mRNA Expression Levels Of Runx2 Research Articles

Kaempferol stimulates WNT/β-catenin signaling pathway to induce differentiation of osteoblasts

Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on stimulating bone mineral density and bone formation. Among them, kaempferol has been reported to assist bone formation in vitro and in vivo, but its precise mechanism of action for stimulating bone forming abilities of osteoblasts remained elusive. In SaOS-2 osteoblasts, treatment of kaempferol increased early and late osteogenic parameters significantly, including alkaline phosphatase (ALP) activity, collagen synthesis, and mRNA expression levels of Runx2, osterix, osteopontin and bone sialoprotein. Interestingly, kaempferol promoted osteoblastic differentiation via the activation of the WNT signaling pathway. The stimulation of SaOS-2 cells by kaempferol resulted in an increased activity of WNT signaling responsive reporter construct, Axin-2, and, subsequently, stabilization of WNT signaling mediated transcription factor β-catenin, probably leading to the activation of WNT-targeted genes for osteogenesis. In corroboration, the kaempferol-induced ALP activity was fully abolished by FH 535, an inhibitor of WNT signaling pathway. Kaempferol mediated activation of WNT signaling pathway through estrogen signaling pathway, as the application of ICI 182,780 (an inhibitor for estrogen receptors) markedly inhibited kaempferol-induced WNT signaling activation and osteogenic marker like ALP activity in SaOS-2 cells. Immunohistochemical studies in drill-hole defect model showed increased expression of Runx2 and β-catenin staining after kaempferol treatment. Thus, it may be concluded that kaempferol stimulates estrogen signaling followed by WNT signaling pathway activation to achieve its potential for bone-sparing effects.

Effect of estradiol on high glucose‑induced osteoblast injury.

Estradiol (E2) serves an important role in the changes of postmenopausal bone turnover rate and the development of osteoporosis. The present study aimed to investigate the effects of E2 on high glucose (HG)-induced osteoblast injury. Cell Counting Kit-8 was used to determine cell viability. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting was used to analyze the mRNA and protein expression levels of osteocalcin, Runt-related transcription factor 2 (Runx2), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO1). Flow cytometry was performed to analyze apoptosis. The results revealed that cell viability was lower in cells treated with HG (100, 200 or 300 mg/dl) compared with the control group. Cell viability was decreased in cells treated with 200 mg/dl HG on days 3, 5 and 7. In addition, cell viability was increased by 0.1 µM E2. E2 with HG co-treatment increased cell viability, osteocalcin and Runx2 mRNA expression levels and nuclear Nrf2 and HO1 protein expression levels compared with the HG-only group. All these changes, with the exception of Runx2, were reversed by silencing Nrf2 expression using small interfering (si)RNA (siNrf2). Additionally, apoptosis was reduced by E2 in HG-treated cells, which was reversed by siNrf2 transfection. These results demonstrated that E2 may prevent HG-induced osteoblast injury by activating Nrf2/HO1 signaling pathways.

MicroRNA-153 inhibits cell proliferation, migration, invasion and epithelial-mesenchymal transition in breast cancer via direct targeting of RUNX2.

A number of microRNAs (miRNAs) are involved in the development and malignant progression of numerous types of human cancer including breast cancer. The underlying regulatory mechanism of miRNA-153 (miR-153) in breast cancer progression remains largely unknown. The present study demonstrated that miR-153 expression levels were significantly reduced in breast cancer tissue samples and cell lines, compared with adjacent healthy tissue samples and normal human breast cell line MCF-10A. In addition, low miR-153 expression was associated with advanced clinical staging and metastasis in patients with breast cancer. However, no association with age, subtype or differentiation was identified. Furthermore, patients with breast cancer with low miR-153 expression had poor prognosis, compared with patients with breast cancer with high miR-153 expression. Overexpression of miR-153 reduced proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in breast cancer SK-BR-3 and BT-549 cells. Runt-related transcription factor 2 (RUNX2), which was revealed to be significantly upregulated in breast cancer, was verified as a target gene of miR-153 in SK-BR-3 and BT-549 cells by luciferase reporter gene assay. High RUNX2 expression was associated with advanced clinical staging as well as distant and lymph node metastasis in patients with breast cancer. However, no association with age, subtype or differentiation was identified. Additionally, an inverse correlation between miR-153 and RUNX2 mRNA expression levels was observed in breast cancer tissues. RUNX2 overexpression reduced the suppressive effects of miR-153 on the proliferation, migration, invasion and EMT of SK-BR-3 and BT-549 cells. The present study indicated that miR-153 may serve a role in breast tumor growth and metastasis via direct targeting of RUNX2. The miR-153/RUNX2 axis may be used as a potential therapeutic target in breast cancer treatment.

Arbutin promotes MC3T3‑E1 mouse osteoblast precursor cell proliferation and differentiation via the Wnt/β‑catenin signaling pathway.

