Journal: Breast Cancer Research : BCR

Article Title: Osteoblasts are “educated” by crosstalk with metastatic breast cancer cells in the bone tumor microenvironment

doi: 10.1186/s13058-019-1117-0

Figure Lengend Snippet: EOs are present in patient samples of bone metastatic breast cancer. Human patient samples of bone metastatic breast cancer were stained using multi-plex immunofluorescence for RUNX2 (green), osteocalcin (OCN, red), IL-6 (purple), and alpha-SMA (yellow). Left panel—osteoblast identification: white arrows show osteoblasts positive for both RUNX2 and OCN. Middle panel—“uneducated” and “educated” osteoblast identification: blue arrows show “uneducated” osteoblasts alpha-SMA and IL-6 positive; yellow arrows show “educated” osteoblasts alpha-SMA high, but IL-6 low; purple arrows show “educated” osteoblasts IL-6 high, but alpha-SMA low. Right panel—“educated” osteoblast identification: green arrows show “educated” osteoblasts both IL-6 and alpha-SMA low, DAPI positive. T, tumor; arrows, osteoblast. DAPI, nuclear stain. Scale bar = 50 μm

Article Snippet: The slides were incubated overnight at 4 °C with either mouse anti-human osteocalcin (R&D Systems, 10 μg/ml), rabbit anti-human RUNX2 (Abcam, 1:500), mouse anti-human alpha-smooth muscle actin (1:100, Abcam), rabbit anti-human alkaline phosphatase (1:100, Abcam), rabbit anti-human osteopontin (1:500, Abcam), or goat anti-human IL-6 (40 μg/mL, R&D Systems), Next, slides were incubated for 1 h at room temperature with either donkey anti-mouse 594, chicken anti-rabbit 594, donkey anti-goat 488, or goat anti-rabbit 488 (1:1000, Biotium).

Techniques: Staining, Immunofluorescence

Journal: International journal of molecular sciences

Article Title: Methodology and Characterization of a 3D Bone Organoid Model Derived from Murine Cells.

doi: 10.3390/ijms25084225

Figure Lengend Snippet: Figure 2. Relative expression screening of key osteoblast and osteoclast markers by in response to differentiation media. (A) RUNX2. (B) TRAP. Relative fluorescence intensity associated with protein expression evaluated from MC3T3-E1 and RAW 264.7 which were grown in 2D culture with exposure to media containing mixtures of osteoblast (BMP2) or osteoclast (RANKL/M-CSF) differentiating factors as compared to complete culture media (negative).. Fluorescence intensity for each condition was assessed by flow cytometry. Raw fluorescence data were normalized to a cell line specific unstained control for each group. Statistical significance was evaluated by one-way ANOVA (A,B) or unpaired T test (** = p < 0.01; **** = p < 0.0001). (C) Qualitative assessment of relative gene expression (2−∆∆Ct) of osteoblastogenic (ALPL, Runx2, Sp7, Bglap, Col1a1) and osteoclastogenic (Trap) genes of undifferentiated cell lines (pooled) versus cells differentiated (pooled) in two-dimensional culture for (MC3T3-E1: day 14; RAW 264.7: day 5). Where appropriate, data are summarized as the sample mean, and error bars represent the sample standard deviation.

Article Snippet: Osteocalcin/Bglap (Bglap) ELISA assays (Novus Biologicals, Catalogue# NBP2-68151, Centennial, CO, USA) were performed per the vendor-supplied protocol.

Techniques: Expressing, Fluorescence, Flow Cytometry, Control, Gene Expression, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration

doi: 10.1186/s13287-018-1066-z

Figure Lengend Snippet: In vitro osteogenesis of young and old donor hMDSCs. a BMP2 secretion levels of 6 populations of LBMP2/GFP-transduced cells. b MicroCT 3D images of pellet culture for non-transduced and LBMP2/GFP-transduced hMDSCs. LBMP2/GFP-transduced hMDSCs showed larger mineralized pellets in all groups. c Quantification of mineralized pellet volume showed significantly higher mineralized pellet volume in all LBMP2/GFP-transduced cells compared to each respective non-transduced hMDSC counterpart. Young donor 1 LBMP2/GFP-transduced cells formed larger pellets than old donor 1 LBMP2-transduced cells. Young donor 2 LBMP2/GFP-transduced hMDSCs also formed larger pellets than old donor 2 cells. No significant difference was found between young donor 3 and old donor 3 LBMP2/GFP-transduced cells. Young donor 2 non-transduced hMDSCs also formed larger mineralized pellets than old donor 2 non-transduced hMDSCs. d Von Kossa staining showed that LBMP2/GFP-transduced hMDSCs had more mineralization (as shown in black) than did non-transduced cells. e Osteocalcin immunochemistry revealed the osteogenic differentiation of both non-transduced and LBMP2/GFP-transduced hMDSCs in all groups. Note that highly mineralized parts of the cell pellets often peeled away. * P < 0.05, *** P < 0.001

Article Snippet: Osteocalcin immunohistochemistry using a mouse anti-human osteocalcin primary antibody (MAB1419,1:100, R&D Systems) was also performed, as previously reported [ ].

