MEK1/2 Inhibitor (GDC0623) Promotes Osteogenic Differentiation of Primary Osteoblasts Inhibited by IL-1β through the MEK-Erk1/2 and Jak/Stat3 Pathways

Objective. We evaluated the effects and mechanisms of GDC0623 on osteogenic differentiation of osteoblasts induced by IL-1β. Methodology. Osteoblasts were treated with 20 ng/ml IL-1β and 0.1 µM GDC0623. Cell proliferation levels were evaluated by the cell counting kit 8 (CCK8), EdU assay, and western blotting [proliferating cell nuclear antigen (PCNA) and Cyclin D1]. Osteoblasts were cultured in an osteogenic induction medium for 1–3 weeks after which their differentiations were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, calcium concentration, immunocytochemistry staining, real-time quantitative PCR (RT-qPCR), and immunofluorescence staining. The osteogenesis-associated mechanisms were further evaluated by western blotting using appropriate antibodies. Results. Relative to the control group, IL-1β induced the rapid proliferation of osteoblasts and suppressed their osteogenic differentiations by upregulating the activities of MEK-Erk1/2 as well as Jak-Stat3 pathways and by elevating MMP13 and MMP9 levels. However, blocking of the MEK-Erk1/2 signaling pathway by GDC0623 treatment reversed these effects. Conclusion. Inhibition of Jak-Stat3 pathway by C188-9 downregulated the expression levels of MMP9 and MMP13, activated MEK-Erk1/2 pathway, and inhibited osteogenic differentiation.

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Optimizing Osteogenic Differentiation of Ovine Adipose-Derived Stem Cells by Osteogenic Induction Medium and FGFb, BMP2, or NELL1 In Vitro

Stem Cells International ◽

10.1155/2018/9781393 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Emil Østergaard Nielsen ◽

Li Chen ◽

Jonas Overgaard Hansen ◽

Matilda Degn ◽

Søren Overgaard ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Stromal Cells ◽

Basic Medium ◽

Induction Medium ◽

Control Analysis ◽

Alizarin Red ◽

Osteogenic Induction Medium ◽

Osteogenic Induction ◽

Fibroblast Growth Factor Basic

Although adipose-derived stromal cells (ADSCs) have been a major focus as an alternative to autologous bone graft in orthopedic surgery, bone formation potential of ADSCs is not well known and cytokines as osteogenic inducers on ADSCs are being investigated. This study aimed at isolating ADSCs from ovine adipose tissue (AT) and optimizing osteogenic differentiation of ovine ADSCs (oADSC) by culture medium and growth factors. Four AT samples were harvested from two female ovine (Texel/Gotland breed), and oADSCs were isolated and analyzed by flow cytometry for surface markers CD29, CD44, CD31, and CD45. Osteogenic differentiation was made in vitro by seeding oADSCs in osteogenic induction medium (OIM) containing fibroblast growth factor basic (FGFb), bone morphogenetic protein 2 (BMP2), or NEL-like molecule 1 (NELL1) in 4 different dosages (1, 10, 50, and 100 ng/ml, respectively). Basic medium (DMEM) was used as control. Analysis was made after 14 days by Alizarin red staining (ARS) and quantification. This study successfully harvested AT from ovine and verified isolated cells for minimal criteria for adipose stromal cells which suggests a feasible method for isolation of oADSCs. OIM showed significantly higher ARS to basic medium, and FGFb 10 ng/ml revealed significantly higher ARS to OIM alone after 14 days.

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An Inhibitory Role of Osthole in Rat MSCs Osteogenic Differentiation and Proliferation via Wnt/β-Catenin and Erk1/2-MAPK Pathways

Cellular Physiology and Biochemistry ◽

10.1159/000445590 ◽

2016 ◽

Vol 38 (6) ◽

pp. 2375-2388 ◽

Cited By ~ 12

Author(s):