Arbutin is a natural compound extracted from various plants, including bearberry leaves, that exerts multiple effects including skin whitening, anti-inflammatory and oxidative stress-protective properties. However, the effects of arbutin on osteoblasts remain unknown. The aim of the present study was to investigate the function and the mechanisms of arbutin on the proliferation and differentiation of MC3T3-E1 mouse osteoblast precursor cells in vitro. The proliferation of MC3T3-E1 cells treated with arbutin was assessed using a Cell Counting Kit-8 assay and a 5-ethynyl-2′-deoxyuridine labeling assay. Additionally, cell cycle and apoptosis were examined using flow cytometry analysis. The effects of arbutin on osteoblast differentiation were investigated using alkaline phosphatase (ALP) staining and by examining the mRNA expression levels of collagen type I α1 chain (COL1A1), bone γ-carboxyglutamate protein (BGLAP) and Sp7 transcription factor (SP7). To further investigate the molecular mechanism underlying arbutin function in promoting osteogenesis, the mRNA and protein expression levels of runt-related transcription factor 2 (RUNX2) and β-catenin were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting. Arbutin significantly promoted MC3T3-E1 cell proliferation and increased the ratio of cells in S-phase. Treatment with arbutin increased ALP activity and the mRNA expression levels of COL1A1, BGLAP and SP7 in MC3T3-E1 cells. Furthermore, the protein and the mRNA expression levels of RUNX2 and β-catenin increased significantly following treatment with arbutin. Collectively, the present findings suggested that arbutin was able to promote proliferation and differentiation of MC3T3-E1 cells via the Wnt/β-catenin signaling pathway.

Experimental study on the prevention of bone loss by deferoxamine in type 1 diabetic mice with osteoporosis

Objective To observe the effect of a hypoxia mimicking agent deferoxamine (DFO) on the mineral density, volume, architecture, strength, and metabolism of the bones in type 1 diabetic mice with osteoporosis. Methods Type 1 diabetic mice model was established by intraperitoneal injections of streptozotocin. The mice were divided into control (normal mice), diabetes mellitus, and DFO groups. Micro-CT was used to analyze the bone mineral density, volume, architecture, and strength of the trabecule in the distal part of femurs. Three point bending test was carried out to evaluate the bone strength. Hematoxylin and eosin (HE) staining was performed to observe the alteration in the number of osteoblasts. Real-time PCR was used to detect the mRNA expressions of Runt-related gene 2 (Runx-2), osteoclacin, and tartrate resistant acid phosphatase (TRAP) in tibias. Western blot was used to detect the protein expressions of Hypoxia-inducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF) in tibias. Results There was a decrease in mineral density, volume, strength of bones as well as deteriorated trabecular microarchitecture in diabetic mice as compared to control mice, which were partially improved by DFO treatment. Moreover, DFO treatment increased the number of osteoblasts and mRNA expression levels of Runx-2, osteoclacin, TRAP, as well as protein expression levels of HIF-1α and VEGF(P<0.05). Conclusion Bone loss could be partially prevented by DFO treatment in type 1 diabetic osteoporosis mice, which might be ascribed to increased bone formation via stimulating hypoxia inducible factor singnaling pathway. Key words: Hypoxia mimicking agent; Deferoxamine; Osteoporosis; Diabete, type 1

MiR-143-3p regulates early cartilage differentiation of BMSCs and promotes cartilage damage repair through targeting BMPR2.

The aim of this study was to explore the role of microRNA-143-3p (miR-143-3p) in cartilage injury, and to investigate the possible underlying mechanism. A chondrogenic differentiation cell model was established in bone marrow mesenchymal stem cells (BMSCs). The mRNA expression levels of runt-related transcription factor 2 (RUNX2), miR-143-3p and bone morphogenetic protein 2 (BMPR2) in BMSCs were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) after 0 d, 5 d and 10 d, respectively. Mesenchymal stem cells (MSCs) were transfected with miR-143-3p mimics and its control in accordance with the liposome method. Alcian blue colorimetric assay was used to evaluate proteoglycan deposition of MSCs. Meanwhile, qRT-PCR and Western blot were performed to analyze the expression levels of ACAN and COL2A1. Luciferase reporter gene assay was applied to verify the binding status of miR-143-3p and BMPR2 3'UTR. Also, proteoglycan deposition and the expression of ACAN and COL2A1 were detected after simultaneous transfection of miR-143-3p mimics and BMPR2 overexpression plasmid. 0 d, 5 d and 10 d after inducing cartilage differentiation, the mRNA expression levels of RUNX2 and BMPR2 were markedly increased. However, the expression level of miR-143-3p was significantly decreased with the prolongation of induction period. After transfection with miR-143-3p mimics, the level of miR-143-3p in MSCs was remarkably increased. Alcian blue colorimetric assay and staining assay showed that the deposition of proteoglycans in the mimics group was significantly lower than that of the control group. Meanwhile, after overexpressing miR-143-3p, the levels of cartilage differentiation marker proteins including ACAN and COL2A1 were remarkably reduced. Luciferase report gene assay indicated that miR-143-3p could negatively regulate BMPR2 by binding to its 3'UTR. In addition, overexpression of BMPR2 could strikingly reverse the above effects of overexpressed miR-143-3p. During chondrogenic differentiation, the level of miR-143-3p was decreased. Moreover, miR-143-3p could regulate the differentiation process by targeting BMPR2 in BMSCs.

