Antifungal activity of Ag:hydroxyapatite thin films synthesized by pulsed laser deposition on Ti and Ti modified by TiO2 nanotubes substrates

doi:10.1016/j.apsusc.2013.12.029

Applied Surface Science

Volume 293, 28 February 2014, Pages 37-45

https://doi.org/10.1016/j.apsusc.2013.12.029 Get rights and content

Highlights

•
Antifungal action of Ag doped HA thin layers.
•
Benefic effect of TiO₂ nanotubes modified substrates on antimicrobial activity of deposited layers.
•
Non-cytotoxic activity of PLD HA thin films containing 0.53 ± 0.1 wt.% Ag.

Abstract

Hydroxyapatite (HA) is a widely used biomaterial for implant thin films, largely recognized for its excellent capability to chemically bond to hard tissue inducing the osteogenesis without immune response from human tissues. Nowadays, intense research efforts are focused on development of antimicrobial HA doped thin films. In particular, HA doped with Ag (Ag:HA) is expected to inhibit the attachment of microbes and contamination of metallic implant surface. We herewith report on nano-sized HA and Ag:HA thin films synthesized by pulsed laser deposition on pure Ti and Ti modified with 100 nm diameter TiO₂ nanotubes (fabricated by anodization of Ti plates) substrates. The HA-based thin films were characterized by SEM, AFM, EDS, FTIR, and XRD. The cytotoxic activity was tested with HEp2 cells against controls. The antifungal efficiency of the deposited layers was tested against the Candida albicans and Aspergillus niger strains. The Ti substrates modified with TiO₂ nanotubes covered with Ag:HA thin films showed the highest antifungal activity.

Introduction

Hydroxyapatite [HA, Ca₁₀(PO₄)₆(OH)₂] bioceramic thin layers as coatings for metallic medical implants, which were demonstrated to promote the formation of bone-like apatite on their surface improving the implant fixation and promoting the bone in-growth [1], [2], [3], [4], [5], still remain a research topic of great interest.

As a metal substrate for regenerative medicine applications, such as orthopedic or dental implants, titanium (Ti) is widely preferred due to its desirable properties, such as suitable elastic modulus and mass density, good mechanical strength, corrosion resistance, biocompatibility and low toxicity [2], [6], [7], [8]. However, Ti exhibits a rather poor bioactivity and in order to establish a direct chemical bonding between the implant and the host bone tissue its adequate surface modification was suggested using various routes [9]. One approach to enhance the bioactivity and improve the bone response to implant surface is by depositing bioactive HA thin films on Ti implant surface.

Recently, nanotubular titanium dioxide (TiO₂) layers have attracted an increased attention in comparison to pure Ti due to their ability to enhance the bonding strength between HA coating and metallic substrate [10], [11], [12]. The nanotubes fabrication by electrochemical anodic oxidation of pure Ti in fluoride-containing electrolytes provides strongly adherent layers which are used for various applications, based upon their semiconductive and biocompatible nature [12].

A challenging task in biomedicine is the prevention of microbial infections which determine the loosening of implants from the bone [8], [13]. A promising strategy to prevent the initial microbial adhesion and colonization of biofilms is via introducing antimicrobial bioactive thin films onto implant's surface. Some previous studies have shown that Ag⁺ ions have a broad spectrum of antimicrobial and antifungal properties [14] while maintaining a low cytotoxicity [4]. The Ag⁺ ions are able to penetrate the microbial cell wall and bind to DNA, and thus interfering with the replication process [15], [16], [17]. Currently, special interest is focused on the development of HA coatings doped with silver to minimize microbes’ adhesion [18].

There are various methods to deposit ceramic thin films on metal surfaces, such as plasma spraying [19], magnetron sputtering [20], pulsed laser deposition [1], [21], sol–gel [22], electrochemical or electrophoretic deposition [23], [24], [25]. Pulsed laser deposition (PLD) is a technique that has proved efficient in the fabrication of calcium phosphate films on metallic substrates with excellent coating attachment [1]. Moreover, by a proper choice of the ablation and deposition parameters, it is possible to control the stoichiometry and crystallinity of a wide range of complex materials deposited at room temperature (RT) [1], [2].

