Electrostatic self-assembly approach in the deposition of bio-functional chitosan-based layers enriched with caffeic acid on Ti-6Al-7Nb alloys by alternate immersion

Highlights

• Electrostatic self-assembly resulted in homogeneous and stable coatings.

• Biopolymers-based layers exhibited an improved corrosion resistance.

• Coatings with CA deposited on Ti-6Al-7Nb showed good biocompatibility.

• CSM/AL_CA/CSM showed bacteriostatic effect towards Gram-(−) bacteria.

• Layers with free CA were the most active than these with chemically conjugated CA.

Abstract

Tailoring surface properties by layer-by-layer (LBL) deposition directed on the construction of complex multilayer coatings with nanoscale precision enables the development of novel structures and devices with desired functional properties (i.e., osseointegration, bactericidal activity, biocorrosion protection). Herein, electrostatic self-assembly was applied to fabricate biopolymer-based coatings involving chitosan (CSM) and alginate (AL) enriched with caffeic acid (CA) on Ti-6Al-7Nb alloyed surfaces. The method of CA grafting onto the chitosan backbone (CA-g-CSM) as well as all used reagents for implant functionalization were chosen as green and sustainable approach. The final procedure of surface modification of the Ti-6Al-7Nb alloy consists of three steps: (i) chemical treatment in Piranha solution, (ii) plasma chemical-activation of the Ti alloy surface in a RF CVD (Radio Frequency Chemical Vapour Deposition) reactor using Ar, O₂ and NH₃ gaseous precursors, and (iii) a multi-step deposition of bio-functional coatings via dip-coating method. Corrosion tests have revealed that the resulting chitosan-based coatings, also these involving CA, block the specimen surface and hinder corrosion of titanium alloy. Furthermore, the antioxidant layers are characterized by beneficial level of roughness (R_a up ca. 350 nm) and moderate hydrophilicity (59°) with the dispersion part of conducive surface energy ca. 30 mJ/m². Noteworthy, all coatings are biocompatible as the intact morphology of cultured eukaryotic cells ensured proper growth and proliferation, while exhibit bacteriostatic character, particularly in contact with Gram-(−) bacteria (E. coli). The study indicates that the applied simple sustainable strategy has contributed significantly to obtaining homogeneous, stable, and biocompatible while antibacterial biopolymer-based coatings.

Introduction

Titanium alloys are promising materials in bioengineering and in general in the production of implants for orthopedics [1], [2]. In the last decades, lots of studies were focused on technologies based on the optimization of both chemical and phase composition of titanium-based alloys. However, these were mainly focused on solving existing problems through the appropriate selection of alloy's composing elements. In line with this approach, in the most popular Ti-6Al-4V titanium alloys, vanadium was replaced with other metallic elements, including Ta, Mo, Zr, Sn and Nb, which are non-allergic and non-toxic [3]. The replacement of vanadium, a cytotoxic metal causing allergic and adverse reactions in the human body [4], with niobium results in a more secure alternative to the Ti-6Al-4V alloys resulting in Ti-6Al-7Nb alloys. Nb also guarantees the stabilization of the β phase of the alloy by its miscibility with titanium [5] and does not cause possible inflammation and allergic reactions, thus, a superior biocompatibility is provided [6]. Unfortunately, the presence of Al in titanium-based alloys can cause Alzheimer's disease and inhibit bone growth [7]. Propitiously, the high corrosion resistance, resulting from the affinity of passive metals such as Ti towards oxygen and the formation of inherent thin oxide layers on their surfaces, protects the implant from the biological environment, further corrosion, and from an uncontrolled release of potentially toxic metal ions [8], [9]. Furthermore, various available techniques of surface modification can be applied not only to prevent the release of toxic metals but also to impart totally new surface functionalities (i.e., physicochemical and biological activity). For instance, among many other one can indicate 3D printing, grit-blasting, acid-etching, plasma-spraying, and anodization, all with currently proven clinical efficacy [4], [10], [11]. Noteworthy, such surface treatment approaches are also a conceivable answer to the huge worrying problem in implantology – a possible development of bacterial biofilms and the resulting associated infections. Both have negative impacts on patient's health and usually generate additional costs and/or the need of an additional surgery.