Effect of hydroxyapatite nanoparticles and wedelolactone on osteoblastogenesis from bone marrow mesenchymal stem cells.

Regenerative medicine has a high demand for the development of scaffold materials combined with other osteogenic inducers to generate bioactive composite materials for bone replacement therapies. Previously, we reported that wedelolactone promoted osteoblastogenesis of bone marrow mesenchymal stem cells (BMSCs). In this study, the effect of hydroxyapatites (HAps), bone composite materials we prepared, and the combined effect of wedelolactone and HAps on osteoblastogenesis differentiation was first evaluated. Three kinds of HAps constructed by a rod-like shape with particle size of 25 nm (HAp-1), 37 nm (HAp-2), and 33 nm (HAp-3) did not affect BMSC survival, but induced activity of alkaline phosphatase(ALP), a marker enzyme for osteoblastogenesis. HAp-1 treatment resulted in a more significant increase in the number of ALP staining-positive BMSC, and maintained an extended time for the increased number of ALP staining-positive BMSC. Moreover, HAp-1 combined with wedelolactone induced a higher ALP activity for a longer time than HAp-2 and HAp-3, and also increased the bone mineralization level. Osteoblastogenesis-related marker genes expression including osteorix, osteocalcin, and runx2 were increased after BMSC were treated with HAp-1 for 6 days. Although three kinds of HAps treatment for 9 days increased osteorix mRNA expression, osteocalcin, and runx2 mRNA expression levels were upregulated only by HAp-1. Similarly, only HAp-1 enhanced wedelolactone-induced osteocalcin, osteorix, and runx2 mRNA expression after 9 days treatment. Together, these results suggested that HAps with different sizes generated different effect on osteoblastogenesis. HAp-1 combined with wedelolacone can exert an enhanced effect on osteoblastogenesis, which has potential for treating osteoporosis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 145-153, 2019.

Cellular Response of Anodized Titanium Surface by Poly(Lactide-co-Glycolide)/Bone Morphogenic Protein-2.

The purpose of this study is to examine physical characteristics of and initial biological properties to anodized titanium treated with poly(d,l-lactide-co-glycolide) (PLG) mixed with recombinant human bone morphogenic protein-2 (rhBMP-2). Titanium specimens were prepared in groups of four as follows: group NC was anodized under 300V as control; group PC was anodized then dropped and dried with solution 0.02ml PLG; group D was anodized then dropped and dried with solution 0.02ml PLG/rhBMP-2 (3.75μg per disc); and group E was anodized then coated with 0.02ml PLG/rhBMP-2 (3.75μg per disc) by electrospray. Human osteoblastic-like sarcoma cells were cultured. Cell proliferation and alkaline phosphatase (ALP) activity test were carried out. Runx-2 gene was investigated by the reverse transcription-polymerase chain reaction. Immunofluorescence outcome of osteogenic proteins was observed. After 3days, there were significantly higher proliferations compared rhBMP-2 loaded titanium discs with rhBMP-2 unloaded discs. The ALPase activity on rhBMP-2 loaded titanium discs was significantly higher than in rhBMP-2 unloaded discs. The expression level of Runx2 mRNA presented the highest on the PLG/rhBMP-2-coated surface. PLG polymers mixed with rhBMP-2 might improve proliferation, differentiation and osteogenic protein formation of cells on the anodized titanium.

Oral epithelial cells inhibit the proliferation, mRNA expressions of collagen type I, RUNX2 and BGP, and ALP activity of osteoblast-like cells in coculture

Cell-to-cell interactions play important roles in regulating cell proliferation and differentiation, tissue production, organ construction and ontogenesis. The purpose of this study was to investigate the effects of oral epithelial (OE) cells on osteoblasts’ behavior for proliferation and osteogenic differentiation. Osteoblast-like cells were co-cultured in 6-well-dishes with or without OE cells growing on cell culture inserts. After 1, 3, 7 and 14 d, the characteristics of osteoblast-like cells were evaluated regarding cell proliferation, mRNA expression levels of collagen type I, RUNX2 and bone Gla protein (BGP), and alkaline phosphate (ALP) activity. In cell proliferation assays, the numbers of osteoblast-like cells cultured with or without OE cells increased over time for 2 w, but the proliferation of osteoblast-like cells co-cultured with OE cells plateaued after 7 d. The mRNA expression levels of type I collagen in osteoblast-like cells cultured with or without OE cells tended to decrease over time, but the decrease in osteoblast-like cells co-cultured with OE cells was more dramatic. The mRNA expression levels of RUNX2 and BGP in osteoblast-like cells cultured without OE cells tended to increase over time, but those increases in cells cultured without OE cells were more dramatic than in osteoblast-like cells cocultured with OE cells. The ALP activity of osteoblast-like cells cultured without OE cells increased until 7 d and then decreased at 14 d, but osteoblast-like cells cultured with OE cells underwent little change in ALP activity throughout the 14 d of culture. The ALP activity of osteoblast-like cells cultured without OE cells was significantly higher at 7 and 14 d than in osteoblast-like cells cultured with OE cells. In conclusion, these results indicate that OE cells inhibit the osteogenic differentiation and function of osteoblast-like cells.

