cortical bone and condylar ossification center of the melatonin supplementation group were significantly higher than those in the artificial light at night group (all P<0.01). Western blotting analysis indicated that the expression levels of glucose metabolism and osteogenic-related proteins RUNX2, OSX, ATP6V0D1, and COX Ⅳ, along with the phosphorylation levels of AMPKα1/α2, were significantly higher in the melatonin supplementation group compared to the artificial light at night group (all P<0.01). Conclusions: Artificial light at night can inhibit melatonin secretion in mice, reduce glucose metabolism in mandibular tissues, and affect both intramembranous and chondrogenic ossification activities, ultimately leading to inadequate mandibular development.

Chemerin mediates exercise-induced improvements of bone microstructure and bone mass in diabetes or high fat diet mice.

To clarify the roles and mechanisms of adipokine chemerin in exercise-induced bone improvements in type 2 diabetes mellitus (DM) mice and mice fed on high fat diet (HFD). DM mice were established by HFD+streptozotocin injection, exogenous chemerin was supplemented prior to running, and found that exogenous chemerin reversed 6-week exercise-induced improvements in cancellous bone parameters in DM mice. While adipose-specific chemerin knockout improved microstructure and mass of cancellous bone in HFD mice and further increased exercise-induced bone improvements, accompanied with promoted osteogenesis and inhibited osteoclasis represented as the changes of RANKL, M-CSF, Runx2, Osterix, OPG, ALP and CTSK. These results indicated that reduced chemerin contributed to exercise-induced enhancements in the microstructure and mass of cancellous bone in DM and HFD mice in association with osteogenesis promotion and osteoclasis inhibition, which is beneficial to clarify chemerin's impact on bone remodeling in metabolic diseases at sedentary and exercise states.

Epstein-Barr virus Infection Exacerbates Ulcerative Colitis by Driving Macrophage Pyroptosis via the Upregulation of Glycolysis

Abstract Background Epstein-Barr virus (EBV) infection is associated with clinical symptoms, treatment response, need for surgical intervention, and an enhanced likelihood of lymphoma among patients with ulcerative colitis (UC). However, existing studies have primarily concentrated on the epidemiological and clinical associations between EBV and UC, leaving the mechanisms by which EBV exacerbates colitis poorly understood. Methods Clinical specimens of UC patients with EBV infection and a mouse model of dextran sulfate sodium (DSS)-induced colitis with concurrent murine γ-herpesvirus 68 (MHV-68) infection were utilized to investigate the relationship between EBV infection and macrophage pyroptosis. In vivo, adoptive transfer of MHV-68-induced macrophages and macrophage depletion were performed to elucidate the underlying mechanisms. In vitro, myeloid leukemia mononuclear cells of human (THP-1) and macrophages derived from mouse bone marrow (BMDMs) were stimulated with EBV and MHV-68, respectively, to assess macrophage pyroptosis and glycolysis. Results EBV-induced activation of macrophage pyroptosis was positively correlated with clinical disease activity in UC patients. Furthermore, MHV-68 infection activated pyroptosis by upregulating Gasdermin D, NLRP3, interleukin-1β, and interleukin-18 in colonic tissues and peritoneal macrophages of mice with colitis. In vitro, EBV and MHV-68 also mediated activation of pyroptosis in human THP-1 cells and mouse BMDMs, respectively. Additionally, the adoptive transfer of MHV-68-induced BMDMs aggravated murine colitis, whereas macrophage depletion attenuated MHV-68-induced intestinal injury. Mechanistically, MHV-68 promoted macrophage pyroptosis by upregulating glycolysis, while the glycolysis inhibitor, 2-Deoxy-d-glucose, blocked this process in vitro. Conclusion EBV infection exacerbates UC by driving macrophage pyroptosis through upregulation of glycolysis, indicating a potential therapeutic approach to mitigate EBV-induced intestinal inflammation.

Sex dimorphism in the mouse bone marrow niche regulates hematopoietic engraftment via sex-specific Kdm5c/Cxcl12 signaling.