Hongyang Hu ◽

Min Chen ◽

Guangzu Dai ◽

Guoqing Du ◽

Xuezong Wang ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathways ◽

Western Blotting ◽

Underlying Mechanism ◽

Mapk Signaling ◽

Cell Counting ◽

Nuclear Antigen ◽

Dependent Manner ◽

Alizarin Red ◽

Mapk Signaling Pathways

Background/Aims: Bone marrow-derived mesenchymal stem cells (MSCs) are responsible for new bone formation during adulthood. Accumulating evidences showed that Osthole promotes the osteogenic differentiation in primary osteoblasts. The aim of this study was to investigate whether Osthole exhibits a potential to stimulate the osteogenic differentiation of MSCs and the underlying mechanism. Methods: MSCs were treated with a gradient concentration of Osthole (6.25 µM, 12.5 µM, and 25 µM). Cell proliferation was assessed by western blotting with the proliferating cell nuclear antigen (PCNA) and Cyclin D1 antibodies, fluorescence activated cell sorting (FACS), and cell counting kit 8 (CCK8). MSCs were cultured in osteogenesis-induced medium for one or two weeks. The osteogenic differentiation of MSCs was estimated by Alkaline Phosphatase (ALP) staining, Alizarin red staining, Calcium influx, and quantitative PCR (qPCR). The underlying mechanism of Osthole-induced osteogenesis was further evaluated by western blotting with antibodies in Wnt/β-catenin, PI3K/Akt, BMPs/smad1/5/8, and MAPK signaling pathways. Results: Osthole inhibited proliferation of rat MSCs in a dose-dependent manner. Osthole suppressed osteogenic differentiation of rat MSCs by down-regulating the activities of Wnt/β-catenin and Erk1/2-MAPK signaling. Conclusions: Osthole inhibits the proliferation and osteogenic differentiation of rat MSCs, which might be mediated through blocking the Wnt/β-catenin and Erk1/2-MAPK signaling pathways.

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The Role and Mechanism of Exosomes from Umbilical Cord Mesenchymal Stem Cells in Inducing Osteogenesis and Preventing Osteoporosis

Cell Transplantation ◽

10.1177/09636897211057465 ◽

2021 ◽

Vol 30 ◽

pp. 096368972110574

Author(s):

Ge Yahao ◽

Wang Xinjia

Keyword(s):

Stem Cell ◽

Osteogenic Differentiation ◽

Umbilical Cord ◽

Cell Counting ◽

Control Group ◽

Human Umbilical Cord ◽

Promoting Effect ◽

Alizarin Red ◽

Osteogenic Induction

Mesenchymal stem cell (MSC) exosomes promote tissue regeneration and repair, and thus might be used to treat many diseases; however, the influence of microenvironmental conditions on exosomes remains unclear. The present study aimed to analyze the effect of osteogenic induction on the functions of human umbilical cord MSC (HucMSC)-derived exosomes. Exosomes from standardized stem cell culture (Exo1) and osteogenic differentiation-exosomes (Exo2) were co-cultured with osteoblasts, separately. Cell counting kit-8 assays, alkaline phosphatase and alizarin red staining were used to observe the exosomes’ effects on osteoblast proliferation and differentiation. The levels of osteogenic differentiation-related proteins were analyzed using western blotting. Estrogen-deficient osteoporosis model mice were established, and treated with the two exosome preparations. Micro-computed tomography and hematoxylin and eosin staining were performed after 6 weeks. MicroRNAs in Exo1 and Exo2 were sequenced and analyzed using bioinformatic analyses. Compared with Exo1 group, Exo2 had a stronger osteogenic differentiation promoting effect, but a weaker proliferation promoting effect. In ovariectomy-induced osteoporosis mice, both Exo1 and Exo2 improved the tibial density and reversed osteoporosis in vivo. High-throughput microRNA sequencing identified 221 differentially expressed microRNAs in HucMSC-derived exosomes upon osteogenic induction as compared with the untreated control group. Importantly, we found that 41 of these microRNAs are potentially critical for MSC-secreted exosomes during osteogenic induction. Mechanistically, exosomal miRNAs derived from osteogenic induced-HucMSCs are involved in bone development and differentiation, such as osteoclast differentiation and the MAPK signaling pathway. The expression of hsa-mir-2110 and hsa-mir-328-3p gradually increased with prolonged osteogenic differentiation and regulated target genes associated with bone differentiation, suggesting that they are probably the most important osteogenesis regulatory microRNAs in exosomes. In conclusion, we examined the contribution of osteogenic induction to the function of exosomes secreted by HucMSCs following osteogenic differentiation in vitro and in vivo, and reveal the underlying molecular mechanisms of exosome action during osteoporosis.