Influence of double gene recombinant vector transfected alcohol-induced rabbit stem cells on expressions of adipogenic and osteogenic genes

Objective To investigate the influence of transfection with double gene recombinant vector on the expression of adipogenic and osteogenic genes in alcohol-induced rabbit stem cells. Methods The rabbit bone marrow stem cells (BMSCs) of third generation were randomly divided into 6 groups: (1) normal group (BMSCs given no special treatment); (2) model group (MBSCs given 0.09 mol/L ethanol, no gene transfection); (3) unrelated sequence group (BMSCs givne 10 μl unrelated sequence transfection); (4) siPPARγ group (BMSCs given 10 μl siPPARγ gene vector transfection); (5) exCGRP group [BMSCs given 10 μl calcitonin gene-related peptide (CGRP) gene transfection]; (6) double gene group (BMSCs given 10 μl double gene recombinant vector transfection). Except the normal group, the final mass concentration of 0.09 mol/L ethanol was added to the remaining groups of the cells after transfection and every time the liquid was changed. The expression of PPARγ, CGRP, runt related transcription factor-2 (Runx2) and Osteocalcin mRNA and their proteins was detected at 7th and 14th day of the experiment. Results At 7th day, the expression levels of PPARγ mRNA and its protein in groups of double gene, siPPARγ and normal were low (0.329±0.037 and 0.162±0.013, 0.413±0.045 and 0.174±0.023, 0.376±0.042 and 0.169±0.021), significantly lower than those in model group (0.672±0.079 and 0.871±0.096), unrelated sequence group (0.674±0.083 and 0.823±0.089), and exCGRP group (0.683±0.079 and 0.839±0.091), and the differences were statistically significant (P=0.000). The expression levels of CGRP mRNA and its protein in groups of double gene and exCGRP were high, significantly higher than those in model group, unrelated sequence group, siPPARγ group and normal group, and the differences were statistically significant (P=0.000). The expression levels of Runx2 mRNA, Osteocalcin mRNA and theirs proteins in groups of double gene, exCGRP, siPPARγ and normal were high, those in double gene group were significantly higher than those in model group and unrelated sequence group, and higher than those in groups of exCGRP, siPPARγ and normal, and the differences were statistically significant (P=0.000). At 14th day, the expression levels in all groups were consistent with those at the 7th day. Conclusion Double gene recombinant vector transfected alcohol-induced rabbit stem cells can effectively inhibit the expression of adipogenic gene and up-regulate the expression of osteogenic genes in rabbit stem cells, which is more significant than single gene action. Key words: Double gene recombinant vector; Transfect; Alcohol; Stem cells; Adipogenic genes; Osteogenic genes; Gene expression

Inhibition of neuropeptide Y Y1 receptor induces osteoblast differentiation in MC3T3-E1 cells

Neuropeptide Y (NPY) is a major neural signaling molecule. NPY is produced by peripheral tissues, such as osteoblasts, and binds to the corresponding Y1 receptor that belongs to the G‑protein‑coupled receptor family. Osteoblast‑specific Y1 receptor knockout mice exhibit high bone mass, indicating a role of the NPY‑Y1 receptor axis in the regulation of bone homeostasis. In the bone microenvironment, peripheral nerve fibers and osteoblasts produce NPY. However, the effects of the Y1 receptor on osteoblasts remain unexplored. In the present study, an RNA interference approach was employed to target the Y1 receptor, in order to determine whether it may function to regulate the growth, differentiation and viability of osteoblasts. Knockdown of the Y1 receptor by small interfering RNA (siRNA) lead to induction of alkaline phosphatase (ALP) activity and mineralization in mouse MC3T3‑E1 osteoblast cells. In addition, the mRNA expression levels of ALP, osteocalcin, collagen (I) α1, and bone sialoprotein were significantly increased following transfection of a Y1 receptor siRNA. Furthermore, the mRNA expression levels of Runx2 and osterix were significantly increased; however, no significant alterations in cell proliferation and caspase‑3/7 activity were observed in Y1 receptor siRNA‑transfected cells when compared with non‑targeting controls. The results demonstrate that Y1 receptor inhibition may increase osteoblastic differentiation, which indicates a role of the Y1 receptor in the regulation of osteoblastic differentiation.

