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1.
Stem Cell Res Ther ; 15(1): 33, 2024 Feb 07.
Article in English | MEDLINE | ID: mdl-38321490

ABSTRACT

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.


Subject(s)
Extracellular Vesicles , Mesenchymal Stem Cells , Humans , Rats , Male , Animals , Osteogenesis/genetics , Proteomics , Mesenchymal Stem Cells/metabolism , Rats, Inbred Lew , Bone Regeneration/physiology , Cell Differentiation , Extracellular Vesicles/metabolism
2.
Beijing Da Xue Xue Bao Yi Xue Ban ; 56(1): 9-16, 2024 Feb 18.
Article in Chinese | MEDLINE | ID: mdl-38318890

ABSTRACT

OBJECTIVE: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. METHODS: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. RESULTS: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. CONCLUSION: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.


Subject(s)
Core Binding Factor Alpha 1 Subunit , Osteogenesis , Thiolester Hydrolases , Animals , Humans , Mice , Adipose Tissue/cytology , Cell Differentiation/genetics , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Mice, Nude , Osteogenesis/genetics , RNA, Messenger/metabolism , Stem Cells/metabolism , Ubiquitin-Specific Proteases/genetics , Thiolester Hydrolases/metabolism
3.
J Orthop Surg Res ; 19(1): 146, 2024 Feb 19.
Article in English | MEDLINE | ID: mdl-38369459

ABSTRACT

BACKGROUND AND OBJECTIVE: The pathogenesis of coronal suture craniosynostosis is often attributed to the dysregulated cellular dynamics, particularly the excessive proliferation and abnormal osteogenic differentiation of suture cells. Despite its clinical significance, the molecular mechanims of this condition remain inadequately understood. This study is dedicated to exploring the influence of the Periostin/Bone Morphogenetic Protein 1 (BMP1) axis on the growth and osteogenic maturation of Suture Mesenchymal Stem Cells (SMSCs), which are pivotal in suture homeostasis. METHODS: Neonatal TWIST Basic Helix-Loop-Helix Transcription Factor 1 heterozygous (TWIST1+/-) mice, aged one day, were subjected to adenoviral vector-mediated Periostin upregulation. To modulate Periostin/BMP1 levels in SMSCs, we employed siRNA and pcDNA 3.1 vectors. Histological and molecular characterizations, including hematoxylin and eosin staining, Western blot, and immunohistochemistry were employed to study suture closure phenotypes and protein expression patterns. Cellular assays, encompassing colony formation, 5-ethynyl-2'deoxyuridine, and wound healing tests were conducted to analyze SMSC proliferation and migration. Osteogenic differentiation was quantified using Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) staining, while protein markers of proliferation and differentiation were evaluated by Western blotting. The direct interaction between Periostin and BMP1 was validated through co-immunoprecipitation assays. RESULTS: In the TWIST1+/- model, an upregulation of Periostin coupled with a downregulation of BMP1 was observed. Augmenting Periostin expression mitigated craniosynostosis. In vitro, overexpression of Periostin or BMP1 knockdown suppressed SMSC proliferation, migration, and osteogenic differentiation. Periostin knockdown manifested an inverse biological impact. Notably, the suppressive influence of Periostin overexpression on SMSCs was effectively counteracted by upregulating BMP1. There was a direct interaction between Periostin and BMP1. CONCLUSION: These findings underscore the significance of the Periostin/BMP1 axis in regulating craniosynostosis and SMSC functions, providing new insights into the molecular mechanisms of craniosynostosis and potential targets for therapeutic intervention.


Subject(s)
Craniosynostoses , Mesenchymal Stem Cells , Mice , Animals , Osteogenesis/genetics , Bone Morphogenetic Protein 1/metabolism , Craniosynostoses/genetics , Craniosynostoses/metabolism , Cell Differentiation/genetics , Mesenchymal Stem Cells/metabolism , Disease Models, Animal , Cell Proliferation/genetics , Cells, Cultured
4.
Elife ; 122024 Feb 20.
Article in English | MEDLINE | ID: mdl-38376133

ABSTRACT

Glucocorticoid-induced osteonecrosis of the femoral head (GONFH) is a common refractory joint disease characterized by bone damage and the collapse of femoral head structure. However, the exact pathological mechanisms of GONFH remain unknown. Here, we observed abnormal osteogenesis and adipogenesis associated with decreased ß-catenin in the necrotic femoral head of GONFH patients. In vivo and in vitro studies further revealed that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation of bone marrow mesenchymal cells (BMSCs) by inhibiting ß-catenin signaling in glucocorticoid-induced GONFH rats. Col2+ lineage largely contributes to BMSCs and was found an osteogenic commitment in the femoral head through 9 mo of lineage trace. Specific deletion of ß-catenin gene (Ctnnb1) in Col2+ cells shifted their commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice. Overall, we uncover that ß-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of GONFH and identify an ideal genetic-modified mouse model of GONFH.


