MicroRNA-375 Inhibits Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation by Targeting The-Eleven Translocation-2 in Aplastic Anemia

Aplastic anemia (AA) is a potentially life-threatening disease and the exact mechanism is still unclear. Herein, we aimed to investigate whether microRNA (miRNA)-375 participates in BMSCs osteogenic differentiation in AA. BMSCs were collected from AA patients to detect the expression of miR-375. BMSCs were transfected with plasmids, followed by analysis of osteogenic differentiationrelated genes by western blot and RT-qPCR, Alkaline phosphatase (ALP) activity and the correlation between miR-375 and TET2. miR-375 was significantly upregulated in BMSCs. Transfection of miR-375 inhibitor significantly enhanced BMSCs osteogenic differentiation.On the contrary, upregulation of miR-375 alleviated BMSC osteogenic differentiation. miR-375 was indicated to target and inhibit TET2 in BMSCs, and reduction of TET2 was accompanied with decreased 5-hydroxymethylcytosine and osteogenic gene expression. Interestingly, TET2 knockdown inhibited osteogenic capacity and TET2 upregulation reversed miR-375-mediated inhibition of BMSC differentiation.miR-375 mitigates BMSCs osteogenic ability in AA by inhibiting TET2, indicating a novel miR-375-based therapeutic strategy for AA.

Calcium-Silicate-Incorporated Gellan-Chitosan Induced Osteogenic Differentiation in Mesenchymal Stromal Cells

Gellan-chitosan (GC) incorporated with CS: 0% (GC-0 CS), 10% (GC-10 CS), 20% (GC-20 CS) or 40% (GC-40 CS) w/w was prepared using freeze-drying method to investigate its physicochemical, biocompatible, and osteoinductive properties in human bone-marrow mesenchymal stromal cells (hBMSCs). The composition of different groups was reflected in physicochemical analyses performed using BET, FTIR, and XRD. The SEM micrographs revealed excellent hBMSCs attachment in GC-40 CS. The Alamar Blue assay indicated an increased proliferation and viability of seeded hBMSCs in all groups on day 21 as compared with day 0. The hBMSCs seeded in GC-40 CS indicated osteogenic differentiation based on an amplified alkaline-phosphatase release on day 7 and 14 as compared with day 0. These cells supported bone mineralization on GC-40 CS based on Alizarin-Red assay on day 21 as compared with day 7 and increased their osteogenic gene expression (RUNX2, ALP, BGLAP, BMP, and Osteonectin) on day 21. The GC-40 CS–seeded hBMSCs initiated their osteogenic differentiation on day 7 as compared with counterparts based on an increased expression of type-1 collagen and BMP2 in immunocytochemistry analysis. In conclusion, the incorporation of 40% (w/w) calcium silicate in gellan-chitosan showed osteoinduction potential in hBMSCs, making it a potential biomaterial to treat critical bone defects.

Influence of MXene Particles with a Stacked-Lamellar Structure on Osteogenic Differentiation of Human Mesenchymal Stem Cells

MXenes with a two-dimensional (2D) structure have attracted attention as potential biomedical materials. In this study, Ti3C2 MXene particles with 2D-lamellar structures were intercalated and their potential as a biomaterial was evaluated using human mesenchymal stem cells. Intercalated MXene was characterized in terms of microstructure, phase composition, and size. Cell proliferation experiments with MXene particles confirmed that concentrations >50 μg/mL were cytotoxic, while concentrations <20 μg/mL promoted osteogenic differentiation. Moreover, MXene effectively facilitated the early and late osteogenic gene expression.