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Mechanical Sensing Element PDLIM5 Promotes Osteogenesis of Human Fibroblasts by Affecting the Activity of Microfilaments

Biomolecules ◽

10.3390/biom11050759 ◽

2021 ◽

Vol 11 (5) ◽

pp. 759

Author(s):

Xiaolan Huang ◽

Rongmei Qu ◽

Yan Peng ◽

Yuchao Yang ◽

Tingyu Fan ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Morphological Changes ◽

Human Fibroblasts ◽

Inhibitory Effect ◽

Induction Medium ◽

Sensing Element ◽

Differentiation Potential ◽

Osteogenic Induction Medium ◽

Osteogenic Induction ◽

Related Proteins

Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.

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Bioinformatics analysis and identification of circular RNAs promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells on titanium treated by surface mechanical attrition

PeerJ ◽

10.7717/peerj.9292 ◽

2020 ◽

Vol 8 ◽

pp. e9292

Author(s):

Shanshan Zhu ◽

Yuhe Zhu ◽

Zhenbo Wang ◽

Chen Liang ◽

Nanjue Cao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Human Bone ◽

Cell Counting ◽

Circular Rnas ◽

Control Group ◽

Alizarin Red ◽

Qrt Pcr ◽

Mechanical Attrition

Background To analyze and identify the circular RNAs (circRNAs) involved in promoting the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) on titanium by surface mechanical attrition treatment (SMAT). Methods The experimental material was SMAT titanium and the control material was annealed titanium. Cell Counting Kits-8 (CCK-8) was used to detect the proliferation of hBMSCs, and alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the osteogenic differentiation of hBMSCs on the sample surfaces. The bioinformatics prediction software miwalk3.0 was used to construct competing endogenous RNA (ceRNA) networks by predicting circRNAs with osteogenesis-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The circRNAs located at the key positions in the networks were selected and analyzed by quantitative real-time PCR (QRT-PCR). Results Compared with annealed titanium, SMAT titanium could promote the proliferation and osteogenic differentiation of hBMSCs. The total number of predicted circRNAs was 51. Among these, 30 circRNAs and 8 miRNAs constituted 6 ceRNA networks. Circ-LTBP2 was selected for verification. QRT-PCR results showed that the expression levels of hsa_circ_0032599, hsa_circ_0032600 and hsa_circ_0032601 were upregulated in the experimental group compared with those in the control group; the differential expression of hsa_circ_0032600 was the most obvious and statistically significant, with a fold change (FC) = 4.25 ± 1.60, p-values (p) < 0.05.

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MiR-26b-5p Promotes Osteogenesis of Bone Mesenchymal Stem Cells via Suppressing FGF21

10.21203/rs.3.rs-1135236/v1 ◽

2021 ◽

Author(s):

Bin Wang ◽

Zhenhui Li ◽

Caiyuan Mai ◽

Penglin Mou ◽

Lei Pan

Keyword(s):

Osteogenic Differentiation ◽

Luciferase Reporter ◽

Control Group ◽

Osteogenic Differentiation Medium ◽

Bone Mesenchymal Stem Cells ◽

Alizarin Red ◽

Osteogenic Induction ◽

Direct Target ◽

Inhibition Group ◽

Fgf21 Expression

Abstract Introduction: It has been established that miR-26b-5p actively participate in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs), which is of great value in osteoporosis treatment. Database showed that Fibroblast growth factor(FGF)21 is a potential binding site of miR-26b-5p. This study aimed to investigate the molecular osteogenic mechanisms of miR-26b-5p targeting FGF21 in postmenopausal osteoporosis (PMOP). Methods: 5ml of bone marrow was aspirated from the anterior superior iliac spine in 10 PMOP women during bone marrow puncture. BMSCs were used to establish an in vitro cell model, and BMSCs markers were analyzed by flow cytometry. miR-26b-5p and FGF21 were overexpressed for 48h, and then placed in an osteogenic induction medium for osteogenic induction culture, the expression of RNA was detect using RT-qPCR. Cells from miR-26b-5p group were collected on days 7, 14 and 21 of induction for ALP and alizarin red S staining. On day 7 of induction, RT-qPCR was used to measure Runx2, Osterix (Osx), and target gene FGF21 expression levels in each group. The dual-luciferase reporter gene system was used to verify that FGF21 was a direct target of miR-26b-5p. FGF21 was measured by western blotting in the miR-26b-5p overexpression group and in the miR-26b-5p inhibition group. Results: BMSCs were identified according with the antigenic characteristics. miR-26b-5p expression was significantly upregulated after the expression of miR-26b-5p mimics; however, FGF21 expression was downregulated after FGF21 mimics. After overexpression of miR-26b-5p, the alkaline phosphatase activity and nodules of alizarin red S in the culture medium gradually increased as the induction time increased. RT-qPCR showed that the expressions of master osteogenic factors Runx2 and Osx in the BMSC+ osteogenic differentiation medium group was significantly higher than in the BMSC group, the expressions of the factors in the BMSC+ miR-26b-5p overexpression group was significantly higher than in the control group. Target gene FGF21 expression was significantly lower in the BMSC+ osteogenic differentiation medium group than in the BMSC group, and was significantly lower in the BMSC+ miR-26b-5p overexpression group than in the control group. Luciferase reporter assays demonstrated that FGF21 was a direct target of miR-26b-5p. Finally, western blotting analysis showed that FGF21 expression was significantly downregulated in the miR-26b-5p overexpressed group and upregulated in the miR-26b-5p inhibition group. Conclusion: miR-26b-5p can regulate the osteogenic differentiation of BMSCs and participate in PMOP pathogenesis via suppressing FGF21. The present study provides the basis for further studies on PMOP.