Subject(s)
Mesenchymal Stem Cells , Osteonecrosis , Humans , Adult , Rats , Mice , Animals , Adipogenesis/genetics , Glucocorticoids , beta Catenin/genetics , Osteogenesis/genetics , Femur Head/pathology , Cell Differentiation , Osteonecrosis/pathology , Homeostasis
5.
J Orthop Surg Res ; 19(1): 134, 2024 Feb 12.
Article in English | MEDLINE | ID: mdl-38342889

ABSTRACT

Stimulating bone formation potentially suggests therapeutics for orthopedic diseases including osteoporosis and osteoarthritis. Osteoblasts are key to bone remodeling because they act as the only bone-forming cells. miR-877-5p has a chondrocyte-improving function in osteoarthritis, but its effect on osteoblast differentiation is unknown. Here, miR-877-5p-mediated osteoblast differentiation was studied. Real-time reverse transcriptase-polymerase chain reaction was performed to measure miR-877-5p expression during the osteogenic differentiation of MC3T3-E1 cells. Osteoblast markers, including alkaline phosphatase (ALP), collagen type I a1 chain, and osteopontin, were measured and detected by alizarin red staining and ALP staining. Potential targets of miR-877-5p were predicted from three different algorithms: starBase ( http://starbase.sysu.edu.cn/ ), PITA ( http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html ), and miRanda ( http://www.microrna.org/microrna/home.do ). It was further verified by dual luciferase reporter gene assay. The experimental results found that miR-877-5p was upregulated during the osteogenic differentiation of MC3T3-E1 cells. Overexpression of miR-877-5p promoted osteogenic differentiation, which was characterized by increased cell mineralization, ALP activity, and osteogenesis-related gene expression. Knockdown of miR-877-5p produced the opposite result. Dual luciferase reporter gene assay showed that miR-877-5p directly targeted eukaryotic translation initiation factor 4γ2 (EIF4G2). Overexpression of EIF4G2 inhibited osteogenic differentiation and reversed the promoting effect of overexpression of miR-135-5p on osteogenic differentiation. These results indicate that miR-877-5p might have a therapeutic application related to its promotion of bone formation through targeting EIF4G2.


Subject(s)
MicroRNAs , Osteoarthritis , Humans , Osteogenesis/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Cell Differentiation/genetics , Osteoblasts/metabolism , Luciferases/metabolism , Osteoarthritis/metabolism , Cells, Cultured , Eukaryotic Initiation Factor-4G/metabolism
6.
J Cell Mol Med ; 28(4): e18123, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38353470

ABSTRACT

Blood vessels are essential for bone development and metabolism. Type H vessels in bone, named after their high expression of CD31 and Endomucin (Emcn), have recently been reported to locate mainly in the metaphysis, exhibit different molecular properties and couple osteogenesis and angiogenesis. A strong correlation between type H vessels and bone metabolism is now well-recognized. The crosstalk between type H vessels and osteoprogenitor cells is also involved in bone metabolism-related diseases such as osteoporosis, osteoarthritis, fracture healing and bone defects. Targeting the type H vessel formation may become a new approach for managing a variety of bone diseases. This review highlighted the roles of type H vessels in bone-related diseases and summarized the research attempts to develop targeted intervention, which will help us gain a better understanding of their potential value in clinical application.


