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: 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: Nature Communications

Article Title: Divide-and-conquer strategy with engineered ossification center organoids for rapid bone healing through developmental cell recruitment

doi: 10.1038/s41467-025-61619-y

Figure Lengend Snippet: a , b HE staining of paraffin sections in Defect, Vehicle, Hybrid, and OCO groups at 4 weeks and 8 weeks after calvarial defect (bar = 200 μm at low magnification and bar = 50 μm at high magnification). c , d Safranin-O staining of paraffin sections in Defect, Vehicle, Hybrid, and OCO group at 4 weeks and 8 weeks after calvarial defect (bar = 200 μm at low magnification and bar = 50 μm at high magnification). e Schematic of neurogenesis in the central area and f xo-immunofluorescent staining of CGRP and β-III tubulin expression (bar = 20 μm); g schematic of angiogenesis in the interlayer and h co-immunofluorescent staining of CD31 and COLX expression (bar = 20 μm); i schematic of osteogenesis in the outer layer and j co-immunofluorescent staining of OCN and OSX expression (bar = 20 μm); in Defect, Vehicle, Hybrid, and OCO groups at 4 weeks post-surgery. At least three times of experiments were repeated independently.

Article Snippet: The samples were treated with primary antibodies at 4 °C overnight, including Ki67 (1:250 dilution, ab16667, Abcam, USA), γh2AX (1:250 dilution, ab22551, Abcam, USA), RUNX2 (1:200 dilution, ab76956, Abcam, UK), OSX (1:200 dilution, ab209484, Abcam, UK), Collagen I (1:100 dilution, ab260043, Abcam, UK), OCN (1:50 dilution, MAB1419, R&D Systems, USA), NGFR (1:50 dilution, NBP2-67296, NOVUS, USA), CGRP (1:100 dilution, ab81887, Abcam, UK), beta III Tubulin (1:500 dilution, ab18207, Abcam, UK), Collagen X (1:100 dilution, 14-9771-82, Invitrogen, USA), CD31 (1:100 dilution, ab222783, Abcam, UK), CD200 (1:100 dilution, AF2724, R&D Systems, USA), Thy1 (1:200 dilution, ab181469, Abcam, UK), KRT8 (1:100 dilution, ab53280, Abcam, UK), HAS1 (1:250 dilution, PA5-95599, Invitrogen, USA), MSX1 (1:100 dilution, ab93287, Abcam, UK).

Techniques: Staining, Expressing

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: 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: The Kaohsiung journal of medical sciences

Article Title: Morinda officinalis polysaccharide regulates rat bone mesenchymal stem cell osteogenic-adipogenic differentiation in osteoporosis by upregulating miR-21 and activating the PI3K/AKT pathway.

doi: 10.1002/kjm2.12544

Figure Lengend Snippet: FIGURE 6 MOP improved OP in OVX rats and increased the expression of miR-21. OVX OP model rats were treated with MOP for 4 weeks. (A) The BMD of the isolated lumbar spine was measured by an X-ray BMD detector; (B,C) the levels of BALP and BGP in serum were detected by ELISA; and (D) the expression of miR-21 in rat BMSCs was detected by RT-qPCR. The data were expressed as mean ± standard deviation. Animal experiment N = 6. One-way ANOVA was adopted for data comparisons among groups, followed by Tukey's multiple comparisons test. p value was obtained by a bilateral test, **p < 0.01 and ***p < 0.001. BGP, osteocalcin; BMD, bone mineral density; BALP, bone-derived alkaline phosphatase; BMSC, bone mesenchymal stem cell; MOP, Morinda officinalis polysaccharide; OP, osteoporosis; OVX, ovariectomy

Article Snippet: The levels of serum bone alkaline phosphatase (BALP, CSB-E11188r, CUSABIO, Wuhan, Hubei, China) and osteocalcin (BGP, CSBE05129r, CUSABIO) were detected using ELISA kits in strict accordance with the instructions.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Standard Deviation, Derivative Assay

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: A novel mouse model to study fracture healing of the proximal femur.

doi: 10.1002/jor.24677

Figure Lengend Snippet: FIGURE 4 Immunohistochemical staining of fractured femurs at day 14 (d14) and day 21 (d21) after fracture. A, Representative images of fractured femurs stained for collagen X and quantification of the positively stained area in the whole fracture callus. B, Representative images of fractured femurs stained for runx2 and quantification of the positively stained area in the whole fracture callus. C, Representative images of fractured femurs stained for osteocalcin and quantification of the positively stained area in the whole fracture callus. Scale bar = 200 µm. C, cortex; G, fracture gap. *.05 > P > .01; ***.01 > P > .0001 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Paraffin‐embedded 7‐μm longitudinal sections were prepared for immunohistochemical staining, the following antibodies were used: rabbit anti‐mouse collagen X (ABIN1077945, dilution 1:200; Antibodies Online), rabbit anti‐mouse Runx2 (8486, 1:50; Cell Signaling), rabbit anti‐mouse osteocalcin (orb77248, 1:200; Biorbyt), goat‐anti rabbit IgG‐biotin (sc‐3840, 1:200, Santa Cruz) and horseradish peroxidase‐conjugated streptavidin (Zytomed Systems).

Techniques: Immunohistochemical staining, Staining