Surface characterization of different titanium surfaces. (A) SEM images of surface morphologies of cp-Ti, Ti-NW, and Ti-NW-Zn. Upper panel: ×5000 magnification. Lower panel: ×50000 magnification; (B) XPS wide scan spectra analysis of cp-Ti and Ti-NW-Zn surfaces; (C) XPS high-resolution spectra analysis of Zn 2p; (D) contact angles of cp-Ti, Ti-NW, and Ti-NW-Zn surfaces. Results were presented as mean ± SD (*p < 0.05, **p < 0.01; N = 3); (E) concentrations of Zn ion released from Ti-NW-Zn samples into PBS after 4 h and 1, 4, and 7 days; N = 3.

Angiogenesis of transgenic zebrafish in the group of control, cp-Ti, and Ti-NW-Zn.

Zebrafish fin amputation and regeneration test. (A) Effect of the control group and Ti-NW-Zn surface on osteogenesis; (B) effect of different concentrations of zinc ions on osteogenesis: 0, 1, and 2 ppm.

Survival rate in (A) embryos (N = 10) and (B) adult fish (N = 5).

(A) Proliferation of human umbilical vein endothelial cells HUVEC with different concentrations of zinc ions for 1, 2, and 3 days; (B) proliferation of osteoblast-like MC3T3-E1 cells for 1, 3, and 6 days. Results were presented as mean ± SD (*p < 0.05,**p < 0.01; N = 3).

Cell adhesion ability assay. The adhesion ability of MC3T3-E1 cells was analyzed by counting the stained nuclei with DAPI using a confocal laser scanning microscope (CLSM) after incubation. (A) CLSM images of the cells under different zinc ions concentrations at ×100 and ×200 magnification. (B) Statistical results for adhesive cell numbers (*p < 0.05,**p < 0.01; N = 3).

Cell adhesion ability assay. The adhesion ability of MC3T3-E1 cells was analyzed by counting the stained nuclei with DAPI using a confocal laser scanning microscope (CLSM) after incubation. (A) CLSM images of the cells under different concentrations of CM at ×100 and ×200 magnification. (B) Statistical results for adhesive cell numbers (*p < 0.05,**p < 0.01; N = 3).

Immunolocalization of FAK (red) and nuclei (blue). The merged images show that the treatment of 1 and 2 ppm zinc ions promoted FAK expression significantly. All images are displayed at ×200 magnification.

Immunolocalization of VCAM-1 (red) and nuclei (blue). The merged images show that the treatment of 1 and 2 ppm zinc ions promoted VCAM-1 expression significantly. All images are displayed at ×200 magnification.

Protein expression levels of osteogenic markers Runx2, OSX, and OCN of osteoblast-like MC3T3-E1 cells in different concentrations of CM (Runx2, Runt-related transcription factor 2; OSX, osterix; OCN, osteocalcin). Quantification was performed by ImageJ software. Results are presented as mean ± SD (*p < 0.05, **p < 0.01; N = 3).

Activation of MAPK/ERK signaling by different concentrations of CM. (A) Protein expressions of vital members for the MAPK/ERK signaling pathway in MC3T3-E1 cells treated with different concentrations of CM. (B) Protein expressions of vital members for the MAPK/ERK pathway in MC3T3-E1 cells exposed to CM of 2 ppm at different time points. (C) Histogram showing normalized ratios of p-ERK/ERK. Results are presented as mean ± SD (*p < 0.05,**p < 0.01; N = 3).