Subject(s)
Bone Diseases, Metabolic , Osteoporosis , Humans , Osteogenesis/genetics , Bone and Bones/metabolism , Osteoporosis/metabolism , Fracture Healing , Neovascularization, Physiologic
7.
Cell Death Dis ; 15(2): 136, 2024 Feb 12.
Article in English | MEDLINE | ID: mdl-38346941

ABSTRACT

Histone methylation plays a crucial role in various cellular processes. We previously reported the in vitro function of histone lysine demethylase 7 A (KDM7A) in osteoblast and adipocyte differentiation. The current study was undertaken to investigate the physiological role of KDM7A in bone homeostasis and elucidate the underlying mechanisms. A conditional strategy was employed to delete the Kdm7a gene specifically in osterix-expressing osteoprogenitor cells in mice. The resulting mutant mice exhibited a significant increase in cancellous bone mass, accompanied by an increase in osteoblasts and bone formation, as well as a reduction in osteoclasts, marrow adipocytes and bone resorption. The bone marrow stromal cells (BMSCs) and calvarial pre-osteoblastic cells derived from the mutant mice exhibited enhanced osteogenic differentiation and suppressed adipogenic differentiation. Additionally, osteoclastic precursor cells from the mutant mice exhibited impaired osteoclast differentiation. Co-culturing BMSCs from the mutant mice with wild-type osteoclast precursor cells resulted in the inhibition of osteoclast differentiation. Mechanistic investigation revealed that KDM7A was able to upregulate the expression of fibroblast activation protein α (FAP) and receptor activator of nuclear factor κB ligand (RANKL) in BMSCs through removing repressive di-methylation marks of H3K9 and H3K27 from Fap and Rankl promoters. Moreover, recombinant FAP attenuated the dysregulation of osteoblast and adipocyte differentiation in BMSCs from Kdm7a deficient mice. Finally, Kdm7a deficiency prevented ovariectomy-induced bone loss in mice. This study establish the role of KDM7A in bone homeostasis through its epigenetic regulation of osteoblast and osteoclast differentiation. Consequently, inhibiting KDM7A may prove beneficial in ameliorating osteoporosis. KDM7A suppresses osteoblast differentiation and bone formation through. upregulating FAP expression and inactivating canonical Wnt signaling, and conversely promotes osteoclast differentiation and bone resorption through upregulating RANKL expression. These are based on its epigenetic removal of the repressive H3K9me2 and H3K27me2 marks from Fap and Rankl promoters. As a result, the expression of KDM7A in osteoprogenitor cells tends to negatively modulate bone mass.


Subject(s)
Bone Resorption , Jumonji Domain-Containing Histone Demethylases , Osteoclasts , Animals , Female , Mice , Bone Resorption/genetics , Bone Resorption/metabolism , Cell Differentiation , Epigenesis, Genetic , Histone Demethylases/genetics , Histone Demethylases/metabolism , Homeostasis , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteogenesis/genetics , RANK Ligand/genetics , RANK Ligand/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism
8.
Biochem Biophys Res Commun ; 701: 149583, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38330731

ABSTRACT

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.


Subject(s)
Mesenchymal Stem Cells , Osteogenesis , Animals , Osteogenesis/genetics , Bone Marrow , Basement Membrane , Cartilage/metabolism , Chondrocytes/metabolism , Phenotype , Chondrogenesis/genetics , Organoids , Cell Differentiation
9.
Chem Biol Drug Des ; 103(2): e14431, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38373741

ABSTRACT

Icariin has been shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanism by which Icariin regulates osteogenic differentiation needs to be further revealed. The viability of BMSCs was assessed by cell counting kit 8 assay. BMSC osteogenic differentiation ability was evaluated by detecting alkaline phosphatase activity and performing alizarin red S staining. The protein levels of osteogenic differentiation-related markers, sirtuin 1 (SIRT1), ubiquitin-specific protease 47 (USP47), and Wnt/ß-catenin-related markers were determined using western blot. SIRT1 mRNA level was measured using quantitative real-time PCR. The regulation of USP47 on SIRT1 was confirmed by ubiquitination detection and co-immunoprecipitation analysis. Icariin could promote BMSC osteogenic differentiation. SIRT1 expression was enhanced by Icariin, and its knockdown suppressed Icariin-induced BMSC osteogenic differentiation. Moreover, deubiquitinating enzyme USP47 could stabilize SIRT1 protein expression. Besides, SIRT1 overexpression reversed the inhibiting effect of USP47 knockdown on BMSC osteogenic differentiation, and USP47 knockdown also restrained Icariin-induced BMSC osteogenic differentiation. Additionally, Icariin enhanced the activity of the Wnt/ß-catenin pathway by upregulating SIRT1. Icariin facilitated BMSC osteogenic differentiation via the USP47/SIRT1/Wnt/ß-catenin pathway.


