USP26 Combats Age-Related Declines in Self-Renewal and Multipotent Differentiation of BMSC by Maintaining Mitochondrial Homeostasis.

Age-related declines in self-renewal and multipotency of bone marrow mesenchymal stem cells (BMSCs) limit their applications in tissue engineering and clinical therapy. Thus, understanding the mechanisms behind BMSC senescence is crucial for maintaining the rejuvenation and multipotent differentiation capabilities of BMSCs. This study reveals that impaired USP26 expression in BMSCs leads to mitochondrial dysfunction, ultimately resulting in aging and age-related declines in the self-renewal and multipotency of BMSCs. Specifically, decreased USP26 expression results in decreased protein levels of Sirtuin 2 due to its ubiquitination degradation, which leads to mitochondrial dysfunction in BMSCs and ultimately resulting in aging and age-related declines in self-renewal and multilineage differentiation potentials. Additionally, decreased USP26 expression in aging BMSCs is a result of dampened hypoxia-inducible factor 1α (HIF-1α) expression. HIF-1α facilitates USP26 transcriptional expression by increasing USP26 promoter activity through binding to the -191 - -198 bp and -262 - -269 bp regions on the USP26 promoter. Therefore, the identification of USP26 as being correlated with aging and age-related declines in self-renewal and multipotency of BMSCs, along with understanding its expression and action mechanisms, suggests that USP26 represents a novel therapeutic target for combating aging and age-related declines in the self-renewal and multipotent differentiation of BMSCs.

Targeting MCH Neuroendocrine Circuit in Lateral Hypothalamus to Protect Against Skeletal Senescence.

Neuroendocrine regulation is essential for maintaining metabolic homeostasis. However, whether neuroendocrine pathway influence bone metabolism and skeletal senescence is unelucidated. Here, a central neuroendocrine circuit is identified that directly controls osteogenesis. Using virus based tracing, this study is identified that melanin concentrating hormone (MCH) expressing neurons in the lateral hypothalamus (LH) are connected to the bone. Chemogenetic activation of MCH neurons in the LH induces osteogenesis, whereas inhibiting these neurons reduces osteogenesis. Meanwhile, MCH is released into the circulation upon chemogenetic activation of these neurons. Single cell sequencing reveals that blocking MCH neurons in the LH diminishes osteogenic differentiation of bone marrow stromal cells (BMSCs) and induces senescence. Mechanistically, MCH promotes BMSC differentiation by activating MCHR1 via PKA signaling, and activating MCHR1 by MCH agonists attenuate skeletal senescence in mice. By elucidating a brain-bone connection that autonomously enhances osteogenesis, these findings uncover the neuroendocrinological mechanisms governing bone mass regulation and protect against skeletal senescence.

Quercetin-primed BMSC-derived extracellular vesicles ameliorate chronic liver damage through miR-136-5p and GNAS/STAT3 signaling pathways.

BACKGROUND: Chronic liver damage (CLD) is a long-term and progressive liver condition characterized by inflammation, fibrosis, and impaired liver function, which ultimately lead to severe complications such as cirrhosis or liver cancer. Quercetin (Que), a flavonoid in various plants, possesses anti-inflammatory, antiviral, anti-ischemic, and anticancer properties. Recently, extracellular vesicles (EVs) derived from pretreated bone marrow mesenchymal stem cells (BMSCs) have shown immense potential in treating various diseases, including CLD. Thus, this study evaluated the regulatory effects of Que-preconditioned BMSC-derived EVs (Que-EVs) on LPS-stimulated RAW264.7 cells and their therapeutic effects on mice with CLD. METHODS: Que-EVs and control-EVs were harvested from the cell culture supernatant of BMSCs. The EVs were characterized using western blot, transmission electron microscopy, and nanoparticle tracking analysis. Further, the DIR labeling of EVs was used to detect in vitro and in vivo uptake. Next, LPS pre-stimulated RAW264.7 cells were treated with Que-EVs and control-EVs for 24 h. The relative expression of inflammatory cytokines and macrophage polarization markers genes was assessed using RT-qPCR, and western blot was conducted to evaluate the GNAS, PI3K, ERK, and STAT3 gene and protein expressions in RAW264.7 cells. Furthermore, transfection techniques were employed to induce miR-136-5p inhibition and GNAS overexpression in RAW264.7 cells to validate the role of miR-136-5p in alleviating inflammation through the GNAS/PI3K/ERK/STAT3 pathway. Subsequently, the outcomes were validated via in vitro experiments. RESULTS: Que enhanced miR-136-5p expression in BMSC-EVs. Furthermore, it was shown that EVs delivered miR-136-5p to macrophages, thereby attenuating M1-type macrophage polarisation through the GNAS/PI3K/ERK/STAT3 pathway, reducing liver inflammation, improving liver function and treating CLD.

