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Mesenchymal stem cells (MSCs) have gained tremendous interest due to their overall potent pro‐regenerative and immunomodulatory properties. In recent years, various in vitro and preclinical studies have investigated different priming ("licensing") approaches to enhance MSC functions for specific therapeutic purposes. In this study, we primed bone marrow‐derived human MSCs (hMSCs) with an inflammation cocktail designed to mimic the elevated levels of inflammatory mediators found in serum of patients with severe injuries, such as bone fractures. We observed a significantly enhanced osteogenic differentiation potential of primed hMSCs compared to untreated controls. By RNA‐sequencing analysis, we identified the immediate early response 3 (IER3) gene as one of the top‐regulated genes upon inflammatory priming. Small interfering RNA knockdown experiments established IER3 as a novel positive regulator of osteogenic differentiation. Mechanistic analysis further revealed that IER3 deletion significantly downregulated bone marrow stromal cell antigen 2 (BST2) expression and extracellular signal‐related kinase 1/2 (ERK1/2) phosphorylation in hMSCs, suggesting that IER3 regulates osteogenic differentiation through BST2 and ERK1/2 signaling pathway activation. On the basis of these findings, we propose IER3 as a novel therapeutic target to promote hMSC osteoblastogenesis, which might be of high clinical relevance, for example, in patients with osteoporosis or compromised fracture healing. [ABSTRACT FROM AUTHOR]

Diabetes osteoporosis (DOP) is a chronic metabolic bone disease. This study aimed to identify potential biomarkers of DOP and explore their underlying mechanisms through bioinformatics methods and experimental verification. Bioinformatics methods were used to identify differentially expressed genes (DEGs) for DOP based on GEO data and the GeneCards database. GO and KEGG enrichment analyses were used to search the key pathways. The STRING website was used to construct a protein–protein interaction (PPI) network and identify key genes. Then, 50 mg/mL glucose was used to interveneosteoblasts (OBs).CCK‐8 and Alizarin Red staining were used to investigate the proliferation and differentiation changes in OBs. Flowcytometry was used to investigate apoptosis. The membrane protein chip, WB, and RT‐PCR were used to verify the expression of key targets or pathways about DOP. Forty‐two common genes were screened between DOP‐related targets and DEGs. GO and KEGG enrichment analysis showed that DOP was mainly associated with cytokine‐cytokine receptor interactions, and apoptosis. PPI network analysis showed that TNF, IL1A, IL6, IL1B, IL2RA, Fas ligand (FASLG), and Fas cell surface death receptor (FAS) were key up‐regulated genes in the occurrence of DOP. The experiment results show that 50 mg/mL glucose significantly inhibited OBs proliferation but presented an increase in apoptosis. Membrane protein chip, WB, and RT‐PCR‐verified a significantly active in the expression of TNF/FASLG/FAS pathway. High glucose activated the TNF‐α/FAS/FASLG pathway and induced the inflammatory microenvironment and apoptosis, then impaired osteogenic differentiation of OBs. These may be an important mechanism for the occurrence and development of DOP. [ABSTRACT FROM AUTHOR]

To explore the potential mechanisms which O‐linked‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates osteogenesis, a publicly RNA‐seq dataset was re‐analyzed with literature‐mining and showed the primary targets of O‐GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O‐GlcNAcylation‐regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt‐KO) in MC3T3‐E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria–endoplasmic reticulum (Mito–ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt‐KO cells. Through artificial intelligence (AI)‐predicted cellular structures, the time‐lapse live cells imaging with reactive‐oxygen‐species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt‐KO cells were correlated with the Mito–ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets (Opa1, Gsk3a, Wnt3a, Hif1a, and Hspa9) may be involved in O‐GlcNAcylation‐regulated Mito–ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O‐GlcNAcylation‐regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role. [ABSTRACT FROM AUTHOR]

Osteoporosis (OP) was long viewed as an inevitable process of aging, due to an imbalance between osteoclast bone resorbing and osteoblast bone formation function, leading to a negative balance in bone remodeling. This leads to low bone mass and increased bone fragility putting the patient at risk for fracture. While this view still holds, a better understanding disclosed that OP can occur at any age, as a comorbidity or a complication of many diseases and treatments. Differentiation, maturation, and function of osteoclasts and osteoblasts are affected by many factors from different morbidities: endocrine, metabolic, mechanical and inflammatory. Inflammatory diseases are often complicated by a generalized bone loss that subsequently leads to OP. Factors such as glucocorticoid treatment, immobilization, malnutrition, and insufficient intake of vitamin D play a role. However, the inflammatory process itself is involved and the resulting bone loss is termed immune‐mediated bone loss. Experiments on animals and on humans, in addition to clinical studies, shed light on the role of inflammation in OP. [ABSTRACT FROM AUTHOR]

The neuropeptide kisspeptin and its cognate receptor have been extensively studied in reproductive physiology, with diverse and well‐established functions, including as an upstream regulator of pubertal onset, reproductive hormone secretion, and sexual behavior. Besides classical reproduction, both kisspeptin and its receptor are extensively expressed in bone‐resorbing osteoclasts and bone‐forming osteoblasts, which putatively permits direct bone effects. Accordingly, this sets the scene for recent compelling findings derived from in vitro experiments through to in vivo and clinical studies revealing prominent regulatory interactions for kisspeptin signaling in bone metabolism, as well as certain oncological aspects of bone metabolism. Herein, we comprehensively examine the experimental evidence obtained to date supporting the interaction between kisspeptin and bone. A comprehensive understanding of this emerging facet of kisspeptin biology is fundamental to exploiting the future therapeutic potential of kisspeptin‐based medicines as a novel strategy for treating bone‐related disorders. [ABSTRACT FROM AUTHOR]

Recent evidence suggests an association between age‐related osteoporosis and cellular senescence in the bone; however, the specific bone cells that play a critical role in age‐related osteoporosis and the mechanism remain unknown. Results revealed that age‐related osteoporosis is characterized by the loss of osteoblast Men1. Osteoblast‐specific inducible knockout of Men1 caused structural changes in the mice bones, matching the phenotypes in patients with age‐related osteoporosis. Histomorphometrically, Men1‐knockout mice femurs decreased osteoblastic activity and increased osteoclastic activity, hallmarks of age‐related osteoporosis. Loss of Men1 induces cellular senescence via mTORC1 activation and AMPK suppression, rescued by metformin treatment. In bone morphogenetic protein‐indued bone model, loss of Men1 leads to accumulation of senescent cells and osteoporotic bone formation, which are ameliorated by metformin. Our results indicate that cellular senescence in osteoblasts plays a critical role in age‐related osteoporosis and that osteoblast‐specific inducible Men1‐knockout mice offer a promising model for developing therapeutics for age‐related osteoporosis. [ABSTRACT FROM AUTHOR]

Objectives: To explore the role of fibrocytes in the recurrence and calcification of fibrous epulides. Methods: Different subtypes of fibrous epulides and normal gingival tissue specimens were first collected for histological and immunofluorescence analyses to see if fibrocytes were present and whether they differentiated into myofibroblasts and osteoblasts upon stimulated by transforming growth factor‐β1 (TGF‐β1). Electron microscopy and elemental analysis were used to characterize the extracellular microenvironment in different subtypes of fibrous epulides. Human peripheral blood mononuclear cells (PBMCs) were subsequently isolated from in vitro models to mimic the microenvironment in fibrous epulides to identify whether TGF‐β1 as well as the calcium and phosphorus ion concentration in the extracellular matrix (ECM) of a fibrous epulis trigger fibrocyte differentiation. Results: Fibrous epulides contain fibrocytes that accumulate in the local inflammatory environment and have the ability to differentiate into myofibroblasts or osteoblasts. TGF‐β1 promotes fibrocytes differentiation into myofibroblasts in a concentration‐dependent manner, while TGF‐β1 stimulates the fibrocytes to differentiate into osteoblasts when combined with a high calcium and phosphorus environment. Conclusions: Our study revealed fibrocytes play an important role in the fibrogenesis and osteogenesis in fibrous epulis, and might serve as a therapeutic target for the inhibition of recurrence of fibrous epulides. [ABSTRACT FROM AUTHOR]

