Search Results (146)

Objective: Diabetic kidney disease (DKD) is characterized by podocyte injury and impaired autophagy. Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) exhibit therapeutic potential for DKD, yet their mechanisms remain unclear. This study investigated whether BMSC-Exos restore podocyte autophagy via the miR-143-3p/Bcl-2/Beclin1 axis to delay DKD progression. Methods: A high-glucose (HG)-induced podocyte injury model was established using mouse podocytes (MPC5). Autophagy-related proteins (Beclin1, Bcl-2, LC3) and the injury marker desmin were analyzed by Western blot and immunofluorescence (IF). High-throughput sequencing identified BMSC-Exos-enriched miRNAs, with the miR-143-3p/Bcl-2 targeting relationship validated by dual-luciferase reporter assays. BMSCs transfected with miR-143-3p mimic or inhibitor were used to assess exosomes effects on autophagy and podocin expression. In vivo, DKD mice received tail vein injections of modified BMSC-Exos, followed by evaluation of physiological parameters, biochemical indices, and renal histopathology. Results: BMSC-Exos were successfully isolated and characterized. Fluorescence microscopy confirmed exosomes internalization by HG-treated MPC5 cells. BMSC-Exos upregulated Beclin1 and LC3-II while downregulating Bcl-2 and desmin, indicating enhanced autophagy. High-throughput sequencing revealed miR-143-3p enrichment in BMSC-Exos, and Bcl-2 was confirmed as a direct target of miR-143-3p. Exosomes from miR-143-3p mimic-transfected BMSCs further promoted autophagy and podocin expression. In DKD mice, BMSC-Exos reduced blood glucose, urinary albumin-to-creatinine ratio (UACR), and ameliorated renal damage, whereas miR-143-3p inhibition attenuated these effects. Conclusions: BMSC-Exos deliver miR-143-3p to target Bcl-2, thereby activating Beclin1-mediated autophagy and ameliorating DKD. This study elucidates a novel autophagy regulatory mechanism supporting BMSC-Exos as a cell-free therapy for DKD. Full article

Osteoarthritis (OA) is a progressive joint disorder affecting over 250 million people globally and is characterized by chronic pain and disability. Among its key pathogenic mechanisms are mitochondrial dysfunction and elevated reactive oxygen species (ROS), often triggered by inflammatory mediators such as lipopolysaccharide (LPS). This study evaluates the protective effects of curcumin on mitochondrial function, autophagy, and apoptosis in an in vitro model of OA. Human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes using MesenCult™-ACF medium. Differentiation was confirmed by histological staining for Type II Collagen, Alcian Blue, and Toluidine Blue. LPS was used to induce an OA-like inflammatory response. Mitochondrial membrane potential (ΔΨm) was assessed using Rhodamine 123 staining. Autophagy and apoptosis were evaluated using Acridine orange and propidium iodide staining, respectively. Western blotting was performed to analyze the expression of pro-caspase-3, Bcl-2, Beclin-1, LC3-I/II, and GAPDH. LPS significantly impaired mitochondrial function, limited autophagy, and enhanced apoptotic signaling (reduced pro-caspase-3). Curcumin (25 µM and 100 µM) restored ΔΨm, increased Beclin-1 and LC3-II, and maintained pro-caspase-3 expression, with Bcl-2 showing a non-monotonic response (higher at 25 µM than at 100 µM). Curcumin exerted cytoprotective effects in inflamed chondrocytes by stabilizing ΔΨm, promoting autophagy, and attenuating apoptotic activation, supporting its multi-target therapeutic potential in OA. Full article

