Microbiological and Cellular Evaluation of a Fluorine-Phosphorus-Doped Titanium Alloy, a Novel Antibacterial and Osteostimulatory Biomaterial with Potential Applications in Orthopedic Surgery

Abstract

Joint prosthesis failure is mainly related to aseptic loosening and prosthetic joint infections, both of which are associated with high morbidity and substantial costs for patients and health systems. The development of a biomaterial that is capable of stimulating bone growth while minimizing bacterial adhesion would reduce the incidence of prosthetic failure. We report antibacterial and osteostimulatory effects in a novel fluorine-phosphorus (F-P)-doped TiO₂ oxide film grown on Ti-6Al-4V alloy with a nanostructure of bottle-shaped nanotubes (bNT) using five bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia) and MCT3T3-E1 osteoblastic cells. The interaction between the bacteria and bNT Ti-6Al-4V was complex, as the adhesion of four bacterial species decreased (two staphylococcus species, E. coli, and S. maltophilia), and the viability of staphylococci and S. maltophilia also decreased because of the aluminum (Al) released by bNT Ti-6Al-4V. This released Al can be recruited by the bacteria through siderophores and was retained only by the Gram-negative bacteria tested. P. aeruginosa showed higher adhesion on bNT Ti-6Al-4V than on chemically polished (CP) samples of Ti-6Al-4V alloy and an ability to mobilize Al from bNT Ti-6Al-4V. The cell adhesion and proliferation of MCT3T3-E1 osteoblastic cells significantly increased at 48 and 168 h, as did the matrix mineralization of these cells and the gene expression levels of three of the most important markers related to bone differentiation. According to our results, the bNT Ti-6Al-4V alloy could have clinical application, preventing infection and stimulating bone growth and thus preventing the two main causes of joint prosthesis failure.IMPORTANCE This work evaluates F-P-doped bNT Ti-6Al-4V from microbiological and cellular approaches. The bacterial results highlight that the antibacterial ability of bNT Ti-6Al-4V is the result of a combination of antiadhesive and bactericidal effects exerted by Al released from the alloy. The cell results highlight that F-P bNT Ti-6Al-4V alloy increases osseointegration due to modification of the chemical composition of the alloy resulting from P incorporation and not due to the nanostructure, as reported previously. A key finding was the detection of Al release from inside the bNT Ti-6Al-4V nanostructures, a result of the nanostructure growth during the anodizing process that is in part responsible for its bactericidal effect.

Keywords: Ti-6Al-4V alloy; antibacterial; fluorine; joint prosthesis; osteostimulation; phosphorus.

FIG 1

Al released from bNT Ti-6Al-4V over time with (black points) and without (gray points) DFX. The bar represents the standard deviation.

FIG 2

Adhered bacteria on each material, stained using a Live/Dead BacLight kit. Green indicates viable bacteria, and red indicates dead bacteria. Magnification, ×400.

FIG 3

Results of the experiments to measure bacterial adhesion of the different bacterial species tested: S. aureus 15981, S. epidermidis ATCC 35984, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. maltophilia 13637. (a) Amount of surface covered. (b) Percent viability of adhered bacteria. (c) Abundance of bacteria in solution after incubation. (d) Concentration of Al released from each alloy in the presence of planktonic bacteria (PB) and of Al not recruited by planktonic bacteria (filtered planktonic bacteria, FPB). The bar represents the interquartile range. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by a Wilcoxon test for differences in results between CP Ti-6Al-4V and bNT Ti-6Al-4V).

FIG 4

Neutralization of the bactericidal effect of Al released from bNT Ti-6Al-4V on S. aureus 15981 and S. maltophilia ATCC 13637. (a) Percent viability of adhered bacteria. (b) Abundance of bacteria in solution after incubation. (c) Concentration of Al released from each alloy in the presence of planktonic bacteria (PB) and of Al not recruited by planktonic bacteria (filtered planktonic bacteria, FPB). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (all, Wilcoxon test for differences in results between CP Ti-6Al-4V and bNT Ti-6Al-4V); †, P < 0.001 (Wilcoxon test for differences in results between bNT Ti-6Al-4V and bNT Ti-6Al-4V+DFX).

FIG 5

Results of Al uptake in different siderophore genotypes of P. aeruginosa. The bar represents the interquartile range. The structures below the graph represent the following: 1, pyoverdine chemical structure from P. aeruginosa ATCC 27853 (65); 2, pyoverdine chemical structure from P. aeruginosa ATCC 15962 (65); 3, pyochelin chemical structure from P. aeruginosa (92). Molecules shown in gray cannot be produced by the strains. **, P < 0.01 (Wilcoxon test for differences in results between CP Ti-6Al-4V and bNT Ti-6Al-4V).

FIG 6

Results of Al mobilization and Al recruitment by P. aeruginosa ATCC 27853 in planktonic (a) and adhered (b) bacteria after 24 h of incubation on bNT Ti-6Al-4V. The bar represents the interquartile range. *, P < 0.05 (Wilcoxon test for differences in results between CP Ti-6Al-4V and bNT Ti-6Al-4V).

FIG 7

MCT3T3-E1 adhesion at 6 h (a), with images for CP Ti-6Al-4V (b) and bNT Ti-6Al-4V (c) samples. The bar represents the standard deviation. ***, P < 0.001 (Wilcoxon test for differences in results between CP Ti-6Al-4V and bNT Ti-6Al-4V).

FIG 8

MCT3T3-E1 proliferation at 48 and 168 h (a), matrix mineralization at 120 h of culture (b), and gene expression of angiogenic (VEGF) and osteoblastic (BGLAP and RunX2) markers (c) at 120 h in the presence of CP Ti-6Al-4V and bNT Ti-6Al-4V. The bar represents the standard deviation. *, P < 0.05 (Wilcoxon test for differences in results between CP Ti-6Al-4V and bNT Ti-6Al-4V).