Modulating macrophage phenotype based on implant surface characteristics, including topography and chemistry, has been employed to enhance osseointegration and long‐term functional outcomes for titanium (Ti)‐based implants. An excessive and/or prolonged M1 macrophage response can lead to damaging immune‐inflammatory reactions, negatively influencing the fate of the implant, and hence, modulating these responses via nanoscale implant surface modification is an emerging paradigm. Herein, an anodized titanium implant surface based on single‐step electrochemical anodization, with preserved underlying microfeatures and superimposed nanopores (50 and 70 nm), compared with irregular rough and microrough (machined‐like) surfaces, is investigated for its effect on the functions of primary macrophages in vitro. Significantly reduced macrophage proliferation and increased tissue‐reparative M2 phenotype polarization are confirmed for the nanopores, which are more pronounced for 70 nm diameter. Moreover, osteoclastogenesis is reduced while osteogenic differentiation of osteoblasts is enhanced for the nanopores (higher for 70 nm pores). Advanced nanoengineered Ti implants can enhance titanium implant tissue integration by modulating the inflammatory response at the implant–cell interface. [ABSTRACT FROM AUTHOR]

Critically‐sized segmental bone defects represent significant challenges requiring grafts for reconstruction. 3D‐printed synthetic bone grafts are viable alternatives to structural allografts if engineered to provide appropriate mechanical performance and osteoblast/osteoclast cell responses. Novel 3D‐printable nanocomposites containing acrylated epoxidized soybean oil (AESO) or methacrylated AESO (mAESO), polyethylene glycol diacrylate, and nanohydroxyapatite (nHA) were produced using masked stereolithography. The effects of volume fraction of nHA and methacrylation of AESO on interactions of differentiated MC3T3‐E1 osteoblast (dMC3T3‐OB) and differentiated RAW264.7 osteoclast cells with 3D‐printed nanocomposites were evaluated in vitro and compared with a control biomaterial, hydroxyapatite (HA). Higher nHA content and methacrylation significantly improved the mechanical properties. All nanocomposites supported dMC3T3‐OB cells' adhesion and proliferation. Higher amounts of nHA enhanced cell adhesion and proliferation. mAESO in the nanocomposites resulted in greater adhesion, proliferation, and activity at day 7 compared with AESO nanocomposites. Excellent osteoclast‐like cells survival, defined actin rings, and large multinucleated cells were only observed on the high nHA fraction (30%) mAESO nanocomposite and the HA control. Thus, mAESO‐based nanocomposites containing higher amounts of nHA have better interactions with osteoblast‐like and osteoclast‐like cells, comparable with HA controls, making them a potential future alternative graft material for bone defect repair. [ABSTRACT FROM AUTHOR]

Runt‐related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2‐deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown‐based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme‐like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)‐mediated proteasome degradation. Wild‐type Hakai but not Hakai‐C109A inhibited Smurf2 protein levels through proteasome‐mediated degradation. These findings underscore Hakai's functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2‐mediated ubiquitin‐proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis. [ABSTRACT FROM AUTHOR]

Objectives: To explore the effect of protein arginine methyltransferase 5 (PRMT5) on tooth extraction sockets healing, we established an extraction sockets model in osteoblast‐conditional Prmt5 knockout mice. The results provided clues for promoting extraction sockets healing in clinical settings. Materials and Methods: Maxillary first molars were extracted from 6 to 8‐week‐old mice to establish an extraction fossa model. Microcomputed tomography (Micro‐CT), histology, and immunostaining assays were performed on samples harvested at 3‐, 7‐, and 14‐day post‐extraction. Prmt5‐silenced cell lines were employed to explore the regulatory mechanisms underlying the osteigenic differentiation. Results: PRMT5 expression was higher in the early stage of socket healing. Micro‐CT analysis showed that the percentage of new bone in the extraction sockets was lower in OC‐Cre; Prmt5fl/fl mice than in the control group, consistent with Masson staining. We found that, Prmt5 deficiency delayed the osteogenesis during extraction socket healing, which might be achieved through the decrease of H4R3me2s in the Sp7 promoter region. Conclusion: PRMT5 in osteoblasts may promote the differentiation of osteoblasts by regulating the Sp7 promoter H4R3me2s and participate in the healing of tooth extraction sockets. [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]

Phenotypic assays detect small‐molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule‐target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4‐g]quinoline (PQ) pseudo‐natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA‐box binding protein‐associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains. [ABSTRACT FROM AUTHOR]

Aims: Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor‐kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3‐E1 osteoblast‐like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.‐LPS) and mechanical stress. Methods: To investigate the effect of P.g.‐LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3‐E1 cells with P.g.‐LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. Results: The RANKL/OPG ratio significantly increased in MC3T3‐E1 cells cultured in a medium containing P.g.‐LPS and undergoing mechanical stress compared to cells treated with P.g.‐LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.‐LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.‐LPS and mechanical stress. MC3T3‐E1 cells treated with P.g.‐LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast‐like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. Conclusion: These findings suggest that P.g.‐LPS and Piezo1‐mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts. [ABSTRACT FROM AUTHOR]

The regenerative ability of limb bones after injury decreases during aging, but whether a similar phenomenon occurs in jawbones and whether autophagy plays a role in this process remain unclear. Through retrospective analysis of clinical data and studies on a mouse model of jawbone defects, we confirmed the presence of delayed or impaired bone regeneration in the jawbones of old individuals and mice. Subsequently, osteoblasts (OBs) derived from mouse jawbones were isolated, showing reduced osteogenesis in senescent osteoblasts (S‐OBs). We observed a reduction in autophagy within both aged jawbones and S‐OBs. Additionally, pharmacological inhibition of autophagy in normal OBs (N‐OBs) led to cell aging and decreased osteogenesis, while autophagic activation reversed the aging phenotype of S‐OBs. The activator rapamycin (RAPA) increased the autophagy level and bone regeneration in aged jawbones. Finally, we found that fatty acid‐binding protein 3 (FABP3) was degraded by autolysosomes through its interaction with sequestosome 1 (P62/SQSTM1). Autophagy inhibition within senescent jawbones and S‐OBs led to the excessive accumulation of FABP3, and FABP3 knockdown partially rescued the decreased osteogenesis in S‐OBs and alleviated age‐related compromised jawbone regeneration. In summary, we confirmed that autophagy inhibition plays an important role in delaying bone regeneration in aging jawbones. Autophagic activation or FABP3 knockdown can partially rescue the osteogenesis of S‐OBs and the regeneration of aging jawbones, providing insight into jawbone aging. [ABSTRACT FROM AUTHOR]

Bioinspired titanium implant nanotexturing achieved via advanced techniques such as lithography, hydrothermal and laser patterning enables modulation of cell functions toward enhanced bioactivity and antibacterial efficacy. However, such costly, multi‐step methods limit clinical translation and benchtop implant modification. For the first time, single‐step, cost‐effective, and translatable electrochemical anodization is reported to fabricate bioinspired nanopillar‐like textures on micro‐rough titanium. In‐depth surface characterization confirms the formation of novel nanostructures, namely nanoscale Spinules, Daggers, Papillae, Spikes, and Flames, exhibiting varied roughness and wettability. Next, in separate experiments, primary human osteoblasts and polymicrobial salivary biofilm are cultured on the implant substrates. Nanotextured surfaces show high protein adhesion and maintain the proliferation, adhesion, and spreading of osteoblasts. Interestingly, all nanotextures exhibit superior antibiofilm abilities compared to control surfaces. Bioactive and antibacterial implant surface nano‐texturing achieved via single‐step anodization has the potential for clinical translation as the next generation of orthopedic and dental implant surface modification. [ABSTRACT FROM AUTHOR]