Techniques: In Vitro, Staining

Journal: Journal of Nanobiotechnology

Article Title: Hypoxia preconditioning of adipose stem cell-derived exosomes loaded in gelatin methacryloyl (GelMA) promote type H angiogenesis and osteoporotic fracture repair

doi: 10.1186/s12951-024-02342-6

Figure Lengend Snippet: GleMA loaded with hypo-ADSC-Exos enhanced local microvascular network formation and osteoporotic fracture healing in vivo via targeting SPRY1. A Schematic illustration of the process of in vivo treatment using hypoxia-pretreated ADSC-derived exosomes. B SEM images of GelMA loaded with different exosomes. C , D Degration and protein released ratio of GelMA loaded with exosomes. E , F Representative X-Ray images and morphometric analysis of osteoporotic fractures after hypo-ADSC-Exo treatment on 4 week and 8 week. G , H Micro-CT images of osteoporotic fractures after hypo-ADSC-Exo treatment and morphometric analysis of new bone volume (BV), tissue volume (TV), and bone mineral density (BMD). n = 6. I Haematoxylin and eosin (H&E) staining on the bone fracture region after PBS, ADSC-Exo and hypo-ADSC-Exo treatment. n = 6. J , N Micro-CT 3D reconstruction images of angiographic images of fracture area after hypo-ADSC-Exo treatment. Scale bar, 20 μm. K , L Immunofluorescence staining and quantitative analysis results showed the CD31 and EMCN staining on the bone fracture region. Scale bar, 50 μm. n = 6. M The serum levels of VEGFA, OCN, BALP and CTX-1. n = 6. O SPRY1 immunohistochemistry staining images and quantitative analysis on the bone fracture region after PBS, ADSC-Exo and hypo-ADSC-Exo treatment. Scale bar, 50 μm. n = 6. P VEGFA immunohistochemistry staining images and quantitative analysis on the bone fracture region after PBS, ADSC-Exo and hypo-ADSC-Exo treatment. Scale bar, 50 μm. n = 6. (Data are presented as the means ± SD; *p < 0.05; **p < 0.01; ***p < 0.001)

Article Snippet: The serum levels of CTX-1, BALP, OCN and VEGF were examined by ELISA using a CTX-1 ELISA kit (NBP2-69074, Novus), OCN ELISA kit (NBP2-68151, Novus), BALP ELISA kit (CSB-E09033h), and VEGF ELISA kit (ab100751, Abcam).

Techniques: In Vivo, Derivative Assay, Micro-CT, Staining, Immunofluorescence, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: A Mixture of Cervus elaphus sibiricus and Glycine max (L.) Merrill Inhibits Ovariectomy-Induced Bone Loss Via Regulation of Osteogenic Molecules in a Mouse Model

doi: 10.3390/ijms24054876

Figure Lengend Snippet: Modulatory effects of BPX on bone formation and resorption markers in serum and modulation of BMP pathways in the femur. The levels of bone resorption markers, such as TRAP ( A ) and Ca ( B ), and bone formation markers, such as osteocalcin ( C ) and ALP ( D ), in serum were analyzed by ELISA. The mRNA expression of BMP-2, BSP-1, and OSX were assessed ( E ), and the protein levels of p -p38, p -smad 1/5/8, smad 4, and Runx2 were assessed by western blot ( F , G ) in the femurs of OVX-induced mice. All band intensities were quantified by Image J. The mice were divided into groups according to the treatment: the Sham, OVX, and BPX (OVX + BPX) groups. The data are expressed as the mean ± SD. # p < 0.05, ## p < 0.01 compared with the Sham group; * p < 0.05, ** p < 0.01 compared with the OVX group.

Article Snippet: Serum levels of bone turnover markers, that is, osteocalcin (OC) (E-EL-M0864, Elabscience, Houston, TX, USA), alkaline phosphatase (ALP), and tartrate-resistant acid phosphatase (TRAP) (MK301, Takara, Japan), and the biochemical marker calcium (E-BC-K103-M, Elabscience, Houston, TX, USA) were measured using an enzyme-linked immunosorbent assay (ELISA) kit.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Calcified Tissue International

Article Title: Deletion of Coagulation Factor IX Compromises Bone Mass and Strength: Murine Model of Hemophilia B (Christmas Disease)

doi: 10.1007/s00223-021-00872-x

Figure Lengend Snippet: Selected skeletal parameters in juvenile (10-week-old) and adult (20-week-old) factor IX knockout (FIX KO) mice compared to their wild-type (WT) littermates

Article Snippet: Serum osteocalcin was measured using a commercial mouse osteocalcin ELISA kit (Quidel Corporation, CA, USA).

Techniques: Knock-Out, Activity Assay