Fig. 4: The preparation and gradient properties of the Ti surface with a gradient in AMP density along the vertical direction.

a Schematic diagram depicting the preparation of the AMP-functionalized gradient Ti surface. A Ti substrate, chemically modified with silane-PEG2000-MAL, was first vertically oriented in an FITC-labeled thiolated AMP solution with a concentration of m (m = 1, 20, or 50 μM). Then, the solution phase was allowed to evaporate in a fume hood for 210 min. AMP was conjugated to the immersed section of the Ti substrate by a click reaction between double bonds in MAL and thiol groups in thiolated AMP. During the evaporation, the solution level was reduced to increase the concentration of the solution phase. As a result, a lower section of the Ti substrate would have been exposed to the AMP solution with a higher concentration and for a longer time and thus be modified with more AMP, making AMP density increasing vertically downward and generating a vertical gradient in the AMP density. The resultant gradient surface was denoted as Ti-Grad-mAMP, in which m represents the concentration of the AMP solution. For convenience, the surface was artificially divided into ten bands, numbered 1–10 from top to bottom, with a band width of 1 mm. Thus, it is expected that the AMP density is increasing from band 1 to 10. b The FITC fluorescence images of Ti-Grad-20AMP. The images of the ten bands were collected individually and lined up as they were originally on the Ti substrate (n = 3, scale bar, 200 μm). c The MFI and d the AMP density (calculated by the fluorescence method¹¹) of each band of Ti-Grad-20AMP. In each band, we randomly selected three points to calculate the MFI (n = 3). e Peak area of N1s high-resolution spectra of each band on Ti-Grad-20AMP, confirming the increase in the AMP density from band 1 to 10 (n = 4). Data are displayed as mean ± SD and analyzed by GraphPad Prism software.