Objectives: Periodontal ligament stem cells (PDLSCs) are essential for the treatment of bone diseases because of its great potential to differentiate into osteoblasts. Remarkably, increasing long‐non‐coding RNAs (lncRNAs) have been reported to be involved in the osteogenic differentiation of PDLSCs. Maternally expressed 8, small nucleolar RNA host gene (MEG8) is implicated in multiple diseases. This study intended to unearth the potential role of MEG8 and unveil the mechanism in PDLSCs undergoing osteoblastic differentiation. Materials and Methods: MEG8 expression was measured by quantitative real‐time PCR (RT‐qPCR) during osteogenic differentiation of PDLSCs into bone cells. Functional assays were used to uncover the biological function of MEG8. Besides, RNA pulldown, RNA‐binding protein immunoprecipitation (RIP), and luciferase reporter assays were used to explore the molecular mechanism of MEG8. Results: MEG8 was apparently overexpressed in osteogenically differentiated PDLSCs. Moreover, MEG8 deficiency suppressed the osteoblastic differentiation of PDLSCs. Furthermore, MEG8 modulated the expression of transcription factor 4 (TCF4) by scavenging microRNA‐495‐3p (miR‐495‐3p) and microRNA‐485‐3p (miR‐485‐3p) through the competing endogenous RNA (ceRNA) mechanism, further stimulating the Wnt/β‐catenin pathway. Conclusion: MEG8 stimulates the capacity of PDLSCs for osteogenic differentiation through a ceRNA mode. [ABSTRACT FROM AUTHOR]

Infected bone defects (IBDs) are the common condition in the clinical practice of orthopaedics. Although surgery and anti‐infective medicine are the firstly chosen treatments, in many cases, patients experience a prolonged bone union process after anti‐infective treatment. Epimedium‐Curculigo herb pair (ECP) has been proved to be effective for bone repair. However, the mechanisms of ECP in IBDs are insufficiency. In this study, Effect of ECP in IBDs was verified by micro‐CT and histological examination. Qualitative and quantitative analysis of the main components in ECP containing medicated serum (ECP‐CS) were performed. The network pharmacological approaches were then applied to predict potential pathways for ECP associated with bone repair. In addition, the mechanism of ECP regulating LncRNA MALAT1/miRNA‐34a‐5p/SMAD2 signalling axis was evaluated by molecular biology experiments. In vivo experiments indicated that ECP could significantly promote bone repair. The results of the chemical components analysis and the pathway identification revealed that TGF‐β signalling pathway was related to ECP. The results of in vitro experiments indicated that ECP‐CS could reverse the damage caused by LPS through inhibiting the expressions of LncRNA MALAT1 and SMAD2, and improving the expressions of miR‐34a‐5p, ALP, RUNX2 and Collagen type І in osteoblasts significantly. This research showed that ECP could regulate the TGF‐β/SMADs signalling pathway to promote bone repair. Meanwhile, ECP could alleviate LPS‐induced bone loss by modulating the signalling axis of LncRNA MALAT1/miRNA‐34a‐5p/ SMAD2 in IBDs. [ABSTRACT FROM AUTHOR]

Objectives Materials and Methods Results Conclusions The purpose of this study was to determine whether indoxyl sulfate (IS) is involved in alveolar bone deterioration and to elucidate the mechanism underlying alveolar bone loss in chronic kidney disease (CKD) patients.Mice were divided into the control group, CP group (ligature‐induced periodontitis), CKD group (5/6 nephrectomy), and CKD + CP group. The concentration of IS in the gingival crevicular fluid (GCF) was determined by HPLC. The bone microarchitecture was evaluated by micro‐CT. MC3T3‐E1 cells were stimulated with IS, and changes in mitochondrial morphology and ferroptosis‐related factors were detected. RT‐PCR, western blotting, alkaline phosphatase activity assays, and alizarin red S staining were utilized to assess how IS affects osteogenic differentiation.Compared with that in the other groups, alveolar bone destruction in the CKD + CP group was more severe. IS accumulated in the GCF of mice with CKD. IS activated the aryl hydrocarbon receptor (AhR) in vitro, inhibited MC3T3‐E1 cell osteogenic differentiation, caused changes in mitochondrial morphology, and activated the SLC7A11/GPX4 signaling pathway. An AhR inhibitor attenuated the aforementioned changes induced by IS.IS activated the AhR/SLC7A11/GPX4 signaling pathway, inhibited osteogenesis in MC3T3‐E1 cells, and participated in alveolar bone resorption in CKD model mice through ferroptosis. [ABSTRACT FROM AUTHOR]

Hypercholesterolaemia is a systemic metabolic disease, but the role of organs other than liver in cholesterol metabolism is unappreciated. The phenotypic characterization of the Tsc1Dmp1 mice reveal that genetic depletion of tuberous sclerosis complex 1 (TSC1) in osteocytes/osteoblasts (Dmp1‐Cre) triggers progressive increase in serum cholesterol level. The resulting cholesterol metabolic dysregulation is shown to be associated with upregulation and elevation of serum amyloid A3 (SAA3), a lipid metabolism related factor, in the bone and serum respectively. SAA3, elicited from the bone, bound to toll‐like receptor 4 (TLR4) on hepatocytes to phosphorylate c‐Jun, and caused impeded conversion of cholesterol to bile acids via suppression on cholesterol 7 α‐hydroxylase (Cyp7a1) expression. Ablation of Saa3 in Tsc1Dmp1 mice prevented the CYP7A1 reduction in liver and cholesterol elevation in serum. These results expand the understanding of bone function and hepatic regulation of cholesterol metabolism and uncover a potential therapeutic use of pharmacological modulation of SAA3 in hypercholesterolaemia. [ABSTRACT FROM AUTHOR]

