Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

Journal: Molecular Medicine Reports

Article Title: Effectiveness of combined salmon calcitonin and aspirin therapy for osteoporosis in ovariectomized rats

doi: 10.3892/mmr.2015.3637

Figure Lengend Snippet: Combined treatment (sCT+ASA) enhances the OPG/RANKL expression ratio in OVX rats. Effects of ASA, sCT and combined treatment (sCT+ASA) on mRNA levels in femur bone marrow cells from ovariectomized (OVX) rats. Gene expression was analyzed by reverse transcription-quantitative polymerase chain reaction. Values are expressed as the mean ± standard deviation. P-values were calculated by analysis of variance with Student-Newman-Keuls analysis. (A) OPG; (B) RANKL; (C) mRNA expression ratio of OPG/RANKL. ASA, aspirin; sCT, salmon calcitonin; OVX, ovariectomized; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor κB ligand; mRNA, messenger RNA.

Article Snippet: The antibodies used were as follows: OPG primary antibody (1:80; BA1475; Wuhan Boster Biological Technology Co. Ltd., Wuhan, China; rabbit anti-rat OPG), RANKL primary antibody (1:80; BA1323; Wuhan Boster Biological Technology Co. Ltd.; rabbit anti-rat OPG ligand) and rabbit anti-rat biotinylated secondary antibody (SP-0023; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Effectiveness of combined salmon calcitonin and aspirin therapy for osteoporosis in ovariectomized rats

doi: 10.3892/mmr.2015.3637

Figure Lengend Snippet: Combined treatment significantly enhances the OPG/RANKL ratio in the proximal tibia of OVX rats. Effects of ASA, sCT and combined treatment (sCT+ASA) on protein expression levels in the proximal tibia of OVX rats. Specimens were observed at magnification, x250. Antigens were detected using an avidin-biotin-peroxidase system and visualized with 3,3′-diaminobenzidine. Protein levels were measured by densitometry. Values are expressed as the mean ± standard deviation. P-values were calculated by analysis of variance, with Student-Newman-Keuls analysis). (A) OPG; (B) RANKL; (C) OPG/RANKL protein ratio. Positive staining for OPG and RANKL was mainly localized in the chondrocytes of the epiphyseal growth plate and trabeculae of bone. ASA, aspirin; sCT, salmon calcitonin; OVX, ovariectomized; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor κB ligand.

Article Snippet: The antibodies used were as follows: OPG primary antibody (1:80; BA1475; Wuhan Boster Biological Technology Co. Ltd., Wuhan, China; rabbit anti-rat OPG), RANKL primary antibody (1:80; BA1323; Wuhan Boster Biological Technology Co. Ltd.; rabbit anti-rat OPG ligand) and rabbit anti-rat biotinylated secondary antibody (SP-0023; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Avidin-Biotin Assay, Standard Deviation, Staining

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: Mice were treated as described in Figure 2. a.) Representative images of 5 μm slices of distal femur stained for TRAP (20x magnification)(red shading; n=4 Vh, 5 TBT). b.) Oc/B.Per.: number of TRAP-positive osteoclasts per nm of trabecular perimeter, excluding cortex. Trab. Perimeter: total perimeter of trabecular bone (um). c.) Quantification of serum TRAP by ELISA (n = 12 Vh, 11 TBT 10 mg/kg). Whole humerus bone mRNA expression (n = 12 Vh, 11 TBT 10 mg/kg) of d.) Osteoclast differentiation markers Nfatc1, Apc5 and Ctsk, and e.) Intercellular communication proteins Rankl/Opg (osteoblast to osteoclast), Ct1 and Sema4d (osteoclast to osteoblast). Data are presented as mean ± SE. *p < 0.05 **p < 0.01, Mann-Whitney.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: Primary bone marrow macrophages were isolated from female C57BL/6J mice, induced to differentiate to osteoclasts with M-CSF and RANKL (see Methods), and after 24 hrs treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (Rosi, 500 nM, PPARγ agonist), LG100268 (LG268, RXR agonist 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) for a total of 6 days of differentiation. a.) mRNA expression of osteoclast differentiation markers. n=10–17 independent cultures. Data are presented as mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. b.) Representative images of TRAP-positive, multinucleated (3 or more nuclei) cells (Scale bar = 400 μm) n=10 independent cultures. c.) Representative images of von Kossa stained mineral surface after 3 days of incubation with differentiated osteoclasts. n=5 independent cultures.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Isolation, Expressing, Control, Staining, Incubation

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: a.) Mice were treated as described in Figure 2. Whole humerus bone mRNA expression of LXR-dependent genes Abca1 and Srebp1c. Data are presented as mean ± SE. n = 11–12 individual mice. **p<0.01, Mann-Whitney b.) Osteoclast cultures were prepared as described in Figure 5 and treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (Rosi, 500 nM, PPARγ agonist), LG100268 (LG268, RXR agonist 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) and analyzed for mRNA expression Abca1 and Srebp1c. n=10–17 independent cultures. Data are presented as mean ± SE. *p<0.05, **p<0.01, ***p<0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. c.) Primary bone marrow macrophages were isolated from male and female LXRα +/+, +/− and −/− mice, induced to differentiate into osteoclasts with M-CSF and RANKL, treated after 24 hrs with Vh (DMSO) or TBT (50 nM) for 4 days, and analyzed for mRNA expression of Ctsk. n=4 independent cultures. Data are presented as mean ± SE. Two-way ANOVA. d.) Osteoclast cultures were prepared as described in b and treated with Vh (DMSO) or TBT (50 nM) with or without GSK2033 (GSK, LXR antagonist, 0.8 μM) and analyzed for mRNA expression Abca1, Srebp1c and Ctsk. n=5 independent cultures. Data are presented as mean ± SE. Two-way ANOVA.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Expressing, MANN-WHITNEY, Control, Isolation

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: a.) Mice were treated as described in Figure 2. Whole humerus bone mRNA expression the RXR-dependent gene Mafb. Data are presented as mean ± SE. n = 11–12 individual mice. **p<0.01, Mann-Whitney b.) Osteoclast cultures were prepared as described in Figure 5 and treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (500 nM), LG100268 (LG268, 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) and analyzed for mRNA expression of Mafb. Data are presented as mean ± SE. n=10–17 independent cultures. *p<0.05, **p<0.01, *** p<0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. Osteoclast cultures were prepared as described in b and treated with Vh (DMSO) or TBT (50 nM) with or without HX531 (RXR antagonist, 1 μM) and analyzed for mRNA expression Mafb and Ctsk. n=5 independent cultures. Data are presented as mean ± SE. Two-way ANOVA.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Expressing, MANN-WHITNEY, Control