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. 2019 Nov 1:4:346-357.
doi: 10.1016/j.bioactmat.2019.10.006. eCollection 2019 Dec.

Micro/nano-structured TiO2 surface with dual-functional antibacterial effects for biomedical applications

Xiang Ge  1   2 , Chengzu Ren  1 , Yonghui Ding  3   4 , Guang Chen  1 , Xiong Lu  5 , Kefeng Wang  6 , Fuzeng Ren  7 , Meng Yang  2 , Zhuochen Wang  8 , Junlan Li  1 , Xinxin An  9 , Bao Qian  10 , Yang Leng  2
Affiliations

Micro/nano-structured TiO2 surface with dual-functional antibacterial effects for biomedical applications

Xiang Ge et al. Bioact Mater. .

Abstract

Implant-associated infections are generally difficult to cure owing to the bacterial antibiotic resistance which is attributed to the widespread usage of antibiotics. Given the global threat and increasing influence of antibiotic resistance, there is an urgent demand to explore novel antibacterial strategies other than using antibiotics. Recently, using a certain surface topography to provide a more persistent antibacterial solution attracts more and more attention. However, the clinical application of biomimetic nano-pillar array is not satisfactory, mainly because its antibacterial ability against Gram-positive strain is not good enough. Thus, the pillar array should be equipped with other antibacterial agents to fulfill the bacteriostatic and bactericidal requirements of clinical application. Here, we designed a novel model substrate which was a combination of periodic micro/nano-pillar array and TiO2 for basically understanding the topographical bacteriostatic effects of periodic micro/nano-pillar array and the photocatalytic bactericidal activity of TiO2. Such innovation may potentially exert the synergistic effects by integrating the persistent topographical antibacterial activity and the non-invasive X-ray induced photocatalytic antibacterial property of TiO2 to combat against antibiotic-resistant implant-associated infections. First, to separately verify the topographical antibacterial activity of TiO2 periodic micro/nano-pillar array, we systematically investigated its effects on bacterial adhesion, growth, proliferation, and viability in the dark without involving the photocatalysis of TiO2. The pillar array with sub-micron motif size can significantly inhibit the adhesion, growth, and proliferation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Such antibacterial ability is mainly attributed to a spatial confinement size-effect and limited contact area availability generated by the special topography of pillar array. Moreover, the pillar array is not lethal to S. aureus and E. coli in 24 h. Then, the X-ray induced photocatalytic antibacterial property of TiO2 periodic micro/nano-pillar array in vitro and in vivo will be systematically studied in a future work. This study could shed light on the direction of surface topography design for future medical implants to combat against antibiotic-resistant implant-associated infections without using antibiotics.

Keywords: Micro/nano-structured surface; Non-invasive treatment; Photocatalytic bactericidal property; Titanium dioxide; Topographical bacteriostatic activity.