Objectives: To investigate the characteristics of monoclonal human urine-derived stem cells (hUSCs) obtained through different culture protocols and compare their therapeutic effects on renal ischemia–reperfusion injury in mice. Methods: Monoclones of hUSCs derived from the urine of healthy volunteers were isolated and cultured using two different culture media. Flow cytometry, qRT-PCR and RNA sequencing were employed to characterize each monoclonal clone of multipotent stem cells across multiple passages. To evaluate their therapeutic effects on unilateral renal ischemia–reperfusion injury in BALB/c mice, 5 × 10⁵ hUSCs from each monoclonal clone were intravenously administered to mice via the tail vein, followed by assessments using Masson staining, qRT-PCR and renal tissue transcriptomics analysis. Results: Four monoclonal strains were successfully isolated from four fresh urine samples of a healthy young male volunteer: three cultured in EGM-MV medium and one in our modified medium. All four strains demonstrated stable expression of mesenchymal stem cell-related markers over eight passages of expansion. Bioinformatics analysis of multiple cell transcriptome datasets revealed that these four cell strains are more closely related to kidney tissue than to bone marrow mesenchymal stem cells (BMSCs), adipose-derived mesenchymal stem cells (ADMSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), and urothelial cells. Additionally, significant differences were observed in the expression of genes associated with kidney development among the four monoclonal strains. Furthermore, the therapeutic effects of different monoclonal clones on renal ischemia–reperfusion injury in mice showed notable variability. Conclusions: The isolated monoclonal urine-derived stem cells in this study were showed closer transcriptomic similarity to renal progenitor cells than to other mesenchymal stem cell types and possessed differential therapeutic effects on acute kidney injury. Full article

This study aims to compare the osteogenic potential of premixed hydraulic calcium silicate-based sealers (HCSSs) with an epoxy resin-based sealer in human bone marrow-derived stem cells (hBMSCs). Three HCSSs (White Endoseal MTA, One-Fil, EndoSequence BC Sealer) were compared with AH Plus Jet, an epoxy resin-based sealer. Disk-shaped specimens were prepared using sterilized Teflon tubes and immersed in osteogenic medium to create eluates. hBMSCs were cultured in each eluate, and osteogenic potential was assessed by alkaline phosphatase (ALP) activity (n = 6), Alizarin Red-S (ARS) staining (n = 6), quantitative real-time polymerase chain reaction (qPCR) (n = 3), and Western blot analysis. Statistical analyses were conducted using SPSS (version 24.0), with significance set at p < 0.05. All experimental groups exhibited higher ALP activity than the control on day 4. ARS staining of HCSSs differed significantly from AH Plus Jet on day 14 (p < 0.05), while White Endoseal MTA exhibited the highest intensity. qPCR revealed that EndoSequence BC Sealer induced the highest SMAD1 expression on day 4, while One-Fil and EndoSequence BC Sealer significantly upregulated RUNX2 expression compared with AH Plus Jet (p < 0.05). Western blotting confirmed that EndoSequence BC Sealer induced the highest RUNX2 protein expression. Collectively, premixed HCSSs promoted superior mineralization and RUNX2 expression compared to conventional resin-based sealer in hBMSCs. Full article

Graphene-based nanomaterials, including graphene oxide (GO) and graphene quantum dots (GQDs), exhibit exceptional properties, which might facilitate the functional modification of TiO₂ nanotubes (NTs) for enhanced rapid osseointegration. This study investigated the effects of GO/GQD-deposited TiO₂-NTs on cell proliferation, osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs), and early osseointegration in male 6-week-old Sprague Dawley (SD) rats. TiO₂-NTs (control group) were fabricated on titanium substrates via anodic oxidation. GO and GQDs were electrochemically deposited onto the TiO₂-NTs using cyclic voltammetry with 0.5 mg/mL GO and 0.1 mg/mL GQD dispersions to form NT-GO and NT-GQDs. In vitro assays evaluated cell adhesion, proliferation, and osteogenic differentiation. Implants were randomly inserted into one femoral epiphysis of nine rats (n = 3), and osseointegration was evaluated using micro-computed tomography and sequential fluorescence labeling at 2, 4, and 6 weeks post-implantation. Statistical analysis was conducted using ANOVA. Cyclic voltammetry successfully synthesized NT-GO and NT-GQDs, with Raman spectra confirming D and G bands. Both NT-GO and NT-GQDs exhibited superior cell adhesion, proliferation, and enhanced osteogenic differentiation compared with TiO₂-NTs. Notably, the NT-GQDs significantly promoted new bone formation in vivo. The integration of graphene nanomaterials onto TiO₂-NTs improves biocompatibility and accelerates osteogenesis, suggesting a promising strategy for enhancing osseointegration in orthopedic and dental implants. Full article