Role of oxytocin in bone.

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

Metformin acts on miR-181a-5p/PAI-1 axis in stem cells providing new strategies for improving age-related osteogenic differentiation decline.

A general decline in the osteogenic differentiation capacity of human bone marrow mesenchymal stem cells (hBMSCs) in the elderly is a clinical consensus, with diverse opinions on the mechanisms. Many studies have demonstrated that metformin (MF) significantly protects against osteoporosis and reduces fracture risk. However, the exact mechanism of this effect remains unclear. In this study, we found that the decreased miR-181a-5p expression triggered by MF treatment plays a critical role in recovering the osteogenic ability of aging hBMSCs (derived from elderly individuals). Notably, the miR-181a-5p expression in hBMSCs was significantly decreased with prolonged MF (1000 µM) treatment. Further investigation revealed that miR-181a-5p overexpression markedly impairs the osteogenic ability of hBMSCs, while miR-181a-5p inhibition reveals the opposite result. We also found that miR-181a-5p could suppress the protein translation process of plasminogen activator inhibitor-1 (PAI-1), as evidenced by luciferase assays and western blots. Additionally, low PAI-1 levels were associated with diminished osteogenic ability, whereas high levels promoted it. These findings were further validated in human umbilical cord mesenchymal stem cells (hUCMSCs). Finally, our in vivo experiment with a bone defects rat model confirmed that the agomiR-181a-5p (long-lasting miR-181a-5p mimic) undermined bone defects recovery, while the antagomiR-181a-5p (long-lasting miR-181a-5p inhibitor) significantly promoted the bone defects recovery. In conclusion, we found that MF promotes bone tissue regeneration through the miR-181a-5p/PAI-1 axis by affecting MSC osteogenic ability, providing new strategies for the treatment of age-related bone regeneration disorders.

Mechanism study of BMSC-exosomes combined with hyaluronic acid gel in the treatment of posttraumatic osteoarthritis.

Objective: To explore the mechanism and efficacy of gel in the treatment of posttraumatic osteoarthritis (PTOA), combined with hyaluronic acid (HA) and bone marrow mesenchymal stem cell exosomes (BMSC-EXOs), and to explain its role in alleviating oxidative stress damage induced by mitochondrial reactive oxygen species (ROS). Methods: How is the therapeutic potential of toa influenced by bone marrow mesenchymal stem cells-EXO to be evaluated both in vitro and in vivo. In vitro, BMSC-EXOs were extracted and characterized from rat specimens and labeled with Dil. Rat primary chondrocytes were then isolated to create a cellular PTOA model. BMSC-EXOs + HA group, BMSC-EXOs + HA + 740Y-P group, model group, BMSC-EXOs group, HA group, and control group were included in the cell group, and the function of cartilage matrix and the level of oxidative stress could be evaluated. Cartilage matrix integrity and oxidative stress can be assessed by grouping rats. At the same time, a rat model of ptosis can be established by excision of the anterior cruciate ligament, and joint rehabilitation, with pro-inflammatory and Enzyme-linked immunosorbent assay (ELISA) can be used to determine anti-inflammatory markers. Result: sThe combined use of BMSC-EXOs and HA gel was found to significantly reduce oxidative stress in chondrocytes and PTOA rat models, improving cartilage mechanical properties more effectively than BMSC-EXOs alone. Conclusion: BMSC-EXOs combined with HA gel offer a promising treatment for PTOA by modulating damage caused by mitochondrial ROS-induced oxidative stress.

