Search Results (244)

Reparative tertiary dentinogenesis requires the recruitment and odontogenic differentiation of dental pulp stem cells (DPSCs). Extracellular vesicles (EVs) as bioactive molecules have gained attention in regenerative medicine for their ability to mediate tissue repair through intercellular communication, influencing cell recruitment, proliferation, and differentiation. This study aimed to evaluate the effects of EVs on DPSC homing and odontogenic differentiation for dentin regeneration. DPSC-derived EVs were cultured in either growth (EV-G) or odontogenic differentiation (EV-O) conditions and isolated using a modified precipitation method. EVs were characterized by nanoparticle tracking analysis, scanning electron microscopy, antibody array, and cellular uptake assay. Treatment with 5 × 10⁸ EVs/mL significantly enhanced DPSC chemotaxis and proliferation compared with a no-treatment control and a lower dosage of EV (5 × 10⁷ EVs/mL). Gene expression and biochemical analyses revealed that EV-O up-regulated odontogenic markers including collagen type 1A1 (COL1A1), runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP). EV-O enhanced dentin regeneration by approximately 55% over vehicle controls in a rabbit partial dentinotomy/pulpotomy model. We identified key microRNAs (miR-21-5p, miR-221-3p, and miR-708-3p) in EV-O involved in cell homing and odontogenesis. In conclusion, our EV-based cell homing and odontogenic differentiation strategy has significant therapeutic potential for dentin regeneration. Full article

High-altitude pulmonary edema (HAPE) is a severe condition associated with high-altitude environments, and its molecular mechanism has not been fully elucidated. This study systematically analyzed the DNA methylation status of HAPE patients and healthy controls using reduced-representation bisulfite sequencing (RRBS) and 850K DNA methylation chips, identifying key differentially methylated regions (DMRs). Targeted bisulfite sequencing (TBS) revealed significant abnormalities in DMRs of five genes, azurocidin 1 (AZU1), growth factor receptor bound protein 7 (GRB7), mannose receptor C-type 2 (MRC2), RUNX family transcription factor 3 (RUNX3), and septin 9 (SEPT9). The abnormal expression of AZU1 was validated using peripheral blood leukocytes from HAPE patients and normal controls, as well as rat lung tissue, indicating its potential importance in the pathogenesis of HAPE. To further validate the function of AZU1, we conducted experimental studies using a hypobaric hypoxia injury model in Human Umbilical Vein Endothelial Cells (HUVEC). The results showed that AZU1 was significantly upregulated under hypobaric hypoxia. Knocking down AZU1 mitigates the reduction in HUVEC proliferation, angiogenesis, and oxidative stress damage induced by acute hypobaric hypoxia. AZU1 induces cellular oxidative stress via the p38/mitogen-activated protein kinase (p38/MAPK) signaling pathway. This study is the first to elucidate the mechanism of AZU1 in HAPE via the p38/MAPK pathway, offering novel insights into the molecular pathology of HAPE and laying a foundation for future diagnostic and therapeutic strategies. Full article

Mechanical stresses affect a variety of cellular events in relation to the frequency, magnitude, and duration of the stimuli applied. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) are pluripotent stem cell aggregates and comprise all somatic cells. Numerous studies have highlighted the effects of mechanosignals on stem cells, whereas their impact on EBs has been barely investigated. Here, we examined how cyclic tensile stress affects the behavior of EBs to differentiate into mineralized osteocytes by applying 2% elongation at 0.5 Hz frequency for 1 h once or 1 h every other day for 5 or 14 days in osteogenic medium. EBs that expressed undifferentiated markers, Oct4 and Sox2, were differentiated into mineralized cells, along with the accumulation of runt-related transcription factor 2 (RUNX2) and β-catenin in osteogenic medium. The application of tensile force inhibited EB’ mineralization via the downregulation of bone sialoprotein, osteocalcin, osterix, and RUNX2. While the transfection with si-β-catenin did not affect the osteogenic potency of EBs at a significant level, treatment with 10 μM of PD98059, but not of SP600125 or SB203580, diminished the mineralization of EBs and the expression of RUNX2 and RUNX2-regulated osteoblastic genes. The level of phosphorylated extracellular signal-regulated kinase-1 (p-ERK1) rather than p-ERK2 was more apparently diminished in tension-applied EBs. The transfection with si-ERK1, but not with si-ERK2, suppressed the mineralization of osteogenic medium-supplied EBs and the expression of osteoblast-specific genes. Collectively, this study demonstrates that tensile stress inhibits osteogenic potency of EBs by downregulating ERK1-mediated signaling and osteogenic gene expression. Full article

