Differentiation Of Marrow Mesenchymal Stem Cells Research Articles

MicroRNA-425-5p modulates osteoporosis by targeting annexin A2

BackgroundStudies have shown that the decrease of osteogenic differentiation of bone marrow mesenchymal stem cells (MSC) is an important mechanism of osteoporosis. The object of this study was to explore the role and mechanism of microRNA miR-425-5p in the differentiation of MSC.MethodsThe expression of miR-425-5p in MSC was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cell proliferation, cell cycle and apoptosis were detected by CCK-8 colorimetry and flow cytometry. The expression of TNF were detected by ELISA.ResultsOur data show that MiR-425-5p could modulate TNF-induced cell apoptosis, proliferation, and differentiation. ANXA2 is also the target of miR-425-5p and ANXA2 was involved in TNF-induced MSC cell apoptosis, proliferation, and differentiation. In addition, MiR-425-5p enhanced osteoporosis in mice.ConclusionMiR-425-5p might serve as a potential therapeutic target for the treatment of osteoporosis.

RIPK1 Coordinates Bone Marrow Mesenchymal Stem Cell Survival by Maintaining Mitochondrial Homeostasis via p53.

Survival of mesenchymal stem cells in the bone marrow is essential for bone marrow microenvironment homeostasis, but the molecular mechanisms remain poorly understood. RIPK1 has emerged as a critical molecule of programmed cell death in tissue homeostasis. However, little is known about the regulation of RIPK1 on bone marrow mesenchymal stem cells (MSCs). Here, we have investigated for the first time the role of RIPK1 in bone marrow MSCs. We have found that RIPK1 knockdown suppressed proliferation, differentiation, and migration in bone marrow MSCs. Furthermore, RIPK1 knockdown resulted in the opening of mitochondrial permeability transition pore (mPTP) and mtDNA damage, leading to mitochondrial dysfunction, and consequently induced apoptosis and necroptosis in bone marrow MSCs. Moreover, we identified that the p53-PUMA axis pathway was involved in mitochondrial dysfunction in RIPK1-deficient bone marrow MSCs. Together, our findings highlighted that RIPK1 was indispensable for bone marrow MSC survival.

Resveratrol facilitates bone marrow mesenchymal stem cells (BMSCs) differentiation to prevent osteoporosis via restraining of secreted frizzled-related protein 1 expression

Secreted frizzled-related protein 1 (SFRP1) is associated with cell differentiation, and its expression can be modulated by resveratrol. However, their impacts on bone marrow mesenchymal stem cells (BMSCs)-induced osteogenesis and ovariectomy-triggered bone loss remain unclear. Therefore, we in this study aimed to dissect the regulation of resveratrol on SFRP1, along with its sequential effects on differentiation and osteoporosis prevention of BMSCs. The SFRP1 expression in the ovariectomized (OVX) mice-originated bone tissues, BMSCs and bone marrow-derived macrophages (BMMs), during their differentiation towards osteoblasts and chondrocytes, was quantified by qRT-PCR and Western-blot. SFRP1-siRNA was applied for studying its influence on osteogenesis of BMSCs. Additionally, we evaluated the impacts of resveratrol on OVX mice and SFRP1 expression. SFRP1 was significantly up-regulated in the OVX mice-derived bone tissues and BMSCs, but gradually decreased during osteogenesis. Its expression was not significantly changed in BMSCs during their differentiation towards osteoclasts or in BMMs. The knockout of SFRP1 significantly improved mineralization potentiality, alkaline phosphatase activity and expression of several osteoblast-specific genes. Moreover, the bone loss was ameliorated in OVX mice treated with resveratrol, whose therapeutic effects were achieved by facilitating the expression of osteogenesis-associated genes while suppressing the SFRP1 expression. We also observed that the SFRP1 exerted a negative effect on osteogenesis of BMSCs and estrogen deficiency-induced osteoporosis, enabling itself to be an indicator of osteogenesis and also a molecular target for PMOP treatment. Resveratrol is a suppressor of SFRP1that can be applied as an active ingredient for treating PMOP.