Targeting Bacteria-Induced Ferroptosis of Bone Marrow Mesenchymal Stem Cells to Promote the Repair of Infected Bone Defects.

The specific mechanisms underlying bacteria-triggered cell death and osteogenic dysfunction in host bone marrow mesenchymal stem cells (BMSCs) remain unclear, posing a significant challenge to the repair of infected bone defects. This study identifies ferroptosis as the predominant cause of BMSCs death in the infected bone microenvironment. Mechanistically, the bacteria-induced activation of the innate immune response in BMSCs leads to upregulation and phosphorylation of interferon regulatory factor 7 (IRF7), thus facilitating IRF7-dependent ferroptosis of BMSCs through the transcriptional upregulation of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4). Moreover, it is found that intervening in ferroptosis can partially rescue cell injuries and osteogenic dysfunction. Based on these findings, a hydrogel composite 3D-printed scaffold is designed with reactive oxygen species (ROS)-responsive release of antibacterial quaternized chitosan and sustained delivery of the ferroptosis inhibitor Ferrostatin-1 (Fer-1), capable of eradicating pathogens and promoting bone regeneration in a rat model of infected bone defects. Together, this study suggests that ferroptosis of BMSCs is a promising therapeutic target for infected bone defect repair.

Granulocyte colony-stimulating factor mediates bone loss via the activation of IL-1ß/JNK signaling pathway in murine Staphylococcus aureus-induced osteomyelitis.

Staphylococcus aureus (S. aureus)-induced bone loss is a significant challenge in the treatment of osteomyelitis. Our previous study was the first to confirm that granulocyte colony-stimulating factor (G-CSF) mediates S. aureus-induced bone loss. However, the underlying mechanism remains unknown. The objective of this study was to elucidate this. We found G-CSF mediated BMSC senescence and increased IL-1ß concentration of serum and bone marrow in mice after S. aureus infection. Furthermore, we demonstrated that G-CSF promoted the expression of IL1b in murine bone marrow-derived neutrophils. Notably, we identified that IL-1ß mediated BMSC (bone marrow mesenchymal stromal cell) senescence in mice after S. aureus infection. Importantly, IL-1ß neutralizing antibody effectively alleviated BMSC senescence and bone loss caused by S. aureus infection in mice. In terms of molecular mechanism, we found IL-1ß induced BMSC senescence by JNK/P53 and JNK/BCL2 pathways. Collectively, G-CSF promotes IL-1ß production which induces BMSC senescence via JNK/P53 and JNK/BCL2 pathways, leading to S. aureus-induced bone loss. This study identified novel targets for preventing and treating S. aureus-induced bone loss in osteomyelitis.

Mg-Cross-Linked Alginate Hydrogel Induces BMSC/Macrophage Crosstalk to Enhance Bone Tissue Regeneration via Dual Promotion of the Ligand-Receptor Pairing of the OSM/miR-370-3p-gp130 Signaling Pathway.

Macrophages play a pivotal role in the crosstalk between the immune and skeletal systems, while Mg-based biomaterials demonstrate immunomodulatory capabilities in this procedure. However, the mechanism of how Mg2+ promotes osteogenesis through the interplay of bone marrow-derived mesenchymal stem cells (BMSCs) and macrophages remains undescribed. Here, we demonstrated that a Mg-cross-linked alginate hydrogel exerted a dual enhancement of BMSCs osteogenic differentiation through the ligand-receptor pairing of the OSM/miR-370-3p-gp130 axis. On the one hand, Mg2+, released from the Mg-cross-linked hydrogel, stimulates bone marrow-derived macrophages to produce and secrete more OSM. On the other hand, Mg2+ lowers the miR-370-3p level in BMSCs and in turn, reverses its suppression on gp130. Then, the OSM binds to the gp130 heterodimer receptor and activates intracellular osteogenic programs in BMSCs. Taken together, this study reveals a novel cross-talk pattern between the skeletal and immune systems under Mg2+ stimulation. This study not only brings new insights into the immunomodulatory properties of Mg-based biomaterials for orthopedic applications but also enriches the miRNA regulatory network and provides a promising target to facilitate bone regeneration in large bone defects.