Treatment for acute liver failure (ALF) is constrained by shortages of liver transplant donors and immune rejection. Porcine bone marrow mesenchymal stem cells (pBMSCs) demonstrate clinical potential in xenotransplantation due to their abundant availability, low immunogenicity, and strong proliferative activity. This study is the first to investigate the reparative effects and mechanisms of pBMSCs derived from Luopan Mountain pigs in a D-galactosamine (D-GalN)-induced ALF rat model. The results demonstrated that tail-vein transplantation of pBMSCs significantly improved survival rates in ALF rats; reduced serum ALT, AST, and TBIL levels; enhanced hepatic glycogen metabolism; and mitigated histopathological liver damage. Additionally, pBMSC transplantation upregulated serum HGF, IGF-1, and VEGF levels while inhibiting hepatocyte apoptosis. Mechanistic studies indicate that pBMSCs promote liver function recovery and regeneration by activating the PI3K/Akt/mTOR signaling pathway and suppressing its key negative regulator, PTEN, by regulating the expression of key genes involved in inflammation, fibrosis, proliferation, and apoptosis. This study provides crucial experimental evidence for the use of pBMSCs in treating acute liver failure (ALF) and lays the groundwork for its clinical translation in the field of xenotransplantation. Full article

Concentrated growth factor (CGF) is an autologous blood-derived product widely used in regenerative medicine due to its high concentration of growth factors and platelets. In this study, the ability of primary stem cells isolated from human CGF to differentiate into adipocytes, endothelial cells, and neuronal-like cells was evaluated in vitro. CGF primary cells (CPCs) were obtained from CGF fragments and characterized after one month in culture. These cells were positive for the surface markers CD105, CD45, CD31, and CD14, and also expressed mRNA levels of the stemness markers Nanog and Oct3/4 comparable to human bone marrow mesenchymal stem cells (BMSCs). Results showed that, following appropriate differentiation protocols, CPCs, similarly to BMSCs, were able to differentiate into adipogenic, endothelial, and neuronal lineages, acquiring specific phenotypic and molecular markers. Adipogenic induction resulted in lipid accumulation and the upregulation of key genes, including PLIN2, FABP4, CD36, and FASN. Under pro-endothelial conditions, the cells exhibited increased expression of endothelial markers, eNOS, VEGFR-2, and CD31. Neuronal induction promoted the expression of β-tubulin III, Nestin, and Neurofilament. Overall, this work highlights the remarkable plasticity of CPCs and supports their potential application in multilineage regenerative therapies. Full article

Scaffold architecture is a key determinant of cell behavior and tissue regeneration in bone tissue engineering, yet the influence of pore size under dynamic culture conditions remains incompletely understood. This study aimed to evaluate the effects of scaffold pore size on osteogenic differentiation of porcine bone marrow-derived mesenchymal stem cells (pBMSCs) cultured in a rotational oxygen-permeable bioreactor system (ROBS). Three-dimensionally (3D) printed beta-tricalcium phosphate (β-TCP) scaffolds with pore sizes of 500 µm and 1000 µm were seeded with pBMSC and cultured for 7 and 14 days under dynamic perfusion conditions. Gene expression analysis revealed significantly higher levels of osteogenic markers (Runx2, BMP-2, ALP, Osx, Col1A1) in the 1000 µm group, particularly at the early time point, with the later-stage marker Osteocalcin (Ocl) rising faster and higher in the 1000 µm group, after a lower expression at 7 days. ALP activity assays corroborated these findings. Despite having lower mechanical strength, the 1000 µm scaffolds supported a homogeneous cell distribution and high viability across all regions. These results suggest that larger pore sizes enhance early osteogenic commitment by improving nutrient transport and fluid flow in dynamic culture. These findings also support the use of larger-pore scaffolds in bioreactor-based preconditioning strategies and underscore the clinical importance of promoting early osteogenic differentiation to reduce in vitro culture time, an essential consideration for the timely preparation of implantable grafts in bone tissue engineering. Full article

