Fabrication and Characterization of Electrospun Cardiac Patches Functionalized with Microbiota-Derived Postbiotics and Decellularized Neonatal Porcine Myocardial Extracellular Matrix for Cardiac Repair

Figures

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  Figure 1. Decellularization and characterization of neonatal porcine myocardial ECM. (A) Surgically isolated neonatal porcine hearts. (B) Native myocardial tissue before decellularization. (C) Macroscopic appearance of dECM after decellularization. (D) DNA quantification of native myocardial tissue and dECM (**** p < 0.0001 versus native tissue). (E) Masson’s Trichrome staining of native myocardial tissue (scale bar = 50 μm). (F) H&E staining of dECM (scale bar = 50 μm). (G) Immunofluorescence staining of dECM showing COL1A1 (green) and DAPI (blue), scale bar = 100 μm). (H) Fourier Transform Infrared spectra of native myocardial tissue and dECM.

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  Figure 2. Antibacterial activity of postbiotics against MRSA. (A) Agar well diffusion assay showing inhibition zones. (B) Broth microdilution assay performed in triplicate demonstrating 20 mg/mL as the MIC value (+, only MRSA; − only media). (C) SYTO 9/PI-based Live/Dead fluorescence staining demonstrating time-dependent antibacterial effects of sub-MIC and MIC-10 concentrations. Green fluorescence indicates viable bacterial cells, whereas red fluorescence represents dead bacterial cells (scale bar = 200 μm).

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  Figure 3. Cytotoxic effects of postbiotics on H9c2 cells assessed by MTT assay (ns: non-significant, p > 0.05; * p < 0.05, ** p < 0.05, ***; p < 0.001 versus control).

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  Figure 4. Effects of postbiotics on LPS-induced inflammatory response in H9c2 cells. (A) Determination of non-cytotoxic concentration of LPS. (B) Relative gene expression of pro-inflammatory (TNF-α, IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines together with (C) ELISA quantification of inflammatory mediators following postbiotic pretreatment. (ns: non-significant, p > 0.05; ** p < 0.01 versus control; **** p < 0.0001 versus control; #### p < 0.0001 versus model).

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  Figure 5. Protective effects of postbiotics against H2O2-induced oxidative stress in H9c2 cells. (A) Determination of the IC50 concentration of H2O2. (B) Annexin V–FITC/PI flow cytometry dot plots showing the distribution of viable, early apoptotic, late apoptotic, and necrotic cell populations. (C) Biochemical analysis of antioxidant defense markers (ns: non-significant, p > 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus untreated group; ## p < 0.01, ### p < 0.001 versus H2O2-treated group).

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  Figure 6. Immunofluorescence images showing COL1A1-positive signals (green) and DAPI-stained nuclei (blue) in untreated and postbiotic-treated groups (scale bar = 20 μm).

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  Figure 7. Fabrication and morphological characterization of electrospun hybrid cardiac patches. (A) Homogeneous electrospinning precursor solution containing gelatin, myocardial dECM, and postbiotics. (B) Schematic illustration of the electrospinning process used for fabrication of hybrid cardiac patches (Figure created using BioRender.com). (C) Macroscopic appearance of the fabricated electrospun patch following crosslinking. (D) SEM micrographs of electrospun hybrid patches containing 5% gelatin with either 1% or 2% myocardial dECM and postbiotics (red arrows indicate fused fiber regions and bead-like structural irregularities).

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  Figure 8. Functional characterization and biocompatibility of electrospun hybrid cardiac patches. (A) Representative stress–strain curves of electrospun cardiac patches with (orange) and without (blue) postbiotic and dECM incorporation. (B) Antibacterial activity of patches against MRSA evaluated by disk diffusion assay (red arrows indicate patches). (C) Live/Dead fluorescence images of H9c2 cells on the electrospun cardiac patches (green, viable cells; red, dead cells; scale bar = 200 μm).