Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: NMEVs effectively attenuated palmitic acid-induced senescence in C2C12 cells. (A) Schematic diagram of cell culture and treatment. (B) qRT-PCR analysis of the expression of senescence markers p53, cdkn1a, and cdkn2a in each group (n = 3). (C-C‴) Western blotting for the expression of senescence markers p53, cdkn1a (p21), and cdkn2a (p16) in each group with relative quantification (n = 6). (D-D′) Immunofluorescence staining for the DNA damage marker γH2AX with quantitative analysis (Scale bar, 100 μm; n = 6 for each group). (E-E′) Immunofluorescence staining for p16 with quantitative analysis (Scale bar, 100 μm; n = 6). (F-F′) Immunofluorescence staining for p21 with quantitative analysis (Scale bar, 100 μm; n = 6). (G-G′) Flow cytometry analysis of relative reactive oxygen species (ROS) levels (n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant. Rel. fold, relative fold; T.Ar, total area.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining, Marker, Flow Cytometry

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: NMEVs alleviated palmitic acid-induced mitochondrial dysfunction and lipid deposition. (A) Relative ATP synthesis rates in each group (n = 6). (B) qRT-PCR analysis of MT-CO1 expression in each group (n = 3). (C) qRT-PCR analysis of MT-ND1 expression in each group (n = 3). (D) qRT-PCR analysis of MT-CO3 expression in each group (n = 3). (E) qRT-PCR analysis of D-loop expression in each group (n = 3). (F) Mitochondrial complex V activity in C2C12 cells of each group (n = 6). (G) Measurement of oxygen consumption rate (OCR) in C2C12 cells of each group (n = 4). (H-H′) Transmission electron microscopy (TEM) assessment of mitochondrial quantity with quantitative analysis (Scale bar, 500 nm; n = 3). (I-I′) Western blotting for PGC-1α expression in each group with relative quantification (n = 6). (J-J′) Immunofluorescence staining for SDHA with quantitative analysis (Scale bar, 100 μm; n = 6). (K-K′) Immunofluorescence staining for EdU with quantitative analysis (Scale bar, 100 μm; n = 6). (L-L′) Representative images of BODIPY staining in each group with quantitative analysis (Scale bar, 20 μm; magnified Scale bar, 5 μm; n = 6). (M-M′) Representative images of Oil Red O staining in each group with quantitative analysis (Scale bar, 20 μm; magnified scale bar, 5 μm; n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Activity Assay, Transmission Assay, Electron Microscopy, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: NMEVs reduced C2C12 senescence and lipid accumulation by enriching miR-542-3p to stabilize mitochondrial function. (A) PCA plot showing sample homogeneity of AMEVs and NMEVs (n = 3). (B) Heatmap showing the top 20 significantly upregulated and downregulated microRNAs. (C) qRT-PCR analysis of the expression of the top 12 significantly upregulated microRNAs (n = 3). (D) qRT-PCR analysis of miR-542-3p expression in C2C12 cells after transfection with NMEVs, mimic, or NMEVs + inhibitor (n = 3). (E-E′) Immunofluorescence staining for p16 with quantitative analysis (Scale bar, 50 μm; n = 6). (F-F′) Immunofluorescence staining for p21 with quantitative analysis (Scale bar, 50 μm; n = 6). (G-G′) Immunofluorescence staining for γH2AX with quantitative analysis (Scale bar, 50 μm; n = 6). (H-H′) Immunofluorescence staining for EdU with quantitative analysis (Scale bar, 50 μm; n = 6). (I-I′) Immunofluorescence staining for SDHA with quantitative analysis (Scale bar, 50 μm; n = 6). (J-J‴) Western blotting for p16, p21 and PGC-1α expression in each group with relative quantification (n = 6). (K) Relative ATP synthesis rates in each group (n = 6). (L) qRT-PCR analysis of MT-CO1 expression in each group (n = 3). (M) qRT-PCR analysis of MT-ND1 expression in each group (n = 3). (N) qRT-PCR analysis of MT-CO3 expression in each group (n = 3). (O) qRT-PCR analysis of D-loop expression in each group (n = 3). (P-P′) Representative images of BODIPY staining in each group with quantitative analysis (Scale bar, 20 μm; magnified Scale bar, 10 μm; n = 6). (Q-Q′) Representative images of Oil Red O staining in each group with quantitative analysis (Scale bar, 20 μm; magnified Scale bar, 10 μm; n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Immunofluorescence, Staining, Western Blot, Quantitative Proteomics

