Inhibited bacterial biofilm formation and improved osteogenic activity on gentamicin-loaded titania nanotubes with various diameters

Wen-tao Lin, Hong-lue Tan, Zhao-ling Duan, Bing Yue, Rui Ma, Guo He, Ting-ting Tang, Wen-tao Lin, Hong-lue Tan, Zhao-ling Duan, Bing Yue, Rui Ma, Guo He, Ting-ting Tang

Abstract

Titania nanotubes loaded with antibiotics can deliver a high concentration of antibiotics locally at a specific site, thereby providing a promising strategy to prevent implant-associated infections. In this study we have fabricated titania nanotubes with various diameters (80, 120, 160, and 200 nm) and 200 nm length via electrochemical anodization. These nanotubes were loaded with 2 mg of gentamicin using a lyophilization method and vacuum drying. A standard strain, Staphylococcus epidermidis (American Type Culture Collection 35984), and two clinical isolates, S. aureus 376 and S. epidermidis 389, were selected to investigate the anti-infective ability of the gentamicin-loaded nanotubes (NT-G). Flat titanium (FlatTi) and nanotubes with no drug loading (NT) were also investigated and compared. We found that NT-G could significantly inhibit bacterial adhesion and biofilm formation compared to FlatTi or NT, and the NT-G with 160 nm and 200 nm diameters had stronger antibacterial activity because of the extended drug release time of NT-G with larger diameters. The NT also exhibited greater antibacterial ability than the FlatTi, while nanotubes with 80 nm or 120 nm diameters had better effects. Furthermore, human marrow derived mesenchymal stem cells were used to evaluate the effect of nanotubular topographies on the osteogenic differentiation of mesenchymal stem cells. Our results showed that NT-G and NT, especially those with 80 nm diameters, significantly promoted cell attachment, proliferation, spreading, and osteogenic differentiation when compared to FlatTi, and there was no significant difference between NT-G and NT with the same diameter. Therefore, nanotube modification and gentamicin loading can significantly improve the antibacterial ability and osteogenic activity of orthopedic implants.

Keywords: bacteria adhesion; biofilm formation; gentamicin; osteogenic activity; titania nanotubes.

