Electrodeposition of Chitosan on Ti-6Al-4V Surfaces: A Study of Process Parameters

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Introduction
Metal implants as bone-anchored prostheses have been employed since 1965 for dental treatments, and today they are also used in orthopedic applications 1,2 .This type of prosthesis offers multiple benefits that can be summarized in a better body image, comfort, and better mobility of the affected zone 3 .However, some problems arise in their use, especially when the prosthesis interacts with bone and is subjected to important mechanical loads.Infections, periprosthetic bone fracture, device breakage, and implant loosening are among the most common complications related to prostheses [4][5][6] .Infections are reported in 73% of the cases, while peri-prosthetic bone fracture in 60%, device breakage in 53%, and implant loosening in 60% 3 .These problems are produced by many causes, but most importantly, a poor integration between bone and the metal surface seems to be the main source of difficulties 4,7,8 .Thus, it is imperative to enhance prostheses osseointegration to reduce post-operative complications, and thereby, improve overall people´s quality of life.
Certain parameters must be considered to tackle this problem.Topography, chemistry, and wettability of the prosthesis surface play an important role in the bone-metal bond 9 .Surfaces can be modified and adapted to improve osseointegration.Bioactive organic and inorganic coatings on prostheses have been widely investigated and represent a good alternative to enhance osseointegration 10,11 .One of those, that has gained considerable attention, is the use of biopolymers.Chitosan (CS) has been studied as a potential biopolymeric coating for application on metal prostheses, especially on titanium alloys 12 .This polysaccharide, obtained by chitin de-acetylation, has suitable properties for the application, including biocompatibility, biodegradability, antibacterial and wound healing activity, and non-significant toxicity.Additionally, CS-coated surfaces support cell adhesion, proliferation and differentiation at the bone tissueimplant interphase, which is fundamental in the process of osseointegration 13 .
Several techniques have been previously employed to deposit CS coatings on implants, including solvent casting, immersion coating (simple absorption), layer-by-layer deposition, electrochemical methods, and electrophoretic deposition [14][15][16][17][18] .Electrodeposition techniques have great advantages compared to other methods due to the higher deposition speed, shorter processing time, and the possibility of controlling film thickness.Furthermore, complex shapes can be uniformly coated by electrodeposition, as well as on selected specific areas of the substrate 19,20 .CS electrodeposition has been applied onto titanium surfaces (both as pure Ti and Ti-6Al-4V) in combination with different bioactive molecules, such as antibiotics, hydroxyapatite and other biopolymers [21][22][23] .
Results from these studies have shown stable coatings with important antibacterial activity, and the ability to support cell proliferation and differentiation.However, the assessment of different important electrodeposition parameters, such as time, *e-mail: jalvarezb@usfq.edu.ecvoltage and CS concentration, has not been carried out on these surfaces.The electrodeposition of CS on gold surfaces have demonstrated that these variables have significant implications in coating thickness and morphology 24,25 .Moreover, to our knowledge, there are no reports on the adhesion assessment of the electrodeposited CS to the metallic surface, nor has it been found any data on coating´s pore size, and, in turn, the relationship with electrodeposition process parameters.
Consequently, the aim of this work was to analyze the effects of chitosan concentration, time and voltage on the chitosan electrodeposition process onto Ti-6Al-4V surfaces, particularly coating´s morphology and adhesion behavior.Experimental data on deposition, coating thickness, microstructure, adhesion, and swelling tests are presented, including variations in dependence of the mentioned variables.

Chitosan preparation
Chitosan with a medium molecular weight (800 KDa) and 98% deacetylation degree was used (Sigma-Aldrich, Catalog Number 448877).Solutions of chitosan at 0.1, 0.5 and 1.0 wt/v% CS were prepared in 1 v/v% acetic acid, and the pH was corrected to 5.5.Subsequently, the solutions were filtered and stored at 4 °C until use.

Surface preparation
Cylindrical samples of Ti-6Al-4V with a diameter of 6.7 mm and a thickness of 2.5 mm were used for electrodeposition.The discs were gritblasted with aluminum oxide particles in a pressure blast cabinet (BNP Double 220) with a distance of 0.1 m between the nozzle and the sample surface, and air pressure of 0.3 MPa 26 .The Ra-roughness obtained was 3.13 µm for all samples.Prior to the CS electrodeposition, samples were cleaned with 70% ethanol in a BRANSON 1800 ultrasound device for 15 min.

Absorption coating
A group of reference samples were coated through adsorption by introducing them in a 1.0 wt/v% chitosan solution for 20, and 30 min at 25 °C.After coating, the samples were rinsed, frozen and lyophilized for further analyses.

