Introduction. Promoting bone mass homeostasis keeps skeleton away from osteoporosis. a-Ketoglutarate (a-KG) is an indispensable intermediate of tricarboxylic acid cycle (TCA) process for cellular energy production. a-KG mitigates cellular senescence, tissue degeneration, and oxidative stress. We investigated whether a-KG affected osteoblast activity or osteoporosis development. Method. Serum and bone specimens were biopsied from 26 patients with osteoporosis or 24 patients without osteoporosis who required spinal surgery. Ovariectomized or aged mice were fed 0.25% or 0.75% a-KG in drinking water for 8 – 12 weeks ad libitum. Bone mineral density, trabecular/cortical bone microarchitecture, mechanical strength, bone formation, and osteoclastic erosion were investigated using mCT, material testing device, in vivo calcein labelling, and TRAP histochemical staining. Serum a-KG, osteocalcin, and TRAP5b levels were quantified using ELISA kits. Bone-marrow mesenchymal cells and macrophages were incubated osteogenic and osteoclastogenic media. Histone H3K27me3 levels and enrichment were investigated using immunoblotting and chromatin precipitation-PCR. Result. Serum a-KG levels in patients with osteoporosis were less than controls; and were correlated with T-scores of hips (R2 = 0.6471, P < 0.0001) and lumbar spine (R2 = 0.7235, P < 0.001) in osteoporosis (AUC = 0.9941, P < 0.001). a-KG supplement compromised a plethora of osteoporosis signs in ovariectomized or aged mice, including bone mass loss, trabecular bone microarchitecture deterioration, and mechanical strength loss. It elevated serum osteocalcin levels and decreased serum TRAP5b. a-KG preserved caclein-labelling bone formation and repressed osteoclast resorption. It reversed osteogenic differentiation of bone-marrow stromal cells and reduced osteoclast formation in ovariectomized mice. Mechanically, a-KG attenuated H3K27 hypermethylation and Runx2 transcription repression, improving mineralized matrix production in osteogenic cells. Conclusion. Decreased serum a-KG is correlated with human and murine osteoporosis. a-KG reverses bone loss by repressing histone methylation in osteoblasts. This study highlighted a-KG supplement as a new biochemical option for protecting osteoporosis

Introduction. Bioactive glasses (BGs) promote osteogenic differentiation of bone progenitor cells by releasing therapeutically active ions. The well-described 45S5-BG (in mol%: SiO. 2. 46.13; P. 2. O. 5. 2.60; CaO 26.91; Na. 2. O 24.35) was supplemented with CaF. 2. and NaF being added to the batch at nominal 5 (F5-BG) and 25 mol% (F25-BG), respectively. While the effect on physical and chemical properties has already been characterized, the biological properties require further studies. This study investigates the effects of fluoride-supplemented BGs on the osteogenic and angiogenic properties of human bone marrow mesenchymal stromal cells (BMSCs) in vitro. Method. BMSCs were co-cultured with melt-derived 45S5-BG, F5-BG, or F25-BG in ascending concentrations (1, 2 and 3 mg/ml). At 7 days, cell number was determined by 4,6-diamidine-2-phenylindole (DAPI) staining and cell viability by fluorescein diacetate (FDA) assay. The osteogenic potential of the BGs was evaluated through alkaline phosphatase (ALP) gene expression and activity, along with bone morphogenetic protein-2 (BMP2) gene expression and protein concentration. Vascular endothelial growth factor (VEGF) gene expression and protein concentration assessed angiogenic potential. As control, BMSCs were cultured without BG exposure. Result. All BGs significantly promoted cell number and viability, with F25-BG showing the highest count at 3 mg/ml. Osteogenic markers showed a significant decrease in ALP gene expression and activity, especially at higher concentrations. All BG groups demonstrated increased BMP2 protein concentration and gene expression compared to the control, with higher BG and fluoride concentrations correlating with greater increases in BMP2. VEGF gene expression increased in all analysed BGs. The fluoride-free BG group had the highest VEGF protein concentrations, while the F25 BG group showed the highest VEGF gene expression. Conclusion. The fluoride-substituted BGs exhibit excellent cytocompatibility, enhance BMSC proliferation and positively affect BMP2 gene expression and levels, suggesting their potential for osteogenic differentiation. Further research is necessary to assess their proangiogenic effect and potential advantages over 45S5-BG