Subject(s)
Flavonoids , Mesenchymal Stem Cells , Osteogenesis , Sirtuin 1 , Humans , beta Catenin/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Flavonoids/pharmacology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Osteogenesis/drug effects , Osteogenesis/genetics , Sirtuin 1/genetics , Sirtuin 1/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Proteases/metabolism , Gene Knockdown Techniques
10.
ACS Appl Mater Interfaces ; 16(7): 8442-8458, 2024 Feb 21.
Article in English | MEDLINE | ID: mdl-38335323

ABSTRACT

The disruption of host-microbe homeostasis and uncontrolled inflammatory response have been considered as vital causes for developing periodontitis, subsequently leading to an imbalance between the bone and immune system and the collapse of bone homeostasis. Consequently, strategies to modulate the immune response and bone metabolization have become a promising approach to prevent and treat periodontitis. In this study, we investigated the cooperative effects of Nel-like molecule type 1 (Nell-1) and gold nanoparticles (AuNPs) on macrophage polarization, osteoclast differentiation, and the corresponding functions in an experimental model of periodontitis in rats. Nell-1-combined AuNPs in in vitro studies were found to reduce the production of inflammatory factors (TNF-α, p < 0.0001; IL-6, p = 0.0012), modulate the ratio of M2/M1 macrophages by inducing macrophage polarization into the M2 phenotype, and inhibit cell fusion, maturation, and activity of osteoclasts. Furthermore, the local application of Nell-1-combined AuNPs in in vivo studies resulted in alleviation of damages to the periodontal and bone tissues, modulation of macrophage polarization and the activity of osteoclasts, and alteration of the periodontal microbiota, in which the relative abundance of the probiotic Bifidobacterium increased (p < 0.05). These findings reveal that Nell-1-combined AuNPs could be a promising drug candidate for the prevention and treatment of periodontitis. However, Nell-1-combined AuNPs did not show organ toxicity or impair the integrity of intestinal epithelium but alter the gut microbiota, leading to the dysbiosis of gut microbiota. The adverse impact of changes in gut microbiota needs to be further investigated. Nonetheless, this study provides a novel perspective and direction for the biological safety assessment of biomaterials in oral clinical applications.


Subject(s)
Gastrointestinal Microbiome , Metal Nanoparticles , Periodontitis , Rats , Animals , Gold/pharmacology , Osteogenesis/genetics , Metal Nanoparticles/therapeutic use , Periodontitis/drug therapy , Macrophages
11.
Bone Res ; 12(1): 10, 2024 Feb 20.
Article in English | MEDLINE | ID: mdl-38378678

ABSTRACT

Fibrous dysplasia (FD) is a rare, disabling skeletal disease for which there are no established treatments. Growing evidence supports inhibiting the osteoclastogenic factor receptor activator of nuclear kappa-B ligand (RANKL) as a potential treatment strategy. In this study, we investigated the mechanisms underlying RANKL inhibition in FD tissue and its likely indirect effects on osteoprogenitors by evaluating human FD tissue pre- and post-treatment in a phase 2 clinical trial of denosumab (NCT03571191) and in murine in vivo and ex vivo preclinical models. Histological analysis of human and mouse tissue demonstrated increased osteogenic maturation, reduced cellularity, and reduced expression of the pathogenic Gαs variant in FD lesions after RANKL inhibition. RNA sequencing of human and mouse tissue supported these findings. The interaction between osteoclasts and mutant osteoprogenitors was further assessed in an ex vivo lesion model, which indicated that the proliferation of abnormal FD osteoprogenitors was dependent on osteoclasts. The results from this study demonstrated that, in addition to its expected antiosteoclastic effect, denosumab reduces FD lesion activity by decreasing FD cell proliferation and increasing osteogenic maturation, leading to increased bone formation within lesions. These findings highlight the unappreciated role of cellular crosstalk between osteoclasts and preosteoblasts/osteoblasts as a driver of FD pathology and demonstrate a novel mechanism of action of denosumab in the treatment of bone disease.TRIAL REGISTRATION: ClinicalTrials.gov NCT03571191.


