Research ArticleMATERIALS SCIENCE

Regulation of macrophage polarization through surface topography design to facilitate implant-to-bone osteointegration

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Science Advances  02 Apr 2021:
Vol. 7, no. 14, eabf6654
DOI: 10.1126/sciadv.abf6654
  • Fig. 1 Surface characterizations of TiO2 honeycomb–like nanostructures and the morphological changes of macrophages on corresponding surfaces.

    (A) Surface morphologies of different samples under SEM; Scale bars, 10 μm (first panel) and 1 μm (second panel). (B) Diameter distribution of different TiO2 honeycombs. (C) XRD patterns of different samples. a.u., arbitrary units. (D) Contact angles of different samples. (E) Cell morphologies of macrophages on different samples; Scale bars, 10 μm (first panel), 5 μm (second panel) and 1 μm (third panel). (F) Quantitative cell spreading area based on SEM images. (G) Quantitative cell aspect ratio based on SEM images. n = 3 independent experiments per group; *P < 0.05 and **P < 0.01.

  • Fig. 2 In vitro immune responses of macrophages on various nanostructures.

    (A) Fluorescence microscopy images of CD206, iNOS, and nucleic staining of macrophages on different samples. (B) Quantitative fluorescence intensity of CD206 and iNOS of macrophages on different samples. (C) Quantitative cell nucleus aspect ratio based on fluorescence microscopy images. (D) Polarization of macrophages was evaluated by the expressions of CCR7 (M1) and CD206 (M2) using flow cytometry. (E) Relative mRNA expression levels of M1 macrophage–related genes IL-1β and TNF-α. (F) Relative mRNA expression level of M2 macrophage–related genes IL-4 and IL-10. (G) Relative mRNA expression level of osteogenic-related genes BMP-2 and OSM. (H) ELISA analyses of proinflammatory cytokines IL-1β and TNF-α. (I) ELISA analyses of anti-inflammatory cytokines IL-4 and IL-10. (J) ELISA analyses of pro-osteogenic cytokines BMP-2 and OSM. n = 3 independent experiments per group; *P < 0.05 and **P < 0.01.

  • Fig. 3 In vitro osteogenic differentiation behaviors of MSCs on various nanostructures.

    (A) ALP activity of MSCs cultured on different samples without macrophage cytokines. (B) ALP activity of MSCs cultured on different samples with macrophage cytokines collected from corresponding samples. (C) Relative expression level of ALP, Runx2, and OCN of MSCs cultured on different samples with macrophage cytokines collected from corresponding samples. (D) Optical images of Alizarin Red staining of MSCs cultured on different samples with macrophage cytokines collected from corresponding samples. Photo credit: Y.Z., The University of Hong Kong. (E) Quantitative analysis of Alizarin Red staining. n = 3 independent experiments per group; *P < 0.05 and **P < 0.01.

  • Fig. 4 In vivo implant-to-bone osteointegration of HC-90 and HC-5000 surfaces.

    (A) Protein absorption on implant surfaces after 8 hours of surgery. (B) 2D and 3D micro-CT images of tissue at the site of implantation. (C) Quantitative analysis of the newly formed bone volume around the implants (BV/TV). (D) Van Gieson’s picrofuchsin staining of tissue around the implants. (E) Bone-implant contact ratio. (F) Representative immunohistochemical images of CD146, Runx2, and BMP-2 in defect areas, and semiquantification of positively stained cells. (G) Representative immunohistochemistry staining of CD68, CD163, and iNOS in defect areas and semiquantification of positively stained cells. n = 3 independent experiments per group; *P < 0.05 and **P < 0.01.

  • Fig. 5 Bioinformatic analysis of macrophage gene expression on various nanostructures.

    (A) Heatmap of Pearson correlation between samples. (B) Venn diagram of the number of differentially expressed genes in different TiO2 honeycombs compared with unpatterned Ti. (C to H) Volcano plot of transcriptomic analysis of differentially expressed genes. n = 3 independent experiments per group.

  • Fig. 6 Mechanistic analysis of macrophage polarization induced by TiO2 honeycomb–like nanostructures.

    (A) GO analysis of all genes in macrophages cultured on HC-90 versus unpatterned Ti. (B) Enriched KEGG pathways of HC-90 versus unpatterned Ti. TH17, T helper 17; IgA, immunoglobulin A; RIG I, retinoic acid-inducible gene I, NF-κB, nuclear factor κB. (C) Heatmap analysis of differentially expressed genes involved in cell adhesion, cytoskeleton arrangement, and mechanotransduction. (D) Scheme illustration of the mechanism of macrophage polarization on the nanostructures. n = 3 independent experiments per group.

Supplementary Materials

  • Supplementary Materials

    Regulation of macrophage polarization through surface topography design to facilitate implant-to-bone osteointegration

    Yizhou Zhu, Hang Liang, Xiangmei Liu, Jun Wu, Cao Yang, Tak Man Wong, Kenny Y. H. Kwan, Kenneth M. C. Cheung, Shuilin Wu, Kelvin W. K. Yeung

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