Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet: EM-523 promotes bone regeneration and suppresses osteoclastogenesis in vitro (A) MC3T3-E1 cells were cultured in osteogenic differentiation medium treated with control solvent or indicated macrolides. (A, left panel) Representative images of mineralization nodules from osteoblasts, stained with Alizarin Red S (ARS) staining after 26 days of the osteogenic differentiation assay. (A, middle panel) The mineralization area in each culture was quantified and represented as a percentage of the total area. (A, right panel) Quantification of the ARS staining using 10% cetylpyridinium chloride (CPC) on day 26 of the osteogenic differentiation assay. The absorbance was measured at 560 nm. The control group was incubated with an osteogenic differentiation medium only. (B) MC3T3-E1 cells were cultured in osteogenic differentiation medium treated with ethanol, ERM, or EM-523. (B, left panel) Representative images of mineralization nodules from osteoblasts, stained with ARS after 20 days of the osteogenic differentiation assay. (B, right panel) The mineralization area in each culture was quantified and presented as a percentage of the total area. The control group was incubated with an osteogenic differentiation medium only. (C) hPDLCs were cultured in osteogenic differentiation medium with control solvent or indicated macrolides. (C, left panel) Representative images of hPDLCs cultures, stained with alkaline phosphatase stain after 26 days of the osteogenic differentiation assay. Scale bars, 200 μm. (C, right panel) The ALP-positive region of each culture was quantified and represented as a percentage of the total area. (D) Gene expression analysis of hPDLCs on day nine of the osteogenic differentiation assay using qPCR. RUNX2 , SP7 , and BGLAP , as representative markers for early, middle, and late osteogenic markers, respectively. Data were normalized to GAPDH mRNA and plotted relative to ethanol-treated control, set as 1. (E) hMSCs were cultured in osteogenic differentiation medium with ethanol or indicated macrolides (10 μg/mL). (E, upper panel) Representative images of mineralization nodules from osteoblasts, stained with Alizarin Red S after 26 days of the osteogenic differentiation assay. (E, middle left panel) The mineralization area in each culture was quantified and presented as a percentage of the total area. (E, middle right panel) Quantification of the Alizarin Red S staining using 10% CPC on day 26 of the osteogenic differentiation assay. (E, lower panel) Gene expression analysis using qPCR of human MSCs on day nine of the osteogenic differentiation assay for the expression of the indicated genes. Data were normalized to GAPDH mRNA and plotted relative to ethanol control, set as 1. (F) Bone marrow cells were collected from WT mice for the osteoclastogenesis assay. Cells were treated with DEL-1-Fc (1 μg/mL), equal molar amount of Fc control, control solvent, or indicated macrolides for one week. (F, upper panel) Representative images of the culture well from each group after TRAP staining are shown. Histological images of TRAP + MNCs. Scale bars, 200 μm. (F, lower left panel) The average size of TRAP + MNCs was measured using the ImageJ software. (F, lower right panel) The percentage of TRAP + area per total area was measured using the ImageJ software. (G) Primary osteoblastic progenitor cells were collected from Del1 −/− mice. Primary osteoblastic progenitor cells were cultured in osteogenic differentiation media administered with DEL-1-Fc, equal molar amount of Fc control, control solvent, or indicated macrolides for 26 days. (G, upper panel) Representative images of mineralization nodules from osteoblasts, stained with Alizarin Red S after 26 days of the osteogenic differentiation assay. (G, lower panel) Gene expression analysis was completed on day nine of the osteogenic differentiation assay using qPCR. Runx2 , Sp7 , and Bglap , as representative markers for early, middle, and late osteogenic markers, respectively. Data were normalized to GAPDH mRNA and plotted relative to ethanol control, set as 1. (H) Bone marrow cells were collected from Del1 −/− mice for the osteoclastogenesis assay. Cells were treated with DEL-1-Fc, equal molar amount of Fc control, control solvent, or indicated macrolides for one week. (H, upper panel) Representative images of the culture well from each group after TRAP staining were shown. Histological images of TRAP + MNCs. Scale bars, 200 μm. (H, lower left panel) The average size of TRAP + MNCs was measured using the ImageJ software. (H, lower right panel) The percentage of TRAP + area per total area was measured using the ImageJ software. Data are means ± S.D. (A–C, n = 4 sets of cultures/group; D, n = 5 sets of cultures/group; E middle and lower panel, n = 4 sets of cultures/group; F–H, n = 5 sets of cultures/group). ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA and Bonferroni’s test.

Article Snippet: The cell lines used in this study were murine macrophage cell lines RAW264.7 TIB-71 (ATCC), murine osteoblastic progenitor cell line MC3T3-E1 (The RIKEN Bioresource Center, RCB1126), human periodontal ligament cells (hPDLCs, CC-7049, Lonza) and human iPSC-derived mesenchymal stem cells (hMSCs, R1098, Cellular Dynamics International).

Techniques: In Vitro, Cell Culture, Solvent, Staining, Differentiation Assay, Incubation, Expressing, Software

Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet:

Article Snippet: The cell lines used in this study were murine macrophage cell lines RAW264.7 TIB-71 (ATCC), murine osteoblastic progenitor cell line MC3T3-E1 (The RIKEN Bioresource Center, RCB1126), human periodontal ligament cells (hPDLCs, CC-7049, Lonza) and human iPSC-derived mesenchymal stem cells (hMSCs, R1098, Cellular Dynamics International).

Techniques: Recombinant, Construct, Transfection, Staining, Chromatin Immunoprecipitation, Magnetic Beads, Luciferase, SYBR Green Assay, Generated, Binding Assay, Expressing, Plasmid Preparation, Software, Imaging, Microscopy, Fluorescence, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet:

Article Snippet: The cell lines used in this study were murine macrophage cell lines RAW264.7 TIB-71 (ATCC), murine osteoblastic progenitor cell line MC3T3-E1 (The RIKEN Bioresource Center, RCB1126), human periodontal ligament cells (hPDLCs, CC-7049, Lonza) and human iPSC-derived mesenchymal stem cells (hMSCs, R1098, Cellular Dynamics International).

Techniques: Recombinant, Construct, Transfection, Staining, Chromatin Immunoprecipitation, Magnetic Beads, Luciferase, SYBR Green Assay, Generated, Binding Assay, Expressing, Plasmid Preparation, Software, Imaging, Microscopy, Fluorescence, Real-time Polymerase Chain Reaction

Journal: International Journal of Bioprinting

Article Title: 3D-Printed scaffolds based on poly(Trimethylene carbonate), poly(ε-Caprolactone), and β-Tricalcium phosphate

doi: 10.18063/ijb.v9i1.641

Figure Lengend Snippet: Gene expression assays on rBMSCs in poly(ε-caprolactone) (PCL)/β-tricalcium phosphate (TCP), poly(trimethyl carbonate) (PTMC)/TCP, and PTMC/PCL/TCP scaffolds with 25% TCP content. Abbreviations: PCL/TCP-7, PCL/25%TCP scaffolds for 7 days; PTMC/TCP-7, PTMC/25%TCP scaffolds for 7 days; PTMC/PCL/TCP-7, PTMC/PCL/25%TCP scaffolds for 7 days; and PTMC/PCL/TCP-14, PTMC/PCL/25%TCP scaffolds for 14 days.

Article Snippet: The osteoblast cell lines, clonal murine cell line of immature osteoblasts derived from mice (MC3T3-E1) and rat bone marrow mesenchymal stem cells (rBMSCs), were provided by the China Center for Type Culture Collection of Wuhan University, China[ ].

Techniques: Gene Expression