F-doped TiO2 microporous coating on titanium with enhanced antibacterial and osteogenic activities

Abstract

To enhance bacterial resistance and osteogenesis of titanium (Ti) -based implants, TiO₂/calcium-phosphate coatings (TiCP) doped with various amounts of fluorine (F) (designated as TiCP-F1, TiCP-F6, and TiCP-F9) were prepared on Ti by micro-arc oxidation. The F doped TiCP coatings possess a microporous structure (pore size of 3-4 μm in average diameter) which is evenly covered by nano-grains of 30-60 nm in size. Successful F incorporation into TiCP was determined by X-ray photoelectron spectroscopy, and it shows weak influence on the microstructure, phase compositions, surface roughness and wettability of TiCP. All the coatings bonded firmly to the Ti substrates and showed enduring high adhesion strength in biological circumstances. The bacterial resistance and osteogenesis of the coatings were evaluated by implanting testing materials in vitro and in an infected rabbit model caused by bacteria. Both the in vitro and in vivo results indicated that TiCP and TiCP-F1 were of much higher osteogenic activity compared with Ti but lacking of bacterial resistance, whereas TiCP with high F addition (TiCP-F6 and TiCP-F9) exhibited both dramatically improved bacterial resistance and osteogenesis. In summary, TiCP-F6 possessed the best antibacterial and osteogenic activities, especially exhibited excellent osseointegration efficacy in the infected rabbit model.

Figure 1

(A) SEM images of TiCP, TiCP-F1, TiCP-F6, and TiCP-F9 with EDX pattern and higher magnification image inserted, (B) Cross-sectional morphology and elemental profiles of TiCP-F9, (C) XRD patterns of the coatings.

Figure 2

XPS spectrum (A) and high-resolution spectra of Ti2p (B), O1s (C), Ca2p (D), P2p (E), and F1s (F) detected from the surface of TiCP-F9.

Figure 3

Cumulative release profiles of (A) F, (B) Ca, and (C) P from the coatings into physiological saline solutions, and (D) adhesion strength of the coatings before and after immersion in physiological saline solutions of different durations.

Figure 4

Antibacterial rates of the coatings as well as Ti after immersion in PBS for 1, 14 and 28 days against (A) E. coli and (B) S. aureus; Fluorescence images of adhered (C) E. coli and (D) S. aureus on the coatings as well as Ti after immersed PBS for 28 days, the dead bacteria appear red while the live ones are green. SEM images of (E) E. coli and (F) S. aureus incubated for 12 h on Ti and the coatings after immersed PBS for 28 days, with the corresponding higher magnification images of the circled regions inserted. Data are presented as the means ± SD, n = 4. ***p < 0.001 compared to Ti; ^†††p < 0.001 compared to TiCP; ^§§§p < 0.001 compared to TiCP-F1.

Figure 5

(A) Protein adsorption onto the coatings as well as Ti after 24 h of immersion in a-MEM containing 10% FBS, (B) LDH amount released by MSCs after 3 days of incubation, (C) MSCs adhesion measured by CCK-8 after 1, 5, and 24 h of culture, (D) MSCs proliferation measured by CCK-8 after 3, 7, and 14 days of culture, (E) SEM images of MSCs after culturing for 3 days on the coatings as well as Ti. Data are presented as mean ± SD, n = 5. *p < 0.05 and **p < 0.01 compared to Ti; ^†p < 0.05 and ^††p < 0.01 compared to TiCP; ^§p < 0.05 and ^§§p < 0.01 compared to TiCP-F1; ^&p < 0.05 and ^&&p < 0.01 compared to TiCP-F6.

Figure 6

(A) Osteogenesis related gene expression of Runx2, BSP, ALP, OPN, Col-I, and OCN, (B) intracellular protein expression of OPN and OCN as well as ALP activity, and quantitative results of collagen secretion (C) and ECM mineralization (D) of MSCs incubated on the coatings as well as Ti for 3, 7 and 14 days. Data are presented as the means ± SD, n = 4. **p < 0.01 and ***p < 0.001 compared to Ti; ^†p < 0.05 and ^††p < 0.01 compared to TiCP; ^§p < 0.05 and ^§§p < 0.01 compared to TiCP-F1; ^&p < 0.05 and ^&&p < 0.01 compared to TiCP-F6.

Figure 7

(A) Counting of the dislodged adhered bacteria after Ti and the coated wires rolling over SBA. (B) Amount of CFU in the wires surrounding femurs, quantified in pulverized bone from operated femur. (C) Histological observations of the implant/bone interface after 8 weeks of implantation in the infected rabbit model, where the tissues stained in red are the newly formed bone. (D) Percentage of bone-to-implant contact (BIC) and (E) pull-out force of the coated wires as well as Ti after 8 weeks of implantation. Data are presented as the means ± SD, n = 4. *p < 0.05 and **p < 0.01 compared to Ti; ^†p < 0.05, ^††p < 0.01 and ^†††p < 0.001 compared to TiCP; ^§p < 0.05, ^§§p < 0.01 and ^§§§p < 0.001 compared to TiCP-F1; ^&p < 0.05 and ^&&p < 0.01 compared to TiCP-F6; ^##p < 0.01 compared to TiCP-F9.