Adipose-derived mesenchymal stem cells-derived exosomes containing nano-pearl powder water-soluble matrix promote osteogenic differentiation of MC3T3-E1 cells

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. We reported previously that glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor gamma-2 (Ppargamma2) expression and blocks adipocyte differentiation. Here we show that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppargamma2 and C/ebpalpha were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, our results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair.

Statement of Retraction Qiang Wang, Xiaoxing Xie, Dehua Zhang, Feng Mao, Shaobo Wang, Yi Liao- 2022, Saxagliptin enhances osteogenic differentiation in MC3T3-E1 cells, dependent on the activation of AMP-activated protein kinase α (AMPKα)/runt-related transcription factor-2 (Runx-2), Bioengineered 13(1), doi: 10.1080/21655979.2021.2008667 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Background Bone Marrow Mesenchymal Stem Cells-Exosomes (BMSC-Exo) possess the ability to facilitate bone remodeling, and this mechanism has always been of great interest in the field. Our study aimed to elucidate the impacts of BMSC-Exo on MC3T3-E1, the murine embryonic osteogenic progenitor cells, and the interaction behind. Methods We initially extracted and characterized exosomes from BMSCs. Following treatment with GW4869, a compound that inhibits exosome production and release, BMSCs produced exosomes (BMSC-Exo). These were subsequently combined in culture with MC3T3-E1 cells. Upon an application of Phalloidin and PKH26 staining, we observed morphology of the cellular actin fibers and the uptake of exosomes. To evaluate the osteogenic potential of the cells, we utilized Alizarin Red S (ARS) and Alkaline Phosphatase (ALP) staining. Additionally, we measured expressions of osteogenic factors RUNX2, ALP, OSX, OCN, and OPN through qRT-PCR and Western blot analyses. Afterwards, we intervened with BMSC-Exo with a lentivirus over-expressing miR-122-5p and co-cultured it with MC3T3-E1 cells. To further assess osteogenic differentiation, we conducted additional ARS & ALP staining, along with qRT-PCR and Western blot assays. With the help of dual-luciferase reporter assay, we found that miR-122-5p interacts specifically with SPRY2. Ultimately, we treated MC3T3-E1 cells with a lentivirus over-expressing miR-122-5p and a plasmid over-expressing OE-SPRY2. Osteogenic differentiation was then assessed using ARS & ALP staining, qRT-PCR, and Western blot. Results Our laboratory outcomes demonstrated that exosomes derived from BMSC-Exo are instrumental in the advancement of calcified nodule genesis within MC3T3-E1 cells, concurrently amplifying the transcriptional and translational expressions of osteogenic markers (RUNX2, ALP, OSX, OCN, and OPN). These excreted exosomes from the BMSCs modified by a miR-122-5p-over-expressing lentivirus are found to further accelerate osteogenic differentiation of the cells. Moreover, our application of dual-luciferase reporter gene system has elucidated a specific interplay between miR-122-5p and SPRY2. Furthermore, overexpressing of SPRY2 negates the miR-122-5p-induced osteogenic differentiation. Conclusions BMSC-Exo facilitates osteogenic differentiation in MC3T3-E1 cells by suppressing SPRY2, a process mediated by miR-122-5p.