Evidence is accumulating that osteal macrophages, in addition to bone‐resorbing osteoclasts and bone‐forming osteoblasts, participate vitally in bone remodeling process. Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin‐6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodeling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We have reported that bone morphogenetic protein‐4 (BMP‐4) stimulates OPG synthesis in MC3T3‐E1 osteoblast‐like cells, and that SMAD1/5/8(9), p38 mitogen‐activated protein kinase (MAPK), and p70 S6 kinase are involved in the OPG synthesis. The present study aims to investigate the effect of OSM on the synthesis of OPG stimulated by BMP‐4 in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP‐4 in MC3T3‐E1 cells. Neither the BMP‐4‐induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK was affected by OSM. On the other hand, the phosphorylation of p70 S6 kinase stimulated by BMP‐4 was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP‐4‐stimulated OPG synthesis via inhibition of the p70 S6 kinase‐mediated pathway in osteoblast‐like cells. Significance Statement: Oncostatin M (OSM), a cytokine of the interleukin‐6 superfamily, modulates bone remodeling. Osteoprotegerin (OPG) secreted from osteoblasts regulates osteoclastogenesis. We previously showed that bone morphogenetic protein‐4 (BMP‐4) activates SMAD1/5/8(9), p38 mitogen‐activated protein kinase, and p70 S6 kinase, resulting in OPG synthesis in osteoblast‐like MC3T3‐E1 cells. In the present study, we investigated the effects of OSM on the BMP‐4‐induced synthesis of OPG in these cells. Our results strongly suggest that OSM suppresses OPG synthesis by inhibiting the p70 S6 kinase‐mediated pathway in osteoblasts. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to investigate the effect of Stat3 on the osteoblast‐mediated bone healing in the inflammatory lesion. Methods: The conditional knockout of Stat3 in osteoblasts (Stat3 CKO) was generated via the Cre‐loxP recombination system using Osterix‐Cre transgenic mice. The calvarial bone inflammatory lesions were established on both Stat3 CKO and wild‐type mice, then harvested to assess the bone healing. In response to lipopolysaccharide (LPS) stimulation, osteoblasts from Stat3 CKO and wild‐type mice were subjected to examine the formation of calcium deposits, the expression of osteogenic markers (i.e., Runx2, OPN, COL1A1), and osteoclast‐related markers (i.e., RANKL, OPG). The EdU and transwell assays were performed to assess the proliferation and migration of the cells. Results: A decrease in bone mass and an increase in osteolysis were found in the inflammatory lesions on Stat3 CKO mice when compared with the control. More osteoclastic‐like cells and an increased expression of RANKL were observed in Stat3 CKO mice. Both mRNA and protein expressions of Stat3 and osteogenic markers in the lesions were significantly decreased in Stat3 CKO mice. After co‐cultured with osteogenic medium, the Stat3‐deficient osteoblasts were found with a significant decrease in calcium deposits and the expression of osteogenic markers, and with a significant increased expression of RANKL. The impaired ossification of Stat3‐deficient osteoblasts was even more pronounced with the presence of lipopolysaccharides in vitro. The most decrease in cell proliferation and migration was found in Stat3‐deficient osteoblasts in response to LPS. Conclusions: Loss of Stat3 in osteoblasts impaired bone healing in an inflammatory microenvironment. [ABSTRACT FROM AUTHOR]

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/β‐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. [ABSTRACT FROM AUTHOR]

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform‐3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV‐mediated mineralization process and its impact on bone formation. Using differentiated MC3T3‐E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3−/−) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, μCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3−/− mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast‐mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization‐related disorders. [ABSTRACT FROM AUTHOR]

A few ubiquitin ligases have been shown to target Runx2, the key osteogenic transcription factor and thereby regulate bone formation. The regulation of Runx2 expression and function are controlled both at the transcriptional and posttranslational levels. Really interesting new gene (RING) finger ubiquitin ligases of which RNF138 is a member are important players in the ubiquitin‐proteasome system, contributing to the regulation of protein turnover and cellular processes. Here, we demonstrated that RNF138 negatively correlated with Runx2 protein levels in osteopenic ovariectomized rats which implied its role in bone loss. Accordingly, RNF138 overexpression potently inhibited osteoblast differentiation of mesenchyme‐like C3H10T1/2 as well primary rat calvarial osteoblast (RCO) cells in vitro, whereas overexpression of catalytically inactive mutant RNF138Δ18‐58 (lacks RING finger domain) had mild to no effect. Contrarily, RNF138 depletion copiously enhanced endogenous Runx2 levels and augmented osteogenic differentiation of C3H10T1/2 as well as RCOs. Mechanistically, RNF138 physically associates within multiple regions of Runx2 and ubiquitinates it leading to its reduced protein stability in a proteasome‐dependent manner. Moreover, catalytically active RNF138 destabilized Runx2 which resulted in inhibition of its transactivation potential and physiological function of promoting osteoblast differentiation leading to bone loss. These findings underscore the functional involvement of RNF138 in bone formation which is primarily achieved through its modulation of Runx2 by stimulating ubiquitin‐mediated proteasomal degradation. Thus, our findings indicate that RNF138 could be a promising novel target for therapeutic intervention in postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

High fat diets overwhelm the physiological mechanisms for absorption, storage, and utilization of triglycerides (TG); consequently TG, TG‐rich lipoproteins (TGRL), and TGRL remnants accumulate, circulate systemically, producing dyslipidemia. This associates with, or is causative for increased atherosclerotic cardiovascular risk, ischemic stroke, fatty liver disease, and pancreatitis. TGRL hydrolysis by endothelial surface‐bound lipoprotein lipase (LPL) generates metabolites like free fatty acids which have proinflammatory properties. While osteoblasts utilize fatty acids as an energy source, dyslipidemia is associated with negative effects on the skeleton. In this study we investigated the effects of TGRL lipolysis products (TGRL‐LP) on expression of a stress responsive transcription factor, termed activating transcription factor 3 (ATF3), reactive oxygen species (ROS), ATF3 target genes, and angiopoietin‐like 4 (Angptl4) in osteoblasts. As ATF3 negatively associates with osteoblast differentiation, we also investigated the skeletal effects of global ATF3 deletion in mice. TGRL‐LP increased expression of Atf3, proinflammatory proteins Ptgs2 and IL‐6, and induced ROS in MC3T3‐E1 osteoblastic cells. Angptl4 is an endogenous inhibitor of LPL which was transcriptionally induced by TGRL‐LP, while recombinant Angptl4 prevented TG‐driven Atf3 induction. Atf3 global knockout male mice demonstrated increased trabecular and cortical microarchitectural parameters. In summary, we find that TGRL‐LP induce osteoblastic cell stress as evidenced by expression of ATF3, which may contribute to the negative impact of dyslipidemia in the skeleton. Further, concomitant induction of Angptl4 in osteoblasts might play a protective role by reducing local lipolysis. [ABSTRACT FROM AUTHOR]

Objectives: The objectives of the study was to discuss the effect and mechanism of fluctuant glucose (FG) on implant osseointegration in type 2 diabetic mellitus (T2DM). Materials and Methods: Rats were divided into control, T2DM and FG group, and the implants were inserted into their femurs. Micro‐CT and histological analysis were used to evaluate the effect on osseointegration in vivo. And we investigated the effect of different conditions (normal, control, high glucose, and FG medium) on rat osteoblast in vitro. Then transmission electron microscope (TEM) and Western blot were used to evaluate the endoplasmic reticulum stress (ERS) response. Finally, 4‐PBA, an inhibitor of ERS, was added into different conditions to observe the functions of osteoblast. Results: In vivo, Micro‐CT and histological analysis showed that the percentage of osseointegration in FG rats were lower than other two group. In vitro, the results demonstrated that the adhesion of the cells becomes worst, and osteogenic ability was also severely impaired in FG group. In addition, FG could induce more serious ERS and 4‐PBA could improve the dysfunction of osteoblasts induced by FG. Conclusion: Fluctuant glucose could restrain the implant osseointegration in T2DM, and the effect was more obvious than consistent high glucose by a possible mechanism of activation ERS pathway. [ABSTRACT FROM AUTHOR]

Objective: Cell pyroptosis is implicated in progressive bone loss in dental inflammatory diseases. We induced caspase‐3/Gasdermin E (GSDME)‐mediated pyroptosis in osteoblast‐like cells and evaluated the effects on osteogenesis. Materials and Methods: Osteoblast‐like cells were treated with various concentrations of sodium butyrate (NaB) to identify the most appropriate for inducing caspase‐3/GSDME‐mediated pyroptosis. Cells were divided into control, NaB and NaB+Ac‐DEVD‐CHO (specific caspase‐3 inhibitor) groups. Pyroptosis level was evaluated by immunofluorescence, morphological observation, flow cytometry, lactate dehydrogenase (LDH) release assays, mRNA and protein levels of pyroptosis‐related markers. Then, inflammation level, osteoprotegerin (OPG) and receptor activator of nuclear factor‐κB ligand (RANKL) expression and osteogenic function were detected. Results: Treatment with 10 mM NaB increased caspase‐3 expression, GSDME cleavage, LDH release and the number of pyroptotic cells, with morphologic changes, indicating GSDME‐mediated pyroptosis induction. The pyroptosis‐related changes were abolished by caspase‐3 inhibition. Caspase‐3/GSDME‐mediated pyroptosis triggered the expression of inflammatory cytokines and RANKL, downregulated alkaline phosphatase (ALP) activity, mineralisation level, mRNA and protein levels of multiple osteogenic markers. These effects were partly reversed by Ac‐DEVD‐CHO. Conclusion: Caspase‐3/GSDME‐mediated pyroptosis induced by NaB activated the inflammatory response, reduced osteogenic differentiation and disturbed OPG/RANKL axis, leading to osteogenic dysfunction in osteoblast‐like cells. [ABSTRACT FROM AUTHOR]

