Atomic layer deposition of nano-TiO2 thin films with enhanced biocompatibility and antimicrobial activity for orthopedic implants

Abstract

Titanium (Ti) and its alloys have been extensively used as implant materials in orthopedic applications. Nevertheless, implants may fail due to a lack of osseointegration and/or infection. The aim of this in vitro study was to endow an implant surface with favorable biological properties by the dual modification of surface chemistry and nanostructured topography. The application of a nanostructured titanium dioxide (TiO₂) coating on Ti-based implants has been proposed as a potential way to enhance tissue-implant interactions while inhibiting bacterial colonization simultaneously due to its chemical stability, biocompatibility, and antimicrobial properties. In this paper, temperature-controlled atomic layer deposition (ALD) was introduced for the first time to provide unique nanostructured TiO₂ coatings on Ti substrates. The effect of nano-TiO₂ coatings with different morphology and structure on human osteoblast and fibroblast functions and bacterial activities was investigated. In vitro results indicated that the TiO₂ coating stimulated osteoblast adhesion and proliferation while suppressing fibroblast adhesion and proliferation compared to uncoated materials. In addition, the introduction of nano-TiO₂ coatings was shown to inhibit gram-positive bacteria (Staphylococcus aureus), gram-negative bacteria (Escherichia coli), and antibiotic-resistant bacteria (methicillin-resistant Staphylococcus aureus), all without resorting to the use of antibiotics. Our results suggest that the increase in nanoscale roughness and greater surface hydrophilicity (surface energy) together could contribute to increased protein adsorption selectively, which may affect the cellular and bacterial activities. It was found that ALD-grown TiO₂-coated samples with a moderate surface energy at 38.79 mJ/m² showed relatively promising antibacterial properties and desirable cellular functions. The ALD technique provides a novel and effective strategy to produce TiO₂ coatings with delicate control of surface nanotopography and surface energy to enhance the interfacial biocompatibility and mitigate bacterial infection, and could potentially be used for improving numerous orthopedic implants.

Keywords: antimicrobial activity; atomic layer deposition; fibroblast; nanostructure; osteoblast; titanium dioxide.

Figure 1

SEM images of (A) Ti control, (B) Ti-TiO₂ (190°C), (C) Ti-TiO₂ (160°C), and (D) Ti-TiO₂ (120°C) and (E) EDAX spectra of Ti samples. Scale bars are 200 nm. Abbreviations: O, oxygen; SEM, scanning electron microscopy; Ti, titanium; TiO₂, titanium dioxide.

Figure 2

AFM images and RMS roughness values (in nm) of (A) Ti control, (B) Ti-TiO₂ (190°C), (C) Ti-TiO₂ (160°C), and (D) Ti-TiO₂ (120°C). Abbreviations: RMS, root-mean-square; Ti, titanium; TiO₂, titanium dioxide; AFM, atomic force microscopy.

Figure 3

XRD patterns of the Ti samples with different TiO₂ coatings. Abbreviations: A, anatase; Ti, titanium; TiO₂, titanium dioxide; XRD, X-ray diffraction.

Figure 4

(A) S. aureus, (B) E. coli, and (C) MRSA growth on Ti samples with different TiO₂ coatings after 24 hours of culture. Data represent mean ± SD, N=3. *p<0.05 compared with Ti control (labeled as Ti). Abbreviations: S. aureus, Staphylococcus aureus; E. coli, Escherichia coli; MRSA, methicillin-resistant Staphylococcus aureus; Ti, titanium; TiO₂, titanium dioxide; CFU, colony-forming unit.

Figure 5

Fluorescent micrographs of S. aureus cultured for 2 hours and 24 hours on Ti and Ti-TiO₂ (160°C) samples. SYTO^® 9 and propidium iodide were used to stain live (green) and dead (red) bacteria cells, respectively. Scale bars are 100 μm. Abbreviations: S. aureus, Staphylococcus aureus; Ti, titanium; TiO₂, titanium dioxide.

Figure 6

Fluorescent micrographs of E. coli cultured for 2 hours and 24 hours on Ti and Ti-TiO₂ (160°C) samples. SYTO^® 9 and propidium iodide were used to stain live (green) and dead (red) bacteria cells, respectively. Scale bars are 100 μm. Abbreviations: E. coli, Escherichia coli; Ti, titanium; TiO₂, titanium dioxide.

Figure 7

Fluorescent micrographs of MRSA cultured for 2 hours and 24 hours on Ti and Ti-TiO₂ (160°C) samples. SYTO^® 9 and propidium iodide were used to stain live (green) and dead (red) bacteria cells, respectively. Scale bars are 50 μm. Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; Ti, titanium; TiO₂, titanium dioxide.

Figure 8

The amount of adsorbed casein protein and total proteins on sample surfaces after 24 hours of culture in a 1.7% casein solution and 3% TSB medium. Data represent mean ± SD, N=3. *p<0.05 compared with Ti control (labeled as Ti) in the same tested solution. Abbreviations: TSB, tryptic soy broth; Ti, titanium; TiO₂, titanium dioxide.

Figure 9

Osteoblast adhesion on Ti samples with different TiO₂ coatings after 4 hours of culture. Data represent mean ± SD, N=3. *p<0.05 compared with Ti control (labeled as Ti). Abbreviations: Ti, titanium; TiO₂, titanium dioxide.

Figure 10

Osteoblast proliferation on Ti and Ti-TiO₂ (160°C) samples. Data represent mean ± SD. N=3. *p<0.05 compared with Ti at the same time period, **p<0.05 compared with the same sample (Day 1). Abbreviations: Ti, titanium; TiO₂, titanium dioxide.

Figure 11

Fibroblast adhesion and growth on Ti and Ti-TiO₂ samples. Data represent mean ± SD. *p<0.05 compared with Ti control at the same time period, **p<0.05 compared with the same sample (4 hours). Abbreviations: Ti, titanium; TiO₂, titanium dioxide.

Figure 12

(A) Osteoblast and (B) fibroblast cell density (after 4 hours of culture) was directly proportional to the surface energy on Ti controls and Ti-TiO₂ samples. Error bars represent SD. Abbreviations: Ti, titanium; TiO₂, titanium dioxide.