2.2 Macrophage polarization

Raw264.7 cells were seeded on the surface of each sample (1×10⁴ cells/well). After 1, 3, and 9 days, macrophages were fixed with the Gluta fixative, dehydrated with graded ethanol, ion sputtered, and then observed using the FE-SEM (Hitachi S-4800, Japan). For each group of samples, 5 fields of view were selected at 3, 6, 9, and 12 o’clock in the center and around the samples. The spread area and longest diameter of the macrophages were measured using ImageJ software. In addition, F-actin was stained using phalloidin-Alexa Fluor 488 (1:1,000; C2201S, Beyotime, China) and incubated for 30 min, followed by incubation in mounting medium with DAPI (ab104139, Abcam, United States) and was visualized using the Olympus FV3000 confocal microscope. The cell proliferation activity of the three groups of samples was detected using the CCK-8 method.

After 7 days, the expression of M1 markers (Mhc2, Inos, and Il-6) and M2 markers (Cd163 and Cd206) was evaluated using qRT-PCR. Primer sequences are shown in Supplementary Table S1. The TNF-α level in the cell culture was detected using an ELISA kit (EK0527, Boster, China). Meanwhile, the expression of M1 markers (iNOS and CD86) and M2 markers (CD163, CD206, and Arg-1) was evaluated using Western blot. Antibodies and dilutions included anti-CD86 (1:200; ab112490, Abcam, United States), anti-iNOS (1:1,000; ab178945, Abcam, United States), anti-CD206 (1:1,000; ab64693, Abcam, United States), anti-CD163 (1:1,000; ab182422, Abcam, United States), and anti-ARG1 (1:5,000; ab233548, Abcam, United States). The anti-β-actin antibody (1:5,000, ab6276, Abcam, United States) was used for quantifying the loading amount of the sample. In addition, the expression of M2 polarization markers (CD206 and CD163) was observed by immunofluorescence. Antibodies and dilutions included anti-CD206 (1:500; ab64693, Abcam, United States) and anti-CD163 (1:1,000; ab182422, Abcam, United States). All stained samples were visualized using the Olympus FV3000 confocal microscope.