Norisoboldine, a Natural Alkaloid from Lindera aggregata (Sims) Kosterm, Promotes Osteogenic Differentiation via S6K1 Signaling Pathway and Prevents Bone Loss in OVX Mice.

SCOPE: Norisoboldine (NOR) is a major isoquinoline alkaloid component in the traditional Chinese herbal plant Lindera aggregata (Sims) Kosterm, with previously reported anti-osteoclast differentiation and antiarthritis properties. However, the roles of NOR on osteoblasts, bone marrow mesenchymal stem cells (BMSCs), and osteoporosis in vivo have never been well established. METHODS AND RESULTS: This study investigates the ability of NOR to improve bone formation in vitro and in vivo. Osteoblasts and BMSCs are used to study the effect of NOR on osteogenic and adipogenic differentiation. It finds that NOR promotes osteogenic differentiation of osteoblasts and BMSCs, while inhibiting adipogenic differentiation of BMSCs by reducing the relative expression of peroxisome proliferator-activated receptor Î³ (Ppar-γ) and adiponectin, C1Q and collagen domain containing (Adipoq). Mechanistic studies show that NOR increases osteoblast differentiation through the mechanistic target of rapamycin kinase (mTOR)/ribosomal protein S6 kinase; polypeptide 1 (S6K1) pathway, and treatment with an mTOR inhibitor rapamycin blocked the NOR-induced increase in mineral accumulation. Finally, the study evaluates the therapeutic potential of NOR in a mouse model of ovariectomy (OVX)-induced bone loss. NOR prevents bone loss in both trabecular and cortical bone by increasing osteoblast number and phospho-S6K1 (p-S6K1) expression in osteoblasts. CONCLUSION: NOR effects in enhancing osteoblast-induced bone formation via S6K1 pathway, suggesting the potential of NOR in osteoporosis treatment by increasing bone formation.

Regulation of metabolic microenvironment with a nanocomposite hydrogel for improved bone fracture healing.

Bone nonunion poses an urgent clinical challenge that needs to be addressed. Recent studies have revealed that the metabolic microenvironment plays a vital role in fracture healing. Macrophages and bone marrow-derived mesenchymal stromal cells (BMSCs) are important targets for therapeutic interventions in bone fractures. Itaconate is a TCA cycle metabolite that has emerged as a potent macrophage immunomodulator that limits the inflammatory response. During osteogenic differentiation, BMSCs tend to undergo aerobic glycolysis and metabolize glucose to lactate. Copper ion (Cu2+) is an essential trace element that participates in glucose metabolism and may stimulate glycolysis in BMSCs and promote osteogenesis. In this study, we develop a 4-octyl itaconate (4-OI)@Cu@Gel nanocomposite hydrogel that can effectively deliver and release 4-OI and Cu2+ to modulate the metabolic microenvironment and improve the functions of cells involved in the fracture healing process. The findings reveal that burst release of 4-OI reduces the inflammatory response, promotes M2 macrophage polarization, and alleviates oxidative stress, while sustained release of Cu2+ stimulates BMSC glycolysis and osteogenic differentiation and enhances endothelial cell angiogenesis. Consequently, the 4-OI@Cu@Gel system achieves rapid fracture healing in mice. Thus, this study proposes a promising regenerative strategy to expedite bone fracture healing through metabolic reprogramming of macrophages and BMSCs.