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: Asxl2 and Eef1a1 served as downstream target genes of miR-542-3p. (A) Prediction of downstream target genes of miR-542-3p using multiple target gene prediction software. (B) qRT-PCR analysis of Asxl2 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (C) qRT-PCR analysis of Eef1a1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (D) qRT-PCR analysis of Lrrc59 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (E) qRT-PCR analysis of Gabarap expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (F) qRT-PCR analysis of Ap3d1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (G) qRT-PCR analysis of Arhgap5 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (H) qRT-PCR analysis of Kcmf1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (I) qRT-PCR analysis of Pten expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (J) qRT-PCR analysis of Ube2e1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (K-K″) Western blotting for Asxl2 and Eef1a1 expression in C2C12 cells after transfection with miR-542-3p mimic, with relative quantification (n = 3). (L-L′) Dual-luciferase reporter assay verifying the direct targeting binding relationship between miR-542-3p and Asxl2 (n = 3). (M-M′) Dual-luciferase reporter assay verifying the direct targeting binding relationship between miR-542-3p and Eef1a1 (n = 3). (N-N″) Western blotting for Asxl2 and Eef1a1 expression in neonatal and aging muscle tissues (n = 3). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Software, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Quantitative Proteomics, Luciferase, Reporter Assay, Binding Assay

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: miR-542-3p suppressed Eef1a1 to ameliorate PA-induced mitochondrial dysfunction and cellular senescence. (A-A′) Western blotting for Eef1a1 expression after PA induction, followed by transfection with miR-542-3p mimic and Eef1a1 overexpression plasmid (Eef1a1 OE ), with relative quantification (n = 3). (B) Schematic diagram illustrating Eef1a1 regulation of lipid storage via AMPK. (C-C′) Western blotting for AMPK and p-AMPK expression in neonatal and aging muscle tissues with relative quantification (n = 3). (D-D′) Western blotting for AMPK and p-AMPK expression after PA induction, followed by transfection with miR-542-3p mimic and Eef1a1 overexpression plasmid (Eef1a1 OE ), with relative quantification (n = 3). (E-E″) Representative images of p16 and p21 staining in each group with quantitative analysis (Scale bar, 50 μm; n = 6). (F) Relative ATP synthesis rates in each group (n = 6). (G) qRT-PCR analysis of MT-CO1 expression in each group (n = 3). (H) qRT-PCR analysis of MT-ND1 expression in each group (n = 3). (I) qRT-PCR analysis of MT-CO3 expression in each group (n = 3). (J) qRT-PCR analysis of D-loop expression in each group (n = 3). (K) Mitochondrial complex V activity in C2C12 cells of each group (n = 6). (L-L′) Representative images of SDHA staining in each group with quantitative analysis (Scale bar, 50 μm; n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative Proteomics, Staining, Quantitative RT-PCR, Activity Assay

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Establishment and characterization of a fibrotic macrophage-muscle fibrosis model. a) Brightfield image of unstimulated RAW264.7 cells in the top chamber of a RAW-C2C12 coculture system, b) Brightfield image of C2C12 cells in the bottom chamber under basal coculture conditions, c) Fluorescence image of C2C12 cells in the bottom chamber exhibiting myotube morphology, d) Fluorescence image confirming the absence of fibrotic marker expression in C2C12 cells under basal conditions, e) Brightfield image of LPS-activated RAW264.7 cells in the top chamber, f) Brightfield image of C2C12 cells in the bottom chamber displaying a myofibroblast-like morphology post-LPS activation, g) Fluorescence image of phenotypic transition of C2C12 cells to myofibroblast-like cells, h) Fluorescence image demonstrating fibrotic protein expression in LPS-treated C2C12 cells (magenta arrow indicates CD38 cells). Immunostaining images: In panels c) and g), TGF-β, DAPI, and actin are colored red, blue, and green, respectively. In panels d) and h), COL1, α-SMA, CD38, and DAPI are colored red, blue, magenta, and green, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Fluorescence, Marker, Expressing, Activation Assay, Immunostaining