Introduction. Osteoporosis accounts for a major risk factor of fracture-associated disability or premature death in the elderly. Enhancement of bone anabolism for slowing osteoporosis is highly demanding. Exerkine fibronectin type III domain containing 5 (FNDC5) regulates energy metabolism, inflammation, and aging. This study was aimed to investigate whether Fndc5 signaling in osteoblasts changed estrogen deficiency-mediated bone loss or microarchitecture deterioration. Method. Female osteoblast-specific Fndc5 transgenic mice (Fndc5Tg), which overexpressed Fndc5 under the control of key osteoblast marker osteocalcin promoter, were given bilateral ovariectomy to induce estrogen deficiency-mediated osteoporosis. Bone mass, microstructures, and biomechanical properties were quantified using μCT imaging and material testing. Dynamic bone formation was traced using fluorescence calcein. Osteogenic differentiation and adipocyte formation of bone-marrow mesenchymal cells were investigated using von Kossa staining and Nile red staining, respectively. Serum osteocalcin, CTX-1 and TRAP5b levels were quantified using designated ELISA kits. Mitochondrial respiration was investigated using Seahorse Extracellular Flux Analyzer. Result. Fndc5Tg mice developed relatively higher bone mass and microarchitecture than wild-type mice. Fndc5 overexpression attenuated the losses of bone mineral density and trabecular network, including trabecular volume, thickness, and trabecular number, and improved cortical thickness and porosity in ovariectomized mice. Gain of Fndc5 function preserved biomechanical characteristics (maximum load, breaking force, and energy), serum bone formation marker osteocalcin levels, and bone formation rate, whereas it reduced serum bone resorption makers CTX-1 and TRAP5b levels, osteoclast overburden, and marrow adiposis. In vitro, Fndc5 reversed the estrogen deficiency-mediated mineralized matrix underproduction and adipocyte formation of bone-marrow mesenchymal cells, and inhibited osteoclast formation in osteoporotic bone. Mechanistically, Fndc5 activated AMPK signaling, promoting mitochondrial respiration and ATP production to enhance osteoblastic activity. Conclusion. Fndc5 signaling exerted bone-protective actions delaying estrogen deficiency-mediated osteoporosis. This study highlighted a new molecular remedial option for osteoporosis development by manipulating Fndc5 functions

Aims. This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. Methods. A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process. Results. Mean callus volume was larger in the elastic fixation group (1,755 mm. 3. (standard error of the mean (SEM) 297)) than in the stiff fixation group (258 mm. 3. (SEM 65)). Pathological observation found that the expression levels of osterix (OSX), collagen, type I, alpha 1 (COL1α1), and alkaline phosphatase (ALP) in the callus of the elastic fixation group were higher than those of the stiff fixation group. The protein sequence of the callus revealed 199 DEPs, 124 of which were highly expressed in the elastic fixation group. In the in vitro study, it was observed that a stress of 200 g led to upregulation of thrombospondin 1 (THBS1) and osteoglycin (OGN) expression in bone marrow mesenchymal stem cells (BMSCs). Additionally, these genes were found to be upregulated during the osteogenic differentiation process of the BMSCs. Conclusion. Elastic fixation can promote fracture healing and osteoblast differentiation in callus, and the ability of elastic fixation to promote osteogenic differentiation of BMSCs may be achieved by upregulating genes such as THBS1 and OGN. Cite this article: Bone Joint Res 2024;13(10):559–572

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Rotator cuff tear (RCT) is the leading cause of shoulder pain, primarily associated with age-related tendon degeneration. This study aimed to elucidate the potential differential gene expressions in tendons across different age groups, and to investigate their roles in tendon degeneration.

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

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This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation.

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The aim of this study was to determine the fracture haematoma (fxH) proteome after multiple trauma using label-free proteomics, comparing two different fracture treatment strategies.

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Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells.

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This study aimed to explore the biological and clinical importance of dysregulated key genes in osteoarthritis (OA) patients at the cartilage level to find potential biomarkers and targets for diagnosing and treating OA.

Aims. To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. Methods. In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed. Results. The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p. Conclusion. Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway. Cite this article: Bone Joint Res 2024;13(2):52–65

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Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice.