Preliminary study on the mechanism by which exosomes derived from human exfoliated deciduous teeth improve the proliferation and osteogenic inhibitory effect of glucocorticoid-induced BMSCs.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is a disease characterized by a collapsed femoral head caused by the overuse of glucocorticoids. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is an important pathological feature of SONFH. In this study, we investigated whether exosomes from SHEDs (stem cells from human exfoliated deciduous teeth) have a therapeutic effect on glucocorticoid-induced inhibition of proliferation and osteogenesis in BMSCs, and elucidated the underlying mechanisms involved. METHODS: Primary dental pulp cells were isolated and cultured from human deciduous tooth pulp, SHEDs were isolated and purified by the limiting dilution method and exosomes were isolated from the supernatants of SHEDs by ultracentrifugation. The cell surface markers CD31, CD34, CD45, CD73, CD90 and CD105 were detected by flow cytometry. A Cell-Counting-Kit-8 assay was used to detect cell activity. ALP and Alizarin Red staining were used to identify osteogenic differentiation ability, and exosomes were identified using transmission electron microscopy, NanoFCM and Western blotting. PKH67 fluorescence was used to track the uptake of exosomes by BMSCs. Transcriptome analysis combined with quantitative real-time PCR was used to explore the underlying mechanism involved. RESULTS: Exosomes secreted by SHEDs can be endocytosed by BMSCs, and can partially reverse the inhibitory effects of glucocorticoids on the viability and osteogenic differentiation of BMSCs. Transcriptome sequencing analysis revealed that the differentially expressed mRNAs regulated by SHED-derived exosomes were enriched mainly in signaling pathways such as the apoptosis pathway, the PI3K-Akt signaling pathway, the Hippo signaling pathway and the p53 signaling pathway. qPCR showed that SHED-derived exosomes reversed the dexamethasone-induced upregulation of HGF and ITGB8 expression and the inhibition of EFNA1 expression, but further increased the dexamethasone-induced downregulation of IL7 expression. In conclusion, SHED-derived exosomes partially reversed the inhibitory effects of glucocorticoids on BMSC proliferation and osteogenesis by inhibiting the expression of HGF, ITGB8 and IL7, and upregulating the expression of EFNA1.

Effects of bone marrow mesenchymal stem cell-conditioned medium on the proliferation and migration of liposarcoma cells.

INTRODUCTION: Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells. MATERIALS AND METHODS: We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay. RESULTS: Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression. CONCLUSIONS: These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Research on and Model Analysis of Flexural Mechanical Properties of Basic Magnesium Sulfate Cement Concrete Beams.

This study presents a comprehensive investigation into the mechanical properties of Basic Magnesium Sulfate Cement Concrete (BMSC) in comparison to Ordinary Portland Cement Concrete (OPC) within reinforced concrete components. The main objective is to evaluate BMSC's applicability for practical engineering purposes, with a focus on its with early high strength, improved toughness, and superior crack resistance compared to conventional concrete. Experimental procedures involved fabricating beam specimens using OPC concrete with a C40 strength grade, alongside BMSC beams with varying strength grades (C30, C40, and C50). These specimens underwent bending resistance tests to analyze crack patterns and mechanical characteristics. The findings reveal that BMSC beams demonstrate enhanced bending and tensile properties at equivalent strength grades compared to OPC beams. Particularly, cracking mainly occurred at the mid-span region of BMSC beams, characterized by narrower cracks, indicating superior crack resistance. However, it was noted that the toughness of BMSC beams decreases as the strength grade increases. The maximum mid-span deflection of the BMSC test beam was smaller than that of the OPC test beam, which was 3.8 mm and 2.6 mm, respectively. The maximum crack width of the OPC beam was 4.7 times that of the BMSC beam. To facilitate practical implementation, the study developed calculation models for estimating the crack bending distance and ultimate bending distance in BMSC beams, offering valuable tools for engineering design and optimization. Overall, this research provides significant insights into the mechanical behavior of BMSC, presenting potential advantages for structural engineering applications.

Extracellular vesicles derived from bone marrow mesenchymal stem cells ameliorate chronic liver damage via microRNA-136-5p.

