A bioactive hydrogel patch accelerates revascularization in ischemic lesions for tissue repair

Figures

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  Figure 1. The MgAP elastic patch exhibited appropriate physical properties for treating ischemic diseases. (a) FTIR spectrum of MgAP. (b) Dependence of G′, G′′, and the G′′/G′ ratio (loss factor) of MgAP on the frequency of oscillation. (c) Stress–strain curves of cyclic compressive loading–unloading tests on MgAP. (d) Photograph of MgAP adhering firmly to the wet surfaces of skeletal (left) and myocardial (right) muscle. (e) Stress–displacement curve from the adhesion test according to a modified method from ASTM standard C907-17, with an inset schematic of the test. (f) Strength and toughness of the material–epicardium interface. (g) SEM images of freeze-dried MgAP. (h–k) SEM maps of elemental C, O, and Mg in MgAP. (l) Mg2+ concentrations in the immersion solution after soaking MgAP for different time periods

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  Figure 2. MgAP exhibited satisfactory biosafety and enhanced in vitro angiogenesis. (a) Live/dead cell staining was conducted after 7 days of the coculture of HUVECs with the MgAP patch extracts. (b) CCK-8 assay results showing the viability of cells cultured with the MgAP patch extracts for 1 or 3 days. (c) HUVECs were subjected to tube formation assays to assess angiogenic capability. (d and e) Quantitative analysis of tube formation assay results. (Calcein-AM: Calcein-acetoxymethyl ester, used to stain live cells; PI: Propidium iodide, used to stain dead cells). All data are expressed as the means ± SDs; ns: nonsignificant, P > 0.05; **P < 0.01. Statistical analysis for comparisons between two groups was performed using Student two-tailed t-test

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  Figure 3. The MgAP elastic patch improved LV systolic function in the rat MI model. (a) Experimental design of the MI treatment model. (b) Doppler echocardiography in M-mode of the left ventricle at the fourth week posttreatment. (c–h) Echocardiographic evaluation of cardiac function in the sham group (n = 5), the MgIV group, the MgAP treatment group, and the untreated MI group (n = 7–9) at the first week and fourth week. The data are representative of independent experiments. All data are expressed as the means ± SDs; ns: nonsignificant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analysis for comparisons between two groups was performed using Student two-tailed t-test. For comparisons involving more than two groups, statistical significance was assessed using a one-way ANOVA followed by an LSD test

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  Figure 4. MgAP improved cardiac strain and synchrony in the rat MI model. (a) Left: schematic diagram of radial and longitudinal deformation of the left ventricle. Right: variation tendency of different myocardial segments shown by speckle-tracking echocardiography. (b) Left ventricular parasternal longitudinal speckle-tracking echocardiography at the fourth week posttreatment. The length of the indicator line represents the degree of displacement. (c) Heatmap of the radial displacement of the ventricular wall. (d–g) Radial and longitudinal strain and strain rates of the left ventricular wall at the fourth week in each group (n = 6). (h) Representative three-dimensional diagram of radial strain. (i) Representative three-dimensional diagram of longitudinal strain. (j) Representative schematic diagrams of OWD in the fourth week. (AW: The anterior wall area of left ventricle; PW: The posterior wall area of left ventricle). The data are representative of independent experiments. All data are expressed as the means ± SDs; ns: nonsignificant, P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical significance was calculated by one-way ANOVA with the LSD test

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  Figure 5. MgAP treatment mitigated adverse myocardial remodeling in the rat MI model. (a) Representative Masson trichrome staining of the heart at the fourth week; (b) analysis of the fibrotic area; and (c) mean fibrotic density. (d and e) qPCR was used to detect the mRNA expression of collagen I/collagen III. (f) Representative H&E staining of the heart at the fourth week and (g) analysis of left ventricular wall thickness. (h and i) DAPI/TUNEL staining of the MI area at the fourth week and quantitative analysis. The data are representative of independent experiments. All data are expressed as the means ± SDs; ns: nonsignificant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical significance was calculated by one-way ANOVA with the LSD test

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  Figure 6. Transcriptomic analysis and validation of MgAP-mediated repair mechanisms. (a) Heatmap of differentially expressed genes between the MI + MgAP group and the MI group. (b) Volcano map of all the expressed genes in the MgAP group and MI group (n = 2). (c) Representative GO analysis of significant variation in biological process (BP), cell component (CC), and molecular function (MF) terms in the MgAP group. (d) Representative images of immunofluorescence staining and quantitative analysis of DAPI, CX43, and cTnT in cardiac sections from animals in the MI and MI + MgAP groups at the fourth week. (e) Representative images of immunofluorescence staining and quantitative analysis of DAPI, vWF, and α-SMA in cardiac sections from animals in the MI and MI + MgAP groups. (f) Western blot analysis of cardiac CX43 and CD31 protein levels in the MI group and MI + MgAP group after 4 weeks of treatment. (CX43: Connexin 43; CD31: Cluster of differentiation 31). All data are expressed as the means ± SDs; ns: nonsignificant, P > 0.05; *P < 0.05. Statistical analysis for comparisons between two groups was performed using Student two-tailed t-test

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  Figure 7. The MgAP patch enhanced angiogenesis to repair ischemic tissue. (a) A Doppler laser scanner was used to quantify blood flow by imaging both limbs in rats before (Day 0) and after treatment (Day 14). (b) Increase in the perfusion ratio between Day 14 and Day 0 (∆perfusion ratio). (c and d) Representative images of immunofluorescence staining and quantitative analysis of DAPI and α-SMA in the lower LI sites of animals in the LI and LI + MgAP groups. (e) Representative CD31 and VEGF immunohistochemical staining of limb muscles in the LI and LI + MgAP rat models. All data are expressed as the means ± SDs; ns: nonsignificant, P > 0.05; ***P < 0.001; ****P < 0.0001. Statistical analysis for comparisons between two groups was performed using Student two-tailed t-test