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Figures

Image 1
Graphical abstract
Scheme 1
Scheme 1
Schematic illustration for the photocatalytic bactericidal process of a bacterium on TiO2 surface. (a) An intact bacterium; (b) Bacterial membrane permeability will be affected by an organic material oxidization effect generated from the TiO2 photocatalysis, but this permeability change is reversible. The arrows indicate the sites where the permeability has been changed; (c) All cell wall layers will be further destroyed, which will make the bacterium leak small molecules and ions (like K+). Bacterial damage will be irreversible from this stage; (d) Leakage of higher molecular weight components such as RNA and protein; (e) Degradation of bacterial internal components such as nucleoid; (f) Finally, the bacterium will be completely mineralized to H2O, CO2, and minerals.
Fig. 1
Fig. 1
Schematic illustration for the preparation processes of Si pillar arrays (Si scaffold) and TiO2 pillar arrays (Si scaffold + TiO2 thin film).
Fig. 2
Fig. 2
(a) A single chip of Si scaffold which consists of ten areas including one flat area and nine patterned areas; (b) Every two areas are insulated by a fence of which width is 4 μm and height is the same as that of pillar (3 μm). Pillar array motif size = pillar width (W) = pillar length (L) = spacing (S) between two pillars; (c) SEM angular views of each area.
Fig. 3
Fig. 3
(a) Si scaffold with ten areas including one flat area and nine patterned areas; (b) Si scaffold coated with a TiO2 thin film; (c) Morphology of a flat Si substrate ( × 50,000, scale bar = 100 nm); (d) Morphology of a TiO2 thin film on a flat Si substrate ( × 50,000, scale bar = 100 nm); (e) Morphology of the side-wall of a Si pillar ( × 50,000, scale bar = 100 nm); (f) Morphology of the side-wall of a Si pillar coated with a TiO2 thin film ( × 50,000, scale bar = 100 nm); (g) Cross-section view of a TiO2 thin film shows that the film thickness is around 52 nm ( × 150,000, scale bar = 100 nm); (h) XPS spectra indicate that the thin film (either on the pillar array or on the flat surface) contains the components of Ti and O; (i) TF-XRD patterns confirm that the crystalline phase of the thin film (either on the pillar array or on the flat surface) is rutile; (j) WCAs of one flat and nine patterned TiO2 areas. Error bar = SD. Inset: illustrating the measuring method of WCA which is smaller than 90°.
Fig. 4
Fig. 4
Characteristics of S. aureus and E. coli after culturing for 30 min on the TiO2 periodic micro/nano-pillar arrays. (a) Typical CLSM images of S. aureus on one flat area and nine patterned areas, respectively; (b) Quantitative statistical analysis of relative S. aureus occupied area percentage which is normalized by that on TiO2_Flat. Error bar = SEM; (c) Typical CLSM images of E. coli on one flat area and nine patterned areas, respectively; (d) Quantitative statistical analysis of relative E. coli occupied area percentage which is normalized by that on TiO2_Flat. Error bar = SEM. One asterisk (*) indicates significant difference at p < 0.05, two asterisks (**) indicate significant difference at p < 0.01 in t-tests.
Fig. 5
Fig. 5
Characteristics of S. aureus and E. coli after culturing for 12 h on the TiO2 periodic micro/nano-pillar arrays. (a) Typical CLSM images of S. aureus on one flat area and nine patterned areas, respectively; (b) Quantitative statistical analysis of relative S. aureus occupied area percentage which is normalized by that on TiO2_Flat. Error bar = SEM; (c) Typical CLSM images of E. coli on one flat area and nine patterned areas, respectively; (d) Quantitative statistical analysis of relative E. coli occupied area percentage which is normalized by that on TiO2_Flat. Error bar = SEM. One asterisk (*) indicates significant difference at p < 0.05, two asterisks (**) indicate significant difference at p < 0.01 in t-tests.
Fig. 6
Fig. 6
Characteristics of S. aureus and E. coli after culturing for 24 h on the TiO2 periodic micro/nano-pillar arrays. (a) Typical CLSM images of S. aureus on one flat area and nine patterned areas, respectively; (b) Quantitative statistical analysis of relative S. aureus occupied area percentage which is normalized by that on TiO2_Flat. Error bar = SEM; (c) Typical CLSM images of E. coli on one flat area and nine patterned areas, respectively; (d) Quantitative statistical analysis of relative E. coli occupied area percentage which is normalized by that on TiO2_Flat. Error bar = SEM. One asterisk (*) indicates significant difference at p < 0.05, two asterisks (**) indicate significant difference at p < 0.01 in t-tests.
Fig. 7
Fig. 7
Antibacterial mechanism of periodic micro/nano-pillar array (S. aureus). S. aureus on a flat surface: (a) (b) (c) SEM top views (with false color) corresponding to bacteria at Phase 1, 2, and 3. The arrows in (b) indicate binary fission positions. The hexagon in (c) illustrates the shape of a bacterium in the middle of a close-packed bacterial colony. S. aureus in a pillar array: (d) (e) (f) SEM top views (with false color) corresponding to bacteria at Phase 1, 2, and 3. The arrows in (e) and (f) indicate binary fission positions. SEM actual observation proves that the pillar array could inhibit the normal elongation & binary fission (e) and colonization (f) of S. aureus.
Fig. 8
Fig. 8
Antibacterial mechanism of periodic micro/nano-pillar array (E. coli). E. coli on a flat surface: (a) (b) (c) Hypothesis models corresponding to a bacterium at Phase 1, 2, and 3; (a’) (b’) (c’) SEM top views (with false color) corresponding to a bacterium at Phase 1, 2, and 3. The arrow in (c’) indicates a binary fission position. E. coli in a pillar array: (d) (e) (f) Hypothesis models corresponding to a bacterium at Phase 1, 2, and 3; (d’) (e’) (f’) SEM top views (with false color) corresponding to a bacterium at Phase 1, 2, and 3. SEM actual observation proves that the pillar array could inhibit the normal elongation (e’) and binary fission (f’) of E. coli as predicted by the hypothesis models in (e) and (f), respectively.
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