Acute myeloid leukemia (AML) proliferation is significantly influenced by the interactions between leukemia blasts and the bone marrow (BM) microenvironment. Specifically, bone marrow mesenchymal stem cells (BMSCs) derived from AML patients (AML-MSCs) are known to support leukemia growth and facilitate disease progression. Studies have demonstrated that the transfer of mitochondria from MSCs to AML blasts not only aids in disease progression but also contributes to chemotherapy resistance. Furthermore, BM stromal cells can trigger a metabolic shift in malignant cells from mitochondrial respiration to glycolysis, which enhances both growth and chemo-resistance. This study focuses on identifying transcriptional and metabolic alterations in AML-MSCs to uncover potential targeted therapies for AML. We employed RNA sequencing and microarray analysis on MSCs cocultured with leukemic cells (MLL-AF9) and on MSCs isolated from both non-leukemic and MLL-AF9 leukemic mice. The Gene Set Enrichment Analysis (GSEA) indicated a significant downregulation of gene sets associated with oxidative phosphorylation and glycolysis in AML-MSCs. Furthermore, coculture of MSCs from wild-type mice (WT-MSCs) and a healthy donor individual (HD-MSCs) with AML cells demonstrated reduced oxidative phosphorylation and glycolysis. These metabolic changes were consistent in AML-MSCs derived from both leukemic mice and patients. Our results indicate that AML cells diminish the metabolic capacity of MSCs, specifically targeting oxidative phosphorylation and glycolysis. These findings suggest potential metabolic vulnerabilities that could be exploited to develop more effective therapeutic strategies for AML. Full article

Background/Objectives: Herein, we investigated the effect of platelet-rich fibrin (PRF) treatment combined with no-ozone cold plasma (NCP) on growth factor levels, rat bone-marrow stem cell (rBMSC) proliferation, and alkaline phosphatase (ALP) activity in the early stage of differentiation into osteoblasts. Methods: The PRF used in the experiment was prepared by collecting blood from the jugular vein of rats, followed by centrifugation. The obtained PRF was treated with NCP, and the cell culture media were conditioned with the PRF extracts alone or with NCP-treated PRF extracts. Three different experimental groups were defined: no treatment (NT); cell culture media extracted from PRF (PRF); and cell culture media extracted from PRF treated with NCP (PRF + NCP). Enzyme-linked immunosorbent assays were performed to determine the levels of transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) AB. Water-soluble Tetrazolium-1 assay was performed to measure cell proliferation in rBMSCs. To analyze cell differentiation into osteoblasts, ALP staining and real-time PCR were performed. Results: Growth factor levels increased in response to treatment (TGF-β: p < 0.001, PDGF AB: p < 0.05), and the cell proliferation rate increased with treatment (145.29% and 150.05% for PRF and the PRF + NCP groups, respectively, relative to the NT group, p < 0.001). Evaluation of the ALP staining intensity and mRNA expression levels showed that the ALP activity was highest in the PRF + NCP group (p < 0.001). Conclusions: Our results confirmed that NCP treatment enhanced the release of several different growth factors contained in PRF to the culture media and that treatment with PRF and NCP increased the proliferation of rBMSCs and their differentiation into osteoblasts. Full article