Objectives: We aimed to investigate bone metastasis induced by Notch signalling pathway dysregulation and to demonstrate that SPARC is a potential therapeutic target in adenoid cystic carcinoma (AdCC) with Notch dysregulation. Materials and Methods: This retrospective study enrolled 144 AdCC patients. RNA‐sequencing and enrichment analyses were performed using 32 AdCC samples. Osteonectin/SPARC and the Notch activation indicator Notch intracellular domain (NICD) were detected using immunohistochemistry. Cell proliferation and migration assays were conducted using stably NICD over‐expressing cells. The effect of SPARC on osteoclast differentiation in NICD cells was investigated using western blotting, quantitative reverse transcription PCR, tartrate‐resistant acid phosphatase staining and resorption assays. Results: RNA‐sequencing analysis showed that genes down‐regulated in Notch‐mutant AdCCs, such as SPARC, were enriched in ossification and osteoblast differentiation. Most (75/110, 68.2%) Notch1‐wild‐type AdCCs showed SPARC over‐expression, whereas 30 out of 34 (88.2%) Notch1‐mutant tumours showed low SPARC expression. SPARC over‐expression was then found negatively to be correlated with NICD expression in 144 AdCCs. NICD over‐expression promoted cell growth, migration and osteoclast differentiation, which could be partly reversed by exogenous SPARC. Conclusions: Notch activation in AdCC contributes to bone metastasis through SPARC inhibition. The study results suggest that SPARC may represent a prognostic biomarker and potential therapeutic target. [ABSTRACT FROM AUTHOR]

Fracture of the alveolar bone resorption is a common complication in orthodontic treatment, which mainly caused by extreme mechanical loading. However, the ferroptosis with orthodontic tooth movement(OTM) relationship has not been thoroughly described. We here analysed whether ferroptosis is involved in OTM‐associated alveolar bone loss. Mouse osteoblasts (MC‐3T3) and knockdown glutathione peroxidase 4 (GPX4) MC‐3T3 were stimulated with compressive force loading and ferrostatin‐1 (Fer‐1, a ferroptosis inhibitor), and the changes in lipid peroxidation morphology, expression of ferroptosis‐related factors and osteogenesis levels were detected. After establishing the rat experimental OTM model, the changes in ferroptosis‐related factors and osteogenesis levels were reevaluated in the same manner. Ferroptosis was involved in mechanical stress regulating osteoblast remodelling, and Fer‐1 and erastin affected osteoblasts under compression force loading. Fer‐1 regulated ferroptosis and autophagy in MC‐3T3 and promoted bone proliferation. GPX4‐dependent ferroptosis stimulated the YAP (homologous oncoproteins Yes‐associated protein) pathway, and GPX4 promoted ferroptosis via the YAP‐TEAD (transcriptional enhanced associate domain) signal pathway under mechanical compression force. The in vivo experiment results were consistent with the in vitro experiment results. Ferroptosis transpires during the motion of orthodontic teeth, with compression force side occurring earlier than stretch side within 4 h. GPX4 plays an important role in alveolar bone loss, while Fer‐1 can inhibit the compression force‐side alveolar bone loss. GPX4's Hippo‐YAP pathway is activated by the lack of compression force in the lateral alveolar bone. [ABSTRACT FROM AUTHOR]

The pathophysiology of osteoporosis is significantly influenced by the impaired functioning of osteoblasts, which is particularly caused by oxidative stress. Nevertheless, the underlying mechanisms responsible for this phenomenon are still not well understood. The objective of this study was to elucidate the impact of fibroblast growth factor 7 (FGF7) on the behavior of osteoblasts under conditions of oxidative stress. The osteoblast-like MC3T3 cells were pretreated with recombinant FGF7 in the presence of oxidative stress induced by hydrogen peroxide (H2O2). We first provided the evidence that the endogenous FGF7 was significantly increased in osteoblasts in response to the increased H2O2 levels. Recombined FGF7 demonstrated a remarkable capacity to resist the detrimental effects of H2O2-induced oxidative stress, including the increase in cell apoptosis, decrease in osteoblast viability, and impairment in osteogenic differentiation capacity, on osteoblasts. Furthermore, we extensively explored the mechanism underlying these protective effects and discovered a remarkable modulation of reactive oxygen species (ROS) homeostasis in H2O2-treated cells following the pronounced expression of FGF7, which significantly differed from the control group. Additionally, we observed that FGF7 exerted partial preservation on both the morphology and function of mitochondria when exposed to oxidative stress conditions. Furthermore, FGF7 exhibited the ability to enhance the activation of the p38/MAPK signaling pathway while concurrently suppressing the JNK/ MAPK signaling pathway in response to oxidative stress. These results underscore the promising role and underlying mechanisms of FGF7 in preserving osteoblast homeostasis in the face of oxidative stress. [ABSTRACT FROM AUTHOR]

Endosteal stem cells are a subclass of bone marrow skeletal stem cell populations that are particularly important for rapid bone formation occurring in growth and regeneration. These stem cells are strategically located near the bone surface in a specialized microenvironment of the endosteal niche. These stem cells are abundant in young stages but eventually depleted and replaced by other stem cell types residing in a non‐endosteal perisinusoidal niche. Single‐cell molecular profiling and in vivo cell lineage analyses play key roles in discovering endosteal stem cells. Importantly, endosteal stem cells can transform into bone tumor‐making cells when deleterious mutations occur in tumor suppressor genes. The emerging hypothesis is that osteoblast‐chondrocyte transitional identities confer a special subset of endosteal stromal cells with stem cell‐like properties, which may make them susceptible for tumorigenic transformation. Endosteal stem cells are likely to represent an important therapeutic target of bone diseases caused by aberrant bone formation. [ABSTRACT FROM AUTHOR]

It has been known that periodontal ligament‐associated protein‐1 (PLAP‐1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP‐1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP‐1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP‐1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP‐1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF‐β1)/Smad3 in inflammatory environments. We further found the KO of PLAP‐1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF‐β1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP‐1/Asporin KO mice was promoted through TGF‐β1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP‐1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP‐1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF‐β1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis. [ABSTRACT FROM AUTHOR]

Objective: Periodontitis is characterized by alveolar bone injury and absorption, with high incidence and poor treatment effect. Proliferation, migration, differentiation and apoptosis of osteoblasts are identified as key factors during the regeneration of alveolar bone tissue processes. Periodontal ligament stem cells (PDLSCs) have been proved to be a possible candidate for the treatment of periodontitis due to its multiple advantages, such as increasing the regenerative capacity of bone tissue. However, the effect of exosomes derived from PDLSCs (PDLSC‐Exo) on osteoblasts remains to be further studied. Methods and Materials: In this work, cell proliferation, migration, osteogenic differentiation, and H2O2‐induced apoptosis were detected after cells were exposed to PDLSC‐Exo by CCK‐8, scratch wound assay, alizarin red S and alkaline phosphatase staining, real‐time PCR, flow cytometry, tunel assay, and so on. Moreover, the activation of PI3K/AKT and MEK/ERK signaling pathways was evaluated by western blotting. Results: We found that PDLSC‐Exo are capable of promoting hFOB1.19 cell proliferation, migration and osteogenic differentiation, inhibiting H2O2‐induced apoptosis, and activating the PI3K/AKT and MEK/ERK signaling pathways. Conclusion: These results suggest that PDLSC‐Exo may be a promising therapeutic for osteoblastic damage. [ABSTRACT FROM AUTHOR]