Chronic liver damage (CLD) encompasses a spectrum of conditions and poses a significant global health challenge, affecting millions of individuals. Currently, there is a deficiency of clinically validated therapeutics with minimal side effects. Emerging evidence underscores the significant potential of extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) as a promising therapeutic method for CLD. This study aimed to evaluate the influence of BMSC-EVs containing microRNA-136-5p (BMSC-EVs-miR-136-5p) on macrophage polarization during chronic liver injury and elucidate the mechanisms associated with the GNAS/PI3K/ERK/STAT3 axis. Surface markers of BMSCs were detected via Immunofluorescent Staining. Subsequently, EVs were harvested from the BMSC culture medium. In vivo fluorescence imaging was employed to locate the BMSC-EVs. Additionally, fluorescence microscopy was used to visualize the uptake of DIR-labeled BMSC-EVs by RAW264.7 cells. Various methods were employed to assess the impact of BMSC-EVs on the expression levels of inflammatory factors (IL-1ß, IL-6, IL-10, and TNF-α), M1/M2 macrophage markers (iNOS and Arg-1), and members of inflammation-related signaling pathways (GNAS, PI3K, ERK, and STAT3) in RAW264.7 cells co-cultured with BMSC-EVs. Loss-of-function approaches targeting miR-136-5p in RAW264.7 cells were subsequently utilized to validate the role of BMSC-EVs-miR-136-5p. The Luciferase Reporter Assay indicates that GNAS was identified to be a target of miR-136-5p, and miR-136-5p demonstrating increased within BMSC-EVs compared to Raw264.7-EVs. BMSC-EVs-miR-136-5p mitigated CCl4-induced liver inflammation and improved liver function by Suppressing the GNAS/STAT3 Signaling. Notably, miR-136-5p suppressed lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. BMSC-EVs-miR-136-5p alleviates CLD by activating M2 polarization through the GNAS-mediated PI3K/ERK/STAT3 axis. Accordingly, the members of this axis may serve as therapeutic targets.

BMSC-Derived Exosomes Attenuate Rat Osteoarthritis by Regulating Macrophage Polarization through PINK1/Parkin Signaling Pathway.

OBJECTIVE: Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) may modulate the M1/M2 polarization of macrophages during osteoarthritis (OA). However, the underlying mechanisms of BMSC-Exos in this process still need to be elucidated. In this study, we explored the role of BMSC-Exos in the polarization of macrophages in vitro and the OA rats in vivo. METHODS: The effects of BMSC-Exos on RAW264.7 cells were determined, including the production of reactive oxygen species (ROS) and the protein expression of Akt, PINK1, and Parkin. We prepared an OA model by resecting the anterior cruciate ligament and medial meniscus of Sprague-Dawley (SD) rats. Hematoxylin-eosin (H&E) and safranin O-fast green staining, immunohistochemistry and immunofluorescence analyses, and the examination of interleukin 6 (IL-6), interleukin 1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10) were performed to assess changes in cartilage and synovium. RESULTS: BMSC-Exos inhibited mitochondrial membrane damage, ROS production, and the protein expression of PINK1 and Parkin. Akt phosphorylation was downregulated under lipopolysaccharide (LPS) induction but significantly recovered after treatment with BMSC-Exos. BMSC-Exos alleviated cartilage damage, inhibited M1 polarization, and promoted M2 polarization in the synovium in OA rats. The expression of PINK1 and Parkin in the synovium and the levels of IL-6, IL-1ß, and TNF-α in the serum decreased, but the level of IL-10 increased when BMSC-Exos were used in OA rats. CONCLUSION: BMSC-Exos ameliorate OA development by regulating synovial macrophage polarization, and one of the underlying mechanisms may be through inhibiting PINK1/Parkin signaling.

BMSC-Derived Exosomes Carrying miR-26a-5p Ameliorate Spinal Cord Injury via Negatively Regulating EZH2 and Activating the BDNF-TrkB-CREB Signaling.

