Macrophage polarization in 2D and 3D cultures to M1. (A) Procedure for macrophage polarization in 2D and 3D cultures. Macrophages in the 3D scaffolds were polarized to either M1 or M2 phenotypes at 1, 7, and 14 days. (B) Expression levels of TNF-α, IL-6, and IL-1β genes in M0, M1, and M2 macrophages to validate macrophage M1 polarization under 2D (top) or 3D (bottom) culture at days 1, 7, and 14. (C) TNFα concentration in the conditioned medium by M0, M1, and M2 on days 1, 7, and 14 under 2D (left) and 3D (right) cultures, which was determined by ELISA. Each value represents the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with either 2D M0 or 3D M0 cultures. 2D, two-dimensional; TNFα, tumor necrosis factor alpha; IL, interleukin; ELISA, enzyme-linked immunosorbent assay.