The toxic effects of nanoparticles‐silver oxide (Ag2O) limited its use. However, loading Ag2O nanoparticles into titanium dioxide (TiO2) nanotubes (Ag2O‐TiO2‐NTs) has more efficient biological activity and safety. The aim of this study was to observe the effect of Ag2O‐TiO2‐NTs on osteogenic differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) and its mechanism. The enzyme activity of lactate dehydrogenase (LDH) and the expression of RUNX family transcription factor 2 (Runx2), OPN, OCN in BMSCs were detected by quantitative real time polymerase chain reaction. At 14 days of induction, the mineralization ability and alkaline phosphatase (ALP) activity of cells in each group were observed by Alizarin Red S staining and ALP staining. In addition, the protein levels of tumor necrosis factor‐α (TNF‐α) and β‐catenin in BMSCs of each group were observed by western blot. After 14 days of the induction, the mineralization ability and ALP activity of BMSCs in the Ag2O‐TiO2‐NTs group were significantly enhanced compared with those in the Ag2O and TiO2 groups. Western blot analysis showed that the BMSCs in the Ag2O‐TiO2‐NTs group exhibited much lower protein level of TNF‐α and higher protein level of β‐catenin than those in the Ag2O and TiO2 groups.Ag2O‐TiO2‐NTs enhance the osteogenic activity of BMSCs by modulating TNF‐α/β‐catenin signaling. [ABSTRACT FROM AUTHOR]

Small extracellular vesicles (sEVs) released by acute myeloid leukaemia (AML) cells have been reported to influence the trilineage differentiation of bone marrow‐derived mesenchymal stem cells (BM‐MSCs). However, it remains elusive which biological cargo from AML‐sEVs is responsible for this effect. In this study, sEVs were isolated from cell‐conditioned media and blood plasma using size‐exclusion chromatography and ultrafiltration and characterized according to MISEV2018 guidelines. Our results demonstrated that AML‐sEVs increased the proliferation of BM‐MSCs. Conversely, key proteins that are important for normal haematopoiesis were downregulated in BM‐MSCs. Additionally, we revealed that AML‐sEVs significantly reduced the differentiation of BM‐MSCs to osteoblasts without affecting adipogenic or chondrogenic differentiation. Next, LC‐MS/MS proteomics elucidated that various proteins, including Y‐box‐binding protein 1 (YBX1), were upregulated in both AML‐sEVs and BM‐MSCs treated with AML‐sEVs. Clinically relevant, we found that YBX1 is considerably upregulated in most paediatric AML patient‐derived sEVs compared to healthy controls. Interestingly, sEVs isolated after the downregulation of YBX1 in AML cells remarkably rescued the osteoblastic differentiation of BM‐MSCs. Altogether, our data demonstrate for the first time that YBX1 containing AML‐sEVs is one of the key players that disrupt the normal function of bone marrow microenvironment by reducing the osteogenic differentiation of BM‐MSCs. [ABSTRACT FROM AUTHOR]

Extensive injuries to bone tissue are still considered a significant clinical challenge; therefore, developing new bone tissue engineering (BTE) strategies is still necessary. This work aims to construct and characterize a chitosan‐gelatin/hydroxyapatite‐based (CG/H) scaffold to provide well‐design support for mesenchymal stem cell (MSC) growth and differentiation to osteoblasts. First, the CG/H scaffolds are construct by freeze‐drying. Then, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, x‐ray diffraction, water uptake, and degradation profiles evaluate the material's surface. In addition, the CG/H morphological, biochemical, and MSC adhesion processes and growth behavior are also assess, indicating reasonable adhesion rates to the surface, low material cytotoxicity, and excellent alkaline phosphatase activity compared to control on the cellular framework. Based on these results, we obtain a highly biocompatible scaffold and that can support osteoblast differentiation. Finally, the in vivo studies demonstrate the CG/H scaffold with MSC adhere is capable of differentiating into osteoblasts, and the application of this scaffold is able to significantly enhance the closure of the bone lesion. Therefore, the CG/H scaffold has potential clinical application for bone regeneration. [ABSTRACT FROM AUTHOR]

Aim: To investigate the relationship between interleukin‐17 (IL‐17), ferroptosis and osteogenic differentiation. Materials and Methods: We first analysed the changes in ferroptosis‐related molecules in experimental periodontitis models. The effects of erastin, a small‐molecule ferroptosis inducer, and IL‐17 on alveolar bone loss and repair in animal models were then investigated. Primary mouse mandibular osteoblasts were exposed to erastin and IL‐17 in vitro. Ferroptosis‐ and osteogenesis‐related genes and proteins were detected. Further, siRNA, immunofluorescence co‐localization and immunoprecipitation were used to confirm the roles of the nuclear factor erythroid‐2‐related factor 2 (NRF2) and phosphorylated signal transducer and activator of transcription 3 (p‐STAT3), as well as their interaction. Results: The levels of NRF2, glutathione peroxidase 4 and solute carrier family 7 member 11 were lower in the ligated tissues than in normal periodontal tissues. Alveolar bone loss in an in vivo experimental periodontitis model was aggravated by erastin and alleviated by IL‐17. In vitro, IL‐17 ameliorated erastin‐inhibited osteogenic differentiation by reversing ferroptosis. Altered NRF2 expression correlated with changes in ferroptosis‐related molecules and osteogenesis. Furthermore, the physical interaction between NRF2 and p‐STAT3 was confirmed in the nucleus. In IL‐17 + erastin‐stimulated osteoblasts, the p‐STAT3–NRF2 complex might actively participate in the downstream transcription of ferroptosis‐ and osteogenesis‐related genes. Conclusions: IL‐17 administration conferred resistance to erastin‐induced osteoblast ferroptosis and osteogenesis. The possible mechanism may involve p‐STAT3 directly interacting with NRF2. [ABSTRACT FROM AUTHOR]

Background: Mandibular condylar hypoplasia negatively affects patient's facial appearance and dentofacial function. Objective: To investigate the effect of local injection of the drug abaloparatide (ABL), an analogue of parathyroid hormone related protein (PTHrP), on promoting lengthening of the mandibular condyle. Methods: Thirty adolescent male Sprague–Dawley rats were randomly divided into two groups, which received the injection of ABL or normal saline (the control) every 3 days in the temporomandibular joint (TMJ) cavity. Cone‐beam computed tomography and immunohistochemistry assays were performed at 2, 4 and 6 weeks since the injection. Mandibular condylar chondrocytes (MCC) and pre‐osteoblasts were treated with ABL or PBS, followed by the CCK‐8 detection, IC50, real‐time PCR assay, Western Blot and immunofluorescence staining. Results: In vivo, compared with the control, the ABL group significantly increased the mandibular condylar process length (by 1.34 ± 0.59 mm at 6 weeks), the thickness of the cartilage layer, and enhanced the matrix synthesis. The ABL group had significant up‐regulation of SOX 9, COL II, PTHrP and PTH1R, down‐regulation of COL X in the cartilage, up‐regulation of RUNX 2, and unchanged osteoclastogenesis in the subchondral bone. In vitro, the intra‐TMJ injection of ABL promoted the MCC proliferation, with up‐regulated expression of chondrogenic genes, and enhanced osteogenic differentiation of the pre‐osteoblasts. Conclusions: Intra‐TMJ injection of abaloparatide promotes mandibular condyle lengthening in the adolescent rats via enhancing chondrogenesis in the mandibular condylar cartilage and ossification in the subchondral bone. [ABSTRACT FROM AUTHOR]