Beta-Catenin Limits Osteogenesis on Regenerative Materials in a Stiffness-Dependent Manner

Targeted refinement of regenerative materials requires mechanistic understanding of cell-material interactions. The nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffold is a porous biomaterial that promotes regenerative healing of calvaria defects in vivo without addition of exogenous growth factors or progenitor cells, suggesting its potential as an off-the-shelf implant for reconstructing skull defects. In this work, we evaluate the relationship between material stiffness, a tunable MC-GAG property, and activation of the canonical Wnt (cWnt) signaling pathway. Primary human bone marrow-derived mesenchymal stem cells (hMSCs) were differentiated on two MC-GAG scaffolds varying by stiffness (non-crosslinked, NX-MC, 0.3 kPa vs. conventionally crosslinked, MC, 3.9 kPa). hMSCs exhibited increased expression of activated β-catenin, the major cWnt intracellular mediator, and the mechanosensitive YAP protein with near complete subcellular colocalization in stiffer MC scaffolds. Small molecule Wnt pathway inhibitors reduced activated β-catenin and YAP protein quantities and colocalization, osteogenic differentiation, and mineralization on MC, with no effects on NX-MC. Concomitantly, Wnt inhibitors increased BMP4 and phosphorylated Smad1/5 (p-Smad1/5) expression on MC, but not NX-MC. Unlike non-specific Wnt pathway downregulation, isolated canonical Wnt inhibition with β-catenin knockdown increased osteogenic gene expression and mineralization specifically on the stiffer MC. β-catenin knockdown also increased p-Smad1/5, Runx2, and BMP4 expression only on the stiffer MC material. Our data indicates stiffness-induced activation of the Wnt and mechanotransduction pathways promotes osteogenesis in MC-GAG scaffolds. However, activated β-catenin is a limiting agent and may serve as a useful target or readout for optimal modulation of stiffness in skeletal regenerative materials.

Histone modifications centric-regulation in osteogenic differentiation

Histone modification critically contributes to the epigenetic control of gene expression by changing the configuration of chromatin and modifying the access of transcription factors to gene promoters. Recently, we observed that histone acetylation and crotonylation mediated the expression of endocytosis-related genes and tumor-related immune checkpoint genes by regulating the enrichment of signal transducer and activator of transcription 3 on these gene promoters in Alzheimer’s disease and tumorigenesis, suggesting that histone modification plays an important role in disease development. Furthermore, studies performed in the past decade revealed that histone modifications affect osteogenic differentiation by regulating the expression of osteogenic marker genes. In this review, we summarize and discuss the histone modification-centric regulation of osteogenic gene expression. This review improves the understanding of the role of histone modifications in osteogenic differentiation and describes its potential as a therapeutic target for osteogenic differentiation-related diseases.

MicroRNA treatment modulates osteogenic differentiation potential of mesenchymal stem cells derived from human chorion and placenta

Mesenchymal stem cells (MSCs) are important in regenerative medicine because of their potential for multi-differentiation. Bone marrow, chorion and placenta have all been suggested as potential sources for clinical application. However, the osteogenic differentiation potential of MSCs derived from chorion or placenta is not very efficient. Bone morphogenetic protein-2 (BMP-2) plays an important role in bone development. Its effect on osteogenic augmentation has been addressed in several studies. Recent studies have also shown a relationship between miRNAs and osteogenesis. We hypothesized that miRNAs targeted to Runt-related transcription factor 2 (Runx-2), a major transcription factor of osteogenesis, are responsible for regulating the differentiation of MSCs into osteoblasts. This study examines the effect of BMP-2 on the osteogenic differentiation of MSCs isolated from chorion and placenta in comparison to bone marrow-derived MSCs and investigates the role of miRNAs in the osteogenic differentiation of MSCs from these sources. MSCs were isolated from human bone marrow, chorion and placenta. The osteogenic differentiation potential after BMP-2 treatment was examined using ALP staining, ALP activity assay, and osteogenic gene expression. Candidate miRNAs were selected and their expression levels during osteoblastic differentiation were examined using real-time RT-PCR. The role of these miRNAs in osteogenesis was investigated by transfection with specific miRNA inhibitors. The level of osteogenic differentiation was monitored after anti-miRNA treatment. MSCs isolated from chorion and placenta exhibited self-renewal capacity and multi-lineage differentiation potential similar to MSCs isolated from bone marrow. BMP-2 treated MSCs showed higher ALP levels and osteogenic gene expression compared to untreated MSCs. All investigated miRNAs (miR-31, miR-106a and miR148) were consistently downregulated during the process of osteogenic differentiation. After treatment with miRNA inhibitors, ALP activity and osteogenic gene expression increased over the time of osteogenic differentiation. BMP-2 has a positive effect on osteogenic differentiation of chorion- and placenta-derived MSCs. The inhibition of specific miRNAs enhanced the osteogenic differentiation capacity of various MSCs in culture and this strategy might be used to promote bone regeneration. However, further in vivo experiments are required to assess the validity of this approach.

