Engineering Immunomodulatory Biomaterials for Regenerating the Infarcted Myocardium

Figures

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  FIGURE 1. Soft and biodegradable alginate hydrogels are produced by internal crosslinking with calcium carbonate. (A) Images of AC2.5, AC5, and AC10 hydrogels, scale bar = 5 mm. (B) Wet weight variation (swelling) of hydrogels compared to their initial weight at day 0. (C) Dry weight variation (mass loss) of hydrogels compared to the initial dry weight at day 0. (D) Rheological characterization of storage (G′) and loss (G″) moduli of AC5 and AC10 by frequency sweep test at 0.5% strain and T = 23°C and (E) shear rate dependence of shear stress (filled circles) and viscosity (open triangles) at T = 23°C.

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  FIGURE 2. BSA is released from AC5 and AC10 in 5 days. (A) Representative confocal laser scanning microscopy images of AC5 (left) and AC10 (right) hydrogels loaded with BSA-FITC protein acquired after 24 h in phosphate buffered saline (PBS). Scale bar: 100 μm. (B) Quantification of BSA content in PBS supernatant collected from hydrogels at different incubation times. Data are represented as cumulative percentage release relative to the initial loading dose ± SD (n = 3).

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  FIGURE 3. Optimized combinations of immunomodulatory cytokines released from AC10 hydrogels promote monocytes polarization toward healing macrophage phenotypes. (A) Isolated human CD14 + monocytes were cultured for 10 days in the presence of medium containing cytokines obtained from solid hydrogels after 10 days of incubation. (B) Representative optical light microscopy showing inflammatory macrophages phenotypes characterized by the round morphology (white arrowheads) and anti-inflammatory macrophage subsets with elongated morphology (red arrows). Scale bar = 50 μm. (C) Confocal images obtained from the specified conditions. Expression of M2-macrophage markers, CD163 (green) and CD206 (red) was evaluated. Scale bar = 50 μm. Nuclei were counterstained with Hoechst 33342 (blue). Rectangles in panel (C) represent cells in magnified area, where merged CD163/CD206/nuclei signals are shown (Scale bar = 20 μm). (D) Number of CD163 + or CD206 + cells was expressed as total positive cell per total nuclei number per analyzed field (ns = p > 0.05, **p < 0.01; ***p < 0.001, n = 3).

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  FIGURE 4. The efficacy of the immunomodulatory biomaterials is tested in a predictive ischemic skin flap model. (A) Digital images of ischemic wounds at 0, 3, 7, and 14 days. Wounds received (1 and 2) no hydrogels, (3) unloaded alginate hydrogels, or (4) alginate hydrogels loaded with 2 μg CSF-1 and 2 μg IL4. Tissue necrosis is visible at day 14 in wounds 1 and 2. Scale bar = 5 mm. (B) Representative hematoxylin/eosin staining of wound tissues treated with AC10 hydrogels unloaded or loaded with 2 μg CSF-1 and 2 μg IL4 at 1, 4, 7, and 14 days post ischemia (scale bar = 1 mm). Hydrogels are identified with a blue circle. Some of the hydrogels detached from the slides during the histological processing (for example, wound image at day 1). The black squares on the H&E staining of loaded AC10 hydrogels indicate the area represented at higher magnification on the right of each sample (scale bar = 200 μm. (C) Immune cells stained with CD68 (pan macrophage marker) and CD206 (reparative macrophage phenotypes) at the wound edges at 4 days post ischemia, scale bars = 20 μm. A 30 μm × 45 μm magnified area is shown in the bottom-left inset of each image. (D) Kinetics of wound closure for the four…

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  FIGURE 5. Infarcted rat hearts receiving the immunomodulatory therapy show improvement in cardiac function and polarization of macrophages in the ischemic wound. (A) Cross section of a heart labeled with picrosirius red/fast green allow identification of the infarct area in red (I) and of the remote healthy tissue (R). Quantification of infarct area relative to the left ventricle area for all groups is shown on the right. Data are represented as mean ± standard deviation. N = 3 hearts were evaluated for all groups except AC10 unloaded- 15 days (n = 2) and AC10 loaded- 15 days (n = 10). (B) Cardiac function evaluated by fractional shortening (%) and ejection fraction (%) over 15 days. Each symbol represents the fractional shortening or ejection fraction of an individual rat. Data are expressed as mean ± standard deviation. (C) CD68 and cTnT staining for assessing macrophages and cardiomyocyte morphology in the infarct and remote area of the heart at day 15, scale bars = 100 μm.

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  FIGURE 6. Identification of macrophage subtypes in the infarct area shows the presence of reparative phenotypes in hearts treated with CSF-1 and IL4 loaded alginate hydrogels. Representative images of the infarct scar treated with unloaded AC10 hydrogels (A) or cytokine-loaded AC10 hydrogels (B) show macrophages stained with antibodies against CD68 (pan macrophage marker: left column), CD80 (inflammatory subsets: top row of A,B) and CD206 (reparative subsets: bottom row of A,B), scale bars = 20 μm. A 25 μm × 35 μm magnified area is shown in the bottom-left inset of each image. (C) Quantification for the macrophage phenotypes normalized to CD68 + cells was performed on n = 198 ± 47 CD68 + cells per group (*p < 0.05).