The bone marrow (BM) niche is critical in regulating hematopoiesis, and sexual dimorphism and its underlying mechanism in BM niche and its impact on hematopoiesis are not well understood. We show that male mice exhibited a higher abundance of leptin-receptor-expressing mesenchymal stromal cells (LepR-MSCs) compared to female mice. Sex-mismatched co-culture and BM transplantation showed that the male BM niche provided superior support for in vitro colony formation and in vivo hematopoietic engraftment. The co-transplantation of male stromal cells significantly enhanced engraftment in female recipients. Single-cell RNA sequencing revealed that the lower expression of the X-linked lysine H3K4 demethylase, Kdm5c, in male MSCs led to the increased expression of Cxcl12. In MSC-specific Kdm5c knockout mouse model, the reduction of KDM5C in female MSCs enhanced MSC quantity and function, ultimately improving engraftment to the male level. Kdm5c thus plays a role in driving sexual dimorphism in the BM niche and hematopoietic regeneration. Our study unveils a sex-dependent mechanism governing BM niche regulation and its impact on hematopoietic engraftment. The finding offers potential implications for enhancing BM transplantation efficacy in clinical settings by harnessing the resource of male MSCs or targeting Kdm5c.

Factor XIII-A Transglutaminase Contributes to Neutrophil Extracellular Trap (NET)-mediated Fibrin(ogen) Network Formation and Crosslinking.

Neutrophil extracellular traps can contribute to thrombosis via stabilization of fibrin network, which is normally conducted by plasma transglutaminase, Factor XIII-A as part of coagulation cascade. The possible presence and activity of FXIII-A in neutrophils or during NETosis are unknown. Here, we investigated potential presence of FXIII-A in neutrophils and participation in NET-fibrin(ogen) interaction in vitro. METHODS: Data mining of human and mouse F13A1/F13a1 mRNA expression in whole-body scRNA sequence atlases was conducted. F13a1 mRNA and protein expression was assessed in isolated mouse bone marrow neutrophils. NETosis was induced using 12-phorbol 13-myristate acetate (PMA), and the transglutaminase activity was assessed with 5-(biotinamido)pentylamine incorporation to plasma fibronectin and a fluorescence-fibrin(ogen)-based activity assay using ATTO488-Cadaverine. Externalization of FXIII-A and its interaction with neutrophil extracellular trap (NET) markers, namely, decondensed DNA, CitH3, and MPO, were examined with immunofluorescence microscopy. NET-fibrin(ogen) interaction was investigated with and without serum and/or transglutaminase inhibitor, NC9. Effect of soluble fibrinogen and fibrin(ogen) network on NETosis was also assessed. Data mining of RNAseq atlases showed F13A1/F13a1 expression in adipose tissue, blood, and bone marrow neutrophils. mRNA expression and protein production were confirmed in isolated neutrophils where expression was comparable to that of macrophages and monocytes. FXIII-A was externalized and active as a transglutaminase and colocalized with NET markers during NETosis. FXIII-A transglutaminase activity promoted NET-fibrin(ogen) interaction and entrapment of neutrophils within fibrin(ogen) matrix. Soluble fibrinogen or fibrin(ogen) network did not induce NETosis. This study identifies neutrophils as a source of FXIII-A and suggests its role in stabilizing NET-fibrin(ogen) matrix structures.

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography.

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast.

It has been well validated that chronic psychological stress leads to bone loss, but the underlying mechanism remains unclarified. In this study, we established and analyzed the chronic unpredictable mild stress (CUMS) mice to investigate the miRNA-related pathogenic mechanism involved in psychological stress-induced osteoporosis. Our result found that these CUMS mice exhibited osteoporosis phenotype that is mainly attributed to the abnormal activities of osteoclasts. Subsequently, miRNA sequencing and other analysis showed that miR-335-3p, which is normally highly expressed in the brain, was significantly downregulated in the nucleus ambiguous, serum, and bone of the CUMS mice. Additionally, in vitro studies detected that miR-335-3p is important for osteoclast differentiation, with its direct targeting site in Fos. Further studies demonstrated FOS was upregulated in CUMS osteoclast, and the inhibition of FOS suppressed the accelerated osteoclastic differentiation, as well as the expression of osteoclastic genes, such as Nfatc1, Acp5, and Mmp9, in miR-335-3p-restrained osteoclasts. In conclusion, this work indicated that psychological stress may downregulate the miR-335-3p expression, which resulted in the accumulation of FOS and the upregulation of NFACT1 signaling pathway in osteoclasts, leading to its accelerated differentiation and abnormal activity. These results decipher a previously unrecognized paradigm that miRNA can act as a link between psychological stress and bone metabolism.