MiR-486-3p promotes osteogenic differentiation of BMSCs by targeting CTNNBIP1 and activating Wnt/β-catenin pathway

Background Dysfunction in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) leads to bone loss/osteoporosis. CTNNBIP1 (Catenin beta interacting protein 1) is an inhibitor of Wnt/β-catenin signaling, whose role in osteogenesis remains elusive. This study aims to reveal the effects of miR-486-3p/CTNNBIP1 in osteogenesis. Methods Bone marrow samples from control and osteoporosis patients were collected and ovariectomy was performed on mice and levels of CTNNBIP1 and miR-486-3p levels were assessed. Dual-luciferase reporter assay was used to confirm their interactions. MiR-486-3p mimics/inhibitor or CTNNBIP1 overexpression lentivirus were transfected in human BMSCs (hBMSCs) and then osteogenic assay was performed. Alizarin red S (ARS) and Alkaline phosphatase (ALP) intensity with osteogenic gene expression including Runx2, Alp, Bglap and OCN was measured. Key proteins in Wnt/β-catenin pathway including active β-catenin, Bcl-2 and Cyclin D1 were assessed. Results CTNNBIP1, an inhibitor of Wnt/β-catenin signaling was upregulated while miR-486-3p was downregulated in ovariectomized (OVX) mice. CTNNBIP1 was confirmed as a target of miR-486-3p. MiR-486-3p overexpression promoted but miR-486-3p knockdown suppressed osteogenic differentiation and Wnt/β-catenin pathway. Rescue experiments further elucidated that the negative effects of CTNNBIP1 overexpression on osteoblastic differentiation and canonical Wnt signaling could be reversed by miR-486-3p mimics. Conclusion This study demonstrated that miR-486-3p sponges CTNNBIP1 thus activating Wnt/β-catenin signaling pathway to promote osteogenesis of BMSCs.

Arbutin ameliorates glucocorticoid-induced osteoporosis through activating autophagy in osteoblasts

Chronic long-term glucocorticoid use causes osteoporosis partly by interrupting osteoblast homeostasis and exacerbating bone loss. Arbutin, a natural hydroquinone glycoside, has been reported to have biological activities related to the differentiation of osteoblasts and osteoclasts. However, the role and underlying mechanism of arbutin in glucocorticoid-induced osteoporosis are elusive. In this study, we demonstrated that arbutin administration ameliorated osteoporotic disorders in glucocorticoid dexamethasone (Dex)-induced mouse model, including attenuating the loss of bone mass and trabecular microstructure, promoting bone formation, suppressing bone resorption, and activating autophagy in bone tissues. Furthermore, Dex-stimulated mouse osteoblastic MC3T3-E1 cells were treated with arbutin. Arbutin treatment rescued Dex-induced repression of osteoblast differentiation and mineralization, the downregulation of osteogenic gene expression, reduced autophagic marker expression, and decreased autophagic puncta formation. The application of autophagy inhibitor 3-MA decreased autophagy, differentiation, and mineralization of MC3T3-E1 cells triggered by arbutin. Taken together, our findings suggest that arbutin treatment fends off glucocorticoid-induced osteoporosis, partly through promoting differentiation and mineralization of osteoblasts by autophagy activation.