Electrodeposition
Chitosan coating electrodeposition was performed at room temperature with an Agilent E3634A DC Power Supply with a digital multimeter by applying voltages of 1.5 and 3.0 V. Time periods and voltages are presented in Table 1.A copper disc (6.7 mm diameter and 5.0 mm thick) was used as the anodic electrode and the Ti-6Al-4V disc as the working electrode.
Three different solutions with chitosan concentrations of 0.1, 0.5, and 1.0 wt/v% at a pH of 5.5 were used.All samples were frozen and lyophilized prior to morphology analyses.
In Figure 1, a diagram of the electrolytic cell used in this study is represented.Ti-6Al-4V and Cu appear submerged in the chitosan solution as cathode and anode electrode, respectively.

Morphology and thickness analyses
Surface morphology was examined by scanning electron microscopy, SEM-JEOL JSM-IT300, at 5-20kV and 50 Pa.For thickness measurements, samples were embedded in an epoxy resin (EpoThin TM 2, Buehler), and cross-sections were produced by cutting the chitosan-coated Ti-6Al-4V with a diamond blade.The cutting was performed transversally to the coating, at different time intervals, at a cutting speed of 500 rpm and feed rate of 0.15 mm/min to not affect the deposited chitosan layer.Energy-dispersive X-ray spectroscopy (EDS) was performed on sample cross-sections to analyze CS presence and measure coating thickness.Pore size and coating thickness were determined with SEM images by  processing them in the software ImageJ.Three samples were analyzed for each condition, and the mean size ± standard deviation and the pore size distribution were obtained.

FTIR Analysis
Native and electrodeposited chitosan samples were analyzed in a Fourier transformed infrared spectrometer; model Cary 630 FTIR-ATR (Agilent Technologies), in the region between 500 and 4000 cm -1 .

Scratch test
The adhesion of the different coatings was studied through the scratch test.The test was performed using a Rockwell AJ-227 Scratch Tester, with a 200-μm radius diamond tip.A normal force was applied on the surface at a constant rate of increase as the indenter moved 3.5 μm distance with a velocity of 10 mm/min.The applied load was increased progressively from 0 to 35 N. Three scratches, separated by at least 1 mm, were created on each type of sample, in at least two specimens.Normal and tangential forces were measured through each scratching path.With these forces, the coefficient of friction of each coating was calculated to assess the adhesion to the substrate.Additionally, a substrate without chitosan coating was used as a blank.

Swelling test
The ability of the chitosan coatings to absorb water was evaluated with the swelling test as a way of estimating coating stability under physiological conditions.Three samples for each condition were initially weighed and later immersed in 5 ml of phosphate buffered saline (PBS, pH 7.4) at 37 °C.The weight of each test sample was measured at different times: 0, 0.25, 0.5, 1, 2, 3, and 24 hours.Swelling degree was calculated through Equation 1.
( ) Where, % SD corresponds to the swelling degree, and and to the weight of the tested samples before and after immersing into PBS, respectively.Swelling degree was reported as the (average ± standard deviation) and plotted against time.Significant differences were determined through ANOVA and Tukey pair-wise comparison, with a 95% confidence level.

Electrodeposition of chitosan on Ti-6Al-4V
CS is a stimuli-responsive and film-forming biopolymer, and, because of this, it has been used for metallic implant coatings to improve osseointegration 27 .Electrodeposition, on the other hand, is one of the methods used to deposit chitosan on metal surfaces; however, generally, unlike in the present study, CS is electrodeposited with co-factors, such as other polymers, nanostructures, biomolecules, or dyes, among others [28][29][30][31] .The mechanism of CS electrodeposition is the dehydrogenation (or deprotonation) of the backbone that allows the formation of a film 32 .
By applying a current density near to the cathode electrode, redox reactions take place.The electrons of the cathode react with the CS in solution and produce hydroxide ions that increase the pH.The primary amines of the chitosan are deprotonated, forming an insoluble hydrogel film on the substrate 33,34 .It is well known that CS forms an insoluble film on Ti-6Al-4V under high pH values over 5-6, while under low pH values, chitosan is soluble and remains in solution 32,35 .Thus, in the present study, the effect of different electrodeposition conditions, particularly voltage, chitosan concentration, and time, was assessed to use this technique in future applications of implant coatings.
In order to assess possible changes in CS molecular structure due to electrodeposition, an FTIR analysis was carried out on native chitosan and 1.0% CS-30 min coating, and the spectra are shown in Figure 2. Characteristic CS peaks were observed around 1580 cm -1 due to -NH 2 stretching, while C=O vibrations were present at 1140 cm -1 .Peaks at 1020 and 1140 cm -1 are attributed to symmetric and asymmetric C-O-C vibrations 36 .These peaks were maintained in both samples, implying that electrodeposition did not significantly alter the CS backbone molecular structure.A similar behavior was observed on coatings at different CS concentrations and electrodeposition times (data not shown).

