Protein Expression Levels Of RUNX2 Research Articles

The osteogenic effects of sappanchalcone in vitro and in vivo.

The utilization of natural products to enhance the function of periodontal ligament cells (PDLCs) has emerged as a popular area of research. Recent investigations have demonstrated that sappanchalcone (SC) possesses pharmacological properties such as anti-inflammatory and osteoprotective effects. This study aims to explore the impact of SC on the in vivo and in vitro osteogenic differentiation ability of PDLCs. Cell proliferation was quantified using the CCK-8 assay, while gene expression levels were assessed through qRT-PCR analysis. Osteoblast differentiation capacity was evaluated by employing Alizarin red staining (ARS), alkaline phosphatase (ALP) staining and western blot (WB) analysis. A rat model of periodontitis was established utilizing the tether-wire method. Micro-CT imaging and hematoxylin and eosin (HE) staining were employed to evaluate alveolar bone resorption. Masson's trichrome staining was utilized to observe fiber alignment, whereas immunohistochemistry (IHC) techniques were applied for detecting osteogenic and inflammatory factors. The results from the CCK-8 assay indicate no observed cytotoxicity for concentrations of 1, 5, or 10 nM for SC treatment (p < .05), while qRT-PCR analysis demonstrates a significant decrease in inflammatory factors such as MMP-1 and IL-6 with treatment by SC (p < .05). Additionally, western blotting reveals an increase in protein expression levels of Runx2 and OPN within PDLCs treated with SC compared to control groups (p < .05), which is further supported by ARS and ALP staining indicating an increase in mineralized nodules formation along with elevated ALP content within these cells following treatment with this compound (p < .05). Finally, both HE staining as well as micro-CT imaging suggest potential benefits associated with using this compound including slowing alveolar bone resorption while simultaneously promoting junctional epithelium proliferation. Our in vitro and in vivo findings suggest that SC can effectively enhance the inflammatory response of PDLCs and promote their osteogenic differentiation ability under inflammatory conditions, indicating its potential as a promising therapeutic agent for improving periodontal inflammation and bone formation.

Embelin enhances the osteogenic potential of LPS-induced periodontal ligament stem cells by activating AMPK and SIRT1

Purpose: To investigate the role of embelin in periodontal ligament stem cells (PDLSCs) in an in vitro model of periodontitis.Methods: Lipopolysaccharide (LPS)-stimulated PDLSCs was used to construct a periodontitis cell model. PDLSCs in the treatment group were pretreated with different concentrations of Embelin, and CCK-8 and TUNEL staining were used to analyze cell viability and apoptosis. Enzyme-linked immunosorbent assay (ELISA) kits were used to evaluate the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1) while reactive oxygen species levels were assessed by 2',7'- dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Subsequently, osteogenic marker, ALP activity and protein expression levels of Runx2, OCN and BMP-2 in PDLSCs were evaluated by western-blot assay; AMPK and SIRT1 levels were also determined using Western blot assay.Results: Embelin pretreatment inhibited PDLSCs apoptosis, inflammatory factors, and oxidative stress, but up-regulated ALP, Runx2, OCN, and BMP-2 levels (p &lt; 0.05). In addition, AMPK phosphorylation and SIRT1 protein levels were regulated by embelin (p &lt; 0.05).Conclusion: Embelin exerts anti-inflammatory, anti-oxidative and osteogenic differentiation effects in LPS-induced PDLSCs cells in vitro by activating AMPK/SIRT1 signaling. Therefore, the compound has potentials for use in the management of periodontitis.

Effect of recombinant human fibroblast growth factor 21 on the mineralization of cementoblasts and its related mechanism.