Osteoarthritis (OA) is a prevalent and debilitating joint disorder that affects a substantial proportion of the global population, underscoring the urgent need for therapeutic strategies that extend beyond symptomatic management. Although mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality, their clinical application remains constrained by several inherent limitations. This study explores a cell-free alternative by investigating the therapeutic potential of exosomes derived from bone marrow (BMSCs), adipose tissue (ADSCs), and umbilical cord (UMSCs) MSCs in mitigating OA pathogenesis, utilizing both in vitro and ex vivo models. Exosomes from each MSC source were isolated and characterized through nanoparticle tracking analysis, transmission electron microscopy, and Western blotting to confirm their identity and purity. Subsequently, their chondroprotective, anti-inflammatory, and regenerative properties were systematically assessed through evaluations of cell viability, expression profiles of inflammatory and chondroprotective markers, and chondrocyte migration assays. The results demonstrate that all three types of MSC-derived exosomes (MSC-Exos) exhibit low cytotoxicity while significantly suppressing proinflammatory markers and enhancing the expression of chondroprotective genes. Notably, BMSC-Exos and UMSC-Exos displayed superior efficacy in attenuating inflammation, promoting cartilage protection, and inhibiting chondrocyte apoptosis. Furthermore, all MSC-Exos markedly enhanced chondrocyte motility, a critical component of cartilage repair. Collectively, these findings support the therapeutic promise of MSC-Exos, particularly those derived from BMSCs and UMSCs, as a targeted, cell-free approach for the treatment of OA compared to ADSCs. By modulating inflammation, promoting cartilage regeneration, and preventing chondrocyte apoptosis, MSC-Exos may serve as a viable and scalable alternative to current MSC-based therapies for this widespread degenerative disease. Full article

Objectives: Traditional 2D cell cultures on flat surfaces fail to replicate 3D environments, affecting cellular morphology and function. Various 3D techniques (e.g., spheroids, organoids, organs-on-chips, 3D bioprinting) have been used for disease modelling and drug testing, but their application in hard tissues remains challenging. This study aimed to develop a biocompatible 3D culture method for bone tissue organoids using human bone marrow-derived stem cells (hBMSCs) and hydrogels. Methods: hBMSCs were isolated from human jawbone marrow. The control group was cultured under 2D conditions, whereas the experimental group was cultured in a 3D hydrogel environment. In vitro analyses, including flow cytometry and RNA sequencing, were performed. Quantitative data were statistically analysed at a 0.05 level of significance. Results: hBMSCs cultured in 3D hydrogel conditions indicated enhanced reproducibility, increased cell viability, and significant osteogenic differentiation. Genes such as MMP-13, LPL, and SP7 showed substantially higher expression in 3D cultures, with protein-level confirmation by Western blot. These findings suggest that 3D culture more effectively supports the natural growth and differentiation of hBMSCs. Conclusions: Culturing hBMSCs in a 3D environment more closely mimics in vivo conditions, thus promoting the expression and activity of critical proteins involved in hBMSC differentiation. Full article

Background and Objectives: Aging-related bone loss still lacks interventions. As bone marrow-derived mesenchymal stem cells (BMSCs) undergo aging, R-loop-induced DNA replication stress impairs the osteogenic ability of BMSCs. High-mobility group A-2 (Hmga2) acts as a DNA-binding protein, and the understanding of its underlying mechanisms is crucial for developing effective preventive and therapeutic strategies. Materials and Methods: Aging mice were used as the experimental model, and mouse BMSCs were isolated from their femurs. Hmga2 was achieved through specific gene delivery methods. R-loop formation was detected using dot blotting, chromatin immunoprecipitation (ChIP), and DNA–RNA immunoprecipitation (DRIP) assays. Osteogenic differentiation was evaluated. Results: R-loops were highly accumulated in aging BMSCs. Notably, the key regulator Hmga2 reversed the accumulation of R-loops in aging BMSCs. Hmga2 overexpression significantly decreased the senescence and improved the osteogenic differentiation of aging mBMSCs. Mechanistically, R-loop-forming sequence (RLFS) regions were confirmed in key osteogenesis-related genes, including runt-related transcription factor 2 (Runx2). Hmga2 bound to the RLFS region of Runx2 and promoted its expression by reducing the R-loop level. More, Hmga2 treatment delivered via the AAV system effectively decreased bone loss in aging mice and increased the serum bone turnover biomarkers and collagen remodeling. Conclusions: Our study demonstrates that Hmga2 acts as an activator of aging BMSCs, significantly promoting their osteogenic ability by eliminating the aging-induced DNA replication stress caused by R-loops. Our findings provide new insights into the mechanisms of aging-related bone loss, suggesting that Hmga2 may be a new strategy for alleviating the bone loss phenotype in aging individuals. Full article