Apigenin inhibits NLRP3 inflammasome activation in monocytes and macrophages independently of CD38.

CD38, a regulator of intracellular calcium signalling, is highly expressed in immune cells. Mice lacking CD38 are very susceptible to acute bacterial infections, implicating CD38 in innate immune responses. The effects of CD38 inhibition on NLRP3 inflammasome activation in human primary monocytes and monocyte-derived macrophages have not been investigated. Apigenin is a naturally occurring flavonoid known to inhibit CD38. However, apigenin has also been proposed to inhibit the extracellular ATP receptor P2XR7, an upstream activator of NLRP3. In this study we aimed to investigate whether apigenin attenuates NLRP3 inflammasome activation in human monocytes and monocyte-derived macrophages through CD38 inhibition. LPS-primed human monocytes and monocyte-derived macrophages were treated with apigenin, the CD38 inhibitor 78c, antagonists of CD38 second messengers (8-br-ADPR and 8-br-cADPR) or the ATP hydrolase, apyrase, prior to NLRP3 activation with ATP, monosodium urate crystals (MSU) or nigericin. IL-1β and TNF secretion and mRNA expression, as well as N-terminal gasdermin-D formation were quantified. Ca2+ mobilization was determined by live confocal microscopy. NLRP3 activity was also compared in WT and CD38-/- mouse bone marrow-derived macrophages (BMDMs) with and without CD38 inhibitors. Apigenin significantly inhibited IL-1β release from LPS-primed monocytes and macrophages activated with ATP, MSU, or nigericin. CD38 inhibition with 78c also attenuated NLRP3-dependent IL-1β release. Apigenin was a potent inhibitor of Ca2+ flux from the endoplasmic reticulum to the cytosol in human monocyte-derived macrophages. Apyrase attenuated IL-1β release induced by ATP or MSU, but not by nigericin. However, the NLRP3 inflammasome is not compromised in CD38-/- bone marrow-derived macrophages compared to corresponding WT cells, and apigenin moderated IL-1β release in both genotypes. Our data support that apigenin attenuates NLRP3 activation independently of CD38. Our results also suggest that MSU crystals activate NLRP3 through autocrine or paracrine ATP signalling.

NR2F6 regulates stem cell hematopoiesis and myelopoiesis in mice.

Nuclear receptors regulate hematopoietic stem cells (HSCs) and peripheral immune cells in mice and humans. The nuclear orphan receptor NR2F6 (EAR-2) has been shown to control murine hematopoiesis. Still, detailed analysis of the distinct stem cell, myeloid, and lymphoid progenitors in the bone marrow in a genetic loss of function model remains pending. In this study, we found that adult germline Nr2f6-deficient mice contained increased percentages of total long-term and short-term HSCs, as well as a subpopulation within the lineage-biased multipotent progenitor (MPP3) cells. The loss of NR2F6 thus led to an increase in the percentage of LSK+ cells. Following the differentiation from the common myeloid progenitors (CMP), the granulocyte-monocyte progenitors (GMP) were decreased, while monocyte-dendritic progenitors (MDP) were increased in Nr2f6-deficient bone marrow. Within the pre-conventional dendritic progenitors (pre-cDCs), the subpopulation of pre-cDC2s was reduced in the bone marrow of Nr2f6-deficient mice. We did not observe differences in the development of common lymphoid progenitor populations. Our findings contrast previous studies but underscore the role of NR2F6 in regulating gene expression levels during mouse bone marrow hematopoiesis and myelopoiesis.

FiberO for an automated quantitative analysis of fibers orientation and organization in biological fibrous tissues.