Background and Objective: Gallic acid (GA) possesses various beneficial functions including antioxidant, anticancer, anti‐inflammatory as well as inhibiting osteoclastogeneis. However, effects on osteogenic differentiation, especially in human ligament periodontal (hPDL) cells, remain unclear. Thus, the aim of this study was to evaluate the function of GA on osteogenesis and anti‐inflammation in hPDL cells and to explore the involved underlying mechanism. Methods: Porphyromonas gingivalis lipopolysaccharide (Pg‐LPS) treatment was used as a model for periodontitis. ROS production was determined by H2DCFDA staining. Trans‐well and wound healing assays were performed for checking the migration effect of GA. Alizarin red and alkaline phosphatase activity (ALP) assays were performed to evaluate osteogenic differentiation. Osteogenesis and inflammatory‐related genes and proteins were measured by real‐time PCR and western blot. Results: Our results showed that GA‐treated hPDL cells had higher proliferation and migration effect. GA inhibited ROS production‐induced by Pg‐LPS. Besides, GA abolished Pg‐LPS‐induced inflammation cytokines (il‐6, il‐1β) and inflammasome targets (Caspase‐1, NLRP3). In addition, GA promoted ALP activity and mineralization in hPDL cells, lead to enhance osteoblast differentiation process. The effect of GA is related to G‐protein‐coupled receptor 35 (GPR35)/GSK3β/β‐catenin signaling pathway. Conclusion: GA attenuated Pg‐LPS‐induced inflammatory responses and periodontitis in hPDL cells. Taken together, GA may be targeted for therapeutic interventions in periodontal diseases. [ABSTRACT FROM AUTHOR]

Background and Objectives: The utilization of natural products to enhance the function of periodontal ligament cells (PDLCs) has emerged as a popular area of research. Recent investigations have demonstrated that sappanchalcone (SC) possesses pharmacological properties such as anti‐inflammatory and osteoprotective effects. This study aims to explore the impact of SC on the in vivo and in vitro osteogenic differentiation ability of PDLCs. Materials: Cell proliferation was quantified using the CCK‐8 assay, while gene expression levels were assessed through qRT–PCR analysis. Osteoblast differentiation capacity was evaluated by employing Alizarin red staining (ARS), alkaline phosphatase (ALP) staining and western blot (WB) analysis. A rat model of periodontitis was established utilizing the tether‐wire method. Micro‐CT imaging and hematoxylin and eosin (HE) staining were employed to evaluate alveolar bone resorption. Masson's trichrome staining was utilized to observe fiber alignment, whereas immunohistochemistry (IHC) techniques were applied for detecting osteogenic and inflammatory factors. Results: The results from the CCK‐8 assay indicate no observed cytotoxicity for concentrations of 1, 5, or 10 nM for SC treatment (p <.05), while qRT–PCR analysis demonstrates a significant decrease in inflammatory factors such as MMP‐1 and IL‐6 with treatment by SC (p <.05). Additionally, western blotting reveals an increase in protein expression levels of Runx2 and OPN within PDLCs treated with SC compared to control groups (p <.05), which is further supported by ARS and ALP staining indicating an increase in mineralized nodules formation along with elevated ALP content within these cells following treatment with this compound (p <.05). Finally, both HE staining as well as micro‐CT imaging suggest potential benefits associated with using this compound including slowing alveolar bone resorption while simultaneously promoting junctional epithelium proliferation. Conclusions: Our in vitro and in vivo findings suggest that SC can effectively enhance the inflammatory response of PDLCs and promote their osteogenic differentiation ability under inflammatory conditions, indicating its potential as a promising therapeutic agent for improving periodontal inflammation and bone formation. [ABSTRACT FROM AUTHOR]

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG‐35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type‐specific responses, with AMG‐35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG‐35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF‐β1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG‐35 elicits osteoblast differentiation through TGF‐β1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF‐β1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG‐35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment. [ABSTRACT FROM AUTHOR]

The process of bone remodeling occurs and is regulated through interactions between osteoclasts, which resorb bone, and osteoblasts, which generate bone tissue. When the homeostatic balance between these two cell types is dysregulated, this can contribute to abnormal bone remodeling resulting in a loss of bone mass as is observed in osteoporosis (OP) and other forms of degenerative bone metabolic diseases. At present, details of molecular mechanism underlying the development of bone metabolic diseases such as OP remain to be elucidated. Circular RNAs (circRNAs) are small non‐coding RNA molecules with a closed‐loop structure that can regulate the differentiation of osteoclasts and osteoblasts. The present review provides a systematic overview of recent literature on the processes through which circRNAs regulate the dynamic balance between osteoblasts and osteoclasts that ultimately preserve bone homeostasis. It will also give insight that can shape current understanding of the pathogenesis of OP and other bone metabolic diseases to better guide diagnostic and treatment strategies for affected patients. [ABSTRACT FROM AUTHOR]

Labeled with pluripotent potential, the transplantation of bone marrow mesenchymal stem cells (BMSCs) is considered as a promising strategy for treating osteoporosis (OP). Melatonin (MEL) has been investigated to be an essential regulator involved in bone metabolism, as well as BMSCs differentiation. Circular RNAs (circRNAs) are a unique kind of non‐coding RNA and play an important regulatory role in OP. However, whether circRNAs are implicated in the effects of MEL on BMSCs osteogenic differentiation remains largely indeterminate. Expression of circ_0005753 in human BMSCs with MEL treatment, clinical specimens diagnosed with OP, either with ovariectomy (OVX)‐induced mice, was measured by RT‐qPCR. Western blot was conducted to analyze protein levels of osteogenesis‐related molecules (Opg, RUNX2, ALP, BMP4) and TXNIP. RNA immunoprecipitation (RIP) and RNA pull‐down assays were performed to validate the binding relationship among circ_0005753, PTBP1, and TXNIP. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were performed to evaluate osteogenic capacity of BMSCs. OP mouse model was established by ovariectomy, as evaluated pathologic changes via hematoxylin–eosin (HE), Masson, and Immunohistochemistry (IHC) staining. Expression of circ_0005753 was remarkably decreased during MEL‐induced osteogenic differentiation of BMSCs. Interestingly, not only circ_0005753 knockdown significantly promoted osteogenic differentiation of BMSCs, but circ_0005753 overexpression also weakened osteogenic differentiation induced by MEL treatment. Mechanistically, circ_0005753 maintained the stabilization of TXNIP mRNA via recruiting PTBP1. Additionally, reinforced circ_0005753 abrogated MEL‐mediated protective effects on OP pathogenesis in a mouse model. This work shows that MEL facilitates osteogenic differentiation of BMSCs via the circ_0005753/PTBP1/TXNIP axis, which may shed light on the development of a novel therapeutic strategy to prevent OP. [ABSTRACT FROM AUTHOR]