Mechanical stress is known to be a pivotal risk factor in the development of OA. However, the involvement of repetitive compressive loading in mitochondrial dysfunction in chondrocytes remains unclear. The aim of this study was to investigate whether physiologic levels of repetitive mechanical force affect the regulation of energy metabolism and activities of mitochondrial function regulators, sirtuin 1 and nicotinamide adenine dinucleotide (NAD) in chondrocytes, and to clarify any correlation with chondrocyte catabolic activity. Repetitive physiological mechanical stress was applied in a 3D chondrocyte-collagen scaffold construct, and the 3D cultured tissues were collected at different time points by collagenase treatment to collect cellular proteins. Changes in chondrocyte activity (cell proliferation, MMP-13 production), energy metabolism regulator levels (sirtuin 1), mitochondrial function (ATP production, NAD level), and the expression level of the osteogenic and hypertrophic chondrogenic transcription factor, runt-related transcription factor 2 (Runx2), were measured. Treatment with repetitive compressive loading resulted in no significant change in the cell viability of chondrocytes. In the repetitive mechanical loading group, there were statistically significant increases in MMP-13 production and expression of both sirtuin 1 and Runx2 in chondrocytes relative to the non-loading control group. Furthermore, ATP production and NAD activity in mitochondria decreased in the repetitive mechanical loading group. Our present study reveals that in chondrocytes, repetitive compressive loading accelerated sirtuin activation, which requires and consumes NAD within mitochondria, leading to a decrease of NAD and ultimately in reduced mitochondrial ATP production. Additionally, since sirtuin 1 is known to positively regulate Runx2 activity in chondrocytes, the activation of sirtuin 1 by repetitive load stimulation may induce an increase in the expression of Runx2, which promotes the expression of MMP-13, and subsequently enhances MMP-13 production. Our findings indicate that repetitive compression loading-mediated mitochondrial dysfunction plays a pivotal role in the progression of OA, primarily by driving the downregulation of ATP production and promoting the expression of the matrix-degrading enzyme MMP-13. 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

Berberine has been widely studied for its biological functions in various diseases, including cancer, diabetes, and cardiovascular diseases. Nevertheless, structural modifications of berberine have been demonstrated to augment its pharmacological efficacy in specific biological processes, particularly osteogenesis. In this study, we aimed to explore new berberine derivatives with pro-osteogenic activity and molecular mechanisms. Our results demonstrated that compound 13 is the most effective among the tested compounds. Compound 13 significantly enhanced BMP4-induced alkaline phosphatase (ALP) staining and increased the transcriptional activity of osteogenic markers such as ALP, Runt-related gene 2 (Runx2), and Osterix at both the mRNA and protein levels. Furthermore, we found that the Akt and PKC signaling pathways play crucial roles in compound 13-induced osteogenesis via treatment with specific inhibitors. The molecular docking results supported the potential interaction between compound 13 and these kinases. These findings highlighted the regulatory role of compound 13 in osteoblast differentiation via the Akt and PKC signaling pathways. Overall, our study provides compelling evidence that compound 13 is a promising therapeutic candidate for the treatment of osteoporosis, with the potential for further development and optimization to improve bone health and strength. Full article

