Journal: bioRxiv

Article Title: Estrogen Deficiency alters Vascularization and Mineralization dynamics: insight from a novel 3D Humanized and Vascularized Bone Organoid Model

doi: 10.1101/2024.10.09.614903

Figure Lengend Snippet: (a) Development of chondrogenic template by differentiating HBMSCs in gelatin-mtgase constructs (from −42 day to −21 day), (b) Culture of chondrogenic template in vascular (HBMSCs: HUVECs) and non-vascular (BMSCs) gelatin-mtgase constructs under osteogenic differentiation and estrogen supplementation (from −21 day to 0 day), (c) Mineralized vascular and non-vascular models at day 0 (day 43 in culture)and further culture under estrogen supplementation or estrogen withdrawal (from 0 day to 21 day).

Article Snippet: These constructs were primed for 21 days (until day −21) with chondrogenic differentiation media (Dulbecco’s modified Eagle’s medium (DMEM) (Merck), 10 ng/mL transforming growth factor (TGF)-β3 (Invitrogen), 50 μg/mL ascorbic acid (Merck), 4.7 μg/mL linoleic acid-oleic acid (Merck), 100 nM dexamethasone (Merck) and 1× insulin–transferrin–selenium (ITS; Invitrogen)) ( ).

Techniques: Construct

Journal: bioRxiv

Article Title: Estrogen Deficiency alters Vascularization and Mineralization dynamics: insight from a novel 3D Humanized and Vascularized Bone Organoid Model

doi: 10.1101/2024.10.09.614903

Figure Lengend Snippet: (a, b) H&E staining at day 0 (day 43 in culture), chondrogenic template (yellow arrow) confirms integration of the vascular primed gelatin layer (green arrow) with the chondrogenic template through multicellular connections (blue arrows) (scale bar 500 µm). By day 21 immunofluorescence and immunohistochemistry staining confirmed (c, d) the retention and distribution of CD31 + Emcn + type H endothelial cells: CD31 (green), endomucin (red), DAPI (blue) (scale bar 100 µm) at day 21, (e, f, g) extracellular matrix production: collagen II, X and I (scale bar 500 µm), (h, i) osteoblast differentiation towards osteocytes: DMP-I (red), DAPI (blue) (scale bar 100 µm), sclerostin (green), actin (red), DAPI (blue) (scale bar 30 µm), (j) and mineral deposition: Von kossa staining for mineral, mineral nodule (blue arrow) (scale bar 500 µm).

Article Snippet: These constructs were primed for 21 days (until day −21) with chondrogenic differentiation media (Dulbecco’s modified Eagle’s medium (DMEM) (Merck), 10 ng/mL transforming growth factor (TGF)-β3 (Invitrogen), 50 μg/mL ascorbic acid (Merck), 4.7 μg/mL linoleic acid-oleic acid (Merck), 100 nM dexamethasone (Merck) and 1× insulin–transferrin–selenium (ITS; Invitrogen)) ( ).

Techniques: Staining, Immunofluorescence, Immunohistochemistry

Journal: bioRxiv

Article Title: Estrogen Deficiency alters Vascularization and Mineralization dynamics: insight from a novel 3D Humanized and Vascularized Bone Organoid Model

doi: 10.1101/2024.10.09.614903

Figure Lengend Snippet: Day 10 and 21 histology staining and quantification confirmed staining for (a-i) Collagen I (scale bar 500 µm), (j-r) DMP-1 (Red), DAPI (Blue) (scale bar 100 µm), (s-aa) Sclerostin (green), actin (red) DAPI (blue) (scale bar 30 µm), (bb-ii) Von Kossa staining for mineral (scale bar 500 µm), (jj) Total calcium normalized to weight of construct. (OG: outer gel, CT: chondrogenic template). Significant differences indicated with * relative to non-vascular group and # relative to other time point. Two-way ANOVA, */# = p < 0.05, **/## = p < 0.01, ***/### = p < 0.001, ****/#### = p < 0.001.

Article Snippet: These constructs were primed for 21 days (until day −21) with chondrogenic differentiation media (Dulbecco’s modified Eagle’s medium (DMEM) (Merck), 10 ng/mL transforming growth factor (TGF)-β3 (Invitrogen), 50 μg/mL ascorbic acid (Merck), 4.7 μg/mL linoleic acid-oleic acid (Merck), 100 nM dexamethasone (Merck) and 1× insulin–transferrin–selenium (ITS; Invitrogen)) ( ).

Techniques: Staining, Construct

Journal: bioRxiv

Article Title: Estrogen Deficiency alters Vascularization and Mineralization dynamics: insight from a novel 3D Humanized and Vascularized Bone Organoid Model

doi: 10.1101/2024.10.09.614903

Figure Lengend Snippet: Day 10 and 21 histology staining and quantification confirmed staining for (a-i) Collagen I (scale bar 500 µm), (j-r) DMP-I (Red), DAPI (Blue) (scale bar 100 µm), (s-aa) Sclerostin (green), actin (red) DAPI (blue) (scale bar 30 µm), (bb-ii) Von Kossa staining for mineral (scale bar 500 µm), (jj) Total calcium normalized to weight of construct. (OG: outer gel, CT: chondrogenic template) Significant differences indicated with * relative to non-vascular group and # relative to other time point. Two-way ANOVA, */# = p < 0.05, **/## = p < 0.01, ***/### = p < 0.001, ****/#### = p < 0.001.

Article Snippet: These constructs were primed for 21 days (until day −21) with chondrogenic differentiation media (Dulbecco’s modified Eagle’s medium (DMEM) (Merck), 10 ng/mL transforming growth factor (TGF)-β3 (Invitrogen), 50 μg/mL ascorbic acid (Merck), 4.7 μg/mL linoleic acid-oleic acid (Merck), 100 nM dexamethasone (Merck) and 1× insulin–transferrin–selenium (ITS; Invitrogen)) ( ).

Techniques: Staining, Construct