Surface morphology
The SEM images in Figure 3 indicate the morphologies obtained by 0.1, 0.5, and 1.0%CS electrodeposition samples with 1.5 V at 2.5 min.It can be seen that the higher concentration of CS in the electrodeposition solution, the greater amount of CS particles on the surface.Figure 3d shows the fine CS particles that cover the entire surface with 1.0% CS electrodeposition.On the contrary, only a few particles spread on the electrodeposited sample with 0.1 and 0.5%CS (arrows) (Figure 3b and c).
When the voltage is increased to 3 V, important changes can be observed.Figure 4 shows the different morphologies obtained after 20 and 30 min of electrodeposition with three different CS concentrations.Compared to the blank sample (Figure 3a), it is easy to identify the chitosan deposit on the surfaces.There are fine CS particles forming when using chitosan concentrations of 0.1% and 0.5% in 20 min (arrows, Figures 4a and c).A thin, porous CS layer is built up with 1.0% CS at 20 min (Figure 4e).When the electrodeposition time is raised to 30 min for 0.1% to 0.5%  4b and d), there is an increase and accumulation of CS particles, and a non-uniform distribution.In contrast, increasing the electrodeposition time to 30 min, the 1.0% CS solution forms a thick, porous layer, uniformly distributed along the substrate surface (Figure 4f).This behavior can be explained by the fact that a low concentration of CS can reduce the amount of polymer chains near the substrate, and thereby only a few amines can be deprotonated to form a stable insoluble layer 35 .Thus, the formation of continuous layers of chitosan can take place at higher CS concentrations and longer times.

CS (Figures
In contrast to the electrodeposited samples, no visible changes are detected with the 1.0%CS adsorption samples (Figure 5).While electrodeposited samples were coated (Figures 4c through 4f), adsorption samples were not.Apparently, the contact only with the chitosan solution does not generate any interaction with the surface (Figure 5).
According to Liu et al. 32 , Ra et al. 33 , Fernandes et al. 34 , and Benea et al. 35 , CS electrodeposition occurs due to a deprotonation of amine groups caused by the application of a certain current density.Nevertheless, when no current is applied (adsorption), this deprotonation does not take place, and the possibilities for CS deposition are significantly hindered.
As seen in Figures 4e and 4f, the 1.0%CS coating shows a highly porous morphology with a pore size of about 118 µm and 80 µm for 20 and 30 min electrodeposition, respectively (Figure 6).The pores are similar to the typical structure of pure CS hydrogel scaffolds, formed by charge interactions 37 .Due to the fact that electrodeposition occurs through amin deprotonation and loss of solubility close to the surface, it is possible that at greater electrodeposition times, a higher number of molecules undergo these changes, thereby creating a denser structure with a consequent smaller pore size.Porosity is essential in prostheses coatings to facilitate cell migration and proliferation 38,39 .Furthermore, pore size and structure would allow cell proliferation and a favorable arrangement of cells which is particularly convenient for biological applications 37,38 .This porosity would also play an important role in the encapsulation and controlled release of drugs of interest, such as antibiotics and growth and differentiation factors 40,41 .The 1.0% CS layer electrodeposited during 30 min shows an apparent high porosity (Figure 4f) compared to the layer electrodeposited during 20 min (Figure 4e).However, diameter values tended to be smaller for deposition time of 30 min of electrodeposition than 20 min.At higher CS concentrations, the viscosity of the solution increases, and thereby, the pore size decreases 42 .Additionally, in both times, the CS coating had a broad size distribution (Figure 6).In general, the results suggest that the number of pores and size are time and CS concentration dependent.
Coating thickness was measured on the cross-sections of 1.0%CS samples at 20 and 30 min exposures.Figure 7 shows the cross-sections and the EDS analysis of CS coatings.It is observed that the electrodeposited coating with 20-min exposure has a lower thickness than the coating with 30 min exposure.Measured thicknesses are in the range of 14.12 to 20.94 µm for the coatings with 20 min and between 36.09 to 70.72 µm for the coatings with 30 min.EDS maps show a high level of oxygen in the CS coatings (yellow color).The presence of oxygen is due to the fact that chitosan is composed of β-1 → linked 2-acetamido-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-B-D-glucopyranose residues 43 which have a large amount of oxygen in their chains.