Background: The purpose of the current study was to explore whether Pinoresinol diglucoside (PD) could relieve osteoporosis through promoting osteogenic differentiation by activating phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Methods: Firstly, human bone marrow mesenchymal stem cells (hBMMSCs) and mouse embryo steoblast recursor cells (MC3T3-E1) were induced for osteogenic differentiation, and then the cells were subjected to 1 mol/l PD. Then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot assay were used to detect the expression of osteogenic marker genes/proteins such as bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), and Osterix. The activity of alkaline phosphatase (ALP) in cells was also detected. Cell viability was further detected by Cell Count Kit-8 (CCK-8), and cell apoptosis was detected by flow cytometer. Finally, the protein expression of p-AKT and AKT was detected by Western blot assay. Results: The results showed that osteogenic differentiation of hBMMSCs and MC3T3-E1 cells were successfully induced, evidenced by increased BMP2, Runx2, Osterix mRNA expression and ALP activity enhancement. The osteogenic differentiation of MC3T3-E1 cells and hBMMSCs was enhanced by PD administration. At the same time, PD promoted the viability of MC3T3-E1 cells and reduced the MC3T3-E1 cell apoptosis. In addition, PD increased the protein expression of p-AKT and the ratio of p-AKT/AKT in MC3T3-E1 cells, suggesting PI3K/AKT pathway activation. Conclusion: PD promoted osteogenic differentiation of hBMMSCs and MC3T3-E1 cells, and it could promoted osteoblast proliferation and inhibit apoptosis, thereby playing a protective role in osteoporosis.

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.

Osteogenic differentiation is a crucial process of new bone formation. This study aimed to explore the roles and mechanism of SRY-Box Transcription Factor 2 (SOX2) on proliferation and osteogenic differentiation of MC3T3-E1 cells. Bone morphogenetic protein 2 (BMP2) was used to induce the osteogenic differentiation of MC3T3-E1 cells. The expression of SOX2 was determined by quantitative real-time PCR (RT-PCR) at different time points after induction. The SOX2 overexpression plasmids were constructed and transfected into MC3T3-E1 cells. Osteogenic differentiation was evaluated by Alizarin Red S staining and alkaline phosphatase (ALP) assay. The expressions of osteogenic differentiation markers including runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) were detected by western blot assay. Luciferase reporter and CHIP assays were used to confirm that SOX2 regulated the transcriptional activation of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). We found that SOX2 was down-regulated upon BMP2-induced osteogenic differentiation in MC3T3-E1 cells. Overexpression of SOX2 effectively inhibited osteogenic differentiation with decreased ALP activity, calcification, and osteogenic differentiation markers' expression including Runx2, OPN, and OCN. LGR4 was identified as a target of SOX2, and the inhibitory effect of SOX2 on osteogenic differentiation was reversed by knockdown of LGR4. The present study confirmed that SOX2 suppressed osteogenic differentiation of MC3T3-E1 cells through targeting LGR4, which possesses a therapeutic strategy for bone formation and generation.

The majority of experiments show that tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of mesenchymal stem cells and pre-osteoblasts by activated nuclear factor-kappaB (NF-κB) signaling. However, the underlying mechanisms by which NF-κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF-α-activated NF-κB signaling on osteogenic differentiation of pre-osteoblasts. Murine MC3T3-E1 pre-osteoblasts were treated with 10ng/mL of TNF-α. NF-κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF-κB signaling inhibition. EphB4 signaling was activated using ephrinB2-fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription-polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p-NF-κB p65 and NF-κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining. TNF-α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3-E1 cells. Activation of EphB4 signaling by ephrinB2-fc promoted osteogenic differentiation of MC3T3-E1 cells, whereas TNF-α impaired the osteogenic differentiation enhanced by ephrinB2-fc. Pyrrolidine dithiocarbamate blocked the activation of NF-κB signaling induced by TNF-α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF-α. TNF-α inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated NF-κB signaling pathway.

Introduction Tripartite motif 65 (TRIM65) is a crucial regulator of cell differentiation, proliferation, migration, invasion, and carcinogenesis. However, its role in osteoporosis (OP) remains unclear. In this study, we evaluated the role of TRIM65 in regulating osteoblast differentiation and calcification. Materials and Methods The role of TRIM65 during the osteogenic differentiation of MC3T3-E1 cells was evaluated. The expression of COL1A1, RUNX2, and OCN was examined using western blot analysis and immunofluorescence staining. The formation of calcium nodules was evaluated using alizarin red staining. Alkaline phosphatase activity was evaluated using ALP staining. Results TRIM65 expression was significantly elevated during the osteogenic differentiation of bone marrow mesenchymal stem and MC3T3-E1 cells. We demonstrated that TRIM65 overexpression enhanced osteogenic differentiation and promoted bone formation in the MC3T3-E1 cells. Conversely, TRIM65 inhibited the osteogenic differentiation and bone formation of the MC3T3-E1 cells. Mechanistically, we found that TRIM65 knockdown in MC3T3-E1 cells up-regulated the phosphorylated protein expression of PI3K and AKT, which was contrary to the results of the TRIM65-overexpression group. Conclusion Our research suggests that TRIM65 is an important osteogenic differentiation and bone formation regulator and offers a therapeutic application for OP.