To investigate the effect of recombinant human fibroblast growth factor 21 (rhFGF21) on the proliferation and mineralization of cementoblasts and its mechanism. Hematoxylin eosin, immunohistochemical staining, and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21 (FGF21) in rat periodontal tissues and cementoblasts (OCCM-30), separately. Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 under treatment with rhFGF21. Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction. The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The expression levels of genes of transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling pathway in OCCM-30 were detected through PCR array analysis. FGF21 was expressed in rat periodontal tissues and OCCM-30. Although rhFGF21 had no significant effect on the proliferation of OCCM-30, treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction. The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction. Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction. rhFGF21 up-regulated Bmpr1b protein expression in cells. rhFGF21 can promote the mineralization ability of OCCM-30. This effect is related to the activation of the TGFβ/BMP signaling pathway.

Effect of condylar chondrocyte exosomes on condylar cartilage osteogenesis in rats under tensile stress.

Background: Functional orthoses are commonly used to treat skeletal Class II malocclusion, but the specific mechanism through which they do this has been a challenging topic in orthodontics. In the present study, we aimed to explore the effect of tensile stress on the osteogenic differentiation of condylar chondrocytes from an exosomal perspective. Methods: We cultured rat condylar chondrocytes under resting and tensile stress conditions and subsequently extracted cellular exosomes from them. We then screened miRNAs that were differentially expressed between the two exosome extracts by high-throughput sequencing and performed bioinformatics analysis and osteogenesis-related target gene prediction using the TargetScan and miRanda softwares. Exosomes cultured under resting and tensile stress conditions were co-cultured with condylar chondrocytes for 24h to form the Control-Exo and Force-Exo exosome groups, respectively. Quantitative real time PCR(RT-qPCR) and western blotting were then used to determine the mRNA and protein expression levels of Runx2 and Sox9 in condylar chondrocytes. Results: The mRNA and protein expression levels of Runx2 and Sox9 in the Force-Exo group were significantly higher than those in the Control-Exo group (p < 0.05). The differential miRNA expression results were consistent with our sequencing results. Bioinformatics analysis and target gene prediction results showed that the main biological processes and molecular functions involved in differential miRNA expression in exosomes under tensile stress were biological processes and protein binding, respectively. Kyoto Gene and Genome Data Bank (KEGG) pathway enrichment analysis showed significant enrichment of differentially expressed miRNAs in the mTOR signaling pathway. The differentially expressed miRNAs were found to target osteogenesis-related genes. Conclusion: These results suggest that stimulation of rat condylar chondrocytes with tensile stress can alter the expression levels of certain miRNAs in their exosomes and promote their osteogenic differentiation. Exosomes under tensile stress culture conditions thus have potential applications in the treatment of Osteoarthritis (OA).

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway

BackgroundBone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms.MethodshDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria.ResultsThe calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture.ConclusionsThe stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.

Metformin activates Wnt/β-catenin for the treatment of diabetic osteoporosis

BackgroundWith the deepening of social aging, the incidence rate of osteoporosis and diabetes continues to rise. More and more clinical studies show that diabetes is highly correlated with osteoporosis. Diabetes osteoporosis is considered as a metabolic bone disease of diabetes patients. This study aims to explore the role and mechanism of metformin (Met) in diabetic osteoporosis.MethodsMouse MC3T3-E1 cells were treated with Met (0.5 mM) and exposed to high glucose (HG, 35 mM). The cells were cultured in an osteogenic medium for osteogenic differentiation, and the cell proliferation ability was determined using Cell Counting Kit-8; Alkaline phosphatase (ALP) activity detection and alizarin red staining were utilized to evaluate the effect of Met on MC3T3-E1 osteogenic differentiation. Western blot was used to detect the expressions of osteogenesis-related proteins (Runx2 and OCN) as well as Wnt/β-catenin signaling pathway-related proteins in MC3T3-E1 cells.ResultsHG inhibited proliferation and calcification of MC3T3-E1 cells, down-regulated ALP activity, and the expression of Runx2 and OCN in MC3T3-E1 cells. Meanwhile, the activity of the Wnt/β-catenin signaling pathway was inhibited. Met treatment was found to significantly stimulate the proliferation and calcification of MC3T3-E1 cells under HG conditions, as well as increase the ALP activity and the protein expression level of Runx2 and OCN in the cells. As a result, osteogenic differentiation was promoted and osteoporosis was alleviated. Apart from this, Met also increased the protein expression level of Wnt1, β-catenin, and C-myc to activate the Wnt/β-catenin signaling pathway.ConclusionMet can stimulate the proliferation and osteogenic differentiation of MC3T3-E1 cells under HG conditions. Met may also treat diabetic osteoporosis through Wnt/β-catenin activation.