Assessment of coating adhesion
Using scratch testing, the effects of different electrodeposition conditions on coating adhesion to Ti-6Al-4V surfaces can be studied.Figure 8 shows the friction coefficient versus normal force applied on coated samples of different concentrations compared to the blank samples.The coefficient of friction (COF) for the blank samples reached an average value of 0.45, while the coated samples of 1.0% CS-30 min and 1.0% CS-20 min that produced a continuous layer showed COF    lower than 0.3 at low loads.Only these two conditions are presented, provided that a more homogeneous coating was observed under SEM.
Two scenarios were identified.In the case of the 1.0% CS-30 min, the layer was present along the whole track of the scratch test, decreasing the friction.In the case of the 1.0% CS-20 min, the layer was ploughed at about 17 N of load, exposing areas of Ti-6Al-4V to the indenter.In these samples, the COF increases up to about 0.4, which is characteristic of the blank samples.
It is worth noting that, under the conditions of the test, the indenter is producing plastic deformation over the Ti-6Al-4V surface, as predicted by Hertzian theory.This highlights the fact that the CS coatings demonstrated good adhesion for both samples: • 1.0% CS-20 min sample failed at about 17 N, which denotes a failure at mean pressures beyond several GPa (GN/Indenter Contact Area) at the point of contact.• 1.0% CS-30 min sample, at most scratches, did not fail by ploughing, which highlights that the coating was always present in between the indenter and the substrate.
The lower COF of coated samples compared to the blanks is an evidence that the stress applied could not induce its detachment but only a progressive plastic deformation of the substrate 44 .

Coating stability
Swelling tests were performed at 37 °C, in PBS media, as a way to assess the hydrogel coating stability in physiological conditions.This methodology is commonly used to assess hydrogel stability 45 .Furthermore, weight loss through swelling tests have been previously used to evaluate the stability of chitosan-vancomycin electrodeposited coatings 46 .Figure 9 shows the swelling kinetics only for 1.0% CS coatings that formed a continuous hydrogel layer on the Ti-6Al-4V surface.Both coatings (formed after 20 min and 30 min) presented initially significantly high swelling degrees, but immediately they experienced a decrease in their swelling capacity.The 1.0% CS-30 min coating exhibited a slight swelling again after 3 h of testing.Although CS has available free hydroxyl and amino groups that could form strong hydrogen bonds 31,47 , the deposited coatings do not retain moisture and degrade rapidly.This is probably due to CS intermolecular interactions, and a crosslinking agent, such as glutaraldehyde or genipin, would be needed to create a more stable structure 48 .Another possibility is that the CS structure needs to be modified for an auto-crosslinking mechanism 45 .Without a more robust crosslinking, as observed, the coating easily degrades in the presence of electrolytes in aqueous media (PBS); the low swelling degree values also corroborate this fact.In both cases, swelling equilibrium is reached after 3h.The coatings prepared after 30 min of electrodeposition showed higher swelling than the 20 min coatings, according to the statistical analysis.
The overall assessment of process parameters of electrodeposition presented in this study indicates that the higher the CS concentration, higher voltage, and longer periods of time in electrodeposition assure a continuous thicker layer and full coverage of the Ti-6Al-4V.The longer the time of exposure increases the thickness and determines smaller micron-size pores on the CS surface.The pore-structure is of interest for cell proliferation and enhanced osseointegration.Further studies are planned for in-vitro cell-assays and evaluation of biocompatibility of the surfaces.Scratch results highlight a significantly high adhesion between the CS and the substrate.Although this assessment as presented is only qualitative, the technique proves to be sensitive to denote the continuous presence of the coating between the indenter and the substrate at high contact stresses.The swelling tests denote a rapid swelling of the electrodeposited CS.Further research will be carried out to improve this response.The CS by electrodeposition is a promising technique to deposit a highly biocompatible layer.

Conclusions
Chitosan coatings were successfully electrodeposited on Ti-6Al-4V substrates.CS deposition on Ti-6Al-4V is affected by polymer concentration, electrodeposition time, and voltage.Low concentrations of CS limit deprotonation and thereby the formation of a continuous insoluble film.Moreover, by examining the CS coating surface, it was determined that the homogeneity, thickness, and pore size are influenced by electrodeposition time.Likewise, the adhesion behavior is affected by the concentration and electrodeposition time.In the window of testing of this study, it was found that a coating with full coverage of the surface, with micron-size open porosity, and good adhesion is attained using higher voltage, higher CS concentration, and longer periods of electrodeposition time.Therefore, electrodeposition of CS coatings is feasible if these parameters are correctly controlled.Nevertheless, as evidenced by the swelling tests, these coatings lack stability in physiological conditions without using a proper crosslinking agent.Future studies to include CS crosslinking, and in vitro cell adhesion, proliferation, and differentiation tests will allow a complete understanding of the generated coatings.

Figure 1 .
Figure 1.Electrodeposition process: a) Chitosan electrodeposition on Ti-6Al-4V surface, b) Coated layer of chitosan on grit blasted surface of Ti-6Al-4V and pH gradient on the cathode.

Figure 6 .
Figure 6.Pore diameter distribution of the electrodeposited chitosan coatings with 1.0% CS at 20 min and 30 min.

Figure 8 .
Figure 8. Scratch test results: a) Scratch tracks; b) Coefficient of friction as a function of the applied load for blank samples, 1%CS-30 min, and 1%CS-20 min samples.