Corrosion resistance and cytocompatibility studies of zinc-doped fluorohydroxyapatite nanocomposite coatings on titanium implant

Abstract

In this study, zinc-doped fluoridated hydroxyapatite (ZnFHA) nano-composite coatings were deposited on the surface of commercially pure titanium (Ti-cp) via electrolytic deposition technique. The phase composition, crystallite size, microstructure, and elemental composition of coated specimens were characterized using different techniques including Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy and energy dispersive X-ray analysis respectively. The influence of coatings on the corrosion behaviour of the specimens was determined by potentiodynamic polarisation in simulated body fluid. In vitro biocompatibility tests, MTT and alkaline phosphatase (ALP) were also employed to assess the cytocompatibility of ZnFHA coating with osteoblast-like MC3T3-E1 cells. Results demonstrated that zinc and fluoride were successfully incorporated into apatite lattice structure. Co-doping of Zn²⁺ and F⁻ ions decreased hydroxyapatite unit cell volume and grain sizes. Obtained coatings were totally crack-free and dense, which led to decrease in corrosion current densities of Ti-cp in physiological solutions. In addition, cytocompatibility tests demonstrated a good osteoblast proliferation and spreading on ZnFHA. More importantly, results showed that the Zn/F-doping can promote ALP expression, which is a probable effect of a combination of nanostructured surface effects and ion release (Zn²⁺ and F⁻). Overall, the ZnFHA coated Ti-cp possesses favourable corrosion resistance and cytocompatibility for the application as biomedical implant material.

Introduction

Fracture in the bone may cause a serious problem when its influence is on a large scale. Replacement of impaired bone is a main problem in orthopaedic operation. To proceed with such a process, we utilised metallic implants, such as commercially pure titanium (Ti-cp). Ti-cp has received considerable interest and success as a hard tissue implant because of its low density, high strength, cytocompatibility and superior corrosion resistance [1], [2], [3]. However, metallic implants act like foreign objects for the host body, and in some cases, they may be rejected. Moreover, these implants may not promote osteoconductive and osteoblastic phenomena in the host body. Fortunately, the Ti-cp implant osseointegration can be stimulated by surface treatments, such as sand blasting [4], acid etching [5], plasma electrolytic oxidation [6] and hydroxyapatite [HA, Ca₁₀(PO₄)₆(OH)₂] coatings [7]. Amongst these treatments, artificial HA coatings are widely employed to promote bone ingrowth because of their osteoconductivity and bioactivity. In this case, bioactivity may be regarded as the enhanced and chemical bonding of bone to the film surface, i.e., osteoinduction.

However, the low tensile strength, poor mechanical behaviour, relatively slow biological interaction rates and high dissolution rate of HA limit its application in the field of implant materials. Improvement of biological and physicochemical properties of HA can be achieved by doping with ions that are usually present in natural apatites of vertebrate skeletal systems [8]. A wide variety of anions, cations and functional groups, such as Na⁺ [9], Sr²⁺ [10], Zn²⁺ [11], Mn²⁺ [12], SiO₄⁴⁻ [13], F⁻ [14], Cl⁻ [15], and CO₃²⁻ [16], can be introduced into the single phase HA lattice. Amongst these substitutions, zinc and fluorine are widely considered as potential substituents because of their biological relevance [17]. Zn-containing HA has been developed to strengthen its osteoconductive characteristics that are induced by the pharmacological effect of Zn. Zn-HA coatings deposited using an electrochemical process improve the differentiation and proliferation of osteoblasts and can thus potentially increase implant osteointegration [18]. Zn²⁺-substituted HA was synthesised to investigate its microstructure, sintering behaviour, antimicrobial characteristics and in vivo biocompatibility [17]. Zn incorporation into implants was shown to stimulate bone formation around the implant and reduce inflammatory response [17]. Recent studies have shown that Zn-doped HA coating can release trace Zn²⁺ in the degradation process, which is favourable to improve cell proliferation [11], [17], [18].

HA coating suffers from comparatively high dissolution rate in the biological environment of the human body, which is detrimental for long-term implant stability [19]. Fluorine doping is frequently employed to enhance thermal stability and biological properties of HA. Fluorine equally acts as a good nucleation agent for apatite; this element promotes skeletal formation process [20]. Therefore, fluoridated hydroxyapatite [FHA, Ca₁₀(PO₄)₆(OH)_2–xF_x, where 0<x<2] was designed as a probable candidate for HA replacement in orthopaedic applications [14]. FHA shows high phase stability that is caused by OH⁻ replacement by F⁻, which leads to contraction in the a-axis without changing the c-axis. This process causes crystallinity and stability enhancement [19]. Fluoride also promotes osteoblast proliferation and differentiation. Moreover, this element has been applied in osteoporosis treatment [20]. A study reported that HA co-doping with zinc and fluoride improves mechanical and biological properties of HA particles [17]. However, to the best of our knowledge, the deposition of Zn²⁺ and F⁻ (Zn/F) co-doped HA (ZnFHA) coating on Ti-cp has not been sufficiently investigated. Natural bone is composed of nanostructured HA; therefore, the use of apatite coating with nanostructured structure would better mimic the configuration of the bone and also enhance implant biocompatibility. In this study, we used nanostructured ZnFHA coatings.

Numerous papers have reported on the different coating approaches of HA on metal substrates [21], such as electrochemical deposition, biomimetic coating, pulsed laser ablation, ion-beam-assisted deposition, sol–gel and plasma spraying process. Amongst these methods, plasma spraying is the only technique that is approved by the Food and Drug Administration [22]. However, this approach poses the following defects [22]. (1) The high temperature and rapid cooling that are associated with this process yield various chemical phases and lower HA crystallinity. This outcome is a problem as HA dissolution rates increase with decreasing crystallinity. (2) This technique cannot coat porous surfaces or include bioactive agents. In this sense, electrolytic deposition (ED) is a comparatively inexpensive, green and energy saving process, which allows to obtain controlled micro- and nanometric structures [9], [13]. ED is widely used in the processing of bio-ceramic coatings [13], with characteristics superior to conventional deposition techniques (i.e., low process temperature, cost-effective, simple apparatus requirement, probability of fabricating onto porous substrates of heterogeneous structure and complex shape as well as simple control of coating properties). A study reported Zn-substituted FHA on 316 L stainless steel via ED method at room temperature [23]. However, the cytocompatibility and corrosion mechanism of electrodeposited ZnFHA coating on Ti is not clearly elucidated. Hence, the present work is designed to achieve the ZnFHA coating on Ti-cp with improved corrosion resistance and cell–biomaterial interactions.

In this study, an ED technique was adopted to develop Zn-substituted FHA on Ti-cp. The surface morphological and elemental compositions, topographical and phase structural properties, ionic substitution level and thermal stability were assessed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDS). The corrosion resistance of ZnFHA coating was evaluated through potentiodynamic polarisation tests. Finally, cell assay was performed to assess the viability of cultured MC3T3-E1 osteoblasts seeded on pure and substituted HA substrates.