Effects of phenotype. Commercially available size 100 compressed Gelfoam sponge was purchased from Pfizer (New York, NY), cut using a sterile 8-mm-diameter biopsy punch, and prewet in sterile 1X PBS or media. Vascular networks were formed on Gelfoam constructs by seeding a 5:1 ratio of HAMEC-dTom to MSCs directly onto the constructs in 10 μl of EC growth media. The cell-seeded constructs were incubated for at least 30 min in a humidified chamber at 37°C and 5% CO2 to facilitate cell attachment; this process was repeated to achieve seeding on both sides of the constructs. After seeding, all constructs were transferred to clean tissue culture dishes, immersed in a 1:1 solution of EC and MSC coculture media, and incubated to initiate vessel formation. On day 3, the engineered vascular grafts were imaged via confocal microscopy as a baseline measurement of vessel growth. After imaging, 100,000 THP-1–derived M0, M1, or M2a macrophages were seeded directly onto the grafts in 10 μl of coculture media. Control grafts without macrophages were seeded with 10 μl of media alone. All samples were incubated for 30 min to allow cell attachment and subsequently transferred to clean 24-well tissue culture plastic for continued incubation in coculture media, which was previously verified to support THP-1 viability (>99.5%) after 3 days in vitro via trypan blue exclusion. Macrophage media were omitted to prevent changes in vessel development. Changes in vascular network formation were monitored over 10 days in vitro using confocal microscopy. For all studies, n ≥ 3 grafts were included per group.

Effects of temporally modulating phenotype. Gelfoam constructs were preseeded with a 5:1 ratio of HAMEC-dTom and MSCs to initiate vessel formation, and on day 3, the grafts were imaged via confocal microscopy as a baseline measurement of vessel growth. After imaging, 50,000 M1 or M2a macrophages were seeded directly onto the grafts in 10 μl of coculture media. All samples were incubated for 30 min to allow cell attachment and subsequently transferred to clean 24-well tissue culture plastic for continued incubation in coculture media. Grafts were imaged on days 4 and 6. After imaging on day 6, 50,000 M1 or M2a macrophages were again seeded directly onto the grafts in 10 μl of coculture media and incubated for cell attachment. The effects of this sequential seeding were measured on days 7 and 10 using confocal microscopy. Experimental groups included the following: control grafts without macrophages, M1-to-M0, M1-to-M1, M1-to-M2a, M0-to-M2a, M2a-to-M2a, and M1+M2a-to-M1+M2a; n ≥ 3 per group.

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