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Dimethyl Sulfoxide Leads to Decreased Osteogenic Differentiation of Stem Cells Derived from Gingiva via Runx2 and Collagen I Expression

European Journal of Dentistry ◽

10.1055/s-0039-1694904 ◽

2019 ◽

Vol 13 (02) ◽

pp. 131-136 ◽

Cited By ~ 3

Author(s):

Hyunjin Lee ◽

Jun-Beom Park

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dimethyl Sulfoxide ◽

Cellular Growth ◽

Collagen I ◽

Cell Counting ◽

Normal Fibroblast ◽

Control Group ◽

Alizarin Red ◽

Human Gingiva

Abstract Objectives Dimethyl sulfoxide (DMSO) plays various functions, including cellular functions such as cellular growth. The aim of this study was to evaluate the effects of DMSO on the proliferation and osteogenic differentiation of human gingiva-derived stem cells. Materials and Methods Stem cells derived from gingiva were cultured in the presence of DMSO at concentrations ranging from 0.01 to 10%. Statistical Analysis We performed a one-way analysis of variance (ANOVA) with post hoc test to determine the differences between the groups using a commercially available program and the level of significance was 0.05. Results The cells in the control group showed normal fibroblast morphology. The cells treated with 0.01%, 0.01%, 0.1%, and 1% DMSO were morphologically similar to those from the control group on each day. Statistically significant decreases in cell counting kit-8 (CCK-8) values were seen in the 3% and 10% DMSO groups (p < 0.05). A statistically significant decrease in alkaline phosphatase activity was seen in the 3% DMSO group. (p < 0.05). The application of DMSO produced a decrease in alizarin red S staining. The expression of Runx2 and collagen I by immunofluorescence decreased as the dose of lovastatin increased. Conclusion The effects of DMSO on the viability of osteogenic differentiation among stem cells derived from human gingiva were evaluated. Applying DMSO produced decreased cell viability and decreased osteogenic differentiation in this experimental setting. This should be considered when designing and interpreting the data, and a DMSO-free method may be considered for bone regeneration applications.

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miR-217 Suppresses Osteogenic Differentiation of Human Dental Pulp Stem Cells by Down-Regulating Sirtuin 1

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2373 ◽

2020 ◽

Vol 10 (7) ◽

pp. 978-986

Author(s):

Haiquan Yue ◽

Yidan Guo ◽

Juan Song ◽

Ruimin Liu

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Sirtuin 1 ◽

Luciferase Reporter ◽

Induction Medium ◽

Alizarin Red ◽

Human Dental Pulp ◽

Osteogenic Induction

The paper is committed to uncovering the effect of miR-217 on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism. hDPSCs were separated from human dental pulp tissues for measurement of stemness. The osteogenic differentiation of hDPSCs was induced in an osteogenic induction medium. The hDPSCs were transfected with miR-217 mimic, miR-217 inhibitor and/or sh-SIRT1 accordingly. The expressions of miR-217 and SIRT1 were detected in hDPSCs after cell transfection and osteogenic differentiation. Calcium nodules were showed by alizarin red staining. Moreover, the expressions of osteogenic differentiation-related genes were also assessed. The binding of miR-217 to SIRT1 was predicted on starBase and further determined by dual-luciferase reporter assay. Down-regulated miR-217 and up-regulated SIRT1 were found during osteogenic differentiation of hDPSCs. The osteogenic differentiation of hDPSCs was suppressed after transfection of miR-217 mimic or sh-SIRT1 while promoted by miR-217 inhibition. Taken together, miR-217 can suppress osteogenic differentiation of hDPSCs by negatively regulating SIRT1.