Hierarchically structured injectable hydrogels with loaded cell spheroids for cartilage repairing and osteoarthritis treatment

The repair of cartilage lesions faces challenges in the integration of biological and mechanical cues into scaffolds. Herein, injectable hyaluronic acid (HA) hydrogels were developed with inoculation of bone marrow mesenchymal stem cell (MSC) spheroids and short fiber fillers. Kartogenin (KGN)-loaded short fibers (Fk) are assembled into cell spheroids (Fk-CS) as cell growth substrates and depots of cell differentiation modulators. Celecoxib (CXB)-loaded short fibers (FC) in HA hydrogels (HA-FC) benefit mechanical reinforcement and sustained release of anti-inflammatory agent. After inoculation in hydrogels, Fk-CS up-regulate differentiation-related gene expressions (aggrecan, collagen II, and Sox9) and the sustained release of KGN promotes chondrogenic differentiation and extracellular matrix (ECM) production in the entire spheroids. Osteochondral defects and osteoarthritis model are surgically created and treated with intraarticular injection of Fk-CS-loaded hydrogels. Osteochondral defects are fully repaired with integrated surrounding tissues, and the chondrocyte morphology, ECM (sulfated glycosaminoglycane and collagen II) deposition, and compressive moduli are comparable to those of normal cartilage. Osteoarthritis-derived cartilage lesions are repaired by regenerated ECMs, and osteoarthritis progression is retarded by downregulating pro-inflammatory cytokines and proteinase expressions. Thus, hierarchically structured injectable hydrogels and cell spheroids provide biochemical and mechanical cues for MSC differentiation, ECM deposition, cartilage regeneration, and inflammation removal in the treatment of osteochondral defects and osteoarthritis.

Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling.

Cellular senescence and its senescence-associated secretory phenotype (SASP) are widely regarded as promising therapeutic targets for aging-related diseases, such as osteoporosis. However, the expression pattern of cellular senescence and multiple SASP secretion remains unclear, thus leaving a large gap in the knowledge for a desirable intervention targeting cellular senescence. Therefore, there is a critical need to understand the molecular mechanism of SASP secretion in the bone microenvironment that can ameliorate aging-related degenerative pathologies including osteoporosis. In this study, osteocyte-like cells (MLO-Y4) were induced to cellular senescence by 2 Gy γ-rays; then, senescence phenotype changes and adverse effects of SASP on bone marrow mesenchymal stem cell (BMSC) differentiation potential were investigated. The results revealed that 2 Gy irradiation could hinder cell viability, shorten cell dendrites, and induce cellular senescence, as evidenced by the higher expression of senescence markers p16 and p21 and the elevated formation of senescence-associated heterochromatin foci (SAHF), which was accompanied by the enhanced secretion of SASP markers such as IL-1α, IL-6, MMP-3, IGFBP-6, resistin, and adiponectin. When 0.8 μM JAK1 inhibitors were added to block SASP secretion, the higher expression of SASP was blunted, but the inhibition in osteogenic and adipogenic differentiation potential of BMSCs co-cultured with irradiated MLO-Y4 cell conditioned medium (CM- 2 Gy) was alleviated. These results suggest that senescent osteocytes can perturb BMSCs’ differential potential via the paracrine signaling of SASP, which was also demonstrated by in vivo experiments. In conclusion, we identified the SASP factor partially responsible for the degenerative differentiation of BMSCs, which allowed us to hypothesize that senescent osteocytes and their SASPs may contribute to radiation-induced bone loss.

Effect of Phosphatase and Tensin Homolog 12 on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells During Aging by Regulating Transforming Growth Factor Beta/Smad Signaling Pathway