Amniotic membrane mesenchymal stem cells-based therapy improves Bmi-1-deficient mandible osteoporosis through stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption.

Mandible osteoporosis with age is characterized by greater fragility and accompanied with abnormal oral function. Mesenchymal stem cell transplantation can ameliorate osteoporosis. Bmi-1 is a transcriptional repressor which is an important regulator of cell cycle, stem cells self-renewal, and cell senescence. Here, we use a new kind of membrane mesenchymal stem cells (MSCs), amniotic membrane mesenchymal stem cells (AMSCs), to explore therapeutic effects on Bmi-1-deficient caused mandible osteoporosis. Phenotypes of mandibles from 5-week-old Bmi-1-deficient mice with AMSCs-based therapy were compared with age-matched Bmi-1-deficient mandibles without AMSCs-based therapy and wild-type mice. Bmi-1-deficient mice without AMSCs-based therapy displayed mandible osteoporosis accompanied with the rising senescence-associated molecules and imbalance redox homeostasis. Results showed that the alveolar bone volume, cortical thickness, type I collagen and osteocalcin immunopositive areas, mRNA expression levels of alkaline phosphatase, superoxide dismutase, gluathione reductase, and protein expression level of Runx2 were all reduced significantly in Bmi-1-/- mandibles. Protein levels of PPARγ, p16, p21, p53, and redox gene levels of Bnip3l, Cdo1, Duox1, and Duox2 were up-regulated in mandibles from vehicle-transplanted Bmi-1-/- mice. Also, osteoclasts were activated in Bmi-1-/- alveolar bone. Transplanted AMSCs migrated into mandibles and improved all the parameters in Bmi-1-/- mandibles with AMSCs-based therapy. These findings indicate that AMSCs-based therapy could rescue mandible osteoporosis induced by Bmi-1 deficiency through stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption. Our findings implied that AMSCs-based therapy had preventative and therapeutic potential for mandible osteoporosis.

Network pharmacology combined with bioinformatics to investigate the mechanism of Xianlinggubao capsule in the treatment of osteoporosis

BackgroundXianlinggubao capsule (XLGB), a traditional Chinese medicine (TCM) prescription, has been widely applied to treat osteoporosis (OP) in China. However, its active components and mechanism have not been fully elucidated. PurposeThis study aims to elucidate the active components of XLGB and its mechanism for the treatment of OP. MethodsNetwork pharmacology and bioinformatics are combined to elucidate the active components and mechanism of XLGB in the treatment of OP, including the acquisition of differentially expressed genes (DEGs) of OP, screening of active components and their targets of XLGB, construction of the drug-target-disease regulatory network, and analysis of the key subnetwork. Finally, these predicted results were validated by in vitro experiments and literature comparison. ResultsBy using bioinformatics, 2473 DEGs of OP were identified in the GEO datasets analysis (GSE35958). By using network analysis, 180 active compounds in the six herbs of XLGB and 61 OP-related targets were identified, which were significantly enriched in 118 signaling pathways and 108 GO terms. The in vitro studies showed that quercetin, luteolin, apigenin and ursolic acid, the active components of XLGB, could effectively promote the mineralization of MC3T3-E1 cells, and the combined application of ursolic acid and luteolin could significantly increase the mRNA and protein expression level of Runx2, OPN and Col l, and enhance the phosphorylation of p38 and JNK. ConclusionXLGB plays an anti-osteoporosis role in a multi-component, multi-target and multi-pathway manner. The combination of network pharmacology and bioinformatics provides a method for the study of TCM in the treatment of complex diseases.