Figures

Figure 1
Figure 1
Morphological characterization of different diameter nanotubular surfaces using scanning electron microscopy (first row) and drug-loaded nanotubular surfaces (second row). Notes: The scanning electron microscopy images show that the tube diameters were 80, 120, 160, and 200 nm, respectively. The surfaces of the drug-loaded nanotubes retain the nanotubular structure. The magnification level is ×30,000. The scale bar is 1 μm. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes.
Figure 2
Figure 2
Loading efficiency and drug release profiles of gentamicin. Notes: (A) Loading efficiency of gentamicin in different diameter nanotubes. *Denotes a significant difference compared to NT-G80 or NT-G120 (P<0.05). (B) Cumulative drug release profiles from different diameter nanotubes loaded with 2 mg of gentamicin, expressed in μg/mL. After a high initial release, the amount of gentamicin eluted from the nanotubes was nearly constant. A large proportion of the gentamicin was not eluted from the nanotubes. Abbreviation: NT-G, gentamicin-loaded nanotubes.
Figure 3
Figure 3
The number of viable bacteria adhered on the flat titanium, nanotubes with no drug loading, and gentamicin-loaded nanotubes surfaces at 4 hours. Notes: (A) The number of viable bacteria was counted and normalized to the counts from the flat titanium (FlatTi) control for each bacterial strain. *Denotes a significant difference compared to FlatTi (P<0.01). #Denotes a significant difference compared to NT80, NT120, NT160, or NT200 (P<0.01). $Denotes a significant difference compared to NT160 or NT200 (P<0.01). The data are representative of the results from three independent experiments and are expressed as mean ± standard deviation. (B) Representative images of bacteria adhered to the surfaces of various specimens after 4 hours of incubation. The antibacterial properties of the specimens (1) FlatTi, (2) NT80, (3) NT120, (4) NT160, (5) NT200, (6) NT-G80, (7) NT-G120, (8) NT-G160, (9) NT-G200 against (a) American Type Culture Collection 35984, (b) Staphylococcus aureus 376 and (c) S. epidermidis 389. Abbreviations: NT, nanotubes; CFU, colony forming unit; NT-G, gentamicin-loaded nanotubes.
Figure 4
Figure 4
Biofilm formation of the three bacterial strains. Notes: (A) American Type Culture Collection 35984, (B) Staphylococcus aureus 376, and (C) S. epidermidis 389 on flat titanium (FlatTi), nanotubes with no drug loading (NT), and gentamicin-loaded nanotubes (NT-G) surfaces at 24 hours and 48 hours, as detected by the tissue culture plate method. *Denotes a significant difference compared to FlatTi (P<0.01). #Denotes a significant difference compared to NT80, NT120, NT160, and NT200 (P<0.01). $Denotes a significant difference compared to NT160 and NT200 (P<0.01). **Denotes a significant difference compared to NT-G80 and NT-G200 (P<0.01). The data are representative of the results from three independent experiments and are expressed as mean ± standard deviation.
Figure 5
Figure 5
Scanning electron microscope images of American Type Culture Collection 35984 adhesion and biofilm formation on different surfaces. Notes: (1) Flat titanium, (2) NT80, (3) NT120, (4) NT160, (5) NT200, (6) NT-G80, (7) NT-G120, (8) NT-G160, and (9) NT-G200 after (a) 4 hours, (b) 24 hours, and (c) 48 hours incubation. The magnification level is ×3,000. The scale bar is 10 μm. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes.
Figure 6
Figure 6
Confocal laser scanning microscopy analysis of bacterial viability on different surfaces. Notes: (1) Flat titanium, (2) NT80, (3) NT120, (4) NT160, (5) NT200, (6) NT-G80, (7) NT-G120, (8) NT-G160, and (9) NT-G200 incubated with American Type Culture Collection 35984 for (a) 4 hours, (b) 24 hours, and (c) 48 hours. The bacteria were stained with green fluorescent SYTO 9 and red fluorescent propidium iodide which resulted in the live cells appearing green and the dead cells appearing red under confocal laser scanning microscopy. The magnification level is ×400. The scale bar is 50 μm. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes.
Figure 7
Figure 7
Attachment and proliferation of the human marrow derived mesenchymal stem cells on the surfaces of various specimens. Notes: (A) Cell adhesion measured by the colorimetric MTT assay. *Denotes a significant difference compared to fiat titanium (P<0.01). #Denotes a significant difference compared to NT160 and NT200 (P<0.05), and $denotes a significant difference compared to NT-G160 and NT-G200 (P<0.05). (B) Cells stained with DAPI after 12 hours of culturing. The magnification level is ×100. The scale bar is 200 μm. (C) Cell proliferation on various specimens. *Denotes a significant difference compared to flat titanium (P<0.01). #Denotes a significant difference compared to NT160 and NT200 (P<0.01), $denotes a significant difference compared to NT-G160 and NT-G200 (P<0.01), ##denotes a significant difference compared to NT200 (P<0.05), and $$denotes a significant difference compared to NT-G200 (P<0.05). The data are representative of the results from three independent experiments and are expressed as mean ± standard deviation. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes; FlatTi, flat titanium.
Figure 8
Figure 8
Representative images of the human marrow derived mesenchymal stem cells stained with rhodamine phalloidin for the actin filaments (red) and nuclei counterstained with DAPI (blue). Notes: Shown are the cytoskeletal morphologies of the cells on the surfaces of the flat titanium, nanotubes with no drug loading, and gentamicin-loaded nanotubes. The cells on the nanotubular surfaces displayed polygonal and clustering morphology, while those on the flat titanium surface exhibited a spindle and spherical morphology. The scale bar is 50 μm. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes; FlatTi, flat titanium.
Figure 9
Figure 9
The alkaline phosphatase activity assay and alkaline phosphatase staining. Notes: (A) Relative alkaline phosphatase (ALP) activity of the human marrow derived mesenchymal stem cells (hMSCs) after culturing for 7, 10, and 14 days. The ALP activity was normalized by the total protein amounts. *Denotes a significant difference compared to flat titanium (P<0.01), #denotes a significant difference compared to NT160 and NT200 (P<0.01), **denotes a significant difference compared to NT200 (P<0.05), ##denotes a significant difference compared to NT-G200 (P<0.01), $denotes a significant difference compared to NT-G160 and NT-G200 (P<0.01), $$denotes a significant difference compared to NT-120 (P<0.01), and ***denotes a significant difference compared to NT-G120 (P<0.01). The data are representative of the results from three independent experiments and are expressed as mean ± standard deviation. (B) Representative images of ALP staining on (1) flat titanium, (2) NT80, (3) NT120, (4) NT160, (5) NT200, (6) NT-G80, (7) NT-G120, (8) NT-G160, and (9) NT-G200 after (a) 7 and (b) 14 days of culturing. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes; FlatTi, flat titanium.
Figure 10
Figure 10
Representative images of Alizarin Red staining on different surfaces. Notes: (1) Flat titanium, (2) NT80, (3) NT120, (4) NT160, (5) NT200, (6) NT-G80, (7) NT-G120, (8) NT-G160, and (9) NT-G200 after incubation for (a) 21 and (b) 28 days. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes.

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