Aging affects bone marrow mesenchymal stem cells (BMSC) differentiation. PTEN12 regulates cell proliferation and apoptosis. However, whether PTEN12 affects BMSCs osteogenic differentiation during aging is unknown. Two BMSCs derived from Zempster24−/− (senescence) and Zempster24+/+ (normal) mice were cultured in vitro. Real-time PCR analysis was used to analyze PTEN12 expression. PTEN12 siRNA was transfected into senescent Zempster24-/-BMSCs and after 14 days of osteogenic induction, cell proliferation was analyzed by MTT method along with measuring expression of osteocalcin, type I collagen, RUNX2 and OPN by Real time PCR, ALP activity, and TGFβ/smad signaling protein expression by Western blot. Compared to normal BMSCs, PTEN12 level in aging BMSCs was significantly elevated, osteocalcin, type I collagen, RUNX2 and OPN mRNA level was decreased along with reduced ALP activity and TGFβ1 and Smad2 expression (P < 0.05). PTEN12 siRNA transfection into senescent BMSCs significantly down-regulated PTEN12, upregulated osteocalcin, type I collagen, RUNX2 and OPN mRNA, increased ALP activity and TGFβ1 and Smad2 expression (P <0.05). Aging increases PTEN12 level and inhibits BMSCs osteogenic differentiation. Down-regulation of PTEN12 in BMSCs during aging can promote BMSCs osteogenic differentiation by regulating TGFβ/smad signaling pathway.

MiR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3.

Objective Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro. Methods BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin. Results miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly. Conclusions miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.

MicroRNA-365-3p inhibits bone marrow mesenchymal stem cell differentiation into islet-like cell clusters via targeting Pax6 and inhibiting the MEK/ERK pathway.

Diabetes has severe impacts on the health of patients. The differentiation of mesenchymal stem cells (MSCs) into islet-like cell clusters (ICCs) is an effective protocol for the treatment of diabetes. microRNAs (miRs) regulate multiple cellular processes including cell differentiation. This study sought to identify the mechanism of miR-365-3p in the differentiation of bone marrow MSCs (bMSCs) into ICCs. Initially, the differentiation of bMSCs into ICCs was induced. Then, the miR-365-3p expression pattern in the bMSCs and ICCs was detected. Next, the miR-365-3p expression pattern was silenced in bMSCs to assess the effect on differentiation efficiency and measure the expressions of ICC marker genes during the differentiation of bMSCs into ICCs. The miR-365-3p downstream target genes were predicted and verified. Paired box protein 6 (Pax6) was downregulated in bMSCs with silenced miR-365-3p to evaluate the differentiation of bMSCs into ICCs. Furthermore, the Pax6 downstream pathway was evaluated. The differentiation of bMSCs into ICCs was successfully induced. The miR-365-3p expression in bMSCs was higher than that in ICCs. miR-365-3p downregulation in bMSCs facilitated the differentiation of bMSCs into ICCs, as evidenced by elevated releases of insulin and C-peptide in ICCs and elevated expressions of ICC marker genes. Our findings denoted that miR-365-3p targeted Pax6. Inhibition of Pax6 expression annulled the promotion of miR-365-3p downregulation on the differentiation of bMSCs into ICCs. Increased phosphorylation levels of MEK and ERK were identified in ICCs after downregulation of miR-365-3p however they were decreased after downregulation of Pax6. This study supported that miR-365-3p inhibited the differentiation of bMSCs into ICCs via targeting Pax6 and inhibiting the MEK/ERK pathway.

Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway.

ObjectivesTo clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC).Materials and MethodsTooth extraction models of Ctsk knockout mice (Ctsk ‐/‐) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK‐8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk‐/‐ JBMMSC was sent to RNA‐seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis.ResultsWe found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up‐regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA‐seq results showed that coding genes of key enzymes in glycolysis were significantly up‐regulated in Ctsk‐/‐ JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC’s regeneration was blocked subsequently.ConclusionsOur findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.

Molecular Mechanism of Neurotrophic Factor-Activated Long Non-Coding RNA Plasmacytoma Variant Translocation 1 Promoting Mesenchymal Stem Cell Migration and Repair of Fractures