Subject(s)
Denosumab , Fibrous Dysplasia of Bone , Animals , Humans , Mice , Denosumab/pharmacology , Fibrous Dysplasia of Bone/drug therapy , Ligands , Osteoblasts/metabolism , Osteogenesis/genetics
12.
Sci Rep ; 14(1): 4155, 2024 02 20.
Article in English | MEDLINE | ID: mdl-38378776

ABSTRACT

Low level laser treatment (LLLT) is known for its photobiostimulatory and photobiomodulatory characteristics, which stimulate cell proliferation, increase cellular metabolism, and improve cellular regeneration. The objective of the present research was to assess the possible influence of infrared diode laser irradiation on the behaviour, attachment, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) seeded on different types of dental implants. Two distinct types of implants, one subjected to laser surface treatment and the other treated with acid etching, were longitudinally divided into two halves and submerged in six wells culture plates. Both implants were subjected to infrared diode laser treatment, and subsequently, the morphology and attachment of cells were examined using scanning electron microscopy (SEM) after 14 and 21 days. The behaviour of (hPDLSCs) towards two types of implants, when exposed to osteogenic medium and low-level laser therapy (LLLT), was assessed using quantitative real-time polymerase chain reaction to measure the expression of stemness markers and osteogenic markers. The scanning electron microscopy (SEM) demonstrated that the application of infrared diode laser irradiation substantially improved the attachment of cells to both types of implants. The stemness gene markers were significantly down regulated in cells seeded on both surfaces when challenged with osteogenic media in relation to control. At 14 days, early osteogenic markers, were upregulated, while late osteogenic markers, were downregulated in both challenged groups. At the 21-day mark, hPDLSCs seeded on an acid-etched implant exhibited increased expression of all osteogenic markers in response to stimulation with osteogenic media and infra-red diode laser, in contrast to hPDLSCs seeded on a laser surface treated implant under the same conditions. Finally, the findings of our research revealed that when subjected to infrared diode laser, human periodontal ligament stem cells cultured on both types of implants demonstrated improved cellular attachment and differentiation. This suggested that infrared diode laser enhanced the activity of the cells surrounding the implants. Hence, the use of infrared diode laser could be pivotal in improving and expediting the clinical osseointegration process around dental implants.


Subject(s)
Dental Implants , Osteogenesis , Humans , Osteogenesis/genetics , Titanium/pharmacology , Lasers, Semiconductor , Periodontal Ligament , Stem Cells , Cell Differentiation , Cell Proliferation , Cells, Cultured
13.
Mol Med ; 30(1): 8, 2024 Jan 10.
Article in English | MEDLINE | ID: mdl-38200452

ABSTRACT

BACKGROUND: Medial vascular calcification is commonly identified in chronic kidney disease (CKD) patients and seriously affects the health and life quality of patients. This study aimed to investigate the effects of protein arginine methyltransferase 3 (PRMT3) on vascular calcification induced by CKD. METHODS: A mice model of CKD was established with a two-step diet containing high levels of calcium and phosphorus. Vascular smooth muscle cells (VSMCs) were subjected to ß-glycerophosphate (ß-GP) treatment to induce the osteogenic differentiation as an in vitro CKD model. RESULTS: PRMT3 was upregulated in VSMCs of medial artery of CKD mice and ß-GP-induced VSMCs. The inhibitor of PRMT3 (SGC707) alleviated the vascular calcification and inhibited the glycolysis of CKD mice. Knockdown of PRMT3 alleviated the ß-GP-induced osteogenic transfomation of VSMCs by the repression of glycolysis. Next, PRMT3 interacted with hypoxia-induced factor 1α (HIF-1α), and the knockdown of PRMT3 downregulated the protein expression of HIF-1α by weakening its methylation. Gain of HIF-1α reversed the PRMT3 depletion-induced suppression of osteogenic differentiation and glycolysis of VSMCs. CONCLUSION: The inhibitory role of PRMT3 depletion was at least mediated by the regulation of glycolysis upon repressing the methylation of HIF-1α.