The effect of aging on the biological and immunological characteristics of periodontal ligament stem cells

BackgroundPeriodontal ligament stem cells (PDLSCs) have many applications in the field of cytotherapy, tissue engineering, and regenerative medicine. However, the effect of age on the biological and immunological characteristics of PDLSCs remains unclear.MethodsIn this study, we compared PDLSCs isolated from young and adult individuals. PDLSC proliferation was analyzed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) staining, and apoptosis level was detected by Annexin V-PE/7-ADD staining. PDLSC osteogenic/adipogenic/chondrogenic differentiation potentials were assessed by alkaline phosphatase (ALP), Alizarin Red, Oil Red O, Alcian Blue staining, and related quantitative analysis. PDLSC immunosuppressive capacity was determined by EdU and Annexin V-PE/7-ADD staining. To explore its underlying mechanism, microarray, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and western blot analyses were performed to detect differentially expressed genes and proteins in PDLSCs.ResultsOur results demonstrated that with aging, the proliferation and osteogenic/adipogenic/chondrogenic differentiation potential of PDLSCs decreased, whereas apoptosis of PDLSCs increased. Moreover, the immunosuppressive ability of PDLSCs decreased with aging. Compared with PDLSCs from young subjects, analysis of mRNA expression revealed an upregulation of CCND3 and RC3H2, and a downregulation of Runx2, ALP, COL1A1, PPARγ2, CXCL12, FKBP1A, FKBP1B, NCSTN, P2RX7, PPP3CB, RIPK2, SLC11A1, and TP53 in those from adult individuals. Furthermore, protein expression levels of Runx2, ALP, COL1A1, and PPARγ2 in the adult group were decreased, whereas that of CCND3 increased.ConclusionsTaken together, aging influences the biological and immunological characteristics of PDLSCs, and thus, it is more appropriate to utilize PDLSCs from young individuals for tissue regeneration, post-aging treatment, and allotransplantation.

Telomerase enhances osteogenic ifferentiation of sheep bone marrow mesenchymal stem cells (BMSCs) by up-regulating PI3K/Akt pathway in vitro.

Telomerase reverse transcriptase (TERT) vectors were transfected into bone marrow mesen- chymal stem cells (BMSCs) which were then cultured and selected to establish TERT-BMSC cell lines whilst sequencing BMSCs and TERT-BMSCs via transcriptome in this study to explore their regulatory mechanism and effect on osteogenic differentiation after TERT ectopic expres- sion in sheep BMSCs. After sequencing and analysing differential genes, PI3K/Akt signalling pathway related to osteogenic differentiation was investigated. Western blot was used before and after applying the PI3K/Akt signalling pathway inhibitor LY294002 to detect protein expression levels of AKT and p-AKT. On the twenty-first day of osteogenic differentiation, RT-qPCR and Western blot were used to detect mRNA and protein expression levels of RUNX2 and OPN and alizarin red staining was utilised to analyse calcium salt deposition. Results showed that pro- tein expression levels of AKT and p-AKT were significantly up-regulated, mRNA and protein expression levels of RUNX2 and OPN increased and calcium salt deposition increased after ectopic expression of TERT. After applying LY294002, the protein expression of AKT and p-AKT was down-regulated, mRNA and protein expression levels of RUNX2 and OPN were reduced and calcium salt deposition was reduced. These results confirmed the stable integration and expression of the exogenous TERT gene in BMSCs to promote the differentiation of BMSC osteoblasts, which may be mediated by the PI3K/Akt signalling pathway.

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.

MiR-137 affects bone mineral density in osteoporosis rats through regulating RUNX2.

