Antibacterial activity and cytocompatibility of an implant coating consisting of TiO2 nanotubes combined with a GL13K antimicrobial peptide

Abstract

Prevention of implant-associated infections at an early stage of surgery is highly desirable for the long-term efficacy of implants in dentistry and orthopedics. Infection prophylaxis using conventional antibiotics is becoming less effective due to the development of bacteria resistant to multiple antibiotics. An ideal strategy to conquer bacterial infections is the local delivery of antibacterial agents. Therefore, antimicrobial peptide (AMP) eluting coatings on implant surfaces is a promising alternative. In this study, the feasibility of utilizing TiO₂ nanotubes (TNTs), processed using anodization, as carriers to deliver a candidate AMP on titanium surfaces for the prevention of implant-associated infections is assessed. The broad-spectrum GL13K (GKIIKLKASLKLL-CONH2) AMP derived from human parotid secretory protein was selected and immobilized to TNTs using a simple soaking technique. Field emission scanning electron microscope, X-ray diffraction, Fourier transform infrared spectroscopy, and liquid chromatography-mass spectrometry analyses confirmed the successful immobilization of GL13K to anatase TNTs. The drug-loaded coatings demonstrated a sustained and slow drug release profile in vitro and eradicated the growth of Fusobacterium nucleatum and Porphyromonas gingivalis within 5 days of culture, as assessed by disk-diffusion assay. The GL13K-immobilized TNT (GL13K-TNT) coating demonstrated greater biocompatibility, compared with a coating produced by incubating TNTs with equimolar concentrations of metronidazole. GL13K-TNTs produced no observable cytotoxicity to preosteoblastic cells (MC3T3-E1). The coating may also have an immune regulatory effect, in support of rapid osseointegration around implants. Therefore, the combination of TNTs and AMP GL13K may achieve simultaneous antimicrobial and osteoconductive activities.

Keywords: antimicrobial peptide; nanotubes; orthopedic infections; titanium.

Figure 1

FESEM micrographs of TNTs after annealing (A) including a high-magnification inset showing the pore diameter of 80 nm and cross-sectional view showing a length of approximately 2 µm; (B) MNA-TNTs showing the decoration of the TNTs with MNA flakes both in top view and in cross-sectional view; (C) GL13K-TNTs both in top view and in cross-sectional view. Abbreviations: FESEM, field emission scanning electron microscope; MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO₂ nanotubes.

Figure 2

XRD pattern of TNTs (A) before and (B) after annealing at 450°C. Abbreviations: TNTs, TiO₂ nanotubes; XRD, X-ray diffraction.

Figure 3

FTIR spectra of (A) TNTs with no absorption wave; (B) MNA-TNTs with a wave number at 1,534.5 cm⁻¹ attributed to the −NO₂ peak from MNA; (C) GL13K-TNTs with amide peaks at 1,630 and 1,540 cm⁻¹ from GL13K. Abbreviations: FTIR, Fourier transform infrared spectroscopy; MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO₂ nanotubes.

Figure 4

Release kinetics of MNA and GL13K from TNTs. Notes: (A) Cumulative drug release over square root of time; (B) cumulative release of MNA and GL13K are close to the linear regression with square root of time from 30 min. Abbreviations: MNA, metronidazole; TNTs, TiO₂ nanotubes.

Figure 5

Evaluation of antimicrobial activity of the MNA-TNTs, GL13K-TNTs, and TNTs against (A) Porphyromonas gingivalis ATCC 33277 and (B) Fusobacterium nucleatum ATCC 25586, assessed using disk-diffusion assay. Abbreviations: MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO₂ nanotubes; ATCC, American Type Culture Collection.

Figure 6

Fluorescence images of MC3T3-E1 cells incubated for 4 h on (A) TNTs, (B) MNA-TNTs, and (C) GL13K-TNTs. (D) Cell adhesion measured by counting the cell nuclei. *Statistical significance (P<0.05, n=3). Magnification: 100×. Abbreviations: MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO₂ nanotubes.

Figure 7

MC3T3-E1 cells on samples. Notes: (A) Absorbance indicating the proliferation of MC3T3-E1 cells cultured on samples for 24, 72, and 120 h. (B) Normalized ALP activities indicating cell differentiation in samples at 7 and 14 days. *Statistical significance (P<0.05, n=3). Abbreviations: ALP, alkaline phosphatase; d, days; h, hours; MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO₂ nanotubes.

Figure 8

Fluorochrome micrography of MC3T3-E1 cells cultured for 24 and 48 h on (A, D) TNTs, (B, E) MNA-TNTs, and (C, F) GL13K-TNTs (magnification =×320; scale bar =100 µm). Actin is shown in red and cell nuclei are shown in blue. On MNA-TNTs, some cells spread poorly with less polygonal and elongated cell shapes. Abbreviations: MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO₂ nanotubes.

Figure 9

(A) CCK-8 assay used to evaluate macrophage proliferation on samples, *statistical significance (P<0.05, n=3). SEM images of macrophages incubated on (B) TNTs, (C) MNA-TNTs, and (D) GL13K-TNTs for 4 h. Abbreviations: CCK-8, Cell Counting Kit-8; h, hours; MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO₂ nanotubes.