It has been reported that neurotrophic factor (NF) promotes bone marrow mesenchymal stem cells (MSCs) migration to repair fractures. However, whether and how lncRNA PVT1 regulates differentiation induced by neurotrophic factors to promote MSC migration to repair fractures has not been explored. To explore the molecular mechanism of neurotrophic factor activating lncRNA PVT1 to promote MSC migration and repair fractures. Differential expression of neurotrophic factors stimulated by MSCs was analyzed based on microarray lncRNA and lncRNAs was further verified by qRT-PCR. The conditions of promoting MSC migration and osteogenic differentiation were identified by trans-fection of lncRNA PVT1 overexpressed plasmids and inhibitor and the targets of its regulation were confirmed by target gene prediction tools. In this study lncRNA array and qRT-PCR showed that lncRNA PVT1 was significantly down-regulated during neurotrophic factor-induced MSCs differentiation. Transfection of lncRNA PVT1 overexpression plasmid significantly inhibited the expression of osteogenic markers alkaline phosphatase (ALP) and osteopontin (OPN) in MSCs, while transfection of lncRNA PVT1 inhibitor promoted the expression of alkaline phosphatase (ALP) and osteopontin (OPN). lncRNA PVT1 is a negative regulator of MSCs differentiation induced by neurotrophic factors. The distal deletion homologous box 5(DLX5) was identified as the target of lncRNA PVT1 and the relationship between lncRNA PVT1 inhibiting the expression of DLX5 and the osteogenic differentiation of MSCs was verified in MSCs. lncRNA PVT1 negatively regulates the migration and differentiation of MSCs induced by neurotrophic factors by targeting DLX5, providing the foundation for bone repair.

Protective effects of berberine on senile osteoporosis in mice.

The incidence of osteoporosis is positively correlated with age. Berberine has been reported to treat osteoporosis due to its beneficial actions on bone formation. However, the direct effects of berberine on senile osteoporosis remain unclear. The present study investigated the protective effects of berberine on senile osteoporosis in mice and preliminarily evaluated its potential mechanism. 20-month-old male C57BL/6J mice were used as senile osteoporosis mouse model and treated with strontium ranelate (SR) or berberine or solvent control by daily gavage for 2months. Thereafter, bone mass and microstructure parameters were assessed. Histological staining was performed to identify the osteogenic, adipogenic and osteoclastic activity of bone tissue. Moreover, role of cAMP/PKA/CREB signaling pathway in berberine affecting bone marrow mesenchymal stem cells (BMSCs) differentiation was clarified by enzyme-linked immunosorbent assay and western blot analysis. The results showed that the SR-treated group displayed a high trabecular bone mass phenotype. For mice administrated with berberine, cancellous bone mass was upregulated in a dose-dependent manner, as indicated by gradually increased bone mass, trabecular bone volume fraction and trabecular number. Furthermore, berberine promotes osteogenic and inhibits adipogenic differentiation of BMSCs via cAMP/PKA/CREB signaling. Also, bone resorption effect becomes more obvious with increasing dose of berberine in vitro. The present results suggest that berberine exerts potent bone protective effects by promoting bone formation, inhibiting marrow fat accumulation and bone resorption. This effect may be achieved through cAMP/PKA/CREB signaling pathway.

Effects of Melatonin on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Inflammatory Environment by Regulating Mammalian Target of Rapamycin/Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling

Human bone marrow mesenchymal stem cells (BMSCs) differentiation into special cell types is affected by inflammation. Melatonin has various effects such as anti-oxidation and immune regulation. However, melatonin’s effect on BMSCs osteogenic differentiation during inflammation has not been elucidated. Rat BMSCs were isolated and assigned into control group, inflammation group (1 μg/ml lipopolysaccharide, LPS) and melatonin group (100 μM melatonin was added to LPSstimulated BMSCs cells) followed by analysis of BMSCs proliferation by MTT assay, Caspase 3 and ALP activity, expression of Runx2 and OP by Real time PCR, ROS content and SOD activity, TNF-α and IL-1β secretion by ELISA and mTOR/PI3K/AKT signaling protein level by Western blot. LPS action on BMSCs significantly inhibits BMSCs proliferation, promotes Caspase 3 activity, inhibits ALP activity, decreases Runx2 and OP expression and SOD activity, increases ROS content and TNF-α and IL-1β secretion as well as reduced mTOR and p-PI3K level (P <0.05). Melatonin addition significantly reversed the above changes (P <0.05). Melatonin can regulate oxidative stress, inhibit inflammation, and promote BMSCs proliferation and osteogenic differentiation in inflammatory environment by activating mTOR/PI3K/AKT signaling pathway.

