Fig 1: Regulation of neutrophil migration and apoptosis and macrophage polarization by the hybrid biomaterial in vitro. A) Quantitative flow cytometric analysis of neutrophil migration after the different treatments. B) Representative fluorescence images and quantitative analysis of neutrophil (red) migration to the lower chamber of transwell. Scale bars, 50 µm. C) Representative flow cytometry plots and quantitative analysis of neutrophil apoptosis. D) Western blot analysis of apoptosis‐related proteins in neutrophils. E) Western blot analysis of the phenotypic markers in LPS‐stimulated macrophages. Proinflammatory macrophage marker, iNOS; anti‐inflammatory macrophage markers, CD206 and Arg‐1. F) The levels of cytokines released by macrophages measured by enzyme‐linked immunosorbent assay (ELISA). G) Representative fluorescence images of macrophage phenotypes after incubation with different pretreated neutrophils; iNOS (red), CD206 (green), and nuclei (blue). Scale bars, 50 µm. All data were generated from at least three independent experiments and are presented as the means ± standard deviation (SD). Statistical analysis was performed by one‐way ANOVA. ns, not significant; *P < 0.05 and **P < 0.01.
Fig 2: Inflammatory response realignment by Gel@fMLP/SiO2‐FasL in diabetic cutaneous wounds. A) Quantitative analysis of neutrophil infiltration in diabetic cutaneous defects was performed by flow cytometry. B) Representative fluorescence images and the percentage of neutrophils (Ly‐6G+ cells, green) in cutaneous defects. Scale bars, 50 µm. C) Representative flow cytometry plots and quantitative analysis of neutrophil apoptosis in cutaneous defects. D) Quantitative analysis of proinflammatory macrophages in cutaneous defects was performed by flow cytometry. E) Quantitative analysis of anti‐inflammatory macrophages in cutaneous defects was performed by flow cytometry. F) Representative fluorescence images of macrophage phenotypes in cutaneous defects. Scale bars, 50 µm. Quantitative analysis of iNOS+/CD206+ cells in each group. G) Cytokines expression in cutaneous defects was analyzed by ELISA. n = 6 per group. The data are presented as the means ± SD. In (A)–(G), statistical analysis was performed by one‐way ANOVA, except for TGF‐β expression (in (G)) which was analyzed by the Kruskal–Wallis H‐test. ns, not significant; *P < 0.05 and **P < 0.01.
Fig 3: The cABs ameliorate cutaneous inflammation and promote regeneration in vivo.(A) In vivo distribution of DiR-labeled MSNs, AB ghosts, and cABs in mice. Quantitative analysis of the fluorescence intensity around the skin defect (n = 3). (B) Representative images of cABs taken up by macrophages in vivo. Scale bars, 50 μm (top) and 15 μm (bottom). (C) The therapeutic design of the cutaneous defect mice (n = 6). (D) Representative photographs of full-thickness cutaneous wounds treated with different agents over time. Quantitative analysis of the wound closure rate in each group (n = 6). (E) Representative images of hematoxylin and eosin (H&E) staining of the dissected skin tissues in each group. Scale bars, 50 μm. (F) Representative fluorescence images of the macrophage phenotypes in skin. Scale bars, 50 μm. Quantitative analysis of the iNOS+/CD206+ cells in each group (n = 6). (G) Western blot analysis of the phenotype markers in skin tissue. (H) Representative images and the percentage of Ki-67–positive population in the healing skin tissues in each group (n = 6). Scale bars, 50 μm. (I) Representative images of cytokeratin 14 expression in the neoskin tissues in each group. Scale bars, 200 μm. Data are presented as means ± SD. **P < 0.01 and ***P < 0.001 by one-way ANOVA. Photo credit: Geng Dou, State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi Key Laboratory of Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, 710032, China.
Fig 4: Dual-targeting and inflammatory regulation ability of cABs.(A) The expression of adhesion molecules in ABs inherited from T cells. (B) Representative fluorescence images in the untreated/TNF-α–treated HUVECs after administration of MSNs/cABs. Scale bars, 100 μm. The fluorescence intensity of P-selectin/ICAM-1 and MSNs/cABs in each group is presented (n = 3). (C) Representative fluorescence images show PKH26-AB ghosts/RhB-MSNs/RhB-cABs taken up by F4/80-positive macrophages. Scale bars, 20 μm. (D) Time-dependent uptake of cABs by macrophages. Scale bars, 20 μm. (E) Cell-specific uptake of cABs by macrophages. Scale bars, 20 μm. (F) Representative images of cABs (red) engulfed by macrophages (not labeled)/FBs (green) in a coculture pattern. Scale bar, 50 μm. The right panel shows the high magnification of the representative images. Scale bar, 20 μm. (G) Representative fluorescence images of the macrophage phenotypes and the percentage of the iNOS/CD206-positive population (n = 3). Scale bars, 50 μm. (H) Western blot analysis of the phenotype markers of macrophages (n = 3). (I) Detection of the levels of immune factors in the supernatants (n = 3). The results are representative of the data generated by at least three independent experiments and are presented as means ± SD. ***P < 0.001 by one-way analysis of variance (ANOVA).
Fig 5: Treatment of cABs loaded with miR-21/curcumin promoted the polarization of macrophages towards M2 phenotype.(A) Real-time RT-PCR analysis of the expression of miR-21 in recipient cells after different treatments (n = 3). (B) Western blot analysis of the protein levels of PTEN and PDCD4 (n = 3). (C) Representative fluorescence images of the macrophages phenotypes after different treatments (n = 3). Scale bars, 50 µm. (D) Western blot analysis of phenotype markers in LPS-stimulated macrophages after different treatments (n = 3). M1 marker, iNOS; M2 markers, CD206 and Arg1. (E) The detection of the immune factors released by macrophages in different groups by ELISA (n = 3). The proinflammatory factors, TNF-a and IL-6; the anti-inflammatory factors, TGF-ß and IL-10. (F) Representative fluorescence images of the macrophages phenotypes after different treatments (n = 3). Scale bars, 50 µm. (G) Western blot analysis of the phenotype markers in LPS-stimulated macrophages after different treatments (n = 3). (H) The detection of the immune factors released by macrophages in different groups by ELISA (n = 3). All results are representative of the data generated in at least three independent experiments and are presented as means ± SD. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA.
Supplier Page from Abcam for Anti-iNOS antibody [EPR16635] (Alexa Fluor® 647)