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AA enhances COL1A1 biological activity and restores <t>osteoblast</t> function and COL1A1 regulation in the presence of Psl. A) ALP staining (red) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Scale bar: 300 µm. B) Quantification of Mean Intensity of ALP (A.U.). C) Alzarin Red (AR) activity assay of osteo‐spheroids after 7 d with or without additional AA. Scale bar, 400 µm. D) Graph showing quantification of Mean Intensity of AR (A.U.). E) OsteoImaging <t>mineralization</t> assay quantifications of Mean Intensity (A.U.) of osteo‐spheroids incubated with or without additional AA for 7 d. F) Gene expression analysis of osteogenic markers ( BGLAP, DMP1, DLX3, RUNX2 ) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Heatmap demonstrates the ∆CT averages. G) Gene expression analysis of COL1A1‐related genes ( PLOD1, PLOD3, DLX3, P3H1, P3H2, P3H3, LOX, SVCT2, COL1A2, COL22A1, IBSP, P4HA2, P4HA3, IFITM5 ) in osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with or without ascorbic acid (AA), and in the presence (+Psl) or absence (–Psl) of prednisolone (Psl); Heatmap demonstrates the ∆CT averages H) Fold change of top collagen‐related putative metabolites elevated in the presence of endogenous ascorbic acid (+AA) within the 3D matrix of osteo‐spheroids after 7 d of Psl (+Psl) treatment, based on untargeted metabolomics analysis; fold change threshold > 2 with significance; FDR p‐value < 0.05. The data are expressed as mean ± SD; N = 3, n = 3; significant differences: * * p‐value < 0.01; *** p‐value < 0.005 ; **** p‐value < 0.0001.
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AA enhances COL1A1 biological activity and restores osteoblast function and COL1A1 regulation in the presence of Psl. A) ALP staining (red) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Scale bar: 300 µm. B) Quantification of Mean Intensity of ALP (A.U.). C) Alzarin Red (AR) activity assay of osteo‐spheroids after 7 d with or without additional AA. Scale bar, 400 µm. D) Graph showing quantification of Mean Intensity of AR (A.U.). E) OsteoImaging mineralization assay quantifications of Mean Intensity (A.U.) of osteo‐spheroids incubated with or without additional AA for 7 d. F) Gene expression analysis of osteogenic markers ( BGLAP, DMP1, DLX3, RUNX2 ) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Heatmap demonstrates the ∆CT averages. G) Gene expression analysis of COL1A1‐related genes ( PLOD1, PLOD3, DLX3, P3H1, P3H2, P3H3, LOX, SVCT2, COL1A2, COL22A1, IBSP, P4HA2, P4HA3, IFITM5 ) in osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with or without ascorbic acid (AA), and in the presence (+Psl) or absence (–Psl) of prednisolone (Psl); Heatmap demonstrates the ∆CT averages H) Fold change of top collagen‐related putative metabolites elevated in the presence of endogenous ascorbic acid (+AA) within the 3D matrix of osteo‐spheroids after 7 d of Psl (+Psl) treatment, based on untargeted metabolomics analysis; fold change threshold > 2 with significance; FDR p‐value < 0.05. The data are expressed as mean ± SD; N = 3, n = 3; significant differences: * * p‐value < 0.01; *** p‐value < 0.005 ; **** p‐value < 0.0001.

Journal: Advanced Healthcare Materials

Article Title: Ascorbic Acid Modulates Collagen Properties in Glucocorticoid‐Induced Osteoporotic Bone: Insights into Chemical, Mechanical, and Biological Regulation

doi: 10.1002/adhm.202502606

Figure Lengend Snippet: AA enhances COL1A1 biological activity and restores osteoblast function and COL1A1 regulation in the presence of Psl. A) ALP staining (red) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Scale bar: 300 µm. B) Quantification of Mean Intensity of ALP (A.U.). C) Alzarin Red (AR) activity assay of osteo‐spheroids after 7 d with or without additional AA. Scale bar, 400 µm. D) Graph showing quantification of Mean Intensity of AR (A.U.). E) OsteoImaging mineralization assay quantifications of Mean Intensity (A.U.) of osteo‐spheroids incubated with or without additional AA for 7 d. F) Gene expression analysis of osteogenic markers ( BGLAP, DMP1, DLX3, RUNX2 ) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Heatmap demonstrates the ∆CT averages. G) Gene expression analysis of COL1A1‐related genes ( PLOD1, PLOD3, DLX3, P3H1, P3H2, P3H3, LOX, SVCT2, COL1A2, COL22A1, IBSP, P4HA2, P4HA3, IFITM5 ) in osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with or without ascorbic acid (AA), and in the presence (+Psl) or absence (–Psl) of prednisolone (Psl); Heatmap demonstrates the ∆CT averages H) Fold change of top collagen‐related putative metabolites elevated in the presence of endogenous ascorbic acid (+AA) within the 3D matrix of osteo‐spheroids after 7 d of Psl (+Psl) treatment, based on untargeted metabolomics analysis; fold change threshold > 2 with significance; FDR p‐value < 0.05. The data are expressed as mean ± SD; N = 3, n = 3; significant differences: * * p‐value < 0.01; *** p‐value < 0.005 ; **** p‐value < 0.0001.

Article Snippet: Human osteoblasts (HOBs) (PromoCell) were cultured in osteoblast growth medium (GM) (PromoCell) and differentiated in osteoblast mineralization medium (MM) (PromoCell).

Techniques: Activity Assay, Staining, Cell Culture, Mineralization Assay, Incubation, Gene Expression