To study the influence of micro ribonucleic acid (miR)-137 on osteoporosis rats by regulating runt-related transcription factor 2 (RUNX2). A total of 36 Sprague-Dawley rats were randomly assigned to the normal group (n=12), model group (n=12), and inhibitor group (n=12). No treatment was performed in the normal group. The osteoporosis model in rats was prepared in the model group, and miR-137 inhibitor was administered in osteoporosis rats of inhibitor group. Following 12 weeks of intervention, sampling was conducted. The expression of RUNX2 was detected via immunohistochemistry, and its protein expression level was determined via Western blotting. Quantitative Polymerase Chain Reaction (qPCR) was carried out to detect the mRNA level of miR-137. The contents of serum bone Gla protein (BGP) and total alkaline phosphatase (TALP) were measured using enzyme-linked immunosorbent assay (ELISA). Finally, bone mineral density was determined with a dual-energy X-ray absorptiometry instrument. According to the immunohistochemistry detection, the rats in model group and inhibitor group had a notably lower positive expression level of RUNX2 than normal group (p<0.05), and its expression level in the inhibitor group was substantially higher than that in the model group (p<0.05). Western blotting results showed that compared with that in the normal group, the protein expression level of RUNX2 was notably lowered in the model and inhibitor group (p<0.05), which was markedly higher in the inhibitor group than that in the model group (p<0.05). It was found through the qPCR that the expression level of miR-137 was remarkably raised in both model group and inhibitor group compared with that in the normal group, showing statistically significant differences (p<0.05). The rats in the inhibitor group had a remarkably lower expression level of miR-137 than the model group (p<0.05). ELISA results revealed that the model group and inhibitor group had substantially lower contents of serum BGP and TALP than the normal group (p<0.05), and that their contents rose dramatically in the inhibitor group compared with that in the model group (p<0.05). Additionally, based on the measurement of bone mineral density, compared with that in the normal group, bone mineral density declined considerably in the model group and inhibitor group (p<0.05). It was markedly elevated in inhibitor group in comparison with that in the model group (p<0.05). MiR-137 regulates RUNX2 to affect the bone mineral density of osteoporosis model rats.

MicroRNA-19b-3p promotes cell proliferation and osteogenic differentiation of BMSCs by interacting with lncRNA H19

BackgroundTo investigated the role of miR-19b-3p in regulating bone marrow mesenchymal stem cell (BMSC) proliferation and osteoblast differentiation.MethodsThe expression of miR-19b-3p and lncRNA H19 were measured in postmenopausal osteoporosis patients and BMP-22 induced BMSCs using qRT-PCR. MiR-19b-3p mimic or inhibitor was transfected into BMP-2 induced BMSCs. Cell proliferation was measured by BrdU method. Protein expression of RUNX2 and COL1A1 were measured by western blot. PcDNA3.1-lncRNA H19 with or without miR-19b-3p mimic was transfected into BMP-2 induced BMSCs.ResultsThe expression of miR-19b-3p was significantly up-regulated in postmenopausal osteoporosis patients and BMP-2 induced BMSCs. MiR-19b-3p overexpression dramatically elevated, while miR-19b-3p inhibition decreased cell proliferation of BMSCs. Additionally, protein expression levels of RUNX2 and COL1A1, as well as ALP activity were significantly promoted by miR-19b-3p mimic transfection and inhibited by miR-19b-3p inhibitor transfection. LncRNA H19 was obviously down-regulated in postmenopausal osteoporosis patients. H19 overexpression significantly decreased cell proliferation and differentiation by down-regulating miR-19b-3p. Moreover, the expression of miR-19b-3p was inhibited, while H19 elvated in 17β-estradiol (E2) treated BMSCs in a dose-dependent manner.ConclusionThese data were the first to reveal the critical role of H19/miR-19b-3p in postmenopausal osteoporosis, and provided a new therapeutic target for OP.

MicroRNA-1286 inhibits osteogenic differentiation of mesenchymal stem cells to promote the progression of osteoporosis via regulating FZD4 expression.