Background: Osteoporosis has become an inevitable health issue with global aging, and the current drug treatments often have adverse side effects, highlighting the need for safer and more effective therapies. Collagen-derived peptides are promising alternatives due to their favorable safety profile and biological activity. This study aimed to investigate the osteogenic and anti-apoptotic properties of collagen peptide UU1 (GASGPMGPR) in addition to its antioxidant activity. Methods: The effects of UU1 were evaluated in MC3T3-E1 cells by assessing osteogenic markers, including alkaline phosphatase (ALP), Cyclin D1, runt-related transcription factor 2 (Runx2), and Akt/β-catenin signaling. Western blot analysis quantified collagen I, osteocalcin, and phosphorylated Akt levels. Anti-apoptotic effects were measured via p-Akt levels and the Bax/Bcl-2 ratio. Computational molecular docking was performed to explore the molecular mechanism of UU1 via its interaction with epidermal growth factor receptor (EGFR) and collagen-binding integrin. Results: UU1 treatment promoted cell differentiation, with elevated ALP, Cyclin D1, Runx2, and Akt/β-catenin signaling. Notably, at 0.025 mg/mL, UU1 upregulated the levels of collagen I, osteocalcin, and phosphorylated Akt by 2.14, 3.37, and 1.95 times, respectively, compared to the control. Additionally, UU1 exhibited anti-apoptotic effects, indicated by increased p-Akt levels and a reduced Bax/Bcl-2 ratio. Molecular docking analysis suggested that UU1 could assist the dimerization of EGFR, facilitating downstream signaling transductions and activating collagen-binding integrin. Conclusions: These findings highlight UU1 as a multifunctional peptide with antioxidant, osteogenic, and anti-apoptotic properties, positioning it as a promising candidate for anti-osteoporosis applications in the food and pharmaceutical industries. Full article

Aging is associated with the accumulation of cellular damage due to oxidative stress and chronic low-grade inflammation, collectively referred to as “inflammaging”. This contributes to the functional decline in various tissues, including the brain and skeletal system, which closely interplay. Mesenchymal stem cells (MSCs), known for their regenerative potential and ability to modulate inflammation, offer a promising therapeutic approach to counteract aging-related declines. In this study, we investigated the effects of homotaurine (a small molecule with neuroprotective properties) on MSCs and its effects on osteogenesis. We found that homotaurine treatment significantly reduced reactive oxygen species (ROS) levels, improved MSC viability, and modulated key stress response pathways, including the sestrin 1 and p21 proteins. Furthermore, homotaurine promoted osteogenesis and angiogenesis in zebrafish models by enhancing the expression of critical osteogenesis-associated genes, such as those coding for β-catenin and Runt-related transcription factor 2 (Runx2), and increasing the levels of the kinase insert domain receptor-like angiogenesis marker in aged zebrafish. In Parkinson’s disease models using patient-specific midbrain organoids with the leucine-rich repeat kinase 2 G2019S mutation, homotaurine treatment enhanced β-catenin expression and reduced ROS levels, highlighting its potential to counteract the oxidative stress and dysfunctional signaling pathways associated with neurodegeneration. Our findings suggest that homotaurine not only offers neuroprotective benefits but also holds promise as a dual-target therapeutic strategy for enhancing both neuronal and bone homeostasis in aging and neurodegenerative diseases. Full article

We investigated the roles of low-density lipoprotein receptor-related protein (LRP) 4 and its ligand Agrin in the pathophysiology of cartilage degeneration. Immunohistochemical analysis of human normal articular cartilage and cartilage tissues from patients with osteoarthritis (OA) obtained during surgery of the knee joint showed marked LRP4 expression in the early stages of OA, which then decreased with cartilage degeneration, whereas Agrin was consistently increased with cartilage degeneration. In normal human articular chondrocytes (NHACs), mild cyclic tensile strain (CTS) (0.5 Hz, 5% elongation, 2 h) increased the expression of LRP4 and aggrecan (ACAN), while intense CTS (0.5 Hz, 10% elongation, 6 h) increased the expression of Agrin without affecting LRP4 expression. Treatment with recombinant human (rh) Agrin downregulated the mRNA expression of LRP4 and ACAN, but upregulated the expression of LRP5/6, SRY-box transcription factor 9 (SOX9), Runt-related transcription factor 2 (RUNX2), and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4). Immunocytochemistry and Western blot analysis showed that rhAgrin treatment upregulated the expression of β-catenin and SOX9. Agrin knockdown by siAGRN transfection partially reduced the nuclear protein expression of β-catenin, which was increased with intense CTS. LRP4 knockdown by siLRP4 transfection increased the expression of LRP5/6, SOX9, RUNX2, ADAMTS-4, and Agrin. These results suggested that intense CTS increases the expression of Agrin, which might interfere with the role of LRP4 in the inhibition of LRP5/6 and their downstream β-catenin signaling, leading to cartilage degeneration. Full article