BACKGROUND: Spinal cord injury (SCI) is a destructive neurological and pathological state that causes major motor, sensory and autonomic dysfunctions. Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes show great therapeutic potential for SCI. Exosomes derived from miR-26a-modified MSCs promote axonal regeneration following SCI. Our study aims to uncover the mechanisms by which BMSC-derived exosomes carrying miR-26a-5p regulate SCI. METHODS: BMSCs and BMSC-derived exosomes were isolated and characterized by Oil Red O and alizarin red staining, transmission electron microscopy, flow cytometry, nanoparticle tracking analysis and Western blotting. PC12 cells were treated with lipopolysaccharides (LPS), and SCI was established through laminectomy with contusion injury in rats. Annexin-V staining, CCK-8 and EdU incorporation were applied to determine cell apoptosis, viability, and proliferation. Hematoxylin and Eosin, Nissl and TUNEL staining was used to evaluate SCI injury and apoptosis in the spinal cord. Luciferase and chromatin immunoprecipitation assays were applied to evaluate gene interaction. RESULTS: BMSC-derived exosomes facilitated LPS-treated PC12 cell proliferation and inhibited apoptosis by delivering miR-26a-5p. Moreover, BMSC-derived exosomal miR-26a-5p alleviated SCI. Furthermore, miR-26a-5p inhibited EZH2 expression by directly binding to EZH2, and EZH2 inhibited BDNF expression via promoting H3K27me3. Increased phosphorylated CREB enhanced KCC2 transcription and expression by binding to its promoter. Knockdown of miR-26a-5p abrogated BMSC-derived exosome-mediated protection in LPS-treated PC12 cells, but it was reversed by KCC2 overexpression. CONCLUSION: BMSC-derived exosomes carrying miR-26a-5p repressed EZH2 expression to promote BDNF and TrkB expression and CREB phosphorylation and subsequently increase KCC2 expression, thus protecting PC12 cells and ameliorating SCI.

PMAIP1, a novel diagnostic and potential therapeutic biomarker in osteoporosis.

BACKGROUND: Osteoporosis is a common endocrine metabolic bone disease, which may lead to severe consequences. However, the unknown molecular mechanism of osteoporosis, the observable side effects of present treatments and the inability to fundamentally improve bone metabolism seriously restrict the impact of prevention and treatment. The study aims to identify potential biomarkers from osteoclast progenitors, specifically peripheral blood monocytes on predicting the osteoporotic phenotype. METHODS: Datasets were obtained from Gene Expression Omnibus (GEO). Based on the differentially expressed genes (DEGs) and GSEA results, GO and KEGG analyses were performed using the DAVID database and Metascape database. PPI network, TF network, drug-gene interaction network, and ceRNA network were established to determine the hub genes. Its osteogenesis, migration, and proliferation abilities in bone marrow mesenchymal stem cells (BMSCs) were validated through RT-qPCR, WB, ALP staining, VK staining, wound healing assay, transwell assay, and CCK-8 assay. RESULTS: A total of 63 significant DEGs were screened. Functional and pathway enrichment analysis discovered that the functions of the significant DEGs (SDEGs) are mainly related to immunity and metal ions. A comprehensive evaluation of all the network analyses, PMAIP1 was defined as osteoporosis's core gene. This conclusion was further confirmed in clinical cohort data. A series of experiments demonstrated that the PMAIP1 gene can promote the osteogenesis, migration and proliferation of BMSC cells. CONCLUSIONS: All of these outcomes showed a new theoretical basis for further research in the treatment of osteoporosis, and PMAIP1 was identified as a potential biomarker for osteoporosis diagnosis and treatment.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Ameliorate Aging-Induced BTB Impairment in Porcine Testes by Activating Autophagy and Inhibiting ROS/NLRP3 Inflammasomes via the AMPK/mTOR Signaling Pathway.