Subject(s)
Glycerophosphates , Renal Insufficiency, Chronic , Vascular Calcification , Animals , Humans , Mice , Hypoxia , Osteogenesis/genetics , Protein-Arginine N-Methyltransferases/genetics , Renal Insufficiency, Chronic/genetics , Vascular Calcification/etiology
14.
BMC Musculoskelet Disord ; 25(1): 5, 2024 Jan 02.
Article in English | MEDLINE | ID: mdl-38167042

ABSTRACT

BACKGROUND: Osteoporosis (OP) is a progressive metabolic disorder that is difficult to cure clinically. The molecular mechanisms of OP urgently need to be further examined. This study was designed to explore the potential function of circ_0027885 during osteogenic differentiation, as well as the systematic interactions among circ_0027885, miR-203-3p and runt-related transcription factor 2 (RUNX2). METHODS: Relative levels of circ_0027885, miR-203-3p and RUNX2 were analyzed with RT-qPCR and western blotting. Alizarin red staining was performed to detect the mineralization ability under the control of circ_0027885 and miR-203-3p. Dual-luciferase reporter gene assay was conducted to examine the combination among circ_0027885, miR-203-3p and RUNX2. RESULTS: Our research demonstrated that circ_0027885 was significantly increased during hBMSCs differentiation. Overexpression of circ_0027885 notably facilitated osteogenic differentiation and upregulated RUNX2 expression, while knockdown of circ_0027885 reversed the above results. Through prediction on bioinformatics analysis, miR-203-3p was the target binding circ_0027885, and RUNX2 was the potential target of miR-203-3p. Subsequently, these changes induced by the overexpression of circ_0027885 were reversed upon addition of miR-203-3p mimic. CONCLUSIONS: Circ_0027885 could sponge miR-203-3p to regulate RUNX2 expression and alleviate osteoporosis progression.


Subject(s)
Core Binding Factor Alpha 1 Subunit , Mesenchymal Stem Cells , MicroRNAs , Osteoporosis , RNA, Circular , Humans , Cell Differentiation/genetics , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Mesenchymal Stem Cells/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Osteogenesis/genetics , Osteoporosis/genetics , Osteoporosis/metabolism
15.
Bone Res ; 12(1): 2, 2024 Jan 15.
Article in English | MEDLINE | ID: mdl-38221522

ABSTRACT

Reconstruction of irregular oral-maxillofacial bone defects with an inflammatory microenvironment remains a challenge, as chronic local inflammation can largely impair bone healing. Here, we used magnesium silicate nanospheres (MSNs) to load microRNA-146a-5p (miR-146a) to fabricate a nanobiomaterial, MSN+miR-146a, which showed synergistic promoting effects on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). In addition, miR-146a exhibited an anti-inflammatory effect on mouse bone marrow-derived macrophages (BMMs) under lipopolysaccharide (LPS) stimulation by inhibiting the NF-κB pathway via targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), and MSNs could simultaneously promote M2 polarization of BMMs. MiR-146a was also found to inhibit osteoclast formation. Finally, the dual osteogenic-promoting and immunoregulatory effects of MSN+miR-146a were further validated in a stimulated infected mouse mandibular bone defect model via delivery by a photocuring hydrogel. Collectively, the MSN+miR-146a complex revealed good potential in treating inflammatory irregular oral-maxillofacial bone defects.


Subject(s)
MicroRNAs , Nanospheres , Mice , Animals , Humans , MicroRNAs/genetics , Osteogenesis/genetics , Inflammation/drug therapy , Bone Regeneration/genetics , Silicates/pharmacology , Magnesium Silicates/pharmacology
16.
Theranostics ; 14(1): 143-158, 2024.
Article in English | MEDLINE | ID: mdl-38164139

ABSTRACT

Human osteogenic differentiation is a complex and well-orchestrated process which involves a plethora of molecular players and cellular processes. A growing number of studies have underlined that circular RNAs (circRNAs) play an important regulatory role during human osteogenic differentiation. CircRNAs are single-stranded, covalently closed non-coding RNA molecules that are acquiring increased attention as epigenetic regulators of gene expression. Given their intrinsic high conformational stability, abundance, and specificity, circRNAs can undertake various biological activities in order to regulate multiple cellular processes, including osteogenic differentiation. The most recent evidence indicates that circRNAs control human osteogenesis by preventing the inhibitory activity of miRNAs on their downstream target genes, using a competitive endogenous RNA mechanism. The aim of this review is to draw attention to the currently known regulatory mechanisms of circRNAs during human osteogenic differentiation. Specifically, we provide an understanding of recent advances in research conducted on various human mesenchymal stem cell types that underlined the importance of circRNAs in regulating osteogenesis. A comprehensive understanding of the underlying regulatory mechanisms of circRNA in osteogenesis will improve knowledge on the molecular processes of bone growth, resulting in the potential development of novel preclinical and clinical studies and the discovery of novel diagnostic and therapeutic tools for bone disorders.