Pulmonary and extrapulmonary manifestations have been reported following infection with SARS-CoV-2, the causative agent of COVID-19. The virus persists in multiple organs due to its tropism for various tissues, including the skeletal system. This study investigates the effects of SARS-CoV-2 infection, including both ancestral and Omicron viral strains, on differentiating mesenchymal stem cells (MSCs), the precursor cells, into osteoblasts. Although both viral strains can productively infect osteoblasts, precursor cell infection remained abortive. Viral exposure during osteoblast differentiation demonstrates that both variants inhibit mineral and organic matrix deposition. This is accompanied by reduced expression of runt-related transcription factor 2 (RUNX2) and increased levels of interleukin-6 (IL-6), a cytokine that negatively regulates osteoblast differentiation. Furthermore, the upregulation of receptor activator of nuclear factor kappa B ligand (RANKL) strongly suggests that the ancestral and Omicron variants may disrupt bone homeostasis by promoting osteoclast differentiation, ultimately leading to the formation of bone-resorbing cells. This process is dependent of spike glycoprotein since its neutralization significantly reduced the effect of infective SARS-CoV-2 and UV-C inactivated virus. This study underscores the capacity of ancestral and Omicron SARS-CoV-2 variants to disrupt osteoblast differentiation, a process essential for preserving the homeostasis and functionality of bone tissue. Full article

Farnesoid X receptor (FXR), a nuclear receptor, is expressed in calvaria and bone marrow stromal cells and plays a role in bone homeostasis. However, the mechanism of FXR-activated osteoblast differentiation remains unclear. In this study, we investigated the regulatory mechanism underlying FXR-activated osteoblast differentiation using bone morphogenetic protein-2 (BMP-2)-induced mouse ST-2 mesenchymal stem cells. We also synthesized a novel FXR agonist, FLG390, and compared its biological effects in osteoblast differentiation with a known FXR agonist, chenodeoxycholic acid (CDCA). As an FXR agonist, FLG390 accelerated osteoblast differentiation to a comparable extent with CDCA, enhancing alkaline phosphatase (ALP) activity and the expression of osteoblast differentiated-related genes such as ALP, collagen type 1 α1 chain (COL1A1), and runt-related transcription factor 2 (RUNX2). FXR activation elevated the expression of cyclooxygenase (COX)-2 and the production of prostaglandin (PG) E₂ in the early phase of osteoblast differentiation. A selective COX-2 inhibitor and an antagonist of EP4 receptors, one of PGE₂ receptors, partially suppressed FXR-activated osteoblast differentiation. Moreover, treatment with either inhibitor during the first 6 h after initiating osteoblast differentiation repressed FXR-activated osteoblast differentiation to the same extent as did the treatment for 6 d. Therefore, a novel FXR agonist, FLG390, exhibited potency comparable to CDCA. FXR activation promoted the early phase of osteoblast differentiation via the COX-2-PGE₂-EP4 axis, representing a potential target for control of bone metabolism. Full article

Calcium deposition in vascular smooth muscle cells (VSMCs), a form of ectopic ossification in blood vessels, can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that CCAAT/enhancer-binding protein beta (C/EBPβ) potentiates calcium deposition in VSMCs and mouse aorta induced by inorganic phosphate (Pi) or vitamin D₃. Based on cDNA microarray and RNA sequencing data of Pi-treated rat VSMCs, C/EBPβ was found to be upregulated and thus selected for further evaluation. Quantitative RT-PCR and Western blot analysis confirmed that C/EBPβ was upregulated in Pi-treated A10 cells, a rat VSMC line, as well as vitamin D₃-treated mouse aorta. The overexpression of C/EBPβ in A10 cells increased bone runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteopontin (OPN) mRNA in the presence of Pi, as well as potentiating the Pi-induced increase in calcium contents. The Runx2 expression was increased by C/EBPβ through Runx2 P2 promotor. Our results suggest that a Pi-induced increase in C/EBPβ is a critical step in vascular calcification. Full article

