Journal: Bioactive Materials

Article Title: Melatonin-incorporated brain extracellular matrix hydrogel enhances NSCs mitochondrial metabolism to promote neuroregeneration via the AMPK-PGC-1α-NRF1/TFAM axis after spinal cord injury

doi: 10.1016/j.bioactmat.2026.04.006

Figure Lengend Snippet: Melatonin activates AMPK signaling and enhances mitochondrial function in vitro. (A) GO enrichment bar plot of differentially expressed genes (DEGs) between Control and Melatonin-treated NSCs. (B) KEGG pathway enrichment bar plot of DEGs between Control and Melatonin groups. (C) Heatmap of selected DEGs associated with neuronal differentiation and mitochondrial function. DEGs were defined as transcripts with FDR <0.05. (D) Representative Western blots showing phosphorylated AMPK (p-AMPK, Thr172) and phosphorylated ACC (p-ACC, Ser79) in Control, Melatonin, Inhibitor, and Melatonin + Inhibitor groups. (E) Densitometric analysis of p-AMPK/total AMPK and p-ACC/GAPDH ratios. (F) RT-qPCR analysis of Ppargc1a and Tfam expression, normalized to GAPDH and presented as fold change relative to the Control group. (G) Representative Western blots of mitochondrial oxidative phosphorylation (OXPHOS) complexes I-V. (H) Densitometric quantification of OXPHOS complexes I-V, normalized to GAPDH (or the corresponding loading control). (I) Representative JC-1 fluorescence images indicating mitochondrial membrane potential (ΔΨm). (J) Quantification of the red/green JC-1 fluorescence ratio from (I). (K) Schematic representation of the proposed melatonin-AMPK-ACC-PGC-1α-NRF1/TFAM signaling axis driving mitochondrial biogenesis in NSCs. All quantitative data (E, F, H, J) are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Holm–Sidak's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. K created with BioRender.com .

Article Snippet: For AMPK inhibition experiments, BAY-3827 (HY-112083, MedChemExpress, USA), a selective AMPK inhibitor, was used at a final concentration of 2 μM for 24 h. The mitochondrial membrane potential was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2003S, Beyotime Biotechnology, China).

Techniques: In Vitro, Control, Western Blot, Quantitative RT-PCR, Expressing, Phospho-proteomics, Fluorescence, Membrane

Journal: Bioactive Materials

Article Title: Melatonin-incorporated brain extracellular matrix hydrogel enhances NSCs mitochondrial metabolism to promote neuroregeneration via the AMPK-PGC-1α-NRF1/TFAM axis after spinal cord injury

doi: 10.1016/j.bioactmat.2026.04.006

Figure Lengend Snippet: Molecular validation of neural repair and mechanism activation in spinal cord tissue. Western blot and qPCR analyses of spinal cord tissue lysates from Sham, SCI, BEM, NSCs@BEM, and NSCs@MT/BEM groups. (A) Representative Western blots for the neuronal marker TUJ1 and the glial scar marker GFAP. (B) Representative Western blots for phosphorylated AMPK (p-AMPK), phosphorylated ACC (p-ACC), and their respective total proteins. (C) Representative Western blots for the five oxidative phosphorylation (OXPHOS) complex subunits. (D) Densitometric quantification of TUJ1 and GFAP protein levels. (E) Densitometric quantification of the p-AMPK/total AMPK and p-ACC/total ACC ratios. (F) Densitometric quantification of OXPHOS complex protein levels. (G) Relative mRNA expression of neural markers (TUJ1, GFAP, Olig2) and key mitochondrial biogenesis regulators (Ppargc1a, Tfam) determined by qPCR. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Holm–Sidak's multiple comparisons test. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Article Snippet: For AMPK inhibition experiments, BAY-3827 (HY-112083, MedChemExpress, USA), a selective AMPK inhibitor, was used at a final concentration of 2 μM for 24 h. The mitochondrial membrane potential was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2003S, Beyotime Biotechnology, China).

Techniques: Biomarker Discovery, Activation Assay, Western Blot, Marker, Phospho-proteomics, Expressing

Journal: Bioactive Materials

Article Title: Spatiotemporally programming the immune-osteogenic cascade with a dual-immunomodulatory scaffold for functional bone regeneration

doi: 10.1016/j.bioactmat.2026.04.002

Figure Lengend Snippet: Immune regulation and endogenous bone regeneration mechanism investigation. A) Network diagram showing the number of interactions between six subclusters. B) KEGG enrichment analysis of the upregulated DEGs in DIBS group compared to the HA group. C) Circular visualization of related pathway–gene enrichment analysis. D) Heatmap of key gene regulation in specific pathways. E) qRT-PCR validation for key gene expression in specific pathways. F) The interaction networks showing the correlation of representative immunomodulatory genes (CCL2, CCL20, Sfrp1, and Stat3, etc.) with angiogenesis/osteogenesis and macrophage regulation gene sets. G) Flow cytometry analysis and quantification of CCR2 F4/80 macrophage in peripheral blood. H) Immunofluorescence staining analysis of macrophage polarization inside scaffolds (one week after intramuscular implantation). I) Macrophage proliferation assay in a CCR2-dependent manner. J and K) Macrophage polarization assay in a CCR2-dependent manner. Data are represented as means ± SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; $ p < 0.05 (vs group without inhibitor), $$ p < 0.01 (vs group without inhibitor), $$$ p < 0.001 (vs group without inhibitor), $$$$ p < 0.0001 (vs group without inhibitor). ns, not significant.

Article Snippet: Throughout the experimental period, sustained CCR2 inhibition was achieved via daily intraperitoneal injections (2 mg/kg) of the highly selective CCR2 inhibitor RS504393 (Cat. No. HY-15418, MCE).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Flow Cytometry, Immunofluorescence, Staining, Proliferation Assay

Journal: Bioactive Materials

Article Title: Spatiotemporally programming the immune-osteogenic cascade with a dual-immunomodulatory scaffold for functional bone regeneration

doi: 10.1016/j.bioactmat.2026.04.002

Figure Lengend Snippet: Revascularization and osteogenesis are reinforced by M2 macrophage activation via the CCL2/CCR2 pathway. A and B) HUVECs and BMSCs proliferation assay under M2 macrophage activation. Created with BioRender.com . C) Migration assay and quantification of HUVECs. D) Tube formation assay and quantification of HUVECs. E and F) Early and later osteogenic differentiation of BMSC influenced by macrophage-induced microenvironment. Data are represented as means ± SD, ∗ p < 0.05 (vs Control), ∗∗ p < 0.01 (vs Control), ∗∗∗ p < 0.001 (vs Control), ∗∗∗∗ p < 0.0001 (vs Control); $ p < 0.05 (vs group without inhibitor), $$ p < 0.01 (vs group without inhibitor), $$$ p < 0.001 (vs group without inhibitor), $$$$ p < 0.0001 (vs group without inhibitor). ns, not significant.

Article Snippet: Throughout the experimental period, sustained CCR2 inhibition was achieved via daily intraperitoneal injections (2 mg/kg) of the highly selective CCR2 inhibitor RS504393 (Cat. No. HY-15418, MCE).

Techniques: Activation Assay, Proliferation Assay, Migration, Tube Formation Assay, Control