Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Comparative analysis of conventional and brush-assisted bioprinting on cellular behavior. (a) Schematic illustration of shear stress distribution in normal versus brush-assisted printing. (b) Overview of the brush-assisted printing setup. (c) SEM images of collagen fibrils and fluorescence images showing cell viability (live/dead) and cytoskeletal organization (DAPI/phalloidin) of C2C12 myoblasts. Quantification of (d) cell viability post-printing (n = 4), (e) cell metabolic activity (MTT assay, in situ /day 3/day 7, n = 4), (f) nuclei aspect ratio (n = 180), (g) orientation factor (n = 3), and (h) F-actin–positive area at day 3 (n = 10). (i) Comparing normal and brush-assisted printing the mechanotransduction pathways activated by shear stress and collagen alignment. (j) Heatmap of relative gene expression ( YAP, TAZ, AKT1, PIEZO1, PI3K, and CAPN2 ) after 7 days of culture (n = 4). (k) Agarose gel electrophoresis of PCR products from cells cultured on normal versus brush-printed scaffolds for 7 days. (l) Schematic illustration of blocking mechano-sensing ion channel with GsMTx-4. (m) Relative gene expression levels associated with mechanosensing channel and ca 2+ pathway (n = 5), (n) Hippo pathway (n = 5), (o) PI3K-AKT pathway (n = 5). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cell metabolic activity assay); F-actin, Filamentous actin; PI3K , Phosphoinositide 3-kinase; CAPN2 , Calcium-activated neutral protease 2.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Shear, Fluorescence, Activity Assay, MTT Assay, In Situ, Gene Expression, Agarose Gel Electrophoresis, Cell Culture, Blocking Assay, Standard Deviation, Metabolic Assay

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Physical and biological evaluation of bioconstructs reinforced with straight (CSP) and coiled (CCP) PCL fibers. (a) Schematic illustration and optical images of flow tests with Col (collagen-only), CSP, and CCP bioinks, in which droplets were tilted at 90° for 10 min. (b) Quantification of droplet outflow (n = 10). Rheological measurements of bioinks: (c) storage modulus (G′) from frequency sweep (n = 3), (d) G′ and G″ from temperature sweep (n = 3), and (e) G′ and G″ under cyclic stress loading (10 and 200 Pa) showing viscoelastic recovery (n = 3). (f) SEM images of CCP constructs highlighting coiled PCL fiber and aligned collagen fibrils. (g) Optical image, live/dead staining, DAPI/phalloidin staining, and orientation factor analysis of C2C12 cells and PCL microfibers. Quantification of (h) cell viability (live/dead, n = 4), (i) F-actin–positive area (n = 20), and (j) metabolic activity (MTT assay, in situ /day 3/day 7, n = 4). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Construct, Staining, Activity Assay, MTT Assay, In Situ, Standard Deviation

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: In vitro myogenic differentiation of C2C12 cells cultured on Col, CSP and CCP scaffolds. (a) Live/dead staining at in situ , DAPI/phalloidin staining at day 3, and DAPI/ MHC staining at day 14. (b) Quantification of cell viability from live/dead assays (n = 4). (c) Nuclei orientation factor after 7 days of culture (n = 4). (d) Nuclei aspect ratio (n = 20). (e) F-actin positive area (n = 4). (f) Quantification of MHC fusion index (left, n = 5) and MHC maturation rate (right, n = 5). (g) Relative gene expression analysis and (h) agarose gel electrophoresis regarding mechanotransduction-related genes ( CAPN2, PIEZO1, RhoA, YAP, and TAZ ) (n = 4). (i) Western blot analysis of PIEZO1 . (j) Schematic illustrating differentiation progression and major genes involved at each stage. (k) Heatmap and (l) agarose gel electrophoresis of PCR products showing relative expression of myogenic markers ( MYF5, MYOD1, MYOG, MHC, MYH2, and MYH4 ) after 21 days of culture (n = 4). (m) Western blot analysis of MHC . Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MYF5 , Myogenic factor 5; MYOD1 , Myogenic differentiation 1; MYOG , Myogenin; MHC , Myosin heavy chain; MYH2 , Myosin heavy chain 2; MYH4 , Myosin heavy chain 4.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: In Vitro, Cell Characterization, Cell Culture, Staining, In Situ, Gene Expression, Agarose Gel Electrophoresis, Western Blot, Expressing, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Recombinant ADAMTS-1 promotes muscle regeneration accompanied by downregulation of Notch signaling

doi: 10.3892/ijmm.2025.5718

Figure Lengend Snippet: Effect of rADAMTS-1 on MuSCs following BaCl 2 -induced TA muscle injury. Groups included NC, BaCl 2 , and two treatment groups receiving rADAMTS-1 (L) and (H). Daily intraperitoneal injections of rADAMTS-1 were initiated immediately after injury and continued until the designated time point for analysis. (A) Flow cytometry analysis of MuSCs isolated from injured TA muscles, defined by surface marker expression (CD45 − /CD31 − /Sca1 − /VCAM1 + ). (B) Representative gating strategy for MuSCs across experimental groups at different time points. (C) Relative number of MuSCs normalized to the NC group over time. Statistical significance was determined using one-way analysis of variance, followed by Tukey's post hoc test: * P<0.05, ** P<0.01 and *** P<0.001 vs. NC; ## P<0.01 and ### P<0.001 vs. BaCl 2 group. (D) Representative immunofluorescence images of Pax7 + cells in injured TA muscle at different time points (scale bar, 25 μ m). (E) Quantification of Pax7 + cell percentages over time. Statistical significance was determined using one-way analysis of variance with Tukey's post hoc test: * P<0.05 and *** P<0.001 vs. NC; ### P<0.001 vs. BaCl 2 ; ††† P<0.001 vs. rADAMTS-1 (L). ADAMTS-1, a disintegrin and metalloproteinase with thrombospondin motifs 1; rADAMTS-1, recombinant ADAMTS-1; MuSCs, skeletal muscle satellite cells; BaCl 2, barium chloride; TA, tibialis anterior; NC, non-injured control; rADAMTS-1 (L), rADAMTS-1 at 5 mg/kg; rADAMTS-1 (H), rADAMTS-1 at 10 mg/kg; CD, cluster of differentiation; Sca1, stem cell antigen 1; VCAM1, vascular cell adhesion molecule; Pax7, paired box protein.