Subject(s)
Mesenchymal Stem Cells , MicroRNAs , Humans , RNA, Circular/genetics , RNA, Circular/metabolism , Osteogenesis/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Cell Differentiation/genetics , Mesenchymal Stem Cells/metabolism
17.
Int J Mol Sci ; 25(1)2024 Jan 01.
Article in English | MEDLINE | ID: mdl-38203740

ABSTRACT

Adolescent Idiopathic Scoliosis (AIS) is the most common form of three-dimensional spinal disorder in adolescents between the ages of 10 and 18 years of age, most commonly diagnosed in young women when severe disease occurs. Patients with AIS are characterized by abnormal skeletal growth and reduced bone mineral density. The etiology of AIS is thought to be multifactorial, involving both environmental and genetic factors, but to date, it is still unknown. Therefore, it is crucial to further investigate the molecular pathogenesis of AIS and to identify biomarkers useful for predicting curve progression. In this perspective, the relative abundance of a panel of microRNAs (miRNAs) was analyzed in the plasma of 20 AIS patients and 10 healthy controls (HC). The data revealed a significant group of circulating miRNAs dysregulated in AIS patients compared to HC. Further bioinformatic analyses evidenced a more restricted expression of some miRNAs exclusively in severe AIS females. These include some members of the miR-30 family, which are considered promising regulators for treating bone diseases. We demonstrated circulating extracellular vesicles (EVs) from severe AIS females contained miR-30 family members and decreased the osteogenic differentiation of mesenchymal stem cells. Proteomic analysis of EVs highlighted the expression of proteins associated with orthopedic disease. This study provides preliminary evidence of a miRNAs signature potentially associated with severe female AIS and suggests the corresponding vesicular component may affect cellular mechanisms crucial in AIS, opening the scenario for in-depth studies on prognostic differences related to gender and grade.


Subject(s)
Circulating MicroRNA , MicroRNAs , Scoliosis , Adolescent , Child , Female , Humans , Circulating MicroRNA/genetics , MicroRNAs/genetics , Osteogenesis/genetics , Proteomics , Scoliosis/genetics
18.
Mol Biol Rep ; 51(1): 206, 2024 Jan 25.
Article in English | MEDLINE | ID: mdl-38270688

ABSTRACT

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) have been widely recognized as a highly promising option for cell-based tissue engineering therapy targeting osteoporosis. However, the osteogenic differentiation of BMSCs is impeded by the limited viability and diminished capacity for bone formation within the osteoporotic microenvironment. METHODS: In this study, the COL6A3 gene was confirmed through an extensive analysis of the preceding single-cell sequencing database. The generation of an inflammatory microenvironment resembling osteoporotic cell transplantation was achieved by employing lipopolysaccharide (LPS). A lentivirus targeting the COL6A3 gene was constructed, and a Western blotting assay was used to measure the marker proteins of osteogenesis, adipogenesis, and mitophagy. Immunofluorescence was utilized to observe the colocalization of mitochondria and lysosomes. The apoptosis rate of each group was evaluated using the TUNEL assay, and the mitochondrial membrane potential was assessed using JC-1 staining. RESULTS: This investigation discovered that the impaired differentiation capacity and decreased viability of BMSCs within the inflammatory microenvironment were markedly ameliorated upon overexpression of the specific COL6A3 gene. Moreover, the administration of COL6A3 gene overexpression successfully mitigated the inhibitory impacts of LPS on mitophagy and the expression of inflammatory mediators, specifically inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BMSCs. To clarify the underlying mechanism, the role of mitophagy during the differentiation of COL6A3 gene-modified BMSCs in the inflammatory microenvironment was evaluated using the mitophagy inhibitor Mdivi-1. CONCLUSIONS: In the context of lipopolysaccharide (LPS) stimulation, COL6A3 enhances the differentiation of BMSCs into osteogenic and adipogenic lineages through the promotion of mitophagy and the maintenance of mitochondrial health. Our findings may provide a novel therapeutic approach utilizing stem cells in the treatment of osteoporosis.