Oxidative stress (OS) is a common feature of many inflammatory diseases, oral pathologies, and aging processes. The impact of OS on periodontal ligament cells (PDLCs) in relation to oral pathologies, including periodontal diseases, has been investigated in different studies. However, its impact on orthodontic tooth movement (OTM) remains poorly understood. This study used an in vitro model with human PDLCs previously exposed to H₂O₂ to investigate the effects of OS under a static compressive force which simulated the conditions of OTM. Human PDLCs were treated with varying concentrations of H₂O₂ to identify sub-lethal doses that affected viability minimally. To mimic compromised conditions resembling OTM under OS, the cells were pretreated with the selected H₂O₂ concentrations for 24 h. Using an in vitro loading model, a static compressive force (2 g/cm²) was applied for an additional 24 h. The cell viability, proliferation, and cytotoxicity were evaluated using live/dead and resazurin assays. Apoptosis induction was assessed based on caspase-3/7 activity. The gene expression related to bone remodeling (RUNX2, TNFRSF11B/OPG, BGLAP), inflammation (IL6, CXCL8/IL8, PTGS2/COX2), apoptosis (CASP3, CASP8), and autophagy (MAP1LC3A/LC3, BECN1) was analyzed using RT-qPCR. This study suggests an altering effect of previous OS exposure on static-compression-related mechanosensing. Further research is needed to fully elucidate these mechanisms. Full article

In recent years, the demand for orthopedic implants has surged due to increased life expectancy, necessitating the need for materials that better mimic the biomechanical properties of human bone. Traditional metal implants, despite their mechanical superiority and biocompatibility, often face challenges such as mismatched elastic modulus and ion release, leading to complications and implant failures. Polyetheretherketone (PEEK), a semi-crystalline polymer with an aromatic backbone, presents a promising alternative due to its adjustable elastic modulus and compatibility with bone tissue. This study explores the functionalization of sandblasted 3D-printed PEEK disks with the bioactive peptides Aoa-GBMP1α and Aoa-EAK to enhance human osteoblast response. Aoa-GBMP1α reproduces 48–69 trait of Bone Morphogenetic Protein 2 (BMP-2), whereas Aoa-EAK is a self-assembling peptide mimicking extracellular matrix (ECM) fibrous structure. Superficial characterization included X-ray photoelectron spectroscopy (XPS), white light interferometer analysis, static water contact angle (S-WCA), and force spectroscopy (AFM-FS). Biological assays demonstrated a significant increase in human osteoblast (HOB) proliferation, calcium deposition, and expression of osteogenic genes (RUNX2, SPP1, and VTN) on functionalized PEEK compared to non-functionalized controls. The findings suggest that dual peptide-functionalized PEEK holds significant potential for advancing orthopedic implant technology. Full article

KMT2A partial tandem duplication (PTD) involves intragenic KMT2A duplications and has been associated with poorer prognosis. In this study, we evaluated KMT2A PTD in 1277 patients with hematological malignancies using optical genome mapping (OGM). KMT2A PTD was detected in 35 patients with acute myeloid leukemia (AML) (7%), 5 patients with myelodysplastic syndrome (MDS) (2.2%), and 5 patients with chronic myelomonocytic leukemia (CMML) (7.1%). The PTDs varied in size, region, and copy number. An Archer RNA fusion assay confirmed KMT2A PTD in all 25 patients tested: 15 spanning exons 2 to 8 and 10 spanning exons 2 to 10. Most patients exhibited a normal (n = 21) or non-complex (n = 20) karyotype. The most common chromosomal abnormalities included loss of 20q or 7q and trisomy 11/gain of 11q. All patients had gene mutations, with FLT3 ITD and DNMT3A prevalent in AML and DNMT3A and RUNX1 common in MDS and CMML. Among patients who received treatment and had at least one follow-up bone marrow evaluation, 82% of those with de novo AML achieved complete remission after initial induction chemotherapy, whereas 90% of patients with secondary or refractory/relapsed AML showed refractory or partial responses. All but one patient with MDS and CMML were refractory to therapy. We conclude that OGM is an effective tool for detecting KMT2A PTD. Neoplasms with KMT2A PTD frequently harbor gene mutations and display normal or non-complex karyotypes. Patients with KMT2A PTD are generally refractory to conventional therapy, except for de novo AML. Full article