Glucocorticoid-induced osteoporosis is the third epidemic osteoporosis following postmenopausal and senileosteoporosis. According to one study, salidroside made ovariectomized rats' bones strong. Salidroside's potential for treating glucocorticoid-induced osteoporosis remains unproven. This study aimed to investigate the protective effect and mechanism of salidroside on dexamethasone-induced osteogenic differentiation and bone formation in MC3T3-E1 cells and zebrafish. The study proved that salindroside had no harmful impact on MC3T3E1 cells. Salidroside significantly relieved dexamethasone-induced inhibition of ALP (alkaline phosphatase) activity and mineralization in MC3T3-E1 cells, and promoted osteogenic differentiation of cells. Salidroside increased the expression of osteopontin (OPN), runt-related transcription factor 2 (Runx2), osterix (Osx), transforming growth factor-beta (TGF-β) proteins and promoted the phosphorylation of Smad2/3 in MC3T3-E1 cells treated with dexamethasone. In addition, the effect of salidroside in relieving dexamethasone-induced inhibition of osteogenic differentiation in MC3T3-E1 cells can be blocked by TGF-β receptor type I/II inhibitor (LY2109761). At the same time, we found that salidroside significantly alleviated the inhibition of dexamethasone-induced bone formation in zebrafish and promoted the mineralization of zebrafish skulls. LY2109761 reversed the protective impact of salidroside on dexamethasone-mediated bone impairment in zebrafish. These findings suggested that salidroside alleviated dexamethasone-induced inhibition of osteogenic differentiation and bone formation via TGF-β/Smad2/3 signaling pathway.

Lipopolysaccharide (LPS) has been confirmed to be the main inhibitor in osteogenic differentiation, posing a clinical challenge to bone healing, particularly for trauma followed by endotoxinemia/sepsis. However, the molecular mechanism remains ambiguous. miR-23b, which regulates multiple signaling pathways in inflammation, has been shown to be deregulated by LPS. In this study, we examined the LPS-mediated regulation on the expression of miR-23b and Smad 3 in preosteoblast MC3T3-E1 cells. Then we determined the regulation of miR-23b overexpression on the Smad 3 expression and on the LPS-mediated inhibition of bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. Our results demonstrated that LPS significantly downregulated the expression of miR-23b, while upregulating Smad 3 in MC3T3-E1 cells. However, the transfection with miR-23b mimics markedly downregulated the Smad 3 in both mRNA and protein levels, via the specific binding to the 3'-untranslated region (UTR) of Smad 3. Moreover, though LPS markedly downregulated the BMP-2-induced osteogenic differentiation of MC3T3-E1 cells by inhibiting the expression of alkaline phosphatase (ALP), Osteocalcin (OCN), Osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2). The upregulated miR-23b reversed such downregulation of ALP, OCN, OPN and RUNX2 in the MC3T3-E1 cells which were treated both with LPS and BMP-2. In conclusion, our data indicates that miR-23b ameliorates the LPS-mediated inhibition of BMP-2-induced osteogenic differentiation in MC3T3-E1 cells, implying the protective role of miR-23b in the LPS-mediated inhibition of osteogenic differentiation and bone formation.

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.