EFFECT OF CHANGE OF TISSUE INTERFACE STIFFNESS ON OSTEOGENIC DIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS

To investigate the effect of tissue interface stiffness change on the spreading, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and to find the suitable stiffness range for stem cell differentiation. Bone marrow of male Sprague Dawley rats (4 weeks old) were selected to isolate and culture BMSCs by whole bone marrow cell adherent method. The third generation BMSCs (1×105 cells/mL) were inoculated into the ordinary culture dishes covered with polyacrylamide hydrophilic gel (PA) which elastic modulus was 1, 4, 10, 40, and 80 kPa (cells seeded on PA), and ordinary culture dish (75 MPa extreme high elastic modulus) as control. Spreading of cells in different stiffness of PA was observed under light microscope. The elastic modulus values of 4, 10, and 40 kPa PA were selected as groups A, B, and C respectively; the ordinary culture dish (75 MPa extreme high elastic modulus) was used as control group (group D). Cell counts was used to detect the growth conditions of BMSCs, alkaline phosphatase (ALP) kit to detect the concentration of ALP, alizarin red staining technique to detect calcium deposition status, and real-time quatitative PCR technique to detect the expressions of bone gla protein (BGP), Runx2, and collagen type I mRNA. With increased PA stiffness, BMSCs spreading area gradually increased, especially in 10 kPa and 40 kPa. At 1 and 2 days after culture, the growth rate showed no significant difference between groups (P>0.05); at 3-5 days, the growth rate of groups B and C was significantly faster than that of groups A and D (P<0.05), but difference was not statistically significant between groups A and D (P<0.05); at 5 days, the proliferation of group C was significantly higher than that of group B (P<0.05). ALP concentrations were (53.69±0.89), (97.30±1.57), (126.60±14.54), and (12.93±0.58) U/gprot in groups A, B, C, and D respectively; groups A, B, and C were significantly higher than group D, and group C was significantly higher than groups A and B (P<0.05). Alizarin red staining showed that the percentages of calcium nodules was 20.07%±4.24% in group C; group C was significantly higher than groups A, B, and D (P<0.05). The expression levels of BGP and collagen type I mRNA were significantly higher in groups A, B, and C than group D, and in group C than groups A and B (P<0.05). The expression level of Runx2 mRNA was significantly higher in groups B and C than group D, and in group C than group B (P<0.05), but no significant difference was found between groups A and D (P>0.05). PA elastic modulus of 10-40 kPa can promote the proliferation and osteogenic differentiation of BMSCs, and the higher the stiffness, the stronger the promoting effect.

EphB4/ephrinB2 ephrinB1 Interaction Mediated Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Osteogenic Differentiation in Vitro and In Vivo

Background and Objective: Osteoblasts, important of stromal cells in bone marrow microenvironment, maintain HSCs in resting state and protect its' functions. Osteoblasts derived from mesenchymal stem cells (MSCs), which can be differentiated into osteoblast in bone marrow under the regulation of cytokines. Recent studies have indicated that EphB4/ephrinB2 protein participates in the regulation of osteogenesis differentiation of MSCs in bone marrow microenvironment.Our previous study found that EphB4 receptor was over expressed in CML patients and cell lines, which played an important role to change characterize of Imatinib(IM)-resistant in chronic myeloid leukemia cells. Furthermore, we performed experiments to prove that osteogenic differentiation in MSCs from CML-initial patient significantly higher in contrast to normal human MSCs and the change of EphB4 molecules on leukemia cells may transform MSCs functions in vitro. However, the mechanism of these transformations of MSCs in vitro and what is change in vivo were still unclear. Therefore, we hypothesis that the change of EphB4 molecules on leukemia cells might play an important role to osteogenic differentiate in MSCs in vitro and in vivo, which support to leukemia progression and disruption of normal hematopoiesis.Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or CML- chronic phase (CCP) patients’ BM and cultures in Cyagen Bone marrow culture medium at 37 °C, 5% CO2 incubator. In vitro, after stimulated with different concentrations of EphB4-Fc (0, 5, 8, 10 ug/ml) for 21 days, visualized by Alizarin Red staining, MSCs (CCP) produced maximum calcium nodules (P<0.05, n=3) in EphB4-Fc (8 ug/ml) group in contrast with other groups, accompanied by increased ephrinB1 and STAT3 phosphorylation. In vitro osteogenesis condition, after treatment with EphB4-Fc (0, 8 ug/ml) 14 days, MSCs (CCP) incubated with K562 cells. After 48 h, the IC50 (0.842±0.065, P<0.05, P<0.05 ANOVA, n=4) of K562 cells in MSCs+EphB4-Fc (8ug/ml) group increased, S phase cells percentage(56.6±4.01, P<0.05, P<0.01, ANOVA, n=4) increased and cells apoptosis rate(P<0.01, P<0.001, LSD, n=4) declined compared with K562 (control group) and K562+MSCs+EphB4-Fc (0 ug/ml). In vivo, K562-R, K562-R+MSCs (normal) (5:1), K562-R-EphB4-sh, K562-R-EphB4-sh+MSCs (normal) (5:1), MSCs (normal) cells were injected respectively into bone cavity of NOG rat (NOD/SCID/γ c-/-, n=12) rat and blank control group were also established. Examined peripheral blood in rats while hCD45+ cells > 1% is considered as leukemia model. K562-R+MSCs mice were earliest to establish leukemia model (31.75±1.26d) and had the shortest survival time(4.25±1.71d) than other groups. After treatment with IM, survival times of K562-R+MSCs mice were not significantly extended (4.7±3.055 d, pared-samples T test, P>0.05). In bone marrow of K562-R+MSCs mice, RUNX2 mRNA (0.654±0.0278; P < 0.001) over expressed in contrast to other groups. After treatment with IM, expression level of RUNX2 mRNA was significantly increased than non-treatment group. Among four leukemia groups of mice, expression levels VEGF mRNA in bone marrow were no significantly difference and there was no statistical difference existed in treatment group and non-treatment group. The same cells lines above were subcutaneously injected to establish subcutaneous transplantation tumor, respectively, in NOG rat (NOD/SCID/γ c-/-, n=8) rat. K562-R+MSCs tumors were earliest to appear (17.333±1.154 d) and had the biggest tumors volume (13116.27±165.502 mm3, P<0.001) compared to other groups. After mice treated by IM, compared with non-treatment group, K562-R+MSCs tumors had significantly increased in volume (14703.14±309.333mm3, pared-samples T test, P<0.01). VEGF mRNA (0.861±0.0648; P<0.01) in K562-R+MSCs tumor over express than other groups. After treatment, the expression level was no significantly declined (0.796±0.0688, P>0.05). The level expression of RUNX2 mRNA in four groups of subcutaneous transplantation tumors are low and had no statistical difference.Conclusion: Our experiments in vitro and in vivo illustrated that EphB4 molecule on leukemia cells may transform MSCs osteogenic differentiation to change characterize of Imatinib(IM)-resistant in CML through ephrinB1 and STAT3 phosphorylation. DisclosuresLin:Natural Science Foundation of China: Research Funding. Na:Natural Science Foundation of China: Research Funding.

