Supplementary Figure 10: In vitro and in vivo macrophage polarization in response to tissue ECM.

(a) Macrophage phenotype on bone and collagen array spots after 24 hours in polarizing conditions was determined by immunofluorescence staining for Arginase-1 (green) and iNOS (red) (representative of n=3 array spots, scale bar=100 μm). (b) Bone tissue ECM and collagen were hydrated and injected subcutaneously into wild type C57BL/6 mice. Implants were harvested after one week for immunofluorescence staining for iNOS (green) and F4/80 (red) (representative of 4 images from n=2 animals). Scale bars=100 μm. Heat map representation of the effect of 2D tissue ECM substrates on macrophage gene expression. Primary mouse bone marrow derived macrophages were cultured on 2D bone, cardiac, liver, lung, or spleen ECM and compared to uncoated polystyrene in M0 (non-polarized), M1 (LPS + IFNγ), and M2 (IL4) cytokine environments for 24 hours. Relative gene expression of IL1β, iNOS TNFα, Arg1, Fizz1, and IL10 was determined via qRT-PCR, and is displayed as ΔΔCt over the tissue culture plastic (TCP) control in each media condition (triplicate wells of n = 3 macrophage isolations). Hierarchical clustering analysis was conducted for each cytokine environment. Macrophage gene expression was analyzed by one-way ANOVA with a post-hoc Tukey test, and these statistical results are provided in Supplementary Table 1.