Because the chronobiotic and cytoprotective molecule melatonin diminishes with age, its involvement in postmenopausal and senescence pathology has been considered since long. One relevant melatonin target site in aging individuals is bone where melatonin chronobiotic effects mediated by MT1 and MT2 receptors are demonstrable. Precursors of bone cells located in bone marrow are exposed to high quantities of melatonin and the possibility arises that melatonin acts a cytoprotective compound via an autacoid effect. Proteins that are incorporated into the bone matrix, like procollagen type I c‐peptide, augment after melatonin exposure. Melatonin augments osteoprotegerin, an osteoblastic protein that inhibits the differentiation of osteoclasts. Osteoclasts are target cells for melatonin as they degrade bone partly by generating free radicals. Osteoclast activity and bone resorption are impaired via the free radical scavenger properties of melatonin. The administration of melatonin in chronobiotic doses (less than 10 mg daily) is commonly used in clinical studies on melatonin effect on bone. However, human equivalent doses allometrically derived from animal studies are in the 1–1.5 mg/kg/day range for a 75 kg human adult, a dose rarely used clinically. In view of the absence of toxicity of melatonin in phase 1 pharmacological studies with doses up to 100 mg in normal volunteers, further investigation is needed to determine whether high melatonin doses have higher therapeutic efficacy in preventing bone loss. [ABSTRACT FROM AUTHOR]

Osteomyelitis (OM), characterized by heterogeneity and complexity in treatment, has a high risk of infection recurrence which may cause limb disability. Management of chronic inactive osteomyelitis (CIOM) without typical inflammatory symptoms is a great challenge for orthopedic surgeons. On the basis of data analysis of 1091 OM cases, we reported that latent osteogenic decline in CIOM patients was the main cause of secondary surgery. Our research shows that impairment of osteoblasts capacity in CIOM patients is associated with ferroptosis of osteoblasts caused by internalization of Staphylococcus aureus. Further studies show that melatonin could alleviate ferroptosis of osteoblasts in infected states through Nox4/ROS/P38 axis and protect the osteogenic ability of CIOM patients. Knockout of NADPH oxidase 4 (Nox4) in vivo could effectively relieve ferroptosis of osteoblasts in the state of infection and promote osteogenesis. Through a large number of clinical data analyses combined with molecular experiments, this study clarified that occult osteogenic disorders in CIOM patients were related to ferroptosis of osteoblasts. We revealed that melatonin might be a potential therapeutic drug for CIOM patients and provided a new insight for the treatment of OM. [ABSTRACT FROM AUTHOR]

Mouse genetic studies have revealed that Runx2 and Sp7 act as master regulators in skeletal development. Numerous mechanistic studies have also uncovered how these two transcription factors execute key gene regulatory programs for the specification and differentiation of the two skeletal cell lineages, osteoblasts, and chondrocytes. The emergence of next‐generation sequencer (NGS)‐based techniques has shifted the research focus from the local actions of Runx2 and Sp7 to their comprehensive actions on the entire genome in cells of interest. NGS‐based studies have elucidated the gene‐regulatory landscapes of Runx2 and Sp7 in osteoblast and chondrocyte lineages, demonstrating that Runx2 and Sp7 exert their cell‐type‐specific functions through cell‐type‐specific modes of action. NGS‐based techniques are expected to continue evolving and providing high‐resolution, genome‐scale datasets. Ongoing accumulation of such data will enhance our understanding of gene‐regulatory networks in skeletal development and maintenance, and assist in the modeling of skeletal disorders and the development of novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Pediatric patients suffering traumatic brain injuries may require a decompressive craniectomy to accommodate brain swelling by removing a portion of the skull. Once the brain swelling subsides, the preserved calvarial bone flap is ideally replaced as an autograft during a cranioplasty to restore protection of the brain, as it can reintegrate and grow with the patient during immature skeletal development. However, pediatric patients exhibit a high prevalence of calvarial bone flap resorption post‐cranioplasty, causing functional and cosmetic morbidity. This review examines possible solutions for mitigating pediatric calvarial bone flap resorption by delineating methods of stimulating mechanosensitive cell populations with mechanical forces. Mechanotransduction plays a critical role in three main cell types involved with calvarial bone repair, including mesenchymal stem cells, osteoblasts, and dural cells, through mechanisms that could be exploited to promote osteogenesis. In particular, physiologically relevant mechanical forces, including substrate deformation, external forces, and ultrasound, can be used as tools to stimulate bone repair in both in vitro and in vivo systems. Ultimately, combating pediatric calvarial flap resorption may require a combinatorial approach using both cell therapy and bioengineering strategies. [ABSTRACT FROM AUTHOR]

The effect of preosteoblast‐derived exosomes on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) was evaluated in vitro, and bone formation studies were performed in vivo in mice. Preosteoblastic MC3T3‐E1 clone 4 (MC4) cell‐derived exosomes (MC4exo) were characterized with particle tracking, transmission electron microscopy and western blot analysis to validate size, number, shape and phenotypic exosome markers. Exosomes pre‐labelled with PKH67 were incubated with BMMΦ and phagocytosis of exosomes was confirmed. To examine the effect of MC4exo on macrophage polarization, BMMΦ were treated with MC4exo and the expression of pro‐ and anti‐inflammatory cytokines was determined by qPCR. MC4exo treatment upregulated mRNA expression of Cd86, Il1β, Ccl2, Rankl and Nos, and downregulated Cd206, Il10 and Tnfα, suggesting a shift towards pro‐inflammatory 'M1‐like' macrophage polarization. Combination of RANKL and MC4exo increased osteoclast differentiation of BMMΦ in comparison to RANKL alone as analysed by TRAP staining. MC4exo treatment showed no significant effect on calvarial osteoblast mineralization. For in vivo studies, intratibial inoculation of MC4exo (2 × 109 particles in PBS, n = 12) and vehicle control (PBS only, n = 12) was performed in C57Bl/6 mice (8 weeks, male). Micro‐CT analyses of the trabecular and cortical bone compartments were assessed at 4 weeks post‐injection. Tibial sections were stained for TRAP activity to determine osteoclast presence and immunofluorescence staining was performed to detect osteocalcin (Ocn), osterix (Osx) and F4/80 expression. Intratibial inoculation of MC4exo increased the diaphyseal bone mineral density and trabecular bone volume fraction due to increased trabecular number. This increase in bone was accompanied by a reduction in bone marrow macrophages and osteoclasts at the experimental endpoint. Together, these findings suggest that preosteoblast‐derived exosomes enhanced bone formation by influencing macrophage responses. [ABSTRACT FROM AUTHOR]

To improve the wear resistance of articulating metallic joint endoprostheses, the surfaces can be coated with titanium niobium nitride (TiNbN). Under poor tribological conditions or malalignment, wear can occur on these implant surfaces in situ. This study investigated the biological response of human osteoblasts to wear particles generated from TiNbN‐coated hip implants. Abrasive particles were generated in a hip simulator according to ISO 14242‐1/−2 and extracted with Proteinase K. Particle characteristics were evaluated by electron microscopy and energy dispersive x‐ray spectroscopy (EDS), inductively coupled plasma mass spectrometry (ICP‐MS) and dynamic light scattering (DLS) measurements. Human osteoblasts were exposed to different particle dilutions (1:20, 1:50, and 1:100), and cell viability and gene expression levels of osteogenic markers and inflammatory mediators were analyzed after 4 and 7 days. Using ICP‐MS, EDS, and DLS measurements, ~70% of the particles were identified as TiNbN, ranging from 39 to 94 nm. The particles exhibited a flat and subangular morphology. Exposure to particles did not influence cell viability and osteoblastic differentiation capacity. Protein levels of collagen type 1, osteoprotegerin, and receptor activator of nuclear factor κB ligand were almost unaffected. Moreover, the pro‐inflammatory response via interleukins 6 and 8 was minor induced after particle contact. A high number of TiNbN wear particles only slightly affected osteoblasts' differentiation ability and inflammatory response compared to metallic particles. Nevertheless, further studies should investigate the role of these particles in peri‐implant bone tissue, especially concerning other cell types. [ABSTRACT FROM AUTHOR]