IL-17A expressed on neutrophil extracellular traps promotes mesenchymal stem cell differentiation toward bone-forming cells in ankylosing spondylitis.

Ankylosing spondylitis (AS) is an inflammatory disease characterized by excessive bone formation. We investigated the presence of neutrophil extracellular traps (NETs) in AS and how they are involved in the osteogenic capacity of bone marrow mesenchymal stem cells (MSCs) through interleukin-17A (IL-17A). Peripheral neutrophils and sera were obtained from patients with active AS and healthy controls. NET formation and neutrophil/NET-associated proteins were studied using immunofluorescence, immunoblotting, qPCR, and ELISA. In vitro co-culture systems of AS NET structures and MSCs isolated from controls were deployed to examine the role of NETs in the differentiation of MSCs toward osteogenic cells. Analysis was performed using specific staining and qPCR. Neutrophils from patients with AS were characterized by enhanced formation of NETs carrying bioactive IL-17A and IL-1β. IL-17A-enriched AS NETs mediated the differentiation of MSCs toward bone-forming cells. The neutrophil expression of IL-17A was positively regulated by IL-1β. Blocking IL-1β signaling on neutrophils with anakinra or dismantling NETs using DNase-I disrupted osteogenesis driven by IL-17A-bearing NETs. These findings propose a novel role of neutrophils in AS-related inflammation, linking IL-17A-decorated NETs with the differentiation of MSCs toward bone-forming cells. Moreover, IL-1β triggers the expression of IL-17A on NETs offering an additional therapeutic target in AS.

Endometriosis stromal cells induce bone marrow mesenchymal stem cell differentiation and PD-1 expression through paracrine signaling.

Endometriosis is an estrogen-dependent, inflammatory gynecological disorder characterized by the growth of endometrial cells in lesions outside the uterus. Bone marrow-derived cells (BMDCs) engraft lesions and increase lesion size. Do endometriosis cells regulate differentiation of engrafted BMDCs in the pathogenesis and growth of endometriosis? Here, we report endometriosis derived stromal cells promote the differentiation of BMDCs to stromal, epithelial and leukocyte cell fates through paracrine signaling. In-vitro studies demonstrated that both mRNA and protein levels of vimentin, cytokeratin and PD-1 were significantly increased in BMDCs cocultured with stromal cells from endometriosis (ENDO) patients compared to stromal cells from normal endometrium (CNTL). Increased expression of PD-1 has been reported in malignancy where it promotes T cell quiescence and immune tolerance. Increased PD-1 was also confirmed in-vivo where we showed that PD-1 expression was induced in BMDCs engrafted into endometriotic lesions in a murine model of endometriosis. AMD3100, an antagonist for CXCR4 receptor inhibited PD-1 expression in BMDCs suggesting that PD-1 induction requires CXCL12. These results suggest that endometriosis stimulated BMDC differentiation through paracrine signaling and increased T cell PD-1 expression. Increased PD-1 expression may be one mechanism by which endometriosis avoids immune surveillance.

The p53/miR-145a Axis Promotes Cellular Senescence and Inhibits Osteogenic Differentiation by Targeting Cbfb in Mesenchymal Stem Cells.

The osteogenic differentiation capacity of senescent bone marrow mesenchymal stem cells (MSCs) is reduced. p53 not only regulates cellular senescence but also functions as a negative regulator in bone formation. However, the role of p53 in MSCs senescence and differentiation has not been extensively explored. In the present study, we investigated the molecular mechanism of p53 in MSCs senescence and osteogenic differentiation. We found that p53 was upregulated during cellular senescence and osteogenic differentiation of MSCs respectively induced by H2O2 and BMP9. Similarly, the expression of p53-induced miR-145a was increased significantly. Furthermore, Overexpression of miR-145a in MSCs promoted cellular senescence and inhibited osteogenic differentiation. Then, we identified that p53-induced miR-145a inhibited osteogenic differentiation by targeting core binding factor beta (Cbfb), and the restoration of Cbfb expression rescued the inhibitory effects of miRNA-145a. In summary, our results indicate that p53/miR-145a axis exert its functions both in promoting senescence and inhibiting osteogenesis of MSCs, and the novel p53/miR-145a/Cbfb axis in osteogenic differentiation of MSCs may represent new targets in the treatment of osteoporosis.

MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/β-catenin signaling pathway by targeting SCAI.

Osteogenic differentiation is an important process of new bone formation, microRNA-409-3p (miR-409-3p) has been reported to be up-regulated in the osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The present study aimed to investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism. The expression of miR-409-3p in osteoblast (human skull osteoblast, HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to suppressor of cancer cell invasion (SCAI) in MSC-B was investigated by performing a dual-luciferase reporter gene assay. MSC-B was selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase (ALP) activity, Alizarin Red staining, and the expression of osteogenic markers (ALP, osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RUNX2)) in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. Additionally, the Wnt/β-catenin pathway was inhibited by dickkopf-related protein 1 (DKK-1) to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs and showed an increasing time-dependent trend on the 0 and 21st day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3′UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI up-regulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/β-catenin signaling pathway-related genes (Axis inhibition protein 1 (AXIN1), β-catenin, Lymphoid Enhancer Binding Factor 1, Cellular-myelocytomatosis (c-myc) and cyclin D1). Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/β-catenin pathway by down-regulating SCAI expression.

The interaction of miR-181a-5p and sirtuin 1 regulated human bone marrow mesenchymal stem cells differentiation and apoptosis

ABSTRACT Osteoporosis (OP) characterizes a decrease in bone density and bone mass which leads to brittle fractures and serious damages to individuals. In recent years, various researches have proved that miRNAs act pivotally in the onset of bone-related diseases. In our research, we probed into the impact of miR-181a-5P on viability, differentiation, as well as apoptosis of human bone marrow mesenchymal stem cells (hBMSCs). Our study reported that overexpressing miR-181a-5p considerably reduced the cell growth, whereas the miR-181a-5p inhibition showed opposite results. Furthermore, the hBMSCs apoptosis percentage was visually elevated or minimized after overexpressing or silencing miR-181a-5p, respectively. Our data also indicated that miR-181a-5p overexpression significantly inhibited ALP activity, and level of OPN, Runx2 and OCN at mRNA and protein level, whereas miR-181a-5p inhibition presented opposite results. In addition, based on luciferase reporter assay, sirtuin 1 (Sirt1) was confirmed as the target of miR-181a-5p in hBMSCs. Finally, Sirt1 overexpression significantly inhibited the impact of miR-181a-5p mimic on apoptosis and inhibited differentiation, while silencing Sirt1 eliminated the inhibitory effects of miR-181a-5p on apoptosis and promoted differentiation via PI3K/AKT pathway. In conclusion, this work revealed that miR-181a-5p could regulate hBMSCs apoptosis as well as differentiation via regulating Sirt1/PI3K/AKT signaling pathway. Graphi Cal

Biological evaluation of the modified nano-amorphous phosphate calcium doped with citrate/poly-amino acid composite as a potential candidate for bone repair and reconstruction

Large numbers of research works related to fabricating organic–inorganic composite materials have been carried out to mimic the natural structure of bone. In this study, a new modified n-ACP doped with citrate (n-ACP-cit)/poly (amino acids) (PAA) composite (n-ACP-cit/PAA) was synthesized by employing high bioactive n-ACP-cit and the biodegradable and biocompatible PAA copolymer. Its basic structure was characterized by X-ray diffraction spectroscopy, Fourier transformed infrared spectroscopy, and X-ray photoelectron spectroscopy. Moreover, the degradability, bioactivity, biocompatibility, and osteoconductivity of n-ACP-cit/PAA composite were evaluated in vitro and in vivo, using simulated body fluid (SBF) solution soaking test, mouse bone marrow mesenchymal stem cells proliferation and differentiation, morphological observation test, expression of genes associated with osteogenesis, and bone defect model repair test, respectively. The modified n-ACP-cit/PAA composite exhibited a much higher weight loss rate (36.01 wt.%) than that of PAA (23.99 wt.%) after immersing in SBF solution for 16 weeks and the pH values of local environment restored to neutral condition. Moreover, cells co-culturing with composites exhibited higher alkaline phosphatase activity, more calcium nodule-formation, and higher expression levels of osteogenic differentiation-related genes (Bmp-2, Colla I, OCN, OPN, and Runx-2) than that of PAA. Furthermore, the bone defect model repair test revealed that the composite could be intimately incorporated with the surrounding bone without causing any deleterious reaction and capable of guiding new bone formation. Together, these results indicated that the new modified bone repair n-ACP-cit/PAA composite material with specific characteristics may be designed for meeting diverse requirements from biomedical applications.

