Macrophage Spheroids with Chronological Phenotype Shifting To Promote Therapeutic Angiogenesis in Critical Limb Ischemia

Therapeutic angiogenesis becomes an essential approach to rescue ischemia and the cell-based therapy facilitates ischemia recovery via stimulation of paracrine signals or replacement of damaged cells. Macrophages participate in multiple processes of tissue repair, and the M1 and M2 phenotypes play distinct roles in the sequential stages of healing initiation, angiogenesis stabilization, and tissue maturation. In this study, macrophage spheroids (MoSs) are proposed to promote therapeutic angiogenesis in critical limb ischemia (CLI) via chronological shifting from M1 to M2 phenotypes. Uniform-sized MoSs are prepared by using electrosprayed microcapsules as the confined growth template, and fiber fragments are inoculated to achieve a local release of chrysin for macrophage phenotype manipulation. The macrophage polarization shifting is confirmed from the decreased CD86 expressions (M1 marker) from 64.7 to 31.7% and the concurrent increase in CD206 expressions (M2 marker) from 39.3 to 72.4%. Meanwhile, the tumor necrosis factor-alpha secretion from M1 is downregulated by around threefold, whereas the levels of interleukin-10 and transforming growth factor-beta 1 from M2 increase by around threefold. The incubation with conditioned media from the MoS culture could promote the proliferation and migration of endothelial cells (ECs), showing a high healing rate of EC layers. The local treatment of hind-limb ischemia leads to a full recovery from ischemic to normal limbs with a high blood perfusion rate. Histological analysis shows few muscle degeneration and fibrosis areas and high capillary densities. As far as we know, this is the first attempt to develop uniform-sized MoSs and to undergo dynamic phenotypic shifting from early M1 to later M2 for angiogenesis promotion in the CLI treatment.