Bone diseases represent a growing healthcare challenge due to population aging and lifestyle changes. Although bone has a natural regenerative capacity, approximately 10% of fractures fail to heal properly, requiring advanced therapeutic approaches. Bone tissue engineering (BTE) has advanced the use of osteoinductive and osteoconductive biomaterials to support bone regeneration. Among them, Bio-Oss^® Collagen, a composite of bovine hydroxyapatite and collagen, has shown excellent biocompatibility and bioactivity properties. This study analyzes the effect of Bio-Oss^® Collagen on human bone marrow-derived mesenchymal stem cells (hBMSCs), assessing its osteoinductive and immunomodulatory potential. After 7 days of culture, the biomaterial modulated the expression of key genes involved in osteogenesis and chondrogenesis, which are known for their role in bone formation and maturation. At the same time, a downregulation of genes associated with bone resorption was observed. Secretome analysis revealed a controlled release of pro-regenerative cytokines, suggesting a role of the biomaterial in modulating inflammation to promote bone regeneration. Furthermore, immunofluorescence confirmed the high expression of osteocalcin and osteopontin, which are key markers of bone mineralization. These findings indicate that Bio-Oss^® Collagen supports osteogenesis and modulates the immune response, creating a microenvironment favorable for bone regeneration. Full article

Background/Objectives: Mandibular reconstruction following trauma or oncologic resection is crucial for restoring function and aesthetics. While autologous bone grafting remains the gold standard, it presents challenges such as donor site morbidity and graft availability. Bone tissue engineering (BTE) offers an innovative alternative, integrating scaffolds, osteogenic cells, and bioactive factors to regenerate functional bone. This systematic review evaluates BTE strategies for mandibular reconstruction, focusing on critical-sized defects in large animal models and their translational potential for clinical applications. Methods: A systematic review was performed following PRISMA guidelines. Eligible studies involved large animal models and critical-sized mandibular defects treated with at least two BTE components (scaffold, osteogenic cells, or growth factors). Quality and bias assessments were conducted using ARRIVE guidelines and SYRCLE tools. Results: Of the 6088 studies screened, 27 met the inclusion criteria, focusing on critical-sized mandibular defects in large animal models such as pigs, sheep, and dogs. Common scaffolds included β-tricalcium phosphate (β-TCP), poly-lactic-co-glycolic acid (PLGA), and polycaprolactone (PCL), frequently combined with bone marrow-derived mesenchymal stem cells (BMSCs) and growth factors like recombinant human bone morphogenetic protein-2 (rhBMP-2). Preclinical outcomes demonstrated effective bone regeneration, vascularization, and biomechanical restoration. Advanced strategies, including in vivo bioreactors and 3D-printed scaffolds, further enhanced regeneration. However, challenges such as incomplete scaffold degradation, hypoxic conditions within constructs, and variability in growth factor efficacy and dose optimization were observed, emphasizing the need for further refinement to ensure consistent outcomes. Conclusions: BTE shows promise in mandibular reconstruction, achieving bone regeneration and functional restoration in preclinical models of critical-sized defects. However, challenges such as scaffold optimization, vascularization enhancement, and protocol standardization require further investigation to facilitate clinical translation. These findings emphasize the need for refinement to achieve consistent, scalable outcomes for clinical use. Full article