Dense Bone-Derived 3d Bioscaffold: Preparation, Characterization, and Assessment of Its Potential for Bone Marrow Mesenchymal Stem Cells (BMSCS) Growth and Differentiation

Background: Since bone defects can result in different disabilities, many efforts have been made to bone tissue engineering. In this case, scaffolds play an important role as a key element of tissue engineering in providing three-dimensional structure for cell growth in vitro Objective: The aim of the present study was to provide the three-dimensional biological bioscaffold from the bovine femur dense bone and investigate the possibility of its potential for application in tissue engineering as biological 3D ECM bioscaffold via mesenchymal stem cells seeding and differentiation toward bone tissue. Methods: For the preparation of bioscaffolds, after cutting bovine femur bone into small pieces, demineralization and decellularization were done. Bioscaffolds biocompatibility was evaluated using an MTT assay. The morphological and cell adhesion characteristics of Bone marrow mesenchymal stem cells (BMSCs) on the bioscaffolds were evaluated using Scanning Electron Microscopy (SEM) technique. Finally, the cells were treated with an osteogenic differentiation medium and then evaluated for differentiation. Results: Histological studies showed that the use of sodium dodecyl sulfate (2.5%) for 8 h eliminated the cells. Radiography and calcium oxalate test confirmed demineralization. MTT assay and SEM studies showed that the obtained bioscaffolds are biocompatible and could provide an optimum three-dimensional environment for cell adhesion and movement. Moreover, the Alizarin red staining showed a higher differentiation rate for BMSCs. Conclusion: In the present study, bone-derived 3D bioscaffold showed an important role in the growth and differentiation of BMSCs, due to the natural characteristics, cell adhesion properties, and potential to enhance differentiation toward bone tissue. It may have the potential for use as bioscaffold as supporting metrics for maintenance, growth in bone tissue engineering.

EMF frequency dependent differentiation of rat bone marrow mesenchymal stem cells to astrocyte cells

The numerous factors regulate the bone marrow mesenchymal stem cell (BMMSC) self-renewal and differentiation response. We aimed to analyze the influence of electromagnetic field (EMF) as an external inducing factor on rat BMMSC differentiation and proliferation to neuron and astrocyte cells. BMMSCs extracted from the rats femurs and tibias and incubated in a cell-cultured CO2 incubator. After the third passages, the plates selected randomly and then divided into seven groups (Sham exposed, three groups of square, and three groups of sinusoidal waveform EMF (25, 50, and 75 Hz, 400 μT, 1 h/day). The BMMSCs exposed to EMF at the middle of a Helmholtz coil for 7 days. The viable cell counting and proliferation performed by the MTT test and BMMSC differentiation into the neuron and the astrocyte cell was studied by immunocytochemistry staining.The results confirmed BMMSC viability and proliferation rate reduction in sinusoidal 25 Hz, square 50 Hz and sinusoidal 75 Hz EMF groups compare to sham. The maximum BMMSC differentiation to neuron was considered in sinusoidal 50 Hz and 75 Hz EMF groups. The increase of BMMSC differentiation to astrocyte cell was frequency dependent and the most differentiation was shown in square 75 Hz, and sinusoidal 75 Hz EMF groups. In conclusion, the results suggest that both square and sinusoidal EMF could affect BMMSC development and differentiation to neuron and astrocyte cells. Further studies for the consequence of EMF with wider flux density and frequency on BMMSC are recommended.