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TMT-Based Proteomic Explores the Influence of DHEA on the Osteogenic Differentiation of hBMSCs

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.726549 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xiaonan Liang ◽

Mingwei He ◽

Bo Zhu ◽

Yongjia Zhu ◽

Xixi He ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Metabolic Pathways ◽

Osteoblast Differentiation ◽

Bioinformatic Analysis ◽

Induction Medium ◽

Complete Medium ◽

Tandem Mass ◽

Osteogenic Induction Medium ◽

Osteogenic Induction ◽

Underlying Mechanisms

Dehydroepiandrosterone (DHEA) has been revealed to implicate in facilitating osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and inhibiting osteoporosis (OP). However, the underlying molecular mechanism remains largely unknown. Here, we induced osteogenic differentiation of hBMSCs derived from elders using an osteogenic induction medium with or without DHEA. The results showed that osteogenic induction medium (OIM) with DHEA could significantly promote the proliferation and osteogenic differentiation of hBMSCs than OIM alone. By using a Tandem Mass Tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, we screened out 604 differentially expressed proteins (DEPs) with at least one unique peptide were identified [524: OIM vs. complete medium (CM), and 547: OIM+DHEA vs. CM], among these proteins, 467 DEPs were shared in these two different comparative groups. Bioinformatic analysis revealed these DEPs are mainly enriched in metabolic pathways. Interestingly, the expression levels of the DEPs in the metabolic pathways showed a more noticeable change in the OIM+DHEA vs. CM group than OIM vs. CM group. Moreover, the protein-protein interaction (PPI) network analysis revealed that three potential proteins, ATP5B, MT-CYB, and MT-ATP6, involved in energy metabolism, might play a key role in osteogenic differentiation induced by OIM+DHEA. These findings offer a valuable clue for us to better understand the underlying mechanisms involved in osteoblast differentiation of hBMSCs caused by DHEA and assist in applying DHEA in hBMSCs-based therapy for osteogenic regeneration.

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Quercetin Stimulates Bone Marrow Mesenchymal Stem Cell Differentiation through an Estrogen Receptor-Mediated Pathway

BioMed Research International ◽

10.1155/2018/4178021 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 11

Author(s):

Xin-Gang Pang ◽

Yu Cong ◽

Ni-Rong Bao ◽

Yong-Gang Li ◽

Jian-Ning Zhao

Keyword(s):

Bone Marrow ◽

Estrogen Receptor ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Osteogenic Differentiation ◽

Western Blotting ◽

Stem Cell Differentiation ◽

Cell Counting ◽

Mouse Bone Marrow ◽

Alizarin Red

Objectives. The present study aimed to investigate the overall effect of quercetin on mouse bone marrow mesenchymal stem cell (BMSC) proliferation and osteogenic differentiation in vitro. Materials and Methods. BMSCs were treated with different concentrations of quercetin for 6 days. The effects of quercetin on cell proliferation were assessed at predetermined times using Cell Counting Kit-8 (CCK-8) assay. The cells were then treated with quercetin, estrogen, or an estrogen receptor (ER) antagonist (which was also administered in the presence of quercetin or estrogen) for 7 or 21 days. The effects of quercetin on BMSC osteogenic differentiation were analyzed by an alkaline phosphatase (ALP) assay kit, Alizarin Red S staining (ARS), quantitative real-time PCR (qPCR), and western blotting. Results. The CCK-8 and ALP assays and ARS staining showed that quercetin significantly enhanced BMSC proliferation, ALP activity, and extracellular matrix production and mineralization, respectively. The qPCR results indicated that quercetin promoted osterix (OSX), runt-related transcription factor 2 (RUNX2), and osteopontin (OPN) transcription in the presence of osteoinduction medium, and the western blotting results indicated that quercetin enhanced bone morphogenetic protein 2 (BMP2), Smad1, Smad4, RUNX2, OSX, and OPN expression and Smad1 phosphorylation. Treatment with the ER inhibitor ICI182780 blocked the effects of quercetin. Conclusions. Our data demonstrated that quercetin promotes BMSC proliferation and osteogenic differentiation. Quercetin enhances BMP signaling pathway activation and upregulates the expression of downstream genes, such as OSX, RUNX2, and OPN, via the ER.

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