Effect of BMPs and Wnt3a co-expression on the osteogenetic capacity of osteoblasts

In the present study, third‑generation autologous‑inactivated bone morphogenic protein2 (BMP2), BMP4, BMP6, BMP7, BMP9 and Wnt3a lentiviral vectors were constructed and integrated into the genome of MC3T3‑E1 murine mesenchymal stem cells (MMSCs) to produce osteoinductive factor gene‑modified MMSCs. The transfection efficiency of each osteoinductive factor was then determined by detecting the expression levels of runt related transcription factor2 (Runx2) mRNA. The cotransfection with combinations of two lentiviruses was performed, and the expression levels of bone γ‑carboxyglutamate protein and alkaline phosphatase in the MC3T3‑E1 cell culture supernatant were detected. The expression level of Runx2 mRNA was detected by reverse transcription‑polymerase chain reaction, and western blotting was performed to detect the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a. The results demonstrated that the recombinant lentiviruses were successfully transfected into MC3T3‑E1 cells. The relative expression levels of Runx2 mRNA were greatest in the BMP2 group, sequentially followed by the BMP4, BMP9, BMP7, Wnt3a and BMP6 groups. The results of cotransfection of MC3T3‑E1 cells (a total of 8groups) demonstrated that BMP‑2 and BMP‑7 exhibited the highest cotransfection efficiency. Western blot analysis demonstrated that following BMP2 and BMP7 cotransfection of MC3T3‑E1 cells, the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a were increased compared with control cells. In conclusion, the third‑generation lentiviral vectors effectively improved the osteogenic efficiencies of MC3T3‑E1 cells, which provided an important theoretical basis and therapeutic strategy for bone reconstruction and tissue engineering.

Regulatory mechanism of Runt related transcription factor 2 in fracture healing by salidrosid

Objective To compare and evaluate the differences of curative effects and biological mechanisms underlying the posttraumatic bone healing, as well as the study of the regulation of Runt related transcription factor 2 (Runx2) signal pathway mechanism of osteogenesis by using Salidroside. Methods To establish an close osteotomy model of SD rats. 40 rats divide into experimental group and control group. Using microCT to evaluate and compare the curative efficacy about the posttraumatic bone healing. Using the method of enzyme linked immunosorbent assay (ELISA), the serum concentrations of alkaline phosphatase (ALP), osteoprotegerin (OPG) were assessed. Using the method of real-time quantitative polymerase chain reaction (Real-time PCR), the mRNA expression levels of Runx2, hypoxia inducible factor (HIF) 2A, Wnt4, Wnt10b, β-catenin, glycogen synthase kinase (GSK)-3β, T-cell factor (TCF)-1 in callus tissue were determined. Results At 2 and 4 weeks after surgery, the callus bone structural parameters of microCT were significantly improved in experimental group compared to that of control group (P<0.05). In addition, at weeks 2 and 4 after surgery, the mRNA expressions of Runx2 (2 w: 1.83±0.11, 4 w: 2.23±0.11), HIF2A (2 w: 1.37±0.12, 4 w: 1.50±0.12), Wnt4 (2 w: 1.78±0.10, 4 w: 1.66±0.11), Wnt10b (2 w: 1.89±0.12, 4 w: 1.92±0.13), β-catenin (2 w: 1.65±0.08, 4 w: 1.72±0.10) were significantly increased, whereas the mRNA expressions of GSK-3β (2 w: 0.61±0.08, 4 w: 0.56±0.09) were significantly decreased (P<0.05). The serum concentrations of ALP [2 w: (335.34±9.23) IU/L, 4 w: (312.45±10.56) IU/L]P, OPG [2 w: (40.02±2.90) IU/L, 4 w: (45.56±3.20) pg/ml] were significantly increased (P<0.05). Conclusion Compared with the Control group, the Salidroside group make an effect to fracture healing. Key words: Salidroside; Runt related transcription factor 2 signal pathway; Osteogenesis regulation