The aim of this study was to investigate whether microRNA-1286 could inhibit the osteogenic differentiation of human marrow mesenchymal stem cells (hMSCs) by regulating FZD4 expression and promoting the progression of osteoporosis. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of microRNA-1286 in the serum of patients with osteoporosis. Meanwhile, microRNA-1286 expression in different stages of osteogenic differentiation of hMSCs was measured by qRT-PCR as well. After overexpression of microRNA-1286 and FZD4 in hMSCs, the mRNA expression levels of microRNA-1286, alkaline phosphatase (ALP), RUNX2 and osteocalcin (OCN) were detected by qRT-PCR. The protein expression levels of RUNX2 and OCN were detected by Western blot. Meanwhile, alkaline phosphatase (ALP) activity and expression in cells were examined using ALP assay kit and ALP staining method, respectively. Cell mineralized nodules were detected through the alizarin red staining test. Bioinformatics method was used to predict the binding site of microRNA-1286 to FZD4. Subsequent luciferase reporter gene assay was performed to verify whether microRNA-1286 could combine with FZD4. After overexpression or knockdown of microRNA-1286, the mRNA and protein expressions of FZD4 were analyzed using qRT-PCR and Western blot assay, respectively. After the simultaneous overexpression of microRNA-1286 and FZD4 in hMSCs, the mRNA expression levels of ALP, RUNX2 and OCN, ALP activity and content, and cell mineralization ability were successively examined. The expression of microRNA-1286 in the serum of patients with osteoporosis was significantly higher than that of the normal population. Meanwhile, microRNA-1286 expression decreased with the increase of osteogenic differentiation days of hAMSCs. After the overexpression of microRNA-1286, ALP, RUNX2, and OCN levels, ALP activity, RUNX2, and OCN protein levels, as well as mineralized nodule formation were significantly reduced. However, results were reversed when FZD4 was simultaneously up-regulated. Luciferase reporter gene assay results verified that microRNA-1286 could bind to FZD4. After the overexpression of microRNA-1286, the mRNA and protein expressions of FZD4 were found significantly down-regulated. However, results were reversed after knocking down microRNA-1286. Furthermore, the simultaneous overexpression of microRNA-1286 and FZD4 could counteract the inhibitory effect of over-expression of microRNA-1286 on osteogenic differentiation of hMSCs. MicroRNA-1286 can regulate FZD4 expression and inhibit osteogenic differentiation of hMSCs, thereby promoting the development of osteoporosis.

Phosphorylation of porcine bone collagen peptide to improve its calcium chelating capacity and its effect on promoting the proliferation, differentiation and mineralization of osteoblastic MC3T3-E1 cells

Abstract In this study, porcine bone collagen peptide (CP) was phosphorylated with sodium tripoly phosphate (STP) in order to improve its calcium binding capacity and osteogenic activity. Firstly, the phosphorylation process were optimized by an orthogonal experiment. Then, the effects of CP, phosphorylated CP (PCP), CP-calcium chelate (CP-Ca) and PCP-Ca on proliferation, differentiation and mineralization of MC3T3-E1 cells were investigated. The results showed that all of the CP, PCP, CP-Ca and PCP-Ca could enhance the proliferation, differentiation and mineralization of MC3T3-E1 cells by increasing the mRNA expression levels of alkaline phosphatase (ALP), Runt-related transcription factor-2 (Runx2), osteocalcin (OCN), osteopontin (OPN) and collagen type I (Col I), as well as the protein expression levels of Runx2 and β-Catenin. Especially, PCP-Ca had the greatest and significant effect on mineralization of MC3T3-E1 cells (P

LncRNA UCA1 affects osteoblast proliferation and differentiation by regulating BMP-2 expression.

