Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis

Abstract

Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility.

Figure 1

(A) Gene expressions of P. gingivalis (16s RNA) and S. mutans (glucosyltransferase, in short “gtf ”) in the biofilm, **P ≤ 0.01, ***P ≤ 0.001; (B) SEM micrographs and DAPI images of S. mutans and P. gingivalis on surfaces of Ti (a, c, e and g) and Ti-Cu alloy (b,d,f and h) after co-culture for 24 h.

Figure 2

Fluorescent images and 3-D representations of S. mutans and P. gingivalis biofilms on surfaces of Ti and Ti-Cu alloy after incubation at 37 °C for 24 h, thickness of S. mutans biofilm on Ti is 40 μm, and not quantifiable on Ti-Cu alloy, while thickness of P. gingivalis biofilm on Ti is 36 μm, and that on Ti-Cu alloy is 18 μm.

Figure 3

TEM micrographs of inner structures of S. mutans and P. gingivalis, (a–d) treated with Ti; (e–h) treated with Ti-Cu alloy. White and black arrows indicate peptidoglycan layer and cytoplasmic membrane, and red arrows indicate separation of the cell membrane from the cell wall and the release of cellular contents.

Figure 4. Cumulative Cu²⁺ concentration curve released from Ti-Cu alloy in 0.9% NaCl solution at 37 °C.

Figure 5. Viabilities of rBMSCs determined by measurement of the optical density (absorbance at 450 nm) in CCK-8 assay.

Figure 6. Fluorescent images of rBMSCs cultured on the surface of Ti and Ti-Cu alloy for 4 h and 24 h with actin stained with Rhodamine Phalloidin (red) and nuclei stained with DAPI (blue).

Figure 7. Schematic representation of the hypothetical scenario for the antibacterial mechanism of Cu²⁺ from Ti-Cu alloy.