Naringin protects human adipose-derived mesenchymal stem cells against hydrogen peroxide-induced inhibition of osteogenic differentiation

Extensive evidence indicates that oxidative stress plays a pivotal role in the development of osteoporosis. We show that naringin, a natural antioxidant and anti-inflammatory compound, effectively protects human adipose-derived mesenchymal stem cells (hADMSCs) against hydrogen peroxide (H2O2)-induced inhibition of osteogenic differentiation. Naringin increased viability of hAMDSCs and attenuated H2O2-induced cytotoxicity. Naringin also reversed H2O2-induced oxidative stress. Oxidative stress induced by H2O2 inhibits osteogenic differentiation by decreasing alkaline phosphatase (ALP) activity, calcium content and mRNA expression levels of osteogenesis marker genes RUNX2 and OSX in hADMSCs. However, addition of naringin leads to a significant recovery, suggesting the protective effects of naringin against H2O2-induced inhibition of osteogenic differentiation. Furthermore, the H2O2-induced decrease of protein expressions of β-catenin and clyclin D1, two important transcriptional regulators of Wnt-signaling, was successfully rescued by naringin treatment. Also, in the presence of Wnt inhibitor DKK-1, naringin is no longer effective in stimulating ALP activity, increasing calcium content and mRNA expression levels of RUNX2 and OSX in H2O2-exposed hADMSCs. These data clearly demonstrates that naringin protects hADMSCs against oxidative stress-induced inhibition of osteogenic differentiation, which may involve Wnt signaling pathway. Our work suggests that naringin may be a useful addition to the treatment armamentarium for osteoporosis and activation of Wnt signaling may represent attractive therapeutic strategy for the treatment of degenerative disease of bone tissue.

Oral mucosal fibroblasts overexpressing BMP-2 differentiate into osteoblasts and participate in new bone formation during bone regeneration

ObjectiveTransplantation of non-osteogenic cells that express osteoinductive factors is a potential strategy for cell-mediated bone regenerative therapy. In this study, we investigated the effects of transplantation of canine oral mucosal fibroblasts (COFs) overexpressing human bone morphogenetic protein (BMP-2) on bone regeneration. MethodsCOFs isolated from canine buccal mucosa were transfected with BMP-2-containing pCEP4 or pcDNA vectors by electroporation. The effects of BMP-2 on osteoblast differentiation were assessed by measuring the activity of alkaline phosphatase and the expression of osterix and Runx2 in BMP-2-overexpressing COFs. BMP-2-overexpressing COFs were transplanted into bone defects created on calvariae of athymic mice. Bone regeneration was assessed by micro-CT analysis and histological examination. The fates of transplanted cells were traced on histological sections by monitoring the expression of green fluorescence protein (GFP), which was cotransfected with BMP-2. ResultsTransfection with pcDNA–BMP2 or pCEP4–BMP2 increased the numbers of ALP-positive cells and the mRNA expression levels of Runx2 and Osterix. Micro-CT and histological analyses revealed that transplantation of COFs transfected with pCEP4–BMP2 or pcDNA–BMP2 induced regeneration of calvarial bone defects, while COFs transfected with control pCEP4 or pcDNA plasmids failed to achieve bone regeneration. The histological experiments tracing GFP expression revealed that COFs transfected with pCEP4–BMP2 or pcDNA–BMP2 differentiated into osteoblasts and osteocytes in regions of regenerating bone. ConclusionsTransplanted COFs that overexpressed BMP-2 differentiated into osteoblasts and osteocytes during bone regeneration, and participated in new bone formation.