The reactions between the implant surface and the body tissues depend on many functional properties of the alloy, including its mechanical parameters and the granted biological activity of the surface modifications. In this regard, anti-inflammatory and antibacterial drugs have also been incorporated on the implant surface during the implantation process [12], [13], [14]. Most of the methods of surface functionalization are based on mechanical treatments (e.g., machining, grinding, shot peening) or chemical treatments (e.g., plasma etching) that can cause an increase on the surface roughness and guarantee the activation of the surface for further functionalization steps. Furthermore, both physical and chemical methods such as thermal spraying, magnetron sputtering physical vapor deposition and plasma-enhanced chemical vapor deposition techniques result in more hydrophilic substrates [15], [16]. This allows for designing a totally new surface chemistry on the implants, which in many cases is also determined by the nature of the resulting thin layers. The latter idea is realized not only by the choice of the biopolymers (e.g., chitosan (CSM), alginate (AL)) on the substrates but as well by applying different concentrations of additional molecules (e.g., drugs, metal nanoparticles) in their hybrid structures. Moreover, a huge variety of novel functionalities is offered by dynamic or long-term interactions between the film components, solvents, and solutes, and then finally by the release and biological properties of the incorporated molecules.

Construction of multilayer coatings based on layer-by-layer (LbL) self-assembly enables the development of novel structures on the surface of medical devices, among other metal alloys. The practicality and versatility of this approach ensure functionalities with desired physicochemical properties tailored by controlling the molecular arrangement having nanoscale precision. The thickness can be controlled by the appropriate selection of factors known to influence film thickness in immersion assembly, including ionic strength, pH, hydrophobicity, and charge density [17]. Thus, nanoassembly by alternate immersion of charged substrates into solutions of different adsorbates is the typical procedure for the deposition of polyelectrolyte biopolymers, including those previously functionalized. For instance, antioxidant-biomacromolecule conjugates can be employed as new food additives, packaging materials, wound dressings [18], [19], but can also be proposed for the functionalization of biodevices or implants. Phenolic compounds extracted from plants, such as caffeic acid, gallic acid, catechin, tannic acid, ferulic acid, and quercetin have been recently successfully grafted to biopolymers and also various biomacromolecules, for example CSM and its derivatives, gelatin, AL, and inulin [20]. The synthesized antioxidant-polymer conjugates exhibit diverse bioactivities including antioxidant character [18], [21], [22], anticancer [23], [24], [25], [26], antibacterial and anti-biofilm [27], [28], [29], antiviral [30], and allow the functionalization of various surfaces [31], [32].

In this work, a multi-step approach involving electrostatic self-assembly of biopolymer-based films deposited on pre-activated Ti-6Al-7Nb alloys by alternate immersion was proposed. Layer-by layer self-assembly of opposite charge molecules is simple, inexpensive, and prospective method with control over a final coating chemistry, along with reaching to a favourable surface morphology. In particular, such desired parameters as adequate wettability, roughness, and chemical composition can be easily achieved with satisfactory effects. The activation of the Ti-6Al-7Nb surface was performed using a chemical etching process in Piranha solution followed by a plasmochemical treatment in a plasma reactor. Then, bio-functional coatings: i) CSM, ii) CA-g-CSM (CSM conjugated with CA), and iii) CSM/AL_CA/CSM (successive layers of CSM, CA mixed with AL, and CSM) were deposited via the immersion technique. Two different approaches based on CA conjugation onto CSM chains and simple CA combination with AL resulting in CA-g-CSM and CSM/AL_CA/CSM functionalizations, respectively, were introduced for a comparative study of the biological response of the covalently inserted and free CA towards both prokaryotic and eukaryotic cells in vitro. In addition, the influence of all the resulting surface modifications on the different surface parameters such as roughness, hydrophilicity, surface energy, delamination, and as well on the biocompatibility and bacteriostatic/bactericidal activity were studied in detail.