Many biological fibrous tissues exhibit distinctive mechanical properties arising from their highly organized fibrous structure. In disease conditions, alterations in the primary components of these fibers, such as type I collagen molecules in bone, tendons, and ligaments, assembly into a disorganized fibers architecture generating a weak and/or brittle material. Being able to quantitatively assess the fibers orientation and organization in biological tissue may help improve our understanding of their contribution to the tissue and organ mechanical integrity, and assess disease progress and therapy effect. In this work, we present FiberO, a new open-source available software that automatically quantifies fibers orientation, by performing morphological image openings, and fibers organization within the tissue, by determining and plotting their continuity in groups. FiberO performance is here evaluated using second harmonic generation microscopy images of mouse bones and tendons as examples of biological fibrous tissues. FiberO outperformed Directionality and OrientationJ, two open-source plugins available in ImageJ, and FiberFit and CT-FIRE, in the calculation and plotting of fibers orientation in reference images with known fibers orientation. Additionally, FiberO is currently the sole software to date able to accurately track the continuity of aligned fibers, and it quantifies and displays the organized surface(s) in the tissue of interest. FiberO can be used in the wider engineering and science field to investigate the fibers orientation and organization of different natural and synthetic fibrous tissues.

Effect of nanoparticulate CaCO3 on the biological properties of calcium silicate cement

This study aimed to evaluate the effects of nanoparticulate CaCO3 (NPCC) on the biological properties of calcium silicate-based cements (CSCs), including their cytotoxicity, in vitro osteogenic activity, and interactions with rat femur tissue. The average size of NPCC was 90.3±26.0 nm. Cytotoxicity and osteogenic activity assays were performed using mouse bone marrow mesenchymal stem cells (BMSCs). BMSCs exposed to the eluents from CSC alone and CSC containing 2.5% NPCC (CSC-NPCC (2.5%)) for 24 h showed decreased cell viability at an eluent concentration of 75%. In contrast, CSC-NPCCs (5%, 10%, and 20%) did not affect cell viability. Regarding osteogenic activity, CSC-NPCCs (5%, 10%, 20%) enhanced the expression of osteogenic genes, including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), type I collagen (COL-1), and osteocalcin (OCN). Additionally, mineralization in cell cultures was enhanced by CSC-NPCC, indicating that NPCC promoted the osteogenic activity of CSCs. In rat femurs, NPCC accelerates CSC resorption and stimulates bone regeneration at the implantation site. CSC alone occupied 22.2%±3.25% of the total femoral area at the implantation site, whereas CSC-NPCC (20%) occupied only 4%. These histological findings suggest that CSC-NPCC has potential as a biodegradable bone cement for use in bone defect areas that require regeneration.

The protection of nicotinamide riboside against diabetes mellitus-induced bone loss via OXPHOS.

Diabetes mellitus is a global disease that results in various complications, including diabetic osteoporosis. Prior studies have indicated a correlation between low levels of nicotinamide adenine dinucleotide (NAD+) and diabetes-related complications. Nicotinamide riboside (NR), a widely utilized precursor vitamin of NAD+, has been demonstrated to enhance age-related osteoporosis through the Sirt1/FOXO/β-catenin pathway in osteoblast progenitors. However, the impact of NR on bone health in diabetes mellitus remains unclear. In this study, we assessed the potential effects of NR on bone in diabetic mice. NR was administered to high-fat diet (HFD)/streptozotocin (STZ)-induced type 2 diabetic mice (T2DM), and various parameters, including metabolic indicators, bone quality, bone metabolic markers, and RNA sequences, were measured. Our findings confirmed that HFD/STZ-induced T2DM impaired bone microstructures, resulting in bone loss. NR effectively ameliorated insulin resistance, improved bone microarchitecture, and bone quality, reduced bone resorption, enhanced the Forkhead box O (FOXO) signaling pathway, mitigated the nuclear factor kappa B (NF-kB) signaling pathway, and ameliorated the disorder of the oxidative phosphorylation process (OXPHOS) in diabetic mice. In conclusion, NR demonstrated the capacity to alleviate T2DM-induced bone loss through the modulation of OXPHOS in type 2 diabetic mice. Our results underscore the potential of NR as a therapeutic target for addressing T2DM-related bone metabolism and associated diseases. Further cell-based studies under diabetic conditions, such as in vitro cultures of key cell types (e.g., osteoblasts and osteoclasts), are necessary to validate these findings.