The current procedures being applied in the clinical setting to address osteoporosis-related delayed union and nonunion bone fractures have been found to present mostly suboptimal outcomes. As a result, bone tissue engineering (BTE) solutions involving the development of implantable biomimetic scaffolds to replace damaged bone and support its regeneration are gaining interest. The piezoelectric properties of the bone tissue, which stem primarily from the significant presence of piezoelectric type I collagen fibrils in the tissue's extracellular matrix (ECM), play a key role in preserving the bone's homeostasis and provide integral assistance to the regeneration process. However, despite their significant potential, these properties of bone tend to be overlooked in most BTE-related studies. In order to bridge this gap in the literature, novel hydroxyapatite (HAp)-filled osteoinductive and piezoelectric poly(vinylidene fluoride-co-tetrafluoroethylene) (PVDF-TrFE) electrospun nanofibers were developed to replicate the bone's fibrous ECM composition and electrical features. Different HAp nanoparticle concentrations (1–10%, wt%) were tested to assess their effect on the physicochemical and biological properties of the resulting fibers. The fabricated scaffolds displayed biomimetic collagen fibril-like diameters, while also presenting mechanical features akin to type I collagen. The increase in HAp presence was found to enhance both surface and piezoelectric properties of the fibers, with an improvement in scaffold wettability and increase in β-phase nucleation (translating to increased piezoelectricity) being observed. The HAp-containing scaffolds also exhibited an augmented bioactivity, with a more comprehensive surface mineralization of the fibers being obtained for the scaffolds with the highest HAp concentrations. Improved osteogenic differentiation of seeded human mesenchymal stem/stromal cells was achieved with the addition of HAp, as confirmed by an increased ALP activity, calcium deposition and upregulated expression of key osteogenic markers. Overall, our findings highlight, for the first time, the potential of combining PVDF-TrFE and HAp to develop electroactive and osteoinductive nanofibers for BTE. Acknowledgements: The authors thank FCT for funding through the projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), the PhD scholarship (2022.10572.BD) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020)

Critical-sized bone defects remain challenging in the clinical setting. Autologous bone grafting remains preferred by clinicians. However, the use of autologous tissue is associated with donor-site morbidity and limited accessibility to the graft tissue. Advances in the development of synthetic bone substitutes focus on improving their osteoinductive properties. Whereas osteoinductivity has been demonstrated with ceramics, it is still a challenge in case of polymeric composites. One of the approaches to improve the regenerative properties of biomaterials, without changing their synthetic character, is the addition of inorganic ions with known osteogenic and angiogenic properties. We have previously reported that the use of a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50% beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating of the TCP particles can enhance the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) (3). To further support the regenerative properties of these scaffolds, inorganic ions with known angiogenic properties, copper or cobalt, were added to the coating solution. β-TCP particles were immersed in a zinc and copper or zinc and cobalt solution with a concentration of 15 or 45 mM. 3D porous scaffolds composed of 1000PEOT70PBT30 and pure or coated β-TCP were additively manufactured by 3D fibre deposition. The osteogenic and angiogenic properties of the fabricated scaffolds were tested in vitro through culture with hMSCs and human umbilical vein endothelial cells, respectively. The materials were further evaluated through ectopic implantation in an in vivo mini-pig model. The early expression of relevant osteogenic gene markers (collagen-1, osteocalcin) of hMSCs was upregulated in the presence of lower concentration of inorganic ions. Further analysis will focus on the evaluation of ectopic bone formation and vascularisation of these scaffolds after implantation in a mini-pig ectopic intramuscular model

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure

The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods. MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging. The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription. Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic. Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024

Several synthetic polymers have been widely investigated for their use in bone tissue engineering applications, but the ideal material is yet to be engineered. Triazine-trione (TATO) based materials and their derivatives are novel in the field of biomedical engineering but have started to draw interest. Different designs of the TATO monomers and introduction of different chemical linkages and end-groups widens the scope of the materials due to a range of mechanical properties. The aim of our work is to investigate novel TATO based materials, with or without hydroxyapatite filler, for their potential in bone tissue engineering constructs. Initially the biocompatibility of the materials was tested, indirectly and directly, according to ISO standards. Following this the osteoconductive properties were investigated with primary osteoblasts and an osteoblastic cell line. Bone marrow derived mesenchymal stem cells were used to evaluate the osteogenic differentiation and consequently the materials potential in bone tissue engineering applications

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity. Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining. HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts. Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity