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 experimental conditions show increased wound healing in presence of cytokines. N = 4–9 wounds were analyzed at different time points for each group. (E) Fraction of healed wounds for the tested groups at different time points (I = ischemic, NI = non-ischemic). At 21 days, all wounds were completely healed.