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Study of myogenesis through myotube formation. Brightfield images taken using a 10× objective: a) C2C12 RAW, b) C2C12 RAW LPS, c) C2C12 RAW LPS PFD, d) C2C12 RAW LPS- H, e) C2C12 RAW LPS - PH, indicating myotube formation, f) Graph representing the number of myotubes. The data points are presented as mean ± SEM (n = 4). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. The scale bar denotes 50 μm # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Immunofluorescence images showing the expression of myogenesis (MyHC) or fibrogenesis (α-SMA and COL) markers. a) C2C12, b) C2C12 RAW, c) C2C12 RAW LPS, d) C2C12 RAW LPS PFD, e) C2C12 RAW LPS - H, f) C2C12 RAW LPS - PH. Semiquantitative expression analysis of the myogenesis/fibrosis markers. g) MyHC/α-SMA and h) MyHC/α-SMA. I) MyHC/α-SMA/DAPI staining, II) MyHC/COL1/DAPI staining. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. The scale bar denotes 50 μm # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Immunofluorescence, Expressing, Staining, Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Gene analysis of fibrogenesis and myogenesis markers. a) COL1, b) MMP8, c) Acta2, d) LOX, and e) MyoG using qPCR. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: AFM images of cells in contact mode in PBS after treatment. a) C2C12 RAW, b) C2C12 RAW LPS, c) C2C12 RAW LPS PFD, d) C2C12 RAW LPS - H, e) C2C12 RAW LPS - PH, (f) graph representing nanoindentation measurements denoting the stiffness of the cell. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Mitochondrial metabolism study using a Seahorse extracellular analyzer. Mitochondrial stress test, a) Scheme representing the mitochondrial stress test profile, b) OCR graphs of the inflammation model, c) OCR graphs of the treatment conditions, d) Proton leak measurements, e) ATP production linked to O 2 consumption, f) Maximal respiration, and g) Spare capacity. The data points are presented as mean ± SEM. (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Transmission electron micrographs depicting cell morphology and mitochondrial morphology. (a) C2C12 RAW co-culture with a black box indicating the native muscle cell structure. (b) C2C12 RAW LPS, with a black box highlighting C2C12 phenotypically transitioned to myofibroblast-like cell structures. TEM images of mitochondria within single cells are shown for (c) C2C12 RAW, (d) C2C12 RAW LPS, and (e) C2C12 RAW LPS - PH (PH - Pirfenidone loaded hydrogel) treatment. Yellow arrows point to mitochondria. (The data points are presented as mean ± SEM. (n = 4). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # and ns indicates significant and non-significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively. Scale bars in figures (a, b) represent 5 μm, c) 1 μm, d, e) 2 μm, and the insets are of a 500 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Transmission Assay, Co-Culture Assay, Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: a) Comparative transcriptomic profiles of the control (C2C12 RAW), fibrotic (C2C12 RAW LPS), and treatment (C2C12 RAW LPS - PH) groups, b) Volcano plot of DEGs in the fibrotic (C2C12 RAW LPS) vs control (C2C12 RAW) groups, and c) Volcano plot of DEGs in the treated (C2C12 RAW LPS - PH) vs fibrotic (C2C12 RAW LPS) groups.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: A comparison analysis depicting the a) biological functions and b) canonical pathways associated with fibrosis and healthy muscle regeneration in the control (C2C12 RAW) vs. fibrotic (C2C12 RAW LPS) vs. treatment (C2C12 RAW LPS - PH) groups. The intensity of the heatmap color corresponds to the activation z-score due to the involvement of upregulated and downregulated genes. The supplementary table listing the pathways along with their corresponding z-score values is included in the supplementary section (Table ST1).

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Comparison, Control, Activation Assay