The aim of this study was to detect the expression of long non-coding ribonucleic acid (lncRNA) urothelial carcinoma associated 1 (UCA1) in the plasma of patients with osteoporosis (OST), and to investigate its influences on the proliferation and differentiation of osteoblasts and its mechanism. Plasma samples were collected from 52 OST patients treated in our hospital and 30 healthy subjects receiving a physical examination, respectively. The expression level of lncRNA UCA1 in OST patients and healthy subjects were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Furthermore, osteoblast MC3T3-E1 cell lines with a stable knockout of UCA1 in mice were constructed using small-interfering RNA (siRNA). The influence of UCA1 knockout on the proliferation of osteoblasts was detected using cell counting kit-8 (CCK-8) assay. Meanwhile, the proportion of EdU-positive cells in osteoblasts of the control group and UCA1 knockout group was detected using EdU staining. Moreover, the messenger RNA (mRNA) levels of differentiation-related genes, including Runt-related transcription factor 2 (Runx2), Collagen1α1, osteoclast (OC), osteoprotegerin (OPG), osteopontin (OPN) and Osterix (OSX), were detected via RT-PCR. The protein expression level of Runx2 was detected via Western blotting. In addition, osteoblasts were cultured with a bone-derived medium for 14 d. Then, the differentiation status was detected via alizarin red staining and alkaline phosphatase staining. Finally, the expression of bone morphogenetic protein-2 (BMP-2)/(Smad1/5/8) signaling pathway was analyzed using Western blotting. The expression of plasma lncRNA UCA1 was significantly increased in OST patients (p<0.05). Cell experiments revealed that UCA1 siRNA intervention could significantly promote the proliferation and differentiation of osteoblast MC3T3-E1 cell lines. In addition, Western blotting showed that the pro-apoptotic effect of UCA1 might be mediated by the BMP-2/(Smad1/5/8) signaling pathway in osteoblasts. Inhibiting lncRNA UCA1 can promote the proliferation and differentiation of osteoblasts by activating the BMP-2/(Smad1/5/8) signaling pathway in osteoblasts. Therefore, UCA1 is expected to be a new therapeutic target for OST.

Lentivirus‑mediated microRNA‑26a overexpression in bone mesenchymal stem cells facilitates bone regeneration in bone defects of calvaria in mice

Repair of bone defects presents a serious clinical challenge as it is difficult to restore bone function and regenerate bone loss. In the present study, the effects of lentivirus-mediated transfection of bone marrow mesenchymal stem cells (BMSCs) with microRNA (miR)-26a on bone regeneration were investigated in a mouse bone defect repair model. Marker of proliferation Ki67 (Ki67) staining was employed to detect the cell proliferation capacity and alkaline phosphatase (ALP) staining was used to investigate osteogenic differentiation. A mouse model of cranial bone defects was established. β-tricalcium phosphate biomaterials co-cultured with the transfected BMSCs were implanted into the defect areas of mouse models. Micro-computed tomography, and hematoxylin and eosin and toluidine blue staining, were used to detect bone regeneration in the defect areas and the degradation of scaffolds. miR-26a expression, and the mRNA and protein expression of osteogenesis-associated cytokines, were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Separated and cultured BMSCs highly expressed CD29 and CD105, but not CD34 and CD45, as determined by flow cytometry. miR-26a expression and the positive cell rate of Ki67 and ALP staining in BMSCs transfected with pLVTHM-miR-26a were increased. The BMSC and negative control-transfected BMSC groups exhibited increased bone regeneration in the defect areas, increased bone volume of newly formed bones, and elevated mRNA and protein expression of runt-related transcription factor 2 (Runx2) and osteocalcin (OC), compared with the blank group. However, the miR-26a-transfected BMSC group exhibited further increases in bone regeneration and the volume of newly formed bones, and further elevations of the mRNA and protein expression levels of Runx2 and OC. The present findings demonstrated that lentivirus-mediated modification of BMSCs enhanced bone regeneration during the repair of cranial bone defects in mice.

Exposure Duration Is a Determinant of the Effect of Sinusoidal Electromagnetic Fields on Peak Bone Mass of Young Rats.