Subject(s)
Mesenchymal Stem Cells , Osteoporosis , Humans , Osteogenesis/genetics , Lipopolysaccharides/pharmacology , Mitophagy/genetics , Collagen , Collagen Type VI
19.
Cell Transplant ; 33: 9636897231221878, 2024.
Article in English | MEDLINE | ID: mdl-38164917

ABSTRACT

This study compared the proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs) derived from infants with polydactyly and adults with basal joint arthritis. The proliferation rate of adult and infant BMSCs was determined by the cell number changes and doubling times. The γH2AX immunofluorescence staining, age-related gene expression, senescence-associated ß-galactosidase (SA-ß-gal) staining were analyzed to determine the senescence state of adult and infant BMSCs. The expression levels of superoxide dismutases (SODs) and genes associated with various types of differentiation were measured using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Differentiation levels were evaluated through histochemical and immunohistochemical staining. The results showed that infant BMSCs had a significantly higher increase in cell numbers and faster doubling times compared with adult BMSCs. Infant BMSCs at late stages exhibited reduced γH2AX expression and SA-ß-gal staining, indicating lower levels of senescence. The expression levels of senescence-related genes (p16, p21, and p53) in infant BMSCs were also lower than in adult BMSCs. In addition, infant BMSCs demonstrated higher antioxidative ability with elevated expression of SOD1, SOD2, and SOD3 compared with adult BMSCs. In terms of differentiation potential, infant BMSCs outperformed adult BMSCs in chondrogenesis, as indicated by higher expression levels of chondrogenic genes (SOX9, COL2, and COL10) and positive immunohistochemical staining. Moreover, differentiated cells derived from infant BMSCs exhibited significantly higher expression levels of osteogenic, tenogenic, hepatogenic, and neurogenic genes compared with those derived from adult BMSCs. Histochemical and immunofluorescence staining confirmed these findings. However, adult BMSCs showed lower adipogenic differentiation potential compared with infant BMSCs. Overall, infant BMSCs demonstrated superior characteristics, including higher proliferation rates, enhanced antioxidative activity, and greater differentiation potential into various lineages. They also exhibited reduced cellular senescence. These findings, within the context of cellular differentiation, suggest potential implications for the use of allogeneic BMSC transplantation, emphasizing the need for further in vivo investigation.


Subject(s)
Arthritis , Mesenchymal Stem Cells , Polydactyly , Adult , Child , Humans , Bone Marrow , Cell Proliferation , Cell Differentiation , Osteogenesis/genetics , Cells, Cultured , Bone Marrow Cells , Arthritis/metabolism , Polydactyly/metabolism
20.
Proc Natl Acad Sci U S A ; 121(5): e2313656121, 2024 Jan 30.
Article in English | MEDLINE | ID: mdl-38252822

ABSTRACT

Long non-coding RNA (lncRNA) serves as a vital regulator of bone metabolism, but its role in pathologically overactive osteoclast differentiation remains elusive. Here, we identify lncRNA Dancr (Differentiation Antagonizing Non-protein Coding RNA) as a critical suppressor of osteoclastogenesis and bone resorption, which is down-regulated in response to estrogen deficiency. Global or osteoclast-specific Dancr Knockout mice display significant trabecular bone deterioration and enhanced osteoclast activity, but minimal alteration of bone formation. Moreover, the bone-targeted delivery of Dancr by Adeno-associated viral remarkably attenuates ovariectomy-induced osteopenia in mice. Mechanistically, Dancr establishes a direct interaction with Brahma-related gene 1 to prevent its binding and preserve H3K27me3 enrichment at the nuclear factor of activated T cells 1 and proliferator-activated receptor gamma coactivator 1-beta promoters, thereby maintaining appropriate expression of osteoclastic genes and metabolic programs during osteoclastogenesis. These results demonstrate that Dancr is a key molecule maintaining proper osteoclast differentiation and bone homeostasis under physiological conditions, and Dancr overexpression constitutes a potential strategy for treating osteoporosis.


Subject(s)
NFATC Transcription Factors , Osteogenesis , RNA, Long Noncoding , Transcription Factors , Animals , Female , Mice , Homeostasis , Mice, Knockout , NFATC Transcription Factors/genetics , Osteoclasts , Osteogenesis/genetics , RNA, Long Noncoding/genetics , Transcription Factors/genetics
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