We herewith report on the physical–chemical characterization of HA and Ag doped HA [Ca_9.95Ag_0.05(PO₄)₆(OH)₂] thin films, with Ag content of 0.53 ± 0.1 wt.%, synthesized by PLD on pure Ti and Ti modified by TiO₂ nanotubes substrates. The antifungal efficiency of the novel Ag:HA/TiO₂ nanotubes structures was tested against two pathogenic, largely spread, fungal strains: Candida albicans and Aspergillus niger. The specific aim of this manuscript is the identification of the most suitable deposition substrates and optimal processing conditions.

Section snippets

Preparation of Ti modified by TiO₂ nanotubes

Ti plates of 20 mm × 10 mm × 0.25 mm (99.7% purity, Sigma Aldrich) were used as substrates for the growth of nanotubes titanium oxide film. The substrates were degreased in acetone and ethanol in an ultrasonic bath for 30 min and rinsed with deionized water. The anodization was performed under constant mixing in a two-electrode cell. The Ti plate was the working electrode and a Pt plate was used as a counter electrode at a distance of 15 mm in 0.4 wt.% HF solution. The experiments were carried out by

Morphology of Ti plates modified by TiO₂ nanotubes

As known [12], TiO₂ nanotubes fabricated by anodization method consist of patterned, strongly adherent nanostructures with an increased surface area compared to planar Ti. Such nanoscale morphology plays a pivotal role as the bone in-growth takes place preferentially in pores.

Recently, it was demonstrated for a wide variety of materials that nanostructured surfaces are enhancing the osteoblast cellular functions, improving the osteointegration response [28], [29], [30]. Superficial

Conclusions

We have applied PLD to assemble HA and Ag:HA thin films on the surface of pure Ti and Ti modified by TiO₂ nanotubes substrates.

A quasi-stoichiometric target-to-substrate transfer was ascertained by EDS, whilst the restoration of the crystalline status after post-deposition heat-treatment performed at 500 °C in water vapors for 6 h was confirmed by the FTIR and XRD analyses. Thus, benefic effects on the long term stability of these films in biological fluids should be expected. We have shown by

Acknowledgements

This research was financed by the Ministry of Education, Science and Technological Development, Republic of Serbia, under contract No. III – 45019.

C.R., L.D., N.S., A.V. and I.N.M. acknowledge with thanks the financial support of UEFISCDI under the contract ID 304/2011.

G.E.S. gratefully acknowledges the support of UEFISCDI under contract TE49/2011.

All authors are thankfull to Dr. M.C. Chifiriuc and Dr. C. Bleotu for cytotoxicity tests of samples.

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Antifungal activity of Ag:hydroxyapatite thin films synthesized by pulsed laser deposition on Ti and Ti modified by TiO2 nanotubes substrates

Highlights

Abstract

Introduction

Section snippets

Preparation of Ti modified by TiO2 nanotubes

Morphology of Ti plates modified by TiO2 nanotubes

Conclusions

Acknowledgements

Int. J. Biol. Macromol.

Acta Biomater.

Mater. Chem. Phys.

Surf. Coat. Technol.

Surf. Coat. Technol.

Curr. Opin. Solid State Mater. Sci.

Electrochim. Acta

Acta Biomater.

Acta Biomater.

Nanomedicine

Mater. Sci. Eng. C

Acta Biomater.

Biomaterials

Nanomedicine

Thin Solid Films

Colloids Surf. A

Prog. Org. Coat.

J. Dent.

Biomaterials

Acta Biomater.

Thin Solid Films

Mater. Sci. Eng. C

Antifungal activity of Ag:hydroxyapatite thin films synthesized by pulsed laser deposition on Ti and Ti modified by TiO₂ nanotubes substrates

Preparation of Ti modified by TiO₂ nanotubes

Morphology of Ti plates modified by TiO₂ nanotubes