Article Snippet: Primary skeletal muscle cells (cat. no. PCS-950-010; ATCC) were cultured in a complete expansion medium (cat. no. PCS-500-030; ATCC) using a primary skeletal muscle cell growth kit (cat. no. PCS-950-040; ATCC).

Techniques: Flow Cytometry, Isolation, Muscles, Marker, Expressing, Immunofluorescence, Recombinant, Control

Journal: International Journal of Molecular Medicine

Article Title: Recombinant ADAMTS-1 promotes muscle regeneration accompanied by downregulation of Notch signaling

doi: 10.3892/ijmm.2025.5718

Figure Lengend Snippet: Effect of rADAMTS-1 on MuSCs following BaCl 2 -induced TA muscle injury. Groups included NC, BaCl 2 , and two treatment groups receiving rADAMTS-1 (L) and (H). Daily intraperitoneal injections of rADAMTS-1 were initiated immediately after injury and continued until the designated time point for analysis. (A) Flow cytometry analysis of MuSCs isolated from injured TA muscles, defined by surface marker expression (CD45 − /CD31 − /Sca1 − /VCAM1 + ). (B) Representative gating strategy for MuSCs across experimental groups at different time points. (C) Relative number of MuSCs normalized to the NC group over time. Statistical significance was determined using one-way analysis of variance, followed by Tukey's post hoc test: * P<0.05, ** P<0.01 and *** P<0.001 vs. NC; ## P<0.01 and ### P<0.001 vs. BaCl 2 group. (D) Representative immunofluorescence images of Pax7 + cells in injured TA muscle at different time points (scale bar, 25 μ m). (E) Quantification of Pax7 + cell percentages over time. Statistical significance was determined using one-way analysis of variance with Tukey's post hoc test: * P<0.05 and *** P<0.001 vs. NC; ### P<0.001 vs. BaCl 2 ; ††† P<0.001 vs. rADAMTS-1 (L). ADAMTS-1, a disintegrin and metalloproteinase with thrombospondin motifs 1; rADAMTS-1, recombinant ADAMTS-1; MuSCs, skeletal muscle satellite cells; BaCl 2, barium chloride; TA, tibialis anterior; NC, non-injured control; rADAMTS-1 (L), rADAMTS-1 at 5 mg/kg; rADAMTS-1 (H), rADAMTS-1 at 10 mg/kg; CD, cluster of differentiation; Sca1, stem cell antigen 1; VCAM1, vascular cell adhesion molecule; Pax7, paired box protein.

Article Snippet: For differentiation, the cells were treated with a skeletal muscle differentiation tool (cat. no. PCS-950-050; ATCC) upon reaching 90% confluence.

Techniques: Flow Cytometry, Isolation, Muscles, Marker, Expressing, Immunofluorescence, Recombinant, Control

Journal: International Journal of Molecular Medicine

Article Title: Recombinant ADAMTS-1 promotes muscle regeneration accompanied by downregulation of Notch signaling

doi: 10.3892/ijmm.2025.5718

Figure Lengend Snippet: Effects of rADAMTS-1 on proliferation and differentiation of primary skeletal muscle cells. (A) Dose-dependent proliferative effects of rADAMTS-1 (0.001-10 ng/ml) on primary skeletal muscle cells, assessed using the bromodeoxyuridine assay. (B) Dose-dependent effect of rADAMTS-1 (0.001-10 ng/ml) on the differentiation of primary skeletal muscle cells. Quantification of myotube length following differentiation (scale bar, 200 μ m). (C) Western blot analysis showing the expression of myogenic and ADAMTS-1 related markers (MyoD, MyoG, ADAMTS-1, NICD, Hes-1, and β-actin) following rADAMTS-1 treatment at a concentration of 10 ng/ml, indicating its promotion of early-stage myogenic differentiation through inhibition of NICD signaling. Each data point represents an individual measurement; error bars indicate standard deviation. Statistical significance was determined using one-way analysis of variance followed by Tukey's honestly significant difference post hoc test and is indicated as follows: (A) * P<0.05 vs. Con group; (B) ** P<0.01 and *** P<0.001 vs. Con group; (C) * P<0.05 and ** P<0.01 vs. Con group. ADAMTS-1, a disintegrin and metalloproteinase with thrombospondin motifs 1; rADAMTS-1, recombinant ADAMTS-1; MyoD, myoblast determination protein 1; MyoG, myogenin; NICD, Notch intracellular domain; HES-1, hairy and enhancer of split-1; Con, control.

Article Snippet: For differentiation, the cells were treated with a skeletal muscle differentiation tool (cat. no. PCS-950-050; ATCC) upon reaching 90% confluence.

Techniques: BrdU Staining, Western Blot, Expressing, Concentration Assay, Inhibition, Standard Deviation, Recombinant, Control