We proposed a three-step strategy to obtain the optimal therapeutic parameters, which is composed of large-scale screening at cellular level, verification in animal experiments, and confirmation by a clinical trial. The objective of the current study was to test the feasibility of our strategy. Newborn rat calvarial osteoblasts were treated by 50Hz 1.8mT sinusoidal electromagnetic fields (SEMFs) with 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0h/days, respectively. The osteogenic differentiation and maturation of the osteoblast were assayed and compared to obtain the optimal duration. One-month-old growing rats were then treated by the same SEMFs with 0.5, 1.5, and 2.5h/days, respectively, and the peak bone mass was analyzed after 2months. It was found that the optimal exposure duration to promote the osteogenic differentiation and maturation of osteoblasts was 1.5h/days, judging by the increasing degrees of ALP activity, calcified nodules formed, the gene and protein expression levels of Runx-2, BMP-2, and Col-I, as well as the expression levels of signaling proteins of the BMP-2/Smad1/5/8 pathway. The highest increase of peak bone mass after 2months was also obtained by 1.5h/days, judging by the results of X-ray dual-energy absorptiometry, mechanical property analysis, micro-CT scanning, and serum bone turnover marker examinations. The above results indicated that exposure duration is a determinant for the therapeutic effect of EMFs, and the optimal therapeutic effects only can be obtained by the optimal exposure duration.

Mechanical strain promotes osteogenic differentiation of bone mesenchymal stem cells from ovariectomized rats via the phosphoinositide 3‑kinase/Akt signaling pathway.

Osteoporosis has become an overwhelming public health problem worldwide. As an elementary physiological factor to regulate bone formation and regeneration, mechanical strain may be used as a non‑invasive intervention in osteoporosis prevention and treatment. However, little is known regarding the underlying mechanism. The aim of the current study was to investigate the effect of continuous mechanical strain (CMS) on osteogenic differentiation of bone mesenchymal stem cells (BMSCs) from ovariectomized rats (OVX BMSCs). In addition, involvement of the phosphatidylinositol3‑kinase (PI3K)/Akt signaling pathway in biomechanical signal transduction and its function were evaluated. The results demonstrated that OVX BMSCs subjected to CMS exhibited higher alkaline phosphatase (ALP) activity and deeper staining at 24 and 48h. In addition, CMS upregulated the mRNA expression levels of ALP, collagen type I, runt related transcription factor 2 (Runx2), as well as the protein expression level of Runx2 in a time‑dependent manner. The PI3K/Akt signaling pathway was rapidly activated by CMS, with its phosphorylation level reaching its maximum in a short duration and a large quantity of phosphorylated‑Akt remaining in the nucleus. Pre‑treatment with a selective blocker significantly blocked the strain‑induced activation of PI3K/Akt and reduced the commitment of OVX BMSCs into osteoblasts, demonstrating a dominated regulative effect of PI3K/Akt signaling in strain‑induced osteogenesis. These results indicated that CMS induced the early differentiation of OVX BMSCs towards an osteogenic phenotype by activating the PI3K/Akt signaling pathway.

FTY720 enhances osteogenic differentiation of bone marrow mesenchymal stem cells in ovariectomized rats.

Sphingosine-1-phosphate and its structural analog FTY720 (fingolimod) are important in the inhibition of osteoclast differentiation and bone resorption, however, it remains unknown whether they enhance osteogenic differentiation of the bone marrow mesenchymal stem cells (BM‑MSCs). The present study investigated the effect of FTY720 on the osteogenic differentiation of BM‑MSCs from the femurs of the ovariectomized (OVX) rats. Three different concentrations (1, 10 and 100nM) of FTY720 were demonstrated to markedly upregulate mRNA expression levels of Runt‑related transcription factor2 (Runx2) and Sp7 transcription factor (Sp7) at 2weeks, and alkaline phosphatase (ALP) at 3weeks. The osteocalcin (OCN) expression was similar at weeks2 and 3. The protein expression levels of Runx2, Sp7, OCN and ALP induced by three different concentrations of FTY720 were higher than those in the control groups at 3weeks in the OVX and sham groups. The findings of the current study suggested a beneficial effect of FTY720 on bone formation in OVX rats, and provided a potential therapeutic method of FTY720 to prevent alveolar bone resorption in patients with post‑menopausal osteoporosis.