Molecular Cloning and Gene Expression of Mummichog (Fundulus heteroclitus) Runx2 During Embryogenesis

In our previous study, we clarified the toxicity of 2,2'-dipyridyldisulfide [(PS)2], one of photodegradation products of a metal pyrithione that is used as an alternative antifouling paint biocides to organotin compounds in Japan. In early life stage toxicity tests, we exposed the mummichog, (Fundulus heteroclitus) to (PS)2, and the hatched larvae subsequently displayed notochord undulations and skeletal deformities ( Mochida et al., 2012 ). Runx2, a transcription factor of the runt family, is a key regulator in skeletal development in mammals. It is possible that (PS)2 inhibits Runx2 gene expression, inducing the skeletal deformities in mummichog. In the present study, we cloned two Runx2 cDNAs (type I and type II) from mummichog embryos. The deduced amino acid sequences of type I and type II contain an open reading frame encoding 450 and 464 amino acid residues, respectively. The derived amino acid sequence of Fundulus Runx2 type I showed the highest identity (93.8%) with Takifugu Runx2 type I, and Fundulus Runx2 type II showed 94.6% homology with medaka Runx2. The expression level of Runx2 mRNA in the early stage series was measured using a real-time quantitative PCR assay. Expression levels tended to increase in both the blastula-gastrula and the retinal pigmentation stage. To examine the effect of toxic compounds on skeletal formation, mummichog embryos in the late blastula to retinal pigmentation stage were exposed to (PS)2. After exposure to (PS)2 for one week, the expression level of Runx2 mRNA was unchanged. These results suggest that there is no inhibition of Runx2 gene expression due to (PS)2 exposure.

Effect of pioglitazone on transdifferentiation of preosteoblasts from rat bone mesenchymal stem cells into adipocytes

We aimed to examine the effect of pioglitazone on transdifferentiation of preosteoblasts from rat bone marrow mesenchymal stem cells (BMSCs) into adipocytes and investigate its effect on bone metabolism. BMSCs were harvested from the femurs and tibias of a rat, then separated, purified, proliferated for 3 generations and differentiated into preosteoblasts for 5 days and 14 days respectively in the presence of osteogenic medium. Thereafter, the preosteoblasts were cultured for 21 days in the presence of adipogenic medium with and without pioglitazone (1 μg/mL). Partially-differentiated osteoblasts were identified by mineralized nodules with Alizarin red S staining. Transdifferentiated adipocytes were identified by Oil Red O staining. Reverse transcription PCR (RT-PCR) was performed to assay the expression levels of osteogenic markers Runx2 and ALP, and an adipogenic marker PPARγ. Those cells cultured for 5 days did not show mineralized nodules as detected by staining of Alizarin red S, while those cultured for 14 days showed dispersed mineralized centers in the form of brown spots, although without obvious red mineralized nodules. After adipogenic transdifferentiation for 21 days, adipose-drops were found in cells of 5CG and 5EG earlier than those of 14CG and 14EG, and the former showed much more adipocytes separately as detected by Oil Red O staining. Whatever the time was 5 days or 14 days of BMSCs osteogenic differentiation, the cells cultured with pioglitazone showed much more adipocytes than those without pioglitazone. Our experiment showed that the less time it took for BMSCs osteogenic differentiation, a stronger ability remained for BMSCs to transdifferentiate into adipocytes. The mRNA expression levels of Runx2 and ALP were decreased by 1.79 and 1.90 times respectively in 5EG (P< 0.05) as compared with 5CG, and that of PPARγ was increased by 1.31 times in 5EG (P<0.05) as compared with 5CG. The mRNA expression levels of Runx2 and ALP were decreased by 1.45 and 1.54 times respectively in 14EG (P<0.05) as compared with 14CG, and that of PPARγ was increased by 1.39 times in 14EG (P<0.05) as compared with 14CG. It was concluded that pioglitazone stimulated the transdifferentiation of BMSCs into adipocytes. These observations provided a potential mechanism of imbalance in thiazolidinedione induced bone metabolism.

Imatinib inhibits proliferation of human mesenchymal stem cells and promotes early but not late osteoblast differentiation in vitro

Altered bone metabolism has been reported in patients with chronic myeloid leukemia treated with the tyrosine kinase inhibitor imatinib. Several studies have shown that imatinib inhibits the differentiation and activity of osteoclasts in vitro, whereas the effects of imatinib on osteoblast differentiation are less clear. In this study osteoblast differentiation was induced in human mesenchymal stem cells (hMSCs) by treatment with bone morphogenetic protein 2 in vitro. Imatinib inhibited proliferation of hMSCs in a dose-dependent manner. Even though imatinib promoted early osteoblast differentiation assessed by alkaline phosphate activity, mineralization measured by Alizarin Red staining (ARS) was reduced by imatinib. Moreover, the inhibitory effect of imatinib on mineralization was most prominent at low concentrations of imatinib. When we measured the relative mRNA expression levels of Runx2, we found that Runx2 expression was higher in imatinib-treated (5μM) cultures at early time points during differentiation. On the other hand, the expression of Osterix late during differentiation was lower in imatinib-treated (5μM) cultures, corresponding to the ARS results. Thus, the effect of imatinib on osteoblast differentiation is not only dependent on the drug concentration, but indeed also on the maturation stage of the cells. This finding might partly explain why previous studies on the effects of imatinib osteoblast differentiation have shown different results.