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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline <t>phosphatase</t> ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.
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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline <t>phosphatase</t> ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.
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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline <t>phosphatase</t> ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.
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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline <t>phosphatase</t> ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.
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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline <t>phosphatase</t> ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.
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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline <t>phosphatase</t> ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.
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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline <t>phosphatase</t> ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.
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In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline phosphatase ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.

Journal: Bioactive Materials

Article Title: A continuous adhesion-enhanced osteogenic pathway in artificial scaffold drives cellular infiltration and condensed mineralization for rapid bone regeneration

doi: 10.1016/j.bioactmat.2026.02.026

Figure Lengend Snippet: In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline phosphatase ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.

Article Snippet: Cultured cells were washed with PBS and lysed using RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Hudson, NH, USA).

Techniques: In Vitro, Staining, Expressing, Incubation, Cell Culture, Western Blot, Co-Culture Assay