Er-xian decoction drug-containing serum promotes Mc3t3-e1 cell proliferation and osteogenic differentiation via regulating BK channel

Osteoporosis is defined as a bone condition characterized by bone mass reduction, bone micro-architectural and quality deterioration, leading to compromised strength and increased chances of fracture. Evidence have shown an essential role of microRNAs (miRNAs) in various osteogenic differentiation processes. However, the function of miR-15a-5p in the differentiation of osteogenic cells and possible mechanisms remains unclear. The present study explored the expression of miR-15a-5p in human osteoporosis specimens and during the osteogenic differentiation of MC3T3-E1 cells. Functions of miR-15a-5p were determined using miR-15a-5p mimics and inhibitors. Luciferase assay was used to verify the binding of miR-15a-5p and PDCD4 3ʹUTR. Alizarin Red Staining (ARS) and Alkaline phosphatase (ALP) activity were used to determine the miR-15a-5p role in osteogenic differentiation. Finally, Wnt pathway inhibitor was used to determine the miR-15a-5p/PDCD4/Wnt signaling pathway in regulating osteogenic differentiation. We found miR-15a-5p expression was increased in human osteoporosis specimens and during differentiation of MC3T3-E1 cells. PDCD4 was also identified as a target of miR-15a-5p and was found to be involved in osteogenic differentiation. Further, miR-15a-5p mimics attenuated the effects of PDCD4 overexpression. Finally, use of XAV939 (Wnt pathway inhibitor) downregulated osteogenic differentiation in miR-15a5p/PDCD4/Wnt-dependent signaling pathway. In conclusion, miR-15a-5p induced differentiation of osteoblasts and mineralization by modulating osteoblast differentiation factors, mainly OSX, ALP, OCN, and RUNX2, by inhibiting PDCD4 and Wnt signaling pathways. This study provides a modality for the future use of miR-15a-5p in the treatment and prevention of osteoporosis.

Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in osteoblastic differentiation. However, the exact role of STAT3 in osteogenic differentiation of the pre-osteoblastic cell line MC3T3-E1 is still controversial. In this study, we demonstrated that eradication of STAT3 signaling by the inhibitors cryptotanshinone (CPT, a STAT3-specific inhibitor) or STAT3 siRNA both suppressed osteogenic differentiation of MC3T3-E1 cells, with a decrease in alkaline phosphatase (ALP) activity, protein expressions of the osteogenic differentiation markers Collagen I (ColI), ALP, and osteocalcin (OCN), and reduced matrix mineralization capacity at the terminal stage of osteogenic differentiation. However, the inhibition of STAT3 by CPT did not affect MC3T3-E1 cell proliferation. To further clarify the effect of STAT3 on osteogenic differentiation of MC3T3-E1 cells, we forced STAT3 expression and found that this ameliorated osteogenic differentiation. Thus, our results confirmed that STAT3 is a likely positive regulator of osteogenic differentiation in MC3T3-E1 cells. These findings may provide a basis for the development of more efficient and controllable protocols for osteoblastic differentiation and facilitate their use in regenerative medicine. In addition, our results provide novel insights into the effect of the STAT3 antagonist CPT on modulation of osteogenesis.

Background Mechanical stress plays an important role in the maintenance of bone homeostasis. Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone. This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress (FSS) is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells. Methods MC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm2 using a parallel plate flow system. After FSS treatment, cytoskeleton immunohistochemical staining and microRNAs (miRNAs) were detected immediately. Osteogenic gene expression and immunohistochemical staining for collagen type I were tested at the 24th hour after treatment, alkaline phosphatase (ALP) activity assay was performed at 24th, 48th, and 72th hours after FSS treatment, and Alizarin Red Staining was checked at day 12. Results One hour of FSS at 12 dyn/cm2 induced actin stress fiber formation and rearrangement, up-regulated osteogenic gene expression, increased ALP activity, promoted synthesis and secretion of type I collagen, enhanced nodule formation, and promoted terminal differentiation in MC3T3-E1 cells. During osteogenic differentiation, expression levels of miR-20a, -21, -19b, -34a, -34c, -140, and -200b in FSS-induced cells were significantly down-regulated. Conclusion The short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be invovled in FSS-induced pre-osteoblast differentiation.