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

Abstract

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 (MøSs) are proposed to promote therapeutic angiogenesis in critical limb ischemia (CLI) via chronological shifting from M1 to M2 phenotypes. Uniform-sized MøSs 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-α secretion from M1 is downregulated by around threefold, whereas the levels of interleukin-10 and transforming growth factor-β1 from M2 increase by around threefold. The incubation with conditioned media from the MøS 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 MøSs and to undergo dynamic phenotypic shifting from early M1 to later M2 for angiogenesis promotion in the CLI treatment.

Biomaterials-assisted spheroid engineering for regenerative therapy

“…Ran et al . ( 68 ) have generated macrophage spheroids with chrysin-loaded fibers. These fibers not only can serve as a scaffold, but also can release chrysin for regulating the phenotypic transformation of macrophages from pro-inflammatory M1 to anti-inflammatory M2.…”

“…In vivo results have revealed that M2 macrophage polarization of spheroids can induce a full recovery of hind-limb ischemia to normal limbs. It has been also proven that macrophage spheroids transplantation can lead to greater limb salvage than single macrophage administration ( 68 ).…”

Nanofabrication Technologies to Control Cell and Tissue Function in Three-Dimension

“…The use of spheroids is currently under investigation for many cell types. To improve the therapeutic effect of cell spheroids even further, various biomaterials (e.g., fibers and hydrogels) have been developed for spheroid engineering [ 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ]. Note that hydrogels, as artificial 3D cell scaffolds, have attracted great attention in recent years because of their ability to reproduce the biological environment, with their inherent biochemical and physicochemical properties, such as cell adhesion, degradation, and viscoelasticity ( Figure 1 ) [ 1 ].…”