As a pivotal player in spermatogenesis, the blood-testis barrier (BTB) made from junction apparatus coexisting in Sertoli cells (SCs) is impaired with an increase in age and ultimately induces spermatogenic dysfunction or even infertility. It has been corroborated that bone marrow mesenchymal stem cell (BMSC) transplantation can efficiently repair and regenerate the testicular function. As vital mediators of cell-to-cell communication, MSC-derived exosomes (Exos) can directly serve as therapeutic agents for tissue repair and regeneration. However, the therapeutic value of BMSC-Exos in aging-induced BTB damage remains to be confirmed. In this study, we explored that the old porcine testes had defective autophagy, which aggravated BTB disruption in SCs. BMSC-Exos could decrease ROS production and NLRP3 inflammasome activation but enhanced autophagy and tight junction (TJ) function in D-gal-triggered aging porcine SCs and mouse model testes, according to in vitro and in vivo experiments. Furthermore, rapamycin, NAC, MCC950, and IL-1Ra restored the TJ function in D-gal-stimulated aging porcine SCs, while BMSC-Exos' stimulatory effect on TJ function was inhibited by chloroquine. Moreover, the treatment with BMSC-Exos enhanced autophagy in D-gal-induced aging porcine SCs by means of the AMPK/mTOR signal transduction pathway. These findings uncovered through the present study that BMSC-Exos can enhance the BTB function in aging testes by improving autophagy via the AMPK/mTOR signaling pathway, thereby suppressing ROS production and NLRP3 inflammasome activation.

Porcine Mandibular Bone Marrow-Derived Mesenchymal Stem Cell (BMSC)-Derived Extracellular Vesicles Can Promote the Osteogenic Differentiation Capacity of Porcine Tibial-Derived BMSCs.

OBJECTIVE: Existing research suggests that bone marrow-derived mesenchymal stem cells (BMSCs) may promote endogenous bone repair. This may be through the secretion of factors that stimulate repair processes or directly through differentiation into osteoblast-progenitor cells. However, the osteogenic potential of BMSCs varies among different tissue sources (e.g., mandibular versus long BMSCs). The main aim of this study was to investigate the difference in osteogenic differentiation capacity between mandibular BMSCs (mBMSCs) and tibial BMSCs (tBMSCs). MATERIALS AND METHODS: Bioinformatics analysis of the GSE81430 dataset taken from the Gene Expression Omnibus (GEO) database was performed using GEO2R. BMSCs were isolated from mandibular and tibial bone marrow tissue samples. Healthy pigs (n = 3) (registered at the State Office for Nature, Environment, and Consumer Protection, North Rhine-Westphalia (LANUV) 81-02.04.2020.A215) were used for this purpose. Cell morphology and osteogenic differentiation were evaluated in mBMSCs and tBMSCs. The expression levels of toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively. In addition, mBMSC-derived extracellular vesicles (mBMSC-EVs) were gained and used as osteogenic stimuli for tBMSCs. Cell morphology and osteogenic differentiation capacity were assessed after mBMSC-EV stimulation. RESULTS: Bioinformatic analysis indicated that the difference in the activation of the TLR4/NF-κB pathway was more pronounced compared to all other examined genes. Specifically, this demonstrated significant downregulation, whereas only 5-7 upregulated genes displayed significant variances. The mBMSC group showed stronger osteogenic differentiation capacity compared to the tBMSC group, confirmed via ALP, ARS, and von Kossa staining. Furthermore, qPCR and WB analysis revealed a significant decrease in the expression of the TLR4/NF-κB pathway in the mBMSC group compared to the tBMSC group (TLR4 fold changes: mBMSCs vs. tBMSCs p < 0.05; NF-κB fold changes: mBMSCs vs. tBMSCs p < 0.05). The osteogenic differentiation capacity was enhanced, and qPCR and WB analysis revealed a significant decrease in the expression of TLR4 and NF-κB in the tBMSC group with mBMSC-EVs added compared to tBMSCs alone (TLR4 fold changes: p < 0.05; NF-κB fold changes: p < 0.05). CONCLUSION: Our results indicate that mBMSC-EVs can promote the osteogenic differentiation of tBMSCs in vitro. The results also provide insights into the osteogenic mechanism of mBMSCs via TLR4/NF-κB signaling pathway activation. This discovery promises a fresh perspective on the treatment of bone fractures or malunions, potentially offering a novel therapeutic method.