Antibacterial and Osteogenic Functionalization of Titanium With Silicon/Copper-Doped High-Energy Shot Peening-Assisted Micro-Arc Oxidation Technique

Abstract

Antibacterial and osteogenic functionalization of titanium (Ti) implants will greatly expand their clinical indications in immediate implant therapy, accelerate osteointegration, and enhance long-term prognosis. We had recently shown that the high-energy shot peening (HESP)-assisted micro-arc oxidation (MAO) significantly improved the bioactivity and coating stability of Ti-based substrates. In this study, we further functionalized Ti with antibacterial and osteogenic properties by doping silicon (Si) and/or copper (Cu) ions into HESP/MAO-treated coatings. Physicochemical characterization displayed that the doping of Si and Cu in HESP/MAO-treated coatings (Si/Cu-MAO) did not significantly change their surface topography, roughness, crystal structure, coating thickness, bonding strength, and wettability. The results of X-ray photoelectron spectroscopy (XPS) showed that Si and Cu in the Si/Cu-MAO coating was in the form of silicate radical (SiO₃ ^2-) and bivalent copper (Cu²⁺), respectively. The total amounts of Si and Cu were about 13.5 and 5.8 μg/cm², which released about 33.2 and 31.3% within 14 day, respectively. Compared with the control group (MAO), Si doping samples (MAO-Si) significantly increased the cell viability, alkaline phosphatase (ALP) activity, mineralization and osteogenic genes (ALP, collagen I and osteocalcin) expression of MC3T3-E1 cells. Furthermore, the addition of Cu presented good bactericidal property against both Staphylococcus aureus and Streptococcus mutans (even under the co-culture condition of bacteria and MC3T3-E1 cells): the bacteriostatic rate of both bacteria was over 95%. In conclusion, the novel bioactive Si/Cu-MAO coating with antibacterial and osteogenic properties is a promising functionalization method for orthopedic and dental implants, especially in the immediate implant treatment with an infected socket.

Keywords: antibacterial; high-energy shot peening; micro-arc oxidation; osteogenesis; titanium.

FIGURE 1

(A) Cell viability of MC3T3-E1 cells on Si-loaded specimens [MAO, Si1, Si2, and Si3 (Si-MAO)] at 4 day. (B) cell viability of MC3T3-E1 cells on Si/Cu-loaded substrates [Si-MAO, Si-Cu1, Si-Cu1 (Si/Cu-MAO), and Si-Cu3] after 4 day. Error bars represent mean ± SEM for n = 6, *p < 0.05.

FIGURE 2

(A) SEM images of MAO, Si-MAO, and Si/Cu-MAO samples (top and sectional views); statistics of coating thickness (B), surface roughness (C), and critical loads (Lc, D) of MAO, Si-MAO, and Si/Cu-MAO substrates.

FIGURE 3

(A) XPS pattern and the deconvoluted Si2p/Cu2p of Si/Cu-MAO samples; XRD patterns (B) and water contact angles (C) images of MAO, Si-MAO, and Si/Cu-MAO substrates; (D) release profile of Si and Cu from Si/Cu-MAO specimens at 1, 4, 7, 10, and 14 day.

FIGURE 4

(A) Bacteriostasis rate of MAO, Si-MAO, and Si/Cu-MAO samples against S. aureus and S. mutans at 24 h. Error bars represent mean ± SEM for n = 6, **p < 0.01; (B) SEM images of two bacteria on MAO, Si-MAO, and Si/Cu-MAO substrates at 24 h (red arrows represent dead bacteria).

FIGURE 5

3D fluorescent images (Live/Dead staining, A) and dead/total rate (B) of two bacteria on MAO, Si-MAO, and Si/Cu-MAO substrates at 24 h. Error bars represent mean ± SEM for n = 6, **p < 0.01.

FIGURE 6

(A) SEM images of MC3T3-E1 cells on MAO, Si-MAO, and Si/Cu-MAO samples (red arrows represent pseudopodia of MC3T3-E1 cells); (B) cell viability of MC3T3-E1 cells on MAO, Si-MAO, and Si/Cu-MAO substrates at 4 and 7 day. Error bars represent mean ± SEM for n = 6, *p < 0.05, **p < 0.01.

FIGURE 7

ALP activity (A) and mineralization level (B) of MC3T3-E1 cells on MAO, Si-MAO, and Si/Cu-MAO substrates at 4, 7, and/or 14 day; (C) osteogenic gene expression of MC3T3-E1 cells on MAO, Si-MAO, and Si/Cu-MAO substrates at 7 day. Error bars represent mean ± SEM for n = 6, *p < 0.05, **p < 0.01.

FIGURE 8

(A) SEM (red arrows: spreading MC3T3-E1 cells) and Live/Dead staining (red color: dead MC3T3-E1cells; green color: living MC3T3-E1cells) images of co-cultured MC3T3-E1 cells and S. mutans on MAO, Si-MAO, and Si/Cu-MAO substrates at 24 h; ALP activity (B,C) osteogenic gene expression of co-cultured MC3T3-E1 cells on MAO, Si-MAO, and Si/Cu-MAO substrates at 7 day. Error bars represent mean ± SEM for n = 6, **p < 0.01.